| Referring to the―global cancer report2014‖by world health organization (WHO),China accounts for about40%of new gastric cancer incidence. Now, the death due tocancer ranks No1around the world. Traditionally, the most effective treatment of gastriccancer is surgery, by which the tumor can be partly supressed but not cured with somelocal and/or distant metastasis as well as relapse. Chemotherapy and radiotherapy becomesmore and more important for improving the therapy effects. However, the cytotoxic drugsin chemotherapy and radiation from the radiotherapy can not specifically recognizemalignant cells, resulting in serious and intolerable side effects. As we known that themalignant cells are different from those of normal cells with rapid cell division and growth.Therefore, focusing on the tumor microenviroment and finding novel ways for the safe andspecific delivery of therapeutic agent to tumor cells is of great importance and urgency inthe clinic.With the rapid progress nanomaterials and nanotechnology, the combination ofnanotechnology and medicine has generated a new field which is called nanomedicine.These nanomedicine based on novel nanocarriers can improve the dissolution rate of thedrug, enhanced drug targeting, slow-releasing potential, controllability, low cytotoxicityand intelligent preperties and so on. Nanomedicine offers new hope to patients forpromoting the cancer diagnosis more accurate treatment. Specially, the nano gene targetingtherapy (gene therapy) has become a new hot topic in tumor therapy. Recent progress ofgene therapy have showed its high potential in treatment of acquired and inherited diseases.This approach is based on the principle of introducing a guest gene to the host cell forcorrecting, removing or replacing the disable (or mutue) gene by the delivery system.Moreover, nanobased gene therapy can avoid some limitations of chemical drugs includinglow bioavailability, high cost and MDR etc. Currently, vehicles for gene delivery can bedivided into two major groups: viral and non-viral vectors. However, viral vectors havesignificant limitations in clinical application because they may induce an immune responsewhich abolishes the transgene expression. Non-viral vectors such as cationic liposomes andPEI overcome the problem of immune response of the viral vectors, but they still havemany drawbacks including unstable in scrum, complex in components, large particle sizeand unfavorable accumlation rate in tumor tissue etc. Therefore, it is highly desired todevelop a new non-virus gene delivery system with a simple composition, long circulation in blood and specific tumor targeting.As mentioned above, the characteristics of the tumor microenvironment has beencharacterized step by step. The unique properties of tumor are attributed to the highmetabolism of tumor cells which requires much more oxygen, nutrients, gas exchange, andwaste removal. But the heterogeneity structure and distribution of the tumor blood vesselsas well as the blood capillaries slow down the energy exchange between intra-andextratumor. All these result in unique characteristics of tumor, that is, the unnormal tumorblood vessels with gap in200–700nm, the relative high temperature of tumor (t>37℃),and the relative low pH (5～6).Herein, for promoting the gene therapeutic index, we synthesized a thermosensitiveblock copolymer PID118which utilizing the temperature sensitive polymer (poly n-isopropyl acrylamide, PNIPAM). The PNIPAM was tuned by introducing the hydrophilicmonomer DMAAM. By aminolysis and conversion of polymer termini, we got thethermosensitive block copolymer phenyl-PID118-b-PLA71wiht high volume phasetransition temperature (VPTT). Through fine tuning the block length, this copolymer canself-assembly to form nanovesicles with regular core-shell structure in conversion oforganics and aqueous phases. The novel nanovesicles have many advantages such as noimmunogenicity, low cell toxicity, serum stability, and repeated injection. The particle sizeand surface charge are tunable for enhancing EPR effect with VPTT~39℃. So it can bestability in normal body temperature of37℃but dissociation in tumor microenvironmentat T>39℃. This makes the vesicles with function of passive targeting. Additionally, thevesicle shell can be modified by various functional groups with long circulation time, lowclearance from the blood by the reticuloendothelial system (RES). For example, the activetargeting function of the vesicle increased by connected with specific antibodies. Theseadvantages make the polymer vesicles have great potential in the gene therapy of cancerresearch.We condensed the macromolecular plasmid DNA by mix it with protamine and thepolymer PID118-b-PLA71. The transfection experiments are conducted in the condisitonswith or without serum. Comparing with the nano vesicle, we found that the PEI has hightransfection efficiency in serum-free culture medium but low effciency in the mediumcontaining serum. Noted here, gene therapy is always accompanied with manybiomacromolecules. So the in vitro transfection should be conducted in serum containingmecium. On the point, it is interesting to find that the vesicles have similar high transfection efficiency in culture medium no matter with or without serum. This similartransfection efficiency suggests that the intelligent vesicles are stability in vivo without anyinterferenced with serum proteins. Furthermore, we found that termperature has little effecton the transfection of PEI. On the contrary, it is found that temperature has obviouslyeffects on the trasfection of our nanovesicles. Its transfection efficiency was significantlyincreased when the temperature is higher than the phase transition temperature ofPID118-b-PLA71. In additiona, by linking the specific antibody fragments to nanovesicles,the results showed that the transfection efficiency of targeted group is much higher thanthat of the untargeted group. The results indicate that the nano immunevesicles havepassive and active dual targeting function. To evaluate the biodistribution, intratumoraccumulation and anti-tumor activity, we established the tumor-bearing nude mouse model.In vivo imaging and organ section further display that the dual targeting nanoimmunevesicles have obvious tumor growth inhibition ability.Considering the pratictal obstacles appeared in the clinical gene therapy and focusingon the unique properties of tumor microenvironment, in this study, we design and finesynthesis of a thermosensitive block copolymer. A well-define nano vesicle is succesfullyconstructed with temperature regulated passive and antibody tuned active dual targeting.Through the in vitro transferring experiments, in vivo tumor accumulation and anti tumoractivity, we found that the dual targeting nano immunevesicles show significant potentialin cancer gene therapy. It is thus helpful to conclude that such dual targetingimmunovesicles could be useful and powerful for the clinical gene therapy. |
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