| At present,anti-cancer targeting drug delivery therapy has gradually become an attractive method for cancer treatment. On one hand, it is encouraged by the fact that the traditional drug delivery methods have seemed to be relatively inefficient in the clinical application. On the other hand, the clinical application of chemotherapeutics is largely restricted by its intolerable adverse reactions. Thus, looking for an effective drug targeting delivery system has become one of the hotspots in the cancer research. The folate-mediated drug delivery system is just one star of these drug delivery systems. As it is reported, the surface of many tumor cells (such as ovarian cancer, endometrial cancer, brain cancer, skin between the organizations cancer, renal cell carcinoma, and so on) have the high expression of the folate receptors (FR), while they have little or virtually no expression on the surface of the normal cells. Using the endocytosis mediated by FR and the high affinity of FR and folic acid, drugs linked with folic acid can be carried to the specific cells or tissues. Their high specificity and affinity largely increase the efficiency of drug delivery.This paper has designed a kind of ternary targeted drug delivery system, folic acid - bovine serum albumin - doxorubicin (Folate-BSA-DOX) polymer. Folic acid and doxorubicin can be linked simultaneously into the different sites of bovine serum albumin. Using the high affinity of folate and folate receptor, doxorubicin can be more effectively uptaked by tumor cells. The formation of such ternary system as Folate-BSA-DOX, has been comfirmed by UV-visible absorption spectra. Moreover, it is also demonstrated in the cellular apoptosis experiments that the cell apoptpsis rate of Folate-BSA-DOX has reached to 38.8﹪, 30% than that of BSA-DOX. This study has proved that Folate-BSA-DOX targeted drug delivery system is feasible and is effective in practice. In this study, the main contents include :Firstly, the hydroxy group of doxorubicin was oxidized into aldehyde group by the classic sodium periodate oxidation. Secondly, the aldehyde group of doxorubicin and the free amino group of BSA formed the covalent bond by Schiff bases reaction. Then using the bifunctional coupling agent 1 - (3 - dimethyl - aminopropyl) -3 - ethyl carbodiimide (EDC), folic acid was turned into the ester, coupling with the BSA-DOX. Folate-BSA-DOX was separated from free folate active ester by passage through the sephadex column chromatography. Finally, the elution curve of purified Folate-BSA-DOX, folic acid, BSA and doxorubicin measured and monitored by a 756-PC UV-visible spectrophotometer has verified that folic acid was successfully coupled with BSA-DOX. After comprehensively analyzing such factors as reaction time, pH and the dose of folic acid active ester, we determined the opitmal coupling process, and quantitatively tested the coupling rate of the folic acid, doxorubicin and BSA in the conjugates.The specific uptake of Folate-BSA-DOX by Hela cells, folate receptor positive cells, has been observed by the inverted fluorescence microscope. Furthermore, we have considered the impact of the concentration of drugs, the time of administration and the different coupling rate on the uptake of Folate-BSA-DOX by Hela cells. The results showed that the fluorescence intensity was enhanced as the lasting of culture time and the increasing concentration of drugs. In other words, the uptake rate of Folate-BSA-DOX was increased by the lasting of culture time and the increasing concentration of drugs. The use of flow cytometry (FCM) quantitative detection of the uptake of Folate-BSA-DOX by folate receptor-positive cells, further confirmed the targeting and specificity of folic acid polymer. Meanwhile, the cell apoptosis rate of Folate-BSA-DOX was detected by flow cytometry. A significant distinction between drugs, including Folate-BSA-DOX and BSA-DOX, adminstrated after 12 hours, 24 hours, 48 hours respectively, was also demonstrated.In short, the macromolecules, Folate-BSA-DOX, was prepared in this study has verified to be qualified with the specificity for the folate receptor positive cells and the feature of drug-induced cell apoptosis.
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