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DOI: 10.1039/D4TB01307G (Paper) J. Mater. Chem. B, 2024, Advance Article

NIR-responsive nano-holed titanium alloy surfaces: a photothermally activated antimicrobial biointerface†

Denise B. Pistonesi‡ a, Federico Belén‡a, Juan M. Rusob, M. Eugenia Centurióna, M. Gabriela Sicacd, Marcelo F. Pistonesia and Paula V. Messina*a
aDepartment of Chemistry, Universidad Nacional del Sur, INQUISUR – CONICET, B8000CPB, Bahía Blanca, Argentina. E-mail: pmessina@uns.edu.ar; Fax: +54 291 4595160; Tel: +54 291 4595159
bSoft Matter and Molecular Biophysics Group, Department of Applied Physics and iMATUS, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain
cDepartment of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, B8000CPB, Bahía Blanca, Argentina
dDepartment of Health Sciences, Universidad Nacional del Sur, B8000CPB, Bahía Blanca, Argentina

Received 15th June 2024 , Accepted 3rd August 2024

First published on 5th August 2024

Abstract

Among external stimuli-responsive therapy approaches, those using near infrared (NIR) light irradiation have attracted significant attention to treat bone-related diseases and bone tissue regeneration. Therefore, the development of metallic biomaterials sensitive to NIR stimuli is an important area of research in orthopaedics. In this study, we have generated in situ prism-shaped silver nanoparticles (p-AgNPs) in a biomorphic nano-holed TiO2 coating on a Ti6Al4V alloy (a-Ti6Al4V). Insertion of p-AgNPs does not disturb the periodically arranged sub-wavelength-sized unit cell on the a-Ti6Al4V dielectric structure, while they exacerbate its peculiar optical response, which results in a higher NIR reflectivity and high efficiency of NIR photothermal energy conversion suitable to bacterial annihilation. Together, these results open a promising path toward strategic bone therapeutic procedures, providing novel insights into precision medicine.

Introduction

Near infrared light (NIR, 800–1300 nm) is administered in advanced targeted healing approaches.1–4 High yields have been obtained due to its superior penetration capacity and effective transmittance through the biological milieu, as well as its minimal damage toward normal tissues, high spatial/temporal precision and easy-to-remote control properties. Current reports have revealed that NIR light-assisted phototherapies are a potential treatment option for bone pathologies,5,6 for example, as precise and less invasive solutions for fracture healing complications.7 Furthermore, NIR irradiation was shown to exhibit outstanding antioxidant activity and excellent anti-inflammatory effects, decreasing inflammatory cytokines and reducing catabolic proteases, which could effectively alleviate the clinical symptoms of osteoarthritis.8 Simultaneously, NIR thermal osteogenesis9,10 has been scientifically proved in vitro and in vivo, as well as its ability to treat implant-associated infections.11 Therefore, the study of NIR-sensitive materials in bone-related therapies is a very auspicious scientific area where the creation of new materials has been increasingly favoured by the rapid development of nanotechnology. In this line of attack, in a previous work, we have developed an electroforming nano-holed TiO2 coating on the Ti6Al4V alloy.12 This particular metallic–dielectric structure exposed periodically arranged sub-wavelength-sized pores that exhibit higher NIR reflectivity. Following this, we have created {111}-faceted silver nanoplates that exhibit NIR localized surface plasmon resonance (LSPR) extinction spectra.13 In the present investigation, we have merged both assemblies to generate, in one design, specific light absorption and conversion capabilities in the NIR light window.14 Titanium and its alloys are currently the most used materials in orthopaedics due to their excellent biocompatibility and mechanical properties.15 On the other hand, the photocatalytic properties of titanium dioxide16 make it suitable for photodynamic and photothermal therapies. Here we have demonstrated that the use of p-AgNPs as a “plasmonic sensitizer” enables photothermal conversion by NIR activation of our nano-holed TiO2 coating and that it can be exploited against microorganisms. Nanostructured titanium surfaces responsive to NIR radiation have a wide range of promising applications, particularly in the biomedical field.17–19 One of the primary applications is in targeted cancer therapy, where the photothermal effect can be used to precisely heat and destroy cancer cells without damaging the surrounding healthy tissues.20 The antibacterial properties of these surfaces also make them ideal for use in medical implants and devices, reducing the risk of infection and promoting better patient outcomes.17,21 Our goal is to develop a versatile NIR light-sensitive metal platform that will increasingly enable us to meet the demands for customized orthopaedic implants.

Experimental

Materials

For the synthesis of AgNPs in aqueous solution, trisodium citrate (C6H5Na3O7·2H2O, CAS N° 6132-04-3, Biopack), anhydrous glucose (D(+)) (C6H12O6, CAS N° 50-99-7, Cicarelli), silver nitrate (AgNO3, CAS N° 7761-88-8, Biopack) and L-ascorbic acid (C6H8O6, CAS N° 50-81-7, Biopack) were used without further purification. For the preparation of nano-holed TiO2 coatings, Ti6Al4V sheets (1000 μm thick; Ti 89. 754%; Al 6.01%; V 4.06%; C 0.01%; N < 0.01%; O 0.08%; Fe 0.04%; H 0.006%, and others < 0.04%) were purchased from Roberto Cordes S.A., Buenos Aires, Argentina. Hydrofluoric acid (HF, CAS N° 7664-39-3, 48 wt%, Sigma-Aldrich), ortho-phosphoric acid (H3PO4, CAS N° 7664-38-2, 85 wt% water solution, EMSURE®, Merck), acetone (AC, CAS No. 67-64-1, 99%, Sigma-Aldrich) and ethanol (EtOH, Art. No. 9065.1, 96%, Carl Roth) have been used as received. All reagents used here were of analytical grade and all solutions were prepared with ultrapure water, 0.05 μS cm−1 electrical conductivity and 18.2 M Ω cm−1 resistivity at 25 °C, obtained via a water purification systems Millipore Milli Q, model Elix Technology Inside 10, Merck, France.

Fixation of silver nanoplates on nano-holed titanium alloy surfaces

Ti6Al4V disks of 10 mm diameter were machined and successively ground with P180, P240, P400, P600, P800, P1000, P1500, and P2000 grade SiC abrasive paper and mechanically polished using 3 μm diamond paste (8000 Grit, Leader Products). Finally, samples were degreased by sonication in AC, EtOH and MQ water for 20 min, in sequence, and dried in an N2 stream. Anodic TiO2 coatings (a-Ti6Al4V) were electrochemically obtained according to Belen et al.12 Fixation of silver nanoplates on nano-holed titanium alloy (a-Ti6Al4V/p-AgNP) surfaces was performed on the basis of two different protocols. (i) Electrochemically anodized TiO2 coated plates were placed in a container where prism-shaped silver nanoplates (p-AgNPs) were synthesized following a previously optimized green synthesis procedure13 and left in the dark for 3, 7 and 11 h. (ii) On the other hand, 10 mL of p-AgNPs were prepared (14 μg mL−1) and concentrated (80 μg mL−1) in a centrifugal evaporator system (Thermo Scientific Speed Vac SPD11V) at 35 °C. Then, a-Ti6Al4V sheets were submerged in 2 mL of p-AgNPs (14–80 μg mL−1) during 3, 7 and 11 h in the dark. Once the a-Ti6Al4V/p-AgNP sheets were prepared, they were weighed in an OHAUS ANALYTICAL Plus, Model No. AP250D; 210 g–0.1 mg resolution.

Microstructural and morphological characterization

Particle size and morphologies were established by transmission electron microscopy (TEM) using a JEOL 100CX II transmission electron microscope (Jeol Ltd, Tokyo, Japan) operating at 120 kV. A standard procedure of sample preparation was used, in which a droplet of diluted AgNP suspension was deposited and dried at RT on a 200 mesh copper grid covered with a thin layer of transparent carbon film under dust protection. Surface characterization of a-Ti6Al4V and a-Ti6Al4V/p-AgNPs was performed with a high resolution field emission scanning electron microscope (HR-SEM) ZEISS ULTRA PLUS, coupled to an X-ray energy-dispersive (EDX) spectrophotometer that enables qualitative and quantitative elemental chemical microanalyses. Images were acquired with a secondary electron detector (SE2; In lens) operated at an accelerating voltage (EHT) of 3.00 kV and at a working distance (WD) resolution of 2.1 nm. Local load compensation was achieved by injecting nitrogen gas and double-layer carbon adhesive tape was used to mount the samples. Mean nano-hole diameters, aspect ratio, wall thickness, inter-hole distances, Feret's diameter (DF), Feret's angle (Fang), roundness (R), circularity (C) and 3D surface plots were acquired by digitalized image processing using the free software ImageJ.22 All statistical values have been obtained using at least 100 measures. The stability and/or possible dissolution of p-AgNPs on a-Ti6Al4V surfaces were verified by quantitative microanalysis using EDX spectroscopy, after 24 h of immersion in Milli-Q water.

Optical measurements

Diffuse reflectance patterns were carried out using a Thermo Scientific Spectrophotometer, Nicolet® IS50 FT-IR model, provided with near infrared reflectors (CaF2/KBr) and detectors (InGaAs/KBr-DLaTGS). Measurements were performed in the reflectance mode with a spectral range between 16.000 and 4.000 cm−1 (625–2500 nm). Spectra were recorded at 90° to the incident radiation on the disks using the integrated software, at a spectral resolution of 4 cm−1 and by integrating 32 scans. All measurements were carried out at room temperature (RT) employing a custom-made poly-lactic acid (PLA 3D printing filament) sample holder for each angle of incidence radiation, printed on an Adonis 3D printer, Hellbot model, and using a Teflon disk as the blank. Molecular fluorescence spectroscopy analysis was performed using a Shimadzu RF-6000 spectrofluorimeter, in the 3D fluorescence mode with a spectral range between 200 and 900 nm for the excitation and emission wavelengths. The spectra were recorded using the integrated software, at a spectral resolution of 5 nm and a scanning speed of 12[thin space (1/6-em)]000 nm min−1. For the measurements, home-made cells printed in PLA were used, which allowed the disks to be placed at angles of 30, 45 and 60° regarding incident radiation, reflecting it towards the detector. Solid absorbance UV–vis measurements were carried out at RT with a UV-Visible spectrophotometer Evolution 220, Thermo Scientific, provided with an integrating sphere accessory. Spectra were recorded in the absorbance mode with a spectral range between 220 and 1100 nm; data were acquired with a scan rate of 1200 nm min−1 after calibration with the Spectralon diffuse reflectance standard.

Infrared images of a-Ti6Al4V and a-Ti6Al4V/p-AgNP surfaces were acquired with a Canon digital camera, Powershot ELPH 190IS model; before capturing images, the camera infrared filter was removed. A tailored system was assembled, which allowed the disks to be located in a closed place, removing the disturbance of external light according to Belen et al.12 Disks were illuminated using an emitting infrared LED, IR383, λ = 940 nm, controlling the spectral radiant flux,23 Φλ = (1.01–38.14) × 10−3 mW nm−1, by the application of a Uni-T dimmable source, model UTP3313TFL at RT. Images were compared in terms of the spectral irradiance, Eλ, that is a measure of the radian flux, emitted at a specific wavelength arriving at a point per differential area surrounding this point;23 the whole metal disk area, 78.5 mm2, was considered for the calculation of E940.

Plasmon-based heat generation

A physiotherapy NIR 904 nm laser (LB-904, SEAKIT, Buenos Aires, Argentina), which was operated at an emission frequency of 2000 Hz (laser radian flux density, irradiance, E = 83.3 mW cm−2), was applied to carry out photothermal experiments. Silver nanoplate dispersions were placed in glass vials of 11.6 mm diameter and 32 mm height. The laser was adjusted so that the spot could be matched with the area occupied by 700 μL of AgNP dispersion, A ≈ 6.3 cm−2. A similar procedure was performed with a-Ti6Al4V and a-Ti6Al4V/p-AgNP surfaces. Time evolution thermographs were recorded using a digital thermal imaging camera (CEM High Performance High Resolution Thermal Imagers DT-9873B, CEM Instruments India Pvt Ltd).

Static contact angle measurements

Static contact angles between the disks’ surface and a water drop were measured by using a tailored system at RT.12 The used configuration includes a custom-made 3D-sample holder in which the disks were placed at the same height as the digital microscope. Furthermore, at the top of the sample holder, a tube was connected to form the drop. The other end of the tube was connected to a syringe attached to a syringe pump, Thermometric 612 Syringe Pump 2, forming reproducible drops in all measurements. A maximum drop volume of 2.0 μL was deposited over the disk to acquire the image using a digital microscope (Andonstar Digital Microscope) before static contact angle analysis. All images were processed using ImageJ software22 and the contact angle (CA) measurements were done with an accuracy of ±1 using the Drop Snake and LB-ADSA available module.24

Antibacterial ability

Bacterial inhibition growth capacity. Qualitative preliminary screening of the p-AgNP dispersion capacity to inhibit microbial growth was evaluated by a disk diffusion method using a modified Kirby–Bauer technique.25 The method was previously standardized by adjusting the microbial inoculation rate, the volume of the agar medium layer and wells of 5 mm diameter and 50 μL capacity were made with a sterile glass tube. For inhibition of growth, microorganisms from the American Type Culture Collection (ATCC) were considered: Gram-positive, Staphylococcus aureus (ATCC: 25923). Bacteria were stored at −70 °C in 20% (v/v) glycerol at Microbiology Chair, Department of Biology, Biochemistry and Pharmacy of the Universidad Nacional del Sur, Argentina. Before their use, microorganisms were cultured in Mueller–Hinton broth (Britania Laboratories, Argentine, B0216906) at 37 °C and allowed to mature overnight to ensure exponential growth. We have selected Staphylococcus aureus as a microbial model on the basis of its less liability to antibiotic therapy compared with Gram-negative bacteria.26

After culturing and harvesting, the strain was re-suspended to maintain the initial concentration of 108 colony forming units per milliliter (CFU mL−1) by spectroscopic adjustment (λ = 600 nm) of the optical density at 0.15 according to the McFarland scale. The inoculum concentration was also verified by plating in the plate count agar (Britania Laboratories, Argentine, B0211206), incubated at 37 °C for 24 h, using the ISO 4833-1 standard plate count method.27 Following, 50 μL of AgNP dispersions were applied to a hole in the centre of a Mueller–Hinton agar (Britania Laboratories, Argentine, B0213706) plate, previously harvested with bacteria. The agar plates were incubated for 24 h at 37 °C and the diameter of the inhibition zone, measured using ImageJ software,22 was related to the level of antimicrobial activity present in the sample.

Bacteriostatic and bactericidal effects. To study the bacteriostatic effect of the as-prepared p-AgNPs, the minimum inhibitory concentration (MIC) was determined. The standard broth dilution method28 was used to study the antimicrobial efficacy of AgNPs by evaluating the visible growth of microorganisms in Mueller–Hinton broth (Britania Laboratories, Argentine, B0213706). Serial two-fold dilutions of silver nanoparticles in concentrations ranging from 3.1 to 50.0[thin space (1/6-em)]μg mL−1 adjusted with Staphylococcus aureus (ATCC 25923) concentrations (106–102 CFU mL−1) were used to determine the MIC. The MIC endpoint is the lowest concentration of silver nanoparticles where no visible growth is seen in the tubes; thus, the visual turbidity comparison of the tubes was noted, both before and after incubation, to confirm the MIC value. The inhibitory effect was also visualized by plating 100 μL aliquots of each AgNP dispersion (50, 25, 12.5, 6.3 and 3.1 μg mL−1) with different Staphylococcus aureus (ATCC 25923) concentrations (102 to 106 CFU mL−1) inoculated in blood agar platelets (Columbia Agar, Sigma-Aldrich, CAS No. 27688, supplemented with 5% v v-1sheep blood) which were then incubated at 37 °C for 24 h. Meanwhile, bactericidal properties were evaluated through a minimum bactericidal concentration (MBC) test and were defined as the lowest AgNP concentration that completely prevented colony forming units. The MBC was determined by sub-culturing 100 μL aliquots of each broth dilution that inhibit the growth of Staphylococcus aureus (ATCC 25923); i.e., those at or above the MIC in blood agar Petri dishes, incubated at 37 °C for 24 h. In all experiments, bacterial growth without the addition of AgNPs and, AgNP dispersions without inoculated bacteria were taken as positive (C+) and negative (C−) controls, respectively.
Bacteria viability on a-Ti6Al4V/p-AgNP substrates. Staphylococcus aureus (ATCC 25923) were seeded on the substrates at a density of 107 CFU mL−1 (as estimated by the McFarland scale) by diluting the bacterial cultures to an optical density of 0.52 at 562 nm using a spectrophotometer. The bacteria were allowed to adhere at 37 °C throughout 24 h and at the end of the prescribed time period, the supernatant was removed and the discs were washed 3 times with phosphate buffered saline (PBS; 0.15 mol L−1 NaCl, 0.05 mol L−1 KH2PO4, 0.05 mol L−1 K2HPO4; pH 7.2) to eliminate non-adhered bacteria. Finally, 1 mL of PBS was added to a-Ti6Al4V/p-AgNPs substrates, sonicated for 2 min followed by vortexing for 1 min, to loosen the adhered bacteria. To count them, 50 μL of each post-sonication supernatant was collected and counted as previously described in the previous section. The same procedure was applied after irradiating a-Ti6Al4V/p-AgNP substrates at 904 nm throughout 10 min; E = 83.3 mW cm−2. After that, samples were fixed with glutaraldehyde, and dried using an E3000 critical point drying device.29 Finally, each sample was mounted on a stub, coated with a gold layer, and observed using a Carl Zeiss EVO MA10 scanning electron microscope.

Eukaryotic cell viability

The viability of preosteoblast cells MC3T3-E1, cultured at 37 °C for 24 h as described by Ghilini and co-workers,30 on a-Ti6Al4V/p-AgNP substrates (sterilized using UV radiation for 5 h) was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium bromide) assay according to the Madiwal et al.31 protocol. Viable cells with active metabolism reduce MTT into a purple-colored formazan product that can be solubilized and quantified spectrophotometrically at 570 nm. When cells die, they lose the ability to convert MTT into formazan; thus, colour formation serves as a marker of the metabolically active cells. Therefore, this assay was performed to evaluate cellular metabolic activity and, indirectly, their viability. Preosteoblasts were seeded at a density of 4 × 104 cells per well and incubated for 24 h. After incubation, an MTT solution (stock concentration, 5 mg mL−1 in PBS) was added to each well and further incubated for 4 h. Then, the wells were aspirated and 200 μL of dimethylsulfoxide was added to dissolve formazan crystals; absorbance of the resultant solution was recorded at 570 nm using an ELISA plate reader (OD 570). Wells containing a-Ti6Al4V/p-AgNPs substrates with medium but without cells were used as negative controls (C−), while wells containing cells but no substrates served as positive controls (C+).

Statistical analysis

All quantitative assessments were taken at least in triplicate, and results are expressed as mean ± standard deviation (SD). Statistical analysis of data was realized by one factor analysis of variance (ANOVA). Student's t-test and probability values below 0.05 (*p < 0.05) were considered as a significant difference.

Results and discussion

Optoelectronic properties of a-Ti6Al4V/p-AgNPs surfaces

Optimizing the photophysical properties of a material is crucial to generating designs with electro-optical functionality. In this sense, we have tested two strategies for incorporating prism-shaped silver nanoplates (p-AgNPs)13 into the anodized titanium surfaces (a-Ti6Al4V):12 (i) in situ generation and (ii) ex situ deposition of preformed nanoparticles after immersion of a-Ti6Al4V discs in p-AgNP reaction media or p-AgNP dispersions for 3, 7, and 11 h, respectively. Considering that NIR reflectivity is the key point of the peculiar electro-optical response of our previously developed nano-holed titanium alloy surfaces, the effectiveness of the synthesis strategies was evaluated by analyzing the NIR diffuse reflectance spectra of a-Ti6Al4V/p-AgNP samples; the results are shown in Fig. 1a and b. A broad reflection signal centered at 1200 nm was observed along with several higher intensity peaks in the mid-infrared regions (1720, 2137 and 2316 nm) that were associated with the effect of radiation on the TiO2 nano-holed array.12 No significant statistical differences were observed after immersion of the a-Ti6Al4V plates in the dispersion of preformed p-AgNPs, Fig. 1a. Conversely, after 7 h of immersion in p-AgNP reaction media, exacerbations of near- and middle-infrared (MIR) wavelength signals are appreciated, Fig. 1b; no differences are seen by increasing the exposure time to 11 h of treatment. Diffuse reflectance is a useful tool to analyze plasmon structures, as was demonstrated by Victor Ovchinnikov.32 The author reported that the wavelength position of the LSPR extinction spectra correlates with those of diffuse reflectance in a clear way. Considering that the prism-shaped silver nanoplates have an LSRP extinction spectrum in the range of 400–1300 nm,13 the increase in the intensity of the NIR and MIR signals observed in Fig. 1b could be attributed to the coherent oscillation of the conduction electrons of silver nanoplates in situ generated on the electroforming nanostructured substrate. In this way, the radiation emitted by the p-AgNPs should be reflected by the titanium substrate within nanoholes, increasing the intensity of the reflection bands. Fig. 1c shows the reflective results of the a-Ti6Al4V and a-Ti6Al4V/p-AgNP surfaces after application of a λ = 940 nm radiation source and regulation of the spectral radiance; bare Ti6Al4V discs were used as a control. As expected, both a-Ti6Al4V and a-Ti6Al4V/p-AgNP surfaces show a shiny appearance compared to a polished non-reflective Ti6Al4V substrate;12 furthermore, a superior effect at low spectral radiance values, Eλ ≤ 1.96 W m−2 nm−1, of a-Ti6Al4V/p-AgNP surfaces is observed. Hybrid samples display a uniform distribution and improve reflectivity along the entire surface, while for a-Ti6Al4V the reflection properties are lost as progressing away from the light source. To corroborate the effect of anisotropic p-AgNPs, anodized Ti6Al4V discs immersed in sphere-like AgNP reaction medium for 7 h were used as a second control; less reflectivity was observed compared to the original a-Ti6Al4V surface; please refer to ESI† Fig. S1.
image file: d4tb01307g-f1.tif
Fig. 1 NIR diffuse reflectance of a-Ti6Al4V after immersion in (a) ex situ preformed p-AgNP dispersions and (b) in situ p-AgNP reaction media; measurements were registered after incident irradiation at 90° and RT. (c) Optical microphotographs of a-Ti6Al4V/p-AgNPs (synthesized, 7 h) after NIR irradiation, λ = 940 nm, as a function of spectral irradiance, Eλ; Ti6Al4V and a-Ti6Al4V were used as controls. The absorbance of a-Ti6Al4V after immersion in (d) ex situ preformed p-AgNP dispersions or (e) in situ p-AgNP reaction media.

Olivieri et al.33 reported a plasmonic surface that produces electrically controlled reflectance, in which high sensitivity is achieved from confined surface plasmon. Plasmon absorptions can lead to the creation of a hot electron with sufficient energy to overcome the Schottky barrier at the metal/semiconductor interface.34 Herein, by combining the localized surface plasmon resonance (LSPR) in the optically NIR excited silver nanoplates, the valence electrons of TiO2 overcome the energy barrier or cross it to become conduction electrons revealing a reflection increment under such conditions. Accordingly, we have analyzed the spectral absorption behavior of tested materials. Fig. 1d and e, display a strong absorption below a wavelength of about 300 nm in a-Ti6Al4V that is associated with the optical band gap of TiO2.35 Red shifts and signal intensification are perceived after the exposure of the a-Ti6Al4V surface to silver nanoplates. The maximum intensity is observed after immersing the a-Ti6Al4V disc in the p-AgNP reaction for 7 h in total agreement with diffuse reflectance results, Fig. 1b. The optical band gap energy, Eg, of direct (m = 2) and indirect (m = ½) transitions were estimated from the sharply increasing absorption region according to Tauc's and Menth's laws36 by extrapolating the adsorption coefficient (α) to zero in the (αhν)m vs. photon energy (hν) plots.35,37 Adsorption coefficient (α) values were computed using the Kubelka–Munk formalism,37–39 α = (1 − R∞)2/ 2R∞, where R∞ = Rsample/Rreference is the reflectance of an infinitely thick sample with respect to a reference (BaSO4) at each wavelength; the results are shown in Fig. 2a–d. The obtained Eg values of direct transitions, about 3.01–2.81 eV, and of indirect transitions, 2.98–2.78 eV, agree with those values reported in the literature for amorphous TiO2,40,41 while the computed Eg value for prism-shaped AgNP dispersions is about 0.98 eV; Fig. S2 (ESI†). Narrowing of Eg for a-Ti6Al4V/p-AgNP composites agrees with the charge transfer of a type-d electron of Ag to the conduction band of TiO2.42 In addition and according to literature experimental evidence, smaller gaps have been measured for successively disordered systems, such as amorphous TiO2, which explain the presence of lower energy states in Fig. 2b and d. All together with Eg red shift and as a result of the presence of p-AgNPs, an important intensification of the absorption coefficient is observed in the energy regime between 3.5 and 4.5 eV, Fig. 2a and c. This fact is more evident for direct transitions and can be ascribed to a superior density of states in the conduction band.37


image file: d4tb01307g-f2.tif
Fig. 2 Absorption coefficient, α, was used to analyze the absorption edge for direct (a) and (b) and indirect (c) and (d) transitions, respectively.

Increased absorption coefficient values and red-shift towards the NIR band obtained from Ag-content TiO2 structures were previously reported by Mosquera et al.42 The authors explained such phenomena as a combined effect of larger AgNP cluster formation along with their surface plasmon resonance that originated from nanosilver agglomeration. Careful inspection of a-Ti6Al4V/p-AgNP surfaces after immersion for 7 h in prism-shaped nanosilver reaction media was done with high resolution scanning electron microscopy (HR-SEM) being able to correlate electro-optical characteristics with their morphological properties, Fig. 3. Electroformed TiO2 nano-holed surfaces, a-TiAl6V412 and anisotropic prism-shaped nanoplates, p-AgNPs13 are shown in Fig. 3a and b. Hybrid a-TiAl6V4/p-AgNP materials display a slight modification of the a-TiAl6V4 substrate after p-AgNP fixation, Fig. 3c–f. The porous structure is preserved with an increase in the size of the holes, Fig. 3d; no statistically significant differences were detected, Fig. S3 and S4 (ESI†). After analyzing the 3D reconstruction of the surfaces of a-TiAl6V4 and a-TiAl6V4/p-AgNP composites, the regularity and periodicity in the distribution of the pores and holes were also comparable; Fig. S5 and S6 (ESI†). Anisotropic silver nanoparticles are homogeneously deposited on the TiO2 surface, Fig. 3c and e; the presence of p-AgNP small clusters fixed on the substrate was corroborated by EDX measurements, Fig. 3f, agreeing with the band gap interpretations previously reported by Mosquera et al.42 From elemental qualitative microanalysis, no peaks related to elements other than Ti and Ag were perceived, ruling out the presence of Ag2O or other silver derivatives, Fig. S7 and S8 (ESI†). Quantitative analysis was used to determine the silver content to be 4.23 wt%; since the measured weight of the a-TiAl6V4/p-AgNP sheets is 0.30950 ± 0.0385 g, a value of 0.046 g Ag per gram Ti was obtained.


image file: d4tb01307g-f3.tif
Fig. 3 (a) HR-SEM microphotograph of the anodized TiO2 surface (a-TiAl6V4).12 (b) TEM microphotograph of silver prism-shaped nanoplates (p-AgNPs).13 (c) HR-SEM microphotograph of the anodized TiO2 surface after nanosilver fixation (a-TiAl6V4/p-AgNPs). Magnified HR-SEM microphotographs of (d) the a-TiAl6V4 substrate and (e) embedded p-AgNPs. (f) EDX analysis of the a-TiAl6V4/p-AgNP sample; individual Ag and Ti peaks were detected, and C corresponds to the double-coated carbon tape used as sample mounting adhesive.

Irradiative recombination of photogenerated electrons and holes (e−/h+) and the alteration of the local density of states (LDoS) due to the interaction among p-AgNPs and a-Ti6Al4V should modulate fluorescence emissions of the hybrid surface.43 3D photoluminescence (PL) spectra, Fig. 4, were used to corroborate the efficiency of charge trapping, displacement and transference.


image file: d4tb01307g-f4.tif
Fig. 4 3D PL patterns recorded upon incident irradiation of the a-Ti6Al4V (a)–(c) and a-Ti6Al4V/p-AgNP (d)–(f) surfaces at 60, 45 and 30°. Peaks indicated with arrows were expressed as (λEM/λEX).

The anodized Ti6Al4V substrate, as shown in Fig. 4a–c, presents the characteristic high emission band at λEM ≈ 300 nm associated with its thin TiO2 layer band edge emission and with self-trapped excitons localized on TiO6 octahedral and oxygen vacancies.44 Minor peak emissions at larger wavelengths are identified as surface state emissions attributed to the quasi-free recombination at the absorption TiO2 band edge, the shallow-trap state near the absorption band edge, the deep-trap band far below the band edge, and permutation of these effects.44 A hallmark of the original a-Ti6Al4V nanostructure, which acts as a Fabry–Pérot resonator, is the induction of specific oscillations at ≈ 800 nm that respond to the decrease of the incident radiation angle.12 Nano-holed hierarchical assembly was preserved after the in situ generation of nanosilver, Fig. 3, and in this way p-AgNPs do not alter the Fabry–Pérot resonator behavior. However, prism-like silver nanoparticles effectively suppress PL bands due to the charge separation between TiO2 and Ag, Fig. 4d–f. According to literature information,45,46 a decrease in the PL intensity is related to rapid separation sites for photo-generated electrons and holes due to the difference in the energy levels of conduction and valence bands in the two-phase structure of p-AgNPs/TiO2 and to exited electrons that migrate to p-AgNP clusters that prevent the direct recombination.46 This effect is of lower intensity when the radiation incides at 45°; under such conditions p-AgNPs merely affect the bands associated with de-excitation from lower vibronic levels, in the NIR region (≈ 800 nm), while not TiO2 layer band edge emission, Fig. 4e. Further 3D PL patterns are included in the ESI† to demonstrate that the presence of spherical-like AgNPs, which exhibit a surface plasmon in the UV spectral region,13 exerts a drastic intensity reduction and the disappearance of the λEM ≈ 850 nm vibronic band on the original a-Ti6Al4V surface; Fig. S9 (ESI†). This behavior influences its NIR reflectivity which also decreases; Fig. S1 (ESI†).

Near-infrared photothermal conversion

Reductions in the optical band gap of the p-AgNPs/a-Ti6Al4V surfaces, as well as, higher density of states in the conduction band are consistent with surface dopant behaviour of p-AgNPs. Under such conditions electrons come from donor p-AgNPs at the a-Ti6Al4V surfaces and their activation requires illumination by light47, as confirmed through the examination of PL patterns, Fig. 4. Following, we analysed the photothermal conversion ability to validate the NIR light harvesting capacity. Temperature of tested p-AgNP's dispersions increases as the radiation exposure time rises demonstrating the ability of nanoparticles to induce a photothermal (PT) effect, Fig. 5a–c. A concentration-dependent linear behavior was established, providing a rational basis for the determination of optimal dosing regimens, Fig. S10 (ESI†). Photothermal conversion efficiency, η, was determined according to a collective heating model based on the literature.48,49
image file: d4tb01307g-f5.tif
Fig. 5 (a) Local temperature variation, ΔT = T − Tsurr, measured at point (1), P1, inside different concentration prism-shaped AgNP dispersions during 600 seconds of NIR laser irradiation at 904 nm (E = 83.0 mW cm−2) followed by a cooling period (laser off) until Tsurr. (b) Linearized time data from the cooling period versus the negative natural logarithm of driving force temperature (dimensionless parameter, θ) to compute τS. (c) Example of IR thermal images of an 80 μg mL−1 prism-shaped AgNP dispersion exposed to a 904 nm (E = 83.6 mW cm−2) laser along 600 seconds; local temperature measured at point (2), r = 6 mm from the central point (P1).

First, the characteristic time constant for cooling (τs) was determined by the introduction of a dimensionless parameter using the maximum system temperature, Tmax, surroundings temperature, Tsurr, and by plotting the non-dimensionalized temperature driving force, θ, vs. time, t, according to eqn (1), followed by taking the negative reciprocal of the linear fit data slope, eqn (2):

 
image file: d4tb01307g-t1.tif(1)
 
t = −τs[thin space (1/6-em)]ln[thin space (1/6-em)]θ (2)

To determine the heat loss due to external heat flux, Qdis, the linearized form of Newton's Law of Cooling is employed as in eqn (3).

 
Qdis = hS(T − Tsurr) (3)
where h is the heat transfer efficiency, and S is the surface area of the interface between the photothermal material and the external environment. External heat flux in the system, Qdis, expresses heat dissipated from the light absorbed by surroundings and was measured independently using a blank that does not contain the photothermal material: the cuvette and solvent for p-AgNP dispersions or Ti6Al4V discs as appropriate. To solve the heat-transfer coefficient across the entire area of flux (hS), eqn (4) is utilized:
 
image file: d4tb01307g-t2.tif(4)
where, mi and Cp,i are the mass and heat capacity of the different materials that compose the sample, running through index i. The hS term is then utilized to solve the photothermal conversion efficiency at steady state laser irradiation, where the heat dissipation (Qdis) was subtracted from the overall heat generation according to eqn (5).
 
QI = hS(Tmax − Tsurr) − Qdis (5)
QI is the energy influx due to photothermal material absorption described as a function of photothermal conversion efficiency, η, by eqn (6):
 
image file: d4tb01307g-t3.tif(6)
where I is the laser incident power on each sample computed as I = E × A and Aλ is the absorption of photothermal material at laser irradiation λ.

High values were obtained, η = 49.7–65.7%, which are in the acceptable range for efficient biomedical applications,50 and the results are summarized in Table S1 (ESI†). Once the ability of the p-AgNPs to induce the PT effect was established, we tested it on p-AgNPs/a-Ti6Al4V surfaces. It can be appreciated that, in agreement with information exposed in the preceding sections, p-AgNPs/a-Ti6Al4V surfaces obtained by immersion of a-Ti6Al4V discs on p-AgNP reaction media give rise an increase of temperature after NIR laser irradiation while no effect can be seen on bare Ti6Al4V, a-Ti6Al4V or in preformed p-AgNPs (80 μg mL−1) deposited on a-Ti6Al4V discs, Fig. 6a–c. Truthfully, the obtained results are comparable to those obtained with p-AgNP dispersions of the same concentration with η = 35%, Fig. 6b and Table S1 (ESI†).


image file: d4tb01307g-f6.tif
Fig. 6 (a) Local temperature variation, ΔT = T − Tsurr, measured at point (1), P1, inside p-AgNPs/a-Ti6Al4V surfaces during 600 seconds of NIR laser irradiation at 904 nm (E = 83.0 mW cm−2); Ti6Al4V and a-Ti6Al4V discs are used as controls. (b) Comparison between local temperature variation, ΔT = T − Tsurr, measured at point (1), P1, inside the 14 μg mL −1 concentration prism-shaped AgNP dispersion and p-AgNPs (14 μg mL −1)/a-Ti6Al4V surfaces during 600 seconds of NIR laser irradiation at 904 nm (E = 83.0 mW cm−2) followed by a cooling period (laser off) until Tsurr. (c) IR thermal images of Ti6Al4V, a-Ti6Al4V, in situ synthesized p-AgNPs (14 μg mL −1)/a-Ti6Al4V and ex situ deposited p-AgNPs (14 μg mL −1)/a-Ti6Al4V surfaces exposed to a 904 nm (E = 83.6 mW cm−2) laser along 600 seconds; local temperature measured at point (2), r = 6 mm from the central point (P1).

Antibacterial properties

The photothermal conversion effect can be used to develop the next generation of controllable antibacterial nano-platforms [43] and, in particular, to control Staphylococcus aureus, examined here, which forms biofilms on bones, heart valves and any implanted medical device.51 Prism-shaped nanosilver exhibits greater bacteriostatic and bactericidal effects after direct contact with Staphylococcus aureus that contrasts with the control spherical-shaped AgNPs, Fig. S11 (ESI†). The obtained results are in complete agreement with a shape dependent interaction mechanism related to the presence of higher localized Ag0 atoms at prim edges.52 Prism-shaped nanosilver shows a minimum inhibition and bactericidal value of 3.1 μg mL−1 at 105 CFU mL−1 bacterial concentration; results are shown in Fig. 7a, b and Table S2 (ESI†). Once the nanoparticles are blended on the a-Ti6Al4V/p-AgNP (14 μg mL−1) substrate, the antibacterial behavior of the p-AgNP dispersion is reproduced demonstrating a significant decrease in bacterial colonies density and in their adhesion, Fig. 7c and d. One of the causes of bacterial annihilation is the surface charge of the materials that come into contact with them. It is generally considered that the electrostatic interaction between negatively charged bacterial membranes and positively charged nanoparticles increases the antibacterial efficiency. Electrostatic interaction disturbs the surface negativity of the microorganism membrane by alteration of lipid mediated signaling and eventually conduces to its destabilization and decrease.53 According to their isoelectric point 4.4–5.2,54,55 Ti6Al4V, a-Ti6Al4V surfaces were negative at pH = 7.4. In addition, p-AgNPs' zeta potential (ζ-potential) measurements gave a value of −33.4 ± 7.4 mV.13
image file: d4tb01307g-f7.tif
Fig. 7 (a) p-AgNPs against Staphylococcus aureus viability; representative images of Staphylococcus aureus with different bacterial colonies (102–104 CFU mL−1) on blood agar platelets: (1) 25 μg mL−1; (2) 12.5 μg mL−1; (3) 6.2 μg mL−1 and (4) 3.1 μg mL−1. (b) The minimum inhibitory concentration (MIC) endpoint is the lowest concentration of silver nanoparticles where no visible growth is seen in the tubes; thus, the visual turbidity comparison of the tubes was noted, both before and after incubation, to confirm the MIC value. Example of the determination of the MIC for prism-shaped AgNPs against Staphylococcus aureus 105 CFU mL−1 concentration cultured in Mueller Hinton broth at 37 °C for 24 h. (c) Bacteria cultures (107 CFU mL−1) after interaction with Ti6Al4V, a-Ti6Al4V and p-AgNP/a-Ti6Al4V discs. (d) Bacteria adhesion (107 CFU mL−1) after 904 nm (E = 83.3 mW cm−2) irradiation of Ti6Al4V, a-Ti6Al4V and p-AgNP/a-Ti6Al4V discs. Statistical analysis was performed among different materials, n = 4; *p < 0.05; significant differences between the samples are indicated with brackets.

Being electronegative, a-Ti6Al4V/p-AgNP surfaces cause some degree of electrostatic repulsion after interaction with the tested microorganisms (Staphylococcus aureus has a ζ-potential value of about −38 mV56 at 37 °C and physiological pH). Therefore, we did not find any correlation in the reduction of bacterial adhesion with the substrate surface charge. Like the electrostatic interaction, topography and chemical features are additional factors that influence microorganism-surface adhesion and its subsequent viability. In a previous work we have demonstrated that the nano-holed coating exhibits a hydrophobic wetting performance according to the Cassie–Baxter regime, even in the presence of chemically hydrophilic compounds.12 Wetting features of the surfaces exposed a minor contact angle denoting a higher hydrophilic character, after incorporation of p-AgNPs, compared with original bare Ti6Al4V, Fig. 8.


image file: d4tb01307g-f8.tif
Fig. 8 Static water contact angle (CA) measured for (a) bare Ti6Al4V, (b) a-Ti6Al4V, (c) a-Ti6Al4V/p-AgNP (14 μg mL−1).

It is known that the hydrophilicity of the surfaces favours bacterial adhesion; however, in light of the obtained results it is also not possible to correlate the antibacterial effect of a-Ti6Al4V/p-AgNP (14 μg mL−1) surfaces with their highly hydrophilic character. Similarly, a-Ti6Al4V surfaces, despite their strong hydrophobicity, have no antibacterial effect. Furthermore, the stability of the surfaces was tested by immersion over a 24 h treatment period. The initial relative results for Ag and Ti remained consistent, indicating no loss or dissolution of the nanoparticles from the surface, Fig. S7 (ESI†). Once the usual physical and chemical bactericidal effects were ruled out, we tested the effect of heating under NIR irradiation.

Fig. 7d shows that there is a clear reduction of bacterial adhesion and viability after NIR irradiation of a-Ti6Al4V/p-AgNP (14 μg mL−1) materials compared with bare and anodized Ti6Al4V substrates. We have confirmed that after 10 minutes of NIR irradiation, and despite a photothermal conversion efficiency of (η) ≈ 50–65%, dispersions of p-AgNPs alone do not show any improvement in their antibacterial capacity, Fig. S12 (ESI†). Furthermore, anodized Ti6Al4V sheets, lacking any photothermal effect, show 100% of bacterial adherence with or without irradiation, Fig. 7d. Thus, we have postulated that the photothermal action cannot be isolated from other mechanisms involved in antimicrobial action. Literature information reports that inhibition of colony formation can be performed by manipulation of the electronic structure of materials to generate e−/h+ vacancies.57 This mechanism of microorganisms' annihilation fits our previous results, where we have established the generation of e−/h+ vacancies in the electronic structure of the TiO2 semiconductor modulated by the incorporation of the p-AgNPs, Fig. 2 and 4. To further confirm the antibacterial ability of a-Ti6Al4V/p-AgNP (14 μg mL−1) surfaces after NIR irradiation, bacterial colonies were observed on the disc surfaces by SEM, Fig. 9. Compared to the a-Ti6Al4V control, Fig. 9a and b, the number of sphere-shaped Staphylococcus aureus bacteria attached to the a-Ti6Al4V/p-AgNP (14 μg mL−1) substrate was significantly reduced and only isolated bacteria were distinguished. Under NIR irradiation of a-Ti6Al4V/p-AgNPs (14 μg mL−1) surfaces, no bacterial proliferation or adhesion was detected, demonstrating photothermal action to inhibit colony formation. Additionally, an initial pre-inspection revealed that the surfaces do not alter the mitochondrial activity of eukaryotic cells, Fig. S13 (ESI†), which aligns with our previous p-AgNP hemocompatibility results.58


image file: d4tb01307g-f9.tif
Fig. 9 SEM microphotographs of Staphylococcus aureus adhesion (107 CFU mL−1) on (a) and (b) a-Ti6Al4V; (c) and (d) a-Ti6Al4V/p-AgNPs (14 μg mL−1) discs without irradiation and (e) and (f) after the application of 904 nm (E = 83.3 mW cm−2) radiation along 10 minutes. Magnified areas (b), (d) and (f) are seen in red squares; bacteria are indicated with arrows.

Conclusions

In this work, we applied the concept of a “plasmonic sensitizer” by embedding prism-shaped silver nanoparticles (p-AgNPs) within an electroforming nano-holed TiO2 coating on the Ti6Al4V alloy (a-Ti6Al4V). We established optimal reaction conditions for the maximum photo-conversion capacity under NIR irradiation. The ordered nano-holed TiO2 array efficiently immobilized anisotropic plasmonic nanoparticles with NIR localized surface plasmon resonance (LSPR) extinction spectra, improving their optoelectronic properties without significant changes in morphology. The enhanced surface reflective capacity and increased absorption coefficient were demonstrated, validating the improved electronic properties of the a-Ti6Al4V/p-AgNP structure. We evaluated the electro-optical properties of a-Ti6Al4V/p-AgNP surfaces, showing a photothermal conversion efficiency of about 35%, generating a temperature increase comparable to p-AgNP aqueous dispersion. The antibacterial properties under NIR irradiation were significantly superior to either a-Ti6Al4V surfaces or p-AgNP dispersion alone. The results indicate a synergistic interaction between a-Ti6Al4V and p-AgNPs, with the photothermal effect and reduced band gap contributing to the observed antibacterial efficacy.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors disclose no potential conflicts of interest.

Acknowledgements

The authors acknowledge the financial support from Universidad Nacional del Sur [UNS, PGI 24/Q131], Consejo Nacional de Investigaciones Científicas y Técnicas [CONICET, PIP 11220210100126CO] and Agencia Nacional de Promoción Científica y Tecnológica [ANPCyT, PICT-2021-I-A-00108]. J.M.R. thanks Ministerio de Ciencia e Innovación [PID2019-805 111327GB-100] and Xunta de Galicia [ED431B 2022/36]. B.D.P and F.B. have a fellowship of CONICET. P.V.M. is a principal researcher of CONICET.

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Footnotes

† Electronic supplementary information (ESI) available: Additional information is provided related to the morphology, qualitative and quantitative elemental microanalyses, initial screening of eukaryote cellular viability, and optoelectronic and bactericidal properties of a-Ti6Al4V/p-AgNP surfaces. See DOI: https://doi.org/10.1039/d4tb01307g
‡ Denise B. Pistonesi and Federico Belén contributed equally to this work.
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