Nitric oxide releasing compositions and methods for making and using the same

Abstract

In accordance with the purpose(s) of the present disclosure, as embodies and broadly described herein, the disclosure, in one aspect, relates to nitric oxide releasing compositions and a method of making nitric oxide releasing compositions. The compositions can include a nitric oxide releasing compound and other components, such as ethanol, water, a thickening agent, a polyol, and a neutralizer. The compositions described herein can be used to reduce or prevent the growth of microbes on a surface.

Description

T|H Docket: 222105-2280 NITRIC OXIDE RELEASING COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under grant number R01HL151473 awarded by the NIH. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 63 / 623,392, filed on January 22, 2024, the contents of which are incorporated by reference herein in their entireties. BACKGROUND

[0003] Infectious diseases have become an enduring challenge, affecting individuals and communities worldwide. The burden of infectious diseases extends far beyond the immediate health impact. It encompasses strains on healthcare infrastructure, economic losses, and disruption of daily life. According to the US Center for Disease Control and Prevention (CDC), approximately 1.7 million hospitalized patients each year contract Healthcare-Associated Infections (HCAIs) while receiving treatment for other medical conditions. HCAIs are infections that a patient did not present upon admittance the healthcare facility. Over 98,000 of these patients, equating to one in 17 cases, succumb to HCAIs, emphasizing the severity of this issue.

[0004] Effective hand hygiene is one of the most straightforward and scientifically validated methods for reducing the transmission of infectious agents. Numerous studies have indicated that implementing basic infection-control measures, such as regular hand sanitization, can play a pivotal role in preventing HCAIs. When executed correctly and consistently, hand hygiene significantly reduces the risk of HCAIs, community-acquired infections, spread of contagious diseases, and healthcare costs associated with treating HCAIs.

[0005] Hand hygiene can be achieved through handwashing with soap and water or using hand sanitizers. However, hand washing it is not always feasible in various settings, such as healthcare facilities, public transportation, or during emergencies. In such situations, hand sanitizers serve as a convenient and effective alternative due to their portability and ease of use. These sanitizers have proven effective against a broad spectrum of microorganisms, including bacteria and viruses, due to the ability of ethanol to infiltrate microbial membranes,T|H Docket: 222105-2280 denature proteins and impede microbial growth. However, their efficacy depends on several factors, including the type and concentration of active ingredients, application technique, and contact time. Additionally, their rapid evaporation upon application limits their residual activity, necessitating frequent reapplication to maintain protection. Frequent use of hand sanitizers with alcohol has been shown to cause skin dryness, irritation, acute toxicity, and other dermatological side effects. Therefore, there is a need to develop hand sanitizers that meet antimicrobial standards and demonstrate longer-term antimicrobial action compared to alcohol-based gels and other hand sanitization methods. SUMMARY

[0006] In accordance with the purpose(s) of the present disclosure, as embodies and broadly described herein, the disclosure, in one aspect, relates to nitric oxide releasing compositions and a method of making nitric oxide releasing compositions. The compositions can include a nitric oxide releasing compound and other components, such as ethanol, water, a thickening agent, a polyol, and a neutralizer. The compositions described herein can be used to reduce or prevent the growth of microbes on a surface.

[0007] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

[0009] Figure 1A illustrates various sources of hand contamination.

[0010] Figure 1B illustrates the structural composition of viruses and bacteria

[0011] Figure 1C shows a detailed, step-by-step guide to the methodology for creating NO-releasing hand sanitizer (NORel).

[0012] Figure 2A shows a pH analysis of NORel hand sanitizer gel.T|H Docket: 222105-2280

[0013] Figures 2B-2E show the pH stability of NORel gels at (2B) -20oC freezer (2C) 4oC fridge (2D) room temperature (23oC), and (2E) 37oC (physiological temperature), up to 60 d of storage.

[0014] Figure 2F shows pictures of NORel and control gels.

[0015] Figure 2G shows the viscosities of NORel and control gels at 1 rpm speed. Each sample (0.5 mL) was measure by viscometer at RT (23oC). The speed ramp was set up from 1 to 100 rpm with 10 increments, and each speed was held for 10 s with data recorded each second.

[0016] Figure 2H shows average viscosity measurements of NORel and control gels. Data represents mean ± SD, n = 3.

[0017] Figure 3A illustrates the chemical structure of NO donor S-nitroso-N- acteylpenicillamine (SNAP), which can be 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.

[0018] Figure 3B shows a nitric oxide release analysis from NORel gels tested using chemiluminescence method at physiological conditions (37 ºC). All data is presented as mean ± SD (n ≥ 3).

[0019] Figure 3C shows UV-vis spectra of SNAP recorded at 300-600 nm wavelength at various concentrations in PBS-EDTA.

[0020] Figures 3D-3G show the storage stability of NORel gels after 28 d of storage at (3D) -20 ºC, (3E) 4 ºC, (3F) Room temperature (23 ºC), and (3G) 37 ºC. 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. All data are presented as mean ± SD (n ≥ 3).

[0021] Figure 3H shows the cytocompatibility of NORel gels tested towards 3T3 mouse fibroblast cells relative to cell control in a 24 h cell viability assay using MTT cell viability kit. All data is presented as mean ± SD (n ≥ 3).

[0022] Figures 4A-4H show the antimicrobial activity of NORel hand sanitizer gel calculated as log of colony forming unit (CFU) mL-1against (4A) S. aureus, (4B) E. coli, (4C) Methicillin-resistant S. aureus (MRSA), and (4D) C. albicans, and the corresponding log reduction in microbial viability after expsoure to NORel gel and controls for (4E) S. aureus, (4F) E. coli, (4G) Methicillin-resistant S. aureus (MRSA), and (4H) C. albicans, plotted with respect to untreated bacteria control. All data are presented as mean ± SD (n ≥ 3).

[0023] Figure 4I illustrates the experimental design used to evaluate the antimicrobial activity of NORel vs. control alcohol gel using an ex vivo rabbit skin model.

[0024] Figures 4J-4K show (4J) the ex vivo disinfection of an infected rabbit skin using commercial alcohol gel and NORel, and (4K) the corresponding log reduction in bacterial viability after exposure to S. aureus bacteria. All data are presented as mean ± SD (n ≥ 3).T|H Docket: 222105-2280

[0025] Figure 4L shows representative images of Luria-Bertani (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.

[0026] Figure 5A shows 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.

[0027] Figure 5B illustrates the experimental design used to evaluate the persistence activity of NORel vs. control alcohol gel using a rabbit skin model.

[0028] Figures 5C-5D show (5C) the validation of long-term effectiveness of NORel on rabbit skin specimen tested against S. aureus bacteria, and (5D) the corresponding log reduction in bacteria viability 2 h after gel exposure. All data are presented as mean ± SD for n ≥ 3.

[0029] Figure 5E shows a schematic illustration of mechanism by which NO denatures bacteria.

[0030] The drawings illustrate only example embodiments and are therefore not to be considered limiting of the scope described herein, as other equally effective embodiments are within the scope and spirit of this disclosure. The elements and features shown in the drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the embodiments. Additionally, certain dimensions may be exaggerated to help visually convey certain principles. In the drawings, similar reference numerals between figures designate like or corresponding, but not necessarily the same, elements. DETAILED DESCRIPTION

[0031] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0032] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0033] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.T|H Docket: 222105-2280

[0034] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0035] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0036] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0037] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0038] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. Definitions and abbreviations

[0039] In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

[0040] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude theT|H Docket: 222105-2280 presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0041] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a solvent” includes, but are not limited to, mixtures or combinations of two or more such solvents, and the like.

[0042] It should be noted that ratios, concentrations, amounts, rates, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed and “about 5 to about 15” is also disclosed.

[0043] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0044] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range isT|H Docket: 222105-2280 explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0045] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0046] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0047] Disclosed are the components to be used to prepare the compositions disclosed herein as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed,T|H Docket: 222105-2280 specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B- D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0048] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.

[0049] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance and instances where it does not.

[0050] As used herein, the term “biocompatible,” with respect to a substance or fluid described herein, indicates that the substance or fluid does not adversely affect the short-term viability or long-term proliferation of a target biological particle within a particular time range.

[0051] The terms “antimicrobial” and “antimicrobial characteristic” refer to the ability to kill and / or inhibit the growth of microorganisms. A substance having an antimicrobial characteristic may be harmful to microorganisms or microbes (e.g., bacteria, fungi, virus, protozoans, algae, and the like). A substance having an antimicrobial characteristic can kill the microorganism and / or prevent or substantially prevent the growth or reproduction of the microorganism.

[0052] The terms “bacteria” or “bacterium” include, but are not limited to, gram positive and gram negative bacteria. Bacteria can include, but are not limited to, Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anabaena affinis and other cyanobacteria (including the Anabaena, Anabaenopsis, Aphanizomenon, Camesiphon, Cylindrospermopsis, Gloeobacter Hapalosiphon, Lyngbya, Microcystis, Nodularia, Nostoc, Phormidium, Planktothrix, Pseudoanabaena, Schizothrix, Spirulina,T|H Docket: 222105-2280 Trichodesmium, and Umezakia genera) Anaerorhabdus, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella, Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter, Helococcus, Holdemania Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus, Phytoplasma, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia Rochalimaea Roseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum, Spiroplasma, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Tropheryma, Tsakamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella. Other examples of bacterium include Mycobacterium tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B. subtilis, Nocardia asteroides, and other Nocardia species, Streptococcus viridans group, Peptococcus species, Peptostreptococcus species, Actinomyces israelii and other Actinomyces species, and Propionibacterium acnes, Clostridium tetani, Clostridium botulinum,T|H Docket: 222105-2280 other Clostridium species, Pseudomonas aeruginosa, other Pseudomonas species, Campylobacter species, Vibrio cholera, Ehrlichia species, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species Brucella abortus, other Brucella species, Chlamydi trachomatis, Chlamydia psittaci, Coxiella burnetti, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Yersinia pestis, Yersinia enterolitica, other Yersinia species, Escherichia coli, E. hirae and other Escherichia species, as well as other Enterobacteria, Brucella abortus and other Brucella species, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis, Fudobascterium nucleatum, Provetella species, and Cowdria ruminantium, or any strain or variant thereof. The gram-positive bacteria may include, but is not limited to, gram positive Cocci (e.g., Streptococcus, Staphylococcus, and Enterococcus). The gram-negative bacteria may include, but is not limited to, gram negative rods (e.g., Bacteroidaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellae and Pseudomonadaceae).

[0053] The terms “fungus” or “fungi” include, but are not limited to yeasts such as, for example, Candida albicans or other Candida spp. including C. glabrata, C. rugosa, C. parapsilosis, C. tropicalis, or C. dubliniensis Fungi can also include dermatophytes such as, for example, Trichophyton spp. and Microsporum spp. (e.g., T. rubrum, T. interdigitale, T. tonsurans, T. violaceum, T. concentricum, T. schoenleinii, T. soudanense, T. mentagrophytes, T. equinum, T. erinacei, T. verrucosum, M. audouinii, M. ferrugineum, M. canis, M. gypseum, M. nanum, and / or M. cookie).

[0054] The term “antimicrobial effective amount” as used herein refers to that amount of the compound being administered / released that will kill microorganisms or inhibit growth and / or reproduction thereof to some extent (e.g. from about 5% to about 100%). In reference to the compositions or articles of the disclosure, an antimicrobial effective amount refers to that amount which has the effect of diminishment of the presence of existing microorganisms, stabilization (e.g., not increasing) of the number of microorganisms present, preventing the presence of additional microorganisms, delaying or slowing of the reproduction of microorganisms, and combinations thereof. Similarly, the term “antibacterial effective amount” refers to that amount of a compound being administered / released that will kill bacterial organisms or inhibit growth and / or reproduction thereof to some extent (e.g., from about 5% to about 100%). In reference to the compositions or articles of the disclosure, an antibacterial effective amount refers to that amount which has the effect of diminishment of the presence of existing bacteria, stabilization (e.g., not increasing) of the number of bacteria present, preventing the presence of additional bacteria, delaying or slowing of the reproduction of bacteria, and combinations thereof.T|H Docket: 222105-2280

[0055] As used herein, the term “subject” includes humans, mammals (e.g., cats, dogs, horses, etc.), birds, and the like. Typical subjects to which embodiments of the present disclosure may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.

[0056] The terms “treat”, “treating”, and “treatment” are an approach for obtaining beneficial or desired clinical results. Specifically, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (e.g., not worsening) of disease, delaying or slowing of disease progression, substantially preventing spread of disease, amelioration or palliation of the disease state, and remission (partial or total) whether detectable or undetectable.

[0057] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action.

[0058] As used herein, the term “reduce” or “reducing” refers to deceasing the degree or amount of an event. For example, reducing the growth of microbes on a surface can mean lower the growth of microbes on a surface when using the compositions described herein when compared to the amount of microbial growth when the compositions described herein are not used. Discussion

[0059] In accordance with the purpose(s) of the present disclosure, described herein are nitric oxide releasing compositions comprising a nitric oxide releasing compound. The compositions can also include ethanol, water, a thickening agent, a polyol, and a neutralizer. The compositions can be used to reduce or prevent the growth of microbes on a surface, such as the skin of a subject. Methods for preparing and using the compositions are described in further detail below.

[0060] Nitric oxide (NO) is a natural gasotransmitter in the human body with diverse biological functions. It plays a role in the body's defense against pathogens and contributes to various physiological processes. The gaseous nature and short half-life of NO allows it to rapidly penetrate into a wide range of microbial species, including bacteria, viruses, and fungi, regardless of their structural composition, without inducing resistance.T|H Docket: 222105-2280 Nitric Oxide Releasing Compounds

[0061] The nitric oxide releasing compound is a compound that possesses one or more nitric oxide groups, wherein nitric oxide can be released from the compound. In one aspect, the nitric oxide releasing compound is an S-nitrosothiol (RSNO) compound. In a further aspect, the nitric oxide compound is S-nitroso-N-acetylpenicillamine, S-nitroso-glutathione, S- nitroso-N-acetylcysteine, S-nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S-nitrosocysteamine, S-nitroso-3-mercapto-propanoic acid, S-nitroso-N- acetyl-l-cysteine ethyl ester (SNACET), S-nitroso-N-acetyl-L-methionine, S- nitrosomercaptoethanol, or any combination thereof. In another aspect, the nitric oxide releasing compound can be S-nitrosothiol conjugated polymers, S-nitrosothiol modified- dendrimers, S-nitrosothiol modified polysaccharides, S-nitrosothiol modified nano / microparticles, or S-nitrosothiol modified-proteins. The nitric oxide releasing compound can also include other NO-donors such as, for example, nitrates and N-diazeniumdiolates (NONOates).

[0062] In other aspects, the nitric oxide releasing compound includes a modified antibiotic compound including a nitric oxide release agent covalently attached to an antibiotic molecule. Having a single molecule with the combined functionalities of both of a stable NO donor and an antibiotic can be a very efficient approach for combating and preventing biofilm related infections. The modified antibiotic compound can be a synthetic RSNO covalently attached to an antibiotic molecule to create a novel dual functional antimicrobial agent, also referred to as a modified antibiotic compound. In other aspects, the nitric oxide releasing agent is S-nitroso- N-acetylpenicillamine (SNAP), S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S- nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S- nitrosocysteamine, S-nitroso-3-mercapto-propanoic acid, or any combination thereof. In other aspects, the antiobiotic molecule can be ampicillin, vancomycin, gentamicin, cephalexin, or any combination thereof. In further aspects, the modified antibiotic compound includes SNAP covalently bonded to ampicillin, referred to herein as SNAPicillin. SNAP can be represented by the following structure: O O SNAPicillin can beT|H Docket: 222105-2280 O

[0063] In one be formed by covalently attaching a nitric oxideattachment can be formed by mixing the nitric oxide release agent and the antibiotic molecule in a solvent and then nitrosating the mixture. The nitrosation can occur through the excess addition of t-butyl nitrite or an acidified sodium nitrite solution to the mixture. The excess addition can be about a 3 times molar excess of t-butyl nitrate with respect to the quantity of the antibiotic molecule, such as ampicillin. Methods for producing the modified antibiotic compound useful as nitric oxide releasing compounds are described in US Patent No.11,220,516, which is incorporated by reference in its entirety.

[0064] In one aspect, the amount of the nitric oxide releasing compound is from about 0.5 weight percent to about 45 weight percent of the composition or about 0.5 wt%, 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, or 45 wt%, where any value can be a lower and upper endpoint of a range (e.g., 1 weight percent to 40 weight percent). Additional Components

[0065] The nitric oxide releasing compositions can include additional components, such as ethanol, water, thickening agents, polyols, and neutralizers. Other components such as fragrances, antimicrobial agents, and antioxidants can also be included in the compositions. The components can be included in varying amounts in the composition.

[0066] In one aspect, the amount of ethanol included can be from about 50 volume percent to about 90 volume percent of the composition or about 50 vol%, 55 vol%, 60 vol%, 65 vol%, 70 vol%, 75 vol%, 80 vol%, 85 vol%, or 90 vol%, where any value can be a lower and upper endpoint of a range (e.g., 60 volume percent to 85 volume percent).

[0067] In one aspect, the amount of thickening agent in the composition can be from about 0.1 weight to about 1.0 weight percent of the composition or about 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, or 1.0 wt%, where any value can be a lower and upper endpoint of a range (e.g., 0.1 weight percent to 0.5 weight percent). In one aspect, the thickening agent can include a polyacrylic acid, carbomer, xanthan gum, aT|H Docket: 222105-2280 cellulose-derived compound (e.g., hydroxyethyl cellulose and hydroxypropyl cellulose), an acrylic acid-derived polymer (e.g., acrylates / C10-30 alkyl acrylate crosspolymer and polyacrylate crosspolymer-6), glycerin, silica, isopropyl myristate, or any combination thereof. In other aspects, the thickening agent is a polyacrylic acid.

[0068] In one aspect, the amount of polyol in the composition can be from about 0.1 volume percent to about 6.0 volume percent of the composition or about 0.1 vol%, 0.5 vol%, 1.0 vol%, 1.5 vol%, 2.0 vol%, 2.5 vol%, 3.0 vol%, 3.5 vol%, 4.0 vol%, 4.5 vol%, 5.0 vol%, 5.5 vol% or 6.0 vol%, where any value can be a lower and upper endpoint of a range (e.g., 0.1 volume percent to 5.0 volume percent). In one aspect, the polyol is an organic compound having two or more hydroxyl groups. In another aspect, the polyol can include glycerin, xylitol, lactitol, mannitol, sorbitol, erythritol, maltitol, lactitol, isomalt, polyethylene glycol, trimethylolpropane or any combination thereof. In another aspect, the polyol is glycerin.

[0069] In one aspect, the amount of neutralizer in the composition can be from about 0.1 weight percent to about 4.0 weight percent of the composition or about 0.1 wt%, 0.5 wt%, 1.0 wt%,1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, or 4.0 wt%, where any value can be a lower and upper endpoint of a range (e.g., 0.1 weight percent to 2.0 weight percent). In one aspect, the neutralizer can include a substituted amine (e.g., diethanolamine), a hydroxide, an acid, salts of acids (e.g., sodium citrate), or any combination thereof. Examples of hydroxides include, but are not limited to, inorganic hydroxides such as KOH, NaOH, and NH4OH. Examples of acids include, but are not limited to, citric acid, phosphoric acid, acetic acid, and boric acid. In other aspects, the neutralizer includes a tri-substituted amine. In further aspects, the neutralizer includes a tri-alkyl substituted amine. In still further aspects, the neutralizer is a tri-ethyl amine.

[0070] In one aspect, the amount of fragrance in the composition can be from about 0.1 weight percent to about 5.0 weight percent of the composition or about 0.1 wt%, 0.5 wt%, 1.0 wt%,1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, 4.5 wt%, or 5.0 wt%, where any value can be a lower and upper endpoint of a range (e.g., 0.1 weight percent to 1.0 weight percent). In some aspects, the fragrance can include natural oils, synthetic fragrances, or any combination thereof. In some aspects, the fragrance comprises a tea tree oil. A tea tree oil can include a pure tea tree oil (derived from Melaleuca alternifolia), an organic tea tree oil, melaleuca oil, a tea tree oil blend, a water-soluble tea tree oil, tea tree oil products, or any combination thereof. Tea tree oil blends can include tea tree oils combined with other essential oils, carrier oils, or any combination thereof. The tea tree oil blends can be formulated for specific purposes, such as aromatherapy or skin care. Tea tree oil products include tea tree oil incorporated into hair- and skin-care products, such as shampoos, conditioner, soaps, and lotions.T|H Docket: 222105-2280

[0071] Examples of synthetic fragrances include, but are not limited to, hydrocarbons, alcohols, acids, aldehydes, ketones, esters, lactones, ethers, phenols, nitriles, sulfides, halides, and polyfunctional compounds. Examples of synthetic hydrocarbon fragrances can include caryophyllene, β-famesene, limonene, α-pinene, and β-pinene. Examples of synthetic alcohol fragrances can include bacdanol, citronellol, linalool, phenethyl alcohol, and α- terpineol (R=H). Examples of synthetic aldehyde fragrances can include 2-methyl undecanal, citral, hexyl cinnamic aldehyde, isocycolcitral, lilial, and 10-undecenal. Examples of synthetic ketone fragrances can include cashmeran, α-ionone, isocyclemone E, koavone, muscone, and tonalide. Examples of synthetic ester fragrances can include benzyl acetate, 4-t- butylcyclohexyl acetate (cis and trans), cedryl acetate, cyclacet, isobornyl acetate, and α- terpinyl acetate (R=acetyl). Examples of synthetic lactone fragrances can include coumarin, jasmine lactone, muskalactone, and peach aldehyde. Examples of synthetic ether fragrances can include ambroxan, anther, and galaxolide. Examples of synthetic nitrile fragrances can include cinnamonitrile and gemonitrile. Examples of synthetic polyfunctional fragrances can include amyl salicylate, isoeugenol, hedione, heliotropine, lyral, and vanillin.

[0072] Examples of natural oils include, but are not limited to: basil (Ocimum basilicum) oil, bay (Pimento acris) oil, bee balm (Monarda didyma) oil, bergamot (Citrus aurantium bergamia) oil, cardamom (Elettaria cardamomum) oil, cedarwood (Cedrus atlantica) oil, chamomile (Anthemis nobilis) oil, cinnamon (Cinnamomum cassia) oil, citronella (Cymbopogon nardus) oil, clary (Salvia sclarea) oil, clove (Eugenia caryophyllus) oil, cloveleaf (Eufenia caryophyllus) oil, Cyperus esculentus oil, cypress (Cupressus sempervirens) oil, Eucalyptus citriodora oil, geranium maculatum oil, ginger (Zingiber officinale) oil, grapefruit (Citrus grandis) oil, hazel (Corylus avellana) nut oil, jasmine (Jasminum officinale) oil, Juniperus communis oil, Juniperus oxycedrus tar, Juniperus virginiana oil, kiwi (Actinidia chinensis) water, lavandin (Lavandula hybrida) oil, lavender (Lavandula angustifolia) oil, lavender (Lavandula angustifolia) water, lemon (Citrus medica limonum) oil, lemongrass (Cymbopogon schoenanthus) oil, lime (Citrus aurantifolia) oil, linden (Tilia cordata) oil, linden (Tilia cordata) water, mandarin orange (Citrus nobilis) oil, nutmeg (Myristica fragrans) oil, orange (Citrus aurantium dulcis) flower oil, orange (Citrus aurantium dulcis) oil, orange (Citrus aurantium dulcis) water, patchouli (Pogostemon cablin) oil, peppermint (Menthe piperita) oil, peppermint (Menthe peperita) water, rosemary (Rosmarinus officinalis) oil, rose oil, rose (Rosa damascena) extract, rose (Rosa multiflora) extract, rosewood (Aniba rosaeodora) extract, sage (Salvia officinalis) oil, sandalwood (Santalum album) oil, spearmint (Menthe viridis) oil, tea tree (Melaleuca alternifolia) oil, and ylang ylang (Cananga odorata) oil.

[0073] In certain aspects, the compositions described herein can include an antimicrobial agent, where the antimicrobial agent includes an antibiotic agent, an antifungal agent, an antiseptic agent, or any combination thereof. Antiseptic agents can include, but are not limitedT|H Docket: 222105-2280 to, isopropanol, ethanol, idophor, hydrogen peroxide, chlorhexidine, thimerosal, benzalkonium chloride, triclosan, or a hypochlorite. In other aspects, the compositions can also include an antioxidant, where the antioxidant includes tocopherol and its derivatives (including Vitamin E), Vitamin C (ascorbic acid) and its derivatives, Vitamin A (retinol) and its derivatives, resveratrol, carotenoids (such as lutein, lycopene, and betacarotene), cysteine, hydroquinone, delphinidin, coenzyme q10, erythorbic acid, α-lipoic acid, polypodium leucotomos extract, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and epigallocatechin gallate (green tea). Methods of Making and Using the Nitric Oxide Releasing Compositions

[0074] Described herein are methods for making nitric oxide releasing compositions. The method can include: (a) admixing ethanol, water, and a thickening agent to produce a first composition; (b) admixing a polyol with the first composition to produce a second composition; (c) admixing a nitric oxide releasing compound with the second composition to produce a third composition; and (d) admixing a neutralizer with the third composition to produce the nitric oxide releasing composition. In further aspects, step (b) can include admixing a fragrance with the polyol and first composition prior to performing step (c). The Examples provide non-limiting procedures for making the compositions described herein.

[0075] The nitric oxide releasing composition can be characterized by various properties. In some aspects, the nitric oxide composition has a pH of about 5.0 to about 9.0 or about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, where any value can be a lower and upper endpoint of a range (e.g., 6.0 to 8.0). In one aspect, the pH of the nitric oxide releasing composition is suitable for applying the skin of a subject.

[0076] In some aspects, the composition has a viscosity of about 2,000 centipoise to about 5,000 centipoise or about 2,000 cP, 2,500 cP, 3,000 cP, 3,500 cP, 4,000 cP, 4,500 cP, or 5,000 cP, where any value can be a lower and upper endpoint of a range (e.g., 3,000 cP to 4,000 cP). In some aspects, the composition releases nitric oxide for at least about 12 hours, or about 12 hours, about 18 hours, or about 24 hours, where any value can be a lower and upper endpoint of a range (e.g., 12 hours to 24 hours).

[0077] The nitric oxide compositions can be used to reduce or prevent the growth of microbes on a surface by applying the composition to the surface. In some aspects, the composition can be applied to the skin of a subject. The microbes can include bacteria, fungi, viruses, protozoan, or algae. Thus, the nitric oxide releasing compositions can be effective as hand sanitizers and other hygiene products that are topically applied to a subject. Aspects

[0078] Aspect 1. A nitric oxide releasing composition comprisingT|H Docket: 222105-2280 (a) ethanol; (b) a nitric oxide releasing compound; (c) water; (d) a thickening agent; (e) a polyol; and (f) a neutralizer.

[0079] Aspect 2. The composition of Aspect 1, wherein ethanol is from 60 volume percent to 85 volume percent of the composition.

[0080] Aspect 3. The composition of Aspect 1 or 2, wherein the nitric oxide releasing compound is an S-nitrosothiol conjugated polymer, an S-nitrosothiol modified-dendrimer, an S-nitrosothiol modified polysaccharide, an S-nitrosothiol modified nano / microparticle, an S- nitrosothiol modified-protein, a nitrate, an N-diazeniumdiolates (NONOate), or an S- nitrosothiol (RSNO).

[0081] Aspect 4. The composition of Aspect 1 or 2, wherein the nitric oxide release agent is S-nitroso-N-acetylpenicillamine, S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S- nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S- nitrosocysteamine, S-nitroso-3-mercapto-propanoic acid, S-nitroso-N-acetyl-l-cysteine ethyl ester (SNACET), S-nitroso-N-acetyl-L-methionine, or S-nitrosomercaptoethanol.

[0082] Aspect 5. The composition of Aspect 1 or 2, wherein the nitric oxide releasing compound is a modified antibiotic compound comprising a nitric oxide release agent covalently attached to an antibiotic molecule.

[0083] Aspect 6. The composition of Aspect 5, wherein the antibiotic molecule is ampicillin, vancomycin, gentamicin, or cephalexin.

[0084] Aspect 7. The composition of Aspect 5, wherein the modified antibiotic compound comprises S-nitroso-N-acetylpenicillamine covalently attached to ampicillin.

[0085] Aspect 8. The composition of any one of Aspects 1-7, wherein the nitric oxide releasing compound is from about 1 weight percent of the composition to about 40 weight percent of the composition.

[0086] Aspect 9. The composition of any one of Aspects 1-8, wherein the thickening agent comprises a polyacrylic acid, a carbomer, xanthan gum, a cellulose-based compound, acrylates / C10-30 alkyl acrylate crosspolymer, polyacrylate crosspolymer-6, glycerin, silica gel, isopropyl myristate, or any combination thereof.

[0087] Aspect 10. The composition of any one of Aspects 1-8, wherein the thickening agent comprises a polyacrylic acid.

[0088] Aspect 11. The composition of any one of Aspects 1-10, wherein the thickening agent is from about 0.1 weight percent of the composition to about 0.5 weight percent of the composition.T|H Docket: 222105-2280

[0089] Aspect 12. The composition of any one of Aspects 1-11, wherein the polyol comprises glycerin, xylitol, lactitol, mannitol, sorbitol, erythritol, maltitol, lactitol, isomalt, or any combination thereof.

[0090] Aspect 13. The composition of any one of Aspects 1-11, wherein the polyol comprises glycerin.

[0091] Aspect 14. The composition of any one of Aspects 1-13, wherein the polyol is from about 0.1 volume percent of the composition to about 5.0 volume percent of the composition.

[0092] Aspect 15. The composition of any one of Aspects 1-14, wherein the neutralizer comprises a substituted amine, a hydroxide, an acid, salts of acids, or any combination thereof.

[0093] Aspect 16. The composition of any one of Aspects 1-14, wherein the neutralizer comprises a tri-substituted amine.

[0094] Aspect 17. The composition of any one of Aspects 1-14, wherein the neutralizer comprises a tri-alkyl substituted amine.

[0095] Aspect 18. The composition of any one of Aspects 1-14, wherein the neutralizer comprises a tri-ethyl amine.

[0096] Aspect 19. The composition of any one of Aspects 1-18, wherein the neutralizer is from about 0.1 weight percent of the composition to about 2.0 weight percent of the composition.

[0097] Aspect 20. The composition of any one of Aspects 1-19, wherein the composition further comprises a fragrance.

[0098] Aspect 21. The composition of Aspect 20, wherein the fragrance comprises natural oils, synthetic fragrances, or any combination thereof. tea tree oil

[0099] Aspect 22. The composition of Aspect 21, wherein the fragrance comprises pure tea tree oil, organic tea tree oil, melaleuca oil, tea tree oil blends, water-soluble tea tree oil, or any combination thereof.

[0100] Aspect 23. The composition of any one of Aspects 20-22, wherein the fragrance is from about 0.1 weight percent of the composition to about 1.0 weight percent of the composition.

[0101] Aspect 24. The composition of any one of Aspects 20-22, wherein the fragrance is tea tree oil from about 0.1 weight percent of the composition to about 1.0 weight percent of the composition.

[0102] Aspect 25. The composition of any one of Aspects 1-24, wherein the composition further comprises an antimicrobial agent comprising an antibiotic agent, an antifungal agent, an antiseptic agent, or any combination thereof.T|H Docket: 222105-2280

[0103] Aspect 26. The composition of Aspect 25, wherein the antiseptic agent comprises isopropanol, ethanol, idophor, hydrogen peroxide, chlorhexidine, thimerosal, benzalkonium chloride, triclosan, or a hypochlorite.

[0104] Aspect 27. The composition of any one of Aspects 1-26, wherein the composition further comprises an antioxidant.

[0105] Aspect 28. The composition of Aspect 27, wherein the antioxidant comprises Vitamin E, Vitamin C, Vitamin A, resveratrol, carotenoids, cysteine, hydroquinone, delphinidin, coenzyme q10, erythorbic acid, α-lipoic acid, polypodium leucotomos extract, butylated hydroxyanisole, butylated hydroxytoluene, and epigallocatechin gallate.

[0106] Aspect 29. The composition of Aspect 27, wherein the antioxidant comprises Vitamin E or derivatives thereof.

[0107] Aspect 30. A nitric oxide releasing composition produced by the method comprising: (a) admixing ethanol, water, and a thickening agent to produce a first composition; (b) admixing a polyol with the first composition to produce a second composition; (c) admixing a nitric oxide releasing compound with the second composition to produce a third composition; and (d) admixing a neutralizer with the third composition to produce the nitric oxide releasing composition.

[0108] Aspect 31. The composition of Aspect 30, wherein in step (b), further comprising admixing a fragrance with the polyol and first composition.

[0109] Aspect 32. The composition of any one of Aspects 1-31, wherein the composition has a pH of about 6 to about 8.

[0110] Aspect 33. The composition of any one of Aspects 1-32, wherein the composition has a viscosity of about 3,000 centipoise to about 4,000 centipoise.

[0111] Aspect 34. The composition of any one of Aspects 1-32, wherein the composition releases nitric oxide for at least 12 hours.

[0112] Aspect 35. A method for reducing or preventing the growth of microbes on a surface, the method comprising applying the composition of any one of Aspects 1-34 to the surface.

[0113] Aspect 36. The method of Aspect 35, wherein the surface is the skin of a subject.

[0114] Aspect 37. The method of Aspect 35 or 36, wherein the microbe comprises bacteria, fungi, virus, protozoan, or algae. EXAMPLES

[0115] Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure areT|H Docket: 222105-2280 described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. Materials and Methods

[0116] Materials

[0117] N-Acetyl-D-penicillamine (NAP), sodium nit-rite, L-cysteine, sodium chloride, potassium chloride, sodium phosphate dibasic, potassium phosphate monobasic, copper (II) chloride, tea tree oil, Luria-Bertani (LB) broth with agar (Lennox), yeast media and agar, ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline (PBS) (10 mM, pH 7.4), and glycerin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carbomer 940 (Carbopol 940), triethanolamine (TEA), ethyl alcohol (100%), were obtained from VWR (Radnor, PA, USA). Methanol, hydrochloric acid, and sulfuric acid were obtained from Fisher Scientific (Hampton, NH). The bacterial strains S. aureus ATCC 6538, E. coli ATCC 25922, C. albicans ATCC MYA 4441 and 3T3 mouse human fibroblast cells for cell compatibility experiments were purchased from American Type Cultural Collection (ATCC). The clinical isolate of antibiotic-resistant bacterial strain, Methicillin-resistant S. aureus (CDC AR-1003) was obtained from the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin– streptomycin (P / S, 5000 U mL−1) were purchased from VWR (Radnor, PA).3T3 (ATCC CRL- 2522) human fibroblast cells for cell compatibility experiments were obtained from American Type Culture Collection (ATCC, Manassas, VA). All of the liquid buffers and media were sterilized prior to biological studies using autoclave sterilization cycles with saturated steam at 121 °C under 15 psi pressure for 30 minutes.

[0118] Synthesis of NO Donor S-nitroso-N-acetylpenicillamine (SNAP)

[0119] The protocol for synthesizing the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was adapted from a previous publication with slight modifications.20Briefly, the precursor NAP was dissolved in a mixture of two parts water and three parts methanol. Next, 0.7 M of H2SO4and 1.6 M of HCl were added to the solution, and sodium nitrite, pre-dissolved in water, was added drop by drop. The mixture was stirred for 10 min at room temperature (RT, ~23 ºC). To facilitate the formation of SNAP, the mixture was then incubated on ice under continuous nitrogen purging for 8 h, all while being shielded from ambient light. After the 8 h incubation period, a filtration setup was prepared using Whatman cellulose filter paper in a Buchner funnel connected to a vacuum suction. The SNAP crystals were collected on the filter paper through suction filtration. Following collection, the SNAP crystals were rinsed with ice- cold deionized water and left in a vacuum desiccator overnight for drying. Care was taken to protect the samples from exposure to light throughout the entire process. The purity of theT|H Docket: 222105-2280 synthesized SNAP crystals, > 90%, was confirmed using chemiluminescence NOA and a UV- vis calibration curve, with SNAP exhibiting a characteristic spectrum at 340 nm.

[0120] Preparation of NO-releasing (NORel) Hand Sanitizer Gel

[0121] Hand sanitizer gels were prepared by initially dissolving carbomer (105 mg) at room temperature (RT) into rapidly agitated ethanol (12 mL). Subsequently, DI water (7.12 mL) was added, and the mixture was stirred for 15 min at 200 rpm to achieve homogenous dispersion. After ensuring homogeneity, tea tree oil (40 μL) and glycerin (600 μL) were introduced into the blend. To include NO-releasing properties, S-nitroso-N-acetylpenicillamine (SNAP), a tertiary S-nitrosothiol (RSNO) NO donor, was incorporated into the formulations. To demonstrate gel's adaptability, two distinct concentrations of SNAP were integrated into it. NORel1 and NORel2 hand sanitizer gels were formulated by adding 10.5 mg and 31.5 mg of SNAP, respectively. Finally, triethylamine (TEA, 139.4 μL) was included for its coagulation properties, which transformed the liquid mixture into a viscous gel. pH of the final gel was adjusted to be in neutral range using sodium hydroxide (NaOH). A corresponding control without SNAP was prepared as a negative control for the study and labeled as control gel. The resulting gels were transferred into 20 mL glass vials with caps and stored at -20 ºC, shielded from light, until further characterization.

[0122] Characterization of NORel Gel

[0123] pH Measurement of Gel

[0124] The initial pH assessment of NORel gels was conducted to assess the gel's safety. The pH of NORel1 and NORel2 gels was determined using a SevenCompact pH / Ion meter S220-std-kit (Mettler Toledo, Columbus, OH) with the probe directly immersed into each vial. Each sample was given a 10-min equilibration period before testing to reach room temperature and ensure precise pH measurements. The pH probe was rinsed with DI water after each measurement. The final results are presented as the mean ± SD for each formulation (n = 3).

[0125] pH Stability of Gel

[0126] The pH stability of gel was determined by tracking pH changes in hand sanitizer samples over 60 d. NORel gels were stored at various temperatures (-20 ˚C, 4 ˚C, room temperature (23 ˚C), and 37 ˚C) (n = 3) and pH was recorded on day 0, 5, 7, 14, 21, 28, 35, 42, 49, and 60 following the same method as described above. If the congealing agent was to breakdown, changes in pH would be observed in the gel. The final results are reported as the mean ± SD for each formulation, with three measurements obtained for each formulation (n = 3).

[0127] Viscosity of Gels

[0128] The viscosity of hand sanitizer gels (0% Control, NORel1, and NORel2) was determined using a Brookfield Viscometer (DV-II+ Pro, Brookfield Ametek, USA), with a cone- shaped spindle featuring a 0.8° cone angle and a 2.4 cm radius. A CPE-40 spindle cone andT|H Docket: 222105-2280 an appropriate sample cup were utilized to contain and assess the gels. The equipment temperature was maintained at room temperature (~23 °C) through water circulation. For testing, 0.5 mL of gel was placed in the viscometer sample cup, which was then reinserted into the viscometer to begin testing. The speed gradually increased from 1 to 100 rpm in 10 increments, with each speed being sustained for 10 sec and data recorded at one-second intervals. The results are presented as the mean ± SD for each formulation, with three measurements obtained for each (n = 3).

[0129] NO Release Kinetics

[0130] 24 h NO Release Measurements

[0131] The release kinetics of NO for NORel1 and NORel2 were assessed using the Zysense chemiluminescence Nitric Oxide Analyzer (NOA) 280i, a well-established standard (Zysense, Frederick, CO). In this evaluation, NORel1 and NORel2 gels were precisely weighed (120 mg) into separate 1.5 mL Eppendorf tubes and positioned within an amber testing cell, maintaining a physiological temperature of 37 °C. Nitrogen (N2) gas was continuously purged into the sample cell containing NORel gels at a rate of 200 mL min-1, effectively transferring the NO into the reaction chamber. The NOA cell maintained a pressure range of 8.3 to 9.2 torr, while the supply pressure remained between 6.2 and 6.5 psi. NO release from the samples was monitored at multiple time intervals (0, 8, and 24 h), with the samples consistently incubated at 37 ºC between testing points. These investigations were conducted in triplicate, and the NO release levels obtained from the gels were normalized to their mass. The results from the study are normalized to the mass and presented as moles min-1mg-1. The final data are reported as mean ± SD (n ≥ 3).

[0132] Stability of NO donor in Gel

[0133] The stability of SNAP in the NORel1 and NORel2 gel was determined at different storage conditions (-20 ˚C, 4 ˚C, room temperature, and 37 ˚C) using UV-vis spectrophotometer (Cary 360, Agilent Technologies). For this, an aliquot of ~100 mg gel dissolved in 1 mL of 10 mM PBS substituted with 100 µM EDTA was added to a cuvette and read for absorbance at 340 nm wavelength corresponding to the characteristic maxima of SNAP in UV-vis. A 1 mL of PBS-EDTA was used as a blank control. The calibration curve for SNAP in PBS with EDTA served as a reference to translate the absorbance readings from the study into compound concentrations within the samples. To ensure accuracy, the buffer solution's volume remained consistent across all samples throughout the experiment, preventing any variations in readings. The stability of the gels was assessed on day 0, 5, 7, 14, 21, and 28 d and compared to results obtained on day 0. Results from the study are reported as % NO remaining on each testing day relative to the initial amount of NO obtained on day 0.

[0134] In Vitro Cytocompatibility ScreeningT|H Docket: 222105-2280

[0135] Preparation of 3T3 Cells

[0136] Mouse fibroblasts (NIH / 3T3 cells, ATCC CRL-2522) were revived from cryopreserved stocks stored in liquid nitrogen vapor phase. BJ cells were subcultured in minimal essential media (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin (S-P) under a 5% CO2-humidified atmosphere. Cells were grown for up to ten passages before discarding. Cells were grown up to 70% sub-confluency. Afterwards, cells were washed with 1x PBS (Ca2+and Mg2+-free) and treated with 0.25% trypsin for 5 min. After detachment, cells were collected via centrifugation (200 rcf, 5 min) and resuspended in complete media. For cytotoxicity experiments, 12 mm hydrophilic polytetrafluoroethylene inserts (Millicell PICM01250) were pretreated with type I rat-tail collagen (~300 µg per insert) then seeded with BJ cells in the intraluminal space to an initial density of 40,000 cells cm-2in 24-well plates. The extraluminal space was supplemented with 600 µL of complete media with 400 µL aliquoted in the intraluminal side. Cells were grown for 24 h prior to treatment.

[0137] Exposure of Gels to Cells

[0138] The aliquots of the hydrogels (~100 mg) were added to the extraluminal side of inserts with clean media added to both sides. Untreated wells (hereafter referred to as Blank) were also prepared with clean media. Cells were incubated for an additional 24 h. Afterwards, the media was replaced with media supplemented with 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) to a final concentration of 0.5 mg / mL. Cells were incubated for an additional 3 h to facilitate the formation of the formazan precipitate. Supernatant was decanted taking caution to not perturb the crystalline layer. Afterwards, the formazan precipitate was dissolved in dimethyl sulfoxide (DMSO, 350 µL insert-1) and quantified for absorbance at 570 nm with reference wavelength at 630 nm. Relative cellular viability was calculated according to Equation 1. ^^^^^^^^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ^%^ ൌ^^ୟ୬ ^ୈ^౨^^౪^^^౪^^ୟ୬ ^ୈా^^^ౡX 100% (1) Final data is formulation (n = 4independent treatments per formulation).

[0139] In Vitro Antimicrobial Activity of NORel

[0140] Growing Microbial Culture

[0141] Antimicrobial activity of the NORel hand sanitizer was analyzed against S. aureus (Gram-positive), E. coli (Gram-negative), C. albicans (fungal), clinical isolates of methicillin- resistant S. aureus (MRSA, AR-1003) using a 24 h antimicrobial assay. For this, each individual colony of microbial strain was inoculated into growth media (S. aureus, MRSA and E. coli were grown in LB media and C. albicans in Yeast media), and allowed to grow overnight in a bacteria shaking incubator at 150 RPM and 37 ºC. The growth of microbes was measured via optical density (O.D) using UV-vis spectrophotometer (Cary 60, Agilent) at 600 nmT|H Docket: 222105-2280 wavelengths. Once the microbial culture reached mid-log phase, cultures were washed using sterile PBS by centrifuging at 3500 RPM for 7 mins. The washed pellet was re-suspended into fresh PBS buffer and diluted to achieve 106-108cells mL-1as working concentration. Diluted culture was then used for the remainder of the study.

[0142] Exposing NORel to Microbes

[0143] To test the antimicrobial potential of NORel hand sanitizer gel, bacteria cultures were exposed to different gel groups (0% (Control), NORel1 and NORel2. Approximately 100 mg of gel were weighed out and transferred into 1.5 mL Eppendorf tubes (n ≥ 3). Then 1 mL of diluted bacterial / fungal culture was added to the tubes containing gels and incubated for 6 h at 37 ºC, 120 rpm to mimic the physiological conditions. For microbial control group, tubes with bacterial culture were incubated along with gels. After 24 h of incubation, samples were diluted and plated on agar plates using a bacteria spiral plater (Eddy Jet 2, IUL Instruments) and incubated at 37 ºC overnight until the countable colonies were obtained. Colonies of microbes were counted using an automated colony counter (Sphere Flash, IUL Instruments). The % killing of bacteria was determined using Equation 2 where C represents the concentration of viable bacteria in CFU mL-1for treatment groups (0% (Control), NORel1 and NORel2) gels with respect to bacterial culture that received no treatment. %^^^^^^^^^^^^^^ ൌ^^^^௧^^^௧^ௗ^ି^^்^^^௧^ௗ^ ^ ^^^௧^^^௧^ௗ^ X 100% (2)

[0144] Ex-vivo

[0145] Collection of

[0146] All experiments pertaining to use of animal models was conducted in accordance with animal use protocol approved by the University of Georgia Institutional Animal Care and Use Committee. New Zealand white rabbits (Covance Battle Creek, MI) were used for this study. Each rabbit, weighing between 2.7-3.5 kg, was euthanized using Euthasol. The area on the back of each euthanized rabbit was shaved prior to the experiment. A rectangular specimen from an exterior area was separated from the fatty layer and removed and a full thickness skin sample was collected. Prior to collecting the samples, the surface of the skin was disinfected and the samples in size 2 x 2 cm2(n = 14) were collected and rinsed with standard buffered saline. Samples were immediately processed if not stored in -20 °C until the future experiments.

[0147] To evaluate the efficacy of NO-releasing (NORel) hand sanitizer the rabbit skin samples were taken out of the freezer at -20 °C and soaked in Deionized water to thaw the specimen. Once thawed, the samples were cut into fifteen 8 mm diameter circles using a biopsy punch and placed into a 24-well plate. To disinfect the specimens, a 1000 µL of diluted chlorhexidine (CHXD) disinfectant was added to each well. After 15 min, the CHXD was washed out by two 1000 µL aliquots of sterilized PBS. The samples soaked in fresh PBS forT|H Docket: 222105-2280 2-3 h to fully remove the effect of disinfectant. The samples were then removed and briefly rinsed again in fresh PBS and transferred into a new 24-well plate for testing.

[0148] Preparation of Bacterial Culture

[0149] S. aureus bacteria is commonly found on the skin and mucous membranes of humans and animals. While S. aureus is typically harmless on the skin, it can lead to infections if it enters the body through medical devices, breaks or cuts in the skin or mucous membranes. It is particularly opportunistic and can cause infections under various conditions. Therefore, S. aureus was chosen as a model organism to test the ex vivo efficacy of NORel hand sanitizer in preventing the spread of bacteria. Overnight culture of S. aureus was prepared following the same protocol as 2.7.1.

[0150] Disinfection Efficacy of NORel

[0151] To investigate the disinfection efficacy of NORel, 500 µL of S. aureus bacteria (106- 108CFU mL-1) was exposed to an 8 mm skin punch out and incubated at 37 °C, for 12 h. After incubation, bacteria suspension from the skin specimen was removed and the samples were washed with fresh, sterile PBS to remove free floating / loosely adhered bacteria. Gels were weighed and ~120 mg of gel was exposed to skin samples adhered with bacteria and left to act for 6 h at 37 °C. To compare the efficacy of NORel in eradicating viable bacteria, S. aureus control (remained untreated) and a commercial alcohol-based gel was used as corresponding controls. Samples were gently rinsed with sterile PBS to remove loosely adhered or dead cells and resuspended into 1 mL of sterile PBS. To extract viable adhered bacteria after gel exposure, samples were homogenized and vortexed for 60 sec each and plated using bacteria spiral plater (Eddy Jet 2, IUL Instruments) and colonies were enumerated using an automated colony counter (Sphere Flash, IUL Instruments). The %reduction in viable bacterial adhesion was determined using Equation 3 where C represents the concentration of viable bacteria in CFU cm-2for treatment groups (commercial alcohol-based gel and NORel2) gels with respect to bacteria control that received no treatment. %^^^^^^^^^^^^^^^^^^ ൌ ^^^^௧^^^௧^ௗ^ି^^்^^^௧^ௗ^^ ^^^௧^^^௧^ௗ^ X 100% (3)

[0152]

[0153] To assess the prolonged efficacy of NORel, the NORel and commercial alcohol- based hand sanitizer gels were exposed to skin samples first and then exposed to bacteria. This step was done to test the efficiency of sanitizer gel in maintaining a prolonged antimicrobial activity after initial exposure. For this, Gels were weighed and ~120 mg of gel was exposed to pristine skin samples and incubated for 2 h at 37 °C. After the gel was exposed, a 500 µL of S. aureus bacteria (106-108CFU mL-1) was then exposed to the 8 mm skin punch out and incubated at 37 °C, for 6 h. After incubation, bacteria suspension from the skin specimen was removed and the samples were washed with fresh, sterile PBS to removeT|H Docket: 222105-2280 free floating / loosely adhered bacteria. To compare the efficiency of NORel in eradicating viable bacteria, skin samples exposed to S. aureus bacteria that received no antimicrobial treated were used as negative control. Samples were gently rinsed with sterile PBS to remove loosely adhered or dead cells and resuspended into 1 mL of sterile PBS. To extract viable adhered bacteria after gel exposure, samples were homogenized and vortexed for 60 sec each and plated using bacteria spiral plater (Eddy Jet 2, IUL Instruments) and colonies were enumerated using an automated colony counter (Sphere Flash, IUL Instruments). The %reduction in viable bacterial adhesion was determined using Equation 4 where C represents the concentration of viable bacteria in CFU cm-2for treatment groups (commercial alcohol- based gel and NORel2) gels with respect to bacteria control that received no treatment. %^^^^^^^^^^^^^^^^^^ ൌ^^^^௧^^^௧^ௗ^ି^^்^^^௧^ௗ^ ^ ^^^௧^^^௧^ௗ^ X 100% (4) Statistics

[0154] All the findings reported in this study are derived from a minimum sample size of n ≥ 3. Data is reported as mean ± standard deviations (SD) unless stated otherwise. To evaluate the statistical significance among various sample types, a one-way ANOVA was employed and a significance level of p < 0.05 was established as the threshold for determining statistical differences between the test and the control groups. Results and Discussion

[0155] pH Analysis and Stability of NORel

[0156] The pH of a hand sanitizer is crucial for its effectiveness against harmful microorganisms like bacteria and viruses. Most hand sanitizers are formulated within a specific pH range to optimize their antimicrobial activity. Deviating from this range can reduce the sanitizer's pathogen-killing capability. Moreover, hand sanitizers come into direct contact with the skin, and an improper pH can lead to skin irritation, dryness, or redness.24Maintaining a skin-friendly pH level is essential for user comfort. The pH of a hand sanitizer also affects the stability of its active ingredients, such as alcohol-based agents.24Extreme pH conditions can degrade these ingredients, reducing their effectiveness and shelf life.

[0157] In the context of NORel gels, pH analysis was performed upon the initial formulation of the gel and the addition of SNAP. The pH values for the 0%, NORel1, and NORel2 gels were 8.04 ± 0.42, 7.96 ± 0.48, and 7.40 ± 0.16, respectively (Figure 2A). Notably, the incorporation of SNAP led to a reduction in the pH level of the NORel gel, attributable to the acidic nature of the SNAP moiety with carboxylic acid group.20However, the pH of the gel remained within the safe range for skin application, posing no risk of skin imbalances.25T|H Docket: 222105-2280

[0158] To determine the optimal storage conditions for preserving the stability of carbomer and SNAP, NORel1 and NORel2 gels were subjected to storage at various temperatures (- 20˚C, 4˚C, room temperature (~23˚C), and 37˚C) for a duration of 60 d (Figure 2B-E). The pH measurements presented revealed that all prepared formulations maintained a pH within the safe and skin-friendly range of 6.5-8.5 throughout the storage period at all temperatures, demonstrating their suitability for skin application.26-28This pH range ensures that NORel is well-tolerated by the skin, minimizing the risk of irritation or dryness. Moreover, it's worth noting that hand sanitizers are most effective in killing germs within this pH range, and significant deviations from it can compromise their efficacy. In summary, these findings affirm the long- term suitability of NORel formulations for skin use for at least 60 d.

[0159] Viscosity of Gels

[0160] Viscosity is a key parameter in assessing the overall quality and performance of a hand sanitizer. Proper viscosity ensures that the sanitizer can be easily applied to the hands without being too runny or too thick, which could affect user experience and compliance. Hand sanitizers with the right viscosity are easier to dispense and spread evenly across the hands. This is crucial for ensuring complete coverage and effective sanitization, especially in healthcare settings and during disease outbreaks. The rheological properties and viscosity characteristics in response to the addition of SNAP was assessed using a Brookfield Viscometer and to investigate the impact of gel components on NORel’s rheological properties. All hand sanitizer gels (0% Control, NORel1 and NORel2) (Figure 2F), regardless of the amount of SNAP added, initially exhibited a viscosity of approximately 3500 cP at 1 rpm speed (Figure 2G). However, as the speed of the rheometer increased, the viscosity dropped significantly to below 100 cP, reaching as low as 36 cP at 100 rpm. The onset of this viscosity reduction occurred at 12 rpm, indicating a shear-thinning property where viscosity rapidly decreases under shear force. The initial viscosity at 1 rpm showed slight variations depending on the SNAP percentage in the formulation. Specifically, control, NORel1, and NORel2 had initial viscosities of 3516 ± 20, 3596 ± 1, and 3583 ± 20 cP, respectively, suggesting potential viscosity effects attributed to SNAP addition (Figure 2H). The addition of SNAP increased viscosity, likely due to its carboxylic acid group. Overall, the viscosity measurements obtained in this study are consistent with earlier published findings that utilized carbomer and triethylamine in the creation of phenolic gels, which reported viscosity measurements within the range of 3405-4604 cP.29

[0161] In this study, carbomer, a cross-linked polyacrylic acid polymer, was used as a thickening agent to create a three-dimensional network, that can expand upon hydration in aqueous solutions.30However, its maximum viscosity occurs at a pH of approximately 6.5–7.5 and decreases at a pH ≥ 9, rendering it unstable under alkaline conditions.31, 32To address this, triethylamine was employed as a neutralizing agent to stabilize carbomers in NORel gels.T|H Docket: 222105-2280 Hydrogen bonds, which are attractive forces between molecules, have a profound impact on the gel's properties. In this case, glycerin played a crucial role by facilitating the formation of hydrogen bonds among the gel's molecules, thereby enhancing its structural integrity and viscosity. Additionally, the SNAP moiety, with its carboxylic acid and secondary amine groups, played a dual role in the gelation process. Firstly, it contributed to the neutralization of carboxylic acids, akin to TEA. Secondly, SNAP participated in the formation of hydrogen bonds, similar to glycerin.33These combined effects resulted in an increased quantity of hydrogen bonds within the gel. Consequently, when higher concentrations of SNAP were introduced into the gel, the number of hydrogen bonds formed increased, leading to augmented gel strength and viscosity. This intricate interplay of carbomer, TEA, glycerin, and SNAP collectively contributed to the stability, consistency, and overall performance of the gel. These findings align with previous research on Pluronic F127 where the viscosity of gel increased due to the addition of RSNO donor.34With these adjustable physical properties, further investigations into NO release were pursued.

[0162] Nitric oxide Release Kinetics

[0163] In the pursuit of robust infection control measures, the role of NO has gained considerable attention for its potential. Nonetheless, the intrinsic instability of NO donors in solution has presented a substantial hurdle, limiting the ability to achieve sustained and efficacious levels of NO. To overcome this, inventive strategies have emerged, centered on the use of polymeric matrices, that can stabilize and prolong the release of NO from materials. These strategies offer a protective shield to NO donor, preserving NO and prolonging its release characteristics that enables consistent, regulated, and highly efficient release of NO. For this reason, a robust NO donor SNAP was used to formulate the hand sanitizer gel. In the presence of heat, light and metal ions, the thiol bond is cleaved that can readily release NO from the donor (Figure 3A). The NO release from NORel hand sanitizer gel was characterized using a chemiluminescence method (Figure 3B). Analytical balance (Mettler Toledo™ XS105DU, Columbus, OH) was used to measure the mass of the NORel1 and NORel2 gels accurately. The study aimed to assess the adaptability of NORel gel by varying the SNAP content and its impact on NO release under physiologically relevant conditions. Specifically, NORel1 and NORel2 gels, each with a known mass, were subjected to NO release testing within an amber sample cell maintained at a temperature of 37 ºC. The results demonstrated that, on average, NORel2 exhibited the highest NO release rates during the entire 24 h study period. The concentration of SNAP within the gel significantly influenced the general trends in NO release. Notably, at 0, 8, and 24 h, NORel1 released 0.78 ± 0.10, 0.30 ± 0.05, and 0.21 ± 0.07 × 10−10mol min−1mg−1of NO, respectively, while NORel2 released 1.18 ± 0.29, 0.80 ± 0.37, and 0.53 ± 0.05 × 10−10mol min−1mg−1of NO, respectively (Figure 3B). Over time, the levels of NO declined as SNAP decomposed in the gel.T|H Docket: 222105-2280

[0164] Prior studies have examined NO release from hydrogels composed of pluronic F127, alginate, gelatin, and similar materials for antibacterial purposes that showed comparable levels of NO from hydrogels over 24 h.34, 35However, the combination of traditional hand sanitizer components with NO represents a novel approach that has not been explored before.

[0165] In summary, both NORel1 and NORel2 gels have demonstrated their ability to release NO under physiological conditions, indicating the increase in NO release by altering the NO donor amount within the gel. These outcomes validate the feasibility and effectiveness of NORel hand sanitizer gel as a proof-of-concept. Importantly, it should be noted that the duration, quantity, and extent of NO release can be further tailored to specific situational needs by changing the NO donor concentration, varying the type of NO donor, or even by changing the gel composition. The enhanced NO release exhibited by NORel gels, in combination with other antimicrobial agents like ethanol and tea tree oil, holds therapeutic potential, particularly in combatting the proliferation of opportunistic pathogens. This innovative approach could have significant implications in the field of infection control and prevention.

[0166] SNAP stability in NORel Gel

[0167] The stability of SNAP in alcohol-based formulations, in conjunction with carbomer, under various thermal conditions is not well-understood. This lack of understanding could potentially affect the shelf-life of NORel gels. From a commercial perspective, it is crucial to determine the optimal storage conditions for these gels, considering potential clinical applications. To investigate the stability, NORel1 and NORel2 gels were subjected to different temperatures (-20˚C, 4˚C, room temperature (~23˚C), and 37 ˚C) to simulate real-world storage conditions and the percent of NO (%NO) remaining in the gels was monitored over 28 d, relative to NO donor SNAP present in fresh gels on day 0. The absorption spectra of SNAP at 340 nm wavelength was used to analyze SNAP concentrations in gel formulations by dissolving 100 mg of gel in 1 mL of 10mM PBS (pH 7.4) with 100 µM EDTA. A standard curve of SNAP in PBS-EDTA was plotted to quantify the amount of SNAP in the gel (Figure 3C). The molar extinction coefficient of SNAP in PBS-EDTA at room temperature was found to be 1025^M−1cm−1at 340^nm.

[0168] The results from the study (Table 1 and 2) indicated high stability at -20 ºC, with > 90% SNAP remaining in both gels after 28 d of storage (Figure 3D). At 4 ºC, NORel1 remained stable (97.14%), while NORel2 had ~65% SNAP remaining after 4 weeks of storage (Figure 3E). Both gels showed reduced stability at higher temperatures, with NORel1 and NORel2 having 58.54% and 27.60% SNAP remaining at room temperature (Figure 3F) and 44.01% and 25.20% SNAP remaining at 37 ºC, respectively (Figure 3G). These findings align with prior research, suggesting that RSNOs exhibit superior stability at lower temperatures substantially better than cysteine-based NO-donors with short half-lives and limited stabilityT|H Docket: 222105-2280 even at −20 °C.36, 37RSNOs can release NO more rapidly at higher temperatures due to heat- mediated catalysis of the NO donor SNAP, particularly at physiological temperatures.20The observed decrease in stability of NORel2 gel over 28 d of storage with a higher concentration of SNAP, can be linked to the decomposition of RSNOs induced by hydroxyl radicals, with the thiyl radicals as intermediates.38, 39It can be hypothesized that the increased SNAP content in NORel2 gel might have led to greater thiyl radical generation within the gel, subsequently accelerating the catalytic process from SNAP in NORel2 gel compared to the lower SNAP content in NORel1 gel. Although this phenomenon is less pronounced within the initial 24 h testing period with NOA, it becomes increasingly noticeable during prolonged testing. Overall, the findings from this study highlight that NORel gels exhibit optimal stability and potency when stored at lower temperatures, which is advantageous for their potential clinical applications. It's worth highlighting that the incorporation of NO into conventional hand sanitizer agents offers significant advantages in augmenting the antimicrobial effectiveness of the gel. This gel holds great potential for use in clinical settings to combat infectious pathogens, including antibiotic-resistant strains that can lead to severe infections. An important benefit of having multiple antibacterial agents is that even as SNAP in the gel degrades over time, the antibacterial properties stemming from ethanol and tea tree oil persists, ensuring the gel remains effective for an extended duration.T|H Docket: 222105-2280

[0169] Biocompatibility of Gel Table 1. Determination of SNAP stability in NORel1 Hand Sanitizer Gel Time % SNAP remaining in NORel1 (d)Tabe 2. Determnaton o SNAP stab ty n NORe2 Hand Santzer Ge Time % SNAP remaining in NORel2 (d) [0-reeasng an san zer ges were ur er evauae or ocompa ity using an indirect contact cytocompatibility evaluation model with NIH / 3T3 mouse fibroblast cells using our previously reported method.34The NIH / 3T3 mouse fibroblast cell line is a standardized cell line often used for evaluation of the biocompatibility of medical devices in accordance with ISO 10993-5 standards.40In the present studies, the gel sanitizers contained several active ingredients – SNAP, tea tree oil, glycerin, carbomer, triethylamine, and ethanol, which may trigger a secondary cytotoxic effect from exposure. Therefore, it was critical to evaluate the relative cytotoxicity of these formulations against both untreated and commercial alternatives for comparison purposes.T|H Docket: 222105-2280

[0171] As shown in Figure 3F, no statistically significant difference was observed in cellular viability between the control gel formulation and the commercial alcohol-based gel alternative. In contrast, both the NORel1 and NORel2 formulations demonstrated an upward trend in cellular proliferation with respect to the alcohol alternative (p < 0.05), while the NORel1 formulation also demonstrated improve viability compared to the gel control. The increase in viability with the NORel1 and NORel2 formulations is likely attributable to presence of NO donor SNAP, which has previously shown a proliferative effect for fibroblast cells in several previous NO-releasing materials.41-43By promoting fibroblast proliferation, the NO-releasing gels offer a key benefit over the control and commercial gels in performing better against bacteria while concurrently developing cytoprotective effects ideal for topical application.

[0172] Antibacterial Efficacy of NORel Hand Sanitizer

[0173] Antibiotic resistance presents a critical challenge in healthcare, particularly in dealing with chronic infections. In clinical settings, the rise and spread of multi-drug resistant bacterial strains have exacerbated the global antibiotic resistance crisis. These resilient strains possess intricate and enhanced resistance mechanisms, significantly diminishing the effectiveness of even the most potent antibiotics and traditional antibacterial agents. This heightened resistance poses a substantial risk, particularly among vulnerable groups such as the elderly, children, and immunocompromised individuals, who are susceptible to severe complications arising from viral infections (for e.g., secondary bacterial infections). A recent study addressing secondary infections related to COVID-19 unveiled a concerning 15.2% mortality rate among pneumonia patients due to antibiotic-resistant S. aureus bacteria.44Furthermore, approximately 28% of intensive care unit patients with severe SARS-CoV-2 pneumonia were found to be co-infected with bacteria.45Effective hand sanitization stands as a crucial measure to reduce the risk of viral infections and secondary infections, potentially decreasing the need for intensive care and antibiotic treatment. Despite the importance of proper hand hygiene in infection prevention, current alcohol-based hand sanitizers have limitations in their ability to combat a broad spectrum of disease-causing microorganisms, especially antibiotic-resistant strains. Therefore, this study aimed to develop a nitric oxide- releasing (NORel) hand sanitizer gel, enriched with tea tree oil alongside traditional ingredients, to create a potent solution against various pathogens. The broad-spectrum antimicrobial efficacy of NORel gel was assessed against four microbes frequently linked to hospital-acquired infections: S. aureus, E. coli, C. albicans, and a clinical isolate of methicillin- resistant S. aureus (MRSA) using bacteria killing assay. The antimicrobial effectiveness test demonstrated that the prepared formulations of NORel gels had significant antimicrobial activity against Gram-positive, Gram-negative bacteria and C. albicans yeast (Figure 4A-D). The killing efficiencies of each sanitizer gel is reported in Table 3. The control gel, without SNAP, individually led to > 70% reduction in pathogen viability compared to untreated control,T|H Docket: 222105-2280 resulting in log reductions of 0.81 ± 0.04, 1.05 ± 0.12, 1.25 ± 0.10, and 0.56 ± 0.12 against S. aureus, E. coli, MRSA, and C. albicans, respectively. This can be attributed to the antimicrobial efficacy of the ethanol and tea tree oil in gel. The addition of SNAP to gel significantly enhanced its antimicrobial potential, with NORel1 gel achieving log reductions of 1.97 ± 0.04, 1.98 ± 0.09, 1.71 ± 0.04, and 1.04 ± 0.35 against all microbial strains under evaluation.

[0174] Notably, NORel2 gel, containing higher amounts of SNAP, exhibited the highest efficacy among all formulations, achieving log reductions of 2.30 ± 0.27, 2.34 ± 0.17, 2.32 ± 0.15, and 1.59 ± 0.28 against S. aureus, E. coli, MRSA, and C. albicans, respectively, compared to untreated control (Figure 4E-H). These findings are consistent with the NO release levels observed in Figure 3B, where higher levels of NO release correlated positively with the amount of SNAP incorporated into the gel. While numerous antibiotics focus on specific pathways within microorganisms, NO utilizes a multifaceted approach to deactivate microorganisms. It disrupts essential proteins, damages DNA, and affects cell membranes, among other actions.46-49This comprehensive strategy enables NO to exhibit broad- spectrum antimicrobial properties, effective against various bacteria, fungi, and viruses. This characteristic of NO has been consistently proven in prior research, and the outcomes observed with this innovative material are in line with findings from previously published studies.19Table 3. Killing Efficiency of NORel In vitro Killing Efficiency (%) Microorganism(s)

[0175] Disinfection Efficacy of NORel Gel using Ex vivo Infected Rabbit Skin Model

[0176] The antimicrobial effectiveness of NORel gel was further assessed through an ex vivo animal study using rabbit skin specimens. The explanted skin specimen from euthanized rabbits were exposed to S. aureus bacteria for 12 h to replicate real-world conditions, allowingT|H Docket: 222105-2280 bacteria to adhere to the skin. Subsequently, NORel2 gel, chosen due to its superior NO release and in vitro antimicrobial performance compared to NORel1, was applied, and left to incubate on the skin for 6 hours to enable NO action against the bacteria (Figure 4I). In this ex vivo study, NORel2 gel's performance was compared to that of a commercial alcohol-based gel, which served as a secondary test group. Both NORel2 and the alcohol-based gel were assessed against a bacterial control group, which consisted of infected skin samples with no active antimicrobial treatment. Results from this investigation revealed that NORel2 gel exhibited similar antimicrobial effects to the commercial alcohol-based gel containing 62% ethyl alcohol as its active component. Notably, both the commercial alcohol and NORel2 gel demonstrated exceptional bacterial reduction, with >99.99% bacterial eradication (Figure 4J) and reductions of 0.58 ± 0.07 and 5.23 ± 0.33 log in bacterial viability compared to the untreated control, respectively (Figure 4K-L).

[0177] Similar animal models have been employed in previous studies to demonstrate the antimicrobial potential of gels on skin.50Various NO-releasing gels have been developed, serving as versatile platforms for angiogenesis, antimicrobial purposes, wound healing therapies, and formulations in the form of topical lotions.51, 52As an endogenous regulatory molecule, NO serves multiple advantageous roles, including providing localized immunity against infectious pathogens. Until recently, the progress of topical NO treatments was impeded by difficulties in securely storing and effectively delivering NO to infection or inflammation sites. Fortunately, with the advancement of stable NO donors, it has become possible to achieve long-term NO release for various biomedical applications. This study's results underscore that NORel gel preserves its antimicrobial effectiveness when applied to the skin, expanding its applicability beyond liquid-based solutions.

[0178] Persistence Activity of NORel Gel using Ex vivo Rabbit Skin Model

[0179] The effectiveness of hand sanitizer gels largely relies on their active components, typically alcohol-based compounds like ethanol. These ingredients function by disrupting bacterial cell membranes, effectively eliminating the bacteria. Although alcohol-based hand sanitizers are successful in eliminating many types of bacteria and viruses, they face certain limitations, including limited residual activity, a short lifespan upon application, and the potential for skin irritation and dryness with excessive usage. To address these drawbacks, this study introduced a combination of ethanol with other antimicrobial agents, such as NO and tea tree oil, as well as moisturizers and skin conditioners. This strategy aimed to prolong the duration of antimicrobial action and enhance the overall effectiveness of the gel while minimizing skin dryness and irritation (Figure 5A).

[0180] To assess the endurance and sustained effects of NORel gel in comparison to commercially available alcohol-based sanitizer gel, a similar infection model employing rabbit skin specimens was utilized as described in Section 4.7. However, in this scenario, theT|H Docket: 222105-2280 sanitizers were applied to the pristine skin specimens first and allowed to incubate at 37 °C for 2 h to evaluate the gel's persistence post-application (Figure 5B). Following the incubation period, S. aureus bacteria were introduced and allowed to incubate with both NORel and a commercial alcohol-based gel for an additional 6 h at 37 °C. Viable bacteria remaining on the skin were then extracted and quantified using a plate counting method.

[0181] As anticipated, the commercial alcohol-based gel had minimal effect on bacterial viability and proved ineffective in eradicating the bacteria after 2 h of application (Figure 5C). The results revealed that the alcohol gel only resulted in a modest 0.33 ± 0.03 log reduction in bacterial viability (p > 0.05). This outcome can be a result of alcohol's evaporation from the skin samples within 2 h of application. Consequently, when the skin samples were exposed to bacteria, there was no antimicrobial agent present to impede bacterial growth. Conversely, NORel2 gel, enriched with tea tree oil, ethanol, glycerin, and carbomer, displayed significant antimicrobial activity even hours following application. Notably, NORel2 gel demonstrated a 1.66 ± 0.22 log reduction in viable S. aureus bacteria compared to the untreated control and a 1.33 ± 0.39 log reduction compared to the commercial alcohol-based gel (Figure 5D). The persistent activity of NORel hand sanitizer can primarily be attributed to the continuous release of NO from the NORel gel. The gel-like consistency of NORel allows it to create a protective layer on the skin, enabling the release of NO at physiological temperatures. As a result, it sustains a microbe-free environment for a more extended period compared to alcohol gel, which has a limited duration of activity. It is expected that NORel's persistent activity will reduce the frequency of gel applications, thereby mitigating issues such as dryness and irritation often caused by repeated exposure to alcohol on the skin.

[0182] Additionally, NO's vasodilatory, anti-inflammatory, and wound healing properties can offer biocompatible and therapeutic benefits beyond its antimicrobial action. This persistence of NORel is particularly valuable in healthcare settings and other high-risk environments where continuous protection against pathogens is crucial, making it a superior alternative to existing alcohol-based gels. In summary, this study underscores the potential of synergizing NO with other antimicrobial agents such as ethanol and tea tree oil to create an innovative and potent gel. This approach effectively addresses the multifaceted challenges posed by antibiotic-resistant strains and provides enduring protection against a diverse array of pathogens. These findings constitute a valuable contribution to the ongoing endeavors aimed at combating antibiotic resistance and enhancing infection control within healthcare settings and beyond. Conclusions

[0183] While alcohol-based formulations have become ubiquitous in our daily lives, the growing demand for hand sanitizers has raised pertinent questions regarding their effectiveness and potential drawbacks. Alcohol-based sanitizers can offer immediateT|H Docket: 222105-2280 protection; however, they often fall short in providing prolonged defense, necessitating frequent reapplication. Additionally, repeated exposure to alcohol can lead to skin issues like dryness and irritation. To address these concerns, this study formulated a nitric oxide- releasing (NORel) hand sanitizer gel using antimicrobial agents (NO donor, ethanol, and tea tree oil) and moisturizers (glycerin). The NORel gel maintained a neutral pH (~7) and stability under various storage conditions for at least 60 d, indicating the effectiveness of its antimicrobial and moisturizing ingredients. The viscosity of the gel was found to be around 3500 cP matching the standards for sanitizer gels. Chemiluminescence tests demonstrated the adjustable release of NO from the gel by varying the NO donor concentration. NORel1 and NORel2 gels released NO at physiologically relevant levels for at least 24 hours. All gel variants exhibited biocompatibility, confirming their safe nature. NORel gels effectively eliminated bacteria and fungi, including antibiotic-resistant MRSA, with over 90% efficiency compared to untreated controls. Their effectiveness was also confirmed on an infected rabbit skin model that showed more than 99.99% efficacy against S. aureus bacteria. Furthermore, NORel gels demonstrated persistent antimicrobial effects even 2 h after application, in contrast to commercial alcohol-based gels, which showed no significant impact due to their short-lived action and rapid evaporation. This research presents a biocompatible NO-releasing gel with superior antimicrobial properties compared to alcohol-based sanitizers, offering an effective hand hygiene solution, especially in high-risk environments. The NORel gel is expected to provide extended protection, eliminate antibiotic-resistant pathogens, and mitigate skin- related side effects.

[0184] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations and are set forth only for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure. REFERENCES 1. Haque, M.; Sartelli, M.; McKimm, J.; Abu Bakar, M., Health care-associated infections - an overview. Infect Drug Resist 2018, 11, 2321-2333. 2. Collins, A. S., Preventing Health Care-Associated Infections. Agency for Healthcare Research and Quality: Rockville (MD), 2008. 3. 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Claims

T|H Docket: 222105-2280 CLAIMS 1. A nitric oxide releasing composition comprising (a) ethanol; (b) a nitric oxide releasing compound; (c) water; (d) a thickening agent; (e) a polyol; and (f) a neutralizer.

2. The composition of claim 1, wherein ethanol is from 60 volume percent to 85 volume percent of the composition.

3. The composition of claim 1, wherein the nitric oxide releasing compound is an S- nitrosothiol conjugated polymer, an S-nitrosothiol modified-dendrimer, an S- nitrosothiol modified polysaccharide, an S-nitrosothiol modified nano / microparticle, an S-nitrosothiol modified-protein, a nitrate, an N-diazeniumdiolates (NONOate), or an S- nitrosothiol (RSNO).

4. The composition of claim 1, wherein the nitric oxide release agent is S-nitroso-N- acetylpenicillamine, S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S- nitrosocysteine, S-nitrosopenicillamine, S-nitroso-B,D-glucose, S-nitrosocaptopril, S- nitrosocysteamine, S-nitroso-3-mercapto-propanoic acid, S-nitroso-N-acetyl-l-cysteine ethyl ester (SNACET), S-nitroso-N-acetyl-L-methionine, or S-nitrosomercaptoethanol.

5. The composition of claim 1, wherein the nitric oxide releasing compound is a modified antibiotic compound comprising a nitric oxide release agent covalently attached to an antibiotic molecule.

6. The composition of claim 5, wherein the antibiotic molecule is ampicillin, vancomycin, gentamicin, or cephalexin.

7. The composition of claim 5, wherein the modified antibiotic compound comprises S- nitroso-N-acetylpenicillamine covalently attached to ampicillin.

8. The composition of claim 1, wherein the nitric oxide releasing compound is from about 1 weight percent of the composition to about 40 weight percent of the composition.

9. The composition of claim 1, wherein the thickening agent comprises a polyacrylic acid, a carbomer, xanthan gum, a cellulose-based compound, acrylates / C10-30 alkylT|H Docket: 222105-2280 acrylate crosspolymer, polyacrylate crosspolymer-6, glycerin, silica gel, isopropyl myristate, or any combination thereof.

10. The composition of claim 1, wherein the thickening agent is from about 0.1 weight percent of the composition to about 1.0 weight percent of the composition.

11. The composition of claim 1, wherein the polyol comprises glycerin, xylitol, lactitol, mannitol, sorbitol, erythritol, maltitol, lactitol, isomalt, or any combination thereof.

12. The composition of claim 1, wherein the polyol is from about 0.1 volume percent of the composition to about 5.0 volume percent of the composition.

13. The composition of claim 1, wherein the neutralizer comprises a substituted amine, a hydroxide, an acid, salts of acids, or any combination thereof.

14. The composition of claim 1, wherein the neutralizer comprises a tri-substituted amine.

15. The composition of claim 1, wherein the neutralizer comprises a tri-alkyl substituted amine.

16. The composition of claim 1, wherein the neutralizer comprises a tri-ethyl amine.

17. The composition of claim 1, wherein the neutralizer is from about 0.1 weight percent of the composition to about 2.0 weight percent of the composition.

18. The composition of claim 1, wherein the composition further comprises a fragrance.

19. The composition of claim 18, wherein the fragrance comprises natural oils, synthetic fragrances, or any combination thereof.

20. The composition of claim 19, wherein the fragrance comprises pure tea tree oil, organic tea tree oil, melaleuca oil, tea tree oil blends, water-soluble tea tree oil, or any combination thereof.

21. The composition of claim 18, wherein the fragrance is from about 0.1 weight percent of the composition to about 1.0 weight percent of the composition.

22. The composition of claim 18, wherein the fragrance is tea tree oil from about 0.1 weight percent of the composition to about 1.0 weight percent of the composition.

23. The composition of claim 1, wherein the composition further comprises an antimicrobial agent comprising an antibiotic agent, an antifungal agent, an antiseptic agent, or any combination thereof.T|H Docket: 222105-2280 24. The composition of claim 23, wherein the antiseptic agent comprises isopropanol, ethanol, idophor, hydrogen peroxide, chlorhexidine, thimerosal, benzalkonium chloride, triclosan, or a hypochlorite.

25. The composition of claim 1, wherein the composition further comprises an antioxidant.

26. The composition of claim 25, wherein the antioxidant comprises Vitamin E, Vitamin C, Vitamin A, resveratrol, carotenoids, cysteine, hydroquinone, delphinidin, coenzyme q10, erythorbic acid, α-lipoic acid, polypodium leucotomos extract, butylated hydroxyanisole, butylated hydroxytoluene, and epigallocatechin gallate.

27. A nitric oxide releasing composition produced by the method comprising (a) admixing ethanol, water, and a thickening agent to produce a first composition; (b) admixing a polyol with the first composition to produce a second composition; (c) admixing a nitric oxide releasing compound with the second composition to produce a third composition; and (d) admixing a neutralizer with the third composition to produce the nitric oxide releasing composition.

28. The composition of claim 27, wherein in step (b), further comprising admixing a fragrance with the polyol and first composition.

29. The composition of claim 1, wherein the composition has a pH of about 6 to about 8.

30. The composition of claim 1, wherein the composition has a viscosity of about 3,000 centipoise to about 4,000 centipoise.

31. The composition of claim 1, wherein the composition releases nitric oxide for at least 12 hours.

32. A method for reducing or preventing the growth of microbes on a surface, the method comprising applying the composition of any one of claims 1-31 to the surface.

33. The method of claim 32, wherein the surface is the skin of a subject.

34. The method of claim 32, wherein the microbe comprises bacteria, fungi, virus, protozoan, or algae.

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