Topical Administration Of Mucoadhesive And Thermo-sensitive Hydrogels With Cyclosporine A Loaded PEG-PCL Micelles To Alleviate Colitis In TNBS-induced Colitis Mouse Model

Background:Inflammatory bowel disease is a chronic disease characterized by super facial inflammation of the rectal and colonic mucosa,which involves the rectum in 95%cases and may be extended continuously to the more proximal part of the colon.Cyclosporine A（Cs A）is a useful immunosuppressive agent for steroid-refractory ulcerative colitis.However,the clinically used vehicle for i.v.administration of cyclosporine A,Cremophor EL,has adverse effects on hepatobiliary physiology.Drug absorption and instability in the harsh environment of the upper gastrointestinal tract（GIT）of oral administration usually leads to serious systemic toxicity,insufficient targeting of inflammation sites,and lacking drug residence in colon.Objective:To improve the efficacy of inflammatory bowel diseas,a rectal delivery system-PEG-PCL micelles with mucoadhesive and thermo-sensitive CS-P407-P188hydrogels was developed,which can lead to a superior therapeutic effect via inhibiting drug leakage and prolonging drug retention time.Method:PEG_2k-PCL_x micelles were prepared by film hydration method.The particle size,encapsulation efficiency and cytotoxicity of the micelles were characterized in detail as well as cellular uptake efficiency and specificity（RAW264.7,LPS-RAW264.7,and Caco-2 cell）were studied to filter the most favorable PEG_2k-PCL_x polymer.The selected PEG_2k-PCL_3.5kmicelles were fabricated in the presence and absence of Cs A.In addition,several methods for characterizing micelles formulations were described,including measurement of particle size and distribution,Zeta potential,morphological characteristics,encapsulation efficiency and stability.Cs A-PEG-PCL/CS hydrogels can be achieved through physical cross-linking method.Then this formulation was subjected to in vitro drug release and rheological parameters study.The in vivo,in vitro,ex vivo distribution or adhesion characteristics were evaluated by IVIS imaging system.Colitis mouse model was induced by rectal administration TNBS（50%ethanol,100mg/kg）.The mice’s general conditions were observed,and the severity of colitis was assessed by using disease activity index（DAI）score.After the treatment,blood samples were collected,organs（spleen,kidney,and colon）were excised and weighed.Colonic mucosal pathological changes were viewed under microscope after hematoxylin-eosin（H&E）and immunohistochemical stain（IHC）.The underlying anti-inflammatory efficacy and mechanisms were further identified by biochemistry,WB and q PCR analysis respectively.Rusults:The selected PEG_2k-PCL_3.5k polymer micelles were spherical in shape with a mean diameter of 42.3±0.7 nm and narrow size distribution（PDI<0.1）.PEG_2k-PCL_3.5kpolymer micelles exhibited a nearly neutral potential of-6.35±0.50 m V.A higher drug-loading content of 17.4%and an encapsulation efficiency of 95.2%were achieved with goog biocompatible and noncytotoxic below 1 mg/m L.More importantly,the cellular uptake results indicated that PEG_2k-PCL_3.5k polymer micelles showed the most optimal uptake efficiency.Cs A-PEG-PCL/CS thermo-sensitive hydrogels revealed a quick gelation at 37℃.Curve fitting results manifested that about 80%of Cs A micelles were dispersed from the thermo-sensitive hydrogels at first 2 h and driven by a sustained manner during the following time intervals.The distribution（in vivo,in vitro,ex vivo）results illuminated that the Cs A-PEG-PCL/CS thermo-sensitive hydrogels preferentially adhered to the inflamed mucosa.This hydrogels significantly relieved TNBS-induced disease activities index（DAI）,colon length shortening,colon/body weight ratio as well as colonic pathological damage.The MPO,NO and ROS generation in the TNBS-induced colitis tissues were also suppressed by the hydrogels.Furthermore,compared to other Cs A formulations,Cs A-PEG-PCL/CS thermo-sensitive hydrogels tempestuously decreased the inflammatory state and reduced the expression of the TNF-α,IL-1β,COX-2 and i NOS2.Exploration of the potential mechanisms demonstrated that Cs A-PEG-PCL/CS thermo-sensitive hydrogels significantly induced Nrf2,NQO1 and HO-1 by inhibiting oxidative stress.Conclusions:The biocompatible and hydrophilic polymer micelles was designed to embed the interior of CS-P407-P188 thermo-sensitive hydrogel which can lead to a superior therapeutic effect via the longer drug retention time as well as reduce system toxicity.