In chemico categorization of magnetite-, hydroxyapatite-, and Ag-derived hybrid nanobiomaterials based on the surface oxidative reactivity: implications of doping and coating

Abstract

In chemico tests are important tools that complement in silico, in vitro and in vivo approaches to predict the toxicological impact of nanomaterials (NMs). Here, we apply a recently proposed in chemico methodology, based on the evaluation of the number, nature and properties of reactive surface sites of NMs, to a series of magnetite-, hydroxyapatite- and silver-based hybrid nanobiomaterials (NBMs). The properties of the NBMs were examined using methanol chemisorption followed by temperature-programmed surface reaction (MeOH-TPSR), dithiothreitol (DTT) oxidation, cyclic voltammetry in biologically relevant media, and electron paramagnetic resonance (EPR) spectroscopy in a series of relevant media as a spin trap. The resulting data were critically compared and correlated with the available in vitro data of the NBMs’ hazard. Our findings reveal significant differences in the oxidative potential of these hybrid NBMs. Iron (Fe) doping in hydroxyapatite (HA) introduced new redox-active surface sites, leading to increased oxidative reactivity via ROS-independent mechanisms, as evidenced by higher DTT depletion and Fenton-like activity compared to HA. Conversely, titanium (Ti) doping modified HA's surface by introducing acidic active sites, reducing its oxidative capacity. Coating Fe₃O₄ with poly(ethylene glycol)-poly(lactic-co-glycolic) acid (PEG-PLGA) enhanced the oxidative reactivity without ROS generation, suggesting a surface-driven process. In contrast, hydroxyethyl cellulose (HEC) coating significantly reduced the high reactivity of uncoated silver (Ag). This study underscores the importance of determining the NBMs’ reactivity profile for safe biomedical use, highlighting how specific coatings and dopants can transform oxidative surface properties.

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Introduction

The integration of nanotechnology and biotechnology has enabled the development of advanced hybrid nanobiomaterials (NBMs) for applications in medical devices, diagnostics, and tissue regeneration.¹ To address clinical challenges like pathogen adhesion and biofilm formation, coatings or doping strategies are proposed to modify the surfaces of common NBMs,² such as magnetite,^3,4 hydroxyapatite (HA),^5,6 and silver^7,8 nanoparticles, altering the number and nature of their surface sites, which in turn changes their oxidative reactivity.^9,10 This reactivity can be a double-edged sword; it may induce toxic effects,¹¹ but it can also serve as an antimicrobial agent¹² with potential applications in areas like food packaging¹³ or a redox regulator in neurodegenerative diseases.¹⁴

Toxicity testing for NBMs, often referred to as nanosafety, is complicated by the growing diversity of their property variants. Nanosafety assessment has traditionally relied on in vivo tests, which are not only time-consuming and resource-intensive, but also increasingly misaligned with global efforts to reduce animal testing.^15,16 Therefore, new approach methodologies (NAMs), based on in silico,^17,18in chemico^19,20 and in vitro^11,21–25 approaches, have appeared as promising alternatives^26–28 unified by a singular objective to facilitate the prediction of the potential hazard or toxicity of nanomaterials, while simultaneously reducing animal testing and the costs of assays.^29,30 This endeavor necessitates the comprehensive characterization of various properties and the establishment of robust correlations between them.^31,32

Assessing not only the physicochemical but also the biological identity of NBMs, i.e., their characteristics and properties in physicochemical and biological media, is crucial for determining the likelihood of NBMs initiating hazardous events and how their intensity is modulated.^33,34 Though effects attributable to particle composition, size and morphology cannot be disregarded, those related to surface chemistry-dependent phenomena are predominant, particularly in metal and metal oxide nanoparticles.^19,35 A spectrum of complementary cellular and acellular techniques can be employed to derive specific descriptors, ideally correlating with in vitro^25,36 or in vivo^37–39 adverse effects, thereby helping to determine the modes of action (MoA) and thus providing extrapolating criteria for the safe-and-sustainable-by-design (SSbD) development of NBMs.³² Additionally, these descriptors can be used for the identification and development of redox-active nanomaterials, of increasing interest for therapeutic purposes in the nanomedicine field.⁴⁰ Thus, surface charge, surface chemistry or surface defect-dependent oxidation or reduction processes^41–44 are important descriptors that should be evaluated in relevant exposure media to better correlate with biological responses, as any surrounding conditions may alter these properties.⁴⁵ Obviously, characterization techniques that quantify the number and nature of reactive surface sites and reactive oxygen species (ROS) production provide valuable descriptors for assessing the redox reactivity, which involves electron transfer during reduction and oxidation reactions,^46,47 and correlating it with oxidative stress damage in cells, one of the most accredited mechanisms driving nanomaterials’ toxicity.^48–50

The aim of this work is to analyze the surface property modifications induced by metal doping in nano-HA or by organic coatings on Ag- and Fe₃O₄-derived NBMs, as case studies representative of commonly used metal-containing NBMs. An in chemico multi-technique methodology is proposed for quantifying the number and nature of reactive surface sites and evaluating redox reactivity. The methodology involves gas-phase methanol chemisorption followed by temperature-programmed surface reaction (MeOH-TPSR) for reactive site quantification and categorization; liquid-phase probe reactions such as dithiothreitol (DTT) oxidation, for general oxidative potential assessment, and salicylic acid hydroxylation, for OH radical generation; spin-trap methods to identify free radicals by electron paramagnetic resonance (EPR) analysis; and cyclic voltammetry to determine surface-driven oxidation processes.

Experimental

Materials and reagents

The NBMs analyzed in this study include two lab-scale doped hydroxyapatites (FeHA and TiHA),⁵¹ a commercial magnetite with a bioresorbable organic coating of poly(ethylene glycol) and poly(lactic-co-glycolic) acid from Colorobbia Italia SpA (Fe₃O₄_PEG-PLGA),³⁴ and lab-scale silver nanoparticles coated with a shell of hydroxyethyl cellulose (AgHEC).^52,53 These NBMs were supplied through the BIORIMA EU project materials repository and were selected based on their biomedical relevance, extensive prior characterization, and commercial availability. Hydroxyapatite, magnetite, and silver are examples of NBMs already used in biomedical applications, and functionalization strategies such as coating and doping are commonly employed to enhance their biocompatibility and efficacy. These materials are categorized as biomimetic and/or bioresorbable and are capable of eliciting specific cellular responses at the molecular level.^54,55 In addition, CuO, HA, Fe₃O₄, and Ag NP reference materials were purchased from Sigma-Aldrich for comparison. Dulbecco's Modified Eagle's Medium (DMEM) with fetal bovine serum (FBS) was prepared by mixing DMEM without sodium pyruvate with 10%v/v FBS (both from Thermo Fisher Scientific) at room temperature.

Physicochemical characterization

The physicochemical and biological characteristics of the target samples were assessed by performing analyses in a powder or liquid suspension state. For the liquid-phase experiments, the powdered nanomaterials (HA, Ag, Fe₃O₄, TiHA, and FeHA) were dispersed according to the NanoGenoTox protocol,⁵⁶ whereas the colloidal samples (Fe₃O₄_PEG-PLGA and AgHEC) were first diluted in Milli-Q water to a concentration of 256 ppm and then diluted in the selected media.

Specific surface area. All nanomaterials were pretreated by degassing for 16 h at 120 °C prior to measurement using Micromeritics ASAP 2020 nitrogen adsorption isotherm equipment. The specific surface area was calculated using the BET equation.

Hydrodynamic diameter and zeta potential. The hydrodynamic diameter (d) and polydispersity index (PDI) of the nano-suspensions in all media as well as the zeta potential (ζ) were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements, respectively, using a Zetasizer Nano instrument ZSP (model ZEN5600, Malvern Instruments, UK). Instrument settings are detailed in the ESI,† while ζ data with the relative standard deviation (RSD, %) were obtained by averaging the signal of three measurements.

Dissolution. Static dissolution measurements were performed to assess the release of Fe^2+/3+ and Ag⁺ from the nanoparticles at established incubation time points. The experimental procedure is schematized in Fig. S1.† The dispersion was diluted in the relevant media (at concentrations of 10–50 mg L⁻¹ (ppm)) and incubated in a thermal bath for 1 h and 24 h at 37 °C. The ion release rate was calculated as the ratio between the dissolved ions after ultrafiltration and the total iron (Fe-based NPs and Fe^2+/3+) or silver (Ag-based NPs and Ag⁺) present in the initial suspension, expressed as percentage. The results from ICP-OES were reported as the average of three independent measurements with the relative standard deviation (RSD).

Surface and redox reactivity

Reactive site quantification and categorization using methanol as a probe molecule. Reactive site quantification and classification were performed by gas-phase methanol chemisorption and temperature-programmed surface reaction (TPSR) experiments as described elsewhere.^19,20 Briefly, 250–500 mg of nanomaterials (mesh size <100 μm) diluted with 500 mg of SiC was placed in a fixed-bed reactor. The sample was pretreated at 450 °C in an air flow of 150 mL min⁻¹ for 35 min to remove moisture and impurities. Then, the sample was cooled down to 100 °C, purged with Ar (100 mL min⁻¹), and fed with 2000 ppm methanol in argon with 5% helium (100 mL min⁻¹). Upon saturation, the sample was purged with 100 ml min⁻¹ Ar and heated to 450 °C at 10 °C min⁻¹ under Ar. The reaction products were detected by mass spectrometry (see the details in the ESI†), defining the reactivity of NBMs’ surface sites; dimethyl ether (DME) was desorbed for acid sites, CO₂ was desorbed for basic or highly redox (over-oxidation) and formaldehyde was desorbed for redox sites. Equivalent blank tests were performed with 500 mg SiC without the NBMs.

Dithiothreitol (DTT) consumption as a probe for oxidative potential assessment. To quantify the oxidative potential of the NBMs, dithiothreitol consumption experiments were performed as described elsewhere.^19,57 Briefly, 3 mL of 200 μg mL⁻¹ NM dispersion in 1 mM phosphate buffer was incubated for 1 h at 37 °C and 500 rpm with 3 mL of 100 μM DTT water solution. Then, the nanomaterials were removed by filtration and the filtrate was mixed with an equal volume of 1 mM Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid)) to quantify the non-oxidized DTT at 412 nm by UV-vis spectrophotometry (calculation details are provided in the ESI†). As a negative control, the same procedure was followed without the NMs to evaluate and subtract from the results the spontaneous oxidation of DTT. 30 wt% H₂O₂ was used as a positive control to calculate the normalized index of oxidant generation (NIOG).^19,58 Additionally, CuO nanoparticles obtained from Sigma-Aldrich (ref: 544868) were used as a reference to compare the reactivity with a well-characterized engineered nanomaterial (NIOG ≈ 1).
Statistical analysis. The DTT depletion values were used to classify the reactivity of the NBMs using K-means clustering applied to the NIOG and significant differences were confirmed between the NBMs for all parameters (NIOG, OP-area and OxTOF) using t-tests (p < 0.05).

Salicylic acid as a probe for OH radical generation. Salicylic acid is an OH radical scavenger that can be used for its indirect detection if the sample does not have any other radical scavenger and all the OH radicals generated by the nanomaterial react with salicylic acid to give dihydroxybenzoic acid hydroxylation products (Fig. S2†).^59,60 Due to the low production of OH radicals, it was not possible to detect the decrease in the concentration of salicylic acid, so it was estimated from the hydroxylation reaction yield using OH radicals produced by 10 μM hydrogen peroxide subjected to UV irradiation. The production of OH radicals was evaluated in suspensions containing the nanomaterial under investigation at a concentration of 100 ppm, 10 mM phosphate buffer (pH 7.4), 50 μM salicylic acid, and 10 μM hydrogen peroxide. The suspensions were kept under magnetic stirring for 24 h, filtered, and subsequently analyzed using a HPLC system equipped with a C18 column and a fluorescence detector. The OH radical formation rate was obtained using eqn (1):where ν_OH represents the OH radicals produced in one hour per liter of solution (nM h⁻¹) and ν_2,5-DHBA is the 2,5-dihydroxybenzoic acid produced in one hour per liter of solution (nM h⁻¹).⁶¹ Finally, f_(2,5-DHBA) is obtained as the ratio between the molecular weights of 2,5-DHBA and salicylic acid (eqn (2)).

Electronic paramagnetic resonance (EPR) monitoring of free radical species. The generation of free radical species was monitored by electron paramagnetic resonance (EPR) spectroscopy (Miniscope 100 EPR spectrometer, Magnettech, Berlin, Germany). Instrument settings: microwave power: 7 mW, modulation amplitude: 1G, scan time: 80 s, and two scans. Different probe reactions were performed to decipher free radical generation. For the production of superoxide and hydroxyl radicals, 0.5/6.8 mg of each sample were suspended in 730 μL of a buffer solution (14 mM potassium phosphate buffer pH 7.4) containing 60 mM DMPO (5,5-dimethyl-1-pyrroline-N-oxide, Enzo Life Sciences, Inc.) and the suspension was constantly stirred. For reactivity mediated by ROS and surface reactive sites, 1/12 mg of each sample were suspended in 2 mL of phosphate buffer containing 50 mM TEMPONE-H (1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine, Enzo Life Sciences, Inc.) and the suspension was constantly stirred. Finally, for the Fenton reaction, 0.5/6.8 mg of each sample were suspended in 730 μL of a buffer solution (14 mM potassium phosphate buffer pH 7.4) containing 60 mM DMPO and 27 mM H₂O₂ and the suspension was constantly stirred. The EPR spectra of all the samples and reactions were recorded in 50 μL of the suspension withdrawn after 60 min. For FeHA and Fe₃O₄_PEG-PGLA, the experiments were repeated after incubation of the samples at 37 °C for 24 h in DMEM + 10% FBS under agitation (0.5 mg ml⁻¹), followed by isolation through three centrifugation/dispersion cycles in PBS.

Simulations of the spin trapping/EPR spectroscopy experimental signals were performed by using the software Winsim2002 (version 0.98, National Institute of Environmental Health Science, National Institutes of Health, USA). The hyperfine splitting constants obtained from the simulation optimization were compared with those reported in the literature (NIESH S.T.B.D. database).

Cyclic voltammetry for the assessment of redox potential. The redox potentials of target materials were quantified from the cyclic voltammograms obtained from a conventional 3-electrode electrochemical cell controlled with a potentiostat. The NBMs were anchored at the top of a glassy carbon electrode (GC) by pressing them toward it.⁶² A modified GC was used as the working electrode and the reference electrode was a calomel electrode (SCE) with a Pt counter electrode. The redox potentials of the NMs were calculated from the reduction and oxidation peaks that appeared in the cyclic voltammogram. Reduction and oxidation peaks were recorded in an aqueous electrolyte (10⁻² M K₂SO₄) and in DMEM.

Results

Dissolution test and colloidal behavior

The results of static dissolution tests and colloidal characterization conducted in water and in complete medium (DMEM + FBS) for different NBM concentrations are reported in the ESI (Table S1 for Fe-based NBMs and Table S2 for Ag-based NBMs†).

The three iron-based magnetic NBMs (Fe₃O₄, Fe₃O₄_PEG-PLGA and FeHA) exhibit poor solubility in complete medium with concentrations below the limit of detection (0.01 mg L⁻¹). Uncoated Fe₃O₄ showed a strong tendency to agglomerate, despite the actual particle size being <200 nm. This behavior may negatively affect its dissolution and dispersion properties, potentially limiting its bioavailability. Moreover, it exhibited size variability and a concentration-dependent behavior. In complete medium, the average particle size of Fe₃O₄ initially decreased due to agglomerate disaggregation or partial dissolution. However, after 24 h of incubation, particle coarsening occurred, likely due to the saline environment. Fe₃O₄_PEG-PLGA in water at neutral pH showed a stable hydrodynamic behavior, with a small particle size (70 nm) and a negative surface charge (ζ = −50 mV), consistent with the PEG-PLGA coating. In DMEM + FBS, however, the ζ value leveled off to −10 mV and a concentration-dependent increase in size was observed at higher concentrations (50 ppm), indicating reduced colloidal stability. FeHA exhibited a stable hydrodynamic diameter of 250–300 nm in water with a negative ζ value. In DMEM + FBS, protein adsorption improved dispersion and reduced the particle size at low concentrations (10 ppm), but after 24 h, coarsening occurred, leading to reduced colloidal stability. The ζ value also shifted to around −13 mV, which is consistent with the typical behavior in complete medium (Table S1†). Concentration-dependent iron dissolution was noticed with no release at 50 ppm, but ca. 5% iron release from FeHA at 10 ppm after 24 h was observed, suggesting that lower concentrations may promote slight dissolution in biological media.

Two silver-based NBMs were tested for their colloidal and dissolution behavior, Ag and AgHEC. Dissolution of Ag NPs in complete culture medium was equivalent to that of AgHEC at 50 ppm (0.2%), but more pronounced at lower concentrations (1.7% vs. 0.8% at 10 ppm) with a slight increase after 24 h of incubation. In water, the commercial uncoated silver exhibited larger hydrodynamic diameters than AgHEC, synthesized in liquid suspension, a typical behavior of dry nanopowders, which tend to aggregate. Despite the dispersion effect expected in DMEM + FBS, Ag particle coarsening was observed, together with a decrease in the value of ζ, probably ascribable to the higher ionic strength of the medium. In DMEM + FBS, the particle size of AgHEC decreased significantly and a negative ζ value was observed. Lower standard deviations and moderate PDI values indicated a more stable suspension compared to Ag NPs.

Number and nature of surfaces sites

The specific surface area was analyzed using N₂ adsorption isotherms for all powder nanomaterials (Fig. 1, left) but for three samples, namely Ag, due to its low porosity and, thus, low N₂ adsorption, and the coated Fe₃O₄_PEG-PLGA and AgHEC NBMs, because the polymeric coating would collapse in the dry state. TiHA presented the highest specific surface area (95 m² g⁻¹), followed by FeHA (77 m² g⁻¹), whereas HA (18 m² g⁻¹) and Fe₃O₄ (6 m² g⁻¹) had the lowest surface areas. Fe- and Ti-doped HA exhibited a high methanol chemisorption (0.23 and 0.19 mmol g⁻¹, respectively), comparable to the Fe₃O₄ reference material, with the highest value despite its low surface area. The trend is thus reversed for the active site surface density (Fig. 1, right); HA exhibits more active sites per unit of surface than the doped hydroxyapatites.

Fig. 1 Left, specific surface area obtained using the N₂ adsorption isotherm (BET). Middle, number of sites per gram. Right, active site surface density obtained by a combination of methanol chemisorption and BET data.

MeOH-TPSR was used to assess the nature of reactive surface sites for four different nanomaterials. HA (Fig. 2A) has low, mainly redox and basic reactivity with small amounts of carbon dioxide and formaldehyde detected as reaction products. Doping modifies the picture; Ti introduces acid reactivity, not detected in undoped HA, as reflected by the dimethyl ether signal (Fig. 2B). In addition, a decrease in the amount of formaldehyde is observed. Conversely, the amounts of formaldehyde and carbon dioxide significantly increase in the Fe-doped HA (Fig. 2C), which reflects a marked enhancement of redox and basic reactive surface sites, along with some acidity, indicated by a small dimethyl ether signal. In contrast, Fe₃O₄ (Fig. 2D) showed just basic surface sites, desorbing CO₂ with a peak maximum at 337 °C. This relatively high temperature for maximum formation of carbon dioxide is closely associated with a limited reactivity of the surface sites, discarding oxidative reactions as the origin. Previous studies reported that for CuO, for example, maximum conversion to CO₂ was observed at a temperature as low as 221 °C,¹⁹ which was attributed to methanol over-oxidation.

Fig. 2 Temperature-programmed surface reaction products of pre-adsorbed methanol for HA (A), TiHA (B), FeHA (C) and Fe₃O₄ (D).

Oxidative potential

So far, several different pathways resulting in oxidative stress are known for nanomaterials. The most common mechanism involves redox-active NMs, which can unbalance the redox homeostasis of cells by generating ROS, degrading biomolecules by reaction with surface active sites, or consuming antioxidant molecules.⁵⁸ In this paper, we have estimated (1) the ROS generation by the salicylic acid method and by EPR spectroscopy, (2) the presence of reactive surface sites by methanol-TPSR, and (3) the ability to consume antioxidants by the DTT assay.

Thiol oxidation. Dithiothreitol (DTT) is a probe that can be used to monitor the capability of NMs to oxidize thiols.^63–65 Thiols like glutathione (GSH) or cysteine (Cys), with high-affinity binding sites of metals, such as zinc or iron, are largely involved in the protection of cells from oxidative stress.⁶⁶ Oxidation of thiols occurs through a one-electron oxidation reaction, giving sulfur-centered radicals that dimerize in disulfide:

R–SH → R–S˙ + H⁺ + e⁻

2 R–S˙ → R–SS–R

The oxidative potentials of different NBMs evaluated by the DTT assay according to different descriptors (see the ESI, section 2.3 and Table S3†) are presented in Fig. 3. K-means clustering classified the NBMs into low, moderate, and high reactivity groups, while pairwise comparisons between the related NBMs (e.g., FeHA/TiHA vs. HA, Ag vs. AgHEC, or Fe₃O₄vs. Fe₃O₄_PEG-PLGA) were performed using t-tests (p < 0.05). Among the HA-based nanomaterials, both dopants increased the overall reactivity as measured using the NIOG parameter (0.20 for TiHA and 0.26 for FeHA). However, only FeHA showed a statistically significant difference compared to HA (NIOG ≈ 0.10). For iron-based nanomaterials, the PEG-PLGA coating of Fe₃O₄ significantly enhanced the oxidative potential as compared to the uncoated Fe₃O₄. This unexpected effect of the polymeric coating might be related to the higher exposed surface of the coated magnetite, which does not aggregate as the coated one. The trend was reversed for silver-based materials, as uncoated Ag NPs’ reactivity was one of the highest in the series, whereas Ag coated with HEC presented one of the lowest oxidative potential values, justifying its use as a safe-by-design strategy for the development of Ag-based antimicrobial agents.^52,53

Fig. 3 Oxidative potential obtained by the DTT assay. DTT depletion normalized by a positive control, NIOG (left), DTT oxidation rate normalized per area (middle), and per reactive site, OxTOF (right). The statistical significance of pairwise comparisons was evaluated using t-tests (*, p < 0.05) between the related NBMs (FeHA/TiHA vs. HA, Ag vs. AgHEC, or Fe₃O₄vs. Fe₃O₄_PEG-PLGA).

DTT oxidation rates (mol DTT s⁻¹) were normalized based on two parameters: physical surface area (μmol DTT s⁻¹ m⁻²) and reactive site number (DTT OxTOF, s⁻¹) – nanomaterials for which the BET surface area could not be measured were excluded from this analysis. When normalized per surface area, the thiol oxidation rates of all tested NBMs were relatively slow compared to the CuO positive reference (Fig. 3, middle panel). When comparing the DTT OxTOF, this difference was reduced, particularly for FeHA (Fig. 3, right). Therefore, although the overall surface reactivity of the tested NBMs is low compared to CuO, the reactivity per reactive site is not negligible, with FeHA demonstrating one-third of CuO's thiol group oxidation rate. FeHA exhibited markedly higher reactivity compared to HA (and even Fe₃O₄), reflecting the influence of the dopant on the oxidative properties of hydroxyapatite-based nanomaterials.

Finally, K-means clustering analysis of DTT NIOG values classified Fe₃O₄_PEG-PLGA as a highly reactive NBM, alongside the control CuO. Ag stood out as a moderately reactive NBM, while Fe₃O₄, HA, TiHA, FeHA, and AgHEC were grouped as low-reactivity NBMs.

ROS generation. The ability to generate hydroxyl or superoxide radicals from water or dissolved oxygen was evaluated using the EPR spectroscopy/spin trapping technique and salicylic acid test. As expected for the NBMs, none of the tested samples generated ROS, as inferred from the results of both analyses, except for Fe₃O₄_PEG-PLGA that was able to oxidize salicylic acid to 2,5-dihydroxybenzoic acid in small amounts (data not shown). To further assess the possible reactivity of the NBMs, the probe molecule TEMPONE-H was used as a spin trap. This probe reacts with both ROS and surface reactive sites, generating the radical TEMPONE that is detected by EPR spectroscopy as a typical three line-signal:

TEMPONE-H → TEMPONE˙ + H⁺ + e⁻

It is therefore suitable to monitor a general reactivity at the surface/water interphase. In this case, Fe₃O₄_PEG-PLGA and FeHA were found to generate the TEMPONE radical, but not Fe₃O₄, TiHA and HA (Fig. 4A). The trend is in line with the oxidative potential inferred from the reactivity profiles (Fig. 2) and DTT depletion tests (Fig. 3). Note that TiHA is inert also under simulated solar irradiation, as previously reported.³⁵ This suggests that Ti ions are not photoreactive or are not in contact with the solvent since they are embedded in the HA matrix.

Fig. 4 EPR spectra. (A) Surface reactivity assessed using the TEMPONE-H probe molecule as a spin trap; (B) Fenton-like reactivity with the addition of hydrogen peroxide assessed using DMPO as a spin trap. Conditions: NBMs in phosphate buffer at pH 7.4.

Due to the possible release of redox active ions, the experiments on Fe-based NBMs were repeated by testing the presence of free radicals in the supernatant obtained by centrifugation or filtration of the NBMs after incubation. Free radicals were found on the supernatant of FeHA, suggesting the role of dissolved iron ions, but not in the supernatant of Fe₃O₄_PEG-PLGA. This agrees with the dissolution experiments (Table S1†).

We investigated the formation of ROS also in the presence of H₂O₂. NBMs that are inert in water might still produce ROS in phagolysosomes once eventually phagocyted by immune system cells. In fact, in this cellular compartment, hydrogen peroxide is abundant and the presence of redox active metal ions (e.g., Fe²⁺) may catalyze the generation of hydroxyl radicals through Fenton-like^67,68 mechanisms:

H₂O₂ + e⁻ → OH⁻ + OH˙

Fig. 4B shows the EPR spectra of the NBMs in the presence of hydrogen peroxide. Among the samples, only FeHA showed the typical four-peak signal of hydroxyl radicals generated in the presence of the spin trap DMPO, but with intensity significantly lower that of the positive control, indicating the presence of redox-active iron, as confirmed by cyclic voltammetry measurements in PBS (Fig. 5).

Fig. 5 Cyclic voltammograms of the NBMs deposited on a glassy carbon electrode. Top: (A) Ag-HEC and (B) Fe₃O₄_PEG-PLGA, both in water (black) and in DMEM medium (red). Bottom: FeHA in (C) water and (D) DMEM. Scan rate: 10 mV s⁻¹; supporting electrolyte: 10⁻² M K₂SO₄.

Direct oxidation capacity. Redox active NBMs can react with biological molecules also by direct exchange of electrons, not mediated by radicals.⁶⁵ We investigated the extent of this mechanism, which adds to radical-mediated oxidation. Fe₃O₄ is the unique NBM in the series producing radicals, while FeHA induced the formation of ROS only in the presence of H₂O₂. Fig. 3, however, indicates that DTT is also consumed by the NBMs that do not generate ROS. Fig. 5 shows the cyclic voltammograms recorded for AgHEC, FeHA and Fe₃O₄_PEG-PLGA both in water and DMEM. The oxidation and reduction peaks of Ag-HEC (Fig. 5A) are eye-catching; the formation of a bio-corona when the particles are exposed to DMEM does not quench the redox activity of the particles, which, evidently, are able to exchange electrons also in the presence of a layer of biological species at their surface. Some redox activity is also visible for Fe₃O₄-PEG-PLGA (Fig. 5B); a clear oxidation peak appears in water at around −0.2 V (black trace), while the relative reduction gives rise to a barely visible shoulder centered at −0.4 V. In DMEM (red trace), cyclic voltammetry exhibits only very low redox activity. Clearly, the redox behavior of this coated magnetite can be attributed only to the Fe ions at its surface. The presence of active Fe ions at the surface can also be hypothesized for the oxidizing activity of FeHA in DTT assay. For this doped hydroxyapatite, large, yet visible, oxidation and reduction bands were observed in water (red trace, Fig. 5C), while just a shoulder on the reduction potential region was observed in DMEM (red trace, Fig. 5D). All in all, these measurements revealed the possibility that the NBMs oxidize thiols also through direct interaction, which adds to the radical mediated oxidation.

Discussion

Fe and Ti doping alters the number and nature of surface reactive sites in HA-based NBMs

The late formation of formaldehyde and CO₂ in MeOH-TPSR for HA is indicative of redox and basic surface sites with low reactivity, compared to other metal oxides and multicomponent systems that under the same conditions over-oxidized methanol to CO₂ in their highly reactive redox sites, as inferred by relatively lower temperatures of CO₂ release (e.g., 221 °C for CuO and 231 °C for CuFe₂O₄).¹⁹ Importantly, the low reactivity exhibited by undoped HA confirmed its established biocompatibility, which justified its widespread use in medical applications.⁵¹

Metal doping in HA NBMs led to a decrease in the active site surface density from 5 to 2 active sites per nm² in both TiHA and FeHA. The surface area increase observed in the doped HA NBMs aligns with previous reports⁶⁹ and the trends seen in doped nano-oxides, where synthesis conditions and dopant incorporation promote smaller crystallites, enhanced porosity, and modified agglomeration.⁷⁰ In terms of surface reactivity, Fe doping shifted HA's reactivity towards more oxidative pathways; MeOH-TPSR tests revealed a slight redox activity, in line with FeHA's ability to oxidize DTT and TEMPONE-H, and to produce hydroxyl radicals by a Fenton-like reaction. Complementary CV data also revealed redox activity attributed to the presence of redox-active iron ions within the HA matrix. Consequently, the reactivity of FeHA involves two distinct mechanisms: surface-mediated heterogeneous reactivity and ion-mediated homogeneous reactions due to dopant release. The observed reactivity correlates with the reported ability of this material to induce intracellular ROS release in HCT116 cells, albeit no significant DNA damage was observed.⁷¹

Ti-doped HA maintained a low basic reactivity similar to that of HA, as seen by the CO₂ bands in MeOH-TPSR, but also exhibited acidic surface sites, as evidenced by the DME signal (Fig. 2E). In line with this, TiHA was poorly reactive toward DTT, in agreement with previous findings on similar TiO₂-HA nanomaterials.¹⁹ The absence of reactivity under simulated solar irradiation further supports TiHA's inert behavior in the liquid phase, as Ti ions appear to be effectively embedded within the HA matrix, in line with previous studies in TiHA-based NBMs.⁷² The low oxidative potential correlates with the absence of a notable increase in 53BP1 foci or micronucleus formation, as reported by Fontaine et al.⁷¹ The enhanced acidic surface reactivity is not translated into significant adverse effects.⁷¹

Surface coatings regulate the oxidative reactivity of hybrid magnetite- and Ag-derived NBMs

Surface coatings transform the oxidative potential of NBMs. The reactivity of iron oxide nanoparticles has been extensively studied in the literature, and it has been related to oxidative stress, as observed in cells exposed to these materials.⁷³ The role of the iron redox state, however, has been found to be decisive for the toxicological effects; while Fe₂O₃ did not present any cytotoxic concentration up to 100 μg mL⁻¹ for A549 cells,¹¹ superparamagnetic Fe₃O₄ nanoparticles induced cell DNA damage.⁷⁴ Additionally, the oxidative stress induced by Fe₃O₄ has been reported both by in vivo^75–78 and in vitro^79–82 studies. In previous works, a Fenton reaction mechanism involving iron ions was suggested as a hypothesis, where hydrogen peroxide interacts with Fe²⁺, promoting its oxidation into Fe³⁺ and leading to localized oxidative stress, rather than diffusive ROS-mediated damage to cellular components such as DNA.⁷¹ However, this topic has been a critical point of discussions. In a recent review, Muranov indicated that under physiological conditions iron transport is regulated by carrier proteins, minimizing the presence of free iron ions in the cytoplasm. However, in pathological states where the carrier protein function is impaired, iron ions may enter the cytosol. At near-neutral pH, typical of the cytosol, these ions form insoluble hydroxides, preventing the classical Fenton reaction. Nevertheless, these iron hydroxides can catalyze hydrogen peroxide decomposition, producing hydroxyl radicals in a Fenton-like reaction.⁸³ This reaction can be used for medical purposes, e.g., in metal-based NBMs for tumor therapy.⁸⁴

Distinct reactivity was observed for the uncoated and PEG-PLGA-coated Fe₃O₄ nanomaterials. Interestingly, DTT oxidation using Fe₃O₄_PEG-PLGA was comparable to that of highly reactive nanomaterials such as CuO^85,86 (positive control). This coated magnetite also showed significant salicylic acid hydroxylation, but did not produce detectable ROS. This pattern suggests that the oxidative properties of Fe₃O₄_PEG-PLGA are likely surface-based rather than mediated through ROS, highlighting the effect of the PEG-PLGA coating on Fe₃O₄'s surface.⁷¹ In line with our results, previous studies concluded that exposure of HCT116 cells to Fe₃O₄_PEG-PLGA led to a concentration-dependent increase of intracellular ROS content, but the absence of genotoxic effects.⁷¹ Finally, Fe₃O₄ PEG-PLGA and Fe₃O₄ were tested in vivo in recent studies, showing no significant risks to the soil invertebrate model species Enchytraeus crypticus and Folsomia candida, as determined by the standard 28 day OECD test and an extended 56 day exposure version.⁸⁷

In the case of the silver-based nanomaterials, uncoated Ag nanoparticles exhibited high intrinsic reactivity, as evidenced by substantial DTT depletion, aligning with previous studies on Ag's redox properties.²⁷ The reported gastrointestinal toxicity of uncoated silver nanoparticles was evaluated in human intestinal cells, producing significant cytotoxicity, increased ROS production, and DNA damage with only minor alterations in toxicity following simulated gastrointestinal digestion.²⁸ In AgHEC, the hydroxyethyl cellulose coating may stabilize Ag, modulating surface reactivity. This phenomenon is reflected in reduced toxicity, lower ROS generation, and less DNA damage than Ag, thanks to the protective coating that mitigated harmful interactions. Similarly, studies on the exposure of the alveolar epithelial cell line A549 to Ag and AgHEC reported significantly higher cytotoxicity, oxidative stress and cellular uptake by uncoated Ag nanoparticles, which led to greater ROS production.⁸⁸ Collectively, these observations underscore the role of oxidative stress as a driving mechanism of toxicity for Ag nanomaterials. The high oxidative potential of uncoated Ag, which correlates strongly with adverse biological outcomes, supports the hypothesis of a reactivity-triggered toxicity model for silver-based nanomaterials.²⁷

In chemico approaches are required to deeply understand the complexity of hybrid nanobiomaterials

The oxidative potential of NBMs represents a critical factor in assessing their safety and applicability in biomedical contexts. While previously established methods, such as those detailed by Zhang et al. (2012), evaluated the oxidative potential of single-component materials (referred to as single metal nano-oxides) through their well-defined electrochemical properties, these approaches often lack robustness when applied to hybrid NBMs.⁸⁹ Hybrid NBMs, by design, integrate multiple components to enhance stability, biocompatibility, or functionality, inherently complicating their surface reactivity profiles. This complexity necessitates the integration of in chemico methodologies to complement existing paradigms. Thus, our findings underscore that the surface reactivity, influenced by doping or coating strategies, is related to the nanomaterial composition, but cannot always be directly correlated with biological outcomes due to the involvement of additional factors such as cellular uptake and protein corona formation. However, in the frame of risk assessment, these in chemico reactive descriptors remain invaluable for identifying key initiating events in adverse outcome pathways (AOPs) and grouping approaches.^32,45,50 Furthermore, the concentration of reactive sites has been proposed as a refined dose metric in nanotoxicology²⁰ based on the premise that not only does the same mass not correspond to the same surface area, but also not all physical surface areas correspond to chemically active sites.³⁵ Consequently, when surface reactivity-driven toxicity is the primary adverse effect, this metric may provide a more accurate assessment of toxicological responses, rather than the mass, the traditional dose metric, or the surface area, as lately suggested.

Finally, the increasing focus on reducing animal testing has driven the development of NAMs, including in silico studies – computational methods designed to predict the toxicological behavior of nanomaterials.^90,91 These methods aim to identify toxicity patterns without requiring in vivo testing, relying instead on a few basic, easily measurable, and standardized properties, such as composition, structure, specific surface area, or reactivity.⁹² However, these methods still suffer from a significant lack of datasets for proper training. Studies like ours, which generate extensive datasets, are particularly important for establishing correlations between physicochemical properties, variations in composition and nanomaterial characteristics, and their relationship with biocompatibility – mainly related to the interactions at the nano–bio interface.⁹³ Therefore, the in chemico NAM proposed has been described in detail in the ESI,† adding a series of steps to analyze new hybrid NBMs.

Conclusions and outlook

A methodology is presented that facilitates the development of safer NBMs, thereby enhancing their biocompatibility, by better understanding their differential surface and reactive properties. The in chemico strategy followed in this work leads to the conclusion that (1) doping of HA NBMs with Ti or Fe results in a more reactive surface with less active site surface density and, in the case of Fe doping, increased oxidative potential and (2) organic coatings modulate NBMs’ reactivity, lowering the surface reactivity in the case of AgHEC, whereas the reactive-driven mechanism is modified in the case of Fe₃O₄_PEG-PLAG.

In terms of dose metrics, the determination of the number of surface sites and the description of the active site surface density aim to propose a refined dose for the assessment of NBMs. In line with our previous works, this approach focuses on evaluating the reactive surface sites of NBMs rather than relying exclusively on their mass or total surface area. By adopting this perspective, it has the potential to advance the field of nanotoxicology, enabling more precise and reliable risk/reactive assessments of new hybrid NBMs.

Data availability

The data supporting the findings of this study are available throughout the article and in the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors are grateful for the financial support from the European Commission through H2020 projects BioRiMa (GA760928) and NanoinformaTIX (GA814426).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5nr00709g

‡ Current address: CNR-Institute for Photonics and Nanotechnologies (CNR-IFN), Milan, 20133, Italy.

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