Investigation Of The Efficacy Of Gene Treatment On Endometriosis And Its Possible Mechanisms Mediated By Targeting Nanoparticle

BackgroundEndometriosis is a common disease in which the endometrial tissue with growth function appears in other parts of the body outside the uterine cavity,which affects the woman physical and moral integrity seriously.The pathogenesis of endometriosis remains unclear although its incidence rate tends to be increased.The prevalence is more than 15%among the women in reproductive age.It is considered to be a beginning disorder,but it exhibits the features that malignancy really holds,for instance it is aggressive,invasive and angiogenesis.The efficacy of the current treatment including medical and surgical therapy is still not good though many treatments were carried out,the side effect is big,and medicine expense is high.So it is requisite to study the pathogenesis of endometriosis and explore a better cost effective treatment to fundamentally solve the main clinical problems of endometriosis.Gene therapy is based on the gene transfer to achieve the purpose of treatment by introducing foreign genes into the human body.The completion of the human genome project will lay a solid foundation for gene therapy,and it has become one of the world's most active research fields.Gene therapy drugs will have a profound impact on the pharmaceutical industry.The latest large number of research has reported on gene therapy for refractory diseases such as cancer,we also believe that gene therapy can also be used for the fundamental treatment of endometriosis.Such as the use of siRNA interference technology to silent water channel gene expression,reduce or inhibit the expression of aquaporin,which is expected to become a new method of treatment of endometriosis.However,one of the biggest problems with gene therapy technology is finding a safe and effective gene vector system.Non-viral vectors have attracted the attention of researchers because of its simple preparation,no immunogenicity and high security.In the transmission of genes,the cationic carrier is used most commonly,mainly liposomes and microparticles.These studies have achieved some success in varying degrees,but the success rate of gene therapy is proportional to its number,which must be delivered as much as possible to cytoplasm in order to obtain the desired biological effects.Thus,it is critical for the non-viral vector carrying the therapeutic gene to accurately identify the target cell and express specifically in the target cell.We designed a new nano-vector targeting endometriosis with structural ligand modification on the surface of nano-carrier based on specific receptor on ectopic endometrium,observed its effect on rat endometriosis and explored its toxic and side effects and related mechanisms to explore an effective and safe method for the treatment of endometriosis.Part I Preparation and in vitro properties study of targeted nano-drug delivery systemObjectiveTo screen and optimize the targeting drug delivery system.MethodsPreparation of chitosan-stearic acid grafted chitosan oligosaccharide(CSO-SA)with carbodiimide as crosslinking agent.The degree of amino acid substitution of grafts was determined by trinitrobenzene sulfonic acid method and the critical concentration of grafts in water was determined by pyrene fluorescence method.The CSO-SA/PEDF complex nano-drug delivery system was prepared by using plasmid DNA(PEDF)as the reporter gene.The surface morphology was measured by transmission electron microscopy.Particle size and surface potential of the CSO-SA/PEDF nano-drug delivery system were measured by particle size and surface potential analyzer.The particle size and surface potential of the CSO-SA/PEDF nano-drug system were obsered.The adhesion degree of the cationic grafted micelles and plasmid DNA was investigated by gel retardation assay.The biological stability of the SA/PEDF nano-drug delivery system was studied.Cytotoxicity was determined by MTT assay.ResultsThe CSO-SA was obtained by chemical grafting method.The degree of amino acid substitution was 13%,and its critical concentration in water was 0.025 mg·mL-1.When N/P was 9.6,the particle size of CSO-SA/PEDF nano-drug delivery system was 135.6 nm and the potential was 6.4 ± 0.1 mV.Transmission electron microscopy observation showed the nano-drug system was spherical,uniform size.In vitro cell experiments showed that cytotoxicity was significantly lower than LipofectamineTM2000 and transfection efficiency was high.ConclusionsThis new type of gene carrier initially shows the efficient and low toxicity characteristics.Part II Gene therapy of endometriosis introduced by polymeric micelles with glycolipid-like structureObjectiveA polymeric gene delivery system composed of lipid grafted chitosan micelles(CSO-SA)was designed and mediate the pigment epithelium derived factor to treatmen endometriosis.And preliminary mechanism was researched.MethodsThe CSO-SA/PEDF gene nanoprotective system was prepared in the first part.A rat model of experimental endometriosis was established by autologous endometrial transplantation to healthy female not pregnant SD rats.The rats were injected with nano-vector-mediated PEDF gene and normal saline.After 2 weeks,the effect on the size of endometriosis lesions and the pathological morphology were observed.The effect of PEDF on the proapoptotic effect of ectopic lesion cells and the inhibition of angiogenesis was analyzed by comparing the changes of apoptosis,VEGF expression and microvessel density in ectopic lesion.At the same time,the histopathological and ultrastructural changes of the organs of the uterus and ovary were observed.ResultsIt showed that the CSO-SA/PEDF nanoparticles gene therapy caused decrease in the sizes of the endometriotic lesions and atrophy and degeneration of ectopic endometrium significantly.The apoptotic index of endometriosis was significantly increased in CSO-SA/PEDF treatment group.However,the pathological morphology of the uterus and ovarian tissue in the CSO-SA/PEDF treatment group was not significantly different,and no significant abnormality was observed in the ultrastructural observation.ConclusionsThe gene delivery system of CSO-SA/PEDF could be used as an effective therapy approach for endometriosis.Part ? The efficacy and mechanism of targeting nano-vector-mediated AQP2-siRNA in the treatment of endometriosisObjectiveTo research on a new candidate for endometriosis treatment with fewer side effects,a new polymeric nanoparticle gene delivery system consisted of polyethylenimine grafted chitosan oligosaccharide(CSO-PEI)with hyaluronic acid(HA)and small interfering RNA(siRNA)was designed.MethodsPreparation of polyethylenimine grafted chitosan oligosaccharide(CSO-PEI)by chemical synthesis.AQP2-siRNA gene nano-drug delivery system was constructed by the method of positive and negative electrical adsorption,and different N/P gene nano-drug delivery system was prepared according to the gene concentration.The hyaluronic acid(HA)was physically encapsulated by the common incubation method.The biological stability of nano-drug delivery system was studied by investigating the adhesion of cationic graft to AQP2-siRNA by gel retardation analysis.The surface morphology was observed by atomic force microscopy.The particle size and surface potential of the CSO-PEI/siRNA were measured by the particle size and surface potential analyzer.The cytotoxicity was determined by MTT assay.Fluorescence grafting was used to observe the uptake of cells.The distribution of nano-drug delivery system in model animals was observed by fluorescence microscopy.The rats were injected with(CSO-PEI/siRNA)HA nano-drug delivery system and nonnal saline.After 2 weeks,the effect on the size of endometriosis lesions and the pathological morphology were observed.To investigate the effect of CSO-PEI/siRNA on the apoptosis of ectopic lesion cells and the inhibition of angiogenesis by comparing the ectopic focus apoptosis,microvessel density and the changes of CD44 in ectopic cells.At the same time,the histopathological and ultrastructural changes of the organs of the uterus and ovary were observed.Results1.(CSO-PEI/siRNA)HA gene nano-drug system has good physical stability and uniform distribution of physical morphology.2.(CSO-PEI/siRNA)HA gene nano-drug system has small cytotoxicity and strong uptake efficiency.3.(CSO-PEI/siRNA)HA gene nano-drug delivery system can specifically target differentiated CD44 through HA ligand.4.(CSO-PEI/siRNA)HA gene nano-drug delivery system in vivo endometrial cyst site distribution was significantly higher than the control group.5.(CSO-PEI/siRNA)HA gene nano-drug delivery system treatment group ectopic lesion size was significantly smaller than the control group,while the histomorphology of the treatment group showed signs of atrophy.6.The vWF labeled MVD was significantly lower in(CSO-PEI/siRNA)HA nano-drug delivery system group than in control group,but there was no significant difference between a-SMA labeled MVD.7.(CSO-PEI/siRNA)HA gene nano-drug delivery system was significantly higher than that of the control group.8.There are no obvious abnormalities in(CSO-PEI/siRNA)HA gene nano-drug delivery system treatment group of uterine and ovarian tissue pathological morphology and ultrastructural observation.9.The expression of CD44 in the different tissue was significantly higher than that in the normal endometrium.The expression of CD44 was significantly decreased when the(CSO-PEI/siRNA)HA gene was administered.Conclusions(CSO-PEI/siRNA)HA gene delivery system might be served as an effective method for the treatment of endometriosis.(CSO-PEI/siRNA)HA gene nano-drug delivery system has no obvious side effects on the reproductive organs.The treatment may be mainly through the specific targeting of internal parts,inhibition of ectopic lesion angiogenesis and promote ectopic uterus Endometrial cell apoptosis to achieve.