Studies On Trans-membrane Drug Transportation Mediated By Self-assembling Peptide Nanotubes And Anti-tumor Activities

Malignant tumor is one of the principal diseases that threaten the health of human beings. Chemotherapy is the most important treatment strategy as well as operation and radiotherapy. However, the effect of chemotherapy is not always good enough due to decreased sensitivity to chemotherapeutics and multidrug resistance (MDR) of cancer cells. It is an urgent task to search for new ways to elevate the sensitivity of cancer cells to chemotherapeutics and overcome MDR. Cell membrane is a natural barrier for the entrance of chemotherapeutics, and the active efflux by P-glycoprotein aggravates the situation. If there are some suitable approaches to break through the membrane and accelerate the entrance of chemotherapeutics at high concentration, the efflux of P-glycoprotein would be weakened, and the effectiveness of chemotherapeutics would be highly strengthened killing tumor cells before resistance generates. In this thesis, the trans-membrane channels in lipid bilayers based on self-assembling of peptide nanotubes were studied and the channel-mediated transport of small-molecule drugs was explored as well as the its effect on tumor cell proliferation and solid tumor growth.The contents of this thesis include synthesis of the cyclic peptides, preparation and characterization of the peptide nanotubes, channel-mediated transportation of small-molecule hydrophilic drugs based on peptide nanotubes in liposomes, the transport of 5-FU mediated by peptide nanotubes in cells, the anti-tumor cell activity of 5-fluorouracil mediated by the peptide nanotubes in vitro and S180 solid tumor in vivo, and molecular dynamics simulations of peptide nanotubes.According to the structure and characteristics of the cyclic peptide, cyclo[-(Trp-D-Leu)₄-Gln-D-Leu-], a two-step synthesis strategy was employed. Firstly, the linear decapeptide was assembled by standard Boc chemistry on solid phase, and the target peptide was cyclized in solution secondly. The total yield of cyclic decapeptide was 30-35% with a purity of over 95%. The product structure was identified by HPLC, LC/MS and ¹H-NMR. After the synthesis of cyclic peptide, the crystalline of self-assembled peptide nanotubes was prepared in solution and characterized. The interior cavity of the nanotubes is hydrophilic allowing transport of water-soluble small molecules, whereas the exterior can be modified to be hydrophobic by selection of amino acid residues of cyclic peptides. The peptide nanotubes prepared by cyclo[-(Trp-D-Leu)₄-Gln-D-Leu-] have a high hydrophobic external surface and can easily self-assemble in lipid bilayers into nanotubes. On liposome (LUV) models containing 5-fluorouracil, tegafur, cisplatin and cytarabine, the channel-mediated transport of peptide nanotubes located in the lipid membrane was confirmed. The size of drugs and the internal diameters of nanotubes were the main influential factors on the transport rate. 5-Fluorouracil, tegafur and cisplatin can be transported through the artificial channels. Both their first-order rate constant k and accumulated transport percent at 90 min showed positive correlation to the does of cyclic peptide. Their transport mediated by peptide nanotubes was fitted first-order kinetics. Membrane permeability of 5(6)-carboxyfluorescein liposomes confirmed that peptide nanotubes did not destroy the lipid membrane but formed hydrophilic channels to transport small-molecule drugs.An anti-tumor drug can only play its role when it entered into the tumor cell and it will be hampered because of the membrane barrier. The uptake of 5-FU in BEL7402 showed that peptide nanotubes can form hydrophilic channels to let 5-FU pass through quickly into the interior of tumor cells instead of diffusion through intact membrane. When the amount of 5-FU uptake increased, the inhibition appeared faster. When more cyclic peptide was added, more channels would be generated and more drug would be transported into the cells.Based on the results of artificial membrane drug transport, BEL7402 and Hela cells were selected to investigate the effect of antitumor activities of 5-FU mediated by self-assembling nanotubes on cell membrane. It was shown that the sensitivity of BEL7402 and Hela to 5-FU were both enhanced as mediated by peptide nanotubes. Meanwhile, it can also be concluded that the synergistic action between 5-FU and peptide nanotubes to the tumor cells was not selective. Cell morphology and flow cytometry confirmed that the peptide nanotubes has the cytotoxity to tumor cells and can induce apoptosis. When the combination of 5-FU and peptide nanotubes was selected, the inhibition of 5-FU mediated by peptide nanotubes has been significantly enhanced.The inhibition of 5-FU mediated by cyclic peptide nanotube on S180 mice were studied in vivo. After intratumorally injecting the radio-labeled cyclic peptide and observing the image in vivo, it can be concluded that the cyclic peptide was localized in the tumor, which was optimal to enhance its efficacy and reduce toxicity. The antitumor effect of 5-FU mediated by cyclic peptide nanotubes was strengthened after intratumoral injection. The combination of the cyclic peptide and 5-FU exhibited additive effect at low concentrations, while exhibited synergistic effect at higher concentrations. Solid tumor slicing showed that 5-FU could penetrate the tumor cells through the artificial channel quickly, and the effect would be strengthened as the dosage of cyclic peptide increased.Molecular dynamics is a nice tool to describe the transport of molecular on the basis of molecular mechanics. Three topoisomers PDB structures, ortho-, meta- and para-nanotubes, were constructed with the Insight II and the structure stability and influential factors were studied by molecule dynamics simulation. The relative position of Gln residuals had important influences to the stability and the state in cell membrane of peptide nanotubes. ortho-nanotubes was chosen for steered molecule dynamics (SMD) and the procedure of 5-FU transport mediated by peptide nanotubes was explored by SMD. Results showed that the molecule of 5-FU "jumped" through the subunits of cyclic peptide nanotubes and 5-FU could go straight through the nanotube successfully only when it was perpendicular to the planar of cyclic peptide. If 5-FU was parallel to the planar, more tensile force would be needed to plumb up the 5-FU and let it pass through the cyclic peptide.