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oxidized alginate hydrogel for drug delivery application
International Journal of Biological Macromolecules 50 (2012) 1299–1305
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application Xingyi Li a , Xiangye Kong b , Zhaoliang Zhang a , Kaihui Nan a , LingLi Li a , XianHou Wang c , Hao Chen a,∗ a
Institute of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical College, 270 Xueyuan Road, Wenzhou 325027, China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, No. 1, Keyuan 4th Road, Chengdu 610041, China Department of Lymphoma, Sino-US Center for Lymphoma and Leukemia, Tianjin Medical University Cancer Hospital and Institute, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin 300060, China b c
a r t i c l e
i n f o
Article history: Received 12 February 2012 Received in revised form 5 March 2012 Accepted 12 March 2012 Available online 20 March 2012 Keywords: Hydrogel Biocompatibility In vitro In vivo Drug delivery
a b s t r a c t In this paper, covalently cross-linked hydrogel composed of N,O-carboxymethyl chitosan and oxidized alginate was developed intending for drug delivery application. In vitro/vivo cytocompatibility and biocompatibility of the developed hydrogel were preliminary evaluated. In vitro cytocompatibility test showed that the developed hydrogel exhibited good cytocompatibility against NH3T3 cells after 3-day incubation. According to the results of acute toxicity test, there was no obvious cytotoxicity for major organs during the period of 21-day intraperitoneal administration. Meanwhile, the developed hydrogel did not induce any cutaneous reaction within 72 h of subcutaneous injection followed by slow degradation and adsorption with the time evolution. Moreover, the extraction of developed hydrogel had nearly 0% of hemolysis ratio, which indicated the good hemocompatibility of hydrogel. Based on the above results, it may be concluded that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel with non-cytotoxicity and good biocompatibility might suitable for the various drug delivery applications. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In recent years, numerous implant biomaterials including synthetic and natural materials have been widely used in the biomedical and pharmaceutical field [1–3]. Hydrogels are a class of polymers very similar to soft tissue for their high water content, the mechanical properties (low modulus and elasticity), softness, oxygen permeability and excellent biocompatibility. According to the resource of materials, hydrogels can be divided into two classes: synthetic materials based hydrogel and natural materials based hydrogel. In the case of synthetic materials based hydrogels, there are presence of some disadvantages including inflammatory reactions, material migration as well as the difficulty of removal and so on. Recently, much attention have been oriented to the biocompatible, biodegradable hydrogels made from natural polymers that are susceptible to enzymatic degradation [4–6]. Chitosan, the second abundant source in nature after cellulose, is an aminopolysaccharide obtained by the deacetylation of chitin [7–10]. It is composed of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues. There are many parameters
∗ Corresponding author. Tel.: +86 577 88833806; fax: +86 577 88833806. E-mail addresses: [email protected], [email protected] (H. Chen). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2012.03.008
influencing the properties of chitosan including molecular weight (MW), degree of deacetylation (DD) and etc. [8]. Chitosan is water insoluble, but can easily dissolve into some acidic aqueous solution, such as acetic aqueous solution and etc. In order to improve the water solubility of this versatile cationic polysaccharide, several strategies are being made to modify chitosan including PEGtylation, carboxymethylation and etc. realizing its full potential application [7,11]. In recent years, an increasing number of in situ gel systems based on chitosan and its derivatives have been viewed in the literature for various pharmaceutical and biomedical applications [6,12]. Alginic acid is mostly encountered as a high molecular weight linear copolymer composed of (1–4)-linked -d-mannuronic acid (M units) and ␣-l-guluronic acid (G units) monomers. The neutralized form, sodium alginate, as a common thickening agent, has been widely used in the food industry and biomedical field [13]. Chitosan exists as a cationic polyelectrolyte yet such solution are not compatible with aqueous solutions of sodium alginate, which is an anionic polyelectrolyte. Our previous study has demonstrated that the novel chitosan covalent hydrogel based on N,O-carboxymethyl chitosan and oxidized alginate could be gained by simple mixing these two components with an expected weight ratio [14]. Although numerous studies have demonstrated that chitosan and alginate were non-cytotoxic, biodegradable, biocompatible suitable for further various drug delivery applications, its hydrogels
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should be carefully checked before its further various drug delivery applications [7,11]. In this paper, our studies are focused on the cytotoxicity and biocompatibility evaluation of this novel hydrogel by means of the in vitro cytocompatibility, acute cytotoxicity, subcutaneous implant test, skin irritation test, and hemolysis test. 2. Materials and methods 2.1. Materials N,O-carboxymethyl chitosan (the degrees of substitution of carboxymethyl groups on both the amino (N-position) and primary hydroxyl (O-position) sites were approximately 85%) and oxidized alginate (the oxidation degree of alginate was about 27.8%) were successfully synthesized by our previous study [14]. All other chemicals used in this paper were analytic grade. Distilled water from Milli-Q water system was used to prepare the aqueous solutions. 2.2. Preparation of N,O-carboxymethyl chitosan/oxidized alginate auto-gelling system A calculated weight of oxidized alginate and N,O-carboxymethyl chitosan were dissolved into 20 ml distilled water to form 6% (w/w) and 4% (w/w) solutions, respectively. The solutions were filtered and stored at 4 ◦ C overnight for the further usage. Auto gelling solutions were prepared as follows: 2 ml of N,O-carboxymethyl chitosan and oxidized alginate solutions with weight ratio of 1:2 were mixed at room temperature with gentle stirring to form homogeneous solution. After that, the auto-gelling solution was placed at 37 ◦ C for 30 min to form the N,O-carboxymethyl chitosan/oxidized alginate hydrogel. 2.3. In vitro cytocompatibility test The NH3T3 cell was used to assess the in vitro cytocompatibility of hydrogels. The NH3T3 cell was obtained from ACTT (USA) and cultured with DMEM(A) medium at 37 ◦ C and 5% CO2 . N,O-carboxymethyl chitosan and oxidized alginate were sterilized by cobalt-ray prior the test. Briefly, N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution was first prepared by follows: 5 ml of N,O-carboxymethyl chitosan solution and 5 ml of oxidized alginate solution was mixed using a vortex mixer at room temperature. Immediately after mixing, the mixture was poured into a 24-well cell culture dish at 0.6 ml or 0.2 ml for completely or partly covering the bottom of the well, respectively. After incubation at 37 ◦ C for 30 min, the resultant gels adhered to the bottom of wells was rinsed with DMEM medium for three times. NH3T3 cells suspended in the DMEM medium were seeded into each well at 5.0 × 105 cells/well. After 24 h, 48 h and 72 h incubation, the morphology of cells surrounding gel was observed using an optical microscope (Olympus, Japan). In order to further evaluation the cytocompatibility of N,O-carboxymethyl chitosan/oxidized alginate hydrogel, NH3T3 cells (5.0 × 105 cell/well) was pre-mixed with N,O-carboxymethyl chitosan solution and then gelled with oxidized alginate solution at 37 ◦ C for 30 min. Subsequently, the cell supporter was incubated with DMEM medium for 24 h, 48 h and 72 h, respectively. Finally, the morphology of cell inside the hydrogel was observed with an optical microscope (Olympus, Japan). 2.4. Acute toxicity test Ten male and 10 female BALB/c mice, 6 weeks of age (18–22 g), were used to evaluate the acute toxicity of N,O-carboxymethyl chitosan/oxidized alginate hydrogel. All experimental protocols and animal care complied with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources,
Table 1 Skin irritation test: criteria of classification of the cutaneous reactions. Cutaneous reaction
Score
Results
None Sporadic or patchy erythema Moderate confluent erythema Severe erythema and edema
0 1 2 3
Normal Irritation Irritation Irritation
and were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical College. Twenty mice were randomly divided into two groups: a treatment group and a control group, five male and five female mice for each group. The mice of treatment group were injected with 50 ml/kg N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution into the abdominal cavity once, and the mice of control group were injected with 50 ml/kg saline solution into the abdominal cavity once. All the animals were observed continuously for 21 days after the administration, including the general conditions (the activity, energy, hair, feces, behavior pattern, other clinical signs, etc.), body weight, and mortality. At specific time point (7 day, 14 day and 21 day), three mice from each group were sacrificed by cervical dislocation, and its major organs including heart, liver, spleen, lung and kidney were removed and fixed in 10% formaldehyde solution. Finally, the fixed organs were embedded in paraffin, sectioned and stained with hematoxylin–eosin for the histopathologic examination. 2.5. Subcutaneous injection test Twenty four BALB/c mice, 6 weeks of age (18–22 g), were employed for subcutaneous injection of N,O-carboxymethyl chitosan/oxidized alginate hydrogel to evaluate the in vivo degradation behavior. Twenty four mice were randomly divided into two groups: a treatment group and a control group, twelve mice for each group. The mice from treatment group were injected with 0.5 ml N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution in back subcutaneous tissue and the mice from control group were injected with 0.5 ml saline solution in back subcutaneous tissue. Three mice from each group were sacrificed at 1, 3, 5 and 7 day and the injection site were opened with a surgical scissors for observation the state of hydrogel. Meanwhile, the tissue around with the injected site were carefully removed and subsequently fixed in 10% buffered formaldehyde, stained with hematoxylin–eosin for further histopathological examination. 2.6. Skin irritation test Three healthy New Zealand albino rabbits (2.3–2.5 kg), were used to evaluate the skin irritation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel. Briefly, 0.5 ml of N,Ocarboxymethyl chitosan/oxidized alginate auto-gelling solution was directly subcutaneous injected on the left back skin next to backbone and 0.5 ml of saline solution was directly subcutaneous injected on the right back skin next to backbone for control. The cutaneous reaction surrounding the injection site was evaluated at 24 h, 48 h and 72 h, using the criteria has been reported in Table 1. 2.7. Hemolysis test According to guide of biological evaluation of medical device (SFDA, China), the hemolysis of N,O-carboxymethyl chitosan/oxidized alginate hydrogel was carefully checked. Initially, 2 ml of N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution was placed in an test tube at 37 ◦ C for 12 h to form the hydrogel. Subsequently, 20 ml of sterile saline was added into test tube with incubation at 37 ◦ C for another 72 h and then the top of
X. Li et al. / International Journal of Biological Macromolecules 50 (2012) 1299–1305
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Fig. 1. The morphology of NH3T3 cells contact with N,O-carboxymethyl chitosan/oxidized alginate hydrogel as function with time.
leaching solution was collected and filtered with 0.22 m membrane for further usage. A 8 ml blood samples was freshly collected from three female normal rabbits into an anticoagulin tube and gently mixed. The pooled blood was diluted with 10 ml saline solution for the further usage. The hemolysis test was performed by method as the following: Briefly, 10 ml of hydrogel extraction, distilled water and saline solution were respectively poured into 50 ml test tube and placed at 37 ◦ C equilibrium for 30 min. After that, 0.2 ml of diluted blood was added to each test tube with gent shake and the resultant solution was placed at 37 ◦ C incubation for another 60 min. Finally, the absorbance of samples was recorded with a UV-visible spectrometer (UV-8000, Shanghai Metash Instrument Co. Ltd.) at 545 nm, and the hemolysis ratio was calculated by the following formula: Hemolysis ratio (%) =
Ahydrogel extraction − Asaline solution Adistilled water − Asaline solution
× 100%
3. Results and discussion 3.1. Gel formation and in vitro cytocompatibility test The novel covalently cross-linked chitosan based hydrogel was formed by simple mixing N,O-carboxymethyl chitosan solution with oxidized alginate solution at room temperature. Because of
the coexistence of amino, hydroxyl, and carboxymethyl groups associated with N,O-carboxymethyl chitosan chain, the plentiful aldehyde and hydroxyl groups along the oxidized alginate chain, the Schiff base as well as hydrogen bond formation were expected after blending N,O-carboxymethyl chitosan and oxidized alginate solutions, yet resulting in the sol–gel transition of system as a function with time. And the more detailed description on the formation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel has been presented in our previous study [14]. Although numerous studies have demonstrated that the chitosan as well as alginate were non-cytotoxic, biodegradable and biocompatible polymer, its derivates should be carefully checked before its further application. The preliminary study on the in vitro cytotoxicity has been demonstrated that the developed hydrogel was non-cytotoxic against NH3T3 cells [14]. Herein, the further study on the cytocompatibility of hydrogel was performed by observing the morphology of NH3T3 cells surrounding or inside the hydrogel as a function with time. As depicted in Fig. 1, it clearly observed that the NH3T3 cells surrounding the gels adhered to, spread and grew on the cell culture dish with the same morphology as those on normal cell culture dishes after 24 h, 48 h and 72 h of seeding, indicating that the developed hydrogel was non-cytotoxic against surrounding NH3T3 cells. However, for the cell of seeding on the completely covered hydrogel, no cells were observed to spread and grew on the hydrogel and the major of cells were found floating and formed cell cluster after
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Fig. 2. Hematoxylin and eosin staining of major organs (cardiac muscle, liver, spleen, lung and kidneys) after intraperitoneal administration of 50 ml/kg N,O-carboxymethyl chitosan/oxidized alginate hydrogel.
24 h, 48 h and 72 h of seeding, indicating that the surface of hydrogel was disadvantage for the cell adhesion and proliferation. However, the result of a lower adhesion of cells on the surface of chitosan derivative based hydrogel was not specific for N,O-carboxymethyl chitosan/oxidized hydrogels. It has been found to be difficult for cells to adhere to and grow on other chitosan and chitosan derivates based hydrogels [15,16]. This might be explained by the excessive hydrophilicity of hydrogel surface was disadvantage for the cell adhesion and proliferation [17,18]. Except that, the NH3T3 cells were also encapsulated into the hydrogel during the hydrogel formation for evaluation the cell adhesion and proliferation in DMEM medium without differentiation factors up to 3 days. According to Fig. 1, it revealed that NH3T3 cells with normal morphology were well spread and grew inside the hydrogel, further supporting that the developed hydrogel with excellent cytocompatibility had the potential application in cell/scaffold. Therefore, we speculated that the covalent cross-linking between N,O-carboxymethyl chitosan and oxidized alginate did not compromise cell viability. Meanwhile, the porous structure of hydrogel provided a pathway that sufficient nutrients and oxygen from the medium could be delivered to the cells inside the hydrogel, yet supporting the cell growth and proliferation, which is in accordance with previous
report [1]. Based on the above studies, it might be concluded that the developed hydrogel with a better cytocompatibility as compared to other injectable hydrogel systems based on methacrylated chitosan might have great potential application in the drug delivery and tissue engineering [19]. 3.2. Acute toxicity test According to the previous study, the acute toxicity test was performed by observing the state of rats as a function with time. No death of all rats was occurred during the whole 21-day periodical study, and no toxic response was found in mice. The rats exhibited normal energy, normal behavior, free movement, and shining hair. The mice were sensitive to sound, light, and other stimulations. There was no flare and no ulceration in the skin. They had no salivation or vomit, no mouth or nose dryness or edema, no running nose or eye secretion. The body weight of rats from N,Ocarboxymethyl chitosan/oxidized alginate hydrogel group showed no significance difference compared with that from the saline solution control group (data not shown). Furthermore, the major organs of rats from each group at specific time point was stained with hematoxylin–eosin for histopathologic examination, as presented
X. Li et al. / International Journal of Biological Macromolecules 50 (2012) 1299–1305
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Fig. 3. In vivo gel-formation and remove process of N,O-carboxymethyl chitosan/oxidized alginate hydrogel as function with time.
in Fig. 2. From Fig. 2, we could clearly observe that cardiac myocytes from hydrogel groups are clear and arranging in good order, and no hemorrhage, necrosis, or inflammatory exudate was observed at 7, 14 and 21 days after administration. For the liver from hydrogel groups, no hepatocellar degeneration or necrosis, and no neutrophil, lymphocyte, and macrophage infiltration was observed not matter at 7 days or 21 days. The normal microstructure of spleen, lung and kidneys tissue from hydrogel groups were also observed no matter at 7 days or 14 and 21 days. All these results suggested that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel was non-toxic for the major organs after 21 days administration suitable for the further various drug delivery application.
early stage [20,21]. Because of the plenty of aminoglucose unite of N,O-carboxymethyl chitosan in the hydrogel, the numerous neutrophil infiltration in the hydrogel might be induced by the N,O-carboxymethyl chitosan component in the hydrogel, which is in accordance with the report of Matsunaga et al. [20]. With the time proceeding, the neutrophil infiltration in the hydrogel was gradually decreased. Seven days later, no neutrophil cells were observed in tissue at injection. However, there was a new fibrous capsule surrounding hydrogels, indicating that N,O-carboxymethyl chitosan/oxidized alginate hydrogel could be slowly degraded or adsorbed by neutrophil cells after S.C. injection. 3.4. Skin irritation test
3.3. Subcutaneous implant test In order to investigate the in vivo destiny as well as biodegradability of the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel, we observed the remove process of hydrogel in situ after S.C. injection as a function with time, as presented in Fig. 3. According to Fig. 3, it clearly observed that the transparent hydrogel was formed in situ after 2 h of S.C. injection, indicating that the quick gelation could take place as placing in physiological temperature and pH condition. Five days later, the transparent hydrogel became opaque gradually combined with the volume decrease as the time proceeding, indicating the degradation or adsorption of hydrogel might be occurred. Seven days later, the beige hydrogel in situ was still observed suggesting the slow degradation or adsorption of hydrogel. The histopathologic examination was also employed to evaluate microscopic changes of the tissue surrounding the inject site. As shown in Fig. 4, it clearly observed that there is numerous neutrophil infiltration in the hydrogel at day 1 after injection, indicating that the degradation or adsorption of hydrogel was occurred. As well known to us, chitosan monomer can induce the migration of polymorphonuclear leukocytes and macrophages in the applied tissue at the
Before humans can be exposed to such substances, the tendency of new chemicals to cause skin irritation must be carefully checked. Assessment of skin irritation potential is an important part of any comprehensive toxicology programme for new chemicals and consumer products. Even today, the final preclinical safety assessment of chemicals is largely based on animal experiments [22]. In this paper, the New Zealand albino rabbit was employed to evaluate the skin irritation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel, and the result were summarized in Table 2. According to Table 2, we could find that the S.C. injection of hydrogel on the rabbit back skin did not induce any cutaneous reaction compared with that of S.C. injection of saline solution (negative control) in all the rabbit at 24, 48 and 72 h of administration. This means that the developed hydrogel was a non skin irritation system, yet could be served as drug carrier for the transdermal/local drug delivery system without any skin irritation. 3.5. Hemolysis test Generally, in vitro erythorocyte-induced hemolysis is considered to be a simple and reliable measure for estimating
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Fig. 4. Hematoxylin and eosin stained sections tissue samples from injection site after N,O-carboxymethyl chitosan/oxidized alginate hydrogel subcutaneous injection (400×).
blood compatibility of materials [23]. The behavior of chitosan formulations in vivo can be predicted by investigating the degree of hemolysis [23,24]. Previous study suggested that the blood compatibility of chitosan and its derivates could be evaluated in terms of hemolysis [25]. Notara et al. [26] has investigated the hemocompatibility of chitosan-alginate physical gel and the result showed that chitosan-alginate physical gel has excellent hemocompatibility suitable for further drug delivery application. Herein, the hemocompatibility of N,O-carboxymethyl chitosan/oxidized alginate hydrogel was preliminary evaluated by a simple colorimetry. As presented in Fig. 5, the blood sample incubation with hydrogel extraction solution showed no evidence of hemolysis, while the obvious hemolysis was observed in distilled water group. More specifically, as presented in Table 3, we could find that the sample from the N,O-carboxymethyl chitosan/oxidized alginate hydrogel group had about 0.97% of hemolysis ratio, which was far smaller than 5% international standard, indicating that the N,Ocarboxymethyl chitosan/oxidized alginate hydrogel has excellent hemocompatibility suitable for various drug delivery applications.
Fig. 5. Hemolysis test on (a) distilled water, (b) saline solution and (c) hydrogel extractions.
Table 2 Cutaneous reactions in rabbit after administration of 0.5 ml N,O-carbomethyl chitosan/oxidized alginate hydrogel as a function with time. Treatment and rabbit (3)
0.5 ml saline solution 1 (right) 2 (right) 3 (right) 0.5 ml N,O-carboxymethyl chitosan/oxidized alginate hydrogel 1 (left) 2 (left) 3 (left)
Time (h) 24
48
72
0 0 0
0 0 0
0 0 0
Table 3 Results of the hemolysis test for N,O-carboxymethyl chitosan/oxidized alginate hydrogel extraction solutions. Groups
0 0 0
0 0 0
0 0 0
Distilled water Saline solution Hydrogel extraction Hemolysis ratio (%)
Samples
Total of hemolysis ratio (%)
1
2
3
0.5257 0.1265 0.1336 1.78
0.5572 0.1226 0.1246 0.46
0.5456 0.1205 0.1247 0.55
0.93 ± 0.73
X. Li et al. / International Journal of Biological Macromolecules 50 (2012) 1299–1305
4. Conclusion In this paper, in vitro and in vivo compatibility of N,Ocarboxymethyl chitosan/oxidized alginate hydrogel was carefully evaluated by means of in vitro cytocompatibility, acute cytotoxicity, subcutaneous injection test, skin irritation test, and hemolysis test before its further various drug delivery applications. In vitro cytocompatibility test revealed that the developed hydrogel was non-cytotoxic and well cytocompatibility against NH3T3 cells after 3 days of seeding. Acute cytotoxicity test suggested that the developed hydrogel was non-toxicity for major organs suitable for the various drug delivery applications, while subcutaneous injection test revealed that the N,O-carboxymethyl chitosan/oxidized alginate hydrogel could slowly degraded or adsorbed after S.C. injection with time evolution. On the other hand, N,O-carboxymethyl chitosan/oxidized alginate hydrogel did not induce any cutaneous reaction after 72 h of the subcutaneous injection in rabbit model and did not cause any hemolysis after co-incubation with the blood solution. All these results strongly suggested that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel was a safe, non-toxic carrier with well in vitro and in vivo compatibility suitable for the various drug delivery applications. Acknowledgement This work was supported by Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents. References
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Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application Xingyi Li a , Xiangye Kong b , Zhaoliang Zhang a , Kaihui Nan a , LingLi Li a , XianHou Wang c , Hao Chen a,∗ a
Institute of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical College, 270 Xueyuan Road, Wenzhou 325027, China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, No. 1, Keyuan 4th Road, Chengdu 610041, China Department of Lymphoma, Sino-US Center for Lymphoma and Leukemia, Tianjin Medical University Cancer Hospital and Institute, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin 300060, China b c
a r t i c l e
i n f o
Article history: Received 12 February 2012 Received in revised form 5 March 2012 Accepted 12 March 2012 Available online 20 March 2012 Keywords: Hydrogel Biocompatibility In vitro In vivo Drug delivery
a b s t r a c t In this paper, covalently cross-linked hydrogel composed of N,O-carboxymethyl chitosan and oxidized alginate was developed intending for drug delivery application. In vitro/vivo cytocompatibility and biocompatibility of the developed hydrogel were preliminary evaluated. In vitro cytocompatibility test showed that the developed hydrogel exhibited good cytocompatibility against NH3T3 cells after 3-day incubation. According to the results of acute toxicity test, there was no obvious cytotoxicity for major organs during the period of 21-day intraperitoneal administration. Meanwhile, the developed hydrogel did not induce any cutaneous reaction within 72 h of subcutaneous injection followed by slow degradation and adsorption with the time evolution. Moreover, the extraction of developed hydrogel had nearly 0% of hemolysis ratio, which indicated the good hemocompatibility of hydrogel. Based on the above results, it may be concluded that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel with non-cytotoxicity and good biocompatibility might suitable for the various drug delivery applications. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In recent years, numerous implant biomaterials including synthetic and natural materials have been widely used in the biomedical and pharmaceutical field [1–3]. Hydrogels are a class of polymers very similar to soft tissue for their high water content, the mechanical properties (low modulus and elasticity), softness, oxygen permeability and excellent biocompatibility. According to the resource of materials, hydrogels can be divided into two classes: synthetic materials based hydrogel and natural materials based hydrogel. In the case of synthetic materials based hydrogels, there are presence of some disadvantages including inflammatory reactions, material migration as well as the difficulty of removal and so on. Recently, much attention have been oriented to the biocompatible, biodegradable hydrogels made from natural polymers that are susceptible to enzymatic degradation [4–6]. Chitosan, the second abundant source in nature after cellulose, is an aminopolysaccharide obtained by the deacetylation of chitin [7–10]. It is composed of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues. There are many parameters
∗ Corresponding author. Tel.: +86 577 88833806; fax: +86 577 88833806. E-mail addresses: [email protected], [email protected] (H. Chen). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2012.03.008
influencing the properties of chitosan including molecular weight (MW), degree of deacetylation (DD) and etc. [8]. Chitosan is water insoluble, but can easily dissolve into some acidic aqueous solution, such as acetic aqueous solution and etc. In order to improve the water solubility of this versatile cationic polysaccharide, several strategies are being made to modify chitosan including PEGtylation, carboxymethylation and etc. realizing its full potential application [7,11]. In recent years, an increasing number of in situ gel systems based on chitosan and its derivatives have been viewed in the literature for various pharmaceutical and biomedical applications [6,12]. Alginic acid is mostly encountered as a high molecular weight linear copolymer composed of (1–4)-linked -d-mannuronic acid (M units) and ␣-l-guluronic acid (G units) monomers. The neutralized form, sodium alginate, as a common thickening agent, has been widely used in the food industry and biomedical field [13]. Chitosan exists as a cationic polyelectrolyte yet such solution are not compatible with aqueous solutions of sodium alginate, which is an anionic polyelectrolyte. Our previous study has demonstrated that the novel chitosan covalent hydrogel based on N,O-carboxymethyl chitosan and oxidized alginate could be gained by simple mixing these two components with an expected weight ratio [14]. Although numerous studies have demonstrated that chitosan and alginate were non-cytotoxic, biodegradable, biocompatible suitable for further various drug delivery applications, its hydrogels
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should be carefully checked before its further various drug delivery applications [7,11]. In this paper, our studies are focused on the cytotoxicity and biocompatibility evaluation of this novel hydrogel by means of the in vitro cytocompatibility, acute cytotoxicity, subcutaneous implant test, skin irritation test, and hemolysis test. 2. Materials and methods 2.1. Materials N,O-carboxymethyl chitosan (the degrees of substitution of carboxymethyl groups on both the amino (N-position) and primary hydroxyl (O-position) sites were approximately 85%) and oxidized alginate (the oxidation degree of alginate was about 27.8%) were successfully synthesized by our previous study [14]. All other chemicals used in this paper were analytic grade. Distilled water from Milli-Q water system was used to prepare the aqueous solutions. 2.2. Preparation of N,O-carboxymethyl chitosan/oxidized alginate auto-gelling system A calculated weight of oxidized alginate and N,O-carboxymethyl chitosan were dissolved into 20 ml distilled water to form 6% (w/w) and 4% (w/w) solutions, respectively. The solutions were filtered and stored at 4 ◦ C overnight for the further usage. Auto gelling solutions were prepared as follows: 2 ml of N,O-carboxymethyl chitosan and oxidized alginate solutions with weight ratio of 1:2 were mixed at room temperature with gentle stirring to form homogeneous solution. After that, the auto-gelling solution was placed at 37 ◦ C for 30 min to form the N,O-carboxymethyl chitosan/oxidized alginate hydrogel. 2.3. In vitro cytocompatibility test The NH3T3 cell was used to assess the in vitro cytocompatibility of hydrogels. The NH3T3 cell was obtained from ACTT (USA) and cultured with DMEM(A) medium at 37 ◦ C and 5% CO2 . N,O-carboxymethyl chitosan and oxidized alginate were sterilized by cobalt-ray prior the test. Briefly, N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution was first prepared by follows: 5 ml of N,O-carboxymethyl chitosan solution and 5 ml of oxidized alginate solution was mixed using a vortex mixer at room temperature. Immediately after mixing, the mixture was poured into a 24-well cell culture dish at 0.6 ml or 0.2 ml for completely or partly covering the bottom of the well, respectively. After incubation at 37 ◦ C for 30 min, the resultant gels adhered to the bottom of wells was rinsed with DMEM medium for three times. NH3T3 cells suspended in the DMEM medium were seeded into each well at 5.0 × 105 cells/well. After 24 h, 48 h and 72 h incubation, the morphology of cells surrounding gel was observed using an optical microscope (Olympus, Japan). In order to further evaluation the cytocompatibility of N,O-carboxymethyl chitosan/oxidized alginate hydrogel, NH3T3 cells (5.0 × 105 cell/well) was pre-mixed with N,O-carboxymethyl chitosan solution and then gelled with oxidized alginate solution at 37 ◦ C for 30 min. Subsequently, the cell supporter was incubated with DMEM medium for 24 h, 48 h and 72 h, respectively. Finally, the morphology of cell inside the hydrogel was observed with an optical microscope (Olympus, Japan). 2.4. Acute toxicity test Ten male and 10 female BALB/c mice, 6 weeks of age (18–22 g), were used to evaluate the acute toxicity of N,O-carboxymethyl chitosan/oxidized alginate hydrogel. All experimental protocols and animal care complied with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources,
Table 1 Skin irritation test: criteria of classification of the cutaneous reactions. Cutaneous reaction
Score
Results
None Sporadic or patchy erythema Moderate confluent erythema Severe erythema and edema
0 1 2 3
Normal Irritation Irritation Irritation
and were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical College. Twenty mice were randomly divided into two groups: a treatment group and a control group, five male and five female mice for each group. The mice of treatment group were injected with 50 ml/kg N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution into the abdominal cavity once, and the mice of control group were injected with 50 ml/kg saline solution into the abdominal cavity once. All the animals were observed continuously for 21 days after the administration, including the general conditions (the activity, energy, hair, feces, behavior pattern, other clinical signs, etc.), body weight, and mortality. At specific time point (7 day, 14 day and 21 day), three mice from each group were sacrificed by cervical dislocation, and its major organs including heart, liver, spleen, lung and kidney were removed and fixed in 10% formaldehyde solution. Finally, the fixed organs were embedded in paraffin, sectioned and stained with hematoxylin–eosin for the histopathologic examination. 2.5. Subcutaneous injection test Twenty four BALB/c mice, 6 weeks of age (18–22 g), were employed for subcutaneous injection of N,O-carboxymethyl chitosan/oxidized alginate hydrogel to evaluate the in vivo degradation behavior. Twenty four mice were randomly divided into two groups: a treatment group and a control group, twelve mice for each group. The mice from treatment group were injected with 0.5 ml N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution in back subcutaneous tissue and the mice from control group were injected with 0.5 ml saline solution in back subcutaneous tissue. Three mice from each group were sacrificed at 1, 3, 5 and 7 day and the injection site were opened with a surgical scissors for observation the state of hydrogel. Meanwhile, the tissue around with the injected site were carefully removed and subsequently fixed in 10% buffered formaldehyde, stained with hematoxylin–eosin for further histopathological examination. 2.6. Skin irritation test Three healthy New Zealand albino rabbits (2.3–2.5 kg), were used to evaluate the skin irritation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel. Briefly, 0.5 ml of N,Ocarboxymethyl chitosan/oxidized alginate auto-gelling solution was directly subcutaneous injected on the left back skin next to backbone and 0.5 ml of saline solution was directly subcutaneous injected on the right back skin next to backbone for control. The cutaneous reaction surrounding the injection site was evaluated at 24 h, 48 h and 72 h, using the criteria has been reported in Table 1. 2.7. Hemolysis test According to guide of biological evaluation of medical device (SFDA, China), the hemolysis of N,O-carboxymethyl chitosan/oxidized alginate hydrogel was carefully checked. Initially, 2 ml of N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution was placed in an test tube at 37 ◦ C for 12 h to form the hydrogel. Subsequently, 20 ml of sterile saline was added into test tube with incubation at 37 ◦ C for another 72 h and then the top of
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Fig. 1. The morphology of NH3T3 cells contact with N,O-carboxymethyl chitosan/oxidized alginate hydrogel as function with time.
leaching solution was collected and filtered with 0.22 m membrane for further usage. A 8 ml blood samples was freshly collected from three female normal rabbits into an anticoagulin tube and gently mixed. The pooled blood was diluted with 10 ml saline solution for the further usage. The hemolysis test was performed by method as the following: Briefly, 10 ml of hydrogel extraction, distilled water and saline solution were respectively poured into 50 ml test tube and placed at 37 ◦ C equilibrium for 30 min. After that, 0.2 ml of diluted blood was added to each test tube with gent shake and the resultant solution was placed at 37 ◦ C incubation for another 60 min. Finally, the absorbance of samples was recorded with a UV-visible spectrometer (UV-8000, Shanghai Metash Instrument Co. Ltd.) at 545 nm, and the hemolysis ratio was calculated by the following formula: Hemolysis ratio (%) =
Ahydrogel extraction − Asaline solution Adistilled water − Asaline solution
× 100%
3. Results and discussion 3.1. Gel formation and in vitro cytocompatibility test The novel covalently cross-linked chitosan based hydrogel was formed by simple mixing N,O-carboxymethyl chitosan solution with oxidized alginate solution at room temperature. Because of
the coexistence of amino, hydroxyl, and carboxymethyl groups associated with N,O-carboxymethyl chitosan chain, the plentiful aldehyde and hydroxyl groups along the oxidized alginate chain, the Schiff base as well as hydrogen bond formation were expected after blending N,O-carboxymethyl chitosan and oxidized alginate solutions, yet resulting in the sol–gel transition of system as a function with time. And the more detailed description on the formation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel has been presented in our previous study [14]. Although numerous studies have demonstrated that the chitosan as well as alginate were non-cytotoxic, biodegradable and biocompatible polymer, its derivates should be carefully checked before its further application. The preliminary study on the in vitro cytotoxicity has been demonstrated that the developed hydrogel was non-cytotoxic against NH3T3 cells [14]. Herein, the further study on the cytocompatibility of hydrogel was performed by observing the morphology of NH3T3 cells surrounding or inside the hydrogel as a function with time. As depicted in Fig. 1, it clearly observed that the NH3T3 cells surrounding the gels adhered to, spread and grew on the cell culture dish with the same morphology as those on normal cell culture dishes after 24 h, 48 h and 72 h of seeding, indicating that the developed hydrogel was non-cytotoxic against surrounding NH3T3 cells. However, for the cell of seeding on the completely covered hydrogel, no cells were observed to spread and grew on the hydrogel and the major of cells were found floating and formed cell cluster after
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Fig. 2. Hematoxylin and eosin staining of major organs (cardiac muscle, liver, spleen, lung and kidneys) after intraperitoneal administration of 50 ml/kg N,O-carboxymethyl chitosan/oxidized alginate hydrogel.
24 h, 48 h and 72 h of seeding, indicating that the surface of hydrogel was disadvantage for the cell adhesion and proliferation. However, the result of a lower adhesion of cells on the surface of chitosan derivative based hydrogel was not specific for N,O-carboxymethyl chitosan/oxidized hydrogels. It has been found to be difficult for cells to adhere to and grow on other chitosan and chitosan derivates based hydrogels [15,16]. This might be explained by the excessive hydrophilicity of hydrogel surface was disadvantage for the cell adhesion and proliferation [17,18]. Except that, the NH3T3 cells were also encapsulated into the hydrogel during the hydrogel formation for evaluation the cell adhesion and proliferation in DMEM medium without differentiation factors up to 3 days. According to Fig. 1, it revealed that NH3T3 cells with normal morphology were well spread and grew inside the hydrogel, further supporting that the developed hydrogel with excellent cytocompatibility had the potential application in cell/scaffold. Therefore, we speculated that the covalent cross-linking between N,O-carboxymethyl chitosan and oxidized alginate did not compromise cell viability. Meanwhile, the porous structure of hydrogel provided a pathway that sufficient nutrients and oxygen from the medium could be delivered to the cells inside the hydrogel, yet supporting the cell growth and proliferation, which is in accordance with previous
report [1]. Based on the above studies, it might be concluded that the developed hydrogel with a better cytocompatibility as compared to other injectable hydrogel systems based on methacrylated chitosan might have great potential application in the drug delivery and tissue engineering [19]. 3.2. Acute toxicity test According to the previous study, the acute toxicity test was performed by observing the state of rats as a function with time. No death of all rats was occurred during the whole 21-day periodical study, and no toxic response was found in mice. The rats exhibited normal energy, normal behavior, free movement, and shining hair. The mice were sensitive to sound, light, and other stimulations. There was no flare and no ulceration in the skin. They had no salivation or vomit, no mouth or nose dryness or edema, no running nose or eye secretion. The body weight of rats from N,Ocarboxymethyl chitosan/oxidized alginate hydrogel group showed no significance difference compared with that from the saline solution control group (data not shown). Furthermore, the major organs of rats from each group at specific time point was stained with hematoxylin–eosin for histopathologic examination, as presented
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Fig. 3. In vivo gel-formation and remove process of N,O-carboxymethyl chitosan/oxidized alginate hydrogel as function with time.
in Fig. 2. From Fig. 2, we could clearly observe that cardiac myocytes from hydrogel groups are clear and arranging in good order, and no hemorrhage, necrosis, or inflammatory exudate was observed at 7, 14 and 21 days after administration. For the liver from hydrogel groups, no hepatocellar degeneration or necrosis, and no neutrophil, lymphocyte, and macrophage infiltration was observed not matter at 7 days or 21 days. The normal microstructure of spleen, lung and kidneys tissue from hydrogel groups were also observed no matter at 7 days or 14 and 21 days. All these results suggested that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel was non-toxic for the major organs after 21 days administration suitable for the further various drug delivery application.
early stage [20,21]. Because of the plenty of aminoglucose unite of N,O-carboxymethyl chitosan in the hydrogel, the numerous neutrophil infiltration in the hydrogel might be induced by the N,O-carboxymethyl chitosan component in the hydrogel, which is in accordance with the report of Matsunaga et al. [20]. With the time proceeding, the neutrophil infiltration in the hydrogel was gradually decreased. Seven days later, no neutrophil cells were observed in tissue at injection. However, there was a new fibrous capsule surrounding hydrogels, indicating that N,O-carboxymethyl chitosan/oxidized alginate hydrogel could be slowly degraded or adsorbed by neutrophil cells after S.C. injection. 3.4. Skin irritation test
3.3. Subcutaneous implant test In order to investigate the in vivo destiny as well as biodegradability of the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel, we observed the remove process of hydrogel in situ after S.C. injection as a function with time, as presented in Fig. 3. According to Fig. 3, it clearly observed that the transparent hydrogel was formed in situ after 2 h of S.C. injection, indicating that the quick gelation could take place as placing in physiological temperature and pH condition. Five days later, the transparent hydrogel became opaque gradually combined with the volume decrease as the time proceeding, indicating the degradation or adsorption of hydrogel might be occurred. Seven days later, the beige hydrogel in situ was still observed suggesting the slow degradation or adsorption of hydrogel. The histopathologic examination was also employed to evaluate microscopic changes of the tissue surrounding the inject site. As shown in Fig. 4, it clearly observed that there is numerous neutrophil infiltration in the hydrogel at day 1 after injection, indicating that the degradation or adsorption of hydrogel was occurred. As well known to us, chitosan monomer can induce the migration of polymorphonuclear leukocytes and macrophages in the applied tissue at the
Before humans can be exposed to such substances, the tendency of new chemicals to cause skin irritation must be carefully checked. Assessment of skin irritation potential is an important part of any comprehensive toxicology programme for new chemicals and consumer products. Even today, the final preclinical safety assessment of chemicals is largely based on animal experiments [22]. In this paper, the New Zealand albino rabbit was employed to evaluate the skin irritation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel, and the result were summarized in Table 2. According to Table 2, we could find that the S.C. injection of hydrogel on the rabbit back skin did not induce any cutaneous reaction compared with that of S.C. injection of saline solution (negative control) in all the rabbit at 24, 48 and 72 h of administration. This means that the developed hydrogel was a non skin irritation system, yet could be served as drug carrier for the transdermal/local drug delivery system without any skin irritation. 3.5. Hemolysis test Generally, in vitro erythorocyte-induced hemolysis is considered to be a simple and reliable measure for estimating
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Fig. 4. Hematoxylin and eosin stained sections tissue samples from injection site after N,O-carboxymethyl chitosan/oxidized alginate hydrogel subcutaneous injection (400×).
blood compatibility of materials [23]. The behavior of chitosan formulations in vivo can be predicted by investigating the degree of hemolysis [23,24]. Previous study suggested that the blood compatibility of chitosan and its derivates could be evaluated in terms of hemolysis [25]. Notara et al. [26] has investigated the hemocompatibility of chitosan-alginate physical gel and the result showed that chitosan-alginate physical gel has excellent hemocompatibility suitable for further drug delivery application. Herein, the hemocompatibility of N,O-carboxymethyl chitosan/oxidized alginate hydrogel was preliminary evaluated by a simple colorimetry. As presented in Fig. 5, the blood sample incubation with hydrogel extraction solution showed no evidence of hemolysis, while the obvious hemolysis was observed in distilled water group. More specifically, as presented in Table 3, we could find that the sample from the N,O-carboxymethyl chitosan/oxidized alginate hydrogel group had about 0.97% of hemolysis ratio, which was far smaller than 5% international standard, indicating that the N,Ocarboxymethyl chitosan/oxidized alginate hydrogel has excellent hemocompatibility suitable for various drug delivery applications.
Fig. 5. Hemolysis test on (a) distilled water, (b) saline solution and (c) hydrogel extractions.
Table 2 Cutaneous reactions in rabbit after administration of 0.5 ml N,O-carbomethyl chitosan/oxidized alginate hydrogel as a function with time. Treatment and rabbit (3)
0.5 ml saline solution 1 (right) 2 (right) 3 (right) 0.5 ml N,O-carboxymethyl chitosan/oxidized alginate hydrogel 1 (left) 2 (left) 3 (left)
Time (h) 24
48
72
0 0 0
0 0 0
0 0 0
Table 3 Results of the hemolysis test for N,O-carboxymethyl chitosan/oxidized alginate hydrogel extraction solutions. Groups
0 0 0
0 0 0
0 0 0
Distilled water Saline solution Hydrogel extraction Hemolysis ratio (%)
Samples
Total of hemolysis ratio (%)
1
2
3
0.5257 0.1265 0.1336 1.78
0.5572 0.1226 0.1246 0.46
0.5456 0.1205 0.1247 0.55
0.93 ± 0.73
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4. Conclusion In this paper, in vitro and in vivo compatibility of N,Ocarboxymethyl chitosan/oxidized alginate hydrogel was carefully evaluated by means of in vitro cytocompatibility, acute cytotoxicity, subcutaneous injection test, skin irritation test, and hemolysis test before its further various drug delivery applications. In vitro cytocompatibility test revealed that the developed hydrogel was non-cytotoxic and well cytocompatibility against NH3T3 cells after 3 days of seeding. Acute cytotoxicity test suggested that the developed hydrogel was non-toxicity for major organs suitable for the various drug delivery applications, while subcutaneous injection test revealed that the N,O-carboxymethyl chitosan/oxidized alginate hydrogel could slowly degraded or adsorbed after S.C. injection with time evolution. On the other hand, N,O-carboxymethyl chitosan/oxidized alginate hydrogel did not induce any cutaneous reaction after 72 h of the subcutaneous injection in rabbit model and did not cause any hemolysis after co-incubation with the blood solution. All these results strongly suggested that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel was a safe, non-toxic carrier with well in vitro and in vivo compatibility suitable for the various drug delivery applications. Acknowledgement This work was supported by Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents. References
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