T|H Docket: 222105-2270 NITRIC OXIDE RELEASING SPONGES AND METHODS FOR MAKING AND USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 63/609,028, filed on December 12, 2023, the contents of which is incorporated by reference herein in their entireties. BACKGROUND [0002] Within the broad spectrum of biomedical engineering applications, medical sponges find extensive utility. Yet, a significant area in this field that remains relatively underexplored is the role of these sponges in addressing microbial infections and their antimicrobial potential. Sponges have been incorporated with various antimicrobial components; however, the demonstrated antimicrobial effectiveness of these sponges lack substantiated evidence against microbes other than bacteria such as fungi or viruses. This aspect holds significant importance in the field of tissue engineering and anti-infection applications. Moreover, it is important to note that the primary objective of most of these sponges does not solely revolve around achieving antibacterial efficacy. Thus, there is a need for medical sponges that provide a broad spectrum of anti-microbial activity. SUMMARY [0003] Described herein are nitric oxide-releasing sponges comprising a sponge material and a nitric oxide releasing compound. The sponges described herein can eradicate a variety of microbes on medical devices. In other embodiments, the sponges can be used in wound healing applications to prevent or limit infection. [0004] 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.
T|H Docket: 222105-2270 BRIEF DESCRIPTION OF THE DRAWINGS [0005] 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. [0006] Figure 1 shows a hydrophilic PDMS sponge fabrication scheme. After mixing each component for 2 min, the resulting solution was compacted into glass molds and cured in an oven at 100 °C for 15 h. Molds were added to hot water to dissolve the salt template for at least 8 h. Sponges were also fabricated without the addition of PDMS-b-PEO using the same steps. [0007] Figure 2 shows impregnation of SNAP into the matrix of PDMS sponges using THF as the solvent (top) and swelling of SNAP into hydrophilic PDMS sponges using 70% IPA (bottom). [0008] Figures 3A-3B show hydrophilic modification of PDMS sponges. (A) UATR-FTIR subtraction spectra of 77% porous PDMS and PDMS-b-PEO sponges. Peaks at 965cm
-1 can be assigned to CH
2 rocking and C-O-C stretch, while peaks at 857cm
-1 correspond to CH
2 rocking and C-O-C bending, and peaks at 767cm
-1 correspond to CH
2 rocking.
1 (B) Absorption of 10 µL droplet of water on the surface of PDMS and hydrophilic modified (PEO) 82% porous sponge showing the fast absorption of the droplet on the hydrophilic sponge surface (n = 1). [0009] Figures 4A-4D show PDMS sponge porosity and SNAP characterization. (A) Porosity of PDMS sponges where NaCl dissolved represents the concentration of salt in the initial sponge fabrication. Data represents the mean ± SEM (n = 3). (B) Porosity comparison of PDMS sponges reveals no effect of incorporating surfactant on porosity. Checkered bars represent control PDMS sponges and solid bars represent hydrophilic PDMS sponges. (C) Mechanical properties of hydrophilic sponge types reveal a correlation between porosity and compressive strength. Increasing porosity causes a reduction in compressive strength due to the increased ratio of void space within the sponge. (D) Increasing porosity increases the absorption capacity of hydrophilic sponges and shows no significant increase after 15 min. Solid, checkered, and striped bars represent 15, 30, and 60 min swelling in 70% IPA. Data represents the mean ± STD (n ≥ 3). [0010] Figure 5 shows the respective SEM images of hydrophilic and SNAP-impregnated PDMS. No significant difference in pore size compared to control PDMS sponges is observed with scale bars corresponding to 500 µm (n = 1). [0011] Figures 6A-6G shows: (A) SNAP loading of PDMS sponges using THF demonstrates the increased loading capacity of SNAP into the polymer matrix as porosity increases. (B) SNAP leaching of PDMS sponges over 24 h supports the SNAP loading results with a faster rate of leaching observed as porosity increases. (C) Nitric oxide release from
T|H Docket: 222105-2270 SNAP-impregnated PDMS sponges under moist conditions exhibited sustained release of NO for 14 d. Data represents the mean ± SEM (n = 3). (D) Cumulative production of NO from SNAP-impregnated sponges in physiological conditions exhibited increased release rates as porosity increases. (E) SNAP incorporation into hydrophilic sponges using 70% IPA at different time points reveal an increasing trend with increasing porosity and no significant increase after 15 min. (F) SNAP delivery from hydrophilic sponges over 24 h exhibit parallel patterns across different sponge types. The assessment of SNAP released in a catheter model offers vital insights into SNAP stability within a hospital-like environment. Data represents the mean ± STD (n = 3). (G) SNAP delivery set up. Secure connection of the luer cap-containing sponge releases SNAP and IPA as it is compressed by the luer connector. [0012] Figures 7A-7D show bacteria CFUs of control and SNAP-impregnated PDMS sponges against adhered (A) and planktonic (B) E. coli and adhered (C) and planktonic (D) S. aureus after 4 h exposure to bacteria. Data are presented as mean of two biological replicates ± SEM (n = 6). * Indicates significance: *** (p<0.001), **** (p<0.0001). [0013] Figures 8A-8J show zone of inhibition comparison between 70% IPA and SNAP- IPA swollen sponges. Quantitative data showcasing inhibited zones of each microbe: (A) E. coli, (B) P. aeruginosa, (C) S. aureus, (D) S. epidermidis, (E) C. albicans. Striped bars represent 70% IPA swollen sponges and solid bars represent SNAP-IPA swollen sponges. Complementing representative images of each microbe tested: (F) E. coli, (G) P. aeruginosa, (H) S. aureus, (I) S. epidermidis, (J) C. albicans, demonstrating significant differences upon SNAP addition. Image labels indicate where control sponges (C), 70% IPA swollen sponges (I), and SNAP-IPA swollen sponges (S) were placed. Disrupted microbial growth areas represent where control sponges were placed with unquantifiable zones. Data represents the mean ± STD (n ≥ 3). * Indicates significance: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001). [0014] Figures 9A-9F show microbial viability after 4 h exposure to 82% porous PDMS sponges (control, 70% IPA swollen, and SNAP-IPA swollen). Viable planktonic microbes presented as CFU per mm
3 of sponge: (A) E. coli, (B) S. aureus, (C) C. albicans. Log
10 reduction in viable planktonic (D) E. coli, (E) S. aureus, (F) C. albicans. Data represents the mean ± STD (n = 4). * Indicates significance: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001). [0015] Figures 10A-10F show microbial viability of contaminated luer connectors after 30 min exposure to SNAP-IPA 82% porous sponges. Viable adhered microbes presented as CFU per mL: (A) E. coli, (B) S. aureus, (C) C. albicans. Log
10 reduction in viable adhered (D) E. coli, (E) S. aureus, (F) C. albicans. Data represents the mean ± STD (n ≥ 3). * Indicates significance: * (p<0.05), ** (p<0.01), **** (p<0.0001). Ø indicates results were below the detection limit of the instrument.
T|H Docket: 222105-2270 [0016] Figures 11A-11E show the characterization of SNAP-swelled medical-grade foam dressings using 70% IPA as the solvent. (A) Porosity of Optifoam and Hydrasorb foam dressings. (B) Weight percent of SNAP loaded into Optifoam and Hydrasorb foams using 70% IPA. (C) Absorption capacity of Optifoam and Hydrasorb revealing a capacity plateau at 15 min for both foams. Solid, striped, and checkered bars represent 70% IPA swelling times of 15, 30, and 60 min. (D) Comparative absorption capacities of Optifoam and Hydrasorb showing an increase in absorption capacity for both foams when SNAP is introduced to the swelling solvent. Stiped and solid bars represent 70% IPA and SNAP-IPA as the swelling solutions. (E) Comparative absorption capacities of hydrophilic PDMS sponges showing no significant difference in absorption capacity when SNAP is introduced to the swelling solvent. Stiped and solid bars represent 70% IPA and SNAP-IPA as the swelling solutions. Data represents the mean ± STD (n ≥ 3). * Indicates significance: *** (p<0.001), **** (p<0.0001). [0017] Figures 12A-12B show the zone of Inhibition of Optifoam and PDMS sponges. (A) Zone of inhibition of Optifoam reveals significant microbial growth inhibition of SANP-IPA compared to IPA. (B) Zone of inhibition of hydrophilic PDMS sponges against drug-resistant C. glabrata shows no significant increase when SNAP is added to 70% IPA. Stiped and solid bars represent 70% IPA and SNAP-IPA as the swelling solutions. Data represents the mean ± STD (n ≥ 3). * Indicates significance: * (p<0.05), **** (p<0.0001). [0018] 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 [0019] 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. [0020] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. [0021] 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
T|H Docket: 222105-2270 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. [0022] 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. [0023] 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. [0024] 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. [0025] 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. [0026] 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 [0027] In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.
T|H Docket: 222105-2270 [0028] 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 the 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. [0029] 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. [0030] 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. [0031] 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’”. [0032] 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
T|H Docket: 222105-2270 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 is 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. [0033] 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. [0034] 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. [0035] 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
T|H Docket: 222105-2270 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, 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. [0036] 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. [0037] 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. [0038] 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. [0039] 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. [0040] 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,
T|H Docket: 222105-2270 Camesiphon, Cylindrospermopsis, Gloeobacter Hapalosiphon, Lyngbya, Microcystis, Nodularia, Nostoc, Phormidium, Planktothrix, Pseudoanabaena, Schizothrix, Spirulina, 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,
T|H Docket: 222105-2270 Peptococcus species, Peptostreptococcus species, Actinomyces israelii and other Actinomyces species, and Propionibacterium acnes, Clostridium tetani, Clostridium botulinum, 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). [0041] 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). [0042] 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,
T|H Docket: 222105-2270 preventing the presence of additional bacteria, delaying or slowing of the reproduction of bacteria, and combinations thereof. [0043] 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. [0044] 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. Nitric Oxide Releasing Sponges [0045] In accordance with the purpose(s) of the present disclosure, described herein are nitric oxide releasing sponges comprising a sponge material and a nitric oxide releasing compound. Methods for preparing and using the compositions described is described in detail below. Sponge Material [0046] The sponge material used to produce the nitric oxide releasing sponges can be composed of any material typically used to produce sponges for biomedical applications. In one aspect, the sponge material has a water uptake of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% and up to 90%. [0047] In one aspect, the sponge material comprises a polyether urethane, a polyether, a polyesters, a polysaccharide, or a carbon-based foam. In another aspect, the sponge material comprises viscose, cellulose, chitosan, alginate or hybrid thereof, graphene oxide, collagen, polyvinyl alcohol, keratin, silk, gelatin, melamine, or aloe. In one aspect, the sponge material comprises viscose (rayon). [0048] In one aspect, the sponge material comprises a carbon-based foam. In one aspect, the carbon-based foam comprises a melamine-formaldehyde foam. In another aspect, the carbon-based foam comprises a polymer such as polypyrrole with graphene oxide incorporated throughout the polymer. In another aspect, the carbon-based foam can be functionalized with metals. In one aspect, the carbon-based foam can be a metal-organic
T|H Docket: 222105-2270 framework alone or in combination with graphene oxide. Examples of metal-organic frameworks include those derived from chitosan and Al, Fe, Cr, and Zr. [0049] In one aspect, the sponge material comprises a polyurethane. In one aspect, the polyurethane can be prepared by using an isocyanate-capped polyether prepolymer. Isocyanate-capped polyether prepolymers such as those disclosed in U.S. Pat. No.4,137,200. These prepolymers have a defined average isocyanate functionality greater than 2. These prepolymers may be capped with aromatic isocyanates, such as, methylene diphenyl isocyanate (MDI), or mixtures of MDI with toluene diisocyanate (TDI) and/or polymeric forms of MDI. Isocyanate-capped polyether prepolymers which have been found suitable for use in the practice of the present invention include prepolymers sold by Hampshire Chemical Company the HYPOL® trademark. Examples include HYPOL FHP 3000, HYPOL FHP 2002, HYPOL FHP 3000, HYPOL FHP 4000, HYPOL FHP 5000, HYPOL X6100, HYPOL JT 6000, and HYPOL hydrogel. The HYPOL FHP 4000 and HYPOL FHP 5000 prepolymers are derived from methylene diisocyanate. The free amino groups formed by the hydrolysis reaction react with unhydrolyzed isocyanate groups to form urea groups which crosslink, with additional ioscyanurate group to form urethane and stabilize the foam, while entrapping a part of the excess water in the cell walls, where it acts to impart hydrophilic properties to the foam. [0050] In one aspect, the polyurethane is a hydrophilic water absorbing material such as, for example, Optifoam® (a high-water uptake polyether urethane manufactured by Medline, Northfield, IL) and Hydrasorb, medical-grade hydrophilic absorbent foam sponge (Qosina, Ronkonkoma, NY). In another aspect, the polyurethane can also include other materials such as metals and graphene oxide. [0051] In one aspect, the sponge material comprises a polysiloxane represented by the general structure where R is C1 to C10 alkyl group. In one aspect, the polysiloxane is polydimethyl siloxane. In another aspect, the polysiloxane is Sylgard 184 from Ellsworth Adhesives. [0052] In one aspect, the sponge further comprises a hydrophilic surfactant. The hydrophilic surfactant can be incorporated into the sponge by contacting the sponge material with the surfactant during the production of the sponge or after the sponge has been formed and subsequently contacting the sponge with the surfactant. Non-limiting procedures for producing sponges with hydrophilic surfactants is provided in the Examples.
T|H Docket: 222105-2270 [0053] In one aspect, the hydrophilic surfactant can be an anionic surfactant such as, for example, sodium dodecylbenzenesulfonate. In another aspect, the hydrophilic surfactant can be an amphoteric surfactant such as, for example, cocamidopropyl betaine and betaines derived from fatty acids. In another aspect, the hydrophilic surfactant can be a quaternary ammonium surfactant such as, for example, cetyltrimethylammonium (cetrimonium) bromide (CTAB) and cetrimonium chloride. [0054] In another aspect, the hydrophilic surfactant is lauryl sulfate, stearamido propyldimethyl-B-hydroxyethyl ammonium nitrate, an alkylaryl polyethoxylated glycol ether, a polyoxyethylene sorbitan monooleate, or any combination thereof. [0055] In one aspect, the surfactant comprises a polyalkylene oxide. In one aspect, the polyalkylene oxide is a C1-C6 polyalkylene oxide such as, for example, polyethylene oxide, polypropylene oxide, polybutylene oxide, and the like. [0056] In one aspect, the hydrophilic surfactant comprises a polyalkylene oxide end- capped with a siloxane group. In one aspect, the hydrophilic surfactant comprises a polyethylene oxide end-capped with a siloxane group. In another aspect, the hydrophilic surfactant comprises a polyethylene oxide end-capped with polydimethyl siloxane. In another aspect, the hydrophilic surfactant is poly(dimethylsiloxane-b-ethylene oxide), methyl terminated (PDMS-b-PEO). [0057] The amount of the hydrophilic surfactant can vary depending upon the sponge material that is selected. In one aspect, the surfactant is from about 0.1 weight percent to about 5.0 weight percent of the sponge material, or about 0.1 weight percent, 0.5 weight percent, 1.0 weight percent, 1.5 weight percent, 2.0 weight percent, 2.5 weight percent, 3.0 weight percent, 3.5 weight percent, 4.0 weight percent, 4.5 weight percent, or 5.0 weight percent, where any value can be a lower and upper endpoint of a range (e.g., 2.0 weight percent to 3.5 weight percent). Nitric Oxide Releasing Compounds [0058] The nitric oxide releasing compound is a compound that possesses one or more nitric oxide groups, wherein nitric oxide is subsequently released from the compound. In one aspect, the nitric oxide releasing compound is an S-nitrosothiol compound. In another 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. [0059] In another aspect, the nitric oxide releasing compound can be S-nitrosothiol conjugated polymers, modified- dendrimers; S-nitrosothiol modified polysaccharides, S- nitrosothiol modified nano/microparticles, or S-nitrosothiol modified-proteins. In another
T|H Docket: 222105-2270 aspect, the nitric oxide releasing compound can include NO-donors such as, for example, nitrates, N-diazeniumdiolates (NONOates), and S-nitrosothiols (RSNOs). [0060] In one aspect, 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 a stable NO donor and an antibiotic can be a very efficient approach for combating and preventing biofilm related infections. [0061] The modified antibiotic compound can be a synthetic RSNO (e.g. S-nitroso-N- acetylpenicillamine (SNAP)) covalently attached to an antibiotic molecule (e.g. ampicillin) to create a novel dual functional antimicrobial agent, also referred to as a modified antibiotic compound. In other aspects, the nitric oxide releasing compound can be such as S-nitroso- glutathione, and S-nitroso-N-acetylcysteine, S-nitrosocysteine, S-nitrosopenicillamine, S- nitroso-B,D-glucose, S-nitrosocaptopril, S-nitrosocysteamine, and S-nitroso-3-mercapto- propanoic acid. [0062] In one aspect, the antibiotic molecule is ampicillin. In other embodiments, the antibiotic molecule can be vancomycin, gentamicin, cephalexin, and the like. [0063] In one aspect, the modified antibiotic compound when comprising SNAP and ampicillin is referred to herein as SNAPicillin. The terms “modified antibiotic compound” and “SNAPicillin” are intended to be used interchangeably, although “modified antibiotic compound” can also include other combinations of nitric oxide release agents and antibiotic molecules as can be appreciated by one of skill in the art. The structure of SNAPicillin is provided below. [0064] In one be formed by covalently attaching a nitric oxide
attachment can be formed by mixing the nitric oxide release agent and the antibiotic molecule in a solvent 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 3 times molar
T|H Docket: 222105-2270 excess of t-butyl nitrate with respect to the ampicillin quantity. 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. [0065] In one aspect, the amount of the nitric oxide releasing compound is from about 0.1 weight percent to about 25 weight percent of the composition or about 0.1 weight percent, 0.5 weight percent, 1 weight percent, 2 weight percent, 4 weight percent, 6 weight percent, 8 weight percent, 10 weight percent, 12 weight percent, 14 weight percent, 16 weight percent, 18 weight percent, 20 weight percent, 22 weight percent, 24 weight percent, or 25 weight percent, where any value can be a lower and upper endpoint of a range (e.g., 4 weight percent to 14 weight percent). Additional Components [0066] The sponges described herein can include one or more additional components depending upon the application of the sponge. The amount of the additional component can also vary as well depending upon the application. [0067] In one aspect, the sponges described herein can include a catalyst. Not wishing to be bound by theory, the catalyst can react with the nitric oxide releasing compound and modify the rate of release of NO. In one aspect, the catalyst comprises copper, selenium, or any combination thereof. [0068] In another aspect, the sponges described herein include metal nanoparticles (e.g., silver, zinc, copper, etc), antibiotics, antimicrobials, bactericides, antiseptics (e.g., isopropanol, ethanol, idophor, hydrogen peroxide, chlorhexidine, thimerosal, or a hypochlorite), antimicrobial peptides, quaternary ammonium compounds, and any combination thereof to enhance the antimicrobial properties of the sponge. Methods for Making the Nitric Oxide Releasing Sponges [0069] Described herein are methods for making nitric oxide releasing sponges. In one aspect, the sponge is produced by the process comprising contacting the sponge material with the nitric oxide releasing compound in a solvent. [0070] In one aspect, the sponge material is submersed in a solution of the nitric oxide releasing compound and the solvent. In one aspect, the solvent is an organic solvent that possesses antiseptic properties. In one aspect, the solvent is an alcohol such as, for example, comprising methanol, ethanol, isopropanol, or any combination thereof. In another aspect, the solvent is an aqueous alcohol, where the alcohol is from about 10 weight percent to about 90 weight percent of the aqueous solution, or is about 10 weight percent, 20 weight percent, 30 weight percent, 40 weight percent, 50 weight percent, 60 weight percent, 70 weight percent, 80 weight percent, or 90 weight percent, where any value can be a lower and upper endpoint of a range (e.g., 60 weight percent to 80 weight percent).
T|H Docket: 222105-2270 [0071] In certain aspects, when a hydrophilic surfactant is used, the sponge material is first contacted with the hydrophilic surfactant prior to contacting the sponge with the nitric oxide release compound. The Examples provide non-limiting procedures for loading the nitric oxide releasing compound into the sponge material. [0072] The properties of the nitric oxide releasing sponge can vary depending upon the application of the sponge. In one aspect, the sponge has a porosity of from about 50% to about 95%, or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, where any value can be a lower and upper endpoint of a range (e.g., 60% to 75%). In another aspect, the sponge has a compressive modulus of about 15 kPa to about 150 kPa, or about 15 kPa, 25 kPa, 50 kPa, 75 kPa, 100 kPa, 125 kPa, or 150 kPa, where any value can be a lower and upper endpoint of a range (e.g., 25 kPa to 125 kPa). In another aspect, the sponge releases nitric oxide for at least 14 days, or 14 days, 21 days, or 28 days, where any value can be a lower and upper endpoint of a range (e.g., 14 days to 21 days). Methods of Use [0073] In one aspect, the sponges described herein can be applied to or incorporated into any article where it is desirable to reduce or prevent a microbial infection or growth. In one aspect, the sponge described herein can be incorporated into a luer cap or lock. For example, as shown in Figure 6F, a nitric oxide releasing sponge 1 can be placed in a luer cap 2 or over the opening of the luer cap 2. The luer cap with sponge can then be secured to a luer connector 3 (e.g., connector of a catheter). Upon connecting the luer cap with sponge to the luer connector, nitric oxide is released on the luer connector as well as other components (e.g., an antiseptic such as an alcohol). Ultimately, microbes are killed on the surface of the luer connector and/or their growth is prevented. [0074] In other aspects, the sponges described herein can be used in wound healing applications. Biomedical sponges are commonly used in surgical procedures and other procedures to enhance wound healing. The sponges described herein can be effective in reducing or preventing microbial infection in a subject while enhancing wound healing. Aspects [0075] Aspect 1. A nitric oxide releasing sponge comprising a sponge material and a nitric oxide releasing compound. [0076] Aspect 2. The sponge of Aspect 1, wherein the sponge material has a water uptake of at least 10%. [0077] Aspect 3. The sponge of Aspect 1, wherein the sponge material comprises a polyether urethane, a polyether, a polyesters, a polysaccharide, or a carbon-based foam. [0078] Aspect 4. The sponge of Aspect 1, wherein the sponge material comprises viscose, cellulose, chitosan, alginate or hybrid thereof, graphene oxide, collagen, polyvinyl alcohol, keratin, silk, gelatin, melamine, or aloe.
T|H Docket: 222105-2270 [0079] Aspect 5. The sponge of Aspect 1, wherein the sponge material comprises viscose (rayon). [0080] Aspect 6. The sponge of Aspect 1, wherein the sponge material comprises a polysiloxane or a polyurethane. [0081] Aspect 7. The sponge of Aspect 1, wherein the sponge material comprises polydimethylsiloxane. [0082] Aspect 8. The sponge of any one of Aspects 1-7, wherein the sponge further comprises a hydrophilic surfactant. [0083] Aspect 9. The sponge of Aspect 8, wherein the hydrophilic surfactant comprises an anionic surfactant, an amphoteric surfactant, or a quaternary ammonium surfactant. [0084] Aspect 10. The sponge of Aspect 8, wherein the hydrophilic surfactant comprises lauryl sulfate, stearamido propyldimethyl-B-hydroxyethyl ammonium nitrate, an alkylaryl polyethoxylated glycol ether, a polyoxyethylene sorbitan monooleate, a polyalkylene oxide, or any combination thereof. [0085] Aspect 11. The sponge of Aspect 8, wherein the hydrophilic surfactant comprises a polyalkylene oxide. [0086] Aspect 12. The sponge of Aspect 8, wherein the hydrophilic surfactant comprises a polyalkylene oxide end-capped with a siloxane group. [0087] Aspect 13. The sponge of Aspect 8, wherein the hydrophilic surfactant comprises a polyethylene oxide. [0088] Aspect 14. The sponge of Aspect 8, wherein the hydrophilic surfactant comprises a polyethylene oxide end-capped with a siloxane group. [0089] Aspect 15. The sponge of Aspect 8, wherein the hydrophilic surfactant comprises a polyethylene oxide end-capped with polydimethyl siloxane. [0090] Aspect 16. The sponge of any one of Aspects 8-15, wherein the hydrophilic surfactant is from about 0.1 weight percent to about 5.0 weight percent of the sponge material. [0091] Aspect 17. The article of any one of Aspects 1-16, wherein the nitric oxide releasing compound is a S-nitrosothiol conjugated polymer, a S-nitrosothiol modified- dendrimers; a S-nitrosothiol modified polysaccharide, a S-nitrosothiol modified nano/microparticle, a S-nitrosothiol modified-protein, a nitrate, a N-diazeniumdiolates (NONOate), or a S-nitrosothiol (RSNO). [0092] Aspect 18. The article of any one of Aspects 1-16, 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.
T|H Docket: 222105-2270 [0093] Aspect 19. The sponge of any one of Aspects 1-16, wherein the nitric oxide releasing compound is a modified antibiotic compound comprising a nitric oxide release agent covalently attached to an antibiotic molecule. [0094] Aspect 20. The sponge of Aspect 19, wherein the antibiotic molecule is ampicillin, vancomycin, gentamicin, or cephalexin. [0095] Aspect 21. The sponge of Aspect 19, wherein the modified antibiotic compound comprises S-nitroso-N-acetylpenicillamine covalently attached to ampicillin. [0096] Aspect 22. The sponge of any one of Aspects 1-21, wherein the nitric oxide releasing compound is from about 0.1 weight percent of the composition to about 25 weight percent of the sponge. [0097] Aspect 23. The sponge of any one of Aspects 1-22, wherein the sponge further comprises a catalyst. [0098] Aspect 24. The sponge of Aspect 23, wherein the catalyst comprises copper, selenium, or any combination thereof. [0099] Aspect 25. The sponge of any one of Aspects 1-24, wherein the sponge further comprises an antimicrobial agent comprising an antibiotic agent, an antifungal agent, an antiseptic agent, or any combination thereof. [0100] Aspect 26. The sponge of Aspect 25, wherein the antiseptic comprises isopropanol, ethanol, idophor, hydrogen peroxide, chlorhexidine, thimerosal, or a hypochlorite. [0101] Aspect 27. The sponge of any one of Aspects 1-26, wherein the sponge has a porosity of from about 50% to about 95%. [0102] Aspect 28. The sponge of any one of Aspects 1-26, wherein the sponge has a compressive modulus of about 15 kPa to about 150 kPa. [0103] Aspect 29. The sponge of any one of Aspects 1-26, wherein the sponge releases nitric oxide for at least 14 days. [0104] Aspect 30. The sponge of any one of Aspects 1-29, wherein the sponge is produced by the process comprising contacting the sponge material with the nitric oxide releasing compound in a solvent. [0105] Aspect 31. The sponge of Aspect 30, wherein the sponge material is submersed in a solution of the nitric oxide releasing compound and the solvent. [0106] Aspect 32. The sponge of Aspect 30 or 31, wherein the solvent comprises an alcohol. [0107] Aspect 33. The sponge of Aspect 32, wherein the solvent comprises water in combination with an alcohol comprising methanol, ethanol, isopropanol, or any combination thereof.
T|H Docket: 222105-2270 [0108] Aspect 34. The sponge of any one of Aspects 30-33, wherein the sponge material is contacted with a hydrophilic surfactant prior to contacting with the nitric oxide releasing compound. [0109] Aspect 35. An article comprising the sponge of any one of Aspects 1-34. [0110] Aspect 36. The article of Aspect 35, wherein the article comprises a luer cap or lock. [0111] Aspect 37. A method for treating or preventing a microbial infection on an article, the method comprising applying the sponge of any one of Aspects 1-34 to the article that is exposed to a microbe. [0112] Aspect 38. The method of Aspect 37, wherein the article comprises a medical device comprising at least one luer fitting, wherein the sponge is applied to the luer fitting. [0113] Aspect 39. The method of Aspect 38, wherein the sponge is incorporated in a luer cap. [0114] Aspect 40. The medical device of Aspect 38 or 39, wherein the medical device comprises a catheter. [0115] Aspect 41. A method for treating or preventing a microbial infection in a wound of a subject, the method comprising applying the sponge of any one of Aspects 1-34 to the wound. [0116] Aspect 42. The method of any one of Aspects 37-41, wherein the microbe comprises bacteria, fungi, virus, protozoan, or algae. EXAMPLES [0117] Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are 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. [0118] Materials and Methods [0119] Materials [0120] Poly(dimethylsiloxane) (PDMS) base and curing agent (Sylgard 184) were purchased from Ellsworth Adhesives. BD DIFCO
TM yeast mold (YM) broth, BD DIFCO
TM YM agar, concentrated hydrochloric acid (conc. HCl, 12.1 M), crystalline sodium chloride, and ethanol (100%) were purchased from Fisher Scientific, Inc. Poly(dimethylsiloxane-b-ethylene oxide), methyl terminated (PDMS-b-PEO) was purchased from Polyscience (Warrington, PA). S-nitroso-N-Acetyl-penicillamine (SNAP) was obtained from PharmaBlock (USA). Luria- Bertani (LB) broth, LB agar, sodium chloride, phosphate buffered saline (PBS), sodium nitrite
T|H Docket: 222105-2270 (>99.0%), methanol (>99.8%), concentrated sulfuric acid (conc. H
2SO
4, 18 M), ethylenediaminetetraacetic acid (EDTA), isopropanol (IPA), and tetrahydrofuran (THF) were purchased from Sigma-Aldrich (St. Louis, MO). Stemless, non-vented, clear male luer caps, female luer lock to barb connectors, and Medical-Grade Hydrophilic Absorbent Foam Sponges (Hydrasorb) were purchased from Qosina (Ronkonkoma, NY). Silicone tubing was purchased from Helix Mark. Medline Optifoam® Basic Polyurethane Foam Dressing was purchased from Cardinal Health (Dublin, OH). Staphylococcus aureus (S. aureus, ATCC 6538), Staphylococcus epidermidis (S. epidermidis, ATCC 35984), Escherichia coli (E. coli, ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853), and Candida albicans (C. albicans, ATCC MYA4441) were obtained from the American Type Culture Collection (Manassas, VA). Candida glabrata (C. glabrata AR Bank #0325) was obtained from the Centers for Disease Control and Prevention (CDC) AR isolate bank. All buffers and media were sterilized in an autoclave at 121 °C, 100 kPa (15 psi) above atmospheric pressure for 30 min. [0121] S-nitroso-N-acetylpenicillamine (SNAP) Synthesis: [0122] A slightly modified approach based on an existing method was used to synthesize SNAP with a purity greater than 90%.
2 First, 5 g N-acetylpenicillamine (NAP) was combined with 60 mL methanol, 5 mL H
2SO
4 and 20 mL HCl while continuously stirring until NAP was fully dissolved. Stirring was discontinued and 5 g sodium nitrite in 40 mL deionized (DI) water was gradually added dropwise to the NAP solution. The reaction mixture was then chilled on ice and subjected to continuous nitrogen purging for 8 h without agitation. Resulting SNAP crystals were collected through vacuum filtration and allowed to dry overnight in a light- protected desiccator. The purity of crystals was determined using a chemiluminescence Nitric Oxide Analyzer (NOA 280i, Sievers, Boulder, CO) and SNAP > 90% purity was used in the subsequent materials and studies. [0123] Polydimethylsiloxane Sponge Fabrication: [0124] Sponge formulation methods were kept consistent with the amount of sodium chloride (NaCl) incorporated ranging from 16 g to 64 g. The PDMS base and curing agent were mixed for 2 min at a ratio of 10:1. Then, NaCl crystals were stirred in for 2 min until combined, resulting in a wet, sand-like mixture. This mixture was compacted into cylindrical glass molds and cured at 100 °C for 15 h. Once cured, the PDMS sponges with varying salt concentrations were removed from the oven and placed in hot water for a minimum of 8 h to dissolve the salt template with the water being replaced every few hours. Sponges were removed from their molds and dried in an oven at 100 °C for 2 h. Dried sponges were repeatedly rinsed with ethanol and dried until no salt was visible on the surface. Sponge samples are referenced according to their corresponding porosities based on the amount of NaCl incorporated during fabrication, including 16, 24, 32, 40, 48, 56, and 64 g NaCl.
T|H Docket: 222105-2270 [0125] Sponges were also rendered hydrophilic through the introduction of PDMS-b-PEO, an amphiphilic surfactant (Figure 1). The surfactant was incorporated into sponge formulations before the addition of NaCl and after mixing PDMS with its curing agent for an additional 2 min. The complex interplay between the surfactant, NaCl concentration, and successful PDMS curing was examined, however the addition of surfactant was kept constant (1.25 wt% compared to PDMS base) to reduce the number of variables in the study. [0126] For the remainder of the studies, PDMS sponges are mentioned based on the corresponding porosities, PDMS sponges without surfactant: 60, 66, 70, 77, 83, 88, 89% (Figure 4A); hydrophilic PDMS sponges: 60, 68, 72, 77, 82% (Figure 4B). Data are presented together, and color coded throughout the figures to maintain clarity. [0127] Universal Attenuated Total Reflectance-Fourier Transform Infrared (UATR- FTIR) Spectroscopy: [0128] A Spectrum Two spectrometer from PerkinElmer (Greenville, SC) was used to perform FTIR spectroscopic measurements for the hydrophilic 77% sponge. The reflection mode with the UATR accessory was employed, providing a resolution of 4 cm
-1 within the range of 4000 to 650 cm
-1 to detect the PEO surfactant added to the sponge. A total of 128 scans were performed to enhance the signal-to-noise ratio. Samples were dried prior to testing and 3 independently prepared specimens were analyzed for the 77% formulation as a representative sponge type. Measurements were carried out for both PDMS and hydrophilic samples to identify any functionalized differences. The final spectra were baseline corrected. [0129] Water Absorption of Hydrophilic Sponges [0130] Hydrophilic modification of the PDMS sponge was assessed by depositing a 10 µL droplet of DI water on both the hydrophobic (without PEO) and hydrophilic (with PEO) sponges (n=1). Video recordings using an Ossila Contact Angle Goniometer (Sheffield, UK) were used to capture the droplets on the sponge surfaces, and snapshots were extracted to monitor the evolution of droplet morphology over time. [0131] Porosity: [0132] The porosity of sponge samples (4.5 mm diameter and 5 mm thick) was determined through a water replacement method. As PDMS is hydrophobic, sponge samples were submerged in ethanol for 4 h for complete swelling. Swollen sponges were then placed in water that was replaced every hour for 4 h then left for 24 h to allow the water to replace the ethanol within the sponges. The porosity of sponge samples was calculated using the mass per volume density before and after swelling using Equation 1. ^
^^^^^^^^^^^^^^^ ^%^ ൌ ^1 െ ^ ^
ೞ ^ ∗ 100% (1)
T|H Docket: 222105-2270 Where p is the density of the sponge and p
s is the density of the swollen sponge after immersing in distilled water for 24 h. Exact methods were repeated for sponges containing surfactant. [0133] Compressive Strength of Hydrophilic Sponges: [0134] Uniaxial compression testing was performed using a Mark-10 Series 5 force gauge (Mark-10, Copiaque, NY). Hydrophilic sponges (60, 68, 72, 77, 82% porous) were compressed to 50% strain at a rate of 1.3 mm min
-1. The mechanical testing was completed with cylindrical samples (approximately 8 mm in diameter and 8 mm in height). The diameter and thickness of each sample were measured at multiple points along its length with an accuracy of 0.025 mm. The minimum value of the cross-sectional area, along with the length of each sample, was recorded for further analysis. A total of 5 samples were compressed for each sponge formulation. [0135] Absorption capacity of Hydrophilic Sponges: [0136] Sponge samples (4.5 mm diameter and 5 mm thick) were immersed in a 1.5 mL microcentrifuge tube containing 1 mL of 70% IPA for either 15, 30, or 60 min. The maximum absorption capacity of the hydrophilic sponges (60, 68, 72, 77, 82% porous) was evaluated by measuring the change in mass of the samples using Equation 2. ^
^^^^^^^^^^^^^^^ ^^^^^^^^^^ ^%^ ൌ ^ ^ ି^ బ ^
బ ∗ 100% (2)
[0137] Scanning Electron Microscopy: [0138] To examine the surface morphology and pore size of the various PDMS sponges (PDMS sponges without surfactant: 60, 66, 70, 77, 83, 88, 89% porous; hydrophilic PDMS sponges: 60, 68, 72, 77, 82% porous), microscopy techniques were employed. Samples with an approximate diameter of 6 mm were prepared for imaging by applying a 10 nm gold- palladium coating using a Leica sputter coater (Leica Micro-systems). Scanning electron microscopy (SEM, FEI Teneo, FEI Co.) was used to capture high-resolution images of the cross-sectional morphology and porosity of the different sponge formulations (PDMS, SNAP- impregnated PDMS, and hydrophilic PDMS). A total of 30 sites were analyzed for each sample formulation using Gaussian Least squares fitting and ImageJ imaging software (National Institutes of Health, Bethesda, MD) to obtain pore size distributions. [0139] SNAP Incorporation [0140] Two methods were employed to incorporate SNAP into PDMS sponges with varying porosities to examine the difference in characteristic properties. [0141] First, a solution of SNAP in THF (25 mg mL
-1) was used to swell the PDMS sponge samples (4.5 mm diameter, and 5 mm thick) for 24 h to facilitate maximum swelling into the
T|H Docket: 222105-2270 PDMS polymer matrix (Figure 2). Samples were then dried for 24 h in a dark environment within a fume hood and stored in a -20 °C freezer until further use. [0142] For the hydrophilic PDMS sponges (4.5 mm diameter, and 5 mm thick), a SNAP solution was prepared by dissolving SNAP in 70% v/v IPA (85 mg mL
-1) using a vortex. Sponge samples were submerged in 0.6 mL of the SNAP-IPA solution within 1.5 mL microcentrifuge tubes and were used immediately after swelling in subsequent experiments (Figure 2). [0143] SNAP Loading [0144] The extent of SNAP loaded into the PDMS sponges (60, 66, 70, 77, 83, 88% porous) was assessed by measuring the concentration of SNAP in each sample using a UV- vis spectrophotometer (Cary 60, Agilent Technologies). Sponges were placed into 10 mL of THF for 1 h to fully extract the SNAP before adding 1 mL of solution to a quartz cuvette. The absorbance was measured at 340 nm where the S-NO group of SNAP emits an absorbance maxima.
3 Using a standard curve of SNAP in THF, the concentration of SNAP was determined and converted to a weight percent of the sponge. The molar absorptivity of SNAP in THF was determined to be 951 M
-1cm
-1 at the absorbance maxima. [0145] Hydrophilic sponges (60, 68, 72, 77, 82% porous) were allowed to swell in the SNAP-IPA solution for 15, 30 or 60 min. Their weight was recorded before being placed in a fume hood covered from light for 24 h to allow the IPA to fully evaporate. The dried SNAP sponges were transferred to 10 mL of fresh 70% IPA for 4 h to extract the SNAP completely. The concentration of SNAP in each sample was determined using a UV-vis spectrophotometer. The molar absorptivity of SNAP was determined to be 875 M
-1cm
-1 using a standard curve of SNAP in 70% IPA. A quartz cuvette was used to measure the absorbance of the samples at 340 nm. A 15 min swelling period was used for subsequent experiments involving hydrophilic sponges swelled with SNAP-IPA. [0146] SNAP Diffused out of PDMS Sponges [0147] Diffusion of SNAP from the SNAP-impregnated PDMS sponges (60, 66, 70, 77, 83%, 88% porous) was quantified by measuring the concentration of SNAP in 1 mL of 10 mM PBS, pH 7.4, with 100 µM EDTA. Samples were stored at 37 °C, and after each extraction (1, 4, 8, 12, and 24 h), PBS with EDTA was replenished. The amount of SNAP leached in the 1 mL samples was determined using a UV-vis spectrophotometer. Using a standard curve of 10 mM PBS with 100 µM EDTA, the molar absorptivity of SNAP at 340 nm was observed to be 1025 M
-1cm
-1. [0148] Real-Time Nitric Oxide Release and SNAP Delivery in a Simulated Catheter Model [0149] Nitric oxide release from SNAP-impregnated sponges (60, 66, 70, 77, 83, 88% porous) was analyzed using an NOA. In this method, NO was continuously purged from the
T|H Docket: 222105-2270 samples and directed into an internal reaction chamber, where it reacted with ozone, forming excited nitrogen dioxide. The relaxation of this excited state released a photon, detected by a photomultiplier tube. The intensity of the photon’s light was directly proportional to the amount of NO released from the sample, allowing for precise measurements.
4, 5 The quantification of NO was expressed in parts per billion (ppb) and used to determine the NO surface flux by normalizing the samples to their weights (x10
-10 mol mg
-1 min
-1). To simulate a moist environment, the sponge samples were gently wrapped in gauze and exposed to 200 µL of PBS, pH 7.4, with 100 µM EDTA, applied dropwise around the gauze. Tegaderm was used to wrap the samples, enabling NO to escape while maintaining a moist environment. The internal cell temperature of 37 °C was maintained using a water bath. After establishing a baseline measurement with a tall amber cell chamber, the sponges were introduced to the chamber, and NO release was monitored until a plateau value was achieved, approximately after 1 h. Samples were stored in a 37 °C incubator under moist conditions, and measurements were taken at 0, 1, 3, 5, 7, 9, and 14 d to evaluate the NO release kinetics over time. [0150] Nitric oxide release from SNAP-impregnated sponges (60, 66, 70, 77, 83, 88% porous) under physiological conditions was also analyzed using an NOA. In this study, the sponge samples were submerged in 3 mL of PBS, pH 7.4, with 100 µM EDTA at 37 °C inside of a tall amber cell chamber after achieving a baseline measurement. The samples were removed after 4 h and the cumulative NO release (nmol) of each sample was calculated. [0151] To investigate the release of SNAP from hydrophilic PDMS sponges (60, 68, 72, 77, 82% porous) in a relevant setting, SNAP-IPA swollen sponges underwent a simulated catheter setup using silicone tubing (outer diameter = 1.96 mm) equipped with a luer connector and cap. Luer connectors were attached to one end of the 3 in silicone tubing, which was then filled with 10 mM PBS, pH 7.4, with 100 µM EDTA. The opposite end of the tubing was clamped, and SNAP swelled sponges were carefully placed inside luer caps and securely fastened onto the luer connectors. Sponges were removed from the caps after 0.5, 1, 4, or 24 h and dried in a fume hood covered from light to ensure the complete removal of IPA. Following the same technique used for SNAP loading, the dried sponges were subjected to SNAP extraction, and the resulting solution was analyzed for absorbance using a UV-vis spectrophotometer. For optimal fit within the luer cap, sponges were cut out using a 1/4-inch diameter punchout and trimmed to thickness of 5 mm. Before and after swelling in SNAP- IPA, the samples were weighed to determine the theoretical amount of SNAP incorporated into the sponges using Equation 3. ^
^^^^^^^ ^^^^ ^^^^^^ ൌ ^^ ^ ି^ బ ^ ^
∗ ^^ (3)
T|H Docket: 222105-2270 Where mf is the swollen weight of the sponge, m0 is the initial weight of the sponge, p is the density of 70% IPA in mg mL
-1 at room temperature, and C is the concentration of SNAP in IPA in mg mL
-1. Using the concentration of SNAP determined from the absorbance measurements and the values calculated from Equation 3, the amount of SNAP remaining in the samples (n=3) was determined. Subsequently, the total amount of SNAP delivered was calculated expressed as nmol mg-1. [0152] Growth of Microbial Cultures: [0153] A single isolated colony of S. aureus, S. epidermidis, E. coli, and P. aeruginosa was inoculated in LB media and incubated at 37 °C for 15 h at 150 rpm. Microbial cultures were resuspended in 10 mL of fresh media and placed back into the shaking incubator. The growth of microbes was assessed overtime by measuring the optical density (OD) of the suspensions at 600 nm using a UV-vis spectrophotometer. A mid-log phase of each bacteria was extracted from the suspensions by centrifuging the cultures at 4400 rpm for 7 min. Suspensions were adjusted to an OD of 0.1 using PBS, corresponding to ~10
7 colony-forming units mL
-1 (CFU), for further analysis. Similarly, C. albicans and C. glabrata were grown under the same conditions using YM media, with an initial incubation period of 20 h. [0154] Bacterial Adhesion and Planktonic Assay of PDMS Sponges [0155] A 4 h adhered bacterial viability experiment was conducted to assess the antibacterial activity of the PDMS sponges impregnated with SNAP. Control (no SNAP) and SNAP sponges (60, 66, 70, 77, 83, 88% porous) were submerged in a 1.5 mL microcentrifuge tube containing a 1 mL sample of adjusted bacterial suspension (0.1 OD). After incubating for 4 h at 37 °C, sponges were placed into a 15 mL conical vial containing 1 mL of sterile 10 mM PBS. Samples were homogenized and vortexed for 1 min each followed by serial dilutions. Diluted samples were spread on LB agar plates using spiral plater (Eddy Jet 2, IUL Instruments) and incubated for 24 h. The grown colonies were enumerated using an automated colony counter (Sphere Flash, IUL Instruments) to calculate the number of viable CFUs per mg of sponge. Percent reduction in viable bacteria compared to respective controls was calculated using Equation 4. Additionally, Equation 5 was employed to determine the log reduction of viable bacteria exhibited by each sponge type compared to their respective controls without SNAP. ^
^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ^%^ ൌ ^ ುವಾೄ ି ^ ೄಿಲು ^
ುವಾೄ ൈ 100% (4) ^^^^^^ ^^^^^^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ൌ ^^^^^^ ^ ುವಾೄ ^^ ^ ^ ^ (5)
T|H Docket: 222105-2270 [0157] A 4 h planktonic bacterial viability assay was conducted to assess the antibacterial activity of the PDMS sponges impregnated with SNAP. Control (no SNAP) and SNAP sponges (60, 66, 70, 77, 83, 88% porous) were submerged in a 1.5 mL microcentrifuge tube containing a 1 mL sample of adjusted bacterial suspension (0.1 OD). After incubating for 4 h at 37 °C, the samples were removed from the exposure inoculum. The resulting solutions were appropriately diluted and 50 µL were spread on LB agar plates using a cotton applicator and incubated at 37 °C for 24 h. The grown colonies were enumerated to calculate the number of viable CFUs per mL of solution and normalized to the weight of the sponge. Percent and log reductions in viable bacteria compared to respective controls were calculated using Equation 4 and Equation 5, where C represents the concentration of viable bacteria in CFU mg-1 mL-1. [0158] Zone of Inhibition Assay of Hydrophilic Sponges [0159] The antimicrobial efficacy of the hydrophilic PDMS sponges (60, 68, 72, 77, 82% porous) was evaluated against E. coli, S. aureus, S. epidermidis, P. aeruginosa, C. albicans. For this, a 50 µL sample of adjusted microbial suspension (0.1 OD) of each microbial type was evenly spread onto LB or YM agar plates using a cotton applicator. The plates were allowed to air dry at room temperature for 5 min. Unmodified, IPA swelled, and SNAP-IPA swelled sponges were placed equidistant on the microbial agar plates. Plates were incubated at 37 °C for 24 h and the diameter of the zone around the sponges was measured to assess the inhibited growth of the microbe. The results of the zone of inhibition study were recorded by measuring the diameter (cm) of the regions where microbial growth was absent around the respective sponge samples. [0160] Evaluation of Antimicrobial Efficacy via Contact-killing Study of Hydrophilic Sponges [0161] To further investigate the antimicrobial activity of the hydrophilic PDMS sponge (82% porous), 70% IPA and SNAP-IPA swelled sponges were evaluated using a 4 h contact- killing study. For this, unmodified, 70% IPA swelled, and SNAP-IPA swelled sponges, were added to 1.5 mL microcentrifuge tubes containing 1 mL of 0.1 OD microbial suspension (E. coli, S. aureus, and C. albicans). Samples were incubated at 37 °C for 4 h to let the SNAP and IPA act on bacteria and fungi. After 4 h of incubation, samples were discarded from the solutions and the remaining viable microbes in the suspension were serially diluted and spread on LB agar for E. coli and S. aureus or YM agar for C. albicans using a bacteria spiral plater (Eddy Jet 2, IUL Instruments). Microbial plates were further incubated at 37 °C for 24 h to allow colonies to grow for CFU counting. The viable colony forming units (CFU) from the study were enumerated using an automated bacteria colony counter (Sphere Flash, IUL Instruments). The percentage and log reductions for each microbe were calculated using Equations 4 and 5 and normalized to an average swollen volume after 15 min in 70% IPA.
T|H Docket: 222105-2270 [0162] In situ Disinfection of Needleless Connectors using Infection Model and Hydrophilic Sponges [0163] To evaluate the microbial disinfection efficacy of the SNAP-IPA swelled hydrophilic sponges (82% porous), female luer connectors were pre-infected with microbes. This configuration was designed to mimic the conditions experienced in clinical environments. The female luer connectors were first exposed to 75 µL of adjusted microbial suspension (0.1 OD600) of E. coli, S. aureus, and C. albicans in media for 6 h at 37 °C. After exposure, the media from the connectors was discarded, and connectors were briefly rinsed with 75 µL of sterile PBS to remove any unadhered microbes. The connectors were allowed to dry at room temperature for 10 min before introducing SNAP-IPA swelled sponges (1/4 in diameter, 5 mm thick) into luer caps, securing them firmly onto the connectors. After an incubation period of 30 min at 37 °C, the caps were removed from the luer connectors, and the connectors were transferred to 15 mL vials containing 3 mL of PBS. Each connector was homogenized and vortexed for 1 min to extract the remaining viable adhered microbes on the caps. The resulting solutions were serial diluted and plated onto an LB or YM agar plate using bacteria spiral plater (Eddy Jet 2, IUL Instruments). Microbial plates were then incubated at 37 °C for 24 h to facilitate efficient colony growth for CFU counting using an automated bacteria colony counter (Sphere Flash, IUL Instruments). The percent and log reductions for viable microbes were determined through Equations 4 and 5 and normalized based on the treated suspension volume. Control connectors were subjected to the same process; however, no disinfecting cap was added to the connectors after the initial microbial exposure. [0164] Statistical Analysis [0165] Data are expressed as mean ± standard error of the mean (SEM) or standard deviation (STD) unless stated otherwise. All analysis calculations were conducted using Prism 9.1 (GraphPad Software, San Diego, CA). A standard one-way analysis of variance (ANOVA) was used to perform statistical comparisons among the treatment groups by evaluating the average values. Multiple comparisons were conducted to assess the differences between the sample group averages. Values of p < 0.05 were considered statistically significant. Results and Discussion: [0166] Fabrication of PDMS Sponges [0167] PDMS sponges were fabricated utilizing crystalline NaCl as the porogen, following similar methods reported in previous studies.
6, 7 Upon immersion in hot water, cured PDMS incorporated with NaCl underwent a dissolution process, leading to the dissolution of NaCl particles and the creation of void spaces. This resulted in a porous network of interconnected tunnels within the PDMS material, giving rise to sponge-like characteristics characterized by porosity. Notably, the concentration of porogen in the formulation played a crucial role in determining the porosity of the resulting sponges.
T|H Docket: 222105-2270 [0168] To enhance the swelling property of the naturally hydrophobic sponge, PDMS was combined with the PDMS-b-PEO surfactant to produce hydrophilic sponges with varying porosities. The formulation choices were made to ensure a diverse range of test types while maintaining uniform hydrophilicity across the experimental groups. UATR-FTIR analysis was performed on both PDMS and hydrophilic PDMS sponges, and the subtraction spectra exhibited minimal differences, except in the fingerprint region. This can be attributed to the structural similarities between PDMS and hydroxy-terminated PDMS, as well as the low concentration of the surfactant used. However, similar peaks observed in the subtraction spectra can be attributed to the incorporation of PDMS-b-PEO (Figure 3A).
1 [0169] During the salt-template removal process, interactions were observed among surfactant and NaCl crystals, which affected the curing of PDMS at specific concentrations and resulted in the disintegration of the sponge templates upon exposure to water. Higher concentrations of surfactant have been shown to inhibit the proper cuing of PDMS,
8 however its interaction with NaCl crystals may also affect the curing ability of PDMS. In aqueous solutions containing PDMS-b-PEO, NaCl dissociates into its ionic components, leading to the formation of ion pair complexes. This reduces the solubility of PDMS-b-PEO in water due to the increased hydrophobicity of the PEO chains in the presence of ion-paired complexes, ultimately affecting the behavior of micelle formation and the critical micelle concentration (CMC).
9 Due to the observed interactions between the different components during fabrication, a comprehensive investigation was conducted to assess different sponge formulations comprising different salt and surfactant concentrations. [0170] The primary aim of this investigation was to identify the maximum concentration of PDMS-b-PEO that would yield a well-structured sponge across multiple formulations. The addition of 0.05 g of PDMS-b-PEO was able to generate a curable PDMS sponge in the presence of diverse salt concentrations. However, a notable correlation emerged between the concentration of surfactant and salt in the formulation and the successful curing of the PDMS sponge. This was particularly evident in the 82% porous sponge formulation, which displayed inadequate PDMS curing when 0.1 g of surfactant was introduced. Furthermore, a consistent pattern was observed across the remaining sponges in which the 77 and 72% sponge formulations exhibited analogous shortcomings upon the addition of 0.2 g and 0.4 g of surfactant, respectively. Surfactant concentrations below 0.05 g were not investigated to prevent reduction in the hydrophilic properties of the sponges. Similarly, 0.1 g of surfactant was not used to ensure an adequate range of sample types (60, 68, 72, 77, 82% porous sponges). Nonetheless, the hydrophilicity of the PDMS sponge was observed by placing a droplet of water on the surface (Figure 3B). The quick absorption of the water droplet was observed across multiple locations on the sponge’s surface, further confirming the successful
T|H Docket: 222105-2270 incorporation of the surfactant. The slight water absorption observed with the control sponge after 14 min can be attributed to the porous nature of the material’s surface. [0171] Physical Characterization of PDMS Sponges [0172] PDMS sponges were fabricated using various amounts of NaCl to create 7 sponges, each possessing distinct levels of porosity. The ratio of void space to PDMS polymer in each sponge type was determined by analyzing the change in density following a 24 h water swelling process. The porosity demonstrated an increasing trend with the addition of higher quantities of NaCl to the initial formulation (Figure 4A). The corresponding porosity values exhibited a gradual rise, ranging from 59.62% to 89.32%, and the addition of the surfactant had no significant impact on sponge porosity (Figure 4B). [0173] The compressive modulus of the hydrophilic sponges is a highly porosity-controlled property that characterizes the material’s stiffness and resistance to deformation under compressive load. To evaluate the mechanical properties of the various sponge formulations, uniaxial compression testing was conducted. The sponges underwent a 50% strain at a rate of 1.3 mm min
-1, and stress-strain relationships were analyzed. The 60% porous sponge exhibited a compressive modulus of 105 kPa, indicating their ability to withstand compressive forces (Figure 4C). As expected, an increase in porosity resulted in a reduction in the materials stiffness and compressive modulus due to a larger pore volume within the material.
7 The 82% porous sponge displayed a significantly lower modulus of 19.8 kPa compared to the 60% porous sponge (p < 0.0001), indicating a decrease in stiffness of the material. [0174] Hydrophilic sponges reached their maximum swelling capacity (Figure 4D) after 15 min in 1 mL 70% IPA solution. The absorption capacity of each sponge was assessed by measuring the change in weight of the sponge following various soaking durations. The absorption capacity of sponges ranged from 101 to 362% for the 60 and 82% porous sponges after 15 min, respectively, demonstrating the influence of porosity on swelling behavior. [0175] The observed increase in porosity with higher concentrations of NaCl is consistent with previously reported findings in which the increase in concentration of porogen increases the overall porosity of the material.
1, 10 The measured porosity values closely align with those previously reported for PDMS sponges fabricated with various NaCl concentrations.
7 Although a higher concentration of PDMS-b-PEO in the sponge formulation can slightly hinder porosity,
11 the limited amount of surfactant used ensures that sponge porosity remains mostly unaffected. These findings, along with the relevant peaks identified in the UATR-FTIR analysis, demonstrate that PDMS sponges can be made hydrophilic without significantly obstructing their porosity. These findings also indicate that the manipulation of the porogen concentration enables precise control over the sponge’s porosity. [0176] The trend in compressive modulus values for the sponges highlights the tunability of their mechanical properties. This trend has been observed in previous literature, which
T|H Docket: 222105-2270 reported similar compressive modulus values for PDMS sponges of comparable porosities, resulting from sugar particles as the porogen.
12 These findings demonstrate that the choice of porogen used in fabricating PDMS sponges does not significantly impact their compressive modulus, as long as the porosity is comparable. [0177] Despite the well-established use of 70% IPA as a disinfecting agent, there is a noticeable absence of relevant data in the literature concerning its absorption characteristics in various polymers, specifically with PDMS. The incorporation of various components into PDMS sponges may lead to slight variations in their absorption capacities. Previous research demonstrated that a PDMS/carbonized bacterial cellulose sponge exhibited an IPA absorption capacity of 392%, which closely aligns with the absorption capacity of the 82% porous sponge with 70% IPA.
13 While the absorption capacity of PDMS sponges with IPA as the swelling solvent, especially with 70% IPA, has not been extensively studied, absorption capacities in the range of hydrophilic PDMS sponges using other alcohols, such as methanol and ethanol, have been examined with capacities reaching around 300%.
7, 14 [0178] Scanning Electron Microscopy and Pore Size [0179] Scanning electron microscopy was used to examine the cross-sectional morphology of the sponges, providing further insight on their porosity and macroscopic network structure, both of which are critical factors influencing their swelling behavior and gas exchange capabilities with the surrounding environment. The SEM imaging analysis demonstrated the highly porous nature of the sponges (Figure 5), with varying concentrations of salt used to alter their porosity. The average pore size in each sponge closely aligns with previously reported studies that fabricated PDMS sponges using NaCl as the porogen.
7, 15 [0180] To investigate the pore characteristics, pore size distributions were determined by analyzing 30 different sites from each sponge formulation. The addition of surfactant or SNAP to the PDMS sponge had no significant impact on the average pore size (Table 1). As all sponge types underwent an identical fabrication process, no significant changes in the macroscopic network structure were observed across the formulations. This observation implies that the change in porosity can be attributed to an increase in the quantity of pores rather than alterations in pore sizes. Table 1: Representative pore size distribution of PDMS, SNAP-impregnated, and hydrophilic (PEO) sponges. Data represents the mean ± STD (n = 1). Sponge Porosity Control Pore Size PEO Pore Size SNAP Pore Size (%) (µm) (µm) (µm) 60 248.2 ± 108.9 216.4 ± 80.94 219.4 ± 75.35
T|H Docket: 222105-2270
[0181] Furthermore, the average pore size of each sponge type closely aligns with previously reported studies that fabricated PDMS sponges using NaCl as the porogen.
7, 15 Given the marginal rise in porosity when shifting from a NaCl to PDMS ratio of 14:1 to 16:1, further investigation of the 89% porous sponge was deemed unnecessary. [0182] Loading and NO Release Characteristics of Sponges: [0183] PDMS Sponges Impregnated with SNAP using THF [0184] The SNAP loading ability of PDMS sponges was evaluated using an impregnation method, which ensured direct incorporation of SNAP into the PDMS polymer matrix, making it readily accessible.
16 The SNAP concentration of 25 mg mL
-1 was selected based on prior evidence in silicone tubing, demonstrating its ability to yield physiologically relevant NO release in both in vitro and in vivo settings, with the added benefit of promoting fibroblast cell proliferation.
5, 17 It was observed that as the porosity of the sponge increased, the capacity to load SNAP also rose correspondingly (Figure 6A). [0185] The diffusion of SNAP from PDMS sponges was monitored over a 24 h period using PBS with EDTA as the extracting medium. This investigation aimed to assess how porosity influences the rate of SNAP diffusion from the sponge and to explore the sponge’s ability to serve as a drug carrier while studying its diffusion kinetics. Conducting SNAP diffusion in a solution allows for pinpointing a critical time point for examining the sponge’s antibacterial capabilities. The 77-88% porous sponges exhibited less than 10% of available SNAP remaining after 24 h, effectively diffusing high quantities of SNAP from their matrices (Figure 6B). Notably, the 83 and 88% porous sponges exhibited exceptional diffusibility within 4 h. In a mere additional 4 h, the 77% porous sponge diffused a comparable amount of SNAP, with 13% of its total SNAP content available after 8 h. In contrast, the 60, 66, and 70% porous sponges displayed slower diffusion kinetics, with only 40, 55, and 72% of their initially loaded SNAP being extracted within the 24 h timeframe. [0186] Nitric oxide release from PDMS sponges was assessed under moist conditions to simulate a natural environment, which is relevant to potential applications such as wound healing. In such scenarios, a moist environment is commonly present, and understanding the release kinetics under these conditions is essential for evaluating the practical utility of the sponges. While exposed to moist conditions, the SNAP sponges demonstrated sustained NO release over 14 d (Figure 6C) using an NOA. Among the sponge types, the 83 and 88%
T|H Docket: 222105-2270 porous sponges consistently exhibited the highest rates of NO release throughout the study duration, maintaining physiological release levels up until the third day.
18 The trends observed in NO release within the different sponge types were strongly correlated with the SNAP concentration present in the sponges, highlighting the role of porosity and SNAP loading in governing the release kinetics. [0187] The porous nature of the PDMS sponge material significantly enhanced SNAP impregnation efficiency compared to previous studies using various silicone-based materials. These prior works achieved a mere ~5 wt% loading with a 125 mg mL
-1 SNAP solution in THF.
16, 19 However, the 88% porous sponge exhibited a significant increase in SNAP impregnation (~23 wt%), surpassing previous reports of SNAP loading in various polymers.
20 Increasing the quantity of NaCl in the PDMS prepolymer leads to a higher concentration of closely packed NaCl particles within the cured structure. This results in the formation of a greater number of interconnected tunnels when the NaCl dissolves. These additional tunnels facilitate a more uniform dispersion of the SNAP solution throughout the PDMS matrix, enabling SNAP to be embedded in a larger portion of the PDMS matrix as well as within the sponge’s pores. [0188] This enhancement in SNAP loading underscores the clear advantage of the porous sponge structure, enabling superior drug incorporation capabilities and presenting promising opportunities for drug delivery applications. The observed increase in loading capacity may lead to more efficient drug delivery systems, allowing for higher concentrations and extended- release profiles. Moreover, the tunability of the sponge’s porosity opens up possibilities for tailoring drug delivery properties, optimizing drug release kinetics, and further enhancing therapeutic outcomes. [0189] The discrepancy in SNAP diffusion of PDMS sponges can be attributed to the variations in porosity among the sponges, with the higher porosity of the 83 and 88% porous sponges facilitating faster liquid penetration and more efficient SNAP extraction from the polymer matrix. Conversely, the less porous sponges were able to retain higher quantities of SNAP for longer periods. The observed change in color during the study provided a visual indicator of SNAP extraction. Sponges that diffused all of their SNAP content reverted to their original white color, with no visible green pigmentation remaining. These results provide initial insight into the diffusion activity of PDMS sponges with varying porosity. However, similar behavior has been reported with variations in drug releasing behavior in porous materials.
21 These findings open up a broad spectrum of potential applications in humid conditions, ranging from the need for sustained, low-quantity NO release over a prolonged period, to instances requiring rapid, high-volume NO bursts. Extended NO release can prove to be beneficial for certain biomedical devices that are intended for long-term clinical use, such as tunneled dialysis catheters, providing prolonged hemocompatibility and biocompatibility to prevent
T|H Docket: 222105-2270 thrombosis and infection.
22 It is worth noting that SNAP can be covalently immobilized to PDMS, creating a tight bond within the material matrix to prevent diffusion out of the polymer.
23 Additionally, SNAP incorporated materials have been coated with one or multiple thin layers of the polymer to reduce SNAP diffusion.
2 These approaches offer potential strategies to prolong the retention of SNAP within sponge materials, particularly addressing the challenge posed by less porous sponges that contain low amounts of the donor molecule. This provides advantages for applications discussed earlier, specifically those that require a continuous release of NO donors, such as addressing antithrombosis concerns in long-term catheters and similar medical contexts. [0190] The presence of void space within the PDMS sponge directly impacts the ability of SNAP to interact with liquid and facilitate NO release. In practical scenarios like a wound healing study, the 60-70% porous sponges would encounter challenges in effectively absorbing exudate from the surrounding environment. These challenges stem from both PDMS’s hydrophobic nature and their comparatively lower porosity, making it a challenge to achieve liquid absorption without oversaturation. Previously reported NO release from different sponge materials, such as collagen,
24 have shown shorter release durations. The observed NO release over 14 d showcases the potential of PDMS sponges for applications requiring prolonged NO release, such as wound healing and long-term infection prevention in catheters, where continuous and controlled release of NO is beneficial for promoting tissue regeneration, antithrombosis, and antimicrobial activity. Recently, a PDMS sponge-based catheter was developed with the ability to prevent thrombosis, inflammation, and bacterial adherence.
25 These properties can be enhanced with the incorporation of NO donors like SNAP to prolong the antithrombotic and antiinfection properties of the sponge catheter. The superior NO release performance of the 83 and 88% porous sponges highlights the significance of porosity in determining the release kinetics. The porous structure facilitates a more extensive diffusion pathway for NO, enabling higher and sustained release rates. These findings underscore the importance of optimizing the sponge’s porosity to achieve desired release characteristics for specific applications. To increase the NO release levels and potentially prolong release rates, higher concentrations of SNAP can easily be loaded into the sponge.
16 Overall, the study provides valuable insights into the NO release behavior of SNAP- impregnated PDMS sponges. [0191] Nitric oxide release from SNAP sponges was assessed under physiologically relevant conditions for 4 h based on the significant diffusion of SNAP after 4 h from the 83% and 88% porous sponges. Given that many biomedical devices come into contact with bodily fluids, including blood, sponge samples were exposed to 37 °C PBS to evaluate their NO release kinetics under physiological conditions. This assessment is crucial for determining the sponges' potential utility in medical applications. While exposed to these conditions, the
T|H Docket: 222105-2270 SNAP sponges demonstrated a steady increase in NO release over the 4 h. The cumulative NO release from samples was calculated over the entire 4 h period (Figure 6D). Among the sponge types, the 88% porous sponge exhibited the highest rates of NO release throughout the study duration, accumulating a total of 714 nmol of NO, respectively. The trends observed in NO release within the different sponge types were strongly correlated with the SNAP concentration present in the sponges, highlighting the role of porosity and SNAP loading in governing the release kinetics. The continuous release of NO from the sponges correlates with the diffusion of SNAP in solution signifying that NO is being quickly depleted from the sponges. [0192] The presence of void space within the sponge directly impacts the ability of SNAP to interact with liquids and facilitate NO release. In practical scenarios like a wound dressing application, the 60-70% porous sponges would encounter challenges in effectively absorbing exudate from the surrounding environment. These challenges stem from both PDMS’s hydrophobic nature and their comparatively lower porosity, making it a challenge to achieve liquid absorption without oversaturation. In contrast, the 83% and 88% porous sponges may not be ideal for applications that require sustained NO release for extended antimicrobial activity. [0193] By the end of the study duration, the 83% and 88% sponges lost much of their green pigmentation, correlating with the amount of NO released. The porous structure facilitates a more extensive diffusion pathway for NO, enabling higher release rates. These findings highlight the importance of optimizing the sponge’s porosity to achieve desired release characteristics for specific applications. Overall, the study provides valuable insights into the NO release behavior of SNAP-incorporated PDMS sponges. [0194] Previously reported NO release from different sponge materials, such as collagen, have shown short release durations.
24 These collagen sponges, incorporated with S- nitrosoglutathione, released approximately 5.4 nmol of NO in almost 2 h, releasing levels of NO comparable to that of the endothelial to promote wound healing.
26 The SNAP-incorporated sponges released significantly higher amounts of NO, with even the 60% porous sponge releasing approximately 159 nmol of NO. The observed NO release over 4 h showcases the potential of PDMS sponges for applications requiring high quantities of NO release in a short period of time, such as disinfection of medical devices and infection prevention in open wounds. Recently, PDMS sponges have been modified and used in applications such as medical devices, conductive materials, and horticulture.
7, 25, 27 These applications can be further improved by incorporating NO donors like SNAP, which enhance the material's antithrombotic and antibacterial properties. The trend in NO release performance, ranging from 60% to the 88% porous sponge, highlights the significance of porosity in determining the release kinetics.
T|H Docket: 222105-2270 [0195] Hydrophilic PDMS Sponges Swelled with SNAP using 70% IPA [0196] To optimize SNAP loading in the sponges, the solubility of SNAP in 70% IPA was investigated. A concentration of 85 mg mL
-1 of SNAP in 70% IPA was employed for further examination, as higher concentrations led to rapid evaporation and precipitation. Quantification of SNAP in each sponge type was accomplished by weighing the swelled sample and then extracting all of the loaded SNAP into fresh 70% IPA before analysis using UV-vis. Sponges reached a maximum SNAP loading ability (Figure 6E) after 15 min in a SNAP-IPA solution. Similarly, as porosity increased, the SNAP loading capacity of sponges also increased, ranging from ~4.12 to 7.68 wt% for the 60 and 82% porous sponges after 15 min. No significant increase in SNAP loading was observed beyond 15 min for all sponge types. [0197] Polydimethylsiloxane sponges have been used for drug loading and delivery purposes, however, the incorporation of a RSNO in a sponge has limited exploration. In a recent study, a wound healing cold-pressed collagen sponge was loaded with GSNO to release NO as it absorbed exudate from the surrounding wound.
24 However, the specific quantity of GSNO loaded into the sponge was not quantified, making the quantity of SNAP loaded into these PDMS sponges a novel addition to the literature. While direct comparison of SNAP loading ability, or any NO donor loading ability, of PDMS sponges with existing research is challenging, it is worth noting that SNAP has been successfully incorporated into various other polymers and biomedical devices with enhanced stability and long-term NO- release.
28 Previous investigations have shown that generating porous structures in polyurethane polymer films results in an enhanced loading of SNAP, as compared to solid samples devoid of pores.
29 This study yielded similar findings, indicating that the introduction of porosity enhances the PDMS’s capacity to incorporate more SNAP compared to other solid silicone substrates lacking pores. As mentioned previously, a silicone polymer tubing achieved ~5 wt% of SNAP loading with a 125 mg mL
-1 concentration of SNAP in THF.
30 The 77 and 82% porous sponges were capable of loading ~7.77 and 7.68 wt% of SNAP (p > 0.05), exhibiting ~53%% more SNAP loaded into the polymer matrix compared to the silicone tubing. [0198] The SNAP loading ability of hydrophilic PDMS sponges demonstrates that increasing sponge porosity facilitates greater incorporation of SNAP into the polymer material. Notably, there is a saturation point in SNAP loading after 15 min of swelling. For example, the 60% porous sponge exhibited a ~0.61 wt% rise in SNAP loading from 15 min to 60 min (p > 0.05), while the 82% porous sponge actually showed a slight reduction in SNAP loading by ~0.97 wt% (p > 0.05). For this reason, subsequent experiments followed a 15 min swelling period. This trend among sponge types signifies that the maximum incorporation of SNAP into the sponge pores and PDMS matrix happens quickly and becomes saturated within 15 min of exposure.
T|H Docket: 222105-2270 [0199] Sponges swelled in the SNAP-IPA solution for 15 min were immediately placed into luer caps and secured onto luer connectors attached to a catheter model. The quantity of SNAP delivered to the luer connectors was tested under real-world conditions by monitoring the amount of SNAP remaining after 0.5, 1, 4, and 24 h following methods provided above to quantify SNAP loading in hydrophilic sponges. All sponge formulations exhibited a similar trend in SNAP delivery (Figure 6F) over the 24 h period. The results depict a strong correlation between sponge porosity and SNAP delivery. The delivery of SNAP from hydrophilic sponges was evaluated within a simulated catheter setup (Figure 6G). This model was designed to replicate real-world conditions commonly encountered in healthcare settings, particularly when using catheters equipped with injection ports. The focus of this study was to understand the release of SNAP from sponges with varying porosities by evaluating the amount of SNAP remaining in them over a 24 h period. While disinfecting caps have shown effectiveness over multiple days,
31, 32 the ability to disinfect the hub regions of catheter access ports quickly and effectively was specifically investigated, considering that these ports are frequently accessed multiple times a day in healthcare settings. [0200] The 82% porous sponge was able to release 368 nmol mg
-1 of SNAP within 24 h, releasing significantly higher levels compared to the other sponge types (p < 0.05). These values were calculated using the theoretical amount of SNAP loaded into each sponge and the quantity of SNAP remaining in each sponge. The high levels of SNAP delivered to the luer connector was observed by the lack of green pigmentation in the sponges after the designated period of time. The amount of SNAP released was not observed to increase significantly over time in any of the sponge types. This can be attributed to the compression of the sponges when attached to luer connectors causing a fast release of SNAP and IPA. Among the sponge formulations, the 82% porous sponge exhibited the highest SNAP release due to its higher porosity and heightened SNAP concentration. [0201] The release of SNAP from hydrophilic sponges showed a high initial release rate with no significant increase after 30 min. This initial burst is observed due to the sponge being compressed in order to release the antimicrobial agents. The amount of SNAP remaining in the sponge was used to determine the quantity of SNAP released into the luer connector with the 60% porous sponge releasing 229 nmol mg
-1 and the 82% porous sponge releasing 366 nmol mg
-1, respectively, within the first 30 min. Due to the specific application of this technology, literature lacks leaching of NO donors via squeezing of the material. However, literature has shown that porous films loaded with ~19.6 and 14.0 wt% SNAP released ~18 and 35% of SNAP when subjected to a PBS solution at 37 °C.
33 Although PDMS sponges were subjected to a different environment, the 60 and 82% porous sponges released significantly higher quantities of SNAP, releasing ~94.94 and 97.25% of their total SNAP loaded. Quantifying the leaching of SNAP from PBS was not investigated, as the sponges
T|H Docket: 222105-2270 are not expected to come into contact with the body or any solutions, such as a catheter lock solution. While a previously reported NO-releasing sponge demonstrated NO release kinetics using GSNO as the NO donor and an NOA,
24 it is worth noting that an NOA was not used to examine the NO release kinetics of SNAP swelled sponges due to the potential impact of IPA evaporation on the instrument. In addition, the application of this technology lies in the sponge’s ability to release IPA and NO when compressed which could not be conducted within an NOA analysis. [0202] Antibacterial Properties of SNAP-Impregnated PDMS Sponges [0203] The antibacterial efficacy of NO-releasing sponges was assessed in a 4 h bacterial study, using E. coli and S. aureus as representative Gram-negative and Gram-positive bacteria. These bacterial strains were selected for their relevance in biomedical contexts; E. coli is commonly linked to urinary tract and surgical site infections, while S. aureus is a leading cause of wound and implant-related infections.
34 The choice of microbes was based on their existing antibiotic-resistant strains, highlighting the potential for the emergence of further resistant variants.
35 Consequently, there is a pressing need for alternative treatment methods that can effectively eliminate microbes without the risk of resistance development. The application of NO ensures that antibiotics can be reserved for the most severe cases. Therefore, this study aimed to evaluate the sponges’ effectiveness in preventing bacterial colonization on the surface and inhibiting further attachment of planktonic bacteria. This targeted approach provides potential applicability of the NO-releasing sponges in biomedical settings, where the prevention and treatment of infections caused by Gram-negative and Gram-positive bacteria are of paramount importance. [0204] During the bacterial adherence study, bacteria were exposed to control and SNAP- impregnated sponges for 4 h, during which high rates of NO release were observed from the88% porous sponge (Figure 6D). The results revealed an increase in bactericidal activity against both E. coli (Figure 7A) and S. aureus (Figure 7C) with higher sponge porosity. Bacterial viability reduction was assessed by calculating the CFUs mg
-1 and comparing SNAP- impregnated sponges with their corresponding PDMS controls (p < 0.05 for all sponge types). The 60% porous sponge exhibited a 1.48-log reduction in viable E. coli, while the 88% porous sponge demonstrated a 2.45-log reduction. Similarly, the 60% porous sponge displayed a 1.07-log reduction in S. aureus viability, whereas the 88% porous sponge exhibited a 2.64-log reduction in bacterial viability, respectively. [0205] Simultaneously, viable planktonic bacteria were investigated after being exposed to control and SNAP-incorporated sponges for 4 h, during which high SNAP diffusibility was observed from the 83% and 88% porous sponges (Figure 6B). Correspondingly, an increase in bactericidal activity against both E. coli (Figure 7B) and S. aureus (Figure 7D) with higher sponge porosity was observed. Bacterial viability reduction was assessed by calculating the
T|H Docket: 222105-2270 CFUs/mL normalized to the sponge weight (mg-1) and comparing SNAP-incorporated sponges with their corresponding controls without SNAP (p < 0.05 for all sponge types). A 1.78- and 2.74-log reduction in E. coli and S. aureus viability was observed after exposure to the 60% porous sponge, while the 88% porous sponge exhibited 2.38- and 5.02-log reductions in viable E. coli and S. aureus viability, respectively. [0206] The observed bactericidal activity against both E. coli and S. aureus yielded intriguing insights into the role of porosity in bacterial eradication. Despite noticing variations in SNAP loading, leaching, and NO release among the different sponge types, the bactericidal activity did not reveal statistically significant differences across all sponges (Tables 2-5). This suggests that the bactericidal effect of the SNAP-impregnated sponges may be primarily driven by the concentration of NO rather than the porosity of the sponges. These findings align with prior research, indicating that the concentration of NO achieved in porous materials plays a pivotal role in determining their antimicrobial efficacy against various bacterial strains.
33 Table 2 Significance values of adhered E. coli antibacterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7A). **** (p < 0.0001), *** (p < 0.001), ** (p < 0.01), ns (not significant). Adhered E. coli Killing Significance Values
a e gn cance vaues o pan onc . co an acterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7B). **** (p < 0.0001), ns (not significant). Planktonic E. coli Killing Significance Values Sponge
T|H Docket: 222105-2270
Table 4: Significance values of adhered S. aureus antibacterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7C). **** (p < 0.0001), ns (not significant). S. aureus Killing Significance Values
Table 5: Significance values of planktonic S. aureus antibacterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7C). **** (p < 0.0001), *** (p < 0.001), * (p < 0.05), ns (not significant). S. aureus Killing Significance Values Sponge
T|H Docket: 222105-2270 [0207] Notably, a discernible trend persisted as sponge porosity increased. Specifically, the 83% and 88% porous sponges consistently demonstrated significant bactericidal activity against E. coli when compared to the other sponge types with the 88% porous sponge preventing more E. coli adhesion than the 83% porous sponge. Similarly, the 70% and 77% porous sponges exhibited analogous bacterial killing efficiency, showing significance over the 60% and 66% porous sponges. In the case of S. aureus, the 83% and 88% porous sponges exhibited significantly higher bactericidal activity compared to the other sponge types. Following a similar trend to that of E. coli, the 70% and 77% porous sponges demonstrated significant killing when compared to the 60% and 66% porous sponges. Although similar trends are seen within both bacterial strains and experiments, an overall higher killing efficiency is seen against S. aureus than E. coli, with an emphasis on the planktonic bactericidal activity. This divergence in antibacterial performance between bacterial strains can be explained by the distinct cell wall and membrane compositions of Gram-negative E. coli and Gram-positive S. aureus bacteria.
36 These disparities likely result in varying requirements for effective bactericidal activity, with certain sponge porosities proving more effective against one bacterial strain over the other. Importantly, this explanation also sheds light on the overall increase in bactericidal activity exhibited by all sponges against S. aureus when compared to E. coli. [0208] As porous scaffolds have been shown to facilitate cell adhesion and proliferation due to more attachment sites,
37 the porous nature of the PDMS sponge would be expected to act similarly. As the porosity of the sponge increased, more bacterial growth was observed on control samples in both adhered and planktonic studies. Bacterial adhesion to the sponge surface aided in the increased concentration of CFU in adhesion and in the free-floating bacteria. The variations observed between the two bacteria can be correlated to the bacteria having different mechanisms of adhesion and proliferation.
38 Nonetheless, this increase in both E. coli and S. aureus adhesion to control sponges highlights the significant bactericidal effect of incorporating SNAP into the sponge. Although more bacteria adhered to the control sponges, the increasing amount of SNAP loaded into the sponge significantly inhibited adhesion to the sponge as porosity increased. These findings reveal the complex interplay between NO release, bacterial strains, and sponge porosity, emphasizing the importance of understanding the specific mechanisms of action and concentration requirements for effective bactericidal activity. [0209] These findings reveal the complex interplay between NO release, bacterial strains, and sponge porosity, emphasizing the importance of understanding the specific mechanisms of action and concentration requirements for effective bactericidal activity. The significant bactericidal activity exhibited by the NO-releasing sponges highlights their potential for applications in various biomedical settings, where preventing and treating infections caused
T|H Docket: 222105-2270 by E. coli and S. aureus are critical. The 83% and 88% porous sponges displayed superior antibacterial adhesion activity compared to other NO-releasing materials. In comparison, NO- releasing PDMS films, using a similar SNAP swelling process (25 mg mL
-1), exhibited log reductions of 0.77 and 1.42 against E. coli and S. aureus, respectively, after 24 h of exposure, respectively.
39 Moreover, silicone catheters swelled with SNAP (50 mg mL
-1) exhibited 1.96- and 1.70-log reductions in viable adhered E. coli and S. aureus, respectively, and 0.74- and 1.11-log reductions against planktonic E. coli and S. aureus following a 24 h exposure.
40 It is worth noting that the 70-88% porous SNAP-impregnated sponges achieved log reductions equivalent to or exceeding those of previously reported SNAP-incorporated materials against both E. coli and S. aureus These studies provide valuable insights into the potential of NO- releasing PDMS sponges as highly effective antibacterial materials against common bacterial pathogens. These findings highlight the significance of fine-tuning NO concentration and sponge porosity to obtain the desired bactericidal efficacy tailored for specific applications. Further research endeavors can center on enhancing the mechanisms of NO action, tailoring them to specific uses such as wound healing, infection prevention, and medical device disinfection. By understanding and refining the intricate interplay between NO release, bacterial strains, and sponge porosity, these sponges can be further developed for targeted and highly effective antimicrobial applications in diverse biomedical contexts. [0210] The antibacterial study provides valuable insights into the potential of NO-releasing PDMS sponges as highly effective antimicrobial materials against common bacterial pathogens. These findings highlight the significance of fine-tuning NO concentration and sponge porosity to obtain the desired bactericidal efficacy tailored for specific applications. Further research endeavors can center on enhancing the mechanisms of NO action, tailoring them to specific uses such as wound healing, infection prevention, and medical device disinfection. By understanding and refining the intricate interplay between NO release, bacterial strains, and sponge porosity, these sponges can be further developed for targeted and highly effective antimicrobial applications in diverse biomedical contexts. [0211] Antimicrobial Properties of Hydrophilic PDMS Sponges in a Zone of Inhibition Assay [0212] Antimicrobial agents like 70% IPA are renowned for their efficacy in combating microbial infections.
31 Given the rapid formation and colonization of biofilms, particularly on surfaces like catheters,
41 the introduction of NO as an additional diffusive agent can complement the action of 70% IPA, potentially strengthening efforts to prevent and eradicate biofilms. In this initial investigation, a standard zone of inhibition (ZOI) test was conducted to examine the antimicrobial effects of SNAP-IPA and 70% IPA alone compared to pristine samples with no antimicrobial activity. Hydrophilic sponges were swelled with either 70% IPA or SNAP-IPA for 15 min before being subjected to microbial agar plates and compared to
T|H Docket: 222105-2270 control sponges without either antimicrobial agent incorporated. It was expected that due to the low porosity (Figure 4B), and corresponding SNAP loading (Figure 6D) and release (Figure 6E), the 60% porous sponge would show limited antimicrobial activity and was excluded from the antimicrobial studies. The quantitative results revealed consistent trends across sponge types, microbes, and antimicrobial agents (Table 6). [0213] Table 6. Zone of inhibition diameter measurements for each sponge formulation on various microbes. Z f I hibiti P % ± ± ± ± ges
exhibited a significantly heightened microbiocidal activity against all the tested microbes compared to the use of 70% IPA alone. The antimicrobial efficacy displayed noteworthy variations between the two experimental groups, with 100% of sponges showcasing substantial disparities against a broad spectrum of microbes, including Gram-negative E. coli and P. aeruginosa, as well as Gram-positive S. aureus, S. epidermidis, and C. albicans (Figure 8A-D). This variability in antimicrobial performance can be attributed to their distinct membrane properties.
36 The SNAP-IPA sponges exhibited a progressively improved microbial inhibition effect as the sponge’s porosity increased. However, this advancement in
T|H Docket: 222105-2270 antimicrobial impact was not observed for the IPA sponges, implying that the microbiocidal capacity of 70% IPA might be restricted (even though the absorption capacity increased, Figure 4D). [0215] Bacterial growth inhibition using the 82% porous sponge resulted in inhibition diameters of 2.12 cm and 3.61 cm against S. aureus and S. epidermidis, respectively, while for E. coli and P. aeruginosa, diameters measured 1.66 cm and 1.20 cm, respectively (Figure 4F-I). The larger inhibition zones observed against Gram-positive bacteria (S. aureus and S. epidermidis) compared to Gram-negative bacteria (E. coli and P. aeruginosa) were anticipated, given that the outer membrane of Gram-negative bacteria makes it more challenging for NO to permeate the cell.
42 The antibacterial attributes of NO stem from its byproducts including but not limited to nitrogen dioxides and peroxynitrites, which induce nitrosative and oxidative stress on various microbial components.
43 Nonetheless, these potent mechanisms of microbial eradication are not always sufficient against some microbes, such as Candida albicans, which possess an inducible NO defense mechanism.
44 [0216] Prior research has indicated that NO alone may lack antifungal properties,
42 potentially due to variations in the concentration of the NO donor employed. While the observed antifungal activity might stem from a synergistic interplay between NO and 70% IPA, it may be that the enhanced antifungal effect is primarily attributed to the elevated concentration of SNAP within the sponges. Several factors support this perspective. Despite 70% IPA displaying antifungal activity against Candida species,
45 the lack of significant differences among the various sponge types suggests that an increase in 70% IPA loading is not the primary driver behind this fungal inhibition (Figure 4E). Notably, the significant disparities observed within the SNAP-IPA sponges, specifically the 82% porous sponge’s greater fungal inhibition compared to the other sponge types (Figure 4J), strongly suggests that the concentration of NO within the sponges governs their antifungal activity. Moreover, the 68% porous sponge, containing the lowest SNAP content, fails to demonstrate enhanced antifungal activity compared to 70% IPA alone providing further evidence of the importance of NO concentration in dictating the sponge’s antifungal potential. [0217] This consistent pattern of antimicrobial activity observed with 70% IPA swollen sponges against C. albicans holds true across all the tested microbes. Remarkably, there is no discernible enhancement in microbial inhibition against any of the tested microbes as the sponge’s porosity and 70% IPA absorption capacity increase. The application of 70% IPA has been examined in a similar ZOI study, showing that the inhibited zone diameters closely resemble those observed in sponges saturated in 70% IPA, specifically concerning Gram- positive bacteria and fungi.
46 These findings provide crucial insight into the antimicrobial efficacy of NO and its donor molecule, SNAP, particularly considering that the antifungal potential of NO has shown limited ability to significantly hinder fungal growth. Further
T|H Docket: 222105-2270 investigations delving into the precise interaction and collaborative mechanism of SNAP and 70% IPA may unveil intriguing insights and potentially assume a pivotal role in modulating the antifungal properties of SNAP, thereby enhancing its viability for diverse biomedical applications. [0218] Contact-killing Antimicrobial Efficacy of Hydrophilic PDMS Sponges: [0219] Planktonic microorganisms that come into contact with medical devices have the potential to rapidly establish biofilms.
47 Recognizing the critical importance of the initial hours in the biofilm formation process, a 4 h exposure study was conducted against E. coli, S. aureus, and C. albicans, as representative Gram-negative bacteria, Gram-positive bacteria, and fungi. These microorganisms are linked to a wide range of medical device related infections with E. coli associated with urinary tract infections, S. aureus with implant-related infections, and C. albicans with left ventricular assist device (LVAD) and catheter-related infections.
34, 48 The 82% porous sponge was selected to be examined in this study due to its significant antimicrobial activity observed in the ZOI study. After subjecting sponge samples (control, 70% IPA swollen, SNAP-IPA swollen) to microbial suspension for 4 h, the reduction in viable CFUs was determined and normalized to an average swollen sponge volume (Figure 9A-C). In both E. coli and C. albicans, 70% IPA had no significant effect in killing the microbes in solution and only exhibited a slight reduction in viable S. aureus. The improved microbicidal activity of the NO-releasing sponge provided increased killing against all microbes compared to control and 70% IPA swelled sponges exhibiting a 0.53, 3.00, and 0.67 log reduction in viable E. coli, S. aureus, and C. albicans, respectively (Figure 9D-F). The increased killing ability observed with S. aureus compared to the other microbes, as discussed previously, can be attributed to the difference in membranous properties and specific defense mechanisms each microbe withholds. [0220] Biofilm formation involves several critical stages, including microbial dispersion in its planktonic state, adhesion to surfaces, and proliferation, resulting in the development of a protective biofilm encased in an extracellular polymeric matrix that shields the cells from antibiotic and disinfectant treatments.
47 Since microbial adhesion and biofilm formation mark the final stages leading to infections associated with medical devices, existing literature primarily focuses on the anti-adherence and biofilm eradication capabilities of NO in combination with other antimicrobial agents.
49 Surprisingly, the antimicrobial efficacy of NO against planktonic microorganisms has not received extensive attention in research. [0221] The results of this study align with previously reported literature in which NO’s bactericidal activity against planktonic forms of E. coli and S. aureus was examined using a 125 mg mL
-1 concentration of SNAP. Following a 24 h exposure to bacteria, the SNAP films demonstrated ~1.20 and 2.26 log reductions in viable E. coli and S. aureus.
50 A more pronounced bactericidal effect on S. aureus compared to E. coli was seen in both studies,
T|H Docket: 222105-2270 further highlighting Gram-positive bacteria’s heightened susceptibility to NO compared to Gram-negative bacteria. Previous literature highlights the synergistic antimicrobial effects achieved by NO releasing technology with other antimicrobial agents.
42 However, the combination of SNAP with the antifungal agent amphotericin B does not significantly enhance antifungal activity compared to amphotericin B alone.
39 In contrast, the combination of SNAP with 70% IPA demonstrated a substantial improvement in antifungal activity compared to 70% IPA alone (p < 0.01). [0222] The inclusion of 70% IPA may impact the bactericidal activity of NO in solution. However, as 70% IPA is commonly employed as a disinfectant for removing adhered microbes, relevant data on the antimicrobial agent's ability to kill microbes in solution is scarce. The lack of significant microbicidal activity associated with 70% IPA might be attributed to its further dilution in solution, causing it to lose its microbicidal properties. Nonetheless, observations suggest that 70% IPA does not significantly hinder microbial growth or promote the killing of microbes in solution, leaving NO as the primary driver of substantial antimicrobial activity in the sponge. [0223] In Situ Microbial Disinfection Study of Hydrophilic PDMS Sponges: [0224] Microbes can proliferate and disseminate within the inner lumen of catheters, stemming from potential sources of contamination like the skin surrounding catheter insertion sites and needleless connectors. These connectors are universally used for patients in need of vascular access devices; however, they can be easily contaminated with the constant handling of them and without consistent disinfection.
51 The passive disinfection method of a protective cap has demonstrated a substantial reduction in the risk of CRBSIs resulting from needleless connector contamination when compared to active disinfection techniques such as wiping.
52 E. coli, S. aureus, and C. albicans, were adhered to needleless connectors as they are commonly observed to generate biofilm and cause infections in medical devices.
53 As the combination of SNAP and 70% IPA was shown to significantly inhibit and kill a wide range of microbes compared to 70% IPA alone, the SNAP-IPA swollen sponge was expected to exquisitely disinfect pre-contaminated needleless connectors. After allowing a thorough amount of time for each microbe to adhere to the connectors, 82% porous sponges swollen with SNAP-IPA were added to luer caps commonly used to protect these connectors. When the cap was secured onto the contaminated luer connectors, the sponge was compressed, leaching out the antimicrobial contents within. The dimensions of the sponges used in this study are consistent with those used in the SNAP delivery study, ensuring uniform NO release for this particular application. As there was no notable increase in SNAP delivery observed beyond the initial 30 min for the 82% porous sponge (Figure 6E) the disinfection study was conducted for 30 min, during which viable microbial CFUs mL
-1 were assessed (Figure 10A- C).
T|H Docket: 222105-2270 [0225] As expected, remarkable decontamination ability of the 82% porous sponge was observed, resulting in a 4.11, 2.72, and 5.60 log reduction in viable E. coli, S. aureus, and C. albicans (Figure 10D-F). The antimicrobial study directly investigated the contact killing effectiveness of SNAP-IPA in a neutral environment. In contrast, the planktonic study assessed the sponge’s performance in a microbial solution. By eliminating any potential interference from PBS and the antimicrobial agents, the disinfecting sponge demonstrated its capability to effectively eliminate adhered microbes from luer connectors by releasing NO and 70% IPA upon compression. These results indicate that the release of NO from the sponge occurs more quickly when compressed. [0226] The exceptional antifungal efficacy of the SNAP-IPA combination suggests a complementary effect. The use of 70% IPA as a disinfectant stem from its ability to precipitate cell wall surface proteins in microorganisms. The process of diluting IPA serves two crucial purposes: first, it allows for a slower, controlled permeation, and second, it hinders the rapid evaporation of alcohol, therefore facilitating its penetration into the cell.
54 This dilution process also creates a concentration differential that enhances the alcohol’s potency in penetrating the cell. Inside the cell, it effectively denatures enzymatic and structural proteins, ultimately leading to the demise of the microorganism.
46 As the alcohol disrupts the cell wall of microbes, it may facilitate the entry of NO into the cell, allowing it to exert its antimicrobial effects more effectively against all the tested microbes, with a particular impact on C. albicans. [0227] The use of alcohols as disinfectants has proven to be ineffective against spore- forming bacteria, notably Bacillus and Clostridium.
55 Further exploration of this promising combinational disinfectant may yield effectiveness in inhibiting and eradicating these resilient bacterial strains that also cause HAIs.
56 An investigation into this combinational disinfectant can be pursued to elucidate the precise interaction between NO and 70% IPA and its impact on various bacterial and fungal strains, particularly spore-forming bacteria. Moreover, potential enhancements in NO-donor solubility and the antimicrobial effectiveness of the disinfectant can be explored through the consideration of other alcohols, varying concentrations, and different NO donors like GSNO. Overall, the disinfection properties exhibited by NO and 70% IPA are remarkable, and their continued exploration holds the potential to further mitigate the risk of CRBSIs and other HAIs beyond current disinfection practices. [0228] Broad Applicability [0229] The broad antimicrobial effectiveness exhibited by PDMS sponges swollen with SNAP-IPA solution proved to be a novel combination to combat bacterial and fungal proliferation. To further evaluate the potential of SNAP-IPA, analogous characterization methods to those used for the hydrophilic PDMS sponges were employed to assess two hydrophilic medical-grade foam dressings. These dressings, namely optifoam basic
T|H Docket: 222105-2270 hydrophilic polyurethane foam dressings (Medline, Northfield, IL) and hydrasorb, medical- grade hydrophilic absorbent foam sponge (Qosina, Ronkonkoma, NY), were immersed in the same SNAP-IPA solution (85 mg mL
-1) and analyzed using techniques described earlier. The observed trend between porosity (Figure 11A) and SNAP loading (Figure 12B) of the foams resembles that of the PDMS sponges. As the porosity of the polyurethane foams increases from 85.0% for hydrasorb to 90.0% for optifoam, the quantity of SNAP loaded into the foams increases correspondingly reaching weight percentages of 8.00 wt% for hydrasorb and 8.55 wt% for optifoam, respectively. This marks an advancement from the maximum SNAP loading observed in hydrophilic SNAP sponges (7.68 wt%). [0230] Investigations into the absorption capacity of the two foams at 15, 30, and 60 min unveiled an absorption plateau after 15 min, mirroring the trend observed in PDMS sponges (Figure 11C). Notably, after 15 min, both foams exhibited significantly greater absorption capacity than any PDMS sponge, with optifoam (1975.03%) outperforming hydrasorb (1753.34%) as well. Upon further examination, the introduction of SNAP to the swelling solution showed an enhanced absorption capacity for both foams (Figure 11D), reaching 2566.49% for optifoam and 2069.43% for hydrasorb, respectively. However, this trend was not observed with the PDMS sponges (Figure 11E), in which the absorption capacity remained consistent with both swelling solutions. [0231] Given Optifoam’s enhanced SNAP loading potential, its antimicrobial properties were investigated. A zone of inhibition study was conducted for optifoam (Figure 12A), encompassing the same microbial strains as assessed with PDMS sponges with the addition of a drug-resistant Candida glabrata (C. glabrata) strain (AR Bank # 0325 CDC & FDA Antibiotic Resistance Isolate Bank, Atlanta, GA). As anticipated, optifoam demonstrated comparable, if not superior, performance against the tested microbes relative to the PDMS sponges. Remarkably, Optifoam swollen with SNAP-IPA exhibited substantial inhibition of C. glabrata growth compared to 70% IPA, a result that was notably absent with the PDMS sponges (Figure 12B). [0232] The substantial enhancement in the 70% IPA swelling capacity of the two foams, in comparison to the PDMS sponges, can be attributed to an increase in porosity, which surpassed that of the PDMS sponges, but also due to the difference in distinct material properties. Both Optifoam and Hydrasorb are fashioned from polyurethane, contrasting with the PDMS composition of the fabricated sponges. The difference in material characteristics and chemical composition provides the medical-grade foams with a heightened 70% IPA absorption ability, an achievement beyond the reach of PDMS sponges with heightened porosity. Isocyanates, fundamental constituents in polyurethane synthesis,
57 display an elevated reactivity toward nucleophiles.
58 The nucleophilic nature of the nitroso (NO) group present in SNAP potentially spurs chemical reactions or bonding with the isocyanate groups
T|H Docket: 222105-2270 inherent to polyurethanes. This interaction has the potential to induce changes in the polyurethane’s properties, including its swelling behavior. Consequently, these interactions likely play a role in the observed increase in absorption capacity upon introducing SNAP to the 70% IPA solution. [0233] The PDMS sponges, in contrast, did not showcase analogous behavior, thus reinforcing the notion that the absorption elevation within the foams is primarily a consequence of their divergent material composition. Although absorption capacities were deduced based on the sample weights, it is important to note that the incorporation of SNAP does not fully account for the observed rise in sponge weight after swelling for 15 min. Calculations revealed that optifoam and hydrasorb experienced a 70% IPA absorption increase of 342% and 115% when swelled in the SNAP-IPA solution, respectively, after subtracting the weight of SNAP loaded into each sponge. This difference between polyurethan sponges may be due to the difference in manufacturing methods used by each company. Nonetheless, these results underscore that the introduction of SNAP to the swelling solvent generates a nuanced scenario. In the case of PDMS sponges, the absorption capacity of 70% IPA remains unaffected in the presence of SNAP. This reduction may be attributed to the specific interaction between SNAP and PDMS, potentially tempering the sponge’s ability to swell, and effectively resulting in SNAP and IPA competing for available space within the sponge’s structure. [0234] The higher SNAP loading observed in optifoam, as compared to hydrasorb, mirrors the patterns previously noted in PDMS sponges. As optifoam exhibited increased SNAP loading due to its enhanced porosity, hydrasorb was not evaluated for its antimicrobial properties. The ZOI assessment encompassed identical parameters, including sample dimensions, swelling durations, and incubation periods. As anticipated, the heightened SNAP loading of optifoam resulted in significant restraint of microbial growth across all examined strains, surpassing the inhibitory potential of 70% IPA alone. Notably, this enhancement also yielded a substantial suppression of a secondary drug-resistant Candida species, an effect unachievable through the maximal SNAP concentrations in the PDMS sponges. These findings promote prior microbial results, indicating that increased SNAP concentration not only amplifies the antibacterial properties but the antifungal properties of 70% IPA as well. However, while these results substantiate the correlation between SNAP concentration and microbial growth inhibition, the precise mechanisms governing the observed relationship warrant further investigation. Nonetheless, based on the results, this consistent trend emerges as a significant advancement in the field of biomedical engineering. It not only sheds light on the practical feasibility of SNAP-IPA infused sponges as efficacious disinfecting medical devices but also highlights their potential to restrict microbial proliferation. [0235] Conclusions
T|H Docket: 222105-2270 [0236] Polydimethylsiloxane sponges with tunable porosity and NO-releasing properties were fabricated using a simple and sustainable template extraction technique with hot water and NaCl as the porogen. Examination of the pore structures through SEM imaging revealed that the addition of SNAP or surfactant to the material did not affect the porosity or macroscopic pore structure. The ability to modify the porosity of the sponges was demonstrated through the compressive modulus of the hydrophilic sponges, as well as the loading and leaching of SNAP using different solvents. A reduction in compressive strength, higher SNAP loading, and faster diffusion of SNAP was observed as the porosity increased. The release behavior of NO under moist conditions further confirmed the relationship between porosity and NO release from DMS sponges whereas hydrophilic sponges swelled with SNAP- IPA released high quantities of SNAP in 30 min. Antibacterial performance of SNAP- impregnated PDMS sponges correlated with SNAP diffusion in solution. The exceptional antimicrobial activity observed by the 77 and 82% porous hydrophilic sponges against all tested microbes demonstrated their potential for antibacterial applications. The ability of these sponges to effectively combat the adherence and colonization of both Gram-positive and Gram-negative bacteria is of significant importance in preventing device-associated infections, which are a persistent challenge in clinical settings. More importantly, the combined antimicrobial effect of 70% IPA and NO proved to be a significant finding, showing an improvement in fungal growth inhibition. [0237] This work highlights the potential of NO-releasing sponges as promising devices for biomedical applications, offering a sustainable and customizable approach to address the critical need for antibacterial materials. The findings of this study provide valuable insights into the relationship between porosity, NO release, and antibacterial activity in PDMS sponges. The ability to tune the porosity of the sponges and achieve desired antimicrobial properties and NO release kinetics paves the way for the development of advanced materials with tailored functionalities, enhancing the efficacy of biomedical devices, wound healing strategies, and infection control measures. This work contributes to the broader field of biomaterials research and holds significant potential for clinical applications, ultimately improving patient outcomes and advancing the field of biomedical engineering. [0238] 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.
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T|H Docket: 222105-2270 (54) Global, M. Why 70% Isopropyl Alcohol Is a Better Disinfectant Than 99% Isopropyl Alcohol When it Comes to COVID-192022. (accessed. (55) Moorer, W. Antiviral activity of alcohol for surface disinfection. International journal of dental hygiene 2003, 1 (3), 138-142. DOI: 10.1034/j.1601-5037.2003.00032.x. (56) Jenkins, D. R. Nosocomial infections and infection control. Medicine 2017, 45 (10), 629- 633. (57) Helena Janik, M. S., Justyna Kucinska-Lipka.9 - Polyurethanes; William Andrew Publishing, 2014. DOI: https://doi.org/10.1016/B978-1-4557-3107-7.00009-9. (58) de Souza, F. M.; Kahol, P. K.; Gupta, R. K. Introduction to polyurethane chemistry. In Polyurethane Chemistry: Renewable Polyols and Isocyanates, ACS Publications, 2021; pp 1-24.
T|H Docket: 222105-2270 Where p is the density of the sponge and ps is the density of the swollen sponge after immersing in distilled water for 24 h. Exact methods were repeated for sponges containing surfactant. [0133] Compressive Strength of Hydrophilic Sponges: [0134] Uniaxial compression testing was performed using a Mark-10 Series 5 force gauge (Mark-10, Copiaque, NY). Hydrophilic sponges (60, 68, 72, 77, 82% porous) were compressed to 50% strain at a rate of 1.3 mm min-1. The mechanical testing was completed with cylindrical samples (approximately 8 mm in diameter and 8 mm in height). The diameter and thickness of each sample were measured at multiple points along its length with an accuracy of 0.025 mm. The minimum value of the cross-sectional area, along with the length of each sample, was recorded for further analysis. A total of 5 samples were compressed for each sponge formulation. [0135] Absorption capacity of Hydrophilic Sponges: [0136] Sponge samples (4.5 mm diameter and 5 mm thick) were immersed in a 1.5 mL microcentrifuge tube containing 1 mL of 70% IPA for either 15, 30, or 60 min. The maximum absorption capacity of the hydrophilic sponges (60, 68, 72, 77, 82% porous) was evaluated by measuring the change in mass of the samples using Equation 2. ^^^^^^^^^^^^^^^^ ^^^^^^^^^^ ^%^ ൌ ^ ^ ି^ బ ^బ ∗ 100% (2)
T|H Docket: 222105-2270 Where mf is the swollen weight of the sponge, m0 is the initial weight of the sponge, p is the density of 70% IPA in mg mL-1 at room temperature, and C is the concentration of SNAP in IPA in mg mL-1. Using the concentration of SNAP determined from the absorbance measurements and the values calculated from Equation 3, the amount of SNAP remaining in the samples (n=3) was determined. Subsequently, the total amount of SNAP delivered was calculated expressed as nmol mg-1. [0152] Growth of Microbial Cultures: [0153] A single isolated colony of S. aureus, S. epidermidis, E. coli, and P. aeruginosa was inoculated in LB media and incubated at 37 °C for 15 h at 150 rpm. Microbial cultures were resuspended in 10 mL of fresh media and placed back into the shaking incubator. The growth of microbes was assessed overtime by measuring the optical density (OD) of the suspensions at 600 nm using a UV-vis spectrophotometer. A mid-log phase of each bacteria was extracted from the suspensions by centrifuging the cultures at 4400 rpm for 7 min. Suspensions were adjusted to an OD of 0.1 using PBS, corresponding to ~107 colony-forming units mL-1 (CFU), for further analysis. Similarly, C. albicans and C. glabrata were grown under the same conditions using YM media, with an initial incubation period of 20 h. [0154] Bacterial Adhesion and Planktonic Assay of PDMS Sponges [0155] A 4 h adhered bacterial viability experiment was conducted to assess the antibacterial activity of the PDMS sponges impregnated with SNAP. Control (no SNAP) and SNAP sponges (60, 66, 70, 77, 83, 88% porous) were submerged in a 1.5 mL microcentrifuge tube containing a 1 mL sample of adjusted bacterial suspension (0.1 OD). After incubating for 4 h at 37 °C, sponges were placed into a 15 mL conical vial containing 1 mL of sterile 10 mM PBS. Samples were homogenized and vortexed for 1 min each followed by serial dilutions. Diluted samples were spread on LB agar plates using spiral plater (Eddy Jet 2, IUL Instruments) and incubated for 24 h. The grown colonies were enumerated using an automated colony counter (Sphere Flash, IUL Instruments) to calculate the number of viable CFUs per mg of sponge. Percent reduction in viable bacteria compared to respective controls was calculated using Equation 4. Additionally, Equation 5 was employed to determine the log reduction of viable bacteria exhibited by each sponge type compared to their respective controls without SNAP. ^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ^%^ ൌ ^ ುವಾೄ ି ^ ೄಿಲು ^ುವಾೄ ൈ 100% (4) ^^^^^^ ^^^^^^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ൌ ^^^^^^ ^ ುವಾೄ ^^ ^ ^ ^ (5)
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[0181] Furthermore, the average pore size of each sponge type closely aligns with previously reported studies that fabricated PDMS sponges using NaCl as the porogen.7, 15 Given the marginal rise in porosity when shifting from a NaCl to PDMS ratio of 14:1 to 16:1, further investigation of the 89% porous sponge was deemed unnecessary. [0182] Loading and NO Release Characteristics of Sponges: [0183] PDMS Sponges Impregnated with SNAP using THF [0184] The SNAP loading ability of PDMS sponges was evaluated using an impregnation method, which ensured direct incorporation of SNAP into the PDMS polymer matrix, making it readily accessible.16 The SNAP concentration of 25 mg mL-1 was selected based on prior evidence in silicone tubing, demonstrating its ability to yield physiologically relevant NO release in both in vitro and in vivo settings, with the added benefit of promoting fibroblast cell proliferation.5, 17 It was observed that as the porosity of the sponge increased, the capacity to load SNAP also rose correspondingly (Figure 6A). [0185] The diffusion of SNAP from PDMS sponges was monitored over a 24 h period using PBS with EDTA as the extracting medium. This investigation aimed to assess how porosity influences the rate of SNAP diffusion from the sponge and to explore the sponge’s ability to serve as a drug carrier while studying its diffusion kinetics. Conducting SNAP diffusion in a solution allows for pinpointing a critical time point for examining the sponge’s antibacterial capabilities. The 77-88% porous sponges exhibited less than 10% of available SNAP remaining after 24 h, effectively diffusing high quantities of SNAP from their matrices (Figure 6B). Notably, the 83 and 88% porous sponges exhibited exceptional diffusibility within 4 h. In a mere additional 4 h, the 77% porous sponge diffused a comparable amount of SNAP, with 13% of its total SNAP content available after 8 h. In contrast, the 60, 66, and 70% porous sponges displayed slower diffusion kinetics, with only 40, 55, and 72% of their initially loaded SNAP being extracted within the 24 h timeframe. [0186] Nitric oxide release from PDMS sponges was assessed under moist conditions to simulate a natural environment, which is relevant to potential applications such as wound healing. In such scenarios, a moist environment is commonly present, and understanding the release kinetics under these conditions is essential for evaluating the practical utility of the sponges. While exposed to moist conditions, the SNAP sponges demonstrated sustained NO release over 14 d (Figure 6C) using an NOA. Among the sponge types, the 83 and 88%
T|H Docket: 222105-2270 porous sponges consistently exhibited the highest rates of NO release throughout the study duration, maintaining physiological release levels up until the third day.18 The trends observed in NO release within the different sponge types were strongly correlated with the SNAP concentration present in the sponges, highlighting the role of porosity and SNAP loading in governing the release kinetics. [0187] The porous nature of the PDMS sponge material significantly enhanced SNAP impregnation efficiency compared to previous studies using various silicone-based materials. These prior works achieved a mere ~5 wt% loading with a 125 mg mL-1 SNAP solution in THF.16, 19 However, the 88% porous sponge exhibited a significant increase in SNAP impregnation (~23 wt%), surpassing previous reports of SNAP loading in various polymers.20 Increasing the quantity of NaCl in the PDMS prepolymer leads to a higher concentration of closely packed NaCl particles within the cured structure. This results in the formation of a greater number of interconnected tunnels when the NaCl dissolves. These additional tunnels facilitate a more uniform dispersion of the SNAP solution throughout the PDMS matrix, enabling SNAP to be embedded in a larger portion of the PDMS matrix as well as within the sponge’s pores. [0188] This enhancement in SNAP loading underscores the clear advantage of the porous sponge structure, enabling superior drug incorporation capabilities and presenting promising opportunities for drug delivery applications. The observed increase in loading capacity may lead to more efficient drug delivery systems, allowing for higher concentrations and extended- release profiles. Moreover, the tunability of the sponge’s porosity opens up possibilities for tailoring drug delivery properties, optimizing drug release kinetics, and further enhancing therapeutic outcomes. [0189] The discrepancy in SNAP diffusion of PDMS sponges can be attributed to the variations in porosity among the sponges, with the higher porosity of the 83 and 88% porous sponges facilitating faster liquid penetration and more efficient SNAP extraction from the polymer matrix. Conversely, the less porous sponges were able to retain higher quantities of SNAP for longer periods. The observed change in color during the study provided a visual indicator of SNAP extraction. Sponges that diffused all of their SNAP content reverted to their original white color, with no visible green pigmentation remaining. These results provide initial insight into the diffusion activity of PDMS sponges with varying porosity. However, similar behavior has been reported with variations in drug releasing behavior in porous materials.21 These findings open up a broad spectrum of potential applications in humid conditions, ranging from the need for sustained, low-quantity NO release over a prolonged period, to instances requiring rapid, high-volume NO bursts. Extended NO release can prove to be beneficial for certain biomedical devices that are intended for long-term clinical use, such as tunneled dialysis catheters, providing prolonged hemocompatibility and biocompatibility to prevent
T|H Docket: 222105-2270 thrombosis and infection.22 It is worth noting that SNAP can be covalently immobilized to PDMS, creating a tight bond within the material matrix to prevent diffusion out of the polymer.23 Additionally, SNAP incorporated materials have been coated with one or multiple thin layers of the polymer to reduce SNAP diffusion.2 These approaches offer potential strategies to prolong the retention of SNAP within sponge materials, particularly addressing the challenge posed by less porous sponges that contain low amounts of the donor molecule. This provides advantages for applications discussed earlier, specifically those that require a continuous release of NO donors, such as addressing antithrombosis concerns in long-term catheters and similar medical contexts. [0190] The presence of void space within the PDMS sponge directly impacts the ability of SNAP to interact with liquid and facilitate NO release. In practical scenarios like a wound healing study, the 60-70% porous sponges would encounter challenges in effectively absorbing exudate from the surrounding environment. These challenges stem from both PDMS’s hydrophobic nature and their comparatively lower porosity, making it a challenge to achieve liquid absorption without oversaturation. Previously reported NO release from different sponge materials, such as collagen,24 have shown shorter release durations. The observed NO release over 14 d showcases the potential of PDMS sponges for applications requiring prolonged NO release, such as wound healing and long-term infection prevention in catheters, where continuous and controlled release of NO is beneficial for promoting tissue regeneration, antithrombosis, and antimicrobial activity. Recently, a PDMS sponge-based catheter was developed with the ability to prevent thrombosis, inflammation, and bacterial adherence.25 These properties can be enhanced with the incorporation of NO donors like SNAP to prolong the antithrombotic and antiinfection properties of the sponge catheter. The superior NO release performance of the 83 and 88% porous sponges highlights the significance of porosity in determining the release kinetics. The porous structure facilitates a more extensive diffusion pathway for NO, enabling higher and sustained release rates. These findings underscore the importance of optimizing the sponge’s porosity to achieve desired release characteristics for specific applications. To increase the NO release levels and potentially prolong release rates, higher concentrations of SNAP can easily be loaded into the sponge.16 Overall, the study provides valuable insights into the NO release behavior of SNAP- impregnated PDMS sponges. [0191] Nitric oxide release from SNAP sponges was assessed under physiologically relevant conditions for 4 h based on the significant diffusion of SNAP after 4 h from the 83% and 88% porous sponges. Given that many biomedical devices come into contact with bodily fluids, including blood, sponge samples were exposed to 37 °C PBS to evaluate their NO release kinetics under physiological conditions. This assessment is crucial for determining the sponges' potential utility in medical applications. While exposed to these conditions, the
T|H Docket: 222105-2270 SNAP sponges demonstrated a steady increase in NO release over the 4 h. The cumulative NO release from samples was calculated over the entire 4 h period (Figure 6D). Among the sponge types, the 88% porous sponge exhibited the highest rates of NO release throughout the study duration, accumulating a total of 714 nmol of NO, respectively. The trends observed in NO release within the different sponge types were strongly correlated with the SNAP concentration present in the sponges, highlighting the role of porosity and SNAP loading in governing the release kinetics. The continuous release of NO from the sponges correlates with the diffusion of SNAP in solution signifying that NO is being quickly depleted from the sponges. [0192] The presence of void space within the sponge directly impacts the ability of SNAP to interact with liquids and facilitate NO release. In practical scenarios like a wound dressing application, the 60-70% porous sponges would encounter challenges in effectively absorbing exudate from the surrounding environment. These challenges stem from both PDMS’s hydrophobic nature and their comparatively lower porosity, making it a challenge to achieve liquid absorption without oversaturation. In contrast, the 83% and 88% porous sponges may not be ideal for applications that require sustained NO release for extended antimicrobial activity. [0193] By the end of the study duration, the 83% and 88% sponges lost much of their green pigmentation, correlating with the amount of NO released. The porous structure facilitates a more extensive diffusion pathway for NO, enabling higher release rates. These findings highlight the importance of optimizing the sponge’s porosity to achieve desired release characteristics for specific applications. Overall, the study provides valuable insights into the NO release behavior of SNAP-incorporated PDMS sponges. [0194] Previously reported NO release from different sponge materials, such as collagen, have shown short release durations.24 These collagen sponges, incorporated with S- nitrosoglutathione, released approximately 5.4 nmol of NO in almost 2 h, releasing levels of NO comparable to that of the endothelial to promote wound healing.26 The SNAP-incorporated sponges released significantly higher amounts of NO, with even the 60% porous sponge releasing approximately 159 nmol of NO. The observed NO release over 4 h showcases the potential of PDMS sponges for applications requiring high quantities of NO release in a short period of time, such as disinfection of medical devices and infection prevention in open wounds. Recently, PDMS sponges have been modified and used in applications such as medical devices, conductive materials, and horticulture.7, 25, 27 These applications can be further improved by incorporating NO donors like SNAP, which enhance the material's antithrombotic and antibacterial properties. The trend in NO release performance, ranging from 60% to the 88% porous sponge, highlights the significance of porosity in determining the release kinetics.
T|H Docket: 222105-2270 [0195] Hydrophilic PDMS Sponges Swelled with SNAP using 70% IPA [0196] To optimize SNAP loading in the sponges, the solubility of SNAP in 70% IPA was investigated. A concentration of 85 mg mL-1 of SNAP in 70% IPA was employed for further examination, as higher concentrations led to rapid evaporation and precipitation. Quantification of SNAP in each sponge type was accomplished by weighing the swelled sample and then extracting all of the loaded SNAP into fresh 70% IPA before analysis using UV-vis. Sponges reached a maximum SNAP loading ability (Figure 6E) after 15 min in a SNAP-IPA solution. Similarly, as porosity increased, the SNAP loading capacity of sponges also increased, ranging from ~4.12 to 7.68 wt% for the 60 and 82% porous sponges after 15 min. No significant increase in SNAP loading was observed beyond 15 min for all sponge types. [0197] Polydimethylsiloxane sponges have been used for drug loading and delivery purposes, however, the incorporation of a RSNO in a sponge has limited exploration. In a recent study, a wound healing cold-pressed collagen sponge was loaded with GSNO to release NO as it absorbed exudate from the surrounding wound.24 However, the specific quantity of GSNO loaded into the sponge was not quantified, making the quantity of SNAP loaded into these PDMS sponges a novel addition to the literature. While direct comparison of SNAP loading ability, or any NO donor loading ability, of PDMS sponges with existing research is challenging, it is worth noting that SNAP has been successfully incorporated into various other polymers and biomedical devices with enhanced stability and long-term NO- release.28 Previous investigations have shown that generating porous structures in polyurethane polymer films results in an enhanced loading of SNAP, as compared to solid samples devoid of pores.29 This study yielded similar findings, indicating that the introduction of porosity enhances the PDMS’s capacity to incorporate more SNAP compared to other solid silicone substrates lacking pores. As mentioned previously, a silicone polymer tubing achieved ~5 wt% of SNAP loading with a 125 mg mL-1 concentration of SNAP in THF.30 The 77 and 82% porous sponges were capable of loading ~7.77 and 7.68 wt% of SNAP (p > 0.05), exhibiting ~53%% more SNAP loaded into the polymer matrix compared to the silicone tubing. [0198] The SNAP loading ability of hydrophilic PDMS sponges demonstrates that increasing sponge porosity facilitates greater incorporation of SNAP into the polymer material. Notably, there is a saturation point in SNAP loading after 15 min of swelling. For example, the 60% porous sponge exhibited a ~0.61 wt% rise in SNAP loading from 15 min to 60 min (p > 0.05), while the 82% porous sponge actually showed a slight reduction in SNAP loading by ~0.97 wt% (p > 0.05). For this reason, subsequent experiments followed a 15 min swelling period. This trend among sponge types signifies that the maximum incorporation of SNAP into the sponge pores and PDMS matrix happens quickly and becomes saturated within 15 min of exposure.
T|H Docket: 222105-2270 [0199] Sponges swelled in the SNAP-IPA solution for 15 min were immediately placed into luer caps and secured onto luer connectors attached to a catheter model. The quantity of SNAP delivered to the luer connectors was tested under real-world conditions by monitoring the amount of SNAP remaining after 0.5, 1, 4, and 24 h following methods provided above to quantify SNAP loading in hydrophilic sponges. All sponge formulations exhibited a similar trend in SNAP delivery (Figure 6F) over the 24 h period. The results depict a strong correlation between sponge porosity and SNAP delivery. The delivery of SNAP from hydrophilic sponges was evaluated within a simulated catheter setup (Figure 6G). This model was designed to replicate real-world conditions commonly encountered in healthcare settings, particularly when using catheters equipped with injection ports. The focus of this study was to understand the release of SNAP from sponges with varying porosities by evaluating the amount of SNAP remaining in them over a 24 h period. While disinfecting caps have shown effectiveness over multiple days,31, 32 the ability to disinfect the hub regions of catheter access ports quickly and effectively was specifically investigated, considering that these ports are frequently accessed multiple times a day in healthcare settings. [0200] The 82% porous sponge was able to release 368 nmol mg-1 of SNAP within 24 h, releasing significantly higher levels compared to the other sponge types (p < 0.05). These values were calculated using the theoretical amount of SNAP loaded into each sponge and the quantity of SNAP remaining in each sponge. The high levels of SNAP delivered to the luer connector was observed by the lack of green pigmentation in the sponges after the designated period of time. The amount of SNAP released was not observed to increase significantly over time in any of the sponge types. This can be attributed to the compression of the sponges when attached to luer connectors causing a fast release of SNAP and IPA. Among the sponge formulations, the 82% porous sponge exhibited the highest SNAP release due to its higher porosity and heightened SNAP concentration. [0201] The release of SNAP from hydrophilic sponges showed a high initial release rate with no significant increase after 30 min. This initial burst is observed due to the sponge being compressed in order to release the antimicrobial agents. The amount of SNAP remaining in the sponge was used to determine the quantity of SNAP released into the luer connector with the 60% porous sponge releasing 229 nmol mg-1 and the 82% porous sponge releasing 366 nmol mg-1, respectively, within the first 30 min. Due to the specific application of this technology, literature lacks leaching of NO donors via squeezing of the material. However, literature has shown that porous films loaded with ~19.6 and 14.0 wt% SNAP released ~18 and 35% of SNAP when subjected to a PBS solution at 37 °C.33 Although PDMS sponges were subjected to a different environment, the 60 and 82% porous sponges released significantly higher quantities of SNAP, releasing ~94.94 and 97.25% of their total SNAP loaded. Quantifying the leaching of SNAP from PBS was not investigated, as the sponges
T|H Docket: 222105-2270 are not expected to come into contact with the body or any solutions, such as a catheter lock solution. While a previously reported NO-releasing sponge demonstrated NO release kinetics using GSNO as the NO donor and an NOA,24 it is worth noting that an NOA was not used to examine the NO release kinetics of SNAP swelled sponges due to the potential impact of IPA evaporation on the instrument. In addition, the application of this technology lies in the sponge’s ability to release IPA and NO when compressed which could not be conducted within an NOA analysis. [0202] Antibacterial Properties of SNAP-Impregnated PDMS Sponges [0203] The antibacterial efficacy of NO-releasing sponges was assessed in a 4 h bacterial study, using E. coli and S. aureus as representative Gram-negative and Gram-positive bacteria. These bacterial strains were selected for their relevance in biomedical contexts; E. coli is commonly linked to urinary tract and surgical site infections, while S. aureus is a leading cause of wound and implant-related infections.34 The choice of microbes was based on their existing antibiotic-resistant strains, highlighting the potential for the emergence of further resistant variants.35 Consequently, there is a pressing need for alternative treatment methods that can effectively eliminate microbes without the risk of resistance development. The application of NO ensures that antibiotics can be reserved for the most severe cases. Therefore, this study aimed to evaluate the sponges’ effectiveness in preventing bacterial colonization on the surface and inhibiting further attachment of planktonic bacteria. This targeted approach provides potential applicability of the NO-releasing sponges in biomedical settings, where the prevention and treatment of infections caused by Gram-negative and Gram-positive bacteria are of paramount importance. [0204] During the bacterial adherence study, bacteria were exposed to control and SNAP- impregnated sponges for 4 h, during which high rates of NO release were observed from the88% porous sponge (Figure 6D). The results revealed an increase in bactericidal activity against both E. coli (Figure 7A) and S. aureus (Figure 7C) with higher sponge porosity. Bacterial viability reduction was assessed by calculating the CFUs mg-1 and comparing SNAP- impregnated sponges with their corresponding PDMS controls (p < 0.05 for all sponge types). The 60% porous sponge exhibited a 1.48-log reduction in viable E. coli, while the 88% porous sponge demonstrated a 2.45-log reduction. Similarly, the 60% porous sponge displayed a 1.07-log reduction in S. aureus viability, whereas the 88% porous sponge exhibited a 2.64-log reduction in bacterial viability, respectively. [0205] Simultaneously, viable planktonic bacteria were investigated after being exposed to control and SNAP-incorporated sponges for 4 h, during which high SNAP diffusibility was observed from the 83% and 88% porous sponges (Figure 6B). Correspondingly, an increase in bactericidal activity against both E. coli (Figure 7B) and S. aureus (Figure 7D) with higher sponge porosity was observed. Bacterial viability reduction was assessed by calculating the
T|H Docket: 222105-2270 CFUs/mL normalized to the sponge weight (mg-1) and comparing SNAP-incorporated sponges with their corresponding controls without SNAP (p < 0.05 for all sponge types). A 1.78- and 2.74-log reduction in E. coli and S. aureus viability was observed after exposure to the 60% porous sponge, while the 88% porous sponge exhibited 2.38- and 5.02-log reductions in viable E. coli and S. aureus viability, respectively. [0206] The observed bactericidal activity against both E. coli and S. aureus yielded intriguing insights into the role of porosity in bacterial eradication. Despite noticing variations in SNAP loading, leaching, and NO release among the different sponge types, the bactericidal activity did not reveal statistically significant differences across all sponges (Tables 2-5). This suggests that the bactericidal effect of the SNAP-impregnated sponges may be primarily driven by the concentration of NO rather than the porosity of the sponges. These findings align with prior research, indicating that the concentration of NO achieved in porous materials plays a pivotal role in determining their antimicrobial efficacy against various bacterial strains.33 Table 2 Significance values of adhered E. coli antibacterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7A). **** (p < 0.0001), *** (p < 0.001), ** (p < 0.01), ns (not significant). Adhered E. coli Killing Significance Values
a e gn cance vaues o pan onc . co an acterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7B). **** (p < 0.0001), ns (not significant). Planktonic E. coli Killing Significance Values Sponge
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Table 4: Significance values of adhered S. aureus antibacterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7C). **** (p < 0.0001), ns (not significant). S. aureus Killing Significance Values
Table 5: Significance values of planktonic S. aureus antibacterial data. Comparing log reduction values of SNAP-impregnated sponges from their respective controls (Figure 7C). **** (p < 0.0001), *** (p < 0.001), * (p < 0.05), ns (not significant). S. aureus Killing Significance Values Sponge