| Ulcerative Colitis(UC)is a severe,lifelong inflammatory bowel disease affecting the colon and rectum.Despite the emergence of new therapeutic approaches,remission rates remain relatively low,leading to surgical interventions for many patients.Studies have indicated a notable decrease in butyrate levels among UC patients,and exogenous butyrate supplementation may offer a new perspective for UC intervention.However,in the delivery vehicles for exogenous butyrate,challenges such as limited conversion efficiency,inadequate odor masking,and rapid intestinal clearance still persist.Current research trends are increasingly focusing on bioadhesive carriers that combine colonic targeting with mucosal adhesiveness.The aim is to enhance butyrate’s retention time in the colon,optimize its bioconversion and absorption,and thereby provide superior therapeutic benefits for conditions like UC.Based on this,this study developed a thiolated pectin microsphere carrier that can effectively encapsulate and deliver butyrate,while also possessing colonic targeting and mucosal adhesiveness.This microsphere system remains structurally intact in the upper digestive tract and,upon reaching the colon,is slowly metabolized by the intestinal microbiota,thereby releasing butyrate.Importantly,the thiol groups on the microsphere carrier can form disulfide bonds with intestinal mucus,prolonging the retention time of butyrate in the colon.In mice with UC,this microsphere system effectively increased the butyrate concentration in the colon,modulating intestinal homeostasis by regulating intestinal immunity,repairing the intestinal barrier,and reshaping the intestinal microbiota,showing promising therapeutic effects.The main research content and conclusions are as follows:(1)Preparation and characterization of thiolated pectina)Commercial pectin(PEC)was chosen as the base material.Utilizing the principle of amide bond formation,cysteine was covalently linked through an amide bond to produce thiolated pectin(PSH).By varying the ratio of added cysteine,three different thiolated pectins with varying thiol contents were prepared,labeled as PSH06,PSH09,and PSH15,showing thiol contents of 517,687,and 732μmol/g,respectively.Their thiol introduction was validated through EDX,FTIR,NMR spectroscopy.b)The influence of thiol functionalization on the molecular characteristics of pectin was investigated.The thiolated pectins retained the chain-like molecular structure of commercial pectin.Furthermore,thiolated pectins with different thiol contents showed no significant difference in molecular weight and monosaccharide composition compared to commercial pectin.c)The interaction of thiolated pectins with different thiol contents and natural mucin was studied.Rheological results suggested that thiol functionalization considerably augmented the interaction between pectin and mucin.The higher the thiol content,the higher the corresponding viscosity,stress,storage modulus,and loss modulus.Compared to commercial pectin PEC,the initial viscosity of mixtures of thiolated pectins PSH06,PSH09,and PSH15 with natural mucin increased by 25.7 times,32.3 times,and 49.5 times,respectively.(2)Construction of thiolated pectin microsphere system for butyrate colontargeted deliveryBased on the aforementioned research conclusions,PSH15 was chosen as the raw material.a)The effects of different proportions of thiolated pectin(0.5%,1%,2%w/v)and oxidizing agents(0.5%,1%,2%v/v)on the gelation properties of the thiolated pectin were investigated.Results showed that a higher proportion of PSH15(2%w/v)and a lower proportion of oxidizing agent(0.5%v/v)yielded a more compact gel network structure,with the highest viscosity,storage modulus,and loss modulus.b)Under the conditions of 2%w/v PSH15 and 0.5%v/v oxidizing agent,different-sized microspheres(1.5±0.50,5.5±1.32,22±3.64μm)were produced using the reverse-phase emulsification method by controlling shear forces during homogenization.c)Before encapsulating in the microsphere carriers,sodium butyrate was embedded in nanoparticles.Specifically,zein nanoparticles loaded with sodium butyrate(SB@Z)were prepared using the solvent evaporation method.Under optimized conditions,the average particle size of SB@Z was 597.0±11.6nm,with an encapsulation efficiency of 60.3±2.0%and a drug loading of 13.1±0.4%.d)Subsequently,the SB@Z nanoparticles were embeded into thiolated pectin.The experimental results revealed that larger microsphere size(22±3.64μm)were more efficient for encapsulating SB@Z nanoparticles.The final microsphere encapsulating the nanoparticle composite was denoted as SB@Z@PSH,with a sodium butyrate loading rate of 5%.e)This microsphere system could significantly mask the specific odor of butyrate and exhibited favorable interactions with natural mucus.(3)Exploration of in vivo and in vitro release patterns of the thiolated pectin microsphere system for butyrate colon-targeted deliveryIn this chapter,we comprehensively studied the in-vivo and in-vitro release behaviors of the thiolated pectin-based microsphere delivery system for colon-targeted butyrate release:a)Using in vitro simulated digestion,we examined the protective effect of the microsphere system on sodium butyrate in the upper gastrointestinal tract.Results showed that after 120 minutes of gastric digestion,the release rate of butyrate from SB@Z@PSH was less than 10%,and after 360 minutes of small intestine digestion,the release rate was less than 30%.b)Combining in vitro fermentation experiments and simulating the colonic environment in an anaerobic workstation,we studied how the microsphere system could be metabolized by gut microbiota to release sodium butyrate.Results showed a significant reduction in the molecular weight,total sugar,and monosaccharide content of SB@Z@PSH microspheres over fermentation time,with a significant increase in butyrate production.This confirms that the SB@Z@PSH microsphere system can be gradually metabolized by the gut microbiota.16s RNA analysis also indicated that the SB@Z@PSH microsphere system can significantly promote the growth of beneficial bacteria like Bacteroides and Faecalibacterium while inhibiting harmful bacteria like Escherichia-Shigella and Desulfovibrio.c)Lastly,using fluorescent molecular imaging in the mouse gastrointestinal tract,we further confirmed that this delivery carrier can effectively prevent the release of fluorescent molecules in the upper digestive tract,target them to the colon,and release them gradually.Simultaneously,the adhesive properties of the carrier effectively slowed down the intestinal clearance of Cy5,enhancing the retention time of Cy5 in the colonic tissue.(4)Regulation of intestinal homeostasis in ulcerative colitis mice by the thiolated pectin microsphere system for butyrate colon-targeted deliveryAn acute model of colitis in mice was established through induction with Dextran Sulfate Sodium(DSS).We systematically examined the alleviating effects of the SB@Z@PSH microsphere system on UC by assessing the apparent symptoms,intestinal inflammation,intestinal barrier function,gut microbiota,and SCFAs levels.The findings are as follows:a)The SB@Z@PSH microsphere system reduced the DAI,alleviated the weight loss in mice,increased the survival rate of mice with colitis,and diminished colon shortening.b)The SB@Z@PSH microsphere system markedly ameliorated inflammatory cell infiltration,crypt destruction,and epithelial injury induced by DSS.It downregulated the levels of pro-inflammatory cytokines and upregulated the anti-inflammatory cytokine.c)The SB@Z@PSH microsphere system contributed to the restoration of intestinal barrier function.It decreased the intestinal permeability indices,promoted the secretion and expression of tight junction proteins like ZO-1,Claudin-1,and Occludin,and repaired the mucus layer of the colon tissue in UC mice.d)Furthermore,after DSS induction,the structure of the mouse gut microbiota became disordered,leading to the enrichment of various pathogens such as Streptococcus and Escherichia-Shigella.The SB@Z@PSH microsphere system optimized the gut microbiota structure in UC mice,significantly increasing the relative abundance of Akkermansia and Dubosiella.e)Intervention with the SB@Z@PSH microsphere group markedly increased the concentration of butyrate in the cecum of UC mice.Accordingly,the concentrations of acetate,propionate,and total SCFAs also increased. |
