Elsevier

Carbohydrate Polymers

Volume 103, 15 March 2014, Pages 110-118
Carbohydrate Polymers

Cytocompatible injectable carboxymethyl chitosan/N-isopropylacrylamide hydrogels for localized drug delivery

https://doi.org/10.1016/j.carbpol.2013.12.017Get rights and content

Highlights

  • First synthesis of injectable hydrogels from carboxymethyl chitosan/PNIPAm.

  • These hydrogels showing good pH/thermo-responsive swelling behavior.

  • Great promise of these hydrogels as localized drug delivery systems.

  • Excellent cytocompatibility of the hydrogels via live/dead and alamarBlue test.

Abstract

Cytocompatible injectable hydrogels with pH and temperature sensitivity based on carboxymethyl chitosan-graft-poly (N-isopropyl acrylamide)-glycidyl methacrylate (CMCS-PNIPAm-GMA) were prepared by UV crosslinking, and these hydrogels as localized drug carriers for anticancer drug and anti-inflammatory drug were also investigated. The chemical structure of CMCS-PNIPAm-GMA and of their hydrogels was characterized by FT-IR and NMR. The effect of PNIPAm grafting percentage, pH and temperature on the swelling ratio of the hydrogels was studied, demonstrating the pH/temperature-responsive nature of the hydrogels. The morphology of the hydrogels before and after swelling was observed by scanning electron microscope. 5-Fluorouracil and diclofenac sodium as model drugs were encapsulated into the hydrogels in situ. Moreover, the effect of pH and temperature on the release of these drugs was discussed. The cytocompatibility of the macromonomer CMCS-PNIPAm-GMA and their hydrogels was studied with dog bone marrow mesenchymal stem cells by using Alamar blue measurement and Live/Dead assay kit. All the results indicated that these degradable injectable hydrogels are good candidates for localized delivery systems of drugs.

Introduction

Biocompatible and biodegradable injectable hydrogels formed via in situ chemical polymerization or via the sol–gel phase transition have gained more and more attention recently (Du et al., 2012, Ko et al., 2013, Tan and Marra, 2010). They have been found wide application in biomedical fields, being used as drug delivery systems, cell carriers and scaffolds for tissue regeneration (Delair, 2012, Li et al., 2012a, Li et al., 2012b, Nakai et al., 2012, Yu and Ding, 2008). The hydrogel precursors loaded with bioactive molecules such as drugs or growth factors were flowable aqueous solutions before injection, and they rapidly changed into gel under physiological conditions once injected. Therefore, injectable hydrogels containing drugs can be operated in a minimally invasive manner, causing less pain for patients. Following gelation, these matrices become specific-site drug delivery depots for pharmaceutics. Many methods have been developed for fabrication of in situ forming injectable hydrogels, such as thermal gelation, ionic complex, self-assembly, chemical crosslinking and photopolymerization (Overstreet et al., 2012, Yu and Ding, 2008). Among these methods, photopolymerization is one of the green techniques, which has good spatial and temporal control over the gelation process under sufficiently mild or physiological conditions (Ifkovits and Burdick, 2007, Zhang et al., 2011). Photopolymerization technique has already been employed to synthesize functional polymers and hydrogels for biomedical applications by us (Guo et al., 2011a, Guo et al., 2011c, Guo et al., 2012) and others (Di Biase et al., 2011, Ortiz et al., 2010). It has been reported that the UV or visible light could penetrate the surface of the skin to form in situ hydrogels after the macromer solution was injected subcutaneously (Anseth et al., 2002, Elisseeff et al., 1999a, Elisseeff et al., 1999b). This means the hydrogels can be formed in vivo by transdermal photopolymerization with minimally invasive implantation (Elisseeff et al., 1999a, Elisseeff et al., 1999b, Lin et al., 2013). Moreover, photocrosslinking of water soluble macromonomers can avoid the presence of the unreacted residual monomers and the use of organic solvent in the drug loading process (Ito, 1998).

During the past decades, various naturally and synthetically derived materials were utilized to prepare injectable hydrogels for drug delivery systems (Kim et al., 2012, Rahman et al., 2012). Biopolymers have been extensively used as injectable hydrogels for drug carries due to their excellent biocompatibility and biodegradability (Barbucci et al., 2010, Gao et al., 2010, Zhou et al., 2011). Chitosan is one of the most abundant biopolymers. It contains one amino group and two hydroxyl groups in the repeating glucosidic residue (Bernkop-Schnurch and Dunnhaupt, 2012, Guo et al., 2011b, Hu et al., 2013, Yu et al., 2011). However, chitosan can just dissolve in acid solution, which greatly restricts its application. Various chitosan derivatives have been therefore developed to increase its water solubility at physiological pH condition (Faizuloev et al., 2012, Guo et al., 2007b, Guo et al., 2008b). Carboxymethyl chitosan (CMCS) is not only soluble in water, but also has excellent chemical, physical and biological properties including low toxicity, biocompatibility and film, gel-forming capabilities. It has been widely used in biomedical fields such as drug delivery carrier, antimicrobial material, gene delivery system and tissue engineering (Guo et al., 2008a, Jayakumar et al., 2010, Upadhyaya et al., 2013). Furthermore, it shows good pH and ion sensitivity in aqueous solutions because it simultaneously contains amino groups (single bondNH2) and carboxyl groups (single bondCOOH) (Chen et al., 2004, Guo et al., 2007c).

Smart hydrogels or intelligent hydrogels which change their various properties upon environmental stimuli including pH or/and temperature, ionic strength, light, electric field and magnetic field, have found wide application in biomedical and pharmaceutical areas (Ajazuddin et al., 2012, Guo et al., 2007a, Qiu and Park, 2012, Schmalz et al., 2012). Among these systems, pH and temperature responsive hydrogels have been most extensively studied because these two factors are crucial to human body and can be easily controlled both in vitro and in vivo conditions (Carreira et al., 2010, Li et al., 2012a, Li et al., 2012b, Mao et al., 2011). Poly(N-isopropylacrylamide) (PNIPAm) is one of the most well-known temperature sensitive polymers with a lower critical solution temperature (LCST) at 32 °C around body temperature (Iijima and Nagasaki, 2006, Wei et al., 2009). The combination of CMCS and PNIPAm has been investigated by some researchers (Chen et al., 2010, Zhang et al., 2009) and our group (Guo & Gao, 2007). Zhong's group prepared thermo-responsive core-shell microgels based on PNIPAm and CMCS by emulsion polymerization (Chen et al., 2010). We synthesized interpenetrating polymer networks based on CMCS and PNIPAm by using ammonium persulphate as initiator and N,N′-methylenebisacrylamide as crosslinker, and studied their controlled release of coenzyme A (Guo & Gao, 2007). However, in situ forming injectable hydrogels based on CMCS and PNIPAm have not been reported.

The aim of this work is to develop cytocompatible pH/thermo-responsive injectable hydrogels based on carboxylmethyl chitosan and poly(N-isopropyl acrylamide) by photocrosslinking for localized drug delivery. We first grafted PNIPAm onto CMCS and then used ring opening reaction of epoxy group from glycidyl methacrylate (GMA) to introduce double bonds to the CMCS-PNIPAm copolymer. The CMCS-PNIPAm-GMA hydrogels were obtained from the aqueous solution of the macromer CMCS-PNIPAm-GMA in situ by photocrosslinking reaction. The chemical structure, swelling behavior, morphology, drug delivery and cell cytotoxicity of the injectable hydrogels were investigated. By developing intelligent injectable hydrogels as localized drug delivery systems from CMCS and PNIPAm, we are taking next step toward the practical applications of these hydrogels.

Section snippets

Materials

Chitosan (CS, molecular weight 100000–300000) with a degree of deacetylation of 86% and N-isopropylacrylamide (NIPAm) were obtained from J&K Scientific Ltd. Monochloroacetic acid, ammonium persulphate ((NH4)2S2O8, APS), glycidyl methacrylate (GMA), photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), diclofenac sodium (DCS) and 5-flurouracil (5-Fu) were all purchased from Aldrich and were used as received. Sodium hydroxide (NaOH), isopropanol, anhydrous ethanol and

Synthesis of the injectable hydrogels by photocrosslinking

The NIPAm was grafted to CMCS main chain by using APS as an initiator. The grafting efficiency was between 32.4% and 41.4%, and the grafting percentage was 80.1%, 134.5% and 172.4% for samples G1, G2 and G3, respectively (Table 1), indicating that the grafting percentage onto the CMCS increased with the increase of the NIPAm concentration. Therefore, we could control the grafting percentage of NIPAm onto the CMCS main chain in a wide range by using different APS amounts in the reaction system.

Conclusions

We demonstrated that the feasibility by employing cytocompatible pH/temperature-responsive injectable hydrogels composed of carboxymethyl chitosan (CMCS), poly (N-isopropyl acrylamide) (PNIPAm) and glycidyl methacrylate (GMA) as localized drug delivery system for anticancer drug and anti-inflammatory drug. The injectable hydrogels were synthesized by photocrosslinking of the CMCS-PNIPAm-GMA aqueous solution, and the anticancer drug 5-fluorouracil (5-Fu) and anti-inflammatory drug diclofenac

Acknowledgment

The authors gratefully acknowledge Xi’an Jiaotong Univerisity for financial support of this work.

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