iranian journal chitosan

7/29/2019 Iranian Journal Chitosan

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Iranian Polymer Journal

20 (5), 2011, 445-456

alginate;

drug delivery system;burst release;

ionic gelation;

tripolyphosphate.

(*) To whom correspondence to be addressed.

E-mail: [email protected]

A B S T R A C T

Key Words:

Reinforcement of Chitosan Nanoparticles Obtained

by an Ionic Cross-linking Process

Morteza Hasanzadeh Kafshgari, Mohammad Khorram*, Mobina Khodadoost,

and Sahar Khavari

Department of Chemical Engineering, University of Sistan and Baluchestan,

P.O. Box: 98161/164, Zahedan, Iran

Received 22 February 2010; accepted 27 November 2010

The reduction in burst release of a new drug nanocarrier system was achieved by

ionic gelation of nanoparticles made of chitosan and tripolyphosphate. Capability

of calcium alginate for reinforcement of the chitosan-tripolyphosphate nano-

particle matrix was studied. Bovine serum albumin (BSA) was loaded into nanoparticles

as a model drug. The final particle size, polydispersity index, entrapment efficiency and

the release rate of BSA were optimized in relation to variations in parameters such as

sodium alginate concentration, theoretical loading of BSA and degree of deacetylation

of chitosan. It was found that increases in sodium alginate concentration resulted in

smaller particle size, higher polydispersity index, lower entrapment efficiency and lower

release rate of BSA. The same parametric changes were observed for the theoretical

loading of BSA, with the exception of drug release which was followed by higher rate.

Nanoparticles of chitosan-tripolyphosphate with a high degree of deacetylation led to

production of smaller particle size, higher polydispersity index and entrapment

efficiency, and a faster drug-release profile. The experimental data of this study

revealed that burst release decreased significantly in reinforced chitosan-tripoly-

phosphate nanoparticles.

INTRODUCTION

Application of chitosan, a cationicpolysaccharide, has been exten-

sively investigated in various fields

of applications, such as biotechno-

logy [1], as a pharmaceutical

excipient in oral drug formulations

[2], waste water treatment [3], cos-

metics [4] and food science [5].

These broad fields of applications

are due to chitosan's unique set of

properties. Favourable biologicalproperties [6], low toxicity [7],

biodegradability [8], proper

mucoadhesive properties [9], abs-

orption enhancer across intestinalepithelium and mucosal sites for

drugs, peptides and proteins [10],

metal complexation [11], antimi-

crobial activity [12], good homo-

static properties [13], and accept-

able anti-cancerous properties [14]

are the most important set of prop-

erties. Among all applications of

chitosan drug delivery systems are

widely recognized as a promisingresearch field [15]. Tablets, micro-

spheres, microgels and nano-

particles are the different forms

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of chitosan-based drug delivery devices that have

been studied. In the past decade, chitosan nano-

particles have been extensively investigated because

they can control drug release rate, prolong the

duration of therapeutic effectiveness, and deliverdrugs to their specific sites in the body [16]. Chitosan

nanoparticles exhibit superior activity that can be

attributed to their small and quantum size

effect [17].

Common methods to prepare chitosan nano-

particles are ionic gelation, coacervation or precipi-

tation, emulsion-droplet coalescence, reverse

micellar, and self-assembly chemical modification

[14]. The ionic gelation process is commonly used to

prepare chitosan nanoparticles because it is a verysimple and mild method [18]. This process can be

performed either by chemical or physical cross-

linking. It is worth noting that chitosan has a high

density of amine groups in its backbone and the

amine groups are protonized to form -NH3+ in acidic

solution. These positively charged groups in chitosan

can be chemically cross-linked with dialdehydes such

as glutaraldehyde [19] and ethylene glycol diglycidyl

ether [20], or physically cross-linked with multivalent

anions derived from sodium tripolyphosphate (TPP)

[21], citrate [22,23] and sulphate [24]. Both

glutaraldehyde and ethylene glycol diglycidyl ether

are toxic and can cause irritation to mucosal

membranes [25]. Physically cross-linked chitosan

gels have been used in drug delivery systems due to

their enhanced biocompatibility over chemically

cross-linked chitosan [26].

Non-toxicity and quick gelling ability of TPP are

the important properties that make it a favourable

cross-linker for ionic gelation of chitosan [14]. In

addition, TPP has been also recognized as an

acceptable food additive by the US Food and Drug

Administration [27]. Moreover, the process of ionic

gelation of chitosan with TPP as a cross-linker is

feasible for the scale-up of entrapment in a particle

processing operation [28]. Chitosan nanoparticles

prepared by TPP as an anionic cross-linker are

homogeneous, and possess positive surface charge

that make them suitable for mucosal adhesion

applications [14].

The properties of ionically cross-linked chitosanare influenced by electrostatic interactions between

the anionic cross-linker and chitosan. This interaction

depends on the variables such as: anionic molecular

structure, its charge density and molecular concen-

tration, pH of chitosan solution, and physical

properties of chitosan, i.e., molecular weight anddegree of deacetylation (DDA) [25]. Several studies

have investigated the effect of these variables on the

properties of ionically produced chitosan, its drug

entrapment efficiency and drug release behaviour

[25].

Advantages of protein delivery systems based on

chitosan-TPP nanoparticles are reported in many

research works performed in the past few years [29].

A shortcoming of chitosan-based nanoparticles as

protein release system is that such particles release30-70% of the protein within 3-6 h placed in release

environment. This is due to the mechanism of burst

release that has been considered as a slow release in

many studies [30].

Burst release management is a major challenge in

the development of drug delivery systems because it

may lead to inefficient delivery and significant

toxicity hazards. The degree of burst release general-

ly depends upon the nature of the polymer, drug

molecular structure and its molecular weight, poly-

mer/drug ratio, relative affinities of the drug and

polymer and the aqueous phase [31].

Many methods, including blending with other

polymers, grafting with monomers, varying the

chemistry of the polymer [32], adding excipients into

polymer phase [33], employing new polymers [34],

encapsulating particulate forms of the drugs into

microparticles, preparation of novel biopolymer/

inorganic material composites [35] and spray/

freezing [36] have been applied to improve the burst

release properties of the polymeric carriers.

Polymer coating is another burst release control

method. Tavakol et al. [37] have studied this method

for N,O-carboxymethyl chitosan (NOCC) beads

coated with chitosan. They produced beads of NOCC

and alginate with ionotropic gelation method and

then, coated the beads with chitosan. The effect of

coating and drying methods on the swelling and

release behaviour of the prepared beads has been

investigated. The results of this work indicate that

burst release has not been observed in chitosancoated beads. Using additional barrier layers is

Reinforcement of Chitosan Nanoparticles Obtained ... Hasanzadeh Kafshgari M et al.

Iranian Polymer Journal / Volume 20 Number 5 (2011)446
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naturally an efficient method to manipulate drug

release profiles and reduce the burst release, though it

is inherently an expensive method due to the

additional materials employed and difficulties

associated with controlling the barriers quality. Chenet al. [30] have reported nanoparticles based on

N-trimethyl chitosan chloride (TMC) and TPP. They

used BSA as a model protein entrapped into the

particles with sodium alginate as a modifier of TMC

nanoparticles. They showed that burst release of BSA

can be reduced from 60% to 45%.

The main objective of this work was to reduce the

burst release of BSA-loaded chitosan nanoparticles

prepared by a one-step simple method. Very few

studies have focused on high loadings (10-15%) ofproteins in micro/nanospheres with low burst release.

Due to the nature of BSA, an optimum BSA delivery

system must undergo burst release as low as possible

while keeping drug loading as high as possible. The

design of an optimum system is the main objective in

this study. Comparison of the release test results of the

current work with other similar works clearly shows

higher efficiency of BSA-loaded chitosan nano-

particles by the method developed in this study. While

this work and the study carried out by Chen et al. [30]

are both using ionic gelation method, two important

differences are to be taken into account. Chen et al.

[30] usedN-trimethyl chitosan as a polymeric carrier

without any ionotropic cross-linker. In this work

chitosan is used as a polymeric carrier and CaCl2 as

an ionotropic cross-linker to further reinforce the

chitosan/alginate composite matrix. In order to

decrease burst release, chitosan nanoparticles with

two different degrees of deacetylation were modified

using calcium alginate, and the effects of sodium

alginate concentration on particle size, entrapment

efficiency and BSA release profile were investigated.

EXPERIMENTAL

Materials

Medium molecular weight chitosan (Fluka, The

Netherlands) was used as an encapsulating polymer.

According to its specification analysis, the molecular

weight was 400 kDa. The degree of deacetylation wasabout 83% as determined by potentiometric titration.

Alginic acid as its sodium salt (medium viscosity,

3500 cps, 2% (w/v) aqueous solution at 25C from

Sigma-Aldrich, The Netherlands) was used as a rein-

forcing polymeric agent. Sodium tripolyphosphate as

ionic cross-linker was supplied by Sigma-Aldrich.Calcium chloride was provided by Merck Chemical

Co., Germany, and used as ionotropic cross-linking

agent. As a model protein molecule for entrapment

and control release studies, BSA was obtained from

Merck Chemical Co., Germany. The other chemicals

were analytical grade reagents and used as received.

Purification of Chitosan

The chitosan was dissolved in 2% (v/v) acetic acid

solution which was filtered to remove the residues ofinsoluble particles. A two-molar solution of NaOH

was added to the filtrate to obtain purified chitosan in

the form white precipitate. The precipitated chitosan

was washed thoroughly using double distilled water,

and then dried at 40C for 48 h. Further deacetylation

of chitosan occurred during the purification process.

Determination of Chitosan Degree of Deacetylation

The degree of deacetylation numerical values of the

unpurified and purified chitosan samples were

determined by potentiometric titration of chitosan

(25 mL of 0.2 M HCl) against 0.1 M NaOH using the

following equation [38]:

(1)

where, V is the volume of alkali consumed in the

titration of amino groups present in the chitosan

samples, CNaOH is the concentration of sodium

hydroxide used for titration, MChitosan is the mass of

chitosan sample, and 16 and 0.0994 are the molecular

weight and theoretical molar mass of the amino

groups, respectively.

Preparation of Chitosan Nanoparticles

Chitosan nanoparticles were prepared on the basis of

ionic gelation of chitosan in presence of TPP.

Unpurified and purified medium molecular weight

chitosan were dissolved in 1% (v/v) acetic acid

solution at the final concentration of 0.5% (w/v). Two

different concentrations of 20% and 50% (w/w) BSA,based on chitosan and alginate, were added each into

447Iranian Polymer Journal / Volume 20 Number 5 (2011)

Reinforcement of Chitosan Nanoparticles Obtained ...Hasanzadeh Kafshgari M et al.

0994.0

1610(%)

3

=

Chitosan

NaOH

M

CVDDA
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a separate chitosan solution prior to nanoparticles

formation. The pH of chitosan-BSA solution was

adjusted to 5.9 by 2 N NaOH solution. Then, 10 mL

of TPP solution (0.5% w/v) prepared in double

distilled water was added into the chitosan-BSAsolution (50 mL) through an insulin syringe needle at

the speed of 60 mL/h under magnetic stirring at room

temperature. The pH of chitosan-BSA solution

reached 5.5 after adding TPP solution. The procedure

described so far results in producing unreinforced

chitosan nanoparticles.

Reinforced chitosan nanoparticles were prepared

by the same procedure, except that various amounts

of sodium alginate (10% and 20% w/v) were

dissolved in the TPP solution before adding thechitosan-BSA solution. Chitosan nanoparticles were

formed upon adding the TPP solution to chitosan-

BSA solution and mixing. After ten minutes of

mixing these two solutions, 0.9 g calcium chloride

was added to the suspension which was stirred

for 20 min. Finally, the nanoparticles were

isolated by centrifugation at 6000 rpm for 45 min.

The supernatant was stored for further analysis.

The precipitate was suspended in double distilled

water, centrifuged again and dried at 37C. The

dried chitosan nanoparticles were suspended

in milli-Q distilled water for characterization tests

where appropriate; or directly used for in vitro

Table 1. Formulations of the prepared chitosan-TPP

samples loaded with BSA.

release experiments.

Formulations of all chitosan-TPP samples that

were used to investigate the effect of BSA loading,

alginate concentration, and degree of deacetylation

on nanoparticles and in vitro release profiles arepresented in Table 1.

Characterization Tests of Nanoparticles

The chemical interactions between chitosan,

alginate, TPP and BSA were established through

FTIR spectrometry. FTIR Spectra of chitosan, TPP

and BSA prepared nanoparticles were recorded on a

460 Plus Jasco Inc FTIR spectrophotometer

(Maryland, USA) within KBr pellets. The instrument

was operated with resolution of 4 cm-1

andfrequency range of 400-4000 cm-1.

The mean particle size and size distribution of

nanoparticles were determined by dynamic light

scattering (DLS) using a Malvern Zetasizer Nano-

ZS-ZEN 1600 (Malvern Instruments Co., UK). The

analysis was carried out at a scattering angle of 90 at

a temperature of 25C using nanoparticles dispersed

in de-ionized distilled water. Every sample measure-

ment was repeated 10 times. Particle size distribution

of the nanoparticles is reported as a polydispersity

index (PDI). The morphology of dried nanoparticles

was observed by transmission electron microscopy

(TEM). For TEM analysis, about 3 mL ethanol was

added to the prepared chitosan nanoparticles, and

dispersed in Sonicator (Power Sonic 405, Hwashin

Technology Co., Korea) for 5 min, and one droplet of

the nanoparticles solution was dripped onto copper

grids. The samples were dried by natural air flow

and then analyzed using TEM without any further

treatment or coating.

To determine the entrapment efficiency (EE) of

BSA into nanoparticles, the amount of free BSA in

supernatant was analyzed with UV-Vis spectro-

photometer at 595 nm using the Bradford protein

assay. The supernatant of non-loaded chitosan (DDA:

83.5% or 96.4%) nanoparticle suspension was used

as a blank. The BSA entrapment efficiency (EE) was

calculated using the following equation:

(2)

Iranian Polymer Journal / Volume 20 Number 5 (2011)448


inBSAfreeaddedBSAtotalEE = [((%)

100]/) addedBSAtotaltsupernatan

Sample Alginate concentration

(w/v%)

Theoretical

loading (w/w%)

DDA

(%)

III

III

IV

V

VI

VII

VIII

IX

X

XI

XII

0.00.1

0.2

0.0

0.1

0.2

0.0

0.1

0.2

0.0

0.1

0.2

2020

20

50

50

50

20

20

20

50

50

50

83.5483.54

83.54

83.54

83.54

83.54

96.38

96.38

96.38

96.38

96.38

96.38
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In Vitro Drug Release

The dried BSA-loaded nanoparticles were resuspend-

ed in 25 mL of 0.1 M phosphate buffer solution (PBS,

pH 7.4), then incubated at 37C while stirred at

100 rpm. At specific time intervals, 0.5 mL of thesample was removed and replaced with fresh PBS.

The withdrawn samples were centrifuged at

6000 rpm for 5 min. The BSA released from the

nanoparticles and present in the supernatant was

measured by Bradford protein assay. The blank

sample consisted of non-loaded chitosan nano-

particles resuspended in PBS. Triplicate samples were

analyzed for each measurment.

RESULTS AND DISCUSSION

Chitosan TPP nanoparticles are formed by ionic

cross-linking (ionic gelation) of oppositely charged

ions of chitosan and TPP. However, the preparation of

reinforced chitosan-TPP nanoparticles has been

attributed to the three following phenomena: (a) ionic

gelation technique between positively charged

chitosan and negatively charged TPP and BSA, (b)

electrostatic interaction between the positively

charged -NH3+ of chitosan and negatively charged

-COO- of alginate, and (c) ionotropic gelation process

between negatively charged alginate and calcium ion

(Ca+2) [39]. In this study, calcium ion is used as a

cross-linking agent to strengthen and stabilize the

particles.

The ratio between chitosan and TPP is critical and

does control the size and PDI of the nanoparticles. It

is shown that by increasing the chitosan/TPP ratio,

smaller nanoparticles can be produced [16]. TPP is a

polyfunctional cross-linking agent and can create five

ionic cross-linking points with amino groups of

chitosan. Thus, in this study the ratio of 5/1 (w/w) was

selected as a suitable chitosan/TPP ratio [16].

The ionic interaction between chitosan and TPP is

dependent on the solution pH. This is due to the

variation in ionization degree of chitosan and TPP.

Other researchers [25] showed that a pH range 3-6

was a suitable range for ionic gelation of chitosan

with TPP. The pH of 5.9 is selected in the present

work because BSA (PI = 4.8) is negatively charged atthis pH and electrostatic interaction between the

negatively charged BSA and positively charged

chitosan causes greater entrapment of BSA into the

nanoparticles.

FTIR was used to confirm the incorporation of

calcium alginate into the chitosan matrix and loadingof BSA in the prepared nanoparticles. The FTIR

spectra of chitosan, alginate, BSA, BSA-loaded

chitosan nanoparticle (sample IV) and BSA-loaded

chitosan-alginate nanoparticles (sample VI) are

presented in Figure 1.

Three characteristics absorption bands observed in

chitosan (spectrum a in Figure 1), at 3413, 1672 and

1317 cm-1, as in the given order are due to the N-H,

amide I and amide III groups present in chitosan.

According to Yuan et al. [40] the peak of 3413 cm-1

corresponds to stretching vibration of N-H in pure

chitosan. It is noticeable that this peak has shifted to

lower wavenumbers centered at 3395 cm-1 and it is

broader and stronger in chitosan particles (samples IV

and VI, spectra d and e in Figure 1). This is an

evidence of molecular interaction between these

Figure 1. FTIR Spectra of: (a) chitosan, (b) alginate, (c)

BSA, (d) BSA-loaded chitosan nanoparticle (sample IV) and(e) BSA-loaded chitosan-alginate nanoparticle (sample VI).


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groups and sodium tripolyphosphate and also due to

hydrogen bonding involvement. Primary amines also

show sharp peak between 3500 and 3400 cm-1 which

could be attributed to the asymmetric and symmetric

stretching of the N-H bonds [40]. The peak in purechitosan and chitosan particles appears broad in this

region due to the contribution of O-H stretching peaks

and hydrogen bonding [41].

FTIR Spectra of BSA showed peaks around 1655,

1534 and 3309 cm-1, reflecting the acetylamino I,

acetylamino II and (NH2) groups, respectively [42].

Acetylamino I at 1655 cm-1 and acetylamino II at

1534 cm-1 in BSA (spectrum c in Figure 1) overlapped

1672 cm-1 of amide I and 1597 cm-1 in chitosan, so

more intensive peaks appeared for the BSA-loadedchitosan nanoparticles (sample IV, spectrum d in

Figure 1).

The characteristic peaks of sodium alginate

included O-H at 3397 cm-1, COO- (asymmetric) at

1617, COO- (symmetric) at 1429 and 1028 cm-1 for

C-O-C stretching. For the BSA-loaded chitosan

nanoparticle of sample IV, the stretching vibration of

-OH and -NH2 at 3392 cm-1 became broader.

Spectrum e in Figure 1 is an indication of intra-

molecular and intermolecular hydrogen bonds which

were formed and enhanced between chitosan and

alginate molecules [43].

The mean particle size, PDI and EE of the

prepared samples are presented in Table 2. PDI is a

Table 2. Mean particle size and entrapment efficiency of

chitosan-TPP nanoparticles loaded with BSA.

measure of homogeneity in dispersed systems and

ranges from 0 to 1. Homogeneous dispersion has PDI

value close to zero while PDI values greater than 0.3

suggest high heterogeneity [44]. Except the three

samples (samples I, IV and XII) other prepared

nanoparticles have PDI values lower than 0.3

(Table 2).

Morphological studies of nanoparticles were

conducted by TEM. The TEM images such as those

presented in Figure 2 indicate that nanoparticles are


450 Iranian Polymer Journal / Volume 20 Number 5 (2011)

Sample Mean particle

size (nm)

PDI Entrapment

efficiency (%)

I

II

III

IV

V

VI

VII

VIII

IX

X

XI

XII

396

116

459

615

255

459

295

255

190

459

220

122

0.162

0.572

0.188

0.179

0.201

0.226

0.075

0.123

0.631

0.274

0.261

0.523

70.54

71.55

75.14

60.97

59.48

55.4

84.55

75.21

71.12

80.74

69.16

68.56

(a) (b)

Figure 2. TEM Photographs of samples: (a) IV and (b) VI.
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not spherical in shape. The size of nanoparticles

based on TEM was smaller than the size determined

by DLS. This can be attributed to the dehydration of

the nanoparticles in the process of sample preparation

required for TEM. It is worth noting that DLS givesthe size of swollen particles. Swelling ratio of some

selected samples of the nanoparticles was measured

according to the method proposed by Denkbas et al.

[45].

About 0.050 g of the nanoparticles was inserted

into a tube with a diameter of 5 mm with a height of

100 mm. The level of the beads in the tube was

marked before filling it with 1 mL of distilled water

and the tube was left for 24 h. After 24 h, the height

of the beads in the solution was measured. Thepercentage of swelling was calculated based on the

following equation:

(3)

where S is the percentage of swelling, ht is the height

of swollen beads (cm) at specific time t and ho is the

initial height of the beads (cm).

The obtained results for samples IV, V and VI

based on three independent measurements for eachsample were 119.3% 20.3, 223% 67 and 146.3%

18.8, respectively.

Effect of Alginate Concentration on

Physicochemical Properties of Nanoparticles

As can be seen in Table 2, mean particle size

decreases as sodium alginate concentration increases.

By contrast, PDI of the prepared nanoparticles

increases as more sodium alginate was added to the

gelation system. In ionic gelation process, TPP as a

cross-linking agent forms hydrogen bond with free

amine groups of BSA and chitosan. Sodium alginate

competes with TPP to form electrostatic complex

with chitosan and BSA. While smaller mean particle

size can be attributed to strong inter-chain reactions

between chitosan and alginate, as more sodium

alginate causes local aggregation and increases PDI

[30]. In other words, the reduction in particle size

determined by DLS at swollen state in presence of

calcium alginate in the network is due to higher

degree of cross-linking of the whole network. Higher

concentration of sodium alginate results in reduced

entrapment efficiency (Table 2). This may be due to

competition of anionic alginate and BSA to form

interchain bonds with cationic chitosan.

BSA Loading in Relation to the Physicochemical

Properties of Nanoparticles

The general pattern which can be deduced from the

experimental data given in Table 2 is that, lowering

theoretical loading of BSA leads to formation of

smaller nanoparticles. BSA loading of nanoparticles

is based on two different phenomena such as

entrapment into particles and adsorption on the

particle surface. As mentioned earlier, BSA

molecules are negatively charged under the solution

conditions used in this study. BSA entrapment intoparticles is due to interactions between oppositely

charged chitosan and BSA, and formation of

hydrogen bonds between the TPP and BSA.

Additional adsorption of BSA on particle surface may

also occur [14] that leads to higher mean particle size.

PDI of the prepared nanoparticles increases with

theoretical BSA of higher loading. Also, the increase

of the particle size and PDI by increased BSA can be

attributed to the increased viscosity of the chitosan/

BSA solution and ionic interaction, respectively. Fewdata from Table 2 regarding the effect of BSA loading

on entrapment efficiency of nanoparticles are

presented in Figure 3. Sample preparation for

samples I and IV were the same except for the

theoretical BSA loading that was higher for sample

IV compared to sample I. The same sample prepara-

tion was applied to sample pair VIII and XI. It can be

Figure 3. Effect of theoretical BSA loading on entrapment

efficiency.



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ot

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concluded from Figure 3 that higher theoretical BSA

loading results in lowering entrapment efficiency. It is

worth noting that data from Table 2 other than those

presented in Figure 3 also support this conclusion.

The decrease of entrapment efficiency with initiallyincreased drug content is due to increased chemical

potential of the drug in chitosan/BSA solution. This

conclusion is in agreement with reported studies such

as the one carried out by Xu et al. [46] and it is in con-

trast to other research reports attesting the opposite

effect of drug loading on entrapment efficiency [14].

DDA in Relation to the Physicochemical Properties

of Nanoparticles

The effects of degree of deacetylation on particle sizeand entrapment efficiency of BSA were investigated

and the obtained experimental data are presented in

Table 2. The data show that samples which were

prepared with higher DDA chitosan (purified

chitosan, DDA 96.4%) have smaller mean particle

size than those prepared with unpurified chitosan

(DDA 83.5%).

In this study, purification process was conducted

by NaOH. It is anticipated that further deacetylation

of chitosan molecules may have occurred under

strong caustic condition. This means that more amine

groups are presented in purified chitosan of high

DDA, which can be transformed into -NH3+ at acidic

solution. The ionic gelation process is dependent on

the number of positively charged groups (-NH3+) left

on the dissolved chitosan molecules. Due to sufficient

positively charged groups, the process of nano-

particles production occurs more easily for chitosan

with high DDA leading to smaller particles [47].

Entrapment of negatively charged BSA molecules is

also occurring more efficiently in the presence of

positively charged amine groups. Sample preparation

for samples I and VII was the same except for the

DDA that was higher for sample VII compared to

sample I. The same sample preparation is applicable

to sample pairs II-VIII, III-IX, IV-X, V-XI and VI-

XII. The presented data in Table 2 for these pairs

reveal that entrapment efficiency of BSA increases at

higher DDA.

In Vitro Release StudyExperimental data on BSA release studies are

presented in Figure 4. The data show that both BSA

release rate and especially burst release are reduced

significantly with higher sodium alginate concentra-

tion used in preparation of nanoparticles. Sustained

drug release from the nanoparticles is important inmany fields of applications. BSA release follows a

biphasic pattern, characterized by initial burst release

followed by a period of slower release. According to

the presented data in Figure 4, fractional BSA

released for sample IV (chitosan-TPP nanoparticles

without alginate) and sample VI (reinforced chitosan-

TPP with alginate) in 30 min are 10.5% and 3.5%,

respectively. Also, these quantities in 6 h reach 14%

and 11.8% for samples IV and VI, respectively. In

addition, fractional drug released for sample IV is35.7% at 98 h while this quantity for sample VI is

26.8% at 98 h. These results prove that reinforcement

of chitosan-TPP nanoparticles with CaCl2 can

reduce both burst release and release rate of BSA.

Reinforcement of chitosan-TPP matrix with

calcium alginate may be considered the cause for

lower burst effect and slower release rate.

Modification of N-trimethyl chitosan chloride

(TMC)-TPP matrix by sodium alginate studied by

Chen et al. [30] also showed that sodium alginate

demonstrates the same result on burst effect and

release rate as calcium alginate in this study. The

amine groups in chitosan interact with TPP through

ionic bonding. The interaction between chitosan

matrix and alginate and ionotropic gelation of sodium

alginate with CaCl2 in reinforced nanoparticles may

Figure 4. Effect of sodium alginate concentration on BSA

release profile from samples: () IV, () VI and (S) XIII.Release medium: T = 37C0.2, pH 7.4.


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also contribute to enhanced cross-linking density of

the matrix. Increased cross-linking density may lead

to lower diffusion of BSA from the reinforced matrix

which consequently decreases the burst release and

rate of release. To investigate the effect of sodiumalginate on drug release profile, without its

conversion to calcium alginate network, a new sample

designated as XIII, was prepared. Preparation of

samples XIII and VI were the same except that CaCl2was not used in preparation of sample XIII. The

release profile of this sample is shown in Figure 4. As

can be seen, the burst release and release rate of BSA

is lower in sample VI compared to sample XIII.

Fractional BSA release profiles for two theoretical

BSA loadings, 20% and 50% corresponding toloading capacity of 4.13% 0.04 and 9.75% 0.32,

are presented in Figure 5. Release profiles show that

when loading capacity of BSA increases from 4.13%

0.04 to 9.75% 0.32, total BSA release at 6 h

increases from 7.5% to 15.8%. The nanoparticles

prepared with higher loading capacity of BSA have

greater overall release. At higher BSA loading, BSA

binding on chitosan molecule is weaker, and some of

the loaded BSA molecules are adsorbed onto the

surface of particles. Weak binding and adsorption of

BSA on the particle surfaces leads to higher release

rate. This result is in agreement with reports by other

researchers [14].

Effect of DDA on the release profile is shown in

Figure 6. The results show that BSA release rate and

burst release increase with higher DDA. As it was

Figure 5. Effect of theoretical BSA loading on BSA release

profile from samples: () IX and () XII. Release medium:T = 37C0.2, pH 7.4.

Figure 6. Effect of chitosan degree of deacetylation on BSA

release profile from samples: () VI and () XII. Release

medium: T = 37C0.2, pH 7.4.

stated above, higher DDA causes significant

reduction in mean particle size, thus increasing the

ratio of surface area to volume of nanoparticles, that

consequently increases the release rate and burst

release.

CONCLUSION

Reinforced chitosan-TPP nanoparticles loaded with

BSA were prepared by ionic gelation. Calcium

alginate was used as a reinforcing agent. Size of the

prepared nanoparticles ranged from 116 to 615 nm

with PDI lower than 0.3. TEM Analysis demonstrated

that nanoparticles were not spherical. At higher

sodium alginate concentration smaller mean particle

size was obtained. High concentration of sodium

alginate solution resulted in lower entrapment

efficiency. Higher degree of deacetylated chitosan

lowered particle size and improved the entrapment

efficiency. This study showed that reinforcement of

chitosan-TPP nanoparticles with calcium alginate sig-

nificantly lowered the burst release and consequently

the BSA release rate. Increased theoretical loading of

BSA and degree of deacetylation of chitosan led to

increased burst release and release rate.

ACKNOWLEDGEMENT

The authors are grateful to the financial supports from


http://www.sid.ir/


10/12

the Vice-Chancellery for Research and Technology

Affairs of University of Sistan and Baluchestan.

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