Single-Atom Catalysts for Biotherapy Applications: A Systematic Review

Single-atom catalysts (SACs), as atomically dispersed metal active sites anchored or coordinated on suitable supports, demonstrate large potential for use in therapeutic applications. SACs have structural features similar to those of natural enzyme, while exhibiting remarkable catalytic activity, desirable stability, and excellent selectivity. This systematic review aims to synthesize evidence on SACs’ biotherapy applications. Three databases (PubMed/MEDLINE, ISI Web of Science, and ScienceDirect) were searched to identify the studies that investigated the therapeutic efficacy of SACs. A total of 12 studies that fulfilled the inclusion criteria were included and reviewed, and the key findings were qualitatively synthesized. Overall, various SACs were investigated for biotherapy applications, including anticancer, anti-infection (antibacterial), and anti-inflammatory applications; brain trauma therapies, and oxidative-stress cytoprotection applications. All of the included studies showed that the synthesized SACs demonstrated superior therapeutic effects compared with their respective controls. Among the 12 studies reviewed, 11 studies showed satisfied biocompatibility of the applied SACs, whereas minimal cytotoxicity was reported in 1 study. Collectively, the reviewed studies indicated that SACs exhibited considerable promise in the field of biotherapy. Additional studies are needed for a better understanding of the effect of SACs in the treatment of various diseases.


Introduction
Single-atom catalysts (SACs), a novel class of catalysts in which all of the isolated, catalytically active metal atoms are stabilized by a support, were first proposed by Zhang and coworkers in 2011 [1]. Subsequently, the state-of-the-art SACs have been proven to exhibit superb performance in various chemical reactions on the basis of the optimized use of metal atoms with well-defined active centers [2][3][4]. Taking advantage of their properties, including but not limited to their extraordinary catalytic activity, outstanding selectivity, desirable stability, and 100% atom utilization, SACs offer the merits of both heterogeneous and homogeneous catalysts, and thus have attracted extensive attention in regard to chemical catalysis in the petrochemical industry, environmental protection, energy conversion, chemical transformation, and biomedical applications [5][6][7][8][9]. A considerable amount of evidence has demonstrated that SACs are powerful and effective in many typical heterogeneous catalytic reactions, such as electrochemical reactions, water-gas shift reactions, and hydrogenation reactions [10][11][12]. Moreover, SACs are low cost, abundant, and environmentally friendly resources [13,14].
In 2018, SACs were introduced into the field of biotherapy [15]. Despite the sizeable amount of evidence in industrial, energy, and ecological applications, the potential of SACs in biotherapy is much less understood. To the best of the authors' knowledge, there is no systematic review on SACs in

Biocompatibility of the SACs
The biocompatibility/biosafety of the synthesized SACs is one of the critical factors for their biotherapy application; thus, the biocompatibility of SACs was reported in all of the included studies. In general, the vast majority of the studies (11 out of 12) reported the favorable biocompatibility of SACs. Minimal cytotoxicity was reported in 1 remaining study. The cytocompatibility studies included a CCK8 assay [17,18,23,27] and/or Live/Dead assay [24] or an MTT assay [15,20,22,25] or a Cell Titer-Fluor assay [26], while studies conducted by Huo and coworkers [19,21] revealed the biocompatibilities of SAF NCs via the hematoxylin and eosin (HE) staining of major organs dissected

Biocompatibility of the SACs
The biocompatibility/biosafety of the synthesized SACs is one of the critical factors for their biotherapy application; thus, the biocompatibility of SACs was reported in all of the included studies. In general, the vast majority of the studies (11 out of 12) reported the favorable biocompatibility of SACs. Minimal cytotoxicity was reported in 1 remaining study. The cytocompatibility studies included a CCK8 assay [17,18,23,27] and/or Live/Dead assay [24] or an MTT assay [15,20,22,25] or a Cell Titer-Fluor assay [26], while studies conducted by Huo and coworkers [19,21] revealed the biocompatibilities of SAF NCs via the hematoxylin and eosin (HE) staining of major organs dissected from mice in different therapeutic groups. In regard to cytocompatibility, the single-atom catalyst Nanomaterials 2020, 10, 2518 5 of 17 of SAs/NC with bifunctional antioxidative enzymes for oxidative-stress cytoprotection exhibited the minimal cytotoxicity, which was reported by Ma and coworkers [15].

Application of SACs in Cancer Treatments
SACs were applied as a cancer therapy in five studies (Table 1) [21,23,24,26,27]. These studies targeted breast cancer (four studies) and liver cancer (one study). HeLa cells (two studies), 4T1 tumor cells (three studies), HEK 293 cells (one study), and HepG-2 cells (one study) were used in their in vitro assays. For studies targeting breast cancer, 4T1 tumor-bearing mice (three studies) and HeLa-bearing tumor mice (one study) were used. In addition, a mouse model based on xenografted HepG-2 tumors was used in a study focused on liver cancer. In general, all of the five studies reported that the synthesized SACs, which exhibited desirable biosafety, were highly efficient in both the in vitro and in vivo cancer treatments. PEGylated single-atom Fe-containing nanocatalysts (PSAF NCs), a metal-organic framework (MOF) rich in porphyrin-like single-atom Fe(III) (P-MOF), and single-atom Ru supported by a Mn 3 [Co(CN) 6 ] 2 MOF (OxgeMCC-r SAE) outperformed the saline or phosphate-Buffered Saline (PBS) control in inhibiting the growth profiles of breast tumors (Figure 2a-e) [21,24,26]. It is worth mentioning that both OxgeMCC-r SAE and P-MOF enhanced the therapeutic outcome of photodynamic therapy (PDT) in breast cancer via two different mechanisms. The OxgeMCC-r SAE exhibited a high loading capacity of Ce6 photosensitizer. On the other hand, the spin state of Fe(III) in P-MOF under NIR would facilitate the generation of singlet oxygen, which promoted PDT. Moreover, they also reported that P-MOF could serve as an agent for a photothermal therapy (PTT) cancer treatment, as well as photoacoustic imaging (PAI) of breast tumors. In addition, Gong et al. found that carbon dot-supported atomically dispersed gold (MitoCAT-g) significantly suppressed tumor growth in subcutaneous and orthotopic patient-derived xenograft hepatocellular carcinoma models without adverse effects or toxicity, when compared with the saline contro1 (Figure 2f,g) [23]. Recently, a study conducted by Lu and coworkers reported that the bioinspired Cu-HNCS could generate two types of reactive oxygen species (ROS) (O 2 • − and •OH) through two parallel reactions, which significantly enhanced the efficacy of the parallel catalytic of tumors in in vitro and in vivo (Figure 2h-j) [27].

Application of SACs in Anti-Infection Therapies
There were four studies on the application of SACs for anti-infection therapies ( Table 2) [17][18][19][20]. Among them, three studies targeted wound disinfection applications [17][18][19], and one study examined sepsis management [20]. These four studies consistently showed that the synthesized SACs demonstrated superior antibacterial activity without, or at least very little, toxicity. In comparison to the control group, single-atom nanozymes with carbon nanoframe-confined FeN 5 (FeN 5 SA/CNF), zinc-based single atom nanozyme (PMCS SAzyme), and single iron atoms anchored in nitrogen-doped carbon (SAF NCs) exhibited very high statistically significant differences in inhibiting the growth of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli)/Staphylococcus aureus (S. aureus) in vitro, and significantly promoted wound healing without toxicity in vivo. Cao and coworkers reported that the synthesized Co/PMCS could significantly reduce the number of bacteria in the liver, lung, kidney, intestines, and blood of E. coli-induced bacteremia in mice, as well as reducing the white blood cells (WBC), ROS, alanine transaminase (ALT), RNS, IL-6, TNF-α, Lym, Neu, and urea, in organs and blood, when compared with those of the PBS-treated control group. Additionally, they found that all the blood indexes of lipopolysaccharide (LPS)-induced sepsis mice in the Co/PMCS-treated group were reduced to normal. In other words, Co/PMCS could significantly eliminate the systematic production of reactive oxygen and nitrogen species (RONS) and lower proinflammatory cytokine levels compared with those of the PBS-treated group, thereby indicating that Co/PMCS could promote the recovery of multiple organ functions in sepsis mice (Figure 3) [20].

Application of SACs in Anti-Infection Therapies
There were four studies on the application of SACs for anti-infection therapies ( Table 2) [17][18][19][20]. Among them, three studies targeted wound disinfection applications [17][18][19], and one study examined sepsis management [20]. These four studies consistently showed that the synthesized SACs demonstrated superior antibacterial activity without, or at least very little, toxicity. In comparison to Neu, and urea, in organs and blood, when compared with those of the PBS-treated control group. Additionally, they found that all the blood indexes of lipopolysaccharide (LPS)-induced sepsis mice in the Co/PMCS-treated group were reduced to normal. In other words, Co/PMCS could significantly eliminate the systematic production of reactive oxygen and nitrogen species (RONS) and lower proinflammatory cytokine levels compared with those of the PBS-treated group, thereby indicating that Co/PMCS could promote the recovery of multiple organ functions in sepsis mice ( Figure 3) [20].

Other Applications of SACs
SACs were also applied for the noninvasive treatment of neurotrauma (Table 3). Yan and coworkers synthesized single-atom Pt/CeO 2 as a therapeutic agent with good biocompatibility to treat brain trauma. It was reported that Pt/CeO 2 could significantly decrease inflammatory factors such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in vitro. Moreover, it could significantly reduce the wound size and area and the expression of inflammatory cytokines and excess peroxidation, while recovering the superoxide dismutase SOD) and MMP-9 levels and activating astrocytes and microglia; these actions lead to a decrease in the overall neuroinflammation in traumatic brain injury (TBI) mice compared with that in the untreated group ( Figure 4) [25].  Two other studies reported that SACs could efficiently protect HeLa cells from damage by cellular oxidative stress by scavenging intracellular ROS, thereby providing an opportunity for an ROS-related disease treatment (Table 3) [15,22]. It was indicated that both the synthesized single atom catalysts (Fe-N/C SACs) and N-doped porous carbon (Fe-SAs/NC) exhibited a higher ability to remove excess ROS that were generated from cells under oxidative stress compared with that of the control group (Figure 4).

Discussion
This is the first systematic review attempting to collate all empirical evidence on the effect of SACs in biotherapy applications. All of the included studies indicated that SACs had been successfully introduced in biotherapy fields. The results of this systematic review suggested that various types of SACs were synthesized, which exhibited exceptional performance in antitumor, wound disinfection, anti-inflammation, antibacterial, trauma therapy, and oxidative-stress cytoprotection applications. In addition, the vast majority of the synthesized SACs (90%) exhibited satisfied biocompatibility. These results indicated that SACs could be a promising candidate for therapeutic applications. The characterization of SACs, along with their therapeutic effects, mechanisms, limitations, and prospects are discussed below.

Formatting of Mathematical Components
SACs have received a great deal of attention since they offer unique advantages in regard to their high activity and selectivity for various catalytic reactions. Isolated single atoms act as the centers of the catalytically active sites of SACs [28][29][30]. It was reported that single-atom dispersion is a very important factor that contributes to the great performance of SACs when used in a wide variety of practical applications [30,31]. Nevertheless, SACs could not be clearly characterized owing to the limits of instrument resolution in the last few years. Until recently, the rapid development of characterization techniques, including STM, HAADF-STEM, EXAFS, and XANES, have been applied to precisely characterize SACs [29,32]. In the present review, HAADF-STEM and/or STEM were used to identify and confirm the single atoms of the synthesized SACs in all included studies. In addition, EXAFS or XANES were applied to elucidate more detailed information regarding the binding mode, coordination environment, and oxidation states of the single atom. It is worth mentioning that any identified studies that lacked the identification of their synthesized SACs were not included in this review. For instance, a study investigating the effect of an Fe-N-C artificial enzyme in a cancer treatment was excluded because the authors only suggested that Fe was atomically dispersed in Fe-N-C without key characterization evidence [33].

Structures of the SACs
In the present review, we found that several types of SACs were developed for biomedical applications due to the structural similarity between natural enzymes and SACs. For these SACs, both noble metals (Pt, Ru, and Au) and non-noble metals (Fe, Co, Cu, and Zn) were used as single atoms. Among them, an Fe single-atom catalyst was the most commonly used single atom in the included studies. Iron is essential for most life on the planet, playing a crucial role in cellular metabolism and ROS production. Therefore, Fe-containing catalysts are generally regarded as one of most promising candidates for the production of abundant toxic •OH radicals.
Apart from the single atom, the support material, which serves as the ligands of the active metal centers, is another crucial factor for the catalytic performance of SACs. Furthermore, the support could stabilize the single-metal atoms and actively participate in catalytic reactions [25]. In the present review, several types of supports were synthesized, including, carbon-based materials, MOFs, Mn 3 [Co (CN 6 ) 6 ] 2 , and CeO 2 . It was noted that carbon-based materials were one of the most commonly used supports of synthesized SACs, similar to SACs in the field of energy, chemical, environmental, and industrial fields. In other words, SACs supported by carbon exhibited excellent promise in the catalysis field, which is mainly due to their specific features, including but not limited to their superb chemical and mechanical dependability, good electrical and thermal conductivity, variable structural and morphological combinations, tunable porosity and surface properties, high specific surface area, easy handling, and low production cost.
Typically, the supports of SACs are divided into two categories: 2D and 3D supports. The overwhelming majority of the studies (10 out of 12) synthesized 2D-supported SACs, and the single-atom loading ratios varied from 0.25 to 3.8 wt.%, which were higher than the carbon sphere support (0.18%) but much less than the 3D carbon-dot support (15.3 wt.%) in one study. However, Zhang and coworkers, who first introduced SACs, reported that 2D supports provided a better nanoplatform than their 3D-structured counterparts in regard to strengthening the metal-support interactions via chemical modifications [32]. The interactions between single metals and supports determine the loading ratio of a single metal and the performance of catalysts, thereby playing a vital role in stabilizing single-metal atoms and increasing activity and selectivity for the development of high-loading of SACs. In our review, one study was related to the 3D-supported SACs. In addition, different supports could offer different anchoring sites to stabilize single-atom metals. Unfortunately, it is unreasonable to conclude whether 3D-supported SACs are better for the biotherapy applications. In future studies, SACs with high metal-loading ratios on appropriate supports with outstanding stability, activity, and selectivity need to be developed for biotherapy applications.

Biotherapeutic Effect of SACs
In line with the superb performance in the energy, chemical, environmental, and industrial fields, all of the included studies in the present review consistently indicated that the synthesized SACs had superior therapeutic effects on several medical diseases and conditions when compared with that of the control. SACs have been applied for treating cancer (liver and breast tumors), infection wounds, sepsis and bacteremia, and brain trauma. In addition, SACs have also been used for oxidative-stress cytoprotection. It was suggested that SACs have great potential to become the next generation approach for the treatment of various medical disorders. However, there is currently a very limited number of studies published on the biomedical applications of SACs. In other words, no more than four published studies employed SACs for treating each type of the aforementioned medical condition. This might be because biotherapy application is a recent application of SACs. Therefore, additional studies that target various medical disorders with methodological rigor are needed to confirm the excellent performance of SACs in medical applications.

Mechanism of Biotherapy Applications Biotherapeutic Effect of SACs
To guide the rational design of high-performance SACs, the underlying therapeutic mechanisms of SACs on wound disinfection, cancer treatment, brain trauma therapy, and oxidative stress cytoprotection have been comprehensively elucidated in all the included studies. In general, reactive oxygen species (ROS) are chemically reactive chemical species containing oxygen, such as superoxide (O 2 − ), singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ), nitric oxide (NO), and highly reactive hydroxyl and monoxide radicals ( • OH, NO • ), and peroxynitrite (ONOO − ), and these ROS play an essential role in the therapeutic applications of SACs. It was suggested that ROS could react with large amounts of biomolecules, including lipids, proteins, RNA, and DNA. In regard to cancer treatments, the synthesized SACs, including PSAF NCs, Au/CDs, P-MOF, OxgeMCC-r SAE, and Cu-HNCS, displayed excellent oxidase-like and/or peroxidase-like activities in acidic environments. The H 2 O 2 molecule was adsorbed on the single-metal active sites in the SACs, and then the activated H 2 O 2 molecules (H 2 O 2 * ) decomposed into 2OH * via the single-metal catalysts, which decreased the reaction energy barrier of homolysis (showed a lower reaction energy barrier than that of heterolysis). Interestingly, the bioinspired Cu-HNCS could concurrently catalyze the decomposition of both O 2 and H 2 O 2 to O 2 •− and • OH, respectively, thus leading to a satisfactory efficacy of this cancer therapy. Similarly, the mechanism of synthesized SACs, such as FeN 5 SA/CNF, PMCS SA, and SAF NCs for anti-infection, was to produce ROS during the catalytic reduction of oxygen, and these ROS were able to seriously impair the membrane integrity of bacteria to enhance antibacterial efficiency. In contrast, the mechanism of single-atom Pt/CeO 2 for the brain trauma treatment was to scavenge RONS, exhibiting multienzyme activities such as peroxidase (POD)-like, catalase (CAT)-like, and oxide (OXD)-like catalytic activities. Similarly, the mechanism of synthesized SACs, such as Fe-N/C SACs, and Fe-SAs/NC for oxidative-stress cytoprotection, was to eliminate ROS and excess H 2 O 2 .
During these biotherapy applications, single-metal atoms commonly behaved as heterogeneous catalysts and influenced the direction and activity of chemical reactions, thereby serving as excellent functional biomimetic enzymes and regulating the generation or elimination of ROS in various microenvironments.

Limitations and Perspectives
Despite their exciting and encouraging beginning, the biotherapy applications of SAC is still in its infancy. Several issues must be addressed to obtain an in-depth understanding of single-atom catalysis and eventually achieve the rational design of SACs for specific therapeutic applications. In the future, precisely controlling the metal-loading ratio and modifying the active sites of SACs will minimize the immune defense and improve the in vivo/in vitro catalytic efficiency. In addition, the biocompatibility of SACs is still a major obstruction for their biological applications. Despite that 11 out of 12 included studies reported that SACs exhibited satisfactory biocompatibilities in the biotherapy applications, the mechanisms of the biocompatibilities were not investigated and discussed. Furthermore, long-term biocompatibility evaluation of the SACs in the biotherapy field has not been explored. Theoretically, SACs might lead to prolonged blood circulation time and undesirable immune responses since SACs have a relatively larger size and higher stability than biomolecules. Therefore, more efforts are needed to regulate the size of SACs for obtaining more promising biotherapy applications with outstanding biocompatibility in the future. To date, only Fe-, Zn-, Co-, Pt-, Au-, and Ru-based SACs have been verified in the biotherapy applications; interestingly, other noble metal-based SACs, such as Pd, Ag, have yet to be evaluated. The development of other supports, such as metallic oxides and polymers, should also be further investigated.

Conclusions
The biotherapy application of SACs is a prospective research frontier. The included studies indicated that the synthesized SACs were effective in anticancer therapy, anti-infection (antibacterial and anti-inflammation) therapy, brain trauma therapy, and oxidative-stress cytoprotection. Furthermore, it is anticipated that remarkable improvements can be made in designing highly efficient SACs for various biotherapy applications and to achieve the desired goal of facilitating the clinical translation of SACs.