Attaching nanozymes to regulate probiotics’ metabolism and function

Attaching nanozymes to regulate probiotics’ metabolism and function

        Fascinating natural systems have sparked remarkable breakthroughs in the creation of functional materials with diverse uses. In particular, nanozymes are an ideal marriage of enzymes and nanotechnology. Nanozymes are artificial enzymes based on nanomaterials that have shown better stability, numerous activities, and lower costs than natural enzymes. Since the finding of ferromagnetic nanoparticles with intrinsic horseradish peroxidase (HRP)-like activity in 2007, massive research on nanozymes has been continuously emerging in the following decade. They can mimic not only the ROS-producing enzymes of peroxidase (POD), oxidase (OXD), and haloperoxidase (HPO), but also the antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Therefore, they can interfere with the body's innate redox homeostasis, and serve as potential therapeutics for a range of diseases.

        In 2017, I began my Ph.D. at Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, working with Prof. Jinsong Ren and Prof. Xiaogang Qu to create a new generation of nanozymes for bio-applications. Both Prof. Ren and Prof. Qu are knowledgeable and kind. They taught me bit by bit and led me into the fascinating field of nanozymes. When I started my project, I learned that the catalytic activities of nanozymes are much inferior to enzymes, always leading to unsatisfactory outcomes. Thus, I seek to improve the catalytic activity of traditional nanozymes to achieve better therapeutic outcomes. Inspired by the concept of roughness-enhanced bacterial adhesion and defect-improved catalytic activity, I fabricated defect-rich adhesive nanozymes, which could capture bacteria and in situ generate enormous amounts of highly toxic ROS to kill bacteria as opposed to the smooth nanozymes (Fig.1). The intensified antibacterial performance was derived from the defect-rich edges, which induced higher intrinsic POD-like activity compared to pristine nanozymes. Density functional theory (DFT) calculations further disclosed that the high catalytic performance was probably due to the lower adsorption energies of H2O2 and desorption energies of OH*, as well as the larger exothermic process for the whole reaction.

Fig. 1 (a) One-step formation of defect-rich, adhesive nanozymes and (b) their use for improved bacterial capture and elimination.

        Although promising, the catalytic activity of nanozymes is still less than satisfactory. Then, with the rise of single-atom catalysts (SACs), which have demonstrated outstanding performance in diverse catalytic reactions due to their highly active metal site in the form of atomic dispersion, I attempted to construct SAC nanozymes (SAzymes). Thus, SAzymes of Co/PMCS, featured with atomically dispersed coordinatively unsaturated active Co-porphyrin centers, were fabricated to rapidly obliterate multiple RONS to alleviate sepsis, defeating traditional nanozymes (Fig.2). Specifically, Co/PMCS could eliminate ·OH and H2O2 by mimicking SOD, CAT, and GPx, while removing ·OH via the oxidative-reduction cycle with markedly higher activity than nanozymes. It could also scavenge ·NO through the formation of a nitrosyl-metal complex. Eventually, Co/PMCS reduced proinflammatory cytokine levels, protected organs from damage, and conferred a distinct survival advantage over the infected sepsis mice. Encouragingly, the emergence of SACs greatly improved the catalytic performance of nanozymes and enhanced their therapeutic potential. Thereafter, tremendous efforts have been devoted to fabricating various SAzymes with different metal elements to treat cancers or bacteria.

Fig. 2 (a) Illustration of the formation of Co/PMCS and (b) their use as multi-antioxidant SAzymes for sepsis management that defeats nanozymes.

        In 2019, after I earned my Ph.D., I began to think about whether the highly active SAzymes could regulate microbial metabolism and function since they can mimic the innate natural enzyme system in cells. Under the supervision of Prof. Xiaoyuan Chen at the National University of Singapore and Prof. Zhengwei Mao at Zhejiang University, I proposed to create an anti-inflammatory artificial enzyme-armed probiotic and investigated its therapeutic effects on intestinal inflammation and microbiota dysbiosis. I feel blessed that they gave me much help and inspiration during the postdoctoral period. Prof. Chen always told me to work on something simple yet effective for actual applications and encouraged me to communicate with industrial people about probiotics’ applications to learn the key issues. Meanwhile, whenever I needed help, Prof. Mao was the first to come forward, especially in the process of establishing mice and canine models. Besides the support from Chen’s and Mao’s groups, this idea was initially inspired by the outstanding works about "photosynthetic biohybrids" of Prof. Peidong Yang at UC Berkeley. When I first read their paper "Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production" (DOI: 10.1126/science.aad3317) during my Ph.D. period, I was very excited that nanomaterials could improve bacterial CO2 fixation in the energy field. Since then, I have always wondered if nanozymes could enhance bacterial function through chemical regulation for bioapplications. After gaining more knowledge about microorganisms, I noticed that most probiotics are anaerobic due to their lack of antioxidant enzymes, such as CAT and SOD, leading to extreme susceptibility in an O2 atmosphere or oxidative microenvironment, thus reducing their probiotic function. Especially in the treatment of inflammatory bowel disease (IBD), probiotics are usually employed as adjuvants to regulate microbiota dysbiosis while anti-inflammatory drugs primarily manage inflammation. During therapy, the hostile inflammatory intestines would damage the probiotics, reducing their function and even inducing their death. Would antioxidant nanozymes be a promising candidate to overcome the deficiencies of strictly anaerobic probiotics?

Fig. 3 (a) Fabrication of artificial enzymes-armed probiotics of BL@B-SA50 and (b) multiple antioxidant performance of BL@B-SA50.

        Based on my expertise in nanozymes, I fabricated the highly active antioxidant SAzymes and attached them to probiotics via a flexible click reaction, finally obtaining the anti-inflammatory BL@B-SA50 ensemble (Fig.3). SAzymes of B-SA could mimic the antioxidant enzymes of SOD and CAT to scavenge O2·- and H2O2, and meanwhile function as antioxidant biomolecules to eradicate ·OH, robustly and rapidly relieving the inflammatory symptoms and thereby shielding commercial probiotics from hostile stressors. Furthermore, in current IBD standard practices, metabolic instability and limited targeting of clinical drugs result in unsatisfactory therapeutic outcomes. To address this, SAzymes are stable in the poor gastrointestinal environment, promising to replace clinical drugs. Furthermore, to improve the targeting and residence time of anti-inflammatory SAzymes ingredients, we chose the probiotic Bifidobacterium Longum (BL), which could not only confer a health benefit on the host but also possess superior intestinal colonization ability among probiotics, ensuring the persistent antioxidant therapy of SAzymes at the disease site. Finally, the SAzymes-armed probiotics of BL@B-SA50 reduced ROS levels, inhibited proinflammatory cytokine production, restored the intestinal barrier functions, and increased the richness and diversity of gut microbiota in murine models of ulcerative colitis (UC) and Crohn’s disease (CD). Most importantly, to demonstrate the clinical translatability of BL@B-SA50, their therapeutic potential in beagle dogs challenged with colitis was also demonstrated.

        I would like to finally highlight the collaborative effort that brought this project to a successful finish. I thank all the people who helped us along the journey, and we take this opportunity to make a call to the community to join forces and combine expertise to explore new biomimetic catalytic nanomaterials and employ them for regulating the metabolism and function of various microorganisms.

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