TUNABLE SYNTHESIS OF GELATIN NANOPARTICLES …

www.wjpps.com Vol 4, Issue 07, 2015.

1365

Suvidya et al. World Journal of Pharmacy and Pharmaceutical Sciences

TUNABLE SYNTHESIS OF GELATIN NANOPARTICLES

EMPLOYING SOPHOROLIPID AND PLANT EXTRACT, A

PROMISING DRUG CARRIER

Zeinab Ghasemishahrestani1, Mihir Mehta

2, Priti Darne

2, Aishwarya Yadav

1, Asmita

Prabhune*2

, Suvidya Ranade*1

.

1Department of Chemistry, Savitribai Phule Pune University, Pune-411007.

2CSIR-National Chemical Laboratory, Pashan, Pune-411008.

ABSTRACT

Biodegradable polymer nanoparticles are being explored as drug

delivery vehicle for various diseases till date. We report for the first

time the green synthesis of tunable gelatin nanoparticles by

nanoemulsion method using Cassia fistula leaf extract and

Sophorolipids synthesized by a non-pathogenic yeast Candida

bombicola ATCC 22214 a glycolipid biosurfactant. Sophorolipid and

plant extract helped in compaction and reduction in size of gelatin

nanoparticles with effective loading of Quercetin so as to get spherical

nanoparticles with size ranging between 20-60 nm. SEM, TEM and

DLS results are in agreement for the size of nanoparticles. Sustained

drug release pattern was seen in vitro at pH 7.4. These drug loaded nanoparticles were further

characterized and tested for cytotoxicity on MCF7 cancer cell line. The bare and drug loaded

nanoparticles displayed differential cytotoxicity towards MCF7 cancer cells. These biogenic

nanoparticles are biocompatible and found to be good candidates for sustained drug delivery

in diseases like cancer.

KEYWORDS: Biodegradable, Gelatin, Cassia fistula, Sophorolipids, Quercetin,

Cytotoxicity.

1. INTRODUCTION

In recent years the research has been focused on developing drug delivery systems using

biocompatible or biodegradable polymers. The biodegradable nanoparticles have wide range

WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS

SSJJIIFF IImmppaacctt FFaaccttoorr 55..221100

VVoolluummee 44,, IIssssuuee 0077,, 11336655--11338811.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357

Article Received on

14 May 2015,

Revised on 05 June 2015,

Accepted on 26 June 2015

*Correspondence for

Author

Dr. Suvidya Ranade

Department of Chemistry,

Savitribai Phule Pune

University, Pune- 411007.


1366


of biomedical applications (Kumari 2012; Nitta 2013). These are especially used in

therapeutics, tissue engineering etc. The macromolecular materials to which drug molecule is

attached, adsorbed, entrapped or encapsulated are being used for drug delivery applications.

The biodegradable polymers like chitosan, alginate (Ciofani 2008; Gan 2005) guar gum

(Soumya 2010), hydroxyethyl cellulose, Gellan gum, CMC, dextrans(Wu 2013) and gelatin

are being used for this purpose (Naidu 2011; Jahanshahi 2008;Soopimath 2001; Rokhade

2006). The selection of matrix constituent is dependent on many factors including size,

properties of the drug, charge and permeability; degree of biodegradability, biocompatibility,

toxicity and antigenicity (Kreuter 1994; Mahapatro 2011). For the synthesis of such

biodegradable nanoparticles conventional methods utilize toxic chemicals like polyvinyl

alcohol, polyethylene glycol, etc as stabilizers (Kumari 2012). One needs to look for green

method of synthesis and use of stabilizers which are biogenic and non-toxic for developing

the drug delivery system.

Proteins are the class of natural molecules that have unique functionalities and potential

applications in biological as well as material fields. Protein nanoparticles are biodegradable

non-antigenic, metabolizable and easily amenable for surface modification and covalent

attachment of drugs. Currently the research is focused on preparation of protein nanoparticles

like albumin (Steinhauser 2008; Ulbrich 2009), gelatin (Kumari 2012; Jahanshahi 2008;

Mahapatro 2011; Zillies2008; Ofokansi 2010; Babaei 2008), gliadin and legumin (Jahanshahi

2008; Arangoa 2001; Mirshahi 2002).

The gelatin nanoparticles are being explored for various therapeutic applications. Interest in

use of gelatin as biodegradable materials are based on the facts that: gelatin is FDA approved,

biodegradable, non-toxic, easy to crosslink and to modify chemically and has therefore an

immense potential to be used for the preparation of colloidal drug delivery systems such as

nanoparticles. Gelatin is obtained by hydrolysis of collagen which is found as the major

component of skin, bones and connective tissue is one of the protein materials (Azarmi 2006;

Jahanshahi 2008). Gelatin being polyampholytic in nature has broad molecular weight range

and thus is difficult to prepare stable monodispersed nanoparticles. Different research groups

have used various methods for synthesis of gelatin nanoparticles till date, such as

nanoprecipitation (Nitta 2013; Lee 2012)coacervation, desolvation (Naidu 2011;Coesters

2000;Sailaja 2012), emulsion/solvent evaporation (Khan 2013; Penning 1993) complex

coacervation (Azimi 2013; Yeh 2005; Gan 2011; Leong 1982), cross linking with


1367


polyethylimine and glutaraldehyde (Kuo 2011; Leo 1997). Each method has its own

advantages and disadvantages. The gelatin nanoparticles are being explored for variety of

biomedical applications such as carriers for ophthalmic drugs, easy adherence to inflamed

occular cells than normal cells (Jahanshahi 2008; Das 2005; Nakaoka 1995). Gelatin

nanoparticles have been used in therapeutic applications for diabetes, breast cancer, bladder

cancer etc. (Nitta 2013; Babu 2012; Zhao 2012; Jain 2012; Lu 2011).

In the present study we have synthesized gelatin nanoparticles using Cassia fistula leaf

extract by Nanoemulsion method for the first time. The extract was used as the stabilizing

agent. Cassia fistula is the traditional Indian medicinal plant described to be useful against

skin diseases, liver troubles, tuberculosis and its use in the treatment of rheumatism,

haematemesis, pruritus, leucoderm and diabetes (Bahorun 2005; Panda 2010). The stem bark

extract of this plant has been already explored for synthesis of metal nanoparticles (Daizy

2012).

As we obtained bigger size particles of few hundred nanometers in experiments conducted

prior to this study using Cassia fistula leaf extract, we used a biosurfactants sophorolipid to

help in the compaction and size reduction of gelatin nanoparticles.

Quercetin has anticancer potential in case of lung cancer, oral cancer; human colon and

breast cancer cell lines reported by various groups (Zhang 2012; Chen 2013; Zheng 2012).

Being hydrophobic in nature the drug has low aqueous solubility, negligible skin

permeability and reduced stability. These limitations were overcome by the use of

amphiphilic, biodegradable and non-toxic sophorolipid which helped in solubilization and

better drug entrapment. In addition to its biosurfactant property it has been reported to have

anti-cancer activity by Joshi-Navare et al. where they have induced cell differentiation in

human glioma cell LN-229 (2011). In this study sophorolipid was used to reduce the size of

gelatin nanoparticles for effective loading and delivery of the water insoluble Quercetin, a

known anticancer agent. These Quercetin loaded Gelatin nanoparticles (GNP) were tested on

MCF 7 cancer cells for analysis of anticancer potential.

2. MATERIALS AND METHODS

2.1. Chemicals and Reagent: All reagents and chemicals used in the study were of

analytical grade and were used as received unless noted. Preparation of plant extract (PE) and

synthesis of nanoparticles by nanoemulsion method has been given in supplementary

material.


1368


2.2. Synthesis of Bare gelatin nanoparticles

It has been reported earlier that surfactants play major role in stabilizing emulsions formed

during NP synthesis. Gelatin type A (25mg) (Himedia, India) was solubilized in 5 ml

deionized water at 50 °C in water bath followed by sonication for 15 minutes with a pulse

time of 10 seconds. 2 mg Sophorolipid in 1mL dichloromethane (Merck, India) was added

drop wise onto the gelatin solution with constant stirring. Plant extract (PE) (3ml) was

supplemented as a natural emulsifier and stabilizing agent. Sonication was done for 40

minutes with pulse of 10 seconds. The organic solvent (DCM) was evaporated by purging

with inert gas. The NPs formed were separated by centrifugation at 10,000 rpm for 30

minutes at 10°C and washed two times with deionized water. The pellet was reconstituted in

1 ml deionized water.

2.3. Entrapment of Quercetin in Gelatin Nanoparticles

To check the functionality of synthesized gelatin NPs, anticancer molecule Quercetin was

loaded. Quercetin (Himedia, India) entrapped gelatin NPs were synthesized by dissolving 25

mg Gelatin in 5 ml deionized water with heating at 50 °C. Quercetin (2.5 mg) was partially

dissolved in 5 ml deionized water and added to the above solution at 37oC. The rest of

method was same as bare gelatin nanoparticle synthesis where sophorolipid is added in DCM

along with PE, sonicated and purged of solvent. These NPs were then separated by

centrifugation at 10,000 rpm for 30 min at 10°C and washed twice with deionized water. UV-

Vis spectra were taken on UV-1800 Shimadzu Corp.

2.4.Characterization

Particle sizing and analysis of the synthesized nanoparticles were studied using SEM, EDS

(Philips P720E instrument) and TEM (Philips CM 200 SAIF IIT, Mumbai and - TECNAI

G2-20, FEI Dept. of Physics SPPU).

2.4.1. SEM analysis

The size and surface morphology was studied using SEM. 10 µl of dilute solution of gelatin

nanoparticles was loaded on silicon wafer and allowed to evaporate overnight. Samples were

observed at different magnifications.

2.4.2. TEM analysis

2 µl of sample was loaded on carbon coated copper grid and allowed to dry overnight. TEM

and EDAX analysis was done on the same instrument.


1369


2.4.3. DLS Analysis

The size and polydispersity of the synthesized nanoparticles was determined by DLS

(dynamic light scattering) measurements on Brookhaven Instrument model 90 Plus Particle

Size Analyzer at National Chemical Laboratory. The scattering angle was set at 90 degrees

and a monochromatic light source was used.

2.5. Sophorolipid production

Sophorolipid was synthesized using a non-pathogenic yeast Candida bombicola ATCC

22214 as described by Singh et al 2013 (Singh 2013).

2.6. Quantification of Quercetin loaded Gelatin Nanoparticles

Entrapment efficiency of Quercetin on the synthesized gelatin nanoparticles was analyzed

using UV-Vis spectroscopy as per the protocol given by Azimi et al (2013). During the

process of separation of nanoparticles by centrifugation, the absorbance of supernatant

solution was observed at 368 nm to determine the amount of free Quercetin which in not

entrapped in gelatin nanoparticles. The calibration curve for Quercetin was prepared by

plotting the concentration of standard Quercetin (0.01–0.1 mg/ml) vs. absorbance at 368 nm.

The encapsulation efficiency (EE) was calculated using the following formula (Azimi 2013):

% Encapsulation Efficiency (EE) = (Amount of drug entrapped/ total amount of drug used in

the formulation)*100.

2.7. Drug release assay: Quercetin loaded gelatin nanoparticles solution (1 ml) was taken in

dialysis bag (10kD) and suspended in 100 ml of 0.1 M PBS at pH 7.4 respectively. The NPs

suspension was continuously stirred on a magnetic stirrer (50 rpm) at 37°C. The samples (1

ml each) were withdrawn at pre-selected time intervals (0hr, 0.5hr, 1hr, 2hr, 3hr, 4hr, 5hr

upto 72 hr) and their absorbance was measured at 368 nm by UV-Vis spectrophotometer.

Amount of released Quercetin was quantified with the help of standard curve (Naidu 2011).

The % cumulative release of Quercetin was calculated by using following equation

Cumulative release (%) = (amount released after time t/total amount entrapped in

nanoparticles)*100

2.8.MTT Assay

Cell Proliferation analysis was done by colorimetric method for determining the number of

viable cells based on bio-reduction of a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-


1370


5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTT salt) by metabolically

active MCF- 7 cells. These cells were seeded (104 cells/well) in 96-well plates. Various

concentrations of gelatin nanoparticles were added in the medium (20-100, 240-1200 µg/ml).

After 24, 48 and 72 hr of exposure to each samples, 20 μl of MTT reagent were added. Plates

were incubated in a humidified 37°C incubator with 5% CO2 for 2 hr. To this 100 μl of

solubilizer (2% SDS in 80% aqueous DMSO) was added and absorbance was recorded at 570

nm on a plate reader.

3. RESULT AND DISCUSSION

3.1. Synthesis of gelatin nanoparticles

Nanoprecipitation and Nanoemulsion are the most widely used methods. Saeed Ahmed Khan

et al. have reported the gelatin nanoparticles using nanoprecipitation method in the size range

200-300 nm, with polydispersity 0.13 (2013). Ethirajan et al. have reported the gelatin

nanoparticles using nanoemulsion method in the size range 206-306 nm (2008). Vijayakumar

Naidu et al. have reported gelatin nanoparticles using modified nano precipitation in the size

range 110-130nm (2010). Bahareh Azimi et al. have reported gelatin nanoparticles using two

step de-solvation in the size 199 nm and polydispersity 0.250 (2013).

When compared with reported data, the nanoparticles synthesized by us were of small size

having least polydispersity. The components of plant extract acted as stabilizing agents for

gelatin nanoparticles whereas sophorolipid which is an amphiphilic molecule showed

compaction and size reduction. This also resulted in spherical shape and reduced

polydispersity of nanoparticles.

It was found in our earlier experiments where only plant extract was used with DCM, the

nanoparticles obtained were larger than 300 nm which are obviously not suitable as drug

carriers. Also they were irregular shaped and tend to agglomerate as can be seen in Fig.1a. To

reduce the size of nanoparticles sophorolipid was introduced as a biodegradable, amphiphilic

molecule in addition to the above mixture. The amphiphilic sophorolipid has both polar and

non-polar groups which facilitates them to lower the interfacial energies by reducing the

surface tension. In our case, we have used milder physical force like sonication to induce

vesicle formation which in turn helped in nanomerization of gelatin particles.


1371


Fig. 1: (a) Plant extract induced bare gelatin nanoparticles (b) Plant extract and

sophorolipids induced bare gelatin nanoparticles

3.2.Particle Sizing and Analysis

The medicinally important plant Cassia fistula was used as stabilizer for the synthesis of

biodegradable gelatin nanoparticles. Initial experiments were carried out using PE along with

DCM as organic solvent (Fig.1a). The bare PE stabilized gelatin NPs obtained by this method

were approximately of 300 nm size. Hence in the next experiment sophorolipid was included

along with PE and DCM which roughly gave spherical nanoparticles of 30-60 nm as seen

from FE-SEM results in Fig. 1b. TEM analysis confirmed the size of these particles and as

seen in Fig. 2a and 2b, sophorolipid and PE stabilized bare gelatin nanoparticles were

spherical shaped and having rough surface with size range of 20-40 nm. Moreover Fig. 2c

and 2d revealed these drug loaded gelatin nanoparticles having size ranging from 30-60 nm,

while the morphology remained unchanged.

The DLS analysis was carried out to know the hydrodynamic diameter and polydispersity.

The plant extract and sophorolipid induced gelatin nanoparticles showed average diameter of

32.8 nm with polydispersity 0.06 the more detailed results are given in Fig 3, table 1.

Table 1. DLS analysis data of gelatin nanoparticles

Sample Effective diameter (nm) Polydispersity

Gel_II (DCM, PE, SL, gelatin) 328.7 0.065

Gel_IIQ (DCM, PE, SL,

Gelatin, Q)

365.5 0.285

a b


1372


Fig.2: TEM images of bare (a, b) and drug loaded (c, d) Gelatin nanoparticles(e) SAED

Selected Area Electron Diffraction pattern shows the bright spots in circular rings

indicating nanocrystalline nature of gelatin nanoparticles.

Fig.3: DLS data of (a) Bare and (b) Drug loaded gelatin nanoparticles.

3.3. EDS analysis

The elemental analysis of gelatin nanoparticles was done by EDS. The bare nanoparticles

showed presence of C, N and O and no metal ions while drug loaded nanoparticles showed


1373


presence of C, O, and P. (table 2 a, b) . This indicates the presence of protein gelatin and the

organic phase on the surface.

Table 2. (a).EDS analysis of bare gelatin nanoparticles and (b) drug loaded gelatin

nanoparticles

3.4. Encapsulation of Quercetin in gelatin NPs and its Characterization

Quercetin has been shown to possess pharmacological properties; however use of Quercetin

in pharmaceutical field is limited due to low aqueous solubility, poor skin permeability,

negligible bioavailability and instability before reaching the systemic circulation (Alessi

2012; Sahoo 2010). These problems were addressed by initially reducing the size of gelatin

particles from ~300 nm to under 70 nm enabled by nanoemulsion, followed by increasing

solubility of Quercetin employing sophorolipid as a biosurfactant. The drug loading

efficiency of Quercetin was studied by UV-Vis spectrophotometry monitoring the λmax of

quercetinat 368 nm. The spectra were compared with that of pure Quercetin. The amount of

encapsulated Quercetin was calculated as explained in the experimental part.

The initial concentration of Quercetin added was 0.5 mg/ml. UV-Visible spectra analysis

revealed that when sophorolipid was used along with PE and DCM for synthesis of

nanoparticles, 0.082 mg/ml quercetin was obtained in the supernatant amounting to

0.418mg/ml of the entrapped drug. Thus the encapsulation efficiency was calculated and

found to be 97% respectively.

Further the drug release pattern was studied for gelatin nanoparticles. The drug loading

efficiency and release depend very much on lipophilicity and solubility of drug. Better results

have been obtained in the presence of lipid surfactants as reported by Cheow et al (2011).The

drug release also depends upon chemical nature of drug and its interaction with polymer used

for the nanoparticles synthesis (Mahapatro 2011). During the synthesis of gelatin


1374


nanoparticles PE acted as emulsifier and helped in Quercetin dissolution thus stabilizing the

gelatin nanoparticles. Sophorolipid increased the wettability and dispensability of Quercetin,

and thus helped in quercetin entrapment within the gelatin nanoparticles (Karan 2011).

3.5. Drug release assay of Quercetin

The in vitro release assay was done in phosphate buffered saline at pH 7.4 in order to mimic

the physiological conditions in human body. The amount of drug released was calculated by

using the UV- visible spectra. The initial drug release was rapid which gradually reduced

with time. This may be because of some amount of Quercetin being adsorbed on the surface

of gelatin NPs which was released rapidly within short time followed by sustained drug

release (Mahapatro 2011). Nanoparticles synthesized using plant extract, DCM and

sophorolipid showed the drug release around 40-44% within an hour and then remaining drug

release was observed up to 60 hours as seen in Fig. 4. This indicated sustained drug release at

physiological pH.

Fig. 4: Drug release profile of Quercetin loaded gelatin nanoparticles.

Drug release depends on polymers used, size of nanoparticles, surface properties, nature of

the drug, swelling of particles etc.(Kumar 2010). V.Kumar Naidu et al. have studied drug

release of gelatin nanoparticles synthesized by nanoprecipitation method, the drug loading

efficiency was found to be 85% and drug release was 75% at 15 hr, which is comparable to

our results. Further studies on animal cell lines for its biological activity were continued and

their MTT assay showed corresponding results.


1375


3.6. MTT assay

The percentage cell survival rate of MCF 7 was calculated by incubating the cells along with

the synthesized gelatin nanoparticles using sophorolipid and PE (both bare and drug loaded)

for 24, 48 and 72 hrs. Pure Quercetin was used as control. For drug loaded gelatin

nanoparticles the concentration range was from 240 to 1200 μg/ml for SL plus PE induced

gelatin nanoparticles. As calculated earlier, the effective concentration of Quercetin was 20-

100µg/ml respectively considering the drug loading efficiency. Keeping this in mind, the

concentration of pure Quercetin used as a control was 20 -100 µg/ml.

Fig. 5: Percent cell survival of MCF7 cells after incubating with bare and drug loaded

gelatin nanoparticles for 24 hrs. (1-5 indicates concentration of bare, drug loaded

gelatin nanoparticles and pure quercetin, 20-100, 240-1200, 20-100 µg/ml respectively).

Drug loaded gelatin nanoparticles in concentration range of 20-100 µg /ml showed no effect

on MCF 7 cells viability after 24 hr of incubation. It was because the effective drug

concentration was very less which was 10 times lesser than the concentration of

nanoparticles. In order to increase the effective drug concentration keeping in mind drug

loading efficiency we increased 10 times the concentration of nanoparticles in the range of

240-1200 µg/ml which were carrying effective drug concentration 20-100 µg/ml. There was

some activity on cells with this concentration range which can be seen from Fig. 5. The cell

survival decreased to 61.59% for 1200 µg/ml of drug loaded nanoparticles. The IC 50 value

was found to be1320 µg/ml (i.e. 110 µg/ml of Quercetin loaded).The cell proliferation was

found to increase a little after 48 hr and 72 hr as seen from Fig. 5. This indicates that drug

release is at peak within first 24 hr in MCF 7 cell line, reducing the proliferation of cancer

cells. Later there is little to no release of drug showing no effect on cell survival. This may be


1376


due to the degradation of particles or because of reduced drug release after 24 hr which was

in agreement with our anticipated results.

Bare nanoparticles (20-100 µg) were tested for percent cell survival, where cell viability

decreased to 64% at 100 µg concentration. The IC 50 value obtained was 140 µg/ml for

gelatin nanoparticles as given in Fig. 6. The bare nanoparticles also showed toxicity to

MCF7cells at 24 hr as they have sophorolipid on /in the surface, and is known to have

anticancer activity (Joshi-Navare 2011; Fu 2008).

Fig. 6: MTT assay of bare gelatin nanoparticles and Quercetin loaded nanoparticles

after (a) 48 hours (b) 72 hours.

Quercetin at 100 µg /ml concentration showed 64% cell survival at 24 hr while at 48 and 72

hr the inhibition was less (cells survival 75%, 88% respectively). The IC 50 value was found

to be 112 µg /ml for drug. Since the IC50 of plant extract and sophorolipid induced drug

loaded nanoparticles is less than that of pure drug indicated more effectiveness of the drug

delivery (100, 110 µg/ml respectively). Similarly the percent cell survival is lower than that

of pure drug (61 and 64% respectively) at highest concentration 100 µg used, indicating that

the gelatin nanoparticles are the better drug delivery system for MCF 7 cells.


1377


4. CONCLUSIONS

We are reporting for the first time the synthesis of gelatin nanoparticles by totally green

approach. The gelatin nanoparticles synthesized by Nanoemulsion using plant extract, DCM

and Sophorolipid were found to be more suitable for biological activity compared to other

types which were synthesized elsewhere. The particles are obtained by totally biogenic

method where sophorolipid and plant extract of Cassia fistula play role in stabilization,

compaction and Quercetin loading. These were stable nanoparticles with average size 32.8

nm (bare), 36 nm (drug loaded) and least polydispersity. The SEM and TEM analysis is

comparable for size, shape and surface nature of the particles. Gelatin NPs obtained were

spherical shaped with rough surface and crystalline nature. The drug loading efficiency is

84% and release assay showed that gelatin NPs has much sustained drug release pattern.

These may be considered as good vehicles for delivery of drugs. MTT assay showed toxicity

of bare as well as drug loaded nanoparticles to MCF 7 cells at 24 hr, due to the presence of

sophorolipid and Quercetin in bare and drug loaded gelatin NPs respectively. The IC 50 and

percent cell survival indicate that the gelatin nanoparticles is better drug delivery system and

hence more effective than quercetin alone towards MCF7 cells.

ACKNOWLEDGEMENT

Authors are thankful to UGC for the funding under project (UGC-UPE nanobiotech ref 226 A

(2)), we are also thankful to Prof Smita Zinzarde and Pranaya Joshi, Institute of

Bioinformatics and Biotechnology, Savitribai Phule Pune University for carrying out the

MTT assay. Authors are thankful to Department of Chemistry, Department of Physics SPPU,

CIF SPPU and National Chemical Laboratory for instrumentation facility. SAIF Mumbai for

TEM.

REFERENCES

1. Alessi P, Cortesi A, Zordi D, Gamse T, Kikic I, Moneghini M, Solinas D (2012)

Supercritical Antisolvent Precipitation of Quercetin Systems: Preliminary Experiments.

Chem Biochem Eng Q, 26(4): 391.

2. Arangoa MA, Campanero MA, Renedo MJ, Ponchel G, Irache JM (2001) Gliadin

Nanoparticles as Carriers for the Oral Administration of Lipophilic Drugs. Relationships

between Bioadhesion and Pharmacokinetics. Pharm. Res 18: 1521.

3. Azarmi SH, Huang Y, Chen H, McQuarrie S, Abrams D, Roa W, Finlay WH, Miller GG,

Löbenberg R (2006) Optimization of a two-step desolvation method for preparing gelatin


1378


nanoparticles and cell uptake studies in 143B osteosarcoma cancer cells. J. Pharm.

Pharmaceut. Sci. 9(1): 124-132.

4. Azimi B, Nourpanah P, Rabiee M, Arbab S (2013) Producing Gelatin Nanoparticles as

Delivery System for Bovine Serum Albumin. Iranian Biomedical Journal 18(1): 34.

5. Babaei Z, Jahanshashi M, Sanati MH (2008) Fabrication and evaluation of gelatin

nanoparticles for delivering of anti - cancer drug. Int. J. Nanosci. Nanotech 4: 1.

6. Babu A, Jeyasubramanian K, Gunasekaran P, Murugesan R (2012) Gelatin nanocarrier

enables efficient delivery and phototoxicity of hypocrellin B against a mice tumour

model. J. Biomed. Nanotechnol. 8: 43–56.

7. Bahorun T, Neergheen VS, Aruoma OK (2005) Phytochemical constituents of Cassia

fistula. African Journal of Biotechnology 4(13): 1530.

8. Chen SF, Nien S, Wu CH, Liu CL, Chang YC, Lin YS (2013) Reappraisal of the

anticancer efficacy of quercetin in oral cancer cells. Journal of the Chinese Medical

Association 76: 146.

9. Cheow WS, Hadinoto K (2011) Factors affecting drug encapsulation and stability of

lipid–polymer hybrid nanoparticles. Colloids and Surfaces B: Biointerfaces 85:214.

10. Ciofani G, Raffa V, Menciassi A, Dario P (2008) Alginate and chitosan particles as drug

delivery system for cell therapy. Biomed Microdevices 10: 131.

11. Coesters CJ, von Briesen KLH, Kreuter J (2000) Gelatin nanoparticles by two step

desolvation a new preparation method, surface modifications and cell uptake. J.

Microencapsul. 17: 187–193.

12. Daizy P, Saipriya K (2012) Biochemical analysis of Cassia fistula aqueous extract and

phytochemically synthesized gold nanoparticles as hypoglycemic treatment for diabetes

mellitus. International Journal of Nanomedicine 2(7): 1189.

13. Das S, Banerjee R, Bellare J (2005) Aspirin Loaded Albumin Nanoparticles by

Coacervation: Implications in Drug Delivery. Trends Biomater. Artif.Organs. 18(2):203.

14. Ethirajan A, Schoeller K, Musyanovych A, Zeiner U, Landfester K (2008) Synthesis and

optimization of gelatin nanoparticles using the miniemulsion process. Biomacromolecules

9: 2383.

15. Gan Q, Wang T, Cochrane C, McCarron P (2005) Modulation of surface charge, particle

size and morphological properties of chitosan–TPP nanoparticles intended for gene

delivery. Collods and Surfaces B: Biointerfaces 44: 65.

16. Gan Z, Ju J, Zhang T, Wu D (2011) Preparation of rhodamine B fluorescent

poly(methacrylic acid) coated gelatin nanoparticles. J. Nanomater.


1379


17. Jahanshahi M, Babaei Z (2008) Protein nanoparticle: A unique system as drug delivery

vehicles. American journal of Biotechnology 7(25): 4926.

18. Jahanshahi M, Sanati MH, Babaei Z (2008) Optimization of parameters for the

fabrication of gelatin nanoparticles by the Taguchi robust design method. Journal of

Applied Statistics vol. 35(12): 1345–1353.

19. Jain A, Gulbake A, Jain A, Shipli S, Hurkat P, Jain A, Jain SK (2012) Development of

surface-functionalised nanoparticles for FGF2 receptor-based solid tumour targeting.

J.Microencapsulation 29: 95.

20. Joshi-Navare K, Shiras A, Prabhune A (2011) Differentiation-inducing ability of

sophorolipids of oleic and linoleic acids using a glioma cell line. Biotechnol.J. 6.

21. Karan M, Sahoo NG, Li L (2011) Dissolution enhancement of quercetin through

nanofabrication, complexation, and solid dispersion. Colloids and Surfaces B:

Biointerfaces 88: 121.

22. Khan SA, Schneider M (2013) Improvement of Nanoprecipitation Technique for

Preparation of Gelatin Nanoparticles and Potential Macromolecular Drug Loading.

Macromolecular Bioscience 13: 455.

23. Kreuter J Nanoparticles In colloidal drug delivery systems(1994) J. K. Ed. Marcel

Dekkar: New York, 1994; 219-342.

24. Kumari A, Kumar V, Yadav SK (2012) Plant Extract Synthesized PLA Nanoparticles for

Controlled and Sustained Release of Quercetin: A Green Approach.PLOS One 7.

25. Kuo WT, Huang HY, Chou MJ, Wu MC, Huang YY (2011) Surface modification of

gelatin nanoparticles with polyethylenimine as gene vector. J Nanomaterials.

26. Lee EJ, Khan SA, Park JK, Lim KH (2011) Studies on the characteristics of drug-loaded

gelatin nanoparticles prepared by nanoprecipitation. Bioprocess Biosyst Eng. 35(12):

297-307.

27. Leo E, Angela Vandelli M, Cameroni R, Forni F (1997) Doxorubicin-loaded gelatin

nanoparticles stabilized by glutaraldehyde: Involvement of the drug in the cross-linking

process. Int. J. Pharm. 155: 75–82.

28. Leong YS, Candau F (1982) Inverse microemulsion polymerization. J. Phys. Chem. 86

(13): 2269–2271.

29. Lu Z, Yeh TK, Wang J, Chen L, Lyness G, Xin Y, Wientjes MG, Bergdall V, Couto G,

Berger FA, Kosarek CE, Au JL (2011) Paclitaxel gelatin nanoparticles for intravesical

bladder cancer therapy. J. Urol. 185: 1478.


1380


30. Mahapatro A, Singh DK (2011) Biodegradable nanoparticles are excellent vehicle for site

directed in-vivo delivery of drugs and vaccines. Journal of nanobiotechnology 9: 55.

31. Mirshahi T, Irache JM, Nicolus C, Mirshahi M, Faure JP, Gueguen J, Hecquet and

Orecchion AM. Adaptive Immune Responses of Legumin Nanoparticles. J. Drug Target

10(8): 625.

32. Naidu BV, Paulson AT (2011) A New Method for the Preparation of Gelatin

Nanoparticles: Encapsulation and Drug Release Characteristics. Journal of Applied

Polymer Science 121: 3495.

33. Nakaoka R, Tabata Y, Ikada Y (1995). Potentiality of gelatin microspheres as

immunological adjuvant. Vaccine 13(7): 653-661.

34. Nitta SK, Numata K (2013) Biopolymer-Based Nanoparticles for Drug/Gene Delivery

and Tissue Engineering. Int.J.Mol.Sci.14:1629.

35. Ofokansi K, Winter G, Fricker G, Coesters C (2010) Matrix-loaded biodegradable gelatin

nanoparticles as new approach to improve drug loading and delivery. European Journal of

Pharmaceutics and Biopharmaceutics 76(1):1.

36. Panda SK, Brahma S, Dutta SK (2010) Selective Antifungal Action of Crude Extracts of

Cassia fistula L. A Preliminary Study on Candida and Aspergillus Species. Malaysian

Journal of Microbiology 6(1): 62 – 68.

37. Penning JP, Ijkstra HD, Pennings AJ (1993) Preparation and properties of absorbable

fibres from L-lactide copolymers. Polymer 34(5): 942.

38. RokhadeAp, Agnohotri SA, Patil SA, Mallikarjuna NN, Kulkarni PV, Aminabhavi TM

(2006) Semi-interpenetrating polymer network microspheres of gelatin and sodium

carboxymethyl cellulose for controlled release of ketorolac tromethamine. Carbohydrate

Polymers 65: 243.

39. Sahoo NG, Kakran M, Shaal LA, Li L, Muller RH, Pal M, Tan LP (2010) Preparation and

characterization of quercetin nanocrystals. J Pharm Sci. 100: 2379–2390.

40. Sailaja AK, Amareshwar P (2012) Preparation of gelatine nanoparticles by desolvation

technique using acetone as desolvating agent. J. Pharm. Res. 5: 1854–1856.

41. Singh PK, Mukherji R, Joshi-Navare K, Banerjee A, Gokhale R, Nagane S, Prabhune A,

Ogale S (2013) Fluorescent sophorolipid molecular assembly and its magnetic

nanoparticle loading: a pulsed laser process. Green Chemistry 15: 943.

42. Soopimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE (2001) Biodegradable

polymeric nanoparticles as drug delivery devices.J.Controlled Release 70: 1.


1381


43. Soumya RS, Gosh S, Abraham ET (2010) Preparation and characterization of guar gum

nanoparticles. International Journal of Biological Macromolecules 46: 267.

44. Steinhauser IM, Langer K, Strebhardt KM, Spankuch B (2008) Effect of trastuzumab-

modified antisense oligonucleotide-loaded human serum albumin nanoparticles prepared

by heat denaturation. Biomaterials (29): 4022-4028.

45. Ulbrich K, Hekmatara T, Harbart E, Kreuto J (2009) Transferrin- and transferrin-receptor

antibody-modified nanoparticles enable drug delivery across the blood-brain barrier

(BBB).Eur J Pharm Biopharm, 71: 251.

46. Wu F, Zhou Z, Su J, Wei L, Yuan W, Jin T (2013) Development of dextran nanoparticles

for stabilizing delicate proteins. Nanoscale Research Letters 8: 197.

47. Yeh TK, Lu Z, Wientjes MG, Au JL (2005) Formulating paclitaxel in nanoparticles alters

its disposition. Pharm Res. 22(6): 867-74.

48. Zhang H, Zhang M, Yu L, Zhao Y, He N, Yang X (2012) Antitumor activities of

quercetin and quercetin-50, 8-disulfonate in human colon and breast cancer cell lines.

Food and Chemical Toxicology 50: 1589.

49. Zhao YZ, Li X, Lu CT, Xu YY, Lv HF, Dai DD, Zhang L, Sun CZ, Yang W, Li XK,

Zhao YP, Fu HX, Cai L, Lin M, Chen LJ, Zhang M (2012) Experiment on the feasibility

of using modified gelatin nanoparticles as insulin pulmonary administration system for

diabetes therapy. Acta Diabetol. 49: 315-325.

50. Zheng SY, li Y, Jiang D, Zhao J, Ge JF (2012) Anticancer effect and apoptosis induction

by quercetin in the human lung cancer cell line A-549. Molecular Medicine Report 5:

822.

51. Zillies JC, Zwiorek K, Hoffman F, Vollmar A, Anchordoquy TJ, Winter G, Coesters C

(2008) Formulation development of freeze-dried oligonucleotide-loaded gelatin

nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics 70: 514.

TUNABLE SYNTHESIS OF GELATIN NANOPARTICLES …

Documents

Transcript of TUNABLE SYNTHESIS OF GELATIN NANOPARTICLES …