Research Article

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Optimization of physicochemical parameters

influencing the fabrication of protein-loaded
chitosan nanoparticles
Aim: In the development of controlled-release protein therapeutics, the high encapsulation of proteins
into biodegradable nanoparticles with uniform size in an anhydrous process along with an excellent
redispersion is of practical interest. The objective of this work was to study the physicochemical and
in vitro release properties of chitosan nanoparticles with different molecular weights (low, medium and
high) using bovine serum albumin (BSA) as a model protein for developing nanoparticle formulations
that were stable and reproducible after lyophilization. Materials & methods: The BSA-loaded chitosan
nanoparticles were prepared by an ionic gelation method using pentasodium tripolyphosphate as the
polyanions. The physicochemical properties and in  vitro release kinetics of the nanoparticles were
evaluated along with Fourier transform infrared spectroscopy studies. Furthermore, the nanoparticles
were freeze-dried for long-term stability in the formulation. To optimize the size of the freeze-dried
nanoparticles after redispersion, various types of lyoprotectants (natural and synthetic) were tested in
varying concentration in the process of lyophilization. Results: The dynamic light scattering measurements
revealed the increase in size of chitosan nanoparticles with the increase in molecular weight of chitosan
with no significant change, irrespective of the concentration of BSA entrapped. In addition, the
entrapment efficiency of the nanoparticles increased with the increasing molecular weight of chitosan
and BSA concentration. By contrast, the redispersity of the freeze-dried samples resulted in further
increase of the mean diameter of the nanoparticles. Conclusion: Among the various types of
lyoprotectants (natural and synthetic) examined, sucrose proved to be very effective in reducing the
size of freeze-dried nanoparticles on redispersion without significant change in surface charge of
nanoparticles. Finally, the in  vitro release kinetics of BSA from nanoparticles of different molecular
weights of chitosan, with and without sucrose, was evaluated and found to depend upon the molecular
weight of chitosan.

KEYWORDS: chitosan n freeze-drying n lyoprotectants n nanoparticles Mallaredy Vandana

n redispersibility n sucrose
The development of recombinant therapeu-
tic proteins and peptides has enhanced tech-
nological advancements for the successful
form­u lation of proteins as therapeutic agents.
However, various problems such as short cir-
culating half-life of peptides, immunogenecity,
extensive hydrolysis of proteins and peptides
in the gastrointestinal tract, proteolytic attack
by several endo- and exo-peptidases and rapid
elimination of peptides and/or proteins with
molecular weights (MW) of less than 30 kDa
through processes such as renal ultrafiltration
mask the therapeutic potential of the peptides
and proteins [1–3] . Thus, the optimal delivery
and efficiency of these therapeutic agents can
be accomplished effectively either by polyethyl-
ene glycol (PEG)ylation or nanoparticle form­
ulation as a protein delivery system that can
be administered into the body system through
various routes, such as parental, oral, ocular,
nasal and pulmonary [4] . PEGylation involves
& Sanjeeb K Sahoo†
†
Author for correspondence:
chemical conjugation of proteins/peptides to Laboratory for Nanomedicine,
PEG, leading to an increase in the shelf-life of Institute of Life Sciences,
the therapeutic proteins/peptides along with a Nalco Square,
reduction in the plasma clearance rate and pre- Chandrasekharpur,
vention from immunogenicity [5,6] . However, Bhubaneswar, Orissa, India
some problems still persist in PEGylation, such Tel.: +91 674 230 2094;
as increased polydispersity in high-MW PEG Fax: +91 674 230 0728;
conjugates, difficulty in removal of unreacted sanjeebsahoo2005@gmail.com
PEG from low-MW PEG conjugates and unde-
sirable interactions of PEG with active sites of
the proteins, which reduces the bioactivity of
these therapeutic agents [7,8] . Owing to these
difficulties, uses of polymeric nanoparticle
formulation for protein/peptide encapsula-
tion have been most extensively investigated
[9,10] . The protein-loaded nanoparticles, which
possess nano-size and a polymeric nature,
have efficient transport, sustained release and
protection from gastrointestinal tract deg-
radation in the body after administration.
Various polymers of synthetic nature, such as

10.2217/NNM.09.54 © 2009 Future Medicine Ltd Nanomedicine (2009) 4(7), 773–785 ISSN 1743-5889 773
Research Article Vandana & Sahoo
poly(d,l‑lactide-co-glycolide), poly(d,l‑lactic
acid), polyethylene oxide, and natural ori-
gin, such as chitosan and gelatin, have been
approved by the FDA for the preparation of
nanoparticles. In the midst of the range of
polymers available, natural polysaccharides
such as chitosan are the most suitable for pro-
tein delivery since they avoid the use of harm-
ful organic solvents and high shear stress in the
fabrication of the nanoparticles [11–13] .
Chitosan (b[1→4] 2‑amino 2‑deoxy a-d-
glucan) is a modified natural cationic carbo­
hydrate polymer prepared by the partial
N‑deacetylation of chitin, a natural copolymer
of glucosamine and N‑acetyl glucosamine,
which is derived from crustacean shells such as
crabs, shrimps and lobsters [14,15] . Chitosan can
be degraded into glucosamine by general lyso-
zymes in the body and subsequently excreted as
carbon dioxide via the glycoprotein synthetic
pathway [16] . The percentage of glucosamine
units in the chitosan is known as its degree
of deacetylation and N‑deacetylated chitosan
has a high density of amine groups, permitting
strong electrostatic interactions with proteins
that carry an overall negative charge at neu-
tral pH conditions [17] . Furthermore, positively
charged chitosan nanoparticles have an added
advantage of electrostatically interacting with
the mucin on the mucosal surface and hence
improving its bioavailability [18] . As a result,
chitosan has been recognized as a promising
natural polymer for protein and peptide thera-
peutics that can be administered either orally
or parentally.
There are several methods employed in the
preparation of the chitosan nanoparticles: ionic
gelation, desolvation, ionic complexion and
microemulsions. The preparation of chitosan
nanoparticles using ionic gelation in mild con-
ditions by reversible electrostatic interactions
between positively charged chitosan chains
and polyanions employed as cross-linkers,
mainly pentasodium tripolyphosphate (TPP),
is the most widely accepted method [4,11,19,20] .
Various studies have shown that the physico-
chemical properties of the nanoparticles not
only affect the absorption and release processes
but also govern the interaction of the particles
with different biological compounds (proteins
or membranes) of the tissue where they are
introduced into the biological environment
[21] . Nonetheless, some critical factors, such as
stability of the protein in the nanoparticulate
system during long-term storage and redis-
persion of the nanoparticles in the solutions
774 Nanomedicine (2009) 4(7)
during administration, direct the potential
efficacy of the drug-loaded nanoparticles in
industrial aspects. Thus, a stable formulation
can be designed to avoid the loss of biological
activity and provide enhanced redispersion of
nanoparticles with high entrapment efficiency
and improved features of size for administra-
tion as a drug through parental, oral, ocular,
nasal and/or pulmonary routes [4] . Freeze-
drying or lyophilization with the choice of
an appropriate lyoprotectant is an ideal way
to retain the long-term stability and better
redispersion of nanoparticles developed on an
industrial scale.
Therefore, the objectives of this work have
been to develop nanoparticle formulations that
can optimize the physicochemical properties of
protein-loaded different-MW chitosan nanopar-
ticles, which were stable and reproducible using
various lyoprotectants. Also, to confirm the
above, studies were done on the protein-release
behavior of the chitosan nanoparticles having
different MWs with and without the presence
of the lyoprotectants.
Materials & methods
„„ Materials
Chitosan with three different MWs were
obtained from Sigma-Aldrich (Munich,
Germany) with a deacetylation degree of
80–85% as reported elsewhere [22] . TPP, bovine
serum albumin (BSA) and PEG with different
MWs of 8000  Da and 10000  Da were pur-
chased from Sigma-Aldrich. PEG with MWs
2000 Da and 6000 Da along with D + ‑sucrose
was availed from Fluka (Germany). D + ‑glucose,
D + ‑mannose and D + ‑mannitol were obtained
from Hi-Media (India). Micro Bicinchoninic
Acid [BCA] Protein Assay Reagent was pur-
chased from Thermo Fisher Scientific, Inc.
(USA). The rest of the chemicals and reagents
used were from Sigma-Aldrich.
„„ Preparation of BSA-loaded
chitosan nanoparticles
Bovine serum albumin-loaded chitosan
nanoparticles were prepared according to the
method based on the ionic gelation of chito-
san and TPP, with the concentration adjusted
to get a chitosan/TPP ratio of 3:1 [23] . Briefly,
17.5 mg of chito­san was dissolved in 10 ml of
1% (v/v) acetic acid and the pH of the solution
was adjusted to 5.0. Various concentrations of
BSA (0.5, 1.0, 1.5 and 2.0 mg/ml) were dis-
solved in 1 ml of 0.1 N NaOH, and later mixed
with 1  ml of aqueous TPP solution with a
future science group
 Fabrication of protein-loaded chitosan nanoparticles Research Article
concentration of 5.82 mg/ml and incubated for „„ Fourier transform infrared
25 s. Thereafter, the incubated BSA–TPP solu- spectroscopy analysis
tion was added drop-wise into chitosan solu- Fourier transform infrared spectroscopy (FTIR;
tion under constant magnetic stirring at room Perkin Elmer [MA, USA], FTIR Spectrometer,
temperature. The colloidal solution was kept SPECTRUM RX I) was used to characterize
for overnight stirring. Later, the colloidal sus- the surface composition of the different form­
pension of the BSA-loaded chitosan nanopar- ulations of chitosan nanoparticles. The spec-
ticles obtained from overnight stirring was tra were detected in KBr disks over a range of
centrifuged using SIGMA 3K30 (Germany) at 3800–350 cm-1.
40,000 g and 10°C for 30 min. The process
of centri­f ugation was repeated three times to „„ Encapsulation efficiency
remove the unencapsulated BSA present on measurement
the surface of nanoparticles. Each time, the The encapsulation efficiency (EE) of BSA-loaded
pellets were dispersed in Milli Q water and nanoparticles was determined through an indi-
the supernatant was pooled and kept aside for rect method by using the supernatant obtained
entrapment-efficiency measurement. Finally, during the BSA-loaded nanoparticle preparation,
the pellets were frozen at -80°C and then as mentioned above. The supernatant was ana-
lyophilized using a lyophilizer (LABCONCO lyzed by micro-BCA protein assay to determine
Corporation, USA) for 48 h at temperature of free BSA concentration with reference to the ear-
‑48°C and 0.05 mbar to get a powdered form lier protocol [24] . Briefly, 500 µl of the supernatant
of the BSA-loaded nanoparticles. The same obtained from centri­f ugation was mixed with
method was applied for different-MW chito- equal amount of phosphate buffer saline (PBS;
san nanoparticles (low, medium, and high) for 0.02 M, pH 7.4) followed by the addition of 1 ml
the encapsulation of various concentrations of of the working solution of micro-BCA assay. The
BSA (0.5, 1.0, 1.5 and 2.0 mg/ml).
CH2OH H NH3+
„„ Particle size & z‑potential analysis H
Dynamic light scattering (DLS) was used to H H OH H
measure the hydrodynamic diameter (diameter O O Chitosan
in nanometer range [d. nm]) and laser Doppler OH H H OH H
anemometry (LDA) was used to determine O
z‑potential (mV). DLS and LDA analyses were H NH3+ CH2OH
performed using a Zetasizer Nano ZS (Malvern
Instruments, Malvern, UK). The DLS mea-
surements were done with a wavelength of O
532 nm at 25°C and an angle detection of 90°. HO P O
Approximately 1  mg of the lyophilized sam-
O
ple was dissolved in 1  ml Milli  Q water and
100 µl of this solution was further diluted to HO P O
1 ml for the measurements of particle size and TPP
O
z‑potential. All measurements were performed
in triplicate. HO P O

O
„„ Morphological analysis of
chitosan nanoparticles
Morphological characteristics of the nano­ H NH3+ CH2OH
particles were examined using a high-resolu- O
tion transmission electron microscope (TEM) OH H H H
(Philips FEI, Inc., OR, USA). The lyophilized O O
OH OH
BSA-loaded chitosan nanoparticles with and O
without sucrose were analyzed under a TEM. To
CH2OH H NH3+
improve the contrast, the samples were treated
with 1% uranyl acetate solution for 10  min,
deposited on a carbon-coated film 150‑mesh Figure 1. Chitosan–pentasodium tripolyphosphate ionic gelation
copper grid, and allowed to dry before TEM interaction during nanoparticle formation.
TPP: Pentasodium tripolyphosphate.
examination. Images were taken at 100 kV.

future science group www.futuremedicine.com 775

Research Article Vandana & Sahoo

1136.43

1651.62 1242.00
1393.74

1537.96
700.21
1165.92

2925.72
3316.01 1453.64
T (%)

1579.34 1879.73 798.66

1316.19 894.16 535.43
1636.11
1540.54
2923.37
3399.67
1540.41 1516.10
1636.29 537.46

3409.83 798.31
1560.21
893.67

3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450
cm-1

50

45 1313.32

1636.54 1391.76
40
1541.19 894.55
700.23
35

30

25
T (%)

20
1076.06 700.31
15 1380.21

3414.01 1313.10
10 893.67
1540.41
5
1636.29
0
3409.33 1053.96
-10
3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600
cm-1

Figure 2. Fourier transform infrared spectroscopy spectra. (A) Bovine serum albumin (BSA; top), chitosan–pentasodium
tripolyphosphate nanoparticles (middle) and BSA-loaded chitosan nanoparticles (bottom). (B) BSA-loaded chitosan nanoparticles before
lyophilization (top) and after lyophilization (bottom).

solution was then incubated for 60 min at 60°C. The BSA EE of the nanoparticles was
The samples were later cooled and read at 562 nm calculated from the following equation:
using UV-Visible Spectrophotometer (BioTek® EE (%) = ([total amount of BSA - free amount
Instruments, Inc., USA). of BSA]/total amount of BSA) × 100.

776 Nanomedicine (2009) 4(7) future science group

 Fabrication of protein-loaded chitosan nanoparticles
„„ Freeze-drying of nanoparticles &
redispersion of freeze-dried samples
For this experiment, we used chitosan nanopar-
ticles with a chitosan:TPP ratio of 3:1 and BSA
(1  mg/ml) of different MWs (low, medium
and high). We thereby studied the effects of
different lyoprotectants with different con-
centrations on the nanoparticles prepared
as mentioned above. Before the particles are
lyophilized, the centrifuged nanoparticles were
divided into two groups. One group was freeze-
dried with different synthetic lyoprotectants
such as PEG  2000, PEG  6000, PEG  8000,
and PEG 10,000 in concentrations of 20 mg/
ml. To the other group, different natural lyo-
protectants such as sucrose, glucose, mannose
and mannitol with concentrations of 20 mg/
ml were added. The samples were frozen at
‑80°C and then lyophilized using a lyophi-
lizer (LABCONCO Corporation, USA) for
48 h at temperature of ‑48°C and 0.05 mbar.
After lyophilization, approximately 1  mg of
the freeze-dried samples were redispersed in
1 ml of Milli Q water with vortexing and the
particle size was analyzed with the same dilu-
tion as mentioned earlier. According to the data
obtained, it was conferred that PEG 2000 and
sucrose gave better results among the various
synthetic and natural lyoprotectants, respec-
tively. Thus, to further assess the effect of
PEG  2000 and sucrose on BSA-loaded chi-
tosan nanoparticles, an increasing concentra-
tion of these lyoprotectants was added to the
nanoparticle solution and freeze-dried using
similar parameters. Later, these lyophilized
samples were redispersed as described above,
followed by particle-size ana­lysis.
„„ BSA release from the
nanoparticles in vitro
The BSA-loaded chitosan nanoparticles were
redispersed in PBS (0.02  M, pH  7.4) and
incubated at 37°C with constant shaking at
150 rpm in Wadegati Orbit Shaking Incubator
(Wadegati Labequip Pvt. Ltd., India). At peri-
odic time intervals, individual samples were
centrifuged using SIGMA 1–15K (Germany)
at 13,800  rpm for 10  min at 25°C and the
supernatant was removed. The exact volume
of supernatant removed was replaced by the
same amount of fresh PBS (0.02 M, pH 7.4).
The amount of the protein in the supernatant
of release medium was evaluated by micro-BCA
protein assay as described in the Materials &
Methods section. All measurements were
­performed in triplicate.
future science group
Research Article
„„ Statistical analysis
All of the experiments were performed in tripli-
cate. Data were expressed as mean values ± stan-
dard deviation and were analyzed by Student’s
t‑test; the level of significance was a p-value
below 0.05.
Results & discussion
„„ Characterization of BSA-loaded
chitosan nanoparticles
In view of our aim to study different parameters
related to protein-loaded chitosan nanoparticles,
BSA as a model protein was selected for encap-
sulation in different-MW chitosan nanoparticles
in this experiment. The chitosan nanoparticles
were prepared in mild conditions by the ionic
gelation method [23,24] . TPP in the aqueous solu-
tion is ionized, which results in its dissociation
to OH - and TPP ions (HP3O104- and P3O105‑).
Chitosan, being a weak polybase upon dissolv-
ing in aqueous acidic solution, forms cations that
have –NH3 + groups at the end. The gelation pro-
cess occurs by the addition of an alkaline phase
(pH 5) to form inter- and intramolecular cross-
linkage between TPP phosphates and chitosan
–NH3 + group [25–30] (Figure 1) . In this study FTIR
was used as a tool to analyze the physical stability
and the possible interaction of BSA with chito-
san–TPP nanoparticles. Figure 2A (top) shows the
FTIR spectra of BSA with characteristic peaks
of BSA for Acetylamino  I at 1651  cm-1, II at
1537 cm-1 and III at 1242 cm-1 with 3316 cm-1
for stretching vibration of (–NH2) [31] . In the
FTIR spectra of chitosan nanoparticles shown
in Figure  2A (middle), the carbonyl-stretching
600 ‡
500
Size of NP (d.nm)
*
400
300
200
100
0
LMW MMW HMW
chitosan chitosan chitosan
Figure 3. Comparison of the size of
different-molecular-weight chitosan
nanoparticles. Data presented are
mean ± standard deviation (n = 3).
*p < 0.05 MMW chitosan versus LMW chitosan.
‡
p < 0.005 HMW chitosan versus LMW chitosan.
HMW: High molecular weight; LMW: Low
molecular weight; MMW: Medium molecular
weight; NP: Nanoparticle.
www.futuremedicine.com 777
Research Article Vandana & Sahoo

Table 1. z‑potential (in mV) of the spectral band at 3300–3500 cm-1 indicates

different-molecular-weight
chitosan nanoparticles.
Type of NPs Without BSA With BSA
LMW chitosan 42.9 ± 6.6 39.4 ± 5.1
MMW chitosan 46.2 ± 5.8 44.2 ± 2.6
HMW chitosan 43.6 ± 5.5 40.6 ± 4.0
Data presented are mean ± standard deviation (n = 3).
BSA: Bovine serum albumin; HMW: High molecular
weight; LMW: Low molecular weight; MMW: Medium
molecular weight; NP: Nanoparticle.
(amide  I band) at 1636  cm-1, amine-bending
(amide  II band) at 1540  cm-1 and the broad
band ascribed to stretching vibration of –NH2
and –OH group at 3300–3500  cm-1 can be
observed. The results indicate the cross-linkage
between TPP ions and the –NH3 + groups of chi-
tosan along with hydrogen bonding due to inter-
action of chitosan and TPP [32–35] . The bands at
1000–1200 cm-1 are attributed to the symmetric
stretch of C–O–C of the saccharide structure
of chitosan [36] .However, in the FTIR spectra of
BSA-chitosan nanoparticles as shown in Figure 2A
(bottom) no band-shift could be detected. But
some bands of chitosan nanoparticles overlapped
with those of BSA, which resulted in more inten-
sive peaks of the carbonyl (1636 cm-1) and amine
bands (1540 cm-1). In addition, the widening of
120
100 ‡
‡
‡ for getting similar results in the z‑potential of
* our formulation  [35,38,39] . The particle size and
*
Entrapment efficiency (%)
enhanced hydrogen bonding, possibly due to the
interaction of BSA with non-cross-linked amino
groups of chitosan [35] . Also, the FTIR spectra
shown in Figure 2B (top and bottom) reveals that
BSA maintains its stability even after its incor-
poration into the chitosan nanoparticles due to
the presence of acetylamino I and II bands – that
is, carbonyl (C=O–NHR) and amine-group
(–NH2) bands at 1636 cm-1 and 1540 cm-1 peaks,
respectively, with the same intensity before and
after lyophilization.
The BSA-loaded chitosan nanoparticles thus
obtained using different-MW chitosan exhibited
an increasing size with similar z‑potential. The
data obtained by DLS measurements showed
the formation of these nanoparticles in the
range of 300–350 nm for the low-MW chitosan,
400–450 nm for medium-MW chitosan and
550–600 nm for high-MW chitosan as shown
in Figure 3. As the MW of chitosan increases, the
particle size of chitosan nanoparticles become
larger, possibly attributable to the increasing
chain length of chitosan [35,37] . However, the
z‑potential measurements showed that all three
different-MW chitosan nanoparticles have
more-or-less similar z‑potential in the range of
40–50 mV, as in Table 1. This can be explained
as when the degree of deacetylation is approxi-
mately the same for different-MW chitosan, the
number of functional groups on the chitosan
remains the same, which may be the reason

‡
*
* z‑potential have a crucial impact on the fate of
80
the protein-delivery system. Depending on the
particle size, the surface-to-volume ratio and
60 specific surface area varies, which directly cor-
relates to the dissolution and bioavailability of
40 the proteins [40,41] . On the other hand, stabil-
ity and bioadhesion of the particulate system
depends upon the z‑potential or surface charge
20 of the nanoparticles [42] .
Using the ionic gelation method, BSA load-
0 ing was optimized. Analysis was conducted for
0.5 1 1.5 2 the entrapment efficiency of the different-MW
BSA in NP (mg/ml) chitosan nanoparticles loaded with different
LMW MMW HMW concentrations of BSA. There was an observed
increase in the entrapment of BSA with the
increasing MW of chitosan and concentration
Figure 4. Entrapment efficiency of different-molecular-weight chitosan NPs of BSA, as shown in Figure 4. These results were
with increasing concentration of BSA. Data presented are mean ± standard
deviation (n = 3).
similar to studies by Lim et al. and Ko et al., who
*p < 0.05 MMW chitosan versus LMW chitosan. found that the increase in MW of chitosan leads
‡
p < 0.005 HMW chitosan versus LMW chitosan. to higher viscosity of the chitosan solution, form-
BSA: Bovine serum albumin; HMW: High molecular weight; LMW: Low molecular ing strong and highly cross-linked chitosan–TPP
weight; MMW: Medium molecular weight; NP: Nanoparticle. chains that can effectively entrap more of the

778 Nanomedicine (2009) 4(7) future science group

 Fabrication of protein-loaded chitosan nanoparticles
drug. As a cross-link and condensing agent, TPP
forms further hydrogen bonding with free –NH2
groups on both chitosan and protein macromol-
ecules, resulting in more compact protein–chi-
tosan nanoparticles [43,44] . Additional adsorp-
tion of protein macromolecules on the surface
of the formed particles may occur in sequence,
leading to additional protein loading on the par-
ticles [35,38] . However, the entrapment efficiency
of BSA remained more than 80% in all cases,
signifying the effectiveness of the entrapment of
proteins in the chitosan nanoparticles. Moreover,
the concentration of the protein along with the
MW of the chitosan influences the release of
therapeutic proteins, as studies have shown that
proteins cross-link with chitosan during ionic
gelation [35,45] .
„„ Influence of freeze-drying
& redispersability of BSA-loaded
chitosan nanoparticles
The protein-loaded chitosan nanoparticles syn-
thesized by an ionic gelation method degrade
with time when dissolved in aqueous media,
due to physical instability (aggregation/parti-
cle fusion/protein degradation) and/or chemi-
cal instability (hydrolysis of polymer materials
forming nanoparticles [46–48] . Such disintegra-
tions are observed due to the mild conditions
adopted during the synthesis of chitosan–TPP
nanoparticles, rendering them to behave as a
metastable system, hence restricting the neces-
sity of fresh aqueous solutions prepared only
when required. However, freshly preparing
the particles at the point of requirement is not
recommended in large-scale synthesis. To over-
come this limitation, the BSA-loaded different-
MW chitosan nanoparticles were freeze-dried
or lyophilized. For pharmaceutical aspects, the
freeze-dried nanoparticles should have the abil-
ity to be stable for a long time and reconstitute or
redisperse in solution without aggregation dur-
ing parental administration. Our studies showed
that these nanoparticles had a poor dispersion
in aqueous solution and an increased particle
size after lyophilization, as confirmed by DLS
measurements shown in Figure 5.
The most commonly used process for long-
term stability and storage is freeze-drying [49] .
Freeze-drying, also known as lyophilization, is a
technique involving the freezing of the colloidal
suspension of nanoparticles followed by elimina-
tion of their water content by sublimation under
reduced pressure. Freezing is a very aggressive
step of freeze-drying of colloidal solution [50] .
During this process, the colloidal suspension of
future science group
Research Article
700
*
600 *
*
500
Size of NP (d.nm)
400
300
200
100
0
LMW chitosan MMW chitosan HMW chitosan
Before lyophilization After lyophilization
Figure 5. Comparison of the particle size of different-molecular-weight
chitosan nanoparticles before and after lyophilization. Data presented are
mean ± standard deviation (n = 3).
*p< 0.05 different-molecular-weight chitosan NPs after lyophilization versus
before lyophilization.
HMW: High molecular weight; LMW: Low molecular weight; MMW: Medium
molecular weight; NP: Nanoparticle.
nanoparticles in a very dilute state are transferred
to a highly concentrated state with the crystal-
lization and sublimation of water, resulting in
a change of pH. �����������������������������
This highly concentrated par-
ticulate system may induce aggregation and in
some cases irreversible fusion of nanoparticles.
Furthermore, the crystallization of ice may exer-
cise a mechanical stress on nanoparticles, leading
to their destabilization [51] .
In the case of protein-encapsulated nanopar-
ticles, various studies have revealed that, as the
solute from the proteins starts to freeze, pure
water separates as ice and the proteins are likely
to undergo physical stress due to ice, air and the
salts that are excluded from ice crystals. This
leads to increased high-salt/solute concentra-
tion, which may denature the protein or induce
partial unfolding, resulting in aggregation on
redispersion [52,53] . The nanoparticles thus
obtained during the process of lyophilization
are in the form of a dry powder that is easy to
handle and store.
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Research Article Vandana & Sahoo

Table 2. Comparison of mean particle size of different-molecular- 2000 and sucrose with an increasing concentration
weight chitosan nanoparticles loaded with bovine serum albumin on the nanoparticles, both lyoprotectants were
(1 mg/ml) using synthetic lyoprotectants (20 mg/ml). added separately to BSA-loaded different-MW
chitosan nanoparticles. The results demonstrated
Type of chitosan Mean particle size (d. nm)
that the nanoparticles after lyophilization with
NP with BSA
(1 mg/ml) PEG 2000 PEG 6000 PEG 8000 PEG 10,000 PEG 2000 had inconsistent sizes with an increas-
ing concentration, while the formulation with
LMW 466.0 ± 4.7 654.0 ± 5.3 671.0 ± 2.3 825.0 ± 4.0
sucrose showed reduction in size (Figures 6A–C) .
MMW 580.0 ± 2.6 348.7 ± 4.5 460.8 ± 5.6 898.0 ± 1.5
There are variety of lyoprotectants used for
HMW 771.0 ± 6.1 758.0 ± 5.9 712.4 ± 3.5 916.3 ± 6.0
conserving the integrity of the colloidal sus-
Data presented are mean ± standard deviation (n = 3).
BSA: Bovine serum albumin; HMW: High molecular weight; LMW: Low molecular weight; pension of protein-loaded nanoparticles dur-
MMW: Medium molecular weight; NP: Nanoparticle; PEG: Polyethylene glycol. ing freeze-drying, such as carbohydrates, poly-
ols, PEGs and various water-soluble polymers.
„„ Effect of lyoprotectants on Several mechanisms such as water-replacement
nanoparticle size during theory, the single amorphous state immobiliza-
freeze-drying tion mechanism and the scavenger mechanism
To optimize fabrication of improved redispersion have been proposed to explain the mode of action
and stability of freeze-dried BSA-loaded nanopar- of the lyoprotectants during freeze-drying [54–57] .
ticles, the addition of suitable lyoprotectants is But the most widely accepted theories regard-
necessary. Experiments with various lyoprotect­ ing the action of lyoprotectants are the water-
ants of carbohydrate origin, such as D+ ‑sucrose, replacement theory and vitrification theory.
D+ ‑glucose, D+ ‑mannose, D+ ‑mannitol and PEG Water-replacement theory studies have shown
with different MWs such as PEG 2000 Da, PEG that lyoprotectants such as disaccharides serve as
6000 Da, PEG 8000 Da and PEG 10,000 Da were a ‘water substitute’ when the hydration shell of the
evaluated in the BSA-loaded chitosan nanopar- proteins and nanoparticles are removed during
ticle formulation. A particular amount of the the drying process upon lyophilization. Water is
aforementioned lyoprotectants were added to the hydrogen-bonded to the surface of the proteins
BSA-loaded chitosan nanoparticle solutions in and nanoparticles during the drying process. It
each vial to be lyophilized. The experiment dem- is replaced by the lyoprotectants that protect the
onstrated that the effect of PEG with increasing native conformation of the protein along with
MW did not exhibit consistent results for the the structural entity of the nanoparticles from
freeze-dried chitosan nanoparticles compared dehydration stress [51,56,58,59] . By contrast, the vit-
with natural lyoprotectants, which reduced the rification mechanism states that the glassy state of
size of the nanoparticles considerably, as shown the lyoprotectants maintains the individual par-
in Tables 2 & 3. The nanoparticles lyophilized with ticles in a state of low molecular mobility, thereby
PEG could not completely redissolve in water even reducing the surface tension of the particles dur-
after sonication, whereas the nanoparticles redis- ing freeze-drying, a factor responsible for the sta-
solved completely without the need for sonication bility of the nanoparticles. Among the range of
when lyophilized with natural lyoprotectants. different lyoprotectants, sucrose is considered as
However, PEG 2000 showed satisfactory results the most versatile and the best nonspecific stabi-
among the synthetic lyoprotectants and sucrose lizer. Generally, sucrose is considered a classical
showed superior results among the natural lyo- amorphous lyoprotectant and forms glassy state
protectants. To further evaluate the effect of PEG with the protein in lyophilization [58] . Vermuri
et al. have studied the effect of sucrose as a lyopro-
Table 3. Comparison of mean particle size of different-molecular- tectant in the freeze-drying of liposomes and solid
weight chitosan nanoparticles loaded with bovine serum albumin lipid nanoparticles and found that it maintains
(1 mg/ml) using natural lyoprotectants (20 mg/ml). uniformity and redispersibility by decreasing the
osmotic activity of water of crystallization and
Type of chitosan Mean particle size (nm)
NP with BSA
favors the glassy state of the lyophilized formula-
(1 mg/ml) D + ‑sucrose D + ‑glucose D + ‑mannose D + ‑mannitol tion [60] . To confirm the same, studies were done
using sucrose as a lyoprotectant in our BSA-
LMW 218.0 ± 2.3 221.0 ± 3.2 295.3 ± 1.2 470.1 ± 5.1
loaded different-MW chitosan nanoparticles.
MMW 285.0 ± 1.2 290.0 ± 5.2 294.0 ± 3.3 383.0 ± 4.8
As shown in Figure 6, there was no considerable
HMW 431.0 ± 7.0 440.0 ± 2.6 503.0 ± 8.0 577.0 ± 7.1
size difference in the formulation as measured by
Data presented are mean ± standard deviation (n = 3).
BSA: Bovine serum albumin; HMW: High molecular weight; LMW: Low molecular weight; DLS. Furthermore, the effect of sucrose on the
MMW: Medium molecular weight; NP: Nanoparticle. formulation was studied by analyzing the TEM

780 Nanomedicine (2009) 4(7) future science group

 Fabrication of protein-loaded chitosan nanoparticles Research Article
images. Figure 7 shows the TEM images indicating
800
that the size range of BSA-loaded low-MW chi- ‡ ‡

tosan nanoparticles with sucrose is around 200– 700

300 nm, similar to size of the low-MW chitosan
nanoparticles before lyophilization. The shapes of 600 ‡

Size of NP (d.nm)
the particles are approximate to spheres, smooth 500 ‡
‡

with an almost homogeneous structure for the

nanoparticles. As stated above, sucrose being 400
hydrophilic helps in dispersion without affect- 300
ing the structural entity of nanoparticles in the
aqueous medium. Conversely, PEG polymers are 200
a series of nonionic polyhydroxyl compounds that
100
are miscible with water and display colligative
properties in solution. They act as a steric stabi- 0
5 10 20 30 50
lizer by protecting the surface of the nanoparticles
Concentration of lyoprotectant (mg/ml)
during the freeze-drying process [61] .
Therefore, it can be concluded that use of
sucrose as lyoprotectant for BSA-loaded chito- 1000 ‡ ‡

san nanoparticles is very effective in controlling 900

the size of nanoparticles without affecting the 800 ‡

physicochemical characteristics after redispersion

Size of NP (d.nm)

700 ‡
of the freeze-dried samples. 600
‡

500
„„ In vitro release kinetics
In order to investigate the feasibility of chito- 400
san nanoparticles as drug carriers for therapeu- 300
tic proteins, an in vitro release study was done 200
on the different-MW chitosan nanoparticles 100
loaded with BSA. In addition, a sequential time 0
frame of TEM images of BSA-loaded chitosan 5 10 20 30 50
nanoparticles during release studies reported by Concentration of lyoprotectant (mg/ml)
Gan et al. was taken into consideration for pro- 900
viding an insight to the protein-release mecha- *
800
nism. The TEM images demonstrated that the
BSA-loaded chitosan nanoparticles maintained 700 *
their shape, size and integral structure during
Size of NP (d.nm)

600
the burst release of BSA, whereas consider- *
*
able particle swelling with loss of density and 500
*
increase in particle size after burst release of 400
protein resulted in loss of physical integrity of
the particle [22] . Also, a comparative study of 300
FTIR spectra, as shown in Figure 8 (top and bot- 200
tom) of the BSA-loaded nanoparticles before
100
and after release (from first 12 h) was carried
out. The spectra indicated the decreased inten- 0
5 10 20 30 50
sity in main characteristic bands of carbonyl Concentration of lyoprotectant (mg/ml)
(C =O–NHR) and amine group (–NH 2 ) at
1636 cm-1 and 1540 cm-1, respectively, from the Sucrose PEG 2000
BSA-loaded chitosan nanoparticles during the
release process. Figure 6. Comparison of polyethylene glycol 2000 and sucrose of different
In relation to the above-mentioned protein- concentrations. (A) Low-molecular-weight chitosan NPs loaded with bovine
serum albumin (BSA) (1 mg/ml). (B) Medium-molecular-weight chitosan NPs
release mechanism, the other parameter respon- loaded with BSA (1 mg/ml). (C) High-molecular-weight chitosan NPs loaded with
sible for the slow release of BSA from nanoparticles BSA (1 mg/ml). Data presented are mean ± standard deviation (n = 3).
is due to the isoelectric point effect of BSA in the *p < 0.05 PEG 2000 versus sucrose.
release medium, having a pH  7.4. The release ‡
p < 0.005 PEG 2000 versus sucrose.
profiles of the BSA-loaded chitosan nanoparticles NP: Nanoparticle; PEG: Polyethylene glycol.

future science group www.futuremedicine.com 781

Research Article Vandana & Sahoo

was done on the BSA-loaded chitosan nanoparti-

100 nm 100 nm
Figure 7. Transmission electron microscope images of (A) low-molecular-
weight chitosan nanoparticles loaded with bovine serum albumin
(1 mg/ml) and (B) low-molecular-weight chitosan nanoparticles
loaded with bovine serum albumin (1 mg/ml) with sucrose (20 mg/ml)
as lyoprotectant.
with different MWs were conducted at pH 7.4.
At this pH, BSA, having the isoelectric point
value  4.8, is negatively charged, and therefore
its interaction with the –NH3+ group of chitosan
is highly favored. This perhaps is also a factor
responsible for the slow release of BSA from the
chitosan nanoparticles. All of the nanoparticles
showed a small burst release in an increasing mode
with decreasing MW of chitosan (approximately
35–40% BSA in low-MW chitosan, 15–20% BSA
in medium-MW chitosan and 10–15% BSA in
high-MW chitosan nanoparticles) in the first
12 h, followed by slow release at a constant but
different rate (Figure 9A). Also, a release-profile study
cles with sucrose as a lyoprotectant during lyophi-
lization (Figure 9B) . This was done to investigate
whether the use of sucrose has any significant
effect on the release of the nanoparticles. From
the results it was revealed that the release of BSA
in the presence of sucrose as a lyoprotectant was
moderately different from BSA-loaded nanoparti-
cle without sucrose in the formulation. As depicted
in Figure 9B, the slight increase of the burst release
(approximately 1–2%) was possibly attributable to
the increased hydrophilicity of the nanoparticles
in release media due to the presence of sucrose.
As evident from the decrease in intensity of
the characteristic bands in FTIR spectra shown
in Figure 8 (top and bottom), the burst release in
the first 12 h can be attributed to rapid surface
desorption of BSA from a large surface area pro-
vided by huge numbers of nanoparticles to the
external medium mimicking the physiological
pH, here PBS (pH 7.4). Then, a slow release of
BSA from the chitosan nanoparticles as shown in
Figures 9A & B of an in vitro release study, is due to
the slow release of protein macromolecules from
the pores of the nanoparticles. The porosity in
the nanoparticle structure is due to sw­elling, with
loss of density and increase in particle size, as
apparent from the TEM studies of Gan et al. [22] .
But the release rate of proteins from the high-
MW chitosan nanoparticles was slow compared
with those of medium- and low-MW chitosan
nanoparticles, as shown in Figures 9A & B. These
results indicate that the increased cross-linking
between chitosan–TPP complexes with increas-
ing MW of chitosan may lead to the higher vis-
cosity of chitosan solution due to the formation

62
1696.48
1319.28 894.99 800.46
1541.96
50
2960.89
1381.62 1077.99 700.29
40
558.79
T (%)

30
700.28
2929.78
20
894.55 559.69
1318.82
10 3420.09 1541.19
1696.54 1381.76

0 3414.01 1076.06
-5
3800 3000 2000 1500 1000 500
cm-1

Figure 8. Fourier transform infrared spectroscopy spectra of bovine serum albumin-loaded

chitosan nanoparticles after release (in first 12 h) (top) and before release (bottom).

782 Nanomedicine (2009) 4(7) future science group

 Fabrication of protein-loaded chitosan nanoparticles Research Article
of a relatively strong matrix of nanoparticles 100
[23,44] . The nanoparticle matrix is also diffu-
90
sion-controlled and obeys the Higuchi equation

Cumulative % release of BSA

[62] . Studies confirmed that the rate of release is 80
directly proportional to the size of nanoparticles. 70
Release from smaller-sized nanoparticles is faster 60
than those from large-sized nanoparticles owing 50
to smaller diffusion path length for the drug and
40
larger surface area of contact of smaller particles
with the dissolution medium [63] . 30
20
Conclusion 10
This work shows a systematic study on the
0
physico­chemical characteristics of protein-encap- 0 1 2 3 4 5 6 7 8
sulated chitosan–TPP nanoparticles formed by Time (days)
the ionic gelation technique. The study ana- LMW MMW HMW
lyzed different parameters, such as particle size,
z‑potential, entrapment efficiency and in vitro 100
release of the protein-loaded different-MW chito- 90
Cumulative % release of BSA

san nano­particles, that proved these nanoparticles 80

to be an ideal protein/drug delivery vehicle.
By contrast, the postpreparative steps, such as
stability and redispersion of the nanoparticles, have
certain limitations that make the final product not
viable. The most common method to curb these
factors is lyophilization or freeze-drying. This pro-
cess involves various stresses during freezing and
drying. The stability and phase transitions of the
lyophilized chitosan–TPP particles are effectively
controlled by the use of lyoprotectants. Stability of
the BSA-loaded different-MW chitosan nanopar-
ticles was achieved using synthetic lyoprotectants
such as PEG 2000  Da, PEG 6000  Da, PEG
8000 Da and PEG 10,000 Da, along with those
of natural origin such as D + ‑sucrose, D+ ‑glucose,
D+ ‑mannose, D+ ‑mannitol. The BSA-loaded chi-
tosan nanoparticles were effectively stabilized by
D + -sucrose during lyophilization. Furthermore,
the TEM examination and in vitro release stud-
ies of the chitosan nanoparticles with sucrose as
lyoprotectants showed similar results as shown
by BSA-loaded chitosan nanoparticles with no
lyophilization. Essentially, a systemic fabrication
of the preparation conditions for protein encapsu-
lation with desired physicochemical properties by
using suitable lyoprotectants can make chitosan
nanoparticles a potential candidate as a delivery
carrier for therapeutic proteins and peptides for a
sustained period.
Summary
The significant advancement in recombinant bio-
technology has initiated the use of proteins and
peptides as therapeutic agents. But the challenges,
such as short half-life, immunogenecity and deg-
radation, have hampered their successful role in
70
60
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8
Time (days)
Figure 9. Release kinetics of bovine serum albumin from different-
molecular-weight chitosan nanoparticles (A) without and (B) with sucrose
(20 mg/ml) as lyoprotectant.
BSA: Bovine serum albumin; HMW: High molecular weight; LMW: Low molecular
weight; MMW: Medium molecular weight.
therapeutics. The application of polymer nano-
technology, using natural polymers such as chito-
san in the form of nanoparticles, has proved to be
potential tool for the delivery of these proteins via
a safer route – either oral or parental. The system-
atic fabrication conditions using different-MW
c­hitosan and BSA as model proteins were done by
optimizing the different parameters such as par-
ticle size, z‑potential, entrapment efficiency and
in vitro release to obtain the desired physicochemi-
cal properties for enhanced encapsulation and sus-
tained release of the protein. The use of a suitable
lyoprotectant such as sucrose further improves the
use and storage of chitosan nano­particles for the
delivery of proteins for oral or parental adminis-
tration purposes. Thus, an ideal delivery vehicle
for therapeutic proteins and peptides can be fab-
ricated by optimizing the various physicochemical
properties of chitosan nanoparticles.

future science group www.futuremedicine.com 783

Research Article Vandana & Sahoo

Financial & competing interests disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a finan‑
cial interest in or financial conflict with the subject matter
or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or
pending, or royalties.
No writing assistance was utilized in the production of
this manuscript.

Executive summary
ƒƒ Natural nanoparticles, such as chitosan, synthesized for delicate protein molecules with high encapsulation efficiency are
enormously appealing.
ƒƒ To date, most of the protein-based drug formulations are either suspensions in a ready-to-use form or lyophilized products for redispersion.
ƒƒ The fabrication of chitosan nanoparticles loaded with proteins involves no harmful organic solvents and is done under mild conditions.
ƒƒ The chitosan polymer is available in a range of different molecular weights and studies showed that low-molecular-weight chitosan was
an ideal protein-delivery device, having a desirable release profile.
ƒƒ For clinical purposes the protein-loaded chitosan nanoparticles are freeze-dried with lyoprotectants to have a stabilized formulation with
conserved physicochemical properties and good redispersability for an oral or parental administration.
ƒƒ The choice of a suitable lyoprotectant and its concentration is crucial during lyophilization to ensure maximum stability of the
nanoparticles. Thus, various synthetic and natural lyoprotectants were analyzed for BSA-loaded chitosan nanoparticles.
ƒƒ Addition of a natural lyoprotectant such as sucrose provides a desirable release profile similar to that of the chitosan nanoparticles
devoid of it.

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