Study On The Probiotic Delivery System With The Functional Coating To Enhance Intestinal Colonization For The Treatment Of Enteritis

With the further study of intestinal microbiota,more and more evidence indicates that intestinal microbiota homeostasis is essential for the maintenance of host health.The imbalance of intestinal flora is closely related to the occurrence and development of many diseases,such as diabetes,obesity,hypertensive heart disease,inflammatory bowel disease（IBD）and some cancers.The orally delivered probiotics can effectively regulate intestinal flora and have important application value for the prevention and treatment of diseases.However,oral probiotics are exposed to complex gastrointestinal conditions such as gastric acid,bile salts and enzymes,which can significantly reduce the activity and colonization efficiency of probiotics.In addition,the large amount of reactive oxygen species（ROS）and depleted mucous layer in the pathological microenvironment further limit the survival and colonization of probiotics in the intestinal tract.Currently,a variety of materials are used to encapsulate probiotics for oral delivery,providing a survival advantage for probiotics.However,these attempts have succeeded only in protecting bacteria from gastrointestinal insults under physiological conditions,and the survival and colonization of probiotics in intestinal pathological conditions still remain extremely challenging.Therefore,it is urgently needed to develop new strategies to endow probiotics with the ability to resist both the physiological and pathological environments of the gastrointestinal tract,increase their intestinal colonization,and thus enhance the treatment of diseases by oral probiotics.Based on this,inspired by the fact that probiotics colonized and grew in the mucus layer under physiological conditions,this project constructed a new probiotic delivery system.Escherichia coli Nissle 1917（EcN）was coated with tannic acid and mucin via layer-by-layer technology,and the EcN@TA-Ca²⁺@Mucin was constructed.By resisting the harsh physiological environment of the gastrointestinal tract and spontaneously regulating the pathological microenvironment of inflammatory diseases,the system effectively improves the colonization of probiotics in the intestinal tract and has a good therapeutic effect on colitis.The specific research contents are as follows:1.Preparation and characterization of EcN@TA-Ca²⁺@Mucin.EcN@TA-Ca²⁺@Mucin was synthesized by modifying the probiotic EcN with tannic acid and mucin by layer-by-layer coating technique.Then we characterized the prepared EcN@TA-Ca²⁺@Mucin.The dynamic light scattering（DLS）results showed that the size of the coated probiotics was 1140±23.18 nm,and the zeta potential was-17.2±0.66 m V.The transmission electron microscope（TEM）images showed a distinct extra shell with a thickness of about 200 nm on the coated EcN,in sharp contrast to the smooth edges of the EcN.The results of flow cytometry and confocal laser scanning microscopy（CLSM）also confirmed the probiotics were successfully coated.In addition,the results of bacterial growth curve and cell counting kit-8（CCK-8）method confirmed that the coating does not affect the bioactivity and proliferation ability of EcN.2.Tolerance ability of EcN@TA-Ca²⁺@Mucin to the simulated gastrointestinal environment in vitro.First,the tolerance of EcN@TA-Ca²⁺@Mucin to the physiological environment of the gastrointestinal tract was investigated by bacterial colony counting,TEM,bacterial live and dead staining and bacterial growth curve.The results showed that EcN@TA-Ca²⁺@Mucin can effectively resist the harsh physiological environment of the gastrointestinal tract,such as gastric acid,bile salts,etc.,due to the existence of the coating,and still retains the ability to grow and proliferate.Flow cytometry and CLSM proved that the most mucin coating of EcN@TA-Ca²⁺@Mucin remained stable on the surface of probiotics after exposure to the harsh conditions in the gastrointestinal tract.In addition,in the pathological environment of some inflammatory diseases such as ulcerative colitis,the abundant reactive oxygen species and damaged mucus layer are also not conducive to the colonization and growth of probiotics.Therefore,we further investigated the ability of EcN@TA-Ca²⁺@Mucin to regulate the simulated pathological environment of diseases such as colitis.The results of bacterial colony count,TEM,bacterial live and dead staining,and electron spin resonance（EPR）experiments demonstrated that EcN@TA-Ca²⁺@Mucin can effectively remove ROS,thereby reducing the damage of ROS to EcN.Since the mucin coating can interact with mucus through hydrogen bonds,disulfide bonds,etc.,the effect of EcN@TA-Ca²⁺@Mucin on the intrinsic properties of mucus was investigated by plate rheometer.The results demonstrate that the interaction of EcN@TA-Ca²⁺@Mucin with mucus can significantly increase the apparent viscosity and storage modulus of mucus,i.e.,increase the viscoelasticity of mucus.Finally,the movement trajectory of EcN@TA-Ca²⁺@Mucin in mucus was tracked by video microscopy and the mucus penetration of EcN@TA-Ca²⁺@Mucin was observed by CLSM,and the results proved the motility and penetration abilities of EcN@TA-Ca²⁺@Mucin in mucus were greatly reduced due to the presence of the coating.3.Study on the enhanced colonization and safety of EcN@TA-Ca²⁺@Mucin in the intestine of healthy miceIn healthy mice,the colonization ability and safety of EcN@TA-Ca²⁺@Mucin were evaluated in vivo.First,the survival rate of EcN@TA-Ca²⁺@Mucin in mice after oral administration was investigated by bacterial plate colony counting.It can be found that the survival rate of EcN@TA-Ca²⁺@Mucin in mice is greatly improved due to the protective effect of the coating,which was about 10 times that of the EcN group.The in vivo retention ability of EcN@TA-Ca²⁺@Mucin in mice was investigated through mouse intestinal retention experiments,long-term intestinal colonization experiments and 3D mucus-adhesive observation experiments.The results demonstrated that due to the strong homology interaction between mucus and mucin in the functional coating,the rate of EcN@TA-Ca²⁺@Mucin being excreted by intestinal peristalsis was significantly reduced,and the retention time of EcN@TA-Ca²⁺@Mucin in the intestine was up to 6 days.Therefore,it had a better intestinal colonization effect than the EcN group.In addition,After oral administration of EcN@TA-Ca²⁺@Mucin three times,the hematoxylin and eosin（H&E）staining results of mouse tissues,the levels of inflammatory factors in serum,and blood routine indexes were not significantly different from those of healthy mice,which proved that EcN@TA-Ca²⁺@Mucin has no obvious side effects while colonizing efficiently.4.Study on the therapeutic effect of EcN@TA-Ca²⁺@Mucin on colitis mice in vivoFinally,the therapeutic effect of EcN@TA-Ca²⁺@Mucin was investigated by constructing a sodium dextran sulfate（DSS）-colitis mouse model.After 5 days of treatment,the body weight changes and colon length results of mice showed that the body weight of mice treated with EcN@TA-Ca²⁺@Mucin quickly regained and the colon length in the EcN@TA-Ca²⁺@Mucin group was similar to that in healthy mice.Representative images of H&E staining and in situ terminal transferase labeling（TUNEL）staining showed that EcN@TA-Ca²⁺@Mucin treatment can effectively protect the colonic epithelium from pathological damage.The results of ROS staining demonstrated that EcN@TA-Ca²⁺@Mucin could effectively scavenge ROS when it reaches the site of colitis.The results of immunofluorescence staining of zonula occludens-1（ZO-1）protein and occludin-1 protein,alcian blue/periodic acid Schiff（AB/PAS）staining and intestinal permeability experiments all proved that EcN@TA-Ca²⁺@Mucin can effectively repair the intestinal barrier.Moreover,the plate colony count and H&E staining results of the heart,liver,spleen,lung and kidney demonstrated that the translocation of EcN@TA-Ca²⁺@Mucin to the distal organs was significantly reduced,and there was no inflammatory cell infiltration in the distal organs of mice.The above results proved that EcN@TA-Ca²⁺@Mucin has a good therapeutic effect on colitis mice.In conclusion,in this study,this project constructed a probiotics delivery system(EcN@TA-Ca²⁺@Mucin)with functional nanocoating by modifying EcN with tannin acid and mucin through a layer-by-layer coating technique.Due to the existence of the coating,EcN@TA-Ca²⁺@Mucin can not only effectively resist stomach acid and bile salts,but also reduce the damage of ROS to probiotics in the pathological microenvironment,and enhance intestinal adhesion,thus having a better intestinal retention effect.Based on this,in the colitis mouse model,EcN@TA-Ca²⁺@Mucin can effectively scavenge ROS,repair the damaged mucosal barrier in the pathological microenvironment,and significantly increase the therapeutic effect of probiotics on colitis.Furthermore,due to the modification of mucin,the distal translocation of bacteria is reduced,enhancing the safety of oral delivery of probiotics.In conclusion,this oral probiotic delivery system provides a simple and efficient strategy to enhance colonization and treatment of probiotics in the intestinal pathological microenvironment,which is expected to provide a novel perspective for applying the intestinal colonization of probiotics to treat a variety of diseases.