Photo-activatable Nano-delivery System For Combined PDT And Gene Therapy

In recent years,gene therapy has become a new means of cancer treatment.The delivery of anti-cancer gene is mainly dependent on effective and safe delivery vectors.Cationic polymer is a widely used non-viral vector,while the coordination between its transfection efficiency and material toxicity is still a great challenge.Although the cationic polymers with high molecular weight can effectively encapsulate genes,enter cells,and transfect genes,the transfected genes are difficult to release into cytoplasm,resulting in limited transfection efficiency and high toxicity after transfection.Relatively,cationic polymers with low molecular weight have low cytotoxicity but poor gene encapsulation capacity and low transfection efficiency.Besides,in the process of gene delivery,the extremely acidic lysosomal environment is another important barrier.The development of stimuli-responsive vectors can solve the contradiction between transfection efficiency and material toxicity.Light is a kind of exogenous stimulation with strong tunability.The location,intensity,duration and wavelength of light irradiation can be accurately controlled by light source.Compared with ultraviolet and blue-violet light,near-infrared or red light has the advantages of strong tissue penetration and high safety.Under short and low energy light,photosensitizers can produce nonlethal dose of ROS,which can be used to break lysosomal membrane to achieve lysosomal escape.In addition,under long-time and high-energy light irradiation,a large number of ROS can be generated to directly kill cancer cells and play the role of photodynamic therapy（PDT）.Based on the above understanding,the first chapter of this thesis is a brief overview of gene therapy,gene delivery barrier,cationic polymer carriers,stimuli-responsive vectors,and PDT.In the second chapter,we designed a ternary complex.The ternary complex’s inner layer was composed of ROS-responsive reversibly crosslinked PEI and encapsulated therapeutic p53 plasmid DNA,while the outer layer was photosensitizer-bound hyaluronic acid（HAP）.The assemble into final ternary complex with particle size of 180 nm was achieved via electrostatic adsorption.Under low-density far-red light(661 nm,5 m W cm^-2,8 min),nonlethal dose of ROS was produced to destroy the lysosomal membrane and degrade TK-PEI to promote the release of p53 gene.Thus,the expression of anticancer gene p53 was promoted.It could be seen from fluorescence imaging that compared with the non-irradiated group,about 50% complex successfully escaped from lysosome and about 40% DNA was released from the carrier to the cytoplasm.After transfection,long-time continuous illumination（30 min）produced lethal dose of ROS to kill cancer cells,to achieve the synergistic anti-tumor effect of PDT and gene therapy.In vivo melanoma model showed that,compared with single gene therapy or single PDT,the treatment group with two-stage illumination（light-enhanced gene therapy and PDT）had the best anti-tumor effect.PDT possesses the advantages of controllability,non-invasiveness and non-aggressiveness,while it can also trigger the stress protection mechanism of cancer cells,including increased expression of heat shock protein,various survival factors and angiogenesis signal molecules.As a result,cancer cells’ tolerance to PDT is enhanced,and the effect of PDT therapy is greatly hindered.In the third chapter,we chose PKM2 siRNA as the therapeutic gene,whose target was pyruvate kinase（the rate-determining step in glycolysis）.This chapter describes that,by means of delivering PKM2 siRNA to silence pyruvate kinase,a series of waterfall effects are induced,including inhibition of glycolysis,reduction of ATP level,reduction of heat shock protein synthesis and enhancement of PDT.The inner layer of the ternary complex described in this chapter was composed of ROS-responsive branched polyamidoamine and encapsulated PKM2 siRNA,and further coated with outer layer（photosensitizer-bound HAP）via electrostatic adsorption.The particle size of the final ternary complex（TK-PAA/HAP/si PKM2）was about 150 nm.After treating MDA-MB-231 cells（human breast cancer cells）with TK-PAA/HAP/si PKM2 for 48 hours,the level of ATP was significantly reduced to ⁶5%,while the group with unordered siRNA did not have obvious change.Compared with the unilluminated group（single gene therapy）and the unordered siRNA group（single PDT）,the TK-PAA/HAP/si PKM2 ternary complex under light showed strongest cytotoxicity.