Preparation Of Hepatocarcinoma Targeted Doxorubicin And Its Application Both In Vitro And In Vivo

Hepatocellular carcinoma (HCC) was one of the most common malignancies in our country. Due to its high degree of malignancy, rapid progress, easy relapse and metastasis, HCC severely threatened people’s health and life. Therefore, how to effectively diagnosis and treatment hepatocarcinoma was now becoming the urgent requirement to resolve the issue.Life of the patients could be prolonged by traditional chemotherapy, radiation therapy and surgery, and the life quality could be improved in certain extent, whilst the effect of these treatments was very limited. The effect of liver transplant, developed in recent years, was also limited because of the very fewer sources. Therefore, targeted therapy for liver cancer as an effective specificity treatment became a hotspot of researched and showed good application prospect in future.Targeted therapy was the therapy that took along the toxic substances in cells selectively as a bullet (such as nuclein, chemotherapy drugs, toxins) to the site of the tumor cells through high specificity carrier and killed them without damaging the normal tissues. The most remarkable feature of the targeted drugs was that the drugs could be taken to the target areas effectually to get a high drug concentration, and eventually effect on the pathological tissues, organs and cells. This could prolong action time, increase the drug concentration, reduce doses of administered drugs, and decrease the toxic and side-effect of the drugs to get a high potency and low side-effect therapeutic effect. However, many technical issues remained unsolved in the clinical application of targeted therapy, such as the carrier, coupling of the carrier and bullet. This might limit the development of targeted therapy in hepatocellular carcinoma. Therefore, looking for more effective and safe carriers would be an emphasis of targeted therapy research at present.In this paper, some specifically binding peptides to hepatocarcinoma tissues were screened by using in vivo phage display technology (amino acid sequence is AGKGTPSLETTP, Chinese Patent:200410017048.X). Making the most use of specifically binding peptides’lower molecular weight and immunogenic to make up the shortcomings of monoclonal antibody and to establish a feasible peptides system for diagnosis and targeting therapy to hepatocarcinoma.Doxorubicin, a classic of antitumor antibiotic drug, was widely applied in acute lymphocytic leukemia, prostate Cancer and hepatocarcinoma, and showed good therapeutic effects. However, the biggest problem in doxorubicin’s clinical application was that doxorubicin had significant side effects of myelosuppression, cardiotoxicity and neurotoxic effects. These toxic and side-effects threatened the patient’s health seriously and limited doxorubicin’s application terriblely in clinic.In this study, doxorubicin was conjugated to hepatocarcinoma-specific peptide through chemical methods. This conjugation increased the drugs concentration of tumor tissue, enhanced the lethality to hepatocarcinoma, reduced the dose of drugs, and inhibited the side effects evidently.This paper was divided into three parts:a) Conjugation of hepatocarcinoma-specific peptide A54and doxorubicinDoxorubicin was conjugated to hepatocarcinoma-specific peptide A54by chemical methods. According to the process and principle, two different types of synthetic methods were undertaken, and three products were obtained:DOX-A54-1、 DOX-A54and DOX-A54H. Subsequently, cell viability was investigated by MTT cell proliferation assay after three drugs treatment.b) Identification the bioactivity of DOX-A54H in vitroTo further study the binding ability and mechanism of DOX-A54H, cells were treated with DOX-A54H when compared with DOX-A54and DOX-A54M, respectively. A54M was the binding motif mutated peptide, proline and serine mutating alanine in its amino acid sequence. Cell viability was investigated by MTS cell proliferation assay after the drug treatment. Then, the binding characteristics of these conjugates were measured by fluorescence microscope. Furthermore, the apoptosis or necrolysis of the treated cells were confirmed by flow cytometric analysis and LIVE/DEAD assay.c) Antitumor activity of DOX-A54H in vivoThe in vivo antitumor activity of DOX-A54H was also investigated in nude mice bearing BEL-7402human hepatocarcinoma cell. Doxorubicin in different concentrations was injected into nude mice via i.v. to investigate the best dosage of drugs. The mice were treated with DOX-A54H and related drugs via i.v.. The survival rate, volume of tumor and weight of the mice was detected. After several times injection, nude mice were killed and serum was collected. CREA, BUN, TBIL, ALT, AFP, CEA were detected by automatic biochemical analyzer.From the data, it was concluded that:(1) Three conjugates DOX-A54-1, DOX-A54and DOX-A54H were constructed successfully. The proliferation of DOX-A54H treated cells was restrained significantly in vitro by MTT assay. The concentrations of drug and the values of OD570showed an obvious dose-effect relationship. Its IC50was2.27μM.(2) DOX-A54H was found to exhibit selective binding and specific cytotoxicity to hepatocyte cell HL-7702, hepatocarcinoma cell BEL-7402, SMMC-7721and SK-Hepl in vitro. Flow cytometric analysis and LIVE/DEAD assay indicated the DOX-A54H can specifically kill hepatocellular carcinoma by inducing the necrolysis but not apoptosis of cancer cells.(3) The treatment of tumor-bearing nude mice with DOX-A54H tumors caused inhibition of tumor growth to a certian extent in vivo. These results suggested the DOX-A54H could be used as an effective agent, which might lay a foundation for further application in targeting therapy to hepatocarcinoma.