Intra-articular Administration of Chitosan Thermosensitive In Situ Hydrogels Combined With Diclofenac Sodium–Loaded Alginate Microspheres

Journal of Pharmaceutical Sciences 105 (2016) 122e130

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org

Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Intra-articular Administration of Chitosan Thermosensitive In Situ Hydrogels Combined With Diclofenac SodiumeLoaded Alginate Microspheres Xiaole Qi 1, Xiaoxue Qin 1, Rong Yang 1, Jiayi Qin 1, Wenyan Li 1, Kun Luan 2, Zhenghong Wu 1, *, Li Song 1, * 1 2

Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing 210009, PR China The Second Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210029, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 April 2015 Revised 20 May 2015 Accepted 9 November 2015

The aims of this study were to prepare fine intra-articulareadministrated chitosan thermosensitive hydrogels combined with alginate microspheres and to investigate the possibility of those hydrogels as a drug delivery system for promoting the anti-inflammation effect. Diclofenac sodium containing alginate microspheres was prepared by a modified emulsification and/or gelation method and then dispersed into injectable thermosensitive hydrogels, consisting of chitosan and b-glycerophosphate. The final combined hydrogels were evaluated in terms of their morphology properties, rheological properties, in vitro drug release, and in vivo biocompatibility and pharmacodynamics behaviors. The optimized formulation exhibited sol-gel transition at 31.72 ± 0.42
C and quickly turned into gel within 5 min, with sustained drug release characteristics followed Ritger-Peppas equation, which could prolong the in vitro drug release to 5 days. In addition, the anti-inflammation efficacy of the combined hydrogels in rabbits with experimental rheumatoid arthritis was higher than that of drug solution and pure chitosan hydrogels. Those results demonstrated that these combined hydrogels could become a potential drug delivery system for improving the therapeutic effect of diclofenac sodium and suggested an important technology platform for intra-articular administration. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Keywords: drug delivery systems chitosan hydrogels microspheres controlled release

Introduction Lifelong progressive drug administration is necessary for some chronic diseases, such as rheumatoid and pseudorheumatism arthritis, which can cause continuing inflammation, pain, even disability in joints. Although treatment with nonsteroidal antiinflammatory drugs (NSAIDs) along with disease-modifying antirheumatic drugs, steroid hormone, and biologics drugs could help suppressing the symptoms of those diseases,1,2 they always lead to many unavoidable side effects including gastrointestinal toxicity, cardiovascular risk, and anaphylaxis.3-5 Moreover, a few drugs could be transported to the inflamed joint via the systemic

This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2015.11.019. Xiaole Qi and Xiaoxue Qin contributed equally to this work. * Correspondence to: Zhenghong Wu and Li Song (Telephone: þ008615062208341; Fax: þ0086-025-83179703). E-mail addresses: [email protected] (Z. Wu), [email protected] (L. Song).

circulation after peroral or parenteral administration, owing to their high degree of binding to plasma proteins. Hence, intra-articular injectable treatment has attracted more and more attention in improving the therapeutic efficiency of arthritis, via directly injecting those drugs into the target morbid joint to achieve drug enrichment and minimize the probability of drug exposure to normal tissue.6 However, the most intra-articular injection was usually administrating the free form of drugs, which can escape rapidly from the synovium into the systematic circulation and thus need frequent injection.7 In human patients with knee rheumatoid arthritis, it was found that the NSAIDs paracetamol, salicylate, and diclofenac sodium (DS) had intra-articular mean elimination half lives of 1.1, 2.4, and 5.2 h, respectively.8 Therefore, various sustained release systems for intra-articular injection, which were retained effectively within the joint, phagocytosed or sequestered by the synovial cells within the synovium, had been widely investigated to prolong the drug retention time and improve the therapeutic effect.9-11 Turker et al.12,13 studied a DS-loaded lipogelosome formulation to prolong the retention time of DS in the arthritic knee joint of the rabbit, which was

http://dx.doi.org/10.1016/j.xphs.2015.11.019 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

demonstrated by gamma scintigraphic imaging studies. Besides, Tuncay et al.14,15 incorporated DS into albumin and poly (lactide-coglycolide) microspheres, of sufficient size to impede physical flux, which can also increase the residence time of drugs in the joints. But, initial burst release was observed in those studies, and the size of particles had serious influence on the retention time. Currently, in situ hydrogels have showed promising potential in drug delivery,16-18 tissue engineering,19 and cell encapsulation.20 Those hydrogels are injectable fluids of minimally invasion but underwent a gelation to form any shape at a specific site after administrated into the body, responded to ionic cross-linking, changes in pH or temperature.21 Among them, thermosensitive in situ hydrogels have been widely investigated in biomedical applications.22,23 Chitosan, a nature polysaccharide obtained from deacetylation from chitin,24 was the most popular material for preparing in situ thermosensitive hydrogels with distinct advantages, such as biocompatibility, biodegradability, nontoxicity, muco-adhesion, and antibacterial peculiarity. Chenite et al.25,26 first reported a neutral (pH 6.8-7.2) thermosensitive solution based on chitosan and/or b-glycerophosphate (b-GP) combinations, which could remain liquid at or below room temperature but transited into gel on heating at physiological temperature (37
C). The mechanism of gelation was considered as a competition among intermolecular interactions including electrostatic repulsion, attraction, and hydrogen bonding. However, the chitosan thermosensitive hydrogels were fragile with low mechanical strength and the contained drug (especially smallemolecular-weight hydrophilic drugs) could not sustain release for long, which limits their further application. To overcome those problems, the chitosan thermosensitive hydrogels were combined with other polymers (such as alginate, poloxamer, polyvinyl alcohol, etc.) or sustained systems (such as liposomes, microspheres, nanoparticles, etc.). As reported by Ruel Gariepy et al.,27 thermosensitive chitosan and/or b-GP hydrogels containing drug-loaded liposomes were able to sustain carboxyfluorescein release for more than 2 weeks, whereas the release was completed within 24 h when carboxyfluorescein was simply mixed with chitosan and/or b-GP hydrogels. Tang et al.28 reported that the thermosensitive chitosan and/or polyvinyl alcohol hydrogels containing nanoparticles could slow the suitable drug release (such as propranolol and DS) and reduce the burst release although the nanoparticles did not reinforce the gel strength. Moreover, there is no report yet that incorporated alginate microspheres into chitosan and/or b-GP thermosensitive hydrogels for intra-articular injection. The objectives of this work were to incorporate alginate microspheres into chitosan and/or b-GP thermosensitive hydrogels for intra-articular injection and to investigate the possibility of those hydrogels as a drug delivery system (DDS) for promoting the arthritis therapy effect. DS, one of the most widely used effective NSAIDs for the treatment of rheumatoid arthritis and osteoarthritis, was selected as the model drug. As well known, gastrointestinal toxicity is the main side effect of DS through oral adminisration.29 Those combined hydrogels were designed to be directly injected into the target morbid joint to achieve drug enrichment and minimize the probability of drug exposure to normal tissue. Meanwhile, those drug-loaded microspheres were embedded in the hydrogels to prolong drug retention time and reduce the initial drug burst release. Our final combined hydrogels could remain liquid at room temperature and quickly turn into gel at 37
C, which were easily introduced into the joint and then became a “drug depot.” In this work, we firstly prepared the DS-loaded alginate microspheres-chitosan and/or b-GP hydrogels combination system for intra-articular administration. The preparation and characterization of DS-loaded alginate microspheres and chitosan and/or b-GP hydrogels were investigated and discussed. And the in vitro

123

drug release, in vivo biocompatibility, and pharmacodynamics studies of this new DDS were evaluated for its feasibility of intraarticular administration. Materials and Methods Materials DS was purchased from Wuhan Zhiqi Biochemical Co., Ltd. Alginate of molecular weight (2.19  105), G/M ratio (0.2), and medium viscosity (386 mPa.s) was gifted from Qingdao Huanghai Biological Pharmaceutical Co., Ltd. Poloxamer 407 was obtained from BASF, German. Chitosan (M 100000, deacetylation degree 95.95%) was purchased from Zhejiang Aoxing Biotechnology Co., Ltd. Complete Freund's adjuvant (CFA) was obtained from SigmaAldrich. Sialic acid (SA) kit was purchased from Nanjing Jiancheng biological research institute. All other materials were of reagent grade and used as received without further purification. Preparation of DS-Loaded Alginate Microspheres Alginate microspheres were prepared by an emulsification and/ or external gelation method as described in previous work with a little modification.30-32 Briefly, 0.2 g of DS was dissolved in 10 mL 1% poloxamer 407 solution and then added 0.2 g of alginate sodium to form a water phase. Ten milliliters of water phase were added into 30-mL liquid paraffin with 2.5% surfactant using XHF-D highspeed disperser (4000 rpm) for 3 min to form emulsion A. Emulsion B was formed by the same way with 5.0 mL of 25% CaCl2 solution and 1.0 mL ethanol and then added into emulation A under continuous magnetic stirring for 1 h. The microspheres were washed by ethanol, acid lotion (0.05 M CaCl2 and 0.1 M acetic acid), and deionized water to remove the residual oil successively. To determine the drug content, 10 mg of DS-loaded microspheres were dissolved in 10 mL of phosphate buffers (PBS) at pH 7.4 under sonication for 3 h. The samples were filtered (0.45-mm pore size) after dissolution, and then the subsequent filtrate was analyzed by UV-752 spectrophotometer at 275 nm (the standard curve was A ¼ 0.0308 C þ 0.00755, r2 ¼ 0.9998). The drug loading was calculated using the following equations:

The mass of DS in microspheres ¼

Absorbance  0:00755  Volume 0:0308

Drug loading ð%Þ ¼

(1)

the mass of DS in microspheres  100% the mass of microspheres (2)

Particle Size Analysis of Alginate Microspheres The mean diameter and size distribution of the microspheres were measured by Laser Diffraction Size Analyzer (Mastersizer 2000S; Malvern Instruments Ltd., Malvern, Worcestershire, UK). The microspheres were dispersed in distilled water and ultrasonically treated for 1 min before each measurement. Differential Scanning Calorimetry and X-ray Diffraction Analysis of Alginate Microspheres X-ray diffraction (XRD) (D8 Advance X-ray Diffractometer, 3.000-40.000
2q range, step 0.02
, and step time 0.3 s) and differential scanning calorimetry (DSC) analysis (NETZSCH DSC 204,

124

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

10
C min1 in the 30
C-350
C temperature range) were used to assess the preparation of alginate microspheres. The analysis was carried out on the DS, empty microspheres, DS-loaded microspheres, and physical mixture of DS and empty microspheres. Preparation of Chitosan Thermosensitive Hydrogels Combined With Alginate Microspheres The chitosan and/or b-GP thermosensitive hydrogels were prepared according to the previous works.33,34 Chitosan was dissolved in 0.1-M acetic acid under continuous magnetic stirring overnight at room temperature to prepare 2.0% chitosan solution. And then, 0.35 mL of 60% b-GP solution was dropped into 1 mL of the chitosan solution with continuous stirring for 10 min at an ice bath. The resultant solutions were then stored at 4
C. Accurate weight of DS-loaded microspheres was dispersed in the aforementioned solutions to form the finally alginate microspheres-chitosan complex hydrogels. For superior character of the complex hydrogels, different combinations of alginate microspheres were carried out for experiments (Table 1). Gelation Time Evaluation of Chitosan Thermosensitive Hydrogels The gelation time of chitosan hydrogels was assessed by testtube inverting method.16,35 In short, 2 mL of chitosan and/or b-GP solution combined with different contents of alginate microspheres were added into a tube and then incubated in a water bath at 37 ± 0.5
C. The gelation time was measured by inverting the tube every 30 s. The time was regarded as the sol-gel transition time when the matrix could not flow while the tube was inverted. Rheological Measurements of Chitosan Thermosensitive Hydrogels Rheology study was carried out on Physica MCR 301 (Anton paar, Austria). The samples were measured in triplicate by using the following tests, and liquid paraffin was added to the surface of the sample to prevent evaporation of solvent. The steady shear sweep test was carried on the dynamic shear mode for determination of flow behavior and viscosity of chitosan and/or b-GP hydrogels. It was assessed in 3 different temperatures, 20
C, 30
C, and 35
C. The shear rates were set between 0.01 and 100 s1. The temperature sweep test was performed in the dynamic oscillation mode to determine the sol-gel temperature of chitosan and/or b-GP hydrogels. The complex viscosity (h*) of the blank chitosan and/or b-GP hydrogels, DS-loaded chitosan and/or b-GP hydrogels (DSH), and DS microspheres-loaded chitosan and/or bGP hydrogels (DSMH) was recorded. All measurements were carried out at a constant frequency (1 Hz) and strain amplitude (0.05%) so as not to destroy the 3-dimensional network structure of the hydrogels. The temperature, ranged from 15
C to 45
C, was increased 1
C every minute. The sol-gel temperature was defined as the points where the h* was obviously increased.

Morphologic Analysis The shape and surface morphology of alginate microspheres and chitosan hydrogels were observed by a scanning electron microscope (SEM, HITACHI S-3000N, Tokyo, Japan). The blank hydrogels and DS microspheres-loaded hydrogels were prepared by the aforementioned method and then incubated at 37 ± 0.5
C for 1 h. The samples of hydrogels were obtained after lyophilization for 48 h and then coated by ion sputter gold under vacuum. In addition, alginate microspheres were completely dispersed in the ethanol, which was volatilized before ion sputter. Then, the surface and structure morphology were investigated, and the pictures were taken. In Vitro Drug Release of DS-Loaded Microspheres In this work, in vitro drug release from DS-loaded microspheres was measured by a dialysis membrane method. DS-loaded microspheres of 25 mg were put in a dialysis bag (cutoff MW 8-14  103) and immersed in 50 ml of PBS (pH 7.4), which was shaken at 100 rpm and 37 ± 0.5
C. At specific time points, 5-mL buffer samples were removed and replaced by an equal volume of the fresh buffer to maintain a constant volume. The samples were analyzed by UV752 spectrophotometer at 275 nm. In Vitro Drug Release of Chitosan Thermosensitive Hydrogels In this article, in vitro drug release from DS or DS microspheresloaded chitosan hydrogels was measured by a membraneless dissolution model.16,36 One milliliter of complex hydrogels loaded with DS or with various concentrations of DS microspheres (10, 20, 25 mg/mL) was placed into a tube with inner diameter 10 mm and incubated at 37 ± 0.5
C for 1 h. Then, 10 mL of PBS (pH 7.4) was added, and the tube was shaken at 100 rpm and 37 ± 0.5
C. At specific time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, and 120 h), the entire medium was collected and replaced by 10 mL of fresh PBS. The samples were analyzed by UV-752 spectrophotometer at 275 nm. In Vivo Biocompatibility Studies in Rats All studies were conducted in accordance with the principles of Laboratory Animal Care and were approved by the Department of Laboratory Animal Research at China Pharmaceutical University.37 The usage of animals was agreed by China Pharmaceutical University Animal Management and Ethics Committee. Male SpragueDawley rats, 200-250 g, purchased from Qinglong Mountain Animal Centre (Nanjing, China), were used for in vivo biocompatibility studies. Blank chitosan hydrogels (0.1 mL), blank microspheresloaded hydrogels, and DS microspheres-loaded hydrogels (0.5 mg of DS) were respectively injected into the left knee joint. Meanwhile, 0.1 mL of sterile saline was injected into the right knee joint of the same animal as control. After 3 days, the rats were sacrificed, and joints were dissected and then fixed into 10% formalin,

Table 1 Typical Composition of Chitosan Thermosensitive Hydrogels Combined With Different Amount of Alginate Microspheres (w/v %) Formulation

1 2 3 4

Alginate Microspheres

Chitosan Hydrogels

Alginate Sodium

Poloxamer 407

DS

Chitosan

b-GP

Alginate Microspheres

DS

e 0.50 1.00 1.25

e 0.25 0.50 0.62

e 0.25 0.50 0.63

1.48 1.48 1.48 1.48

15.56 15.56 15.56 15.56

e 1 2 2.5

0.5 e e e

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

embedded in paraffin wax. Sections (5 mm) were cut, stained with eosin and hemotoxylin and evaluated for the inflammatory changes. In Vivo Anti-inflammation Experiments in Rabbits With Experimental Rheumatoid Arthritis New Zealand rabbits, weighing 2.5 ± 0.2 kg, were provided by Qinglong Mountain Animal Centre. The experimental rheumatoid arthritis was induced by CFA.37 The time of induction was defined as 0 day. Arthritis was induced in both knee joints by injecting a certain quantity of CFA (0.5 mL/kg). All the injections were done under anesthesia (5% chloral hydrate, 1.5-2 mL/kg) to avoid the pain and stress to animals. The knee joints diameter was measured to assess the development of arthritis by Vernier Calipers. Drug treatment was started at 7 days after the induction. Each knee joint of the rabbits was injected intra-articularly with DS-containing preparations at a dose of 2.5 mg/0.5 mL once a week and lasted for 3 weeks. There were 4 groups (n ¼ 4): (1) DS-saline solutions, (2) DSH, (3) DSMH, and (4) saline as the control. The diameter of each knee joint was measured per day at the second week, whereas per week at the last 2 weeks, which was defined as Diameter. Swelling rate and relative swelling rate are defined as the following Equations 3 and 4, respectively:

Swelling rate ð%Þ ¼

Diameter1 Diameter0  100% Diameter0

Relative swelling rate ð%Þ ¼

(3)

Swelling rate1  Swelling rate0 Swelling rate0

 100%

(4)

where, Diameter0 is the diameter of joints before induction, and Diameter1 is the diameter of joints at the certain day after induction; Swelling rate0 is the swelling rate of the joints before treatment, and Swelling rate1 is the swelling rate of joints at the certain day after treatment. Blood samples were collected from each rabbit at the moment before induce, treatment, and sacrifice. Plasma was extracted and stored at 20
C for further analysis. The content of SA was determined by SA kit. Samples of 100 mL (plasma, 1 mmol/L SA solution, or deionized water), 200 mL of reagent I, and 4 mL of reagent II were added into Eppendorf tube of 10 mL and incubated into a water bath at 100
C for 15 min. The supernatant was obtained after centrifuging (3500 rpm, 10 min) and then measured by a UV detector at 560 nm. The content of SA in plasma was calculated using the following Equation 5:

The content of SA ðmg=LÞ ¼

Asample  Ablank  Cstandard Astandard  Ablank  Mstandard

(5)

where, Asample is the absorbance of plasma samples; Astandard is the absorbance of SA solution; Ablank is the absorbance of deionized water; Cstandard is 1 mmol/L; Mstandard is 309.3. Statistical Analysis All results were expressed as mean ± SD. One-way analysis of variance followed by Dunnett's multiple test using SPSS 19.0 software was applied to evaluate the differences between groups, which were considered statistically significantly different for p < 0.01 and statistically different for p < 0.05.

125

Results and Discussion Preparation of Alginate Microspheres Alginate microspheres were prepared by a modified emulsification and/or external gelation method in this work. Alginate microspheres show high porous structure, and drugs may be leaked in the process of preparation, especially in the process of washing, causing the low incorporation of drugs. The poloxamer 407, used in this work, could solubilize DS via forming micelles and fill the pores of alginate microspheres, and then could effectively increase the drug loading of microspheres, which was detected to be 25.29 ± 2.72%. Compared to the microspheres without poloxamer 407 (the drug loading was 8.18 ± 2.08%), the drug loading of microspheres with poloxamer 407 was detected to be 25.29 ± 2.72%. Intra-articular injection of drug-loaded microspheres has been widely studied by many researchers.37-40 The size has significant effects on the retention time of microparticles from the joints, and the most suitable size is between 1 to 10 mm. In this article, the size approximately ranged from 1 to 25 mm, with an average of 10.744 ± 1.246 mm, was measured by a Laser Diffraction Size Analyzer. Although the size of the microspheres was larger than the most suitable size, the microspheres prepared in this work could be administrated to the joints because they were finally dispersed into chitosan gels, instead of injecting only. DSC and XRD Analysis of Alginate Microspheres The alginate microspheres were characterized by DSC (Fig. S1a) and XRD (Fig. S1b). The DSC curve of DS showed a sharp melting peak at 281.9
C, whereas the blank microspheres exhibited a broad band. There were 2 fusion peaks at 271.1
C (the forward peak) and 285.3
C (the backward peak) in the physical mixture, as the possible consequence of interactions between drug and the surface of blank microspheres. However, the drug endothermic peak was disappeared in the DSC curve of alginate microspheres loading with DS, which showed the drug-loaded microspheres were well integrated and the drug was successfully dispersed in the excipients. The XRD pattern of pure DS showed several typical peaks in the region from 3
to 30
, whereas there was no diffraction peak in the blank microspheres. The drug typical peaks can be seen in the pattern of physical mixture but reduced, which were vanished in the drug-loaded microspheres. This provides the evidence of the successful formation of drug-loaded microspheres, which is supported by DSC results. Preparation of Chitosan Thermosensitive Hydrogels Combined With Alginate Microspheres The chitosan and/or b-GP hydrogels were prepared by the method described in the previous item. As presented in the Figure 1, the blank chitosan and/or b-GP solution was a homogeneous and clear liquid at room temperature (Fig. 1a), whereas microspheres-loaded chitosan solution was opaque (Fig. 1c). Both of those solutions became a nonflowing gel rapidly at around body temperature (Fig. 1b and 1d). As summarized in Table 2, effects of alginate microspheres contents on the thermosensitivity of chitosan hydrogels were estimated by adding different amount of microspheres into chitosan hydrogels. It was found that chitosan hydrogels combined with or without alginate microspheres were neutral solutions at room temperature and could quickly turn into gel within 5 min at 37
C. Moreover, with the increase of alginate microspheres content in the formulation, gelling time of chitosan and/or b-GP hydrogels decreased accordingly. It might be a probable consequence of increased viscosity and

126

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

Figure 1. The appearance of chitosan hydrogels at different temperature. (a and b) Photographs of blank hydrogels at room temperature and 37
C, respectively; (c and d) Photographs of microspheres-loaded hydrogels.

intensity of the complex formation, caused by the interaction between alginate, poloxamer 407, and chitosan. Considering the drug content, 20 mg/mL of alginate microspheres was chosen to be the final formation.

pseudoplastic flow at all test conditions, and the 3-dimensional network of chitosan and/or b-GP hydrogels was gradually destroyed by increased shear rate. The viscosity of the chitosan and/ or b-GP hydrogels was smaller at 20
C than that at 35
C, which demonstrated that the chitosan and/or b-GP hydrogels were in sol

Rheological Measurements of Chitosan Thermosensitive Hydrogels The results of rheological studies by steady shear sweep test and temperature sweep test are shown in Figure 2. The flow behavior and viscosity profiles of chitosan and/or b-GP hydrogels were investigated using steady shear sweep test at room temperature (20 ± 0.5
C), sol-gel temperature (30 ± 0.5
C), and body temperature (35 ± 0.5
C). Viscosity profiles obtained from this test shown in Figure 2d, indicating that chitosan and/or b-GP hydrogels exhibited

Table 2 Effects of Alginate Microspheres Contents on the Thermosensitivity and pH of Complex Chitosan Hydrogels (n ¼ 3) Formulation

pH

1 2 3 4

7.00 7.01 7.00 7.01

Sol-Gel Transition Time (min) ± ± ± ±

0.01 0.006 0.01 0.015

3.5 3.0 2.75 2.50

± ± ± ±

0.25 0.50 0.25 0.25

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

127

Figure 2. The rheological measurements of chitosan and/or b-GP hydrogels. Dependence of complex viscosity (h*) on temperatures for the blank chitosan hydrogels (a), DS-loaded chitosan hydrogels (b), and DS microspheres-loaded chitosan hydrogels (c) are investigated. (d) Logarithmic plot of viscosity against shear rate of chitosan and/or b-GP hydrogels.

state at room temperature and turned into gel state at 35
C. In addition, the sol-gel transition temperature was measured by the temperature sweep test and defined as the points where the complex viscosity (h*) was obviously increased. As seen in Figure 2a-2c, the complex viscosity was low in the state of solution and increased exponentially with temperature, which suggested the sol-gel transition was taken place. The sol-gel transition temperature of blank chitosan and/or b-GP hydrogels (Fig. 2a), DSH (Fig. 2b), and DSMH (Fig. 2c) was detected to be 32.63 ± 0.30, 32.15 ± 0.57, and 31.72 ± 0.42
C, respectively. Comparing to blank hydrogels, DS, or DS microspheres-loaded hydrogels had no significant effects on the phase transition temperature. All hydrogels had a transition temperature below 37
C which ensured the sol-gel transition progress in vivo. Morphologic Analysis by SEM The shape and surface morphology of alginate microspheres and chitosan hydrogels were observed by SEM. Figure 3a shows the surface morphology of alginate microspheres and Figure 4b and 4c show the microstructure of chitosan and/or b-GP hydrogels. From

the micrograph, the microspheres showed good sphere structure, and the chitosan hydrogels generally exhibited irregular porous microstructures. The surface of blank hydrogels (Fig. 3b) were smooth and exquisite, whereas DS microspheres-loaded hydrogels (Fig. 3c) were rough and thick. It is clear that alginate microspheres were embedded in the porous structure of chitosan hydrogels and could stay spherical after the incorporation. In Vitro Drug Release To determine in vitro drug release of DS from DS-loaded microspheres and the chitosan and/or b-GP hydrogels with or without alginate microspheres, the cumulative release (%) of DS was measured at certain time. Figure 4a shows the cumulative release profiles of DS from the DS-loaded microspheres, which was sustained released for 48 h. As seen in Figure 4b, there was a sustained slow release of DS from the chitosan hydrogels. The release profiles of DSH consisted of a quick release in the first 12 h and sustained released for 48 h. However, the drug release from DSMH was slower and longer than that from DSH, which lasted for 120 h. In addition, the effect of alginate microspheres loadings on the DS release

Figure 3. SEM of alginate microspheres (a), blank chitosan hydrogels (b), and microspheres-loaded chitosan hydrogels (c).

128

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

and diffusion, and when n < 0.45, the release of drug was mainly diffusion controlled. As summarized in Table 3, the diffusion index (n) of DSH was larger than that of DSMH. And with the increase of alginate microspheres content in the hydrogels, the value of n was decreased accordingly. In particular, when the content of microspheres was 25 mg/mL, the release mechanism of drugs was more likely following Higuchi model. It may indicate that the more microspheres added, the more proportion of drugs released by diffusion. This may be due to the increased viscosity and intensity of chitosan hydrogels for adding the microspheres, which could induce the erosion rate of hydrogels to be slower. In Vivo Biocompatibility Studies in Rats Unlike the systemic administration, intra-articular injection may cause interaction between the in situ gel and components in articular. For this reason, in vivo biocompatibility must be thoroughly studied to prove that the drug carriers do not cause inflammation or immune response. In this work, the in vivo biocompatibility study was investigated in male Sprague-Dawley rats. After injection, the gait of rats was changed visually, which disappeared after 1 day. After the rats were sacrificed and their furs and skins removed around the knee joints, there were no soft lumps found on the surface of joints. Histopathology studies were performed 3 days after the injection, results of which showed that only slight inflammatory infiltrates (proliferation of synovium) were observed after injection of complex hydrogels loaded with or without microspheres (Fig. S2). The alginate microspheres-loaded hydrogels did not cause gross inflammatory changes in the synovium, appearing to be biocompatible with articular.

Figure 4. In vitro drug release profiles. (a) In vitro drug release profiles of DS-loaded microspheres in PBS 7.4 (n ¼ 3); (b) In vitro drug release profiles of DSH and DSMH which contained different contents of microspheres (10, 20, 25 mg/mL) in PBS 7.4 (n ¼ 3). #DSMH (20 mg/mL): the optimal formulation.

profiles was evaluated. With the increase of alginate microspheres content in the hydrogels, the DS release rate decreased accordingly. This result demonstrated that the release rate of DS from the complex hydrogels was affected by alginate microspheres loadings, as a consequence of increased viscosity and intensity of chitosan hydrogels. The drug release curves were assessed by zero-order, first-order, Higuchi, and Ritger-Peppas equation, respectively. Table 3 summarizes the release model parameters of DS-loaded microspheres, DSH, and DSMH. According to the R2, the Ritger-Peppas equation was the optimal equation. The diffusion index (n) of DS-loaded microspheres, DSH, DSMH (10 mg/mL microspheres), DSMH (20 mg/mL microspheres), and DSMH (25 mg/mL microspheres) were 0.765, 0.616, 0.498, 0.532, and 0.601, respectively. According to Ritger,41 when 0.45
In Vivo Anti-inflammation Experiments in Rabbits With Experimental Rheumatoid Arthritis After the intra-articular injection with CFA, the rabbits had decreased activity level, reduced appetite, and dull hairs, whereas the control group injected with saline behaved normally. One week after the induction of arthritis, the knee joints showed sustained redness and heavy swelling. The diameter of the arthritic joint was changed from 2.079 ± 0.069 cm to 2.731 ± 0.122 cm (Fig. 5a), with the swelling rate 31.34 ± 3.68%, whereas the change of control joints was slight. The curative effects were estimated by the diameter of knee joints, which was measured per day at the second week and per week at the last 2 weeks. Changes in swelling rate and relative swelling rate of arthritis joints calculated by Equations 2 and 3 were shown in Figure 5b and 5c. After treatment for 3 weeks, the swelling rate of DS solution, DSH, and DSMH groups was reduced to 23.31 ± 3.10%, 21.79 ± 2.66%, and 17.98 ± 2.22%, respectively, and the relative swelling rate was changed to 81.63 ± 10.77%, 69.44 ± 10.20%, and 63.89 ± 9.74%, respectively. However, comparing to the treatment groups, the swelling rate and relative swelling rate of saline groups

Table 3 Models Fitting for Release Mechanism of Drugs (n ¼ 3) Model

Zero-order model First-order model Higuchi model Ritger-Peppas a

R-Square DS-Loaded Microspheres

DSH

DSMHa 10 mg/mL

20 mg/mL

25 mg/mL

0.764 0.945 0.928 0.992 (n ¼ 0.765)

0.692 0.913 0.884 0.997 (n ¼ 0.616)

0.755 0.921 0.922 0.993 (n ¼ 0.498)

0.859 0.910 0.969 0.991 (n ¼ 0.532)

0.947 0.843 0.967 0.956 (n ¼ 0.601)

DSMH: Chitosan hydrogels combined with different contents of DS-loaded microspheres (10, 20, 25 mg/mL).

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

129

Figure 5. In vivo pharmacodynamics studies on rabbits. (a) Diameter-time profiles of knee joints after the induction of rheumatoid arthritis; (b) the swelling rate, and (c) relative swelling rate-time profiles of knee joints after the treatment by intra-articulareinjecting saline, DS solution, DSH, and DSMH. Data are shown as mean ± SD, comparisons were made between adDS solution, DSH, and DSMH groups versus saline groups; bdDSH and DSMH groups versus DS solution groups. Symbols represent statistical significance: *p < 0.05, **p < 0.01. (d) SA lever in plasma after the treatment. Data are shown as mean ± SD, comparisons were made between DS solution, DSH, and DSMH groups versus saline groups and symbols represent statistical significance: *p < 0.05.

were slightly increased to 33.62 ± 2.77% and 105.94 ± 11.05%, respectively. There were significant differences between treatment and control groups (p < 0.01). Differences were also observed between DSMH and DS solution groups (p < 0.05) but no differences between DSH and DS solution. Among the treatment groups, DSMH seemed to effectively reduce the swelling of arthritis joints and had superior anti-inflammation effects. Furthermore, changes of joints swelling in 1 week after intra-articulareinjecting DS-contained preparations were studied on the day 7 to day 14 after the first injection, providing more details of anti-inflammation activities. From Figure 5c, we could see that the relative swelling rate of DSH and DSMH groups was reduced slowly with date. But the DS solution groups were decreased quickly in the first 2 days and then increased to near the former swelling lever before treatment. This might demonstrate that the preparations of DSMH and DSH could effectively sustain drug releasing and increase their retention time in the joints after intra-articular injection. In addition, the joints swelling of DSMH groups was decreased slower than DSH, but lasted for longer, which suggested that the combination chitosan hydrogels with alginate microspheres would be more effective in drug sustained-releasing than pure hydrogels. Unfortunately, the experimental rheumatoid arthritis was not cured after 3-week treatment by intra-articulareinjecting DScontained preparations, one reason of which might be that antigen-induced arthritis is difficult to treat within 21 days.15 Furthermore, at day 28, after the rabbits were sacrificed and their furs and skins removed around the knee joints, we unexpectedly found several small soft lumps protruding from the surface of joints. This might be the undegraded chitosan hydrogels which turned into semisolid at physiological temperature, indicating that the combination system could not be

biodegraded completely within at least 7 days. The developed in situ gel system combined alginate microspheres with long residence time in the joints seemed to be suitable for sustained release of loading drugs. However, the biodegradability of DDS cannot be ignored. Thus, the treatment of intra-articulare injecting the combination system once a week might be inappropriate and should adjust according to both curative effects and biodegradability. In consideration of aforementioned discussions, a novel treatment strategy that DS solution is firstly intra-articular injected to relieve the joints swelling rapidly, followed by injecting DSMH to maintain the curative effects might be feasible, which needs further studies. In close correlation with treatment effectiveness, changes in the level of inflammation-related markers could also demonstrate the progress of inflammation. Sialic acids (SA), acetylated derivatives of neuraminic acid, has been reported as a parameter of several diseases, such as cancer,42 myocardial infarction,43 and inflammation.44 Elevated SA lever was observed during inflammatory processes and was reported to be a useful maker of rheumatoid arthritis.45 In this work, blood samples were collected before induction, treatment, and sacrifice, and SA was measured by SA kit. The lever of SA was increased to 1166.32 ± 100.32 mg/L after the induction of rheumatoid arthritis, whereas the normal lever was 772.33 ± 67.52 mg/mL. After the treatment, differences were observed (Fig. 5d) between DSMH, DSH groups, and saline groups (p < 0.05), but no difference between DSMH and DSH groups. Conclusions In this work, an intra-articular DDS was prepared based on thermosensitive chitosan hydrogels that can spontaneously form

130

X. Qi et al. / Journal of Pharmaceutical Sciences 105 (2016) 122e130

a “drug depot” in the articular after injection and increase the retention time of drugs. The thermally responsive hydrogels could remain liquid at or below room temperature, making it convenient to use by injecting, but transited into gel at physiological temperature within 5 min. Loaded with alginate microspheres, the combined hydrogels showed increased viscosity and reinforced intensity, and had sustained drug releasing for 5 days in vitro studies. In vivo biocompatibility studies in rats showed that only slight synovium proliferation was caused by the combined system, appearing to be biocompatible with articulars. The results of pharmacodynamics revealed that the combined system seemed to effectively reduce the swelling of arthritis joints and had superior anti-inflammation effects than drug solution, although the experimental rheumatoid arthritis was not cured after 3-week treatment. In conclusion, the alginate microspheres-loaded chitosan hydrogels prepared in our work seem to be an interesting and promising way for intra-articular injection for the treatment of articular diseases. As for the treatment strategies by this combination system, there are still needs for further long-term studies. Acknowledgments This work was financially supported by the Fundamental Research Funds for the Central Universities (Program No. 2015ZD007, No. 2015PT052), the National Natural Science Foundation of China (No. 81402859), and the Natural Science Foundation of Jiangsu Province (No. BK20130663). References 1. Blackburn WD. Management of osteoarthritis and rheumatoid arthritis: prospects and possibilities. Am J Med. 1996;100(2):S24-S30. 2. Scott DL, Shipley M, Dawson A, Edwards S, Symmons DPM, Woolf AD. The clinical management of rheumatoid arthritis and osteoarthritis: strategies for improving clinical effectiveness. Br J Rheol. 1998;37(5):546-554. 3. Scarpignato C, Hunt RH. Nonsteroidal antiinflammatory drug-related injury to the gastrointestinal tract: clinical picture, pathogenesis, and prevention. Gastroenterol Clin North Am. 2010;39(3):433-464. 4. Howard PA, Delafontaine P. Nonsteroidal anti-inflammatory drugs and cardiovascular risk. J Am Coll Cardiol. 2004;43(4):519-525. 5. Brogden RN, Heel RC, Pakes GE, Speight TM, Avery GS. Diclofenac sodium: a review of its pharmacological properties and therapeutic use in rheumatic diseases and pain of varying origin. Drugs. 1980;20(1):24-48. 6. Larsen C, Ostergaard J, Larsen SW, et al. Intra-articular depot formulation principles: role in the management of postoperative pain and arthritic disorders. J Pharm Sci. 2008;97(11):4622-4654. 7. Butoescu N, Jordan O, Doelker E. Intra-articular drug delivery systems for the treatment of rheumatic diseases: a review of the factors influencing their performance. Eur J Pharm Biopharm. 2009;73(2):205-218. 8. Owen SG, Francis HW, Roberts MS. Disappearance kinetics of solutes from synovialfluid after intraarticular injection. Br J Clin Pharmacol. 1994;38(4):349-355. 9. Elron-Gross I, Glucksam Y, Biton IE, Margalit R. A novel diclofenac-carrier for local treatment of osteoarthritis applying live-animal MRI. J Control Release. 2009;135(1):65-70. 10. Saravanan M, Bhaskar K, Maharajan G, Pillai KS. Development of gelatin microspheres loaded with diclofenac sodium for intra-articular administration. J Drug Target. 2011;19(2):96-103. 11. Elron-Gross I, Glucksam Y, Margalit R. Liposomal dexamethasone-diclofenac combinations for local osteoarthritis treatment. Int J Pharm. 2009;376(1-2): 84-91. 12. Turker S, Erdogan S, Ozer ZY, et al. Scintigraphic imaging of radiolabelled drug delivery systems in rabbits with arthritis. Int J Pharm. 2005;296(1-2):34-43. 13. Turker S, Erdogan S, Ozer YA, Bilgili H, Deveci S. Enhanced efficacy of diclofenac sodium-loaded lipogelosome formulation in intra-articular treatment of rheumatoid arthritis. J Drug Target. 2008;16(1):51-57. 14. Tuncay M, Calis S, Kas HS, Ercan MT, Peksoy I, Hincal AA. In vitro and in vivo evaluation of diclofenac sodium loaded albumin microspheres. J Microencapsulation. 2000;17(2):145-155. 15. Tuncay M, Calis S, Kas HS, Ercan MT, Peksoy I, Hincal AA. Diclofenac sodium incorporated PLGA (50:50) microspheres: formulation considerations and in vitro/in vivo evaluation. Int J Pharm. 2000;195(1-2):179-188. 16. Li X, Kong X, Zhang J, et al. A novel composite hydrogel based on chitosan and inorganic phosphate for local drug delivery of camptothecin nanocolloids. J Pharm Sci. 2011;100(1):232-241.

17. Khodaverdi E, Tafaghodi M, Ganji F, Abnoos K, Naghizadeh H. In vitro insulin release from thermosensitive chitosan hydrogel. AAPS PharmSciTech. 2012;13(2):460-466. 18. Kim S, Nishimoto SK, Bumgardner JD, Haggard WO, Gaber MW, Yang Y. A chitosan/beta-glycerophosphate thermo-sensitive gel for the delivery of ellagic acid for the treatment of brain cancer. Biomaterials. 2010;31(14): 4157-4166. 19. Cho JH, Kim SH, Park KD, et al. Chondrogenic differentiation of human mesenchymal stem cells using a thermosensitive poly(N-isopropylacrylamide) and water-soluble chitosan copolymer. Biomaterials. 2004;25(26):5743-5751. 20. Dang JM, Sun DDN, Shin-Ya Y, Sieber AN, Kostuik JP, Leong KW. Temperatureresponsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells. Biomaterials. 2006;27(3):406-418. 21. Ruel-Gariepy E, Leroux JC. In situ-forming hydrogelsdreview of temperaturesensitive systems. Eur J Pharm Biopharm. 2004;58(2):409-426. 22. Suh JKF, Matthew HWT. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials. 2000;21(24): 2589-2598. 23. Agnihotri SA, Mallikarjuna NN, Aminabhavi TM. Recent advances on chitosanbased micro- and nanoparticles in drug delivery. J Control Release. 2004;100(1): 5-28. 24. Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci. 2006;31(7):603-632. 25. Chenite A, Buschmann M, Wang D, Chaput C, Kandani N. Rheological characterisation of thermogelling chitosan/glycerol-phosphate solutions. Carbohydr Polym. 2001;46(1):39-47. 26. Ruel-Gariepy E, Chenite A, Chaput C, Guirguis S, Leroux JC. Characterization of thermosensitive chitosan gels for the sustained delivery of drugs. Int J Pharm. 2000;203(1-2):89-98. 27. Ruel-Gariepy E, Leclair G, Hildgen P, Gupta A, Leroux JC. Thermosensitive chitosan-based hydrogel containing liposomes for the delivery of hydrophilic molecules. J Control Release. 2002;82(2-3):373-383. 28. Tang Y, Zhao Y, Li Y, Du Y. A thermosensitive chitosan/poly(vinyl alcohol) hydrogel containing nanoparticles for drug delivery. Polym Bull. 2010;64(8): 791-804. 29. Todd PA, Sorkin EM. Diclofenac sodium. A reappraisal of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy. Drugs. 1988;35(3): 244-285. 30. Zhu AM, Chen JH, Liu QL, Jiang YL. Controlled release of berberine hydrochloride from alginate microspheres embedded within carboxymethyl chitosan hydrogels. J Appl Polym Sci. 2011;120(4):2374-2380. 31. Maysinger D, Krieglstein K, FilipovicGrcic J, Sendtner M, Unsicker K, Richardson P. Microencapsulated ciliary neurotrophic factor: physical properties and biological activities. Exp Neurol. 1996;138(2):177-188. 32. Moebus K, Siepmann J, Bodmeier R. Alginate-poloxamer microparticles for controlled drug delivery to mucosal tissue. Eur J Pharm Biopharm. 2009;72(1): 42-53. 33. Chenite A, Chaput C, Wang D, et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials. 2000;21(21):2155-2161. 34. Xing J, Qi X, Jiang Y, et al. Topotecan hydrochloride liposomes incorporated into thermosensitive hydrogel for sustained and efficient in situ therapy of H22 tumor in Kunming mice. Pharm Dev Technol. 2014:1-8 [Epub ahead of print]. 35. Khodaverdi E, Ganji F, Tafaghodi M, Sadoogh M. Effects of formulation properties on solegel behavior of chitosan/glycerolphosphate hydrogel. Iran Polym J. 2013;22(10):785-790. 36. Bhardwaj R, Blanchard J. Controlled-release delivery system for the a-MSH analog Melanotan-I using poloxamer 407. J Pharm Sci. 1996;85(9):915-919. 37. Thakkar H, Sharma RK, Mishra AK, Chuttani K, Murthy RSR. Efficacy of chitosan microspheres for controlled intra-articular of celecoxib in inflamed joints. J Pharm Pharmacol. 2004;56(9):1091-1099. 38. Thakkar H, Sharma RK, Mishra AK, Chuttani K, Murthy RR. Albumin microspheres as carriers for the antiarthritic drug celecoxib. AAPS PharmSciTech. 2005;6(1):E65-73. 39. Lu Y, Zhang G, Sun D, Zhong Y. Preparation and evaluation of biodegradable flubiprofen gelatin micro-spheres for intra-articular administration. J Microencapsulation. 2007;24(6):515-524. 40. Bozdag S, Calis S, Kas HS, Ercan MT, Peksoy I, Hincal AA. In vitro evaluation and intra-articular administration of biodegradable microspheres containing naproxen sodium. J Microencapsulation. 2001;18(4):443-456. 41. Ritger PL, Peppas NA. A simple equation for description of solute release. I. Fickian and non-fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs. J Control Release. 1987;5(1):23-26. 42. Erbil KM, Jones JD, Klee GG. Use and limitations of serum total and lipid-bound sialic acid concentrations as markers for colorectal cancer. Cancer. 1985;55(2): 404-409. 43. Crook M, Haq M, Haq S, Tutt P. Plasma sialic-acid and acute-phase proteins in patients with myocardial-infarction. Angiology. 1994;45(8):709-715. 44. Citil M, Gunes V, Karapehlivan M, Atalan G, Marasli S. Evaluation of serum sialic acid as an inflammation marker in cattle with traumatic reticulo peritonitis. Rev Med Vet. 2004;155(7):389-392. 45. Mohan SK, Priyav V. Serum total sialic acid, lipid peroxidation, and glutathione reductase levels in patients with rheumatoid arthritis. Turk J Med Sci. 2010;40(4):537-540.