Cysteine conjugated chitosan based green nanohybrid hydrogel embedded with zinc oxide nanoparticles towards enhanced therapeutic potential of naringenin

https://doi.org/10.1016/j.reactfunctpolym.2020.104480Get rights and content

Abstract

This article reports the role of l-Cysteine (CYS) conjugated nanohybrid hydrogel carrier for the enhanced therapeutic delivery of the Naringenin (NRG). CYS was effectively conjugated with chitosan through the amidation reaction followed by the incorporation of phyto-synthesised zinc oxide nanoparticles (ZNPs). Dialdehyde cellulose (DAC), a green crosslinker derived from Sugarcane Bagasse (SCB) crosslinked the modified chitosan. Taguchi method optimised the NRG loading in the hydrogel carrier. The hybrid material was characterized using 1H and 13C NMR, FTIR, XRD, SEM and swelling studies. The hybrid hydrogel, loaded with 86.09% of NRG was further subjected to drug release studies at varying pH and NRG loading concentrations. Maximum release of 72.78% was obtained for 1 mg/mL of initial drug concentration at pH 5. CYS conjugation with chitosan stabilized the hydrogel and enabled a sustained release of NRG drug. Kinetic modelling predicted the NRG release to follow a non-Fickian diffusion along with polymer erosion. The antimicrobial activity was studied against the Staphylococcus aureus and Trichophyton rubrum strains. Biocompatibility assay of the materials with L929 cells revealed significant cell viability. The NRG delivery using the developed nanohybrid hydrogel exhibited a two-fold increase in cytotoxicity towards A431 human skin carcinoma cells compared to NRG delivery without carrier.

Introduction

Biopolymers based drug carriers are excellent in biocompatibility, biodegradability, cost-effectiveness and eco-affability. These properties make them suitable in the field of pharmaceutical industry towards drug formulation and delivery. Among the biopolymer sources, chitosan which is an amino-polysaccharide of glucosamine and N-acetylglucosamine units and a derivative of chitin, the second most abundant biopolymer next to cellulose is highly preferred in the field of drug delivery [1]. Chitin present in certain fungal cell walls and the exoskeleton of crustaceans such as shrimps and crabs is derivatized to chitosan by the alkaline deacetylation process [2]. In addition to the traditional biopolymer characteristics, chitosan possesses good mucoadhesion, antimicrobial activity, hemostatic and wound healing properties. Its defined chemical structure rich in hydroxyl and amino groups provides greater scope for chemical modifications of the polymer. Appropriate modifications with desired functional groups reinforce unique physicochemical and biological properties to the polymer. Some chitosan derivatives discussed in literature include quaternized chitosan, N alkyl chitosan, carboxyl alkyl chitosan, acyl chitosan, thiolated chitosan, sulphonated chitosan and phosphorylated chitosan [3]. Quaternized chitosan showed improvement in the antimicrobial activity of chitosan whereas the thiolation process improved the muco-adhesive characteristics [4,5]. The carboxyl chitosan with both carboxyl and amino groups is of high interest as a drug carrier towards controlled or sustained drug-delivery process [6].

The functionalized chitosan polymer thus can be used for the preparation of various drug dosage forms with such as microcapsules, nanoemulsions, nanoparticles and hydrogels etc. The development of such hybrid carrier with different functional constituents synergize the efficient drug encapsulation and delivery, thereby reducing the half maximal inhibitory concentrations values (IC50 or ID50) with enhanced therapeutic potential of the concerned drug. In this regard, hybridisation of the carrier using inorganic metal/metal oxide nanoparticles is of a great therapeutic benefit [7]. Hydrogel based drug carriers are preferred for their stimuli based sensitivity, epidermal biocompatibility and eco-friendly degradation [8,9]. These hybrid hydrogels are prepared from the biopolymers such as chitosan using various crosslinking techniques. DAC based crosslinking was recently discussed as an economic and non-toxic substitute to the conventional chemical crosslinkers such as glutaraldehyde, glyoxal, sodium tripolyphosphate etc. [[10], [11], [12]]. Recent studies on carrier mediated delivery of polyphenol drugs reported the increase in bioavailability of the phyto-derived drugs, which also improved the Absorption, Distribution, Metabolism, and Elimination (ADME) profile and the therapeutic efficacy of the drugs, ultimately [13,14].

In the present work, chitosan biopolymer was conjugated with CYS amino acid. The presence of thiol group in CYS in addition to the amino and carboxyl group enforce multiple functional group characteristics to the modified chitosan for better drug loading. The CYS conjugation was reported to improve the solubility of the carrier and also the mucoadhesiveness, enzyme inhibitory properties including the other biomolecular interactions [15]. CYS as the key role precursor of the antioxidant Glutathione (GSH), protects erythrocytes from oxidative damage and exhibits a cumulative effect towards the Ultraviolet (UV) induced radical scavenging, anti-coagulative and antimicrobial activities [16]. Thus CYS conjugated chitosan (CH-CYS) was chosen as a polymer base in the present work for the development of a green nanohybrid hydrogel. The developed hydrogel carrier was compared with pure chitosan based system described in our previous work for their enhanced physicochemical and therapeutic properties [17]. The CH-CYS was further hybridized with ZNPs, that are phyto-synthesised from muskmelon seeds extract. The addition of these inorganic nanoparticles are expected to increase the electrostatic interactions between the drug - carrier macromolecules for effective drug encapsulation and sustained drug delivery. The CH-CYS-ZNPs polymer matrix was crosslinked using green crosslinker DAC, which was prepared by peroxidation of the treated SCB. The newly developed hybrid hydrogel carrier (CYS-CHGZ) was directly compared with the pure chitosan – cellulose/ZNPs carrier (CHGZ) for their physicochemical characteristics and drug delivery properties. NRG, with strong anti-inflammatory and anti-oxidant characteristics and a principal dietary flavanone, which is highly abundant in citrus fruits was chosen as the model drug [18]. The efficiency of NRG loading in the above nanohybrid hydrogel was optimised and the release kinetics were studied. Biological studies including biocompatibility, antimicrobial and anticancer (skin carcinoma) activities were performed to study the nature of the hybrid hydrogel in NRG drug delivery.

Section snippets

Materials

Muskmelon seeds and SCB were collected from a juice stalls in the nearby locality. Low molecular weight chitosan (85% degree of deacetylation), CYS and NRG were procured from Sigma–Aldrich Chemie, Steinheim, Germany. Sodium hydroxide, sodium chlorite, glacial acetic acid and silicon oil were supplied by Loba Chemie Pvt. Ltd., Mumbai, India. Hydrogen peroxide, sodium metaperiodate, triethylamine, zinc acetate dihydrate, potassium chloride, hydrochloric acid, acetone, potassium dihydrogen

Formulation of CYS-CHGZ

A diagramatic representation for the formulation of organic/inorganic functionalized nanohybrid hydrogel based drug carrier system is represented in Scheme 1. The amidation reaction between the amine groups of chitosan and carboxyl groups of CYS yielded the CYS conjugated CH-CYS. Triethylamine, a deprotonating base activated the amine groups of chitosan. In addition, EDC activated the carboxylic acid groups of CYS and together they enhanced the conjugation of CYS on the backbone of chitosan [24

Conclusion

This work revealed the prospective application of cysteine-chitosan conjugation for the development of a completely bio-derived hydrogel based drug carrier. Various characterisation techniques performed for the developed drug – carrier formulation ensured the ZNPs embedment, CYS conjugation, hydrogel formation, stability and drug loading. While ZNPs based nanohybridisation improved the drug loading and release properties, the CYS conjugation resulted in the controlled release of the loaded NRG.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Declaration of Competing Interest

None.

Acknowledgments

Dr. P. Uma Maheswari acknowledges DST-WOS-A (SR/WOS-A/CS-75/2016), India for financial support.

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