International Journal of Biological Macromolecules 50 (2012) 1299–1305Contents lists available at SciVerse ScienceDirectInternational Journal of BiologicalMacromoleculesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /i j b i o m acCytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery applicationXingyi Li a ,Xiangye Kong b ,Zhaoliang Zhang a ,Kaihui Nan a ,LingLi Li a ,XianHou Wang c ,Hao Chen a ,∗aInstitute of Biomedical Engineering,School of Ophthalmology &Optometry and Eye Hospital,Wenzhou Medical College,270Xueyuan Road,Wenzhou 325027,ChinabState Key Laboratory of Biotherapy and Cancer Center,West China Hospital,West China Medical School,Sichuan University,No.1,Keyuan 4th Road,Chengdu 610041,China cDepartment 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,Chinaa r t i c l ei n f oArticle history:Received 12February 2012Received in revised form 5March 2012Accepted 12March 2012Available online 20 March 2012Keywords:HydrogelBiocompatibility In vitro In vivoDrug deliverya b s t r a c tIn 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 bio-compatibility of the developed hydrogel were preliminary evaluated.In vitro cytocompatibility test showed that the developed hydrogel exhibited good cytocompatibility against NH3T3cells 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 72h of subcutaneous injection followed by slow degra-dation 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.IntroductionIn 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,oxy-gen 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 reac-tions,material migration as well as the difficulty of removal and so on.Recently,much attention have been oriented to the biocompat-ible,biodegradable hydrogels made from natural polymers that are susceptible to enzymatic degradation [4–6].Chitosan,the second abundant source in nature after cellu-lose,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.:+8657788833806;fax:+8657788833806.E-mail addresses:dragonhaochen@ ,chehao@ (H.Chen).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 solu-tion,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 sys-tems 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 neutral-ized form,sodium alginate,as a common thickening agent,has been widely used in the food industry and biomedical field [13].Chi-tosan 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 mix-ing these two components with an expected weight ratio [14].Although numerous studies have demonstrated that chitosan and alginate were non-cytotoxic,biodegradable,biocompatible suit-able for further various drug delivery applications,its hydrogels0141-8130/$–see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.ijbiomac.2012.03.0081300X.Li et al./International Journal of Biological Macromolecules50 (2012) 1299–1305should be carefully checked before its further various drug deliv-ery applications[7,11].In this paper,our studies are focused on the cytotoxicity and biocompatibility evaluation of this novel hydro-gel by means of the in vitro cytocompatibility,acute cytotoxicity, subcutaneous implant test,skin irritation test,and hemolysis test.2.Materials and methods2.1.MaterialsN,O-carboxymethyl chitosan(the degrees of substitution of car-boxymethyl groups on both the amino(N-position)and primary hydroxyl(O-position)sites were approximately85%)and oxidized alginate(the oxidation degree of alginate was about27.8%)were successfully synthesized by our previous study[14].All other chem-icals 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 systemA calculated weight of oxidized alginate and N,O-carboxymethyl chitosan were dissolved into20ml distilled water to form6%(w/w) and4%(w/w)solutions,respectively.The solutions werefiltered and stored at4◦C overnight for the further usage.Auto gelling solutions were prepared as follows:2ml of N,O-carboxymethyl chi-tosan and oxidized alginate solutions with weight ratio of1:2were mixed at room temperature with gentle stirring to form homoge-neous solution.After that,the auto-gelling solution was placed at 37◦C for30min to form the N,O-carboxymethyl chitosan/oxidized alginate hydrogel.2.3.In vitro cytocompatibility testThe NH3T3cell was used to assess the in vitro cytocompat-ibility of hydrogels.The NH3T3cell was obtained from ACTT (USA)and cultured with DMEM(A)medium at37◦C and5%CO2. N,O-carboxymethyl chitosan and oxidized alginate were ster-ilized by cobalt-ray prior the test.Briefly,N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution wasfirst prepared by follows:5ml of N,O-carboxymethyl chitosan solution and5ml of oxidized alginate solution was mixed using a vortex mixer at room temperature.Immediately after mixing,the mixture was poured into a24-well cell culture dish at0.6ml or0.2ml for com-pletely or partly covering the bottom of the well,respectively.After incubation at37◦C for30min,the resultant gels adhered to the bottom of wells was rinsed with DMEM medium for three times. NH3T3cells suspended in the DMEM medium were seeded into each well at5.0×105cells/well.After24h,48h and72h incuba-tion,the morphology of cells surrounding gel was observed using an optical microscope(Olympus,Japan).In order to further evalua-tion the cytocompatibility of N,O-carboxymethyl chitosan/oxidized alginate hydrogel,NH3T3cells(5.0×105cell/well)was pre-mixed with N,O-carboxymethyl chitosan solution and then gelled with oxidized alginate solution at37◦C for30min.Subsequently,the cell supporter was incubated with DMEM medium for24h,48h and 72h,respectively.Finally,the morphology of cell inside the hydro-gel was observed with an optical microscope(Olympus,Japan).2.4.Acute toxicity testTen male and10female BALB/c mice,6weeks of age(18–22g), 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,Table1Skin irritation test:criteria of classification of the cutaneous reactions.Cutaneous reaction Score Results None0Normal Sporadic or patchy erythema1Irritation Moderate confluent erythema2Irritation Severe erythema and edema3Irritationand were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical College.Twenty mice were ran-domly divided into two groups:a treatment group and a control group,five male andfive female mice for each group.The mice of treatment group were injected with50ml/kg N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution into the abdomi-nal cavity once,and the mice of control group were injected with 50ml/kg saline solution into the abdominal cavity once.All the animals were observed continuously for21days after the adminis-tration,including the general conditions(the activity,energy,hair, feces,behavior pattern,other clinical signs,etc.),body weight,and mortality.At specific time point(7day,14day and21day),three mice from each group were sacrificed by cervical dislocation,and its major organs including heart,liver,spleen,lung and kidney were removed andfixed in10%formaldehyde solution.Finally,the fixed organs were embedded in paraffin,sectioned and stained with hematoxylin–eosin for the histopathologic examination.2.5.Subcutaneous injection testTwenty four BALB/c mice,6weeks of age(18–22g),were employed for subcutaneous injection of N,O-carboxymethyl chi-tosan/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 with0.5ml N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solu-tion in back subcutaneous tissue and the mice from control group were injected with0.5ml saline solution in back subcutaneous tis-sue.Three mice from each group were sacrificed at1,3,5and7day and the injection site were opened with a surgical scissors for obser-vation the state of hydrogel.Meanwhile,the tissue around with the injected site were carefully removed and subsequentlyfixed in10%buffered formaldehyde,stained with hematoxylin–eosin for further histopathological examination.2.6.Skin irritation testThree healthy New Zealand albino rabbits(2.3–2.5kg),were used to evaluate the skin irritation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel.Briefly,0.5ml of N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution was directly subcutaneous injected on the left back skin next to backbone and0.5ml 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 24h,48h and72h,using the criteria has been reported in Table1.2.7.Hemolysis testAccording to guide of biological evaluation of medical device(SFDA,China),the hemolysis of N,O-carboxymethyl chi-tosan/oxidized alginate hydrogel was carefully checked.Initially, 2ml of N,O-carboxymethyl chitosan/oxidized alginate auto-gelling solution was placed in an test tube at37◦C for12h to form the hydrogel.Subsequently,20ml of sterile saline was added into test tube with incubation at37◦C for another72h and then the top ofX.Li et al./International Journal of Biological Macromolecules50 (2012) 1299–13051301Fig.1.The morphology of NH3T3cells contact with N,O-carboxymethyl chitosan/oxidized alginate hydrogel as function with time. leaching solution was collected andfiltered with0.22␮m mem-brane for further usage.A8ml blood samples was freshly collected from three femalenormal rabbits into an anticoagulin tube and gently mixed.Thepooled blood was diluted with10ml saline solution for the furtherusage.The hemolysis test was performed by method as the follow-ing:Briefly,10ml of hydrogel extraction,distilled water and salinesolution were respectively poured into50ml test tube and placedat37◦C equilibrium for30min.After that,0.2ml of diluted bloodwas added to each test tube with gent shake and the resultant solu-tion was placed at37◦C incubation for another60min.Finally,theabsorbance of samples was recorded with a UV-visible spectrome-ter(UV-8000,Shanghai Metash Instrument Co.Ltd.)at545nm,andthe hemolysis ratio was calculated by the following formula:Hemolysis ratio(%)=A hydrogel extraction−A saline solutionA distilled water−A saline solution×100%3.Results and discussion3.1.Gel formation and in vitro cytocompatibility testThe 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 for-mation 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 demon-strated that the developed hydrogel was non-cytotoxic against NH3T3cells[14].Herein,the further study on the cytocompati-bility of hydrogel was performed by observing the morphology of NH3T3cells surrounding or inside the hydrogel as a function with time.As depicted in Fig.1,it clearly observed that the NH3T3cells surrounding the gels adhered to,spread and grew on the cell cul-ture dish with the same morphology as those on normal cell culture dishes after24h,48h and72h of seeding,indicating that the devel-oped hydrogel was non-cytotoxic against surrounding NH3T3cells. However,for the cell of seeding on the completely covered hydro-gel,no cells were observed to spread and grew on the hydrogel and the major of cells were foundfloating and formed cell cluster after1302X.Li et al./International Journal of Biological Macromolecules 50 (2012) 1299–1305Fig.2.Hematoxylin and eosin staining of major organs (cardiac muscle,liver,spleen,lung and kidneys)after intraperitoneal administration of 50ml/kg N,O-carboxymethyl chitosan/oxidized alginate hydrogel.24h,48h and 72h 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 NH3T3cells were also encapsulated into the hydrogel during the hydrogel for-mation for evaluation the cell adhesion and proliferation in DMEM medium without differentiation factors up to 3days.According to Fig.1,it revealed that NH3T3cells with normal morphology were well spread and grew inside the hydrogel,further support-ing that the developed hydrogel with excellent cytocompatibility had the potential application in cell/scaffold.Therefore,we specu-lated 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 previousreport [1].Based on the above studies,it might be concluded that the developed hydrogel with a better cytocompatibility as com-pared 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 testAccording to the previous study,the acute toxicity test was per-formed 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 exhib-ited normal energy,normal behavior,free movement,and shining hair.The mice were sensitive to sound,light,and other stimula-tions.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,O-carboxymethyl chitosan/oxidized alginate hydrogel group showed no significance difference compared with that from the saline solu-tion 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 presentedX.Li et al./International Journal of Biological Macromolecules50 (2012) 1299–13051303Fig.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 at7, 14and21days after administration.For the liver from hydrogel groups,no hepatocellar degeneration or necrosis,and no neu-trophil,lymphocyte,and macrophage infiltration was observed not matter at7days or21days.The normal microstructure of spleen, lung and kidneys tissue from hydrogel groups were also observed no matter at7days or14and21days.All these results suggested that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel was non-toxic for the major organs after21days admin-istration suitable for the further various drug delivery application.3.3.Subcutaneous implant testIn order to investigate the in vivo destiny as well as biodegrad-ability 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 trans-parent hydrogel was formed in situ after2h 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 degrada-tion 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 exam-ination 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 day1after injection,indicating that the degradation or adsorption of hydrogel was occurred.As well known to us, chitosan monomer can induce the migration of polymorphonu-clear leukocytes and macrophages in the applied tissue at the 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 newfibrous 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 testBefore 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 con-sumer products.Even today,thefinal 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 Table2.According to Table2,we couldfind 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 at24,48and72h 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 testGenerally,in vitro erythorocyte-induced hemolysis is con-sidered to be a simple and reliable measure for estimating1304X.Li et al./International Journal of Biological Macromolecules 50 (2012) 1299–1305Fig.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 com-patibility of chitosan and its derivates could be evaluated in terms of hemolysis [25].Notara et al.[26]has investigated the hemocom-patibility of chitosan-alginate physical gel and the result showed that chitosan-alginate physical gel has excellent hemocompati-bility suitable for further drug delivery application.Herein,the hemocompatibility of N,O-carboxymethyl chitosan/oxidized algi-nate hydrogel was preliminary evaluated by a simple colorimetry.As presented in Fig.5,the blood sample incubation with hydro-gel 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,O-carboxymethyl chitosan/oxidized alginate hydrogel has excellent hemocompatibility suitable for various drug delivery applications.Table 2Cutaneous reactions in rabbit after administration of 0.5ml N,O-carbomethyl chi-tosan/oxidized alginate hydrogel as a function with time.Treatment and rabbit (3)Time (h)2448720.5ml saline solution 1(right)0002(right)0003(right)0.5ml N,O-carboxymethyl chitosan/oxidized alginate hydrogel 1(left)0002(left)0003(left)Fig.5.Hemolysis test on (a)distilled water,(b)saline solution and (c)hydrogel extractions.Table 3Results of the hemolysis test for N,O-carboxymethyl chitosan/oxidized alginate hydrogel extraction solutions.GroupsSamplesTotal of hemolysis ratio (%)123Distilled water 0.52570.55720.5456Saline solution0.12650.12260.12050.93±0.73Hydrogel extraction 0.13360.12460.1247Hemolysis ratio (%)1.780.460.55X.Li et al./International Journal of Biological Macromolecules50 (2012) 1299–130513054.ConclusionIn this paper,in vitro and in vivo compatibility of N,O-carboxymethyl chitosan/oxidized alginate hydrogel was carefully evaluated by means of in vitro cytocompatibility,acute cytotoxic-ity,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 NH3T3cells after 3days of seeding.Acute cytotoxicity test suggested that the devel-oped 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 algi-nate hydrogel could slowly degraded or adsorbed after S.C.injection with time evolution.On the other hand,N,O-carboxymethyl chi-tosan/oxidized alginate hydrogel did not induce any cutaneous reaction after72h 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.AcknowledgementThis work was supported by Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents.References[1]R.Jin,L.S.Moreira Teixeira,P.J.Dijkstra,M.Karperien,C.A.Van Blitterswijk,Z.Y.Zhong,J.Feijen,Biomaterials30(2009)2544–2551.[2]H.Zitter,H.Plenk Jr.,J.Biomed.Mater.Res.21(1987)881–896.[3]S.Ozawa,S.Kasugai,Biomaterials17(1996)23–29.[4]N.Bhattarai,J.Gunn,M.Zhang,Adv.Drug 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