Biocompatible antibiotic-coupled nickel-titanium nanoparticles as a potential coating material for biomedical devices

Abstract

The challenges facing metallic implants for reconstructive surgery include the leaching of toxic metal ions, a mismatch in elastic modulus between the implant and the treated tissue, and the risk of infection. These problems can be addressed by passivating the metal surface with an organic substrate and incorporating antibiotic molecules. Nitinol (NiTi), a nickel-titanium alloy, is used in devices for biomedical applications due to its shape memory and superelasticity. However, unmodified NiTi carries a risk of localized nickel toxicity and inadequately supports angiogenesis or neuroregeneration due to limited cell adhesion, poor biomineralization, and little antibacterial activity. To address these challenges, NiTi nanoparticles were modified using self-assembled phosphonic acid monolayers and functionalized with the antibiotics ceftriaxone and vancomycin via the formation of an amide. Surface modifications were monitored to confirm that phosphonic acid modifications were present on NiTi nanoparticles and 100% of the samples formed ordered films. Modifications were stable for more than a year. Elemental composition showed the presence of nickel, titanium, and phosphorus (1.9% for each sample) after surface modifications. Dynamic light scattering analysis suggested some agglomeration in solution. However, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy confirmed a particle size distribution of <100 nm, the even distribution of nanoparticles on coverslips, and elemental composition before and after cell culture. B35 neuroblastoma cells exhibited no inhibition of survival and extended neurites of approximately 100 μm in total length when cultured on coverslips coated with only poly-l-lysine or with phosphonic acid-modified NiTi, indicating high biocompatibility. The ability to support neural cell growth and differentiation makes modified NiTi nanoparticles a promising coating for surfaces in metallic bone and nerve implants. NiTi nanoparticles functionalized with ceftriaxone inhibited Escherichia coli and Serratia marcescens (SM6) at doses of 375 and 750 μg whereas the growth of Bacillus subtilis was inhibited by a dose of only 37.5 μg. NiTi-vancomycin was effective against B. subtilis at all doses even after mammalian cell culture. These are common bacteria associated with infected implants, further supporting the potential use of functionalized NiTi in coating reconstructive implants.

Keywords: Antibacterial activity; Biocompatibility; Ceftriaxone; Metallic implants; Nanoparticles; Neural differentiation; Nitinol; Phosphonic acid; Vancomycin.

Fig. 1

Structure of Antibiotics used to modify NiTi nanoparticles with labeled amine.

Fig. 2

Schematic of COOHPA on NiTi nanoparticles functionalized with ceftriaxone as an antibiotic.

Fig. 3

Infrared spectra of CH region of NiTi nanoparticles modified with (A) 12-NH₂PA, (B) COOHPA, and (C) ODPA 18 months after preparation.

Fig. 4

IR spectra of the modifications (black spectra) steps for NiTi compared to unbound solid (red spectra): (A) IR of the carboxylic acid region of the carboxylic terminated phosphonic acid-modified sample (step 1), (B) EDC/NHS formation (step 2 and 3), (C) ceftriaxone attachment (step 4), an (D) vancomycin attachment. Both antibiotic samples were rinsed three times.

Fig. 5

XPS analysis of NiTi nanoparticles and their modifications. (A) Compositional survey scans of the original (red), COOHPA-modified (blue), NHS-ester (magenta), and antibiotic-added (purple for ceftriaxone and brown for vancomycin) NiTi nanoparticles. (B) High-resolution P2p spectra for COOHPA-modified samples showing phosphorus from COOHPA. (C) S2p spectra for samples with ceftriaxone, illustrating the antibiotic's attachment. (D) Cl2p spectra for vancomycin-modified samples, confirming the presence of the antibiotic.

Fig. 6

XPS characterization of unmodified and phosphonic acid-modified NiTi nanoparticle coverage on poly-K coated coverslips. (A) Compositional survey scans of bare coverslip (red), unmodified NiTi-coated coverslip (blue), and coverslips coated with 12-NH₂PA (green), COOHPA (purple), or ODPA (brown)-modified NiTi nanoparticles. (B–D) High-resolution Ni2p, Ti2p, and P2p spectra.

Fig. 7

SEM images at 600 × with corresponding EDS of a site representation of each poly-K-coated coverslips with nanoparticles before cell culture of (A) unmodified NiTi, (B) NiTi modified with ODPA, (C) NiTi modified with 12-NH₂PA (D) NiTi modified with COOHPA (E) NiTi modified with ceftriaxone, and (F) NiTi modified with vancomycin. Nickel (blue color mapping) and titanium (orange color mapping) with the microscope can be detected for each coverslip analyzed.

Fig. 8

SEM images at 20,000 × of each poly-K-coated coverslips with nanoparticles before cell culture of (A) unmodified NiTi, (B) NiTi modified with ODPA, (C) NiTi modified with 12-NH₂PA (D) NiTi modified with COOHPA (E) NiTi modified with ceftriaxone, and (F) NiTi modified with vancomycin.

Fig. 9

SEM images at 600 × with corresponding EDS of a site representation of each poly-K-coated coverslips with nanoparticles after cell culture of (A) unmodified NiTi, (B) NiTi modified with ODPA, (C) NiTi modified with 12-NH₂PA (D) NiTi modified with COOHPA (E) NiTi modified with ceftriaxone, and (F) NiTi modified with vancomycin. Nickel (blue color mapping) and titanium (orange color mapping) with the microscope.

Fig. 10

NiTi substrates with various modifications do not alter the neurite extension properties of differentiating B35 cells. Cells labeled for F-actin (green) and nuclei (blue) were grown on coverslips coated with (A) poly-K only or (B) NiTi, (C) NiTi-COOHPA, (D) NiTi-COOHPA-ceftriaxone, (E) NiTi-COOHPA-vancomycin. (F) Mean total neurite length per cell measurements indicate no significant differences between cells grown on different surface coatings (G) A live/dead assay confirms that cell survival is comparable between cells grown on NiTi-coated surfaces and on poly-K alone. Mean + SD. p > 0.05. One-way ANOVA with Tukey's multiple comparisons.

Fig. 11

Inhibition (mm diameter) of the tested bacterial strains by NiTi functionalized with (A) ceftriaxone or (B) vancomycin for doses of 37.5, 375 and 750 μg. NiTi functionalized with either antibiotic (75 μg) and collected from coverslips after mammalian cell culture also effectively inhibited the growth of B. subtilis.