| Ovarian cancer is among the top five causes of cancer deaths in women. Ovarian cancer, like most solid cancers, initially spread from the primary site via tumor’s surrounding lymphatics before hematological dissemination. The overall incidence of retroperitoneal positive nodes was54.3%. It has long been known that lymphatic node involvement is one of the most important prognostic factors in ovarian cancers and the regional lymph node status is the primary parameter determining treatment strategies. Positive lymph nodes were found in4.2%of patients at stage I,35.7%at stage II,41.3%at stage III, and87.5%at stage IV. With regard to grading, positive lymph nodes were found in4.4%of G1, in21.6%of G2, and in49.1%of G3. However, the detailed mechanism of lymphogenous metastasis remains unknown and so far no effective treatment for ovarian cancer with lymphatic metastasis.Standard treatment of these patients consists of cytoreduction and systemic chemotherapy with paclitaxel along with a platinum agent. Systemic chemotherapy take an important role in the course of treatment, because optimal cytoreduction is not always achievable in the clinic. Nevertheless, there are some inevitable limitations in systemic chemotherapy. A high percentage of patients (25%-77%) are found to have metastatic nodes after chemotherapy. In particular, positive nodes are detected in17%-40%of patients at second-look laparotomy. Conventional chemotherapeutic agents are distributed nonspecifically, the dose achievable within the tumor are limited and also resulting in excessive toxicities.Targeted therapy has emerged as one approach to overcome the lack of specificity of conventional chemotherapeutic agents. Targeted drug delivery system can be categorized into active and passive targeting preparations. A drug delivery system that depends on passive targeting mechanisms inevitably faces limitations to its specificity. One approach suggested to overcome this limitation is the inclusion of a targeting ligand or antibody in the drug delivery system.Our previous studies had demonstrated that nanoparticle loading paclitaxel had the potential to allow the effective sustained release of the drug inside the lymph nodes by passive targeting. Then follicle stimulating hormone peptide-modified nanoparticles delivery system (FSHP-NP) was constructed in our another study, and we confirmed that it was capable of delivering more drugs into follicle stimulating hormone receptor (FSHR) positive cells with a high selectivity through specific endocytosis mediated by FSHR. It is feasible that the follicle stimulating hormone peptide-modified nanoparticles delivery system can enhance the concentration of drugs in lymph node metastasis of ovarian cancer while avoiding toxicity in normal organs theoretically. The aim of this study is to evaluation the efficacy of this drug delivery system in the treatment of ovarian cancer with lymph node metastasis and its toxic effects of other normal organs.The first part of this study detected the expression of FSHR by Polymerase Chain Reaction (PCR) analysis and immunocytochemistry in NuTu-19, a Fischer344rat derived epithelial ovarian cancer cell line. The results showed that FSHR positively expressed in NuTu-19cell, it was feasible that follicle stimulating hormone peptide (FSHP) could be selected as targeting head of the drug delivery system to make a drug accumulate in the target cells by FSHR mediated specific endocytosis.In the second part, nanoparticles (NP) were prepared with maleimide-PEG-PLA and MPEG-PLA(Methoxyl PEG-polylactic acid), which were synthesized by ring opening polymerization. FSHP-NP was prepared by conjugating with FSH β81-95peptide. And then6-coumarin, a lipophilic fluorescent probe and PTX, the chemotherapeutic drug, were incorporated into nanoparticles. And they were prepared via an emulsion/solvent evaporation method. The resulting nanoparticles were characterized in terms of particle size, zeta potential, drug encapsulation efficiency (EE) and drug loading capacity (DLC).The aim of the third part was to establish an ovarian cancer with highly lymphatic metastasis in an immunocompetent animal. Female Fischer344rats were injected with1×107NuTu-19cells into footpad or by intraperitoneal (ip) to establish ovarian cancer models, and evaluated their metastatic ability to lymph nodes. In the footpad group, the incidence of metastasis in the ipsilateral popliteal lymph nodes was100%. While in the ip group, no metastatic lesions were found in lymph nodes. And the survival time of the footpad group (18.429±1.112weeks) significantly longer than that of the ip group (10.286±0.505weeks). Ovarian cancer with highly lymphatic metastasis model can be successfully established by inoculating NuTu-19cells into footpad of the female Fischer344rats. The model was simple, efficient and reproducible and it was used in the subsequent sections.In the fourth part, Cellular association of coumarin-6loaded FSHP-NP was detected by fluorescence microscopy and flow cytometry was used to quantify cellular uptake of coumarin-6loaded FSHP-NP. The results showed that, by comparing with FSHR negative SKOV-3cells, cellular uptake of FSHP-NP was significantly higher than that of the unmodified NP in FSHR-positive NuTu-19cells. And uptake of FSHP-NP by NuTu-19cells was the time-and concentration-dependent. Then biodistribution were performed to determine the targeting properties of PTX loaded FSHP-NP in tumor-bearing rats. PTX solution for injection was rapidly cleared from the systemic circulation after intravenous administration.In contrast, following its encapsulation in nanoparticles (both NP-PTX and FSHP-NP-PTX), much higher concentration and longer circulation time were achieved. In the lymph node metastasis, PTX solution for injection showed the lowest PTX disposition in lymph node. NP-PTX showed higher drug accumulation. FSHP-NP-PTX, modified with FSH peptide which has its specific receptor expressed in the cell membrane of NuTu-19cells, showed the highest PTX accumulation.In the fifth part, PTX was incorporated into nanoparticles and anti-tumor efficacy was evaluated in vitro and in vivo. In vitro, cytotoxicity of various paclitaxel formulations, PTX, NP-PTX and FSHP-NP-PTX, to NuTu-19cells were examined. The cell viability was evaluated using the SRB assay. It was shown that FSHP-NP-PTX caused dramatically higher cytotoxicity than NP-PTX and PTX at0.1μM and1μM. And FSHP-NP-PTX showed higher cytotoxicity over PTX after24h,48h and72h incubation. While after48h incubation, FSHP-NP-PTX showed higher cytotoxicity over NP-PTX. Anti-proliferation ability of the formulations followed the order:FSHP-NP-PTX> NP-PTX> PTX.In vivo, anti-tumor efficacy of FSHP-NP-PTX was evaluated in rats bearing ovarian cancer xenograft and compared with that of commercial PTX and NP-PTX. The rats were intravenously administered with PTX, NP-PTX, FSHP-NP-PTX (PTX dose of4.5mg/kg) every week for seven consecutive injections. Rats received saline was used as control. At the experimental terminal, the volume and weight of ipsilateral popliteal lymph nodes in different groups followed the order: FSHP-NP-PTX<NP-PTX<PTX<Saline. And the median survival time of rats treated with FSHP-NP-PTX (25.00±1.23week) was significantly longer than those of rats treated with physiological saline (18.71±1.05week)(P<0.01), commercial PTX(20.86±0.70week)(P<0.01) and NP-PTX (22.71±0.78week)(P<0.05) through log-rank analysis, respectively.In order to estimate the side effects of the PTX formulations, body weight of the rats was recorded during the treatment. At the experimental terminal, the average body weight of the rats in all groups showed slow increase without significant difference among the groups. And the result of blood routine test, liver and kidney function test showed that there was no significant difference among the groups. The results showed no increase in side effects with this drug delivery system.In summary, the results of this study indicated that the FSHP mediated recognition and internalization significantly facilitated the cellular association of FSHP-NP in NuTu-19cells, and the FSHP-NP loading paclitaxel improved the cytotoxicity in vitro. In vivo, the biodistribution analysis demonstrated that PTX loaded FSHP-NP could significantly increase the concentration of PTX in lymph node metastasis. And it could enhance antitumor efficacy of chemotherapeutic drugs without increasing side effect.Thus it was expected to become a selective drug carrier for ovarian cancer with lymphatic metastasis. |
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