Synthesis And Biological Evaluation Of Hepatocarcinoma Targeted Drug A54-GFLG-DOX

Hepatocellular carcinoma, is called HCC for short. It is the third highest cause of common malignant cancer mortality after gastric cancer, esophageal cancer. Every year the number of people in China die from HCC is about 11 million of the world 45% of HCC deaths. It's a serious threat to the physical and psychological health of our citizens. The onset of HCC and the initial symptom is always not obvious, and it's found in the follow-up of hepatopathy, or in the general survey of physical examination applying AFP and B-type ultrasonic inspection. At this time it lack of sign of tumor itself in the patient's physical examination, this period is known as subclinical. However, when patients come with obvious clinical symptoms, the condition often have been in the late fall. Clinical surgery, radiotherapy and chemo-therapy combining with Chinese medicine treatment methods are always used in clinical. But the cure rate is low because of the spread of the cancer cell, and signi-ficant side effects have caused by the radiotherapy and chemotherapy. And the effect of allogeneic transplantation therapy is limited due to lacking of the number of donor.Compared with the traditional method of treatment, the tumor targeted thrapy has the advantage that it can use specific molecular as carrier which can take the drugs to the tumor tissue or organ, it can improve the transmission efficiency of anticancer drugs and the specificity to the tumor tissue, so as to achieve more effective treatment. It has become a focus for international scholars to explore and reseach, and brings about new hope and dawn for the treatment of HCC.Doxorubicin, having a highly anti-tumor activity and broad anti-cancer effect of amphiphilic anthracycline antibiotics, is currently one of the most used chemotherapy drugs. As a cyclical non-specific drug, DOX major plays a role in the nucleus by inhibiting the synthesis of RNA and DNA, particularly in RNA, it can kill cancer cells in any period of cell cycle. However, this anti-cancer drug with broad spectrum, can produce a wide rang of biochemical effects to the body, especially causing severe cardiactoxicity, inhibiting bone marrow function of hematopoiesis, and leading to severe heart failure. These side effects make the DOX be greatly restricted in the clinical application.In this paper, some specifically binding peptides to hepatocarcinoma tissues were screened by using in vivo phage display technology, which were named A54, it's composed by 12 amino acids, and the sequence is AGKGTPSLETTP. Compared with traditionally taking monoclonal antibody as carrier, A54 can enrich in the cancer site for its lower molecular weight lower immunogenic, and better penetrability.In this study, DOX was conjugated with A54 by means of chemical methods, with a short linker--GFLG, which can be degradation by Cahepsin B, in middle. We get hepatocarcinoma targeted drug A54-GFLG-DOX. This drug can be carried to the tumor site by A54, and release DOX by cutting GFLG in the degradation of Cathepsin B in the lysosome. There is more DOX entering into the nucleus site, so as to achieve more effective anti-cancer effect.This paper is divided into three parts:1) Conjugation of hepatocarcinoma-specific targeted drug A54-GFLG-DOXFirstly, we got A54-GFLG (AGKGTPSLETTP-GFLG) by chemical synthesis, then DOX was conjugated to A54-GFLG by chemical methods, and got A54-GFLG-DOX at last.Two other products were also synthesized as control, one is A54-DOX, it was synthesized by conjugating A54 with DOX directly without linker peptide GFLG. The other is A54M-DOX, the A54 in which was mutated by changing proline (P), serine (S) into two alanine (A).Finally, the several products mentioned above were identified with high-perfor-mance liquid chromatography (HPLC), purity of these products are higher than 95%.2) Identification activity of A54-GFLG-DOX in vitro. a. Cytotoxicity testing of A54-GFLG-DOX in vitro.We evaluated the cytotoxicity of A54-GFLG-DOX in BEL-7402 and SMMC-7721 by compared with DOX, A54-DOX, A54M-DOX using MTS cell proliferation assay. The results showed that A54-GFLG-DOX retained the cytotoxicity of DOX and showed good dose-dependent.b. Binding ability of A54-GFLG-DOX in vitro.The binding characteristics of drug with liver cancer cells as well as the amount of drug into the nucleus were measured by fluorescence microscope. The drugs mentioned above were incubated with BEL-7402 and SMMC-7721 and photographed by fluorescence microscopy after 30min or 12h to identify the biological activity of the several synthetic drugs. It was found that the gathering situation of the several drugs were no significant differences after 30min treatment. As time went by, the mount of A54-GFLG-DOX was the most after 24h.3) Identification activity of A54-GFLG-DOX in vivo and physiological testing.a. Identification activity of A54-GFLG-DOX in vivoWe established animal model of liver tumor-bearing nude mice by injecting BEL-7402 subcutaneous. The antitumor activity of A54-GFLG-DOX was investigated in Human Liver Cancer Xenograft Models by injecting drugs via caudal vein, taking DOX, A54-DOX, A54M-DOX and PBS as control. We evaluated the in vivo biological activity of the drugs by monitoring tumor volume and weight. The results showed that, compared to A54-DOX and A54M-DOX group, A54-GFLG-DOX retained anti-tumor activity of DOX, but A54-GFLG-DOX significantly reduced the drug side effects of DOX.b. Influence of drugs to biochemical indicators in nude blood.After a period of drug injection, we took the blood of nude mice, the white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT) of the whole blood were detected by automatic biochemical analyzer. Also the serum was isolated, the serum alanine aminotransferase (ALT), total protein (TP), albumin (ALB), alkaline phosphatase (AKP), blood urea nitrogen (BUN) and creatinine (CREA) were detected. We evaluated the anti-tumor activity through changes in these biochemical indexes.4) Biodistribution of A54-GFLG-DOX in vivo.Heart, liver, spleen, lung, kidney and tumor were taken out to remove protein after grinding with a homogenizer after 2h above the injection drugs, then supernatant was collected by centrifugation. Residues of drugs in the supernatant were detected by high-performance liquid chromatography (HPLC) technique. So as to further confirm that targeting ability of A54-GFLG-DOX. The results showed that, A54-DOX and A54-GFLG-DOX in the tumor site were significantly enriched compared with DOX and A54M-DOX, indicating that the coupling the A54 with DOX kept the ability of targeting tumor. Accumulation of A54-GFLG-DOX in tumor sites was more obvious than A54-DOX.From the data, it can conclude that:1) The Hepatocarcinoma Targeted Drug A54-GFLG-DOX was constructed successfully.2) A54-GFLG-DOX was found to exhibit obvious cytotoxicity to hepatoma cell line BEL-7401 and SMMC-7721 by cell toxicity test and selective binding ability to tumor cell by fluorescence microscopy.3) In vivo experimental result showed that, A54-GFLG-DOX had significant inhibition of tumor. The weight changes of nude mice revealed that the side effect of A54-GFLG-DOX is smaller than that of DOX.4) The content of drugs residues in organizations which was detected by HPLC revealed that the residue of A54-GFLG-DOX is the highest, next is A54-DOX, indicating that the specificity of A54 binding to tumor cell is very stable with good prospects.