M13mp18 İskele İpliğinden DNA Origami Nanodemetlerin Geliştirilmesi Ve İlaç Taşıyıcı Olarak Kullanımının İncelenmesi

Abstract

Currently, in nanotechnology and biomedical field DNA nanotechnology is more and more used instead of conventional methods and important studies are carried out. Complementary base pairing of DNA has taken it to the next level in genomic studies. DNA origami is a multidisciplinary field and advanced nanomaterials are developed. DNA nanostructures have many advantages such as being modular, biocompatible, biodegredable and giving low immune response over polymer and lipid based drug delivery agents. In the common technique DNA origami, long single stranded DNA is folded by over hundreds of 'staple' strands via thermal annealing and 2D and 3D DNA nanostructures are created.

In nanomedicine and biomedical field, utilized from DNA origami nanostructures in generating solutions for antimicrobial resistance. Various antibiotics are used against infectious diseases and the pathogens cause infections after surgeries. However pathogen agents develop resistance to antibiotics, it cause many challenges in therapy, after surgery and leads to financial burden in healthcare system. In cancer therapy conventional methods such as drug, surgery, chemotherapy, radiotherapy and phototherapy can kill normal cells as well. Moreover, particularly in cancer therapy many therapy methods weakens the patients' immune system and patient's resistance to infections. In this type of patients, effective antibiotic use improve the success of chemotheraphy too.

Lantibiotics are post translational modified polycyclic peptite antibiotics. A member of lantibiotics class, nisin is FDA approved, shows high activity, low cytotoxicity, low possibility to develop bacterial resistance and has many advantages over other antibiotics against antimicrobial resisted epidemic nosocomial pathogens. Because nisin shows double mode of action, by binding one of cell wall initiators lipid II, it disrupts cell wall formation and it forms pores in cell membrane and leads cell to lysis.

In the first part of the thesis, a) bare nisin, b) DNA nanostructures were built by m13mp18 scaffold strand and electrostatically loaded with nisin, c) bare DNA nanostructures were built by m13mp18 scaffold strand.

Antimicrobial activities of these 3 samples against pathogen agents methicillin resistant Staphylococcus aureus (MRSA ATCC 43300) and vancomycin resistant Enterococcus faecalis (VRE ATCC 51299) were investigated. In the concentration of 2500 nM, a) by bare nisin 97% of methicillin resistant Staphylococcus aureus (MRSA ATCC 43300) bacteria was inhibited and by loading onto DNA nanoparticles (DNA-NP) 95% of inhibition achieved. In 2500 nM concentration a) bare nisin inhibited up to 96% and b) by loading nisin onto DNA nanoparticles up to 91% of vancomycin resistant Enterococcus faecalis (VRE ATCC 51299), c) for both pathogens bare DNA nanoparticles (DNA-NP) has not exhibited any inhibition.

In the second part of the study, IC50 values calculated against methicillin resistant Staphylococcus aureus (MRSA) for nisin as 1051 nM and for nisin loaded DNA-NP as 1567 nM. And against vancomycin resistant Enterococcus faecalis (VRE), IC50 values found for nisin and nisin loaded DNA NP are 1303 nM and 1387 nM, respectively. By using nisin against MRSA and VRE, highest inhibition values obtained as 97% and 96% compared with minimum inhibition concentration (MIC) values in the literature and found compatible.

In the last part of the thesis, in vitro cytotoxic activity of nisin loaded DNA based drug delivery system investigated against human breast cancer cells (MCF-7) and mouse fibroblast cells (L929). Bare nisin in 2.5 µM concentration and nisin loaded DNA nanostructures showed cytotoxic effect in 45% and 43% of MCF-7 cells, respectively. And against L929 cells in 2.5 µM concentration, bare nisin and nisin loaded DNA nanostructures showed cytotoxic effect in only 8% and 17% of the cells, respectively.

Furthermore as a result of the study, it is believed that the developed nisin loaded DNA nanostructures will be used in both against nosocomial multi resistant pathogens thanks to their antimicrobial effects and in cancer therapy with their selective anticancer activity.