International Journal of Pharmaceutics 430 (2012) 318 327

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International Journal of Pharmaceutics

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Pharmaceutical Nanotechnology

Chitosa nketripoly

Soa A. P iarLaboratory of P essalo

a r t i c l

Article history:Received 1 FebReceived in reAccepted 2 ApAvailable onlin

Keywords:ChitosanPoly(ethylene Bovine serum Poly(glycolic aTripolyphosphNanoparticles

copolar wechnmic aapsuiamethe crially copold of T

1. Introduction

Polymeric nanoparticles have been widely investigated as car-riers for drug delivery using many different materials and methodsfor the preAmong thethat are mato their gooHowever, thprotein dru2009). In rerials is usedoffer site-spmolecular w2010; Howmers, activeusing naturbeen largelunique prop2008). Despmain drawbchitosan cation (Werle

CorresponE-mail add

Chemical modication through graft copolymerization is quitepromising as it provides a wide variety of molecular character-istics (Yao et al., 2007). The poly(ethylene glycol) (PEG) graftingof chitosan cope with the major problem of nanoparticles which

0378-5173/$ http://dx.doi.oparation and size control (Rao and Geckeler, 2011).m, much attention has been paid to the nanoparticlesde of synthetic biodegradable aliphatic polymers dued biocompatibility (Papadimitriou and Bikiaris, 2009).ese nanoparticles are not ideal carriers for hydrophilicgs because of their hydrophobic properties (Shu et al.,cent years, nanotechnology of polycation based mate-

in creating evolved drug delivery systems since it mayecic and/or time-controlled delivery of small or largeeight drugs and other bioactive agents (Park et al.,

ard, 2009). Besides the commonly used synthetic poly- research is focused on the preparation of nanoparticlesal hydrophilic polymers like chitosan (CS). Chitosan hasy favored as a potential nanoparticle carrier due to itserties (Dash et al., 2011; Hamidi et al., 2008; Liu et al.,ite the superiority of chitosan as biomaterial, it has theack of poor solubility in water. However water soluble

n be easily synthesized with proper chemical modica- et al., 2009).

ding author. Tel.: +30 2310 997812; fax: +30 2310 997667.ress: dbic@chem.auth.gr (D.N. Bikiaris).

is their rapid elimination from the blood stream through phago-cytosis after intravenous administration and recognition by themacrophages of the mono-nuclear phagocyte system. PEGylationof chitosan nanoparticles can increase their physical stability andprolong their circulation time in blood by reducing the removalby the reticuloendothelial system. PEGylated chitosan nanoparti-cles have been investigated as nanocarriers (Yang et al., 2008). Atthe same time chitosan nanoparticles are usually prepared by ionicgelation method using tripolyphosphate (TPP). The ionic gelationmethod has received much attention in recent years for the prepa-ration of nanocarriers for low molecular drugs (Papadimitriou et al.,2008). Recently some studies are referred to the use of TPP asionic crosslinking agent for PEG-g-CS materials in order to createnanoparticles for encapsulation of macromolecular drugs (Zhanget al., 2008a; Csaba et al., 2009) Another technique is the creation ofself-assembled polyelectrolyte complexes (PECs) which have beenrecently investigated for protein delivery (Park et al., 2010; Amidiet al., 2010). Oppositely charged polyelectrolytes can form stableintermolecular complexes. Recently poly(glutamic acid) is used aspolyion for the creation of PEC chitosan nanoparticles as proteindelivery systems. (Tang et al., 2010; Keresztessy et al., 2009; Imotoet al., 2010; Peng et al., 2009). Poly(glutamic acid) is an unusualanionic, natural polypeptide, water soluble, biodegradable and edi-ble and with its good tissue afnity gains interest for biological

see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.ijpharm.2012.04.004n-g-PEG nanoparticles ionically crossliphosphate as protein delivery systems

apadimitriou, Dimitris S. Achilias, Dimitrios N. Bikolymer Chemistry and Technology, Department of Chemistry, Aristotle University of Th

e i n f o

ruary 2012vised form 1 April 2012ril 2012e 10 April 2012

glycol)albumincid)ate

a b s t r a c t

In the present study chitosan graftedand studied using PEG with molecuized using 1H NMR, FTIR and WAXD ttripolyphosphate (TPP) and poly(gluta2:1, 3:1 and 4:1, w/w) for the nanoencare spherical in shape with a mean dthe molecular weight of the PEG and to affect the release rate of BSA especcontaining PEG5000, compared with was used as crosslinking agent, instea/ locate / i jpharm

d with poly(glutamic acid) and

is

niki, 541 24 Thessaloniki, Greece

lymers with poly(ethylene glycol) (CS-g-PEG) were preparedeights 2000 and 5000 g/mol. The materials were character-iques. These polyelectrolytes were ionically crosslinked withcid) (PGA) at different polymer/crosslinking agent ratios (1:1,lation of bovine serum albumin (BSA). Prepared nanoparticlester ranging from 150 to 600 nm. The size depends mainly toosslinking agent used. The PEG molecular weight also seemsthe rst burst effect which appears to be high in copolymersymer prepared with PEG2000, and it is also higher when PGAPP.

2012 Elsevier B.V. All rights reserved.

S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327 319

applications (Tsao et al., 2011). These kind of nanoparticles are pre-pared by electrostatic complexation of poly(glutamic acid) (PGA)and chitosan. PGA is selected as a negatively charged crosslink-ing agent as it has been shown that nanoparticles containing thispolymer have the capacity to target hepatocytes (Lin et al., 2005).

Based on the aforementioned comments, the main idea ofthis study targeted anan alginateby forming(CPCDs), wepichlorohypolycondenattraction bof its polymunder simuorder to crethe blood smodicatioproduced wnanocarrierBSA. Nanopusing two acid). To ouuntil now rcrosslinkingnanocarrierthe in vitroThe main gthe materiamolecular wCS-g-PEG/TTherefore, tdeterminedstudied.

2. Materia

2.1. Materi

Chitosandeacetylatioand tripolMonometh(mPEG2000supplied al(average mserum albuAcros Organin this stud

2.2. Prepara

PEG-aldature, by thet al., 1984anhydrate dimethylsu6% CHCl3 ature (20 C)diethyletheand repreciration of PEwere used,

2.3. Preparation of CS-g-PEG materials

The preparation of CS-g-PEG materials was performed by themethod of Harris (Harris et al., 1984) which was partially modi-ed by the (0.5 g) was d

) andde (mise d1998de so

Afte for actioorocg antely,

12 4he pHe in

eton. AftEG.

arac

NucleNMRed w

(CDw/v)

Fouri speer, mmouand , in a400

Wideay d

usiwith54 n

epar

opar casedditi

in tmagnf, aqlar wncened ation)t to tith aed byiscarther was to prepare modied chitosan nanoparticles asd protection drug delivery systems for proteins. In/chitosan nanoparticle system, insulin was protected

complexes with cationic -cyclodextrin polymershich were synthesized from -cyclodextrin (-CD),

drin (EP) and choline chloride (CC) through a one-stepsation (Zhang et al., 2010). Due to the electrostaticetween insulin and CPCDs, as well as the assistanceeric chains, CPCDs could effectively protect insulin

lated gastrointestinal conditions. In the present study inate drug carriers that will not be rapidly eliminated fromtream, poly(ethylene glycol) was used for the chemicaln of chitosan, creating graft copolymers. The materialsere fully characterized and subsequently used as drugs for the encapsulation of peptide/protein drug such asarticles were produced by the ionic gelation methoddifferent crosslinking agents, TPP and poly(glutamicr knowledge, no other research has been publishedeferring to the use of poly(glutamic acid) as ionically

agent for CS-g-PEG materials, in order to create proteins. The nanoparticles were fully characterized. Moreover

release of BSA from the nanoparticles was studied.oal of the present work was to identify the effect ofls ratio and crosslinking agent, together with the PEGeight on the physicochemical characteristics of the

PP and CS-g-PEG/PGA nanoparticles loaded with BSA.he encapsulation efciency, yield, drug loading were

and release prole of BSA was also performed and

ls and methods

als

with high molecular weight (MW: 350 000 g/mol,n degree >75% and viscosity 8002000 cp)

yphosphate were supplied by Aldrich chemicals.ylated PEG (mPEG) with molecular weights 2000 g/mol) and 5000 g/mol (mPEG5000), respectively, wereso by Aldrich chemicals. Poly(glutamic acid) (PGA)olecular weight 15 00050 000 g/mol) and bovinemin (BSA) were purchased from Sigma Aldrich andics, respectively. All other materials and reagents used

y were of analytical grade.

tion of PEG-aldehyde (mPEG-CH O)

ehyde was prepared, as previously reported in the liter-e oxidation of PEG with DMSO/acetic anhydrate (Harris; Sugimoto et al., 1998). For this reason 5 ml of aceticwas added under N2 atmosphere to 32 ml anhydrouslfoxide containing 10 g mPEG (MN = 2000 g/mol) andnd the mixture was stirred for 9 h at room tempera-. The reaction mixture was then poured into 400 mlr. The precipitate was ltered, dissolved in chloroformpitated twice by the use of diethylether. For the prepa-G-aldehyde two different molecular weights of mPEG2000 and 5000 g/mol.

(40 mlaldehydropwet al., aldehypH = 6.stirredthe retion (Glterinalternacut-offuntil tfrom thand acreactedCS-g-P

2.4. Ch

2.4.1. 1H

obtainroformof 5% (6 kHz.

2.4.2. FTIR

trometsmall aticles) tabletsof 450scans.

2.4.3. X-r

formedFlex II ( = 0.1

2.5. Pr

NanIn thisupon aor PGAunder In briemolecuious copreparcentraamountions wcollectwere dfor furuse of novel synthetic procedures (Yao et al., 2007). CSissolved in a mixture of aqueous 2% acetic acid solution

methanol (20 ml) and an aqueous solution of PEG-PEG-CH O) with MW = 2000 g/mol (2.9 g) was added

uring stirring for 30 min at room temperature (Sugimoto; Muslim et al., 2001). Then the pH of chitosan/PEG-lution was increased by gradually adding Na2CO3 untilr 1 h NaCNBH3 (0.183 g) was added and the mixture was5 h at 55 C. The precipitate was obtained by pouringn mixture into a saturated ammonium sulfate solu-hovceva et al., 2005). The material was collected byd dialyzed against aqueous 0.05 M NaOH and water

for 96 h using a dialysis cellulose membrane bag (mw00 g/mol), with a frequent change of the solutions used,

of the external water phase reached 7. The materialner solution was freeze-dried and washed with ethanole in order to remove the remaining mPEG that did noter drying in vacuum the obtained white powder was

terization of PEG-aldehyde and CS-g-PEG materials

ar magnetic resonance (NMR) and 13C NMR spectra of prepared materials wereith a Bruker AMX 400 spectrometer. Deuterated chlo-Cl3) was used as solvent in order to prepare solutions. The number of scans was 10 and the sweep width was

er transform-infrared spectroscopy (FT-IR)ctra were obtained using a Perkin-Elmer FTIR spec-odel Spectrum 1000. In order to collect the spectra, ant of each sample was mixed with KBr (1 wt% nanopar-compressed to form tablets. The IR spectra of thesebsorbance mode, were obtained in the spectral region0 cm1 using a resolution of 4 cm1 and 64 co-added

angle X-ray diffractometry (WAXD)iffraction measurements of the samples were per-ng an automated powder diffractometer Rigaku Mini

BraggBrentano geometry (2), using CuK radiationm) in the angle 2 range from 5 to 55.

ation of CS-g-PEG nanoparticles loaded with BSA

ticles were prepared by a simple ionic-gelation method. the nanoparticles were self-assembled instantaneouslyon of two different cross-linking agents an aqueous TPPhe presence of BSA into an aqueous CS-g-PEG solutionetic stirring at room temperature (Sonaje et al., 2010).

ueous CS-g-PEG (which was prepared using differenteights of PEG, PEG2000 or PEG5000) solutions at var-trations, such as 0.5, 1, 1.5 and 2.0 mg/ml, were rst

t pH = 3.5. Aqueous TPP or PGA (0.5 mg/ml nal con- was premixed with BSA stock solution (2 mg nal drughe samples) and added into the aqueous CS-g-PEG solu-

rate of 1 ml/min. The obtained nanoparticles (NPs) were centrifugation at 32 000 rpm for 50 min. Supernatantsded and NPs were resuspended in deionized (DI) waterstudies.

320 S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327

2.6. Characterization of CS-g-PEG materials and nanoparticles

2.6.1. Morphological characterization of nanoparticlesTransmission electron microscopy (TEM) was used to examine

the morphomicrographwere obtainat 120 kV.

The morformed usi(JMS-840). probe curre

2.6.2. Size mThe part

was determInstrumentZEN3600. Tred laser anon-invasivtechnologydistilled wakept at 37

2.7. Evalua

The druin previous(free drug) trometry (Sas describeMarkwell eproduced uple was me

The drugof the nanoequations:

%LC = totalnan

%AE = totatot

2.8. In vitro

BSA rele37 C in phdeterminedsupernatanof free BSA ous paragrasamples we

3. Results

3.1. Synthe

Chitosandistributed glucosaminand amino normal conprepare thesince PEG icould be prPEG was u

4000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Fig. 1

mole in tor to, MNsful gas

ride oto (raturbe a n of

be sdehyd to

wasr ths 20o pre

N-P dem

as Bimarf a reducing agent (Gorochovceva et al., 2005). Borch reactions a protic solvent or addition of an equivalent amount of anhitosan is soluble in acidic media and thus these conditionso be the most suitable for modication of chitosan. Duringond step of the procedure, Schiff base is created and reduc-action follows, leading to the CS-g-PEG graft copolymers. Inses of the produced copolymers the reducing agent is added

reaction mixture dropwise in a time period of 1 h in order torecipitation of chitosan due to high alkalinity of NaCNBH3.viously reported the precipitation of chitosan during theion process of Schiff base by NaCNBH3 would suppress theh reduction of Schiff-base (Sugimoto et al., 1998). The exces-aCNBH3 changes rapidly the pH of the solution from acidicline leading to the precipitation of chitosan in the aqueoust. The adjustment of pH to 6.5 with the use of Na2CO3 beforeuction of Schiff base, leads to a smooth transfer of the pH

cidic to alkaline with the dropwise addition of NaCNBH3, anduently to the protection of chitosan from precipitation. Alsol pH suppresses the degradation of Schiff-base (Yao et al.,logy of the nanoparticles prepared in this study. TEMs of nanoparticle samples deposited on copper gridsed with a JEOL 120 CX microscope (Japan), operating

phological examination of nanoparticles was also per-ng a scanning electron microscope (SEM) type JEOLOperating conditions were: accelerating voltage 20 kV,nt 45 nA and counting time 60 s.

easurements of nanoparticlesicle size distribution of CS-g-PEG/drug nanoparticlesined by dynamic light scattering (DLS) using a Malvern

(Worcestershire, United Kingdom) Zetasizer Nano ZShis model is equipped with a 633 nm wavelengthnd a detector at 173 angle position measuring thee back-scatter with moving optics (NIBS is a patented). A suitable amount of nanoparticles was dispersed inter (pH = 7) creating a total concentration 1 and wasC under agitation at 100 rpm.

tion of drug encapsulation

g-loaded nanoparticles were centrifuged as described paragraph and the amount of non-entrapped drugwas measured in the clear supernatant using UV spec-himadzu PharmaSpec UV-1700) with Lowry methodd in the literature (Nikiforidis and Kiosseoglou, 2010;t al., 1978). The corresponding calibration curves weresing the supernatant of blank nanoparticles. Each sam-asured in triplicate.

loading capacity (LC) and association efciency (AE)particles were calculated according to the following

drug free drugoparticle weight

100 (1)

l drug free drugal drug amount

100 (2)

drug release

ase was determined by incubating nanoparticles atosphate buffer (pH = 7.4) under mild agitation. At pre-

time intervals, samples were centrifuged and thet was removed and replaced by fresh buffer. The amountwas determined by the method reported in the previ-ph using UVVis spectrometry. In each experiment there analyzed in triplicate.

and discussion

sis and characterization of CS-g-PEG

is a linear polysaccharide composed of randomly-(14)-linked d-glucosamine and N-acetyl-d-

e. In its repeating using it contains mainly hydroxylgroups. However, these are very difcult to react atditions with the hydroxyl end groups of PEG in order to

grafted material (CS-g-PEG). Furthermore, in this cases a bifunctional reagent crosslinked macromoleculesepared. In order to avoid this mono methyl ether ofsed, which contains only one hydroxyl group in the

Ab

sorb

an

cemacroweightbehaviweightsuccesether wanhydSugimtempemight creatioAs canPEG-alascribeof PEG

Afteweightorder tfor thetion isknownof a prence orequireacid. Cseem tthe section reboth cato the avoid pAs prereductsmootsive Nto alkasolventhe redfrom aconseqneutra1994).1000200030000

Wavelength (cm-1

)

1742c m-1

mPEG 2000

mPEGCH=O 2000

. FTIR spectra of (a) mPEG2000 and (b) mPEG-CH O 2000.

cular chain. To study the effect of PEGs molecularhe properties of the grafted material and mainly its

BSA release, PEG having number average molecular, 2000 and 5000 g/mol was used. Except this, for arafting reaction of PEG into chitosan, PEG monomethyloxidized in a rst stage to PEG-aldehyde with aceticand DMSO according to the method described byHarris et al., 1984; Sugimoto et al., 1998). The roome was preferred for the oxidation reaction since therechance of excessive reaction (Muslim et al., 2001). ThePEG-aldehyde was veried by the use of FTIR (Fig. 1).een the most characteristic peak to the spectrum ofde is the one that appears at 1742 cm1, which isthe aldehyde groups in which the hydroxyl end groups

transformed after the oxidation reaction.e successful synthesis of mPEG-CH O with molecular00 and 5000 g/mol, PEG can be reacted with chitosan inpare the grafted materials. The synthetic method usedEGylation of chitosan by the use of reductive amina-onstrated in Fig. 2. One step-reductive amination, alsoorch reduction, is the reaction between an amino groupy or secondary amines and aldehyde group in the pres-

S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327 321

Fig. 2. Synthetic route via the two-step reaction for the synthesis CS-g-PEG.

During the grafted reaction it is possible some reagents andmainly mPEG-CH O do not react with chitosans amino groups.Separation and purication of the grafted copolymers is a rathercomplicated procedure. As previously reported (Sugimoto et al.,1998) the use of dialysis membrane is ineffective as a part ofunreacted aldehyde of mPEG cannot be removed from the prod-ucts. Having this in mind the salting out method was used(Gorochovceva et al., 2005). According to this technique the graftedcopolymers created a concentrated gel-like upper phase undersalting out Finally the residual am

Graftinguse of FTIRmPEG5000 shown in Fi

To the schitosan anof PEG at 1appear morChitosan choverlappedOn the con1650 cm1

4000,0

0,2

0,4

0,6

0,8

1,0

1,2

Ab

sorb

an

ce

Fig. 3. FTIR sp(d) chitosan.

II, respectively, show a quite different behavior. The absorbanceof Amide I, at 1650 cm1, seems to remain unaffected, a fact thatseems to be reasonable as this peak is attributed to the stretchingvibration of the C O group of the acetylated amino groups ofchitosan, which is not affected during the modication procedureof chitosan. Contrary the absorbance of Amide II, 1560 cm1, isalmost disappeared from the spectra of the grafted chitosan. Thischaracteristic group is attributed to the bending vibration of N Hgroup of chitosan (NH2), and this constitutes strong evidence of the

degree of reaction between the free NH2 groups of chitosane aldical (c) c

of line anidrrespng toG50.3 a. CSo be and unreacted mPEG-CH O remained in the solution.materials were dialyzed against water to remove themonium sulfate used for the salting out method.

of mPEG aldehyde on chitosan was conrmed by the spectroscopy. Comparative IR spectra of chitosan,and the synthesized mPEGCH O and CS-g-PEG areg. 3.pectra of grafted chitosan the characteristic peaks ofd mPEG can be identied. The characteristic peaks110 cm1 (C O stretch) and 2886 cm1 (C H stretch)e intense than those of chitosan at the grafted materials.aracteristic bands appear at 3420 cm1 (O H stretch

with N H stretch) and 2879 cm1 (C H stretch).trary other two characteristic peaks of chitosan atand 1560 cm1, that correspond to Amide I and Amide

3420cm-1

1110 cm-1

288 6cm-1

extendand th

Typ5000, patterncrystal(Nunth(2) cospondiof mPEdeg 19and 27seem t

y (

cou

nts

)1000200030000

2879cm-1 1560cm-1

165 0cm-1

Chitosan

CS-g-PEG 50 00

mPEGCH=O 5 000

cm-1

PEG 5 000

ectra of (a) mPEG 5000, (b) mPEGCH O 5000, (c) CS-g-PEG 5000 and

Inte

nsi

t

Fig. 4. X-ray p(c) chitosan anehyde group of mPEGCH O (Bhattarai et al., 2005).XRD patterns of (a) mPEG5000 and (b) mPEG-CH Ohitosan, (d) CS-g-PEG5000 are shown in Fig. 4. XRDneat CS showed that is in an amorphous to partiallystate. This observation is in accordance with Nunthanid

et al., 2001) who reported a peak at approximately 10

onding to hydrated crystals and one at 18 (2) corre- anhydrous crystals. On the other hand the XRD pattern00 has two strong characteristic crystalline peaks at 2nd 23.6 and two weak crystalline peaks at 2 deg 26

-g-PEG5000 pattern gives also two obvious peaks thatthe characteristic peaks of mPEG but broadened due to

d) CS-g-PE G 50005040302010

a) mPEG 5000

b) m PEGCHO 5000

2 theta (deg)

c) CH ITOSAN

owder diffraction patterns of (a) mPEG5000, (b) mPEGCH O 5000,d (d) CS-g-PEG5000.

322 S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327

2,5

CS-g-PEG 500 0

3,36pp m

CS-g-PEG 200 0

Fig

the disrupt(Deng et al.

The chathrough thestitution (Dproton sign(Fig. 5). Thton signal ocalculated bto chitosanring). The D

DS = I3.363 I2

Accordinthat the DSPEG2000 an

3.2. Preparaloaded with

Grafted corder to beformation owater solubmain chainwith increathe avoidanadsorption

In the pinter-ionic particles winteraction in chitosan the formatigels preparionic enviroas previousof nanoparsame solutiadded to CSway smalle

these systems tends to be narrowest (Hajdu et al., 2008). Also isvery important the pH that is being used during the preparationof nanoparticles especially in the samples were PGA is used ascrosslinking agent. In our case it was chosen to be pH = 3.5 and

becaboxyIn the to fre wable ed. Impl

al conn the

mate 1 athe pcien

the amp

on tmodn th

m Taamicresu. All

is obslinks use

be stole

tionomp

casnkingallerslinkease e mo

nanoounrafteed thalle

ribu4,03,53,0ppm

2,6ppm

. 5. 1H NMR spectra of CS-g-PEG2000 and CS-g-PEG5000.

of PEG crystalline structure from amorphous chitosan, 2007)racterization of the grafted materials is completed1H NMR spectra in order to calculate the degree of sub-S) of mPEG moiety (Yang et al., 2008). The characteristicals of CS-g-PEG appeared in the range of 3.54.0 ppme peak at around 3.36 ppm is attributed to the pro-f methoxyl of CS-g-PEG and therefore the DS could bey comparing the ratio of mPEG protons at 3.36 ppm

protons around 2.6 ppm (CH carbon 2 of glucosamineS could be calculated by the following equation:

ppm

.6ppm 100% (3)

g to this equation and the 1H NMR spectra it was found of mPEG moiety was 43% and 75% for CS-g-PEG withd PEG5000, respectively.

tion and characterization of CS-g-PEG nanoparticles BSA

hitosan materials were synthesized as shown above, in

this is the cartively. capablstructu

In Tproduceach sathe nwhile igraftedin Tablsize of tion efdenotesame sdependof the betwee

Froby dynesting 640 nmtion. Itto crosagent iit maysmall minteracmore ccles. Incrosslithe smas crosa decrAlso thon theand amchain gbe statgive smit is att used as a vehicle for the delivery of drugs through thef polyion complex. These materials are composed of ale hydrophilic mPEG side chain and a cationic chitosan. The mPEG side chain endows the chitosan moleculesed solubility and has functional advantages such asce of the reticuloendothelial system (RES) and protein(Jeong et al., 2006).resent study, nanoparticles were prepared by a novelgelation method in aqueous medium (pH = 3.5) andere obtained spontaneously during the process. Ionicbetween the positive charge ions of the amino groupsand the negative charge groups of TPP or PGA can lead toon of inter-ionic polymer complexes. Polyionic hydro-ed by ionic gelation have the advantage of creating annment that favors the stabilization of bioactive agentsly reported (Zhang et al., 2008a). During the processticle preparation it was chosen the BSA to be in theon with the crosslinking agent and this solution to be-g-PEG as previous studies having reported that in thisr particle can be obtained and the size distribution of

materials aIt is likely tthe PEG endprocedure, et al., 2008PEG graftedof the nanoDS of CS-g-43%, givingcharacter. Alies in a qucrosslinkingdenite cordegree of cthat there iof the degrcombined wciency, it cwith the uslated moreuse the pKa values of the amino groups of chitosan andl groups of PGA are approximately 6.5 and 2.9, respec-is range chitosan and PGA are ionized and consequentlyorm polyelectrolyte complexes, which result in a matrixith a spherical shape (Sonaje et al., 2010).1 are demonstrated the nanoparticle samples that weren the rst column is demonstrated the name used fore (where the numbers 0.5, 1.0, 1.5 and 2.0 correspond tocentration mg/ml of the grafted chitosan to the sample)

second column is demonstrated the weight ratio of theerial to the crosslinking agent in the nal sample. Alsore summarized the results of the nanoparticle yield, therepared nanoparticles, the loading capacity and associa-cy of the grafted copolymers of chitosan. The valuesstandard deviation between the measurements of thele (i.e. measurements error). These parameters mainlyhe polymer nature and physicochemical characteristicsel drug used as well as from the probable interactionse polymer matrices, the crosslinking agent and the drug.ble 1 and the nanoparticle sizes that were measured

light scattering (DLS) many different and quite inter-lts may arise. The mean diameter varied from 170 to

nanoparticle samples show a unimodal size distribu-served that in all samples as the ratio of grafted polymering agent increases, which means the less crosslinkingd, the nanoparticle size seems to increase. Consequentlyated that the crosslinking agent, regardless of its naturecule like TPP or macromolecule like PGA, creates ionics with the amino groups of chitosan which leads to aact and stable conformation for CS-g-PEG nanoparti-e of the samples A and B where TPP is used as the

agent, seems that the ratio of CS-g-PEG:TPP 2:1 gives nanoparticle sizes. On the contrary when PGA is useding agent the nanoparticle size seems to increase withof the degree of crosslinking without any exceptions.lecular weight of PEG seems to play an important roleparticle size. Thus, in the samples with the same kind

t of crosslinking agent but with a different length of PEGd on chitosan (samples A with B and C with D) it canat the nanoparticles created with a shorter PEG chainr nanoparticle size. This is reasonable and expected asted to the core-shell structure that the grafted chitosanre able to create during the nanoparticles preparation.hat PEG covers the chitosan core to form a shell, since-group migrates to the surface of nanoparticles during

particularly because of the hydrophilicity of PEG (Zhanga). The higher molecular weight of PEG leads to longer

chains resulting in bigger outer shell and hence sizeparticles. This observation is reinforced by the fact thatPEG 5000 is 75% unlike DS of CS-g-PEG 2000 which is

to CS-g-PEG 5000 a bigger size and a more stealthlso it is observed that nanoparticle yield for all samplesite satisfactory level, 6480%. Moreover, use of TPP as

agent results in high yield but with no signicant andrelation in terms of the yield sequence values and therosslinking. When PGA is used, it can clearly be stateds an increase in nanoparticle yield despite the decreaseee of crosslinking. If the above mentioned results areith the values of loading capacity and association ef-

an be stated that the decrease of crosslinking degreee of PGA gives space to BSA in order to be encapsu-

efciently in CS-g-PEG nanoparticles. BSA (Ip = 4.7) is

S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327 323

Table 1Concentration and characteristics of BSA-loaded CS-g-PEG nanoparticles.

Sample code CS-g-PEG2000/TPP (w/w) Yield Diameter (nm) Loading capacity (%) Association efciency (%)

A2000 0.5 1:1 82.2 2.5 382 3 6.20 79.0A2000 1.0 2:1 74.5 2.9 177 7 3.39 55.5A2000 1.5 3:1 79.2 2.6 309 8 2.73 57.5A2000 2.0 4:1 82.1 0.4 328 5 2.93 74.5

CS-g-PEG5000/TPP (w/w)B5000 0.5 1:1 79.5 2.4 554 7 2.61 64.5B5000 1.0 2:1 77.6 1.4 497 9 4.49 44.0B5000 1.5 3:1 79.8 1.5 541 5 4.36 62.0B5000 2.0 4:1 84.0 3.8 609 4 2.45 92.0

CS-g-PEG2000/PGA (w/w)C2000 0.5 1:1 54.8 2.9 304 6 8.77 51.0C2000 1.0 2:1 63.4 0.8 456 8 8.52 69.5C2000 1.5 3:1 68.4 2.5 562 8 5.92 64.0C2000 2.0 4:1 67.8 6.3 643 9 6.46 84.0

CS-g-PEG5000/PGA (w/w)D5000 0.5 1:1 60.0 5.8 400 6 8.75 56.5D5000 1.0 2:1 64.2 1.5 401 6 7.60 63.5D5000 1.5 D5000 2.0

negatively manufacturinteraction If the Ip of pat the crossrelease proa lower valuwill appearchitosan anthe polymeticles capabtime releasexpected torelease.

Scanninmicroscopyples are shofor the restBSA-loadedspherical shis not in agrtering showTEM microgdeterminedin the prepsince the naDLS experimwith waterals are also

ed Therynamecau

ig. 8 sent

absens ther inf the

ity, aals isve bof CSsslin

2007nt (sof CSith tles A

betted ccrystnopa3:1 65.5 3.2 525 5 4:1 71.7 1.7 521 5

charged at the pH that the experiment of nanoparticlee takes place (pH = 3.5) which favors its electrostaticwith the positively charged amino groups of chitosan.rotein is different, e.g. the protein is positively chargedlinking reaction condition, the LC, AE and consequentlyles should be quite different. In this case it is expectede for LC and AE as the possible electrostatic interaction

weak (or even no interaction will be possible betweend BSA) and unable to form strong interactions withr matrix and eventually create well dened nanopar-le of protecting the active agent (BSA). At the samee prole would be also quite different as it would be

give a signicant burst effect at pH value 7.4 for drug

g electron microscopy (SEM) and transmission electron (TEM) photographs from different nanoparticle sam-wn in Figs. 6 and 7. These images are also representative

of the samples. SEM micrographs established that the nanoparticles of CS-g-PEG copolymers had a discreteape with sizes ranging from 200 up to 300 nm, a fact thateement with the measurements of dynamic light scat-n in Table 1. The size of the nanoparticles based on theraphs was about 100 nm or more, smaller than the size

by DLS. This was mainly due to the process involvedaration of the sample and it could have been expected

perform2008).hydrodTEM b2008)

In Fare preto the samplehowevterns ointensmaterithat hagroup the croet al., ing agepeaks ison w(sampactionsof grafof the BSA nanoparticles were dispersed in an aqueous phase for theents, and chitosan has the ability to swell in contact

, PEG is water soluble polymer and CS-g-PEG materi- soluble in water, while the TEM experiments were

molecular wis not affectg-PEG wheas a result

Fig. 6. SEM micrographs of CS-g-PEG nanoparticles (A) A208.39 90.07.21 92.0

in dry samples (Aktas et al., 2005; Papadimitriou et al.,efore the size determined by laser light scattering was aic diameter and was larger than the size measured by

se of solvent effect as previously reported (Yang et al.,

XRD patterns of the prepared BSA loaded nanoparticlesed. As can be seen, BSA is completely amorphous duece of any characteristic peaks. In CS-g-PEG/BSA loaded

e characteristic peaks of CS-g-PEG are recorded, which all samples are broadened, in comparison with the pat-

neat materials. Furthermore, these peaks have lower fact that justies that the crystal structure of grafted

disrupted probably due to the electrostatic interactionseen developed between the positively charged amino-g-PEG materials and the negative charged groups ofking agents TPP and PGA as previously reported (Lin). Also is obvious that when PGA is used as crosslink-amples D and C) the intensity of the two characteristic-g-PEG materials is decreased even more in compar-he samples where TPP is used as crosslinking agentand B). This may indicate that the intermolecular inter-ween PGACOO groups and NH2+ or NH+ groupshitosan may be stronger leading to a greater disruptalline structure. At the same time the XRD patterns ofrticles prepared with the grafted polymers, where the

eight of PEG is 5000 g/mol, show that the crystallinity

ed as much as in case of the nanoparticle samples of CS-re PEG molecular weight is 2000 g/mol. This may comeof the different degree of substitute of the two grafted

00 1.0 and (B) C2000 0.5.

324 S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327

Fig. 7. TEM image of CS-g-PEG loaded BSA nanoparticles of the sample A2000 1.0.

materials and as it was calculated above is lower in PEG2000 (43%)compared with PEG5000 (75%).

In Figs. 912 is demonstrated the in vitro release prole of BSAprotein from nanoparticles. In all cases the overall release processcan be characterized as a biphasic procedure.

The initial burst effect, for all samples, in the rst almost 5 h,varies betweffect may the surfacediffused rapthe release Also the sursoluble andthermore thweight. Aftthe dominathrough chet al., 2008)or degradatwas not coeroded or ddue to the i

Inte

nsity

a.u

Fig. 8. X-ray pionically cross

1801601401201008060402000

20

40

60

80

100

A 20 00 0,5A 20 00 1A 20 00 1,5A 20 00 2

rele

ase

%

time (hrs)

Fig. 9. The in vitro release prole of BSA from CS-g-PEG nanoparticles with PEG MW2000 and TPP as ionic crosslinker (sample A).

1801601401201008060402000

0

0

0

80

100

B 5000 0,5B 5000 1B 5000 1,5B 5000 2

time (hrs)

The in vitro release prole of BSA from CS-g-PEG nanoparticles with PEG0 and TPP as ionic crosslinker (sample B).

80

00een 10 and 30% of the encapsulated BSA. This burstbe attributed to the desorption of the protein close to

during preparation of the nanoparticles, which thenidly when the nanoparticles came into contact withmedium, as previously reported (Zhang et al., 2008a).face of nanoparticles is consisted of PEG, which is water

this contributes to the observed initial burst effect. Fur-e rate of BSA release is affected by the PEG molecular

er the burst release period, the rate of release fell asnt release mechanism was changed to drug diffusionitosan matrix as previously reported (Papadimitriou. Furthermore BSA was released slowly due to swellingion of the polymer. The remaining BSA in nanoparticlesmpletely released until the particles were completelyissolved in release medium, which might have been

nteraction between the remaining BSA and the few free

2

4

6

rele

ase

%

Fig. 10. MW 500

150403020102 deg

D 5000 2.0

C 2000 1 .0

B 50 00 2 .0

A 2000 0.5

BSA

owder diffraction patterns of BSA and CS-g-PEG loaded nanoparticleslinked with TPP and PGA.

1801601401201008060402000

20

40

60

C 2000 0,5C 2000 1 C 2000 1,5C 2000 2

rele

ase

%

time (hrs)

Fig. 11. The in vitro release prole of BSA from CS-g-PEG nanoparticles with PEGMW 2000 and PGA as ionic crosslinker (sample C).

S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327 325

1801601401201008060402000

20

40

60

80

100

D 5000 0,5D 5000 1D 5000 1,5D 5000 2

rele

ase

%

time (hrs)

Fig. 12. The in vitro release prole of BSA from CS-g-PEG nanoparticles with PEGMW 5000 and PGA as ionic crosslinker (sample D).

amino groups on the chitosan segments (Zhang et al., 2008a,b) as itis previously established from the shift of the characteristic peaks inFT-IR spectra, mentioned above. At this point we have to point outthat the different DS of CS-g-PEG 2000 and 5000 plays also its rolein drug release of BSA. It is logically expected that CS-g-PEG 5000would give a more intense burst effect as the DS of this material is75%. Moreover, it is important to be stated that it is not possible tocontrol the DS for these materials, as previously reported, in orderto have exactly the same materials, with the same DS (Muslim et al.,2001; Gorochovceva et al., 2005; Sugimoto et al., 1998).

Except molecular weight of PEG the crosslinking agents play alsoan important role to the release behavior of BSA. When TPP is usedas ionic crosslinker the release of BSA from nanoparticles seemsto be slower (samples A and B, Figs. 9 and 10) toward the resultsobtained when PGA is used as crosslinking agent (samples C and D,Figs. 11 and 12). Probably this occurs because of the smaller molec-ular size of TPP, in comparison with PGA. Thus TPP has the ability topenetrate easily through the macromolecular chains of CS-g-PEG,during the nanoparticle formation, creating a more stable networkof ionic interactions among the polymer matrix and the crosslink-ing agent. The aforementioned behavior may also be attributed tothe low loading capacity of nanoparticles with TPP as it is shown inTable 1, which means that a lower amount of BSA exists to the outersurface of the nanoparticles and thus the release mainly occurs asa procedure of diffusion from the inner part of the nanoparticlesrather than from the surface. Due to these differences it can be saidthat TPP may has higher efciency as crosslinking agent, comparedwith PGA.

Another characteristic of these release proles is that as theratio of crosslinking agent is increased, independently TPP or PGAis used; the release rate seems to increase. This may be attributedto the fact that the less extent the ionic crosslinking of CS-g-PEGis, the more are the free amino groups of chitosan that can interactwith the negative charged groups of BSA. As has been previouslyreported the reason for the slow drug release form the polyion com-plex micelles might be the strong ion complex between the aminogroup of chitosan and the carboxyl group of drug (Jeong et al., 2006).

Furthermore, the kinetics of drug delivery was investigatedusing semi-empirical models. More detailed mathematical mod-els could be used in order to elucidate the exact drug releasemechanism, though this probably would require additional exper-imental data. In order to identify the kinetic parameters, datareported in Figs. 912 were re-evaluated in order to be presented

1001

10

A200 0 B500 0 C200 0 D500 0

Mt/M

oo

1

10

100

Mt/M

oo100101

Time (h)

1001011

10

100

A200 0 B500 0 C200 0 D500 0

Time (h)

Mt/M

oo

1

10

100

Mt/M

oo

Fig. 13. Plots of Mt/M versus time for the samples A2000, B5000, C2000 an100101

Time (h)

A2000 B5000 C2000 D5000

100101

Time (h)

A200 0 B500 0 C200 0 D500 0

d D5000 at 0.5 (a), 1.0 (b), 1.5 (c) and 2 (d).

326 S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327

Table 2Kinetic rate constants and release exponent according to Eq. (4) for all formulations.

Sample code Initial drugreleased

Linear region (h) k n R2

A2000 0.5 A2000 1.0A2000 1.5 A2000 2.0 B5000 0.5 B5000 1.0B5000 1.5B5000 2.0C2000 0.5 C2000 1.0 C2000 1.5 C2000 2.0 D5000 0.5 D5000 1.0D5000 1.5 D5000 2.0

in the usuadenote cumtime, respereasons in regions weinitial highburst effectseen in thelations, excfor 4 h (TabIn the secolinear depein a loglogpas equatioapplied acc

MtM

= k t

Here, k characteristmight be infrequently uwhich is a ssecond law.for spheres,cation for dthe solely rdelivery sysics are obsecorrespondSiepmann, 2ulation to eobey Fickianrelease mecwhile in all observed wThis is an inpotentially

Finally, fwith the am

4. Conclus

PEG grausing Borchspectroscop

PEG5000. These materials are able to prepare nanoparticles via ion-ically crosslinking procedure, using TPP and PGA as crosslinkingagents, in the presence of BSA, as macromolecular model drug. TPPdue to its high reactivity produces nanoparticles with lower parti-

s, con nanking

relealeadse abffectrisonncrevent

nces

., Andr. Preppase ., Mas

system2.i, N., Rn as anrol. R., Kpso, Mems: Eed. N., Chisemi-1014, Qi, Hethoxm. Edvceva(ethylace. Eu., Bodn. Nan

. Collo M., Azv. Rev.M., Sthesis m. Ch, K.A.,d nan., Kidespon(g-glu712.I., Kimim, K.rans r348ssy, Zly, J.(%)

10 272 3.63 0.75 0.9984 472 1.05 1.05 0.9975.5 472 1.67 0.91 0.9999 472 1.87 0.81 0.9982 272 2.14 1.02 0.989

11 272 7.76 0.60 0.9875.5 272 5.89 0.67 0.9925 172 10.8 0.46 0.9968.5 272 6.4 0.66 0.991

12 272 9.12 0.54 0.9898.5 272 8.32 0.56 0.9851.0 272 3.24 1.14 0.9946 272 9.0 0.61 0.9674.5 272 11.2 0.54 0.9584 0.512 20.4 0.67 0.978

10 272 20.9 0.42 0.979

l form of Mt/M versus time. The symbols Mt and Mulative amount of drug released at time t and innite

ctively. These data are plotted in Fig. 13ad. For kineticall different experimental conditions three distinctivere identied. The rst, where the amount presents an

and rather constant value is attributed to the initial described previously. This period, as it can be clearlyse gures, lasts for approximately 2 h in most formu-ept for A2000 1, A2000 1.5 and A2000 2 where it lastsle 2). The initial amount released is at most 1011%.nd stage lasting almost until 72 h in most samples, andence of Mt/M versus time appears when plotted

scale. In this interval, the well-known, so-called Pep-n, or power law (Siepmann and Siepmann, 2008) wasording to Eq. (4):

n (4)

is a constant incorporating structural and geometricics of the system and n is the release exponent, whichdicative of the mechanism of drug release. This is a verysed and easy-to-apply model to describe drug releasehort time approximation of the exact solution of Ficks

A release exponent of 0.5 in Eq. (4) for thin lms or 0.43 as those used in this investigation, can serve as an indi-iffusion-controlled drug release. If polymer swelling iselease rate controlling mechanism and in the case of atem with lm geometry, zero order drug release kinet-rved corresponding to a release exponent of n = 1. Theing value of n for spheres is equal to 0.85 (Siepmann and

cle sizechitosacrosslirate oftution have thburst ecompawhile iused, e

Refere

Aktas, Y2005a cas

Amidi, Mery 598

BhattaratosaCont

Csaba, NAlonsystBiom

Dash, Mtile 981

Deng, L.of mPoly

Gorochopolysurf

Hajdu, I2008acid

Hamidi,Deli

Harris, JSyntPoly

Howardbase

Imoto, TpH-rpoly10, 2

Jeong, YS., Kall-t95, 2

KereszteBorb008). Then according to the values obtained from sim-xperimental data, only B5000 2 and D5000 2 seem to

diffusion. Polymer swelling seems to be the main drughanism in A2000 1, A2000 1.5, B5000 0.5 and C2000 2,the rest samples a so-called anomalous transport wasith release exponent ranging in between 0.43 and 0.85.dication of overlapping of different types of phenomena,including drug diffusion and polymer swelling.or time periods higher than 72 h a plateau is reachedount of drug released reaching its nal constant value.

ions

fted into chitosan backbone was easefully prepared reduction process, as was veried by NMR and FTIRy. The grafted percent is 43% for PEG2000 and 75% for

for targeteLin, Y.H., Chun

ration of nevaluation1104111

Lin, Y.H., Mi, FPreparatioinsulin de

Liu, Z., Jiao, Y.,ticles as dr

Markwell, M.Aprocedureples. Anal.

Muslim, T., MoSynthesis hydr. Poly

Nikiforidis, C.Vproteins b

Nunthanid, J., erties and143157.mpared to PGA. The release proles of BSA from graftednoparticles reveal that the PEG content as well as the

agent used and the crosslinking ratio determines these. Longer PEG chain as well as greater degree of substi-

to faster drug release from nanoparticles. TPP seems toility to create a more stable network leading to a slower

regardless the nanoparticle size, which is smaller in with the nanoparticles prepared with the use of PGA,asing degree of ionic crosslinking, regardless the agentually increases the release rate of BSA.

ieux, K., Alonso, M.J., Calvo, P., Grsoy, R.N., Couvreur, P., C apana, Y.,aration and in vitro evaluation of chitosan nanoparticles containing

inhibitor. Int. J. Pharm. 298, 378383.trobattista, E., Jiskoot, W., Hennink, W.E., 2010. Chitosan-based deliv-s for protein therapeutics and antigens. Adv. Drug Deliv. Rev. 62,

amay, H.R., Gunn, J., Matsen, F.A., Zhang, M., 2005. PEG-grafted chi- injectable thermosensitive hydrogel for sustained protein release. J.

elease 103, 609624.ing-Hggrd, M., Fernandez-Megia, E., Novoa-Carballal, R., Riguera, R.,.J., 2009. Ionically crosslinked chitosan nanoparticles as gene deliveryffect of PEGylation degree on in vitro and in vivo gene transfer. J.anotech. 5, 162171.ellini, F., Ottenbrite, R.M., Chiellini, E., 2011. Chitosan A versa-synthetic polymer in biomedical applications. Prog. Polym. Sci. 36,.., Yao, C., Feng, M., Dong, A., 2007. Investigation of the propertiesy poly(ethylene glycol)/chitosan graft co-polymers. J. Biomater. Sci.. 18, 15751589., N., Naderi, A., Dedinaite, A., Makuska, R., 2005. Chitosan-N-ene glycol) brush copolymers: Synthesis and adsorption on silicar. Polym. J. 41, 26532662.r, M., Filipcsei, G., Hartmann, J.F., Darczi, L., Zrnyi, M., Borbly, J.,oparticles prepared by self-assembly of chitosan and poly--glutamicid. Polym. Sci. 286, 343350.adi, A., Raei, P., 2008. Hydrogel nanoparticles in drug delivery. Drug. 60, 16381649.ruck, E.C., Case, M.G., Paley, M.S., Vanalstine, J.M., Brooks, D.E., 1984.and characterization of poly(ethylene glycol) derivatives. J. Polym. Sci.em. Ed. 22, 341352.

2009. Delivery of RNA interference therapeutics using polycation-oparticle. Adv. Drug Deliv. Rev. 61, 710720.a, T., Matsusaki, M., Akashi, M., 2010. Preparation and uniquesive properties of novel biodegradable nanocapsules composed oftamic acid) and chitosan as weak polyelectrolytes. Macromol. Biosci.77., S.H., Jung, T.Y., Kim, I.Y., Kang, S.S., Jin, Y.H., Ryo, H.H., Sun, H.S., Jin,K., Ahn, K.Y., Jung, S., 2006. Polyion complex micelles composed ofetinoic acid and poly (ethylene glycol)-grafted-chitosan. J. Pharm. Sci.2360.., Bodnr, M., Ber, E., Hajdu, I., Zhang, M., Hartmann, J.F., Minko, T.,, 2009. Self-assembling chitosan/poly--glutamic acid nanoparticlesd drug delivery. Colloid. Polym. Sci. 287, 759765.g, C.K., Chen, C.T., Liang, H.F., Chen, S.C., Sung, H.W., 2005. Prepa-anoparticles composed of chitosan/poly-gamma-glutamic acid and

of their permeability through Caco-2 cells. Biomacromolecules 6,2..L., Chen, C.T., Chang, W.C., Peng, S.F., Liang, H.F., Sung, H.W., 2007.n and characterization of nanoparticles shelled with chitosan for orallivery. Biomacromolecules 8, 146152.

Wang, Y., Zhou, C., Zhang, Z., 2008. Polysaccharides-based nanopar-ug delivery systems. Adv. Drug Deliv. Rev. 60, 16501662..K., Haas, S.M., Bieber, L.L., Tolbert, N.E., 1978. Modication of Lowry

to simplify protein determination in membrane and lipoprotein sam- Biochem. 87, 206210.rimoto, M., Saimoto, H., Okamoto, Y., Minami, S., Shigemasa, Y., 2001.and bioactivities of poly(ethylene glycol)-chitosan hybrids. Carbo-m. 46, 323330.., Kiosseoglou, V., 2010. Competitive displacement of oil body surfacey Tween 80 Effect on physical stability. Food Hydrocol., 16.Puttipipatkhachorn, S., Yamamoto, K., Peck, G.E., 2001. Physical prop-

molecular behavior of chitosan lms. Drug. Dev. Ind. Pharm. 27,

S.A. Papadimitriou et al. / International Journal of Pharmaceutics 430 (2012) 318 327 327

Papadimitriou, S., Bikiaris, D., 2009. Novel self-assembled core-shellnanoparticles based on crystalline amorphous moieties of aliphaticcopolyesters for efcient controlled drug release. J. Control. Release 138,177184.

Papadimitriou, S., Bikiaris, D., Avgoustakis, K., Karavas, E., Georgarakis, M., 2008.Chitosan nanoparticles loaded with dorzolamide and pramipexole. Carbohydr.Polym. 73, 4454.

Park, J.H., Saravanakumar, G., Kim, K., Kwon, I.C., 2010. Targeted delivery of lowmolecular drugs using chitosan and its derivatives. Adv. Drug Deliv. Rev. 62,2841.

Peng, S.F., Yang, M.J., Su, C.J., Chen, H.L., Lee, P.W., Wei, M.C., Sung, H.W., 2009.Effects of incorporation of poly(g-glutamic acid) in chitosan/DNA complexnanoparticles on cellular uptake and transfection efciency. Biomaterials 30,17971808.

Rao, J.P., Geckeler, K.E., 2011. Polymer nanoparticles: preparation techniques andsize-control parameters. Prog. Polym. Sci. 36, 887913.

Shu, S., Zhang, X., Teng, D., Wang, Z., Li, C., 2009. Polyelectrolyte nanoparticles basedon water-soluble chitosanpoly (l-aspartic acid)polyethylene glycol for con-trolled protein release. Carbohydr. Res. 344, 11971204.

Siepmann, J., Siepmann, F., 2008. Mathematical modeling of drug delivery. Int. J.Pharm. 364, 328343.

Sonaje, K., Lin, K.J., Wang, J.J., Mi, F.L., Chen, C.T., Juang, J.H., Sung, H.W., 2010. Self-assembled pH-sensitive nanoparticles: a platform for oral delivery of proteindrugs. Adv. Funct. Mater. 20, 36953700.

Sugimoto, M., Morimoto, M., Sashiva, H., Saimoto, H., 1998. Preparation and charac-terization of water-soluble chitin and chitosan derivatives. Carbohydr. Polym.36, 4959.

Tang, D.W., Yu, S.H., Ho, Y.C., Mi, F.L., Kuo, P.L., Sung, H.W., 2010. Heparinizedchitosan/poly(g-glutamic acid) nanoparticles for multi-functional delivery ofbroblast growth factor and heparin. Biomaterials 31, 93209332.

Tsao, C.T., Chang, C.H., Lin, Y.Y., Wu, M.F., Wang, J.L., Young, T.H., Han, J.L., Hsieh, K.H.,2011. Evaluation of chitosan/-poly(glutamic acid) polyelectrolyte complex forwound dressing materials. Carbohydr. Polym. 84, 812819.

Werle, M., Tacheuchi, H., Bernkop-Schnurch, A., 2009. Modied chitosans for oraldrug delivery. J. Pharm. Sci. 98, 16431656.

Yang, X., Zhang, Q., Wang, Y., Chen, H., Zhang, H., Gao, F., Liu, L., 2008. Self-aggregatednanoparticles from methoxy poly(ethylene glycol)-modied chitosan: synthe-sis; characterization; aggregation and methotrexate release in vitro. ColloidsSurf. B. Biointerfaces 61, 125131.

Yao, K., Peng, T., Xu, M., Yuan, C., Goosen, M.F.A., Zhang, Q., Ren, L., 1994. pH-dependent hydrolysis and drug release of chitosan/polyether interpenetratingpolymer network hydrogel. Polym. Int. 34, 213219.

Yao, Z., Zhang, C., Ping, Q., Yu, L.L., 2007. A series of novel chitosan derivatives:Synthesis, characterization and micellar solubilization of paclitaxel. Carbohydr.Polym. 68, 781788.

Zhang, X.G., Teng, D.Y., Wu, Z.M., Wang, X., Wang, Z., Yu, D.M., Li, C.X., 2008a. PEG-grafted chitosan nanoparticles as an injectable carrier for sustained proteinrelease. J. Mater. Sci. Mater. Med. 19, 35253533.

Zhang, X.G., Zhang, H.J., Wu, Z.M., Wang, Z., Niu, H.M., Li, C.X., 2008b. Nasal absorptionenhancement of insulin using PEG-grafted chitosan nanoparticles. Eur. J. Pharm.Biopharm. 68, 526534.

Zhang, N., Li, J., Jiang, W., Ren, C., Li, J., Xin, J., Li, K., 2010. Effective protection andcontrolled release of insulin by cationic -cyclodextrin polymers from algi-nate/chitosan nanoparticles. Int. J. Pharm. 393, 212218.

Chitosan-g-PEG nanoparticles ionically crosslinked with poly(glutamic acid) and tripolyphosphate as protein delivery systems1 Introduction2 Materials and methods2.1 Materials2.2 Preparation of PEG-aldehyde (mPEG-CHO)2.3 Preparation of CS-g-PEG materials2.4 Characterization of PEG-aldehyde and CS-g-PEG materials2.4.1 Nuclear magnetic resonance (NMR)2.4.2 Fourier transform-infrared spectroscopy (FT-IR)2.4.3 Wide angle X-ray diffractometry (WAXD)

2.5 Preparation of CS-g-PEG nanoparticles loaded with BSA2.6 Characterization of CS-g-PEG materials and nanoparticles2.6.1 Morphological characterization of nanoparticles2.6.2 Size measurements of nanoparticles

2.7 Evaluation of drug encapsulation2.8 In vitro drug release

3 Results and discussion3.1 Synthesis and characterization of CS-g-PEG3.2 Preparation and characterization of CS-g-PEG nanoparticles loaded with BSA

4 ConclusionsReferences