Investigation On Probiotic Spore-based Oral Delivery System For Colitis And Colon Cancer Therapy

Colitis and colitis-associated colon cancer（CAC）are systemic gastrointestinal diseases that seriously threaten human health.These diseases are usually caused by the interaction of multiple complex factors,such as intestinal microbiota,mucosal barrier,environment and immune system.And their prevention and treatment are still great challenges.In recent years,with the interaction among probiotics,host and intestinal microbiota gradually clarifying,more and more researchers have recognized that oral probiotics can positively regulate intestinal microbiota,enhance intestinal mucosal barrier,and competitively inhibit or eliminate pathogenic bacteria.Unfortunately,most probiotics cannot tolerate the harsh stomach environment after oral administration,and consequently the number of effective viable bacteria delivered to the intestinal tract cannot be guaranteed.Notably,spores,the dormant life forms of probiotics,are encased in a thick hydrophobic protein coat with high resistance.After oral administration,they can resist the complex and changeable gastrointestinal tract（GIT）conditions and maintain good physiological activity.These characteristics of spores can provide an ideal platform for the preparation of therapeutic nanodrugs and probiotic agents,which is expected to become a breakthrough for the prevention and treatment of colitis and CAC.In order to effectively enhance the stability of nanoparticles in the GIT and improve the bioavailability of oral drugs and colonization efficiency of probiotics in the intestinal tract,we constructed different types of oral delivery systems based on probiotic spores,and carried out a systematical investigation in vitro and in vivo.The details are as follows:1.A probiotic spore-based oral self-assembled nanodrug for colon cancer therapyThe probiotic spores are produced by Bacillus coagulans（BC）in nutrient-starved environment.After oral administration,BC spores can pass through the gastric acid environment and be delivered to the intestine.When spores bind to some nutrients in the intestinal microenvironment,the process of germination would be triggered with the disintegration of their hydrophobic protein coat,regenerating vegetative cells and colonizing in the intestine.In this study,we utilized the above mentioned natural characteristic of BC to construct an autonomous nanodrug“generator”（DOX/SOR/Spore-DA）by modify spores with deoxycholic acid（DA）and loading with anti-tumor drugs doxorubicin hydrochloride（DOX）and sorafenib（SOR）.This system could spontaneously form therapeutic NPs in the intestine after oral administration because of spore germination.The experimental results showed that the biological activity of spores was hardly affected after modification and drug loading,which was a key condition for the self-assembly of disintegrated hydrophobic protein with DA and drugs to form drug-loaded NPs.The drug loading efficiency of DOX and SOR in DOX/SOR/Spore-DA was about 11.5%and 25.7%,respectively,which could satisfy the formation of therapeutic NPs.The result of germination in simulated GIT environment showed that intestine was the most suitable region for spore germination.The TEM results of spore and DOX/SOR/Spore-DA after being incubated with growth medium showed that DOX/SOR/Spore-DA could germinate and autonomously form DOX/SOR/Spore-DA NPs with uniform size and good dispersion,while spores alone could not form NPs after 6 h incubation.The results of Caco-2 cell uptake and transepithelial transport showed that DOX/SOR/Spore-DA NPs were internalized by Caco-2 cells via apical sodium-dependent bile acid transporter（ASBT）-mediated endocytosis.And then NPs entered lysosomes and escaped,enhancing their transepithelial transport efficiency and increasing basolateral drug content which was 2.2 times higher than that in DOX/SOR group.Additionally,in vitro antitumor cell experiments showed that the cell survival rate of DOX/SOR/Spore-DA NPs was only 42.4%,which was significantly lower than that of other groups,indicating that DOX/SOR/Spore-DA NPs could efficiently inhibit the growth of tumor cells.The results of intestinal tissue uptake,in vivo intestinal villi absorption and in situ intestinal circulation all proved that DOX/SOR/Spore-DA could self-assembly form DOX/SOR/Spore-DA NPs in vivo,and improve oral bioavailability of drugs by enhancing the cell uptake and transport efficiency of epithelial cells.And the relative bioavailability of DOX/SOR/Spore-DA group was increased by about 9.4 times compared with the oral DOX/SOR group.In vivo anti-tumor efficacy study showed that,compared with saline group,the tumor growth inhibition rate of DOX/SOR/Spore-DA group was about 79.6%,showing a substantial antitumor effect.2.Construction of the spore coat-based oral probiotics delivery system and its treatment of colitis and colitis-associated colon cancerThe intestinal dysbacteriosis and gut mucosal barrier impairment are the main pathological features of colitis and CAC.Although the oral probiotic agents have significant advantages in the treatment of colitis and CAC,the viability and colonization efficiency of probiotics are still affected by excipients and production processing-related factors.Additionally,spores,as a way of oral delivery of probiotics,also suffer from the inherent limitation that their germination efficiency can not be accurately controlled in vivo.Therefore,how to take best advantage of the high resistant spore coat to enhance oral delivery and intestinal colonization efficiency of probiotics have become key factors to improve the therapeutic effect of probiotic agents on colitis and CAC.Based on this,BC was selected as a model strain in this study.The spore coat was separated and innovatively prepared into a type of versatile spore coat nanomaterial（CN）.And then CN was adsorbed on BC surface as a protective“armor”to prepare CN@BC.The universality of this method was verified by Bacillus subtilis（BS）and Bacillus Licheniformis（BL）,respectively.And then in vitro results indicated that CN retained the integrity of spore coat components and had high GIT environmental stability.Furthermore,CN could inhibit H₂O₂-induced oxidative damage,repair intestinal epithelial mucosal barrier,provide the extra active substances for explosive proliferation of probiotics,promote the competitive colonization and proliferation of probiotics etc.Finally,the biosafety and preventive and therapeutic effects of CN@BC were systematically evaluated in healthy mice,DSS-induced colitis mice model and AOM/DSS-induced CAC mice prevention model,respectively.These results showed that CN@BC with good biosafety could resist gastric acid environment and rapidly colonize in the colonic region of healthy mice after 48 h administration.Notably,CN@BC hardly damaged the intestinal mucosal barrier and caused intestinal inflammatory reaction.Besides,CN@BC could inhibit inflammatory progression by decreasing the expression levels of typical pro-inflammatory cytokines such as IL-1β,IL-6 and TNF-αand blocking the IL-6-STAT3 pathway which is closely related to tumorgenesis.Furthermore,CN@BC could also significantly enhance the expression levels of ZO-1 and Occludin（known for the restoration of intestinal mucosal barrier）,so as to achieve the superior therapeutic effect of colitis and CAC.Most noteworthy,the results of 16S r DNA amplicon sequencing and Tax4fun functional annotation cluster thermal map showed that CN@BC could observably improve the species richness and diversity of intestinal available bacteria,and reduce the relative abundance of species that associated with tumorigenesis and development.CN@BC-treated group showed a similar composition and function of microbiota with the healthy mice,which demonstrated that CN@BC had good treatment and prevention effect for CAC mice model.