| Background:Ovarian cancer is a malignant disease with the highest fatality rate among all femalegenital system tumors, which seriously threatens women's health and quality of life. Sincethis disease is with latent onsets, and there has been a lack of effective medical screeningmethods against it up to now, as many as70%of diagnosed patients nowadays have alreadyreached the advanced stage of the disease. Epithelial ovarian cancers, most of which aresensitive to chemotherapies, account for85%~90%of malignant ovarian cancers. Thecurrent standard clinical therapy to epithelial ovarian cancers is an optimal primarycytoreductive surgery (shortened as OPCS), followed by systemic chemotherapies withmedicines combined from paclitaxol and platinum. However, because of side effects fromthe medicines or their dissolutions and tumor cells' constantly increasing tolerance of themedicines, actual effectiveness of these chemotherapies are virtually limited. Many clinicalresearchers are exploring better chemotherapy medicines with improved performance,longer-lasting effectiveness, reduced side effects, and better bioavailability, in the purpose ofprolonging patients'lives and improve their life qualities.Paclitaxol (commercialized as Taxol, and shortened as PTX) is a primary chemotherapymedicine against ovarian cancers. To improve its low water solubility, majority of itsclinically applied injections are dissolved by a mixed solution containing polyoxyethylenecastor oil and anhydrous ethanol in the volume portion of1:1. However, both paclitaxolitself and the solution can lead to severe allergy, hemolysis, neurotoxicity and other adversereactions, which have affected the medicines' actual performance. This fact has raised anurgent need for an improvement on paclitaxol's bioavailability and a reduction of its sideeffects against human health.This research uses the copolymer of two-amphiphilic biodegradable triblockpolyethylene glycol poly (acid carbonate, shortened as PEG-b-PLA), which has no toxiceffects on cells, as the medicine carrier to covalent bond with paclitaxol, to form polymerpaclitaxel nano micelle microspheres(M(PTX)), and mix M(PTX) with PEG-b-PLAcontaining folic acid (FA) or gonadotropin releasing hormone (LHRH-M(PTX)) accordingto a certain portion. FA-M(PTX) or LHRH-M(PTX) can form―core-shell‖structure in water soluble medium, while bundling the fat-soluble paclitaxol inside of the sphere. The―core-shell‖structure reduces the adverse reactions of pure paclitaxol and its menstruum,while improves the stability and constant release function of the medicine. Organizationalcharacteristics of high expression of folate receptor in tumor tissue and ovarian tissue withhigh expression of gonadotropin releasing hormone enable the targeted medicine-deliveringperformance, which is supported by the highly efficient biological characteristics of ligand-receptor specificity, and guided by FA or LHRH.Objective:To explore FA-M(PTX) and LHRH-M(PTX)'s cytotoxic effects and the ability toinduce apoptosis to SKOV3cells from epithelial ovarian cancer on cell level and In vivoanimals. To discover FA-M(PTX)'s inhibiting effects against tumor growth in tumor-bearinganimals.Method:Synthesize polymer micelle medicine with solution polymerization, and monitor itsbiological releasing behavior with high performance liquid chromatography (HPLC). Testthe polymer micelle medicine's cytotoxic effects against SKOV3cells with tetrazoliummethod on cell level; and detect the medicine's apoptosis inducing abilities on these cellswith flow cytometry (FCM). Use nude mice as tumor animal model for SKOV3from humanovarian cancers and monitor the born tumor's tolerance to polymer micelle medicine.Observe polymer micelle medicine's influence over SKOV3-bearing mice's survival timeand its inhibiting effects against the mice's tumor growth; Research polymer micellemedicine's tumor killing effects with Immunohistochemical methods, and test its apoptosisinducing effects on tumor cells in model mice with both Immunohistochemical methods andflow cytometry (FCM).Result:1. Formulated PTX bonding polymeric medicine with2.5PTX on each polymer chainby the condensation of PTX and two amphiphilic block copolymer PEG-b-P(LA-co-MCC).Result of micelles medicine's in vitro release experiment has shown that PTX micelles'cumulative release has reached49.1%in24hours,70.5%in72hours, and79.7%in144hours, which proves that the release of PTX micelles is faster in24hours than later. Thisresult has confirmed our anticipation of the experiment. PEG-b-P(LA-co-MCC/PTX) mixedwith FA-PEG-b-PLA or LHRH-PEG-b-PLA in accordance with the weight ratio of9:1, thenwe got FA-M(PTX) and LHRH-M(PTX). The particle size of this three kinds of micelles, the M (PTX), the FA-M (PTX) and the LHRH-M (PTX) were95nm,110nm and130nm,respectively. Two conjugated polymer micelle drugs of them are also spherical, which wereconducive to the accumulation of the drug-loaded micelles targeted.2. On cell level, micellar carrier from this research has shown no toxic effects or sideeffects to cultured cells. The cultured cells keep in spindles or stars shapes with cores in thecenter, pseudopodium stretching and connecting to each other, and cells are proliferatingvigorously. Contact inhibition is seen in cell density areas. Single use of paclitaxol onSKOV3cells in24hours has shown obvious cytotoxicity: cells have significantly shrunk,rounded and become smaller in size. Cell divisions have reduced, coarse pellents haveappeared in cytoplasm, and part of the cells has been destructed and their contents escaped.In24hours of the usage of paclitaxol micelles, folic acid paclitaxel micelles andgonadotropin paclitaxel micelles, cell toxicity has only appeared to part of the SKOV3cells;in72hours, majority of the cells died. At the concentration of10g/ml of paclitaxol, all kindsof paclitaxol preparations have shown no differentiation in inhibiting cells in24hours. Asthe concentration decreases, three parathion types' inhibiting rates have appeared lower thanthat of pure paclitaxol. In48hours and later, paclitaxol micelles' inhibition against ovariancancer cells have shown to be obviously weaker than that of paclitaxol in the sameconcentration. But as the concentration and working time of the medicine increase, inhibitingrates against SKOV3cells of all the three types of paclitaxol micelles tend to become similar,which has reached between78%and87%. Monitoring result of the four paclitaxolmedicines'apoptosis-inducing effects on SKOV3cells in72hours has shown that, apoptosisrate of M(PTX),FA-M(PTX) and LHRH-M(PTX) have been significantly higher than thatof PTX group(P<0.05).3. Animal experiment in vivo: nude mice from M(PTX) and FA-M(PTX) treatmentgroup have shown no adverse reactions, and this is the same as mice from saline treatmentgroup. However, nude mice injected with clinical PTX have shown adverse reactionsincluding screaming, biting and agitations. From observation on survival experiments,M(PTX) and FA-M(PTX) allow the median survival time (55days and69days respectively)of tumor-bearing animals to significantly exceed that of control animals (49days) and thoseunder PTX treatment (50.5days). Volume measurement control group, PTX group, M(PTX)group, FA-M(PTX) group's tumor growth rate I nude mice are87.6%,10.3%,6.7%and-27%respectively. The result from each treatment groups is very different from that of salinecontrol group. The tumor growth rate of FA-M(PTX) has appeared significantly lower than that of PTX and M(PTX) treatment groups. The tumor weigh inhibitory rates of PTXtreatment group, M(PTX) treatment group and FAC-M(PTX) treatment group are33.3%,57.7%and70%respectively, which has proven that M(PTX) and FA-M(PTX) treatmentgroups have demonstrated notably better tumor weight inhibiting effects than PTX treatmentgroup. Expression of apoptotic protein rate derived from Immunohistochemical testing hasshown that, in accordance with the order of normal saline, PTX, M(PTX), then FA-M(PTX),Bax protein's positive expression rates are12.5%,50.0%,66.6%and77.8%respectively;those for Bcl-2protein are75.0%,62.5%,44.4%and22.2%; and those for Caspase-3proteinare25.0%,62.5%,66.6%and77.8%. Expressions from all these groups are significantlydifferent from each other.Conclusion:1. Macromolecule bonding medicine demonstrates slow continuous releasing process inwater soluble medium2. Same as paclitaxol, targeted paclitaxol micelles are with significant cytotoxicityagainst ovarian cancer cells, which can lead to the death of tumor cells.3. On cell level, the cytotoxicity of M(PTX), FA-M(PTX) and LHRH-M(PTX) are notas strong as pure paclitaxol.4. In comparison with PTX, PA-M(PTX) is with lower toxic and side effects, and higheranimal tolerance.5. FA-M(PTX) is more effective in inhibiting ovarian cancer cells, and it cansignificantly prolong the survival time of animals.6. In comparison with pure paclitaxol, FA-M(PTX) is more effective in inducingapoptosis to ovarian cancer cells... |
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