Targeted Treatment Of Murine Type Ⅲ Prostatitis With PH/ROS-Responsive Nanomedicine Loaded With Dexamethasone

BackgroundsProstatitis is a common urological disease that affects nearly half of all males at some point in their lives.TypeⅢprostatitis,also known as chronic prostatitis/chronic pelvic pain syndrome（CP/CPPS）,accounts for more than 90%of cases of prostatitis.It can cause patients to experience pelvic pain and discomfort,urinary abnormalities,sexual dysfunction,and even depression,greatly affecting the quality of life of those affected.Due to the incomplete understanding of its etiology and the recurrent symptomatology,currently there is a lack of effective treatment methods in clinical practice.Studies have shown that autoimmunity plays an important role in the pathogenesis of typeⅢprostatitis,and corticosteroids with anti-inflammatory and certain immunosuppressive effects may be an important choice for treating typeⅢprostatitis.Multiple clinical studies have shown that steroidal anti-inflammatory drugs can improve NIH-CPSI and quality of life scores in patients with typeⅢprostatitis,and may also improve depression scores.Therefore,the"2022 Europe Association of Urology Chronic Pelvic Pain Syndrome Guideline"lists steroidal anti-inflammatory drugs as the first-line drugs for relieving symptoms of typeⅢprostatitis.However,long-term use of steroidal anti-inflammatory drugs can lead to serious side effects,and therefore they are not widely used in clinical practice.Given the recurrence and refractory nature of typeⅢprostatitis,further research is needed on how to achieve targeted delivery of steroid anti-inflammatory drugs to reduce their distribution in normal tissues,thereby reducing the toxic side effects of the drugs and enhancing their therapeutic efficacy.In recent years,nanomedicine has seen extensive development in the diagnosis and treatment of inflammatory diseases.Nanosystems can be used to deliver drugs and modulate the inflammatory microenvironment to treat such diseases.However,there has been little research on the application of nanomedicine in the treatment of type III prostatitis.Previous work by the research group has shown that typeⅢprostatitis exhibits oxidative stress,with high levels of ROS in the inflammatory tissue.Additionally,there are a large number of activated macrophages infiltrating the inflamed prostate tissue,which exhibit overexpression of folate receptors.In addition,chronic inflammatory sites are acidic,and nuclear bodies（p H5-6）and lysosomes（p H 4-5）in macrophages also have lower p H values,providing an appropriate microenvironment for controlled release and targeted delivery of therapy for typeⅢprostatitis.Our project aims to utilize the microenvironmental characteristics of typeⅢprostatitis inflammatory tissue to synthesize folate-modified p H/ROS-responsive nanomedicine loaded with dexamethasone,which can target the delivery of drugs to the inflammatory tissue and perform responsive release.The targeted effect and anti-inflammatory effect of this nanomedicine will be evaluated at both the cellular level and in animal models,with the goal of addressing the problem of the significant toxic side effects of long-term use of steroid anti-inflammatory drugs and potentially providing a new strategy for the treatment of typeⅢprostatitis.Objectives1.Based on the microenvironmental characteristics of inflammation site in typeⅢprostatitis,a p H/ROS-responsive nanomedicine loaded with dexamethasone modified with folic acid was constructed.Its physicochemical properties were extensively evaluated,as well as its suitability for both in vitro and in vivo applications.2.Detailed study of the targeted effect and anti-inflammatory effect of nanomedicine in treating TypeⅢprostatitis at the cellular and animal levels,by implementing targeted drug delivery and controlled release through nanosystems,in order to reduce the toxic side effects of steroid drugs and provide a new strategy for treating TypeⅢprostatitis.Methods1.Preparation and characterization of p H/ROS responsive nanomedicine loaded with dexamethasone.Cinnamaldehyde and 4-（hydroxymethyl）phenylboronic acid pinacol ester alcohol were used to modifyα-cyclodextrin to synthesize a p H/ROS dual-responsive carrier.Blank NPs were then prepared,as well as Dex/CA-Oxi-αCD NPs and Dex/FA-CA-Oxi-αCD NPs.Use Malvern particle size analyzer and TEM to detect the particle size,surface potential,PDI,and morphology characteristics of nanomedicine.Use HPLC to detect the drug loading amount of nanomedicine,as well as its release efficiency in different media.2.Evaluation of biological effects and anti-inflammatory effects of nanomedicine in vitro.Cultivate RAW264.7,mouse prostate epithelial cells,and human prostate stromal cells.Use CCK-8 assay to detect the cytotoxicity of nanomedicine.Use CLSM to observe the uptake of Cy5-labeled nanomedicine by inflammatory cells.Use H₂O₂ detection kit to test the ability of nanomedicine to eliminate H₂O₂ in inflammatory cells inside and outside the cell.Use q RT-PCR,Western blot and other methods to detect the expression of cell inflammatory factors TNF-α,IL-1β,and IL-17A.3.Distribution and anti-inflammatory effect evaluation of nanomedicine in vivo.Establish an experimental autoimmune prostatitis（EAP）mouse model,inject Cy5-labeled nanomedicine via tail vein and use a small animal imaging system to detect the distribution of nanomedicine in vivo,and use CLSM to observe the entry of nanomedicine into the prostatic glandular cavity.Treat the EAP model with nanomedicine,and evaluate the biological safety of nanomedicine by blood routine,liver and kidney function parameters,and observation of major organ pathological changes.Test the pelvic pain of mice with Von-Frey filaments,and observe the pathological changes of mouse prostate tissue with H&E staining.In order to detect the expression of inflammatory cytokines TNF-α,IL-1β,and IL-17A in mouse blood and prostate tissue,ELISA kits and Western blot were utilized.Additionally,the expression of 5-HT1A receptors and SERT in the mouse hippocampus,as well as the expression of GFAP in the L5-S1 spinal cord segment,were detected using immunohistochemistry.Results1.Successfully prepared p H/ROS responsive nanomedicine loaded with Dex.TEM results showed that the nanomedicine had uniform spherical structures with particle size distribution in the range of 160-180 nm,good dispersion,low surface potential and excellent stability.HPLC analysis revealed that the drug loading was about 6%.The drug release experiment demonstrated that the Dex nanomedicine could efficiently release drugs in acidic and/or ROS-rich microenvironments.2.Cell toxicity experiments showed that nanomedicine has lower cell toxicity compared to free Dex.Cell engulfment experiments showed that FA-modified Dex nanomedicine can be effectively engulfed by LPS-stimulated macrophages,prostate epithelial cells,and prostate stromal cells.Additionally,nanomedicine can effectively reduce the concentration of H₂O₂ in inflammatory cells intracellular and extracellular.q RT-PCR and WB methods confirmed that inflammatory cytokines TNF-α,IL-1β,and IL-17A in inflammatory cells were significantly down-regulated after treatment with Dex nanomedicine.3.In vivo imaging showed that FA-modified nanomedicine can target prostate tissue and accumulate in the prostate gland.In vivo therapeutic experiments demonstrated that the nanomedicine has good biological safety through detecting mouse blood routine,liver and kidney function,and observing major organ pathological changes.Dex nanomedicine can down-regulate the expression of inflammatory factors TNF-α,IL-1β,and IL-17A in mouse blood and prostate tissue,relieve the infiltration of inflammatory cells in prostate tissue,and alleviate pelvic pain symptoms.Immunohistochemistry confirmed that the nanomedicine can reduce the expression of 5-HT1A receptors and SERT in the brains of EAP model,as well as the expression of GFAP in the L5-S1 spinal cord segment.Conclusion1.p H/ROS-responsive nanomedicine loaded with Dex has been successfully prepared,which exhibits stable properties,high drug-loading capacity,and excellent p H/ROS-responsive efficacy.It is suitable for further in vitro and in vivo experiments.2.In vitro cell experiments have demonstrated that Dex nanomedicine exhibit lower cytotoxicity,and FA-modified nanomedicine can be efficiently engulfed by inflammatory prostate cells.Dex nanomedicine can effectively eliminate H₂O₂ concentrations of inflammatory prostate cells intracellular and extracellular and significantly reduce the expression of inflammatory factors in inflammatory cells.3.In vivo animal experiments have shown that FA-modified nanomedicine can target the site of prostate inflammation and have a longer circulation time in the body.Dex nanomedicine has good in vivo biosafety,can alleviate pelvic pain symptoms in mice,reduce the infiltration of inflammatory cells in prostate tissue,downregulate the expression of inflammatory factors in the blood and prostate tissue of model mice,and may alleviate depression in mice.SignificanceThis project aims to develop p H/ROS-responsive nanomedicine loaded with Dex,and investigate their targeting and therapeutic effects both in vitro and in vivo.The goal is to address the problem of high toxicity associated with long-term use of steroid anti-inflammatory drugs,and potentially provide a new strategy for the treatment of Type Ⅲ prostatitis.