| Background and PurposeThe incidence rate of hepatocellular Carcinoma(HCC)ranks sixth In the world.It is difficult to treat HCC,with high mortality and insensitivity to radiotherapy,so it can only be treated with chemotherapy.However,the current clinical chemotherapy drugs have two serious problems:weak or no targeting and the rapid formation of Multiple Drug Resistance(MDR),which lead to poor therapeutic effect and poor prognosis of HCC.Based on the above problems,how to improve the targeting of current clinical chemotherapy drugs and MDR reversal is an effective approach and hope for the future treatment of all cancers,especially HCC.Doxorubicin(DOX)is a first-line chemotherapy drug commonly used in the clinical treatment of HCC.It is considered to be one of the most effective chemotherapy drugs for HCC due to its powerful cancer cell killing ability.However,DOX has strong toxicity to heart and other proliferating cells,especially to heart,which will lead to serious myocardial damage and heart failure.Liposome is a kind of common nano carrier in clinical.It has good biocompatibility,can effectively contain a variety of chemotherapy drugs,and has the ability of slow and controlled release.It has a good prospect of clinical cancer treatment.Currently,Lipo-DOX has been marketed as a liposome pharmaceutical preparation.Due to the water-solubility of liposomes,Lipo-DOX is easier to fuse with the cell membrane,so that more DOX can enter the tumor cells,increase the concentration in the cells,and improve the killing efficiency of DOX on cancer cells.However,because Lipo-DOX has no obvious targeting and is still highly toxic,cancer cells can also quickly establish MDR.HCSP4,a hepatocellular carcinoma targeting polypeptide,has been screened out in the laboratory in the early stage,and a series of HCC cell specificity and sensitivity tests have been conducted to confirm its targeting of HCC cells.Combined with literature reports and database results analysis,it was predicted that miR101 could inhibit MDR of chemotherapy drugs,and the precursor of miR101(pre-miR101)as its plasmid expression carrier.So we will liver targeting polypeptide HCSP4 links outside the liposomes,miR101 wrapped in clinical chemotherapy drugs DOX liposomes and carrying into HCSP4-Lipo-DOX-miR101 liposome system,and in vitro transfection HepG2 HCC cells and DOX resistant HCC cell HepG2/ADR,studied the liposome drug preparations poisonous to the cells of HCC cells,proliferation,athletic ability,the biological effects of apoptosis and damage.The purpose of this study is to provide new ideas for the development of new targeted chemotherapy drugs for liver cancer and to accumulate experimental basis,so as to solve the bottleneck of current cancer clinical chemotherapy.The main features and innovations of this study are as follows:(1)targeting of HCC;(2)MDR inhibitory application of miR101;(3)simultaneously integrate the targeting of HCC and the inhibitory characteristics of MDR into a drug delivery system.MethodsBased on the above situation,this study attempted to construct a HCC targeting drug delivery system HCSP4-Lipo-DOX-miR101 successfully meeting the requirements of in vivo and in vitro application by taking the HCSP4 targeting peptide of HCC cells obtained in this room as the guiding element and miR101 which we screened based on the database as the MDR inhibiting element.(1)Scanning electron microscopy,transmission electron microscopy and particle size diffraction analyzer were used to conduct quality control analysis on the prepared liposome system;(2)pcDNA6.2-gw/EnGFP plasmid was used instead of pcDNA6.2-gw/miR101 expression plasmid to transfect HCC cells,and the targeting of liposome system hcsp4-lipo-dox-mir 101 on HepG2 of HCC cells was detected under inverted fluorescence microscope,and the transfection efficiency was determined to determine the optimal transfection conditions.(3)Toxicity of HCC cells,especially dox-resistant HCC cells,was detected by MTT and cell cloning;(4)The effects of liposomes on apoptosis of two types of HCC cells were detected by DAPI staining of the nucleus,Mitoview633 staining of the mitochondria and DNA ladder experiments.(5)The movement and proliferation capacity of HepG2 and HepG2/ADR cells after transfection of HCC cells with liposomes was detected by the scratch method and Transell assay.(6)Cell cycles of HCC cells HepG2 and HepG2/ADR were detected by flow cytometry;(7)The cytoskeletal morphology of two types of HCC cells,especially dox-resistant HCC cells,was detected and observed by scanning electron microscopy,coomassie blue staining and-tubulin assay.(8)MDR related genes used chemotherapy drugs were analyzed through network database.ResultsBased on the above experiments,the following results were obtained in this study:(1)the drug loading system of hcsp4-lipo-dox-mir101 was successfully prepared.The liposomes were spherical in shape,evenly distributed,uniform in size,particle size between 100-135nm,and the drug encapsulation rate was 90%,which met the requirements of in vitro experiments.(2)in vitro experimental results confirmed that HCSP4-Lipo-DOX-miR101 had a good targeting effect on HCC cells.(3)MTT and cell cloning experiments confirmed that HCSP4-Lipo-DOX-miR101 significantly enhanced DOX's lethalcy against HCC cells.(4)DAPI staining of the nucleus,Mitoview633 staining of the mitochondria and DNA ladder experiments confirmed that HCSP4-Lipo-DOX-miR101 significantly promoted the apoptosis of HCC cells.(5)the ability of HCSP4-Lipo-DOX-miR101 to significantly inhibit the movement and proliferation of HCC cells was confirmed by the scratch test and Transell assay.(6)flow cytometry confirmed that HCSP4-Lipo-DOX-miRl01 blocked the HCC cell cycle in the G0/G1 phase.(7)HCSP4-Lipo-DOX-miR101 significantly inhibited the development of motion-related microstructures of HCC cells through scanning electron microscopy,coomassie blue staining,and t-tubulin staining.(8)According to the analysis of network database,the drug resistance related genes of adriamycin are ABHD2,ANPEP,CHSY1,GDPGP1,RCCD1,ACAN,etc;the drug resistance related genes of 5-fluorouracil are CDH3,DVL2,FBX039,MPDU1,SAT2,SENP3,etc;the drug resistance related genes of cisplatin are MTUS2,AC064874.1,AGAP1,C8orf49,GATA4.ConclusionsThe liposome system prepared by us,hcsp4-lipo-dox-mir101,is specific to cultured HCC cells,especially HepG2 cells.Hcsp4-lipo-dox-mir101 has obvious cytotoxic and malignant inhibitory effects on HCC cells HepG2,especially dox-resistant HCC cells HepG2/ADR.On the basis of highly targeted HCC mediated by HCSP4,the therapeutic efficiency and toxicity of HCC were significantly improved.MDR of mir101-mediated DOX was significantly inhibited or.In the presence of these two key guiding elements,targeted liposome drug delivery system hcsp4-lipo-dox-mir 101 may achieve high efficacy,low toxicity,anti-mdr,anti-recurrence and other effects in the treatment of HCC.However,this is the bottleneck problem in the treatment of liver cancer and other cancers. |
PDF Full Text Request