Construction Of Gold Nanorods System For Targeted Delivery Of HIF-1? And Combined Photothermal Therapy In The Treatment Of Breast Cancer

Objective:Gene-targeted therapy has become a hot topic in current cancer therapy research.Hypoxia-inducible factor-1??HIF-1??is a key regulator of tumor growth and adaptation to hypoxic conditions,and gold nanorods?GNRs?,as a multifunctional nanomaterial,gold nanorods?GNRs?are not only a photothermal agent but also a good gene carrier.Our study aims to construct a novel HIF-1?siRNA targeting introduction system,GNR-MUA-PEI-FA?GMPF?/siHIF-1?,to determine its biological safety and biological characteristics,combine the photothermal effect of GNR with the synergistic treatment of mice bearing breast cancer,and preliminarly explore its possible mechanisms.Methods:1.The classical seed-mediated growth method was used to prepare hexadecane-based trimethylammonium chloride?CTAB?-coated gold nanorods.The positively charged PEI was used to increase the transfection efficiency of cells,and it was also used to connect folic acid,which acts as a tumor targeting molecule.The plasmonic resonance,specific groups of folic acid,and the size,shape,and degree of the dispersion of gold nanomaterials were detected by ultraviolet spectrum,nuclear magnetic resonance?1H NMR?,and transmission electron microscopy?TEM?,respectively.2.The modified gold nanorods and GAPDH siRNA were mixed and incubated at different mass ratios.The effect of carrying and releasing si RNAs was observed by gel retardation assays.3.CCK-8 method was used to detect the biological toxicity of modified gold nanomaterials on 4T1 of mouse breast cancer cells.Flow cytometry was used to detect the transfection efficiency of GMPF/cy3-siGAPDH and to detect the tumor targeting of folate acid.4.Using 808nm near-infrared laser irradiation for 2min,4min,6min,8min,and10min,the temperature changes of the gold nanomaterials were measured.4T1 cells were irradiated with different degrees of near-infrared laser for 5 minutes.After 24hours,the photothermal effect of the material was detected by CCK-8.5.Different concentrations of cobalt dichloride were used as hypoxia-inducing agents to simulate hypoxia in vitro,and detect the HIF-1?protein in 4T1 cells after24h stimulation.6.Western blot was used to detect the silencing effect of GMPF/siHIF-1?transfected 4T1 cells.7.Apply a mixture of 50%glycerol,10%DMSO,and GMPF/cy3-siGAPDH to the transplanted tumors of breast cancer mice.Prepare frozen sections and observe the efficiency of their entry into the skin by fluorescence microscopy.8.To establish a tumor-bearing mouse model of breast cancer,and apply anti-tumor therapy in combination with near-infrared light irradiation by topical application of GMPF/siHIF-1?to tumor skin.Observe and record the effect on tumor growth.9.Western blot analysis detected the protein levels of HIF-?and MMP-2 in mouse tumors.Immunohistochemistry was used to detect the changes of microvessel density?MVD?,CD34⁺MVD,and CD146⁺MVD markers.The TUNEL method was used to detect the apoptosis of murine breast cancer.Results:1.The absorption peak of the GMPF ultraviolet spectrum of gold nanorods modified by MUA-PEI-FA had an obvious red shift,and the size and dispersal of the particles before and after the modification are not significantly changed,as confirmed by transmission electron microscopy.2.Gel block experiments showed that when the mass ratio of GMPF to siRNA was 6:1,and the GMPF was completely bound to the si RNA.Flow cytometry results showed that when the mass ratio was 20:1,the transfection efficiency reached a higher level,indicating that increasing the concentration of GMPF promoted an increase in transfection efficiency.However,when human osteoblasts were transfected with the same conditions,GMPF failed to increase transfection efficiency,demonstrating the targeting effect of GMPF on tumor cells.3.Compared with GNRs,GMPF significantly reduced the survival rate of 4T1cells,and had higher biocompatibility and biosafety levels.4.The temperature rises from the initial 28°C to 47°C after irradiation of GMPF for 10 minutes with near-infrared light.With the increase of power,the photothermal effect of GMPF was gradually enhanced.5.When CoCl₂ was used to stimulate 4T1 cells for 24h,the expression of HIF-1?protein was increased.When the concentration of CoCl₂ was 100?mol/L,the expression of HIF-1?protein was the highest.6.The transfection of 4T1 cells with GMPF/siHIF-1?can effectively reduce the expression of HIF-1?protein.7.Observed under the fluorescence microscope,GMPF/cy3-siGAPDH can effectively penetrate the skin and enter the tumor.8.The results of animal experiments showed that the gold nanorod-mediated HIF-1?siRNA targeting system combined with light has the best inhibitory effect on murine mammary carcinoma.The tumor growth rate was significantly inhibited,and the tumor quality was the lightest?P<0.05?.The results showed that the expression of MMP-2 protein was inhibited in the mouse tumors after silencing HIF-1?;immunohistochemistry results showed that CD34⁺MVD in the tumor tissue of the GMPF/siHIF-1?group and the GMPF/siHIF-1?combined with light group.The expression of CD146⁺MVD was significantly decreased?P<0.001?,and the apoptosis rate of tumor tissue in GMPF/siHIF-1?combined with light was higher than that of other groups?P<0.01?.Conclusions:1.The size,morphology,and dispersion of nanorods modified by MUA-PEI-FA of GNRs and prepared by Au seeds-mediated growth have no significant changes on the material.These nanorods can significantly reduce the material's biological toxicity,and specifically target tumor cells to effectively carry siRNA for transfection in addition to producing a photothermal effect under the excitation of near-infrared light.2.The novel gold nanomaterial GMPF/siHIF-1?has a transdermal effect.After being smeared on the skin,it can carry siRNAs into the target cell and silence the target gene.3.The combined photothermal effect of GMPF/siHIF-1?can effectively silence HIF-1?in tumor tissues of mice bearing 4T1 breast cancer,reduce the expression of MMP-2 protein in breast cancer tissue,reduce the generation of tumor blood vessels,and promote the apoptosis of tumor cells--creating a synergistic therapeutic effect on mouse breast cancer treatment.