New Polypeptide-adriamycin-miR-liposome Targeted Therapy For Liver Cancer And Its Anti-drug Resistance Effect

Background and PurposeHepatocellular carcinoma(HCC)is the sixth most common malignant tumor in the world and has a high mortality rate.It is second only to gastric cancer in rural areas in some remote areas of China,especially in the southern coastal areas.Liver cirrhosis is the basis for the development of HCC.New cases occur each year in patients with HCC,and the global incidence continues to increase.At present,the clinical treatment of HCC is facing a huge bottleneck problem:the vast majority of patients have lost the opportunity for surgical treatment.Moreover,regardless of postoperative chemotherapy and conservative treatment of non-surgical patients,chemotherapy must be used as the main and only clinical treatment.However,current clinical chemotherapeutic drugs have presented a huge bottleneck in the rapid development of efficacy,toxicity,and multidrug resistance(MDR)due to the following two points.There are two basic reasons for this problem;First,most drugs do not have cancer cell/tissue targeting or lack of targeting;second,these drugs cannot effectively inhibit the formation of MDR.The above causes the clinical chemotherapy characteristics of poor drug efficacy and great toxicity.In particular,the clinical cure rate(the clinical standard survival period)and the short-term mortality rate of patients with high-grade malignant tumors such as HCC are still high.Based on the above,how to improve the targeting and anti-MDR characteristics of current clinical anticancer first-line chemotherapy drugs is an effective way and hope for the development of cancer,especially HCC,in the future.Doxorubicin(DOX)is the first-line broad-spectrum antitumor drug currently used in DNA in clinical practice.This drug is considered to be one of the most effective HCC chemotherapeutics because of its strong cancer cell killing ability.However,the toxicity of DOX,especially of the heart and other proliferating cells,is very prominent.Later,several brands of DOX liposomes(Lipo-DOX)were developed and marketed.This drug is able to greatly increase the concentration of DOX in cells due to its ease of fusion with the cell membrane,thereby significantly enhancing cytotoxicity of the DOX against cancer cells.However,since the drug is still very toxic due to its lack of significant targeting,MDR will be established very quickly.Therefore,solving the problem of targeted and MDR reversal is a problem that needs to be solved urgently for chemotherapeutic drugs including Lipo-DOX,and it is also one of the effective ways to make breakthrough progress in cancer clinically.Based on the above-mentioned targeting and reversal of MDR,the following drug carrier system was designed and its partial in vitro efficacy was completed.We screened the HCC-targeting 12-peptide HCSP4 in advance and performed a series of in vitro and in vivo validation of human HCC cell/tissue including clinical specimens.The results showed that HCSP4 has very good binding specificity for HCC and Sensitivity,therefore,can completely solve the HCC targeting problem with HCSP4 as a guiding element! With regard to anti-MDR,which is the MDR reversal problem,combined with literature and our own database analysis and prediction,we believe that a miRNA(the specific name is temporarily inconvenient for disclosure)has the potential to reversibly transform the drug MDR.We succeeded in using this miRNA precursor(pre-miRNA)has constructed its plasmid expression vector.In this study,the DOX liposome drug delivery system(HCSP4-Lipo-miRNA-DOX)with MDR reversion characteristics targeting HCC was constructed using the above peptides and miRNAs.It is assumed that this system may have high HCC targeted and obvious MDR reversal characteristics,so as to achieve the efficacy of DOX with high efficacy and low toxicity,no or weak MDR HCC treatment! This design itself has a high theoretical basis and originality,and it is also very much in line with the actual needs of the current worldwide cancer clinical.The liposomal properties of the prepared HCSP4-Lipo-miRNA-DOX were studied by particle size,dispersion coefficient,drug entrapment efficiency,and targeted detection.The results showed that the expected range of properties was achieved.In order to study the therapeutic effect of HCSP4-Lipo-miRNA-DOX on HCC and evaluate its ability to reverse MDR of DOX,this study investigated the liposomal drug delivery system on human HCC after transfection of cells with HCSP4-Lipo-miRNA-DOX.Biological effects of HepG2 and DOX-resistant human HCC HepG2/ADR cells on cytotoxicity,proliferation,motility,and apoptosis.In particular,HepG2/ADR was used to evaluate the MDR reversal effect of DOX on HCC cells.Methods1.The target sequence of the miRNA was determined on-line by mirbase(http://www.mirbase.org),the insert was designed,and it was inserted into the pcDNA6.2-GW vector by synthesis and annealing to obtain pcDNA6.2-GW/miRNA.2.The HCSP4-modified liposomes were prepared by thin film ultrasonic dispersion method.The DOX drug was loaded by ammonium sulfate gradient method.miRNAs were linked to the properties of cationic liposomes interacting with negatively charged plasmids,resulting in a novel HCSP4-targeted miRNA doxorubicin liposome,HCSP4-Lipo-miRNA-DOX.3.The use of laser particle size analyzer,transmission electron microscope,scanning electron microscope,fluorescence spectrophotometer and other techniques to detect the high drug encapsulation efficiency of novel liposomes HCSP4-Lipo-miRNA-DOX and other liposome characteristics.4.The pcDNA6.2-GW/EmGFP plasmid was used instead of pcDNA6.2-GW/miRNA to optimize the transfection efficiency of the HCSP4-Lipo-miRNA-DOX system.The targeting of HCSP4 was evaluated by transfection of human gastric cancer SGC7901 and human hepatoma HepG2.5.Transfer HCSP4-Lipo-miRNA-DOX into HepG2 and HepG2/ADR,use wild-type cells and HCSP4-Lipo-DOX as controls,the effects of HCSP4-Lipo-miRNA-DOX on cell killing,proliferation,locomotion,and apoptosis of HepG2 and HepG2/ADR were detected by MTT assay,injury repair assay,flow cytometry,cell and nuclear staining.6.Several miRNA-targeting genes that are associated with MDR formation have been detected and the experiments have been very successful.The preliminary molecular mechanism of HCSP4-Lipo-miRNA-DOX reversal of MDR was obtained! The results and contents of this part will be stated in the follow-up study materials of this study.This paper does not include the contents of this part of the statement.Results1.The miRNA expression plasmid was successfully constructed and sequenced correctly.2.The HCSP4-Lipo-miRNA-DOX system was successfully prepared and its DOX drug entrapment efficiency was high(90%),the shape of the liposome was spherical,the size was uniform,the distribution was uniform,and the particle size was ±130nm,which was in line with future in vivo applications.3.The HCSP4-Lipo-miRNA-DOX system was transfected into HepG2 cells with a transfection efficiency of 80%.HCSP4-targeted liposomes had good targeting.4.MTT,injury repair,DAPI staining,Coomassie brilliant blue staining,cell plate cloning,flow cytometry results suggest that HCSP4-Lipo-miRNA-DOX liposome itself is a safe biomaterial,the killing power of HepG2 and HepG2/ADR cells was significantly enhanced,which significantly enhanced the ability of DOX to inhibit cell proliferation and migration,promoted DOX-induced apoptosis of HCC cells,and arrested the cell cycle at G1 phase.ConclusionsPreliminary experimental results show that the HCSP4-Lipo-miRNA-DOX liposome drug delivery system is a novel HCC specific therapeutic drug delivery system.The cytotoxicity of HCSP4-Lipo-miRNA-DOX on HepG2 cells was stronger than that of HCSP4-Lipo-DOX,and the killing power of HepG2/ADR cells was significantly stronger than that of HCSP4-Lipo-DOX.According to the above experimental results,HCSP4-Lipo-miRNA-DOX has a good therapeutic effect on HCC in vitro,and more importantly,it has a significant inhibitory effect on the MDR formation of DOX during HCC treatment,so that DOX drugs can continue to exert their killing effect.This is of great significance and attractive social and economic benefits for improving the clinical effect of DOX on the treatment of HCC,reducing toxicity,and inhibiting the recurrence rate after HCC treatment.The design of HCSP4-Lipo-miRNA-DOX has a world-wide originality.Our above preliminary results suggest that it is of great value for the treatment of HCC and deserves further follow-up studies.At the same time,the experimental design and results have important implications for the research and development of other cancer types and chemotherapy drugs.