Abstract:
The main overall goal of this study is to investigate the application of modified chitosan as a potential vector for apoptotic gene delivery to gonadotropin-releasing hormone receptor (GnRHR)-expressing cells (i.e. testicular cells and mammary cancer cells). This study reported Gonadotropin Releasing Hormone-modified Chitosan (GnRH-CS) nanoparticle as a promising vector for targeted gene delivery, and a GnRH peptide was used for active targeting of a transgene in GnRHR expressing cells. The physiological characterizations of the prepared nanoparticle were investigated by proton nuclear magnetic resonance spectroscopy (1H NMR), Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and Nanosizer. The prepared GnRH-CS was able to condense DNA to form positively charged nanoparticles. In both two-dimensional (2D) and three-dimensional (3D) cell cultures systems, this alternative gene transfer strategy could specifically deliver the reporter genes to a transiently transfected Human Embryonic Kidney cell line model system expressing GnRHR, mouse-derived spermatogonia cells (GC-1 cell line) and mammary cancer cells (MDA-MB-436). Importantly, GnRH-CS exhibited higher transfection activity and lower cell toxicity compared to unmodified CS at the same ratio. From in vitro to in vivo study, Tumor Necrosis Factor alpha (TNF-α) was exploited as a therapeutic gene delivered by GnRH-CS in order to induce testicular cell death in male rat via intra-testicular injection. Significant differences in a reduction in testicular volume on day 7 (P<0.001) were found in all treated groups, compared to the pre-treatment volume. The testicular volume continued to reduce on day 14 (P<0.05) and 28 (P<0.05) in GnRH-CS/TNF-alpha and GnRH-CS groups, respectively. On day 35, after castration, testicular weight and volume of dissected testis were significantly lower in GnRH-CS/TNF-alpha and GnRH-CS groups (P<0.001). Serum testosterone levels did not differ (P>0.05) throughout the observation period. Ultrasonographically and histopathologically, GnRH-CS/TNF-alpha and GnRH-CS induced testicular degeneration and death while TNF-alpha and control groups showed normal findings. GnRH-CS/TNF-alpha treated animals showed higher severity degree of testicular degeneration compared to GnRH-CS treated animals. Moreover in pilot study using mammary gland tumor-bearing nude mice showed promising results in tumor suppression when TNF-α was delivered by the GnRH-CS. It was possible that our study might be apply for non-surgical sterilization and mammary cancer treatment in the future.
