The Study Of A Prodrug Of Cisplatin For The Targted Therapy Against Hepatocellular Carcinoma

Objective Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in clinic. Chemotherapy plays an important role in the treatment of HCC, especially for the terminal case. Cisplatin or cis-diamminedichloroplatinum(II)(CDDP) is broad spectrum chemotherapeutic drug, which is used frequently in the prescription.However, the application of CDDP has been limited due to its defects, such as short half-life, low water solubility and high toxicity. Thus, in order to overcome the above drawbacks, we designed a polymeric prodrug of cisplatin based on pullulan for the targeted therapy against HCC.Contents Our main contents were divided into three parts. The first part was the preparation and characterization of polymeric prodrug CDDP-SUPA. The second part was the in vitro study of CDDP-SUPA, including release profiles, cellular toxicity, the interaction of genomic DNA, the influence of apoptosis and cell cycle. The third part was the in vivo study of CDDP-SUPA, including acute toxicity in normal Babl/c mice,the distribution and anti-tumor activity in tumor-bearing mice.Methods1. The preparation and characterization: Polymeric prodrug CDDP-SUPA was synthesized by two-step esterizations. First, pullulan was reacted with succinic anhydride to obtain pullulan monosuccinate(SUPA). Next, CDDP was conjugated to SUPA to form prodrug of CDDP-SUPA. The chemical structure of CDDP-SUPA was confirmed by IR and 1H NMR. The degree of substitution(DS)of succinyl group in SUPA was determined by the p H-metric titration method.The weight content of CDDP in CDDP-SUPA was determined by atomic absorption analysis.2. The in vitro studies: The in vitro CDDP releases from CDDP-SUPA were studied by dynamic dialysis method. The cytotoxicity of CDDP-SUPA in HCC Hep G2 cells was assessed by CCK-8 assay. After incubation with genomic DNA,polymerase chain reaction technique(PCR) was used to amplify the reporter gene and the amplified product was then detected using agarose gel electrophoresis to evaluate the influence of CDDP-SUPA on the DNA replication. The effects of CDDP-SUPA on the apoptosis and cell cycle of Hep G2 cells were analyzed by the flow cytometry.3. The in vivo animal studies: The acute toxicity of CDDP-SUPA was investigated in normal Balb/c mice and the sections of their livers and kidneys were stained using hematoxylin and eosin(H&E) to observe the histopathological changes.The distribution of CDDP-SUPA in MHCC-97 H tumor-bearing nude mice was evaluated with in vivo imaging technique. The in vivo antitumor activity of CDDP-SUPA was studied in MHCC-97 H tumor-bearing nude mice via tail vein injection. The tumor sizes of nude mice bearing MHCC-97 H cells were measured and the sections of tumors were stained by H&E staining for the further histological analysis.Results1. The polymeric prodrug of CDDP-SUPA was successfully synthesized and its chemical structure was confirmed. The DS of succinyl group in SUPA was 38.2%.The weight content of CDDP in CDDP-SUPA was up to 18.7%.2. Compared with free CDDP, CDDP-SUPA significantly delayed CDDP releases in both p H 7.4 phosphate buffer saline(PBS) and p H 7.4 PBS containing 10%human serum.3. CDDP-SUPA exhibited a significantly higher toxicity in HCC HepG2 cells than that in lung cancer A549 cells. CDDP-SUPA notably inhibited the DNA replication in a concentration-dependent manner. CDDP-SUPA could significantly induce the apoptosis of Hep G2 cells and also activate S phase arrest of the cell cycle.4. Compared with free CDDP, CDDP-SUPA significantly prolonged the survival times of mice at the CDDP doses of 15 μmol/kg and 30 μmol/kg. No histopathlogical alterations were observed in the kidneys and livers of mice treated with CDDP-SUPA.5. At 24 h after administration of Cy5.5-labled CDDP-SUPA(CDDP-SUPA-Cy5.5),the fluorescent signals were almost observed only in the liver and tumor tissues.Compared with free CDDP, CDDP-SUPA significantly inhibited the tumor growth at the CDDP doses of 3.5 and 7.0 μmol/kg.Conclusions In this study, a pullulan-based polymeric prodrug, CDDP-SUPA, with a relatively high CDDP content was successfully synthesized and characterized. CDDP-SUPA exhibited high selectivity for HCC cells and effectively inhibited the proliferation of HCC Hep G2 cells. Compared with free CDDP, CDDP-SUPA had low in vivo toxicity and obviously prolonged the survival times of Babl/c mice. After administration via tail vein injection, CDDP-SUPA mainly distributed in the liver and tumor tissues and evidently inhibited the tumor growth in MHCC-97 H tumor-bearing nude mice. Thus,we deduced that CDDP-SUPA, as a polymeric prodrug, has the excellent properties and exhibits a high potential to be applied in the targeted therapy against HCC.