Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles.

Abstract

Chitosan nanoparticles (CS NP) with various formations were produced based on ionic gelation process of tripolyphosphate (TPP) and chitosan. They were examined with diameter 20-200 nm and spherical shape using TEM. FTIR confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles. Factors affecting delivery properties of bovine serum albumin (BSA) as model protein have been tested, they included molecular weight (Mw) and deacetylation degree (DD) of chitosan, the concentration of chitosan and initial BSA, and the presence of polyethylene glycol (PEG) in encapsulation medium. Increasing Mws of chitosan from 10 to 210 kDa, BSA encapsulation efficiency was enhanced about two times, BSA total release in PBS (phosphate buffer saline) pH 7.4 in 8 days was reduced from 73.9 to 17.6%. Increasing DD from 75.5 to 92% promoted slightly the encapsulation efficiency and decelerated the release rate. The encapsulation efficiency was highly decreased by increase of initial BSA and chitosan concentration; higher loading capacity of BSA speeded the BSA release from the nanoparticles. Adding PEG hindered the BSA encapsulation and accelerated the release rate.

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Effect of molecular structure of chitosan on protein delivery
properties of chitosan nanoparticles
Yongmei Xu, Yumin Du?
Department of Environmental Science, Wuhan University, Wuhan 430072, PR China
Received 8 June 2002; received in revised form 15 September 2002; accepted 30 September 2002
Abstract
Chitosan nanoparticles (CS NP) with various formations were produced based on ionic gelation process of
tripolyphosphate (TPP) and chitosan. They were examined with diameter 20?/200 nm and spherical shape using TEM.
FTIR confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles.
Factors affecting delivery properties of bovine serum albumin (BSA) as model protein have been tested, they included
molecular weight (Mw) and deacetylation degree (DD) of chitosan, the concentration of chitosan and initial BSA, and
the presence of polyethylene glycol (PEG) in encapsulation medium. Increasing Mws of chitosan from 10 to 210 kDa,
BSA encapsulation efficiency was enhanced about two times, BSA total release in PBS (phosphate buffer saline) pH 7.4
in 8 days was reduced from 73.9 to 17.6%. Increasing DD from 75.5 to 92% promoted slightly the encapsulation
efficiency and decelerated the release rate. The encapsulation efficiency was highly decreased by increase of initial BSA
and chitosan concentration; higher loading capacity of BSA speeded the BSA release from the nanoparticles. Adding
PEG hindered the BSA encapsulation and accelerated the release rate.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Chitosan; Bovine serum albumin (BSA); Nanoparticles
1. Introduction
The hydrophilic nanoparticles have received
much attention to deliver therapeutic peptide,
protein, antigen, oligonucleotide and gene by
intravenous, oral, and mucosal administration
(Janes et al., 2001a). The information has empha-
sized the importance of size, and revealed the
advantages of nanoparticles over the microspheres
(Meclean et al., 1998), it has been observed that
the number of nanoparticles that cross the epithe-
lium is greater than the number of microspheres.
Chitosan is a biodegradable and bioadhesive
polysaccharide. It has been shown that chitosan
is non-toxic and soft tissue compatible in a range
of toxicity tests (Aspden et al., 1997). It has been
widely used in pharmaceutical research and in
industry as a carrier for drug delivery and as
biomedical material (Mao et al., 2001). Chitosan
was selected for nanoparticles because of its
recognized mucoadhesivity and ability to enhance
? Corresponding author. Tel.: ? /86-27-8768-6402; fax: ? /86-
27-8768-6402
E-mail address: duyumin@whu.edu.cn (Y. Du).
International Journal of Pharmaceutics 250 (2003) 215?/226
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the penetration of large molecules across mucosal
surface (Illum, 1998).
More recently, researches have attempted to
study chitosan nanoparticles as follows: prepara-
tion, modification, properties of loading various
drugs and their physiological characters, such as
chitosan nanoparticles coated PLGA (Vila et al.,
2002) and PEO-PPO (Calvo et al., 1997), nano-
particles loaded insulin (Fernandez-Urrusuno et
al., 1999), DNA (Mao et al., 2001) and anticancer
drug doxorubicin (Janes et al., 2001b). But some
important factors affecting drug properties have
not always been investigated, such as basic mole-
cular parameters of chitosan, molecular weight
(Mw) and deacetylation degree (DD), were seldom
evaluated in protein delivery system of nanoparti-
cles. Janes stated the Mws of chitosan might have
a role in the protein or peptide release in the review
about chitosan nanoparticles as delivery systems
for macromolecules (Janes et al., 2001a), but the
relative paper has not been reported. The intro-
duction of a second ingredient may increase their
versatility in terms of the encapsulation and
delivery of proteins and their susceptibility to
interact with biological surface. Polyethylene gly-
col (PEG) coated nanoparticles have been found
to be important potential therapeutic application
for controlled release of drugs and site-specific
drug delivery (Quellec et al., 1998). Few studies
have attempted to investigate chitosan nanoparti-
cles coated with PEG. Therefore, we investigate a
series of factors affecting delivery properties, the
influence of Mw and DD of chitosan, concentra-
tion of chitosan and initial bovine serum albumin
(BSA), and PEG introduction are all evaluated.
The purpose of the current study was to
examine the influence of a number of factors on
the encapsulation of a model protein BSA and
release properties during incubation in phosphate
buffer saline (PBS) of pH 7.4, and investigate the
physicochemical structure of nanoparticles by
FTIR and TEM. A series of chitosan nanoparti-
cles with various molecular parameters were pre-
pared, effect of Mw and DD of chitosan on
protein delivery was studied systematically. Effect
of initial BSA and chitosan concentration and
PEG presence was also evaluated. Thus, we can
modulate their encapsulation capability and re-
lease rate by adjusting the molecular and forma-
tion parameters.
2. Material and method
2.1. Material
Chitosan (CS) from a shrimp shell was pur-
chased from Yuhuan Ocean Biochemical Co.
(Zhejiang, China), DD was 92%, and Mw was
210 kDa. Chitosan of DD 92% with different Mws
(115, 80, 48, 10 kDa) and chitosan of Mw 210 kDa
with different DDs (75.2, 85.5, 92%) were prepared
according the reference (Qin et al., 2002; George
and Frances, 2001). The Mws were measured by a
gel permeation chromatography (GPC), the DDs
were determined by elemental analysis. BSA with
Mw 68 kDa, PEG with Mw 20 kDa, and
Coomassie brilliant blue G250 were purchased
from Sigma Chemical Co. (USA). All other
chemicals were of reagent grade.
2.2. Preparation of chitosan nanoparticles and BSA
loaded nanoparticles
Chitosan with various Mw and DD was dis-
solved in acetic aqueous solution at different
concentration. The concentration of acetic acid
in aqueous solution was, in all case, 0.5 times
higher than that of chitosan. Then, tripolypho-
sphate (TPP) was dissolved in distilled water at 0.7
or 1.0 mg/ml. Finally, 2 ml of TPP solution was
added to 5 ml of the chitosan solution, opalescent
suspension was formed spontaneously under mag-
netic stirring at room temperature, and was further
examined as nanoparticles.
In some preparations PEG of 10 mg/ml was
dissolved in the chitosan solution before adding
TPP solution. The BSA loading nanoparticles
were formed upon incorporation of TPP solution
to chitosan solution containing various concentra-
tion of BSA. The detailed formation conditions
were shown in the corresponding legends and
figures.
The factors tested included BSA initial concen-
tration (0.1, 0.2, 1, 1.5, 2 mg/ml), Mws of chitosan
(210, 115, 80, 48, 10 kDa), DDs of chitosan (75.2,
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85.5, 92%), and chitosan concentration (1, 1.5, 2,
2.5, 3 mg/ml). When we evaluated one factor, we
only altered its own parameters, and kept the
parameters of other factors constant. Without
specific depiction, the parameters of chitosan
were as follows: concentration 2 mg/ml, DD 92%
and Mw 210 kDa; initial concentration of BSA
was 1.5 mg/ml; TPP concentration was 1 mg/ml.
2.3. Morphology and structure characterization of
nanoparticles
The morphology and particles size measure-
ments of the nanoparticles were performed by
TEM-100CXII. Chitosan nanoparticles separated
from suspension were dried by a freeze dryer, their
FTIR were taken with KBr pellets on Perkin?/
Elmer Spectrum one FTIR.
2.4. Determination of BSA encapsulation efficiency
of nanoparticles
Encapsulation efficiency and loading capacity of
nanoparticles with the different formation were
determined by ultra-centrifugation of samples at
20,000?/g and 15 8C for 30 min, the amount of
free BSA was determined in clear supernatant by
UV spectrophotometry at 280 nm using super-
natant of non-loaded nanoparticles as basic cor-
rection. The BSA loading capacity (LC) of
nanoparticles and the BSA encapsulation effi-
ciency (AE) of the process were calculated from
Eqs. (1) and (2) indicated below:
LC?(A?B)=C?100
AE?(A?B)=A?100
(1)
(2)
A, total amount of BSA; B, free amount of BSA;
C, nanoparticles weight.
2.5. BSA release from the nanoparticles in vitro
The in vitro BSA release profiles of chitosan
nanoparticles were determined as follows. The
BSA loaded chitosan nanoparticles separated
from 18 ml suspension were placed into test tubes
with 6 ml of 0.2 mol/l PBS of pH 7.4, and
incubated at 37 8C under stirring. At appropriate
intervals samples were ultra-centrifuged, and 4 ml
of the supernatant were replaced by fresh medium.
The amount of BSA released from the nanoparti-
cles was evaluated by modified Coomassie Brilli-
ant Blue protein assay (Pierce, Inc, New York,
NY, USA). The calibration curve was made using
non-loaded BSA nanoparticles as correction.
3. Results and discussion
3.1. Physicochemical characterizations of
nanoparticles
TEM of the nanoparticles and their surface
morphology are shown in Fig. 1, they are about
20 nm in size and spherical in shape. Compared
with chitosan nanoparticles, the surfaces of nano-
particles contained PEG exhibits fluffy. It has been
previously reported that the incorporation of PEG
in gel system is through intermolecular hydrogen
bonding between the electro-positive amino hydro-
gen of chitosan and the electro negative oxygen
atom of PEG, thus forming a CS/PEG semi-
interpenetration network (Kim and Lee, 1995).
So we suppose that PEG is attached to the
nanoparticles surface.
FTIR spectra of chitosan nanoparticles and
chitosan matrix are shown in Fig. 2. A band at
3434/cm has been previously attributed to ?/NH2
and ?/OH group stretching vibration in chitosan
matrix (Yu et al., 1999). In chitosan nanoparticles
a shift from 3434 to 3399/cm is shown, and the
peak of 3399/cm becomes wider, this indicates
hydrogen bonding is enhanced (Yu et al., 1999). In
nanoparticles the shoulder peak of 1644/cm dis-
appears and a new sharp peak 1630/cm appears,
and the 1602/cm peak of ?/NH2bending vibration
shifts to 1534/cm. Knaul observed the similar
result in the study of chitosan film treated with
phosphate (NaH2PO4), and attributed it to the
linkage between phosphoric and ammonium ion
(Knaul et al., 1999). So we suppose that the
tripolyphosphoric groups of TPP are linked with
ammonium group of chitosan, the inter and
intramolecular action are enhanced in chitosan
nanoparticles.
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3.2. Encapsulation of BSA within nanoparticles
3.2.1. Effect of BSA concentration and PEG
introduction
BSA encapsulation efficiency from 10 to 90%
was significantly affected by the initial BSA
concentration in Fig. 3, the lower the concentra-
tion, the higher the encapsulation efficiency.
However, the protein loading was enhanced dra-
matically from 26 to 47% by increasing the initial
BSA concentration from 0.2 to 2 mg/ml in Fig. 4.
On the other hand, protein encapsulation effi-
ciency of chitosan nanoparticles was decreased
with the addition of PEG in chitosan solution,
their loading capacity was also decreased. As
reported previously (Kim and Lee, 1995), inter
molecular hydrogen bonding can be formed be-
tween the electro negative oxygen atom of PEG
and the amino groups of chitosan in gel system.
The acid group of BSA and oxygen atom of PEG
may compete in their interaction with chitosan
amino groups, so the possibilities of an interaction
between the BSA and the chitosan are reduced, the
entanglement of PEG chains with the chitosan
molecules hinders the encapsulation of BSA into
the nanoparticles.
3.2.2. Effect of chitosan concentration
When TPP concentration was 1 mg/ml, decreas-
ing chitosan concentration down to 0.5 mg/ml,
Fig. 1. TEM of CS (a) and CS/PEG (b) nanoparticles (Mw?/48 kDa, CS 1.5 mg/ml, TPP 0.7 mg/ml).
Fig. 2. FTIR of chitosan nanoparticles (1) and chitosan (2).
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aggregates with large diameter were formed;
increasing chitosan concentration up to 4 mg/ml
made encapsulation extremely difficult. Formation
of nanoparticles is only possible for some specific
concentration of chitosan and TPP. As for gela-
tion between TPP solution of 1 mg/ml and
chitosan solution of 1?/3 mg/ml, we usually
observed that some opalescent suspension was
Fig. 3. The influence of BSA initial concentration on encapsulation efficiency (CS 1.5 mg/ml, TPP 0.7 mg/ml, n?/4).
Fig. 4. The influence of BSA initial concentration on loading capacity (CS 1.5 mg/ml, TPP 0.7 mg/ml, n?/4).
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