Enzyme microencapsulation and its application for overcoming multidrug resistance in breast cancer treatment

Cancer chemotherapy is largely hampered by the lack of selectivity of therapeutic agents, the development of multidrug resistance (MDR) and insufficient drug concentration available in solid tumors. In order to overcome these barriers for solid tumor chemotherapy, a novel strategy of using a reactive oxygen species (ROS)-generating enzyme, glucose oxidase (GOX), encapsulated in hydrogel microspheres formulation was developed and investigated.;GOX was encapsulated in alginate/chitosan microspheres (ACMS) using an emulsification-internal gelation, followed by GOX adsorption and polyelectrolyte coating method. High GOX loading and encapsulation efficiency were achieved when the pH of the adsorption medium was lower than the isoelectric point (pI) of GOX. The activity of GOX in ACMS was maintained and showed sustained production of H2O2 compared to free GOX (FR-GOX).;The distribution of GOX within ACMS was inhomogeneous with higher concentrations near the surface of ACMS as detected by confocal laser scanning microscope (CLSM) and x-ray photoelectron spectroscopy (XPS). GOX was loaded into calcium alginate (CaAlg) gel beads via electrostatic interactions between GOX and CaAlg. GOX retained its integrity after adsorption to CaAlg and coating of the CaAlg gel beads with chitosan. Fourier transform infrared (FT-IR) and differential scanning calorimeter (DSC) results indicated the formation of a CaAlg-GOX-chitosan complex, which stabilized GOX.;The in vitro cytotoxicity of GOX encapsulated in ACMS (ACMS-GOX) was evaluated using murine breast cancer EMT6/AR1.0 cells, which overexpress P-glycoprotein (P-gp) and wild type EMT6/WT parent cells. The mechanism of the cytotoxicity of ACMS-GOX was investigated by using various extracellular and intracellular ROS inhibitors. ACMS-GOX exhibited similar cytotoxicity to EMT6/AR1.0 cells as to EMT6/WT cells, in effect by-passing the MDR phenotype of EMT6/AR1.0 cells. Extracellular H2O2 and intracellular hydroxyl radicals were found to play a critical role in the cytotoxicity of ACMS-GOX towards EMT6/AR1.0 cells.;In summary, the results from this Thesis demonstrate that the ACMS and the fabrication methods are suitable for microencapsulation of proteins like GOX. The enzymatic activity of GOX was maintained by encapsulation into ACMS. ACMS-GOX is effective in circumventing P-gp mediated MDR mechanism in the studied breast cancer cells. The combination treatment of ACMS-GOX with intracellular antioxidant inhibitors enhanced the cytotoxicity.