| Bladder cancers are one of the most common malignant tumors in human.The treatment of bladder cancers is based on pathology,stages and metastasis.At present,gene therapy is also being used in the treatment of bladder cancers.The combination of gene therapy with nanocarriers is still unclear and lack of relevant clinical trials.Photodynamic therapy(PDT)is a new method to treat tumor diseases with photosensitive drugs and laser activation.PDT is a cold photochemical reaction consist of oxygen,photosensitizer and visible light(commonly used laser).Firstly,the photosensitizer is selectively absorbed and stored within the tumor tissues.Then,the photosensitizer is activated by local irradiation with an appropriate wavelength of light.In previous study,it was found that prolyl 3-hydroxylase family member 4(P3H4),a protein coding gene,plays an irreplaceable role in a variety of physiological and pathological processes.It was highly expressed in bladder uroepithelial carcinoma,and knockdown of P3H4 gene can significantly inhibit bladder cancer cell proliferation,migration and invasion,as well as tumor growth at the animal level.Based on our previous works,a novel nano complex for PDT encapsulating siP3H4,was constructed to reduce P3H4 expression in bladder cancer cells and tissues and inhibit the genesis and development of bladder cancers through laser irradiation.This paper provides a solid theoretical basis for the diagnosis and treatment of bladder cancers in the future.Part ⅠConstructiron of nanocomplexes encapsulating siP3H4 for PDTObjective:Gene therapy using siRNA as a drug molecule has attracted much attention in the biomedical field in recent years.This study aimed to construct an integrated nanomedicine encapsulating siP3H4 to achieve a combination ofgene therapy,photodynamic therapy and immunotherapy.Methods:Using a chemical modification method,the hydrophobic structures and tumor-specific targeting groups RGD were modified at the ends of polyarginineto encapsulate the photosensitizer Ce6 and siP3H4 molecules,respectively.The agarose gel electrophoresiswas performed to verify that siP3H4 and CH3-R9-RGD polypeptide were assembled to form a nanocomplex structure,whose characterization was verified by DLS and transmission electron microscopy.The ability of cells to uptake the nanocomplexes,as well as the permeability and delivery efficiency of the photosensitizer Ce6,were analyzed by laser scanning confocal immunofluorescence microscopy Thereafter,the drugloading capacityof Ce6 in the nanocomplexes was subsequently measured and the delivery efficiency of nanocomplexes encapsulating si-P3H4 and Ce6 in bladder cancer cells was verified.Combined with the immunofluorescence technique,the ability of the nanocomplexes to produce 1O2 in the cells was verified.Not only that,the effects of laser irradiation and pH on the drug release of nanocomplexes were also investigated.Finally,the mechanism for targeted delivery of miRNAs by the self-assembled nanocomplexes was investigated.Results:The target carrier compounds were successfully synthesized through thesynthesis of target carrier compound 1a and target carrier compound 1b,and the acetylenic linkage modification,and their molecular weights and structures were determined.The nanocomplexes were self-assembled successfully from siP3H4,commercial photosensitizer ce6 and CH3-R9-RGD peptide,and the immunofluorescence imaging showed that the nanocomplexes could successfully encapsulatesi-P3H4 and Ce6,thus targetedly deliver si-P3H4 and Ce6 to bladder cancer cells simultaneously.Ce6 in the nanocomplex could cause an increase in intracellular reactive oxygen species(ROS)level.In addition,the targeted nano-drug delivery system could control the drug release by laser irradiation,and the interaction between cRGD and cRGD receptors is crucial inthe delivery process ofmiRNAs by the self-assembled nanocomplex.Conclusion:The above experiments demonstrate that the nanocomplexes encapsulating siP3H4 for PDT can be successfully prepared,and the novel nanocomplexes can targetedly deliver si-P3H4 and Ce6 into bladder cancer cells and induce ROS production in bladder cancer cells.the mechanism for targeted delivery of miRNAs by nanocomplex may be related to the interaction between cRGD and cRGD receptors.Part ⅡBiological effects of novelnanocarriers for PDT on bladder cancersObjective:Previous studies and the experiments in part Ⅰ verified the biological effects of P3H4 on bladder cancers and successfully prepared novel nanocomplexes encapsulating siP3H4 for PDT.respectively;they also confirmed that P3H4 could exert a carcinogenic effecton bladder cancer,and the novelnanocarriers for PDT could targetedly deliver siP3H4 into bladder cancer cells,inhibit the expression of P3H4 in vitro andinduce the production of tumor-killing ROS.However,the biological effects of the novel nanocomplexes for PDT on bladder cancer in vivo and in vitro remain uncertain,therefore,in this part,we aimed to explore the inhibitory effect of the novel nanocomplexes encapsulating siP3H4 for PDT on bladder cancers in vitro and in vivo and its effect on the expression of P3H4 in vitro and in vivo.Methods:The cytotoxicity of the novel nanocarriers for PDT on bladder cancers was examined in vitro by co-culturing the novel materials with bladder cancer cells.The killing effects of nanocomplexes on tumor cells in different groups were measured by flow cytometry,and apoptosis-related indicators were determined.Meanwhile the in vitro expression levels of P3H4 and CRT in bladder cancer cells of different groups uptaking different amount of nanocomplexes were detected by q-PCR and WB.The mouse tumor models were constructed to determine the in vivo localization statuses of the nanocomplexes and their inhibitory effects on tumor growth in different groups.The expression levels of P3H4 and Ki67 in the tumors within mice were detectedby WB and HE staining,and the expression level of CRT in the tumor tissues was also detected.Finally,the inhibitory effects of nanocomplexes on the distal tumors of mice in different groups were detected,and the expression levels of related apoptotic proteins such as Ki67 and P3H4 in the distal tumors of mice were determined by WB and HE staining.Results:In different groups,different amount of nanocomplexes were co-cultured with bladder cancer cells,the results revealed that the cancer targeted peptides almost had no toxicity to bladder cancer cells and exhibited a good compatibility,When the material was irradiated by a laser,the novel nanocomplexes encapsulating photosensitizer exerted a killing effect on the vitality of bladder cancer cells,which had the greatest effect on cancer cell apoptosis in the CH3-R9-cRGD&ce6&siP3H4 group.Similarly,q-PCR and WB results showed that the nanocomplexes produced an inhibitory effect on the expression levels of P3H4 and CRT in bladder cancer cells in vitro,indicating that the nanocomplexes could exert a killing effect on bladder cancer through photosensitization in vitro,and inhibit the expression of the target protein P3H4,and thus had a good carcinostatic effect.The nanocomplexes accumulated mainly in the bladders of bladder cancer mice and had a good localization effect.Thus exerting a significant inhibitory effect on tumor growth in mice and inhibiting the expression of apoptosis-related proteins such as Ki67,CRT and P3H4.Similar effects were observed indistal tumors of mice,where the growth of distal tumors and expression levels of P3H4 and Ki67 were inhibited to different degrees.Conclusion:Both in vivo and ex vivo experimental results indicate that the novel nanocomplexes for PDT can inhibit the expression of P3H4 in bladder cancer cells and tissues in vivo and in vitro,promote cell apoptosis and inhibit the genesis and development of bladder cancer,and have a good biocompatibility. |
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