Study On The Effect Of Oral Delivery Lactobacillus Reuteri With Sodium Alginate-Chitosan Microgel On Colon Cancer

Colorectal cancer(CRC)incidence is increasing in recent years due to intestinal flora imbalance,making oral probiotics a hotspot for research.However,numerous studies related to intestinal flora regulation ignore its internal mechanisms without indepth research.Here,we developed a probiotic microgel delivery system(L.r@(SACS)2)through the layer-by-layer encapsulation technology of Alginate(SA)and Chitosan(CS)to improve gut microbiota dysbiosis and enhance anti-tumor therapeutic effect.Short chain fatty acids(SCFAs)produced by L.r have direct anti-tumor effects.Additionally,it reduces harmful bacteria such as Proteobacteria and Fusobacterium,and through bacteria mutualophy increases beneficial bacteria such as Bacteroid and Firmicutes,which produce butyric acid.By binding to the G protein-coupled receptor 109A(GPR109A)on the surface of colonic epithelial cells,butyric acid can induce apoptosis in abnormal cells.Due to the low expression of GPR109A in colon cancer cells,MK-6892 can be used to stimulate GPR109A.With butyrate production increased,the activated GPR109A could bind more butyrate,which further promote apoptosis of cancer cells and trigger antitumor responses.It appears that the oral administration of L.r@(SA-CS)2 microgels may provide a treatment option for CRC by modifying the gut microbiota.ObjectiveOral administration of L.r increases the production of SCFAs in the gut,regulates the gut flora,reduces the presence of harmful bacteria in the gut,and increases the presence of beneficial bacteria,especially beneficial bacteria that produce butyric acid.2.Combined with MK-6892’s ability to stimulate the poorly expressed butyric acid receptor GPR109A in colon cancer cells,the combination with butyric acid promotes apoptosis in colon cancer cells and triggers an anti-tumor response,further expanding the use of butyric acid receptor agonists in the treatment of colon cancer.Methods1.Preparation and characterization of L.r@(SA-CS)2 micro gel.The morphology of micro gel was observed;Measure its particle size and potential change;The 24 h bacterial growth curve was recorded with the enzyme marker at the wavelength of 600 nm;Bacterial activity was detected by AlarmarBlue;Baclight fluorescence staining was used to observe the survival rate of bacteria after encapsulation.2.Function verification of the microgel against gastrointestinal environment.The protective ability of the microgel against bacteria was tested by simulating gastroenteric fluid,bile salts and antibiotics in vitro,and then the mice were inlavated with the gel to test the resistance ability in vivo.3.To investigate the mechanism of CT26 apoptosis induced by microgel in colon cancer.Use CCK8 kit and Annexin V-FITC/PI apoptosis detection kit to detect the killing ability of bacteria and bacterial supernatant to CT26 cells.Western Blot detection of apoptotic protein expression:LC-MS analysis of bacterial supernatant was performed to determine the main components.4.To evaluate the regulatory effect of oral microgels on intestinal flora.After oral administration of L.r,16S rDNA was used to detect the regulation of bacterial flora in mouse feces.CT26 tumor-bearing mouse model was established,and whether the anticancer effect of L.r depended on intestinal flora was detected by complex antibiotic clearance test.The fecal dilution smear count measures the metabolism of bacteria in the body.5.To evaluate the anti-tumor effect of microgel combined with MK-6892 in vivo.CCK8 was used to detect the killing effect of sodium butyrate(NaBu),MK-6892 and their combination on CT26 cells.Western blotting was used to detect the expression of apoptotic protein after combination.The mouse model of CT26 bearing tumor was established to investigate the inhibitory effect of microgel combined with MK-6892.After treatment,the body weight of the mice was recorded,and the tumor changes were recorded by imaging in vivo.After 21 days,the tumor tissues and main organs were taken out for hematoxylin-eosin(H&E)and Ki67 staining to evaluate the pathological changes of colon and tumor.The expression of GPR109A receptor and apoptotic protein in tumor tissues was detected by Western blot.6.In vivo safety assessment and effect on immune system.Periodic orbital blood collection for blood routine and blood biochemical detection;ELASA kit was used to detect the change of serum immune factors TNF-α,IL-6,IFN-γ;HE evaluated the damage to major organs.Results1.L.r@(SA-CS)2 microgel was successfully constructed,and the growth and vitality of bacteria were not affected when the material was wrapped in the second layer.2.L.r@(SA-CS)2 microgel has obvious resistance to in vivo and in vitro gastrointestinal environment.3.By producing short chain fatty acids,bacteria cause the increase of apoptosis-promoting proteins Caspase-3 and Bax in tumor cells,and the decrease of apoptosis-inhibiting protein Bcl-2,which leads to apoptosis.4.Oral L.r@(SA-CS)2 microgel can regulate the intestinal flora of mice,reducing harmful bacteria,and increasing beneficial bacteria.L.r relies on interactions with other flora to achieve antitumor effects.5.CT26 in vitro tumor bearing mouse model was established.Oral administration of L.r@(SA-CS)2 microgel regulated the flora and increased the production of butyrate in vivo,and combined with GPR109A receptor agonist MK-6892 had the strongest effect on tumor inhibition.6.L.r@(SA-CS)2 microgel can induce anti-tumor immune response in vivo,and has good biological safety.ConclusionsThe L.r@(SA-CS)2 microgel delivery system was successfully constructed by layer-by-layer assembly using the electrostatic interaction between SA and CS.Microgels coated with L.r exhibit good gastrointestinal tolerance and significantly improve oral availability.2.L.r promotes apoptosis of tumor cells through the production of SCFAs,while remodeling the gut flora,reducing harmful bacteria in the gut and increasing beneficial bacteria that produce butyric acid through bacterial flora interactions.3.Microgels in combination with butyric acid receptor agonists further enhance anti-tumor effects and have been shown to be highly effective in inhibiting in situ colon cancer.The biocompatible L.r@(SA-CS)2 microcapsules provided an idea for oral probiotics to treat solid tumors,and further expanded the application of MK in tumor therapy.