Nitric Oxide Release and Antibacterial Efficacy Analyses of S-Nitroso- N-Acetyl-Penicillamine Conjugated to Titanium Dioxide Nanoparticles

Abstract

Therapeutic agents can be linked to nanoparticles to fortify their selectivity and targeted delivery while impeding systemic toxicity and efficacy loss. Titanium dioxide nanoparticles (TiNPs) owe their rise in biomedical sciences to their versatile applicability, although the lack of inherent antibacterial properties limits its application and necessitates the addition of bactericidal agents along with TiNPs. Structural modifications can improve TiNP's antibacterial impact. The antibacterial efficacy of nitric oxide (NO) against a broad spectrum of bacterial strains is well established. For the first time, S-nitroso-N-acetylpenicillamine (SNAP), an NO donor molecule, was covalently immobilized on TiNPs to form the NO-releasing TiNP-SNAP nanoparticles. The TiNPs were silanized with 3-aminopropyl triethoxysilane, and N-acetyl-d-penicillamine was grafted to them via an amide bond. The nitrosation was carried out by t-butyl nitrite to conjugate the NO-rich SNAP moiety to the surface. The total NO immobilization was measured to be 127.55 ± 4.68 nmol mg^-1 using the gold standard chemiluminescence NO analyzer. The NO payload can be released from the TiNP-SNAP under physiological conditions for up to 20 h. The TiNP-SNAP exhibited a concentration-dependent antimicrobial efficiency. At 5 mg mL^-1, more than 99.99 and 99.70% reduction in viable Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria, respectively, were observed. No significant cytotoxicity was observed against 3T3 mouse fibroblast cells at all the test concentrations determined by the CCK-8 assay. TiNP-SNAP is a promising and versatile nanoparticle that can significantly impact the usage of TiNPs in a wide variety of applications, such as biomaterial coatings, tissue engineering scaffolds, or wound dressings.

Keywords: NO donor; antibacterial; biomedical application; nitric oxide; surface modification; titanium dioxide nanoparticles.

Figure 1.

Synthesis route of TiNP–APTES, TiNP–NAP, and TiNP–SNAP. Acid-treated TiNPs were reacted with APTES using a reflux system under a nitrogen gas atmosphere at 120 °C for 12 h (a), followed by NAP thiolactone reaction at RT with vigorous stirring for 24 h (b). Finally, nitrosation reaction with t-butyl nitrite at RT for 1 h led to the production of SNAP-immobilized to TiNPs (c). Toluene was used as the solvent for all the reactions.

Figure 2.

Representative FTIR spectra of TiNP, TiNP–APTES, TiNP–NAP, and TiNP–SNAP.

Figure 3.

Representative ¹³C CP/MAS NMR spectra of (a) TiNP–APTES and (b) TiNP–NAP. (a) Three characteristic NMR signals were observed at 10.26, 22.84, and 42.99 ppm for the three propyl carbon atoms of the APTES moiety (marked as 1, 2, and 3). (b) Two characteristic carbonyl carbon atom peaks for the NAP thiolactone unit were observed at 171 and 179 ppm along with other characteristic carbon peaks.

Figure 4.

DLS (black bars) and zeta potential (red line) measurements of TiNPs at different stages of surface modification (n = 3).

Figure 5.

In vitro, NO release kinetics of the TiNP–SNAP obtained from a 1 mg mL⁻¹ colloidal suspension of the nanoparticles in PBS containing 100 μM EDTA at 37 °C within the amber NOA sample cell. Data represent as the mean ± SD (n = 3).

Figure 6.

Bactericidal efficacy analysis of TiNP–SNAP against (a) Gram-positive S. aureus and (b) Gram-negative E. coli. The concentration of bacteria (control) without exposure to nanoparticles is presented by solid black bars. Striped black and solid red bars are used to show the concentration of viable bacteria after 24 h exposure to TiNP and TiNP–SNAP, respectively. Data are presented as the mean values ±SD (n = 4), where ** = p ≤ 0.01 and *** = p ≤ 0.001 for TiNP vs TiNP–SNAP.

Figure 7.

Cytocompatibility of TiNP and TiNP–SNAP leachates in media toward NIH 3T3 mouse fibroblast cells. The viability of cells was assessed using a CCK-8 cell viability kit. The cells exposed to the TiNP and TiNP–SNAP extracts depicted by striped black and solid red bars, respectively, exhibited cell viability similar to that of the untreated control cells in DMEM that were not exposed to TiNPs, depicted in solid black bars, highlighting the non-toxic nature of the particle leachate. Data are presented as mean ± SD for n = 6, normalized to the untreated cell control.