| Other Abstract | Lung cancer (LC) is the most common type of cancer in China. Chemotherapy is the main treatment method for lung cancer. But just like other cancers, multi-drug resistance(MDR) of tumors is an important factor limiting LC treatment after long-term medication. The use of siRNA to inhibit over-expressed P-glycoprotein (P-gp) in cancer cells is one of the effective ways to solve MDR. However, one key challenge for the applications of siRNA is the safe and effective delivery of siRNA to the tumor and sufficient cytosolic transport. In addition, the shortcomings of chemotherapeutic agents, such as poor solubility, easy degradation, and toxicity, limit chemotherapeutics application. One of the effective ways to solve these problems is to develop new drug delivery systems (DDS). Polymer micelles which have a special "shell-core"structure, is a perfect carrier to transport hydrophobic drugs and siNRA, so we chose it for our research. In this paper, six amphiphilic peptide analogs named M1~6 were synthesized with NPS as the parent peptide for fatty acid modification. The study showed that under the premise of good biological safety, M2, M3, M4 and M6 not only have strong cell membrane penetration ability but also rapidly self-assemble to form nanomicelles of "hydrophilic outer shell and hydrophobic inner corein aqueous solution at very low concentration. Thus we think they can be used as drug carriers. The secondary structures of M2, M3, M4, and M6 were determined by circular dichroism measurements to be the &alpha-helix. According to the corresponding relationship, we think that the structure of &alpha-helix is one of the key factors for the self-assembly ability of amphiphilic peptides. The characterization of nano-micelles is determined by dynamic light scattering technology (DLS) and the results showed size distribution of particles formed by M2, M3, M4, and M6 is between 100 and 200 nm. And the results of Zeta-potential measurement indicate that the structure nanomicelles is very stable and there are a large amount of positive charges in the hydrophilic shell. We observed the morphology of nanomicelles by TEM, and found that the nanomicelles formed by M2 and M6 are nanoparticle, while M3 and M4 are nanofiber. Finally, on the basis of self-assembly ability, the infiltration efficiency and cytotoxicity, M4 and M6 are selected for the binding and delivery of P-gp siRNA in vitro. According to the results of agarose electrophoresis, both M4 and M6 could bind siRNA, and the M4 showed better binding ability. Considering the safety of cell plasma membrane, we finally chose M4 as the carrier of P-gp siRNA, and performed the delivery experiment in A549 cells. The results of confocal and cell flow cytometry showed that M4 can carry siRNA into cells effectively, and showed a higher delivery efficient than Lipo2000. Meanwhile, the results of real-time quantitative PCR experiments showed that after M4 carried P-gp siRNA into A549 cells, the content of P-gp related mRNA was obvious reduced in A549 cells within 24h and 48h, which means the expression of P-gp was inhibited. However, its inhibitory effection did not show corresponding advantages compared with Lipo2000, we speculated that there are two reasons. First, the endosomal escape efficiency of M4 is weaker than Lipo2000, which causes a large amount of siRNA is degraded by lysosomes. The second reason is that the excessive binding between M4 and siRNA affects the release of siRNA in the cytoplasm and and prevents its function. Based on our speculations, firstly, we used a variety of endocytosis inhibitors to confirm the entry mechanism of M4, and provide basis for further studySecondly, according to the special REDOX conditions in tumor cells, M4 was further modified into M4-S-S-C18. Compared with M4, the main peptide chain of M4-S-S-C18 is connected to stearic acid C18 through disulfide bonds of two cysteine (Cys). It is expected that the large amount of reduced glutathione present in tumor cells can quickly break disulfide bonds and destroy the integrity of micelles, so as to accelerate the release of siRNA. In addition, the polymer micelles have a special "shell-core"structure, which can be used as an effective carrier for the transport of insoluble anticancer drugs. Therefore, while performing siRNA delivery experiments we also tried encapsulation experiments of Adriamycin by M4 and M4-S-S-C18. |
