Investigation On The Tumor Targeting Drug Delivery System, Enabling Insulin As Multi-Drug Carrier

Coupling of the ligand with its receptor has the characteristics of high specificity, selectivity and affinity. Recent researches showed that the cytomembrane of many-tumor cells have higher expression of insulin receptors with increased affinity compared to normal cells, and therefore, using insulin as the vector for active targeted drug delivery system was expected to carry anticancer drug to specific tumor cells, which can either enhance anticancer effect or reduce serious side effects. The fact that insulin maintained receptor-coupling competence after it was conjugated with some compounds through covalent bond makes the assumption more feasible. But, the small molecular weight (5.8 kDa) limits the possible sites for drug conjugation. And this also leads to the very low drug/carrier molar ratio. Thus, dendritic molecules were employed in order to address this problem. This linker unit is required to enable several drug molecules to be attached to one residue of the insulin and to increase the drug/carrier molar ratio.5-Fluorouracil (5-Fu), a broad spectrum anti cancer drug, was used as the model drug. The insulin-5-Fu conjugates was obtained through a dendritic spacer - tetrakis[(carboxyethoxy)methl]methane. And hepatocellular carcinoma was used as targeting objective in this investigation. The active tumor cell targeting characteristics of Insulin-5-Fu conjugate (Ins-5-Fu) were also investigated.The intermediate and final products were characterized by melting point, UV, IR, ¹H NMR, MS and HPLC. The molar ratio of 5-Fu to insulin was 9.06:1. The ratio of free 5-Fu content to total 5-Fu content was 0.53%.In vitro stability trials showed that the pH values could affect prodrug stability. Prodrugs were prone to hydrolysis in slightly basic environment (more than 40% of free 5-Fu released),while relatively stable in acidic environment(less than 10% of free 5-Fu released), at 37℃within 120 h, and accelerated hydrolysis was observed Under the action of some enzymes in plasma within 48 h (about 50% of free Mit released).In vivo study on tumor-bearing mice (H22 hepatocellular carcinoma) showed that, compared with Mit, the conjugates showed better tumor-targeting efficiency. Pharmacokinetic analysis in plasma indicated that AUC_lns-5-Fu was1.36 times that of AUC_5-Fu, t_1/2βof Ins-5-Fu was1.40 times of that of 5-Fu, which means slower drug clearance in blood of Ins-5-Fu than 5-Fu. In tumor, AUC_Ins-5-Fu was 1.01 times of AUC_5-Fu; t_1/2Ins-5-Fu was 0.44 times of t_1/25-Fu; CLIns-5-Fu was 1.25 times of CL5-Fu; CeIns-5-Fu was1.25 times of Ce5-Fu. These results mean the higher initial drug concentration and higher clearance in tumor of Ins-5-Fu compared with 5-Fu, which lead to short-lived higher drug concentration of Ins-5-Fu compared with 5-Fu. Good tumor targeting effect of the conjugate was showed by the parameters as follows: Re=1.01, Ce=1.54, R%T =2.42. In organs of heart, liver, spleen, lung and kidney, Re were 0.26, 1.01, 0.10, 0.59 and 1.44 respectively; Ce were 0.31, 1.00, 0.31, 0.78, and 2.33 respectively; R%T were 0.52, 5.61, 0.10, 1.58 and 1.15 respectively. On the whole, these parameters reflected the distributions of the conjugates in tumor, kidney and liver. While the clearance of Ins-5-Fu was faster than 5-Fu in kidney, and the kidney clearance was the fastest in all organs of Ins-5-Fu group, which lessened the poisonous side effect of the conjugate to the kidney compared with the free drug. But the slight poisonous side effect to liver existed. In conclusion, the conjugate, compared with the free drug, had longer t_1/2 in plasma, fine targetibility to tumor tissue and lessened drug distribution in heart, spleen, lung.Fluorescence Imaging, MTT method, Flow Cytometry were used to inspect tumor cell suppression effects and mechanism of Insulin-5-Fu conjugate. These results show conjugate entered into hepatocellular carcinoma more rapidly than 5-Fu, and the cell suppression was similar with 5-Fu. The anti-tumor effects result from the suppression of G₁ to S transfer process.The tumor inhibition, living period, and the pathological study in vivo revealed that Ins-5-Fu could inhibit tumor growth, while the living period was shortened. The further tissue section studies of heart, liver, spleen, lung, kidney showed that the liver was abnormal. This may be due to liver toxicity of Ins-5-Fu conjugate.In sum, the conjugate prepared, with improved stability in vitro, had certain targetibility to the tumor and kidney, lessened poisonous side effect to the heart, spleen, lung, enhanced tumor cell suppression, while it may cause liver poisonous side effect afer long term treatment.