| Objective To investigate the inhibition of the growth of cells with corresponding receptors in cytology by the active targeting drug delivery system luteinizing hormone-releasing hormone (LH-RH)-methotrexate (MTX) compound, free MTX and MTX liposomes.Methods We select MTX as the model drug and [D-Lys~6] LH-RH as the carrier. The targeting compound LH-RH-MTX was incorporated with MTX, APA and [D-Lys~6] LH-RH. Then the preparative method was optimized. The crude products were analyzed,purified and quantified by Beckman reversed-phase HPLC and semipreparative HPLC after recrystal and lyophilization, and its molecular weight determined by ESI-MS. MTT assay was applied to examine the inhibition ratios of the purified LH-RH-MTX compound, free MTX and MTX liposomes to the nasopharyngeal darcinoma cell line HNE-1,the oophoroma cell line SKOV-3 and the prostatic carcinoma cell line PC-3.Results Using APA to prepare LH-RH-MTX targeting compound, we obtained the crude products with purity of 98% and the yield of 61.69%, determined by semipreparative HPLC. The molecular weight was 1268.70m/z by ESI-MS analysis, The fomular weight of LH-RH-MTX is 1268m/z. The results showed that MTX liposomes,free MTX and LH-RH-MTX could inhibit the growth of nasopharyngeal darcinoma HNE-1 cells,oophoroma SKOV-3 cells and prostatic carcinoma PC-3 cells. The inhibition ratios were from 0.10% to 97.73% with the tendency of concentration dependention. In paticular to prostatic carcinoma PC-3 cells, the ratio of LH-RH-MTX was up to 97.73% at the concentration of 200.0ng/ml after administration for 12h, and those of free MTX and MTX liposomes were 49.90% and 54.25%, respectively. To oophoroma SKOV-3 cells, the inhibition ratios of LH-RH-MTX, free MTX and MTX lipsomes were 48.46%, 8.47% and 26.81%, respectively; and to nasopharyngeal darcinoma HNE-1 cells, the inhibition ratios were 38.92%, 7.41% and 17.17%, respectively. To the same cell the IC50 order was: LH-RH-MTX≤MTX liposomes≤free MTX.Conclusions The method of incorporating APA and [D-Lys6] LH-RH to prepare LH-RH-MTX targeting compound was simple and more scientific. The yield and purity were high by HPLC purification and quantification. The molecular weights of products were verified by ESI-MS analysis. The number of LH-RH receptors expressed on Nasopharyngeal darcinoma cells is few, are 80% on oophoroma cells and 86% on prostatic carcinoma cells. The results revealed that LH-RH-MTX,free MTX and MTX liposomes could inhibit the growth of nasopharyngeal darcinoma HNE-1 cells,oophoroma SKOV-3 cells and prostatic carcinoma PC-3 cells by MTT method. The inhibition order was prostatic carcinoma PC-3 cells≥oophoroma SKOV-3 cells≥nasopharyngeal darcinoma HNE-1 cells. Furthermore, the inhibition order was LH-RH - MTX≥MTX liposomes≥free MTX, with the tendency of concentration dependention. The inhibition ratio was the highest after administration for 12h. LH-RH-MTX had better inhibition of tumor cell growth than MTX liposomes and free MTX coupling with LH-RH receptors specifically. All pointed out that LH-RH as carrier on TDDS to carry out target release and enhance the bioavailability of drug was feasible.
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