Application Of Chitosan-based Nanomaterials In Targeted Therapy Of Bladder Cancer

Objective:Chitosan(chitosan,CS)nano materials has become commonly used transport carrier targeted therapy of cancer because of its excellent biological properties.CD44 is the receptor of hyaluronic acid HA,which is highly expressed in bladder cancer.We prepared a new type of chitosan nanomaterials modified by HA to package cy3-labeled siRNA,and transfected the bladder cancer T24 cells with the Bcl-2 oncogene as the target.A series of experiments were conducted to demonstrate the inhibitory effect of sCSNPs-HA on bladder cancer T24 cells,to evaluate the feasibility of chitosan nanomaterials as siRNA delivery carriers,and to provide new ideas for the treatment of advanced bladder cancer.Methods:Chitosan(CS)and sodium triphosphate(TPP)were prepared by ionic crosslinking chitosan nanometer materials(chitosan nanoparticles,CSNPs).Then sCSNPs was prepared by CSNPs package containing cy3-labeled siRNA targeting Bcl-2 oncogene,and CSNPs and sCSNPs were combined with hyaluronic acid(HA)to prepare CSNPs-HA and sCSNPsHA.The average particle size,dispersion(PDI)and Zeta potential of the four materials were measured by laser particle size analyzer for 6 consecutive days to test their stability.The SEM(scanning electron microscopy,SEM)was used to observe the preparation of chitosan nanoparticles on the surface of nanomaterials structure.The inclusion capacity of CSNPs and CSNPs-HA on siRNA was evaluated by spectrophotometry.The preparation method of chitosan nanomaterials was continuously optimized by changing the ratio of CS to TPP,the preparation temperature,the frequency and time of ultrasound.MTT experiment was used to evaluate the cytotoxicity of chitosan nanomaterials CSNPs and CSNPs-HA.sCSNPs and sCSNPs-HA were transfected into bladder cancer T24 cells by using CSNPs and CSNPs-HA containing cy3-labeled siRNA.The transfection effect was observed by fluorescence microscope and the fluorescence data were analyzed.MTT experiment and scratch experiment were used to detect the proliferation and migration of bladder cancer T24 cells in different groups.qRT-PCR and Western Blot were used to detect the differences in Bcl-2 mRNA levels and expression levels of related regulatory proteins in bladder cancer T24 cells,and to evaluate the effect to silence the expression of Bcl-2 of sCSNPs and sCSNPs-HA.Results:The average particle size of chitosan nanometer material obtained by the optimal design was 120 nm,the dispersion degree was less than 1,and it had good dispersion.The parameters obtained for 6 consecutive days did not change much,indicating its stability was good.Under the scanning electron microscope,the nanoparticles are typically spherical,with a round shape and good dispersion.CSNPs and CSNPs-HA had strong ability to package siRNA.MTT experiments showed that CSNPs and CSNPs-HA cytotoxicity of chitosan nanomaterials met the standards of biomedical material.Fluorescence images and fluorescence data proved that the transfection effect of chitosan nanomaterials was better than that of Hiperfect.Scratch test showed that sCSNPs-HA group can significantly reduce the migration ability of bladder cancer T24 cells.MTT experiment showed that s CSNPsHA group could significantly inhibit the proliferation of bladder cancer T24 cells.qRT-PCR and Western Blot showed that s CSNPs-HA group had a significant inhibitory effect on the expression of Bcl-2 gene at mRNA and protein levels.Conclusion:This research successfully constructed the HA modified chitosan nanomaterials(sCSNPs-HA)containing cy3-labeled siRNA.The optimized ion crosslinking method was used to prepare the nanoparticles with ideal size,shape and dispersion.CSNPs and sCSNPs-HA met the standards of biomedical materials,and the transfection effect was better than the recognized transfection reagent.MTT experiment and scratch experiment proved that transfection of bladder cancer T24 cells with s CSNPs-HA can silence Bcl-2 gene and significantly reduce the proliferation and migration capacity of T24 cells.qRTPCR and Western Blot showed that s CSNPs-HA group had a significant inhibitory effect on the expression of Bcl-2 gene at mRNA and protein levels,and thus promoted the apoptosis of tumor cells,providing a new theory for targeted therapy of bladder cancer gene.