Follicle Stimulating Hormone Receptor Targeted Nanoparticulate Chemotherapeutic Drug Delivery System For Ovarian Cancer

Ovarian cancer is one of the most common gynecologic malignancies and is the leading cause of death from gynecologic malignancies.In recent years,great development has taken place in the way of operation and medication.However,the 5 year survival rate always fluctuates at the level of 25%-30%.One of the reasons lies in that it is hard to be diagnosed at an early time,which is partly due to a shortage of the effective and in-time diagnosing measures.The majority of ovarian cancer patients are diagnosed at an advanced stage,at which time cytoreductive surgery combination chemotherapy is the routine therapeutic approach.However,neither cytoreductive surgery nor radiation treatment is effective to control this vicious disease thoroughly. Therefore,chemotherapy is to take an important role in the course of treatment. Nevertheless,there exist some inevitable limitations to the measure of systemic chemotherapy。That is,the distribution of chemotherapeutic drugs has no selectivity and specificity,that is to say,majority of chemotherapeutic drugs locate at the non-focal sites,which lowers the real dose of chemotherapeutic drugs and besides results in serious dose limiting side effects.Based on these limitations that listed above, the disease can not be effectively prevented and survival rate can not be greatly improved.In that case,the problem of how to improve the efficacy of medication on one part and lower side effect,i.e.,to explore targeted therapy with precise selectivity has become one of the most important subject in the field of cancer research.Targeted therapy has a high degree of selectivity,which is competent in recognition and binding with the specific molecule of tumor focus.Not only can it improve the killing effect of drugs on tumor cells but also the side effect of drugs to other unrelated organs can be greatly lowered.Therefore,targeted therapy is superior to other kind of tumor biological treatment.By targeted power,target-oriented drug delivery system can be categorized into two kinds,active and passive targeting preparations.By comparing the two kinds,passive targeting preparation is hard to achieve at the targeted foci.For instance,nanoparticles and liposomes are firstly endocytosed by macrophage-monocyte system and with the degradation of carriers and the release of drugs,the majority of drugs are concentrated to the organs as liver and spleen. However,by using the cartier with surface modification,active targeting preparations are capable to deliver drugs to targeting cells so as to concentrate drugs to the targeting area.While there still a problem needs solving,that is the lipidization or chemical delivery system has a high requirement to the physico chemical characteristics of drugs. At present,monoclonal antibody-oriented delivery system is commonly used,which is prepared by tumor associated antigen or cytokine.Due to the fact that the antigen is tumor-specific in laboratory animals and tumor-associated in human body,the specificity in clinical practice is low.What is more,the preparation of humanized fusion antibody is quite complicated.In the recent research of receptor mediated targeting treatment,most receptors chosen express in some normal tissues other than gonad.So for ovarian cancer,its selectivity is still not high enough.The ideal antitumor target-oriented drug delivery system should only aim at those tumor cells of interested,rather than those normal cells with the same targeting sites.To solve the problems listed above,our project constructed a novel target-oriented drug delivery system for ovarian cancer based on the etiology and biochemical characteristics of ovarian cancer.That was follicle stimulating hormone peptide modified nanoparticulate system(FSHP-NP),which was mediated by follicle stimulating hormone receptor(FSHR).The aim of our project was to overcome the limitations of current treatment of ovarian cancer.This novel therapeutic system used nanoparticles modified with polyethylene glycol(PEG) as drug carrier.In this way endocytosis of macrophage-monocyte system could be decreased.And the drug delivery capacity was enhanced.In addition,physico chemical characteristics of drugs were overshaded instead of nanoparticle properties.Ovary is the target organ of gonadotropin,whose receptor,FSHR,usually localized in the reproductive system.But it is reported that ovarian surface epithelium(OSE),most ovarian cancer cell lines and tissues express FSHR mRNA and protein.In this case,we selected follicle stimulating hormone peptide(FSHP) as targeting head,which could deliver more drugs to the interested sites with a high selectivity so as to enhance efficacy and minimize side effect.This process was implemented by FSHR mediated specific endocytosis.In order to evaluate the feasibility of using FSHR as the target of ovarian cancer therapy,the first part of this paper detected the expression of FSHR and luteinizing hormone receptor(LHR) by immunocytochemistry,immunohistochemistry and Western blot in several human ovarian cancer cell lines,Chinese hamster ovary cells, human hepatic cancer cell line,human ovarian cancer tissues,human ovarian cancer xenografts and main organs of nude mice.The results showed that FSHR and LHR positively expressed in human ovarian cancer cell lines Caov-3 and OVCAR-3,human ovarian cancer tissues(FSHR is 56.67%;LHR is 30.00%),and Caov-3 xenografts of nude mice.Except uterus and ovary,the expression of FSHR and LHR were negative in heart,liver,spleen,lung and kidney of nude mice.Besides,human hepatic cancer cell line BEL-7402 and human ovarian cancer cell line SKOV-3 were negative,either. Both our results and previous literatures have indicated that the distribution of gonadotropin receptor was relative specific in body and thus demonstrated it was feasible to use it as the therapeutic target of ovarian cancer treatment.The aim of the second part was find out the very FSH binding fragment that can recognized FSHR with great specificity in ovarian cancer cells and have no stimulating effect on cellular growth.We firstly prepared FSH 13 1-15,33-53,51-65 and 81-95 amino acid fragments and labeled with biotin or fluorescein isothiocyanate(FITC). And then the affinity between FSHP and FSHR was analyzed using enzyme-linked immunosorbnent assay(ELISA),fluorescence microscopy and flow cytometry.Finally, their influence on ovarian cancer cells growth was detected by MTT assay.The results showed that the four polypeptide fragments all recognized and bound with FSHR in Caov-3 cells,of which FSHβ33-53 and FSHβ81-95 fragments had the ability to specifically recognize Caov-3 cells with a higher affinity.What is more,the four polypeptide fragments had no significant influence on the growth of human ovarian cancer cell line Caov-3.Based on the above,FSHβ33-53 and FSHβ81-95 was chosen as the targeting head in our research.In the third part,FSHP-modified nanoparticles were prepared by incorporating maleimide into one end of PEG-PLA copolymer and using its thiol group binding reactivity to conjugate with FSHβ33-53 and FSHβ81-95.The mean size of the well-prepared nanoparticles was below 100 rim,and its zeta potential was about -25 mV.Even after the surface modification with FSHP,the size didn't increase significantly,either.X-ray photoelectron spectroscopy showed that N element of the surface of NP mainly came from those peptides modified,which demonstrated that FSHβ33-53 and FSHβ81-95 were covalently coupled to the surface of nanoparticles.In order to evaluate the capacity of FSHP-NP for drug delivery into ovarian cancer cells,in the fourth part,a lipophilic fluorescent probe with high sensitivity,6-coumarin, and the chemotherapeutic drug,paclitaxel(PTX) were incorporated into nanoparticles. The concentrations of 6-coumarin and PTX in FSHR positive Caov-3 cells and FSHR negative SKOV-3 cells were determined by fluorescence microscopy,flow cytometry, and high performance liquid chromatography(HPLC).The results showed that,by comparing with SKOV-3 cells,Caov-3 cells had a higher uptake for FSHP33-NP and FSHP81-NP,and the uptake amount of both FSHP33-NP and FSHP81-NP was significantly higher than NP with no FSHE Besides,the uptake of FSHP33-NP and FSHP81-NP was the time-,concentration- and temperature-dependent.The drug-targeting efficiency of the nanoparticles was ranked as the followings: FS HP33-NP＞FSHP81 -NP＞NP.In the fifth part,the chemotherapeutic drug,PTX,was incorporated into FSHP-NP and its preliminary evaluation was performed in vitro and in vivo.The inhibitory effects of PTX loaded FSHP-NP on human ovarian cancer cells were determined by MTT assay.To analyze antitumor effect in vivo,nude mice models of human ovarian cancer were established and randomly divided into five groups administrated with physiological saline,commercial PTX,NP-PTX,FSHP33-NP-PTX and FSHP81-NP-PTX,respectively.The inhibitory effects of the four groups on Caov-3 cells ranked as the following:FSHP33-NP-PTX＞FSHP81-NP-PTX＞NP-PTX＞PTX. The IC50 of FSHP-NP-PTX was two times lower than NP-PTX,and ten times lower than free PTX.When the accumulated amount of PTX was 30 mg per kg body weight, cell cycles of tumor xenografts were significantly arrested.And the inhibition rates of tumor volume and weight were both about 69%in FSHP-NP-PTX group,about 2 times higher than NP-PTX and 3.5 times higher than commercial PTX.The results showed that PTX loaded FSHP-NP had an enhanced anti-tumor effect both in vitro and in vivo,and the efficacy of chemotherapeutic drugs in the same dose could be improved with the help of FSHP-NP drug delivery system.In conclusion,the novel FSHP-NP drug delivery system,constructed in this project, was capable of delivering more drugs into FSHR positive cells with a high selectivity through specific endocytosis mediated by FSHR.It facilitated the concentration of drugs to foci so as to enhance antitumor efficacy of chemotherapeutic drugs and minimize side effect in unrelated normal organs.Thus,our research established a strong experimental foundation for the treatment against ovarian cancer.