The Radiosensitizing Effects Of Folate-functionalized And CDDP Loading Polymeric Micelles For The Treatment Of Lung Cancer

Cisplatin (CDDP) is one of the most frequently used anti-tumor drugs in clinical. Combining with other drugs CDDP can significantly improve the prognosis of multiple cancers, including NSCLC and SCLC. CDDP is also a kind of radiosensitizer widely used in stage IV NSCLC concurrent with local irradiation. But CDDP lack the ability of targeting, it kills both the tumor cells and normal cells. Although it increases the therapeutic effect of radiation in chemoradiation, the side effect is also increased. One of the solutions is to construct new drug delivery systems, such as polymeric micelles. Polymeric micelles have the properties of high water solubility, high drug loading rate, low toxicity, and tumor targeting, including passive targeting EPR effect, and active targeting, which means conjugate tumor.targeting moieties such as folate to the surface of micelles. Then the micelles can conjugate with the highly expressed specific receptors of tumor, delivering the drug to the tumor site. In this research the tumor-targeting drug delivery system FA-PEG-g-PAsp-CDDP and mPEG-g-PAsp-CDDP micelles were fabricated. In vitro, we compared the cytotoxicity of FA-PEG-g-PAsp-CDDP, mPEG-g-PAsp-CDDP and CDDP in the FR over-expressing cell lines. In vivo, we compared their anti-tumor efficacy in tumor-bearing nude mice when the drugs were administered along or concurrent with radiation. The acute side effect of the micelles was also measured. The purpose of this research was to provide theoretical basis and experimental evidence to the clinical use of FA-PEG-g-PAsp-CDDP.Methods:1. FA-PEG-NH2was prepared by activating the y-carboxyl group in folic acid with DCC/TEA. And then, FA-PEG-g-PAAsp was synthesized through ring-opening of PSI with FA-PEG-NH2in DMF, then with potassium aminomalonate, L-aspartic acid in the mixed solvent of water and triethyiamine. At last, FA-PEG-g-PAsp-CDDP was synthesized by the electrovalent bond between CDDP and FA-PEG-g-PAAsp. mPEG-g-PAsp-CDDP was synthesized according to the similar procedure.2. The two polymeric micelles we synthesized were characterized by multiple methods:the content of platinum was measured by ICP-AES, the content of folate was measured by UV-vis, the diameter, distribution and ζ potential was measured by DLS, and their morphology was observed by TEM.3. In CCK-8test, the cytotoxicity of FA-PEG-g-PAsp-CDDP, mPEG-g-PAsp-CDDP and CDDP was compared in the FR over-expressing cell lines SPC-A-1.4. The tumor-bearing nude mice model was fabricated. Then56nude mice were randomized into8groups, in which4groups were assigned to the chemotherapy test, the other4groups were assigned to the concurrent chemoradiation test. The tumor suppression and radiosensitizing effects of FA-PEG-g-PAsp-CDDP, mPEG-g-PAsp-CDDP and CDDP were compared in the chemotherapy test and concurrent chemoradiation test, the body weight change was also measured.5.30ICR mice were equally randomized into3groups.11mg/kg, CDDP of the LD50dose, FA-PEG-g-PAsp-CDDP and mPEG-g-PAsp-CDDP of the same content of CDDP, was administered by the tail vein. The survival rate of the mice was observed7days later.Results:1. The polymeric micelles FA-PEG-g-PAsp-CDDP and mPEG-g-PAsp-CDDP were successfully synthesized.2. The drug loading rate of FA-PEG-g-PAsp-CDDP was15.2%, the the mass fraction of folate was9.59%, and the drug loading rate of mPEG-g-PAsp-CDDP was17.9%. The diameter of both micelles was about100nm. They were well-distributed and negatively charged. TEM showed that FA-PEG-g-PAsp-CDDP had the core/shell structure.3. In the culture medium having over-dose folate, FA-PEG-g-PAsp-CDDP and mPEG-g-PAsp-CDDP had similar cytotoxicity. In the culture medium having normal concentration folate, the cytotoxicity of FA-PEG-g-PAsp-CDDP was higher than mPEG-g-PAsp-CDDP, and the IC50of FA-PEG-g-PAsp-CDDP was only1/3of the latter (P<0.05). But in vitro, CDDP had significantly higher tumor-suppressing effect than the two types of micelles.4. In chemotherapy test in vivo, FA-PEG-g-PAsp-CDDP group had tendency of higher tumor-suppressing effect than CDDP, but no significant difference (P>0.05); and both groups had higher tumor-suppressing effect than mPEG-g-PAsp-CDDP and blank control group (P<0.05). In concurrent chemoradiation test in vivo, FA-PEG-g-PAsp-CDDP had higher tumor-suppressing effect than CDDP (P<0.05), and both groups had higher tumor-suppressing effect than mPEG-g-PAsp-CDDP and radiation-only group (P<0.05). In chemotherapy and concurrent chemoradiation test, the body weight of nude mice of CDDP had distinct decrease, up to about10%; the body weight of FA-PEG-g-PAsp-CDDP and mPEG-g-PAsp-CDDP nearly had no change.5.7days after the delivery of LD50dosage of CDDP,6of10mice in the CDDP died but all the mice in FA-PEG-g-PAsp-CDDP and mPEG-g-PAsp-CDDP survived.Conclusion:1. The folate receptor targeting polymeric micelles were successfully synthesized. They could self-assemble into spherical shape micelles in water and had good physical character fitting the requirement of new drug delivery systems. 2. The inducing of folate group significantly enhanced the tumor-suppressing effect of FA-PEG-g-PAsp-CDDP.3. When administered along, FA-PEG-g-PAsp-CDDP and CDDP had not significant different in tumor-suppressing, and both could suppress the growth of tumor effectively.4. FA-PEG-g-PAsp-CDDP had significantly better radiosensitizing effect than CDDP.5. FA-PEG-g-PAsp-CDDP reduced the acute side effects when had similar or even better therapeutic effect of CDDP.