i

HYDROGEL FROM LINUM USITATISSIMUM L.: ISOLATION,

MODIFICATION, CHARACTERIZATION AND

PHARMACEUTICAL APPLICATIONS

A DISSERTATION SUBMITTED

TO

THE UNIVERSITY OF SARGODHA, SARGODHA

IN PARTIAL FULFILLMENT OF THE REQUIREMENT

FOR

THE DEGREE OF DOCTOR OF PHILOSOPHY

IN

PHARMACEUTICS

BY

MUHAMMAD TAHIR HASEEB

COLLEGE OF PHARMACY

FACULTY OF PHARMACY

UNIVERSITY OF SARGODHA, SARGODHA

Session 2010-2015

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DEDICATED TO

MY PARENTS

iii

APPROVAL CERTIFICATE

It is solemnly described that the dissertation titled ―Hydrogel from Linum usitatissimum L.:

Isolation, modification, characterization and pharmaceutical applications‖ submitted by

Muhammad Tahir Haseeb in the partial fulfillment of the requirement for the award of

degree of DOCTOR OF PHILOSOPHY in Pharmaceutics is hereby approved.

Supervisor 1: __________________ Supervisor 2: _______________

Prof. Dr. Sajid Bashir Dr. Muhammad Ajaz Hussain

Dean Associate Professor

College of Pharmacy Department of Chemistry

Faculty of Pharmacy University of Sargodha, Sargodha

University of Sargodha, Sargodha

External examiner: __________

Dean: _____________________

Prof. Dr. Sajid Bashir

College of Pharmacy

Faculty of Pharmacy

University of Sargodha, Sargodha

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DECLARATION

I declare that the work described in this thesis was carried out by me under the supervision of

Prof. Dr. Sajid Bashir, Dean Faculty of Pharmacy and Dr. Muhammad Ajaz Hussain,

Associate Professor, Department of Chemistry, University of Sargodha, Sargodha, Pakistan,

in partial fulfillment of the requirement for the degree of ―Doctor of Philosophy in

Pharmaceutics‖. I certify that the main content of this thesis accounts for my own research

and has not previously been submitted for a degree at any educational institution. Further, it

is submitted that the material taken from other sources has been properly acknowledged.

MUHAMMAD TAHIR HASEEB

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ACKNOWLEDGEMENTS

All praises are for Almighty Allah, the most kind, magnificent and merciful who gave me

strength, power, knowledge and above all good health to complete this work amicably.

My deepest gratitude goes to my supervisors Prof. Dr. Sajid Bashir, Dean, Faculty of

Pharmacy and Dr. Muhammad Ajaz Hussain, Associate Professor Department of Chemistry

for their continuous support, constant encouragement and invaluable guidance throughout the

course of my research work. Particularly, rigorous, meticulous, serious and responsible

academic attitude of Dr. Muhammad Ajaz Hussain who has always been my role model. I

am also thankful to the Higher Education Commission (HEC) of Pakistan for granting

scholarship under ―Indigenous 5000 PhD Fellowship‖ and ―IRSIP‖ programs.

I would like to acknowledge Prof. Dr. Soon Hong Yuk, Dean, College of Pharmacy, Korea

University, Sejong, Republic of Korea for his cooperation and support in conducting the

research work in his laboratory under IRSIP funded by HEC for six months. I am also

thankful to him for providing research facilities and, analyses and biological and

cytotoxicity studies of some research samples. I also want to pay my gratitude to Dr. Eun

Hee Lee, Associate Prof., College of Pharmacy, Korea University, Sejong, Republic of

Korea for helpful discussion. I am grateful to Mr. Nisar Ul Khaliq, Mr. Ameeq Ul Mushtaq

and Mr. Bishal Adhikari for their cooperation and valuable scientific discussion.

I am deeply indebted to Prof. Dr. Wolfgung Tremel and Dr. Muhammad Nawaz Tahir,

Institute of Inorganic and Analytical Chemistry, Johannes Guttenberg University, Mainz,

Germany for taking TEM analyses.

I am very thankful to Dr. Muhammad Sher, Deputy Manager, Instruments Lab., University

of Sargodha, Pakistan for providing necessary facilities for the characterization of research

vi

samples and fruitful discussion. I also acknowledge the advice of all of my teachers in

accomplishing this research work.

I sincerely extend my gratitude to my PhD fellows: Ms Alia Erum, Mr. Muhammad Umer

Ashraf, Mr. Muhammad Amin, Mr. Khawar Abbas, Mr. Azhar Abbas, Mr. Gulzar

Muhammad and Mr. Muhammad Nauman for their cooperations and discussions.

Appreciation is due to Trison Research Laboratories (Pvt.) Ltd., Sargodha, Pakistan for

providing the facility of pharmaceutical equipment for preparation of tablet formulations.

I would also like to thank staff of all laboratories including Mr. Farhan Khan, Mr. Atta-ur-

Rehman, Mr. Laeeq, Mr. Amir Latif, Mr. Raheem, Mr. Ashfaq, Mr. Waqas, Mr. Liaqat, Mr.

Nasir, Mr. Sohail Armaghan, Mr. Muhammad Umair and Mr. Naveed for helping me in

research experimentation.

I would also like to extend my heartiest gratitude to my beloved wife Mrs. Fatima Akbar

Sheikh for her untiring efforts and moral support during my PhD studies. Furthermore, I

would also like to acknowledge the cherishing company and inspiring smiles of my beloved

son Muhammad Moeez Tahir and my sweet daughter Amna Tahir which enabled me to

overcome the bad moments during the research.

Last but not the least, special thanks to my parents who are the torch bearer for the whole of

my life, who grew me up in a way that I am able to stand before the world and face every

person and situation with a humble confidence.

MUHAMMAD TAHIR HASEEB

vii

ABBREVIATIONS

∆G Gibbs free energy

∆H Enthalpy

∆S Change in entropy

AFM Atomic force microscopy

Ag NPs Silver nanoparticles

ALSH Acetylated linseed hydrogel

ALT Alanine aminotransferase

ANOVA Analysis of variance

AST Aspartate aminotransferase

ATCC American type culture collection

AUC Area under curve

BS Bletilla striata

CDCl3 Deuterated chloroform

CMC Carboxymethyl cellulose

COX Cyclooxygenase

DAPI 4ʹ,6-diamidino-2-phenylindole

DLP-NPs DTX-loaded LSH Pluronic F-68 nanoparticles

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DLS Dynamic light scattering

DMAc Dimethylacetamide

DMAP 4-dimethylaminopyridine

DMF Dimethylformamide

DMSO Dimethyl sulfoxide

DMSO-d6 Dimethyl sulfoxide-deuterated

DS Diclofenac sodium

DSb Degree of substitution

DSC Differential scanning calorimetry

DTX Docetaxel

Ea Activation energy

EPR Enhanced permeability and retention

FE-SEM Field emission scanning electron microscopy

FTIR Fourier transform infrared

FWO Flynn-wall and Ozawa

GCMS Gas chromatography mass spectrometry

GIT Gastrointestinal tract

GLP Good laboratory practice

ix

GPC Gel permeation chromatography

HDL High density lipoprotein

HEC Hydroxyethyl cellulose

HMQC Heteronuclear multiple-quantum correlation

HPC Hydroxypropyl cellulose

HPCMC Hydroxypropyl carboxymethyl cellulose

HPMC Hydroxypropylmethyl cellulose

HSQC Heteronuclear single-quantum correlation

IPDT Integral procedural decomposition temperature

IPN Interpenetrating polymer network

ITS Index of thermal stability

KDa Kilodalton

LDL Low density lipoprotein

LiCl Lithium chloride

LSH Linseed hydrogel

MALLS Multi angle laser light scattering

MBA N,Nʹ-methylene bisacrylamide

MCF-7 Michigan cancer foundation-7

x

MCH Mean corpuscular hemoglobin

MCHC Mean corpuscular hemoglobin concentration

MCV Mean corpuscular volume

MMTS Maximum mean total score

MSC Model selection criterion

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

NMR Nuclear magnetic resonance

NPs Nanoparticles

NSAIDs Non-steroidal anti-inflammatory drugs

OECD Organization for Economic Co-operation and Development

PDII Primary dermal irritation index

PEG Polyethylene glycol

PG Phellinus gilvus

pHEMA Poly(2-hydroxyethyl methacrylate)

PLG Polylactic-co-glycolic acid

PVA Polyvinyl alcohol

PXRD Powder X-ray diffraction

RP-HPLC Reverse-phase high performance liquid chromatography

xi

SEC Size exclusion chromatography

SEM Scanning electron microscopy

SGF Simulated gastric fluid

SIF Simulated intestinal fluid

SIPN Semi-interpenetrating polymer network

TBA Thiobarbituric acid

TBAF Tetrabutylammonium fluoride

TEM Transmission electron microscopy

Tg Transition temperature

TGA Thermogravimetric analysis

TOCSY Total correlation spectroscopy

USFDA United States Food and Drug Administration

USP United States Pharmacopeia

UV Ultra violet

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LIST OF FIGURES

Fig. 1.1. Structure of diclofenac sodium.

Fig. 1.2. Structure of caffeine.

Fig. 1.3. Structure of diacerein.

Fig. 1.4. Structure of docetaxel.

Fig. 3.1. FTIR spectrum of LSH.

Fig. 3.2. 1H NMR (600 MHz, ppm, 40 °C) spectrum of LSH in DMSO-d6 showing

repeating unit between 3.11-5.61.

Fig. 3.3. PXRD spectrum of LSH.

Fig. 3.4. Reaction scheme for the synthesis of acetylated LSH.

Fig. 3.5. FTIR spectra of LSH, ALSH 1, ALSH 2 and ALSH 3.

Fig. 3.6. 1H NMR (600 MHz, ppm, DMSO-d6, 40 °C) spectrum of ALSH 3 (DSb 2.91).

Fig. 3.7. 1H

1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91).

Fig. 3.8. 1H

1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91)

showing correlation of sugar region.

Fig. 3.9. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3.

Fig. 3.10. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing

correlation of acetyl methyl region.

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Fig. 3.11. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing

correlation of sugar region.

Fig. 3.12. Overlay of TG and DTG curves of LSH (a, c) and ALSH (b, d), respectively

recorded at multiple heating rates.

Fig. 3.13. Overlay of 2DTG curves of LSH (a) and ALSH 3 (b) recorded at multiple

heating rates.

Fig. 3.14. Overlay of TG (a) and DTG (b) curves of LSH and ALSH 3 recorded at 10 °C

min-1

showing stability imparted to ALSH 3.

Fig. 3.15. α vs. T graph of thermal degradation of first (a) and second (b) step of LSH at

multiple heating rates and Flynn-Wall-Ozawa (FWO) plot between log β and

1000/T (K-1

) for calculation of Ea of first degradation (c) and second degradation

(d) step at several degree of conversion for LSH.

Fig. 3.16. α vs. T graph of thermal degradation of ALSH 3 at multiple heating rates (a) and

Flynn-Wall-Ozawa (FWO) plot between log β and 1000/T (K-1

) for calculation

of Ea at several degree of conversion for ALSH 3.

Fig. 3.17. Swelling capacity (a) and second order swelling kinetics (b) of LSH in buffer of

pH 1.2, pH 6.8 and 7.4 and in deionized water (D.W).

Fig. 3.18. Swelling capacity of LSH in deionized water at different temperatures.

Fig. 3.19. Swelling capacity of LSH in different conc. of NaCl and KCl (a) and swelling-

shrinking (on-off switching) behavior of LSH; at pH 7.4 (basic) and pH 1.2

(acidic) (b), in deionized water and normal saline (0.9% NaCl solution) (c) and

in deionized water and ethanol (d), respectively.

xiv

Fig. 3.20. SEM images of lyophilized sample of LSH showing porous and elongated

structure.

Fig. 3.21. FTIR spectra of LSH, PVP and diacerein alone, physical mixture of LSH with

diacerein and physical mixture of LSH, diacerein and PVP.

Fig. 3.22. FTIR spectra of LSH, PVP and caffeine alone, physical mixture of LSH with

caffeine and physical mixture of LSH, caffeine and PVP.

Fig. 3.23. FTIR spectra of LSH, PVP and diclofenac sodiume alone, physical mixture of

LSH with diclofenac sodiume and physical mixture of LSH, diclofenac sodiume

and PVP.

Fig. 3.24. Swelling capacity (a) and swelling kinetics (b) of LSH tablet (FH) at different

pH and in deionized water and swelling photographs (radial and axial view) of

FH formulation at pH 6.8 (c).

Fig. 3.25. Swelling capacity of FH, FC1, FC2, and FC3 at pH 1.2 (a), 6.8 (b), 7.4 (c), and

DI water (d) and swelling photographs (radial and axial view) of FC3

formulation at pH 6.8 (e).

Fig. 3.26. Swelling kinetics of LSH tablet (FH) and LSH-caffeine tablets (FC1, FC2 and

FC3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).

Fig. 3.27. Swelling capacity of FH, FD1, FD2, and FD3 at pH 1.2 (a), 6.8 (b), 7.4 (c) and

deionized water (d) and swelling behavior of FD3 formulation at pH 6.8

expressed in photographs (radial and axial view) (e).

Fig. 3.28. Swelling kinetics of LSH tablet (FH) and LSH-diacerein tablets (FD1, FD2 and

FD3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).

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Fig. 3.29. SEM images of broken surface of FH tablet (a), broken surface of FD3 tablet (b)

and cross section of swollen then freeze dried tablet formulation FD3.

Fig. 3.30. Equilibrium swelling of LSH tablet (FH), LSH-caffeine tablet (FC3) and LSH-

diacerein tablet (FD3) in different molar concentrations of salt solutions; NaCl

(a) and KCl (b).

Fig. 3.31. Stimuli responsive swelling and deswelling behavior of LSH tablet (FH), LSH-

caffeine tablet (FC3) and LSH-diacerein tablet (FD3) at basic (pH 7.4) and

acidic (pH 1.2) environment (a), in deionized water and normal saline (b) and

deionized water and ethanol (c), respectively.

Fig. 3.32. Swelling capacity of LSH based DS tablets in water (a), drug (DS) release study

from LSH matrix tablets in SGF and SIF (b) and photographs showing swelling

response (aerial and axial view) of D3 formulation in water (c).

Fig. 3.33. Caffeine release from LSH-caffeine tablet in different media; pH 6.8 (a), pH 7.4

(b), deionized water (c) and physiological pH and transit time of gastrointestinal

tract (d).

Fig. 3.34. Diacerein release from LSH-diacerein tablet in different media; pH 6.8 (a), pH

7.4 (b), deionized water (c) and physiological pH and transit time of

gastrointestinal tract (d).

Fig. 3.35. Size distribution of DLP-NPs with different drug loadings: 1% (a), 2% (b), 3%

(c); FESEM image of DLP-NPs (formulation with 1% DTX loading) (d); size

distribution of LSP (1 wt% aqueous solution) (e); size distribution calculated

from FESEM (f) and Zeta potential of 1% DLP-NPs (g).

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Fig. 3.36. PXRD (a) and FTIR (b) spectra of LSH, DTX, Pluronic F-68 and three

formualtions of DLP-NPs.

Fig. 3.37. Docetaxel release from different formulations of DLP-NPs.

Fig. 3.38. In vitro cytotoxicity of LSH, DTX and DLP-NPs at various concentrations.

Statistical significance is shown by * p < 0.05, performed by student‘s t-test for

comparison.

Fig. 3.39. Cellular uptake images of DLP-NPs (20x, a) and (40x, b).

Fig. 3.40. Schematic illustration showing synthesis of LSH mediated Ag NPs.

Fig. 3.41. Photographs of LSH-Ag+ mixture (20 mmol AgNO3) showing color change

with passage of time.

Fig. 3.42. UV/Vis spectra of LSH mediated Ag NPs: 10 mmol (a), 20 mmol (b) and 30

mmol solution of AgNO3 (c) at different reaction times and cumulative

graphical representation (d) showing increase in absorption of Ag NPs solutions

with increase in concentration and reaction time.

Fig. 3.43. TEM images of Ag NPs isolated from 10, 20 and 30 mmol LSH-Ag+

solution having size range from 10-25 nm (a), 10-30 nm (b) and 10-35 nm (c),

respectively.

Fig. 3.44. PXRD spectra: LSH (a), Ag NPs embedded LSH film (b) and isolated Ag NPs,

10 mmol (c), 20 mmol (d) and 30 mmol (e).

Fig. 3.45. UV/Vis spectra of Ag NPs synthesized from aqueous solution of AgNO3 (20

mmol) and LSH recorded after 10 h, 01, 15, 30 days and 06 months storage (a);

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PXRD spectrum of Ag NPs taken after 06 month storage of LSH-Ag NPs film

(b); Aqueous solution prepared from stored LSH-Ag NPs film (c); see through

and foldable LSH-Ag NPs film (d); TEM image of Ag NPs (10-30 nm) isolated

from LSH-Ag NPs film stored for 06 months under dark (e).

Fig. 3.46. Antimicrobial activity (a) and graphical representation of zone of inhibition of

Ag NPs (20 mmol) against different bacterial and fungal strains (b).

Fig. 3.47. Schematic illustrations of wound treatment with Ag NPs embedded LSH wound

dressing patch (a) and also showing its main parts (b).

Fig. 3.48. Collagen contents of epithelialized wound tissue of various groups after 15th

day. Statistical significance from control group is expressed by * p < 0.05.

xviii

LIST OF TABLES

Table 2.1. Composition of different formulations to evaluate the sustained release

behavior of diclofenac sodium from LSH tablet.

Table 2.2. Tablet formulation design to evaluate the sustained release behavior of

caffeine.

Table 2.3. Constituents of various tablet formulations to study sustained release behavior

of diacerein.

Table 2.4. Group scheme for acute oral toxicity study of LSH in mice.

Table 3.1. Reaction parameters and results of the synthesis of acetylated LSH.

Table 3.2. Mean thermal decomposition temperatures, weight loss % and char yield % of

LSH at multiple heating rates.

Table 3.3. Mean thermal decomposition temperatures, weight loss % and char yield % of

ALSH 3 at various heating rates.

Table 3.4. Thermal kinetics and thermodynamic parameters of LSH.

Table 3.5. Thermal kinetics and thermodynamic parameters of ALSH 3.

Table 3.6. Physical properties of LSH.

Table 3.7. Pre-compression parameters of diclofenac sodium formulations (Mean ± SD).

Table 3.8. Pre-compression parameters of caffeine formulations (Mean ± SD).

Table 3.9. Pre-compression parameters of diacerein formulations (Mean ± SD).

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Table 3.10. Post-compression parameters of DS containing tablets (Mean ± SD).

Table 3.11. Post-compression parameters of caffeine containing tablets (Mean ± SD).

Table 3.12. Post-compression parameters of prepared tablets containing diacerein (Mean ±

SD).

Table 3.13. Mathematical data of power law.

Table 3.14. Values of drug release kinetics models for LSH-caffeine formulations at pH

6.8, 7.4 and deionized water.

Table 3.15. Values of drug release kinetics models for LSH-diacerein formulations at pH

6.8, 7.4 and deionized water.

Table 3.16. Drug loading and encapsulation efficiency of different formulations.

Table 3.17. Wound area (mm2) and wound closure (%) after selected day intervals.

Table 3.18. Scores for grading the primary eye irritation study of LSH.

Table 3.19. Clinical observations of acute oral toxicity and dermal testing of LSH.

Table 3.20. Biochemical blood analysis of control and LSH treated mice.

Table 3.21. Liver, kidney and lipid profile of mice treated with LSH.

Table 3.22. Absolute mean organ weight (g) of mice after oral administration of LSH.

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CONTENTS

ABSTRACT 1

1. INTRODUCTION 4

1.1. Polysaccharides as a biomaterial 4

1.1.1. Hydrogels 7

1.1.2. Linseed 10

1.1.3. Modification of polysaccharides 18

1.1.4. Stimuli responsive properties of polysaccharidal hydrogels 21

1.1.5. Toxicological studies of polysaccharides 25

1.2. Polysaccharides based drug delivery systems and pharmaceutical applications 27

1.2.1. Sustained release of NSAIDs from polysaccharidal materials 27

1.2.2. Polysaccharides based NPs as anticancer drug delivery system 34

1.2.3. Polysaccharides mediated synthesis and application of Ag NPs 37

1.2.4. Polysaccharides based antiseptic dressing 43

1.3. Characterization techniques 47

1.3.1. Fourier transform infrared spectroscopy 47

1.3.2. Nuclear magnetic resonance spectroscopy 47

1.3.3. Thermal analysis 48

1.3.4. Electron microscopic analysis 48

1.3.5. Powder X-ray diffraction 49

1.3.6. MTT assay 49

1.3.7. Drug release models 49

1.3.7.1. Zero order 49

1.3.7.2. First order 50

1.3.7.3. Higuchi model 51

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1.3.7.4. Hixson-Crowell model 51

1.3.7.5. Korsmeyer-Peppas model 52

1.4. Background and significance of the study 53

1.5. Aims and objectives 54

2. MATERIALS AND METHODS 56

2.1. Materials 56

2.2. Measurements 57

2.2.1. Fourier transform infrared spectroscopy 57

2.2.2. 1H NMR spectroscopy 57

2.2.3. Heteronuclear single quantum correlation spectroscopy 57

2.2.4. 1H

1H TOCSY NMR spectroscopy 57

2.2.5. UV-Vis spectrophotometry 58

2.2.6. Thermogravimetric analysis 58

2.2.7. Field emission scanning electron microscopy 58

2.2.8. Transmission electron microscopy 58

2.2.9. Powder X-ray diffraction 59

2.3. Acetylation of linseed hydrogel 59

2.3.1. Acetylation of LSH 59

2.3.2. Calculation of degree of substitution 61

2.3.3. Thermogravimetric analysis and degradation kinetics of LSH and

ALSH 61

2.4. Dynamic swelling and stimuli responsive on-off switching of LSH 62

2.4.1. Isolation of LSH 62

2.4.2. Physical properties of LSH 62

2.4.3. Preparation of buffer solutions of different pH 64

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2.4.4. Evaluation of pH responsive property of LSH 65

2.4.5. Swelling kinetics 66

2.4.6. Thermoresponsive swelling capacity of LSH in deionized water 66

2.4.7. Evaluation of salt solution-responsive properties of LSH 67

2.4.8. Evaluation of pH responsive on-off switching of LSH 67

2.4.9. Evaluation of saline responsive on-off switching of LSH 67

2.4.10. Evaluation of on-off switching of LSH in water and ethanol 68

2.5. Development of sustained drug delivery system 68

2.5.1. Formulation design 68

2.5.1.1. Drug excipient compatibility study 68

2.5.1.2. Preparation of tablets 69

2.5.1.3. Pre-compression evaluation 71

2.5.1.4. Post-compression evaluation 71

2.5.2. Dynamic swelling and stimuli responsive evaluation of LSH based

tablet formulations 73

2.5.2.1. pH responsive swelling of LSH containing tablets 73

2.5.2.2. Swelling kinetics 73

2.5.2.3. Evaluation of salt solution responsive swelling 74

2.5.2.4. Stimuli responsive swelling-deswelling (on-off) behavior 74

2.5.3. Evaluation of drug release behavior 74

2.5.3.1. In-vitro drug release studies 74

2.5.3.2. Drug release kinetics 76

2.5.3.3. Drug release mechanism 77

2.5.4. Scanning electron microscopy analysis 78

2.6. Docetaxel loaded LSH-Pluronic NPs 79

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2.6.1. Preparation of NPs 79

2.6.2. Encapsulation efficiency and drug loading 79

2.6.3. Particle size and morphology 80

2.6.4. X-ray diffraction analysis 80

2.6.5. In vitro drug release study 81

2.6.6. Cytotoxicity and cellular uptake behaviour 81

2.6.7. Statistical analysis 82

2.7. Nanobiotechnological application of LSH mediated Ag NPs 83

2.7.1. Preparation of AgNO3 solution and LSH suspension 83

2.7.2. Green synthesis of Ag NPs 83

2.7.3. Film formation 83

2.7.4. UV spectrophotometric analysis 83

2.7.5. Powder X-ray diffraction 84

2.7.6. Transmission electron microscopy 84

2.7.7. Antimicrobial activity 84

2.7.8. Wound healing studies 85

2.7.8.1. Design of wound dressing 85

2.7.8.2. Wound healing study design 85

2.7.8.3. Collagen estimation 86

2.8. Acute toxicological evaluation of LSH 87

2.8.1. Study design 87

2.8.2. Acute oral toxicity 88

2.8.3. Primary eye irritation 88

2.8.4. Acute dermal toxicity 89

2.8.5. Primary dermal irritation study 89

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2.8.6. Body weight gain study 90

2.8.7. Food and water consumption 90

2.8.8. Hematology and clinical biochemistry 90

2.8.9. Gross necropsy and histopathology 90

2.8.10. Statistical analysis 91

3. RESULTS AND DISCUSSION 92

3.1. Isolation and characterization of LSH 92

3.1.1. Isolation of LSH 92

3.1.2. FTIR spectroscopy 92

3.1.3. 1H NMR spectroscopy 93

3.1.4. PXRD 94

3.2. Synthesis and characterization of LSH-acetates 95

3.2.1. FTIR spectroscopy 96

3.2.2. 1H NMR spectroscopy 97

3.2.3. 1H

1H TOCSY spectroscopy 98

3.2.4. HSQC spectroscopy 98

3.2.5. Isoconversional thermal analysis of LSH and LSH-acetates 101

3.2.5.1. Thermal analysis 101

3.2.5.2. Degradation kinetics 104

3.2.5.3. Thermodynamic analysis 106

3.3. Dynamic swelling and stimuli responsive on-off switching of

superabsorbent LSH 107

3.3.1. Physical properties of LSH 107

3.3.2. Swelling capacity of LSH in deionized water and at different

physiological pH 108

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3.3.3. Swelling kinetics 108

3.3.4. Thermoresponsive swelling capacity of LSH 109

3.3.5. Saline responsive swelling of LSH 110

3.3.6. Responsive swelling-deswelling (on-off switching) behavior of LSH

at basic and acidic pH 110

3.3.7. Responsive swelling-deswelling (on-off switching) behavior of LSH

in deionized water and in NaCl solution (0.9%) 111

3.3.8. Responsive swelling-deswelling (on-off switching) behavior of LSH

in deionized water and ethanol 111

3.3.9. Field emission scanning electron microscopy 112

3.4. Evaluation of LSH as a novel controlled release and stimuli responsive

oral drug delivery system 113

3.4.1. Drug-excipients compatibility study 113

3.4.2. Pre-compression evaluation of tablet formulations 117

3.4.3. Post-compression evaluation of tablet formulations 118

3.4.4. Swelling response of LSH containing tablet formulations

at different pHs 120

3.4.4.1. Swelling response and swelling kinetics of LSH tablets 120

3.4.4.2. Swelling response and swelling kinetics of LSH-caffeine

tablets 121

3.4.4.3. Swelling response and swelling kinetics of LSH-diacerein

Tablets 122

3.4.5. Swelling morphology of LSH containing tablets 125

3.4.6. Morphological analysis of LSH containing tablets by SEM 127

3.4.7. Salt solution responsive swelling of LSH containing tablet

xxvi

formulations 128

3.4.8. Swelling-deswelling response of LSH tablet formulations against

external stimuli 129

3.4.8.1. Swelling-deswelling response in basic and acidic pH 129

3.4.8.2. Swelling-deswelling response in deionized water and normal

saline solution 130

3.4.8.3. Swelling-deswelling response in deionized water and ethanol 132

3.4.9. In-vitro drug release studies 132

3.4.9.1. DS release studies and release mechanism 132

3.4.9.2. Caffeine and diacerein release studies 134

3.4.9.3. Drug release kinetics and mechanism 138

3.5. Docetaxel loaded LSH-Pluronic NPs 142

3.5.1. Preparation and characterization of DLP-NPs 142

3.5.2. Particle size and morphological analysis 142

3.5.3. XRD and FTIR analysis of DLP-NPs 144

3.5.4. In vitro drug release study from DLP-NPs 145

3.5.5. Cytotoxicity and cellular uptake behaviour of DLP-NPs 146

3.6. Nanobiotechnological application of LSH 149

3.6.1. Green synthesis of Ag NPs 149

3.6.2. Characterization of Ag NPs 150

3.6.2.1. UV spectrophotometry 150

3.6.2.2. Transmission electron microscopy of isolated Ag NPs 152

3.6.2.3. Powder X-ray diffraction 153

3.6.2.4. Storage of Ag NPs in LSH thin film 154

3.6.2.5. Antimicrobial activity of Ag NPs 156

xxvii

3.6.2.6. Wound healing studies 157

3.7. Acute toxicological evaluation of LSH 160

3.7.1. Acute oral toxicity study in mice 160

3.7.2. Primary eye irritation 160

3.7.3. Acute dermal toxicity 161

3.7.4. Primary dermal irritation study 162

3.7.5. Body weight gain study 162

3.7.6. Food and water consumption 162

3.7.7. Haematology and clinical biochemistry 164

3.7.8. Gross necropsy and histopathology 164

CONCLUSIONS 166

REFERENCES 168

LIST OF PUBLICATIONS 211

1

ABSTRACT

Linseed hydrogel (LSH) was isolated from linseeds (Linum usitatissimum L.) using hot water

extraction method, characterized and used in various formulation designs. Characterization of

LSH was carried out using Fourier transform infrared (FTIR), powdered X-ray diffraction

(PXRD), nuclear magnetic resonance (NMR), scanning electron microscopy (SEM) and

thermogravimetric analysis (TGA). LSH was also modified by acetylation and structures

obtained were thoroughly characterized.

Stimuli responsive swelling of LSH was evaluated at gastrointestinal pHs (1.2, 6.8 and 7.4)

and in deionized water and also in different molar concentrations of NaCl and KCl solutions.

Swelling-deswelling (on-off) response of LSH against environmental conditions was also

observed. LSH has shown high swelling at pH 6.8, 7.4 and deionized water while negligible

swelling was seen at pH 1.2 indicating potential of LSH as intestine targeting drug delivery

system. Swelling behaviour of LSH at various pHs of gastrointestinal tract (GIT) has

followed the second order kinetics. Inverse relation between swelling of LSH and molar

concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 M) of NaCl and KCl were observed.

Moreover, the water swollen LSH when immersed in normal saline, shrinking was observed.

A more abrupt shrinking of water swollen LSH was observed on immersing in ethanol.

Similarly, swelling-deswelling response was also observed in buffer of pH 7.4 and 1.2,

respectively. These results have revealed that LSH is a smart material and can be used to

make intelligent drug delivery systems.

High swelling and water holding capability of LSH were used to develop the sustained

release formulation of diclofenac sodium. Drug release data from LSH tablets was compared

with commercially available product (Voltral®

) and found better results. It was observed that

the release of diclofenac sodium from LSH matrix tablets was dependent on the concentration

2

of LSH and followed the anomalous transport mechanism. Therefore, LSH can be used as a

release retarding agent in sustained release formulation.

LSH-caffeine and LSH-diacerein tablets were prepared to analyze the stimuli (pH, salt

solution and ethanol) responsive swelling and swelling-deswelling (pH 7.4/1.2, water/normal

saline and water/ethanol) behaviour of LSH when used in tablet formulation. Although,

stimuli responsive properties of LSH remain the same even after compression in tablet form

but less swelling capacity was observed after compression. This might be due to the packing

arrangements of LSH and also less exposed area to the swelling medium in tablet form as

compared to powder form. LSH appeared as a novel material for stimuli responsive and pH

dependent release of NSAIDs in gastrointestinal tract.

The elongated porous structure arranged in uniformly distributed layers were seen in FE-

SEM analysis of swollen then freeze dried powder sample of LSH. Similar pattern of porous

channels was also observed even in tablet formulations of LSH. High swelling and water

holding capability of LSH are due to these porous channels.

Docetaxel loaded LSH Pluronic F-68 nanoparticles (DLP-NPs) were synthesized by core

shell formation. Drug loaded core of LSH was protected and stabilized by Pluronic F-68. Size

and morphological analysis of DLP-NPs was performed by dynamic light scattering (DLS),

PXRD and TEM. Results indicated that DLP-NPs are spherical in shape having size range of

220-335 nm. In vitro drug release study has shown a prolong release pattern for more than 4

days. Cell viability study of LSH and DLP-NPs has proved even better results when

compared with free docetaxel. Cell uptake behaviour of DLP-NPs was monitored using Nile

red and high concentration of DLP-NPs was accumulated in the cytoplasmic region of the

cell. Therefore, DLP-NPs have shown a promising anticancer drug delivery system.

LSH was used as a reducing and capping agent for the green synthesis of Ag NPs. Aqueous

suspension of LSH were mixed with silver nitrate solution and exposed to sunlight.

3

Formation of Ag NPs was monitored by noting the colour of solution and through UV

spectrophotometer. UV absorptions were observed from 410-437 nm. TEM images revealed

the formation of spherical Ag NPs in the range of 10-35 nm. Face centered cubic array of Ag

NPs was confirmed by characteristic diffraction peaks in PXRD spectrum. Significant

antimicrobial activity was observed when microbial cultures (bacteria and fungi) were

exposed to the synthesized Ag NPs. Wound healing studies revealed that Ag NPs

impregnated in LSH thin films could have potential applications as an antimicrobial dressing

in wound management procedures.

Acute toxicity study of LSH was conducted on albino mice and albino rabbits. Three groups

of mice were exposed to a single oral dose of LSH (1, 5 and 10 g/kg). For eye irritation study

and dermal toxicity study, rabbits were exposed to LSH. After day 14, the haematological

and biochemical testing were performed and the values obtained were within the normal

range. Furthermore, the histopathological evaluation of the vital organs has not shown any

abnormalities. After acute toxicity study, LSH was found safe up to the dose of 10g/kg of the

body weight of the animal.

Overall, LSH has shown itself as a highly swellable and smart biomaterial having stimuli

responsive swelling-deswelling properties both in powder form and tablet formulation.

Furthermore, the preparation of DTX loaded LSH NPs has proved its utilization in the

development of novel drug delivery system for cancer treatment. Ag NPs embedded LSH

matrix is a new biocomposite for wound dressing and wound healing. Therefore, LSH has

proved as a potential material with wide range of pharmaceutical applications.

4

1. INTRODUCTION

1.1. Polysaccharides as a biomaterial

Polysaccharides are a group of carbohydrate which is a combination of more than ten

monosaccharides attached with each other through glycosidic linkages. Depending on the

type of polysaccharides, these monosaccharides are arranged either in branched or linear

form. Nature and properties of polysaccharides are greatly dependent on the composition of

the building block, molecular weight and type of branching. Polysaccharides are abundant in

nature, e.g., starch, cellulose, glycogen, chitin, arabinoxylan, galactomannan, pectin, alginate,

guar gum and inulin, etc. Some microbes (bacteria, fungi and algae) secrete various

polysaccharides, i.e., pullulan, dextran, xanthan gum and gellan gum. Polysaccharides are

widely available, abundant in nature, inexpensive, biodegradable, biocompatible, safe and

highly stable, nontoxic and diverse in structure and properties (Hovgaard and Brondsted,

1996).

Polysaccharides are diverse in their properties. Chitin, pectin and inulin are only fermented

by different bacteria present in the intestine and colon, hence used for the site specific

delivery of different drugs (Englyst et al., 1987; Salyers et al., 1977; Tozaki et al., 1997;

Rubinstein, 1990). These polysaccharides are used in film formation for the coating of dosage

forms (Coffin and Fishman, 1993). Polysaccharides are widely used as a coating agent for

effective and site specific drug delivery system, matrix systems, prodrugs and dry coating.

Colonic bacteria secrete glycosidase enzymes that are responsible for the cleavage of

glycosidic bond present in the polysaccharide. This hydrolytic cleavage at gastric pH and

intestinal pH provided the basis to fabricate the hydrolysable (ester/amide) prodrug using

dextran as an important polysaccharide (Hovgaard and Brondsted, 1996; Hussain et al.,

2011).

5

Moreover, hydrolysable prodrugs have been formulated using a number of polysaccharides,

i.e., dextran (Larsen et al., 1991; Hussain et al., 2011), cellulose (Kumar and Negi, 2014),

pullulan (Hussain et al., 2013; Hussain and Heinze, 2008), HPMC (Hussain et al., 2009),

HEC (Amin et al., 2015; Abbas et al., 2016), HPC (Hussain 2008; Hussain et al., 2014;

Hussain et al. 2015).

HPMC based sponges containing curcumin were prepared by lyophilization with the aim to

increase its solubility and bioavailability. Curcumin was released from these sponges within 2

h and area under curve (AUC) was greater than 5-fold when compared with simple powder

formulation. In vitro drug release and bioavailability study from these sponges has proved

their utilization as an effective delivery system for water insoluble curcumin (Petchsomrit et

al., 2016).

Arabinoxylan extracted from Ispaghula seed husk was used as drug carrier for sustained

release tablet formulation (Iqbal et al., 2011a) and as a mediator for the synthesis of silver

and gold nanoparticles (Amin et al., 2013). Carboxymethylation and ethylation was carried

out to modify the swelling and solubility of arabinoxylan (Saghir, 2008; Saghir, 2009).

A polysaccharide, agarose, extracted from marine red algae has a wide range of application in

biological sciences. Monosaccharide units are attached with each other through

galactopyranose linkage and exhibited resistance against chemical and enzymatic

degradation. Agarose solution is converted into gel at 40 °C due to the formation of helices

which further shaped into bundles (Aymard et al., 2001).

Dextran is composed of linear and branched chains attached by α-1,6 glycosidic and α-1,4

glycosidic linkages and produced by different lactic acid bacteria. Depending on the

molecular weight (20-40 KDa), dextran have shown short and long term antithrombotic

effects (Qiao et al., 2009). Pullulan is a water soluble exopolysaccharide produced by fungus

6

Aureobasidium pullulans. Pullulan is odorless, tasteless, non-toxic, biodegradable, non-

antigenic, biocompatible and non-immunogenic. Due to these properties, it is widely used in

pharmaceutical, food and paper production industries (Leathers, 2003; Shingel, 2004; Singh

et al., 2008b)

Starch is a long and branched polymer mainly a combination of amylose (20-25%) and

amylopectin (75-80%). Starch being a biocompatible, biodegradable, non-toxic, non-irritant

and cheap is used in pharmaceutical, agrochemical, food, paper and packing industries.

Starch has been used as drug carrier either modified or unmodified form (Kim et al., 2003;

Abbas et al., 2015).

Carrageenan, a biopolymer, is composed of sulphated forms of 3,6-anhydro-D-galactose and

D-galactose. Six different forms of carrageenan polysaccharides are extracted and three of

them, κ, ι and λ, are important comprising 22, 32 and 38% of sulphate group. Carrageenan gel

has wide range of application in pharmaceutical and food industries due to its temperature

responsive properties, water solubility and non-toxic nature (Daniel-da-Silva et al., 2007).

Gum acacia is extracted from the stems of acacia tree and composed of acidic polysaccharide

having arabinose (27%), glucuronic acid (16%), galactose (44%), rhamnose (13%) and

peptides (2-3%) (Al-Assaf et al., 2009). Due to emulsifying properties and controlled release

behavior, gum acacia is extensively used in pharmaceutical industry (Ali et al., 2009; Nishi

and Jayakrishnan, 2007).

Polysaccharides obtained from plant origin are versatile in nature and having a wide range of

application. Locust bean gum was used to develop mucoadhesive macromolecule with the

help of sodium alginate for the delivery of aceclofenac. Optimized formulation was able to

extend the drug release up to 10 h. Particle size was found in the range from 1.328 ± 0.11 to

1.428 ± 0.13 µm (Prajapati et al., 2014).

7

1.1.1. Hydrogels

Hydrogel is a three dimensional polymeric network which can absorb and retain a large

amount of water (Buwalda et al., 2014). Hydrogels are covalently or non-covalently

crosslinked. These physically or chemically crosslinking are responsible for water

penetrability in hydrogel and water holding capacity of the hydrogel which lead to the

development of various devices for biomedical applications. Hydrogels are designed or

obtained from natural, synthetic polymers or combination of both. Natural polymers are

composed of polysaccharides, natural polyesters, nucleic acids and proteins (gelatin,

collagen, silk fibroin and elastin) (Vlierberghe et al., 2011). Therefore, hydrogel has gained

many applications in biomedical field. Two of the important applications are in tissue

engineering and sustained drug delivery from several days to months (Hoare and Kohane,

2008; Kabanov and Vinogradov, 2009).

Hydrogels of synthetic (PEG, pHEMA and PVA) and semisynthetic origins (derivatives of

cellulose) are used in biomedical fields especially in the sustained and targeted delivery of

various drugs (hydrophilic or hydrophobic drugs, protein based drugs etc.) (Ford et al., 1985).

Generally, hydrogels are biocompatible due to their water holding ability and

physicochemical resemblance with the extracellular fluid both by mechanically and

compositionally. Biodegradability of hydrogels was taken place through hydrolytic, altering

in pH or temperature and enzymatic pathways.

Hydrogels are formed by physically cross-linking of polymers chains which is triggered by

environmental factors (temperature, pH or ionic concentration) or physicochemical

interactions (hydrogen bonding, charge condensation or stereocomplexation) (Hoare and

Kohane et al., 2008).

8

Temperature and pH responsive hydrogel was fabricated using β-cyclodextrin, 2-

methylacrylic acid and N,Nʹ-methylene diacrylamide. Synthesized hydrogel was investigated

for the controlled and sustained release of atorvastatin (Samanta and Ray, 2014a). Hydrogel

was found to have high swelling at pH 8.06 while exhibited less swelling at pH ≤ 3.84 and ≥

10.34. Swelling of hydrogel is directly proportional to the temperature of the media. Drug

release from the hydrogel was high (90.5%) at pH 8.06. Solubility of atorvastatin was also

improved after incorporation in hydrogel (Yang et al., 2016). Copolymerization of sodium

alginate and acrylamide with the aid of N,Nʹ-methylene bisacrylamide (MBA) was

successfully achieved and characterization by NMR, FTIR, XRD, thermogravimetric analysis

(TGA) and SEM. Swelling of hydrogel was found to be pH dependent and drug release from

hydrogel followed the kinetic models.

Modified form of chitosan was synthesized and used to separate the heavy metal ions from

aqueous solvents by adsorption through chelation (Kandile and Nasar, 2009). Genipin, a

water soluble crosslinking agent, after reacting with chitosan produce a fluorescent hydrogel.

Chitosan/genipin hydrogel was used for sustained release formulations, cartilage substitutes

due to elasticity, encapsulating agent for the delivery of biological products and wound

healing medications (Muzzarelli, 2009). Superporous hydrogel was prepared with the help of

chitosan to deliver the insulin and other protein or peptide drugs through mucoadhesive

delivery systems (Yin et al., 2007). Synthesis of hydrogel was confirmed by FTIR, NMR,

SEM and DSC analyses. Enhanced loading capacity with more than 90% insulin release

within first hour of the delivery was achieved.

Xanthan and chitosan based hydrogel was prepared by ionic complexation method. Newly

synthesized hydrogel was used as a controlled release material for the delivery of

theophylline which was evaluated for the treatment of chronic pulmonary obstructive disease

9

(Popa et al., 2010). Chitosan based hydrogel was synthesized by copolymerization of MBA

and acrylic acid. Characterization of hydrogel was performed through FTIR, TGA, NMR,

XRD and swelling behaviour. Theophylline and tinidazole release study was carried out and

rapid release was observed at pH 7.6 than at pH 1.5 (Samanta and Ray, 2014b).

Psyllium hydrogel was synthesized through chemical method with the help of polyvinyl

alcohol and used for the controlled release of rabeprazole. Haemo-compatibility of the

synthesized hydrogel was monitored and haemolytic index was found <5%. Due to haemo-

compatibility of hydrogel and antiulcer nature of psyllium, newly synthesized hydrogel is

used as a carrier for sustained delivery of an antiulcer drug (Singh et al., 2012). Psyllium

based hydrogel was used for colon specific delivery of tetracylcline HCl. Ammonium

persulfate (initiator) and N,Nʹ-methylenebisacrylamide (crosslinker) were used in synthesis of

hydrogel. Formation of hydrogel was confirmed by FTIR. Swelling of hydrogel and drug

release study was carried out in different buffers and results have shown the drug release

followed the Fickian diffusion (Singh et al., 2008a).

Cellulose based hydrogel was develop in order to improve the mechanical strength, swelling

properties, biocompatibility and antimicrobial activity necessary for the used in disposable

diapers. Cellulose and quaternized cellulose was crosslinked in an aqueous NaOH/urea

solution to synthesize highly swellable, superabsorbent and biodegradable hydrogel.

Antimicrobial activity against Saccharomyces cerevisiae was excellent and due to the

attraction of anionic microbial membrane by cationic hydrogel leading to distraction of

microbial membrane (Peng et al., 2016).

Hydroxyethyl cellulose and gelatin were blended to prepare microspheres and evaluated as a

sustained release delivery system for theophylline. Formation of IPN was confirmed by

10

FTIR, XRD, DSC and SEM. Equilibrium and dynamic swelling of these microspheres was

determined and drug release followed the non-Fickian diffusion (Kajjari et al., 2011).

1.1.2. Linseed

Flax (Linum usitatissimum L.; Syn: Alsi) is one of the oldest crop which is known to human

being and cultivated for both seeds and fibers. Seeds of Linum usitatissimum L. (Linseed;

Syn: Flaxseeds, Alsi seeds) is a good source of edible oil which is used as a nutritional

supplement. Two varieties of linseed (yellow and brown) are cultivated to get oil, fibers and

polysaccharides. Both varieties have similar constituents including oil, carbohydrates and

proteins. Flaxseeds have shown many benefits and also enhanced the antitumor effect of

tamoxifen (Chen et al., 2007a).

In a study, flaxseed gum was extracted by using hot water and composition of

polysaccharides was determined. During extraction process, temperature of water was

maintained at 85-90 °C and pH from 6.5-7.0. Gum was separated after precipitation with

ethanol and then fractionated through ion exchange chromatography. After thorough analysis

by NMR, gel filtration chromatography and chemical treatment, presence of L-arabinose, D-

xylose, L-fucose, L-rhamnose, D-galacturonic acid and D-glalactose was confirmed.

Rheological properties of the flaxseed gum were also studied and shear thinning behavior

was observed at higher concentration (Cui et al., 1994).

Other study has reported that linseed were mixed with water and stirred at 100, 80, 25 and 4

°C for 2 h to obtain mucilage. Extracted mucilage was separated through filtration and treated

with cetyltrimethylammonium bromide to separate the low and high density polysaccharides.

These polysaccharides were analyzed through GPC, NMR, gas liquid chromatography and

acid hydrolysis. Rhamnose, galactose, arabinose, xylose and galaturonic acid are the main

components of linseed mucilage. It was also determined that the viscosity of linseed mucilage

11

is greatly affected by the pH of water and presence of electrolytes (Fedeniuk and Biliaderis,

1994).

Flaxseed mucilage is mainly composed of arabinoxylane (75%) and studied by SEC/MALLS.

After analysis, it was found that there are three distinctive proportion of arabinoxylane, i.e.,

5000000 g/mol, 200000 g/mol, and 1000000 g/mol. In depth analysis has shown that

formation of aggregates is due to the weak hydrogen bonding and can be reduced by

increasing the temperature. Rheological behavior was also investigated and presence of

hydrogen bond was also confirmed by addition of lyotropic and chaotropic salts (Warrand et

al., 2005a).

Flaxseed was evaluated for the inhibition of the breast tumors in combination with tamoxifen.

Ovariectomized mice induced with breast cancer cell line (MCF-7) were treated with

flaxseeds (5 and 10%) and tamoxifen alone (5 mg/tablet) or in combination for a period of 16

weeks (Chen et al., 2007). At the end of study, tumor was analyzed for apoptosis, cell

proliferation and expression of signal transduction and estrogen related genes. Results have

indicated that flaxseed alone has no impact on the growth of tumor and combination with

tamoxifen reduces the tumor size.

Effect of water extracted flaxseed gum was evaluated as a blood sugar and cholesterol lowing

agent in diabetic patients (Type II). Flaxseed gum was administered to 60 patients for a

period of 3 months. At the end of study, the values of fasting blood sugar, total cholesterol

and low density lipids were dropped from 154 to 136 mg/dl, 182 to 163 mg/dl and 110 to 92

mg/dl, respectively (Thakur et al., 2009).

Water extracted flaxseed gum was dried by adopting different methods (freeze drying,

ethanol precipitation then oven drying at 80 °C, vacuum drying, spray drying, drying in hot

air oven at 80 °C and 105 °C). Color of flaxseed gum was observed after drying as well as in

12

aqueous solution. Effect of drying on Zeta potential, emulsion, gelling and foaming

properties of flaxseed gum was evaluated. It was observed that the method of drying has

pronounced effect on the functional properties of flaxseed gum and appropriate method

should adopt to get potential benefits (Wang et al., 2010).

Rheological properties of flaxseed gum solution were studied after high pressure

homogenization (Wang et al., 2011b). During this process, temperature of solution was

increased and as a result viscosity was decreased. Power law was used to define the relation

of shear rate and shear stress. Conductivity of flaxseed gum solution and gelling temperature

were found to be independent to the homogenization process while clarity of solution was

significantly improved by this process.

Mucilage was extracted from flaxseed and subjected to freeze drying after precipitation with

ethanol (Mazza and Biliaderis, 1989). Composition, foamability, solubility and moisture

sorption properties were determined and compared with guar gum and locust bean gum. High

viscosity of flaxseed mucilage was observed at pH 6.0-8.0 and low in aqueous solution of

NaCl.

Gelling strength of flaxseed gum was studied along with factors effecting on the gel forming

ability (Chen et al., 2006). Gel forming temperature, pH and salt concentration was also

determined. Maximum gel strength was observed at pH 6-9. Addition of sodium ion

decreases the gel strength. Calcium chloride less than 0.3% are used to increase the gel

strength while higher concentration has negative effect. Addition of carrageenan in flaxseed

gum increased the viscosity but lowered the syneresis.

Linseed mucilage was separated by alkali extraction method. Seeds were boiled with sodium

bicarbonate and extracted mucilage was neutralized with acetic acid. Mucilage was air dried

and used to prepare mucoadhesive microspheres for effective delivery of venlafaxine using

13

spray drying method (Nerkar and Gattani, 2011). Microspheres were thoroughly

characterized by DSC, SEM and XRD and evaluated for swelling behavior, mucoadhesion, in

vitro and in vivo drug release and stability study. Results indicated that improved

bioavailability of venlafaxine was observed when compared to traditional oral route.

Flaxseed mucilage was purified by size exclusion and ion exchange chromatography and

three main polysaccharides were separated (Warrand et al., 2003). These polysaccharides

constitute the main component of the mucilage (75%). Molecular weight of these three

polysaccharides was 1.7 × 104 g/mol, 6.5 × 10

5 and 1.2 × 10

6.

Laws of extraction were applied on flaxseed mucilage extraction process (Ziolkovska, 2012).

For this purpose, extraction was carried out under different conditions of temperature (40-100

°C), duration of seed soaking in water (0-1 h), water to seed ratio (5-30 to 1) and mixer

stirring speed (0-240 rpm). Optimum temperature and water to seed ratio to get maximum

flaxseed mucilage are 80 °C and 25:1, respectively. Different mathematical equations were

used to explain the effects of different parameters on the yield of flaxseed mucilage.

Analysis of polysaccharides of flaxseed meal was performed by viscometry and light

scattering techniques. Size exclusion chromatography revealed the presence of small and

large molecular weight fraction (3.1 × 105 Da) and (1.0 × 10

6 Da), respectively (Goh et al.,

2006). SEC analysis of the yellow flaxseed mucilage confirmed three distinct types of

polymers. Molecular weight distribution of these polymers is analyzed by multi laser light

scattering and found high molecular weight (9.3 × 105 and 5.7 × 10

6 g mol

-1) and low

molecular weight (3.2 × 105 g mol

-1) fractions (Warrand et al., 2005b).

Soybean oil (10%, v/v) was emulsified using the combination of flaxseed gum (0.05-0.5%,

w/v) and soybean protein isolate (1%, w/v). Objective was to observe the emulsifying

behavior of soybean protein isolate in the presence of flaxseed gum. Formulated emulsion

14

was analyzed for stability, turbidity, surface charge, particle size and rheology (Wang et al.,

2011c). Initially, zeta-potential and turbidity of emulsion decreased as the concentration of

flaxseed gum was increased (up to 0.1%) but increased at high concentration of gum up to

0.35% (w/v). Particle size decreased with the increase of gum concentration up to 0.1% (w/v)

and increased beyond this concentration. Therefore, protein-based emulsions can be

stabilized with the addition of flaxseed gum.

Mucilage extracted from seven varieties of Italian flax was investigated for their

physiochemical, sensory and functional properties (Kaewmanee et al., 2014). Chemical

composition of polysaccharides was also determined by acid hydrolysis. Presence of

rhamnogalacturonan as a backbone of the polysaccharide was identified by NMR analysis.

Mucilage from all seven varieties was tasteless but varies with respect to viscosity, zeta-

potential, conductivity, sugar content, swelling ability, foaming capacity and emulsifying

properties.

Mucilage extracted from flaxseed hull was fractionated by ion exchange chromatography into

acidic and neutral fractions. Rhamnogalacturonans having two acidic fractions of molecular

weights 1510 kDa and 341 kDa while arabinoxylans, being a neutral fraction, having

molecular weight of 1470 kDa. Acidic and neutral fractions have shown Newtonian and

pseudoplastic flow, respectively. Physical (stability and molecular weight distribution),

chemical (composition of polysaccharide), functional (emulsify properties and surface

tension) and rheological properties (viscosity and critical concentration) of mucilage was

thoroughly examined (Qian et al., 2012b). Structure of rhamnogalacturonans was determined

after methylation and by using 1D/2D NMR spectroscopy. Rhamnogalacturonans is highly

branched structure with degree of branching 0.55 and composed of rhamnogalacturonan-1

backbone connected with homorhamnan and homogalacturonan (Qian et al., 2012a).

15

Four different methods were used to extract mucilage from flaxseed meal and optimize the

extraction process to get maximum and pleasant taste mucilage product (Singer et al., 2011).

In precipitation method, flaxseed meal was mixed with water and raised the pH up to 9 with

the addition of NaOH solution. Mixture was then treated with HCl to decrease the pH and

centrifuge to separate the precipitates. In second method, instead of HCl, ethanol was used to

get precipitates. Boiling water was used to get the mucilage and separated from the flaxseed

meal by ethanol precipitation. Hot water dispersed flaxseed meal was treated with enzymes

and then filter to obtain mucilage. After physicochemical evaluation of these four processes,

second method was considered the most appropriate to extract good quality mucilage for food

industry.

Investigation for the polysaccharides composition among 109 varieties of flaxseed from 12

geographical regions were conducted and found in a range from 3.6-8.0% (Oomah et al.,

1995). After acid hydrolysis, rhamnose, xylose, galactose, glucose, fucose and arabinose

were the main component of flaxseed mucilage. Depending on the variations in yield of

different components of mucilage, desired carbohydrates were obtained from flaxseed

mucilage.

Noodles were prepared with the addition of different concentration of flaxseed mucilage as a

replacement of wheat flour (Kishk et al., 2011). Physicochemical properties and noodle

quality were analyzed and results were compared in term of swelling index, nitrogen and

cooking loss, cooking yield, cooking time and cooking temperature. Good quality noodles

were prepared with 3% flaxseed mucilage and cooking temperature range from 68.2-70 °C.

Results were also analyzed statistically and after sensory evaluation, noodles prepared with

flaxseed mucilage have shown improved texture and pleasant in taste.

16

Effect of daily intake of flaxseed and sunflower seed on lipid profile of postmenopausal

women was studied for six weeks (Arjmandi et al., 1998). On examine the lipid profile at the

end of study, LDL level was significantly reduced in flaxseed treated group as compared to

sunflower seed treated group. Both groups have not shown any effect on HDL and

triglyceride level. LDL lowering ability of these two types of seeds is supposed to be due to

linoleic or α-linolenic acid component.

Emulsion was prepared using whey protein isolate solution in imidazole and soybean oil

under high speed mixing (Khalloufi et al., 2009). pH of emulsion was adjusted to pH 3.5 and

maintained at 4 °C. Various concentrations of flaxseed gum was prepared in imidazole at pH

7.0 and then adjusted to 3.5 before adding in the emulsion. Physicochemical properties of the

synthesized emulsion were investigated by particle size analysis, zeta potential, viscosity and

separation behavior. Results indicated that emulsion was stabilized by negatively charged

flaxseed gum.

Polysaccharide analysis of flaxseed mucilage was carried out after acid hydrolysis at various

molar concentrations and temperature (Emaga et al., 2012). Effect of chemical and enzymatic

hydrolysis was also studied. Hydrolysis with trifluoroacetic acid resulted in less damage to

the sugar components of flaxseed gum as compared to HCl and H2SO4. Moreover, enzymatic

degradation also reduced the destruction of sugars. Monosaccharide composition was

determined but quantification was found to be difficult due to strong bonding between acidic

and neutral fractions.

Flaxseed mucilage is composed of rhamnogalacturonan I and arabinoxylan and in detail study

was performed to analyze the structure of polysaccharides. After acid hydrolysis and

evaluation through GPC and SEC, presence of arabinose, xylose, galactose, glucuronic acid

17

and fucose was verified along with determination and analysis of linkages (Naran et al.,

2008).

Flaxseed mucilage was extracted by adopting three different methods and then analyzed the

mucilage for different physical parameters (Fabre et al., 2015). Ultrasonic treatment of the

hydrated seeds secreted maximum mucilage when compared with other treatments, i.e.,

microwave and magnetic stirring. Monosaccharide composition, galacturonic acid

concentration and viscosity of extracted mucilage from all three methods were evaluated. It

was found that high yield was obtained from ultrasound treatment though the viscosity and

concentration of high molecular weight fraction of mucilage was decreased.

Composite material was prepared from flax fiber and mucilage extracted after treating with

water at 20 °C and 40 °C. In order to prepare a water insoluble biocomposite material,

mucilage was treated with glutaraldehyde, as a crosslinking agent, and glycerol, as a

plasticizer. Water sorption and swelling was reduced after crosslinking while increased the

mechanical strength and rigidity (Alix et al., 2008).

Structure of polysaccharides derived from linseed mucilage was thoroughly characterized by

Muralikrishna et al. in 1987. Neutral fraction is composed of and D-galactose, L-arabinose

and D-xylose (1:3.5:6.2) while in acidic fraction, L-rhamnose, D-galacturonic acid, L-

galactose and L-fucose (2.6:1.7:1.4:1) are the main components. In neutral fraction, galactose

and arabinose are attached with the backbone of xylan. Galactose and fucose are linked as a

side chain unit with D-galactopyranosyluronic acid and rhamnopyranosyl residue

(Muralikrishna et al., 1987).

High molecular weight fractions were separated from flaxseed cake using anion exchange

chromatography. Molecular weights of arabinoxylan (arabinose to xylose ratio, 0.32) and

galactoglucan were 8.46 × 105 and 6.5 × 10

4, respectively. Furthermore, two more fractions

18

were also separated having molecular weights of 3.1 × 105 and 1.3 × 10

5. Rheological study

of the 2% solution of flaxseed gum has shown the insignificant viscosity for use as a

texturing component (Warrand et al., 2005c).

Mucilage extracted from flaxseed was crosslinked with epichlorohydrin and plasticized with

glycerol to prepare a composite material by varying the reaction conditions. Synthesized

composite material has shown very low tendency to swell. Tensile strength and other

parameters were determined and better results were found with the composite material

prepared by using glutaraldehyde as a crosslinking agent (Paynel et al., 2013).

1.1.3. Modification of polysaccharides

Naturally occurring polysaccharides are being widely used in many fields of biomedical,

pharmaceuticals, cosmetic, food industry and bioengineering. To improve some of their

properties, polysaccharides are modified to get the desired results. Therefore, the researchers

are modifying the naturally occurring polysaccharidal material by different ways, i.e.,

copolymerization, grafting, etherification, carboxymethylation, sulphonation, oxidation,

amide formation and esterification etc.

In carboxymethylation, polysaccharides are reacted with monochloroacetic acid/sodium salt

in an alkaline medium. As a result of this simple process, a variety of properties are

introduced in polysaccharide including swellability, pH responsive swelling and stimuli

responsive on-off switching etc. Cellulose (Klemm et al., 2002), chitin and chitosan (Roberts

1992), dextran (Huynh et al., 1998), pullulan (Shingel 2004) and starch (Shogrun 1998) are

successfully carboxymethylated and have number of applications. Arabinoxylan, isolated

from Ispaghula, was treated with sodium monochloroacetate in the presence of NaOH to form

carboxymethylated arabinoxylan (Saghir et al., 2008). The resulted product was verified by

19

FTIR and NMR analyses. Maximum degree of substitution of the carboxymethylated

arabinoxylan was calculated as 1.81 and product was found soluble in water.

Oxidation of polysaccharides is performed to introduce the aldehyde group which is further

used for grafting of different molecules on the polymer chain and for crosslinking reactions

with amines (Rinaudo 2010; Sarymsakov 1975). These modifications reduce the stiffness of

polysaccharides and have potential application in drug delivery. Metaperiodate was used to

oxidize the hydroxyethyl cellulose and methylcellulose to enhance the hemostatic properties

(Gibbons 1956). Flexibility and elasticity of alginate was decreased after oxidation (Gomez et

al., 2007; Andresen et al., 1977). Dextran was also oxidized with sodium periodate and

crosslinked with adipic acid dihydrazide. The resulted hydrogel was evaluated for swelling

and mechanical properties and degradation behaviour (Maia et al., 2005). Carboxyl groups

were introduced in pullulan through oxidation reaction which make it more water soluble

(Spatareanu et al., 2014).

In etherification, polysaccharidal hydrogels reacted with alkylating agents. Cellulose ethers

were prepared in homogeneous reaction condition (Takaragi et al., 1999). Cellulose was first

dissolved in a solution of dimethyl acetamide/Lithium chloride and methyl, hydroxyethyl and

hydroxypropyl ethers were synthesized using epoxide or iodomethane as alkylating agents

(McCormick and Callais, 1987). Pullulan was also transformed into propyl ether and butyl

ether with different degree of substitution when treated with sodium hydroxide and alkyl

bromide in a mixture of DMSO/H2O (Shibata et al., 2002). Carboxymethyl ether of guar

gum, xylan and konjac glucomannan were synthesized with DS value of 0.8, 1.2 and 0.3,

respectively (Lindblad and Albertsson 2004; Petzold et al., 2006). Ethylation of arabinoxylan

was performed with ethyl iodide in the presence of NaOH. Product was soluble in DMSO and

degree of substitution was calculated as 0.61 (Saghir et al., 2009).

20

Generally, esterification involved a reaction between an alcohol and an acid. There are

number of methods used for the esterification (Heinze et al., 2003). Cellulose was treated in

homogenous reaction condition with acid chloride in the presence of a base, triethylamine

(McCormick and Callais, 1987). In another method, acetylation of cellulose was carried out

in an ionic medium using acetic anhydride. Highly substituted cellulose acetate (DS up to 3)

can be prepared by simply treated with acetic anhydride or acetylchloride (Wu et al., 2004).

Starch was also esterified by acyl chloride and pyridine in a solution of DMAc/LiCl at 100 °C

(Grote and Heinze 2005). Xylan acetate was synthesized using acetic anhydride and pyridine

while DMF as a reaction medium (Belmokaddem et al., 2005). Acetylation of alginate and

chitosan was carried out in homogenous reaction conditions using DMSO/TBAF or

DMAc/LiCl as a solvent and acetic anhydride/pyridine or acid halides as acylating agent

(Pawar and Edgar 2011; Badawy et al., 2005).

Polysaccharides possess some unique properties based on the types of glycosidic linkages,

molecular weight (Li and Shah 2014), polymer chain configuration (Hattori et al., 1998),

solubility (Lu et al., 2012) and some special configurations (Wang et al., 2010). Types of

glycosidic linkages and composition of monosaccharides is the key to determine the

properties of polysaccharides. Modification at molecular level in the polysaccharides changed

or altered the properties of these polysaccharides and achieved by chemical (sulfation,

carboxymethylation, phosphorylation, selenization, acetylation, alkylation and acid/alkali

degradation), physical (ultrasonic disruption, microwave exposure and radiation treatment) or

biological (enzymatic) processing (Li et al., 2016). These methods are adopted to increase the

water solubility (Jung et al., 2011; Vasconcelos et al., 2013), reduce molecular mass (Chen et

al., 2013; Parvathy et al., 2005), reduce intrinsic viscosity (Li and Feke 2015; Yan et al.,

2015) and increase thermal stability (Carbinatto et al., 2012).

21

1.1.4. Stimuli responsive properties of polysaccharidal hydrogels

Smart hydrogels have the tendency to respond against different stimuli which may be

physical or chemical, i.e., light (Juodkazis et al., 2000; Tatsuma et al., 2007), pH (Zhang et

al., 2007; Qu et al., 2006: Kim et al., 2006), electric and magnetic field (Kwon et al., 1991;

Satarkar and Hilt, 2008; Wang et al., 2009), temperature (Yoshida et al., 1995; Ju et al., 2006)

and different species (Holtz and Asher, 1997; Chu et al., 2004). On the basis of these stimuli

responsive behaviour, hydrogels are being used in gene and controlled drug delivery systems

(Qiu and Park, 2001; Cheng et al., 2008), soft machines (Calvert, 2008), biosensors (Beebe et

al., 2000), liquid microlenses and bio or chemical separations (Ju et al., 2009; Yang et al.,

2008; Xie et al., 2009), etc.

Potato starch was modified and then used to synthesize semi-interpenetrating polymer

network (SIPN) composite with vary the degree of crosslinking (Dragan and Apopei, 2013).

Synthesized hydrogel was evaluated by FTIR and SEM. Swelling kinetics was determined in

distilled water and stimuli responsive properties of SPIN was observed in water/ethanol,

water/1 M NaCl and at pH 8/pH 1. A superfast swelling was seen in all formulations.

Swelling shrinking behaviour was found to be dependent on the incorporated functional

group and the ratio of the cross-linker.

IPN based on the derivatives of acrylamide and acrylic acid was synthesized by varying the

reaction conditions (Diez-Pena et al., 2002). Swelling response of these hydrogels was found

to be dependent on the nature of polymer, temperature and pH of the swelling media.

Formation of hydrogen bond between amide and carboxyl moiety has shown impact on the

swelling behaviour of hydrogel in acidic environment and water.

Guar gum and acrylic acid were used in different combinations to synthesize a hydrogel and

confirmed by FTIR, DSC and SEM analysis (Huang et al., 2007). Electrostatic interaction

22

between anionic group of acrylic acid and cationic group of guar gum was the main driving

force for the synthesis of hydrogel. Swelling of hydrogel is influenced by the concentration of

hydrophilic group in the composition of hydrogel. Swelling of hydrogel is increased with the

increase in the pH of the media. Hydrogel has shown swelling deswelling behaviour at pH

7.4 and 5.0, respectively. Ketoprofen was loaded in hydrogel and release was monitored at

different pHs. Release was also observed at pH 2.2 for 1 h, pH 6.8 for 4 h and pH 7.4 for 10

h.

Stimuli responsive hydrogel was prepared from poly (acrylic acid) and poly (aspartic acid)

and evaluated for pH, salt and temperature induce swelling (Zhao et al., 2006). It is noted that

the swelling ratio increased by increasing the temperature of the swelling media from 40-60

°C. pH responsive swelling deswelling was also observed and strongly dependent on the

concentration of poly (aspartic acid) in the hydrogel. An inverse relation between the

swelling capacity and concentration of poly (aspartic acid) was observed. Swelling of

hydrogel was high in deionized water as compared to other biological fluids i.e., Hank‘s

solution, glucose, urea, synthetic urine and physiological saline water.

Dynamic swelling and swelling-deswelling behaviour of an anionic hydrogel was monitored

under different conditions of pH and ionic strength (Kare and Peppas, 1995). Dynamic and

equilibrium swelling was determined in acetate buffer with a pH range from 3.2-7.6. Ionic

strength of the swelling media was maintained with the appropriate addition of NaCl. Due to

the ionization of the hydrogel at high pH, water uptake ability of the hydrogel increases

which results in the increased swelling of hydrogel. At pH 4.0, with the increase in ionic

strength from 0.0074-0.08M, dynamic swelling of the hydrogel was not affected. At pH 7.0,

due to the ionization of hydrogel, the increase in ionic strength has shown its impact on the

dynamic and equilibrium swelling.

23

Composite cryogel was synthesized in the presence of chitosan by crosslinking of acrylamide

with N,N'-methylenebisacrylamide (Dragan et al., 2012). Swelling behaviour was observed

from pH 1-12 and it was noted that the composite cryogel has not shown any swelling up to

pH 3 and then suddenly started to swell from pH 4-12 with higher rate. This swelling was due

to the presence of hydrophilic group of the gel whose ionization resulted in the repulsion.

This gel was used for the separation and removal of cationic organic dye.

Psyllium and polyacrylamide based hydrogel was prepared using N,Nʹ-methylenebis-

acrylamide as a crosslinker (Singh, 2007). Swelling trend of the polymeric network was

thoroughly studied at various temperatures, pH and different concentrations of NaCl.

Swelling was found to be dependent on the concentration of the crosslinker. Abrupt swelling

and deswelling was observed in distilled water and NaOH solution. Structural

characterization of hydrogel was investigated through SEM, TGA and FTIR.

Hydrogel having polyampholytic nature was synthesized by interaction between negatively

and positively charged polymers (Mohan and Geckeler, 2007). Tween 80 (non-ionic),

dodecylpyridinium chloride (cationic) and sodium dodecyl sulfate (anionic) were used as a

surfactant to prepare various types of interpenetrated polymer network. High swelling was

observed in hydrogel with cationic surfactant at pH 5-6. Saline responsive swelling was also

seen in various salt solutions.

Chitosan and poly(vinyl alcohol) were cross-linked by glyoxal to produce a gastro retentive

superporous hydrogel for the delivery of rosiglitazone maleate (Vishal and Shivakumar,

2010). Synthesized hydrogel has the ability to swell in acidic pH while deswell in basic pH.

A decrease in swelling was observed while increasing the ionic strength of the swelling

media. Swelling is dependent on the concentration of the chitosan and cross-linker. Release

of rosiglitazone was sustained for 6 h in acidic media.

24

Chitosan was grafted with acrylamide under the influence of a cross-linker,

methylenebisacrylamide (Pourjavadi and Mahdavinia, 2006). Chitosan grafted hydrogel was

evaluated for swelling behavior at various pHs and swelling shrinking response in acidic and

basic media, respectively. Rate of swelling was found to follow second order kinetics.

Swelling capacity was reduced with the increase in the ionic charge of the swelling media.

Superabsorbent hydrogel was prepared by mixing the aqueous solution of starch and

polyacrylonitrile under mild heating through alkaline hydrolysis (Sadeghi and Hosseinzadeh,

2008). Salt and pH responsive swelling was evaluated and found highly swellable material in

basic environment and in NaCl solution. Reversible swelling shrinking behavior at basic and

acid pH, respectively, made it a suitable material for various applications.

Hydrogel based on PEG was synthesized with prime objective for molecular recognition and

swelling deswelling response against different ionic solutions (Tominaga et al., 2013).

Swelling properties of hydrogel can be controlled by changing the composition and

concentration of reactants.

Composite hydrogel was prepared by copolymerization using sodium acrylate, HEC and

medicinal stone (Wang et al., 2011a). Formation of hydrogel and medicinal stone distribution

in the synthesized composite was analyzed by TGA, FTIR, energy dispersive spectroscopy,

FESEM, TEM and elemental map. By increasing the concentration of medicinal stone, the

swelling capacity of hydrogel was increased up to 400%. Hydrogel was evaluated for

swelling deswelling behavior at basic and acidic pH and also in water and normal saline

solution. Due to deswelling potential in surfactant solution, this hydrogel has the ability to

play an important role as absorbing agent.

25

1.1.5. Toxicological studies of polysaccharides

In order to determine the toxic characteristics (if any) of a compound, the initial screening

was performed through acute toxicity study. Toxicity of a compound is evaluated through

three types of studies, i.e., acute or sub-acute (24 h to 28 days), sub-chronic (90 days) and

chronic (6 to 12 months) toxicity studies.

Chitosan was reported as a carrier for nasal drug delivery by Aspden and coworkers and

toxicological study was carried out on different animals (guinea pig, frog, rat and mouse) as

well as on human volunteers. For the development of nasal delivery system based on

chitosan, cilia beat frequency was determined by repeatedly applying chitosan solution as a

marker of nasal toxicity on nasal tissues of guinea pigs for 28 days (Aspden et al., 1997). Rat

nasal perfusion method was employed to investigate the extent of insulin absorbance through

nasal epithelial membrane (Aspden et al., 1996).

Polysaccharide extracted from turmeric was evaluated for acute oral toxicity and

mutagenicity (Velusami et al., 2013). Oral toxicity study was conducted on Wistar rats and

results indicated the safety of turmeric polysaccharides up to the level of 5g/kg body weight.

Mutagenicity testing was assessed by chromosome aberration, bacterial reverse mutation test

and micronucleus testing. Results obtained from mutagenicity and acute oral toxicity studies

have proved the safety of turmeric polysaccharide.

α-Cyclodextrin, being a water soluble dietary fiber was investigated for oral toxicity study

over a period of 13-week at various concentrations (Lina and Bär, 2004). During this study

period, different parameters were considered as a marker of potential toxicity, i.e.,

consumption of food and water, body weights calculation, biochemical and hematological

evaluation, histopathological observations and organ to body weight ratio. Moreover, urine,

feces and ophthalmic examination were also carried out. At the end of study, treatment

26

related mortality has not seen. Mild diarrhea was observed with soft stool. Food and water

intake was slightly increased. Hematological, biochemical and histopathological evaluation

did not show any abnormality. Intake of α-cyclodextrin up to 20% of body weight of rats was

found to be safe.

Meratrim, an extract from flowers and fruits of Sphaeranthus indicus and Garciania

mangostana, respectively were used for weight management (Saiyed, et al., 2015). Safety

assessment of Meratrim intake was evaluated including acute and sub-chronic toxicity and

animal toxicological studies and was carried out on Sprague-Dawley rats. LD50 level was

found to be > 5 and 2 g/kg body weight as determined by acute and sub-chronic toxicity

study. Meratrim was non-irritating to the skin, slightly irritating to eye and non-mutagenic.

No sign of mortality, morbidity and other side effects were observed during the whole study

period. These results when combined with the human clinical trials indicated the safe use of

Meratrim.

Safety of arabinoxylan, isolated from Ispaghula husk, was investigated through acute toxicity

study carried out in mice and rabbits (Erum, et al., 2015). Different doses of arabinoxylan

(1,5 and 10 g/kg body weight) was administered in animals and kept under observation for up

to 14 days. Biochemical, histological and hematological parameters was monitored and did

not show any significant abnormalities. Moreover, effect of arabinoxylan on heart of frog was

also monitored and found safe with respect to heart rate and vascular contraction.

Partially hydrolyzed guar gum (K-13) was examined through acute and subchronic toxicity

studies (Koujitani, et al., 1997). For acute toxicity study, guar gum (6 g/kg) was given to

mice and rice. Various concentrations (0.2, 1 and 5%) of guar gum were used for subchronic

toxicity studies. Mutagenicity study was performed using bacteria by adopting reverse

mutation test. At the end, lethal dose (LD50) was more than 6 g/kg and also no death was

27

found. No significant findings were seen in subchronic toxicity study and also found

mutagenically save.

An exopolysaccharide (Bacterial Cellulose) was isolated from sugarcane molasses and

evaluated for acute toxicity study along with antigenotoxicity, cytotoxicity and genotoxicity

studies (Pinto et al., 2016). Bacterial Cellulose (2 g/kg body weight) was administered as a

single dose to Wistar rats and was observed for any sign of toxicity. Results indicated that

Bacterial Cellulose is not genotoxic, cytotoxic and acutely toxic.

1.2. Polysaccharides based drug delivery systems and pharmaceutical applications

1.2.1. Sustained release of NSAIDs from polysaccharidal materials

Non-steroidal anti-inflammatory drugs (NSAIDs) are prescribed as antipyretic, analgesic and

anti-inflammatory agent. NSAIDs act by inhibiting the enzyme, cyclooxygenase (COX),

which ultimately prevent the synthesis of prostaglandins.

Diclofenac is a phenylacetic acid derivative and its chemical name is 2-(2,6-dichloranillino)

phenylacetic acid synthesized for the first time by Alfred Sallmann and Rudolf Pfister 1973.

Diclofenac is marketed as sodium (Fig. 1.1) or potassium salt for inflammation, pain

associated with kidney or gallstones, fever, dysmenorrhea, arthritis (rheumatoid arthritis and

osteoarthritis), dental pain and endometriosis.

Diclofenac is rapidly absorbed after oral administration and absorption is dependent on dose,

salt form, composition and condition of GIT. Due to short half-life, frequent administration of

diclofenac is required for therapeutic efficiency (Davies and Anderson, 1997). Diclofenac is

also available in enteric coated, extended and sustained release formulations.

28

Controlled release of diclofenac sodium was achieved by preparing microspheres in which

sodium alginate and calcium chloride were used as a polymer and cross linking agent,

respectively (Gohel and Amin, 1998). Prolong release of diclofenac sodium was evaluated

from tablet formulation prepared with the help of polycarbophil and release profile was

compared with a commercially available product, ―Voltaren‖. In vitro and animal trials have

shown that the formulation was bioequivalent to ―Voltaren‖ (Hosny, 1996).

Drug release mechanism (either swelling or erosion of the polymer) from tablet formulation

prepared with poly(D,L-lactic acid) was investigated. It was observed that the release of

diclofenac sodium was governed both by swelling and erosion of the tablets. pH of the media,

interaction between drug and polymer, porosity and nature of the drug (acidic or basic) are

the major factors which made influence on the release of drug from the polymeric matrix

tablets. Extended release of drug is achieved at pH 7.4 as compared to pH 5.4 (Proikakis et

al., 2006).

Fig. 1.1. Structure of diclofenac sodium.

Sustained release formulation of caffeine was prepared with the aim to attain the prolong

alertness for 8-12 h. Drug and poly(ethylene oxide) were mixed to make an erodible tablet for

the sustained release of caffeine. In vitro release study and pharmacokinetic study showed the

initial burst release and then maintained a sustained systemic concentration for 8 h (Tan et al.,

2006).

29

Release retarding potential of karaya gum and xanthan gum was explored using two model

drugs, diclofenac sodium and caffeine, having different aqueous solubilities (Munday and

Cox, 2000). Gum swelling, erosion and drug release behaviour were studied. Drug release

from these two gums is dependent on the solubility of drug, agitation speed and drug to

polymer ratio. Drug release from these gums followed the zero order kinetic which is based

on erosion of the polymer matrix.

Caffeine (Fig. 1.2), being a neutral drug, is the drug choice for most of the researchers

working on the evaluation of new polymers as sustained release matrix. Talukdar and Kinget

evaluated the sustained release potential of xanthan gum using soluble neutral drug

(caffeine), soluble acidic drug (Indomethacin sodium) and insoluble acidic drug

(Indomethacin). Swelling (radial and axial) and drug release behaviour of xanthan gum

formulated tablets were observed in the media having similar physiological ionic strength.

Swelling of tablets and drug release from tablets followed Case I and Case II diffusion,

respectively (Talukdar and Kinget, 1995). Caffeine was also used to find out the release

mechanism from ethylcellulose matrix tablets. Furthermore, release kinetic models were

applied on release data and best fit model was selected (Neau et al., 1999). For the

assessment of core-in-cup drug delivery system as a sustained release matrix, caffeine and

ibuprofen were used as model drugs (Danckwerts, 1994). To develop such a system, core was

prepared with different grades of HPMC while cup shape tablet was made up of

ethylcellulose and carnauba wax. Drug release data of caffeine and ibuprofen have shown the

zero order release for 8 to 23 h depending on the drug to polymer ratio.

Stress and sleep deprivation exhibited negative impact on mood and performance of human

beings (Renner et al., 2007). Administration of different doses of caffeine (100, 200 and 300

mg) to 72 h sleep deprivation Navy trainees resulted in the improvement of visual vigilance,

30

sleepiness, reaction time, mood, memory and alertness. Caffeine (200 mg) was considered the

optimum dose to get rid of stress and problems related to sleeplessness (Lieberman et al.,

2002). Caffeine also enhanced the analgesic effect of acetaminophen when given in a

combination of 130 mg and 1000 mg, respectively. Absorption of acetaminophen was

accelerated by caffeine and relief in pain was observed for longer period of time.

Sorbitan esters containing niosomes were prepared for the effective delivery of caffeine from

dermal drug delivery systems (Khazaeli et al., 2007). Drug entrapment is dependent on the

lipophilicity of surfactants, particle size and charge of niosomes. Caffeine release was

controlled by both the erosion and diffusion mechanism. Due to high encapsulation efficiency

and stability of niosomes, this type of system is better than liposomes for topical

administration of caffeine.

Hydrolytic cleavage of poly(DL-lactic acid) (PLA50) in the presence of caffeine was

thoroughly examined in order to observe the effect of tertiary amine on breakdown of

polyester chains (Li et al., 1996). Caffeine was loaded in polymer by solvent evaporation

technique and mixture was processed into thin film and plate. Degradation of polymer is

effected by caffeine due to its catalytic action in the degradation process.

Pickering emulsion, a new form of emulsion, is prepared and compared with the conventional

w/o emulsion (Frelichowska et al., 2009). In both emulsions, caffeine was used as a

hydrophilic penetrant and effect of interfacial layer on drug release from both types of

emulsions was monitored. Due to the penetration of adsorbed caffeine on silica particles

through stratum corneum, pickering emulsion was considered the most suitable form of

emulsion.

Gastro retentive and sustained release formulation containing sodium alginate was developed

for the delivery of caffeine and chlorpheniramine (Stockwell et al., 1986). Presence of

31

sodium bicarbonate in the tablet formulation made a buoyant system. Drug release is

dependent on the nature and concentration of polymer.

For the treatment of cellulite, two topical formulations were designed containing caffeine as

an active ingredient (Hamishehkar et al., 2015). Caffeine hydrogel and caffeine loaded solid

lipid nanoparticles were prepared and evaluated through the permeation study and

histological study. Caffeine flux value through rat skin was high in case of nanoparticles as

compared to simple hydrogel. Complete breakdown of adipocytes were found in histological

study when treated with caffeine loaded solid lipid nanoparticles.

Fig. 1.2. Structure of caffeine.

Diacerein (Fig. 1.3) belongs to anthraquinone usually prescribed for the treatment of knee or

hip osteoarthritis (Nguyen et al., 1994; Pelletier et al., 2000). In vitro (Martel-Pelletier et al.,

1998) and in vivo (Moore et al., 1998) study has shown that diacerein exhibits its action by

blocking the activity and also production of a protein, interleukin-1 β, which is involved in

destruction of cartilage and commencement of inflammation (Moldovan et al., 2000; Yaron et

al., 1999) whereas, NSAIDs act by interfering with the synthesis of prostaglandin (Pelletier et

al., 1998). Diarrhea is the most common side effect of diacerein due to which daily dose of

diacerein is adjusted to half of the normal dose for first 14 to 28 days of the treatment. After

absorption, diacerein is deacetylated to rhein which is an active metabolite (Debord et al.,

1994).

32

Fig. 1.3. Structure of diacerein.

In a 16 weeks study on 484 patients of knee osteoarthritis, effective and safe dose of

diacerein was found to be 50 mg twice daily (Pelletier et al., 2000). A study was conducted to

observe the effect of diacerein on the degenerative changes in cartilage in osteoarthritis.

Results have proved that diacerein reinforced the repairing process and improved the

condition of joints (Hwa et al., 2001).

To increase the absorption of diacerein after oral administration, gastroretentive and

immediate release formulations were developed and compared with the already marketed

product (Mandawgade et al., 2016). Dissolution profile confirmed the immediate release of

diacerein and in vivo trial in healthy humans showed 1.2 fold and 1.7 fold increase in AUC0-

6h for gastroretentive and immediate release formulation, respectively.

Safety and efficacy of diacerein and piroxicam in the treatment of knee osteoarthritis was

evaluated and results are compared with each other. Piroxicam (20 mg/day) and diacereine

(100 mg/day) were administered to the patients for 16-weeks. At the end of study, diacerein

was found as effective as piroxicam in reducing pain but former has better safety profile

(Louthrenoo et al., 2007). Symptomatic efficacy of diacerein in treating osteoarthritis was

also observed by Bartels et al. during 6 month study. Diacerein was found safe and effective

against osteoarthritis and those patients can also take this medicine having problems with

NSAIDs (Bartels et al., 2010). For the treatment of osteoarthritis knee, combination of

33

diacerein and diclofenac sodium was used and evaluated the efficacy (Singh et al., 2012).

Diacerein and diclofenac sodium were given in a single dose of 50 mg and 75 mg per day,

respectively. Improvement in osteoarthritis knee was observed more rapidly when treated in

combinations of diacerein and diclofenac sodium than to the treatment with only one drug.

Niosomes based drug delivery system was developed for Diacerein to increase its dissolution

and also sustained the release the release of drug (Khan et al., 2015). Formulations were

designed with cholesterol and sorbitan monostearate with varying the ratios using reverse-

phase evaporation technique. Release studies have shown that niosomes sustained the release

of diacerein for 10 h and followed the zero order kinetics. Furthermore, non-Fickian and

anomalous transport mechanism was followed for the diacerein release.

Sustained release formulation of diacerein was developed using HPMC as a release retarding

agent (Meyyanathan et al., 2014). Drug release data was evaluated by kinetic models. Drug

release from the prepared tablets followed the first order kinetics and governed by non

Fickian diffusion. Pharmacokinetic parameters proved the sustained release of diacerein from

matrix tablet with increase in half-life.

Effectiveness of diacerein, glucosamine and NSAIDs in the treatment of osteoarthritis knee

was compared and results indicated that both glucosamine and diacerein are equally effective

but the former has fewer side effects (Kongtharvonskul et al., 2015).

Newly designed solid lipid nanoparticles were developed for the effective and sustained

delivery of diacerein with and without the loading of gold nanoparticles using solvent

emulsification-evaporation technique, hot melt encapsulation and microemulsification

method (Rehman et al., 2015). In vitro drug release study has shown the sustained release of

diacerein for three days through diffusion control mechanism. Release data followed the

Higuchi model and zero order kinetics.

34

1.2.2. Polysaccharides based NPs as anticancer drug delivery system

Nanoparticles are nanocarriers of different shapes and morphologies having the size range

between 1 to 1000 nm (Jung et al., 2000). Now a day, NPs are being used to deliver different

drugs, vaccines, genes and hormones (Pan-In et al., 2014; You and Peng, 2004; Zahoor et al.,

2005). Different materials (synthetic, semisynthetic or natural) and various methods (ionic or

covalent crosslinking, complexation or self-assembly of hydrophilic/hydrophobic groups

after modification) are being employed to synthesize NPs of desired sizes and according to

therapeutic requirements (Liu et al., 2008). Among these materials, polysaccharides have

gained the interest of researchers due to their biodegradable, biocompatible, non-toxic and

non-irritant nature (Lemarchand et al., 2004). Polysaccharides are obtained from various

sources, i.e., animal (chondroitin, chitosan), microbial (xanthan gum, dextran), plant (guar

gum, pectin) and algae (alginate) (Sinha and Kumria, 2001). Moreover, naturally occurring

polysaccharides are abundant in nature and recognized as highly stable, safe and cheap.

Mostly, naturally occurring polysaccharides are hydrophilic due to the presence of amino,

carboxyl and hydroxyl groups which play an important role in the formation of weak bonding

with biological tissues resulting in bio-adhesion and prolonged residence time (Lee et al.,

2000).

Polysaccharides are used to synthesize the drug loaded NPs for the treatment as well as

diagnostic purpose in the management of different tumours. Stable dextran based NPs with a

functional carboxylic acid group was synthesized using graft copolymerization method (Dou

et al., 2005). Reaction was carried out without any organic solvent or surfactant and initiated

from water soluble monomer. These NPs have various potential applications due to the

addition of acidic functional group.

35

Chitosan, a diverse polysaccharide, investigated extensively as a carrier for many drugs and

chemicals for diagnosis and treatment of many diseases. It has been used as a carrier for drug

delivery, cell imaging and gene delivery (Shukla et al., 2013). Chitosan NPs were synthesized

to encapsulate docetaxel and evaluated its efficiency on cancer cell line (Lozano et al., 2013).

Chitosan based NPs were prepared by solvent displacement method with 78% encapsulation

efficiency of docetaxel. Further study has proved the integrity of NPs and also the effective

intracellular delivery of docetaxel. Sustained release of doxorubicin was made possible from

chitosan NPs by Janes and coworkers (Janes et al., 2001). Similarly, paclitaxel and

doxorubicin were also delivered by chitosan NPs which made them therapeutically more

effective and less toxic (Hwang et al., 2008; Mitra et al., 2001). Nanocapsules of chitosan

were used by Lozano et al., for the intracellular transport of docetaxel in A549 and MCF-7

cell lines of human lung carcinoma and breast adenocarcinoma, respectively (Lozano et al.,

2008).

Effect of chitosan NPs were observed on the growth of hepatocellular carcinoma (Qi et al.,

2007). Chitosan NPs were evaluated by TEM, MTT assay, flow cytometry, electrophoresis,

GC/MS, spectrophotometric thiobarbituric acid (TBA) assays. After oral administration of

chitosan NPs in nude mice, size of tumour was measured periodically and morphological

changes in liver was also studied under electron microscope.

Core shell NPs was designed for doxorubicin delivery in which bovine serum albumin

conjugated chitosan act as a core and dextran conjugated chitosan as a shell (Qi et al., 2010).

Prepared NPs are highly stable having diameter of 130-230 nm. Doxorubicin is loaded in NPs

by virtue of hydrophobic and electrostatic interaction between drug, dextran and chitosan.

These NPs have shown less adverse effects of doxorubicin and increase the survival rate of

tumor bearing mice.

36

Docetaxel, an anticancer drug, belongs to taxoid family which is being used for gastric

(Cutsem, 2004), breast (Palmeri et al., 2008), pancreatic (Androulakis et al., 1999) and

urothelial carcinoma (Yafi et al., 2011). Docetaxel is an analog of paclitaxel and an inhibitor

of microtubule polymerization.

Fig. 1.4. Structure of docetaxel.

Biocompatible and biodegradable polymer, polylactic-co-glycolic acid (PLG), was used to

synthesize docetaxel loaded NPs by solvent evaporation. Small size NPs were obtained by

varying the experimental conditions. Release of docetaxel from NPs is dependent on the type

of surfactants used for the synthesis of NPs. Results indicated that PLG NPs can be used for

prolonged release of docetaxel (Keum et al., 2011). Core shell NPs was synthesized for

loading of docetaxel in which mannitol core was protected by PLG shell (Tao et al., 2013).

These NPs have proved antitumor activity against breast cancer.

In order to reduce the toxicity of docetaxel, self-assembly process was used to fabricate

docetaxel loaded albumin nanoparticles with a mean diameter of 150 nm (Tang et al., 2015).

In vivo study has shown better results as compared to Taxotere® in term of hemolysis,

tolerance, EPR effect and antitumor ability.

37

In another study, docetaxel loaded dendritic nanoparticles were prepared and used to evaluate

the in vivo antitumor activity against human breast cancer cell lines (Zhang et al., 2014). This

new type of drug delivery system has shown a promising application as a chemotherapeutic

agent. In order to evaluate the effect of NPs on pharmacokinetic parameters of docetaxel, two

different drug loading NPs were fabricated using soft-lithography technique (Chu et al.,

2013). Plasma concentration and antitumor activity of 9% docetaxel loading NPs was higher

than 20% drug loading NPs.

Poly(lactide-co-caprolactone) and poly(lactide-co-glycolide-co-caprolactone) based

docetaxel loaded NPs were prepared and evaluated for the treatment of prostate cancer

(Sanna et al., 2011). These NPs demonstrated better release properties of docetaxel with

minimum side effects as compared to pure docetaxel.

1.2.3. Polysaccharides mediated synthesis and application of Ag NPs

NPs have gained attraction of many scientists over the last two decades. NPs usually fall in

the range from 1 to 100 nm and their synthesis and application is an emerging scientific field.

There are different physical and chemical methods for the synthesis of NPs which include

photochemical reduction, electrochemical treatments, chemical reduction etc. (Frattini et al.,

2005). Now a day it is possible to synthesize NPs with high stability, longer shelf life, desired

shapes and morphology by adopting different techniques and chemicals (Knoll and

Keilmann, 1999; Sengupta et al., 2005).

Silver is used for the synthesis of ethylene oxide and formaldehyde as a catalyst (Nagy and

Mestl, 1999). Due to its good conductivity, antibacterial activity and chemical stability, it is

widely used in chemical industries (Frattini et al., 2005).

38

There are numbers of reducing agents used for the preparation of silver nanoparticles (Ag

NPs) that include ascorbate, borohydrate, elemental hydrogen and citrate (Lee and Meisel,

1982; Shirtcliffe et al., 1999; Nickel et al., 2000). A cost effective, facile and environment

friendly synthesis of Ag NPs is accomplished in three stages; solvent selection, reducing

agent selections and stability agent for Ag NPs (Raveendran et al., 2003).

Polysaccharides can serve as both capping and reducing agent for the synthesis of Ag NPs. In

starch mediated synthesis of Ag NPs, starch was used as a stabilizing agent while β-D-glucose

played its role as a reducing agent and mixture was exposed to gentle heating. A highly

spherical and monodisperse Ag NPs with diameter range of approximately 20 nm was

synthesized with environment benign material, i.e., starch (Raveendran et al., 2003).

Starch was also used as capping or reducing agent by many researchers while changing other

conditions. Ag NPs can be synthesized by keeping the solution of starch and silver nitrate in

autoclave at 121 °C and 15 psi pressure for 5 min. By this process, spherical shaped NPs in

the range of 10-34 nm was synthesized and can be stored for 3 months even in solution form

at room temperature (Vigneshwaran et al., 2006). Starch was also used to synthesize smaller

NPs with less than 10 nm in diameter (Tai et al., 2008). Solution of NaOH, starch and

glucose was used as an accelerator, capping agent and reducing agent, respectively. Mixture

of all three reagents was kept in spinning disk reactor for not more than 10 min to get these

smaller sized NPs.

Microwave irradiation was employed on the solution of silver nitrate and carboxymethyl

cellulose (CMC) sodium for the synthesis of NPs of uniformed size and with stability for at

least 2 months at ambient temperature (Chen et al., 2008). Soluble starch and basic amino

acids were used as protecting and reducing agents, respectively for the preparation of Ag NPs

under the influence of microwave radiations (Hu et al., 2008). CMC was synthesized from

39

cotton sample and used for the preparation of Ag NPs (Hebeish et al., 2010). Effect of

reaction temperature, pH of solution, concentration of CMC and silver nitrate and exposure

time on the synthesis of Ag NPs was noted. Colloidal suspension of Ag NPs was produced by

increasing the concentration of the reactants.

Oligochitosan was used as a stabilizer in the synthesis of small and biocompatible Ag NPs

when exposed to gamma radiations. Synthesized Ag NPs were in the range from 5-15 nm.

These NPs were stable between pH 1.8-9.0 and also in NaCl solution. Aggregation of Ag NPs

was observed in solution of NaNO3 and NaH2PO4 (Long et al., 2007). Solution of acetic

water, chitosan and silver nitrate were placed in front of gamma radiation to synthesize Ag

NPs. In depth analysis revealed that the size of NPs were found in the range of 4-5 nm which

were protected by chitosan chains (Chen et al., 2007b).

Arabinoxylan, isolated from seed husk of isphagula, a gel forming branched polysaccharide

were used as a reducing and stabilizing agent for the synthesis of Ag NPs. Arabinoxylan was

suspended in distilled water, mixed with silver nitrate solution and kept just below 100 °C

under continuous stirring. Change in color of the mixture was observed within 60 min

indicating the synthesis of Ag NPs. Particle size of the synthesized NPs were determined by

TEM and found in the range of 5-20 nm. Reaction mixture temperature and time,

concentration of arabinoxylan and pH of the mixture are the main determining factors for size

of NPs (Amin et al., 2013).

Gum kondagogu (Cochlospermum gossypium) was used as a reducing and stabilizing agent to

facilitate the synthesis of spherical silver nanoparticles (3 nm). Raman and FTIR

spectroscopy were used to study the mechanism of stabilization and reduction process. Ag

NPs were characterized by TEM, XRD, TGA and UV spectroscopy. These synthesized Ag

40

NPs have shown antibacterial activity against Gram positive and Gram negative bacteria

(Kora et al., 2010).

Polysaccharide (glucoxylan) was separated from the seeds of Mimosa pudica and used as a

reducing and capping agent for the green synthesis of 6 nm sized Ag NPs and 40 nm gold

NPs (Iram et al., 2014). Phyto-toxicity study of synthesized NPs was performed on the

germination of radish seed and did not found any significant effect.

A mixture of aqueous gum acacia and silver nitrate solution was exposed to gamma radiation

for the synthesis of Ag NPs. As a result, NPs of different sizes were obtained by changing the

intensity of radiation. The morphology of Ag NPs was evaluated by TEM, XRD and dynamic

light scattering (DLS). Bonding between NPs and COO─ group present on gum acacia was

confirmed by FTIR (Rao et al., 2010). Sodium alginate solution, as a reducing agent, was

used for the synthesis of Ag NPs in the presence of gamma radiation. NPs were retained their

integrity for 6 months at room temperature (Liu et al., 2009).

Nanocomposite of chitosan with silver and other metals were synthesized in the presence of

NaBH4. The synthesis of NPs was verified by UV spectroscopy and TEM analysis (Huang et

al., 2004).

A facile, cost effective and green synthesis of Ag NPs can be achieved by using gum acacia

as a reducing agent. Aqueous solution of gum acacia and silver nitrate was mixed and placed

at room temperature for 24 h. Formation of Ag NPs was confirmed by observing the change

in color and UV spectroscopy. Further, the shape and size of NPs were determined by XRD

and TEM analysis. These NPs were found stable for 5 months (Mohan et al., 2007).

Chitosan as a polymeric stabilizer was used to synthesize Ag NPs in the presence of PEG and

silver nitrate at 60 °C. Newly formed nanocomposite was characterized through UV-Vis

41

spectroscopy, TEM, FTIR and XRD. Effect of stirring time on the diameter of Ag NPs was

evaluated and it was found that the size of Ag NPs was dependent on the stirring time i.e.,

size increased with increase in stirring time (Ahmad et al., 2011).

Silver nitrate was reduced to form silver nanoparticles in the presence of chitosan. Ag NPs

bounded chitosan solution was evaporated under mild heating condition to form a thin film.

Antibacterial activity of this film was evaluated against different bacteria (Wei et al., 2009).

Dextran was used as a reducing and capping agent to synthesize Ag NPs (Bankura et al.,

2012). Aqueous solution of dextran and silver nitrate was mixed and after addition of NaOH

solution (0.001 M) at room temperature, color change of the solution was observed

considering a sign of the reduction of silver ion into metal. Formation and morphology of Ag

NPs was verified by UV-vis spectroscopy, TEM, XRD and AFM. These NPs was tested

against five different bacteria and inhibition of bacterial growth was found to be inversely

proportional to the concentration of Ag NPs.

Chitosan film was synthesized with embedded Ag NPs using thermal treatment. Dendritic

structure was observed in the chitosan-Ag NPs film (Dongwei et al., 2009). Further

investigation proved its application in surface-enhanced Raman spectroscopy.

Recently, a new approach was adopted to synthesize core-shell nanoparticles in which Ag

NPs and Au NPs acted as core and shell, respectively (El-Naggar et al., 2016). In this reaction

process, curdlan was used as a reducing and capping agent, AgNO3 and HAuCl4 as precursor

for silver and gold and microwave radiation as a source of energy. Formation and

characterization of core-shell NPs was determined by UV-vis spectroscopy, TEM, zeta

potential analysis, XRD, FTIR and atomic force microscopy (AFM). Spherical shaped core-

shell NPs with average diameter of 45 nm was obtained.

42

Ag NPs embedded chitosan film was synthesized by photochemical induce reduction of silver

nitrate in the presence of chitosan solution (Thomas et al., 2009). Film was evaluated by

TEM, TGA and XRD. Due to its antibacterial potential, it can use as wound healing dressing,

antimicrobial packaging material and biomedical implants.

Aqueous solution of hydroxypropylcellulose and silver nitrate was placed in sunlight to

induce the formation of Ag NPs (Hussain et al., 2015). Synthesized NPs were analyzed by

UV-vis spectroscopy, TEM and XRD. Ag NPs embedded HPC film was evaluated through

SEM and AFM. These Ag NPs have shown antimicrobial activity against bacteria and fungi.

The embedded Ag NPs HPC film can be stored for more than one year without any

morphological changes in NPs.

Dialysis process is used to synthesize the Ag NPs of 6 nm in diameter. HPC, ethylene glycol

and silver nitrate were used as a capping agent, solvent support and silver precursor,

respectively (Francis et al., 2010). Different concentrations of silver nitrate were used to get

optimized Ag NPs. After getting through dialysis process, the UV-visible spectrum shifted

from 410 nm to 440 nm indicating the completion of reduction process. After drying the

resulting solution at 80 °C, HPC capped Ag NPs were characterized by TEM, XRD and

FTIR. Skewed distribution of Ag NPs was observed in TEM analysis.

Hydroxypropyl cellulose was prepared by etherification reaction and used for the synthesis of

Ag NPs. Reaction mixture having HPC and silver nitrate solution was kept for 90 min at 90

°C to complete the formation of Ag NPs (Abdel-Halim and Al-Deyab, 2011).

Hydroxypropyl carboxymethyl cellulose (HPCMC) was used to prepare Ag NPs (Abdel-

Halim et al., 2015). Formation of Ag NPs is dependent on the degree of substitution of

HPCMC, concentration of silver nitrate, pH of the reaction medium, reaction time and

temperature.

43

Like other polysaccharides, dextran was used as a stabilizing and capping agent in the

synthesis of Ag NPs (Hussain et al., 2014). Spherical shaped Ag NPs of 50-70 nm in

diameter were obtained when aqueous solution of dextran and silver nitrate were placed in

sunlight. Formed particles were evaluated through TEM, PXRD, SEM, AFM and also

determined the antimicrobial activity.

1.2.4. Polysaccharides based antiseptic dressing

Wound healing is mainly a four phase process which includes inflammation, tissue

regeneration, matrix remodeling and reepithelialization (Cochrane et al., 1999). Chitosan, a

N-deacetylated polysaccharide, was used as a wound healing agent (Kojima, et al., 1998).

Hyaluronic acid, composed of N-acetyl-glucosamine and D-glucuronic acid, has known for its

lubrication, water retention capacity and cell proliferation (Bulpitt and Aeschlimann, 1999;

Hu et al., 2003). Xu et al. used chitosan and hyaluronic acid to fabricate a composite film as a

wound healing dressing and in vivo animal study also has proved its effectiveness (Xu et al.,

2007).

Aloe vera leaf gel has shown wound healing ability when applied topically as well as through

systemic administration. Wound healing ability of Aloe vera gel mainly depends on the

stability of the active constituents which is affected by gel extraction time after harvesting.

Different mechanisms of wound healing from Aloe vera gel were suggested including

migration of epithelial cells, reduction in inflammation, wound moistening and collagen

maturation (Reynolds and Dweck, 1999). Glycoprotein (5.5 kDa), isolated from Aloe vera

has shown excellent wound healing ability in hairless mice (Choi et al., 2001). Effect of Aloe

vera gel was studied on the formation of fibrous tissue, synthesis of collagen and contraction

of wound (Chithra, et al., 1998).

44

Many plants are being used in the treatment of wound. Polysaccharides extracted from

Opuntia ficus-indica L. cladodes were evaluated for their potential as a wound healing agent.

For this purpose, two polysaccharide fractions (low and high molecular weight) were isolated

and applied on the wound of rats. Results have indicated that low molecular weight fraction is

better in wound healing than high molecular weight fraction (Trombetta et al., 2005).

Wound dressing comprising carboxymethylcellulose and alginate was evaluated for their

effectiveness as engulfing and immobilization agent of two bacteria (Pseudomonas

aeruginosa and Staphylococcus aureus) present in wound. SEM examination has shown that

due to viscous nature of CMC gel, it was found a better wound healing agent than alginate gel

(Walker et al., 2003).

Alginic acid in salt form is used in wound dressing due to its high absorbency, gelling and

hydrophilic nature. Upon contact with exudate of wound, ionic interaction takes place

between the calcium ions of alginate and sodium ions present in serum. As a result, a

swellable protective film of alginate is formed (Thomas, 2000). Ionic crosslinking of alginate

makes it an ideal polymer gel for wound healing treatment and tissue engineering (Kuo et al.,

2001; Wang et al., 2003). It was also found that due to slow degradation, alginate wound

dressing is more beneficial than other hydrocolloids (Ichioka et al., 1998).

Calcium alginate has shown some pharmacological activities to speed up the healing process

including proliferation of fibroblast (Doyle et al., 1996), stimulate synthesis of tumour

necrosis factor-α (Thomas et al., 2000) and helping in clotting (Blair et al., 1990). Alginate

wound dressing is biodegradable (Gilchrist and Martin, 1983) and hence used in the

preparation of surgical sutures.

45

Hydrogel dressings are nonreactive, nonadherent and nonirritant to biological tissues. These

dressing produce the cooling effect and reduce the sensation of pain hence increase patient

compliance (Wichterle and Lim, 1960; Moody, 2006).

Aqueous solution of photocrosslinkable chitosan has the ability to convert into an elastic and

insoluble hydrogel in no time (Ishihara et al., 2002). This unique ability of chitosan gel was

used on the mouse whose skin was sliced in two pieces. After UV irradiation on the applied

crosslinked chitosan solution for 90s, the rate of wound healing and contraction was

observed. Histological evaluation also confirmed the epithelialization and tissue formation at

the wound site which make this crosslinked chitosan gel as an excellent dressing in

emergency situations.

Extracted polysaccharides from plants are used in wound healing dressings. Polysaccharides

from Bletilla striata (BS) were crosslinked and evaluated as a wound healing agent in mouse

model. For this purpose, BS hydrogel dressing is applied on the wound and healing process

was found to be fast as compared to vaseline and iodine gauze (Luo et al., 2010).

Polysaccharides were separated from the fruit of Phellinus gilvus (PG) by hot water

extraction process. Different concentrations of polysaccharide solution were applied on the

surgically induced skin wounds and Madecassol® was used for comparison. Wound

contraction diameter and rate of reepithelialization were determined periodically. At the end

of study, it was found that PG has significantly good results as compared to Madecassol®

(Bae et al., 2005).

Seed husk of psyllium was used to extract psyllium hydrogel and then in the preparation of

wound dressing film (Patil et al., 2011). Psyllium hydrogel film was loaded with Povidone

iodine and undergo for in vitro and in vivo evaluation. Psyllium dressing was found to have

antimicrobial activity against different bacteria which was usually present in wounds. After

46

applying on the wound of rat, psyllium dressing has shown better results in terms of wound

closure as compared to Band aid®.

Chitin was used to prepare an antiseptic film which was evaluated as a wound healing

material (Yusof et al., 2003). A real wound was created on the dorsal side of rat and covered

with chitin films of different concentrations. Commercially available wound dressings,

Opsite™ and Easifix™, were used as a control. A rapid healing was observed in chitin film

treated wound as compared to commercially available dressings. Moreover, chitin film

dressing was transparent, durable, biodegradable and non-fragile.

Chitosan was used as a reducing agent in the synthesis of silver nanoparticles (Li et al.,

2013). Mixture of chitosan, silver nanoparticles and poly (vinyl alcohol) was used to prepare

a fibrous mat as a wound healing dressing by electrospinning technique. This composite

material has shown antibacterial activity against E. coli and S. aureus. In vitro and in vivo

study of this composite has proved its effectiveness as wound healing material.

Dextran and poly (vinyl alcohol) was crosslinked to form a hydrogel by freezing-thawing

method (Hwang et al., 2010). Gentamicin was loaded in the hydrogel and this hydrogel film

was placed on the wound of rats and served as a wound healing dressing. Hydrogel film was

evaluated through swelling ratio, water vapor transmission test, mechanical properties,

morphology and thermal analysis. In vivo wound healing analysis confirmed gentamicin

loaded hydrogel film as an excellent wound healing dressing with improved patient

compliance.

Gamma radiation was employed for the synthesis of crosslinked chitosan/poly (vinyl alcohol)

hydrogel (El Salmawi, 2007). After successful evaluation of physical parameters of hydrogel

film, wound healing study was conducted. Hydrogel film was found a physical barrier for the

penetration of microorganisms and hence enhances wound healing.

47

Natural polymers were blended with PVA to improve the mechanical strength and

physicochemical properties of the natural hydrogel. Alginate being biocompatible and

hydrophilic in nature is used in biomedical field. Nitrofurazone was incorporated in hydrogel

composed of sodium alginate and PVA which were crosslinked by freeze-thawing process.

Size reduction of wound was seen after treating with sodium alginate/PVA hydrogel

indicating a new wound healing dressing material (Kim et al., 2008). Another method,

electrospinning, was employed for the synthesis of PVA and calcium alginate hydrogel which

was further used as wound healing dressing. In vivo study on rat has shown the formation of

epithelium without any adverse effect (Tarun and Gobi, 2012).

1.3. Characterization techniques

Polysaccharides are characterized by using various analytical, microscopic and spectroscopic

techniques.

1.3.1. Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is used to characterize the different

functional groups present in a molecule. For solid material, pellet was prepared with the

mixture of KBr and the sample. Dried pellet was scanned through 4000-400 cm-1

in

transmittance or absorbance mode. Absorption peaks obtained are as a result of different

frequencies of bond‘s vibrations. Each functional group has its own characteristic frequency

of vibration. Due to advancement of technology, FTIR is used for both quantitative and

qualitative analysis (Griffiths and de Haseth, 2007).

1.3.2. Nuclear magnetic resonance spectroscopy

To determine the structure of a molecule, nuclear magnetic resonance (NMR) spectroscopy is

proved to be a power full tool. Proton NMR (1H NMR) spectroscopy is used to find the

48

structure of a molecule at the level of hydrogen-1 nuclei. Structure of polysaccharides is

evaluated by 1H NMR and more precisely by Heteronuclear single-quantum correlation

(HSQC) and total correlation spectroscopy (TOCSY). These spectroscopic techniques are

used to investigate the proton and carbon of polysaccharides (Martin and Zektzer, 1988; Lane

and Lefèvre, 1994).

1.3.3. Thermal analysis

Thermal analysis of polymer was performed to find the thermal degradation pattern, thermal

stability, glass transition temperature (Tg) and activation energy (Ea). Thermal analysis can

give the information of the stability of polymer, storage temperature and shelf life of the

polymer if used in dosage forms (Coats and Redfern, 1963). Different methods were

employed to study the kinetics of thermal degradation process. Activation energy, reaction

order (n) and frequency factor (Z) were calculated by using kinetic models. Change in

entropy (∆S), Gibbs free energy (∆G) and enthalpy (∆H) were also determined through

established methods (Evans and Polanyi, 1935).

1.3.4. Electron microscopic analysis

Analysis through scanning electron microscope (SEM) and transmission electron microscope

(TEM) is performed to observe the surface of dried form of polymer after its extraction as

well as the cross section of swollen then freeze dried sample of the polymer. Sample was

placed in front of a beam of high energy electrons whose intensity was controlled with

various mechanisms. Image was received as a result of interaction between these electrons

and the atoms on the surface of sample (Clarke and Eberhardt, 2002). Transmission electron

microscopy (TEM) was used to examine the very small specimen. TEM is very powerful and

capable to observe even a single column of atoms.

49

1.3.5. Powder X-ray diffraction

X-ray spectroscopy is widely used for the characterization of all forms of materials to get the

geometric and electric structure (Guo, 2009). For the measurement of X-ray diffraction

(XRD), sample was placed on goniometer and X-rays was bombarded on the sample. As a

result, reflections or diffraction pattern was recorded as a regularly spaced spots (Kumirska et

al., 2010). Clark and smith were the first scientists who used XRD to investigate the structure

of chitin and chitosan in 1936 (Clark and Smith, 1936).

1.3.6. MTT assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a yellow coloured,

water soluble dye which is reduced to purple coloured formazan, a water insoluble material,

by live cells. MTT assay is a homogeneous colorimetric assay used to assess the cell

metabolic activity (Mosmann 1983). During this assay, MTT after entering into cells passes

the mitochondria and reduce to formazan which is an insoluble product. Cells are further

solubilized in an organic solvent and formazan is measured spectrophotometrically as the

reduction of MTT take place in metabolically active cells, therefore, the detection of activity

is directly related to the viability of the cells. If cell dies, the ability to reduce MTT into

formazan will also losses and is evident from the color of the cell. MTT reduction might be

due to the reducing molecule (NADH) that donates electrons to MTT (Marshall et al., 1995).

1.3.7. Drug release models

Release of drug from sustained release formulation depends mainly on the physicochemical

properties of the polymeric materials used as a release retarding agent.

1.3.7.1. Zero order

50

Slow and continuous release of drug from a controlled drug delivery system which does not

disintegrate or erode is explained by zero order release model (Eq. 1).

(Eq. 1)

where, K0 is the zero order rate constant which can be expressed as concentration per unit

time and Qt is the amount of drug released from the tablet in time t.

To calculate and study the zero order drug release kinetics, a graph is plotted between the

cumulative drug release and time (Narashimhan et al., 1999). Zero order kinetics is used to

describe mechanism of the continuous release of drug from a dosage form at a constant rate

which usually occurs in matrix system having less soluble drugs, osmotic drug delivery

systems and some transdermal systems (Yang and Fassihi, 1996; Freitas and Marchetti,

2005).

1.3.7.2. Firsst order

First order release model is used to explain the release of soluble drug from a swellable

polymeric material (Bravo et al., 2002) and expressed using Eq 2.

(Eq. 2)

where, Q is the residual concentration of drug in tablet after time t, Q0 is the initial loaded

amount of drug in tablet and K1 is the first order rate constant (Gibaldi and Feldman, 1967;

Wagner 1969).

Drug release data is plotted as a log of cumulative percentage of drug remaining in the tablet

against time which is expressed as a straight line having slop of –K/2.303. This model is

applied to explain the dissolution profile of water soluble drugs from porous matrices

(Narashimhan et al., 1999).

51

1.3.7.3. Higuchi model

In 1961 and 1961, Higuchi was the first scientist who proposed a mathematical model to

explain the release of water soluble and poorly soluble drug from semisolid and solid

pharmaceutical dosage forms. Initially, he described the drug release behaviour from a planar

system but later on from porous system and different geometrical shapes. Higuchi model was

derived to consider some assumptions that (i) in a matrix system, the drug concentration is

higher than its solubility in the media, (ii) diffusion of drug from the matrix takes place

unidirectional, (iii) particles of drug are than the thickness of system, (iv) matrix dissolution

and swelling is negligible, (v) rate of drug diffusion remains constant, (vi) perfect sink

condition of the system is maintained. Furthermore, Higuchi modified the model and covered

the system in which the concentration of drug in a matrix is less than its solubility. In this

case the release of drug occurs through the pores and channels of the matrix. Eq. 3 is the

simplest form of Higuchi model (Higuchi, 1961; Higuchi, 1963).

(Eq. 3)

where, Qt is the amount of drug released in time t and KH is the Higuchi rate constant.

In Higuchi model, cumulative drug release (%) was plotted against the square root of time.

Drug dissolution mechanism from transdermal drug delivery system and tablet formulation

containing polymeric matrix can be described by using Higuchi model (Grassi and Grassi,

2005).

1.3.7.4. Hixson-Crowell model

Hixson and Crowell (1931) described an equation showing that the particles‘ regular area is

proportional to the cube root of its volume as shown in Eq. 4.

52

(Eq. 4)

where, Q0 is the initial amount of drug in dosage form, Qt is the amount of drug released

from the dosage form in time t and KHC is the Hixson-Crowell release constant describing

surface-volume relation (Hixson and Crowell, 1931). This equation explained the release of

drug from the dosage form with respect to its surface area and diameter. Drug release data

was plotted as cube root of drug concentration remaining (%) versus time. This model

applies on the dosage form especially tablets when the dissolution process proceed in a plane

which is parallel to the surface of drug and dimensions/geometric shape of tablet remain the

same (Chen et al., 2007).

1.3.7.5. Korsmeyer-Peppas model

Korsmeyer derived an equation explaining the drug release mechanism from a polymeric

drug delivery system. For Korsmeyer-Peppas model, first 60% drug release data was

considered (Korsmeyer et al., 1983) and expressed in Eq. 5.

(Eq. 5)

where, Mt/M∞ is the fraction of drug released at time t and Kp is the Korsmeyer-Peppas

constant. n is the release exponent and its value is calculated from the slop of a graph plotted

between log Mt/M∞ and log t. The value of n indicates the drug release mechanism from a

polymeric matrix system. For a cylindrical (tablet) drug delivery system, release mechanism

is considered as Fickian diffusion (n ≤ 0.45), non-Fickian diffusion (0.45 < n < 0.89), case II

transport (n = 0.89) and super case II transport (n > 0.89) (Korsmeyer et al., 1983; Ritger and

peppas, 1987).

53

1.4. Background and significance of the study

Drug delivery systems usually require more than one ingredient/excipient for effective

delivery of therapeutic agents. These ingredients/excipients are synthetic, semisynthetic or

naturally occurring biomaterials. Due to chemical nature/composition of synthetic and

semisynthetic materials, there is always a chance that on administering, they may stimulate or

initiate the immune system of the body and may start biorejection process. Therefore, the

researchers are switching towards naturally occurring materials due to their biocompatability,

biodegradability, nonimmunogenicity and easy availability. Among naturally occurring

biomaterials, polysaccharides based materials are most important and are being used in

conventional as well as in novel and smart drug delivery systems. Furthermore, swellable

(hydrogellable) polysaccharides based materials have wide range of applications in sustained

and targeted drug delivery systems to improve the bioavailability of therapeutic agents. Due

to immense importance and unique properties of polysaccharides based material, it is utmost

important to introduce novel naturally occurring biomaterials especially water swellable and

pH sensitive polysaccharides as novel drug delivery carriers. Therefore, hydrogellable,

stimuli responsive linseed polysaccharides are one of the most important entities having great

potential for intelligent drug delivery systems.

54

1.5. Aims and objectives

Aim of this research was to evaluate a naturally occurring polysaccharide LSH for

pharmaceutical and biomedical applications. Aims were to study swelling kinetics of LSH in

deionized water and at different physiological pH. Our interests were focused on stimuli

responsive swelling-deswelling (on-off switching) in different solvents, at various ionic

concentrations and pH. Micromeritic properties of LSH will also be taken in to account.

Potential application of LSH as a sustained release oral drug delivery system for caffeine,

diclofenac sodium and diacerein are in focus. Swelling characteristics and swelling-

deswelling response of these drug containing LSH matrix tablet formulations will also be

monitored in various environments. Drug release kinetics and mechanism will be determined

using various kinetic models in order to get deeper insight into the mechanism of drug

release.

Hydrogelable polysaccharides arrange themselves into defined morphologies after swelling in

aqueous media. Therefore, our interests were to study morphological analysis of LSH in

powder form and tablet formulations using SEM to see channeling/pores in the swollen then

freeze dried LSH and its tablets.

Acetylation of LSH will be performed to check if the polysaccharides are modifiable for

esterification reactions. The obtained structures will also be characterized. As the acetylation

may enhance the stability of polysaccharides, therefore our interests were to study thermal

analysis of LSH and acetylated LSH in order to compare their thermal stability.

Aim was focused on the formation of LSH nanoparticles for the loading of docetaxel, a

chemotherapeutic agent. Docetaxel loaded LSH nanoparticles will be evaluated for

encapsulation efficiency and drug loading capability. In addition, morphology of the obtained

NPs will also be evaluated by TEM and PXRD. For an effective delivery system of

55

anticancer drug, in vitro drug release behaviour, cytotoxicity analysis and cellular uptake

studies are also the topics of interest.

Furthermore, role of LSH in the green synthesis of Ag NPs is under investigation. Factors

affecting on the synthesis of Ag NPs and potential of LSH as a storage media for Ag NPs will

be investigated. Antimicrobial activity of Ag NPs embedded LSH film as a wound healing

agent will be tested.

Due to the potential application in oral and topical drug delivery system, it was necessary to

investigate the acute toxicity study of LSH on albino mice and rabbit therefore relevant

toxicological studies will be performed.

Summarizing, it can be stated that we are interested to report a new polysaccharides based

hydrogel for sustained oral drug delivery system, wound healing applications, green synthesis

of Ag NPs and nano-particulate carrier for chemotherapeutic agents.

56

2. MATERIALS AND METHODS

2.1. Materials

Linseeds were purchased from local market, cleaned from superfluous material by passing

through different meshes and stored at ambient temperature in air tight container. Ethanol, n-

hexane, KCl, NaCl, acetone, acetic acid, sodium acetate, CHCl3, formalin, diclofenac sodium

(DS), NaOH, iso propyl alcohol, potassium dihydrogen phosphate and HCl were purchased

from Riedel-de Haën, Germany. NaOH was standardized with oxalic acid before use.

Caffeine, acetic anhydride, DMSO, 4ʹ,6-diamidino-2-phenylindole (DAPI), DMAc, AgNO3,

DMSO-d6 and CDCl3 were purchased from Sigma-Aldrich, USA. Magnesium stearate and 4-

dimethylaminopyridine (DMAP) was obtained from BDH and Alfa Aesar, England,

respectively. Microcrystalline cellulose was procured from Fluka. Simulated gastric fluid

(SGF) and simulated intestinal fluid (SIF) were prepared without enzymes as mentioned in

United States Pharmacopeia (2010). Diacerein (according to the standard of European

Pharmacopoeia) and polyvinylpyrrolidone (PVP) K30 were obtained from Consolidated

Chemical Laboratories Pvt. Ltd., Pakistan. Nylon mesh was used to separate the hydrogel

from linseeds. Pluronic F-68 (Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene

oxide)) triblock copolymer (Mw = 8350; (EO)79(PO)28(EO)79) was purchased from BASF

Corp., Republic of Korea and used as received. Anhydrous form of docetaxel (DTX) and

Tween 80 were obtained from Parling Pharma Tech Co., Ltd. (Shanghai, China) and Sigma

(St. Louis, MO, USA), respectively. Deionized water was used during the whole research

work.

57

2.2. Measurements

2.2.1. Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) was used to characterize linseed hydrogel

(LSH), acetylated LSH (ALSH) and to determine the compatibility of the excipients with

active ingredient in a tablet formulation. Potassium bromide (KBr) was used to prepare the

pellets of the sample and dried at 50 °C for 2 h in vacuum oven before recording the spectra

on FTIR instrument IR Prestige-21 (Shimadzu, Japan).

2.2.2. 1H NMR spectroscopy

1H NMR spectra of LSH were recorded on Avance 600 MHz spectrometer equipped with a

triple-resonance RT probe (Bruker, Billerica, MA, USA). Deuterated solvents (DMSO-d6,

CDCl3) were used to prepare the LSH and ALSH sample (10 mg/mL). The 1H NMR spectra

were processed using TopSpin software.

2.2.3. Heteronuclear single quantum correlation spectroscopy

Heteronuclear single quantum correlation (HSQC) spectra were recorded on Bruker Avance

600 MHz spectrometer and for data analysis, TopSpin software was used. In HSQC

spectroscopy, proton and 13

C spectrum were presented along x-axis and y-axis, respectively.

1H signal is measured in direct dimension while

13C signal is recorded in indirect dimension.

2.2.4. 1H

1H TOCSY NMR spectroscopy

Total correlation spectroscopy (TOCSY) was performed on Bruker Avance 600 MHz

spectrometer and obtained data was analyzed by using TopSpin software. In TOCSY, proton

spectrum was plotted against x-axis while 13

C spectrum was shown along y-axis. 1H and

13C

signals are measured in direct and indirect dimensions, respectively.

58

2.2.5. UV-Vis spectrophotometry

UV-Vis spectra were recorded on UV-1700 PharmaSpec (Shimadzu, Japan). Formation of

Ag NPs was observed through UV-Vis spectrophotometer. Release of drugs from LSH

matrix tablets were also monitored periodically through UV-Vis spectrophotometer.

2.2.6. Thermogravimetric analysis

Thermal decomposition temperature of LSH and ALSH were recorded on SDT Q600 thermal

analyzer (TA Instruments, USA) under nitrogen flowing (100 mL/min) at heating rate of 5,

10, 15 and 20 C/min from 35-800 C. Thermal degradation data was processed by using

Universal Analysis 2000 v 4.2E software.

2.2.7. Field emission scanning electron microscopy

Dried LSH was swollen in deionized water and freeze dried. Morphology of freeze dried

sample was observed under FE-SEM (JEOL JSM-6700F, Japan). For this purpose, samples

were dispersed in deionized water to obtain a solution of 0.1 wt%. Each solution was drop

casted on a carbon mount and dried at 25 °C in a vacuum oven for 24 h prior to analysis.

Texture and cross section area of prepared tablets were investigated using scanning electron

microscope (Quanta 250, FEI, USA).

2.2.8. Transmission electron microscopy

Morphology of the LSH mediated Ag NPs was evaluated by transmission electron

microscopy (TEM). Prepared Ag NPs were first separated by centrifugation and then

examined on a Philips 420 instrument. Sample was drop casted on carbon coated copper

wired TEM grid and instrument was operated at 120 kV. Similarly, Ag NPs embedded

LSH film was air dried and stored in a dark area for six months before analyzing by TEM.

59

In another experiment, stored LSH film impregnated with Ag NPs was dissolved in

deionized water. Ag NPs were isolated by centrifugation and viewed by TEM.

Morphology and particle size determination of Docetaxel loaded LSH nanoparticles were

evaluated using TEM. Freeze dried NPs (0.1 wt%) were dispersed in deionized water.

Solution (5 µL of aqueous solution containing NPs (1 mg/1 mL in distilled water)) was

dropped on the carbon-coated grid and kept it in a vacuum oven for 24 h at 25 °C. Prepared

sample was examined with microscope (Hitachi 7600) operated at 100 kV.

2.2.9. Powder X-ray diffraction

Powder X-ray diffraction (PXRD) spectra of LSH and LSH mediated Ag NPs were recorded

on Bruker D8 Advance (Germany) diffractometer (over a range of 10-80°, 2ϴ), operated at

40 mA and 40 KV.

2.3. Acetylation of linseed hydrogel

2.3.1. Acetylation of LSH

LSH was isolated using hot water extraction method (for isolation procedure, see section

2.4.1). To study the modification ability of LSH, acetylation reaction was selected as a novel

approach (Muhammad et al., 2016). Three reactions were carried out to observe the effect of

reactants on degree of acetylation of LSH. Acetylation reaction was performed using LSH

and acetic anhydride in mole ratio of 1:6 (ALSH 1), 1:12 (ALSH 2) and 1:18 (ALSH 3),

respectively.

ALSH 1:

In a typical reaction condition, LSH (1.0 g, 6.167 mmol) was suspended in DMSO (40 mL)

followed by the addition of acetic anhydride (3.5 mL, 37.002 mmol) using DMAP (40 mg) as

60

a catalyst. The suspension was heated at 80 °C with continuous stirring for 6 h. Acetylated

LSH (ALSH 1) was dried at 50 °C after precipitation and washing with ethanol.

Yield: (1.02 g); DS: 2.11; FTIR: 1749 (COEster), 2963 (CH), 1376 (CH2), 3440 cm-1

(OH),

1042 (COC); 1H NMR (DMSO-d6; 600 MHz; NS 64): 2.05 (CH3), 3.13-5.62 (Repeating unit-

Hs).

ALSH 2:

In another reaction, LSH (1.0 g, 6.167 mmol) was allowed to suspend in DMSO (40 mL).

Acetic anhydride (6.996 mL, 74.01 mmol) and DMAP (40 mg) were added in the suspension

of LSH. Reaction mixture was kept 80 °C with continuous stirring for 6 h. Acetylated

product (ALSH 2) was separated through precipitation in ethanol. ALSH 2 was washed with

ethanol and dried at 50 °C.

Yield: (1.12 g); DS: 2.53; FTIR: 1752 (COEster), 2936 (CH), 1373 (CH2), 3425 cm-1

(OH),

1043 (COC); 1H NMR (DMSO-d6; 600 MHz; NS 64): 2.05 (CH3), 3.13-5.62 (Repeating unit-

Hs).

ALSH 3:

In this reaction, suspension of LSH (1.0 g, 6.167 mmol) was prepared in DMSO (40 mL) and

acetic anhydride (10.5 mL, 111.015 mmol) was added as an acetylating agent. DMAP (40

mg) was used as a catalyst. Reaction assembly was heated for 6 h at 80 °C with continuous

stirring. ALSH 3 was separated and purified by using ethanol. Finally, sample was dried at

50 °C and yield was calculated.

Yield: (1.33 g); DS: 2.91; FTIR: 1738 (COEster), 2926 (CH), 1376 (CH2), 3514 cm-1

(OH),

1044 (COC); 1H NMR (DMSO-d6; 600 MHz; NS 64): 2.06 (CH3), 3.14-5.62 (Repeating unit-

Hs).

61

2.3.2. Calculation of degree of substitution

For the determination of degree of substitution (DSb) of acetylation on LSH by acid-base

titration method, ALSH (100 mg) was stirred with 0.1M NaOH aqueous solution (50 mL) for

complete saponification. The solution was kept neutral at pH 7.0 with 0.01M HCl solution.

After that, 1M NaOH solution (known volume) was mixed with this neutral solution. DSb of

acetylation was calculated after neutralizing excess NaOH with 0.1M HCl solution using

following formula;

(Eq. 6)

where, n.NaOH is the number of moles of NaOH added after saponification, M (RU) is molar

mass of repeating unit of the polymer, Ms is the mass of sample taken and Mr (RCO) is

molar mass of ester functionality.

2.3.3. Thermogravimetric analysis and degradation kinetics of LSH and ALSH

Initial thermal decomposition temperature (Tdi), maximum thermal decomposition

temperature (Tdm) and final thermal decomposition temperature (Tdf) were calculated from

TG curve of LSH and ALSH. Thermal kinetics was calculated using isoconversional

methods. Flynn-wall and Ozawa (FWO) method was used to calculate the kinetic parameters

as described in Eq. 7 (Flynn, 1990; Ozawa, 1965; Sathasivam and Haris, 2012).

ln ln 5.331 1.052( )

a aAE E

Rg RT

(Eq. 7)

where, β is heating rate, A is pre-exponential factor, Ea is activation energy, R is general gas

constant and T is the temperature at the conversion rate (α). The α is calculated by Eq. 8.

o t

o f

W W

W W

(Eq. 8)

where, W0 is initial mass, Wf is final mass and Wt is the mass of sample at any temperature T.

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FWO method is considered as a model free approach because rate of thermal degradation is

dependent on temperature for a fixed extent of conversion. To calculate fixed value of α at

different heating rate, a graph is plotted between ln β and 1000/T which results in straight line

graph. Value of Ea is calculated from the slop.

Kissinger‘s method is based on the assumption that in differential thermal analysis,

temperature required for maximum deflection is the same on which the maximum rate of

reaction is observed (Kissinger, 1957). Kissinger described the method using Eq. 9.

(Eq. 9)

Graph is plotted between ln(β/Tm2) and 1000 Tm

-2 for a constant conversion which gives Ea at

that conversion.

2.4. Dynamic swelling and stimuli responsive on-off switching of LSH

2.4.1. Isolation of LSH

Linseeds were soaked in deionized water for 48 h before heating at 80 °C for 30 min.

Mucilage was extruded from seeds, separated through nylon mesh, de-fatted with n-hexane

and washed thoroughly with deionized water. Mucilage was centrifuged at 4000 rpm for 30

min. to get LSH which was air dried for 24 h and then placed in vacuum oven at 60 °C for

another 24 h. Finally, dried LSH was ground to fine powder, passed through sieve no. 60 and

stored in an air-tight container in desiccator under vacuum. Yield: 6.5% (g/g) of dried seeds.

2.4.2. Physical properties of LSH

Physical properties of LSH were evaluated by determining moisture content, particle size,

angle of repose, bulk density, tap density, Hausner ratio and Carr‘s index (Lachman et al.,

1987). All parameters were measured in triplicate and mean value was expressed with ± SD.

63

Angle of repose

Angle of repose was calculated to determine the flow property of LSH by using fixed funnel

method (Wells, 1988). LSH was permitted to fall through a fixed funnel on a graph paper.

The height (h) and radius (r) of the heap formed was noted and angle of repose (θ) was

calculated by using Eq. 10.

an  h

Tr

(Eq. 10)

Bulk and tap density

Accurately weighed LSH (1.0 g) was taken in graduated cylinder (10 mL) and bulk volume

(Vb) was noted. Tapped volume (Vt) of LSH was noted after 100 tapping. Bulk density (Db)

and tapped density (Dt) was calculated by dividing the weight of LSH with bulk volume (Vb)

and tapped volume (Vt), respectively.

Hausner ratio and Carr’s index

Hausner ratio (Wells, 1988) is the ratio of the bulk density to tap density and it was

calculated using Eq. 11. Carr‘s index (Wells, 1988) is the measure of packing arrangements

of LSH and calculated using Eq. 12.

(Eq. 11)

     100  1  b

t

DCarr sindex C

D

(Eq. 12)

where, in Eq. 11 and 12, H is Hausner ratio, Db is bulk density, Dt is tap density and C is

Carr‘s index.

64

Gelation content

LSH (0.1 g) was allowed to swell in deionized water (10 mL) for 24 h at room temperature.

It was then centrifuged at 4000 rpm for 30 min. Supernatant was decanted and sediment paste

was collected and weighed. Sediment paste was oven dried at 70 ºC under vacuum until

constant weight was obtained. This dried sediment paste is the gelling content of LSH (Ring,

1985; Peerapattana et al., 2010). Percentage of gelling content was calculated using Eq. 13.

    %   100f

i

WGelling content

W (Eq. 13)

where, Wf is the dried weight of the sediment paste and Wi is the weight of wet paste.

Centrifuge retention capacity

Centrifuge retention capacity is commonly known as water retention capacity. Freshly

prepared LSH (1% w/w in deionized water) was centrifuged at 4000 rpm for 30 min to

determine its water retention capacity. Sediment paste of LSH separated by decanting the

upper portion was completely dried at 70 ºC. Water retention capacity was calculated as the

ratio of weight of sediment paste to the weight of dried mass (Ring, 1985; Peerapattana et al.,

2010).

Moisture content

Moisture content of LSH was calculated by recording the weight of LSH before and after

heating at 105 ºC for 1 h in a vacuum oven.

2.4.3. Preparation of buffer solutions of different pH

Swelling study of LSH was carried out in deionized water and pH 1.2, 6.8 and 7.4. Buffer

solution of pH 1.2 was prepared by mixing KCl (250 mL, 0.2 M) and HCl solution (425 mL,

0.2 M) in 1000 mL measuring flask and final volume was adjusted to 1000 mL with

65

deionized water. To prepare buffer solution of pH 6.8, potassium dihydrogen phosphate (250

mL, 0.2 M) and NaOH solution (112 mL, 0.2 M) were taken in 1000 mL measuring flask and

volume was made up to 1000 mL with deionized water. Potassium dihydrogen phosphate

(250 mL, 0.2 M) and NaOH solution (195.5 mL, 0.2 M) were placed in 1000 mL measuring

flask and deionized water was added up to the mark to prepare buffer solution of pH 7.4.

2.4.4. Evaluation of pH responsive property of LSH

LSH was evaluated for its swelling behavior under different environmental conditions. For

this purpose, swelling of LSH was monitored at different pH mimicking the gastrointestinal

tract (GIT) pH. Furthermore, stimuli responsive swelling de-swelling capability of LSH was

also determined by altering the contact media.

Accurately weighed LSH (0.5 g) was placed in each of four cellophane bags and hung in

separate beakers (100 mL each). Buffer solutions of pH 1.2, 6.8, 7.4 and deionized water

were added in the beakers. Cellophane bags containing swollen LSH were removed after

specific time intervals. The bags were hung for some time to remove excess water, accurately

weighed and placed again in respective media for further swelling. Swelling capacity (g/g)

was calculated using Eq. 14. Experiment was performed thrice and mean of all values were

reported.

0 0   ( ) /t cSwelling capacity w w w w (Eq. 14)

where, wt is weight of wet cellophane bag containing swollen LSH, wo is weight of dry LSH

and wc is weight of wet cellophane bag.

Same experiment was performed in a single step for 24 h in order to note swelling capacity

which is another essential physical parameter for swellable inactive pharmaceutical

ingredient.

66

2.4.5. Swelling kinetics

To study the rate of absorbency of LSH, samples (0.5 g) were poured into cellophane bags

and immersed in 50 mL media (pH 1.2, 6.8, 7.4 and deionized water). At specific time

intervals, the water absorbency of the LSH was measured according to the following relation

(Krusic et al., 2006; Malana et al., 2012).

 s d tt

d d

W W WQ

W W

(Eq. 15)

where, Wd is initial weight of dried powder, Ws is swollen weight of powder at time t and Wt

is weight of water penetrated into LSH at time t. The Qt is normalized degree of swelling.

The normalized equilibrium degree of swelling Qe was determined using Eq. 16.

  d ee

d d

W W WQ

W W

(Eq. 16)

where, W∞ is weight of swollen gel at t∞ when swelling rate becomes constant, Wd is initial

weight of dried powder and We is amount of water penetrated into LSH at t∞.

It was possible to analyze kinetics of the swelling process by using values of normalized

degree of swelling (Qt) and normalized equilibrium degree of swelling (Qe) at time t. For

second-order kinetics following equation can be used.

2

1

t e e

t t

Q Q kQ (Eq. 17)

For second order kinetics, a plot of t/Qt vs t should be linear with the slope of 1/Qe and an

intercept of 1/kQe2.

2.4.6. Thermoresponsive swelling capacity of LSH in deionized water

Swelling response of LSH in deionized water was evaluated at different temperatures (30, 40

and 50 °C). LSH (0.5 g) was taken separately in three cellophane bags and hung in separate

beakers (50 mL). Deionized water was added up to the mark and beakers were placed in

67

water baths previously maintained at 30, 40 and 50 °C. Remaining procedure was same as

mentioned in section 2.4.4. Thermoresponsive swelling capacity of LSH was determined

three times and mean was calculated and reported.

2.4.7. Evaluation of salt solution-responsive properties of LSH

Equilibrium swelling (after 24 h) of LSH was measured in different aqueous salt solutions of

NaCl and KCl (0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 M each). LSH (0.5 g) was taken in each

cellophane bag and hung in different beakers (50 mL) having the above mentioned

concentrations of NaCl and KCl. After 24 h, cellophane bags were removed and weighed

after hanging for some time to drain out excess water. Swelling capacity (g/g) was calculated

using Eq. 14. This experiment was performed three times and mean was reported.

2.4.8. Evaluation of pH responsive on-off switching of LSH

Swelling behavior of LSH was observed at pH 7.4 and 1.2. Accurately weighed LSH (0.5 g)

was taken in cellophane bag and hung in a beaker having buffer solution (50 mL) of pH 7.4

for 15 min. Cellophane bag containing LSH was weighed after removing excess medium.

This cellophane bag was hung in another beaker having buffer solution (50 mL) of pH 1.2 for

15 min. After removing excess medium, cellophane bag was weighed and hung in buffer

solution of pH 7.4. Swelling capacity was calculated after predetermined time intervals using

Eq. 14. The on-off switching cycle were performed five times. On-off switching experiment

was repeated three times and mean was expressed graphically.

2.4.9. Evaluation of saline responsive on-off switching of LSH

Saline responsive on-off switching of LSH was determined in deionized water and NaCl

(0.9%, w/v), respectively. LSH (0.5 g) was taken in cellophane bag and hung in a beaker

containing deionized water (50 mL). Cellophane bag was removed after specific time

68

intervals, hung for some time to remove excess water and weighed to calculate the weight of

swollen LSH. After 1 h of swelling study in deionized water, swelling media was replaced

with NaCl (0.9%) and rest of the procedure was same as mentioned in section 2.4.4. This on-

off procedure was repeated four times. On-off procedure was performed three times and

mean was reported graphically.

2.4.10. Evaluation of on-off switching of LSH in water and ethanol

Accurately weighed LSH (0.5 g) was taken in cellophane bag and hung in a beaker.

Deionized water (50 mL) was added and after predetermined time intervals, cellophane bag

was removed from the beaker and weighed after removing the excess water. After 1 h,

deionized water was replaced with ethanol and swelling capacity of LSH was determined

using Eq. 14. This on-off switching as function of solvent nature was measured four times.

Whole experiment was repeated three times and mean was plotted graphically.

2.5. Development of sustained drug delivery stystem

2.5.1. Formulation design

To evaluate LSH as sustained release material, tablets were prepared and undergo for

analysis for sustained release formulation. For this purpose, diclofenac sodium (DS), caffeine

and diacerein were used to evaluate the sustained release potential of LSH. Additionally,

caffeine and diacerein containing formulations were also used to observe the stimuli

responsive behaviour of LSH in tablet formulation.

2.5.1.1. Drug-excipient compatibility study

Purity of isolated LSH and its compatibility with drugs and excipients were evaluated using

FTIR spectroscopy. Samples were mixed with KBr to prepare the pellets for FTIR analysis.

FTIR spectra were recorded on IR Prestige-21 (Shimadzu, Japan) from 4000 to 400 cm 1

.

69

2.5.1.2. Preparation of tablets

Caffeine, DS and diacerein were used to evaluate the potential of LSH as a sustained release

agent for oral drug delivery system. Tablets of LSH with DS, caffeine and diacerein were

prepared by wet granulation method according to the composition mentioned in Table 2.1,

Table 2.2 and Table 2.3, respectively. LSH, DS and microcrystalline cellulose were passed

through sieve no. 40 and mixed thoroughly in pestle and mortar. Dry mixture was kneaded

with polyvinylpyrrolidone (PVP K30) solution (5% w/v in isopropyl alcohol) to get a damp

mass. Wet mass was passed through sieve no. 12 and dried at 40 °C for 6 h. Dried granules

were finally passed through sieve no. 20, lubricated with magnesium stearate and evaluated

for pre-compression parameters. Granules (300 ± 7 mg) were compressed on a rotary press

fitted with 9 mm flat surface punch. Hardness and thickness of tablets were adjusted at 6-7

kg/cm2 and 3.45-3.65 mm, respectively.

Similarly, formulation of caffeine and diacereine were also prepared by wet granulation

method as adopted for DS containing formulation and according to the composition

mentioned in Table 2.2 and Table 2.3, respectively. Compression weight of lubricated

granules was set at 265 ± 5 mg on a rotary tablet machine fixed with flat surface punch of 9

mm diameter.

Prepared tablets of DS, caffeine and diacereine were evaluated for post-compression

parameters and stored in desiccator until further use. To observe the effect of LSH

concentration on the release of these drugs, three different formulations of each drug with

varying the concentration of LSH were prepared.

70

Table 2.1. Composition of different formulations to evaluate the sustained release behavior

of diclofenac sodium from LSH tablet.

Formulation composition (mg/tablet) D1 D2 D3

LSH 75 100 125

Diclofenac sodium 100 100 100

Microcrystalline cellulose 115 90 65

PVP K30 7 7 7

Magnesium stearate 3 3 3

Total weight 300 300 300

Table 2.2. Tablet formulation design to evaluate the sustained release behavior of caffeine.

Formulation composition (mg/tablet) FH FC FC1 FC2 FC3

LSH 100 - 50 75 100

Caffeine - 100 100 100 100

Microcrystalline cellulose 150 150 100 75 50

PVP K-30 10 10 10 10 10

Magnesium stearate 5 5 5 5 5

Total weight 265 265 265 265 265

71

Table 2.3. Constituents of various tablet formulations to study sustained release behavior of

diacerein.

Formulation composition (mg/tablet) FH FD FD1 FD2 FD3

LSH 100 - 50 75 100

Diacerein - 100 100 100 100

Microcrystalline cellulose 150 150 100 75 50

PVP K30 10 10 10 10 10

Magnesium stearate 5 5 5 5 5

Total weight 265 265 265 265 265

2.5.1.3. Pre-compression evaluation

Before feeding into tablet compression machine, lubricated granules of each formulation

(Table 2.1, 2.2 and 2.3) were evaluated for their flow properties and compressibility. For this

purpose, angle of repose, loose and tapped bulk density, Hausner ratio and compressibility

index were determined as described in section 2.4.2. All experiments were performed thrice

and mean values along with ± SD were expressed.

2.5.1.4. Post-compression evaluation

Compressed tablets were evaluated for hardness, thickness, diameter, weight variation and

friability (Lachman et al., 1987). Content uniformity of prepared tablets was also determined

to confirm the uniformly distribution of active ingredient.

Diameter, thickness and hardness test

Prepared tablets were evaluated for diameter, thickness and hardness test through hardness

tester (Pharma Test, PTB 311E, Germany). Hardness or crushing strength is the force

72

required to break the tablet diametrically. Each test was performed on randomly selected ten

tablets from each formulation and mean value along with standard deviation were reported.

Weight variation test

Compressed tablets from each formulation were tested for weight variation test. For this

purpose, twenty tablets were selected randomly and carefully weighing each tablet on

analytical balance (Shimadzu, Japan). The mean weight and standard deviation was

calculated and reported.

Friability test

Ten tablets from each formulation were randomly selected, accurately weighed, placed in a

chamber of friability tester (Pharma Test, PTF 10E, Germany) and allowed to rotate at 25

rpm for 4 min. Tablets were removed from the chamber, gently cleaned from dust particles

and accurately weighed. Friability was calculated in term of percentage weight loss using Eq.

18.

(Eq. 18)

where, wi and wf are the weight of the tablet before and after friability test, respectively.

Friability test was performed thrice and mean was calculated and reported.

Content uniformity

To determine the DS, caffeine and diacerein content in tablet formulations, ten tablets were

picked randomly from each formulation and separately crushed in pestle and mortar.

Accurately weighed crushed powder (300 mg for DS formulation and 265 mg for caffeine

and diacerein formulations) were taken in volumetric flask (50 mL), mixed vigorously with

methanol and make the volume up to the mark. Solution was filtered, properly diluted and

scan through UV spectrophotometer at 276 nm, 273 nm and 254 nm for DS, caffeine and

73

diacerein, respectively. Absorbance was compared with standard sample and content (%) of

DS, caffeine and diacerein was calculated. Mean of three values were taken and standard

deviation was calculated.

2.5.2. Dynamic swelling and stimuli responsive evaluation of LSH based tablet

formulations

Swelling and stimuli responsive behavior of LSH in tablet formulations (Table 2.2 and Table

2.3) were evaluated.

2.5.2.1. pH responsive swelling of LSH containing tablets

Swelling response of caffeine and diacerien containing formulations (Table 2.2 and Table

2.3) was evaluated in acid buffer of pH 1.2, phosphate buffer of pH 6.8 and 7.4 and also in

deionized water at 37 °C for 16 h. Buffer solutions were prepared according to United States

Pharmacopeia (USP) 37. Formulated tablets were undergo pH responsive swelling study

using cellophane bag to keep all the disintegrated fragments (if any) of swollen tablets at one

place. Four tablets of each formulation were taken separately in four cellophane bags and

placed in four beakers (100 mL) containing buffer solutions (pH 1.2, 6.8, 7.4) and deionized

water. After predetermined time intervals, these cellophane bags containing swollen tablets

were taken out of the beaker, hung for some time to drain out excess swelling media,

weighed accurately and placed them again in their respective beakers to continue the

swelling study. Swelling capacity (g/g) was calculated using Eq. 14.

2.5.2.2. Swelling kinetics

Kinetics of swelling process of tablet formulations was calculated from their swelling data in

various media and determined by the procedure as described in section 2.4.5.

74

2.5.2.3. Evaluation of salt solution responsive swelling

Prepared tablets from each formulation (Table 2.2 and Table 2.3) were evaluated for the

swelling response in different molar solutions of NaCl and KCl. Swelling response was

monitored against eight different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5 and 2.0 M) of

aqueous NaCl and KCl solution by adopting the cellophane bag method as described in

section 2.4.7. After 24 h, swelling capacity (g/g) was calculated using Eq. 14.

2.5.2.4. Stimuli responsive swelling-deswelling (on-off) behavior

Stimuli responsive behavior of the prepared tablets was observed in deionized water, normal

saline solution, ethanol and buffer solution of pH 7.4 and 1.2. For this purpose, tablets of

each formulation were first placed in the swelling media (deionized water and pH 7.4) for 1 h

and then transferred to the deswelling media (normal saline, ethanol and pH 1.2) for another

1 h. Swelling capacity was calculated after every 10 min as described in section 2.4.4. The

swelling-deswelling experiments were carried out over four cycles and repeated thrice.

2.5.3. Evaluation of drug release behavior

2.5.3.1. In-vitro drug release studies

In order to evaluate the sustained release potential of LSH, in vitro drug release study from

prepared formulations was carried out in buffer solutions of pH 1.2, 6.8, 7.4 and in deionized

water. Drug release behaviour imitating the pH and transit time through GIT was also

determined. Each experiment was performed thrice, mean was calculated and reported

graphically.

75

In-vitro release study of DS

DS release study from LSH containing tablets were carried out in USP dissolution apparatus

II (Pharma Test, PT-DT7, Germany). Drug release behavior from the prepared tablets were

studied in SGF (900 mL) for 2 h, then tablets were transferred to SIF (900 mL) and release

was monitored for 14 h at 37 °C and 50 rpm. After selected time intervals, sample (1 mL)

was taken out from respective media, filtered through 0.45 µm filter, diluted (if necessary)

and analyzed through UV spectrophotometer (Shimadzu, Japan) at 276 nm. The volume of

release media were immediately replenished with fresh SGF or SIF. This experiment was

repeated for three times and mean values were expressed in terms of cumulative percentage

of drug release. Same experiment was performed with a commercially available sustained

release formulation of diclofenac sodium (Voltral® SR 100 tablet) to compare with LSH

containing formulations.

In-vitro release study of caffeine

In vitro caffeine release from LSH containing tablets were performed by using USP

dissolution apparatus II. Four different media (buffers of pH 1.2, 6.8, 7.4; and deionized

water) were used to evaluate the release behavior of caffeine from LSH containing caffeine

formulation. Buffers of pH 1.2, 6.8 and 7.4 were prepared according to the procedure

mentioned in USP 37. Drug release study was carried out in 900 mL media kept at 37 ± 0.1

°C and 50 rpm. Aliquot (3 mL) was withdrawn from the vessels after predetermined time

intervals (0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16 and 24 h) and immediately replenished with

the same media to maintain the volume at 900 mL. Samples were filtered through 0.45 µm

nylon filter, suitably diluted and analyzed through UV spectrophotometer at 273 nm. Further,

drug release pattern of each formulation mimicking the transit time and pH of the GIT was

76

also monitored. Therefore, drug release study was carried out for 2 h at pH 1.2, then 8 h at

pH 6.8 and finally for 6 h at pH 7.4.

In-vitro release study of diacerein

USP dissolution apparatus II was used to determine the release of diacerein from three

formulations. For this purpose, buffers of pH 1.2, 6.8 and 7.4, and deionized water were

selected as drug release media. Drug release study from each formulation was carried out in

900 mL media maintained at 37 ± 0.1 °C and paddle rotation speed was adjusted to 50 rpm.

Sample (3 mL) was withdrawn from the vessels after preset time intervals (0.5, 1, 1.5, 2, 3, 4,

5, 6, 8, 10, 12, 16 and 24 h) and immediately replaced with the same preheated (37 ± 0.1 °C)

media to maintain the volume at 900 mL. Samples were filtered through 0.45 µm nylon filter,

suitably diluted and analyzed through UV spectrophotometer at 254 nm. Furthermore, drug

release pattern of each formulation was also analyzed in conditions mimicking the transit

time and pH of the gastrointestinal tract. Therefore, drug release study was carried out at pH

1.2 for 2 h, then at pH 6.8 for 8 h and finally at pH 7.4 for 6 h.

2.5.3.2. Drug release kinetics

Dissolution profile of the LSH containing tablets of DS, caffeine and diacerein were analyzed

by using different kinetic models i.e., zero order (Eq. 19), first order (Eq. 20), Higuchi (Eq.

21) and Hixson-Crowell (Eq. 22). The coefficient of determination (R2) was calculated by

plotting a graph between cumulative drug release and time for zero order, log of cumulative

drug remaining and time for first order, cumulative drug release and square root of time for

Higuchi model and cube root of percentage remaining and time for Hixson-Crowell model.

The model having highest value (~1) for coefficient of determination (R2) was considered the

best fitted model.

(Eq. 19)

77

where, Qt is the amount of drug released from the tablet in time t and K0 is the zero order rate

constant.

(Eq. 20)

where, Q is the remaining amount of drug in the tablet after time t, Q0 is the initial amount of

drug in the tablet and K1 is the first order rate constant (Gibaldi and Feldman, 1967; Wagner,

1969).

(Eq. 21)

where, Qt is the amount of drug released in time t and KH is the Higuchi rate constant

(Higuchi, 1961; Higuchi, 1963).

(Eq. 22)

where, Q0 is the initial amount of drug in the tablet, Qt is the amount of drug released from

the tablet in time t and KHC is the Hixson-Crowell release constant (Hixson et al., 1931).

2.5.3.3. Drug release mechanism

The release of drug from a polymeric matrix system is a complex phenomenon and may

involve one or more mechanisms i.e., swelling and/or erosion of polymer, dissolution and/or

diffusion of drug. Drug release mechanism from a polymeric drug delivery system is best

explained by Korsmeyer-Peppas model (Korsmeyer et al., 1983; Ritger and Peppas, 1987) in

Eq. 23.

(Eq. 23)

where, Mt/M∞ is the fraction of drug release at time t, Kp is the Korsmeyer-Peppas constant

and n is the release exponent. The value of n is calculated from the slop of a graph plotted

between log Mt/M∞ and log t. The value of n indicates the release mechanism of the drug

from a polymeric matrix system. In case of cylindrical (tablet) drug delivery system, the

release mechanism is considered Fickian diffusion, non-Fickian diffusion (anomalous

78

transport), case II transport and super case II transport if n ≤ 0.45, 0.45 < n < 0.89, n = 0.89

and n > 0.89, respectively (Korsmeyer et al., 1983; Ritger and peppas, 1987).

To select a best fit model for drug release, a modified Akaike Information Criterion called

Model Selection Criterion (MSC) was used as described by Iqbal et al., 2011a and expressed

in Eq. 24.

2

- -

1

2

- -

1

. -

ln

. -

n

i i obs obs

i

n

i i obs pre

i

w y y

MSC

w y y

(Eq. 24)

where, yi_obs is the ith

observed value of y, y _obs is the mean of observed data points of y and

yi_pre is the ith

predicted value of y. wi is weighting factor and its value is usually equal to 1

for fitting the dissolution data, n is the number of all data points and p is the number of

parameters in the model.

All calculations related to drug release kinetics and mechanism was performed by using

DDSolver, an add-in program for drug dissolution modeling and comparison, as used by

Zhang et al., 2010.

2.5.4. Scanning electron microscopy analysis

Dried LSH was swollen in deionized water and freeze dried. Cross-sections of hydrogel were

obtained by using sharp blade and observed under FE-SEM to determine the morphology of

freeze-dried sample. Sample was drop casted on a carbon mount and dried at 25 °C in a

vacuum oven for 24 h prior to analysis.

Texture and cross section area of prepared tablets were also investigated using SEM. Tablets

were immersed in deionized water to swell followed by freeze drying and then observed

under SEM.

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2.6. Docetaxel loaded LSH-Pluronic NPs

2.6.1. Preparation of NPs

Docetaxel (DTX) loaded LSH-Pluronic F-68 nanoparticles (DLP-NPs) were prepared in two

steps. First, LSH (2 mg) was dispersed in distilled-deionized water (1 mL). DTX (10 mg) was

dissolved separately in ethanol (0.5 mL) and then added in LSH solution drop wise. This

mixture was kept on stirring for 6 h followed by lyophilization to prepare LSH-DTX core. In

second step, lyophilized sample was dispersed in distilled-deionized water and Pluronic F-68

(500 mg) was added with gentle stirring. After 6 h, mixture was lyophilized to complete the

core shell formation of DLP-NPs.

To get purified DLP-NPs, the prepared NPs were dispersed in deionized water and kept under

mild shaking condition at 25 °C. After 5 min, the dispersion of NPs was centrifuged at 3500

rpm for 10 min followed by freeze drying to get ultra-purified NPs.

For cellular uptake experiment, Nile red, a fluorescence dye, was loaded in DLP-NPs. For

this purpose, 100 µL of Nile red (1 mg/mL in ethanol) was mixed at drug loading phase

during the preparation of DLP-NPs (Khaliq et al., 2016).

2.6.2. Encapsulation efficiency and drug loading

Weight amount of DLP-NPs were dissolved in 4 mL distilled water and centrifuged at 3500

rpm for 5 min. The supernatant was taken, mix with methanol in 1:1 (v/v) and passed through

0.22 µm filter. Quantification of the DTX was determined by Agilent Technologies 1260

series HPLC system. The analysis were performed by reverse phase (RP-HPLC) using a Cap

cell-pack C18 column and an acetonitrile/water (60/40, v/v) as a mobile phase over 10 min at

a flow rate of 1.2 mL/min with an injection volume of 5 µL. The eluent was monitored by

UV absorption at 227 nm.

80

Drug loading (DL) and encapsulation efficiency (EE) was calculated using Eq. 25 and Eq.

26, respectively.

(Eq. 25)

(Eq. 26)

where, in Eq. 25 and Eq. 26, wD, wNP and wDi are the weight of drug in nanoparticles, weight

of nanoparticles and weight of drug initially added for incorporation into nanoparticles,

respectively.

2.6.3. Particle size and morphology

Average diameter, size distribution and zeta potential of the DLP-NPs was determined by

dynamic light scattering (Zeta Sizer Nano Series, Malvern, UK) at 632.8 nm and 25 °C. For

this purpose, freeze dried DLP-NPs (1 mg) were dispersed in phosphate buffered saline

(PBS, 1 mL) of pH 7.4 and analyzed by DLS. The morphology of the prepared DLP-NPs was

examined by field emission electron microscopy (FESEM). Freeze dried DLP-NPs (0.1 wt%)

were dispersed in distilled-deionized water. Aqueous solution (5 µL) of DLP-NPs (0.1 wt%)

was dropped on the aluminum stub and kept it in a vacuum oven for 24 h at 25 °C. Prepared

sample was examined with FESEM (Nova NanoSEM 450, FEI) operated at 10 kV and

distance of 5 mm using though lens detector (TLD).

2.6.4. X-ray diffraction analysis

To evaluate the nature (amorphous or crystalline) of Pluronic F-68, LSH, DTX and DLP-

NPs, XRD pattern was determined using Bruker D8 Advance (Germany) diffractometer

operated at 40 mA and 40 kV with Cu Kα radiation. For this purpose, sample was placed in a

quartz sample holder and scanned from 5 to 40° at a scanning rate of 5°/min.

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2.6.5. In vitro drug release study

In vitro drug release study of DTX from the prepared DLP-NPs was performed in dialysis

bag (Oh et al., 2014). DLP-NPs (10 mg) were dispersed in PBS (10 mL), put in dialysis bag

(molecular weight cut off, MWCO: 12000-14000 Daltons, Spectrum®, Rancho Dominquez,

CA, USA) and completely immersed in PBS (pH 7.4, 20 mL) having Tween 80 (0.1% w/v).

Assembly was kept at 37 °C in a water bath and shaken horizontally at 100 rpm. After

predetermined time intervals, aliquot (2 mL) was withdrawn and whole medium was

replaced with fresh PBS (20 mL). Amount of released DTX was determined by reverse-phase

high performance liquid chromatography (RP-HPLC) using C18 column at detection

wavelength of 220 nm. Mobile phase (acetonitrile/water, 60/40 v/v) was run for 15 min at a

flow rate of 1.2 mL/min.

2.6.6. Cytotoxicity and cellular uptake behaviour

Murine SCC-7 (squamous cell carcinoma) cells were cultured in RPMI 1640 media (Gibco,

Grand Island, USA) containing 10% (v/v) FBS (Gibco) and 1% (w/v) penicillin-streptomycin

at 37 °C in a humidified 5% CO2-95% air atmosphere. Cytotoxicity of DLP-NPs, LSH and

free DTX (Taxotere®, a commercially available DTX formulation) was evaluated using MTT

assay. Cells were harvested in 96-well flat bottomed plates at a density of 5×103 cells/well

and allowed to adhere overnight. Cells were washed with PBS and incubated with various

concentrations of DLP-NPs, LSH and free DTX for 24 h. Cells were washed twice with fresh

PBS in order to eliminate any residual drugs. Twenty five microliters of MTT solution (5

mg/mL in PBS) was added to each well and incubated further for 2 h at 37 °C. In each well,

200 µL of DMSO was added and absorbance was measured at 570 nm with the help of

microplate reader (VERSAmax™, Molecular Devices Corp., Sunnyvale, USA). Percentage

cell viability was calculated for free DTX and empty NPs (without loading of DTX) and

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considered as equivalent DTX concentrations and NPs with same concentrations,

respectively. Cell viability was also determined for LSH and only culture medium was kept

as a negative control groups. IC50 was calculated for the drug concentration from the cell

viability data, in which cell growth was inhibited by 50%. Cell viability data was used to

calculate IC50 which is a drug concentration at which 50% inhibition of cell growth was

observed.

To confirm the cellular uptake of Nile red loaded DLP-NPs, murine squamous cell carcinoma

(SCC-7) tumor cells at a density of 1 × 105 were seeded onto a dish which was protected with

cover slip and allowed to attach for 24 h. After that, cells were treated with fresh medium (2

mL) containing Nile red loaded DLP-NPs followed by incubation for 4 h. Cells were washed

twice with PBS (pH 7.4), paraformaldehyde solution (4%) was used to fix these cells. For

nuclear staining, 4ʹ,6-diamidino-2-phenylindole (DAPI, 3 mmol) was added and incubated at

25 °C for 5 min followed by multiple washing with PBS. Intracellular localization of Nile red

loaded DLP-NPs was visualized using an IX81-ZDC focus drift compensation microscope

(Olympus, Tokyo, Japan) adjusted at 640 nm and 553 nm for emission and excitation

wavelengths, respectively.

2.6.7. Statistical analysis

All experiments were performed at least for three times and data values are expressed as the

mean ± SD. Statistical analysis was carried out using one-way ANOVA and value of p < 0.05

was considered significant.

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2.7. Nanobiotechnological application of LSH mediated Ag NPs

2.7.1. Preparation of AgNO3 solution and LSH suspension

Three different concentrations of AgNO3 solution (10, 20, 30 mmol) were prepared by

dissolving 169.87, 339.74 and 509.61 mg of AgNO3, respectively in deionized water (100

mL). LSH (0.2 g) was suspended in deionized water (10 mL) to prepare 2% (w/v) solution.

2.7.2. Green synthesis of Ag NPs

In order to synthesize silver nanoparticle (Ag NPs), freshly prepared solution of AgNO3 (10

mmol, 10 mL) was added to the suspension of LSH (10 mL). The mixture was stirred in a

dark area for 5 min at room temperature to ensure homogenous mixing of AgNO3 with LSH

suspension. This mixture was then placed in sunlight and progress of reaction was monitored

by observing the change of color. Sample (1 mL) of this reaction mixture was also analyzed

with the help of UV/Vis spectrophotometry. Same protocol was adopted for the other AgNO3

(20 and 30 mmol) solutions, respectively.

2.7.3. Film formation

A mixture of AgNO3 (10, 20 and 30 mmol) and LSH was exposed to diffused sunlight for 10

h. This mixture was air dried under dark in a petri dish to produce LSH thin films containing

the embedded Ag NPs. The thin dry films were subsequently characterized using UV/Vis

spectrophotometry, PXRD and TEM.

2.7.4. UV spectrophotometric analysis

Formation of Ag NPs with the passage of time was monitored by UV/Vis spectrophotometry.

Sample (1.0 mL) of the reaction mixture was taken at selected time intervals (0.25, 0.5, 0.75,

1, 2, 4, 6, 8 and 10 h) and analyzed in a range 200 to 800 nm on UV/Vis Spectrophotometer

84

(Optizen POP, Mecasays, Korea). Effect of various reaction conditions (conc. of AgNO3

solution and reaction time) on the progress of reaction was also studied.

2.7.5. Powder X-ray diffraction

LSH suspension containing Ag NPs and isolated Ag NPs were freeze dried and characterized

by PXRD. PXRD spectra were recorded on Bruker D8 Advance (Germany) diffractometer

(over a range of 10-80°, 2ϴ), operated at 40 mA, 40 KV.

2.7.6. Transmission electron microscopy

Morphology of Ag NPs was assessed by TEM. For this purpose, Ag NPs were isolated from

solution by centrifugation and analyzed on a Philips 420 instrument. For recording TEM

images, samples were drop casted on carbon coated copper wired TEM grid and instrument

was operated at 120 kV. The LSH thin films were air dried under dark and stored for 6

months. After the specified storage period, Ag NPs were again analyzed by TEM. In a typical

experiment, stored LSH thin film impregnated with Ag NPs was dissolved in deionized

water, Ag NPs were isolated by centrifugation and viewed by TEM.

2.7.7. Antimicrobial activity

Aqueous solution of the synthesized Ag NPs (10 mg/mL) was used to study the antimicrobial

activity against different bacterial (Gram positive and Gram negative) and fungal strains.

The tested bacterial strains include Streptococcus mutans (S. mutans) American type culture

collection (ATCC) 25175, Staphylococcus epidermidis (S. epidermidis) ATCC 12228,

Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853, Escherichia coli (E. coli) ATCC

25922, Staphylococcus aureus (S. aureus) ATCC 25923, Bacillus subtilis (B. subtilis) ATCC

6633, Actinomyces odontolyticus (A. odontolyticus) ATCC 17929. The fungal strain used in

the antimicrobial assay was Aspergillus niger (A. niger). Mueller Hinton Agar Media (Oxoid

85

Ltd., England) was used as growth medium for bacterial species while fungi were grown on

Sabouraud Dextrose Agar (Hardy Diagnostics, USA). Inoculums (fungal and bacterial

culture in respective media, 10 mL) were incubated at 27-30 °C for 30-37 h for fungal strains

and at 37 °C for 24 h for bacteria. Fungal culture (7 days old) was washed, suspended in

normal saline solution and incubated at 28 °C after being filtered through aseptic glass wool.

The turbidity of inoculums was adjusted by 0.5 Mc Farland Standard.

Disk diffusion method (Bauer et al. 1966) was used to determine the antimicrobial activity of

Ag NPs. In a typical experiment, aqueous solution of the Ag NPs (20 µL) was loaded onto

filter paper discs (Whatman filter paper No. 1, 6 mm in diameter) and implanted on the

surface of the microbial culture plates. These plates were then incubated at 37 °C for 24 h for

bacterial strains and at 30 °C for 36 h for fungal strains. At the end of incubation period, zone

of inhibition was measured in millimeter. Discs loaded with pure DMSO were used as

negative control. All experiments were performed in triplicate and mean values are reported.

2.7.8. Wound healing studies

2.7.8.1. Design of wound dressing

LSH thin layer (dried) containing the embedded Ag NPs was evaluated as a wound dressing

material. Dressing patch consists of two layers. One layer is a cubic porous adhesive (USP)

backing membrane (4 × 4 cm) and the second layer is cubic sterile cotton patch (2 × 2 cm)

immersed in LSH impregnated with Ag NPs (20 mmol) which is implanted as an inner

wound dressing at the center of adhesive membrane.

2.7.8.2. Wound healing study design

Nine healthy male rabbits (2.5 ± 0.2 kg, 4 month old) were selected and divided into three

equal groups, i.e., control group, standard group and test group. All rabbits were caged under

86

standard laboratory conditions for 48 h before experiment and provided standard food and

water ad libitum throughout the whole study. Rabbits were given anesthesia and placed on

surgical table in natural position. Hair from the rear leg of the rabbit was shaved and excised

a circular wound of 6 mm in diameter using a biopsy punch. Wound of control group rabbits

were left open and untreated. A Band-aid®

dressing was applied to the wound of standard

group while the test group was applied with freshly prepared dressing patch made as

described above. The healing attributes were studied by tracing the raw wound area on

tracing paper till the wound was completely epithelialized (Lee and Tong, 1968).

Eighteen healthy male albino rats (170-220 g body weight) were selected and divided into

three groups of six animals each. The animals were given anesthesia and placed on operation

table in natural position. A round seal (3 cm diameter) was marked 3 cm away from the ears

in central dorsal thoracic region of each animal as described in literature (Morton and Malon,

1972). Skin was scratched from the marked area to induce a wound (1.0 cm diameter).

Wound of control group animals was left open and untreated. A Band-aid® dressing was

applied to the wound of standard group while the test group was applied with prepared

dressing patch. The healing attributes were studied by tracing the raw wound area on tracing

paper till the wound was completely epithelialized (Lee and Tong, 1968).

2.7.8.3. Collagen estimation

Collagen contents of the regenerated tissue were measured using a reported method (Lee and

Tong 1968). For this purpose, regenerated tissue of the wound was chopped and suspended

into 0.5 M acetic acid (10% w/v) after being washed with 0.5 M sodium acetate. It was

stirred for 48 h and then centrifuged for 2 h at 5000 × g. Collagen was precipitated by adding

sodium chloride (10% w/v) and filtered on a weighed Whatman filter paper. Amount of

87

collagen was calculated from the difference in the weight of filter paper before and after

filtration.

2.8. Acute toxicological evaluation of LSH

2.8.1. Study design

Swiss albino mice (25-31 g) and albino rabbits (1115-1225 g) of either sex bred were

obtained from the animal house of University of Sargodha, Sargodha, Pakistan and

thoroughly examined for any symptoms of sickness and anomalies. Animals were kept in

clean stainless steel cages in a 12 h photoperiod (light on at 06:00 and off at 18:00) at 22 to

25 °C and 40-70% humidity. Mice and rabbits were fed with standard laboratory diet and had

free access to ordinary tap water. All tests and procedures conformed to the good laboratory

practice (GLP) regulations as described by United States food and drug administration

(USFDA). Furthermore, acute toxicity procedures were in accordance with organization for

economic co-operation and development (OECD) test guidelines and National Institute of

Health for the care and use of laboratory animals. Animals were divided into four groups

(n=5) as described in Table 2.4. The study protocol was approved by pharmacy research

ethics committee of University of Sargodha, Sargodha, Pakistan.

Table 2.4. Group scheme for acute oral toxicity study of LSH in mice.

Group I Group II Group III Group IV

Control

Given only

standard diet

Treated with LSH

(1g/Kg bw) ground

and mixed with diet

Treated with LSH

(2g/Kg bw) ground

and mixed with diet

Treated with LSH

(5g/Kg bw) ground

and mixed with diet

88

2.8.2. Acute oral toxicity

Acute oral toxicity study of LSH was executed in Swiss albino mice following the OECD

guidelines for analysis of chemicals toxicity. At the instigation of the study, the weight

variation of animals involved was kept minimal and should be < ± 20 % of the mean weight

(Lina et al., 2004). After overnight fasting, mice were orally administered 1-5 g/Kg bw/day

of LSH mixed with food as directed in Table 2.4. The dose of LSH was according to criteria

for excipient toxicity testing and was more than estimated daily intake of excipient (George

and Shipp, 2000). The mice were observed in detail for any indication of toxic effect,

mortality, ill health and any reaction to treatment such as changes in fur, skin, mucus

membranes, eyes, behavior pattern, tremors, salivation, diarrhea, sleep and coma within the

first six hours after the treatment and daily for a further period of 14 days.

2.8.3. Primary eye irritation

This study was carried out in Albino rabbits of either sex. Animals were thoroughly

examined for any abnormalities according to Draize scale for eye lesions (Draize, 1944).

Well ground and moistened LSH (10 mg/10 mL) was placed into the conjunctival sac of the

right eye of each rabbit. Upper and lower lids were kept together with fingers for some time

in order to reduce the risk of any loss of the test material. For comparison, the left eye of

each rabbit was remained untreated and used as a control. After LSH administration, eyes

were examined after 1, 24, 48, and 72 hours for any lacrimation, redness, swelling and

irritation in cornea and pupil. Primary eye irritation score was determined with maximum

mean total score (MMTS) as described by Kay and Calandra (Kay and Calandra, 1962).

Ocular lesion was classified according to the Draize scale for scoring eye lesions (Draize et

al., 1944).

89

2.8.4. Acute dermal toxicity

Five white albino rabbits were used for acute dermal toxicity testing. Rabbits were

thoroughly examined for health and skin abnormalities before and after removal of hair from

the back of these rabbits. Thick paste of ground LSH (500 mg) in deionized water was

prepared and applied on a 4 ply gauze pad (4 in × 4 in). Prepared gauze pad was placed on

the skin of rabbit and wrapped with Micropore™ tape (3 inch wide) to avoid displacement of

the pad. Rabbits were allowed to move freely in their respective cages and observed them for

any behavioral changes for next few hours. After 24 h, the pads were carefully removed from

the skin and the site of application was observed for any color changes in skin or presence of

rashes etc. Rabbits were kept under observation for next 14 days and observed for weight

gain or loss, mortality, sign of behavioral changes, any changes in skin, fur, eyes and mucous

membrane, diarrhea, salivation, coma, convulsions and tremors (Saiyad et al., 2015).

2.8.5. Primary dermal irritation study

Five adult, young and non-pregnant female albino rabbits were selected to carry out the

primary dermal irritation study. Hair from the back of rabbits was removed by clipping the

dorsal and trunk area. Skin of rabbits was examined for any abnormalities before and after

the removal of hair and no pre-existing irritation or lesion on the skin was observed. To apply

test material on the skin, LSH (500 mg) was moistened with deionized water to make a thick

paste and applied on an area of approximately 6 cm2. Test area was covered with gauze pad

and protected from dislocation with the help of suitable semi-occlusive dressing. After 4 h,

dressing was removed and test site was gently cleaned from residual test material. Test site

on each rabbits was observed and score was recorded according to the Draize scoring system

(Draize et al., 1944) at 1, 24, 48 and 72 h after removal of dressing. Irritancy was classified

by adding the average edema and erythema scores for 1, 24, 48 and 72 h scoring intervals

90

and dividing by the number of evaluation intervals (Saiyed et al., 2015). The calculated

Primary Dermal Irritation Index (PDII) was classified according to the descriptive rating

(Sreejayan et al., 2010).

2.8.6. Body weight gain study

The body weights of mice were recorded before treatment, at day 1, 2, 3, 5, 7 and 14 of

treatment. Mean body weight was calculated for each group.

2.8.7. Food and water consumption

The amount of food and water consumed by mice before treatment, at day 1, 2, 3, 5, 7 and 14

was measured and compared with control to observe the influence of LSH intake on these

parameters.

2.8.8. Hematology and clinical biochemistry

On day 15, blood samples were collected from the overnight (about 12 h) fasted mice of each

group into lithium heparin or K2-EDTA containing tubes. Blood samples were analyzed for

hemoglobin, white blood cells count, red blood cells count, platelets, monocytes, neutrophils,

lymphocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and

mean corpuscular hemoglobin concentration (MCHC).

Blood plasma was also analyzed for alanine aminotransferase (ALT), asparate

aminotransferase (AST), creatinine, urea, uric acid, cholesterol and triglycerides.

2.8.9. Gross necropsy and histopathology

At the end of day 14, mice of each group were kept fast for next 12 h, weighed and then

sacrificed approximately at the same time. Gross necropsy of external orifices and surfaces,

thoracic, abdominal cavities, organs and tissues were performed for any lesion and

91

abnormality. Organs (heart, liver, lung, kidney, stomach and spleen) were weighed

immediately and then preserved in neutral buffered formalin (10% v/v). Preserved organs

and tissues were sliced to 4-5 µm thickness and histopathology was performed after staining

with hematoxylin-eosin.

2.8.10. Statistical analysis

The difference among the numerical values of control group and different treated groups

were analyzed through one way analysis of variance (ANOVA) followed by Student‘s t test

using statistical analyses software, Minitab 11. The level of significance was set at p < 0.05.

All values were expressed as mean ± S.D.

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3. RESULTS AND DISCUSSION

3.1. Isolation and characterization of LSH

3.1.1. Isolation of LSH

Linseed hydrogel (LSH) was isolated from linseeds using hot water extraction method.

Polysaccharides usually have hydrophilic groups (-OH, -COOH etc.) in their structure which

facilitate the penetration of water molecules in the polymer. As a result, polymer swells by

absorbing more and more water and then extrudes from the pores of the seeds. This swollen

polymer (hydrogel) was separated by filtration using nylon mesh. To purify the hydrogel

from fatty or waxy material, which may be added from the surface or inside of seeds during

heating process, n-hexane was used to wash out such impurities. Finally, hydrogel was

separated by filtration, dried, ground and stored in air tight container under vacuum for

further use.

3.1.2. FTIR spectroscopy

FTIR spectrum of LSH is shown in Fig. 3.1 and characteristic peaks glycosidic linkage (C-O-

C) and stretching vibrations of (C-O-H) pertaining to side groups of polysaccharide are

shown from 1200-1000 cm-1

(Kacurakova et al., 2000). The band observed around 1640 and

1420 cm-1

are attributed to deprotonated (COO─) carboxylic groups of uronic acid (Boulet et

al., 2007). Peaks from 3500-2500 cm-1

are assigned to C-H and O-H stretching.

93

3500 3000 2500 2000 1750 1500 1250 1000 750 500

Wavenumber, cm-1

C-O-C &

C-O-H

COO-

C-H

O-H CH2

Fig. 3.1. FTIR spectrum of LSH.

3.1.3. 1H NMR spectroscopy

Structure of LSH was characterized by 1H NMR spectroscopy (600 MHz, 40 °C) recorded in

DMSO-d6) and spectrum is shown in Fig. 3.2. Rhamnogalacturonan is a major component of

LSH which is a highly branched structure and composed of rhamnose, galacturonic acid,

galactose, fucose and xylose (Naran et al., 2008; Cui et al., 1994). Presence of these

constituents is verified through 1H NMR spectra by comparing the specific signals with

already reported results (Qian et al., 2012a; Oomah et al., 1995; Emaga et al., 2012).

94

2.503.003.504.004.505.005.50

0

500000

1000000

1500000

5.5 5.0 4.5 4.0 3.5 3.0 2.5

ppm

Fig. 3.2. 1H NMR (600 MHz, ppm, 40 °C) spectrum of LSH in DMSO-d6 showing repeating

unit between 3.11-5.61.

3.1.4. PXRD

PXRD spectrum of LSH is shown in Fig. 3.3. It was observed that the spectrum of LSH did

not have any sharp or distinct peak which is a characteristic of a crystalline material.

Therefore, the extracted LSH is free from any form of crystalline impurities and also it is a

confirmation of the amorphous nature of LSH.

95

0

100

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 90

Inte

nsi

ty

2 ϴ

Fig. 3.3. PXRD spectrum of LSH.

3.2. Synthesis and characterization of LSH-acetates

LSH was acetylated with acetic anhydride using 4-dimethylaminopyridine (DMAP) as

catalyst (Muhammad et al., 2016; Heinze et al., 2003). Acetylation reaction mechanism is

demonstrated in Fig. 3.4. Degree of substitution (DSb) of LSH was increased from 2.11-2.91,

when molar ratio of anhydroglucose unit to acetic anhydride was enhanced from 1:6 to 1:18.

The acetylated LSH (ALSH) was found soluble in dimethyl sulfoxide (DMSO), acetone,

dimethyl acetamide (DMAc) and CHCl3. The results, reaction conditions and DSb are shown

in Table 3.1. Acid-base titration after saponification was repeated thrice to obtain DSb and

mean values are discussed.

LSH + Acetic anhydride 80 C, 6 h

DMAP Acetylated LSH

Fig. 3.4. Reaction scheme for the synthesis of acetylated LSH.

96

Table 3.1. Reaction parameters and results of the synthesis of acetylated LSH.

Sample Molar ratioa Yield (g) DSb

b Solubility

ALSH 1 1:6 1.02 2.11 DMSO, acetone, DMAc, CHCl3

ALSH 2 1:12 1.12 2.53 DMSO, acetone, DMAc, CHCl3

ALSH 3 1:18 1.33 2.91 DMSO, acetone, DMAc, CHCl3

aAnhydroglucose unit of LSH:acetic anhydride

bDegree of substitution calculated from acid base titration after saponification

3.2.1. FTIR spectroscopy

FTIR spectroscopic technique was employed for the identification of ester peaks in acetylated

LSH (ALSH). FTIR spectra of ALSH are shown in Fig. 3.5. The pellets of LSH and ALSH

were prepared with KBr and characterized by FTIR spectrophotometer. A prominent peak at

1749 cm-1

, 1752 cm-1

and 1738 cm-1

in ALSH 1, ALSH 2 and ALSH 3, respectively indicates

effective acetylation of LSH. Moreover, broad peak of unreacted OH groups was still

observed at 3440 cm-1

, 3425 cm-1

and 3514 cm-1

in ALSH 1, ALSH 2 and ALSH 3,

respectively. Other peaks at 1042 cm-1

, 1043 cm-1

and 1044 cm-1

showed the presence of

COC in ALSH 1, ALSH 2 and ALSH 3, respectively. Presence of CH2 groups in ALSH 1,

ALSH 2 and ALSH 3 is evident from peaks at 1376 cm-1

, 1373 cm-1

and 1375 cm-1

,

respectively. It was also observed that as the molar concentration of acetic anhydride

increases from 1: 6 to 1:18, DSb also increased from 2.11 to 2.91.

97

Wavenumber, cm-1

4000 3600 3200 2800 2400 2000 1600 1200 800 400

C─O─C

CH2

C=O

C─H

O─H

LSH

ALSH 1

ALSH 2

ALSH 3

Fig. 3.5. FTIR spectra of LSH, ALSH 1, ALSH 2 and ALSH 3.

3.2.2. 1H NMR spectroscopy

The confirmation of successful acetylation was achieved by typical 1H NMR (DMSO-d6; 600

MHz) spectrum of ALSH 3 (Fig. 3.6.) which showed peaks at 2.06 and 3.14-5.62 ppm for

methyl group and polymer repeating units, respectively.

98

1.02.03.04.05.05.0 4.0 3.0 2.0 1.0

1.9001.9502.0002.0502.1002.1 2.0 1.9

Fig. 3.6. 1H NMR (600 MHz, ppm, DMSO-d6, 40 °C) spectrum of ALSH 3 (DSb 2.91).

3.2.3. 1H

1H TOCSY spectroscopy

1H

1H TOCSY spectra of the acetylated LSH (ALSH 3) were recorded in CDCl3 and shown in

Fig. 3.7. Correlation of sugar region present in ALSH 3 was expressed in Fig. 3.8.

3.2.4. HSQC spectroscopy

HSQC spectrum of ALSH 3 was shown in Fig. 3.9. Correlation of sugar region and acetyl

methyl region are more clearly depicted in Fig. 3.10 and Fig. 3.11, respectively.

99

Fig. 3.7. 1H

1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91).

Fig. 3.8. 1H

1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91)

showing correlation of sugar region.

100

Fig. 3.9. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3.

Fig. 3.10. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing correlation

of acetyl methyl region.

101

Fig. 3.11. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing correlation

of sugar region.

3.2.5. Isoconversional thermal analysis of LSH and LSH acetates

3.2.5.1. Thermal analysis

Thermal degradation behavior of LSH and ALSH 3 was evaluated using isoconversional

method at four different temperatures. The degradation of hydrogel and its acetate was

carried out from ambient temperature to 800 °C. The TG curves of LSH and ALSH 3

exhibited 8.492% and 4.421% loss in the mass in the temperature range of 50 to 130 °C. This

mass loss is actually the moisture present in the sample (Iqbal et al., 2011b). TGA of LSH

and ALSH 3 revealed that degradation occurs in two steps and one step, respectively. TG and

DTG curves of LSH and ALSH 3 recorded at different rates were overlaid and are shown in

Fig. 3.12. Overlay curves of 2DTG of LSH and ALSH 3 recorded at different heating rates

were also shown in Fig. 3.13. Results of thermal decomposition temperature, weight loss and

char yield of LSH and ALSH 3 are displayed in Table 3.2 and Table 3.3, respectively. For

102

first degradation step, the average of four different initial (Tdi) and final (Tdf) thermal

degradation temperatures of LSH was approximately 221 and 378 °C, respectively. For

second degradation step, average Tdi and Tdf values were 380 and 503 °C, respectively. The

average loss in mass of LSH for first and second degradation steps was approximately 46.61

and 28.91%, respectively for four different temperatures. However, average Tdm values for

first and second step was 287 and 459 °C, respectively.

0

20

40

60

80

100

45 195 345 495 645

Wei

gh

t, %

Temperature, C

5 °C/min

10 °C/min

15 °C/min

20 °C/min

0

20

40

60

80

100

40 140 240 340 440

Wei

gh

t, %

Temperature, C

5 °C/min10 °C/min15 °C/min20 °C/min

0

0.2

0.4

0.6

0.8

45 195 345 495 645

Der

iv. w

eig

ht,

%/m

in

Temperature, C

5 °C/min

10 °C/min

15 °C/min

20 °C/min

0

0.3

0.6

0.9

1.2

40 140 240 340 440

Der

iv. w

eig

ht,

%/m

in

Temperature, C

5 °C/min

10 °C/min

15 °C/min

20 °C/min

(d) (c)

(b) (a)

Fig. 3.12. Overlay of TG and DTG curves of LSH (a, c) and ALSH 3 (b, d), respectively

recorded at multiple heating rates.

The thermal degradation of ALSH 3 took place in single step and average Tdi, Tdm and Tdf

values of ALSH 3 were 139.5, 337 and 368.5 °C, respectively with mass loss of 83% at Tdf.

The maximum decomposition temperature (Tdm) of ALSH 3 (337 °C) was higher than LSH

(287 °C) indicating greater stability after acetylation (Table 3.2 and Table 3.3). TG and DTG

103

curves of LSH and ALSH 3 at a heating rate of 10 °C min-1

are compared and shown in Fig.

3.14 indicating an increased thermal stability of acetylate LSH.

-0.026

-0.011

0.004

0.019

0.034

145 225 305 385 465 545

2n

d. D

eriv

. w

eig

ht,

%/m

in2

Temperature, C

5 °C/min 10 °C/min15 °C/min 20 °C/min

-0.07

-0.05

-0.02

0.01

0.03

290 315 340 365 390

2n

d. D

eriv

. w

eig

ht,

%/m

in2

Temperature, C

5 °C/min10 °C/min15 °C/min20 °C/min

(a) (b)

Fig. 3.13. Overlay of 2DTG curves of LSH (a) and ALSH 3 (b) recorded at multiple heating

rates.

Table 3.2. Mean thermal decomposition temperatures, weight loss % and char yield % of

LSH at multiple heating rates.

Sample Step Tdi (°C) Tdm (°C) Tdf (°C) Weight loss

% at Tdf

Char yield

Wt. (%)

LSH I 221 287 378 46.61

6.81 at 700 °C II 380 459 503 28.91

Table 3.3. Mean thermal decomposition temperatures, weight loss % and char yield % of

ALSH 3 at various heating rates.

Sample Step Tdi (°C) Tdm (°C) Tdf (°C) Weight loss

% at Tdf

Char yield

Wt. (%)

ALSH 3 I 139.5 337 368.5 83 7.95 at 600 °C

104

0

20

40

60

80

100

45 195 345 495 645

Wei

gh

t, %

Temperature, C

-0.01

0.19

0.39

0.59

0.79

45 195 345 495 645

Der

iv. W

eig

ht,

%/m

in

Temperature, C

(a) (b)

LSH

—— ALSH

LSH

—— ALSH

Fig. 3.14. Overlay of TG (a) and DTG (b) curves of LSH and ALSH 3 recorded at 10 °C min-

1 showing stability imparted to ALSH 3.

3.2.5.2. Degradation kinetics

Flynn-wall, Ozawa (FWO) and Kissinger isoconversional methods were employed to

evaluate different kinetic parameters like energy of activation (Ea) and frequency factor (A).

Kissinger method was used to find the order of thermal degradation reactions (n). The Ea

values of first and second degradation steps for LSH were found to be 118.32 and 162.34 kJ

mol-1

, respectively (Table 3.4). However, the Ea value of only degradation step of LSH was

169.18 kJ mol-1

. The higher value of activation energy for ALSH 3 showed that acetylated

hydrogel is more stable than unacetylated hydrogel. Fig. 3.15 and Fig. 3.16 show the plots of

α vs. T curves of thermal degradation steps for LSH and ALSH 3, respectively at different

heating rates. FWO plots between logβ and 1000/T (K-1

) for each thermal degradation step at

several degree of conversion for LSH and ALSH 3 are shown in Fig. 3.15 and Fig. 3.16,

respectively. In Kissinger method, thermal decomposition of LSH and ALSH 3 was found to

be first order.

105

0

0.2

0.4

0.6

0.8

1

230 265 300 335 370

α

Temperature, C

5 °C/min10 °C/min15 °C/min20 °C/min

0

0.2

0.4

0.6

0.8

1

380 415 450 485 520

α

Temperature, C

5 °C/min10 °C/min15 °C/min20 °C/min

0.6

0.8

1.0

1.2

1.60 1.70 1.80 1.90

log

β

1000/T, K-1

α 0.1

α 0.2

α 0.3

α 0.4

α 0.5

α 0.6

α 0.7

α 0.8

α 0.9

0.6

0.8

1.0

1.2

1.27 1.34 1.41 1.48

log

β

1000/T, K-1

α 0.1

α 0.2

α 0.3

α 0.4

α 0.5

α 0.6

α 0.7

α 0.8

α 0.9

(a) (b)

(c) (d)

Fig. 3.15. α vs. T graph of thermal degradation of first (a) and second (b) step of LSH at

multiple heating rates and Flynn-Wall-Ozawa (FWO) plot between log β and

1000/T (K-1

) for calculation of Ea of first degradation (c) and second degradation

(d) step at several degree of conversion for LSH.

0

0.2

0.4

0.6

0.8

1

180 220 260 300 340 380

α

Temperature, C

5 °C/min

10 °C/min

15 °C/min

20 °C/min0.6

0.8

1.0

1.2

1.55 1.70 1.85 2.00 2.15

log

β

1000/T, K-1

α 0.1

α 0.2

α 0.3

α 0.4

α 0.5

α 0.6

α 0.7

α 0.8

α 0.9 (a) (b)

Fig. 3.16. α vs. T graph of thermal degradation of ALSH 3 at multiple heating rates (a) and

Flynn-Wall-Ozawa (FWO) plot between log β and 1000/T (K-1

) for calculation of

Ea at several degree of conversion for ALSH 3.

106

3.2.5.3. Thermodynamic analysis

TG data of LSH and ALSH 3 was used to calculate various thermodynamic parameters like

∆H, ∆G and ∆S (Table 3.4 and Table 3.5). Area under TG curves was used to calculate

integral procedural decomposition temperature (IPDT) and index of thermal stability (ITS)

values which are important to evaluate the thermal stability. The mean ITS values for LSH

and ALSH 3 was calculated to be 0.41 and 0.49, respectively. The mean ITS value of ALSH

3 is higher than LSH showing that ALSH 3 has greater thermal stability. Moreover, ITS

values for LSH (0.41) and ALSH 3 (0.49) are higher than other reported hydrogels such as

hydrogels from Astragalus gummifer (0.38), Acacia nilotica (0.40), Argyreia speciosa (0.35),

Acacia modesta (0.42), Ocimum basicilicum (0.41), Plantago ovata (0.39), Salvia aegyptiaca

(0.33) and P. ovata husk (0.41). It means LSH and ALSH 3 are thermally more stable than

many reported polysaccharides (Iqbal et al., 2013). Furthermore, the mean IPDT values of

LSH and ALSH 3 are 350 and 295, respectively.

Table 3.4. Thermal kinetics and thermodynamic parameters of LSH.

Sample Method Step R2 n

Ea

(kJ/mol) lnA ∆H ∆S ∆G IPDT ITS

LSH

FWO I 0.979 - 118.32 27.22 113.66 -36.58 134.14

350 0.41 Kissinger I - 0.87 - - - - -

FWO II 0.976 - 162.34 29.38 156.25 -22.67 172.85

Kissinger II - 1.17 - - - - -

Table 3.5. Thermal kinetics and thermodynamic parameters of ALSH 3.

Sample Method Step R2 n

Ea

(kJ/mol) lnA ∆H* ∆S* ∆G* IPDT ITS

ALSH 3 FWO I 0.966 - 169.18 38.97 164.11 61.97 126.30

295 0.49 Kissinger I - 1.03 - - - - -

107

3.3. Dynamic swelling, stimuli responsive on-off switching of superabsorbent LSH

3.3.1. Physical properties of LSH

Physical properties, i.e., particle size, moisture content, angle of repose, bulk density, tapped

density, swelling capacity, Carr‘s index, Hausner ratio and centrifuge retention capacity of

finely ground LSH passed through sieve no. 60 were determined. The results have indicated

that the particle size, moisture content, centrifuge retention capacity, gelling content and

swelling capacity after 24 h are 250 µm, 0.4%, 67.24%, 33.27% and 42.33, respectively

(Table 3.6). Powder flow-ability parameters, i.e., Carr‘s index, Hausner ratio and angle of

repose were noted as 41.07%, 1.70 and 59.87, respectively. These high values indicated

somewhat poor flow properties of LSH. Therefore, lubricating and gliding agents should be

used for the preparation of tablets containing LSH as an excipient.

Table 3.6. Physical properties of LSH.

Physical properties LSH

Moisture content (%) 0.4 ± 0.01

Particle size (µm) ≈ 250

Angle of repose 59.87 ± 0.32

Bulk density (g/mL) 0.33 ± 0.02

Tapped density (g/mL) 0.56 ± 0.03

Carr‘s index (%) 41.07 ± 2.87

Hausner ratio 1.70 ± 0.08

Centrifuge retention capacity (%) 67.24 ± 1.83

Swelling capacity on 24 h (g/g) 42.33 ± 1.16

Gelling content (%) 33.27 ± 0.79

108

3.3.2. Swelling capacity of LSH in deionized water and at different physiological pH

Swelling behavior of LSH at different pH of gastrointestinal tract was evaluated. Buffers

corresponding to gastric (pH 1.2), small intestine (pH 6.8) and large intestine environment

(pH 7.4) were used to study the effects of pH on swelling of LSH. Swelling of LSH was also

evaluated using deionized water. It was noted that LSH swells rapidly at pH 6.8, 7.4 and in

deionized water (Fig. 3.17.a). However, there was negligible swelling at pH 1.2. If we closely

observe the swelling behavior at pH 6.8, 7.4 and deionized water, it is noted that the

maximum swelling observed in deionized water which is a little bit higher than swelling at

pH 7.4 and 6.8. This slight less swelling at pH 7.4 than deionized water may be due to charge

screening effect of excess cations (Na+) that results in anion-anion repulsions due to shielding

of carboxylate anions (Peppas and Mikes, 1986; Pourjavadi et al., 2004).

0

10

20

30

40

50

60

0 500 1000 1500 2000 2500 3000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

pH 1.2 D. W. pH 7.4 pH 6.8

0

10

20

30

40

50

60

70

0 500 1000 1500 2000 2500 3000

t/Q

t

Time, min

D. W. pH 7.4 pH 6.8

(a) (b)

Fig. 3.17. Swelling capacity (a) and second order swelling kinetics (b) of LSH in buffer of

pH 1.2, 6.8 and 7.4 and in deionized water (D.W).

3.3.3. Swelling kinetics

For practical applications of any hydrogel in formulation design, a higher swelling capacity

as well as a higher swelling rate is required. It is also academically well-known that swelling

kinetics for the hydrogels is significantly influenced by different factors such as swelling

109

capacity, size distribution of powder particles and pH (Malana et al., 2012), etc. Keeping in

view the immense importance of hydrogels, swelling kinetics of LSH was studied. Second

order kinetics appeared the best fit on swelling data acquired in water and at different

physiological pH values (Fig. 3.17.b).

3.3.4. Thermoresponsive swelling capacity of LSH

Effect of temperature on the swelling capacity of LSH in deionized water was determined at

30, 40 and 50 °C (Fig. 3.18). Swelling capacity of LSH increased significantly with the

increase in temperature of swelling medium. As the temperature of swelling medium

increased, water penetration capacity of hydrogel also increased due to relaxation of the

polymer chains. This relaxation allowed water molecules to penetrate within the polymer

chains faster and deeper. Moreover, there is a significant difference in the swelling capacity

of LSH against above mentioned temperatures for first 1000 min. As the maximum relaxation

of polymer chains was achieved at about 1500 min, therefore swelling capacity in the later

stage of swelling appeared independent of temperature.

0

10

20

30

40

50

0 500 1000 1500 2000 2500 3000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

30 °C 40 °C 50 °C

Fig. 3.18. Swelling capacity of LSH in deionized water at different temperatures.

110

3.3.5. Saline responsive swelling of LSH

Swelling of naturally occurring polysaccharides depends upon the presence of

hydrophilic/hydrophobic groups, degree of crosslinking and elasticity of network. Beside

this, swelling of hydrogels is greatly affected by varying the salt concentration in swelling

medium (Pourjavadi and Mahdavinia, 2006). Therefore, effects of increase in ionic

concentration of NaCl and KCl on the swelling ratio of LSH were recorded and shown in Fig.

3.19.a. As the molar concentration of aqueous salt solution increases from 0.1-0.5 M, there is

an abrupt decrease in the equilibrium swelling. This reduced swelling in salt solutions might

be due to charge screening effect of excess cations resulting in non-perfect anion-anion

electrostatic repulsions (Peppas and Mikes, 1986). Because of this repulsion, osmotic

pressure difference between the polymer and swelling medium decreases which results in

shrinkage of swollen polymer in aqueous medium (Pass et al., 1997). It was also noted that

lesser swelling in KCl solution was observed due to higher affinity of the hydrogel towards

K+ ion.

3.3.6. Responsive swelling-deswelling (on-off switching) behavior of LSH at basic and

acidic pH

On immersing LSH in pH 7.4 solution, it rapidly swells and on transferring the swollen LSH

to the pH 1.2 solution, it rapidly deswells showing on-off switching and pH sensitive

behavior. This behavior might be due to the presence of COOH groups in the network

structure of LSH polysaccharides. Literature has indicated that LSH contains acidic sugars,

i.e., rhamanose (20.30%) and uronic acid (22.10%) in 84.25% carbohydrate portion of LSH

when isolated at ambient temperature before defatting (Barbary et al., 2009).

In pH 1.2 solution, COOH groups exist as such and make inter and intra molecular hydrogen

bonding with the polysaccharide chains which might be the driving force that allows the LSH

111

to shrink back. While at pH 7.4, the opposite process starts as the COOH groups are

converted into COONa and COO─

groups which are a cause of repulsion among the similar

groups. Due to this electrostatic repulsion property, LSH chains go away from each other and

lose hydrogen bonding amongst polymer chains and suddenly swell. Fig. 3.19b is showing

the pulsatile on-off switching of LSH at basic (7.4) and acidic pH (1.2), respectively. After

five swelling-deswelling cycles, LSH showed pH responsiveness indicating that the hydrogel

is reversibly pH sensitive.

3.3.7. Responsive swelling-deswelling (on-off switching) behavior of LSH in deionized

water and in NaCl solution (0.9%)

Swelling-deswelling behavior of LSH was examined in deionized water and 0.9% NaCl

solution, respectively (Fig. 3.19c). LSH swelled rapidly on contact with deionized water and

deswelled on contact with 0.9% NaCl solution. The reason of this swelling-deswelling

behavior in deionized water and 0.9% NaCl solution is the osmotic pressure difference

between polymer chains of LSH and the surrounding solution. When the swollen LSH comes

in contact with 0.9% NaCl solution, the presence of Na+ in the solution decreases the osmotic

pressure and as a result water molecules move out of the swollen gel and deswelling of LSH

gel occur. Similarly, when this shrunk LSH is transferred to deionized water, Na+ are washed

out and as a result the osmotic pressure is recovered and LSH again swelled.

3.3.8. Responsive swelling-deswelling (on-off switching) behavior of LSH in deionized

water and ethanol

Swelling-deswelling (on-off switching) behavior of LSH was observed in water and ethanol,

respectively as an essential parameter of swelling behavior to external stimuli (Fig. 3.19d). It

was noted that swollen LSH, abruptly deswells in ethanol. The swelling-deswelling cycles in

112

water/ethanol was repeated four times. The rapid deswelling of LSH in ethanol is because of

the fact that ethanol repels water molecules hence cause faster shrinking of hydrogel.

4

5

6

7

8

9

10

11

0 60 120 180 240 300 360 420 480

Sw

elli

ng

cap

acit

y, g

/g

Time, min

12

14

16

18

20

22

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

Sw

elli

ng

cap

acit

y, g

/g

Concentration, M

NaClKCl

1

2

3

4

5

6

7

0 30 60 90 120 150 180

Sw

elli

ng

cap

acit

y, g

/g

Time, min

7

7.5

8

8.5

9

9.5

10

10.5

11

0 60 120 180 240 300 360 420 480

Sw

elli

ng

cap

acit

y, g

/g

Time, min

(a) (b)

(c) (d)

Fig. 3.19. Swelling capacity of LSH in different conc. of NaCl and KCl (a) and swelling-

shrinking (on-off switching) behavior of LSH; at pH 7.4 (basic) and pH 1.2

(acidic) (b), in deionized water and normal saline (0.9% NaCl solution) (c) and in

deionized water and ethanol (d), respectively.

3.3.9. Field emission scanning electron microscopy

To observe the morphology of superabsorbent LSH, field emission scanning electron

microscopy (FE-SEM) was performed and resultant photographs are shown in Fig. 3.20. It is

obvious from the FE-SEM photographs that elongated porous structures are uniformly

distributed on the surface of LSH. This high porosity of LSH is responsible for the faster and

higher swelling of this supper-porous hydrogel.

113

20 µm 20 µm20 µm

Fig. 3.20. SEM images of lyophilized sample of LSH showing porous and elongated

structure.

3.4. Evaluation of LSH as a novel controlled release and stimuli responsive oral drug

delivery system

Isolated LSH was evaluated for the development of a sustained release formulation of

diclofenac sodium (DS) which was further compare with a commercially available product,

Voltral®. Caffeine and diacerein were selected as a neutral drug and NSAIDs, respectively

and used to explore the potential of LSH as a stimuli responsive and intelligent drug delivery

system.

3.4.1. Drug-excipients compatibility study

To find out the compatibility of LSH with active ingredient and other excipients, FTIR

spectra of different combinations were recorded using KBr pellet technique. Fig. 3.21, Fig.

3.22 and Fig. 3.23 have shown the FTIR spectra of diacerein, caffeine and diclofenac sodium

containing formulations, respectively. IR spectrum of LSH mixed with drug and excipients

indicated that LSH was compatible with the ingredients of tablets.

114

3500 3000 2500 2000 1750 1500 1250 1000 750 500

Wavenumber, cm-1

Diacerin + LSH + PVP

LSH

PVP

Diacerin

Diacerin + LSH

Fig. 3.21. FTIR spectra of LSH, PVP and diacerein alone, physical mixture of LSH with

diacerein and physical mixture of LSH, diacerein and PVP.

115

3500 3000 2500 2000 1750 1500 1250 1000 750 500

Wavenumber, cm-1

Caffeine + LSH + PVP

LSH

PVP

Caffeine

Caffeine + LSH

Fig. 3.22. FTIR spectra of LSH, PVP and caffeine alone, physical mixture of LSH with

caffeine and physical mixture of LSH, caffeine and PVP.

116

3500 3000 2500 2000 1750 1500 1250 1000 750 500

Wavenumber, cm-1

LSH

PVP

Diclofenac sodium

Diclofenac sodium + LSH

Diclofenac sodium + LSH + PVP

Fig. 3.23. FTIR spectra of LSH, PVP and diclofenac sodiume alone, physical mixture of LSH

with diclofenac sodiume and physical mixture of LSH, diclofenac sodiume and

PVP.

117

3.4.2. Pre-compression evaluation of tablet formulations

LSH was used in different concentration to prepare the tablets of diclofenac sodium, caffeine

and diacerein by wet granulation method. Lubricated granules from each formulation were

evaluated for their flow property and compressibility through the determination of angle of

repose, loose bulk density, tapped bulk density, Hausner ratio and compressibility index. The

mean of three values were taken and results are summarized for DS, caffeine and diacerein

containing formulations in Table 3.7, Table 3.8 and Table 3.9, respectively. Loose bulk

density and tapped bulk density of the lubricated granules of all formulations were in the

range of 0.502 to 0.753 g/cm3 and 0.625 to 0.833 g/cm

3, respectively. The values of Hausner

ratio, compressibility index and angle of repose were found in the range of 1.091 to 1.245,

8.358% to 17.945% and 21.11° to 31.69°, respectively. The values of pre-compression

parameters of all formulations indicated good flow and compressibility properties (Trivedi et

al., 2008, Wilson et al., 2011, El-Zahaby et al., 2014).

Table 3.7. Pre-compression parameters of diclofenac sodium formulations (Mean ± SD).

Formulation

code

Angle of

repose

n = 3

Loose bulk

density

(g/cm3)

n = 3

Tapped bulk

density

(g/cm3)

n = 3

Hausner‘s

ratio

n = 3

Compressibility

index (%)

n = 3

D1 19.33 ± 0.08 0.642 ± 0.04 0.713 ± 0.03 1.111 ± 0.03 9.958 ± 0.94

D2 23.29 ± 0.05 0.566 ± 0.03 0.633 ± 0.04 1.118 ± 0.06 10.581 ± 1.88

D3 24.78 ± 0.07 0.593 ± 0.05 0.694 ± 0.05 1.171 ± 0.06 14.561 ± 2.38

118

Table 3.8. Pre-compression parameters of caffeine formulations (Mean ± SD).

Formulation

code

Angle of

repose

n = 3

Loose bulk

density

(g/cm3)

n = 3

Tapped bulk

density

(g/cm3)

n = 3

Hausner‘s

ratio

n = 3

Compressibility

index (%)

n = 3

FH 31.69 ± 0.09 0.502 ± 0.01 0.625 ± 0.02 1.245 ± 0.01 19.680 ± 1.91

FC1 23.72 ± 0.11 0.753 ± 0.02 0.833 ± 0.01 1.106 ± 0.03 9.604 ± 0.82

FC2 28.95 ± 0.15 0.608 ± 0.01 0.714 ± 0.03 1.174 ± 0.01 14.846 ± 0.63

FC3 30.53 ± 0.13 0.535 ± 0.01 0.652 ±0.01 1.219 ± 0.01 17.945 ± 2.23

Table 3.9. Pre-compression parameters of diacerein formulations (Mean ± SD).

Formulation

code

Angle of

repose

n = 3

Loose bulk

density

(g/cm3)

n = 3

Tapped bulk

density

(g/cm3)

n = 3

Hausner‘s

ratio

n = 3

Compressibility

index (%)

n = 3

FH 31.69 ± 0.09 0.502 ± 0.01 0.625 ± 0.02 1.245 ± 0.01 19.680 ± 1.91

FD1 21.11 ± 0.05 0.625 ± 0.02 0.682 ± 0.02 1.091 ± 0.01 8.358 ± 1.11

FD2 26.67 ± 0.06 0.603 ± 0.02 0.682 ± 0.01 1.131 ± 0.02 11.584 ± 2.76

FD3 29.99 ± 0.08 0.555 ± 0.01 0.652 ± 0.01 1.175 ± 0.03 14.877 ± 3.03

3.4.3. Post-compression evaluation of tablet formulations

Compressed tablets of all formulations are analyzed through post-compression parameters

and results are tabulated in Table 3.10, Table 3.11 and Table 3.12 for DS, caffeine and

diacerein, respectively. Mean values of hardness, thickness, weight and friability of tablets

119

were in the range of 6.11 to 6.92 Kg/cm2, 3.41 to 3.95 mm, 264.5 to 266.1 mg (in case of

caffeine and diacerein formulations) and 299.5 to 301.3 (DS formulations) and 0.79% to

0.95%, respectively. Mean DS content was calculated as 99.71%, 98.45% and 97.11% for

D1, D2 and D3, respectively. Mean caffeine content in FC1, FC2 and FC3 were found to be

99.02%, 98.51% and 98.06%, respectively. In FD1, FD2 and FD3, the amount of diacerein

was 98.81%, 98.05% and 97.08%, respectively.

Table 3.10. Post-compression parameters of DS containing tablets (Mean ± SD).

Formulation

code

Hardness

(Kg/cm2)

n = 10

Thickness

(mm)

n = 10

Weight

(mg)

n = 20

Friability

(%)

n = 10

Drug content

(%)

n = 10

D1 6.33 ± 0.04 3.83 ± 0.03 301.3 ± 1.11 0.93 ± 0.06 99.71 ± 0.53

D2 6.11 ± 0.02 3.95 ± 0.04 300.8 ± 0.98 0.85 ± 0.04 98.45 ± 0.43

D3 6.64 ± 0.04 3.88 ± 0.02 299.5 ± 0.43 0.90 ± 0.04 97.11 ± 0.55

Table 3.11. Post-compression parameters of caffeine containing tablets (Mean ± SD).

Formulation

code

Hardness

(Kg/cm2)

n = 10

Thickness

(mm)

n = 10

Weight

(mg)

n = 20

Friability

(%)

n = 10

Drug content

(%)

n = 10

FH 6.13 ± 0.05 3.45 ± 0.02 264.5 ± 0.29 0.95 ± 0.01 -

FC1 6.15 ± 0.01 3.63 ± 0.02 266.1 ± 0.68 0.79 ± 0.02 99.02 ± 0.62

FC2 6.72 ± 0.03 3.41 ± 0.02 265.2 ± 0.32 0.84 ± 0.02 98.51 ± 0.91

FC3 6.45 ± 0.02 3.49 ± 0.04 265.9 ± 0.46 0.91 ± 0.01 98.06 ± 0.56

120

Table 3.12. Post-compression parameters of prepared tablets containing diacerein (Mean ±

SD).

Formulation

code

Hardness

(Kg/cm2)

n = 10

Thickness

(mm)

n = 10

Weight

(mg)

n = 20

Friability

(%)

n = 10

Drug content

(%)

n = 10

FH 6.13 ± 0.05 3.45 ± 0.02 264.5 ± 0.29 0.95 ± 0.01 -

FD1 6.92 ± 0.03 3.51 ± 0.03 264.8 ± 0.97 0.87 ± 0.01 98.81 ± 0.53

FD2 6.58 ± 0.03 3.71 ± 0.01 265.4 ± 1.12 0.91 ± 0.01 98.05 ± 0.43

FD3 6.22 ± 0.01 3.75 ± 0.03 265.1 ± 0.58 0.92 ± 0.02 97.08 ± 0.55

3.4.4. Swelling response of LSH containing tablet formulations at different pHs

Swelling response of all formulations (Table 2.2 and Table 2.3,) was observed in deionized

water and at different physiological pH, i.e., 1.2, 6.8 and 7.4. Tablets were fully immersed in

the above said media and swelling capacity was calculated periodically using Eq. 14.

3.4.4.1. Swelling response and swelling kinetics of LSH tablets

Swelling response and swelling kinetics of LSH tablet (formulation FH) is depicted in Fig.

3.24. It was noted that FH swelled more and rapidly in deionized water, at pH 7.4 and 6.8 as

compared to pH 1.2 (Fig. 3.24a). As we move from lower to higher pH, the acidic group (-

COOH) dissociates into ionic form and the ionic repulsion between similar charged ions

allow the water molecules to penetrate into the polymer chains (Amin et al., 2014; Huang et

al., 2007; Wang et al., 2011a). Hence, more swelling was observed with the increase in pH.

However, the less swelling at pH 7.4 than deionized water was due to the charge screening

effect of the more Na+ present in pH 7.4 buffer (Peppas and Mikes, 1986).

121

Furthermore, swelling kinetics of FH in different swelling media was also determined and

shown in Fig. 3.24b. The results of swelling kinetics indicated that the swelling kinetics

followed the second order kinetics.

0

2

4

6

8

10

12

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

pH 1.2 pH 6.8 pH 7.4 DW

0

50

100

150

200

250

0 200 400 600 800 1000t/

Qt

Time, min

pH 1.2 pH 6.8 pH 7.4 DW

(a) (b)

0.5 h 1 h 2 h 4 h 8 h 16 h

(c)

Fig. 3.24. Swelling capacity (a) and swelling kinetics (b) of LSH tablet (FH) at different pH

and in deionized water and swelling photographs (radial and axial view) of FH

formulation at pH 6.8 (c).

3.4.4.2. Swelling response and swelling kinetics of LSH-caffeine tablets

Swelling response of LSH-caffeine formulations FC1, FC2 and FC3 were studied in

deionized water and buffer solutions of pH 1.2, 6.8, 7.4 and results are shown in Fig 3.25. It

is observed that there is very low swelling capacity for all formulations at acidic pH (Fig.

3.25a). Swelling of polymer depends on the nature and ionization of functional groups

present on the polymer chain (Huang et al., 2007). The dissociation of acidic group (-COOH)

present on the LSH polymer chain is not possible at low pH and less swelling was observed.

122

Furthermore, it is also observed that the swelling of these tablets depend on the concentration

of LSH. Formulation FH and FC3 has shown less swelling as compared to FC1 and FC2 as

former two formulations have high amount of LSH in their composition.

Swelling trend of formulations (Table 2.2) at pH 6.8, 7.4 and in deionized water is expressed

in Fig. 3.25b, Fig. 3.25c and Fig. 3.25d, respectively. The results indicated that the swelling

of tablets is increased with the increase in the concentration of LSH in each media.

Swelling kinetics was also determined for all these formulations at pH 1.2, 6.8 and 7.4 and in

deionized water and shown in Fig. 3.26a, Fig. 3.26b, Fig. 3.26c and Fig. 3.26d, respectively.

The results have shown that the data of swelling of these tablets followed the second order

kinetics.

3.4.4.3. Swelling response and swelling kinetics of LSH-diacerein tablets

Swelling response of diacerein containing LSH tablet formulations (Table 2.3) were

evaluated at pH 1.2, 6.8, 7.4 and in deionized water and shown in Fig. 3.27. It was observed

that at pH 1.2 (Fig. 3.27a), there is very less swelling for all the formulations. Moreover, the

swelling does not affect with the presence of diacerein. It is also noted that as the

concentration of LSH increases (FD3) the swelling of tablets will decrease. At pH 6.8 (Fig.

3.27b), an increase in swelling capacity was observed with the increase in the concentration

of the LSH in the tablets. It is obvious that FD1 show less swelling as compared to FD3 due

to the less amount of LSH in FD1. Same pattern of swelling was also observed for all

formulations at pH 7.4 (Fig. 3.27c) and in deionized water (Fig. 3.27d). The formulation

having less amount of LSH has shown lower tendency to swell as compared to the

formulation having high amount of LSH. The rate of swelling is determined through swelling

kinetic and it was noted that all formulations follow the second order kinetics (Fig. 3.28).

123

0

2

4

6

8

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FC1

FC2 FC3

0

2

4

6

8

10

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FC1

FC2 FC3

0

2

4

6

8

10

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FC1

FC2 FC3

0

2

4

6

8

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FC1

FC2 FC3

(a) (b)

(c) (d)

0.5 h 1 h 2 h 4 h 8 h 16 h

(e)

Fig. 3.25. Swelling capacity of FH, FC1, FC2, and FC3 at pH 1.2 (a), 6.8 (b), 7.4 (c), and DI

water (d) and swelling photographs (radial and axial view) of FC3 formulation at

pH 6.8 (e).

124

0

30

60

90

120

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FC1 FC2 FC3

0

30

60

90

120

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FC1 FC2 FC3

0

30

60

90

120

150

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FC1 FC2 FC3

(a) (b)

(c) (d)

0

50

100

150

200

250

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FC1 FC2 FC3

Fig. 3.26. Swelling kinetics of LSH tablet (FH) and LSH-caffeine tablets (FC1, FC2 and

FC3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).

Overall, the swelling indices of LSH containing tablets were lesser as compared to swelling

indices of powder. This lower swelling of LSH tablets as compared to LSH in powder form

can be explained due to decrease in exposed surface area of LSH particles in tablets. In the

tablets form, only the outer surfaces of the tablets are exposed to swelling medium and

swelling medium can only enters from this surface into large tablet matrix. Whereas, LSH

particles in the core of tablets are not directly exposed to media. The swelling process, that

involves the diffusion of swelling medium into the polymeric network and the relaxation of

the polymer chains, starts after the entry of medium in tablets (Razmjou et al., 2013). As the

surface area for the diffusion of medium is small in tablets therefore the swelling indices of

tablets is lower. Contrarily, in case of LSH in powder form, contact area exposed to swelling

125

medium is much greater than tablets that results in entry of more swelling medium in LSH

chains leading to higher swelling. Additionally, when the particles are compressed in tablets,

the interstitial spaces decrease that further contribute to lower swelling of tablets (Bao et al.,

2011). Although swelling indices of LSH were lesser in tablet form than in powder form,

however, LSH tablet still exhibited superabsorbent characteristics with high swelling indices.

3.4.5. Swelling morphology of LSH containing tablets

Tablets of formulations FH, FC3 and FD3 were allowed to swell at pH 6.8 and observed the

morphological changes for 16 h. It was observed that the rate of swelling of all formulations

was little bit different from each other. At the end of 16 h, more fragments of FC3 and FD3

tablets were seen as compared to FH (Fig. 3.24c, Fig. 3.25e and Fig. 3.27e, respectively).

This might be due to the release of entrapped drug which creates micropores in the tablet.

After hydration, these pores spread out and ultimately break the tablet into fragments.

126

0

2

4

6

8

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FD1

FD2 FD3

0

2

4

6

8

10

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FD1

FD2 FD3

0

2

4

6

8

10

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FD1

FD2 FD3

0

2

4

6

8

0 200 400 600 800 1000

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FD1

FD2 FD3

(a)

(d) (c)

(b)

0.5 h 1 h 2 h 4 h 8 h 16 h

(e)

Fig. 3.27. Swelling capacity of FH, FD1, FD2, and FD3 at pH 1.2 (a), 6.8 (b), 7.4 (c) and

deionized water (d) and swelling behavior of FD3 formulation at pH 6.8 expressed

in photographs (radial and axial view) (e).

127

0

30

60

90

120

150

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FD1 FD2 FD3

0

30

60

90

120

150

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FD1 FD2 FD3

0

30

60

90

120

150

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FD1 FD2 FD3

0

50

100

150

200

250

0 200 400 600 800 1000

t/Q

t

Time (min)

FH FD1 FD2 FD3

(a)

(d) (c)

(b)

Fig. 3.28. Swelling kinetics of LSH tablet (FH) and LSH-diacerein tablets (FD1, FD2 and

FD3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).

3.4.6. Morphological analysis of LSH containing tablets by SEM

SEM analysis of FD3 tablet formulation was conducted to observe the morphological

arrangements of LSH in tablet formulation. The SEM micrograph of tablet surface, broken

surface and cross section of swollen then freeze dried FD3 formulation are shown in Fig.

3.29. Intra-particle microscopic spaces were seen on the surface of FD3 and broken surface of

FD3 tablet as shown in Fig. 3.29a and Fig. 3.29b, respectively. Moreover, the cross section of

swollen then freeze dried FD3 tablets revealed the presence of multilayered porous structure

(Fig. 3.29c). This highly porous structure of LSH makes it an ideal material for drug loading

and control release applications.

128

a b c

Fig. 3.29. SEM images of broken surface of FH tablet (a), broken surface of FD3 tablet (b)

and cross section of swollen then freeze dried tablet formulation FD3.

3.4.7. Salt solution responsive swelling of LSH containing tablet formulations

Swelling response of all formulations was studied at various concentrations of NaCl and KCl

to observe the effects of salt solutions on swelling behavior. It was observed that in all

formulations there was an abrupt decrease in the equilibrium swelling from the molar

concentration 0.1-0.5 M but monotonous decrease from 0.5-2.0 M. Equilibrium swelling

capacity of FH, FC3 and FD3 in salt solutions of NaCl and KCl is expressed in Fig. 3.30a and

Fig. 3.30b, respectively as a typical example. Moreover, it was also monitored that the

equilibrium swelling of formulation FC3 and FD3 was less than the equilibrium swelling of

LSH. Same swelling trend was also observed in KCl salt solution. On closely observing the

equilibrium swelling of these formulations in both the media (NaCl and KCl), it was revealed

that all formulations swelled more in NaCl solution as compared to KCl solution. This less

swelling in salt solution is due to the presence of Na+ and K

+ cations. These cations decrease

the osmotic pressure difference between the polymer chains and the surrounding solutions.

As a result the water penetration ability into the tablet and swelling capability is also reduced.

The reduced swelling in the presence of salts solutions may also be explained due to the

neutralization of COO─ ions by Na

+ and K

+ ions that reduce the electrostatic repulsion

(among COO─) responsible for swelling (Pandey et al., 2013). The reason of less swelling in

129

KCl solution as compared to NaCl is due to the high charge density of the K+ as compared to

the Na+.

1

2

3

4

5

6

0 0.25 0.5 0.75 1

Sw

elli

ng

cap

acit

y, g

/g

KCl solution concentration, M

FH FC3 FD3

1

2

3

4

5

6

0 0.25 0.5 0.75 1

Sw

elli

ng

cap

acit

y, g

/g

NaCl solution concentration, M

FH FC3 FD3

(a) (b)

Fig. 3.30. Equilibrium swelling of LSH tablet (FH), LSH-caffeine tablet (FC3) and LSH-

diacerein tablet (FD3) in different molar concentrations of salt solutions; NaCl (a)

and KCl (b).

3.4.8. Swelling-deswelling response of LSH tablet formulations against external stimuli

To explore the potential of LSH as an intelligent biopolymer, swelling-deswelling response

of LSH, LSH-caffeine and LSH-diacerein tablets were observed at pH 7.4 and 1.2, in water

and normal saline solution and in water and ethanol. The evaluation of change in pH on the

swelling and deswelling of these tablets is important due to the acidic and basic environment

of gastrointestinal tract (GIT). Moreover, the presence of NaCl and ethanol in GIT can alter

the swelling capability of LSH containing tablet formulations.

3.4.8.1. Swelling-deswelling response in basic and acidic pH

Swelling-deswelling response of formulated tablets were evaluated at basic (pH 7.4) and

acidic (pH 1.2) environment (Fig. 3.31a). It was noted that all formulations were swelled at

pH 7.4 and rapidly deswelled when shifted to pH 1.2. With the increase of pH, the carboxylic

acid moiety of the polymer converts into its ionized form. The high density of these charged

130

particles creates a strong anion-anion electrostatic repulsion which results in the extension of

polymer chains and water molecules move between the free spaces of polymer chains (Wang,

et al., 2011a; Huang, et al., 2007). When these swelled tablets were placed at pH 1.2, the

protonation of the carboxylate ion (COO─) and formation of strong hydrogen bonding

regained the deswelled form of the tablets. When we compare three formulations, it was

observed that the diacerein containing formulation (FD3) has shown little bit less swelling-

deswelling response due to the presence of acidic drug (diacerein) as compare to FH and FC3

tablets. These findings indicated the swelling-deswelling potential of LSH in tablet

formulations with caffeine and diacerein and also as an oral control release drug delivery

system. The lower swelling at acidic pH (stomach) can protect the release of drug in stomach

while higher swelling at intestinal pH can be utilized to target drug release in intestinal

region. Moreover, caffeine and diacerein have negligible influence on the swelling-

deswelling capability in tablets formulation of LSH.

3.4.8.2. Swelling-deswelling response in deionized water and normal saline solution

Swelling-deswelling response of tablets in deionized water and normal saline solution were

analyzed and results are shown in Fig. 3.31b. This swelling-deswelling behavior of tablets in

water and normal saline solution was due to the difference in the osmotic pressure between

the swollen tablets and surrounding environment. The decrease in the osmotic pressure due to

the presence of sodium ion in the medium was the main force which draws the water

molecules to come out of the swollen tablets. Results have indicated the responsive swelling-

deswelling behavior of these formulations against normal saline solution.

131

0

1

2

3

4

0 60 120 180 240 300 360

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FC3 FD3

(a)

(b)

(c)

0

1

2

3

4

0 60 120 180 240 300 360

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FC3 FD3

0

1

2

3

4

0 60 120 180 240 300 360

Sw

elli

ng

cap

acit

y, g

/g

Time, min

FH FC3 FD3

Fig. 3.31. Stimuli responsive swelling and deswelling behavior of LSH tablet (FH), LSH-

caffeine tablet (FC3) and LSH-diacerein tablet (FD3) at basic (pH 7.4) and acidic

(pH 1.2) environment (a), in deionized water and normal saline (b) and deionized

water and ethanol (c), respectively.

132

3.4.8.3. Swelling-deswelling response in deionized water and ethanol

Swelling-deswelling response of all formulations was observed in deionized water and

ethanol (Fig. 3.31c). Tablets were allowed to swell in deionized water for 1 h and then shifted

to shrinking media i.e., ethanol for the same time period. It was observed that the rate of

shrinking is faster than the rate of swelling in all formulations. This abrupt shrinking in

ethanol was due to the fact that water molecules were quickly replaced by ethanol (Dragan

and Apopei, 2013). Therefore, the presence of ethanol will create hindrance in swelling of

LSH containing tablet formulations and as a result release of drug will be affected.

3.4.9. In-vitro drug release studies

3.4.9.1. DS release studies and release mechanism

To observe the effect of LSH on the release of DS, tablets were place in simulated gastric

fluid (SGF) and simulated intestinal fluid (SIF) for 2 h and 14 h, respectively (Fig. 3.32b).

Drug-hydrogel interaction, swelling ability of hydrogel and drug solubility in dissolution

media are the major influencing factors that control the drug release from polymeric matrix

system (Brazel and Peppas, 1999; Siepmann and Peppas, 2001). In SGF, minimum or

negligible amount of DS, 5.04%, 4.27% and 3.96%, was released from D1, D2 and D3,

respectively. Due to less swelling tendency of LSH at acidic pH (1.2) and insolubility of DS

in acidic environment, low concentration of DS was released from all three formulations.

Contrary to this, in SIF or near neutral/basic environment, both swelling of LSH and

solubility of DS was increased. After 14 h study in SIF, DS release from D1, D2 and D3 was

90.4%, 67.6% and 49.1%, respectively. Moreover, a more sustained release of DS from

tablets was observed with the increase of LSH concentration from 75-100 mg/tablet (Table

2.1). For comparison, drug release profile of DS from LSH containing formulation was

evaluated against commercially available formulation. It was observed that formulations D2

133

and D3 sustained the release of DS even better than already marketed formulation (Fig.

3.32b).

Mainly, the drug release from swellable polymeric system is controlled by swelling and

diffusion mechanism. Release mechanism was found out using Korsmeyer-Peppas model

(Eq. 23). The value of n and kp was calculated from the slop and intercept of the plot of

ln(Mt/M∞) vs ln t, respectively and values are given in Table 3.13. The value of n was found

in the range from 0.762-0.824 for all formulations. Hence, drug release from DS formulation

follows the non-Fickian diffusion (anomalous transport mechanism). Therefore, the swelling

of polymer and diffusion of drug from polymer matrix occurred simultaneously (Korsmeyer

et al., 1983; Ritger and Peppas, 1987).

Swelling capacity of all formulations (D1, D2 and D3) was studied in deionized water and

results are expressed in Fig. 3.32a. It can be seen that swelling of tablets has a direct relation

with the concentration of LSH. An increase in swelling was observed with the increase of

LSH concentration in tablets. Physical condition of the tablet during the swelling process was

noted and shown in Fig. 3.32c. It was observed that the tablet has been able to maintain its

physical state for longer period of time with minimum fragmentation.

Table 3.13. Mathematical data of power law.

Formulation n kp r2

D1 0.762 12.416 0.9277

D2 0.826 7.645 0.9488

D3 0.824 5.556 0.9566

134

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

Cu

mu

lati

ve

dru

g r

elea

se, %

Time, h

Voltral D1

D2 D3

SGF

(b)

0

2

4

6

8

10

0.25 0.5 1 2 4 8 12 16

Sw

elli

ng

cap

acit

y, g

/g

Time, h

D1 D2 D3

(a)

(c)

0 h 1 h 2 h 4 h 8 h

SIF

Fig. 3.32. Swelling capacity of LSH based DS tablets in water (a), drug (DS) release study

from LSH matrix tablets in SGF and SIF (b) and photographs showing swelling

response (aerial and axial view) of D3 formulation in water (c).

3.4.9.2. Caffeine and diacereine release studies

To explore the potential of LSH as a controlled release material, LSH containing tablets were

prepared using caffeine and diacerein. The release profile of caffeine from formulation FC1,

FC2 and FC3 at pH 6.8, 7.4 and in deionized water is shown in Fig. 3.33a, Fig. 3.33b and Fig.

3.33c, respectively. It was observed that the release of caffeine was sustained as the amount

of LSH increased from 50 mg (FC1) to 100 mg (FC3) per tablet in all the media. This

sustained release of drug from FC3 is due to the high swelling and water holding capacity of

LSH present in these tablets. Therefore, it is difficult for soluble drug to come out of the

swelled matrix of LSH. The cumulative drug release after 16 h from FC1, FC2 and FC3 was

89.09 ± 0.93, 74.11 ± 1.05 and 61.31 ± 0.62% at pH 6.8; 99.14 ± 1.25, 86.27 ± 1.11 and

135

67.33 ± 0.93% at pH 7.4 and 99.67 ± 1.71 (after 12 h), 89.52 ± 1.66 and 74.29 ± 1.01% in

deionized water, respectively. Results have indicated that release of caffeine is dependent on

the concentration of LSH in the tablet and inversely proportional relation is observed.

Furthermore, a tablet formulation (FC) was prepared without LSH and it was observed that

caffeine was completely released from the formulation within 2 h in all media.

In vitro drug release studies were performed in dissolution media mimicking the

physiological pH and maximum transit time in the GIT (Fig. 3.33d). Therefore, tablets from

formulations FC1, FC2 and FC3 were exposed to pH 1.2, 6.8 and 7.4 buffer solutions for 2, 8

and 6 h, respectively. Results have indicated that very less amount of drug was released at pH

1.2 (9.13 ± 0.08, 7.51 ± 0.05 and 6.7 ± 0.08% for FC1, FC2 and FC3, respectively), while at

pH 6.8 and 7.4, higher and sustained release behavior was observed. After 14 h, the

cumulative drug release from FC1, FC2 and FC3 was 100.25 ± 1.72, 81.23 ± 0.91 and 66.26

± 1.21%, respectively. Release of very small amount of caffeine at acidic pH is due to the

dissolution of the loosely packed caffeine from the tablet surface. From these results, it is

concluded that LSH based tablet formulations can be used for sustained and site specific

delivery of drug at small and large intestinal pH.

136

0

20

40

60

80

100

0 4 8 12 16 20 24

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FC1

FC2

FC3

FC

0

20

40

60

80

100

0 4 8 12 16 20 24

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FC1

FC2

FC3

FC

0

20

40

60

80

100

0 4 8 12 16 20 24

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FC1

FC2

FC3

FC

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FC1

FC2

FC3

pH 1.2 pH 6.8 pH 7.4

(c)

(a) (b)

(d)

Fig. 3.33. Caffeine release from LSH-caffeine tablet in different media; pH 6.8 (a), pH 7.4

(b), deionized water (c) and physiological pH and transit time of gastrointestinal

tract (d).

Diacerein release from formulations FD1, FD2 and FD3 was observed at GIT pHs and in

deionized water (Fig. 3.34). At pH 1.2, negligible amount of drug was released from all three

formulations (data not shown) because diacerein is insoluble at acidic pH. At pH 6.8, 7.4 and

in deionized water, a sustained release profile of the drug was observed from FD1, FD2 and

FD3. In buffer of pH 6.8, cumulative drug release (after 16 h) from FD1, FD2 and FD3 was

79.28 ± 2.11, 63.6.9 ± 4.86 and 54.31 ± 3.04%, respectively (Fig. 3.34a). At pH 7.4, diacerein

release (after 16 h) from FD1, FD2 and FD3 was 99.71 ± 0.91, 84.45 ± 2.02 and 70.04 ±

2.52%, respectively (Fig. 3.34b). Furthermore, a tablet formulation (FD) was prepared

without LSH and the release of diacerein from the formulation was completed within 3 h at

137

pH 6.8 and 7.4 whereas, in deionized water, diacerein release was completed in 4 h. This

slight delay release in deionized water was due to less solubility of diacerein in deionized

water.

0

20

40

60

80

100

0 4 8 12 16 20 24

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FD1 FD2

FD3 FD

0

20

40

60

80

100

0 4 8 12 16 20 24

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FD1

FD2

FD3

FD

0

20

40

60

80

100

0 4 8 12 16 20 24

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FD1

FD2

FD3

FD

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

Cu

mu

lati

ve

dru

g r

elea

se (

%)

Time (h)

FD1

FD2

FD3

pH 1.2 pH 6.8 pH 7.4

(c)

(a) (b)

(d)

Fig. 3.34. Diacerein release from LSH-diacerein tablet in different media; pH 6.8 (a), pH 7.4

(b), deionized water (c) and physiological pH and transit time of gastrointestinal

tract (d).

After 16 h of drug release study in deionized water, the amount of diacerein released from

FD1, FD2 and FD3 was 68.03 ± 3.42, 52.84 ± 2.3 and 39.19 ± 2.84%, respectively (Fig.

3.34c).

Results have indicated that the release of diacerein from FD1, FD2 and FD3 was dependent

on the solubility of drug as well as on the concentration of LSH. Diacerein, being an acidic

138

drug is poorly soluble in deionized water while soluble in buffers of pH 6.8 and 7.4 due to the

presence of NaOH. Therefore, the cumulative drug release from FD1, FD2 and FD3 at pH 6.8

and 7.4 was high as compared to deionized water. It was also observed that the release of

diacerein was inversely proportional to the concentration of the LSH in the formulations at a

given medium.

Furthermore, to evaluate the release behavior of diacerein from the formulated tablet during

the transit through gastrointestinal tract, formulations were tested at pH 1.2 for 2 h, pH 6.8 for

8 h and pH 7.4 for 6 h (Fig. 3.34d). The results have indicated that there is negligible amount

of drug release in acidic pH which is mainly due to less swelling at such a low pH and also

due to poor solubility of the drug. When these formulations were placed in pH 6.8, a

significant and sustained release of diacerein was observed for next 8 h. In pH 7.4, a slight

increase in the drug release pattern was observed as compared to pH 6.8 due to more

solubility of diacerein at this pH. At the end of 14 h, drug released from FD1, FD2 and FD3

was 95.21 ± 2.91, 81.61 ± 1.91 and 63.91 ± 3.13%, respectively. In-vitro drug release study

suggests that LSH can be used as a potential carrier for small intestine and colon specific

drug delivery of diacerein as well as other NSAIDs. The in-vitro results indicated the

potential of LSH tablets as oral controlled release system where pH difference between the

stomach and intestine can used to trigger drug release. LSH release drug at intestinal pH

suggested that it can be further developed as site specific delivery system in intestine.

Moreover, the lower release in the stomach can also help to avoid the side effect of drugs like

NSAIDs.

3.4.9.3. Drug release kinetics and mechanism

To find out the drug release pattern and mechanism, kinetics models were applied on

dissolution data obtained at pH 6.8, 7.4 and deionized water. The value of coefficient of

139

determination (R2) were calculated from the equations of zero order, first order, Higuchi,

Korsmeyer-Peppas and Hixon-Crowell and given in Table 3.14 and Table 3.15 for caffeine

and diacerein containing LSH tablets, respectively. The value of R2 indicated that the best fit

models for both drugs are first order, Higuchi, Korsmeyer-Peppas and Hixon-Crowell. It

indicates that the release of drug depends on the swelling of polymer as well as diffusion of

the drug. Results have also indicated that caffeine and diacerein release pattern followed the

anomalous transport mechanism (non-Fickian diffusion). MSC analysis of the data indicated

that the most appropriate model in explaining the rate and mechanism of drug release was

Korsmeyer-Peppas model. The values of diffusion exponent n indicate the release mechanism

of the drug from a polymeric matrix system. In case of cylindrical geometry (tablets) drug

delivery system, the release mechanism is considered Fickian diffusion, non-Fickian

diffusion (anomalous transport), case II transport and super case II transport if n ≤ 0.45, 0.45

< n < 0.89, n = 0.89, and n > 0.89, respectively (Korsmeyer et al., 1983; Ritger and Peppas,

1987). The values of n for LSH tablets were in the range of 0.6 to 0.8 indicating that the

release of drug was governed by non-Fickian diffusion (anomalous transport), i.e., both the

swelling of polymers and diffusion of the drug occurred.

140

Table 3.14. Values of drug release kinetics models for LSH-caffeine formulations at pH 6.8,

7.4 and deionized water.

pH 6.8 pH 7.4 Deionized water

FC1 FC2 FC3 FC1 FC2 FC3 FC1 FC2 FC3

Zer

o o

rder

R2 0.7930 0.7310 0.624 0.8721 0.6834 0.6548 0.9039 0.8388 0.6746

K0 6.994 4.801 3.751 8.958 5.366 4.280 10.662 7.139 4.668

MSC 1.408 1.159 0.824 1.875 0.996 0.909 2.142 1.658 0.969

Fir

st o

rder

R2 0.9969 0.9969 0.9299 0.9924 0.9984 0.9712 0.9900 0.9947 0.9893

K1 0.136 0.096 0.065 0.168 0.120 0.080 0.194 0.138 0.093

MSC 5.601 5.617 2.504 4.693 6.287 3.394 4.406 5.080 4.386

Hig

uch

i

R2 0.9676 0.9633 0.9656 0.9557 0.9578 0.9629 0.9352 0.9490 0.9607

KH 22.05 18.067 14.287 24.89 20.302 16.242 26.82 22.36 17.681

MSC 3.262 3.152 3.215 2.935 3.011 3.140 2.536 2.809 3.082

Ko

rsm

eyer

-Pep

pas

R2 0.9995 0.9954 0.9852 0.9993 0.9977 0.9983 0.9991 0.9986 0.9954

KKP 15.42 11.61 11.47 17.05 12.49 11.39 17.23 13.718 11.603

n 0.703 0.735 0.614 0.724 0.776 0.690 0.792 0.773 0.735

MSC 7.132 4.979 3.876 6.707 5.652 5.986 6.426 6.094 4.979

Hix

son-

Cro

wel

l

R2 0.9850 0.9773 0.8700 0.9938 0.9921 0.9276 0.9987 0.9954 0.9583

KHC 0.038 0.027 0.019 0.047 0.034 0.023 0.055 0.039 0.027

MSC 4.03 3.632 1.886 4.902 4.691 2.472 6.416 5.212 3.023

141

Table 3.15. Values of drug release kinetics models for LSH-diacerein formulations at pH 6.8,

7.4 and deionized water.

pH 6.8 pH 7.4 Deionized water

FD1 FD2 FD3 FD1 FD2 FD3 FD1 FD2 FD3

Zer

o o

rder

R2 0.7833 0.8638 0.8284 0.8047 0.8329 0.7303 0.8196 0.8168 0.7286

K0 4.848 4.101 3.090 10.66 6.832 4.561 3.995 3.218 2.497

MSC 1.375 1.840 1.609 1.433 1.623 1.157 1.559 1.543 1.150

Fir

st o

rder

R2 0.9992 0.9987 0.9700 0.9991 0.9993 0.9932 0.9886 0.9687 0.8901

K1 0.095 0.069 0.045 0.204 0.129 0.088 0.067 0.048 0.034

MSC 7.022 6.523 3.353 6.841 7.168 4.830 4.318 3.309 2.055

Hig

uch

i

R2 0.9586 0.9409 0.9530 0.9590 0.9540 0.9584 0.9625 0.9618 0.9774

KH 18.12 15.123 11.47 27.16 21.43 17.16 14.86 11.98 9.403

MSC 3.029 2.675 2.903 2.995 2.911 3.027 3.131 3.110 3.636

Kors

mey

er P

eppas

R2 0.9989 0.9994 0.9894 0.9917 0.9936 0.9966 0.9952 0.9932 0.9908

KKP 10.99 8.326 8.206 19.74 13.71 10.64 10.26 8.828 7.829

n 0.753 0.776 0.640 0.738 0.752 0.754 0.664 0.627 0.577

MSC 5.634 7.093 4.237 4.226 4.612 5.275 5.014 4.678 4.376

Hix

son-

Cro

wel

l

R2 0.9889 0.9877 0.9412 0.9868 0.9906 0.9677 0.9662 0.9380 0.8479

KHC 0.027 0.020 0.014 0.057 0.036 0.025 0.020 0.014 0.010

MSC 4.349 4.246 2.681 4.126 4.499 3.279 3.234 2.627 1.729

142

3.5. Docetaxel loaded LSH-Pluronic NPs

3.5.1. Preparation and characterization of DLP-NPs

Docetaxel (DTX) loaded LSH-Pluronic F-68 NPs (DLP-NPs) was prepared by core shell

formation in which DTX loaded LSH particles act as a core and Pluronic as a shell. For the

synthesis of core, ethanolic solution of DTX was added in the aqueous suspension of LSH

before subjected to lyophilization. LSH has the tendency to shrink in ethanol (Haseeb et al.,

2016) and during this process, DTX was entangled in LSH. Moreover, DTX was also

entrapped in the polymeric network of LSH due to the crosslinking of polymeric chains

during lyophilization (Giannouli and Morris, 2003). Drug loaded LSH core was further

protected by the self-assembly of Pluronic F-68.

3.5.2. Particle size and morphological analysis

Particle size analysis of the synthesized DLP-NPs have shown the mean diameter of 163 nm,

187 nm and 223 nm for formulation with 1%, 2% and 3% DTX loading, respectively (Table

3.16). Size of DLP-NPs increased with the increase in the drug loading amount. Size

distribution of drug loaded DLP-NPs are shown in Fig. 3.35a-c. Fig. 3.35e has shown the size

distribution of aqueous LSH suspension and average diameter of the particles was found

228.6 nm. Zeta potential of 1% drug loaded NPs is shown in Fig. 3.35g and value was −31

mV indicating the formation of well dispersed NPs and having fewer tendencies to aggregate.

143

Inte

nsi

ty (

nm

)

0 200 400 600 800 1000 1200 1400 1600 1800 2000

0

5

10

15

20

Size (d.nm)

Inte

nsity

(%

)

100 200 400 0

Size (nm) 300

0

5

10

15

20

0 200 400 600 800 1000 1200 1400 1600 1800 2000

0

5

10

15

20

25

Inte

nsity

(%

)

Size (d.nm)100 200 400 0 300

Size (nm) In

ten

sity

(n

m)

Inte

nsi

ty (

nm

)

0

5

10

15

20

25

0 200 400 600 800 1000 1200 1400 1600 1800 2000

0

5

10

15

20

25

30In

tens

ity (%

)

Size (d.nm)

0 200 400 600 800 1000 1200 1400 1600 1800 2000

0

5

10

15

20

25

30

Inte

nsi

ty (

%)

Size (d.nm)

25

20

15

10

0

5

30

100 200 400 0 300

Size (nm)

Zeta potential (mV)

To

tal

cou

nts

-100 0 100 200 0

50000

100000

150000

Inte

nsi

ty (

%)

Size distribution by intensity

0.1 1 10 100 1000 10000

10

20

30

Diameter (nm)

(a) (b) (c)

(d) (e)

(f) (g)

Fig. 3.35. Size distribution of DLP-NPs with different drug loadings: 1% (a), 2% (b), 3% (c);

FESEM image of DLP-NPs (formulation with 1% DTX loading) (d); size

distribution of LSP (1 wt% aqueous solution) (e); size distribution calculated from

FESEM (f) and Zeta potential of 1% DLP-NPs (g).

Shape and size of DLP-NPs was observed through the FESEM analysis (Fig. 3.35d).

Spherical morphology of DLP-NPs was observed through FESEM and the size was found to

be 155± 44nm (Fig. 3.35f).

144

Table 3.16. Drug loading and encapsulation efficiency of different formulations.

Formulation Drug loading

(wt%)

Encapsulation efficiency

(wt%)

Average diameter

(nm)

1% (w/w) DTX loading 0.98% ± 0.19 98.12% ± 1.8 163

2% (w/w) DTX loading 1.86% ± 0.85 93.07% ± 2.1 187

3% (w/w) DTX loading 2.35% ± 1.11 78.33% ± 3.5 223

3.5.3. PXRD and FTIR analysis of DLP-NPs

PXRD spectra of DLP-NPs were recorded to find out the texture of these NPs (Fig. 3.36a)

and also the encapsulation ability of DTX and LSH by Pluronic F-68. Being amorphous in

nature, LSH has not shown any peak in the PXRD spectrum. Sharp and distinct peaks were

observed in PXRD spectra of DTX and Pluronic F-68 indicating the crystalline nature of

these samples. PXRD spectrum of DLP-NPs is somewhat similar with the spectrum of

Pluronic F-68 as represented by the characteristics peaks observed at 19° and 24° (Khaliq et

al., 2016). These results indicated that both DTX and LSH were completely surrounded by

Pluronic F-68 and no part of LSH or DTX is left out of the prepared NPs. Moreover, the

spectrum of 3% drug loaded NPs has shown less crystalline in nature as compared with other

formulations (1% and 2% DTX loading) which may be due to inefficient encapsulation of

DTX in LSH core or inability of Pluronic F-68 to completely surround the drug loaded LSH

core. Therefore, some part of LSH remained outside during the self-assembly of Pluronic F-

68.

FTIR spectra of DLP-NPs (1% DTX loading), Pluronic F-68, DTX and LSH are shown in

Fig. 3.36b. Spectrum of DLP-NPs reflecting the presence of all signals of alkyl group and

COC of Pluronic F-68, characteristic peaks of OH, COC and CH2 of LSH and specific signals

of DTX which indicated their presence in prepared DLP-NPs.

145

5 10 15 20 25 30 35 40

Inte

nsi

ty

2 ϴ

LSH Docetaxel Pluronic F-68

1% loading 2% loading 3% loading

40080012001600200024002800320036004000

40080012001600200024002800320036004000

400900140019002400290034003900

Pluronic

DLP-NPs (1% loading)

Docetaxel

LSH

40080012001600200024002800320036004000

Wave number, cm-1

(a) (b)

Fig. 3.36. PXRD (a) and FTIR (b) spectra of LSH, DTX, Pluronic F-68 and three

formualtions of DLP-NPs.

3.5.4. In vitro drug release study from DLP-NPs

Fig. 3.37 has shown the release of DTX from DLP-NPs of all three formulations with

different drug loading. Prolonged release of DTX from these NPs was observed for more than

96 h. Furthermore, in all three formulations, only 30-50% DTX was released in first 24 h and

at the end of 96 h, 42-65% drug was released indicating a good sustained release pattern.

Furthermore, it can be concluded that the integrity of NPs was also maintained for such a

long time period.

Encapsulation efficiency and drug loading was calculated and results are shown in Table

3.16. It was observed that for 1% and 2% drug loading formulations, encapsulation efficiency

was reasonably high with 98% and 93%, respectively. With the increase in drug loading

amount to 3%, encapsulation efficiency was dropped to 78.33%. This may be due to the high

146

concentration of DTX which is not being able to retain within these DLP-NPs. Total drug

loading in DLP-NPs was 0.98%, 1.86% and 2.35% for formulations with 1%, 2%, and 3%

initial loading of DTX, respectively (Table 3.16).

Drug release mechanism from the prepared DLP-NPs was analyzed using Korsmeyer

Peppas equation (Nguyen et al., 2014). Drug release followed the Fickian diffusion if the

value of n < 0.45 and for 1% drug loading formulation, it was calculated to 0.43. Therefore, it

can be concluded that the drug release from these prepared DLP-NPs (1% drug loading) was

mediated by diffusion mechanisms (Ritger and Peppas, 1987). One advantage of diffusion

controlled drug release mechanism over swelling or erosion based system is the prolong

release of drug as the integrity of the system is maintained for longer period of time.

0

20

40

60

80

100

0 12 24 36 48 60 72 84 96

Cu

mu

lati

ve

DT

X r

elea

se, %

Time, h

1 % loading 2 % loading

3 % loading DTX

Fig. 3.37. Docetaxel release from different formulations of DLP-NPs

3.5.5. Cytotoxicity and cellular uptake behaviour of DLP-NPs

MTT assay was used to observe the cytotoxicity of LSH and DLP-NPs. It was observed that

LSH have shown good cell viability (≈ 100%) up to the concentration of 250 µg/mL while at

concentration of 500 µg/mL cell viability was reduced to 82%. These values indicate that

147

LSH possess very low toxicity, hence, biocompatible to the cell culture system. DLP-NPs

have shown no effect on cell viability up to the concentration of 25 µg/mL. Cytotoxicity in

cell culture was seen at a concentration of 125 µg/mL and 52% cells were observed viable

(Fig. 3.38). IC50 values of DLP-NPs and free DTX are 116.994 ± 0.27 and 16.173 ± 0.15

µg/mL, respectively. Whereas, LSH and untreated cells did not show any IC50 value.

0

20

40

60

80

100

0 125 250 375 500

Cell

via

bil

ity, %

Concentration, µg/mL

Untreated cells LSH

DLP-NPs Free DTX *

Fig. 3.38. In vitro cytotoxicity of LSH, DTX and DLP-NPs at various concentrations.

Statistical significance is shown by * p < 0.05, performed by student‘s t-test for

comparison.

Pluronic NPs has the ability to penetrate into SCC-7 tumour cells through endocytosis

process (Rapoport et al., 2002). Therefore, cellular uptake of DLP-NPs in cell culture system

was monitored. Nile red was incorporated in DLP-NPs during drug loading phase and these

NPs were incubated in SCC-7 tumour cells. Cellular uptake of these NPs was examined using

fluorescent microscope and it was revealed that NPs are only taken up by cytoplasm of SCC-

7 tumour cells and nuclear compartment is free from such penetration (Fig. 3.39).

148

Control

0.5 h

4h

2h

1h

Phase DAPI (Nucleus) Nile Red (Cytoplasm) Merge D

LP

-NP

s w

ith

Nil

e R

ed,

20

x

Control

0.5 h

4h

2h

1h

DL

P-N

Ps

wit

h N

ile

Red

, 4

0 x

(a)

(b)

Fig. 3.39. Cellular uptake images of DLP-NPs (20x, a) and (40x, b).

149

3.6. Nanobiotechnological application of LSH

3.6.1. Green synthesis of Ag NPs

On subjecting AgNO3 solutions to diffused sunlight, hydrated electrons are generated in the

system. These electrons can be used to reduce monovalent silver cations (Ag+) to zerovalent

silver atom (Ag0). The Ag

0 atoms so generated usually

have a size ranging in nanometers.

Therefore, UV irradiation can be exploited as a green method for the synthesis of Ag NPs.

This method can also be used to avoid environmentally toxic and hazardous reducing agents.

Green synthesis of Ag NPs was carried out using LSH as a green reducing agent under

diffused sunlight. LSH was also evaluated as a storage medium for the synthesized Ag NPs.

LSH mediated synthesis of Ag NPs was carried out using different concentrations of AgNO3

solutions (10, 20 and 30 mmol). On mixing the reactants, Ag+ in the AgNO3 solution reacted

with LSH to give [Ag(LSH)]+ complex. When the mixture was subjected to diffused sunlight,

Ag+

in the complex were reduced by LSH to produce [Ag(LSH)] precursor. The positive

charge on the surface of Ag NPs was electrostatically stabilized and capped by the negative

charge on polysaccharide hydroxyls which resulted in colloidal stabilization of [Ag(LSH)].

The progress of formation of Ag NPs by irradiation of [Ag(LSH)]+ complex was followed by

noting the color change of reaction mixture with passage of time. The color of LSH and

AgNO3 mixture changed from colorless to reddish brown and finally dark brown within 10 h.

A schematic illustration of synthesis of LSH mediated synthesis of Ag NPs is shown in Fig.

3.40.

150

AgNO3

(aq)

+

LSH

Ag

+

+ +

+ +

+ +

+

O

O

O

O

O

O O

O

O

O

O

O

O

O

O

O

O

O

O O

O

Ag NPs capped by LSH

Fig. 3.40. Schematic illustration showing synthesis of LSH mediated Ag NPs.

3.6.2. Characterization of Ag NPs

3.6.2.1. UV spectrophotometry

LSH suspension (2 mL) was mixed with different conc. of AgNO3 (10, 20, 30 mmol, 2 mL

aliquot each) in dark and then placed in diffused sunlight. Reduction of Ag+ to Ag

0 was

indicated by change in color of reaction mixture and monitored by UV/Vis

spectrophotometry. The conduction electrons of Ag NPs exhibit surface plasmon resonance

(SPR) due to their collective oscillation and this SPR results in strong absorption in visible

region of spectrum (Mock et al., 2002). The wavelengths absorbed for SPR transitions of Ag

NPs are changed by change in reaction time and size of Ag NPs, so reaction mixture showed

a color change as the reaction progressed. The color changed from colorless to reddish brown

and finally dark brown with passage of time from 0.25 to 10 h. Photographs showing the

color change of reaction mixture with progress of reaction are shown in Fig. 3.41.

151

05 Min

30 Min

60 Min

90 Min

120 Min

600 Min

Fig. 3.41. Photographs of LSH-Ag+ mixture (20 mmol AgNO3) showing color change

with passage of time.

The LSH-Ag+ solutions showed UV/Vis absorptions at 410, 415, 419, 425, 426, 428, 430,

431, 436 nm for 10 mmol. Whereas, 412, 416, 422, 426, 428, 430, 431, 434, 436 nm

absorptions were recorded for 20 mmol LSH-Ag+ solution. Likewise, 30 mmol solution

showed UV absorptions at 413, 417, 424, 427, 429, 432, 433, 435 and 437 nm. The

absorption spectra were recorded at reaction time of 0.25, 0.5, 0.75, 1, 2, 4, 6, 8 and 10 h,

respectively. Similar results have been reported in literature where Ag NPs were synthesized

using exogenous reducing agents (El-Sheikh et al., 2013). The absorption spectra of Ag NPs

showed red shift indicating increase in size of Ag NPs with increase in reaction time.

Absorption intensity also showed an increase when reaction time was increased from 0.25 to

10 h which indicated continuous reduction of Ag+ by LSH with passage of time. Results of

UV/Vis analyses are summarized in Fig. 3.42.

152

280 380 480 580 680

Ab

sorb

ance

Wavelength, nm

0.25h

0.5h

0.75h

1h

2h

4h

6h

8h

10h

280 380 480 580 680

Ab

sorb

ance

Wavelength, nm

0.25h

0.5h

0.75h

1h

2h

4h

6h

8h

10h

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.2

0.4

0.6

0.8

1.0

0.2

0.4

0.6

0.8

1.0

280 380 480 580 680

Ab

sorb

ance

Wavelength, nm

0.25h

0.5h

0.75h

1h

2h

4h

6h

8h

10h

405

410

415

420

425

430

435

440

Wav

elen

gth

, n

m

Time, h

10 mmol

20 mmol

30 mmol

(a)

(c)

(b)

(d)

Fig. 3.42. UV/Vis spectra of LSH mediated Ag NPs: 10 mmol (a), 20 mmol (b) and 30 mmol

solution of AgNO3 (c) at different reaction times and cumulative graphical

representation (d) showing increase in absorption of Ag NPs solutions with

increase in concentration and reaction time.

3.6.2.2. Transmission electron microscopy of isolated Ag NPs

Size distribution and morphology of Ag NPs was assessed by TEM. Ag NPs were separated

from LSH and AgNO3 (10, 20 and 30 mmol) solutions by centrifugation and TEM image was

recorded. TEM images confirmed the formation of highly spherical Ag NPs in the size

regimen of 10-25 nm for 10 mmol, 10-30 nm for 20 mmol and 10-35 nm for 30 mmol

AgNO3 solution (Fig. 3.43).

153

500 nm 50 nm50 nm

a) b) c)

Fig. 3.43. TEM images of Ag NPs isolated from 10, 20 and 30 mmol LSH-Ag+ solution

having size range from 10-25 nm (a), 10-30 nm (b) and 10-35 nm (c),

respectively.

3.6.2.3. Powder X-ray diffraction

PXRD (Fig. 3.44) was used to confirm the crystal phase of Ag NPs isolated from AgNO3

(10, 20 and 30 mmol) solutions in the range of 10-80°, 2ϴ. The diffraction peaks centered at

(111), (200), (220) and (311) indicated that Ag NPs had face-centered cubic lattice in all

samples.

154

(e)

(d)

(c)

(b)

(a)

10 20 30 40 50 60 70 80

2θ (Degree)

(311) (220) (200)

(111)

Fig. 3.44. PXRD spectra: LSH (a), Ag NPs embedded LSH film (b) and isolated Ag NPs, 10

mmol (c), 20 mmol (d) and 30 mmol (e).

3.6.2.4. Storage of Ag NPs in LSH thin film

Potential of LSH for the storage of Ag NPs in solution as well as in the form of a thin film

was evaluated. For this purpose, Ag NPs were synthesized by reducing the AgNO3 (20

mmol) solution with LSH over 10 h. UV/Vis spectra were recorded after 10 and 24 h and

sample was stored as dry thin film under dark. Absorption spectra of the isolated Ag NPs

were acquired by dissolving the films in deionized water. Comparable absorption spectra

were obtained after the storage period (15, 30 days, and 06 months). No significant change in

the absorption wavelength and intensity was observed for the stored samples (Fig. 3.45a).

Therefore, it was inferred that the Ag NPs did not undergo agglomeration on storage in LSH

thin films. X-ray diffraction pattern was recorded for the Ag NPs isolated from stored thin

films (06 months). It was revealed that there was no change in diffraction peak of samples

155

after storage. Therefore, it was concluded that LSH could be used for the long term storage of

Ag NPs without affecting their morphology (Fig. 3.45b). Moreover, the stored film was

dissolved in deionized water and Ag NPs were isolated by centrifugation. TEM image of

isolated Ag NPs showed that the NPs retained their morphology and size over the storage

period (Fig. 3.45e). Therefore, it was concluded that the LSH thin films could be used as

storage media for Ag NPs. Results of storage experiments are illustrated in Fig. 3.45.

10 20 30 40 50 60 70 80

2θ (Degree)

(311)(220)

(200)

(111)

After 6

Months

10 h

24 h

15 days

30 days

6 months

0.2

0.4

0.6

0.8

1.0

280 380 480 580 680

Wavelength, nm

Ab

sorb

ance

50 nmc d

d

e

a b

After 6

Months

(c)

10 h

24 h

15 days

30 days

6 months

0.2

0.4

0.6

0.8

1.0

280 380 480 580 680

Wavelength, nm

Ab

sorb

ance

(a)

10 20 30 40 50 60 70 802θ (Degree)

(311) (220)

(200)

(111) (b)

(d) (e)

10 20 30 40 50 60 70 80

2θ (Degree)

(311)(220)

(200)

(111)

After 6

Months

10 h

24 h

15 days

30 days

6 months

0.2

0.4

0.6

0.8

1.0

280 380 480 580 680

Wavelength, nm

Ab

sorb

ance

50 nmc d

d

e

a b

(f)

Fig. 3.45. UV/Vis spectra of Ag NPs synthesized from aqueous solution of AgNO3 (20

mmol) and LSH recorded after 10 h, 01, 15, 30 days and 06 months storage (a);

PXRD spectrum of Ag NPs isolated after 06 month storage of LSH-Ag NPs film

(b); vial containing solution of stored LSH-Ag NPs film in water (c); foldable (d)

and see through (f) Ag NPs embedded LSH thin film; TEM image of Ag NPs

(10-30 nm) isolated from LSH-Ag NPs film stored for 06 months under dark (e).

156

3.6.2.5. Antimicrobial activity of Ag NPs

LSH based Ag NPs were tested for antimicrobial properties. Significant antibacterial and

antifungal activity was observed against S. mutans, S. epidermidis, P. aeruginosa, E. coli, S.

aureus, B. subtilis, A. odontolyticus, and A. niger strains. The cultures of above mentioned

strains showed inhibition zones of 22, 13, 18, 09, 20, 15, 16, and 10 mm on being exposed to

the aqueous solution of Ag NPs synthesized with AgNO3 (20 mmol) solution, respectively.

No antimicrobial activity was noticed for control experiments performed by using LSH

solution and sterile distilled water. However, AgNO3 (0.02 M/20 mmol) solution was found

active against the above mentioned strains. Antimicrobial properties Ag NPs prepared from

10 and 30 mmol AgNO3 solution were also tested. Results of antimicrobial activity of Ag

NPs (20 mmol) is summarized in Fig. 3.46b and average of three readings are reported. As a

typical example, antimicrobial activity of Ag NPs (20 mmol) against A. odontolyticus and S.

aureus is shown in Fig. 3.46a. It is revealed that LSH based Ag NPs show a clear zone of

inhibition against the above mentioned strains while LSH alone did not show any effect on

the microbial cultures.

157

LSH Water DMSO

AgNO3

Activity against

P. aeruginosa

0

5

10

15

20

25

Zo

ne

of

inh

ibit

ion (

mm

)

Microorganisms

(a) (b)

Fig. 3.46. Antimicrobial activity (a) and graphical representation of zone of inhibition of Ag

NPs (20 mmol) against different bacterial and fungal strains (b).

3.6.2.6. Wound healing studies

In order to evaluate wound healing properties of LSH films impregnated with Ag NPs,

excision wounds were created on rear leg of rabbit (Fig. 3.47). Average wound area was

calculated for different groups of rabbits. Wound healing was assessed after measuring the

area of wound (mm2) at regular time intervals. Control group showed a wound closure of

0.29, 1.11, 20.32, 38.76, 62.55 and 83.27 % on 1st, 3

rd, 6

th, 9

th, 12

th and 15

th day, respectively.

The animals treated with standard Band aid® dressing showed closure area of 2.17, 19.45,

63.67, 87.27, 98.49 and 100% while the test group showed 1.41, 17.73, 54.62, 93.21, 99.22

and 100% wound closure on 1st, 3

rd, 6

th, 9

th, 12

th and 15

th day, respectively. Results indicated

that tissue regeneration and percentage wound closure of LSH based wound dressing was

comparable to the standard Band aid® dressing. Results of wound healing study for various

groups are summarized in Table 3.17.

158

Fig. 3.47. Schematic illustrations of wound treatment with Ag NPs embedded LSH wound

dressing patch (a) and also showing its main parts (b).

In order to understand the mechanism of wound healing, collagen content and tensile strength

of the epithelialized wound tissue was measured. The control group showed collagen content

of 38 mg/kg while standard and test groups showed collagen content of 59 and 55 mg/kg,

respectively. Therefore, it was noticed that the standard and test group had higher collagen

content than control group which resulted rapid wound healing in the said groups in contrast

to the control. Tensile strength was also found higher for test and standard groups as

compared to control. Results of collagen content of regenerated tissue for various groups are

depicted in Fig. 3.48. It was inferred that collagen synthesis is initiated at the wound area

through formation of a polypeptide precursor. Inter and intramolecular crosslinking of this

collagen results in rapid tissue regeneration and wound closure.

159

0

10

20

30

40

50

60

70

Control Band aid®

dressing

LSH dressing

Co

llag

en c

on

ten

t (m

g/k

g)

*

Fig. 3.48. Collagen contents of epithelialized wound tissue of various groups after 15th

day.

Statistical significance from control group is expressed by * p < 0.05.

Table 3.17. Wound area (mm2) and wound closure (%) after selected day intervals.

Wound area in mm ± SD (% of wound closure) at day

1st 3

rd 6

th 9

th 12

th 15

th

Control 5.98 ± 0.06

(0.29)

5.93 ± 0.05

(1.11)

4.78 ± 0.03

(20.32)

3.67 ± 0.10

(38.76)

2.25 ± 0.06

(62.55)

1.00 ± 0.02

(83.27)

Band aid®

dressing

5.87 ± 0.04

(2.17)

4.83 ± 0.08

(19.45)

2.18 ± 0.09

(63.67)

0.76 ± 0.05

(87.27)

0.09 ± 0.08

(98.49)

(100)

LSH

dressing

5.91 ± 0.04

(1.41)

4.94 ± 0.07

(17.73)

2.72 ± 0.08

(54.62)

0.41 ± 0.06

(93.21)

0.05 ± 0.08

(99.22)

(100)

160

3.7. Acute toxicological evaluation of LSH

3.7.1. Acute oral toxicity study in mice

In acute toxicity study, a single oral dose of LSH was given to three groups (Group II, III and

IV) of male albino mice (Table 2.4). At the end of day 14, all animals were alive, active and

healthy. No other abnormalities were seen including behavioral changes, pharmacological

adverse effects and gross toxicities during the 14 day study period. Food intake was less after

day 1 as compared to remaining days. Results have indicated that the acute oral lethal dose

(LD50) of LSH is greater than 5 g/kg of body weight (bw) for male albino mice.

3.7.2. Primary eye irritation

To find out the primary eye irritation, LSH was used for a single ocular installation in the eye

of rabbit. Treated eye was not found any corneal opacity and iritis during 72 h study. Eye of

one animal was exhibited conjunctival discharge and cured within 24 h. The score was

assigned 2.0 and 1.0 after 1 h and 24 h, respectively according to Kay and Calandra eye

irritation scale (Kay and Calandra, 1962) (Table 3.18). No other adverse effects and

symptoms were seen. Therefore, the conjunctival discharge was classified as accidental and

overall LSH was found to be nonirritating to the eye.

161

Table 3.18. Scores for grading the primary eye irritation study of LSH.

Studied groups Ocular observations Time duration (h)

01 24 48 72

Group I Corneal opacity 0 0 0 0

Iritis 0 0 0 0

Conjunctivitis 0 0 0 0

Severity (MMTS) 0 0 0 0

Group II Corneal opacity 0 0 0 0

Iritis 0 0 0 0

Conjunctivitis 2 1 0 0

Severity (MMTS) 0.67 0.33 0 0

Group III Corneal opacity 0 0 0 0

Iritis 0 0 0 0

Conjunctivitis 0 0 0 0

Severity (MMTS) 0 0 0 0

Group IV Corneal opacity 0 0 0 0

Iritis 0 0 0 0

Conjunctivitis 0 0 0 0

Severity (MMTS) 0 0 0 0

MMTS (Maximum mean total scores) of 3 animals

3.7.3. Acute dermal toxicity

Acute dermal toxicity study was carried out in male albino rabbits. Single topical application

of three different doses of LSH (1, 2 and 5 g/kg bw) were applied on three groups of rabbits.

162

All animals survived, gained weight and did not found any other clinical findings or

abnormalities during the whole course of study. It is concluded that the acute dermal toxic

dose was greater than 5 g/kg body weight.

3.7.4. Primary dermal irritation study

Primary dermal irritation study was conducted to evaluate the potential of LSH irritancy on a

single topical application. No erythema or edema was found on the skin of treated animal

over the course of 14 days. Primary dermal irritation index was calculated as 0.0 indicating

the LSH as a non-irritating material to the skin.

3.7.5. Body weight gain study

Results of body weight gain study are summarized in Table 3.19. It was noted that there are

gradually increase in mean body weight of the control group during the whole study period.

A decrease in body weight of group II, group III and group IV was observed at day 1 while

mice were gained the body weight at day 7 and 14.

3.7.6. Food and water consumption

Food and water intake of each group was measured on daily basis and results have indicated

that there was not any significant difference in food and water consumption of control group

during the whole study period (Table 3.19). A significant decrease in the food and water

intake was noted for group II, group III and group IV after day 1 when compared to the

control group. At day 7 and 14, amount of food and water consumed by experimental group

had not much different when compared to the control group.

163

Table 3.19. Observations of body weight, water and food intake study of LSH.

Observations Group I Group II Group III Group IV

Signs of illness NIL NIL NIL NIL

Body weight (g)

Pretreatment 26.4 ± 2.2 28.8 ± 1.8 26.1 ± 2.2 27.7 ± 1.5

Day 1 26.5 ± 1.4 28.7 ± 1.5 26.0 ± 1.9 27.4 ± 3.5

Day 2 27.6 ± 1.2 29.1 ± 1.9 26.2 ± 1.1 27.5 ± 3.5

Day 3 28.0 ± 1.8 29.8 ± 1.1 26.5 ± 0.9 27.7 ± 3.5

Day 5 28.4* ± 2.7 30.1 ± 2.2 27.2 ± 2.0 28.8* ± 3.5

Day 7 28.5* ± 2.4 30.4* ± 1.8 27.6* ± 2.8 29.4* ± 2.5

Day 14 28.8* ± 2.0 30.8* ± 2.8 28.4* ± 2.0 29.6* ± 1.8

Water intake (mL)

Pretreatment 10.4 ± 1.0 12.2 ± 0.5 11.1 ± 2.1 11.7 ± 1.5

Day 1 10.8 ± 1.5 12.4 ± 1.5 11.6 ± 1.5 12.3 ± 1.7

Day 2 11.5 ± 1.1 12.6 ± 0.5 12.0 ± 2.1 12.6 ± 1.1

Day 3 12.8 ± 0.8 12.5 ± 1.3 12.3 ± 1.8 12.7 ± 1.1

Day 5 13.1* ± 1.8 13.3 ± 1.5 12.8* ± 1.0 12.9 ± 0.7

Day 7 13.0* ± 2.0 13.7* ± 2.0 13.0* ± 2.5 13.0 ± 2.3

Day 14 13.2* ± 1.5 13.9* ± 1.5 13.2* ± 1.5 13.2* ± 2.5

Food intake (g)

Pretreatment 3.4 ± 1.0 3.1 ± 1.5 3.6 ± 2.1 3.8 ± 1.6

Day 1 2.7 ± 1.5 2.4 ± 2.1 1.8* ± 1.1 1.1* ± 1.7

Day 2 3.2 ± 1.9 2.7 ± 2.1 2.3* ± 0.7 2.0* ± 2.1

Day 3 3.8 ± 1.0 2.9 ± 1.1 3.0 ± 1.2 2.3* ± 1.3

Day 5 3.6 ± 1.3 3.5 ± 1.4 3.2 ± 1.6 3.2 ± 0.7

Day 7 4.0 ± 1.0 4.0 ± 1.9 3.8 ± 0.5 3.6 ± 0.9

Day 14 3.8 ± 1.5 3.8 ± 1.3 4.3 ± 2.1 4.2 ± 1.1

Body weight, water and food intake are expressed as mean ± S.D. *P < 0.05 as significant

difference when compared to control.

164

3.7.7. Haematology and clinical biochemistry

Hematological parameters of each group were found to be normal and they are comparable

with the control group (Group I) as mentioned in Table 3.20. Liver, kidney and lipid profile

was also determined and results are found to be normal. All values are comparable with the

animals of control group as mentioned in Table 3.21.

3.7.8. Gross necropsy and histopathology

Macroscopic analysis mice of each group were performed and did not found any LSH-related

abnormalities (Table 3.22). All vital organs were remove and weighed separately. Results

have indicated that there were not found any significant difference in weight when compared

with the mice of control group.

Table 3.20. Biochemical blood analysis of control and LSH treated mice.

Hematology Group I Group II Group III Group IV

Hb (g/dL) 11.6 ± 0.8 12.1 ± 1.6 13.7 ± 1.6 13.5 ± 1.4

WBCs (103/µL) 4.2 ± 0.3 5.1 ± 0.4 5.9 ± 1.2 6.5 ± 0.5

RBCs (106/µL) 8.28 ± 0.23 8.39 ± 0.45 8.32 ± 0.35 8.15 ± 0.23

Platelets ( 103/µL) 549.7 ± 2.9 667.2 ± 3.9 678.6 ± 5.5 709.6 ± 4.1

Monocytes (%) 2.1 ± 0.02 2.3 ± 0.01 2.5 ± 0.01 2.2 ± 0.02

Neutrophils (%) 12.8 ± 0.5 13.5 ± 0.3 12.9 ± 0.9 13.8 ± 0.02

Lymphocytes (%) 83.3 ± 2.1 82.3 ± 1.5 84.6 ± 2.1 82.9 ± 1.3

MCV 45.8 ± 1.5 45.9 ± 2.1 51.4 ± 1.4 53.5 ± 1.4

MCH 14.5 ± 0.5 16.0 ± 0.8 15.7 ± 1.0 14.4 ± 1.2

MCHC (g/dL) 28.7 ± 0.3 30.4 ± 0.7 29.6 ± 1.5 31.6 ± 1.1

All values are expressed as mean ± S.D.

165

Table 3.21. Liver, kidney and lipid profile of mice treated with LSH.

Biochemical analysis Group I Group II Group III Group IV

ALT (IU/L) 54 ± 5.3 70 ± 3.2 58 ± 5.2 66 ± 2.1

AST (IU/L) 134 ± 8.3 145 ± 6.1 176 ± 7.1 185 ± 5.1

Creatinine (mg/dL) 0.43 ± 0.02 0.42 ± 0.01 0.35 ± 0.04 0.38 ± 0.02

Urea (mg/dL) 62 ± 2.3 53 ± 4.2 48 ± 4.2 59 ± 3.4

Uric acid (mg/dL) 5.6 ± 0.2 4.5 ± 0.05 4.6 ± 0.1 4.8 ± 0.1

Cholesterol (mg/dL) 147 ± 7.2 136 ± 2.3 108 ± 1.1 116 ± 5.2

Triglyceride (mg/dL) 132 ± 9.4 110 ± 2.3 115 ± 5.4 87 ± 4.8

All values are expressed as mean ± S.D.

Table 3.22. Absolute mean organ weight (g) of mice after oral administration of LSH.

Organ Group I Group II Group III Group IV

Heart 0.66 ± 0.05 0.58 ± 0.03 0.57 ± 0.05 0.53 ± 0.01

Liver 6.44 ± 0.37 6.15 ± 0.22 5.52 ± 0.14 5.70 ± 0.32

Lung 0.54 ± 0.12 0.63 ± 0.11 0.67 ± 0.23 0.51 ± 0.11

Kidney 0.98 ± 0.05 1.08 ± 0.07 0.88 ± 0.05 1.52 ± 0.02

Stomach 1.05 ± 0.18 1.79 ± 0.1 1.38 ± 0.4 1.6 ± 0.20

Spleen 0.63 ± 0.12 0.55 ± 0.01 0.61 ± 0.03 0.60 ± 0.12

All weights are expressed as relative organ weights and expressed the mean values ± S.D.

166

CONCLUSIONS

Polysaccharides based hydrogel was isolated from linseeds and characterized. LSH have

shown marked swelling in deionized water and at gastrointestinal tract pH (6.8 and 7.4) while

less swelling was observed at pH 1.2. Therefore, we have studied the stimuli responsive

swelling deswelling of LSH in different solvents and at basic and acid pH. Moreover, we

used said valuable properties of LSH, i.e., marked swelling, high water holding capacity and

stimuli responsible properties of LSH and successfully developed sustained release oral drug

delivery system of diclofenac sodium, caffeine and diacerein. Interestingly, the swelling

properties and swelling deswelling responses of LSH were still observed even in compressed

form, i.e., tablet formulations. Therefore, LSH can be a good candidate for the development

of sustained release formulation of short half-life drugs and also for pH dependent drug

delivery. For future prospect, LSH can be used to protect the active ingredient from the harsh

gastric environment/pH.

Highly porous structure with microscopic channeling was observed in swollen then freeze

dried sample of LSH which is considered the main reasoning of highly swellable nature of

LSH. This provided the basis to use LSH in future oral dosage forms.

LSH based nanoparticles were developed using Pluronic F68 and an anticancer drug,

docetaxel, was loaded. These docetaxel loaded LSH Pluronic (DLP) NPs has not shown any

toxicity and proved its anticancer effectiveness with minimum side effects. Formation of

DLP-NPs has opened up a new door for the delivery of other therapeutic and diagnostic

agents.

LSH appeared as a reducing and capping agent for the green synthesis of Ag NPs. The COO─

and OH groups are responsible for the synthesis of Ag NPs. Spherical shaped Ag NPs

embedded in LSH film has shown a pronounced antimicrobial and wound healing dressing.

Collagen contents and wound healing period of LSH containing wound dressing was

167

comparable with commercially available product. In future, wound healing mechanism of

these dressing can be explored to broaden the wide range of LSH application.

Additionally, LSH was proved safe after acute toxicity study without any significant

observations. Biochemical, haematological and histopathological examination has shown the

nontoxic nature of LSH. Therefore, LSH can be used as a novel drug delivery carrier for oral,

topical and parenteral administration of many drugs.

On the basis of current research work and available literature of related materials, further

research on applications of LSH in tissue engineering and tissue regeneration should be

carried out. Additionally, use of LSH as a selective chemosensor, biosensor and vaccine

adjuvant should be explored. Commercialization of LSH as an inactive pharmaceutical

ingredient should be taken into account as a future aspect of work reported here.

168

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LIST OF PUBLICATIONS

1. Haseeb, M. T., Hussain, M. A., Yuk, S. H., Bashir, S. and Nauman, M. (2016).

Polysaccharides based superabsorbent hydrogel from Linseed: Dynamic swelling, stimuli

responsive on-off switching and drug release. Carbohydr. Polym., 136:750-756.

2. Haseeb, M. T., Hussain, M. A., Bashir, S., Ashraf, M. U., Ahmad, N. (2017). Evaluation

of superabsorbent Linseed-polysaccharides as a novel stimuli-responsive oral sustained

release drug delivery system. Drug Dev. Ind. Pharm. 43:409-420.

3. Haseeb, M. T., Hussain, M. A., Abbas, K., Youssif, B. G. M., Bashir, S., Yuk, S. H.,

Bukhari, S. N. A. (2017). Linseed hydrogel mediated green synthesis of silver

nanoparticles for antimicrobial and wound dressing applications. Int. J. Nanomedicine.

12:2845-2855.

4. Haseeb, M. T., Bashir, S., Hussain, M. A., Ashraf, M. U., Erum, A., Hassan, M. N.

(2018). Acute toxicity study of a polysaccharide based hydrogel from linseed for potential

use in drug delivery system. Braz. J. Pharm. Sci. 54:e17459.

5. Haseeb, M. T., Hussain, M. A., Yuk, S. H., Amin, M., Bashir, S. Acetylation of linseed

hydrogel: Synthesis, characterization, isoconversional thermal analysis and degradation

kinetics. Cell. Chem. Technol. (Accepted on 26.06.2018).

Submitted

1. Haseeb, M. T., Khaliq, N. U., Yuk, S. H., Hussain, M. A., Bashir, S. Linseed

polysaccharides based nanoparticles for controlled delivery of docetaxel: Design, in vitro

drug release and cellular uptake. Journal of Drug Delivery Science and Technology.