International Journal of Pharmaceutics 511 (2016) 560–569

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Stable thermosensitive in situ gel-forming systems based on the

lyophilizate of chitosan/a,b-glycerophosphate salts
Guanghao Wu, Yuan Yuan, Jintian He* , Ying Li, Xiaojing Dai, Baohua Zhao*
College of Life Science, Hebei Normal University, No. 20 Road East of 2nd Ring South, Shijiazhuang City, Hebei Province 050024, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Article history:
Received 27 May 2016 In the present study, lyophilization was attempted to improve the long-term storage of CS/GP
Received in revised form 7 July 2016 thermogelling systems for biopharmaceutical applications. After lyophilization, CS/a,b-GP lyophilizate
Accepted 22 July 2016 could not be dissolved in water, but some metal salts, such as NaCl, CaCl2, and MgCl2 surprisingly
Available online 25 July 2016 facilitated its dissolution. X-ray powder diffraction analysis suggested that calcium ions might
preferentially form salts with a,b-GP, inhibit the transfer of protons from CS to a,b-GP, and then inhibit
Keywords: the aggregation of CS molecules during lyophilization. Comparison of the freshly prepared CS/a,b-GP/
Chitosan salt solutions and the reconstituted solutions from lyophilizates showed that lyophilization clearly
Glycerophosphate
influenced the properties of reconstituted CS/a,b-GP/salt solutions such as gelation time, viscosity, and
Lyophilization
pH. Furthermore, the reconstituted CS/a,b-GP/CaCl2 solutions maintained thermogelling properties and
Hydrogel
Stability formed hydrogels at 37  C within approximately 5 min, but did not form hydrogels at 20  C and 4  C over 2
weeks. The model protein bovine serum albumin (BSA) was further incorporated into the CS/a,b-GP/
CaCl2 system. In vitro release experiments showed the sustained release of BSA from CS/a,b-GP/CaCl2
hydrogels in a pH-sensitive manner, demonstrating that CS/a,b-GP/CaCl2 may be useful as an in situ gel-
forming system.
ã 2016 Elsevier B.V. All rights reserved.

1. Introduction
In situ gel-forming systems are interesting polymeric systems
that exist as flowing aqueous solutions before administration, and
undergo phase transition to form gels in a physiological environ-
ment. Among these systems, thermogelling solutions, which form
semi-solid hydrogels after administration in response to tempera-
ture increases from ambient to physiological, are of particular
interest. Such injectable in situ forming chitosan (CS)-based
hydrogels were first described by Chenite et al. (2000). CS is a
natural polysaccharide derived from partially deacetylated chitin,
and is non-toxic, biodegradable, and biocompatible (Zhou et al.,
2015; Cheung et al., 2015). Recently, chitosan-based hydrogels have
been widely developed for their intelligent delivery of biomacro-
molecules including peptides, proteins, antigens, oligonucleotides,
and genes, and have shown potential to achieve prolonged release
over days to weeks (Kim et al., 2010; Zou et al., 2016; Bhattarai
* Corresponding authors.
E-mail addresses: 576477418@qq.com (J. He), zhaobaohua86178@sohu.com
(B. Zhao).
et al., 2010; Lavertu et al., 2008; Berrada et al., 2005). Furthermore,
hydrogels are reportedly promising injectable scaffolds for cell
encapsulation and tissue engineering, especially for bone and
cartilage repair, due to their high water content and porosity of the
three dimensional matrix (Zhou et al., 2015; Tessmar and
Göpferich, 2007). Glycerophosphate (GP) is a compound that is
naturally found in the body, and is commonly used to treat
abnormalities in phosphate metabolism. To prepare CS-based
hydrogels, GP is usually used to catalyze the sol-to-gel transition of
aqueous CS solutions at physiological pH and temperature (Lavertu
et al., 2008; Filion and Buschmann, 2013). CS/GP thermogelling
systems have been developed as promising gel-forming systems
for biomedical application (Filion and Buschmann, 2013; Supper
et al., 2014a).
In light of their potential biomedical benefits, CS/GP thermog-
elling systems must be storable for extended periods. However, the
storage stability of CS-based thermogelling systems has received
little attention and only few results has been reported (Ruel-
Gariépy et al., 2000; Zhou et al., 2009). Ruel-Gariépy et al. (2000)
found that CS/GP thermogelling solutions lacked long-term
stability at room temperature. Under refrigerated conditions, CS
with a high degree of deacetylation still could not effectively

http://dx.doi.org/10.1016/j.ijpharm.2016.07.050
0378-5173/ã 2016 Elsevier B.V. All rights reserved.
 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569
decrease the gelation rate under refrigerated conditions and CS/GP
solutions gelled within 7 days at 4  C. However, CS with a low
degree of deacetylation could substantially decrease gelation rate,
and CS/GP solutions could remain in solution for at least 3 months
at 4  C. The similar result was reported by Zhou et al. (2009). To
improve the long-term stability of CS-based thermogelling
solutions, gelling agent GP was replaced with glucose-1-phosphate
(G1-P) in a study reported by Supper et al. (2014b). The results
indicated that CS/G1-P solutions can be stable for more than 9
months under refrigerated conditions. It is undoubted that
solution formulations are convenient for administration as they
can be presented as pre-filled syringe products. Moreover,
refrigerated conditions can significantly improve long-term
stability of CS-based thermogelling solutions. However, solution
formulations show obvious disadvantages when they are used to
deliver labile drugs, especially protein drugs, which are unstable in
solution state and are susceptible to denature during long term
storage. As a result, delivery of these labile drugs with CS-based
thermogelling solution formulations will be restricted.
Lyophilization is widely used for pharmaceutical products to
improve the long-term storage stability of labile protein and
peptide drugs (Remmele et al., 2012; Kasper and Friess, 2011).
Freeze-dried formulations not only have the advantage of better
stability, but also provide easy handling (shipping and storage).
Thus, in this study, lyophilization was evaluated for improving the
long-term storage of CS/GP thermogelling systems for biopharma-
ceutical applications. CS/a,b-GP lyophilizate could not be dis-
solved in water, but surprisingly, salts such as NaCl, CaCl2 and
MgCl2 could facilitate reconstitution of the lyophilizate, the
mechanism underlying the dissolution of lyophilizate by salts
was investigated. Subsequently, the effects of lyophilization on the
gelation properties of the reconstituted CS/a,b-GP/salts solution
were systematically studied. The long-term storage stability of the
CS/a,b-GP/CaCl2 system was also evaluated by examining the
appearance, pH, and gelation time of the reconstituted solutions. In
addition, bovine serum albumin (BSA) was employed as a model
protein and incorporated into the CS/a,b-GP/CaCl2 system, after
which its in vitro release from the hydrogel was systematically
investigated.
2. Materials and methods
2.1. Materials
CS with a molecular weight ranging from 8000 to 12,000 Da and
80% deacetylation, BSA, and partially hydrolyzed (87–89% hydro-
lysis) poly(vinyl) alcohol (PVA) with a molecular weight ranging
from 13,000–23,000 Da were purchased from Sigma-Aldrich (St.
Louis, MO, USA). a,b-glycerophosphate disodium salt (a,b-GP)
was obtained from Shanxi Haitai Electronic Materials Co., Ltd.
(Yuncheng, Shanxi, China). The bicinchoninic acid (BCA) protein
assay kit was purchased from Sangon Biotech Co., Ltd (Shanghai,
China). All of the other chemicals used were analytical grade or
better. Ultrapure water was obtained from a MilliQ1 Millipore
filtration system (Millipore, Molsheim, France).
2.2. Preparation of CS, a,b-GP, and salt solutions
A CS solution was obtained by dissolving 220–400 mg CS in
10 mL of 0.1 M lactic acid under mechanical stirring overnight at
room temperature. This solution was cooled to 4  C using an ice
bath. Then, the pH of the CS solution was adjusted to 6.0 by the
dropwise addition of a mixture solution of 0.1 M sodium lactate
and 0.1 M NaOH. a,b-GP and all salt solutions were prepared by
dissolving in 0.1 M lactate buffer at pH 6.0.
561
2.3. Lyophilization process
CS, a,b-GP, and all salt solutions were first chilled to 4  C for
15 min. Then, the a,b-GP and salt solutions were added dropwise
to the CS solution under stirring, and the final CS/a,b-GP/salt
solution was mixed for 20 min. For lyophilization, CS, CS/a,b-GP,
and CS/a,b-GP/salt solutions were deposited into a 5 mL tube. The
tubes were immediately frozen at 80  C overnight and then
placed in a Christ ALPHA 1–2 plus freeze-dryer (MartinChrist,
Germany) with a condenser temperature of 70  C. Lyophilization
was performed at a pressure of 0.1 mbar and a shelf temperature of
40  C for 6 h, followed by secondary drying at 25  C for 12 h. After
removal from the freeze drier, lyophilizate samples were placed in
a desiccator over P2O5 at 4  C until testing.
2.4. X-ray powder diffraction of the lyophilizate
X-ray diffraction patterns of the lyophilizate samples were
recorded using a D8 Advance X-Ray Diffractometer system (Bruker,
Germany). The samples were loaded on the quartz sample-holder
and scanned from 5 to 60 at a rate of 6 /min.
2.5. Determination of gelation time
CS/a,b-GP/salt solutions were reconstituted by adding 3 mL
water to the lyophilizates. Gelation times of freshly prepared
solutions and solutions reconstituted from CS/a,b-GP/salt lyophi-
lizates were determined by a simple test tube inverting method
(Zhou et al., 2008). A total of 3 mL CS/a,b-GP/salt solutions were
added to a 5.0 mL tube in a temperature-controlled bath of
37  0.5  C. In order to observe whether the solution underwent
gelation, the tube was inverted horizontally and the gelation time
was defined as the time when the solution no longer flowed.
2.6. Viscosity determination
A total of 25 mL CS/a,b-GP/salt solution at 4  C was added to a
30.0 mL tube in a temperature-controlled bath. After 10 min, the
solution temperature was constant and the viscosity of the
solution at different temperatures was measured with an NDJ-
8S Digital Viscosimeter (Jinghai Instruments, Shanghai, China).
2.7. pH measurement during the gelation process
The pH values of CS/a,b-GP/CaCl2 solutions were measured as a
function of time at 37  C using a Mettler-Toledo FE20 pH meter
(Mettler-Toledo Instruments Co., Ltd., Shanghai). The cold CS/
a,b-GP/CaCl2 solutions were placed in a temperature- controlled
bath at 37  C and pH values were recorded during the gelation
process.
2.8. Preparation of CS/a,b-GP/CaCl2 thermosensitive hydrogel
The freshly prepared or reconstituted CS/a,b-GP/CaCl2 solution
was deposited into a 5 mL tube at 4  C. Then the cold tubes were
placed in a temperature-controlled bath at a constant temperature
of 37  C. Finally, a CS/a,b-GP/CaCl2 thermosensitive hydrogel was
obtained. The CS/a,b-GP/CaCl2 hydrogel containing BSA was
prepared in a similar manner, except that proteins were dissolved
in CS solution before the a,b-GP and CaCl2 solutions were added.
2.9. Stability of CS/a,b-GP/CaCl2 lyophilizate
Stability tests were performed with CS/a,b-GP/CaCl2 lyophi-
lizate under refrigerated conditions (i.e., 2–8  C) and at room
temperature (i.e., 25  1.0  C). The samples were analyzed after 0,
562 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569

Table 1
Reconstitution of freeze-dried mixture of 1.5% CS, a,b-GP, and various salts.

Code a,b-GP concentration Salt concentration (%) reconstitution

A 0.34% – precipitation
B 0.56% – precipitation
C 0.80% – precipitation
D 0.34% 1.4% NaCl solution
E 0.56% 1.4% NaCl solution
F 0.80% 1.4% NaCl precipitation
G 1.01% 1.4% NaCl precipitation
H 0.80% 1.4% CaCl2 solution
I 1.01% 1.4% CaCl2 solution
J 1.24% 1.4% CaCl2 precipitation
K 0.90% 1.0% MgCl2 solution
L 1.13% 1.0% MgCl solution
M 1.36% 1.0% MgCl2 precipitation

15, 30, 60, and 90 days. At each time point, the following
performance measurements were conducted for each sample:
(1) the formulation was visually inspected for noticeable changes
in physical appearance (i.e., color, state); (ii) the pH value of the
reconstituted solution was determined using a Mettler-Toledo
FE20 pH meter (Mettler-Toledo Instruments Co., Ltd., Shanghai);
and (iii) gelation time at 37  C was determined by a simple test tube
inverting method, as described in Section 2.5. All of the measure-
ments were performed in triplicate.
2.10. In vitro release studies and polymer degradation behavior
BSA release from the CS/a,b-GP/CaCl2 hydrogel was investigat-
ed using an in vitro release assay. The CS/a,b-GP/CaCl2 hydrogel
was loaded with 5.0%, 10.0%, and 20.0% BSA. A total of 2 mL CS/
a,b-GP/CaCl2 solution containing 5.0%, 10.0%, and 20.0% BSA was
added to a 3.0 mL tube at 37  C. After the hydrogel formed, it was
placed in a tube containing 6 mL phosphate-buffered saline (PBS)
at pH values of 5.0, 6.0, and 7.4. The tube was placed in a rocking
incubator (SKY-211D, China) operating at 60 rpm and 37  C. At each
sampling time, solution was withdrawn and replaced with the
same volume of fresh PBS. The amount of BSA released was
measured using the BCA protein assay kit. At predetermined time
intervals, hydrogel was collected and subsequently lyophilized.
The mass loss of hydrogel was evaluated gravimetrically.
3. Results and discussion
3.1. Effects of various salts on the reconstitution of CS/a,b-GP/salt
lyophilizate
Lyophilizate of the CS and GP mixture unexpectedly formed a
precipitate that could not be re-dissolved in water (Table 1 and
Supplementary Fig. 1). During the lyophilization process, water is
removed from the frozen formulation, resulting in dramatic solute
condensation. As a result, CS concentration significantly increases
while the electrostatic repulsion between positively charged CS
chains increases simultaneously. It has been reported that a,b-GP
can act as a proton acceptor, and increases in temperature can
induce the transfer of protons from CS to a,b-GP leading to CS
precipitation and gelation (Filion and Buschmann, 2013; Lavertu
et al., 2008). During the lyophilization process, a,b-GP might also
act as a proton acceptor, catalyzing proton dissociation from CS
amine groups, resulting in an increase in attractive interactions
(e.g., van der Waal’s, hydrophobic interactions) between CS chains
as well as chain aggregation (Fig. 1). Therefore, pH of the

Fig. 1. A schematic diagram showing the preferential interaction between Ca2+ ions and a,b-GP, which inhibited the transfer of protons from CS to a,b-GP and then inhibited
aggregation of CS during lyophilization. The freshly prepared chitosan/a,b-GP/CaCl2 solutions and reconstituted solutions from lyophilizates could quickly form hydrogel at
37  C.
 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569
Fig. 2. X-ray diffraction patterns of lyophilizate of 1.5% CS (A), powder of a,b-GP (B), lyophilizate of 1.01% a,b-GP and 1.6% CaCl2 mixture (C), lyophilizate of 1.5% CS and 1.6%
CaCl2 mixture (D), lyophilizate of 1.5% CS, 1.01% a,b-GP, and 1.6% CaCl2 mixture (E).
reconstituted CS/a,b-GP suspension should decrease while the CS
chains aggregated. In support of this hypothesis, pH of the
supernatant of reconstituted CS/a,b-GP suspension was measured.
The results indicated that pH of the suspension clearly decreased
with an increase in a,b-GP content of the lyophilizate (Supple-
mentary Fig. 2). Moreover, a,b-GP concentration in the superna-
tant of reconstituted CS/a,b-GP suspension was almost identical to
that of freshly prepared CS/a,b-GP solutions (Supplementary
Fig. 3). These results suggest that a,b-GP act as a proton acceptor,
but not a divalent electrostatic cross-linker of chitosan amine
groups during the formation of CS aggregation.
It has been reported that NaCl can inhibit CS/a,b-GP hydrogel
formation (Tsai et al., 2011); thus, various salts including NaCl,
CaCl2, and MgCl2, were evaluated for their ability to inhibit
formation of the CS aggregation during lyophilization. As shown in
Table 1 and Supplementary Fig. 1, NaCl, CaCl2, MgCl2 could inhibit
precipitate formation dependent upon a,b-GP content. To under-
stand the effects of salts on reconstitution of the CS/a,b-GP/salt
system, X-ray diffraction analysis was used to analyze the system’s
structure. In the X-ray diffraction images, CS/CaCl2 lyophilizate
showed broad, small diffraction peaks between 2u of 9.6 and
24.0 , indicating some level of crystallinity (Fig. 2). CS/a,b-GP/
CaCl2 lyophilizate showed sharp diffraction peaks at 2u of 32.2 ,
45.9 , and 57.1 (Fig. 2), suggesting obvious crystallinity. These
sharp peaks were the same as those produced by a,b-GP/CaCl2
lyophilizate (Fig. 2), indicating that there was an obvious
563
interaction between a,b-GP and CaCl2 during the lyophilization
process. The negatively charged a,b-GP and Ca2+ ions may form a
crystalline complex during the lyophilization process. As a result,
formation of the crystalline complex between a,b-GP and Ca2+
ions inhibited transfer of protons from CS to a,b-GP; thus, only a
few CS chains polymerized during the lyophilization process
(Fig. 1). As a result, CS/a,b-GP/CaCl2 lyophilizate could be
redissolved in water. These results suggest that salts might interact
with a,b-GP, inhibit transfer of protons from CS to a,b-GP, and
then inhibit aggregation of CS chains during the lyophilization
process.
3.2. Effects of lyophilization on gelation properties
To understand the effects of lyophilization on gelation behavior,
gelation time and viscosity of freshly prepared CS/a,b-GP/salt
solutions and reconstituted solutions from lyophilizate were
systematically compared. As shown in Fig. 3A and B, gelation
time of reconstituted CS/a,b-GP/NaCl and CS/a,b-GP/CaCl2
solutions was shorter than that of freshly prepared solutions
(P < 0.05). For the CS/a,b-GP/MgCl2 system, gelation time of
reconstituted solutions were shorter than that of the freshly
prepared solutions when temperatures were lower than 40  C;
however, at temperatures above 40  C, gelation time of the
reconstituted solutions was close to that of the freshly prepared
solutions (Fig. 3C). This might be associated with formation of a
564 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569

Fig. 3. Comparison of gelation times of freshly prepared and reconstituted solutions from lyophilizate at different temperatures. (A) solutions containing 1.5% CS, 0.56%
a,b-GP, and 1.6% NaCl; (B) solutions containing 1.5% CS, 1.01% a,b-GP, and 1.6% CaCl2; (C) solutions containing 1.5% CS, 1.13% a,b-GP, and 1.0% MgCl2. Values are expressed as
mean  SD (n = 3).

few CS oligomers during the lyophilization process. After forma-
tion of cross-linked CS oligomers, pH of the reconstituted CS/
a,b-GP/CaCl2 solutions decreased (Supplementary Fig. 4), more CS
amino groups were positively charged, and the CS chains adopted a
more extended conformation (Illum, 1998; Tsaih and Chen, 1997;
Chee et al., 2014). The reconstituted CS/a,b-GP/CaCl2 solutions are
able to more easily form hydrogel than freshly prepared solutions
when the temperature increases. On the other hand, with
formation of CS oligomers, the average molecular weight of CS
increases. An increase in the average molecular weight of CS,
together with the more extended conformation of CS chains,
should lead to an increase in viscosity of the reconstituted CS/
a,b-GP/salts solutions (Illum, 1998; Tsaih and Chen, 1997; Chee
et al., 2014). Measurement of the viscosity of CS/a,b-GP/salts
solutions indicated that viscosity of the reconstituted solutions at
different temperatures was significantly higher than that of freshly
prepared solutions (Fig. 4), which further confirmed our spec-
ulations. The gelation time of reconstituted CS/a,b-GP/MgCl2
 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569 565

Fig. 4. Comparison of the viscosity of freshly prepared and reconstituted solutions from lyophilizates at different temperatures. (A) solutions containing 1.5% CS, 0.56%
a,b-GP, and 1.6% NaCl; (B) solutions containing 1.5% CS, 1.01% a,b-GP, and 1.6% CaCl2; (C) solutions containing 1.5% CS, 1.13% a,b-GP, and 1.0% MgCl2. Values are expressed as
mean  SD (n = 3).

solutions (66.1 min) was too long at 37  C, so this system is not
suitable for pharmaceutical application, and as such, was excluded
from further investigations.
3.3. The thermogelling properties of the reconstituted solutions
CS/a,b-GP/salts solutions reconstituted from lyophilizate must
possess thermogelling properties (i.e., maintain liquid state at
temperatures below 25  C, but can be transformed into a semisolid
hydrogel at body temperature). Thus, the thermogelling properties
of reconstituted CS/a,b-GP/NaCl and CS/a,b-GP/CaCl2 solutions
from lyophilizate were investigated. Specifically, the thermogelling
properties of reconstituted CS/a,b-GP/NaCl solutions containing
0.34% and 0.56% a,b-GP was investigated at 4  C, 20  C, and 37  C
and the results are shown in Fig. 5. During the storage period at 4  C
and 20  C, the viscosity of reconstituted CS/a,b-GP/NaCl solutions
containing 0.34% and 0.56% a,b-GP gradually increased (Fig. 5A1,
B1). The reconstituted CS/a,b-GP/NaCl solution containing 0.34%
a,b-GP continuously remained in a liquid state, but the
reconstituted solution containing 0.56% a,b-GP formed hydrogel
after 6 days and 11 days at 20  C and 4  C, respectively. When the
temperature increased to 37  C, the reconstituted CS/a,b-GP/NaCl
566 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569

Fig. 5. Viscosity changes in solutions reconstituted from lyophilizates with time at 4  C (A1 and A2), 20  C (B1 and B2), and 37  C (C1 and C2). (A1, B1, C1) solutions
reconstituted from lyophilizate containing 1.5% CS, 1.6% NaCl2, and 0.34% or 0.56% a,b-GP; (A2, B2, C2) solutions reconstituted from lyophilizate containing 1.5% CS, 1.6% CaCl2,
and 0.80% or 1.01% a,b-GP. Values are expressed as mean  SD (n = 3).

solution containing 0.34% a,b-GP remained in a liquid state for 4 h
(Fig. 5C1). The viscosity of reconstituted CS/a,b-GP/NaCl solution
containing 0.56% a,b-GP quickly increased at 37  C (Fig. 5C1), and
was transformed into a semisolid hydrogel within 38.6 min. The
results indicated that although the CS/a,b-GP/NaCl solution
containing 0.56% a,b-GP possessed thermogelling properties, it
had difficulty remaining in a liquid state at low temperatures over a
long period of time.
The thermogelling properties of reconstituted CS/a,b-GP/CaCl2
solution containing 0.80% and 1.01% a,b-GP was investigated at
 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569 567

Table 2 The in vitro release of BSA from CS/a,b-GP/CaCl2 hydrogel with

pH and gelation time at 37  C for solutions reconstituted from lyophilizate stored at
different BSA concentrations was evaluated at different pH. As
2–8  C or at 20–25  C. The lyophilizate contained 1.5% CS, 1.6% CaCl2, and 1.01%
a,b-GP. shown in Fig. 6, BSA release rates were significantly influenced by
pH of the release medium and BSA concentration within the
No. of days pHa gelation time at 37  C (min) a
hydrogel. With an increase in pH from 5.0 to 7.4, BSA release rates
   
2–8 C 20–25 C 2–8 C 20–25 C from hydrogels significantly decreased (Fig. 6). At pH 5.0,
1 6.02  0.05 6.01  0.04 5.14  0.17 4.95  0.07 approximately 97% BSA was released from hydrogels containing
15 5.99  0.06 5.96  0.04 4.95  0.11 5.05  0.08 5.0% BSA within 120 min (Fig. 6A). Comparatively, at pH 7.4, release
30 6.05  0.05 5.98  0.05 5.08  0.12 5.16  0.12 of approximately 97% BSA from the same hydrogels took 780 min
60 6.01  0.07 5.98  0.06 4.93  0.19 4.98  0.07
90 5.99  0.05 6.02  0.06 5.02  0.18 5.05  0.06
120 5.96  0.04 6.04  0.05 4.98  0.19 5.08  0.08
a
Mean  S.D., n = 3.

4  C, 20  C, and 37  C. As shown in Fig. 5A2 and B2, reconstituted

CS/a,b-GP/CaCl2 solutions remained in a liquid state for more than
2 weeks at 4  C and 20  C. During the storage period, viscosity of
the reconstituted CS/a,b-GP/CaCl2 solution slightly increased at
the initial stage of storage (1 day), and subsequently tended to be
constant for about 2 weeks (Fig. 5A2 and 5B2). When the
temperature increased to 37  C, reconstituted CS/a,b-GP/CaCl2
solutions containing 0.80% and 1.01% a,b-GP showed completely
different gelation behavior. The reconstituted CS/a,b-GP/CaCl2
solution containing 0.80% a,b-GP continuously remained in a
liquid state and did not form hydrogel even after 110 min (Fig. 5C2).
However, the reconstituted CS/a,b-GP/CaCl2 solution containing
1.01% a,b-GP quickly transformed into a semisolid hydrogel 5 min
after the temperature increased to 37  C. The viscosity of the
reconstituted CS/a,b-GP/CaCl2 solution containing 1.01% a,b-GP
at 37  C continuously increased from 3613 Pa s to 23300 Pa s. after
660 min (Fig. 5C2). These results indicate that reconstituted CS/
a,b-GP/CaCl2 solution containing 1.01% a,b-GP possess the
desired thermogelling properties. Thus, the CS/a,b-GP/CaCl2
solution was selected for subsequent studies.

3.4. Storage stability of CS/a,b-GP/CaCl2 lyophilizate

Long-term storage stability is a crucial parameter in the

pharmaceutical development of an in situ forming thermosensitive
hydrogel system; thus, the storage stability of CS/a,b-GP/CaCl2
lyophilizate was investigated in this study. After being stored for 4
months at 4  C and 25  C, no change in appearance was observed.
The gelation time and pH of reconstituted solutions from CS/
a,b-GP/CaCl2 lyophilizate were studied, and the results are
illustrated in Table 2. The pH of reconstituted solution from
lyophilizate stored at 4  C and 25  C remained at 6.0  0.05
(Table 2). The reconstituted solution from lyophilizate stored at
4  C and 25  C displayed a constant gelation time of 5.04  0.13 min
at 37  C (Table 2).

3.5. In vitro release of model proteins

To evaluate the possibility of CS/a,b-GP/CaCl2 hydrogel as a

promising thermogelling system for protein and peptide delivery, a
model protein BSA was incorporated into the CS/a,b-GP/CaCl2
solution. Then the thermogelling properties of the reconstituted
CS/a,b-GP/CaCl2 solution containing 5.0%, 10.0%, and 20.0% BSA
were investigated. As shown in Supplementary Fig. 5, with an
increase in BSA concentration from 5.0% to 20.0%, gelation time
increased from 8 min to 14.5 min at 37  C. These results showed
that hydrogel could be quickly obtained at 37  C, although addition
of BSA extended the gelation time of CS/a,b-GP/CaCl2 solution
containing BSA. These in vitro data suggest that CS/a,b-GP/CaCl2
solution containing BSA can be quickly converted into hydrogel at Fig. 6. In vitro cumulative release of BSA from hydrogel prepared with a
body temperature, thereby making it potentially useful as a reconstituted solution from CS/a,b-GP/CaCl2 lyophilizate at 37  C. BSA concentra-
tions within the hydrogel were 5.0%, 10.0%, and 20.0%. pH of the release medium
promising thermogelling system for protein and peptide delivery.
was 5.0 (A), 6.0 (B) and 7.4 (C). Values are expressed as mean  SD (n = 3).
568 G. Wu et al. / International Journal of Pharmaceutics 511 (2016) 560–569
Fig. 7. Weight loss of the hydrogel prepared with a reconstituted solution from CS/
a,b-GP/CaCl2 lyophilizate at 37  C. BSA concentration within the hydrogel was
10.0%. pH values of the release medium were 5.0, 6.0, and 7.4. Values are expressed
as mean  SD (n = 3).
Fig. 8. (A) BSA release from the hydrogel plotted against the erosion profile of the
hydrogel at 37  C. The hydrogel was prepared with a reconstituted solution from CS/
a,b-GP/CaCl2 lyophilizate. BSA concentration within the hydrogel was 10.0%. pH of
the release medium was 5.0 and 6.0. (B) The cumulative release of BSA from
hydrogel as a function of the square root of time. BSA concentrations within the
hydrogel were 5.0% and 10.0%. pH of the release medium was 7.4.
(Fig. 6C). For the hydrogel containing 10.0% and 20.0% BSA, the
results were similar (Fig. 6). With an increase of BSA concentration
in the hydrogels, the release rates of BSA significantly decreased at
different pH of the release medium.
To understand the mechanism underlying BSA release from the
hydrogel at different pH, hydrogel erosion was investigated by
assessing mass loss of hydrogel during the in vitro release period;
the results are illustrated in Fig. 7. The mass loss of CS/a,b-GP/
CaCl2 hydrogel containing 10% BSA at pH 5.0 and pH 6.0 was
obviously faster than that at pH 7.4. When comparing mass loss
with corresponding in vitro release of BSA, linear relationships
were established between dissolution of the polymer matrix and
the amount of BSA released from CS/a,b-GP/CaCl2 hydrogel at pH
5.0 and pH 6.0, respectively (Fig. 8A). The results suggest that BSA
release from the CS/a,b-GP/CaCl2 hydrogel at pH 5.0 and pH 6.0
was mainly controlled by erosion of the polymer matrix (Tsaih and
Chen, 1997; Chee et al., 2014). On the contrary, the in vitro release
rate at pH 7.4 was clearly faster than the mass loss rate. To further
understand the release process at pH 7.4, the in vitro BSA release
data were analyzed using the Higuchi model. The release profile
against the square-root of time showed a linear fit with r-squared
values of >0.98 (Fig. 8B), indicating that BSA release from the CS/
a,b-GP/CaCl2 hydrogel at pH 7.4 followed a diffusion-dominant
mechanism (Harding, 1997; Li et al., 2013).
4. Conclusions
In this study, lyophilization was attempted to improve the long-
term storage of CS/GP thermogelling systems for biopharmaceuti-
cal applications. After lyophillization, CS/a,b-GP lyophilizate could
not be dissolved and some metal salts such as NaCl, CaCl2, and
MgCl2 surprisingly facilitated dissolvation of the lyophilizate.
Further investigation indicated that lyophilization clearly influ-
enced the properties of the reconstituted CS/a,b-GP/salt solutions
such as gelation time, viscosity, and pH. However, reconstituted CS/
a,b-GP/CaCl2 solutions from lyophilizates maintained thermogel-
ling property, which could quickly form hydrogel at 37  C, but did
not form hydrogel at 20  C and 4  C longer than 2 weeks.
Furthermore, CS/a,b-GP/CaCl2 lyophilizate could be stably stored
at 25  C for more than 4 months. A model protein BSA was
incorporated into the CS/a,b-GP/CaCl2 system, resulting in a
slightly extended gelation time. In vitro release experiments
showed that BSA could be sustainedly released from CS/a,b-GP/
CaCl2 hydrogel in a pH-sensitive manner, demonstrating that CS/
a,b-GP/CaCl2 lyophilizate may be useful as a stable thermosensi-
tive in situ gel-forming system.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
ijpharm.2016.07.050.
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