Probiotic ‘backpacks’ show promise for treating inflammatory bowel diseases
Authors: Jun Liu, Yixin Wang, William John Heelan, Yu Chen, Zhaoting Li, Quanyin Hu
Reactive oxygen species (ROS), which are by-products of aerobic metabolism, are crucial molecules in physiological processes (1, 2). However, their overproduction will cause oxidative stress, which will amplify inflammatory responses and exacerbate inflammatory disorders, especially in the gastrointestinal (GI) tract, resulting in intestinal mucosal layer damage and pathogen invasion, subsequentially stimulating immune responses, and ultimately leading to the development of inflammatory bowel diseases (IBDs) (3–5). Excessive ROS-induced oxidative stress in the intestine is thought to be a major factor in the pathogenesis and progression of IBDs. Oxidative stress is caused by the imbalance in the generation of reactive species and the host’s antioxidant defense capability (6, 7).
Excessive ROS can cause intestinal endothelial cell damage through inducing lipid peroxidation and DNA mutation, impairing protein functions, altering epithelial permeability, and disrupting intestinal epithelial barriers, eventually leading to the initiation or deterioration of IBDs (8, 9). Furthermore, excessive ROS can also cause dysregulated proinflammatory reactive species–sensitive pathways in immune cells (10, 11) and induce dysbiosis of the gut microbiota (12, 13). Antioxidants are known to scavenge ROS in the intestines and are helpful for the treatment of IBDs (14–16). However, because of their rapid clearance, nonspecific drug biodistribution after systemic administration, and relatively inefficient ROS-scavenging ability, antioxidants demonstrate inconsistent efficacy for treating inflammatory diseases. Furthermore, undesirable drug distribution profiles of antioxidants in normal tissues can cause a variety of adverse effects (17–19).
In addition to excessive ROS in the intestine, IBD is associated with dysbiosis of the gut microbiota in the colonic microenvironment (20–22). In a healthy individual, microbiota provides the host with short-chain fatty acids and essential vitamins while also protecting them from pathogen colonization and invasion. In contrast, a disordered microbiota induces a chronic inflammatory state, increases toxic production, and disrupts the host’s metabolism (23–25). In our previous studies, we found that orally administered probiotics can colonize colon tissues and aid in the restoration of the normal gut microbiome to treat GI tract–related diseases (26, 27). Unfortunately, these probiotics are highly sensitive to the harsh environments in the GI tract, which limits their viability and retention time in the intestine, leading to decreased therapeutic efficacy (28, 29).
In this study, we established a platform that can selectively and sustainably scavenge ROS in inflamed colon tissues while also improving probiotic delivery for gut microbiota homeostasis modulation. This platform could help to restore a normal gut microenvironment and address the fundamental issues for effective IBD therapy. We initially selected poly(propylene sulfide) (PPS), a hydrophobic polymer, to scavenge ROS (30). The sulfur atoms of PPS are easily oxidized by ROS to form a sulfoxide that will be further oxidized to produce a sulfone (31). This inherent PPS reactivity with ROS provides PPS antioxidant properties that can serve as a highly efficient tool for scavenging ROS (32). Moreover, the PPS polymer contains multiple ROS-reacting sites capable of scavenging multiple ROS molecules compared to small antioxidant molecules.
However, the clinical application of PPS is limited because it is highly hydrophobic, which makes it difficult for in vivo applications (33). In this study, we synthesized a hyaluronic acid (HA)–PPS conjugate and self-assembled the HA-PPS nanoparticle (HPN) based on the amphiphilic properties that exist when HA and PPS are combined (Fig. 1A). HA was used to modify PPS and construct HPN because it can improve IBD treatment by modulating immune responses, such as regulation of macrophages, and serving as a potent anti-inflammatory agent (34). In addition, HA, which is often present in synovial fluids and the extracellular matrix, is biocompatible and biodegradable. HA has been widely used in biomedical applications with a great in vivo biosafety profile. The newly generated HPN exhibited improved hydrophilicity and demonstrated cytoprotective effects while maintaining the robust ROS-scavenging activity of PPS. Moreover, it was shown that once the ROS were consumed and the sulfur atoms were oxidized to sulfone, the HPN would self-degrade because of the transformation of PPS from a hydrophobic to a hydrophilic state (fig. S1) (35). This transformation enhances the safety of HPN and opens the door for potential clinical applications.
To improve the oral probiotic delivery to the colon for enhanced bacteriotherapy, we encapsulated probiotics with norepinephrine (NE), which could be auto-oxidized to form a poly-NE film on the probiotic surface to protect it from external environmental assaults (36, 37). Furthermore, the catecholamine group of NE, which was found rich in mussel adhesive foot proteins and responsible for the strong adhesive properties in mussels (38, 39), endowed probiotics with robust mucosa adhesive property and extended retention time of probiotics in the intestine without influencing probiotics’ growth and proliferation for enhanced therapeutic efficacy (Fig. 1B). On the basis of the colon tissue colonization property of probiotics, we further conjugated HPN to the surface of probiotics to effectively deliver the HPN to inflammatory colon tissues for normalizing ROS levels and minimizing off-target side effects. This platform not only has the ability to prolong the retention time of probiotics in the intestine for enhanced bacteriotherapy, but it can also specifically deliver and slow-release HPN in the intestine for improved ROS-scavenging capabilities (Fig. 1C). Last, this platform exhibited the enhanced prophylactic and therapeutic efficacy against dextran sulfate sodium (DSS)–induced mouse colitis.
In summary, given the overproduced ROS levels and disordered microbiota in the intestinal microenvironment of GI tract–related diseases, we prepared a platform of HPN-NE-EcN capable of preventing and mitigating the detrimental effects associated with IBDs. The HPN nanoparticles, self-assembled from the synthesized molecule of HA-PPS, exhibited potent ROS-scavenging capability and could protect colonic epithelial cells, as well as the microbiome, including EcN and E. coli K12 strains from oxidative stress–induced damages. The commensal bacteria of EcN that are beneficial for regulating the balance of intestinal flora were further encapsulated with the NE layer on their surface.
The generated NE layer provides EcN resistance against a variety of environmental assaults to improve viability in oral delivery. Moreover, the mucoadhesive capability of the NE layer endowed the EcN bacteria with prolonged retention time in the intestine for enhanced therapeutic efficacy. Furthermore, the probiotic EcN is an effective colon-targeted carrier due to the natural colon tissue colonization property. Thus, except for the intestinal flora modulation property of EcN, the generated platform of HPN-NE-EcN by conjugating HPN on the surface of EcN via a ROS-responsive linker could also effectively deliver HPN to inflammatory colon tissues for targeted ROS scavenging. Moreover, the prolonged retention time of HPN-NE-EcN due to the mucoadhesive capability of the NE layer decreased the clearance of HPN for persistently scavenging ROS in colon tissues.
Notably, the NE layer–coating strategy can be adapted to encapsulate other live cells for cytoprotection, and the NE layer coating, as well as HPN conjugation, would not affect the growth and proliferation of EcN. Meanwhile, the HPN nanoparticles could self-degrade after completely reacting with ROS due to the physicochemical transformation of PPS from hydrophobic to hydrophilic, and this improves the safety profile of this approach. The synergized effectiveness of HPN-NE-EcN for simultaneously scavenging ROS and modulating microbiota homeostasis in colonic microenvironments provided significantly enhanced prophylactic and therapeutic efficacy against DSS-induced colitis. Because of the studies reported that many E. coli strains including EcN have the possibility of pathogenicity to promote colon cancer (46), the safety of the EcN strain developed in this study needs to be further evaluated before clinical applications, and the strategy needs to be tested with other beneficial bacteria as carriers for further translation.