Preparation And Anticancer Activity Of Poly (γ-glutamic Acid)-Cisplatin Conjugate

Cisplatin [cis-dichlorodiammineplatinum(II), CDDP] is one of the most widely used antitumor drugs for the clinic treatment of many malignancies. At present, among the clinical union chemotherapy schemes, 70-80% of these schemes need cisplatin as the major drug or including cisplatin. However, its excellent clinical record is marred by its severe side effects including kidney toxity, acute nephrotxicity, chronic neurotoxity and serious intestines reactions. Those research results show that the higher concentration of the cisplatin the more side toxic effects to the tissue. Cisplatin can combine with the protein in the blood and lose its activity quickly.Because of these difficulties of the cisplatin, an easily administered CDDP formulation with less toxicity and greater antitumor effect would be extremely valuable. To develop a CDDP formulation that could be administrated systemically and provide prolonged tumor exposure with reduced toxicity, we conjugated CDDP to biosynthesized poly(y-glutamic acid)( γ-PGA). Here We described PGA-CDDP, a water-soluble CDDP derivative. The hydrolyzed γ-PGA has a molecular weight between 45 and 60 kDa, and is a water-soluble, biodegradable, and nontoxic polymer produced by microbial fermentation.Our research contents include four parts as following:1. Preparation of low molecular weight of γ-PGA used as drug carrier:γ-PGA was produced by Bacillus sp. and was further heated at 121℃, 0.1MPa for 20 min. The hydrolyzed γ-PGA used for drug carrier with low molecular weight around 20kD-65kD was obtained.2. Preparation of PGA-CDDP conjugateWe use orthogonal test to find the optimal preparation conditions: the ratio PGA( —COOH):CDDP(mol:mol)=5:1; reaction time 72 h, reaction pH 7.5 and reaction temperature 37℃. In this way, the drug loading efficiency can be arrived at 10-12% by weight.3. Investigation the biological activity of PGA-CDDP conjugate in vitro:The release profile of PGA-CDDP showed that CDDP can be released from the resulting conjugate in PBS and there was initially a burst release during the first 6 h.followed by sustained release. The interaction between PGA-CDDP and DNA was investigated by PCR model. The MTT assay was used to investigate the in vitro anticancer activity of the conjugate. The cell cycle analysis of the conjugate was investigated by FCM assay. These experiment results showed that the PGA-CDDP has obvious antitumor effects and apoptosis effects on tumor cells.4. The research of PGA-CDDP conjugate in vivo:The in vivo toxicity investigation showed that when the normal KM mice injected with PGA-CDDP with the dose of 4 mg/kg, the body weights of the mice increased about 50% in 15 days. However, when the mice were injected with free cisplatin at a dose of 4 mg/kg, the body weight of mice decreased about 40% in 15 days. Furthermore, when the mice treated with free CDDP at the dose of 12 mg/kg, the mice died in three days. However, when the mice treated with PGA-CDDP at the dose of 12 mg/kg, the survival is 100%. These results showed that the PGA-CDDP was less toxic than free CDDP.The antitumor effects of PGA-CDDP conjugate was also investigated in vivo. PGA-CDDP was given as 3 doses at an equivalent CDDP dose of 4 or 12 mg/kg with 2-day intervals between injections to Bcap-37-grafted mice. This treatment showed stronger antitumor activity and was less toxic than CDDP in vivo. Antitumor activity assays demonstrated that the PGA-CDDP conjugate treatment had significantly higher antitumor activity than control PBS treatment (P<0.01). PGA-CDDP also increased the survival of mice bearing Bcap-37 cells with reference to PBS treatment or free CDDP treatment. Furthermore, mice treated with PGA-CDDP (4 mg/kg, administered on day 0 and 5) showed no body weight loss (P>0.05 with respect to PBS treatment), whereas free CDDP treatment at the same dose caused a body weight loss of 20-30% (P<0.001).These findings suggest that PGA produced by microbial fermentation may be used as an effective drug carrier for CDDP and that PGA-CDDP may have potential applications in the treatment of human breast cancer.