| The incidence of inflammatory bowel disease(IBD)is gradually increasing,the course of disease is lengthy,and there is a risk of colon cancer,which has attracted more and more attention.Studies have shown that intestinal microbiota imbalance,immune dysfunction,and intestinal mucosal damage are closely related to the development of IBD.It is difficult to improve the single factor of the above pathogenesis to achieve an ideal therapeutic effect.It is a new idea to improve the efficacy of treating IBD through the multiple effects of regulating flora,maintaining immune homeostasis and repairing damaged intestinal mucosa.At present,the physiological barriers of the gastrointestinal tract(p H barrier,mucus barrier,intestinal epithelial cell barrier,etc.)lead to low in vivo bioavailability of oral IBD therapeutic preparations.However,the strong resistance of probiotic spores to external factors is conducive to solving this problem.Based on the above analysis,this project constructed an intestinal in situ recombination nanomedicine based on probiotic spores,which can not only successfully pass through the gastric acid environment,enhance mucus adhesion and penetration,and promote trans-intestinal epithelial cell transport to overcome oral physiological barriers,but also It can effectively treat IBD through the multiple effects of intestinal flora regulation,immune regulation and intestinal mucosal repair.First,the Aryl hydrocarbon receptor agonist ITE was loaded into poly-N-vinyl caprolactam nanoparticles(PVCL NPs),and ITE/PVCL was chemically modified on the surface of Clostridium butyricum spores(CBs)to construct ITE/PVCL@CBs formulations.The system has the following characteristics: 1)After oral administration,it smoothly passes through the gastric acid environment,CBs germinate after targeting the intestinal site,and the hydrophobic sprout protein(SP)and ITE/PVCL on the surface of CBs are shed together.SP adhered to the surface of ITE/PVCL through chemical bonds and hydrophobic interactions,and reassembled into an ITE/PVCL@SP nanosystem.2)On the one hand,the sulfhydryl group in SP can exchange disulfide bonds with mucin in the intestinal mucus layer,which enhances the mucus adhesion and penetration of the nanoformulation,and improves the uptake and transport capacity of ITE/PVCL@SP by intestinal epithelial cells.On the other hand,a large number of positively charged proteins are enriched on the surface of inflammatory intestinal epithelial cells,and the negative charge of SP helps ITE/PVCL to target and adhere to inflammatory intestinal epithelial cells.Subsequently,ITE activates the aryl hydrocarbon receptors of intestinal cells to play anti-inflammatory,maintain T helper cell 17(Th17)/regulatory T cell(Treg)cell balance,and repair damaged intestinal mucosa.3)CBs germinate into Clostridium butyricum in the gut,which not only regulates intestinal flora homeostasis,but also produces short-chain fatty acids to maintain intestinal mucosal integrity,support the differentiation and expansion of Treg,and improve the inflammatory environment.The nanomedicines constructed in this paper based on the in situ recombination of probiotic spores in the intestine have multiple functions of intestinal flora regulation,immune regulation and intestinal mucosa repair,which are of great significance for the treatment of IBD.1.First,ITE was loaded into PVCL NPs polymer nanoparticles,and ITE/PVCL was chemically modified on the surface of CBs through amide bonds to construct an ITE/PVCL@CBs oral drug delivery system.The successful construction of the nanosystem was demonstrated by scanning electron microscopy and ultraviolet spectroscopy.We found that CBs had a protective effect on ITE/PVCL under the strong gastric acid environment of p H 1.2.After ITE/PVCL@CBs were incubated in vitro,it was proved by scanning electron microscopy and sulfhydryl content detection that SP and ITE/PVCL were shed together and reconstituted into ITE/PVCL@SP nanosystems.In vitro experiments mimicking the positively charged proteins on the surface of inflammatory intestinal epithelial cells show that ITE/PVCL@SP is more likely to adhere to the surface of inflammatory intestinal epithelial cells than ITE/PVCL.2.In the in vitro cell experiment,Caco-2 was selected as the intestinal epithelial cell model,and the cytocompatibility experiment proved that ITE/PVCL@SP has good safety.Using Transwell technology to simulate the intestinal epithelial barrier function,it was found that the sulfhydryl groups in SP could interact through disulfide bonds to enhance the mucus adhesion and penetration of ITE/PVCL@SP,which would improve the uptake and transport of nanoparticles by intestinal epithelial cells.3.In vivo animal experiments,mice with colitis induced by dextran sulfate sodium(DSS)were used as a model to investigate the distribution of ITE/PVCL@CBs in vivo.The results showed that ITE/PVCL@CBs had good colon targeting ability.In the pharmacodynamics experiment,the body weight and colon length of colitis mice were restored to normal mice after ITE/PVCL@CBs treatment.In addition,H&E experiments showed that ITE/PVCL@CBs improved the integrity of colon tissue structure,increased the expression of tight junction proteins,and repaired the damaged intestinal mucosa.Flow sorting experiments proved that ITE/PVCL@CBs restored the intestinal Th17/Treg balance and had a good therapeutic effect on IBD.In the intestinal flora analysis experiment,after ITE/PVCL@CBs treatment,the diversity and richness of the intestinal flora were increased,and the flora structure was developed towards a healthy direction.In conclusion,the constructed ITE/PVCL@CBs can be reassembled in situ into ITE/PVCL@SP nanosystems in the gut,overcoming the oral absorption barrier.And it can effectively treat IBD through the triple regulation effect of regulating the flora,maintaining immune homeostasis and repairing the damaged intestinal mucosa.This addresses the limitations of monotherapy for the treatment of IBD.In addition,the colonic targeting of CBs will alleviate the toxic side effects of long-term drug treatment. |
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