A Detailed Investigation on The Development of Biomolecule Based Multifaceted Therapeutic Nanoformulations and Their Cytotoxic Behaviour in Multiple Cancer Cells

Abstract

In the present thesis, we have developed protein-based multiple therapeutic nanoformulations and evaluated their anticancer efficacy in multiple cancer cells as well as other therapeutic potentials such as antimicrobial, antioxidant, and anti-inflammatory activity. In the first strategy, we prepared hybrid supramolecular nanoassembly (SNLYZ-BLA) of two proteins, bovine α-lactalbumin, and hen egg-white lysozyme to evaluate their cytotoxic effect in multiple cancer cells including breast cancer (MCF-7 and MDAMB231), cervical (HeLa), osteosarcoma (MG 63) and myeloma (B16F10) cells. Both the proteins have already been reported to possess anticancer activity in either native form (lysozyme) or the conjugate state with oleic acid (α-lactalbumin, such as BAMLET or HAMLET). However, their potential as a drug delivery agent and the subsequent anticancer potential has not been much explored. Moreover, we also investigated the anticancer activity of two independent nanoassembly of pure lysozyme (SNLYZ) as well as non-anticancer protein, casein (NSCS), and their curcumin (anticancer phytochemical agent) loaded formulations. Both SNLYZ-BLA and SNLYZ demonstrated exceptional drug (curcumin) loading (SNLYZ-BLA-Cur and SNLYZ-Cur) ability and exhibited excellent cytotoxic effect in both forms (without and with the drug-loaded state) in multiple cancer cells. We further demonstrated significant (>80%) drug release ability of the formulations under low pH conditions. Moreover, after the drug release, the anticancer activity of both reconstituted SNLYZ-BLA and SNLYZ (their drug released state) was investigated and found almost the same as that of freshly prepared. All these supramolecular protein nanoassembly (SNLYZ-BLA and SNLYZ) showed excellent stability under wide pH and temperature. In the second strategy, metal-based nanoparticles such as zinc oxide (ZnONPCS) and silver-gold alloy (AgAuNP) nanoparticles were synthesized via biogenic synthesis method using casein as capping and reducing agent to improve their biocompatibility. Further, curcumin, a phytochemical, was loaded (ZnONPCS-Cur and AgAuNP-Cur) on both nanoparticles, and their anticancer potential was investigated. Results revealed an excellent cytotoxic effect of the formulation and drug delivery system in multiple cancer types. We also demonstrated that both ZnONPCS and ZnONPCS-Cur were biodegradable under acidic conditions, thus making them more applicable as a drug delivery system and therapeutic agent in cancer than AgAuNP or AgAuNP-Cur. Further, we also performed folate based targeting for all the above formulations, which revealed their enhanced cytotoxic effect in cancer cells. The physical and storage stability of all the above formulations were investigated in terms of their commercial potential and was found stable. We also investigated the antioxidant, antimicrobial, and anti inflammatory activity of all the formulations loaded with curcumin. While both SNLYZ-BLA-Cur and SNLYZ-Cur exhibited excellent antibacterial, antioxidant and anti inflammatory activity, the anti-inflammatory activity of SNLYZ-Cur was predominantly due to loaded curcumin. ZnONPCS exhibited excellent antimicrobial activity and photocatalysis of toxic industrial dyes. However, NSCS and AgAuNP loaded with curcumin demonstrated antioxidant activity and anti inflammatory activity due to loaded curcumin, while AgAuNP also showed antibacterial activity due to the presence of nanosilver (Ag). Further, it was also found that the solubility and stability of the poorly soluble drug, curcumin, were significantly increased when loaded on various nanostructure, thereby increased its bioavailability, thus improved their therapeutic use. All the formulations were observed to be biocompatible to healthy human cells (HaCaT cells) and erythrocytes. Finally, we validated the potential of SNLYZ-BLA as a drug delivery system using doxorubicin, a well-known chemotherapeutic agent. We found the excellent drug loading capacity of SNLYZ-BLA for DOX (48.2 mg/g) and its pH-responsive in vitro drug release up to 74 % at pH 5.0, which indicates its potential as an effective drug delivery system. Further, when SNLYZ-BLA-DOXwas applied to cancer cells, it demonstrated better cytotoxicity than when applied alone (DOX and SNLYZ-BLA). More importantly, the undesired cytotoxicity of DOX was reduced to the healthy cells after loading to protein nanoassembly (SNLYZ-BLA).From the overall study, we observed that among all the nanoformulations we investigated, both SNLYZ-BLA and ZnONPCS demonstrated excellent anticancer potential in vitro in multiple types of cancer cells as a therapeutic agent as well as a drug delivery system. However, considering that the hybrid supramolecular nanoassembly (SNLYZ-BLA) is composed of pure biomolecules (proteins), and both proteins were reported to have a little/ignorable immunogenic response, this fact might make them as a potential therapeutic candidate for the
treatment of multiple cancers along with its excellent antibacterial, antioxidant and anti-inflammatory activity.