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International Journal of Pharmaceutics 430 (2012) 318 327
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journa l h omepa g e: www.elsev ier .com
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:
[email protected] (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.
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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