Synthesis Of Novel Gene Vectors Based On Polymers Crosslinked With Cell Penetrating Peptide And Preliminary Study Of Melanoma Cell Apoptosis

Basal cell carcinoma, squamous cell carcinoma and malignant melanoma, are common skin disorders, but fatal diseases. In the case of melanoma, effective therapies are rare. The current treatment methods mainly include traditional immunotherapy, chemotherapy, surgery and so on, and all of them display side effects that restrict the applications. Thus, novel strategies and ideas to discover new possibilities for curing the disorders are required. At present, gene therapy has been focused on applied to many cancer treatments, showing great potential in many studies according to its efficacy and ability to fix the disease at its source, and to eliminate or to prevent hereditary diseases, and it also may be seen in the field of melanoma treatment in the future. The starting point of this thesis is to synthesize a novel gene carrier with good biocompatibility and high transfection efficiency towards melanoma cells, and to carry apoptosis gene into melanoma cells for killing them, funding a possibility of curing malignant melanoma.In this study, polyethyleneimine (PEI) and chitosan-based polymers were chosen as basic materials for synthesizing gene vectors:PEI and CS/TPP (sodiumtripolyphosphate) nanocarriers. The evaluation of these two carriers was conducted and it turned out that these two carriers both had poor transfection efficiency in A375 cells (human melanoma cells). Finally, with the help of trans-activating transcriptional activator (TAT), polyethyleneimine (PEI)-sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate-TAT(PEI-SMCC-TAT) was synthesized successfully with lower cytotoxicity and higher transfection efficiency into A375 cells with comparison with PEI and CS/TPP. Lastly, PST nanocarriers were used to carry apoptosis gene PUMA (p53 upregulated modulator of apoptosis) to study the effect of PUMA, preliminarily evaluating the potential method for killing melanoma cells.The biocompatibility tests indicated that the PEI/DNA complex had higher cytotoxicity than that of PST/DNA complex. And CS/TPP-DNA complex had the best biocompatibility among them. Meanwhile, the size and zeta potential measurements showed that PEI/DNA complex presented the smallest size with small fluctuation around 170 nm at the N/P value from 4 to 50. However, CS/TPP nanoparticles exhibited the largest size (around 350 nm), which could be a cause for low transfection efficiency. Moreover, the size of PST nanocarrier was below 200 nm at N/P of 50, where the highest transfection efficiency into A375 cells appeared. The DNA-binding ability analysis showed that PEI, PST and CS/TPP nanocarriers all had good DNA condensing abilities. Fourier Transform Infrared Spectroscopy (FT-1R) indicated that TAT cell penetrating peptides had been successfully modified with PEI backbones.Furthermore, chitosan was also used to synthesize CS/TPP nanoparticles as gene vectors here. The obtained gene vectors were also evaluated by many measurements including size and zeta potential values, biocompatiblity and so on. CS/TPP-DNA complex exhibited good biocompatibility and larger size than PEI/DNA complex (around 400 nm), indicating the failure try on HEK273T and A375 cell transfection.Finally, a novel gene vector PST was synthesized with a higher transfection efficiency than PEI vectors and a better biocompatibility than those of CS/TPP nanoparticles. Many other factors were also discussed, including the ratio of TAT and SMCC added, N/P ratio of PST and DNA. At last, PST was used to carry PUMA gene into A375 cells to check the effect of the apoptotic gene on melanoma cells.In summary, the PEI, CS/TPP and PST nanocarriers were all studied for finding a best gene vector. And all the study in this work can provide experimental evidences and theoretical basis to researchers to further build a better gene delivery system and a new promising method for melanoma treatment.