Enhancing hand hygiene compliance through the long-lasting antimicrobial effects of nitric oxide-releasing hand sanitizer gel

Abstract

Effective hand hygiene is crucial for reducing the transmission of disease-causing pathogens. While alcohol-based hand sanitizers have become popular, their increased usage during the COVID-19 pandemic raised concerns about their short-lived activity and potential side effects. The increased application of hand sanitizers and harmful side effects has necessitated an effective alternative with prolonged and enhanced antimicrobial properties which could result in a reduced number of sanitizer applications. To address these issues and improve antimicrobial efficacy, this study developed a nitric oxide (NO)-releasing hand sanitizer (NORel) gel enriched with other antimicrobial and moisturizing ingredients like ethanol, tea tree oil, and glycerin. The NORel gel underwent comprehensive analysis, including assessments of pH for 60 d, rheology, NO release, cytocompatibility, and in vitro and ex vivo antimicrobial effectiveness on rabbit skin proving its ability to eliminate over 97% of bacteria and fungi, including antibiotic-resistant strains. One NORel gel formulation, NORel2, demonstrated antimicrobial activity comparable to a commercial alcohol-based gel containing 62% ethyl alcohol, achieving a reduction of more than 5 logs in S. aureus bacteria on a rabbit skin model. Additionally, the NORel gel significantly outperformed the commercial alcohol gel by maintaining its antimicrobial efficacy on infected rabbit skin, showing a persistent activity with a 1.6-log reduction in viable S. aureus 2 h after application. This research introduces a biocompatible NO-releasing gel with superior antimicrobial properties compared to common alcohol-based sanitizers, making it an effective hand hygiene solution to reduce infections, especially in high-risk environments.

Fig. 1. (A) Various sources of hand contamination. (B) Structural composition of bacteria (C) a detailed, step-by-step guide to the methodology for creating NO-releasing hand sanitizer (NORel) gel.

Fig. 2. (A) pH analysis of NORel hand sanitizer gel. pH stability of NORel gels at (B) −20 °C freezer (C) 4 °C fridge (D) room temperature (23 °C) and (E) 37 °C (physiological temperature), up to 60 d of storage. (F) Average viscosity measurements of NORel and control gels. Data represents mean ± SD, n = 3.

Fig. 3. (A) Chemical structure of NO donor S-nitroso-N-acteylpenicillamine (SNAP) non-covalently dispersed with other traditional hand sanitizer ingredients to generate NO-releasing hand sanitizer. NO donor can be catalyzed via heat, light or metal ions to achieve real-time NO release. (B) Nitric oxide release analysis from NORel gels tested using chemiluminescence method at physiological conditions (37 °C). (C) UV-vis spectra of SNAP recorded at 250–600 nm wavelength at various concentrations in PBS–EDTA. Storage stability of NORel after 28 d of storage at (D) −20 °C, (E) 4 °C, (F) room temperature (23 °C) and (G) 37 °C. The retention of NO in NORel gels was measured using the absorbance peak of SNAP at 340 nm using UV-vis spectroscopy normalized to day 0. (H) Cytocompatibility of NORel tested towards 3T3 mouse fibroblast cells relative to cell control in a 24 h cell viability assay using MTT cell viability kit. All data are presented as mean ± SD (n ≥ 3).

Fig. 4. Antimicrobial activity of NORel hand sanitizer gel calculated as log of colony forming unit (CFU) mL⁻¹ against (A) S. aureus, (B) E. coli, (C) methicillin-resistant S. aureus (MRSA), and (D) C. albicans after 6 h exposure. Corresponding log reduction in microbial viability after expsoure to NORel and controls for (E) S. aureus, (F) E. coli, (G) methicillin-resistant S. aureus (MRSA), and (H) C. albicans, plotted with respect to untreated bacteria control. (I) Experimental design used to evaluate the antimicrobial activity of NORel vs. control alcohol gel using an ex vivo rabbit skin model. (J) Ex vivo disinfection of an infected rabbit skin using commercial alcohol gel and NORel. (K) Corresponding log reduction in bacterial viability after exposure to S. aureus bacteria. (L) Representative images of LB agar plates with viable S. aureus bacteria CFU after 6 h of exposure to NORel and commercial alcohol gel in the ex vivo rabbit disinfection model. All data are represented as mean ± SD (n ≥ 3).

Fig. 5. (A) Schematic illustration of difference between standard alcohol containing sanitizer vs. NO-releasing hand sanitizer gel (NORel). Alcohol containing gels with no secondary antimicrobial action evaporate quickly and lack in continuous activity overtime. NO releasing gel with other active ingredients can effectively kill and also exhibit persistent antimicrobial activity over an extended period of time. (B) Experimental design used to evaluate the persistence activity of NORel vs. control alcohol gel using a rabbit skin model with 2 h exposure to the test gel. (C) Validation of long-term effectiveness of NORel on rabbit skin specimen tested against S. aureus bacteria. (D) Log reduction in bacteria viability 2 h after gel exposure. (E) Schematic illustration of mechanism by which NO denatures bacteria. All data is presented as mean ± SD for n ≥ 3.