Ruifang Han,Yu Xiao,Qianqian Bai,Chung Hang Jonathan Choi
Self-therapeutic metal-based nanoparticles for treating inflammatory diseases
Metal-based nanoparticles,Inflammatory disease,Self-therapeutic,Nanomedicine,Delivery,Gold nanoparticles,Clinical translation,Reactive oxygen species
Inflammatory diseases are key contributors to high mortality globally and adversely affect the quality of life. Current treatments include corticosteroids or nonsteroidal anti-inflammatories that may cause systemic toxicity and biologics that may increase the risk of infection. Composite nanoparticles that bear not only the drug payload but also targeting ligands for delivery to inflammation sites at lowered systemic toxicity are established in the nanomedicine field, but their relatively large size often leads to systemic clearance. Metal-based nanoparticles with intrinsic anti-inflammatory properties represent attractive alternatives. They are not only designed to be compact for crossing biological barriers (with the nanoparticle serving as a dual carrier and drug), but also support label-free tracking of their interactions with cells. The review commences with an outline of the common inflammatory diseases, inflammatory pathways involved, and conventional drug-loaded nanoparticles for anti-inflammation. Next, the review features the emerging applications of self-therapeutic metal-based nanoparticles (e.g., gold, coper oxide, platinum, ceria, and zinc oxide) for managing inflammatory diseases in animals over the past three years, focusing on therapeutic outcomes and anti-inflammatory mechanisms. The review concludes with an outlook on the biodistribution, long-term toxicity, and clinical translation of self-therapeutic metal-based nanoparticles.
Self-therapeutic metal-based nanoparticles for treating inflammatory diseases

Inflammatory diseases are key contributors to high mortality globally and adversely affect the quality of life. Current treatments include corticosteroids or nonsteroidal anti-inflammatories that may cause systemic toxicity and biologics that may increase the risk of infection. Composite nanoparticles that bear not only the drug payload but also targeting ligands for delivery to inflammation sites at lowered systemic toxicity are established in the nanomedicine field, but their relatively large size often leads to systemic clearance. Metal-based nanoparticles with intrinsic anti-inflammatory properties represent attractive alternatives. They are not only designed to be compact for crossing biological barriers (with the nanoparticle serving as a dual carrier and drug), but also support label-free tracking of their interactions with cells. The review commences with an outline of the common inflammatory diseases, inflammatory pathways involved, and conventional drug-loaded nanoparticles for anti-inflammation. Next, the review features the emerging applications of self-therapeutic metal-based nanoparticles (e.g., gold, coper oxide, platinum, ceria, and zinc oxide) for managing inflammatory diseases in animals over the past three years, focusing on therapeutic outcomes and anti-inflammatory mechanisms. The review concludes with an outlook on the biodistribution, long-term toxicity, and clinical translation of self-therapeutic metal-based nanoparticles.

Inflammation is characterized by the activation of immune and non-immune cells in response to damage to internal organs (e.g., infection, imbalanced hormone, and dysfunction) or external stimuli (e.g., pathogenic microbes or foreign particles invasion). At the cellular level, the key feature of inflammation is the accumulation of phagocytic cells that engulf and digest foreign bacteria and particles (e.g., neutrophils),. At the tissue level, inflammation is usually associated with redness, swelling, heat and pain, all pathological symptoms that arise from local immune response, vascular inflammation, and other responses to infection or injury. Important microcirculatory events that occur during inflammation increase vascular permeability, leukocytes recruitment and accumulation, and release of inflammatory mediators. Inflammation can be classified into two types: acute inflammation and chronic inflammation. Acute inflammation spans a shorter duration from minutes to hours or to days, depending on the extent of injury. Initially confined to a specific area and arisen due to an inability to rapidly eliminate foreign pathogens, acute inflammation may evolve into autoimmune disorders that attack other normal and healthy organs and subsequently cause chronic inflammation. Examples of acute inflammation include acute pancreatitis, acute kidney injury, and acute asthmatic inflammation. Chronic inflammation entails a longer duration, say months or years. Persistent exposure to any remaining foreign pathogens or bodies that cannot be fully degraded or cleared from the body during acute inflammation stage may trigger chronic inflammation. Examples of chronic inflammation include atherosclerosis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis (Fig. 1A).

Oxidative stress arises from the excessive generation of reactive oxygen species (ROS) and their inefficient clearance from the body. ROS may cause irreversible damage to the fundamental biomolecular building blocks of life (proteins, lipids, and carbohydrates), leading to inflammation. Initiation of oxidation may originate from the light- or heat-induced cleavage of chemical bonds or electron transfer between transition metals and hydroperoxides, yielding radicals [e.g., hydroxyl (), alkyl (), alkoxyl (), hydroperoxyl ()]. Oxidative stress not only can induce diverse inflammatory diseases such as diabetes, asthma, and arthritis, but also can promote multiple inflammatory pathways, including nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) (discussed below).

The Janus kinase/signal transduction and activator of transcription (JAK-STAT) signaling pathway transmits signals received from extracellular receptors to the cell nucleus and is associated with the pathogenesis of inflammatory and autoimmune diseases including RA, psoriasis, and IBD. Many inflammation-associated cytokines, such as interferons and interleukins, employ the JAK-STAT signaling cascade to transduce intracellular signals upon extracellular ligand binding. There are four members of the JAK pathway (JAK1, JAK2, JAK3, and TYK2) and seven members of STAT (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6). Upon the phosphorylation of JAKs, the intracellular receptors (which are docking sites of STAT transcription factors) are also subsequently phosphorylated. Then, the activated STATs translocate to the cell nucleus where they serve as transcription factors to modulate gene expression. An important cytokine that activates JAK-STAT signaling is the interleukin (IL)-6, a key player in multiple diseases of chronic inflammation.

The NF-κB signaling pathway is related to various processes of inflammation, immune response, survival, and apoptosis. The NF-κB family consists of five members, including NF-κB1 (or p50), NF-κB2 (or p52), RelA (or p65), RelB and c-Rel. Activatable by various biochemical stimuli (e.g., pathogen-derived substances, intercellular inflammatory cytokines, and enzymes,), the NF-κB pathway centrally mediates pro-inflammatory processes by regulating the expression of diverse target genes (e.g., cytokines, chemokine, growth factors) to protect the host from infection or tissue damage. However, dysregulation of the NF-κB pathway may cause acute or chronic inflammation disorders such as IBD, asthma, RA, multiple sclerosis, and atherosclerosis,.

The MAPKs signaling pathway involves a family of threonine (Thr) and tyrosine (Tyr) protein kinases that mediate key cellular processes, such as cell survival and apoptosis, proliferation, differentiation, cellular stress, and inflammation. The MAPK family consists of three members, namely extracellular signal-regulated kinases (ERKs), stress-activated protein kinases (SAPKs), and p38. Each MAPK pathway axis has three modules, namely mitogen-activated protein kinase kinase kinase (MAPKKK or MAP3K), mitogen-activated protein kinase kinase (MAPKK or MAP2K), and MAPK. Activation of MAPK occurs upon the association of a protein or peptide with the substrate pocket of p38 MAPK, followed by binding of ATP to the ATP pocket. Once activated, MAPKs phosphorylate and initiate kinases or nuclear proteins (e.g., transcription factor). Pro-inflammatory cytokines [e.g., tumor necrosis factor (TNF)-α and IL-1β] and cellular stresses (e.g., osmotic, hypoxic, and oxidative stress) trigger the SAPKs and p38 signalling cascades, ultimately leading to inflammatory disorders including RA, asthma,, IBD,, and stroke.

Different types of anti-inflammatory therapies are available. Chronic diseases (e.g., IBD, RA, and psoriasis) are conventionally treated with (1) corticosteroids (e.g., prednisolone) that block inflammatory pathways mainly by reversing histone acetylation of activated inflammatory genes, (2) non-steroidal anti-inflammatory drugs (NSAIDs, e.g., aspirin) that inhibit inflammation-related enzymes, or (3) broad immunosuppressants [e.g., methotrexate (MTX)]. Acute diseases (e.g., trauma and acute airway inflammation) are often treated with high dosages of anti-inflammatory drugs. However, corticosteroids can increase the risk of infection while NSAIDs may adversely affect gastric mucosa, renal system, and hematologic system. Biologics that block specific gene targets (e.g., inhibitors of TNF-α) are emerging treatments for inflammatory diseases over the past two decades, thanks to our enhanced understanding in the immunological pathways and processes that underpin the pathogenesis of diseases. For instance, antibodies against TNF-α (anti-TNF-α), anti-IL-17A, anti-IL-12/23p40, anti-phosphodiesterase (PDE)-4, and anti-JAK are effective in treating psoriasis because such therapeutic targets are related to the development of psoriasis. Despite improved efficacy when compared to conventional treatments, the high cost (roughly USD 1000 per injection) and compromise to the immune system hamper the widespread application of biologics.

Over the past two decades, NPs (e.g., liposomes, lipid NPs, metallic NPs, polymeric NPs, and polymer micelles) are garnering attention for treating inflammatory diseases (Fig. 1B, blue curve), because they can be engineered for delivery to different types of biological destinations, thereby addressing the issues of rapid renal clearance and systemic toxicity that are associated with conventional therapies. Liposomes or lipid NPs are mainstream carriers (Fig. 1B, red curve) for delivering a broad range of anti-inflammatory chemical and biological drugs, such as prednisolone, aspirin, MTX, peptides, siRNA, and miRNA. Yet, when the drug payload, targeting ligand, and carrier lipids are all encapsulated, the resultant lipid NPs often have a large size (>100 nm) that disfavour penetration through biological barriers and delivery to the target site. For instance, NPs >15 nm have a low chance of crossing the stratum corneum (SC) to reach skin epidermal cells upon topical application or crossing the blood–brain barrier to reach the brain neurons upon intravenous (I.V.) application. Larger NPs (say >50 nm) are prone to clearance by the liver and spleen upon I.V. injection. Another disadvantage of lipid-based NP carriers is that, without modification by fluorescent dyes or heavy-metal ions, it is technically challenging to assess their mechanism of action and derive insights into their safety profile by elucidating their in vivo interactions with tissues and cells, including distribution, metabolism, and clearance profiles. Metal-based NPs are emerging nanocarriers for anti-inflamm-atory applications (Fig. 1B, red curve). From a drug delivery standpoint, the tunable size and morphology of metal NPs offer the flexibility to optimize a NP design that (1) bypasses different physiological barriers, (2) is compatible with the extracellular environment, and (3) enters defined cell types. From an in vivo imaging standpoint, metal NPs possess unique physical properties (e.g., optical, electrical, acoustic, and magnetic) that support label-free tracking of their distribution by various modalities, such as computed tomography and magnetic resonance imaging. From an ex vivo analysis standpoint, metal NPs are amenable for quantifying their organ-level distribution by inductively coupled plasma mass spectrometry (ICP-MS), visualizing their tissue-level distribution by label-free confocal reflectance imaging, and assessing their cellular or organellar-level distribution by transmission electron microscopy (TEM). Thus, metal-based NPs are attractive platforms for elucidating bio-nano interactions. Notably, some metal-based NPs exhibit self-therapeutic efficacy for treating inflammatory diseases, such as gold NPs, platinum NPs, copper oxide NPs, zinc oxide NPs, and ceria NPs. For example, they can inhibit or slow down oxidation in the inflamed tissues or cells by either trapping free radicals or by reducing the initiation of oxidation. Interestingly, the metal NP core not only serves as a drug carrier but also a therapeutic agent for inflammatory diseases, without the need for loading additional anti-inflammatory drugs or applying an external physical stimulus (e.g., heat, light, and magnetic field). Self-therapeutic anti-inflammatory metal-based NPs have a pharmacologically simple design; contrary to traditional drug-loaded NPs, issues of drug encapsulation and drug release are irrelevant for self-therapeutic NPs. Also, self-therapeutic NPs are potentially cheaper and more clinically translatable than traditional drug-loaded NPs because they do not require the incorporation of extra chemical compounds or biological agents that may complicate large-scale preparation and purification. In this review, we define “self-therapeutic metal-based NPs” as metal-based NPs that can treat or alleviate a disease through their intrinsic materials property, without the need for loading additional therapeutic compounds or biologics and for applying an external physical stimulus. Self-therapeutic metal-based NPs are not materially different than conventional metal-based NPs, but the idea of using their intrinsic anti-inflammatory property for treating inflammatory diseases represents an emerging direction in the field of nanomedicine. Here, we opt for reviewing the preclinical advances on using self-therapeutic metal-based NPs for treating inflammatory diseases over the past three years. Specifically, we include metal-based NPs (e.g., citrated-capped gold NPs) that act as the primary anti-inflammatory therapeutic agent but do not contain known chemical or biological drugs. Note that the self-therapeutic metal-based NPs may also contain an outer coating [e.g., polyethylene glycol (PEG) and dextran] for biocompatibility and colloidal stability in biological medium and/or targeting ligands (e.g., peptides or proteins) for specific cellular delivery, provided that these coating or ligands do not exhibit known anti-inflammatory efficacy. We exclude metal-based NPs that do not play an anti-inflammatory role, for instance NPs that are purely employed for elucidating basic bio-nano interactions, NPs that only serve as carriers of anti-inflammatory agents, NPs that merely act as imaging or contrast agents, and NPs that require an external physical force for treatment (e.g., gold NPs for photothermal therapy and photodynamic therapy). For each type of metal-based NP that meets the above inclusion and exclusion criteria, we delineate its chemical design, the animal models and routes of administration used, the major therapeutic outcomes, and the anti-inflammatory pathways involved. Lastly, we offer our perspectives on the clinical outlook of metal-based NPs as a self-therapeutic treatment. Publications on self-therapeutic metal-based NPs for treating inflammatory diseases from 1/2019-3/2022 are listed in Table 1. Anti-inflammatory mechanisms of self-therapeutic metal-based nanoparticles are listed in Table 2.

Gold (Au) NPs are widely used to examine bio-nano interactions, drug delivery, and photothermal therapy, and interestingly, recent preclinical evidence points to their anti-inflammatory properties. It was reported that AuNPs downregulated the inflammatory response triggered by IL-1β in vitro and in vivo. Furthermore, AuNPs were shown to selectively reduce the production of ROS and some pro-inflammatory cytokines, such as IL-1β, IL-17, and TNF-α, in lipopolysaccharide (LPS)-induced splenocytes. In addition, AuNPs can downregulate cytokines, oxidative stress and fibrosis by modulating the AKT/PI3K (protein kinase B/phosphatidylinositol-3-kinase) and MAPK signaling pathways in a rat model of alcohol-methamphetamine (METH)-induced liver injury. In the past three years, a few studies have further confirmed the role of AuNPs as potential anti-inflammatory agents that suppress inflammatory pathways. In 2019, Dos Santos Haupenthal et al. reported the effects of AuNPs on a rat model of acute pulmonary inflammation induced by LPS. At 12 h after a single intraperitoneal (I.P.) administration of LPS, the rat was treated daily with 20 nm citrate-capped AuNPs (2.5 mg/kg) via I.P. injection for 7 days (1 dose/day). Before efficacy assessment, the researchers evaluated the acute toxicity of gold NPs following the treatment regimen (i.e., 2.5 mg/kg per dose for 7 doses). The results showed that AuNPs did not cause significant changes in the plasma levels of damage and toxicity markers, such as lactate dehydrogenase, ALT/glutamic-pyruvic transaminase, and AST/glutamic-oxaloa-cetic transaminase, indicating that the dose was safe for in vivo administration. Efficacy was evaluated, and the authors found that treatment with AuNPs reversed LPS-induced inflammation with respect to total peritoneal leukocyte count, pulmonary levels of pro-inflammatory cytokines (including interferon-γ, TNF-α, IL-1ꞵ, and IL-6) and alveolar septum fibrosis. In addition, AuNPs decreased the production of oxidants (nitrite and dichlorodihydrofluorescein), reduced oxidative damage (carbonyl and sulfhydryl), and downregulated the activities of superoxide dismutase (SOD) and catalase (CAT). In this study, the authors suggested that AuNPs exerted anti-inflammatory effects due to the suppression of NF-κB [possibly by inhibiting the translocation of NF-κB to the nucleus due to reduction in the phosphorylation of inhibitor of nuclear factor kappa B kinase subunit (IKK)-β]. However, the authors did not explain why they used 20 nm AuNPs, how the AuNPs were transported to the lung, or how AuNPs function in vivo. In addition, the researchers did not study the in vivo distribution, clearance and long-term toxicity of AuNPs. These concerns hamper the translational potential of citrate-capped AuNPs for treating acute liver injury (ALI), even though the AuNPs demonstrated effectiveness in an LPS-induced disease model. Other researchers showed that I.P.-injected gold NPs accumulate in different organs in a size-dependent manner. By injecting citrate-capped AuNPs of 10, 30, or 60 nm in diameter into Wistar rats over 8 days (∼0.2 mg/kg per day), Lopez-Chaves et al. observed a strongly positive correlation between NP size and spleen accumulation but a strongly negative correlation between NP size and kidney accumulation. The route of excretion was also size-dependent; 10 and 30 nm NPs were excreted via feces, whereas 60 nm NPs mostly undergo splenic clearance. Finally, the authors did not observe a significant inflammatory response or change in tissue morphology upon I.P. injection of gold NPs. Recently, we reported a three-component, self-therapeutic AuNP (Fig. 2A) for treating psoriasis in imiquimod (IMQ)-induced mice, which is an established model of psoriasis. Our design contained a gold NP core that allowed for label-free imaging and quantification of intradermal distribution. The second component was a shell of polyethylene glycol (PEG) strands for colloidally stabilizing the Au core upon exposure to skin. The third component was a collection of alkyl chains to boost topical delivery to epidermal cells. To elucidate the effects of NP size and alkylation on in vivo delivery, we coated the citrate-capped AuNPs with a core diameter of x (x = 3, 6, or 13 nm) with a mixture of y mol% (y = 0, 10, or 30) of HS-PEG1000-alkyl (hexyl, dodecyl, or octadecyl) strands and (100-y) mol% of HS-PEG1000-methoxy strands (Fig. 2A). Next, we topically applied NPs with various Au core sizes and alkyl contents onto the skin of mice, followed by using ICP-MS to quantify the amounts of gold NPs in various skin layers, TEM imaging to track the intracellular distribution (Supporting Information Fig. S1A and S1B), and label-free confocal reflectance imaging to track the intradermal distribution of alkylated gold NPs (Fig. S1C). Based on our data, we concluded that an overall NP size <15 nm (3 nm Au core coated with 30 mol% of octadecyl-terminated PEG chains) is optimal for topical delivery to the epidermis of psoriatic skin. Of note, unalkylated PEG-coated Au cores of 13 nm are larger than 20 nm, thereby unable to permeate the SC. Also, PEG-coated Au cores of 3 nm coated with >30 mol% octadecyl chains tend to mildly aggregate to form ∼35 nm clusters due to hydrophobic interactions, also not effective for skin penetration. In short, alkylated gold NPs could cross the SC to enter epidermal cells after topical application. Surprisingly, even though Au3@PEG1000-octadecyl30% NPs were devoid of known chemical and biological drugs, applying these NPs concurrently with IMQ for six days (prevention mode: apply NPs during psoriasis development; 0.2 mg per dose over 6 doses) prevented the development of psoriasis. Histological analysis showed that Au3@PEG1000-octadecyl30% NPs significantly reduced the thickness of the epidermis, keratinocyte hyperproliferation, and T cell infiltration. In addition, the concentrations of psoriasis-related cytokines, such as IL-17, IL-12/23p40, and IL-1ꞵ, were also significantly decreased by AuNP treatment. To understand the antipsoriatic mechanism of alkylated AuNPs, we used RNA-sequencing (RNA-seq) to profile changes in the genes in treated skin. Gene ontology (GO) analysis showed that Au3@PEG1000-octadecyl30% NPs significantly modulated inflammation and immune-related biological processes (Fig. 2B). The inhibited genes (Fig. 2C) included (i) cytokines, that are released during psoriasis pathogenesis (e.g., 65-fold reduction in expression of Il12b, 11-fold reduction in Il17f, and 6-fold reduction in Il1b), (ii) chemokines that are secreted by keratinocytes to guide the infiltrating leukocytes to the inflammation site (e.g., 7-fold reduction in expression of Ccl4, 28-fold reduction in Ccl3, and 2-fold reduction in Ccl20), and other related genes. These DEGs are significantly enriched in the downstream of the IL-17 signaling pathway (Supporting Information Fig. S2), the downstream of the TNF signaling pathway (Supporting Information Fig. S3), the cytokine-cytokine receptor interactions signaling pathway (Supporting Information Fig. S4), the NF-κB signaling pathway (Supporting Information Fig. S5), and MAPK signaling pathway (Supporting Information Fig. S6). These data suggest that our alkylated NPs may reduce psoriasis by blocking the communication between different inflammatory cells. Furthermore, our NPs treated psoriasis (treatment mode: apply NPs after psoriasis development; 0.2 mg per dose over 4 doses) as effectively as standard betamethasone-calcipotriol therapy but with significantly less skin wrinkling and hair loss (Fig. 2D). Both betamethasone (which is a type of steroid) and calcipotriol (a vitamin D analog) are standard therapies for psoriasis. Finally, we also evaluated the long-term toxicity of the AuNPs after treatment. Our results showed that after four weeks, the NPs did not remain in major internal organs or induce severe cutaneous or systemic toxicity. The present NPs not only represent a simple, safe, and effective therapy for psoriasis but also show great potential for other inflammatory skin diseases, such as dermatitis. We did not investigate the effect of NP size on efficacy because only <15 nm NPs managed to penetrate the skin. Other researchers studied the effect of NP size on the efficacy of gold NPs. Upon intraarticular injection of 5, 25, or 75 nm PEG-coated gold NPs into rats with collagen-induced arthritis, Abdel-Hakem et al. found that the 25 nm gold NPs exhibited the most favorable therapeutic outcomes, as evidenced by significantly reduced joint swelling and inflammatory cellular infiltration, decreased bone and cartilage erosion, and prevention of pannus formation. While injection of differently sized gold NPs did not significantly change tissue morphology, injection of only 75 nm NPs led to similar adverse effects to those induced by MTX treatment (as evidenced by significantly higher aspartate transferase (AST) and lower creatinine blood levels).

Copper (Cu) is an essential trace element in humans that plays an important role in many enzymes, such as tyrosinase and Cu–Zn superoxide dismutase. Copper has three oxidation states: Cu(0) (solid metal state), Cu(I) (cuprous ion), and Cu(II) (cupric ion). The different oxidation states of copper are used to design NPs with different properties. Cu NPs have excellent catalytic activity to scavenge H2O2 and O2− due to the strong quantum confinement of electrons in the ultrasmall size regime. Cuprous oxide (Cu2O) NPs possess good catalytic activity and can promote electron transfer reactions to inactivate H2O2 or OH·. Massive ROS production initiates many pathological signaling pathways, especially the NF-κB pathway, resulting in increased production of pro-inflammatory factors such as IL-8 and monocyte chemoattractant protein-1 (MCP-1). By scavenging ROS, Cu-based NPs further inhibit the secretion of pro-inflammatory factors (MCP-1 and IL-8) and inflammatory cell infiltration. Therefore, Cu-based NPs have been designed to treat ROS-induced inflammation. Liu et al. reported a Cu-based NP to scavenge reactive oxygen species and alleviate inflammation-related diseases (Fig. 3). The researchers synthesized Cu-based NPs with a mixture of Cu and Cu2O (3.4:1), which was denoted Cu5.4O NPs (Fig. 3A). The hydrodynamic size of the NP was 4.5 nm, which not only boosted the catalytic efficiency due to the large surface-to-volume ratio but also allowed rapid renal clearance because the NPs could pass through the glomerular filtration barrier. Impressively, these properties of Cu5.4O NPs led to excellent therapeutic effects on three ROS-related pathological conditions, including glycerol-induced acute kidney injury (AKI), acetaminophen-induced acute liver injury (ALI), and diabetic wound healing (Fig. 3B). The authors verified that Cu5.4O NPs could effectively scavenge a broad spectrum of ROS, including H2O2, O2− and , and could functionally mimic many enzymes, including CAT, SOD, and glutathione peroxidase (GPx), in vitro (Fig. 3C). In vivo, the authors performed a safety assessment of Cu5.4O NPs in healthy mice. The results showed that a single injection of Cu5.4O NPs at a 10-fold higher concentration (20 μg/kg) than that used in AKI treatment (2 μg/kg) did not cause significant hemolysis (hemolysis rate <5%). Furthermore, after a single injection of a 2-fold higher concentration (4 μg/kg) than that used in AKI treatment, there was no noticeable tissue damage and no significant change in the levels of serum cytokines (e.g., IL-6, TNF-α) or blood biochemistry markers (e.g., AST, ALT, BUN, and CRE). After repeated daily administration for seven consecutive days, Cu5.4O NPs mainly accumulated in the liver (7.92% ID/g) and kidney (7.97% ID/g), and no obvious toxicity was observed. Pharmacokinetics and biodistribution analysis showed that 70% of injected Cu5.4O NPs could be excreted within 48 h post injection (60% through the kidney and 10% through the intestine). The low working concentration and relatively rapid renal clearance of Cu5.4O NPs suggested their low long-term toxicity. The therapeutic efficacy of Cu5.4O NPs was first tested in mice with glycerol-induced AKI. A single injection of Cu5.4O NPs (2 μg/kg) significantly improved the survival and kidney function of AKI mice, as indicated by the normal levels of BUN and CRE. Such efficacy was similar to that in AKI mice treated with an antioxidant drug (160 mg/kg N-acetyl cysteine). At the molecular level, Cu5.4O NPs significantly reduced SOD activity and the expression of two important kidney injury markers [heme oxygenase-1 (HO-1) and kidney injury molecule-1 (KIM-1)]. RNA-seq analysis revealed that the MARK and TNF signaling pathways were significantly inhibited by Cu5.4O NPs, while several important antioxidant genes were upregulated. These data indicated that Cu5.4O NPs could protect kidney tissues from oxidative stress by inhibiting the excessive production of pro-inflammatory mediators by decreasing ROS levels. Apart from the AKI model, Cu5.4O NPs also exhibited therapeutic efficacy in other ROS-related pathological conditions, including acetaminophen (APAP)-induced acute liver injury and full-thickness skin defects in chronic diabetic mice. Whereas the authors did not examine the distribution or toxicity of Cu5.4O NPs in these two disease models, the toxicity of copper oxide NPs appears to be size-dependent in vitro. Karlsson et al. reported that NPs are more cytotoxic than microparticles possibly because of the higher surface reactivity of NPs that contributes to more release of toxic Cu2+ ions. Wongrakpanich et al. reported that 24 nm CuO NPs were significantly more cytotoxic than 4 nm CuO NPs possibly because 24 nm NPs exhibit more abundant cellular entry that favors intracellular release of Cu2+ ions.

Platinum nanoparticles (PtNPs) can act as strong and stable mimetics of the enzymes SOD and CAT. They can catalyze the reduction of H2O2 to water and molecular oxygen and act in the same manner as the biological enzyme CAT or promote the oxidation of a reduced substrate to decompose H2O2 to water, similar to HRP mimics. PtNPs are also able to act like SOD, catalyzing the dismutation of O2 into molecular oxygen and H2O2. Based on their enzymatic properties, PtNPs have been demonstrated to effectively attenuate oxidative stress-induced inflammation. In LPS-induced RAW264.7 cells, PtNPs suppressed the phosphorylation of extra-cellular regulated kinase 1/2 (ERK1/2) and AKT and inhibited the phosphorylation/degradation of IκB-α, as well as NF-κB transcriptional activity. Apart from cell studies, PtNPs have also been reported to alleviate UV-light-induced epidermal inflammation, cigarette smoke-induced pulmonary inflammation, and neurological impairment in mice. Recently, Zhu et al. reported that PtNPs were a therapeutic agent against dextran sodium sulfate (DSS)-induced colitis in mice. The researchers assessed the therapeutic effect of citrate-capped spherical PtNPs with physical diameters of 5, 30 and 70 nm against DSS-induced colitis in C57BL/6 mice; PtNPs were administered by oral gavage for 8 days during and after exposure to DSS (3% DSS in the drinking water for 5 days). On Day 8, PtNPs prevented inflammation in a size-dependent manner, and 5 nm PtNP-treated mice showed more significant inhibition of body weight loss, disease activity index (DAI), and tight junction protein (including zonula occludens-1, occludin, claudin-4, and heat shock protein 25) loss than mice treated with the larger compartments. In addition, PtNPs attenuated colonic and systemic inflammation caused by DSS, as indicated by reduced myeloperoxidase (marker of neutrophil infiltration) activity and inflammatory cytokine (IL-6 and TNF-α) levels in the colon and reduced cytokine (IL-6 and TNF-α) levels in serum; however, these anti-inflammatory activities exhibited no significant dependence on NP size. From their in vitro studies, the authors proposed that the anti-inflammatory mechanism of PtNPs involved intracellular reactive oxygen species scavenging and Toll-like receptor 4/NF-κB signaling suppression. However, the authors did not investigate the in vivo biodistribution or toxicity of orally administered PtNPs, nor did they explain why 5 nm PtNPs showed better efficacy than other sizes. Also, orally administered 5 nm PtNPs changed the gut microbiota profile, leading to gut dysbiosis. Therefore, probiotic or prebiotic supplementation may be needed in combination with PtNPs to avoid potential adverse effects of the NPs on the gut microbiota. Based on literature precedent, administration of PtNPs might lead to biochemical and morphological changes in different tissues. Adeyemi et al. reported that, in Wistrar rats, oral exposure of PtNPs (12–19 nm) at a daily dose of 10 mg/kg over 30 days significantly reduced organ weights, increased the ratio of low density lipoprotein to high density lipoprotein, and elevated the atherogenic index. Zhang et al. developed ultrasmall polyvinylpyrrolidone platinum NPs (Pt NPs-PVP) as multienzyme mimics and RONS scavengers to alleviate AKI (Fig. 4). The hydrodynamic diameter of Pt NPs-PVP (∼4.5 nm) was smaller than the renal filtration size threshold (∼5.5 nm); therefore, these NPs exhibit high renal accumulation and effective renal clearance. The authors showed the multienzyme mimetic effects [CAT, peroxidase (POD), and SOD] of their Pt NPs-PVP by demonstrating their excellent scavenging capacity toward RONS and that these NPs can protect cells from oxidative stress in vitro. ICP-MS measurements revealed that Pt NPs-PVP rapidly accumulated in the kidneys of both healthy and glycerol-induced AKI mice within 2 h of I.V. injection (Supporting Information Fig. S7B and S7C). The blood circulation half-life of the NPs is 2.27 h, indicating rapid blood elimination. By leveraging the strong NIR absorption of Pt NPs-PVP, the authors employed label-free photoacoustic computed tomography to track their in vivo distribution, showing accumulation in the kidney (Fig. S7D and S7E). Importantly, a single injection of Pt NPs-PVP (500 μg) into AKI mice significantly reduced the serum levels of BUN and CRE and the expression of KIM-1, HO-1, and 8-hydroxy-2-deoxyguanosine (8-OHdG; marker of DNA damage) in renal tissues. The same amount of Pt NPs-PVP (500 μg) more effectively treated acute kidney injury than amifostine (AMF), an antioxidant drug approved by the U.S. Food and Drug Administration (FDA) for preventing AKI (500 μg). Two weeks after I.V. injection of 1 mg of Pt NPs-PVP, there was no obvious body weight loss or tissue damage in major internal organs. The blood markers of kidney function (BUN and CRE) and liver function (ALT and AST) were in the normal range. Overall, Pt NPs-PVP are promising therapeutic agents.

Cerium oxide is a lanthanide metal oxide that is known for its antioxidative and ROS scavenging properties. Ceria nanoparticles (CeNPs) allow for rapid and reversible shuttling between the reduced Ce3+ state and the oxidized Ce4+ state. The CeNPs react with superoxide and hydrogen peroxide in a catalytic manner; smaller NP size and higher Ce3+ to Ce4+ ratio in CeNPs render them powerful antioxidants and ROS scavengers. Past researchers studied the biodistribution of CeNPs by injecting a high dose (50 mg/kg) of citrate-capped CeNPs of different diameters (5, 15, 30 nm) into Sprague–Dawley rats. A large proportion of I.V. and I.P. injected CeNPs passively accumulate in the liver and spleen. In a separate report, CeNPs of other sizes also predominantly accumulate in the liver,. Larger CeNPs are mostly cleared via feces, whereas smaller (<10 nm) NPs are mostly cleared via urine,. Jeong et al. presented a class of CeNPs that used 6-aminohexanoic acid (6-AHA) as a surface stabilizer and were further modified by PEG to improve their stability in biofluids, and these NPs had a hydrodynamic size of ∼38.5 ± 3.1 nm. In vitro experiments showed that 6-AHA-CeNPs successfully scavenged intracellular ROS and suppressed the expression of inducible nitric oxide synthase (iNOS) and IL-1β. The anti-inflammatory property of the 6-AHA-CeNPs was evaluated in a mouse model of systemic inflammatory response syndrome (SIRS), which occurs when local control of inflammation is lost and inflammation spreads throughout the body; 0.5 mg/kg 6-AHA-CeNPs were injected into mice through the tail vein. After injection, 11.2% 6-AHA-CeNPs had accumulated in the lung. This lung accumulation is higher than lung accumulation values obtained from past studies performed on healthy mice, possibly due to the enhanced lung vascular permeability induced by SIRS. Compared with diseased mice, the 6-AHA-CeNP-treated mice exhibited greatly reduced pulmonary histopathologic alterations and decreased neutrophils and active macrophage infiltration in the lungs. Further studies of survival rates and clinical outcomes indicated that 6-AHA-CeNPs showed better protective effect, including higher survival rate and improved clinical outcomes of survivors, than the untreated and ceria nanopowder-treated groups. Recently, Kim et al. reported a citric acid-coated ultrasmall CeNP that could mitigate acute hind paw inflammation. This CeNP had a hydrodynamic size of 3.4 ± 1.1 nm and a negative zeta potential (−19.5 ± 3.2 mV). The ROS scavenging and anti-inflammatory properties were verified by in vitro experiments, and the data showed that CeNPs prevented the increase in ROS levels in LPS-induced macrophages. In addition, the mRNA levels of pro-inflammatory cytokines (TNF-α and IL-1β) were significantly repressed by CeNPs treatment. The in vivo anti-inflammatory effect was evaluated in mice with acute hind paw inflammation. Upon I.V. injection, the biodistribution of CeNPs was investigated in the disease model at 24 h post-treatment. It is found that less than 16% of injected NP in the body after 24 h, indicating good clearance. Most CeNPs were retained in liver and spleen while small amounts of them were found in kidney, lung and the remaining carcass. Importantly, a considerable amount of CeNPs accumulated in the inflamed paw, which was ∼7-fold higher than the contralateral paw. To exam the efficacy, mice that were treated with a single tail vein injection of CeNPs (100 mg Ce/kg) 24 h after complete Freund's adjuvant (CFA) injection showed reductions in paw edema. On the other hand, CeNP treatment significantly reduced pain hypersensitivity, as indicated by the paw withdrawal latency study, and the mice that were treated with CeNPs fully recovered and returned to baseline levels 18 h post injection. Moreover, the authors showed that the anti-inflammatory effect of this CeNP was mediated by suppressing the secretion of pro-inflammatory cytokines (TNF-α, IL-1β and CD68), upregulating IL-10 expression, and inhibiting the migration of macrophages to inflammatory loci. Due to the efficient renal clearance of CeNPs from the body, histological examination of major organs did not reveal noticeable toxicity. Lastly, CeNPs are mostly biocompatible in vivo, as evidenced by similar levels of serum biomarkers for liver and kidney functions (e.g., AST, ALT, total bilirubin, bile acids, and BUN) between saline- and CeNPs-treated groups. A study of dual detoxicated ceria nanozymes (CeNZs) in 2020 by Li et al. showed that this CeNZ was effective for treating drug-induced liver injury (DILI) by not only scavenging ROS but also generating oxygen to inhibit pro-inflammatory macrophages (Fig. 5). CeNZs have a hydrodynamic size of ∼12 nm. Upon I.V. injection of CeNZs (1.5 mg/kg), in vivo biodistribution was examined and revealed that they primarily accumulated in the liver (∼20 ng Ce/mg tissue) and were taken up by hepatocytes and nonparenchymal cells. Genes related to oxidative stress [NADPH oxidase 2 (Nox2), cytochrome p450 family 2 subfamily e member 1 (Cyp2e1)] were found to be downregulated, while antioxidative genes [NAD(P)H quinone dehydrogenase 1 (Nqo1), glutathione peroxidase 1 (Gpx1), and HO-1] were upregulated, in liver cells isolated from CeNZs-injected mice, as shown by the quantitative reverse transcription results. Surprisingly, CeNZs significantly reduced the numbers of CD86+ pro-inflammatory macrophages, while no obvious difference was found in the proportions of anti-inflammatory CD206+ macrophages. In addition, pro-inflammatory cytokines, including IL-1β, TNF-α, and hypoxia inducible factor (HIF)-1α, were prominently decreased by CeNZs. Considering that most DILI patients are diagnosed during the late stage, the therapeutic effect of CeNZs against the late stage of DILI was also investigated and compared with the first-choice antidote N-acetylcysteine (NAC). Flow cytometry revealed that pro-inflammatory CD86+ macrophages in the CeNZs-treated group were significantly decreased 12 h post injection, and DILI mice that received CeNZs showed a more significant reduction in necrotic hepatocytes and a longer survival rate than mice that received NAC treatment. Moreover, CeNZs induced no obvious toxicity to the major internal organs, as shown by histological staining of internal organs at various time points post injection (90 min, Day 1, 3, and 28). Kalashnikova et al. demonstrated albumin-modified and indocyanine green (ICG)-conjugated nanoceria NPs (A-nanoceria) could be used for treating RA and imaging. This multifunctional and theranostic NP was synthesized by biomineralization and bioconjugation chemistry, resulting in an overall size of ∼30 nm by TEM. The inflammatory targeting efficiency of this NP was evaluated in a collagen-induced arthritis (CIA) mice model. Assisted by ICG labeling, sufficient retention of A-nanoceria was observed in swollen and inflamed joints of collagen-induced arthritis mice after tail vein injection (1 mg/kg) by fluorescence imaging, and the researchers showed that the NPs was internalized within the RA site via a secreted protein acidic and rich in cysteine (SPARC) mechanism. The efficacy data showed that A-nanoceria could effectively scavenge ROS, reprogram the M1 macrophages into the M2 phenotype, and deliver oxygen to the ‘starving’ RA tissue to reduce inflammation in the joints. A clinical scoring scheme was also used to compare the efficacy of A-nanoceria and the gold standard treatment MTX. Intriguingly, A-nanoceria achieved similar therapeutic outcomes as MTX, while A-nanoceria benefited from its targeting and accumulation ability, as well as minor side effects compared with MTX (e.g., ulcerative colitis, hepatotoxicity and nephrotoxicity,). Yet, this study did not feature in vivo toxicity data of the NPs. Cheng et al. reported that doses higher than 50 μg/mL CeNPs could induce cytotoxicity in human hepatocellular carcinoma SMMC-7721 cells. One in vitro study by Wang et al. also indicated significant cellular apoptosis when NP concentration exceeded 50 μg/mL; of note, the authors did not observe a significant effect of NP shape on cytotoxicity by measuring the cytotoxicity induced by CeNP-based cubes, octahedra, and rods. Conversely, Forest et al. showed that rod-shaped CeNPs were more toxic than octahedral CeNPs in vitro. By literature precedent, a dose on the order of 0.1 mg of CeNPs per kg of animal body weight is considered safe in vivo, therefore a typical therapeutic dose for in vivo efficacy studies. Hirst et al. reported that repeated dosing of CeNPs (0.5 mg/kg, weekly for 2 or 5 weeks) by oral, intravenous and intraperitoneal administration did not induce obvious toxicity in CD-1 mice.

Zinc oxide (ZnO) is biocompatible and approved by the FDA. Due to its inexpensive production cost, low toxicity, and antioxidant properties, ZnO NPs have found applications in the fields of biosensing and drug delivery. ZnO NPs effectively inhibit the levels of pro-inflammatory cytokines such as iNOS, IL-1β, IL-6 and TNF-α,. An in vitro study revealed that ZnO NPs suppressed the NF-κB pathway in RAW 264.7 macrophages induced by LPS by upregulating the negative regulator A20 (a zinc finger protein). An attractive administration route of ZnO NPs is oral gavage due to its lower toxicity than intravenous injection. Upon oral administration, ZnO NPs (50 nm, 2.5 g/kg; or 100 nm, 500 mg/kg) abundantly accumulate in the liver124, 125, 126. Interestingly, Paek et al. demonstrated that the surface charge of ZnO NPs rather than NP size is the key parameter of biodistribution. The authors showed that l-serine-modified positively charged ZnO NPs (20 and 70 nm, 2 g/kg by oral administration) preferentially accumulate in the lung. The most common excretion route for ZnO NPs (20 and 70 nm) is via feces, whereas some NPs may be excreted via urine,. In 2021, Abdulmalek et al. reported the protective effect of ZnO NPs against type 2 diabetes mellitus (T2DM) in rat models. The researchers orally gavaged 21 nm (TEM) ZnO NPs (10and 50 mg/kg) into a rat model of T2DM. The researchers found that 50 mg/kg ZnO NPs markedly reduced fasting blood glucose and insulin levels, as well as advanced glycation end products (AGEs). ZnO NP-mediated improvements in liver and kidney functions were indicated by reduced serum levels of liver function enzymes [ALT, AST and gamma-glutamyl transferase (GGT)] and kidney function indicators (creatine and urea). The anti-inflammatory effect of ZnO NPs was further investigated in the hepatic and pancreatic tissues of diabetic rats. Compared with the untreated group, the ZnO NPs group showed potent suppression of iNOS, IL-1β and TNF-α expression. In addition, it is reported that T2DM is associated with an increased risk of neurocognitive dysfunction, so these authors also investigated the role of ZnO NPs in attenuating T2DM-promoted hippocampal neurotoxicity in a separate work. Compared with that in diseased rats, 50 mg/kg ZnO NPs was shown to boost the recovery of learning and memory function, resolve redox and inflammatory status, and increase Bcl2 expression in the hippocampus. The researchers examined oxidant and antioxidative biomarkers in the hippocampus, and the data showed that oxidative markers [NO and thiobarbituric acid reactive substances (TBARS)] were decreased, while antioxidant markers [glutathione S-transferases (GST), CAT, GPx, SOD, and glutathione (GSH)] were increased by ZnO NPs. In addition, the hippocampal levels of inflammatory mediators were also studied. ZnO NPs significantly decreased the expression of pro-inflammatory TNF-α and IL-6. Additionally, the authors showed that the p38-MAPK signaling pathway was markedly suppressed in the rats with T2DM, as indicated by the suppression of p-p38MAPK, p-ERK and p-MEK (mitogen-activated protein kinase kinase) after ZnO NPs administration (Fig. 6), which was also considered to be relevant for the regulation of T2DM-induced neuroinflammation in the hippocampus. However, the authors did not report the in vivo biodistribution and toxicity data of the orally administrated ZnO NPs. Their in vitro toxicity data on hepatocytes cell lines suggested no significant cytotoxicity. In addition to applying ZnO NPs alone to treat inflammatory conditions, researchers incorporated them into hydrogels for combined treatment. Lin et al. demonstrated that a ZnO NP-loaded hydrogel (ZnO NP-Gel) could markedly reduce ROS levels and apoptosis in the injured spinal cord during the early stage. ZnO NPs with sizes of 10–50 nm were incorporated into hydrogels and implanted into a severe long-term spinal cord transection mouse model. The data showed that this ZnO NP-gel significantly restored motor function, as indicated by the better performance in footprint analysis compared with the untreated and gel-only groups. Then, the recovery of the spinal cord was assessed by dual immunofluorescence [Tuj1 and glial fibrillary acidic protein (GFAP)] and histology. ZnO NP-gel treatment markedly decreased the expression of Tuj1 and GFAP, as well as the lesion volume in the spinal cord. The in vivo inhibitory effect on ROS was confirmed by the increased expression of antioxidant markers [SOD, GSH, nuclear factor-erythroid factor 2-related factor 2 (Nrf2), and HO-1]. Furthermore, the anti-inflammatory effect of ZnO NP-gel was verified by quantifying the expression of arginase-1 (Arg-1) and iNOS. Seven days after implantation, the level of iNOS decreased significantly, accompanied by an increase in Arg-1. After 28 days of treatment, no obvious change in the tissue morphology of major organs or hematology was observed, and the levels of liver and kidney function indicators (AST, ALT, BUN, and CRE) in the ZnO NPs-Gel-treated group were similar to those in the control group. On the toxicity of ZnO NPs, Wang et al. showed that long-term exposure to 50 and 200 mg/kg ZnO NPs in diets showed minimal toxicity. In general, the toxicity of ZnO NPs depends on their physicochemical properties. On the effect of NP size, Kang et al. demonstrated that smaller ZnO NPs (26 nm) exhibited higher cytotoxicity than larger NPs (62 and 90 nm). The effect of NP surface charge on toxicity is inconsistent. Baek et al. showed that positively charged ZnO NPs induce greater cytotoxicity in A549 cells than negatively charged NPs with the same morphology and size, yet Wingett et al. suggested that negatively charged ZnO NPs exhibited a higher level of cytoxicity in T cells. More studies are needed to verify the in vivo biocompatibility of ZnO NPs with different physicochemical properties.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used for a variety of biomedical applications, mostly for orthopedic implants. A few studies investigated the applications of TiO2 NPs for anti-inflammatory treatments, but few uncovered the mechanism. Only one study indicated that TiO2 NPs could reduce platelet counts in blood and increase thrombin-antithrombin complex levels. Chakrabartty et al. examined the anti-inflammatory and PLA2 activity of TiO2 NPs in a snake venom-induced sterile inflammation mouse model. TiO2 NPs were mostly 5–6 nm in diameter. Edematogenic, hemorrhagic and phospholipase A2 (PLA2) activity induced by the venom and their neutralization by TiO2 NPs were first characterized. In vivo investigation of neutralization was performed by injecting 5 ng/mouse TiO2 NPs into venom (10 μg)-treated mice. Data indicated that TiO2 NPs could neutralize PLA2 activity and protect the mice from necrosis. Anti-inflammatory efficacy was assessed by measuring mouse paw edema, and I.P. injection of TiO2 NPs significantly inhibited venom-induced inflammatory swelling compared with commercial drugs, such as aspirin and indomethacin. Several sterile inflammatory markers, including HMGB1 (high mobility group box protein 1), HSP70 (70 kDa heat shock protein), and IL-6, were also tested, and the TiO2 NP-treated group showed significantly reduced levels of these inflammatory markers. Rhodium (Rh) nanozymes can serve as an anti-inflammatory and antitumor treatments for colitis-associated colorectal cancer due to their reactive oxygen and nitrogen species (RONS)-scavenging properties. Recently, Miao et al. showed that PEG-coated ultrasmall rhodium nanodots (Rh-PEG NDs) not only have a high capacity for RONS elimination but also act as a photothermal therapy. The average hydrodynamic size of Rh-PEG NDs was ∼16.3 nm. First, the researchers demonstrated that subcutaneously administered (10 μg/leg) or I.V. administered (10 mg/kg) Rh-PEG NDs significantly reduced pro-inflammatory cytokines (TNF-α and IL-6) in an LPS-induced mouse tibialis anterior muscle injury model. Furthermore, they assessed the anti-inflammatory effect of Rh-PEG NDs on a DSS-induced acute colitis model. I.V. injection of Rh-PEG NDs (10 mg/kg) not only reversed the changes in colon length, colonic epithelial thickness, and body weight but also downregulated pro-inflammatory cytokines (TNF-α and IL-6). Then, photothermal performance was evaluated in CT-26 tumor-bearing mice. Complete elimination of CT-26 colon tumors without any recurrence was observed for up to 16 days. Ruthenium (Ru) is a preventive antioxidant that shares similar functions with POD, CAT, and SOD. Ru protects cells from oxidative stress by breaking down hydrogen peroxides to H2O and oxygen and dismutating superoxide radicals. In addition, ruthenium complexes have been shown to induce apoptosis via the poly (ADP-ribose) polymerase/ataxia-telangiectasia mutated (PARP/ATM) pathway and inhibit tumor growth. Ruthenium also affects cell behaviors such as proliferation and osteogenic differentiation by activating Akt signaling pathways. Lu et al. reported that the multi-ROS scavenging nanozyme ruthenium oxide polyvinylpyrrolidone (RuO2-PVP) had anti-inflammatory and neuroprotective effects. The size of RuO2-PVP NP was ∼2.73 nm, as confirmed by TEM imaging. The anti-inflammatory effects in the ear inflammation model and the neuroprotective effects on a Parkinson's disease (PD) model were examined. Local injection of RuO2-PVP (10 μL, 600 μg/mL) significantly decreased ROS production and reduced the thickness of the dermis. In addition, in the PD model, the RuO2-PVP-treated (5 μL, 600 μg/mL) group showed substantially enhanced levels of dopamine, as indicated by measuring the expression of tyrosine hydroxylase (TH), and lower levels of ionized calcium binding adaptor molecule-1 (IBA-1), a marker of microglial activation.

This review presents the emerging research landscape of using metal-based NPs for managing various inflammatory diseases over the past three years. Thanks to their intrinsic anti-inflammatory properties, these drug-free metal-based NPs are promising drug candidates for treating or alleviating inflammation, but their advantage in efficacy over existing clinical drugs or traditional drug-loaded NPs remains to be proven. As self-therapeutic metal-based NPs as anti-inflammatory agents is still an emerging direction, more systematic investigations into their biodistribution, excretion, and toxicity, and knowledge in how the physicochemical properties of NPs affect in vivo pharmacological behavior, are sorely needed to catalyze clinical translation. We call upon the community to contemplate the three points below.

To exert an intended therapeutic effect, NPs need to reach the target organ, tissue, and cell effectively. The tunable size, shape, and surface chemistry of NPs allow them to meet the spatial and temporal constraints for bypassing different biological obstacles and accumulating in target inflammation sites. Unfortunately, among the 16 studies mentioned in this review, only half of them reported the biodistribution of NPs at the organ level and few explained their rationale of NP design and pharmacokinetics. Whereas these studies all documented favorable therapeutic outcomes of their respective self-therapeutic NP, a deeper understanding in how the type and loading of targeting ligands (e.g., peptides, antibodies) and the chemical composition of NPs affect the overall distribution of NPs to the inflammation site will improve overall efficacy. Beyond the classical organ- and tissue-level distribution studies, our understanding in the in vivo distribution of NPs to various cell types in a specific organ (say upon inhalation and I.V. injection) remains inadequate. For example, atherosclerosis, an inflammatory condition that underpins stroke and myocardial infarction (both leading causes of death globally), entails multiple plaque cell types, such as smooth muscle cells, endothelial cells, macrophages, and T cells. Developing nanomedicines for atherosclerosis will benefit from knowledge in how fast and how much the injected NPs localize in different cell types inside the atherosclerotic plaque. As the interactions between NPs and the constituent cell types of an organ ultimately affect the overall retention of the NPs in that organ, knowledge in the cell-nano interactions of each cell type will support more efficient nanocarriers with higher retention in the inflamed site and lower diversion to other organs, such as liver and spleen. It is important to understand how different types of metal-based NPs (with different surface modifications) interact with specific cell types in different tissues. In Section 2.1, we discussed how alkylation (specifically attachment of octadecyl chains) boosts the delivery of AuNPs to skin keratinocytes in vitro and in vivo. Recently, our group reported that the catechol and amine groups collectively enhance the binding of I.V.-injected NPs to Kupffer cells that are rich in dopamine receptor D2 (D2DR) in the liver. These data further validate the feasibility of using functional groups to modulate the delivery of NPs to different cell types in vivo, including macrophages. Many inflammatory disorders involve the participation of immune cells, especially macrophages. M1 macrophages primarily act as pro-inflammation regulatora and M2 macrophages assist in anti-inflammation. Differentiated macrophages are actively involved in multiple inflammatory diseases, such as CIA, RA, and DILI. By targeting specific subpopulations of macrophages rather than via random distribution, we envision that metal-based NPs can fully realize their therapeutic potential as anti-inflammation agents.

The major concern over the clinical use of metal-based NPs is toxicity. While some metal ions are essential for health (e.g., iron and copper), other non-essential metal ions are toxic (e.g., cadmium, lead and mercury). Metals may induce toxicity by forming complexes with cellular compounds containing sulfur, oxygen, or nitrogen. The complexes inactivate enzyme systems or modify critical protein structures, leading to cellular dysfunction and death. Gold is relatively non-toxic due to its low aqueous solubility and chemical inertness in biological tissues. In the early 20th century, gold salts were used as a standard treatment for RA, but unfortunately overexposure to gold salts led to a series of adverse effects, such as bone marrow depression, stomach and intestinal bleeding, headaches, vomiting, focal or generalized continuous fine vibrating muscle movements. Besides, deposition of gold salts in connective tissue can cause chrysiasis, characterised by blue-grey to greyish-purple staining of the skin. For these side effects, gold salt therapy was stopped in the 1990s when less toxic drugs (e.g., methotrexate) were developed. Platinum-containing complexes are used in cancer treatment. The first platinum-containing coordination complex as cancer treatment was cisplatin [cis-dichlorodiammineplatinum (II)], initially approved for patient use in 1971. The anti-cancer efficacy of cisplatin propelled further research on other platinum derivatives with a higher activity and lower toxicity, such as carboplatin (1980), oxaliplatin (1985–6), and nedaplatin (1987–8). However, platinum-based chemotherapy may induce leucopenia, nephrotoxicity, and gastrointestinal toxicity. There are still no currently available medications with copper and zinc as active ingredients, and their potential clinical use will require caution. Copper poisoning may cause vomiting, diarrhea, tachycardia, respiratory problems, hemolytic anemia, and gastrointestinal bleeding. Wilson's disease, a genetic disorder that entails the accumulation of copper due to impaired excretion, will cause liver disease and neuropsychiatric symptoms. Zinc is relatively non-toxic, but it may lead to toxic symptoms (nausea, vomiting, epigastric pain, lethargy, and fatigue) when zinc intake becomes excessive. Despite the abundance of clinical data on the toxicity of metal ions, there is a dearth of clinical studies on the toxicity of metal-based NPs. From animal studies, researchers found that nanotoxicity depends on various experimental parameters, including NP composition, concentration, size, shape, coating, and administration route. As the distribution, metabolism, and clearance of metal ions are starkly different than their respective NPs, each type of metal NP should be studied meticulously for a specific application.

There is no known record of clinical trials that use self-therapeutic metal-based NPs for treating inflammatory diseases, based on our search of the Clinicaltrials.gov website. Even though if we relax the search criteria from metal-based self-therapeutic NP only and include metal-containing NPs in which the NP serves non-therapeutic functions (say drug delivery, contrast agent, or biosensor), the search results are still scarce. Among the metal types presented in this review, gold NP represents the most studied in the clinical setting, with 12 completed and ongoing trials as of now. Half of the trials use gold NPs as contrast agents for imaging (computed tomography) ex vivo (e.g., salivary gland tumors; NCT04907422) or chemical handles for quantifying the NP content in tissues. The other half of the trials entail the use of gold NPs as drug carriers that facilitate the in vivo intracellular delivery of drug cargoes, such as nucleic acids for treating recurrent glioblastoma multiforme by antisense therapy (NCT03020017), proinsulin peptides for treating Type 1 diabetes by immunotherapy (NCT02837094), T-cell priming peptides for vaccination against Dengue (NCT04935801) and COVID-19 virus (NCT05113862), and heat for photothermally treating atherosclerosis (NCT01436123). We summarized the recent in vivo clinical trials that involve the use of gold NPs in Supporting Information Table S2. Copper NPs, titanium oxide NPs, and zinc oxide NPs are clinically tested in antibacterial applications. Lastly, there has been no clinical trial for platinum NPs, ruthenium oxide NPs, rhodium NPs, or molybdenum NPs. Researchers should conduct in-depth preclinical investigations before proceeding to clinical trials. In particular, choosing animal disease models that properly mimic human pathological mechanisms will empower more accurate prediction of therapeutic efficacy in human clinical trials. Take our anti-psoriatic gold NPs in Section 2.1 as an example. We may need another disease model (e.g., xenotransplantation model) to validate their efficacy and further optimize NP formulation, dosage, and treatment schedule. Next, we may need to assess their safety in non-rodent species (e.g., minipig). Because psoriasis is a chronic and recurrent disease, human patients may need to apply the gold NPs for months or even years. To mimic human usage more adequately, it will be instructive to evaluate the safety and tolerability of our gold NPs after applying extended doses (e.g., 30 days) in animals, even though our preclinical efficacy data show that six days of consecutive topical application is sufficient to engender an anti-psoriatic outcome in IMQ mice. Finally, for clinical testing, it is crucial to scale up the metal-based NPs per the Good Manufacture Practice (GMP), noting that the guidelines for producing GMP-grade metallic raw materials are unclear for many types of metal NPs. With reference to our anti-psoriatic gold NP, there are at least three challenges that are associated with GMP production of 3 nm gold NPs for clinical studies. Industrial preparation of colloidally stable and chemically pure 3 nm gold NPs requires careful attention to technical details that may not be relevant to the initial stage of laboratory testing. In our work, we employed sodium borohydride (NaBH4) for reducing Au3+ ions to metallic gold because the reaction is simple and rapid, but the resultant gold NPs may be prone to aggregation depending on the concentration of Au3+ added. Another method of preparing stable 3 nm gold NPs is using tannic acid as both the reducing agent and capping agent, but industrial-scale purification of residual tannic acid from water requires ion exchange, ultrafiltration, coagulation, or other processes that complicate production. Feasible routes of synthesizing ultrasmall metal-based NPs are desirable for clinical translation. In our case, we used NaBH4 to prepare 3 nm gold NPs and immediately stabilized them with PEG strands to ensure colloidal stability. Next, supply of GMP-grade raw materials for producing metallic NPs is not common. For instance, although gold-based raw materials [such as chloroauric acid (HAuCl4)] are commercially available for purchase in bulk from the electroplating and catalysis industries, production lines of HAuCl4 at pharmaceutical grade for clinical testing are rare. The typical scale of production of HAuCl4 in the electroplating industry is more voluminous (in hundreds of kilograms) than that in the pharmaceutical industry (in hundreds of grams), making GMP accreditation of this reagent by existing electroplating vendors specifically for pharmaceutical applications not economically attractive. Strategic collaboration between the chemical and pharmaceutical industries is needed to establish a unified goal for clinical translation. Finally, the regulatory guidelines for metal-based NPs are incomprehensive now because these NPs are infrequently tested for humans; there have been only several clinical trials of gold NPs as therapeutics. Currently, nanomedicines are regulated on a case-by-case basis in different countries. The European Medicines Union issued preliminary guidelines for nanomedicine preparations (e.g., iron oxide NPs, polymeric micelles, and liposomes), but they remain at the public consultation stage. In 2022, the FDA published guidelines on the development of drug products in which a nanomaterial is present in the finished dosage form. These guidelines recommend clear descriptions of nanomaterial quality attributes, characterization data and methods used, drug release and dissolution testing, manufacturing processes, and stability. When preparing for clinical studies, the FDA recommends applicants to determine if the nanomaterial-based drug product exhibit significant changes in exposure, safety, and/or effectiveness relative to the free drug. However, these guidelines may not be applicable to the metal-based NPs covered in this review whereby the metal is the main therapeutic component.

This work was in part supported by General Research Funds (Project No.: 14300120 and 14300221, Hong Kong SAR, China) and via the Research Matching Grant Scheme from the Research Grants Council (RGC, China). It was supported by the Chow Yuk Ho Technology Centre for Innovative Medicine and a Vice Chancellor Discretionary Fund from The 10.13039/501100004853Chinese University of Hong Kong (10.13039/501100004853CUHK). Chung Hang Jonathan Choi acknowledges a Croucher Innovation Award from the 10.13039/501100001692Croucher Foundation (China). We thank Prof. Wai Sze Agnes Chan and Prof. Elaine Chow (Department of Medicine and Therapeutics, CUHK) and Dr. Christina Man-tung Cheung (Prince of Wales Hospital, Hong Kong, China) for their insightful discussions on the clinical translation of metal-based nanoparticles.

Chung Hang Jonathan Choi conceived the idea and provided the funding. Ruifang Han, Yu Xiao, and Qianqian Bai drafted the manuscript. Chung Hang Jonathan Choi revised the paper. Ruifang Han and Yu Xiao contributed equally to this work. All authors read and approved the final manuscript.

The authors declare no conflicts of interest.