Cationic Polymer-based Anti-tumor Combinatory Drug Delivery System

Cancer is one of the most notorious illness haunting human-being. Tens of thousands of lives are devoured by it every year, and it is always one of the most popular issues to develop effective treatments against cancer. The oncogenesis and growth of tumor are influenced by several factors, thus it can be futile to inhibit the tumor growth through single treatment. Combinatory treatments which can damage tumor cells through multiple pathways, improve the inhibition effect towards drug-resistant tumor cells, lower the effective dosage, are preferred. It has become a hot spot to research and develop combinatory and/or multi-target therapy in recent years.Polymer-based drug delivery system has a significant advantage on combinatory therapy. First, the drug-loaded vehicles can aggregate in solid tumors through passive targeting effect, increase the drug concentration within tumors, and lower the damage of normal tissue. Second, through the adjustment on the type and molecular weight of functional units,polymer-based delivery system can load different type of drugs simultaneously.Third, the polymer-based delivery system makes it available to control the ratio of the drugs conveniently, thus the adverse effect and instability of the treatment due to the unbalanced ratio can be avoided or alleviated.For the purpose of efficiently inhibit and damage the drug-resistant tumor cell MCF-7/ADR both in vitro and in vivo, a novel cationic polymer-based anti-tumor drug delivery system,PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38, was designed in this study,thus the combinatory treatment of SN38, KLAK peptide and survivin siRNA was realized through attacking the tumor cells from three separate directions,including DNA replication inhibition, mitochondria destruction and silencing the anti-apoptosis gene survivin. The polymer was synthesized through RAFT polymerization, and a total of nine groups of polymers were obtained through the adjustment of molecular weight in each block. After a preliminary screen of the micelles, which were prepared through nanoprecipitation method, we chose 3M, 4M and 9M (The composition of 3M is PEG114-P(MEMA-SS-KLAK)5-PHEMASN3820. Please check the information of 4M and 9M in the text) to carry out further tests on their structure and function.First, we measured the physicochemical characters of 3M, 4M and 9M.After the calculation of polymer molecular weight basing on the 1H NMR and GPC data, we measured the size distribution of the micelles with DLS and TEM. We tested the feasibility to use KLAK as a gene-vehicle on the basis of its cationic feature. The electrophoresis of free siRNA and vehicle-loaded siRNA proved that KLAK could absorb and protect siRNA effectively. We noticed that the absorption of siRNA could significantly influence the surface charge of micelles, and this phenomenon was both related to the neutralization of the positive charge and the secondary structure of KLAK. We observed the maintenance of the polymer-conjugated KLAK's secondary structure by CD,and proved that the absorption of siRNA could strengthen the a-helix signal.In the drug release test, we proved that the KLAK could be released from the micelles significantly in reductive environment, while an acid condition was required for the break of ester-bond between SN38 and the main polymer chain.Second, we measured the function of drug-loaded micelles on cellular level. Because of the electrostatic interaction between the positively-charged micelles and the negatively-charged cell surface, the endocyotosis rate of micelles by drug-resistant cell line MCF-7/ADR is fast, and the flow cytometry data showed that the rate was positively correlated to the amount of the unoccupied amino-group. We also tested the endocytosis pathway of the micelles through the addition of inhibitors, and the result showed that the micelles were mainly internalized through macropinocytosis and caveolae-mediated endocytosis, which may enlighten the possibility that the micelle-contained endosomes may not be fused by lysosome, thus increase the successful transduction rate. The mitochondria damage function of KLAK and the siRNA escape rate after endocytosis were tested through JC-1 kit and 5'-FAM marked siRNA, respectively. Camparing with the result of non-reductive responsiveness NC micelles, we noticed that the detachment of KLAK from the main polymer chain was critical for the its biological function,and that the breakage of disulfide bond may not be occurred in cytoplasm as many studies referred. Then we proved that the micelles could damage the tumor cells more significantly comparing with free drugs, though cytotoxicicity test and apoptosis induction test. The feasibility of transduction with siRNA was also proved on phenotype, mRNA and protein level, through luciferase activity detection, qPCR and western blot, respectively.Finally, we measured the function of the micelles on animal level. The biodistribution results of the micelles injected to mice through tail vein showed that the size and surface charge of the particles may significantly affect the distribution of them in tumor and main organs, thus the absorption of siRNA may alter the biodistribution and intratumoral distribution due to the charge change of micelles. In the anti-tumor test, 3Mf and 3Msur inhibited the growth of MCF-7/ADR solid tumor significantly, while 4Msur and 9Msur showed no visible effect on tumor inhibition because of their relatively poor biodisribution, despite their remarkable cytotoxicicity against tumor cell on cellular level. This indicated that the biodistribution result should have a higher priority to that of cellular test to evaluate the anti-tumor ability of drug-loaded micelles. After marking the blood vessels in tumor slices with fluorescence antibody, we found that the micelles could significantly damage tumoral blood vessels due to the positive charge provide by KLAK, thus enhance the anti-tumor result. We also evaluated the adverse effect of micelles on mice, and no visible influence was witted on the behavior, morphology and main organ function of mice.We synthesized another tri-block polymer,PHPMA-(SS)-PDMAEMA-(SS)-PHEMASN38, and other fluorescence marked polymers, to test the timing of the disulfide-bond breakage, and compare the influence of the disulfide-bond's position on the transduction efficiency. The result shows that: (1) the breakage of disulfide-bond happened right after the endocytosis began, not after the micelles were released into cytoplasm; (2) although the position of the disulfide-bond didn't influenced the transduction significantly, we could conclude that the KLAK peptide had a better efficiency as gene-drug vehicle comparing with traditional cationic polymers, such as PDMAEMA.