WO2019236825A1 - Antimicrobial compositions with wound healing properties - Google Patents

Abstract

Antimicrobial compositions and materials are provided. The compositions include biocompatible material having a synthetic polymer combined with a natural polymer. The combined polymers are crosslinked with an antimicrobial effective amount of a NO-donor. Articles and wound dressings having the biocompatible material are described. Wound dressings can be made by combining a synthetic polymer with a natural polymer to form a polymer mixture and crosslinking the polymer mixture with an antimicrobial effective amount of a NO-donor. The crosslinked polymer mixture can be lyophilized to form a porous film.

Description

ANTIMICROBIAL COMPOSITIONS WITH WOUND HEALING PROPERTIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 62/682,663, having the title“ANTIMICROBIAL COMPOSITIONS WITH WOUND HEALING PROPERTIES”, filed on June 8, 2018, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant K25HL111213 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] The cases of chronic wounds in the United States are as prevalent as the cases of heart failure. Chronic wounds affect approximately 6.5 million patients in the United States (US) annually, causing an estimated economic burden of $25 billion every year. Burn injury cases alone are estimated to be around 1 million, resulting in approximately 35,000 reported deaths every year in the US. Skin scarring is an additional issue associated with inefficient wound healing, which has a market of $12 billion annually. Increasing healthcare cost along with the sharp rise in cases of diabetes and obesity and an aging population are

exacerbating the existing socioeconomic burden caused by wounds.

[0004] Wound healing is a complex biological process that aims to re-establish tissue integrity at the site of injury and involves four main phases: homeostasis, inflammation, proliferation, and tissue remodeling (maturation). The presence of infection-causing agents at the wound site negatively impacts the host immune response, ultimately delaying the natural wound healing process. Depending on the nature of the wound, maturation of the wound may take days, weeks or even a year. Inappropriate progression of the healing process in the case of chronic wounds can often make the total healing time unpredictable.

In addition to the delayed wound healing, a wound infection can potentially cause tissue necrosis, septicemia, and transmission of infectious agents to other patients in hospitals. It can also result in life-threatening complications like pressure sores and diabetic foot ulcers, which can lead to amputation of the affected limb. Among the most serious chronic wound complications, burn wound infections specifically are responsible for 70% of the deaths of all the people with severe burns. Skin infections contribute to 200 million visits to physicians costing over $350 million annually.

[0005] The complexity underlying the wound healing mechanism demands the development of advanced wound dressings which can support the endogenous wound healing mechanism, thus accelerating the overall healing process. Wound dressings act as a temporary barrier between the wound and the external environment, protecting it from further physical damage and bacterial infection while restoring the milieu required for skin regeneration. Wound dressings have a global market worth expected to expand to $10.16 billion in 2020, with compound annual growth rate (CAGR) of 4.5% from 2014 to 2020 as per an analysis that was done by Transparency Market Research.

SUMMARY

[0006] Embodiments of the present disclosure provide materials, compositions, and articles for wound healing, methods of making, and the like.

[0007] An embodiment of the present disclosure includes biocompatible materials that can include a synthetic polymer combined with a natural polymer. The combined polymers are crosslinked with an antimicrobial effective amount of a NO-donor.

[0008] An embodiment of the present disclosure also includes articles that include biocompatible materials as above.

[0009] Another embodiment of the present disclosure includes methods of making a wound dressing, which include combining a synthetic polymer with a natural polymer to form a polymer mixture and crosslinking the polymer mixture with an antimicrobial effective amount of a NO-donor. The crosslinked polymer mixture can be lyophilized to form a porous film.

[0010] An embodiment of the present disclosure includes wound dressings that can include a synthetic polymer combined with a natural polymer, wherein the combined polymers are crosslinked with an antimicrobial effective amount of a NO-donor.

[0011] Other compositions, apparatus, methods, features, and advantages 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 compositions, apparatus, methods, features and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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.

[0013] Figures 1A-1 B provide images of example embodiments of (Figure 1A) Control Alginate-PVA dressing and (Figure 1 B) NO releasing Alginate-PVA-GSNO dressing. [0014] Figure 2 shows real time profiling of NO release from an Alginate-PVA-GSNO wound dressing of the present disclosure, as observed by Nitric Oxide Alanyzer (NOA).

[0015] Figure 3 shows the NO release over time from an embodiment of Alginate-PVA- GSNO wound dressings of the present disclosure. Statistical data is expressed as mean ± standard error of the mean of n=3 samples. Values of p < 0.05 were considered statistically significant.

[0016] Figures 4A-4B provide Surface Electron Microscopy (SEM) images of (Figure 4A) Alginate-PVA and (Figure 4B) Alginate-PVA GSNO wound dressings taken at 300X magnitude.

[0017] Figure 5 is a graphical representation of the bacterial inhibition by a NO releasing Alginate-PVA-GSNO wound dressing as compared to the control alginate-PVA dressing.

[0018] Figure 6 shows the zone of inhibition in S. aureus bacterial lawn formed via nitric oxide diffusion bactericidal effect.

[0019] Figure 7 shows data representing the non-cytotoxic nature of the Alginate-PVA- GSNO films of the present disclosure.

[0020] 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.

DETAILED DESCRIPTION

[0021] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0022] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. 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.

[0023] 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 this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0024] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0025] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, microbiology, material science, and the like, which are within the skill of the art.

[0026] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and articles disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0027] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0028] It must be noted that, 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.

[0029] Abbreviations

[0030] NO, nitric oxide; SNAP, S-nitroso-/\/-acetylpenicillamine; GSNO, S-nitroso- glutathione; PVA, polyvinyl alcohol

[0031] Definitions

[0032] The terms“antimicrobial” and“antimicrobial characteristic” refers to the ability to kill and/or inhibit the growth of microorganisms. A substance having an antimicrobial characteristic may be harmful to microorganisms (e.g., bacteria, fungi, 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.

[0033] The term“antimicrobial effective amount” as used herein refers to that amount of the compound being administered which 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.

[0034] The terms“bacteria” or“bacterium” include, but are not limited to, gram positive and gram negative bacteria. Bacteria can include, but are not limited to, Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus,

Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia,

Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anabaena affinis and other cyanobacteria (including the Anabaena, Anabaenopsis, Aphanizomenon, Camesiphon, Cylindrospermopsis, Gloeobacter

Hapalosiphon, Lyngbya, Microcystis, Nodularia, Nostoc, Phormidium, Planktothrix,

Pseudoanabaena, Schizothrix, Spirulina, Trichodesmium, and Umezakia genera)

Anaerorhabdus, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter, Atopobium,

Aureobacterium, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium,

Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium,

Chryseomonas, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas,

Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella,

Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter, Helococcus, Holdemania Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium,

Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella,

Pediococcus, Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus,

Phytoplasma, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia Rochalimaea Roseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum, Spiroplasma, Staphylococcus,

Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces,

Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema,

Tropheryma, Tsakamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio,

Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella. Other examples of bacterium include Mycobacterium tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B. subtilis, Nocardia asteroides, and other Nocardia species, Streptococcus viridans group, Peptococcus species, Peptostreptococcus species, Actinomyces israelii and other Actinomyces species, and Propionibacterium acnes, Clostridium tetani, Clostridium botulinum, 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).

[0035] "Alginate" as used herein refers to the salts of alginic acid (usually sodium alginate), but it can also refer to alginic acid or derivatives of alginic acid. Alginate, also called algin, is a natural polymer present in the cell walls of brown algae. [0036] General discussion

[0037] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, embodiments of the present disclosure, in some aspects, relate to compositions having bactericidal and bacteria-inhibitive properties, articles including such compositions, and methods of making the compositions.

[0038] The present disclosure includes a composition comprising a synthetic polymer combined with a natural polymer, wherein the combined polymers are crosslinked with a NO-donor. Advantageously, the composition can be used in various applications and articles (e.g. bandages and wound dressings) where bactericidal and antimicrobial activity is needed. In embodiments, the composition also increases cell proliferation, specifically that of fibroblastic cells and shows no cytotoxic effects. When used as a wound dressing, the composition and articles including the composition provide a barrier to inhibit entry of bacteria to the wound, and maintains a moist environment conducive to wound healing.

[0039] Embodiments of the present disclosure include compositions or articles as above, wherein the synthetic polymer can be polyvinyl alcohol (PVA), polycaprolactone (PCL), poly (lactic-co-glycoiic acid) (PLGA), polyg!yco!ic acid (PGA), or combinations thereof. In an embodiment, the synthetic polymer can be PVA.

[0040] Embodiments of the present disclosure include compositions or articles as above, wherein the natural polymer can be sodium alginate, chitosan, cellulose, collagen, chondroitin, gelatin, silk fibroin, eggshell membrane, albumin, wheat bran, arabinoxylan, or combinations thereof. In an embodiment, the natural polymer can be sodium alginate.

[0041] In embodiments, the NO-donor is S-nitroso-glutathione (GSNO). Advantageously, GSNO is conducive to wound repair, and can release NO over a period of days to create a sustained bactericidal effect. In other embodiments, other biocompatible NO-donors could be used, such as S-nitroso-/\/-acetyl-penicillamine (SNAP), but for embodiments in which the composition or articles will be in contact with patient tissue for prolonged periods, or in which wound healing characteristics are used, GSNO is preferred.

[0042] In an embodiment, the composition can include sodium alginate, PVA, and GSNO.

[0043] In various embodiments, the % weight/volume of the natural polymer can be from about 0.1 % to about 50% or about 2.5%. The % weight/volume of the synthetic polymer can be about 0.1 % to about 50%, or about 0.5%. The NO donor can be in the range of from about 1mg/ml-50 mg/ml in a crosslinking solution with CaC .

[0044] In various embodiments, the composition can be lyophilized to form a porous film. Advantageously, a porous structure in the wound dressing provides for gaseous exchange from the wound bed and allows for a controlled water vapor transmission to improve cell proliferation. [0045] In an embodiment, the composition or article (or a coating disposed on a surface of the article) may have an antimicrobial characteristic (e.g., kills at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the microorganisms (e.g., bacteria) on the surface and/or reduces the amount of microorganisms that form or grow on the surface by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, as compared to a similar surface without the coating disposed on the surface). In an embodiment, the composition or article can have an antimicrobial effective amount of an NO donor.

[0046] Embodiments of the present disclosure include articles or substrates as above, where the coating is applied to the surface using e.g. spin coating, spray coating, dip coating, lyophilization, vacuum oven drying, solvent evaporation, solvent swelling, pad application, films with adhesive backing, porous morphology, non-porous morphology, hydrogels, and the like. In embodiments, compositions of the present disclosure can be used in powdered form after freeze drying and crushing. The powdered form can be applied to articles or surfaces.

[0047] The uses of the compositions and articles of the present disclosure are not limited to wound dressings. Other applications can include, but are not limited to, such as food packaging, surgical packing, antimicrobial wipes for cleaning surfaces in hospitals, antimicrobial wipes for cleaning surfaces in households, hydrogels, drug delivery vehicles in the form of encapsulations, cosmetic materials (e.g. antibacterial and skin repair).

[0048] Embodiments of the present disclosure include methods of making a wound dressing, by combining a synthetic polymer with a natural polymer to form a polymer mixture, and crosslinking the polymer mixture with a NO-donor. Natural polymers possess great wound healing potential, but have poor mechanical strength. By combining the natural polymer with a biocompatible synthetic polymer, the materials can be strengthened, and the inherent wound dressing ability can be further enhanced by using a nitric oxide donor to create a synergistic antimicrobial effect. The resulting crosslinked polymer mixture can be lyophilized to form a porous film. In embodiments, the natural polymer can be sodium alginate. In embodiments the NO-donor can be S-nitroso-glutathione (GSNO). In

embodiments, the synthetic polymer can be polyvinyl alcohol (PVA). In embodiments the wound dressing includes sodium alginate, PVA, and GSNO.

Aspects of the Invention

[0049] In one aspect, the present disclosure relates to a biocompatible material comprising a synthetic polymer combined with a natural polymer, wherein the combined polymers are crosslinked with an antimicrobial effective amount of a NO-donor. More specifically, the synthetic polymer can be selected from polyvinyl alcohol, polycaprolactone (PCL), poly (iactic-co-glycolic acid) (PLGA), poiyglycolic acid (PGA), and a combination thereof. The natural polymer can be selected from sodium alginate, chitosan, cellulose, collagen, chondroitin, gelatin, silk fibroin, eggshell membrane, albumin, wheat bran, arabinoxylan, and a combination thereof. In some aspects, the synthetic polymer is polyvinyl alcohol. In some aspects, the natural polymer is sodium alginate. In some aspects, the NO- donor is S-nitroso-glutathione. In some aspects, the crosslinked polymers are lyophilized to form a porous film having pore diameters from about 400-1600 mhi. The % weight/volume of the natural polymer is about from about 0.1% to about 50% and the % weight/volume of the synthetic polymer is about from about 0.1% to about 50%. In some aspects, the NO-donor is from about 1 mg/ml to 50 mg/ml in a crosslinking solution comprising CaCh.

[0050] In another aspect, the present disclosure relates to articles comprising the composition(s) described above. More specifically, the article can be a bandage or wound dressing

[0051] In another aspect, the present disclosure relates to methods of making a wound dressing, comprising combining a synthetic polymer with a natural polymer to form a polymer mixture, and crosslinking the polymer mixture with an antimicrobial effective amount of a NO-donor. In various aspects, the method further comprises lyophilizing the crosslinked polymer mixture to form a porous film. In various aspects, the pores of the porous film have a diameter of about 400-1600 mhi. In various aspects, the synthetic polymer is polyvinyl alcohol. In various aspects, the natural polymer is selected from sodium alginate, chitosan, cellulose, collagen, chondroitin, gelatin, silk fibroin, eggshell membrane, albumin, wheat bran, arabinoxylan, and a combination thereof. In various aspects, the NO-donor is S- nitroso-glutathione.

[0052] In another aspect, the present disclosure relates to wound dressings comprising a synthetic polymer combined with a natural polymer, wherein the combined polymers are crosslinked with an antimicrobial effective amount of a NO-donor. In various aspects, the synthetic polymer is polyvinyl alcohol. In various aspects, the natural polymer is selected from sodium alginate, chitosan, cellulose, collagen, chondroitin, gelatin, silk fibroin, eggshell membrane, albumin, wheat bran, arabinoxylan, and a combination thereof. In various aspects, the NO-donor is S-nitroso-glutathione. In various aspects, the wound dressing comprises a porous film, wherein the pores have a diameter of about 400-1600 mhi. In various aspects, the % weight/volume of the natural polymer is about from about 0.1 % to about 50% and the % weight/volume of the synthetic polymer is about from about 0.1% to about 50%. In various aspects, the NO-donor is from about 1 mg/ml to 50 mg/ml in a crosslinking solution comprising CaCh.

EXAMPLES

[0053] 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.

[0054] An ideal wound dressing should not only be able to offer appropriate mechanical strength, biodegradability, and water transmission but should also be biocompatible in terms of supporting appropriate cellular responses while preventing infection at the wound site. Based on these principles, various wound dressings based on natural or synthetic polymers have been designed over the years. However, both natural and synthetic polymers have their own strengths and limitations and hence a combination of both would be ideal for wound dressing fabrication. A hybrid wound dressing combining natural and synthetic polymers, such as that described herein, will be able to overcome the limitations of the individual class of polymers [11] For example, the combination of alginate, a natural polymer, and polyvinyl alcohol can offer great potential for wound dressing fabrication. Their inherent wound dressing ability can be further enhanced by using a nitric oxide donor.

Sodium alginate (NaAIg) (as well as other natural polymers mentioned above) provides an advantageous matrix material for wound dressing applications [14-16] It is an inexpensive biopolymer that has widely been used in biomedical applications due to its high hydrophilicity and biocompatibility [17, 18] The bioadhesive and biodegradable behavior of alginate films, in addition to maintaining a moist environment, are important characteristics of an effective wound dressing [19, 20] Unfortunately, natural polymers like alginate, despite possessing great wound healing potential, undergo rapid in vivo degradation by proteases due to poor mechanical strength, making it difficult to prolong the diffusion of an encapsulated therapeutic agent [6, 7] Therefore, biocompatible synthetic polymers, such as PVA, due to its chemical and mechanical resistance, can complement alginate for the purpose of wound dressing fabrication. Owing to its biocompatibility with tissue and plasma proteins, PVA is a potential candidate for wound healing applications [21] In the novel wound dressings of the present disclosure, in addition to the base polymers that make the dressings, a therapeutic agent is included to prevent bacterial infection and regulation of the healing during all four phases.

[0055] Nitric oxide is a cellular signaling molecule produced by macrophages and vascular endothelial cells to regulate multiple physiological and pathological processes in mammals including humans. Studies have suggested that endogenous NO regulates vasodilation, inflammation, cell proliferation, the immune response against infection, and tissue remodeling, all of which are pivotal for wound healing [22-25] In addition, NO provides mechanical strength to the new tissues at the healing site via its role in collagen synthesis and deposition at the site of injury [26] As an antibacterial, NO is effective against a wide variety of microorganisms: gram positive and negative bacteria, fungus, yeast, and viruses [27-30] The biggest advantage of NO comes from the fact that it is highly effective against even the antibiotic-resistant strains. Moreover, unlike antibiotics, NO application would not lead to the emergence of resistant bacterial strains owing to its rapid action, short half-life (< 5 sec) and endogenous nature. The gaseous nature of NO allows penetration through the matrix in the biofilm, which gives it an extra advantage over antibiotics and silver-based antibacterial strategies. In the past, NO releasing materials have been shown to positively regulate the wound healing process [13, 31, 32] However, the present disclosure provides the first bio-inspired wound dressing combining alginate-PVA with the nitric oxide donor S-nitrosoglutathione (GSNO). GSNO is an endogenously produced S-nitrosothiol (RSNO) in the body that serves as an important signaling molecule. These functions range from preventing embolization within the vasculature to modulating angiogenesis by promoting vascular endothelial growth factor (VEGF) production within damaged tissue [33, 34] Along with these functions, GSNO has also been shown to be involved in collagen deposition in cutaneous wound repair, further demonstrating its abilities in wound healing [35] The fabrication and characterization of a novel NO-releasing hybrid wound dressing of alginate-PVA and its antibacterial attributes and biocompatibility as a wound dressing are described herein.

Material and Methods

[0056] Materials

[0057] Sodium salts of alginic acid, calcium chloride, ethylenediaminetetraacetic acid (EDTA), sodium chloride, were obtained from Sigma-Aldrich (St. Louis, MO). Polyvinyl alcohol (PVA- 88% hydrolyzed, M.W. approximately 13,000-23,000) was bought from Acros Organics (New Jersey) and glycerol was bought from Fischer Chemicals (Fair Lawn, NJ).LB broth and LB Agar were obtained from Fisher Bioreagents (Fair Lawn, NJ). Dulbecco’s Modification of Eagle’s medium (DMEM) and trypsin-EDTA were purchased from Corning (Manassas, VA). The Cell Counting Kit-8 (CCK-8) was obtained from Sigma-Aldrich (St Louis, MO). The antibiotic Penicillin-Streptomycin (Pen-Strep) and fetal bovine serum (FBS) were purchased from Gibco-Life Technologies (Grand Island NY 14072). L-Glutathione (reduced 98+ %) was purchased from Alfa Aesar (Ward Hill, MA). The bacterial

strains Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 5538) and Mouse fibroblast cell line (ATCC 1658) was originally obtained from American Tissue Culture Collection (ATCC). Autoclaved Phosphate buffered saline (PBS), was used for all in vitro experiments.

[0058] Methods

[0059] Synthesis of GSNO [0060] Synthesis of S-nitroso-glutathione (GSNO) was performed by modifying a standard protocol (T.W. Hart, Some observations concerning the S-nitroso and S- phenylsulphonyl derivatives of L-cysteine and glutathione, Tetrahedron Lett. 26(16) (1985) 2013-2016, which is herein incorporated by reference). Reduced glutathione (900 mg, 2.93 mmol) was first dissolved in 4 mL of Dl water and 1.25 mL of 2M HCI. The solution was allowed to chill in ice for 10 minutes before being nitrosated with an equimolar amount of sodium nitrite. The solution was then covered and allowed to cool in an ice bath for a further 30 minutes. Chilled acetone (5 mL) was then slowly added to the solution and allowed to stir for an additional 10 minutes while still in the ice bath. The GSNO precipitate that formed was then collected by vacuum filtration and further washed with cold acetone and water. The resulting washed product was then allowed to dry under vacuum overnight before being collected and stored in the freezer.

[0061] Engineering NO releasing wound dressings

[0062] Fabrication of the Alqinate-PVA dressings: The NO releasing wound dressings were formulated by the solvent casting method. Sodium alginate (1 g) was slowly added to a conical flask containing 20 mL of deionized water at 40°C for 2 hours. A magnetic stirrer was used to formulate a polymeric dispersion of appropriate consistency. As recommended by existing literature, 0.2 g of PVA was dissolved in 19.6 mL of deionized water, which was simultaneously stirred using a magnetic stirrer and heated to a temperature of 90°C (] S. Patel, D. Shah, S. Tiwari, Bioadhesive films containing fluconazole for mucocutaneous candidiasis, Indian J. Pharm. Sci. 77(1) (2015) 55, herein incorporated by reference). The temperature of the PVA solution was brought down to around 45-50^°C before adding it to the alginate solution. Both polymeric solutions were blended together for 10 minutes to obtain a uniform polymeric dispersion. 0.4 ml of glycerol was added to alginate-PVA mixture to impart flexibility and durability to the wound dressing. Finally, it produced a solvent mixture of 40 mL (total) with 2.5 % (w/v) sodium alginate, 0.5% (w/v) polyvinyl alcohol (PVA) and 1% (v/v) glycerol. The resulting formulation was cast into Petri dishes and was kept in the freezer for 3 hours.

[0063] Crosslinkinq of the wound dressings and Ivophilization: The crosslinking process was carried out by using a solution of GSNO-CaCL_. A 2% (w/v) calcium chloride (CaCh) was prepared by dissolving 0.4 g of CaCL in 20 mL of deionized water. In parallel, 30 mg/mL GSNO solution was prepared by using water as a solvent. Finally, CaCL and GSNO were mixed in a 1 :1 ratio by adding 5 mL of 2% CaCL and 5 mL of GSNO (30 mg/mL) using a vortex mixer to obtain a uniform solution. The final GSNO concentration was 15 mg/mL in the crosslinking solution. The frozen Alginate-PVA polymeric dispersions were soaked in the CaCL-GSNO solution and allowed to crosslink for 20 h. The uniformity of solution distribution around alginate-PVA-GSNO formulation was checked every 1 h of the first 6 h. After 20 h of crosslinking, the crosslinked films were lyophilized for 7 h at -80^°C and < 1.5 mBar pressure Labconco freeze dryer to create a porous wound dressing matrix.

[0064] Lyophilisation is one of the novel methods to create pores in the wound dressing where water crystals formed in the frozen formulation act as a porogen.[38] As the material freeze dries into 2D sheets of the wound dressing, the ice crystals sublimate, leaving pores. No further processing is required, making this a very simple method to develop porous wound dressings. In addition, since the process happens at a temperature of -80^°C, the undesired loss of NO from GSNO via thermal stimulus can be avoided. Figure 1 shows the control (alginate-PVA) and NO releasing (alginate-PVA-GSNO) wound dressings.

[0065] NO release analysis

[0066] The NO release study was performed to investigate the NO flux from the hybrid wound dressing using a Sievers chemiluminescence Nitric Oxide Analyzer (NOA) 280i (Boulder, CO). The NOA has the capability to selectively map NO via the reaction of NO with ozone, thereby reducing intervention from other molecules [27] Small circular films

(diameter of a 5/16^th inch) were punched out and wrapped in wipes (Kimwipes, KIMTECH) and dipped in PBS (pH 7.4) containing EDTA to mimic a moist wound environment prior to using them for NO flux analysis. Once dipped, the film was immediately placed at the bottom of the sample holder. The released nitric oxide was continuously purged from the sample and swept from the headspace using nitrogen as the sweep gas into the chemiluminescence detection chamber. The films were tested for release at the 0 h and 24 h.

[0067] Water permeability, moisture content, and swelling index

[0068] Water permeability: The water permeability of the wound dressings was determined using a standard protocol [27] The wound dressings with a diameter of 2 cm were wrapped around the 10 ml_ glass vial after filling them with 5 g of dehydrated silica. The weight of the vials (n=3 each) with wound dressings were reported and then the vials were placed in a humid environment established with saturated sodium salt (75% relative humidity, 22 ± 2C). The weight of the vials was measured every 24 h for a period of 7 days. A linear curve was plotted between gained weight (dw) and time (d0t) and the water vapor permeability K (kg m rrr² day^-1 Pa^-1) of the wound dressing was calculated using the formula below.

[0069] K = ^dw/d0t

^{L J} Ap*P [0070] dw = weight gain due to moisture retention (kg), 0t= time point (day), dw/d0t = slope between weight gain and time point (day), Ap is the surface area of the dressing (m²), P is the saturation vapor pressure of water at 22° C.

[0071] Moisture content: The moisture content (MC%) was determined by using a recommended protocol [39] After measuring, the thickness wound dressings with a surface area of 3 cm² were weighed and their weights were reported. Thereafter these samples were kept in a vacuum oven for 24 h at 105 °C and their weight was measured again. The MC% was calculated by comparing the weights of dressings before and after drying using the following formula.

[0073] Swelling index (SO: To measure the swelling ratio (SR) of the wound dressings, a recommended protocol was slightly modified [11] Wound dressings of dimension 2 X 2 cm² size (n=3) were weighed and dried in a vacuum oven at 105 °C for an hour. The weight of both control and NO releasing dressings were taken again after drying. Thereafter, the dressings were swelled in 0.1 M phosphate buffer saline (PBS, pH 7.4) at room temperature. After soaking for an hour, the weight of the dressing was measured again. The SI was calculated using the formula below.

[0074]

Weight after soaking— Weight after drying

SI (%) = 100

Weight after drying

[0075] Contact angle

[0076] Measurement of contact angle (Q) allows estimating the solid-liquid interfacial tension that is performed by establishing the tangent (angle) of a liquid drop with a solid surface at the base. This method is popularly used to determine the

hydrophobicity/hydrophilicity of the material. The contact angle of the wound dressing was measured by using Kruss DSA 100 drop shape analyzer. The wound dressing (with and without GSNO) was cut into 12 mm X 12 mm, stuck on top of a glass slide and placed under drop shape analyzer. A single ~1 pi drop was placed on the dressing at three random spots. The initial contact angle values on the dressings were measured from each frame of the recorded files using the sessile drop approximation.

[0077] Morphology of the dressing surface and pores analysis

[0078] Scanning electron microscopy: Scanning electron microscopy (SEM) is a useful tool to understand the surface characteristics of a polymer. In the present study,

microstructure and surface morphology of the wound dressing (before and after NO donor incorporation) were examined using SEM (FEI Inspect F FEG-SEM). A total of three samples of each of the control (without GSNO) and alginate-PVA-GSNO were sputter coated with gold-palladium (10 nm) using a Sputter Coater (Leica EM ACE200) after mounting them on a metal stub. An accelerating voltage of 5 kV was used to capture SEM images of the sample at 100X magnification.

[0079] Porosity measurement: Pore size was determined using Image J software from images taken with light microscopy (Thermo Fisher scientific EVOS™ XL Cell Imaging System). At least 30 pores were used to determine measurements of the diameter each sample.

[0080] In vitro testing of gram-positive and gram-negative bacteria inhibition

[0081] The designed NO releasing alginate-PVA-GSNO dressings were examined for antibacterial efficacy against common bacteria responsible for skin infections: gram-positive Staphylococcus aureus (S. aureus) and gram-negative Pseudomonas aeruginosa (P.

aeruginosa). A modified version of standard bacterial adhesion test was used to pursue this [40, 41] This test allows to quantify the amount of bound viable bacteria on polymer surface and compare the bacterial inhibition in the presence or absence of the antibacterial agents such as NO.

[0082] Preparation of bacterial suspension: A single isolated colony of bacteria was picked from the pre-cultured LB-agar plate and inoculated into 10 mL of Luria Broth (LB) medium in a 50 mL Eppendorf tube and allowed to incubate at 37 °C for 14 h at a radial shaking speed of 120 rpm. After 14 h, the optical density of the bacteria was measured at 600 nm (Oϋboo) using UV-vis spectrophotometer (Thermo Scientific Genesys 10S UV-Vis). This step assures that the bacteria is in actively diving log phase (and not in dead phase) prior to the use in the experiment to keep the experimental condition unbiased. After this step, Bacterial cells were separated from the LB medium by centrifuging the bacterial culture at 2500 rpm for 8 min, the supernatant was discarded and fresh sterile phosphate buffer saline (PBS, pH 7.4) was added. The same procedure was repeated to wash off the traces of LB. This step was repeated twice and the bacterial cells were ultimately suspended in PBS (without any residual traces of LB medium) to be used in the experiment further. The removal of traces of nutrient medium (LB) and suspension of bacterial strains in the PBS assures that the bacteria would not grow back after being killed by the antibacterial agent and thus allow a fair comparison between the control (without GSNO) and alginate-PVA- GSNO wound dressings.

[0083] Bacterial inhibition and its quantification: Prior to exposing the wound dressing to the bacterial suspension, the ODeoowas measured again and diluted with PBS to represent bacterial cells count in the range of 10⁸-10¹⁰ CFU/ml_ to represent the bacterial cell count in infected chronic wounds. Triplicate (n=3) samples (diameter = 2.4 cm) of both the alginate- PVA-GSNO wound dressing and alginate-PVA (control) were exposed for 24 h to 5 mL of bacterial suspension in a 50-mL tube incubated at 37 °C at 120 rpm. After bacterial exposure, the wound dressings were removed from the bacterial suspension and the unbound or loosely bound bacteria were washed off by rinsing the dressings with 2 mL PBS using a pipette. The dressings with the adhered bacteria were then transferred to 2 mL of fresh PBS, homogenized for 30 secs using a vortex mixer in order to detach the bound bacteria into the PBS solution. The resulting bacterial suspension was serially diluted (10^_1 to 10 ⁵) using PBS, plated in pre-made LB agar Petri dishes (LB agar concentration 40 g/L) and post-incubated for 20 h at 37 °C. After 24 h, the colony forming units (CFUs) appeared on the LB agar plate. The CFU were counted while adjusting the dilution factor and amount of bacterial suspension and CFU per weight (mg) of the wound dressings were obtained for both alginate-PVA-GSNO wound dressings and control dressings. The percentage bacterial inhibition was calculated relative to the control using the following formula.

[0085] Zone of inhibition (ZOI) study using agar diffusion method. The ability of the wound dressing material to inhibit bacterial growth beyond the direct point of contact was tested via standard agar diffusion method. [40] As a proof of concept gram positive, S. aureus was used. The strain culture was spread uniformly and aseptically on a premade LB agar petridish. Circular wound dressing disks (diameter: 2.7 cm) of alginate-PVA and alginate- PVA-GSNO were gently placed and pressed on top of bacterial culture. The petridish was then placed in an incubator at 37 °C for 20 h to allow the formation of zone of inhibition (ZOI).

[0086] Cell viability assay

[0087] As per the ISO 10993 standards, the purpose of performing biocompatibility testing on a biomaterial is to investigate its undesirable effects such as cytotoxicity and to validate its fitness for human use. To ensure that the leached-out agents of the wound dressing material do not kill the healthy host cells, cytotoxicity testing in accordance with ISO 10993 standards was conducted on 3T3 mouse fibroblast cells using cell counting kit-8 (CCK-8) assay [41] The manufacturer’s (Sigma-Aldrich) protocol was followed while using CCK-8 kit which utilizes highly water-soluble tetrazolium salt. In the live cells, WST-8 [2-(2- methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt] is reduced by dehydrogenases to give formazan (an orange color product), which can be detected at 450 nm. This means that a noncytotoxic material should generate a higher level of formazan than a relatively cytotoxic material because of WST-8 reduction. A sample of skin substitute weighing 10 mg was added to 10 mL to leach/degrade in the DMEM medium for 24 h.

[0088] Seeding of cells: A culture of the fibroblast cells was grown in a 75 cm² T-flask with Dulbecco modified Eagle’s medium (DMEM) with 4.5 g/L-glutamine, 4.5 g/L glucose,

1 % penicillin-streptomycin and 10% fetal bovine serum (FBS) after thawing a cryopreserved vial of fibroblast cells. The cells were allowed to proliferate in an incubator which provided a physiological condition for their growth (5% C02, 37 °C) until the confluency reached around 80-90%. The cells were detached from the T-flask surface by enzymatically degrading their extracellular matrix layer by treating them with 0.18% trypsin and 5 mM EDTA for 5 min.

Absorbance of the test samples

[0089] % Cell Viability = 100

Absorbance of the control samples

Results and Discussion

[0090] NO release kinetics

[0091] NO release from alginate-PVA-GSNO dressings was tested in real time via chemiluminescence nitric oxide analyzer. RSNO’s like GSNO are capable of passively releasing NO from heat, moisture, and catalytically when in the presence of certain metal ions. GSNO was shown to be stable when placed in the alginate-PVA matrix, as each NO release profile showed a steady release trend over the testing periods: 0 h, 24 h, 48 h, and 72 h at 37°C. The wound dressing showed an initial NO release of 5.01 ± 0.49 ^c 10 ¹¹ mol mg^-1 min^-1 and sustained the NO release over a 72-hour period (0.54 ± 0.Q88 ^c 10^-11 mol mg- ¹ min^-1). This is significant in terms of wound application as open wounds are most prone to infection in the first few hours post injury. Polymers releasing NO of this magnitude has been shown in the past to be effective against a wide variety of gram-positive and gram negative bacteria. [27] In the current study also we have been able to achieve a significant reduction in gram positive and gram negative bacteria. A real-time NO release profile is shown in Figure 2 and the NO release profile over a 72 h period is presented in Figure 3. The ability of these wound dressings to sustain NO release over a 3-day period would also allow avoiding frequent redressing, thus not only reducing the pain caused during redressing, but also would cut down the wound care cost. In Figure 3, statistical data is expressed as mean ± standard error of the mean of n=3 samples. Values of p < 0.05 were considered statistically significant.

[0092] The flexible design process of the wound dressings allows fine-tuning of the NO flux at certain time points by adjusting the amount of GSNO present. Depending on the depth of the wound and the level of infection, in some instances, a large flux of NO may be required for the initial antimicrobial effect, while in different situations it may not be as necessary to avoid cytotoxic effects of higher dose on host cells.

[0093] Surface morphology and pore size analysis

[0094] The surface electron microscopy was used to measure the morphology of the alginate-PVA-GSNO wound dressings relative to the controls. As shown in Figure 4, the surface of the dressing material was not altered in the presence of GSNO, confirming that GSNO has no negative effect on the surface morphology. The porous structure in the wound dressing offers a great advantage in terms of water vapor transmission as well as a gaseous exchange from the wound bed.

[0095] Both the alginate-PVA-GSNO film and the control (alginate-PVA) exhibited macroporous characteristics. The average pore size of the GSNO dressing was

approximately 860 mhi and the average pore size of the control dressing was approximately 900 mhi. The pore sizes for both films ranged from 400-1600 mhi. The non-homogenous, larger pore sizes are comparable to commercial wound dressings such as Cellosorb

Adhesive (Urgo Medical Co) [42]

[0096] Contact angle analysis

[0097] Kruss DA100 Drop Shape Analyzer is a standard tool for contact angle measurement. Contact angle measurement helps to determine the affinity of a polymeric surface with water. In general, polymers with a contact angle less than 90° are considered hydrophilic while the materials with a contact angle less than 10° are considered super- hydrophilic [43, 44] In the present study, when contact angle was measured the instrument could not capture a reading. A plausible explanation for this can be that PVA, alginate, and GSNO are all hydrophilic in nature. Moreover, the porous and hydrophilic nature of the dressing caused immediate absorption of the water droplet from the analyzer.

[0098] From an application point of view, the super-hydrophilic nature of the designed wound dressing should not only result in the active release of NO from the GSNO but will also be useful for maintaining moist conditions in the wound bed, which can enhance the wound healing process [22, 45] In addition, hydrophilic wound dressings regulate water vapor transmission rate and prevent the wound bed from drying, thus increasing adsorption of blood and accelerating the overall healing process [22, 46, 47] Other studies have also shown the role of the hydrophilic surfaces in mammalian cell adhesion and proliferation, all of which are involved in enhancing the wound healing process [10, 47, 48] This correlation between contact angle and vapor permeability is further discussed below in addition to the biological response towards alginate-PVA-GSNO wound dressing.

[0099] Thickness, water vapor permeability, moisture content, and swelling index [0100] The physical properties like thickness, moisture content (MC%), water permeability and swelling index are important parameters that govern the healing potential of a wound dressing.

[0101] The thickness of the film was measured with Digimatic Micrometer (Mitutoyo, Japan) and was found to be 0.29 ± 0.001 mm for control and 0.30 ± 0.006 mm for the films with GSNO. Thereafter, water permeability, MC%, and SR% were calculated and results are presented in Table 1. The water permeability of the films with GSNO was found to be lower than that of the control films (without GSNO). In the past, also we have shown that natural polymer incorporated with NO donors tend to decrease the water permeability due to closer networking between the base polymer and NO donors. [49] The data from the study described herein is in agreement with the published report suggesting that the GSNO incorporation resulted in reducing the water permeability as compared to the control.

Table 1. Physical characterization of the Wound dressing in terms of thickness, Vapor Permeability, Moisture, Swelling Ratio.

[0102] The moisture content (MC%) from the total weight of the wound dressings was calculated based on the differences in weight before and after drying the wound dressing for 24 h at 105 °C. The MC% for the alginate-PVA-GSNO dressings 33.06 ± 2.1 was found to be higher as compared to the control films 23.9 ± 3.7. This is in line with the water permeability results, suggesting that a decrease in water permeability due to the presence of GSNO help them retained more moisture and hence higher MC%.

[0103] The swelling index studies were done for 24 h using PBS (pH 7.0), and the difference in weight of the wound dressings was compared before and after soaking in PBS. The swelling index (SI) of the alginate-PVA-GSNO was higher (64.55 ± 2.26 %) than control films (48.94 ±3.56 %), further strengthening the conclusion that the presence of GSNO resulted in better retaining of the moisture in the NO releasing films as compared to the control. A similar trend resulting in an increase in swelling index after adding a drug

(antibiotics) to an alginate-PVA wound dressing has been observed in another report also [11]·

[0104] Combining all these results and comparing it with contact angles brings an interesting discussion. The contact angle analysis indicated that addition of GSNO significantly increased the hydrophilicity of the alginate-PVA-GSNO films. This resulted in an increase in moisture content and the swelling index of alginate-PVA-GSNO wound dressings as compared to control alginate-PVA dressings. The water vapor permeability was shown to decrease because of increase in water retention by the NO releasing wound dressing. This theoretically should increase the NO release from the wound dressings over time as moisture is one of the triggers that increases NO release from NO donors such as GSNO. This would also have positive consequences when such wound dressings are applied to a wound site where the wound exudates would be absorbed in the alginate-PVA-GSNO dressing resulting in the prolonged supply of NO from the wound dressings. Eventually, an increase in NO flux will benefit the wound healing through its bactericidal effect [13, 22, 50] From a clinical perspective, this is of great advantage in the presence of infection in the wound exudates, which can cause serious clinical challenges including hyperinflammation and delay the healing process [51-53] In addition, an increase in NO flux will also positively impact all four phases of the wound healing process from homeostasis until the final phase of tissue remodeling [22, 53-55] A controlled water vapor transmission rate has been shown to help in the proliferation and regular function of fibroblast cells [56] This concept was validated in the cell viability assay with the mouse fibroblast cells results of which are discussed in detail below. Table 1 shows the observed values (average mean of n=3) for different characterization parameters.

[0105] Antibacterial attributes of the wound dressing

[0106] The wound dressings were tested to see their ability to inhibit both gram-positive and gram-negative bacterial growth. Results showed that the NO releasing wound dressings (with GSNO) showed 99.89 ± 0.4% bacterial inhibition of gram-positive S. aureus and 99.93 ± 0.7% inhibition of gram-negative P. aeruginosa as compared to the control alginate-PVA dressing without the NO donor. In log scale this amount to around 3 log reduction. Figure 5 shows a graphical representation of both gram-positive and gram negative bacterial inhibition by NO releasing wound dressings. The dressings caused ~ 3 log reduction in S. aureus and > 2 log reduction in P. aeruginosa CFU/mg. Statistical data is expressed as mean ± standard error of the mean of n=3 samples. Values of p < 0.05 were considered statistically significant.

[0107] Both of these bacteria contribute to most of the blood and burn infections and are frequently found in hospital settings and cause biofilm formation to resist antibiotics effect. Thus, effective killing of these bacteria is a significant stride towards proving the highly effective and practical use of the designed wound dressings. In the past, we have also shown another NO donor, S-nitroso-/\/-acetyl-penicillamine (SNAP) to be highly effective against a wide variety of bacteria including S. aureus, P. aeruginosa, A. baumanni, and E. coli, which are responsible for wound infections [27, 40, 41] The cellular damage caused by GSNO to the bacterial cells may result from oxidative and nitrosative stress mainly facilitated by oxidation of thiol or nitrosation of thiol groups [57] Another accepted mechanism is the NO-mediated inhibition of enzyme activity in bacterial cells. Lipid peroxidation, nitrosation of amines and thiols in the extracellular matrix, tyrosine nitration in the cell wall and DNA damage are also among the targeted antibacterial mechanism of NO [58]

[0108] In other embodiments, the NO donor can be other than GSNO (such as SNAP However, in the example presented here, GSNO was selected for its ability to imitate the natural NO donor found in humans. S-Nitrosoglutathione (GSNO) is an endogenous S- nitrosothiol (SNO) that plays a critical role in nitric oxide (NO) signaling and is a source of bioavailable NO. Thus chances of an allergic response or cytotoxicity were confirmed to be very low in the experiments described above.

[0109] While killing the bacteria on the surface of wound bed is important, it is equally important to inhibit its entry into the host tissue, which can otherwise result in septicemia and inflammation. The agar diffusion method showed the ability of NO releasing wound dressing to eradicate bacteria beyond the direct point of contact at physiological temperature. The results showed no zone of inhibition (ZOI) around control alginate-PVA wound dressings while a 2.6 cm ZOI was formed with alginate-PVA-GSNO dressing (Figure 6). A similar level of bacteria inhibition has been observed in the past using zone of inhibition study which also utilized the chemistry of alginate-PVA but with an antibiotic, sodium ampicillin instead of GSNO [11]. However, the nature of that study didn’t allow the bacteria killing in log scale while the present study showed a 3 log reduction in bacterial growth showing superiority to studies done with alginate-PVA dressings. Moreover, the problem with antibiotic resistance and cytotoxicity raises an alarming concern and hence GSNO provides better alternative to the existing antimicrobial agents. Nitric oxide, due its short half-life, rapid and nonspecific bactericidal action is not expected to develop resistance in the bacterial strains [59-61]

[0110] Fibroblast proliferation and non-cvtotoxicitv testing

[0111] T reating cells with a non-biocompatible material can result in a variety of cell fates such as a decrease in cell viability, altered metabolism, and necrosis. Therefore, cytotoxicity testing is an important step to demonstrate that the NO releasing wound dressing does not lead to any undesirable biological effects in mammalian cells. In the present study, mouse fibroblast cells were exposed to leachates from the wound dressing for 24 h, and results showed that the NO releasing alginate-PVA-GSNO wound dressings possess no relative cytotoxicity towards the cells. Figure 7 shows the cell viability for alginate-PVA-GSNO relative to the alginate-PVA wound dressings and in the absence of any wound dressing (positive control). This agrees with different reports that also suggest that materials used individually in the fabrication of the wound dressing (PVA, alginate, and GSNO) are non-cytotoxic in nature [62, 63] The films grown in the well plate without any leachate exposure were considered as the positive control and at 100% for cell viability. Statistical data is expressed as mean ± standard error of the mean of n=3 samples. Values of p < 0.05 were considered statistically significant.

[0112] In addition, the degradation products of both the polymers sodium alginate and PVA are not known to cause any cytotoxic, teratogenic or mutagenic effects as claimed by the Material Safety Data Sheet (MSDS) provided by the manufacturer. It is worth noticing that there was a proportional decrease in NO flux with respect to the time instead of random NO flux peaks (which results from NO burst effect caused by leaching). This is important as randomly given NO overdose can otherwise be cytotoxic to mammalian cells. Thus, the NO releasing alginate-PVA-GSNO wound dressing is a potential alternative to overcome the limitation of the commercial wound dressings currently in use. Commercially available advanced wound dressings often contain antibiotics (Septocoll® by Biomet Merck;

Collatamp® by Innocoll) or other antibacterial agents such as silver (e.g., Acticoat® by Smith & Nephew, Actisorb® by J&J and Aquacel® by ConvaTec), or chlorohexidine (Biopatch® by J&J) or iodine (lodosorb® by Smith & Nephew). However, there are growing concerns about the emergence of antibiotic-resistant bacterial strains. Similarly, silver nanoparticles have shown to be genotoxic and cytotoxic. Besides causing these side effects, they also cause a delay in healing if applied indiscriminately to damaged tissue areas, which defies the actual purpose of their application [64-66] The inefficiency of currently available therapeutic agents not only adds to the suffering of the patient but also causes huge healthcare expenses. In the past, published reports including ours have shown other NO releasing materials to possess biocompatibility and hemocompatibility in vitro and wound healing attributes via regulation of TGF-b and epithelization, thus enhancing the overall wound healing process [13, 41 , 67]

[0113] The onset of the proliferative phase that is marked by fibroblasts entering the wound site overlaps with the infection inhibiting inflammatory phase. In this regard, alginate- PVA-GSNO wound dressings were not only effective in controlling infection by up to 99.89 ± 0.4 % but also increased the proliferation of fibroblast cells by >30% as compared to control (without any wound dressing leachate), thus contributing to proliferation and infection control at the same time in vitro. When applied in vivo, the proliferation potential of GSNO combined with physical properties of the wound dressing such as moisture content and WVTR is expected to further enhance the wound healing rate [56] Overall, the NO releasing wound dressing is highly effective in preventing infection while promoting the proliferation of fibroblast cells without causing any cytotoxic effects.

Conclusion

[0114] The present disclosure describes a bio-inspired wound dressing engineered using a natural and synthetic polymer crosslinked with an NO-release agent. The present example of a wound dressing of the present disclosure was fabricated using alginate-PVA-GSNO. The nitric oxide-releasing donor molecule GSNO was incorporated to mimic the natural NO release that occurs during different phases of the wound healing. These wound dressings allowed a controlled release of NO through a slow but sustained release mechanism, which helps avoid cytotoxicity yet ensures delivery of a therapeutic dose to reduce infection at the wound site. Physical characterization (contact angle, moisture content, water vapor transmission, pore size, swelling ratio, and surface morphology), NO release kinetics, and biological characterization (antibacterial efficacy, cell proliferation, and non- cytotoxicity) that the wound dressing possesses provide the desired characteristics needed for enhancing the natural wound healing process. The results showed maintenance of sustained NO flux in a 72-h study analyzed via a chemiluminescence-based NO analyzer. The test performed on both gram-positive and gram-negative bacteria showed up to 3 log reductions over a 24 h period (99.89 ± 0.4 % ), which makes it a very effective way to enhance wound healing process. In the past, attempts have been made to design wound dressings with topical application of antibiotics and silver nanoparticles but at the compromise of bacterial resistance, cytotoxicity, and genotoxicity. The current example demonstrated proliferation of mouse fibroblast cells in vitro upon application of NO releasing wound dressing material resulting in >30% increase in cellular viability while simultaneously establishing the non- cytotoxic nature of the dressing. Moreover, the rapid application, short half-life, and non specific antibacterial action of NO do not allow development of bacterial resistance. The NO releasing wound dressings described herein offer highly effective material.

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[0115] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of“about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment,“about 0” can refer to 0, 0.001 , 0.01 , or 0.1. In an embodiment, the term“about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase“about‘x’ to‘y’” includes“about‘x’ to about‘y’”.

[0116] 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.

Claims

CLAIMS What is claimed is:

1. A biocompatible material comprising a synthetic polymer combined with a natural polymer, wherein the combined polymers are crosslinked with an antimicrobial effective amount of a NO-donor.

2. The biocompatible material of claim 1 , wherein the synthetic polymer is selected from polyvinyl alcohol, polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), and a combination thereof.

3. The biocompatible material of claims 1 or 2, wherein the natural polymer is selected from sodium alginate, chitosan, cellulose, collagen, chondroitin, gelatin, silk fibroin, eggshell membrane, albumin, wheat bran, arabinoxylan, and a combination thereof.

4. The biocompatible material of any of claims 1-3, wherein the synthetic polymer is polyvinyl alcohol.

5. The biocompatible material of any of claims 1-4, wherein the natural polymer is sodium alginate.

6. The biocompatible material of any of claims 1-5, wherein the NO-donor is S-nitroso- glutathione.

7. The biocompatible material of claim 1 , wherein the crosslinked polymers are lyophilized to form a porous film having pore diameters from about 400-1600 mhi.

8. The biocompatible material of any of claims 1-7, wherein the % weight/volume of the natural polymer is from about 0.1% to 50%, the % weight/volume of the synthetic polymer is from about 0.1 % to 50%, and the NO-donor is from about 1 mg/ml to 50 mg/ml in a crosslinking solution.

9. An article comprising the composition of any of claims 1-8.

10. The article of claim 9, wherein the article is a bandage or wound dressing.

11. A method of making a wound dressing, comprising: combining a synthetic polymer with a natural polymer to form a polymer mixture; and crosslinking the polymer mixture with an antimicrobial effective amount of a NO- donor.

12. The method of claim 11 , further comprising lyophilizing the crosslinked polymer mixture to form a porous film.

13. The method of claim 11 , wherein the pores of the porous film have a diameter of about 400-1600 mhi.

14. The method of any of claims 11 or 12, wherein the synthetic polymer is polyvinyl alcohol.

15. The method of any of claims 11-14, wherein the natural polymer is selected from sodium alginate, chitosan, cellulose, collagen, chondroitin, gelatin, silk fibroin, eggshell membrane, albumin, wheat bran, arabinoxylan, and a combination thereof.

16. The method of any of claims 11-15, wherein the NO-donor is S-nitroso-glutathione.

17. A wound dressing comprising a synthetic polymer combined with a natural polymer, wherein the combined polymers are crosslinked with an antimicrobial effective amount of a NO-donor.

18. The wound dressing of claim 17, wherein the synthetic polymer is polyvinyl alcohol.

19. The wound dressing of claim 17, wherein the natural polymer is sodium alginate.

20. The wound dressing of claim 17, wherein the NO-donor is S-nitroso-glutathione.

21. The wound dressing of claim 17, wherein the wound dressing comprises a porous film, wherein the pores have a diameter of about 400-1600 mhi.

22. The wound dressing of any of claims 17-20, wherein the % weight/volume of the natural polymer is about from about 0.1 % to about 50% and the % weight/volume of the synthetic polymer is about from about 0.1% to about 50% .