Silanized and Chitosan Conjugated Graphene Oxide: Synthesis, Characterization and Evaluation of Its Potential as Biomaterial and Drug Delivery Vehicles

In recent years, graphene oxide (GO) has been explored for various application in biomedical engineering that include biosensing, tissue engineering and drug delivery. However, the biomedical application of GO is often being strongly scrutinized because of its potential toxicity. A strategy to surmount this problem is to functionalize GO with bioactive molecules. For this purpose, we have decided to explore three kinds of functionalized GO, namely silanized graphene oxide (SiGO), reduced silanized graphene oxide (rSiGO) and chitosan conjugated graphene oxide (CSGO). Recently, SiGO has shown good performance as reinforcing material. However, its biological properties, especially the cytocompatibility and in vivo compatibility have not been investigated in details. rSiGO, a derivative of SiGO, has not been explored as a biomaterial yet; so we include it in our study. In the case of CSGO, chitosan was chosen considering the promising performances of chitosan-GO composites as biomaterial. All the materials were subjected for comprehensive characterization. The goals of the thesis are: (i) to study the in vitro cytocompatibility and in vivo compatibility of SiGO, rSiGO, and CSGO, and (ii) to evaluate its performance as osteogenic material and drug delivery vehicles. The purpose of setting such aims is to find out the suitability of the aforesaid functionalized GO for its application in tissue engineering and drug delivery. The whole study is divided into three main parts. In the first part, we have reported the synthesis and physico-chemical characterization of five materials namely GO, reduced GO (rGO), SiGO, rSiGO and CSGO. GO was synthesized by modified Hummer’s method. SiGO was synthesized by treating GO with (3-aminopropyl) triethoxysilane (APTES). Both GO and SiGO were reduced through hydrazine treatment to obtain rGO and rSiGO. CSGO was prepared by crosslinking GO with chitosan using EDC-NHS. Physico-chemical characterization of the functionalized GO was done by XRD, FT-IR, Raman spectroscopy, XPS, elemental mapping, SEM and TEM. Silanization led to an increase in the interlayer distance and disorder in the lattice which became more prominent in the case of rSiGO. Chitosan functionalization showed minimal effect on basic architecture of GO. In the second part of the study, in vitro cytocompatibility of the materials was tested in details along with the acute toxicity study in vivo in murine model. Study of the in vitro toxicity of functionalized materials on three different cell lines namely primary human dermal fibroblast (HADF), murine embryonic fibroblast (NIH 3T3) and human osteosarcoma cell lines (MG63) revealed that toxicity of functionalized GO was significantly less than GO. Among all the functionalized derivatives, CSGO was found to be the most cytocompatible. The effect of functionalization was found concentration dependent which was more prominent at higher concentration of the materials. We further showed that in vitro activation of macrophage was less in the case of functionalized GO in comparison to GO. Study of the acute toxicity in the murine model indicated that GO was mildly hepatotoxic at experimental concentration whereas functionalized GO were not.In the last part, we evaluated the performances of these functionalized materials as (i) osteogenic biomaterials, and (ii) as a delivery system for hydrophilic anticancer drug. Analysis of the expression of different osteogenic differentiation markers such as runx2, collagen type I, alkaline phosphatase, osteocalcin, in human mesenchymal stem cell (hMSCs) revealed that CSGO possess the highest osteogenic potential among all the samples. Osteogenic differentiation induced by all other materials was almost same to that of GO. The loading of doxorubicin hydrochloride was almost same in the case of GO and SiGO but the release was higher in the case of SiGO. Further, among all samples, SiGO was found as the best drug delivery system in terms of efficacy when tested in vitro in 2D human hepatocarcinoma model. In the appendix of the thesis, we also reported the reinforcing effect of functionalized GO in polymeric film in the context of tissue engineering application. The study altogether implied that both SiGO and CSGO are more cytocompatible than GO and could be better alternatives of GO for tissue engineering and drug delivery application