Construction Of Photosensitive Gene Carrier And The Application Of Photochemical Internalization In Gene Delivery

Gene therapy has played great roles in many disease treatments including cancer therapy.However,inefficient intracellular gene delivery has limited its further application.Recently,photochemical internalization(PCI)has emerged with the opportunity to overcome endo-lysosomal sequestration in gene delivery,which utilizes photosensitizer(PS)plus light and resulting in reactive oxygen species(ROS)at sub-lethal level to facilitate intracellularly targeted drug delivery.In order to control the release of genes spatiotemporally,two types of nanocarriers based on porphyrin photosensitizers were designed for gene delivery taking the advantage of PCI.One is about asymmetric polyplexnanocapsule loaded with photosentisizer,and the other is about pH-sensitive nanopolyplexes containing PS as one segment of the copolymer for efficient light-assisted gene transfer.The details include:1.Asymmetric polyplex-nanocapsules were prepared based on PEGPCL-PEI triblock copolymers using the simple solvent-injection method.Subsequently,the hydrophobic PS is encapsulated in the hydrophobic layer of nanocapsule by hydrophobic interaction.Agarose gel electrophoresis and fluorescence scanning spectroscopy showed such nanocarriers can effectively condense plasmid DNA and encapsule the PS to form the stable nanocapsules.Transmission electron microscopy(TEM)and dynamic light scattering(DLS)measurements showed that the polyplex-nanocapsules in water had average size in range of 200～280 nm and the negatively charged surface depending on the loading content of PS.MTT assay in Hela celld shows that,neither the nanocapsule carrier nor the laser treatment shows any significant evidence of cytotoxicity.More importantly,these polyplex-nanocapsules can be certainly uptaken by Hela cells,and the loaded genes can be released from the nanocapsule assisted by PCI effect resulting in improved gene transfection efficiency in comparison with the case without laser treatment.2.In order to further improve the combination stability of the PS and gene vector and to achieve more controllable and accurate PCI,a new type of pH-degradable polymer-photosensitizer conjugate(m PEG-Pheo A-l PEI)was prepared as polymeric gene carriers for light-assisted intercellular gene delivery.The m PEG-Pheo A-l PEI is a conjugate containing pH-sensitive hydrazone bond,which could efficiently condense pDNA above N/P ratio of 8 through electrostatic interaction,forming polyplexes with the size less than 290 nm and positive surface charge.At neutral pH corresponding to extracellular and cytosol milieu,the PSs in nanopolyplexes would be quenched,having low ROS generation capability.After endocytosis,the pHresponsive hydrazone bonds were cleaved under mildly acidic pH in endo/lysosomes,which recovers ROS generation of PSs.Consequently,the PCI effect allows the cargo gene to escape from endosomes.Such pHsensitive and light-assisted gene delivery system may remarkably increase the therapeutic potency and specificity of intracellular gene delivery.