DEVELOPMENT OF HIGH CATIONIC NANOTECHNOLOGICAL ADJUVANT SYSTEMS AND DEMONSTRATION OF THEIR EFFICIENCY IN HER2 BREAST CANCER DNA VACCINE MODEL
Project no. 723 · Status: Kapanmış · 2016
Summary
DNA vaccines have found different application areas in recent years, they are used both against infectious agents and different types of cancer. However, DNA vaccines can not stimulate desired immune responses when they are used alone because they are rapidly degraded by serum nucleases and consequently they have short half-lives. Therefore, immune stimulator substances or micro/nano delivery systems are used as adjuvants to increase the immune response elicited by DNA vaccines. In particular, the adjuvant systems created by new nanotechnological approaches are promising in this respect. In project scope; cationic pHEMA-His/PEG, pHEMA-Kitosan/PEG, pHEMA-PEI/PEG and pHEMA-DOTAP/PEG particles were developed. Characterization studies were carried out by shape and dimensional analysis, zeta potential and particle size measurement, and determination of DNA loading capacities. Then, the cytotoxic effects on CHO-K1, MCF-7 and TUBO cells were defined and the concentration values to be used in in vitro transfection studies were determined. Finally, in vitro transfection efficiencies of the pDNA/adjuvant formulations under the control of naked DNA and commercially purchased transfection agent were measured using pIRES2EGFP empty vector and then pIRES2- EGFP/DEC205ScFvMEHer2 DNA vaccine vector, which was developed against Her2 positive breast cancer by fluorescent microscopy and flow cytometry analyzes. As a result, all pHEMA-based adjuvant systems, which can be produced in nano-sizes and in the desired properties, have been shown to increase in vitro transfection efficiency compared to naked DNA by using them in different pDNA/ adjuvant formulation ratios. When compared to the commercially purchased Lipofectamine 2000 agent; it was found that a 2:4 ratio of pDNA/pHEMA-PEI polyplex for CHO-K1 and MCF-7 cells and a 2:6 ratio of pDNA/pHEMA-DOTAP complex for TUBO cells provided more effective transfection. For this reason, it is concluded that these adjuvant formulations are promising candidates as gene delivery agents in large scale transfection studies and in vivo studies.