MMPs, particularly MMP-8, play a crucial role in periodontitis and are closely associated with periodontal status (Arias-Bujanda et al., 2019). Upon selective degranulation of PMNs, latent MMPs are activated by ROS or proteolytic cleavage. In saliva, the predominant forms of MMPs are pro-forms or inactive forms (Koller et al., 2021; Gupta et al., 2023). MMP-8 is primarily expressed by neutrophils and is essential for wound healing and tissue regeneration (Silbereisen et al., 2020). It is present in saliva and serum, making it a promising biomarker that is often detectable before radiographic and clinical signs of gingival and alveolar bone damage (Cennamo et al., 2024). Studies since 2005 have demonstrated the association between MMP-8 and various periodontal diseases, highlighting its predictive capability (Arias-Bujanda et al., 2019; Arias-Bujanda et al., 2020). Several diagnostic kits for periodontitis and peri-implantitis based on MMP-8 have been commercialized (Arias-Bujanda et al., 2019). MMP-8’s good sensitivity and excellent specificity make it the most effective biomarker for diagnosing periodontitis in systemically healthy participants (Arias-Bujanda et al., 2019), with a median sensitivity and specificity of 76.7% and 92.0%, respectively (Arias-Bujanda et al., 2020). In a meta-analysis, MMP-8 was identified as the second-best salivary biomarker, with an estimated sensitivity of 72.5% (Arias-Bujanda et al., 2020).

MMP-8 exists in various molecular forms and activity stages, including latent proforms (proMMP-8), active forms (aMMP-8), and fragmented forms (Arias-Bujanda et al., 2019; Silbereisen et al., 2020). It remains in its latent proMMP-8 form throughout the induction and resolution of gingival inflammation. In patients with periodontitis, salivary MMP-8 is activated and fragmented, and studies have shown that aMMP-8, rather than total MMP-8 levels, correlates strongly with connective tissue destruction (Arias-Bujanda et al., 2019; Silbereisen et al., 2020). Monitoring aMMP-8 enzymatic levels and activation degree could aid in disease progression assessment (Arias-Bujanda et al., 2019). aMMP-8 activity and immunoreactivity are elevated in the gingival tissue, GCF, saliva, and mouth rinse of patients with periodontitis, making them useful for chair-side/point-of-care (PoC) lateral flow immunoassays (Gupta et al., 2023). Alassiri et al. found higher diagnostic sensitivity (76%–90%) and specificity (96%) for aMMP-8 PoC/chair-side tests, with an odds ratio of >72 (Arias-Bujanda et al., 2020).

NE is a serine protease stored in azurophilic granules and is released during bacterial phagocytosis or cell lysis, contributing to host defense by degrading pathogenic bacteria and extracellular matrix proteins (Kayar et al., 2020). This release leads to local tissue damage and regulation of inflammatory and repair processes (Kayar et al., 2021). NE can degrade connective tissue materials, alter cellular functions, and break down serum proteins, exerting potent pro-inflammatory effects (Kayar et al., 2021; Sriwattanapong et al., 2021; Tseng et al., 2022). In periodontitis, neutrophils migrate to inflamed sites and release excessive NE, leading to connective tissue degradation, exacerbation of inflammation, and periodontal tissue loss. Prolonged chronic inflammation results in the loss of periodontal ligament support and alveolar bone, potentially leading to tooth loss (Aral et al., 2020; Tseng et al., 2022). Studies using murine models of periodontitis have shown that NE activity is linked to periodontal bone loss, which can be mitigated using NE inhibitors (Hiyoshi et al., 2022). In human gingivitis, NE levels in the GCF increase, correlating with clinical attachment loss in patients with periodontitis (Hiyoshi et al., 2022). NE levels were significantly higher in patients with aggressive periodontal disease compared to healthy controls (Tseng et al., 2022). NE, the most concentrated proteinase in the GCF during inflammation (Kayar et al., 2020), is linked to the pathogenesis and progression of periodontal disease (Tseng et al., 2022). Cleaves cell adhesion molecules, disrupts gingival epithelial barrier and aggravates periodontitis. Periodontal pathogens such as T.d and P. g can stimulate NE release and evade host proteases (Hiyoshi et al., 2022). NE modulated the expression of cytokines, chemokines, and growth factors during infection (Sriwattanapong et al., 2021). It upregulates placental growth factors and contributes to the inflammatory cascade in periodontal disease. NE activates protease-activated receptors (PAR)-1, PAR-2, and PAR-4, promoting the secretion of IL-1β, TNF-α, and IL-6 by oral epithelial cells (Tseng et al., 2022). Due to its role in extracellular matrix degradation and tissue disruption, NE is a potential diagnostic marker for periodontal disease (Kayar et al., 2020; Kayar et al., 2021). GCF NE activity is a reliable indicator for diagnosing periodontal disease and alveolar bone loss, with a sensitivity and specificity of 84% and 66%, respectively, in one study, and 77% and 61%, respectively, in another (Kayar et al., 2020; Kayar et al., 2021). A recent meta-analysis by Arias-Bujanda et al. found NE to be the second most researched GCF biomarker, with median sensitivity and specificity of 75% and 81%, respectively (Arias-Bujanda et al., 2019).

Metals such as gold (Au), silver (Ag), titanium (Ti), copper (Cu), magnesium (Mg), zinc (Zn), and bismuth effectively inhibit bacterial growth and show significant potential in periodontitis treatment. Platforms using metal NPs ranging from 0.2 to 100 nm in size have been developed as antibacterial agents (Maleki Dizaj et al., 2015; Rudramurthy et al., 2016; Nasiri et al., 2023). NPs with antimicrobial properties include Au, Ag, Ag₂O, titanium dioxide (TiO2), copper oxide (CuO), and zinc oxide (ZnO) (Priyadarsini et al., 2018). The antibacterial effects of metallic NPs can be attributed to three primary mechanisms: ROS production, ion release, and interactions with the cellular membrane (Kırmusaoğlu, 2018)( Figure 3A ). ROS generated by these NPs, such as superoxide anions and hydroxyl radicals (-OH), degrade essential cellular components (DNA, RNA, and proteins) and effectively kill periodontal pathogens (Priyadarsini et al., 2018; Agarwal et al., 2019; Nasiri et al., 2023). Owing to their small size, metallic NPs can penetrate the peptidoglycan layer and damage bacterial cells (Safarov et al., 2019). The metal ions released by NPs can damage bacterial DNA and proteins (Agarwal et al., 2019; Nasiri et al., 2023). Negatively charged bacterial cell surfaces will absorb Cu²⁺, Ag+, and Zn²⁺ released by metallic NPs (Safarov et al., 2019). These ions penetrate the cells and react with sulfhydryl groups on microbial proteins, inhibiting their ability to synthesize proteins or nucleic acids (Agarwal et al., 2019; Nasiri et al., 2023). The effectiveness of NPs in eradicating bacteria depends on the particle size and surface charge. Smaller particle sizes and higher surface-to-volume ratios confer superior antimicrobial efficacy without compromising the mechanical properties of the material, making NPs highly effective in combating bacterial infections in periodontal disease (Agarwal et al., 2019; Nasiri et al., 2023). Periodontitis is primarily driven by pathogenic bacteria organized in biofilms composed of extracellular polymeric substances and microbial flora (Carrouel et al., 2020; Nasiri et al., 2023). These biofilms play a critical role in disease progression, making bacteria within them 1,000 times more resistant to various antibiotic treatments than planktonic bacteria, including penicillin, aminoglycosides, and quinolones (Carrouel et al., 2020; Gao et al., 2023; Nasiri et al., 2023). Furthermore, biofilms enable bacteria to evade the immune system by producing superantigens (Carrouel et al., 2020). Metals, metal oxides, and other NPs have emerged as promising alternatives to combat bacterial infections due to their enhanced antibacterial activity at the nanometer scale. NPs facilitate interaction and penetration of bacterial cells (Carrouel et al., 2020). NP-based photodynamic therapy (PDT) and photothermal therapy (PTT) have shown increasing potential in treating bacterial infections. PDT uses light to produce ROS that destroy bacterial molecules, whereas PTT transforms light into heat, causing bacterial destruction. Combining PDT and PTT enhances efficacy owing to the synergistic effects of ROS and heat (Gao et al., 2023).

The inflammatory response and cell behavior are influenced by material properties such as surface chemistry, topography, microgrooved design, wettability, and the presence of biologically active proteins. These characteristics are crucial for determining the anti-inflammatory efficacy of NPs (Agarwal et al., 2019; Ghinassi et al., 2020). Metal and metal oxide NPs, including Au, Ag, ZnO, TiO₂, CuO, and magnesium oxide, exhibit significant anti-inflammatory properties, making them effective in treating periodontal diseases by targeting both gram-positive and gram-negative bacteria (Agarwal et al., 2019). For NPs to exert anti-inflammatory effects, they must enter cells via ion channels or pores, and their size and concentration influence cellular uptake (Simko et al., 2011). Higher concentrations of small NPs are typically endocytosed, whereas macrophages and neutrophils undergo phagocytosis and macropinocytosis (Mahmoudi et al., 2011; Kuhn et al., 2014; Muñoz et al., 2016; Carrouel et al., 2020) ( Figure 3C ). Gold nanoparticles (GNSs) reduce ROS production, decrease lipopolysaccharide-induced cytokine production (IL-1β, IL-17, TNF-α), and modulate mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase pathways (Agarwal et al., 2019). Silver nanoparticles (AgNPs) decrease levels of vascular endothelial growth factor (VEGF), reduce hypoxia-inducible factor 1α (HIF-1α) expression, prevent mucin (mucus glycoproteins) hypersecretion, suppress pro-inflammatory cytokines (IL-12, TNF-α), and decrease cyclooxygenase-2 (COX-2) gene expression (Agarwal et al., 2019). ZnO NPs block the production of pro-inflammatory cytokines (IL-1β, IL-18), inhibit mast cell proliferation by upregulating p53 and reducing thymic stromal lymphopoietin levels, and suppress lipopolysaccharide-induced COX-2 and inducible nitric oxide synthase expression (Agarwal et al., 2019). TiO2 NPs reduced platelet numbers and increased thrombin–antithrombin levels (Agarwal et al., 2019). These NPs, through modulating the expression of enzymes or genes, offer promising anti-inflammatory strategies for periodontal disease treatment.

In addition to reducing inflammation, periodontal treatment aims to control infection and rebuild tissues such as the cementum, periodontal ligaments, and bone (Zhu et al., 2023). Kiarashi et al. demonstrated 45 nm AuNPs reduce inflammation and improve the periodontal ligament environment by regulating cytokines and macrophage polarization. This results in a microenvironment characterized by low levels of inflammatory cytokines and high levels of reparative cytokines such as bone morphogenetic protein-2. This favorable environment promotes the differentiation of human periodontal ligament cells and periodontal tissue regeneration and halts the progression of periodontitis ( Figure 3B ) (Kiarashi et al., 2024). Graphene, known for its mechanical properties, antibacterial activity, high surface area, and biocompatibility, promotes stem cell differentiation. Graphene oxide (GO) and reduced graphene oxide (rGO) are promising materials for tissue engineering that enhance the proliferation of periodontal ligament stem cells (PDLSCs), which are available for regenerative therapy (Liu C. et al., 2022). Polycaprolactone, used for three dimensional printing due to its favorable properties, can form bone–ligament–cementum complexes in vivo ( Roato et al., 2022). Plant-derived exosome-like nanoparticles (PELNs), which are natural nanocarriers rich in lipids, proteins, RNAs, and other active molecules, can enter mammalian cells and regulate cellular activity. Literature reports indicate the great potential of PELNs in regulating immune function, inflammation, microbiome balance, and tissue regeneration (Zhang Z. et al., 2022). In conclusion, NPs offer significant advantages in periodontal tissue regeneration. Their unique properties, such as high surface-area-to-volume ratio and tunable surface chemistry, enable the development of advanced therapies to enhance tissue repair and overcome traditional treatment limitations.

NZs are garnering significant attention owing to their affordability, excellent stability, scalability, and multifunctionality. Unlike conventional antibiotics, NZs are less likely to induce bacterial resistance (Attar et al., 2019), owing to their favorable membrane permeability and biocompatibility. Their catalytic activities effectively eliminate bacterial biofilms, and their unique physicochemical properties allow for additional functionalities that are not found in natural enzymes. By modulating the composition, size, and shape of the NZs, they can perform multiple functions (Hosseini Hooshiar et al., 2024). As highly effective nanoantibiotics, NZs bridge the gap between biology and nanotechnology by exhibiting broad-spectrum antimicrobial activity with minimal biotoxicity (Yang et al., 2020). NZs act through several mechanisms. First, POD or oxidase mimics convert H₂O₂ or O₂ into ROS such as singlet oxygen (1O2) or -OH (Fang, 2011), leading to irreversible bacterial and biofilm destruction ( Figure 4B ). Second, NZs can transform prodrugs into active antibiotics, thereby enhancing their antibacterial effects. Third, the phosphatase-like activity of NZs can break down bacterial cell membranes, causing bacterial death (Kim and Kim, 2021; Hosseini Hooshiar et al., 2024). Among these, ROS generation has been the most extensively studied. ROS display rapid wide-ranging activity against bacteria and cancer cells without promoting drug resistance. They can also attack the DNA and lipids of dormant bacteria, including “superbugs” and persistent biofilms (Wang et al., 2022) ( Figure 4B ). Their remarkable properties have made them a focal point of research, marking significant advancements in the development of novel antibacterial agents.

Excessive ROS levels can lead to uncontrolled inflammation, which is a key factor in the development of numerous diseases (Gao et al., 2017). Thus, NZs with ROS-scavenging capabilities offer a promising approach for mitigating inflammation and preventing related disorders (Munir et al., 2020). Various NZs exhibit antioxidant properties that help to regulate ROS (Yang et al., 2021)( Figure 4C ). Singh et al. revealed that low-dose cerium NPs efficiently neutralize free radicals in human liver cells, preventing early stage apoptosis and DNA damage without activating the body’s natural antioxidant defenses (Singh and Singh, 2019). CeO₂’s ROS scavenging abilities stem from its ability to mimic superoxide dismutase and catalase, which also proved effective in reducing inflammation when used to coat Ti surfaces for peri-implantitis prevention (Li et al., 2019). Additionally, CeO₂ NPs grown on montmorillonite surfaces successfully suppress inflammatory responses by eliminating ROS such as superoxide (O₂), H₂O₂, and -OH, while lowering pro-inflammatory cytokines and boosting anti-inflammatory cytokines (Zhao et al., 2020). Similarly, vanadium pentoxide nanowires demonstrated strong antioxidant effects by mimicking the enzyme glutathione peroxidase, which allows them to neutralize ROS under physiological conditions (Vernekar et al., 2014). NACu-Cys NZs with superoxide dismutase-like activity eliminate ROS and protect against oxidative damage (Dashtestani et al., 2018). Manganese oxide (Mn₃O₄) NPs, due to their dual oxidation states (Mn²⁺ and Mn³⁺), also displayed enzyme-mimicking properties such as superoxide dismutase and catalase activities, enabling them to effectively scavenge O₂ and -OH (Yao et al., 2018). These NPs are more stable than natural enzymes and surpass the CeO₂ NZs in their ability to eliminate ROS (Miao et al., 2020). Furthermore, Cao et al. introduced ultra-small polyethylene glycol-coated rhodium nanodots, which exhibited impressive scavenging abilities for reactive oxygen and nitrogen species (RONS) and photothermal activity. These nanodots reduced pro-inflammatory cytokine levels ( Figure 4C ), such as TNF-α and IL-6, through their multiple enzyme-like activities against RONS. This led to a significant reduction in inflammation in dextran sulfate sodium-induced colitis model, suggesting its potential for treating inflammatory bowel diseases (Yang et al., 2021).

Among various nanomaterials, GNSs are particularly notable in bioengineering and offer significant advancements in diagnostic tools and treatments for severe diseases. GNSs are recognized for their biocompatibility, stability, low toxicity, and ability to efficiently manipulate energy and matter at the nanoscale level. In diagnostics, GNSs can be engineered to bind to specific biomolecules, enabling highly sensitive and precise disease detection. They are particularly effective in the early diagnosis and prevention of periodontal disease, overcoming the challenge of collecting sufficient fluid for analysis. Their unique physicochemical properties, including size- and shape-dependent optical characteristics, surface chemistry, and biocompatibility, have positioned them at the forefront of early dental disease detection (Zhang S. et al., 2024).

The primary bacterium responsible for periodontitis, P.g, secretes harmful proteases known as gingipains. Detecting gingipain activity in the gingival fluid is promising for early diagnosis and treatment. A previous study developed a nanoplasmonic biosensor using GNPs to detect gingipain proteolytic activity. GNSs were permitted to self-assemble into sub-monolayers within multi-well plates coated with casein or IgG. Proteases in the buffer catalyze the proteolytic degradation of casein as a substrate, resulting in a blue shift of the localized surface plasmon resonance (LSPR) band by 1–2 nm, depending on the concentration and duration. A significant LSPR change was observed only in samples with gingipain activity. The sensor’s limit of detection (LOD) was < 0.1 μg/mL, significantly lower than gingipain concentrations found in patients with severe periodontitis. This biosensor can quickly detect protease activity, thereby facilitating chair-side diagnosis (Svärd et al., 2020). Verma et al. created an electrochemical immunosensor using a composite of GNP and rGO to detect the salivary biomarker interleukin-8 (IL-8) non-invasively, with rapid detection in 9 min and high sensitivity across a range of 500 fg/mL – 4 ng/mL (Verma et al., 2017; Zhang S. et al., 2024). The accurate identification of MMP-8 enzyme is crucial for assessing periodontal disease progression. Tortolini et al. described a novel voltammetric sensor for detecting salivary MMP-8 using a graphene screen-printed electrode enhanced with GNPs and MMP-8-specific antibodies. Graphene is of particular interest owing to its unique electronic properties. When combined with metallic NPs, the electrocatalytic activity of graphene was significantly enhanced, improving the electron transfer pathways. The sensor showed excellent performance with a linear range from 2.5 to 300 ng/mL, an LOD of 1.0 ± 0.1 ng/mL, and a sensitivity of 0.05 μA mL cm² ng^-¹. This disposable and economical biosensor is highly attractive for detecting MMP-8 in human saliva (Tortolini et al., 2024).

AgNPs are important for the development of bioengineering materials. Their unique properties make them invaluable for applications in targeted drug delivery, imaging, and nanotherapeutics. Salivary biomarkers such as ALP and IL-1β are critical in diagnosing periodontal diseases (Kim et al., 2023). ALP, which is detectable in the saliva through secretion, GCF from tissue degradation, and bacterial cells, normally ranges between 25 and 100 U/L. Several strategies exist for ALP assays, including colorimetric, fluorometric, Raman scattering, capillary electrophoresis, and electrochemical biosensors (Hafez et al., 2021). One study developed a multicolorimetric ALP and IL-1β sensing platform based on the geometric modification of a silver nanoplate transducer. ALP dephosphorylates p-aminophenol phosphate (p-APP) to p-aminophenol (p-AP), altering the LSPR of silver nanoplates and changing their color from blue to yellow. This multicolor sensor detects ALP in the range of 0 – 25 U/L with a LOD of 0.0011 U/L and IL-1β from 0 to 250 pg/mL with a LOD of 0.066 pg/mL, significantly lower than the conventional monochromatic ELISA (LOD of 3.8 pg/mL) (Kim et al., 2023). Hafez’s multicolor biosensor modified gold nanobipyramids (AuNBPs) with Ag nanoshells. The nanostructures consisted of an AuNBP core surrounded by an Ag nanoshell. This bioassay leverages the selective catalytic activity of ALP to dephosphorylate the glucose phosphate substrate into glucose, which subsequently reduces Ag ions to form nanoshells on the surface of the AuNBPs. A binary surfactant mixture of cetyltrimethylammonium bromide and Tween 20 was used to achieve a maximal blue shift of the LSPR with a high-resolution multicolor response correlated with the ALP concentration. This surfactant mixture promotes the uniform growth of Ag on the AuNBP surface, resulting in distinctive color changes corresponding to the ALP concentration. The released glucose induces a blue shift in the LSPR from 740 to 550 nm, with visible color transitions from yellowish-brown to green, blue, purple, and red as the ALP concentration increases from 0 to 50 U/L. These spectral changes demonstrated high sensitivity for ALP detection, achieving a low detection limit of 0.085 U/L (Hafez et al., 2021). These advanced sensors significantly improve the detection and monitoring of ALP and IL-1β levels in saliva, aiding in the early diagnosis of periodontal diseases.

Copper oxide nanoparticles (CuO NPs) have shown promise for biosensor development. One study developed a non-enzymatic, highly electrocatalytic H₂O₂ biosensor using an electrode composed of chitosan, black phosphorus nanosheets (BP NSs), and CuO NPs. The integration of CuO and BP provided outstanding electrocatalytic performance, featuring a low detection limit of 30 nM, high sensitivity of 138.00 μA mM^-¹ cm^-², excellent selectivity, reusability, and long-term stability. This sensor effectively measured H₂O₂ levels in saliva and GCF samples, enabling precise differentiation between periodontitis patients and healthy individuals. Furthermore, the device successfully detected H₂O₂ release from macrophages and gingival fibroblasts at the cellular level, highlighting its potential for the early diagnosis of periodontal disease (Wang K. et al., 2021).

Up-converting nanoparticles (UCNPs) are luminescent nanomaterials that convert low-energy photons into high-energy emissions. They possess unique photophysical properties such as resistance to photobleaching, low background autofluorescence, high penetration depth, photostability, low toxicity, and long luminescent lifetime. These attributes make UCNPs highly suitable for applications in biosensing and imaging (Duan et al., 2018; Zhang et al., 2019).

Accurate detection of matrix metalloproteinase-2 (MMP-2) is crucial for the early diagnosis, prognosis, and treatment of oral diseases (Wang et al., 2016; Schmitt et al., 2019; Liu L. et al., 2022; Zhang S. et al., 2022). A fluorescence resonance energy transfer (FRET)-based ratiometric fluorescent nanoplatform (UCNPs@SiO2@Cy3-pep) was developed to detect MMP-2. This platform used SiO2-coated UCNPs modified with a cyanine 3 (Cy3) polypeptide containing an MMP-2-specific peptide substrate (PLGVR). In the FRET system, the UCNPs served as the energy donor and Cy3 as the energy acceptor. The green luminescence of UCNPs@SiO2 was quenched by Cy3, whereas the red luminescence remained unaffected. Upon cleavage of Cy3 at the PLGVR peptide by MMP-2, green luminescence was recovered, allowing the detection of MMP-2 activity. Both in vitro and in vivo experiments confirmed the effectiveness of the nanoplatform for monitoring MMP-2 levels, showing great potential for periodontitis diagnosis (Bi et al., 2023).

The rapid and multiplex detection of biomarkers in the GCF is vital for diagnosing periodontitis progression and severity. Scientists have developed a disk-like lateral flow immunoassay strip (LFIS) using green core–shell UCNPs (G-UCNPs) as luminescent probes. This strip simultaneously detects three periodontitis biomarkers: MMP-8, IL-1β, and TNF-α. The G-UCNP-LFIS platform demonstrated high sensitivity and specificity in spiked GCF, with detection limits of 5.455, 0.054, and 4.439 ng/mL in the standard solutions. It showed stable recovery rates in modified artificial saliva and high correlation with clinical methods (0.995 for MMP-8, 0.976 for IL-1β, and 0.977 for TNF-α). The entire detection process was completed within 30 min, making this tool highly promising for managing and treating periodontitis (He et al., 2021).

Organic fluorescent nanoparticles also play a critical role in the development of fluorescent probes. A coumarin-based fluorescent probe (named ZPS-NO), was developed to detect nitric oxide (NO). Excessive NO can react with molecular oxygen to form highly oxidative compounds such as N₂O₃ or peroxynitrite causing significant cell damage and potentially leading to tumors, inflammation, and other diseases (Zhang Y. et al., 2024). In healthy individuals, salivary NO concentrations range from 10 to 20 μM, whereas patients with oral periodontitis exhibit levels from 50 to 90 μM (Zhang Y. et al., 2022). The ZPS-NO probe, consisting of coumarin and o-phenylenediamine connected via a double bond, functions as a “turn-on” fluorescent probe. The electron-donating property of o-phenylenediamine induces the photoinduced electron transfer (PET) effect, quenching fluorescence. When NO is present, the PET effect is inhibited, resulting in a strong fluorescence “turn-on” effect. The ZPS-NO probe has an ultra-low NO detection limit of 14.5 nM, with detection results statistically comparable to those obtained using a commercial kit, demonstrating good applicability for NO detection in real saliva samples (Zhang Y. et al., 2024). Numerous small molecules and NPs contrast agents have been developed for photoacoustic imaging, with a particular emphasis on activatable probes for molecular imaging. Moore et al. introduced a dual-modal imaging probe, C2A, for detecting gingipain proteases secreted by P.g. This probe utilizes the intramolecular dimerization of peptide-linked cyanine dyes to create fluorescent and photoacoustic off states. Upon cleavage by Arg-specific gingipain (RgpB), the probe showed a 5-fold enhancement in photoacoustic signals and over 100-fold increase in fluorescence, with detection limits of 1.1 nM RgpB and 4.4E4 CFU/mL bacteria. The evaluation of GCF samples from 14 participants with and six without periodontal disease showed a strong correlation with qPCR-based detection of P.g. (Pearson’s r = 0.71), particularly in cases of severe disease progression (Moore et al., 2022). Advancements in NPs have enhanced gingipain detection methods. The existing strategies include nanobody immunoassays (Alhogail et al., 2018), electrochemical biosensors, fluorogenic dipeptides (Kaman et al., 2012), peptide-functionalized magnetic nanobeads, and protein-functionalized GNSs. These methods have reported detection limits of 7.81E6 CFU/mL bacteria, 5E5 CFU/mL bacteria, 1E5 CFU/mL bacteria, 49 CFU/mL bacteria, and 4.3 nM Kgp (CFU/mL not reported) (Kaman et al., 2012; Alhogail et al., 2018).

NPs significantly advance the early diagnosis and dynamic monitoring of periodontitis by targeting enzymes (mentioned above). Their integration into biosensors and fluorescent probes enhances their sensitivity, specificity, and practicality, making them valuable tools for detecting early-stage periodontal disease. These advancements have provided timely and effective management of periodontitis, representing a promising development in the field of oral diagnostics.

The small size of NPs can increase their reactivity and toxicity, leading to cytotoxicity, genotoxicity, reticuloendothelial system toxicity, and altered inflammatory responses. Ensuring the biological safety of nanomaterials is crucial to prevent potential harm to human health. Biocompatible materials such as GNSs are essential for mitigating these adverse reactions (Zhang S. et al., 2024). GNSs are less hazardous to mammalian cells because their antibacterial effects do not rely on ROS (Kiarashi et al., 2024). Their ease of synthesis, excellent biocompatibility, and high potential for functionalization make GNSs valuable for drug delivery, diagnosis, and therapy, including anti-inflammatory and antitumor applications (Zhou et al., 2021). In bone regeneration, GNSs promote osteogenic differentiation of mesenchymal stem cells, inhibit osteoclast formation, and accelerate bone formation (Zhang et al., 2021). They may influence cytoskeletal structures through endocytosis, thereby promoting osteogenic differentiation via autophagy activation (Zhang et al., 2021). GNSs can also facilitate alveolar bone defect repair using cell sheet technology and modulate macrophage phenotypes to support periodontal tissue regeneration (Ni et al., 2019; Zhang et al., 2021). As carriers, GNSs protect polypeptides from protease degradation and facilitate their cellular entry via receptor-mediated endocytosis (Wang S. et al., 2018). Human β-defensin 3 (hBD3), a broad-spectrum antibacterial peptide (Harder et al., 2001), promotes osteogenesis in periodontal tissues (Zhu et al., 2017). GNSs combined with hBD3 can inhibit the secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) and increase OPG expression, potentially reducing alveolar bone resorption. Advances in nanomaterials and tissue engineering have suggested that combining GNSs with hBD3 can effectively treat periodontitis, representing a novel approach to periodontal therapy (Zhou et al., 2021).

AgNPs are highly effective antibacterial agents against both gram-negative and gram-positive bacteria (Siddiqi et al., 2018; Yin et al., 2020). They exert antibacterial effects by increasing the membrane permeability and inducing ROS production through intracellular uptake (Liang et al., 2023). Despite their promise, the clinical use of AgNPs is limited due to toxicity concerns, such as silver accumulation in organs, particularly the liver and spleen (Yin et al., 2020), and oxidative stress and cellular injury from increased ROS production (Siddiqi et al., 2018; Palacios-Hernandez et al., 2020). Ebselen can mitigate these effects by increasing glutathione and superoxide dismutase 2 levels, reducing AgNP-induced ROS, and showing no significant cytotoxicity at concentrations up to 20 μg/mL in human gingival fibroblasts. Combining AgNPs with ebselen enhances their bactericidal efficiency, showing significant effects against planktonic P. g and F. n and demonstrating synergistic anti-biofilm activity on both mono- and multi-species biofilms (Liang et al., 2023). Silver-based biomaterials (AgBMs) exhibit several antimicrobial properties, including the disruption of bacterial cell membranes, enzymes and proteins, as well as damaging genetic material. These properties make AgBMs effective antibacterial agents while maintaining relatively low toxicity. They have been explored for various dental applications including implant coatings, periodontal plaque control, caries prevention, root canal sterilization, denture additives, and anti-inflammatory materials for oral and maxillofacial surgery (Hosseini Hooshiar et al., 2024).

CuS, a metal sulfide semiconductor, has garnered significant attention due to its excellent photodynamic and photothermal properties under near-infrared light irradiation (Mo et al., 2022; Zhang et al., 2023). CuS-based NPs demonstrate strong antibacterial properties and promote wound healing (Liang and Yan, 2019; Wang X. et al., 2021). Yu et al. developed a novel protease-loaded CuS NP that combined the features of PDT, PTT, and protease activity. The experimental results confirmed the photothermal activity and the capacity of the NPs to generate ROS, which formed the basis for their antibacterial function. The CuS@A NPs exhibited high antimicrobial activity against F.n and its biofilms. In vitro assays demonstrated proper hemo- and cyto-compatibility. In a rat periodontitis model, CuS-based NPs effectively inhibited bone resorption and alleviated inflammation, indicating their potential as promising materials for managing periodontitis (Gao et al., 2023).

NZs composed of noble metals, such as platinum, silver, and gold, exhibit significant catalytic activity, aiding bacterial adhesion and cell membrane disruption. Their inherent OXD-like and POD-like properties induce intracellular ROS production, effectively kill bacteria, and promote wound healing (Zheng et al., 2018). Recent research has underscored the challenges of managing pathogenic biofilms, such as those from F.n in periodontitis, with traditional treatments, owing to bleeding, drug resistance, and limited efficacy. NZs represent a novel solution. For example, a bimetallic cluster zyme with enhanced POD-like activity and glucose oxidase exhibited outstanding biocompatibility and antibacterial characteristics. This clusterzyme converts glucose into toxic -OH, effectively inhibiting and eliminating F.n biofilm in vivo. Animal studies demonstrated that bimetallic cluster zyme effectively treated periodontitis in rats, reduced inflammation, and promoted the regeneration of periodontal tissue (Wang et al., 2023). Another study developed a TM/BHT/CuTA hydrogel system comprising Cu-based NZs (Cu-tannic acid coordination nanosheets, CuTA NSs) and a triglycerol monostearate/2,6-di-tert-butyl-4-methylphenol (TM/BHT) hydrogel. This system retains inflammatory sites via electrostatic adsorption and hydrolyzes them in response to increased MMPs, enabling the on-demand release of CuTA NZ. The released CuTA NZ exhibits antibacterial and anti-plaque properties, scavenges ROS by simulating SOD and CAT processes, and regulates the shift of macrophage polarization from M1 to M2 through the Nrf2/NF-κB signaling pathway, reducing inflammation and promoting tissue regeneration (Xu et al., 2023). In addition, FeSN, a histidine-doped FeS₂ with high POD-like activity, demonstrated potent antibacterial activity, low cytotoxicity, and high biocompatibility. The catalytic efficiency of FeSN was approximately 30 times greater than that of FeS₂. In the presence of H₂O₂, FeSN demonstrated strong antibacterial activity against F.n, increased OXD coenzyme levels, and decreased GR and ATP levels in bacterial cells. It effectively reduced biofilm development, inflammation, and loss of alveolar bone in a rodent model (Shen et al., 2023). These studies highlighted the potential of metal-based NZs for treating bacterial infections and periodontal diseases.

CeO₂ NPs exemplify a biological catalyst with high POD-like activity owing to the reversible redox transition between Ce^{4 +} and Ce^{3 +} ions. This transition efficiently facilitated the CeO–H₂O₂ ROS system. The high redox potential and surface-rich oxygen vacancies of nanoceria confer various enzymatic activities, such as those of SOD, CAT, POD, and OXD. These properties enable CeO₂ NPs to mitigate inflammation through their antioxidant properties (Hosseini Hooshiar et al., 2024). Researchers have enhanced the biocompatibility and dispersion of CeO₂ NPs by loading them onto mesoporous silica and modifying them with polyethylene glycol. This formulation protects PDLSCs from oxidative stress induced by periodontitis by modulating ROS levels and promoting osteogenic development (Ren et al., 2021; Cai et al., 2022). CeO₂ NPs with strong CAT- and SOD-like activities demonstrated significant anti-inflammatory and antioxidant effects. CeO₂ NPs were tested as in situ injections for managing periodontitis. Both in vivo and in vitro studies showed that CeO₂ NPs effectively neutralize ROS and suppress inflammatory responses by impeding the MAPK–NFκB signaling pathway. In rodent models, CeO₂ NPs notably decreased alveolar bone resorption, reduced osteoclast activity and inflammation, and supported tissue regeneration (Yu et al., 2022). In summary, CeO₂ NPs exhibit significant potential for clinical application in treating periodontitis. Their ability to scavenge ROS, inhibit inflammatory pathways, and promote tissue regeneration makes them valuable in managing oxidative stress-induced inflammatory diseases (Yu et al., 2022).

Carbon-based nanomaterials, including CDs, carbon nanotubes (CNTs), graphene and its derivatives, carbon nitrides, and fullerenes, are extensively used in the biomedical field due to their biocompatibility, physicochemical properties, and enzyme-mimicking abilities. Wang et al. produced a range of oxidized CNTs (o-CNTs) with a high concentration of oxidized groups, which exhibited superior POD-like activity across a broad pH range. The carbonyl groups on the surface of the o-CNTs serve as active catalytic centers, whereas the carboxyl and hydroxyl groups act as competitive sites. The carboxyl group, owing to its inherent negative charge and tendency to form hydrogen bonds, exhibited a greater inhibition of the catalytic activity than the hydroxyl group. Owing to their enhanced POD-like activity and unique surface properties, o-CNTs have great potential for various biomedical applications (Wang H. et al., 2018; Hosseini Hooshiar et al., 2024).

MOFs are crystalline and porous structures formed by organic linkers bridging metallic nodes and demonstrate outstanding mechanical and chemical characteristics. Recently, MOF-based NZs have proven to be highly promising for biological catalysis and sensing due to their versatile structures, high activities, and robust stabilities. MOFs provide a versatile platform for developing novel NZs with built-in catalytic properties for the treatment and diagnosis of bacterial infections. Ongoing research has led to the development of catalytic systems based on MOF and single atoms, which offer greater potential owing to their three dimensional porous structures and increased number of catalytic sites. Single-atom catalytic systems exhibit higher catalytic activity and lower metal consumption (Ali et al., 2021). In one study, researchers have developed an injectable ointment featuring potent anti-biofilm properties and excellent biocompatibility by employing a single-atom catalysis system based on MOFs. Specifically, they utilized a porphyrin-based MOF (PCN-222) integrated with Pt single atoms. Incorporating single metal atoms such as Pt, Au, Cu, and Ru into PCN-222 improves its OXD-like activity by lowering the adsorption and activation energies of O₂. The resulting PCN-222-Pt spontaneously generated ROS and exhibited strong OXD-like- and POD-like activities. It showed remarkable anti-biofilm efficacy, achieving 98.69% inhibition against S. aureus biofilm and 99.91% inhibition against E. coli biofilm in vitro within just 1 hour. Compared to the clinically prescribed periocline, the injectable PCN-222-Pt ointment demonstrated a lower bone degradation rate, healthier periodontal tissue, and reduced inflammation in the treatment of biofilm-induced periodontitis. This offers a fast, efficient, non-invasive, and practical approach for treating periodontitis without antibiotics (Yu et al., 2021).

NZs are extensively studied nanomaterials that exhibit enzymatic activity. However, challenges such as insufficient substrate selectivity, complex composition, and a low density of active sites have limited their widespread application. Enzymes featuring atomically dispersed active sites, which are known for their exceptional performance, have been used in the field of catalysis. SAzymes are highly regarded by researchers for their optimal atom utilization, cost-effectiveness, well-defined coordination structures, and distinct active sites (Han et al., 2023). They have become a focal point in NZ research because of their high activity, stability, and cost-effectiveness, making them excellent alternatives to natural enzymes. In comparison to conventional NZs, SAzymes exhibit superior catalytic activity and specificity owing to their enhanced atom utilization and accurately defined geometric and electronic structures (Jiang et al., 2023).