Ultrasound-targeted Photodynamic And Gene Dual Therapy For Effectively Inhibiting Triple Negative Breast Cancer By Cationic Porphyrin Lipid Microbubbles Loaded With HIF1?-siRNA

Objective The purpose of this experiment is to prepare cationic porphyrin microbubbles carrying HIF-1 alpha siRNA combined with ultrasound to open the tumors-blood vessel barrier and increase the permeability of tumor cell membranes,increase the accumulation of photosensitizers in tumour cells and realize gene transfection of HIF-1alpha siRNA,construct a vector delivery system at the level of tumour cells,and realize the combination of gene therapy and photodynamic therapy.Methods The cationic porphyrin microbubbles were prepared by ethanol injection.HIF-1 alpha siRNA was modified on the surface of cationic porphyrin lipid microbubbles?CpMBs?by electrostatic adsorption to obtain siHIF@CpMBs.The particle size of siHIF@CpMBs was analyzed by Kurt counter.The potential of siHIF@CpMBs and the particle size of siHIF@CpNPs were detected by ZetaPALS potential analyzer.The morphology of siHIF@CpMBs was observed under fluorescence microscope.The morphology of siHIF@CpNPs was observed by TEM.The absorption and fluorescence of siHIF@CpMBs were detected by ultraviolet spectrophotometer and fluorescence spectrophotometer.Confocal microscopy was used to observe the phagocytosis of FAM-siHIF@CpMBs by MDA-MB-231 cells.SOSG single-line oxygen green fluorescence probe was used to detect the ability of siHIF@CpMBs to produce ¹O₂ under 650±5 nm laser irradiation.qRT-PCR was used to detect the ability of siHIF@CpMBs to knockdown HIF-1a.MTT was used to detect the effect of siHIF@CpMBs on the viability of HUVEC and MDA-MB-231 cells.The model of MDA-MB-231 tumor-bearing BALB/c nude mice was established by subcutaneous modeling.Ultrasound parameters:3-12 MHz,MI 0.104,and siHIF@CpMBs suspension with 200?L concentration of 1 mg/ml was injected into tail vein at the same time.The imaging of siHIF@CpMBs in tumor tissue was observed under two-dimensional gray scale pattern and contrast mode.BALB/c nude mice bearing tumors were randomly divided into three groups?n=5?.Ultrasound parameters:pulse frequency 1.03 MHz,duty cycle 50%,ultrasound intensity 1 W/cm²,intermittent ultrasound emission 30s/30s,duration of 3 minutes.Simultaneously,cy5.5 labeled siHIF@CpMBs were injected into tail vein and compared with in vivo fluorescence system of small animals.BALB/c nude mice bearing tumors were randomly divided into five groups?n=5?.Ultrasound parameters:pulse frequency 1.03 MHz,duty cycle 50%,ultrasound intensity 1 W/cm²,intermittent ultrasound emission 30s/30s,duration 3 minutes.Meanwhile,siHIF@CpMBs or siN.C@CpMBs was injected into tail vein to record the size and weight of tumors every other day.After 20 days of treatment,the heart,liver,spleen,lung and kidney of each group were stained with H&E.Results The particle size of siHIF@CpMBs is mainly concentrated in 1.3?m,the surface Zata potential is-10.57±1.74 mV,and siHIF@CpMBs show spherical structure of about several microns in size under the microscope.FAM-siHIF is successfully adsorbed on CpMBs.After the ultrasound targeted microbubbles destruction?1.03 MHz,50%duty,1 W/cm²,1 min?,the particle size of siHIF@CpNPs is about 100nm with good dispersion.The absorption and fluorescence spectra of CpMBs,siHIF@CpMBs,siHIF@CpMBs+US did not change.When N/P was 15:1,all HIF-1 alpha siRNA adsorbed on the surface of CpMBs.2.Prophyrin and FAM-siHIF were successfully transfected into cells under the ultrasound targeted microbubbles destruction;only siHIF@CpMBs+US+Laser group produced a large amount of ¹O₂;the expression of HIF1 alpha mRNA was detected by qRT-PCR.Compared with other groups,the expression of HIF1 alpha mRNA in siHIF@CpMBs+US+Laser group and siHIF@CpMBs+US-Laser group decreased significantly,the results showed that there were statistical differences?P<0.05?.MTT assay showed that the cell viability of siHIF@CpMBs+US+Laser group was significantly lower than that of other groups?P<0.05?.3.In vivo imaging of siHIF@CpMBs has a good contrast-enhanced ultrasound effect.Semi-quantitative fluorescence analysis of Prophyrin enrichment in mice showed that the concentration of Prophyrin in cy5.5-siHIF@CpMBs+US group was significantly higher than that in cy5.5-siHIF@CpMBs group,while the concentration of Prophyrin in liver was decreased,and the fluorescence intensity of tumor tissue and liver tissue in vitro were 131.9±17.14?5.56±1.75?58.54±5.59?129.97±13.98,respectively.The results showed that there was a statistical difference between the two groups?P<0.05?.4.The tumor recurred in the siN.C@CpMBs+US+Laser group when injected with 200?L of 1 mg/ml siN.C@CpMBs or siHIF@CpMBs in nude mice,but there was no recurrence in the siHIF@CpMBs+US+Laser group during the observation period,and there was statistical difference compared with other treatment groups?P<0.05?.5.After 20 days of treatment,there was no significant organ damage in each group.Conclusion 1.siHIF@CpMBs s has a stable spherical structure.2.siHIF@CpMBs combined with ultrasound increased the tumor-vascular barrier and cell membrane permeability,and increased the concentration of photosensitizers/genes in tumors.3.siHIF@CpMBs enhances the therapeutic effect of TNBC by combining photodynamic therapy with gene therapy.