Carbon-based nanomaterials with higher specific surface area: more expensive but more effective antimicrobials

Abstract

Titanium dental implants are prone to infections by pathogenic bacteria, leading to the formation of biofilm and development of peri-implantitis. Due to their excellent biocompatibility, peroxidase-like activity, and photothermal capabilities, carbon-based nanozymes emerge as a low-risk alternative to traditional antibiotics to treat drug-resistant bacterial infections. However, a crucial question regarding carbon-based nanomaterials with antibacterial properties lies in how to optimize the process parameters to maximize their bactericidal efficacy while minimizing adverse effects such as energy consumption, cytotoxicity, and damage to titanium implants. We have synthesized five groups of carbon-based nanozymes with distinct microstructures through pyrolysis and comprehensively evaluated their performance in five key aspects: energy consumption, photothermal performance, peroxidase-like activity, environmental sensitivity, and cytotoxicity. The results of our experiments provide valuable references for the rational design of carbon-based nanozymes for the treatment of peri-implantitis.

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Donghui Wang

Donghui Wang received his BS degree in Materials Science and Engineering from Sichuan University in 2012, and his PhD degree in Materials Science from the Shanghai Institute of Ceramics, Chinese Academy of Sciences, in 2017. He is currently a professor at Hebei University of Technology. His research focuses on the surface modification of biomedical pure titanium, nitinol and magnesium alloys, and the influence of surface physical and chemical properties on cellular behaviors.

Introduction

Titanium and its alloys are commonly used as the base material for dental implants due to their excellent mechanical properties and good biocompatibility.¹ According to the American Academy of Implant Dentistry, about 3 million Americans have dental implants, and the figure is continuously increasing with about 500 000 new cases per year. Despite the high success rate of dental implant placement, the incidence of peri-implantitis reaches up to 47%,² primarily caused by the formation of bacterial biofilms around the implants, representing a significant complication in clinical practice,³ which can lead to implant loosening and failure.⁴ The key to treat peri-implantitis is to debride the infectious biofilm. Currently, the debridement methods mainly include mechanical debridement,⁵ adjunctive antibiotic therapy,⁶ laser-assisted treatment,^7,8 and adjunctive treatment with high-concentration H₂O₂.⁹ However, these methods are inefficient in clearance of the biofilm,¹⁰ are prone to inducing bacterial resistance,^11–13 and weaken the osteoblast compatibility of titanium implants. Additionally, poor bone integration may create conditions for continued bacterial invasion, leading to a vicious cycle.¹⁴ Therefore, the development of efficient and non-damaging debridement methods for biofilm on titanium implants is of great interest.

In recent years, the emergence of a series of nanomaterials with characteristics such as photothermal,^15–17 photodynamic,¹⁸ chemodynamic,¹⁹ and ultrasonic dynamics²⁰ has provided new possibilities for effective bacterial elimination. These materials can generate heat and radicals by responding to exogenous stimuli (light, magnetic field, microwave, and ultrasound) or chemical substances such as H⁺ and H₂O₂, thereby killing bacteria and avoiding bacterial resistance.²⁰ Among the numerous functional nanomaterials including metal-based nanomaterials,^21,22 polymer-based nanomaterials,²³ MOF-based nanomaterials,²⁴ and carbon-based nanomaterials,^25,26 carbon-based materials have good light absorption at 808 nm and excellent biocompatibility, making them one of the most clinically translatable materials.^27–29 However, optimizing carbon-based materials and maximizing their bactericidal capacity while minimizing energy consumption, cytotoxicity, and damage to titanium implants remain a great challenge.

Recently, researchers have enhanced the enzymatic catalytic activity and bactericidal capacity of carbon-based nanozymes through modification strategies such as microstructural adjustment, surface modification, and element doping. To adjust the microstructure of carbon-based nanozymes, Rostami et al.³⁰ prepared graphene nanoribbons (GNRs) by longitudinal unzipping of carbon nanotubes (CNTs). Compared to CNTs, GNRs exhibit a larger interlayer spacing and a higher I_D/I_G ratio, indicating a higher level of defects. This is beneficial for electron transfer within the material, further enhancing the peroxidase-like activity of GNRs. Ma et al.³¹ applied concentrated HNO₃ oxidation treatment to two-dimensional graphdiyne (GDY). During the oxidation process, the structure of GDY remained intact, and the unique conjugated electronic structure and porous structure of the oxidized product (GDYO) provided abundant transport channels and adsorption sites for substrates, resulting in oxidized GDY sheets with excellent peroxidase-like activity.

To examine the effects of surface modification on the performance of carbon-based nanozymes. Wang et al.³² reported a carbon-based nanozyme (ICG@GO-Apt) composed of aptamers, indocyanine green, and carboxyl-functionalized graphene oxide. Compared to GO, ICG@GO-Apt can specifically adhere to Salmonella typhimurium under near-infrared irradiation and more effectively eradicate its biofilm. Zhang et al.³³ aimed to enhance the antibacterial effect of a multifunctional graphene-based nanozyme (PtCo@G) by modifying its surface with a bacterial targeting molecule, CPB, which carries a hydrophobic alkyl tail. Under acidic gastric conditions, the CPB-modified nanozyme (PtCo@G@CPB) efficiently catalyzes the formation of reactive oxygen species (ROS), demonstrating excellent targeting and antibacterial properties against Helicobacter pylori.

Heteroatom (non-metal) doping can effectively enhance the enzyme-mimetic activities of carbon-based nanozymes. Wang et al.³⁴ reported nitrogen-doped carbon-based nanozymes (N-CNDs) that exhibited superior oxidase-like activity under 365 nm light irradiation. Compared to the undoped carbon-based nanozymes, N-CNDs achieved inactivation rates of 97.91% and 80.02% against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively, under light irradiation. Jiang et al.³⁵ designed a phosphorus-doped carbon-based nanozyme (P-CD). Phosphorus doping imparted higher crystallinity to the nanozymes, leading to enhanced photo-responsive oxidase-like activity. Specifically, 75 μg mL⁻¹ of P-CD killed 98% of E. coli and 50 μg mL⁻¹ of P-CD inhibited 100% of S. aureus under 1 W white light irradiation for 60 minutes. Furthermore, metal atom doping is also an effective strategy to boost the enzyme-like activities of carbon-based nanozymes. Liu et al.³⁶ prepared iron-doped carbon dot (Fe-CD, ∼3 nm) nanozymes. The Fe doping endowed the CDs with photo-enhanced peroxidase-like activity, and the combination of photothermal therapy and ROS generation achieved antibacterial rates of 99.68% and 99.85% against S. aureus and E. coli, respectively.

In the modification strategies for carbon-based nanozymes mentioned above, surface modification and elemental doping introduce new elements, which may pose adverse effects on biocompatibility. However, a strategy involving the modification of carbon-based nanozymes by adjusting their microstructure can circumvent this risk. Herein, we investigate the effects of microstructural changes on the peroxidase-like and photothermal conversion properties of carbon-based materials. We also evaluate their efficacy in removing bacteria and their biofilms. Additionally, we comprehensively assess the materials’ capabilities in terms of energy consumption, photothermal performance, peroxidase-like activity, environmental sensitivity, and cytotoxicity using radar chart scoring. We hope that the relevant data can provide theoretical guidance for the design of carbon-based materials with optimal performance for the eradication of biofilm on titanium implants.

Experimental

Materials and chemicals

Zinc nitrate hexahydrate, 2-methylimidazole, 3,3′,5,5′-tetramethylbenzidine (TMB), O-phenylenediamine (OPD), titanium sulfate (TiSO₄), hydrogen peroxide (H₂O₂, 30 wt%), and acetate buffer were purchased from Aladdin Chemical Reagent Co., Ltd. Alamar blue (AB) was purchased from Thermo Fisher Scientific Co., Ltd. All chemicals were used as received without any further purification. Ultrapure water was obtained using the Milli Q purification system (Merck Millipore, France).

Synthesis

Synthesis of ZIF-8. Zn(NO₃)₂·6H₂O (2.65 g) was dissolved in 40 mL of methanol. Subsequently, this solution was added into a solution of 2-methylimidazole (8 g) in 120 mL of methanol with continuous stirring at 25 °C for 1 h, followed by aging for 24 h. The ZIF-8 product was obtained via centrifugation, washing with methanol three times, and then drying at 60 °C for 24 h.

Synthesis of NPC-T. ZIF-8 (1 g) was pyrolyzed in an Ar atmosphere for 2 h at varying temperatures (600, 700, 800, 900, or 1000 °C). The heating rate was 5 °C min⁻¹. The pyrolysis products were subsequently treated with 1 mol L⁻¹ HCL at 80 °C for 8 h to eliminate Zn element, followed by washing with water and ethanol until the pH of the rinse liquid reached neutrality. The resulting N-doped porous carbons are denoted as NPC-T, with T representing the pyrolysis temperature.

Characterization

The morphologies and dimensions of the NPC-T were characterized using a scanning electron microscope (SEM, S-4800, Japan). X-ray diffraction (XRD) spectra were recorded using a high-throughput Bruker D8 Discover Advance diffractometer (Germany) working in transmission mode and equipped with a focusing Göbel mirror producing CuKα radiation (λ_Cu = 1.5418 Å) and a LynxEye detector. Diffraction patterns were recorded in the 2θ range of 5°–80°. Raman spectra were collected using a Renishaw inVia Reflex spectrometer system (England), equipped with a 532 nm laser excitation source. Nitrogen physisorption was conducted using a specific surface and pore size analyzer (Micromeritics ASAP 2460, USA) at liquid nitrogen temperature (77 K). X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, USA), energy dispersive spectroscopy (EDS, OCTANE PLUS, USA), and inductively coupled plasma-optical emission spectroscopy (ICP-OES, Optima 8300, USA) were used to quantify the elemental contents in the samples. For ICP-OES sample preparation, 25 mg of samples were immersed in 5 mL of 10 wt% HNO₃ to release any Zn element present, and the samples were subsequently diluted to the appropriate ppm range for ICP-OES measurements.

Photothermal performance tests

Photothermal performance tests were carried out at 25 °C. A 1 mL dispersion of NPC-T (1 mg mL⁻¹) was introduced into a 48-well plate and exposed to 808 nm laser irradiation at various power densities (0.25, 0.5 and 1 W cm⁻¹) for 5 min. The real-time temperature of the dispersion was monitored using an infrared thermographic camera (LX-F300, China) at 1-minute intervals. The photothermal stability of NPC-T was evaluated via three laser on/off cycles, and the photothermal conversion efficiency was calculated according to the methods outlined in the ESI.†

Peroxidase-like activity tests

Conventional peroxidase-like activity tests. The analysis of hydroxyl radicals (˙OH) was based on the principle that ˙OH can oxidize the TMB or OPD probe, enhancing the absorbance of TMB at 652 nm or OPD at 424 nm. The peroxidase-like activity tests were carried out in a 0.1 mol L⁻¹ acetate buffer (pH = 3.6) at 25 °C. Typically, 500 μL of a TMB solution (TMB in ethanol, 1.25 mg mL⁻¹)/OPD solution (OPD in ethanol, 0.54 mg mL⁻¹), 50 μL H₂O₂ (10 mmol L⁻¹) and 500 μL of an NPC-T dispersion (1 mg mL⁻¹) were added into acetate buffer, making the total volume of the solution 5 mL. After 30 min, the time-course evolution of the ratio of TMB oxidation product (TMB_ox)/OPD oxidation product (OPD_ox) was obtained by monitoring the absorbance at 652 nm and 424 nm using a UV-vis spectrophotometer (UV-vis, UV2700, Japan). The catalytic performance of NPC-T was further evaluated using a titanium sulfate (TiSO₄) probe. Dispersion of NPC-T (100 μg mL⁻¹), TiSO₄ (10 μg mL⁻¹) and H₂O₂ (100 μM) were mixed at 25 °C. Following a 3-hour incubation, the degradation of TiSO₄ induced by hydrogen peroxide depletion was monitored by the change in absorbance at 405 nm.

Peroxidase-like kinetic tests. Kinetic experiments were conducted using NPC-T and different concentrations of H₂O₂ as the substrate. The experimental procedure was similar to the procedure described above, with a 1 mL test dispersion comprising 100 μL of an NPC-T dispersion (1 mg mL⁻¹), 100 μL of TMB (1.25 mg mL⁻¹) and different volumes of H₂O₂ solution (10 mmol L⁻¹) ranging from 0 to 100 μL. The reaction time was 30 min. The v–[s] and 1/v–1/[s] plots were generated using the Michaelis–Menten fitting method in GraphPad Prism software.

Peroxidase-like environmental sensitivity tests. The impact of pH and temperature on the peroxidase-like activity of NPC-T was investigated within the pH range of 3.6–7.4 and the temperature range of 25–55 °C using TMB as a probe.

In vitro cell cytotoxicity tests

AB was employed to assess the cell toxicity of the samples. Human bone marrow mesenchymal stem cells (hBMSCs), mouse embryonic osteoblasts (MC3T3-E1), and mouse embryonic fibroblasts (L929) were cultured in a 96-well plate with a density of 5000 cells per well and allowed to attach for 24 hours. Subsequently, the old medium was replaced with a fresh culture medium containing NPC-T at various concentrations (0, 100, 250, 500, and 1000 μg mL⁻¹). After an additional 24 hours of culture, 100 μL of 10% AB solution was added to the respective wells and incubated for 4 hours. AB was used as the colorant, the fluorescence intensity of each well was determined using a fluorescence enzyme marker (Varioskan LUX, USA) with a detection wavelength of 560 nm (Ex)/590 nm (Em). All experiments were repeated three times. Cell viability was calculated using the following equation:

In the above formula, F is the fluorescence intensity of cells treated with NPC-T; F₀ is the fluorescence intensity of cells without any treatment; and F_blank is the fluorescence intensity of the medium containing 10% AB.

In vitro antibacterial tests. During antibacterial investigations, E. coli was used as the model Gram-negative bacterial and S. aureus and Streptococcus mutans (S. mutans) as model Gram (+) bacterial cell strains. The E. coli, S. aureus, and S. mutans strains were revived from frozen glycerol stocks by inoculation in Luria-Bertani (LB) or Brian Heart Infusion (BHI) medium overnight at 37 °C in an incubator. The density of the bacterial cells was determined by measuring the absorbance at 600 nm. E. coli, S. aureus and S. mutans were individually divided into the following eight groups: (1) bacteria; (2) bacteria + NIR light; (3) bacteria + H₂O₂; (4) bacteria + NIR light + H₂O₂; (5) bacteria + NPC-T; (6) bacteria + NPC-T + NIR; (7) bacteria + NPC-T + H₂O₂; and (8) bacteria + NPC-T + NIR light + H₂O₂. Groups (2), (4), (6), and (8) were further exposed to NIR laser irradiation (808 nm, 1.0 W cm⁻²) for 5 min. After the NIR irradiation, the procedure was the same as that for groups (1), (3), (5), and (7). The final concentrations of NPC-T, H₂O₂, and bacteria were 250 μg mL⁻¹, 1 mM, and 1 × 10⁶ CFU mL⁻¹, respectively. The total volume of dispersion in each well was 1 mL. After incubation for 15 min, 100 μL of bacterial suspensions of groups (1)–(8) were spread on agar culture plates and incubated at 37 °C for 24 h, and the number of colonies was counted. All experiments were repeated three times.

Biomass staining tests. The bacterial biofilms that underwent different treatments were stained with 0.1% crystal violet staining solution for 20 min. Subsequently, the biofilms were washed with PBS until the wash solution became clear, followed by drying at 25 °C. Then ethanol was applied to decolorize the biofilms. After 30 min, the biomass was monitored spectrophotometrically by measuring the absorbance at 590 nm.

Live/dead staining tests. Cells that underwent different treatments were stained with calcein-AM and propidium iodide (PI) for 30 min, followed by washing with PBS for three times. Fluorescence microscopy was used to observe the live (green fluorescence) and dead (red fluorescence) cells.

Bacterial biofilms that underwent different treatments were stained with N01 and PI for 30 min, followed by washing with PBS for three times. Fluorescence microscopy was used to observe the live (green fluorescence) and dead (red + green = yellow fluorescence) bacteria.

Morphological observation of the bacterial biofilms. Bacterial biofilms inoculated on smooth titanium sheets (1 cm × 1 cm) were fixed with 2.5% glutaraldehyde. After 12 h, the fixed samples were further dehydrated gradually using ethanol solution with increasing ethanol concentrations (30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% and 100%). Finally, the biofilms inoculated on titanium sheets were observed by SEM.

Criteria for radar chart rating. All ratings in this work are categorized as “1”, “2”, “3”, “4”, and “5”, with higher numerical ratings indicating better performance.

Criteria for assessing pyrolysis energy consumption. The group with the lowest energy consumption required for the synthesis is rated as “5”, and, in order, the higher the energy consumption required for synthesis, the lower the rating given.

Criteria for assessing photothermal performance. The group with the highest heating rate after irradiation using 808 nm near-infrared light (1 W cm⁻², 5 min) was rated as “5”, and, in order, the slower the heating rate, the lower the rating given.

Criteria for assessing peroxidase-like activities. The group with the fastest generation rate of ˙OH was rated as “5”, and, in order, the slower the enzyme catalysis rate, the lower the rating given.

Criteria for assessing environmental sensitivity. The group with the fastest generation rate of ˙OH under acidic pH conditions and near-infrared light irradiation was rated as “5”, and, in order, the slower the enzyme catalysis rate, the lower the rating given.

Criteria for assessing biocompatibility. The comprehensive assessment of hBMSCs’ cell viability after treatment with different concentrations of NPC-T (250, 500, and 750 μg mL⁻¹). The group with the highest cell viability is rated as “5”, and, in order, the lower the cell viability, the lower the rating given.

Statistical analysis

All data were given as mean ± standard deviation. Statistically significant differences (P) between groups were analyzed using one-way analysis of variance (ANOVA) in the GraphPad Prism statistical analysis software package. Significant differences between groups were analyzed in each group containing at least three parallel samples. P values < 0.05 are denoted by “*”, P values < 0.01 are denoted by “**”, P values < 0.001 are denoted by “***”, P values < 0.001 are denoted by “****”, and P values < 0.0001 are denoted by “****”.

Results and discussion

Preparation and characterization of NPC-T

The NPC-T samples were prepared by pyrolyzing ZIF-8 crystals at different temperatures (Fig. 1a). The balance between structural order and defects is optimal at higher temperatures: when T was less than 500 °C, the obtained NPC-T would dissolve during the acid etching process, so the pyrolysis starting temperature was set to 600 °C. ZIF-8 crystals were synthesized referring to a previous report,³⁷ resulting in yielded characteristic truncated rhombic dodecahedral crystals with a size of approximately 100 nm, and all NPC-T samples inherited the truncated rhombic dodecahedral morphology of the ZIF-8 precursor (Fig. 1b). As shown in Fig. 1d and e, the XRD patterns revealed the decomposition of ZIF-8 to a carbon phase above 600 °C. This was evidenced by the complete disappearance of the sharp ZIF-8 peaks and the emergence of new broad peaks at 26° and 44°, corresponding to the (0 0 2) and (1 0 0/1 0 1) planes of nanocrystalline graphitic carbon.³⁸ XPS data confirmed the presence of elements C, N, O, and Zn in NPC-T samples (Table S1, ESI†). EDS confirmed that the weight ratio of Zn in all NPC-T samples ranged from 0.74 to 2.96 wt%. ICP-OES determined that the Zn contents in the NPC-T samples were 102.3–197.5 mg mL⁻¹ (Table S3, ESI†). NPC-1000 with the highest pyrolysis temperature has the lowest zinc ion content, which may contribute to the formation of its high specific surface area and defective structure, and improve its peroxidase like catalytic efficiency while improving its biocompatibility. All NPC-T samples exhibited similar adsorption/desorption characteristics (Fig. 1e–i), indicating that the pyrolysis temperature did not significantly alter the structure of the product NPC-T. The Brunauer–Emmett–Teller (BET) specific surface areas of NPC-T were 64.8, 691.4, 771.1, 811.5, and 994.6 m² g⁻¹, respectively. Based on the Barrett–Joyner–Halenda (BJH) method, the cumulative pore volumes were 0.37, 0.55, 0.72, 0.77, and 0.83 cm³ g⁻¹, respectively. The average pore sizes were 34.6, 16.1, 21.8, 27.7 and, 27.5 nm, respectively (Table S4, ESI†). The BET data showed that the cumulative pore volume and average pore diameter of NPC-T did not change significantly with increasing temperature, but the specific surface area of the product increased significantly. Raman spectra of NPC-T samples exhibited two typical peaks at about 1335 cm⁻¹ for the D band (defects in the carbon structure) and 1590 cm⁻¹ for the G band (sp²-hybridized carbon structure unit) in graphitized carbon. Additionally, NPC-600, NPC-700, and NPC-800 showed miscellaneous peaks representing ZIF-8 between the D and G bands, indicating that a pyrolysis temperature of 900 °C is required for complete carbonization of ZIF-8. Based on the Raman spectra, the I_D/I_G values of NPC-T were 0.98, 1.09, 1.29, 1.33, and 1.36, respectively (Fig. 1j), which indicated that the degree of defects in NPC-T obtained at higher temperatures is higher.

Fig. 1 (a) Schematic illustration of the synthesis of NPC-T. (b) SEM images of ZIF-8 and NPC-T. XRD patterns of (c) ZIF-8 and (d) NPC-T. N₂ absorption and desorption curves of (e) NPC-600, (f) NPC-700, (g) NPC-800, (h) NPC-900, and (i) NPC-1000. (j) Raman spectra of NPC-T.

During the high temperature pyrolysis process, ZIF-8 crystals retain their special rhombic dodecahedral morphology, but their organic components have been gradually carbonized and zinc is gradually evaporated, leaving holes on the surface of the material. The higher the pyrolysis temperature of the NPC-T samples, the more the pores formed, the larger the specific surface area, and the greater the degree of defects. When the pyrolysis temperature reaches 900 °C or above, no characteristic peaks attributable to ZIF-8 can be found in the XRD and Raman spectra, which proves that ZIF-8 has been completely carbonized at this temperature.

The energy consumption during the preparation of NPC-T samples is mainly related to the pyrolysis temperature and the heating time, and the heating time is the same for all NPC-T samples with different pyrolysis temperatures, so in this study the ratings of NPC-600, NPC-700, NPC-800, NPC-900, and NPC-1000 are “5”, “4”, “3”, “2”, and “1”, respectively.

Photothermal performance of NPC-T

Satisfactory near-infrared absorption is crucial for effective photothermal reagents. To investigate the photothermal performance of NPC-T, we examined the heating effect of all NPC-T samples under 808 nm laser irradiation (0.25, 0.5 and 1 W cm⁻², 5 min). The photothermal heating curves of NPC-T under 808 nm laser irradiation showed that all samples had power density dependent photothermal heating performance, with the photothermal performance of the samples, except NPC-600, being essentially identical (Fig. 2a–f). Subsequently, the photothermal conversion efficiency of NPC-T was calculated to be 58.46, 67.88, 68.58, 69.15 and 68.15%, respectively (Fig. 2g). The inferior photothermal performance of NPC-600 is attributed to incomplete carbonization, while the superior photothermal conversion efficiency of other NPC-T samples may be attributed to their excellent near-infrared absorption.

Fig. 2 (a) Temperature-elevating curves under 808 nm laser irradiation (1 W cm⁻²). Temperature-elevating curves of (b) NPC-600, (c) NPC-700, (d) NPC-800, (e) NPC-900, and (f) NPC-1000 at different laser powers. (g) Photothermal conversion efficiency of NPC-T. Photothermal stability (three laser on/off cycles) of (h) NPC-600, (i) NPC-700, (j) NPC-800, (k) NPC-900, and (l) NPC-1000.

Excellent photothermal stability is crucial for successful photothermal therapy. Therefore, the photothermal stability of NPC-T was evaluated. After three laser (1 W cm⁻², 5 min) on/off cycles, the amplitude of temperature elevation did not change significantly (Fig. 2h–l), indicating the good photothermal stability of NPC-T.

In conclusion, all NPC-T samples demonstrated excellent photothermal stability, thereby eliminating hierarchy in the radargram. The photothermal conversion efficiency was the main evaluation parameter for each group, and finally, in the photothermal performance section of the radargram, NPC-600, NPC-700, NPC-800, NPC-900, and NPC-1000 received the ratings of “4,” “5,” “5,” “5,” “5,” and “5,” respectively.

Peroxidase-like activity of NPC-T

N-doped nanocarbons are capable of catalysing the peroxidase-like decomposition of H₂O₂ into ˙OH. Initially, the peroxidase-like catalytic activity of NPC-T was evaluated using sufficient concentrations of H₂O₂ and TMB as substrates. TMB_ox serves as an indicator of ˙OH, the production of which induces a color change from colorless to blue (Fig. S1, ESI†).³⁹ The group of H₂O₂ + TMB (control) did not produce blue color or absorption at 652 nm, whereas the groups of NPC-T + H₂O₂ +TMB showed absorption at 652 nm. The absorption peak of NPC-T at 652 nm is more pronounced at higher pyrolysis temperatures, indicating that NPC-T obtained at higher pyrolysis temperatures possessed enhanced peroxidase-like catalytic activity (Fig. 3a). In addition, the peroxidase-like catalytic performance of NPC-T was further tested with OPD (Fig. 3b and Fig. S2, ESI†)⁴⁰ and TiSO₄ probes (Fig. 3c). Similar phenomena were observed with TMB, OPD and TiSO₄ probes, providing comprehensive evidence that NPC-T obtained at higher temperatures displayed exceptional peroxidase-like catalytic activity. In summary, using TMB, OPD, and TiSO₄ as probes, the order of peroxidase-like activity for all NPC-T from strongest to weakest was recognized as: NPC-1000 > NPC-900 > NPC-800 > NPC-700 > NPC-600. Therefore, in the peroxidase-like activity section of the radar chart, the ratings for NPC-600, NPC-700, NPC-800, NPC-900, and NPC-1000 are “1”, “2”, “3”, “4”, and “5”, respectively.

Fig. 3 Peroxidase-like activities and colorimetric images of NPC-T probed with (a) TMB, (b) OPD, and (c) TiSO₄. (d) The v–[s] images of NPC-T. The 1/v–1/[s] images of (e) NPC-600, (f) NPC-700, (g) NPC-800, (h) NPC-900, and (i) NPC-1000.

To delve into the catalytic mechanism of NPC-T, a more detailed kinetic study was therefore performed on all samples. The constant K_m was employed to estimate the binding affinity for a particular substrate, and a lower value of K_m indicates a stronger binding affinity between the enzyme and the substrate. Furthermore, a higher value of V_max proved that the reaction rate is higher. The value of V_max/K_m represents the rate of adsorption/desorption of H₂O₂ from the sample. Michaelis–Menton analysis revealed that the NPC-T obtained at higher pyrolysis temperatures exhibited a swifter rate of adsorption/desorption of H₂O₂ (Fig. 3d–i), indicating that a higher specific surface area facilitates a more efficient reaction of the substrate with the material, thereby amplifying the rate of adsorption/desorption of H₂O₂ on the surface of the sample, and thus increasing its peroxidase-like activity.

Environmental sensitivity of NPC-T

In general, all NPC-T samples could act as peroxidase-like nanozymes, had ˙OH generation ability and showed photothermal performance. It is well-established that the catalytic activity of most natural peroxidases, represented by horseradish peroxidase (HRP), is strongly contingent on temperature and pH. Similarly to HRP, all NPC-T samples also exhibited a strong pH-dependence, with higher catalytic activity observed in acidic environments (Fig. 4a). It was noteworthy that all NPC-T samples demonstrated enhanced enzyme-like activity under 808 nm laser (0.5 W cm⁻², 5 min) irradiation (Fig. 4b), potentially attributed to the elevated temperature. To substantiate this hypothesis, the peroxidase-like activities of all the samples at temperatures of 25, 37, 45, 50, and 55 °C were analyzed in conjunction with the photothermal capacity of the individual samples. Under peroxidase-like catalysis, ˙OH generated from H₂O₂ decomposition oxidizes TMB to form a blue product (TMB_ox) with an absorbance peak at 652 nm. The absorbance at 652 nm correlates positively with the amount of ˙OH (i.e., catalytic activity). Catalytic activity was tested at 25 °C, 37 °C, 45 °C, 50 °C, and 55 °C, corresponding to labels (1)–(5) in the legends of Fig. 4c–h. For each temperature, the colorimetric intensity of the solution after the reaction of NPC-T with H₂O₂ and TMB reflects activity differences. In Fig. 4h, the solution color in the sample vials for each group (left to right) becomes increasingly deeper blue with increasing temperature, indicating higher TMB_ox production and thus enhanced peroxidase-like activity. Analysis suggests that the photothermal conversion ability of NPC-T enables local heating under near-infrared irradiation. Higher temperatures accelerate molecular motion, enhancing H₂O₂ and TMB adsorption/reactivity on the material surface and boosting catalytic efficiency. Kinetic analysis (Fig. 3d–i) shows that, at high temperatures, the enzymatic reaction rate (V_max) of NPC-T increases significantly, while the Michaelis constant (K_m) decreases, indicating stronger substrate binding affinity. This aligns with quantitative colorimetric results, further supporting the conclusion that elevated temperatures enhance activity. The colorimetric results in Fig. 4h complement the findings in Fig. 4b (NIR on/off effect) and Fig. 4c–g (temperature-dependent absorbance curves), and the quantified absorbance data in Table S5 (ESI†). Overall, the peroxidase-like activity of NPC-T increases with temperature from 25 °C to 55 °C (Fig. 4c–h). It was observed that from 25 to 55 °C, the peroxidase-like activity of all NPC-T samples increased with increasing environmental temperature (Fig. 4c–h).

Fig. 4 Peroxidase-like activity of NPC-T (a) in different pH environments and (b) at 808 nm NIR light on/off. Peroxidase-like activities of (c) NPC-600, (d) NPC-700, (e) NPC-800, (f) NPC-900, and (g) NPC-1000 at different temperatures. (h) Colorimetric images of NPC-T at different temperatures. Labels (1)–(5) correspond to reaction temperatures of 25 °C, 37 °C, 45 °C, 50 °C and 55 °C, respectively.

In conclusion, the analysis of the peroxidase-like activities of NPC-T in different pH environments showed that there was no significant difference in the peroxidase-like activities of all NPC-T samples in the pH range of 5.5–7.4, but when the environmental pH was further reduced to 4.5 or even 3.6, the peroxidase-like activities of NPC-T were all improved, and the rates of their enzyme activities were ranked from the highest to the lowest as follows: NPC-1000 > NPC-900 > NPC-800 > NPC-700 > NPC-600. Moreover, the analysis of the peroxidase-like activities of NPC-T at different temperatures reveals that the peroxidase-like activity of all NPC-T increases with temperature, with almost identical increments. Therefore, in the environmental sensitivity section of the radar chart, NPC-600, NPC-700, NPC-800, NPC-900, and NPC-1000 are rated as “1”, “2”, “3”, “4”, and “5”, respectively.

Cytotoxicity of NPC-T

Based on clinical relevance, experimental standardization and international biocompatibility evaluation norms, hBMSCs, MC3T3-E1 and L929 cell lines were selected to test the cytotoxicity of the materials. Taking NPC-1000 as an illustrative case, we explored the cytotoxicity of varying sample concentrations on hBMSCs. It was obvious that there was no significant effect on the cell proliferation of hBMSCs when the concentration of NPC-1000 remained below 250 μg mL⁻¹, with the cell survival rate exceeding 80% (Fig. 5a). Subsequently, a comprehensive investigation was conducted on the proliferation effects of 250 μg mL⁻¹ NPC-T on hBMSCs to assess the cytotoxicity across all sample groups. It was found that more than 80% of hBMSCs could still survive after treatment with NPC-T with a concentration of 250 μg mL⁻¹, showing good biocompatibility (Fig. 5b). Consistent findings were observed in the live–dead staining images (Fig. 5c) of hBMSCs. To further investigate the biocompatibility of NPC-T, we evaluated the cytotoxicity of 250 mg mL⁻¹ of NPC-T on L929 (Fig. S3, ESI†) and MC3T3-E1 (Fig. S4, ESI†), and no significant cytotoxicity was found. Furthermore, we refined the concentration of NPC-T to 500 and 750 μg mL⁻¹, exploring the cell viability of hBMSCs following treatment with elevated concentrations of NPC-T samples (Fig. 5d and e). It was evident that when the concentration of NPC-T samples was increased to 750 μg mL⁻¹, the cell viability of the experimental group was significantly different from that of the control group.

Fig. 5 The viability of hBMSCs treated with (a) different concentrations of NPC-1000 and (b) 250 μg mL⁻¹ NPC-T. (c) Live/dead staining images of hBMSCs treated with 250 μg mL⁻¹ NPC-T. The viability of hBMSCs treated with (d) 500 μg mL⁻¹ and (e) 750 μg mL⁻¹ NPC-T.

In summary, in a comprehensive comparison of the biocompatibility results for all NPC-T samples, the radar chart in the biocompatibility section assigns ratings of “5”, “4”, “3”, “2”, and “2” to NPC-600, NPC-700, NPC-800, NPC-900, and NPC-1000, respectively.

Comprehensive performance evaluation of NPC-T

The comprehensive score of the performance of the NPC-T samples is shown in Fig. 6, where an inverse relationship is observed between the photothermal performance, peroxidase-like activity and environmental sensitivity of the samples and the energy consumption and biocompatibility required for production. Preparation schemes can be chosen based on the key properties required for specific application scenarios.

Fig. 6 Rating scores and radar charts for the performance of each of the NPC-T samples.

Antibacterial properties of NPC-T

NPC-T exhibits peroxidase-like activity, enabling it to catalyze the generation of large amounts of reactive oxygen species (ROS) even at low H₂O₂ concentrations. This catalytic effect significantly enhances the bactericidal ability of H₂O₂, enabling efficient antibacterial performance without relying on high concentrations of H₂O₂. Given the aforementioned exceptional properties of NPC-T, we explored the bactericidal effect of the photothermal cascade-like peroxidase catalysis of NPC-T. Fig. 7a shows the photographs of bacterial colonies of E. coli treated with NPC-T. It was found that all NPC-T samples themselves did not exhibit a significant bactericidal effect on E. coli, which also proved their good biocompatibility. However, after applying 808 nm laser irradiation and low concentration H₂O₂ stimulation to the samples, all NPC-T samples showed obvious antibacterial effects, and the NPC-T obtained with higher temperature treatment exerted more significant antibacterial effects. Fig. 7b shows the photographs of bacterial colonies of S. aureus treated with NPC-T. It was found that all the NPC-T samples themselves did not exhibit a significant bactericidal effect on S. aureus. Individual exposure of the samples to low concentrations of H₂O₂ or 808 nm laser irradiation alone did not significantly enhance resistance to S. aureus. This is attributed to the fact that S. aureus is a heat-resistant bacterial species, and the heat generated by NPC-T under laser irradiation is insufficient to produce a conspicuous bactericidal effect against S. aureus. Additionally, S. aureus possesses carotenoids with antioxidant properties, imparting a degree of resistance to low concentrations of H₂O₂ and ˙OH. However, when we applied both external stimuli at the same time, all NPC-T samples, except NPC-600, showed a significant killing effect against S. aureus, which was attributed to the fact that the concentration of ˙OH produced by them at higher temperatures surpassed the threshold required for S. aureus extermination. Fig. 7c shows the photographs of bacterial colonies of S. mutans treated with NPC-T. It was found that NPC-T itself had no killing effect against S. mutans. The growth of S. mutans was slightly inhibited when applying low concentrations of H₂O₂ and laser irradiation alone to the samples. The growth inhibition of S. mutans by NPC-T became more pronounced after applying both external stimuli simultaneously.

Fig. 7 Colony generation images of NPC-T after co-incubation with (a) E. coli, (b) S. aureus, and (c) S. mutans for 15 min under different conditions.

Biofilm removal properties of NPC-T

Prolonged colonization of the oral cavity by bacteria leads to the formation of biofilms, which adheres to titanium implants causing peri-implantitis and exacerbating bone defects around the implant. Therefore, antimicrobial materials for the treatment of peri-implantitis should be able to remove biofilms. First, we successfully modeled the biofilm using S. mutans as the pathogenic bacterium, which was cultured in a medium containing 1% sucrose for a time period ranging from 4 hours to 2 days. From the crystal violet staining results, it was clearly observed that 4–8 hours was the logarithmic growth phase of the S. mutans colony number. During this period, bacterial biomass exhibited rapid growth, with the growth rate decelerating after 8 hours (Fig. 8a). SEM images (Fig. 8b) and live/dead staining images (Fig. 8c) of the bacterial biofilm acquired after incubation for different times revealed that the results were consistent with the conclusions obtained by the crystal violet staining method.

Fig. 8 (a) Biomass, (b) SEM images, and (c) live/dead staining images of S. mutans biofilms after incubation for different times. (d) The proliferation of hBMSCs on smooth titanium sheets after different treatments. (e) Biomass, (f) SEM images, and (g) live/dead staining images of S. mutans biofilms after different H₂O₂ treatments.

Upon comprehensive consideration, S. mutans could be identified as having developed a dense biofilm after a 1-day incubation period. Therefore, all biofilms utilized in subsequent experiments were cultured for a duration of 1 day. A 3% H₂O₂ solution has been recognized as a potent agent for biofilm removal in the oral cavity, primarily due to its rapid and efficient eradication of bacteria at elevated concentrations.

Nevertheless, heightened concentrations of H₂O₂ were also found to corrode the surface of titanium implant prostheses. Severely corroded titanium surfaces could impede cell proliferation in the vicinity. In contrast, titanium treated with a lower concentration of H₂O₂ (0.5%) had no significant effect on hBMSCs’ proliferation (Fig. 8d). By introducing 1% H₂O₂ in the control group, it can be demonstrated that, with the increase of concentration, the survival rate of cells gradually decreased. Therefore, the ideal biofilm remover should be capable of effectively eliminating bacteria and biofilm at low concentrations of H₂O₂.

To assess the efficacy of NPC-T for S. mutans biofilm removal, samples from each group were co-cultured with biofilm for 15 min, during which they were double-stimulated using 0.5% H₂O₂ and 808 nm laser irradiation. According to the crystal violet staining results of the S. mutans biofilm after treatment by different methods, it was seen that all groups of NPC-T exhibited a notable biofilm cleaning ability. Consistent with the resistance trend observed in free bacteria, NPC-T obtained at higher pyrolysis temperatures demonstrated increased effectiveness in biofilm eradication under dual stimulation, where NPC-1000 was as effective as 3% H₂O₂ for biofilm removal (Fig. 8e). SEM images (Fig. 8f) and live/dead staining images (Fig. 8g) of biofilms treated by different means also corroborated these findings.

Conclusions

In summary, preparation using ZIF-8 as a precursor resulted in NPC-T materials exhibiting peroxidase-like activity dependent on pyrolysis temperature, with higher temperatures enhancing activity. This enhancement is attributed to the larger specific surface area of NPC-T obtained at higher pyrolysis temperatures, which facilitates the adsorption/desorption of H₂O₂ on the material surface. In addition, NPC-T has significant near-infrared absorption ability and can efficiently convert light energy into heat. The generated heat through the photothermal effect further amplifies the peroxidase-like catalytic activity of NPC-T, leading to increased ROS production. Simultaneously, the outcomes of antibacterial experiments reveal that, due to their catalytic and photothermal synergistic effects, all NPC-T samples exhibit pronounced antibacterial effects against E. coli, S. aureus and S. mutans, and demonstrate excellent biofilm-removal effects against biofilms formed by the aggregation of a large number of S. mutans. Most significantly, we created a radar chart encompassing energy consumption for production, photothermal performance, peroxidase-like activity, environmental sensitivity, and cytotoxicity as five scoring criteria for each NPC-T group. This facilitates a clearer comparison of advantages and disadvantages among the samples in each group, offering theoretical guidance for designing carbon-based nanozymes for the treatment of peri-implantitis.

Author contributions

Ye Feng: validation, writing – review & editing. Xin Zhang: data curation, formal analysis, investigation, writing – original draft. Chunyong Liang: funding acquisition, resources. Dan Xia: supervision. Donghui Wang: conceptualization, funding acquisition, supervision, writing – review & editing, project administration. Baoe Li: supervision, funding acquisition, resources. Ting Ma: supervision, validation, visualization.

Data availability

The ESI† is available free of charge via the Internet at https://pubs.acs.org and includes supplementary data (tables and figures) on the composition and elemental contents in each NPC-T sample group, as well as the BET related data of NPC-T. The calculation formula and derivation process of photothermal conversion efficiency are also included in the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors gratefully acknowledge the support from the National Natural Science Foundation of China (52271245 and U21A2055), the Natural Science Foundation of Hebei Province (H2022202007 and E2024202100), and the Natural Science Foundation of Tianjin (24JCYBJC01100).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb02778g

‡ These authors contributed equally to this work.

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