| Breast cancer is an important cause of women 's death.The treatment is mostly surgical treatment,but surgical treatment causes a lot of inconvenience to women's future life,so most patients choose breast-conserving treatment,including radiotherapy and chemotherapy.However,chemotherapy will cause certain side effects to patients.In order to improve the efficacy of chemotherapeutic drugs and reduce their toxicity,we have developed a chemotherapy drug that uses partially acetylated polyamidoamine as a carrier and luteinizing hormone releasing hormone as a target seeker Gene drug targeting co-delivery system.The co-delivery system can target the delivery of the drug to the location of the lesion,increase the concentration of the drug and reduce the toxic and side effects of the drug on normal tissues and organs.Objective:Successfully prepared and characterized LHRH-G5.0NHAC-FUA/miR-205 nano-co-delivery targeted drug delivery system,and studied its anti-breast cancer effect in vivo and in vivo and its mechanism of action.Method: 1.Preparation of LHRH-G5.0NHAC-FUA/miR-205nano-drug co-delivery system: G5.0NHAC is obtained after partial acetylation of the amino group on the surface of the fifth generation PAMAM,and the remaining amino groups are coupled to G5.0NHAC through functionalized PEG to LHRH surface to obtain LHRH-G5.0NHAC,and then 5-fluorouracil-1-acetic acid(FUA)modified with bromoacetic acid was bonded to G5.0NHAC through amide bond,and LHRH-G5.0NHAC-FUA nanoparticles were successfully prepared Target prodrugs.After that,miR-205 mimic and the above prodrugs were electrostatically combined in different proportions to prepare LHRH-G5.0NHAC-FUA/miR-205 nano-medicine co-delivery system.2.Characterization of physical and chemical properties of LHRH-G5.0NHAC-FUA/miR-205 nanometer drug delivery system and its biocompatibility in vitro: The optical properties and chemical structure of LHRH-G5.0 NHAC-FUA/miR-205 were characterized by ultraviolet(UV)spectroscopy and NMR.The optimal mass ratio betweenLHRH-G5.0 NHAC-FUA and miR-205 was determined by gel electrophoresis and cell transfection.The particle size distribution and potential of miR-205 were characterized by nanometer.The drug loading capacity of LHRH-G5.0 NHAC-FUA/miR-205 and its drug release performance at different pH values were determined by ultraviolet spectrophotometry.The biocompatibility of LHRH-G5.0NHAC-FUA/miR-205 was investigated by hemolysis assay.The toxicity of LHRH-G5.0NHAC to normal cells was investigated by MTT.3.In vitro antitumor effect and mechanism of LHRH-G5.0NHAC-FUA/miR-205 nano-delivery co-delivery system:evaluation of LHRH-G5.0NHAC in vitro targeting by microfluorescence photography;human breast cancer Cells MCF-7 and MDA-MB-231 are the research objects of in vitro antitumor experiments.The in vitro tumor proliferation inhibition of LHRH-G5.0NHAC-FUA/miR-205 was characterized by MTT method;the scratch healing experiment was used to evaluate LHRH-G5.0NHAC-FUA/miR-205 ability to inhibit tumor cell migration;Transwell experiment was used to observe the ability of LHRH-G5.0NHAC-FUA/miR-205 to inhibit tumor cell invasion;the effects of LHRH-G5.0NHAC-FUA/miR-205 on tumor cell apoptosis and cycle were detected by flow cytometry.4.LHRH-G5.0NHAC-FUA/miR-205 nanometer delivery co-delivery system in vivo tissue distribution and antitumor effect:MCF-7 breast cancer nude mice experimental animal model was established and randomly divided into abdominal cavity After administration by injection,the changes in body weight and tumor volume were recorded.After 21 days of administration,the tumor-bearing nude mice were dissected and the tumor tissues and heart,liver,kidney and other major organs were removed.To evaluate the toxic and sideeffects of LHRH-G5.0NHAC-FUA/miR-205 on normal organs of animals,another section of tumor and organ tissue sections were stained with VEGF-A antibody to observe the antitumor effect of miR-205.At the same time,blood was taken after 7 days of continuous administration to SD rats,and the main organ-related parameters were measured by an automatic biochemical detector to evaluate the toxic and side effects of LHRH-G5.0NHAC-FUA/miR-205.Results: The compounding ratio of LHRH-G5.0NHAC-FUA is LHRH: G5.0NHAC: FUA=1.5: 1: 300,and the optimal mass ratio of LHRH-G5.0NHAC-FUA to miR-205 is 1024: 1.Under the optimal synthesis conditions,the LHRH-G5.0NHAC-FUA/miR-205 nanometer delivery co-delivery system has a drug loading of 40.76%(n=3),an average particle size of 238.40 nm,and a Zeta potential of 4.40 mV;UV and NMR showed that LHRH-G5.0NHAC-FUA was successfully synthesized;drug release performance results showed that LHRH-G5.0NHAC-FUA/miR-205 had a good sustained-release effect;LHRH-G5.0NHAC-FUA/miR-205 The hemolysis rate is below 5%,which indicates that it has good biocompatibility;the fluorescence photograph results show that LHRH-G5.0NHAC-FUA/miR-205 has good targeting for breast cancer cells,and the competitive binding experiment also shows that LHRH Targeting to breast cancer cells;MTT method results showed that the toxicity of G5.0NHAC obtained after acetylationwas significantly less than that of G5.0 PAMAM,and LHRH-G5.0NHAC-FUA/miR-205 had a synergistic effect on inhibiting breast tumor cell proliferation There is time and dose dependence;the results of scratch healing experiments and Transwell experiments show that LHRH-G5.0NHAC-FUA/miR-205 has the ability to synergistically inhibit cell migration and invasion;experimental results in vitro further illustrate LHRH-G5.0NHAC-FUA/miR-205 has stronger anti-breast cancer effect than other groups.Experimental results in vivo and in vitro show that LHRH-G5.0NHAC-FUA/miR-205 reduces the toxic and side effects of 5-FU on major organs to a certain extent,and has a synergistic effect on breast cancer.Conclusion: LHRH-G5.0NHAC-FUA/miR-205 nano-delivery drug delivery system targeting breast cancer has been successfully prepared.The system has good biocompatibility,safety,sustained release,and has anti-tumor effects in vitro and in vivo. |
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