Development Of Follicle-stimulating Hormone Receptor-mediated Drug Delivery System For Ovarian Cancer

Mortality rate of ovarian cancer is in the fifth at the women cancer, and at top of gynecological malignancies. The majority of patients with ovarian cancer diagnosis at advanced stage, and treatment of advanced ovarian cancer is a very difficult problem. Although the improvement of operation、new drugs come into market and launched of new chemotherapy program, it certain extent and improve the effect of the treatment, but 5 years survival rate has not improved obviously. Chemotherapy is an important treatment for advanced ovarian cancer, but most advanced patients recurrent after treatment or drug resistance, leading to treatment failure. Therefore, it is necessary exploring new treatment from the occurrence and development of ovarian cancer mechanism and its hormonal environment. Our team study before shows, based on the pathological features of ovarian cancer and physiological hormone environment, selected as a specific target site, may be can obtain more efficient therapeutic effect and lower toxic and side reaction.Ovary is the target organ of the hypothalamic pituitary hormones, therefore, drug targeted by mediated hormone receptor, can obtain more in advantages in ovarian cancer treatment. Among them, follicle stimulating hormone receptor (Follicle-stimulating hormone receptor, FSHR) limited distribution in the human body, mostly concentrated in the reproductive system, and also express in human ovarian epithelial carcinoma. Our group previous research shows, nano complex modified with FSHR binding fragment can effectively improve the chemotherapy drug entrapped drug or gene specific delivery capability, FSHR is a more appropriate therapeutic target sites in ovarian cancer.MUC16 is the genetic code by CA125, and CA125 high expression in ovarian cancer tissues and cells we can use MUC16 promoter as second target sites, to guid the gene targeted system express on ovarian cancer cells. Combined with our previous study, introduce MUC16 promoter into shRNA plasmid, regulated the expression of plasmid shRNA, to specific downregulated gene expression of MUC16 positive ovarian cancer cells. At the same time, modify the surface of nano materials entrapped plasmid with FSHR binding fragment, through FSHR receptor mediated and regulated by MUC16 promoter to achieve the dual target. Therefore, this study chooses FSHR peptide binding fragment as target head base, constructed the active targeting drug delivery system of ovarian cancer. In order to further improve the targeted drug delivery system, introduce the in MUC16 promoter to regulate the downstream gene.For tumor cells, stromal cell not only for pure support and nutrition, but also involved in the occurrence、development and metastasis of tumor. Among them, fibroblasts as main component of matrix, it plays the major role in the interaction of cancer cells and stromal cells. It has been confirmed, the tumor stroma fibroblasts in form and function are different from the normal matrix, can promote cells malignant transformation, and interaction with tumor proliferation、invasion and metastasis。In our previous work also confirmed that high express of gro-a in human ovarian carcinoma. Therefore, strategy of inhibit the expression of gro-a or its function, can be effective intervention strategies of tumor stroma. The intervention of tumor stroma cells and inhibit tumor cell, can improve the antitumor efficacy.Growth-regulated oncogene alpha (Gro-a), is the key component in the process of malignant transformation, high expression in ovarian cancer tissues and cells, strongly linked with ovarian cancercells proliferation invasion and metastasis。In order to verify delivery system, select the gro-a shRNA as a model drug, investigate the efficacy of targeted complex in vitro and in vivo.The first part of this study used immunocytochemistry、Western Blot、ELISA and real-time quantitative PCR detecte the expression of FSHR、MUC16 and gro-α, in human ovarian carcinoma cells. The results suggest that human ovarian cancer cell line HEY high express of FSHR、MUC16 and gro-a, so we will used HEY cells for subsequent experiments. While the human ovarian cancer cells SKOV3 low expression or no expression of FSHR, while expression of MUC16 and gro-a, so we used this cell line as control.In the second part of this study designed and synthesis of 4 sequence of gro-a shRNA. Using flow cytometry and fluorescence microscopy to detected the transfection efficiency of different sequences on human ovarian cancer cells. Using ELISA、Western Blot and real-time quantitative PCR to detect the down-regulation of targeted mRNA and protein expression, when it transfected with different sequences of gro-a shRNA, and to screened out the high efficiency sequences. The results suggested that shRNA sequences# 4 compared with the other sequences with better silencing effect, so we choose this sequence for subsequent experiments.In the third part of this study using bio-informatics prediction obtain 4 possible sequences of MUC16 promoter. And then use dual fluorescence reporter gene method to detecte its promoter activation, and then constructed the gro-a shRNA plasmid regulated by MUC16 promoter. Results suggest that the promoter sequence 1 and 2 have higher activation in MUC16 positive human ovarian cancer cell line HEY, while we will constructed the plasmid regulated by this 2 MUC16 promoter sequences.In the fourth part of this study using alpha succinimidyl-ω-propyl malcimide groups-polyethylene glycol and polyethylene iminc branches preparation of different N/P ratio nano complexes, modify with FSH binding fragment, and connected with the plasmid DNA. Using electron microscope detect the nanometer of compound particle and the elucidated structures of nanometer compound by 1H-NMR, using particle size/Zeta potential instrument to measure the particle size and Zeta potential of nano compound. Characterization shows, with the N/P ratio increases, the particle size reduced gradually, Zeta potential the increased from negative charge to the positive charge, gel clectrophoresis experiments show thai when the N/P ratio is 10, can reach 100% encapsulation. When the N/P ratio is 25.2% PEG grafiting nano particle modifie with FSH peptide its particle size is (142.0±3.8) nm, and its Zeta potencial is(24.1±2.4)mV; 2% PEG grafiting nano particle without modifie with FSH peptide its particle size is (123.8±6.0) nm, and its Zeta potencial is (37.7±3.6) mV; 5% PEG grafiting nano particle modifie with FSH peptide its particle size is (168.0±4) nm, and its Zeta potencial is (25.1±3.4) mV; 5% PEG grafiting nano particle without modifie with FSH peptide its particle size is (125.0±4.5) nm, and its Zeta potencial is (35.7±4.6) mV; and MUC16 promoter regulated plasmid nano particle modifie with FSH peptide its particle size is (186.0±4.5) nm, and its Zeta potencial is(30.0±2.3)mV; MUC16 promoter regulated plasmid nano particle without modifie with FSH peptide its particle size is (170.0±4.1) nm, and its Zeta potencial is (33.0±3.3) mV.In the fifth part of this study using ELISA, Western Blot and quantitative real-time PCR assay to study the downregulation effeciency of targeting complexes. The results show that, the grafting amount of PEG is 2% or 5%, FSHR binding fragment modified nano composite could significantly down regulated gro-a expression in FSHR positive ovarian cancer cells. When the drug delivery system import MUC 16 promoter it also can inhibit the expression of gro-a in MUC 16 positive in the ovarian cancer cells. It suggests that FSHR mediated targeting or regulated by MUC 16 promoter targeting drug delivery system can enhance the shRNA down-regulation effect, may be due to based of target group in nano materials to improve induced the ability of drugs into cells.In the sixth part of this study to exam the effect of nano complex on the malignant biological behavior of ovarian carcinoma cells by using CCK-8 assay and transwell assay. The results suggest that, nano comlex modified with FSH polypeptides could significantly inhibit FSHR positive ovarian cancer cells in proliferation, migration and invasion. When the MUC 16 promoter as a second target site, inhibitory effect of nano composite on the biological behavior of malignant cells was significantly enhance, especially on MUC 16 promoter sequence#1.In addition, the nano complex modified with FSH polypeptide decreased toxicity, increase the feasibility for in vivo applications.In the seventh part of this study is preliminary evaluation the efficacy and safetyof this nano conplex in vivo by using the xenograft tumor model of human ovarian carcinoma in nude mice. The results show that, the compound which grafting amount of 2%PEG and modified with FSH polypeptides can inhibit tumor growth in nude mice, and prolong the survival time, and the compound without modified with FSH polypeptides,70% died in acute toxicity in second days after administration. When PEG ratio increased to 5%, nude mice showed no obvious side reactions. Compared with other groups, compound which modified with FSH polypeptides significantly inhibite the tumor growth in nude mice, the tumor inhibition rate about 75.28%. At the end of the experiment, exam the tumor of xenograft model, results indicated that nanocomposite modified with FSH polypeptides could down regulate gro-a mRNA and protein level in tumor tissue.This study indicated that a FSHR-mediated delivery system could mediate the highly selective delivery of shRNA into ovarian cancer cells and that silencing gro-a expression could be a potential choice for ovarian cancer treatment.This study developed a gene drug delivery system modified with FSHR polypeptide and regulated by MUC16 promoter, and choose gro- alpha small hairpin RNA as a model drug, which is important in the interactive dialogue of ovarian cancer cells and stromal cells. In FSHR positive and MUC16 positive ovarian cancer cells, nano system modified with FSH polypeptide or modified with FSH polypeptide and regulated by MUC16 promoter, with the help of specific receptor-mediated binding, can delivering more drugs into cells, enhance the efficacy of the drug. Targeted down regulate gro-α gene can significantly inhibit the growth of ovarian cancer. At the same time, in this study shows that, the initiative to target system which is modification with FSHR can decrease the cytotoxicity of nano materials increased the feasibility of in vivo experimental studies. This study once again confirmed our proposed before, in order to establish efficient targeted therapy system, screening drugs targeted point based on the pathophysiological characteristics of gynecological tumor this result provides a reference method for other tumor targeted therapy.