The Tumor-targeting Intracellular Translocation And Application Of Lycosin-? Modified Gold Nanoparticles

Toxin peptides have attracted worldwide attention as excellent research tools and promising drug leads in pharmacology and neurobiology along with the development of modern medicine.Though the major studies have been focused on the neurotoxins,another group of toxin peptides which possessed membrane activity have been thorough studied and applied in recent years.The cationic peptide lycosin-I isolated from wolf spider Lycosa singorensis venom has been proven to be a novel antimicrobial and anticancer drug candidate in our previous researches.In this work,we explored the action mode and dynamic behavior of lycosin-I on the lipid membranes,and established a promising cancer-targeting delivery system by using lycosin-I functionalizing the gold nanoparticles.In the first chapter,we discussed the application of natural peptides for nanoparticle and drug design.With the development of nanotechnology,a number of nanoparticles have been used in the study of biological medicine.Functionalization with natural peptides of nanoparticles is the most popular modification strategy for enhancing the stabilities of peptides in vivo and improving the efficiency of diverse nanoparticles in the diagnosis and treatment of diseases.The lipid nanoparticles and polymer nanoparticles are two classes of the most common nanoparticles used in nanomedicine.On the other hand,based the structures and bioactivities,the most used natural peptides can be classified as cell penetrating peptides(CPPs)and antimicrobial peptides(AMPs).Many of these CPPs are originally derived from natural proteins which translocate across biological membranes and can be classified into 3 groups,cationic,amphipathic and hydrophobic CPPs,according to their sequences.The modifications of CPPs on the surfaces of nanoparticles will improve the penetration efficiency of nanoparticles to the cells confirmed in previous researches.Like CPPs,most AMPs contain several alkaline amino acids and hydrophobic amino acids,and can interactive lipid membranes.The studies of AMP drug design have been focus on the sequence modification of AMPs for reduce the off-target effect to normal cells and the combination of nanoparticles for increase their bioactivities and duration in vivo.In the second chapter,we quantified the distribution of the stoichiometric composition of anti-cancer peptide lycosin-I on lipid membrane with single molecule spectroscopy.Lycosin-I,a peptide toxin derived from spider venom,has been demonstrated to be a promising candidate for the inhibition of tumor cell growth in-vitro and in-vivo by interacting with and penetrating the cell membrane.Owing to the shortage of efficient characterization strategy,however,there is still lacking of detailed knowledge about the distribution of the stoichiometric composition information of this peptide in solution and on lipid membrane prior to the cellular uptake process,which is fundamentally important for the understanding of the anti-cancer mechanism.In this work,with an objective-type total internal reflection fluorescence microscopy(TIRF),the distribution of the stoichiometric composition of lycosin-I in different solutions as well as on the lipid membrane was explored extensively based on the statistical single molecule fluorescence intensity analysis for the first time.It was found that lycosin-I mainly displayed in a monomer state in diverse physiological solutions regardless of the concentration of the peptide and the incubation time.However,on the lipid membrane,the fraction of small size oligomers increased as a function of time.Fusion of movable peptide molecules to those peptide oligomers with restricted motion on the lipid membrane was also observed.These new insights on the dynamic molecular stoichiometric information of lycosin-I could considerably improve the knowledge on the mechanism of how lycosin-I interacts with cancer cell membrane,which is of great importance for an increased understanding of the anti-cancer mechanism and for their potential as clinical anti-cancer drugs.In the third chapter,we established a stable cancer-targeting delivery system by conjugating lycosin-I on the surface of gold nanoparticles,and the application potentials of functionalized gold nanoparticles in cancer diagnose and treatment have been thorough studied.In our work,the lycosin-I-conjugated gold nanoparticles exhibited the high translocation efficiency to cancer cells in vitro,while remained low efficiency in cellular uptake to noncancerous cells compared to the famous CPP Tat peptide.The mechanism investigates revealed that lycosin-I-gold nanoparticles(LGNP)entered the cells via energy-dependent endocytosis,and accumulated in cell cytoplasm with time prolonging.The safety evaluation results indicated that the uptake of LGNP have minimal effect on cell viability,and can be cleared from the animal circulatory system without any adverse effects to the organs.However,in tumor xenograft model,the LGNP were found largely accumulated in tumor tissues.With the similar modification strategy,we conjugated the lycosin-I on the surfaces of gold nanorods,which were widely used in photothermal therapy.The lycosin-I-gold nanorods(LGNR)shared the high efficiency of intracellular translocation to the cancer cells with LGNP,and effectively kill cancer cells under continuous radiation of 808 nm laser.Together,the established LGNP and LGNR nanoparticle systems possessed great potential in cancer diagnose and treatment.