Investigation of Self-Assembled Nanostructured Protein-Based Therapeutic Approaches in Breast Cancer

Abstract

Development of various therapeutic approaches for the treatment of breast cancer is on the rise. Among them, chemotherapy has been widely practised for the treatment of breast cancer. However, the approach exposes the patient to various kinds of side effects caused by the drugs. Although gene therapy finds a decent approach but it is expensive and associated with delivery problems. In order to avoid the side effects of chemotherapeutic drugs and achieve a cheap and toxicity free therapeutics, the focus has been gradually shifting towards biomolecule-based therapeutics like gene, peptide and proteins as an alternate therapeutic strategy.
In our present investigation, we have developed protein-based various therapeutic strategies in breast cancer which includes two proteins, bovine α-lactalbumin (BLA) and hen egg white lysozyme (LYZ). Both of them are already reported to have antitumor and anticancer activity. However no detail investigation was performed in breast cancer. Moreover, it was also reported for BLA that the specific structural state is responsible for its antitumor or anticancer activity. Here both the proteins were transformed into their self-assembled nanostructure state using simple desolvation technique through the chemical crosslinking process. Both the nanoassembly were characterized in details and their anti-proliferative activity in breast cancer cells was evaluated.
The nanostructures were characterized using FESEM (Field Emission Scanning Electron Microscopy), AFM (Atomic Force Microscopy) and DLS (Dynamic Light Scattering) analysis for size and shape, FTIR (Fourier Transform Infrared Spectroscopy for bonds involved in nanoassembly), Circular dichroism and Fluorescence spectroscopy and conformational change of proteins. Microscopy and DLS analysis confirmed their average size as 300 nm. The stability of both the self-assembled nanostructures of lysozyme (snLYZ) and α-lactalbumin (snBLA) were examined against thermal, pH and protease stress and found to be highly stable. Moreover, both of them demonstrated excellent hemocompatibility in human erythrocytes as well as cytocompatibility in normal human cells like HaCaT and mouse cells like 3T3.
When the nanoassemblies were applied to breast cancer cells MCF-7 and MDA-MB231, they demonstrated high ( > 90% ) cell death at 24 h and almost complete cell death after 48 h of administration. The microscopic images confirmed that the internalization of protein assemblies by MCF-7 cancer cells was the major cause of such cell death. Further, the investigation also confirmed that such death was mediated by reactive oxygen species (ROS) generation.The microscopic image and western blotting results confirmed that apoptosis had no role in cell death mechanism.
The cytotoxic behaviour of snBLA was compared with a well reported anti-cancer agent BAMLET (bovine α-lactalbumin made lethal to tumor cells) in four different cancer cell lines including two breast cancer cell lines MCF-7 and MDA-MB-231. Our synthesized snBLA demonstrated better cell killing potential than BAMLET with much higher hemocompatibility.
Both the nanoassembly demonstrated high Tamoxifen loading (124%) through chemical crosslinking process and release of drug efficiently in a pH-responsive manner. Furthermore the drug release process was completely regulated by conformational change of the protein in nanoassembly.
In our next step we attempted to develop Graphene oxide nanosheets (GOns) and Zinc oxide nanoparticle (ZnONP) based therapeutic strategy in breast cancer to compare with our protein-based therapeutic approach.
Graphene oxide nanosheets (GOns) and Zinc oxide nanoparticles (ZnONP) are being developed as drug carriers, however, such an approach was plagued with problems such as hemotoxicity and cellular biocompatibility. Hence, we made an attempt to develop a therapeutic strategy based on ZnONP and GOns by functionalizing them with BLA protein. The surface functionalization with BLA improved their hemocompatibility in human erythrocytes and cytocompatibility in normal human cells. They also demonstrated high anti-proliferative activity in breast cancer cells via the ROS-based mechanism of cell death. However, considering the consequence like protein degradation in cells and subsequent exposure of GOns and ZnONP to cells may lead to their accumulation in normal cells and tissues that might limit their use as a rational approach.
Therefore, in our present study, we concluded that our developed stable self-assembled nanostructured lysozyme and α-lactalbumin can be used as a rational therapeutic strategy in breast cancer.