Nitric oxide releasing antimicrobial polymers and scaffolds made therefrom and methods relating thereto

Abstract

Several embodiments of NO-releasing structures are disclosed. In some embodiments, the structures are covalently modified to store and release nitric oxide. Some embodiments relate to methods of making and using these structures. The covalently modified polymer structures can be tailored to release nitric oxide in a controlled manner, and are useful for treating various medical conditions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 786,098, filed December 28, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Government Interests This invention was made with Government support under Grant Nos. DK108318 and DE025207 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The subject matter of the present disclosure generally relates to nitric oxide-releasing polymers and scaffolds made therefrom that store and / or release nitric oxide in a controlled manner. Also disclosed are methods for the synthesis of the nitric oxide-releasing polymers and scaffolds made therefrom, and methods for their use as antimicrobial agents in therapeutic methods. [Background technology]

[0004] Bacterial infections pose a major challenge to human health in community and hospital environments. Biofilms are cooperative communities of bacteria encapsulated by an exopolysaccharide (EPS) matrix that protects the bacteria from the host immune response and antibiotics. Summary of the Invention

[0005] Nitric oxide (NO) plays various physiological roles as a signaling molecule and may play an important role in treating or ameliorating pathophysiology, for example, as a therapeutic agent, as disclosed herein. NO as a therapy has been underutilized to date, based at least in part on the limited NO payload of therapeutic compositions, faster than desired NO release rates, and lack of targeted NO delivery. Provided herein are NO-releasing polymers and scaffolds, methods of making such polymers and scaffolds, and methods of treating various pathophysiologies using such polymers and scaffolds that utilize enhanced NO-releasing properties and beneficial physical properties, taking advantage of the rich potential of NO-releasing pharmacological compounds and compositions. In some embodiments, provided herein are compounds and compositions that are highly effective as antimicrobial agents. In some embodiments, provided herein are compounds and compositions that have beneficial antimicrobial properties and physical properties such as viscosity and gelation. In some embodiments, the polymers and / or scaffolds disclosed herein have advantageous activity as viscosity enhancing agents.

[0006] For example, in some embodiments, one or more macromolecular structures are provided that release NO and exhibit strong antimicrobial properties. In some embodiments, the macromolecular structures are polymers. In some embodiments, the polymers can be used as dilute solutions (e.g., for evaporation and inhalation, etc.), while in other embodiments, the polymers can be self-assembled in solution and / or crosslinked to provide scaffolds with advantageous physical properties (including three-dimensional shape, hardness, adhesion, and viscosity). In some embodiments, the polymers retain beneficial antimicrobial activity even as gels and viscous liquids.

[0007] In some embodiments, a polymer is provided having structural units along the chain of the polymer. In some embodiments, one or more of the structural units are functionalized with one or more instances of each of R1, R2, R3, R4, R5, and R6. In some embodiments, R1, R2, R3, R4, R5, and R6 can include functional units that infuse the polymer with desired properties. In some embodiments, R1, R2, R3, R4, R5, and R6 can include NO binding moieties. In some embodiments, R1, R2, R3, R4, R5, and R6 along the polymer chain can include functional units that infuse desired properties. R Each of the six examples (e.g., one or more structural units) may be -OH, -NH2, -OCH3, -C(O)OH, -CH2OH, -CH2OCH3, -CH2OCH2CH2OH, -OCH2C(O)OH, -CH2OCH2C(O)OH, -CH2C(O)OH, -NHC(O)-CH3, -C(O)O((CH2) a O) b -H, -C(O)O((CH2) a O) b -(CH2) c H, -C(O)O(C1-5 alkyl), -C(O)-NH-((CH2) d NH) e -H, -C(O)-NH-((CH2) d NH) e -(CH2) f H, -C(O)-X 1 -((CH2) g X 2 ) h -(CH2) i H, -C(O)-X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l H, -O-((CH2) a O) b -H, -O-((CH2) a O) b -(CH2) c H, -O-(C1-5 alkyl), -NH-((CH2) d NH) e-H, -NH-((CH2) d NH) e -(CH2) f H, and -X 1 -((CH2) g X 2 ) h -(CH2) i H, -X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l H. In some embodiments, each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each of a, b, c, d, e, f, g, h, i, j, k, and l may be independently greater than 10. In some embodiments, X 1 , X 2 , and X 3 Each instance of X is independently selected from -O-, -S-, -NH-, and C(O)NH-. 1 , X 2 , and X 3 At least one example of X is an NO-donor moiety. 1 , X 2 , and X 3 At least one instance of is represented by one of the following: [ka] ; and

[0008] In some embodiments, each instance of R1, R2, R3, R4, R5, or R6 is independently selected from the group consisting of: [ka] In some embodiments, each secondary amine of the above structure may be functionalized with an NO-donor group as disclosed herein.

[0009] In some embodiments, the polymer (or scaffold made therefrom) has a viscosity of about 10 mPa·s or greater at a concentration of 5% w / w at 20° C. In some embodiments, the polymer (or scaffold made therefrom) has a gel stiffness of about 1.0 Nm or greater at a concentration of 5% w / w. In some embodiments, the viscosity may be greater, such as about 10 mPa·s, about 20 mPa·s, about 30 mPa·s, about 50 mPa·s, about 60 mPa·s, about 70 mPa·s, about 80 mPa·s, about 90 mPa·s, about 100 mPa·s, or any viscosity therebetween. In some embodiments, the gel stiffness may be greater, such as about 5 Nm, about 10 Nm, about 20 Nm, about 30 Nm, about 40 Nm, about 50 Nm, about 75 Nm, about 100 Nm, or any gel stiffness therebetween.

[0010] In some embodiments, the polymer is a biopolymer. In some embodiments, the polymer is a polysaccharide. In some embodiments, one or more structural units are represented by a saccharide unit of formula I. [ka] Formula I

[0011] In some embodiments, the saccharide units are representative of carboxymethylcellulose structural units. In some embodiments, the structure of formula I represents the saccharide units of a hyaluronic acid polymer. In some embodiments, the structure of formula I represents the saccharide units of a hydroxyethylcellulose polymer. In some embodiments, the structure of formula I represents the saccharide units of a methylcellulose polymer. In some embodiments, the structure of formula I represents the saccharide units of an alginate polymer. In some embodiments, the structure of formula I represents the saccharide units of a cyclodextrin ring structure.

[0012] In some embodiments, provided herein are NO-releasing carboxymethylcellulose-derived polymeric compounds comprising a unit structure of formula I: [ka] Formula I

[0013] In some embodiments, R1, R2, and R3 are independently -OH, -CH2OH, -OCH2C(O)OH, -CH2OCH2C(O)OH, -C(O)-O-((CH2) a O) b -H, -C(O)-O-((CH2) a O) b -(CH2) c H, -C(O)-O-(C1-5 alkyl), -C(O)-NH-((CH2) d NH) e -H, -C(O)-NH-((CH2) d NH) e -(CH2) f H, -C(O)-X 1 -((CH2) g X 2 ) h -(CH2) i H, -C(O)-X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l H, -O-((CH2) a O) b -H, -O-((CH2) a O) b -(CH2) c H, -O-(C1-5 alkyl), -NH-((CH2) d NH) e -H, -NH-((CH2) d NH) e -(CH2) f H, -X 1 -((CH2) g X 2 ) h -(CH2) iH, -X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l In some embodiments, each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, X 1 , X 2 , and X 3 Each instance of X is independently selected from -O-, -S-, -NH-, and C(O)NH-. 1 , X 2 , and X 3 At least one of the following is represented by one of the following: [ka]

[0014] In some embodiments, the carboxymethylcellulose derived polymeric compound has a viscosity of about 10 mPa·s or greater at a concentration of 5% w / w in water at 20° C.

[0015] In some embodiments, formula I has the stereochemical configuration shown in formula I'. [ka] Formula I'

[0016] In some embodiments, X 1 , X 2 , and X 3 At least one of the following is represented by: [ka]

[0017] In some embodiments, R 1 is -CH2C(O)-X1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l In some embodiments, R2, and R3 are -OH. In some embodiments, each instance of R1, R2, and R3 is independently selected from the group consisting of: [ka]

[0018] In some embodiments, the compound has a viscosity of about 20 mPa·s or greater at 20° C. at a concentration of 20% w / w in water. In some embodiments, the compound is soluble in water at a concentration of 50 mg / ml. In some embodiments, the compound has a total releasable NO pool in the range of 0.1-1.0 μmol NO / mg of compound. In some embodiments, the compound has a NO half-life in the range of 0.1-24 hours. In some embodiments, the compound has a total duration of NO release in the range of 1-60 hours. In some embodiments, the total NO release after 4 hours is in the range of 0.1-1.0 μmol NO / mg of compound. In some embodiments, more than 15% of the repeat units in the compound are monomers of formula I. In some embodiments, the compound has a molecular weight in the range of about 90 kDa to about 700 kDa. In some embodiments, the compound comprises two or more different covalently modified monomers of formula I.

[0019] In some embodiments, provided herein are NO-releasing hyaluronic acid-derived polymeric compounds comprising a unit structure of formula II: [ka] Formula II

[0020] In some embodiments, each instance of R1, R2, R3, R4, R5, and R6 is independently -OH, -NH2, -CH2OH, -C(O)OH, -NHC(O)-CH3, -O-((CH2) a O) b -H, -O-((CH2) a O) b -(CH2) c H, -O-(C1-5 alkyl), -NH-((CH2) d N.H. e -H, -NH-((CH2) d N.H. e -(CH2) f H, -X 1 -((CH2) g X 2 ) h -H, -X 1 -((CH2) g X 2 ) h -(CH2) i H, or -X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l In some embodiments, each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 1 , X 2 , and X 3 Each instance of X is independently selected from -O-, -S-, -NH-, and C(O)NH-. 1 , X 2 , and X 3 At least one of the following is represented by one of the following: [ka]

[0021] In some embodiments, the compound has a viscosity of about 10 mPa·s or greater at 20° C. at a concentration of 5% w / w in water.

[0022] In some embodiments, formula II has the stereochemical configuration shown in formula II'. [ka] Formula II'

[0023] In some embodiments, X 1 , X 2 , and X 3 At least one of the following is represented by: [ka]

[0024] In some embodiments, R 1 is -CH2C(O)-X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l In some embodiments, R1, R2, R3, R4, R5, and R6 are independently selected from the group consisting of: [ka]

[0025] In some embodiments, the compound has a viscosity of about 20 mPa·s or greater at 20° C. at a concentration of 20% w / w in water. In some embodiments, the compound is soluble in water at a concentration of 50 mg / ml. In some embodiments, the compound has a total releasable NO pool in the range of 0.1-1.0 μmol NO per mg of compound. In some embodiments, the compound has a NO half-life in the range of 0.1-24 hours. In some embodiments, the compound has a total duration of NO release in the range of 1-60 hours. In some embodiments, the total NO release after 4 hours is in the range of 0.1-1.0 μmol NO per mg of compound. In some embodiments, the compound has a molecular weight in the range of about 6 kDa to about 90 kDa.

[0026] In some embodiments, the polymer comprises a polyaminoglycoside. In some embodiments, the polyaminoglycoside is a hyperbranched polyaminoglycoside comprising a first aminoglycoside of formula III: [ka] Formula III; In the formula, G 1 is selected from the group consisting of: [ka] In the formula, G 2 is selected from the group consisting of: [ka]

[0027] In some embodiments, R 1 Each instance of is independently selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted polyamino having 1-6 repeat units with an intervening C1-C6 alkyl group, optionally substituted polyether having 1-6 repeat units with an intervening C1-C6 alkyl group, or represents a covalent bond to a linking unit. aEach instance of is independently selected from -H, -OH, and C1-C6 alkyl. 1 At least one example of the present invention shows a covalent bond to one or more linking units selected from the following: [ka] During the ceremony, [ka] indicates attachment to the first aminoglycoside. In some embodiments, W 1 Each instance of R, when present, is independently selected from one or more additional aminoglycosides, or one or more end-capping substituents, and at least one linking unit provides a covalent bridge from a first aminoglycoside to a second aminoglycoside. a Each instance of is independently selected from the group consisting of an optionally substituted C1-C6 alkyl, an optionally substituted polyamino having 1-6 repeat units (having C1-C6 alkyl), or an optionally substituted polyether having 1-6 repeat units (having C1-C6 alkyl). In some embodiments, one or more end-capping substituents, if present, are independently selected from the group consisting of -X 1 -((CH2) h X 2 ) i -(CH2) j It has the formula H.

[0028] In some embodiments, the polymer further comprises an end group selected from the group consisting of: [ka]

[0029] In some embodiments, R 5 Each example is H or -N + (=NO-)O-, and / or represented by: [ka]

[0030] In some embodiments, the polymer comprises an end group selected from the group consisting of: [ka]

[0031] Some embodiments relate to methods of delivering nitric oxide to a subject in need of treatment. In some embodiments, an effective amount of a compound or viscosity inducing agent is administered to the subject. In some embodiments, the effective amount of the compound or viscosity inducing agent is administered as a hydrogel or a hydrogel is formed at the site of administration (e.g., in vivo). In some embodiments, the subject is a patient with a wound and the compound or viscosity inducing agent is administered to aid in wound healing. In some embodiments, the subject is in need of tissue replacement and the compound or viscosity inducing agent is administered as a tissue scaffold or filler and / or tissue regrowth promoter.

[0032] Some embodiments relate to a method of treating a disease state, in which an effective amount of a compound or viscosity inducing agent is administered to a subject in need thereof, the disease state being selected from the group consisting of cancer, cardiovascular disease, microbial infection, platelet aggregation and adhesion caused by exposure of blood to medical devices, pathological conditions resulting from abnormal cell proliferation, transplant rejection, autoimmune disease, inflammation, vascular disease, scar tissue, wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunction, and sexually transmitted diseases.

[0033] Some embodiments relate to a pharmaceutical formulation comprising a compound (eg, a polymer) or a viscosity inducing agent and a pharma- ceutically acceptable excipient.

[0034] Some embodiments relate to a method of reducing or preventing microbial load on a surface. In some embodiments, a compound or viscosity inducing agent is applied to a surface contaminated with multiple microorganisms. In some embodiments, the compound or viscosity produces nitric oxide and induces oxidative and / or nitrosative damage to microbial DNA and membrane structures, thereby preventing or reducing the microbial load. In some embodiments, the multiple microorganisms include one, two, or more of gram-positive bacteria, gram-negative bacteria, fungi, yeast, and viruses. In some embodiments, the surface is an organic surface. In some embodiments, the surface is human skin. In some embodiments, the surface is epithelial tissue. In some embodiments, the surface is a wound surface. In some embodiments, the surface is animal skin. In some embodiments, the application does not induce skin irritation.

[0035] In some embodiments, the surface is an inorganic surface. In some embodiments, the inorganic surface is an exterior or interior surface of a medical device. In some embodiments, application of the compound produces an antimicrobial coating on the exterior or interior surface of the medical device. In some embodiments, the medical device comprises an endoscope.

[0036] In some embodiments, the microbial load includes drug-resistant bacteria. In some embodiments, the microbial load includes microorganisms associated with the presence of one or more of human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, hepatitis, coxsackie virus, shingles, measles, mumps, rubella, rabies, pneumonia, hemorrhagic viral fever, H1N1, prions, parasites, fungi, molds, Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, Group A Streptococcus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, carbapenem-resistant Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, and Burkholderia cepacia. In some embodiments, the microbial load includes methicillin-resistant Staphylococcus aureus. In some embodiments, the microbial load includes carbapenem-resistant Enterobacteriaceae. In some embodiments, the microbial burden comprises Staphylococcus aureus. In some embodiments, the microbial burden comprises Pseudomonas aeruginosa. In some embodiments, the microbial burden comprises Burkholderia cepacia. In some embodiments, the NO donor produces nitric oxide, causing damage to the membrane and / or DNA of the microorganisms, thereby reducing the number of viable microorganisms. In some embodiments, the plurality of microorganisms comprises one or more of viruses, gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof.

[0037] Some embodiments relate to methods of preventing and / or reducing microbial contamination. In some embodiments, the method comprises contacting a surface contaminated with multiple microorganisms (or a surface that may be exposed to microorganisms) with a NO-releasing scaffold. In some embodiments, the NO donor of the scaffold produces NO and causes damage to the membrane and / or DNA of the microorganisms, thereby reducing the number of viable microorganisms and / or preventing colonization or infection of the area with the microorganisms. In some embodiments, the surface comprises an organic surface. In some embodiments of the method, the surface is human skin or animal skin. In some embodiments of the method, the surface is in tissues in or around the mouth (e.g., lips, nostrils, teeth, gums, etc.). In some embodiments, the surface comprises oral mucosa. In some embodiments, the surface is in the lungs. In some embodiments, the surface is an inorganic surface (such as a device). In some embodiments, the surface is an inorganic surface. In some embodiments, the inorganic surface is an external or internal surface of a medical device. In some embodiments, the device is a dental device. Advantageously, in some embodiments of the method, the applying step does not induce skin or tissue irritation. In some embodiments, the plurality of microorganisms comprises one or more of viruses, gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof.

[0038] Some embodiments relate to a method of making any one of the compounds or viscosity inducing agents disclosed herein, comprising selecting a polymer and functionalizing the polymer with an NO-binding moiety. In some embodiments, the polymer is a biopolymer. In some embodiments, the method comprises exposing the compound or viscosity inducing agent to NO to provide an NO-donor compound or viscosity inducing agent.

[0039] It should be understood that the compositions and related methods described in more detail below describe particular actions taken by a practitioner, but may also include instructions for those actions by another party. Thus, an action such as "administration of a NO-donating scaffold" includes "instructions for administration of a NO-donating scaffold." [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows the FTIR analysis of the amine modified CMC.

[0041] [Diagram 2] Figure 2 shows the purity analysis of amine-functionalized hyaluronic acid derivatives. (A) shows the analysis of unreacted starting material, (B) shows the analysis of the amine-modified 6 kDa HA derivative, and (C) shows the analysis of the amine-modified 90 kDa HA derivative via HPLC-ELSD. The amine-modified HA derivative does not contain detectable amounts of EDC and NHS reactants.

[0042] [Figure 3-1] Figure 3 shows representative 1H NMR and 13C NMR spectra of unmodified hyaluronic acid. (A) shows a representative 1H NMR spectrum, and (B) shows a representative 13C NMR spectrum of HA6 in DO. [Figure 3-2] Same as above.

[0043] [Figure 4-1] Figure 4 shows representative 1H NMR spectra of secondary amine functionalized hyaluronic acid: (A) HA6-PAPA, (B) HA90-PAPA, (C) HA6-HEDA, (D) HA90-HEDA, (E) HA6-DPTA, (F) HA90-DPTA, (G) HA6-DETA, and (H) HA90-DETA in DO. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0044] [Figure 5-1]Figure 5 shows representative 13C NMR spectra for secondary amine functionalized hyaluronic acid and a comparison of representative 13C NMR for unmodified and amine modified hyaluronic acid. (A) shows representative 13C NMR for HA6-PAPA, (B) shows representative 13C NMR for HA90-PAPA, (C) shows representative 13C NMR for HA6-HEDA, (D) shows representative 13C NMR for HA90-HEDA, (E) shows representative 13C NMR for HA6-DPTA, (F) shows representative 13C NMR for HA90-DPTA, (G) shows representative 13C NMR for HA6-DETA, and (H) shows representative 13C NMR for HA90-DETA in DO. (I) shows representative 13C NMR for unmodified and amine modified hyaluronic acid. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0045] [Figure 6-1] Figure 6 shows representative UV-Vis spectra of the following secondary amine functionalized and NO-releasing hyaluronic acids: (A) HA6-PAPA, (B) HA90-PAPA, (C) HA6-HEDA, (D) HA90-HEDA, (E) HA6-DPTA, (F) HA90-DPTA, (G) HA6-DETA, and (H) HA90-DETA. Modifications include representative UV-Vis spectra of control (-) hyaluronic acid, and NO-releasing (- -) hyaluronic acid. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0046] [Figure 7-1]Figure 7 shows the real-time NO release profiles and cumulative NO release of 6 and 90 kDa NO-releasing hyaluronic acid. (A-B) show the real-time NO release profiles for the first 30 min of release, and (C-D) show the total cumulative NO release, for (A,C) 6 kDa and (B,D) 90 kDa NO-releasing hyaluronic acid in PBS (10 mM, pH 7.4, 37°C), with modifications including PAPA (- -), HEDA (-), DPTA (· · ·), and DETA (- · -). (E) also shows the real-time measurement of NO release. [Figure 7-2] Same as above.

[0047] [Figure 8] FIG. 8 shows the FTIR spectra of unmodified and amine-modified CMC scaffolds.

[0048] [Figure 9] Figure 9 shows time-based bactericidal assays of 6 kDa NO-releasing HA derivatives against (A) P. aeruginosa and (B) S. aureus. Treatments included HA6-PAPA / NO (blue circles), HA6-HEDA / NO (green squares), HA6-DPTA / NO (red triangles), HA6-DETA / NO (purple diamonds), and no treatment (black crosses). All HA derivatives were prepared at the same dose, 2 mg mL-1 for P. aeruginosa eradication and 16 mg mL-1 for S. aureus eradication.

[0049] [Figure 10-1] Figure 10 shows the antibacterial efficacy of the active ingredients (neomycin or NO) against (A) E. coli, (B) P. aeruginosa, (C) S. aureus, (D) E. faecalis, (E) MDR-P. aeruginosa, and (F) MRSA after treatment with HA6-DPTA / NO (blue circles), HA90-DPTA / NO (green squares), or neomycin sulfate (red triangles). NO dose was calculated from the total NO released over a 4-h exposure time in PBS (10 mM, pH 7.4, 37°C) for HA6-DPTA / NO and HA90-DPTA / NO. [Figure 10-2] Same as above.

[0050] [Figure 11] FIG. 11 shows biofilm viability after treatment of pre-existing biofilms of (A) P. aeruginosa and (B) MDR-P. aeruginosa in solution with equivalent active ingredient doses of neomycin sulfate (solid) or HA6-DPTA / NO (stripes) for 24 h.

[0051] [Figure 12-1] Figure 12 shows the results of in vitro cytotoxicity. (A1) shows the concentration of amine-modified hyaluronic acid derivatives (solid) and NO-releasing hyaluronic acid derivatives (stripes) required to reduce the enzyme activity of L929 mouse fibroblast cells by 50% (IC50). (A2) shows the concentration of amine-modified hyaluronic acid derivatives and NO-releasing hyaluronic acid derivatives required to reduce the enzyme activity of L929 mouse fibroblast cells by 50% (IC50), as well as the inhibitory active ingredient dose of NO-releasing hyaluronic acid. In Figure 12A2, HA6 is the left bar of each pair of bars. (B) shows the dose of NO released from hyaluronic acid derivatives required to reduce enzyme activity by 50%. [Figure 12-2] Same as above.

[0052] [Figure 13-1] Figure 13 shows the antibacterial efficacy of 6 and 90 kDa NO-releasing hyaluronic acid derivatives against E. coli, P. aeruginosa, S. aureus, and E. faecalis. Shown is the antibacterial efficacy of (A-D) 6 kDa and (E-H) 90 kDa NO-releasing hyaluronic acid against (A,E) E. coli, (B,F) P. aeruginosa, (C,G) S. aureus, and (D,H) E. faecalis. Modifications include PAPA (blue circle), HEDA (green square), DPTA (red triangle), and DETA (purple diamond). [Figure 13-2] Same as above. [Figure 13-3] Same as above.

[0053] [Figure 14-1]Figure 14 shows colonies remaining after treatment of E. coli, P. aeruginosa, S. aureus, and E. faecalis with 6 and 90 kDa amine-functionalized (control) hyaluronic acid. (A) E. coli, (B) P. aeruginosa, (C) S. aureus, and (D) E. faecalis colonies remaining after 4 hours of treatment with 6 kDa and 90 kDa amine-modified hyaluronic acid (no NO). Modifications include PAPA (blue), HEDA (green), DPTA (red), and DETA (purple). All modifications were evaluated for (A) E. coli and (B) P. aeruginosa at 8 mg mL-1. Modifications were evaluated for (C) S. aureus and (D) E. faecalis at 16 mg mL-1 unless higher doses were required for eradication by the NO-releasing derivatives. For both S. aureus and E. faecalis, HA6-DETA, HA90-HEDA, and HA90-DETA were evaluated at 32 mg mL-1. For E. faecalis, HA90-PAPA was also evaluated at 32 mg mL-1. Of note, none of the amine-modified HA derivatives were bactericidal (≥3 log reduction) at the concentrations evaluated. [Figure 14-2] Same as above.

[0054] [Figure 15] Figure 15 shows the bacterial kill curves of 6 and 90 kDa DPTA-functionalized and NO-releasing hyaluronic acid against MDR-P. aeruginosa and MRSA. The antibacterial efficacy of 6 kDa (circles) and 90 kDa (squares) DPTA-modified (hollow) and NO-releasing (solid) hyaluronic acid against antibiotic-resistant bacterial strains including multidrug-resistant P. aeruginosa (red) and methicillin-resistant Staphylococcus aureus (blue) is shown.

[0055] [Figure 16-1]Figure 16 shows the anti-biofilm efficacy of 6 and 90 kDa NO-releasing DPTA-modified HA against P. aeruginosa and MDR-P. aeruginosa biofilms. Biofilm viability is shown after 24 hours of treatment of (A-B) P. aeruginosa biofilms and (C-D) MDR-P. aeruginosa biofilms with HA6-DPTA / NO (blue circles), HA90-DPTA / NO (green squares), or neomycin sulfate (red triangles). Alternative profiles of biofilm viability of 6 and 90 kDa NO-releasing DPTA-modified HA and neomycin against P. aeruginosa and MDR-P. aeruginosa biofilms are shown in (E) and (F). [Figure 16-2] Same as above.

[0056] [Figure 17] Figure 17 shows biofilm viability after 24 h exposure of P. aeruginosa biofilms and MDR-P. aeruginosa biofilms to blank (left, grey), control HA6-DPTA (middle, blue), or control HA90-DPTA (right, green) solutions. Solutions of HA were prepared at MBEC24 h for each NO-releasing derivative. Of note, HA90-DPTA was prepared at 32 mg mL-1 for testing of MDR-P. aeruginosa biofilms. This is due to the lack of MBEC24 h for the NO-releasing derivatives.

[0057] [Figure 18] FIG. 18 shows the viability of L929 mouse fibroblast cells after 24 hours of treatment with unmodified 6 kDa (blue, left) and 90 kDa (green, right) hyaluronic acid.

[0058] [Figure 19-1] Figure 19 shows dose-response curves following 24 h treatment of L929 mouse fibroblast cells with amine-modified (hollow) and NO-releasing (solid) HA derivatives. Modifications of 6 kDa (red circles) and 90 kDa (blue squares) HA include (A) PAPA, (B) HEDA, (C) DPTA, and (D) DETA. [Figure 19-2] Same as above.

[0059] [Figure 20] FIG. 20 shows the metabolic activity of HGF-1 cells determined via MTS assay after 24 h exposure to (A) CMC-DETA (solid), CMC-DPTA (diagonal stripes), CMC-HEDA (horizontal stripes), and CMC-PAPA (dots), and (B) CMC-DETA / NO (solid), CMC-DPTA / NO (diagonal stripes), CMC-HEDA / NO (horizontal stripes), and CMC-PAPA / NO (dots).

[0060] [Figure 21-1] FIG. 21 shows the UV-vis absorption spectra in 50 mM NaOH of A) CMC-DETA (solid line) and CMC-DETA / NO (dashed line), B) CMC-DPTA (solid line) and CMC-DPTA / NO (dashed line), C) CMC-HEDA (solid line) and CMC-HEDA / NO (dashed line), and D) CMC-PAPA (solid line) and CMC-PAPA / NO (dashed line). [Figure 21-2] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Some embodiments disclosed herein provide the synthesis and characterization of N-diazeniumdiolate NO donor modified scaffolds and their use in antimicrobial applications. In some embodiments, the scaffold comprises a polymer. In some embodiments, the scaffold comprises a biopolymer. In some embodiments, the scaffold comprises a biocompatible polymer. In some embodiments, the scaffold comprises one or more saccharide units and / or is a polysaccharide. In some embodiments, the scaffold comprises one or more of chitosan, hyaluronic acid (HA), carboxymethylcellulose (CMC), hydroxyethylcellulose, methylcellulose, cellulose, alginate (containing 1,4-linked α-l-guluronic acid (G) and β-d-mannuronic acid (M) units), collagen, gelatin, cyclodextrin (e.g., having 5 (α), 6 (β), 7 (γ), or more α-D-glycopyranosides), aminoglycosides (e.g., kanamycin, streptomycin, tobramycin, gentamicin, neomycin, etc.), elastin, repeating units thereof, structural units thereof, or combinations thereof. In some embodiments, one or more polymers are crosslinked to form the scaffold. In some embodiments, the polymers are not crosslinked to form the scaffold. In some embodiments, the scaffold allows for efficient reduction in viability and / or eradication of microorganisms (e.g., prokaryotic cells, bacteria, protozoa, fungi, algae, amoebae, slime molds, etc., and particularly those microorganisms that express at least some degree of drug resistance) with low toxicity to native tissue and patient cells (e.g., eukaryotic cells, mammalian cells, human cells, etc.).

[0062] Unless otherwise defined, 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 subject matter belongs. The terms used in the description of the subject matter herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the subject matter.

[0063] The term "effective amount" as used herein, as will be known in the art, is that amount of the recited compound that confers a modulating effect, which may be a beneficial effect on a subject suffering from a disorder, disease, or illness, including, for example, improving the condition (e.g., one or more symptoms) of the subject, slowing or reducing the progression of the condition, preventing or delaying the onset of the disorder, and / or changing clinical parameters, diseases, or illnesses. For example, an effective amount may refer to the amount of a composition, compound, or agent that improves the condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In some embodiments, the improvement in the condition may be a reduction in infection. In some embodiments, the improvement may be a reduction in bacterial load (e.g., bioburden) on a surface or in the subject. The actual dosage level of the active ingredient in the active composition of the subject of the present disclosure can be changed so as to administer an effective amount of the active compound to achieve the desired response for a specific subject and / or application.The selected dosage level will depend on various factors, including but not limited to the activity of the composition, formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated.In some embodiments, the minimum dosage is administered, and the dosage is titrated up to the minimum effective amount in the absence of dose-limiting toxicity.The determination and adjustment of the effective dosage, and the evaluation of when and how to make such adjustments are contemplated herein.

[0064] The term "biopolymer" refers to polymeric substances that occur in living organisms, including polynucleotides (e.g., DNA, RNA), polysaccharides (e.g., cellulose), proteins (e.g., polypeptides), glycopeptides, peptidoglycans, and the like.

[0065] "Treate" or "treating" or "treatment" refers to any type of action that imparts a modulating effect, which can be a beneficial effect to a subject afflicted with a disorder, disease, or condition, including, for example, amelioration of the subject's condition (e.g., one or more symptoms), slowing or reducing the progression of a condition, and / or a change in clinical parameters, a disease or condition, a cure of a condition, etc.

[0066] The terms "disrupting" and "eradicating" refer to the ability of the disclosed structures to combat biofilms. A biofilm may be partially eradicated or destroyed, meaning that the cells are no longer attached to each other or to a surface. A biofilm may be completely eradicated, meaning that the biofilm is no longer an interconnected, cohesive, or continuous network of cells to any substantial degree.

[0067] The term "nitric oxide donor" or "NO donor" refers to species and / or molecules that donate, release, and / or directly or indirectly transport nitric oxide species and / or stimulate endogenous production of nitric oxide in vivo and / or increase endogenous levels of nitric oxide in vivo, such that the biological activity of the nitric oxide species is expressed at an intended site of action.

[0068] The terms "nitric oxide releasing" or "nitric oxide donating" refer to a species that donates, releases, and / or directly or indirectly delivers any one (or more) of the three redox forms of nitric oxide (NO+, NO-, NO (e.g., ·NO)) and / or a method that donates, releases, and / or directly or indirectly delivers any one (or more) of the three redox forms of nitric oxide (NO+, NO-, NO). In some embodiments, nitric oxide release is achieved such that the biological activity of the nitric oxide species is expressed at the intended site of action.

[0069] As used herein, the term "microbial infection" refers to bacterial infections, fungal infections, viral infections, yeast infections, as well as other microorganisms, and combinations thereof.

[0070] The "patient" or "subject" treated as disclosed herein is, in some embodiments, a human patient, although it should be understood that the principles of the disclosed subject matter indicate that the disclosed subject matter is effective for all vertebrate species, including mammals, which are intended to be included in the terms "subject" and "patient". Suitable subjects are generally mammalian subjects. The subject matter described herein finds use in research, as well as veterinary and medical applications. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cows, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats or mice), monkeys, and the like. Human subjects include neonatal, infant, juvenile, adult, and geriatric subjects. A subject "in need of" the methods disclosed herein may be a subject experiencing and / or expected to experience a disease state, and the methods and compositions of the present invention are used for therapeutic and / or prophylactic treatment.

[0071] For the general chemical formulas provided herein, if no substituents are shown, one of ordinary skill in the art would understand that the substituents are hydrogen. Bonds that are not connected to an atom but are shown indicate that the position of such substituents is variable. A jagged line, wavy line, or double wavy line drawn through or at the end of a bond indicates that some additional structure is attached at that position. For many additional monomers disclosed herein, although not explicitly shown in the structures, one of ordinary skill in the art of polymers would understand that these monomers can be added to alter the physical properties of the resulting polymeric material, even when elemental analysis does not indicate that such a distinction would be expected. Such physical properties include solubility, charge, stability, crosslinking, secondary and tertiary structure, and the like. Additionally, if no stereochemistry is shown for compounds having one or more chiral centers, all enantiomers and diasteromers are included. Similarly, for any description of an aliphatic or alkyl group, all structural isomers thereof are also included. Unless otherwise indicated, A1-A n The groups shown as and referred to herein as alkyl groups in the general formula provided herein are independently selected from alkyl or aliphatic groups, particularly alkyls having 20 or fewer carbon atoms, and even more typically lower alkyls having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl. The alkyls may be optionally substituted (e.g., substituted or unsubstituted, as disclosed elsewhere herein). The alkyls may be substituted alkyl groups, such as, for example, alkyl halides (e.g., -CX3, where X is a halide, and combinations thereof, either in a chain or attached thereto), alcohols (i.e., aliphatic or alkyl hydroxyls, particularly lower alkyl hydroxyls), or other similarly substituted moieties, such as amino-, amino acid-, aryl-, alkylaryl-, alkyl ester-, ether-, keto-, nitro-, sulfhydryl-, sulfonyl-, sulfoxide-modified alkyl groups.

[0072] The terms "amino" and "amine" refer to nitrogen-containing groups such as NR3, NH3, NHR2, and NHR, where R can be as described elsewhere herein. Thus, as used herein, "amino" can refer to a primary amine, a secondary amine, or a tertiary amine. In some embodiments, R in one of the amino groups can be a diazeniumdiolate (i.e., NONO).

[0073] Whenever a group is described as "optionally substituted," the group can be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as "unsubstituted or substituted" (or "substituted or unsubstituted"), if substituted, the substituents can be selected from one or more of the indicated substituents. If no substituents are indicated, it means that the indicated "optionally substituted" or "substituted" group may be individually substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), cycloalkyl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, amino, monosubstituted amine groups, disubstituted amine groups, monosubstituted amine(alkyl), disubstituted amine(alkyl), diamino groups, polyamino, diether groups, and polyether groups.

[0074] As used herein, "C" is a formula in which "a" and "b" are integers. a ~C b" refers to the number of carbon atoms in the group. A designated group may contain from "a" to "b" carbon atoms, inclusive of "a" and "b". Thus, for example, a "C1-C4 alkyl" or "C1-C4 alkyl" group refers to all alkyl groups having one to four carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. When "a" and "b" are not specified, the broadest range described in these definitions is intended.

[0075] When two "R" groups are described as "taken together," the R groups and the atoms to which they are attached can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic ring. For example, and without limitation, NR a R b R in the group a and R b When are shown as "taken together," it is meant that they are covalently linked to each other to form a ring: [ka]

[0076] As used herein, the term "alkyl" refers to a fully saturated aliphatic hydrocarbon group. The alkyl moiety may be branched or straight chain. Examples of branched alkyl groups include, but are not limited to, isopropyl, sec-butyl, t-butyl, and the like. Examples of straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and the like. An alkyl group may have 1 to 30 carbon atoms (whenever appearing herein, a numerical range such as, for example, "1 to 30" refers to each integer in the given range; for example, "1 to 30 carbon atoms" means that an alkyl group may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms, but this definition also encompasses the occurrence of the term "alkyl" when no numerical range is specified). An "alkyl" group may also be a medium alkyl having 1 to 12 carbon atoms. An "alkyl" group may also be a lower alkyl having 1 to 6 carbon atoms. An alkyl group may be substituted or unsubstituted. By way of example only, "C1-C5 alkyl" indicates that the alkyl chain has from one to five carbon atoms, i.e., the alkyl chain is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl (branched and straight chain), etc. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl.

[0077] As used herein, the term "alkylene" refers to a divalent, fully saturated, straight-chain aliphatic hydrocarbon group. Examples of straight-chain alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene. An alkylene group is: [ka] followed by the number of carbon atoms and optionally followed by "*". For example, [ka] The alkylene group may have 1 to 30 carbon atoms (whenever indicated herein, a numerical range such as "1 to 30" refers to each integer in the given range; for example, "1 to 30 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 30 carbon atoms, but this definition also encompasses the occurrence of the term "alkylene" when no numerical range is specified). The alkylene group may also be a medium alkyl having 1 to 12 carbon atoms. The alkylene group may also be a lower alkyl having 1 to 6 carbon atoms. The alkylene group may be substituted or unsubstituted. For example, the lower alkylene group may be substituted by replacing one or more hydrogens of the lower alkylene group and / or by replacing both hydrogens on the same carbon with a C3-6 monocyclic cycloalkyl group (e.g., [ka] ) can be substituted.

[0078] The term "alkenyl" as used herein refers to a monovalent straight or branched chain radical of 2 to 20 carbon atoms containing a carbon double bond, including, but not limited to, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. An alkenyl group can be unsubstituted or substituted.

[0079] The term "alkynyl" as used herein refers to a monovalent straight or branched chain radical of 2 to 20 carbon atoms containing a carbon triple bond, including, but not limited to, 1-propynyl, 1-butynyl, 2-butynyl, etc. Alkynyl groups can be unsubstituted or substituted.

[0080] As used herein, "cycloalkyl" refers to a fully saturated (no double or triple bonds) monocyclic or polycyclic (such as bicyclic) hydrocarbon ring system. When composed of more than one ring, the rings may be fused, bridged, or joined together in a spiro fashion. As used herein, the term "fused" refers to two rings that share two atoms and one bond. As used herein, the term "bridged cycloalkyl" refers to a compound in which a cycloalkyl contains one or more atomic bonds connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings that share one atom, and the two rings are not connected by a bridge. Cycloalkyl groups can contain 3-30 atoms in the ring, 3-20 atoms in the ring, 3-10 atoms in the ring, 3-8 atoms in the ring, or 3-6 atoms in the ring. Cycloalkyl groups may be unsubstituted or substituted. Examples of mono-cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-1H-phenalenyl, and tetradecahydroanthracenyl, examples of bridged cycloalkyl groups are bicyclo[1.1.1]pentyl, adamantanyl, and norbornanyl, and examples of spirocycloalkyl groups include spiro[3.3]heptane and spiro[4.5]decane.

[0081] As used herein, "cycloalkenyl" refers to a monocyclic or polycyclic (such as bicyclic) hydrocarbon ring system containing one or more double bonds in at least one ring, but if there is more than one, the double bonds cannot form a completely delocalized pi-electron system across all rings (otherwise the group would be "aryl" as defined herein). Cycloalkenyl groups can contain 3-10 atoms in the rings, 3-8 atoms in the rings, or 3-6 atoms in the rings. When composed of more than one ring, the rings may be fused, bridged, or connected together in a spiro fashion. Cycloalkenyl groups may be unsubstituted or substituted.

[0082] As used herein, "aryl" refers to a carbocyclic (all carbon) monocyclic or polycyclic (such as bicyclic) aromatic ring system (including fused ring systems in which two carbocyclic rings share a chemical bond) having a completely delocalized pi-electron system throughout the entire ring. The number of carbon atoms in an aryl group can vary. For example, an aryl group can be any of C6-C 14 Aryl groups, C6-C 10The heteroaryl group may be an aryl group or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene, and azulene. The aryl group may be substituted or unsubstituted. As used herein, "heteroaryl" refers to a monocyclic or polycyclic (such as bicyclic) aromatic ring system (a ring system having a fully delocalized pi-electron system) containing one or more heteroatoms (e.g., 1, 2, or 3 heteroatoms), i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur. The number of atoms in the ring of a heteroaryl group may vary. For example, a heteroaryl group can contain 4 to 14 atoms in the ring, 5 to 10 atoms in the ring, or 5 to 6 atoms in the ring, e.g., 9 carbon atoms and 1 heteroatom; 8 carbon atoms and 2 heteroatoms; 7 carbon atoms and 3 heteroatoms; 8 carbon atoms and 1 heteroatom; 7 carbon atoms and 2 heteroatoms; 6 carbon atoms and 3 heteroatoms; 5 carbon atoms and 4 heteroatoms; 5 carbon atoms and 1 heteroatom; 4 carbon atoms and 2 heteroatoms; 3 carbon atoms and 3 heteroatoms; 4 carbon atoms and 1 heteroatom; 3 carbon atoms and 2 heteroatoms; or 2 carbon atoms and 3 heteroatoms. Additionally, the term "heteroaryl" includes fused ring systems in which two rings, e.g., at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine.The heteroaryl group may be substituted or unsubstituted.

[0083] As used herein, "heterocyclyl" or "heteroalicyclyl" refers to 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, and up to 18-membered monocyclic, bicyclic, and tricyclic ring systems, in which carbon atoms together with 1-5 heteroatoms make up said ring systems. Heterocycles may optionally contain one or more unsaturated bonds positioned in such a way that, however, a fully delocalized pi-electron system does not occur throughout all rings. Heteroatoms are elements other than carbon, including, but not limited to, oxygen, sulfur, and nitrogen. Heterocycles may further contain one or more carbonyl or thiocarbonyl functional groups, such that the definition includes oxo systems, and thio systems, such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be fused, bridged, or joined together in a spiro fashion. As used herein, the term "fused" refers to two rings that share two atoms and one bond. As used herein, the term "bridged heterocyclyl" or "bridged heteroalicyclyl" refers to a compound in which a heterocyclyl or heteroalicyclyl contains one or more atomic bonds connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings that share one atom, and the two rings are not connected by a bridge. Heterocyclyl and heteroalicyclyl groups can contain 3-30 atoms in the ring, 3-20 atoms in the ring, 3-10 atoms in the ring, 3-8 atoms in the ring, or 3-6 atoms in the ring. For example, five carbon atoms and one heteroatom, four carbon atoms and two heteroatoms, three carbon atoms and three heteroatoms, four carbon atoms and one heteroatom, three carbon atoms and two heteroatoms, two carbon atoms and three heteroatoms, one carbon atom and four heteroatoms, three carbon atoms and one heteroatom, or two carbon atoms and one heteroatom. Additionally, any nitrogen in a heteroalicyclic can be quaternized.A heterocyclyl or heteroalicyclic group can be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroalicyclyl" groups include, but are not limited to, 1,3-dioxine, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiine, 1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, cyclohexane, cyclohexane-1,3,5-triazine ... Examples of suitable amines include amines such as amines, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, azepane, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, and benzo-fused analogues thereof (e.g., benzimidazolidinone, tetrahydroquinoline, and / or 3,4-methylenedioxyphenyl). Examples of spiroheterocyclyl groups include 2-azaspiro[3.3]heptane, 2-oxaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-oxaspiro[3.4]octane, and 2-azaspiro[3.4]octane.

[0084] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group connected as a substituent through a lower alkylene group. The lower alkylene and aryl groups of an aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0085] As used herein, "cycloalkyl(alkyl)" refers to a cycloalkyl group connected as a substituent through a lower alkylene group. The lower alkylene and cycloalkyl groups of the cycloalkyl(alkyl) may be substituted or unsubstituted.

[0086] As used herein, "heteroaralkyl" and "heteroaryl(alkyl)" refer to a heteroaryl group connected as a substituent through a lower alkylene group. The lower alkylene and heteroaryl groups of a heteroaralkyl may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, as well as benzo-fused analogs thereof.

[0087] "Heteroalicyclyl(alkyl)" and "heterocyclyl(alkyl)" refer to a heterocyclic or heteroalicyclic group connected as a substituent via a lower alkylene group. The lower alkylene and heterocyclyl of (heteroalicyclyl)alkyl may be substituted or unsubstituted. Examples include, but are not limited to, tetrahydro-2H-pyran-4-yl(methyl), piperidin-4-yl(ethyl), piperidin-4-yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl), and 1,3-thiazinane-4-yl(methyl).

[0088] As used herein, the term "hydroxy" refers to an --OH group.

[0089] As used herein, "alkoxy" refers to the formula -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl) as defined herein. A non-limiting list of alkoxy is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, phenoxy, and benzoxy. Alkoxy may be substituted or unsubstituted.

[0090] As used herein, "acyl" refers to hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), and heterocyclyl(alkyl) connected as a substituent through a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. Acyl may be substituted or unsubstituted.

[0091] As used herein, a "cyano" group refers to a "-CN" group.

[0092] As used herein, the term "halogen atom" or "halogen" means any one of the radioactive stable atoms in column 7 of the periodic table of the elements, such as fluorine, chlorine, bromine, and iodine.

[0093] A "thiocarbonyl" group refers to a "-C(=S)R" group, where R can be the same as defined for O-carboxy. The thiocarbonyl can be substituted or unsubstituted. An "O-carbamyl" group refers to a -C(=S)R group, where R can be the same as defined for O-carboxy. A and R Bmay be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A R B )" group. O-Carbamyl may be substituted or unsubstituted.

[0094] An "N-carbamyl" group is R and R A ROC(═O)N(R A )-" group. N-Carbamyl may be substituted or unsubstituted.

[0095] The "O-thiocarbamyl" group is R A and R B may be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A R B )" group. O-Thiocarbamyl may be substituted or unsubstituted.

[0096] The "N-thiocarbamyl" group is R and R A ROC(=S)N(R A )-" group. N-Thiocarbamyl may be substituted or unsubstituted.

[0097] A "C-amide" group is R A and R B may be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A R B )" group. C-amides may be substituted or unsubstituted.

[0098] An "N-amide" group is one that is selected from R and R A may be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A )-" group. N-amides may be substituted or unsubstituted.

[0099] An "S-sulfonamide" group is R A and R B may be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A R B )" group. S-sulfonamides may be substituted or unsubstituted.

[0100] An "N-sulfonamide" group is R and R A may be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A)-" group. N-Sulfonamides may be substituted or unsubstituted.

[0101] An "O-carboxy" group refers to an "RC(=O)O-" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. O-carboxy can be substituted or unsubstituted.

[0102] The terms "ester" and "C-carboxy" refer to the group "-C(=O)OR", where R can be the same as defined for O-carboxy. Ester and C-carboxy can be substituted or unsubstituted.

[0103] A "nitro" group refers to a "-NO2" group.

[0104] A "sulfenyl" group refers to the "-SR" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The sulfenyl can be substituted or unsubstituted.

[0105] A "sulfinyl" group refers to a "-S(=O)-R" group, where R can be the same as defined for sulfenyl. The sulfinyl can be substituted or unsubstituted.

[0106] A "sulfonyl" group refers to a "SO2R" group, where R can be the same as defined for sulfenyl. The sulfonyl can be substituted or unsubstituted.

[0107] As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen (e.g., monohaloalkyl, dihaloalkyl, trihaloalkyl, and polyhaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl, and pentafluoroethyl. Haloalkyl may be substituted or unsubstituted.

[0108] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more of the hydrogen atoms are replaced by halogen (e.g., monohaloalkoxy, dihaloalkoxy, and trihaloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy, and 2-fluoroisobutoxy. Haloalkoxy may be substituted or unsubstituted.

[0109] As used herein, the terms "amino" and "unsubstituted amino" refer to the group --NH.

[0110] A "monosubstituted amine" group is defined herein as A may be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A " group. R A may be substituted or unsubstituted. Mono-substituted amine groups include, for example, mono-alkylamine groups, mono-C1-C6 alkylamine groups, mono-arylamine groups, mono-C6-C 10 arylamine groups, etc. Examples of mono-substituted amine groups include, but are not limited to, -NH(methyl), -NH(phenyl), etc.

[0111] A "disubstituted amine" group is defined herein as A and R B may be independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). A R B " group. R A and R B can be independently substituted or unsubstituted. Di-substituted amine groups include, for example, di-alkylamine groups, di-C1-C6 alkylamine groups, di-arylamine groups, di-C6-C 10 arylamine groups, etc. Examples of di-substituted amine groups include, but are not limited to, -N(methyl)2, -N(phenyl)(methyl), -N(ethyl)(methyl), and the like.

[0112] As used herein, a "mono-substituted amine(alkyl)" group refers to a mono-substituted amine provided herein connected as a substituent via a lower alkylene group. The mono-substituted amine(alkyl) may be substituted or unsubstituted. Mono-substituted amine(alkyl) groups include, for example, mono-alkylamine(alkyl) groups, mono-C1-C6 alkylamine(C1-C6 alkyl) groups, mono-arylamine(alkyl groups), mono-C6 ... 10 It may include arylamine (C1-C6 alkyl) groups, etc. Examples of mono-substituted amine (alkyl) groups include, but are not limited to, -CH2NH(methyl), -CH2NH(phenyl), -CH2CH2NH(methyl), -CH2CH2NH(phenyl), etc.

[0113] As used herein, a "disubstituted amine(alkyl)" group refers to a disubstituted amine provided herein connected as a substituent via a lower alkylene group. The disubstituted amine(alkyl) may be substituted or unsubstituted. Disubstituted amine(alkyl) groups include, for example, dialkylamine(alkyl) groups, di-C1-C6 alkylamine(C1-C6 alkyl) groups, di-arylamine(alkyl groups), di-C6 ... 10 They may include arylamine (C1-C6 alkyl) groups, etc. Examples of disubstituted amine (alkyl) groups include, but are not limited to, -CH2N(methyl)2, -CH2N(phenyl)(methyl), -CH2N(ethyl)(methyl), -CH2CH2N(methyl)2, -CH2CH2N(phenyl)(methyl), -NCH2CH2(ethyl)(methyl), and the like.

[0114] As used herein, the term “diamino” refers to “—N(R A )R B -N(R C )(R D )" group, where R A , R C , and R D may independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein, where R B connects two "N" groups and (R A , R C , and R D and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 2 A , R B , R C , and R D can independently be further substituted or unsubstituted.

[0115] As used herein, the term "polyamino" refers to "-(N(R A )R B -) n-N(R C )(R D For purposes of illustration, the term polyamino refers to -N(R A ) alkyl-N(R A ) alkyl-N(R A ) alkyl-N(R A ) alkyl-H. In some embodiments, the alkyl of the polyamino is as disclosed elsewhere herein. Although this example has only four repeat units, the term "polyamino" can consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units. R A , R C , and R D may independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein, where R B connects two "N" groups and (R A , R C , and R D and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 2 A , R C , and R D may be independently further substituted or unsubstituted. As described herein, polyamino comprises amine groups having intervening alkyl groups, where alkyl is as defined elsewhere herein.

[0116] As used herein, the term "diether" refers to "-OR B OR A " group, wherein R A may be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein, where R BR connects two "O" groups and can be a substituted or unsubstituted alkylene group. A can independently be further substituted or unsubstituted.

[0117] As used herein, the term "polyether" refers to a repeating -(OR B -) n OR A For purposes of illustration, the term polyether refers to the -Oalkyl-Oalkyl-Oalkyl-Oalkyl-OR A In some embodiments, the alkyl of the polyether is as disclosed elsewhere herein. Although this example has only four repeat units, the term "polyether" may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units. A R may be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. B R can be a substituted or unsubstituted alkylene group. A may be independently further substituted or unsubstituted. As described herein, polyethers include ether groups having intervening alkyl groups, where alkyl is as defined elsewhere herein and may be optionally substituted.

[0118] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may include one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" may include one or more of the same or different alkoxy groups containing one, two, or three atoms.

[0119] As used herein, a radical refers to a species that has a single unpaired electron, such that the species containing the radical can be covalently bonded to another species. Thus, in this context, a radical is not necessarily a free radical. Rather, a radical refers to a specific portion of a larger molecule. The term "radical" can be used interchangeably with the term "group."

[0120] When a range of integers is given, the range includes any number falling within the range, as well as the numbers defining the ends of the range. For example, if the term "an integer from 1 to 20" is used, the integers included in the range are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., up to and including 20.

[0121] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0122] Additionally, as used herein, the term "about" in referring to measurable values ​​such as amounts, doses, times, temperatures, etc. of the compounds or agents of the invention is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. The term "consisting essentially of" (and grammatical variants) shall have its ordinary meaning and shall also mean that the composition or method referred to may contain additional components as long as the additional components do not significantly alter the composition or method. The term "consisting of" (and grammatical variants) shall have its ordinary meaning and shall also mean that the composition or method referred to is closed to the additional components. The term "comprising" (and grammatical variants) shall have its ordinary meaning and shall also mean that the composition or method referred to is open to containing the additional components.

[0123] The subject matter of the present disclosure will be described more fully below. However, those skilled in the art to which the subject matter of the present disclosure belongs, having the benefit of the teachings presented in the foregoing description, will come up with many modifications and other embodiments of the subject matter of the present disclosure described herein. Therefore, it should be understood that the subject matter of the present disclosure should not be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated documents, patents, and similar materials differ from or conflict with this application (including, but not limited to, defined terms, use of terms, techniques described, or similar), this application shall control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety.

[0124] Nitric Oxide Scaffold Nitric oxide, an endogenously produced diatomic free radical, is associated with numerous biological processes including platelet aggregation and adhesion, vasodilation, wound repair, immune response, mediation of angiogenesis, and carcinogenesis. Deficiency of NO may lead to some degree of dysfunction of NO-related physiological systems. Exogenous NO delivery may be an effective strategy for the resolution of biomedical therapies ranging from cardiovascular disease to antimicrobial and anticancer therapy. NO delivery may also be used to achieve antimicrobial activity. However, in some embodiments, the difficulties of NO delivery as a treatment necessitate the use of various synthetic NO donors (e.g., N-diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates) to control NO delivery. N-diazeniumdiolates (NONOates) may be useful as NO donors due to their good stability under physiological conditions and proton-induced NO delivery capability. They have a relatively short biological half-life (seconds) and are inherently reactive. In some embodiments disclosed herein, the NO donor comprises any one of the following nitric oxide releasing moieties: [ka] During the ceremony, [ka] indicates attachment to another atom in the structure (e.g., any of the examples, such as -H, -CH2-, -CH-, etc.). In some embodiments, the NO donor is an N-diazeniumdiolate NO donor. In some embodiments, the NO donor is attached along a linear unit at a secondary amine as disclosed elsewhere herein.

[0125] The synthesis of scaffolds capable of controlled NO storage and release is important to exploit the role of NO in physiology and to develop NO-based therapies. In addition to the effects of NO mentioned above, NO is also a potent antibacterial agent that acts on bacteria via nitrosation and / or oxidative stress. NO is a broad-spectrum antibacterial agent, and in some embodiments, scaffolds that deliver NO can eradicate both bacteria and biofilms, primarily through the formation of reactive NO by-products (e.g., peroxynitrite and nitrous oxide) that cause oxidative and nitrosative damage to microbial DNA and / or membrane structures. Advantageously, the mechanism by which NO exerts its antibacterial effect is broad-spectrum, reducing the risk of bacteria developing resistance. Thus, NO releasers may be good targets for fighting bacterial infections. The antibacterial efficacy of NO releasers may depend on both the NO payload and the associated release kinetics. In some instances, high NO total is a key parameter for effectively evaluating the storage capacity of a good scaffold. Furthermore, in some embodiments disclosed herein, the high density of secondary amine groups confers high NO storage capacity to certain donors. However, too fast NO release and too high NO pools may result in undesirable toxicity to mammalian cells.Therefore, there are challenges to prepare biocompatible NO releasers with high NO pools and low cytotoxicity, and such challenges are addressed, among others, according to some embodiments disclosed herein.

[0126] Some embodiments disclosed herein have one or more of the following advantages: efficient and unique synthetic routes and chemical composition of the resulting polymer constructs. Controllable amounts of secondary amines and a variety of external end groups (e.g., hydroxyl, methyl, hydroxymethyl, and primary amines) can be provided. The NO storage and NO release kinetics of the generated nitric oxide releasing scaffolds can be tailored for specific applications. This tuning is achieved in some embodiments by varying the type and / or number of functionalized monomers of the formulas disclosed herein. In some embodiments, additional functionalization of amines in the generated nitric oxide releasing scaffolds, such as with compounds having different compositions, allows further control over NO release kinetics. In some embodiments, the secondary amine groups directly affect the stability of the N-diazeniumdiolates (or other NO carrier groups), allowing control over both NO storage and release kinetics.

[0127] As disclosed elsewhere herein, nitric oxide not only plays a fundamental role in several important biological processes, but also exhibits functions as an antibacterial or anticancer agent. As disclosed elsewhere herein, various NO donors (e.g., N-diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates) can be used for controlled exogenous NO delivery. N-linked diazeniumdiolates are attractive due to their good stability and easy storage, and spontaneously undergo proton-induced dissociation under physiological conditions to regenerate NO radicals. In some embodiments, progress has been made in the preparation and testing of biocompatible N-diazeniumdiolate-modified scaffolds, including those derived from biopolymers and sugar-derived polymers (e.g., chitosan, hyaluronic acid, CMC, etc.).

[0128] Unlike current treatments, NO, an endogenously produced free radical, eradicates bacteria using a variety of mechanisms, including but not limited to lipid peroxidation, nitrosation of membrane proteins, and DNA damage via reactive oxygen / nitrogen species (e.g., peroxynitrite, dinitrogen trioxide). Due to its multiple bactericidal mechanisms, NO can significantly reduce the risk of fostering bacterial resistance. Furthermore, NO has an improved ability to actively degrade both biofilm matrix and mucus structures, thus allowing for more efficient bactericidal action and mucociliary clearance.

[0129] As disclosed elsewhere herein, some embodiments disclosed herein relate to the use of polymer scaffolds to deliver NO to achieve bactericidal action. In some embodiments, the polymer scaffold is derived from a biopolymer. In some embodiments, the scaffold and / or the biopolymer is water-soluble. In some embodiments, the scaffold and / or the biopolymer is biodegradable. In some embodiments, the polymer scaffold is a hyperbranched structure as disclosed in U.S. Patent Application No. 62 / 737,603, which is incorporated by reference in its entirety for all purposes. In some embodiments, the scaffold is a viscosity enhancing agent.

[0130] In some embodiments, a scaffold, polymer, blend of polymers, etc., has structural units (e.g., repeat units, etc.) along the chain of the polymer. In some embodiments, one or more structural units are functionalized with one or more instances of each of R1, R2, R3, R4, R5, and R6. In some embodiments, each instance of R1, R2, R3, R4, R5, and R6 is independently -OH, NH2, OCH3, C(O)OH, CH2OH, CHOCH3, CHOCH2CH2OH, OCH2C(O)OH, CHOCH2C(O)OH, CH2C(O)OH, NHC(O)CH3, C(O)O((CH2) a O) b H, C(O)O((CH2) a O) b (CH2) cH, -C(O)O(C1-5 alkyl), C(O)NH((CH2) d N.H. e H, C(O)NH((CH2) d N.H. e (CH2) f H, C(O)X 1 ((CH2) g X 2 ) h (CH2) i H, C(O)X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k (CH2) l H, O((CH2) a O) b H, O((CH2) a O) b (CH2) c H, O(C 15 alkyl), NH((CH2) d N.H. e H, NH((CH2) d N.H. e (CH2) f H, X 1 ((CH2) g X 2 ) h (CH2) i H, X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k (CH 2)l H, where a, b, c, d, e, f, g, h, i, j, k, and l are independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; 1 , X 2 , and X 3 Each instance of is independently selected from -O-, -S-, -NH-, C(O)NH-, and 1 , X 2 , and X 3At least one example of is represented by one of the following NO donor groups: [ka]

[0131] For example, underivatized polymer chains having one or more hydroxyl, amino, or carboxyl functional groups can be functionalized and / or derivatized through those functional groups, e.g., adding one or more of R1, R2, R3, R4, R5, and R6. Thus, the methods of the present disclosure are applicable to any biocompatible polymer having one or more of these functional groups pendant from the polymer chain. In some embodiments, the polymer is a biopolymer. In some embodiments, the polymer is a biodegradable polymer. In some embodiments, the polymer is a polysaccharide. In some embodiments, the polysaccharide comprises a polymer derived from chitosan, hyaluronic acid, carboxymethylcellulose, hydroxyethylcellulose, methylcellulose, cellulose, alginate, cyclodextrin, aminoglycosides, or other polysaccharides. In some embodiments, the polysaccharide comprises one or more of the following structures: [ka] [ka] Any one or more of the hydroxyl, amino, or carboxyl functional groups shown above can be functionalized or derivatized through those functional groups, for example, adding one or more of R1, R2, R3, R4, R5, and R6. In some embodiments, any one of the amino groups of the aminoglycoside can be functionalized with a linking unit (disclosed in PCT / IB2018 / 052144, published as WO / 2018 / 178902, which is incorporated herein by reference in its entirety) to tailor the macromolecular structure.

[0132] In some embodiments, the scaffold and / or NO-releasing polymer system comprises one or more structural units represented by Formula I: [ka] Formula I

[0133] In some embodiments, the structural units represented by formula I represent one or more of saccharide units of a cellulose polymer, a hyaluronic acid polymer, an alginate polymer, a chitosan polymer, a carboxymethylcellulose polymer, a hydroxyethylcellulose polymer, a methylcellulose polymer, and / or a cyclodextrin ring structure. In some embodiments, formula I has the stereochemical configuration shown in formula I'. [ka] Formula I' And, the scaffold polymer comprises carboxymethylcellulose.

[0134] In some embodiments, the scaffold and / or NO-releasing polymer system comprises one or more structural units represented by Formula II: [ka] Formula II

[0135] In some embodiments, the structural unit represented by formula I represents one or more of a saccharide unit of a cellulose polymer, a saccharide unit of a hyaluronic acid polymer, and / or a saccharide unit of an alginate polymer. In some embodiments, formula II has the stereochemical configuration shown in formula II': [ka] Formula II' And, the scaffold polymer comprises hyaluronic acid.

[0136] In some embodiments, each instance of R1, R2, R3, R4, R5, and R6 is independently selected from the group consisting of: [ka] In some embodiments, any one of the secondary amines may be functionalized as an NO-donor moiety, including, for example, [ka]

[0137] characteristics In some embodiments, the various structural units (e.g., repeating units), functionalization of the structural units (with various moieties), level of crosslinking (if crosslinked), molecular weight, concentration, or other chemical features of the disclosed scaffolds contribute to the tunability of the properties of the scaffolds disclosed herein. In some embodiments, by varying one or more of these features, one or more of the properties of the scaffold can be tuned. In some embodiments, NO release rate, antimicrobial effect, water solubility, degradation rate, viscosity, gel hardness (if the scaffold forms a gel), viscoelasticity, modulus, etc. can be tuned.

[0138] In some embodiments, the properties of the polymers and / or compositions prepared therefrom can be tailored by adjusting the molecular weight of the polymer used. In some embodiments, the weight average molecular weight in kDa (M w ) is about 2.5, 5.0, 7.0, 10, 15, 30, 50, 100, 200, 500, 750, 1,000, 2,000, 10,000, or a range including and / or spanning the foregoing values, or greater. In some embodiments, the number average molecular weight in kDa (M n) is about 2.5, 5.0, 7.0, 10, 15, 30, 50, 90, 100, 200, 500, 700, 1,000, 2,000, 10,000, or a range including and / or spanning the aforementioned values ​​or more. In some embodiments, the polymers disclosed herein may have n repeat units. In some embodiments, n is about 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, or a range including and / or spanning the aforementioned values ​​or more. In some embodiments, size exclusion chromatography (SEC) can be used to measure the molecular weight of the scaffold structures disclosed herein. In some embodiments, a multi-angle light scattering (SEC-MALS) detector can be used. In some embodiments, the scaffold structure can be characterized using its polydispersity index. The polydispersity index (PDI) is a measure of the distribution of molecular weights in a given polymer sample. The PDI can be calculated by dividing the weight average molecular weight and the number average molecular weight. In some embodiments, the scaffold has a PDI of about 1.05, 1.1, 1.2, 1.3, 1.5, 1.7, 1.8, 1.9, 2.0, or a range including and / or spanning the aforementioned values, or greater.

[0139] In some embodiments, the polymer (or mixture of polymers) may be water soluble and / or miscible with each other. In some embodiments, the scaffold is soluble in water (at about 20° C.) at a concentration of about 1 mg / ml, 10 mg / ml, 20 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml, or a range including and / or spanning the aforementioned values ​​or greater.

[0140] According to some embodiments, different NO-carrying polymers can be combined to prepare an aqueous solution containing a concentration of about 100 μg / mL or more, and can be even higher, such as about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 40 mg / mL or more. The amount of the second polymer in the aqueous composition can be at least about 10% by weight based on the weight of the first polymer, and can be higher (e.g., at least about 20% by weight, at least about 30% by weight, or at least about 50% by weight, on the same basis). The polymers in the aqueous composition are selected such that the polymers are miscible with each other. As mentioned above, a first polymer with antimicrobial activity and a second polymer with antimicrobial activity are considered to be miscible with each other if, upon visual inspection, at least about 90% of the polymer components remain soluble in each other after 24 hours of mixing and maintaining at room temperature in water at a concentration of 1 mg / mL of each polymer. Surprisingly, such mutual miscibility of the water-polymer can be achieved at the 1 mg / ml concentration and molecular weight described herein, despite the expectation of phase separation due to the typical mutual incompatibility of polymers in aqueous solutions. The aqueous compositions described herein can be prepared by mixing the individual polymer components with water, for example, with stirring at room temperature.

[0141] In some embodiments, the polymers (or mixtures of polymers, etc.) disclosed herein have properties characteristic of a viscous fluid and / or gel. In some embodiments, the polymers (or mixtures of polymers, etc.) have a gel point at room temperature (in water or PBS) at a concentration (in w / w%) of about 0.5%, 1%, 2.5%, 5%, 10%, or ranges up to and including and / or throughout the aforementioned values. In some embodiments, the polymers (or mixtures of polymers, etc.) may have a gel point in water. In some embodiments, the polymers gel in water (at about 20° C.) at a concentration of about 0.5 mg / ml, 1 mg / ml, 10 mg / ml, 20 mg / ml, 50 mg / ml, 100 mg / ml, 250 mg / ml, or ranges up to and including and / or throughout the aforementioned values. In some embodiments, in a 5% w / w concentration solution, the polymer has a viscosity (in cPa·s at 20° C.) of about 10, 50, 100, 1,000, 2,000, 5,000, 10,000, or a range including and / or ranging through the aforementioned values, or greater. 3 / kg, 1.0m 3 / kg, 2.0m 3 / kg, 4.0m 3 / kg, 8.0m 3 / kg, or a range including and / or ranging from the aforementioned values.

[0142] In some embodiments, at a concentration of 5% w / w, the polymer has a stiffness of about 1.0 mN, 2.5 mN, 5 mN, 10 mN, 15 mN, 20 mN, 30 mN, 50 mN, or a range including and / or ranging through the aforementioned values, or greater. In some embodiments, at a concentration of 5% w / w, the polymer has a work of adhesion (mN*mm) of about 1.0, 2.5, 5, 10, 15, 20, 30, 50, 100, or a range including and / or ranging through the aforementioned values, or greater. In some embodiments, at a concentration of 5% w / w, the polymer has a storage modulus (G') in Pascals (Pa) of about 250, 500, 1,000, 2,000, 4,000, 5,000, 10,000, or a range including and / or ranging through the aforementioned values, or greater. In some embodiments, at a concentration of 5% w / w in solution, the polymer has an elastic modulus (G") in Pascals (Pa) of about 25, 50, 100, 200, 400, 500, 1,000, 2,000, 5,000, 10,000, or a range including and / or spanning the aforementioned values ​​or greater. In some embodiments, the aqueous composition is characterized by barrier activity as measured by a reduction in the diffusion rate of an anionic dye of more than 2 log at a total scaffold concentration of 40 mg / ml or less.

[0143] In some embodiments, the gels are stable at various temperatures up to 20° C. (e.g., 40° C., 45° C., 55° C., 60° C., 80° C., etc.) and are also stable for extended storage periods (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., days, such as 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., weeks, such as 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., months, such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., or even years (1 year or more)).

[0144] In some embodiments, the viscosity of the composition increases with increasing temperature, as described above. In some embodiments, the viscosity of the composition decreases with decreasing temperature. For example, when the composition is above the gelation temperature, the composition has a relatively high viscosity, as in the form of a gel. In some embodiments, when the composition is cooled below the gelation temperature, the composition decreases in viscosity, as in the form of a liquid. In some embodiments, such polymers disclosed herein may be reversible polymers (e.g., thermoreversible polymers), and the transition from liquid to gel may be reversed when exposed to appropriate conditions. For example, as described above, the compositions of the present disclosure include thermoreversible polymers, and the viscosity of the composition may change depending on the temperature of the composition. In some embodiments, the tunability of the viscosity allows for a tailored composition profile for delivery (e.g., more fluid at the delivery temperature, e.g., more viscous at body temperature).

[0145] In some embodiments, the polymer is characterized by a degree of swelling when exposed to water. In some embodiments, the percent swelling of the polymer disclosed herein is about 100, 250, 500, 1,000, 2,000, 5,000, or a range including and / or spanning the aforementioned values ​​or more. In other words, the polymer may swell or expand 2x, 4x, 5x, 10x, 20x, 50x, 100x, or more.

[0146] In certain embodiments, the polymers disclosed herein have a gelation temperature similar to the normal body temperature of the subject, e.g., similar to human body temperature, 37°C. The gelation temperature refers to the intersection between the plot of elastic modulus and the plot of viscous modulus. In some cases, when the composition is below the gelation temperature, the composition has a relatively low viscosity, such as in the form of a liquid. In some cases, when the composition is above the gelation temperature, the composition increases in viscosity (e.g., polymerizes), such that the composition is in the form of a gel. A composition that transitions from liquid to gel can facilitate administration of the composition to a subject, for example, by facilitating injection of a low-viscosity (e.g., liquid) composition at a temperature below the gelation temperature. After injection of the composition into the target treatment site, the temperature of the composition can increase due to absorption of heat from the surrounding body tissue, such that the composition increases in viscosity (e.g., transitions from liquid to gel or polymerizes), thereby providing structural and / or geometric support to the body tissue at the target treatment site. In some cases, gelation of the composition at the target treatment site may also facilitate retention of the composition at the treatment site by reducing diffusion and / or migration of the composition away from the treatment site. In certain embodiments, the composition has a gelation temperature of 30° C. to 40° C., such as 32° C. to 40° C., including 35° C. to 40° C. In certain examples, the composition has a gelation temperature of 37° C.

[0147] In some embodiments, the methods disclosed herein provide NO-releasing polymers with a NO storage capacity (μmol NO / mg polymer) of about 0.25, 0.4, 0.5, 1.0, 1.5, 2.0, 3.0, or more than ...

[0148] In some embodiments, NO release may occur over about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or a range including and / or spanning the aforementioned values. In some embodiments, the NO release half-life is greater than or equal to about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or a range including and / or spanning the aforementioned values. In some embodiments, NO release may occur for about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or less than or including and / or spanning the above values. In some embodiments, nitrosamine is not present during NO release. As used herein, the phrase "nitrosamine is not present" refers to a level of nitrosamine that is not detectable as determined by UV-vis spectroscopy (or other art-recognized methods).

[0149] In some embodiments, the disclosed scaffolds and / or polymers of the disclosed compositions have a degradation rate per hour in an amylase enzyme exposure assay of less than or equal to about 0.2%, 0.5%, 1.0%, 1.5%, 2.5%, 5.0%, 10%, or a range including and / or spanning all of the aforementioned values.

[0150] In some embodiments, the functionalized NO-releasing polymers of the present disclosure have antimicrobial activity. In some embodiments, the functionalized NO-releasing polymers of the present disclosure provide a bacterial reduction of 90% or more against one or more of Pseudomonas aeruginosa, Staphylococcus aureus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Actinomyces viscosus, and / or Streptococcus mutans in a bacterial viability assay performed under static conditions over a period of 2 hours at a polymer concentration of about 8 mg / ml, 6 mg / ml, 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, or a range including and / or spanning the aforementioned values. In some embodiments, the functionalized NO-releasing polymers of the present disclosure provide a bacterial reduction of 99% or more and / or a 2-3 log reduction in a bacterial viability assay performed under static conditions for 2 hours against gram-positive bacteria at a polymer concentration of about 8 mg / ml, 6 mg / ml, 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, or a range including and / or spanning the aforementioned values ​​or less. In some embodiments, the functionalized NO-releasing polymers of the present disclosure provide a bacterial reduction of 99% or more and / or a 2-3 log reduction in a bacterial viability assay performed under static conditions for 2 hours against gram-negative bacteria at a polymer concentration of about 8 mg / ml, 6 mg / ml, 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, or a range including and / or spanning the aforementioned values ​​or less. In some embodiments, the bacterial reduction is greater than 95%, greater than 98%, or greater than 99%.

[0151] Crosslinking A crosslink is a bond that connects one polymer chain to another (e.g., by covalent or ionic bonds). In some embodiments, polymers that can be crosslinked generally exhibit branches from the backbone. In the presence of a crosslinker, such as calcium cations, negatively charged branches from the same or different chains are attracted to positive cations. The branches that connect the chains together are called "crosslinks." When polymer chains are linked together by crosslinks, they may lose some of their ability to move as individual polymer chains. For example, a liquid polymer (with free-flowing chains) can be converted to a "solid" or "gel" by crosslinking the chains together. This description applies when an anionic polymer, such as sodium alginate, is crosslinked with calcium chloride. The sodium alginate may be kept in solution, but the addition of calcium chloride causes the alginate chains to aggregate or crosslink with the calcium cations, thereby forming an immobilized product. Depending on the embodiment, other crosslinkers may also be used. The generally immobilized product may also generally immobilize other substances that may be present, such as active agents.

[0152] In some embodiments, crosslinks can be formed by chemical reactions initiated by heat, pressure, changes in pH, or radiation. For example, mixing a non-polymerized or partially polymerized resin with certain chemicals called crosslinking agents can result in a chemical reaction that forms crosslinks. Crosslinks can also be induced in normally thermoplastic materials through exposure to radiation sources such as electron beam exposure, gamma irradiation, or UV light. In some embodiments, the polymers disclosed herein can be crosslinked using salts or multifunctional compounds with multiple charges to covalently crosslink the structure (e.g., diamines, triamines, dicarboxylic acids, diepoxides, etc.).

[0153] Calcium chloride, a reagent used in some of the embodiments disclosed herein, provides an example of a simple ionic bond. When calcium (Ca) and chlorine (Cl) are combined, the calcium atoms each lose two electrons to form a cation (Ca 2+), each chlorine atom gains one electron to form an anion (Cl-). These ions then attract each other in a 1:2 ratio to form calcium chloride (CaCl 2) Other cation to anion ratios are possible depending on the materials used. In some embodiments, calcium salts other than calcium chloride may be used as well as other suitable metals, such as other multivalent cations. Similarly, alginates other than sodium alginate may be used, such as potassium alginate and ammonium alginate. Additionally, crosslinking may be used with materials other than alginates (e.g., polysaccharides). In some embodiments, electrostatically crosslinking materials may be used to form a binder suitable for hemostatic applications. In some embodiments, it may be desirable to utilize a converted alginate, i.e., a material that is primarily calcium alginate with partial sodium content, such that at least a portion of the alginate is water soluble.

[0154] Hydrogels can be synthesized by crosslinking each polymer using an appropriate crosslinker selected according to the chemical moieties present along the polysaccharide chain. In some embodiments, amine-containing polymers can be crosslinked with carboxylic acid-containing polymers by simple coupling reactions (e.g., with EDC). For example, hyaluronic acid hydrogels and carboxymethylcellulose hydrogels can be synthesized following the same chemical route, for example, using EDC chemistry. Essentially, amide bonds between the carboxylic acid groups of the polysaccharide and the primary amines of 1,3-diaminopropane (DAP)-crosslinker-can be formed in the presence of EDC. The crosslinker, DAP, can be added to the mixture in a molar ratio of 0.5 to the moles of carboxylic acid of the polymer and to the moles of EDC and NHS.

[0155] composition In some embodiments, the polymer is administered as an aqueous gel, e.g., topically. In some embodiments, the gel contains one or more salts and is isotonic. In some embodiments, the composition can take the form of a tablet or capsule prepared by conventional techniques with pharma- ceutically acceptable excipients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods known in the art. For example, the therapeutic agent may be formulated in combination with hydrochlorothiazide as a pH-stabilized core with an enteric or delayed release coating that protects the therapeutic agent until it reaches the target organ.

[0156] In some embodiments, the composition comprises two or more polymers with a specific ratio (w / w). In some cases, the ratio (w / w) is 1:10, or 1:9, or 1:8, or 1:7, or 1:6, or 1:5, or 1:4, or 1:3, or 1:2, or 1:1, or 2:1, or 3:1, or 4:1, or 5:1, or 6:1, or 7:1, or 8:1, or 9:1, or 10:1. For example, the ratio (w / w) may be in the range of 1:1 to 10:1, such as 2:1 to 10:1, including 3:1 to 10:1, or 4:1 to 10:1, or 4:1 to 9:1, or 4:1 to 8:1, or 4:1 to 7:1, or 4:1 to 6:1. In certain embodiments, the ratio (w / w) is 5:1. The polymers can then be dissolved together in an aqueous solution to provide a gel or solution, hi some embodiments, each polymer of the mixture is provided at a concentration of about 1 mg / ml, 10 mg / ml, 20 mg / ml, 50 mg / ml, 100 mg / ml, 250 mg / ml, or a range up to and including and / or spanning the aforementioned values.

[0157] How to use An unmet need in the field of wound healing, general surgery, and orthopedic surgery is for an antimicrobial material that can form a gel, release NO at the required rate, and degrade during a desired time frame. This tailored degradation rate can be adapted to the healing cycle of each particular condition and / or to the time when the wound is at high risk of infection. Examples of these conditions include treatment of hernias, healing of diabetic foot ulcers, orthopedic repair of tendons, to name just a few. In some embodiments, the compounds and materials disclosed herein target compositions with tunable degradation times.

[0158] Some embodiments provide a method of treating a tissue defect, comprising positioning any of the polymers described herein at, on, or into the tissue defect. In some embodiments, the tissue defect is a wound. Some embodiments provide a method of treating a wound, performing tissue repair, and / or providing tissue and organ replacement. In some embodiments, the first step in treating a tissue defect, treating a wound, and / or tissue replacement and replacement involves identifying a patient in need of an antimicrobial scaffold to aid in the salvage and healing of a tissue defect, wound healing, or a condition in need of tissue replacement.

[0159] A non-limiting list of patients who need an antimicrobial scaffold includes patients suffering from tissue defects. In some embodiments, patients who need an antimicrobial scaffold suffer from wounds, including wounds from burns, skin ulcers, lacerations, bullet holes, animal bites, and other wounds prone to infection. Antimicrobial polymers can also be used to treat diabetic foot ulcers, venous leg ulcers, bedsores, amputation sites, other skin injuries, or other wounds or diseases. Patients who need an antimicrobial scaffold also include patients who need repair and replacement of tendons, ligaments, fascia, and dura mater. The degradable antimicrobial polymers may be used in replacement tissues in procedures including, but not limited to, rotator cuff repair, Achilles tendon repair, leg or arm tendon or ligament repair (e.g., torn ACL), vaginal prolapse repair, bladder sling for urinary incontinence, post-surgery breast reconstruction, hernia treatment, staple or suture reinforcement, bariatric surgery repair, pelvic floor reconstruction, dura repair, gum repair, bone grafting and reconstruction. Additionally, patients in need of antimicrobial scaffolds also include those in need of tissue or organ replacement. In some embodiments, the antimicrobial polymers described herein can be used to replace and / or replace tissues by acting as a filler and / or as an artificial extracellular matrix. In such applications, the antimicrobial scaffolds can be used to support cell and tissue growth. Briefly, cells can be taken from a patient or viable host and seeded onto the antimicrobial scaffold either in vivo or ex vivo. The patient's natural tissue then invades the material, which is then conditioned to degrade, leaving behind only non-infected tissue and cells of natural origin.

[0160] In some embodiments, applications also include the delivery of therapeutic molecules to local sites, use as adhesives or sealants, and joint protection drugs, and in wound healing, among others.The stabilized composition can also be used as tissue fillers, dermal fillers, bone fillers, bulk agents, such as urethral or esophageal bulk agents, and embolization agents, as well as agents for repairing cartilage defects / damage, and agents for enhancing bone repair and / or growth.In some embodiments, the antimicrobial scaffold can be placed in or on a patient to fill a void, such as within a void.

[0161] In some embodiments, a polymer is provided for repairing damaged tissue. In some embodiments, the composition is formulated for administration to a target treatment site in a subject. For example, the composition may be formulated to facilitate administration to a damaged or infected tissue in a subject.

[0162] In some embodiments, after administration of the composition (e.g., antimicrobial scaffold), the composition may increase in temperature by absorbing heat from surrounding body tissue in the subject. In some embodiments, the subject's body temperature is sufficient to increase the viscosity of the composition (e.g., transition from liquid to gel). In some embodiments, the increase in viscosity (e.g., gelation) may result in a three-dimensional network sufficient to provide structural and / or geometric support to body tissue, such as cardiac tissue (e.g., cardiac tissue in an infarcted area). In some embodiments, the composition may be injected in vivo using a syringe or catheter. In some embodiments, the composition may be injected directly into the treatment site, or may be partially preheated in a syringe to increase the viscosity of the composition prior to injection. In some embodiments, a preheated formulation may reduce the likelihood that a less viscous composition may diffuse and / or migrate out of the target tissue area after injection.

[0163] Tooth decay (e.g. dental caries) is another important disease condition affecting 60%-70% of school-age children and the majority of adults in most developed countries. Worldwide, 11% of the total population suffers from severe periodontitis, which leads to tooth loss and systemic diseases such as coronary, cardiovascular, stroke, and adverse pregnancy outcomes. Of the more than 700 microorganisms in the oral cavity, cariogenic bacteria (e.g. Streptococcus mutans, Actinomyces viscosus) and periodontopathogenic bacteria (e.g. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans) play a major role in the initiation and progression of oral diseases. Oral diseases are one of the most common health problems faced by humans. Gram-positive cariogenic bacteria (e.g., Streptococcus mutans, Actinomyces viscosus) and Gram-negative periodontal pathogenic bacteria (e.g., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans) represent the main aggravating factors associated with the development and progression of dental caries and periodontal disease, respectively. Unfortunately, current therapies to combat these pathogens have undesirable side effects. For example, systemic use of antibiotics can result in gastrointestinal disorders and promote bacterial resistance. Chlorhexidine, a common oral antiseptic, can alter taste, stain teeth and tongue, and irritate the buccal mucosa. Polymeric NO delivery vehicles (e.g., silica nanoparticles, gold, etc.) kill Gram-negative periodontal pathogenic bacteria. However, these substances have not been demonstrated to kill Gram-positive cariogenic bacteria at safe concentrations (e.g., concentrations that are bactericidal but non-toxic to mammalian cells). As with these nanomaterials, the lack of biodegradability and potential cytotoxicity of silica nanoparticles also hinder their future for biomedical applications. Current research is also focusing on the use of nanomaterials such as silver, gold, zinc, and copper as alternatives to traditional antibiotics that allow for the promotion of bacterial resistance. However, these nanomaterials may accumulate in the body and cause cumulative toxicity, limiting their future for certain applications. Developing oral therapeutics that can kill these disease-causing bacteria is important in maintaining a healthy oral cavity.In some embodiments, the structures (eg, NO scaffolds and / or polymers) disclosed herein solve one or more of these or other problems.

[0164] In some embodiments, the compositions disclosed herein may be used as eye drop formulations (e.g., artificial tears). In some embodiments, the compositions include a scaffold at about 0.1% to about 1.0% (or concentrations disclosed elsewhere herein). In some embodiments, the mixture includes two or more polymer scaffolds (e.g., an HA-derived scaffold and a CMC-derived scaffold), with the second polymer scaffold being present in an amount of 0.05% to about 0.15% (or concentrations disclosed elsewhere herein).

[0165] Cystic fibrosis (CF) is a genetic disorder characterized by poor mucociliary clearance and chronic bacterial infection. As shown herein, in some embodiments, nitric oxide (NO) has broad-spectrum antibacterial activity against CF-associated bacteria, making it an attractive alternative to traditional antibiotics. Treatment with NO limits bacterial resistance through its multiple bactericidal mechanisms (e.g., induction of nitrosation and oxidative stress). Surprisingly, it has been found that storing NO on a scaffold using one of the disclosed designs improves bactericidal efficacy and reduces systemic cytotoxicity. The treatment is effective against planktonic and biofilm-based pathogens, and cytotoxicity assays on mammalian lung cells show little harm to the cells of treated subjects.

[0166] CF is a debilitating disease characterized by chronic bacterial infection of the lungs, resulting in a short life expectancy of 20 years. A genetic defect in the CF membrane conductance regulator (CFTR) prevents normal transport of ions (e.g., Cl-) into the airway surface liquid, inhibiting water transport. Thus, the airway epithelium becomes dehydrated and produces thick mucus that cannot be efficiently cleared via the mucociliary clearance mechanism. As goblet cells continually excrete mucin into the now dehydrated airways, mucus accumulation accelerates to the point where cilia become damaged or non-functional and are unable to clear mucus from the airways. Planktonic bacteria thrive in this static environment, promoting the formation of complex communities of pathogenic bacteria known as biofilms. The exopolysaccharide matrix produced by these biofilms inhibits oxygen diffusion, creates pockets of anaerobic environments, and alters bacterial metabolism. This combination of a thickened mucus layer and a robust biofilm significantly reduces the antibacterial efficacy of common CF therapies.

[0167] In some embodiments, the microbial load to be reduced and / or eliminated comprises drug-resistant bacteria. In some embodiments, the drug-resistant bacteria comprises carbapenem-resistant Enterobacteriaceae. In some embodiments, the drug-resistant bacteria comprises Methicillin-resistant Staphylococcus aureus. In some embodiments, the microorganisms include human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, hepatitis, coxsackie virus, shingles, measles, mumps, rubella, rabies, pneumonia, prions (such as hemorrhagic viral fever, H1N1), parasites, fungi, molds, yeasts, and bacteria (both gram positive and gram negative), including Candida albicans, Aspergillus niger, E. coli, Pseudomonas aeruginosa, and Staphylococcus aureus, Group A Streptococcus, Pneumococcus, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, P. gingivalis, Aggregatibacter actinomycetemcomitans, A. viscosus, and / or S. mutans, among others, as well as various drug resistant bacteria. The terms microorganism and microbe are intended to be used interchangeably. Microorganisms may include wild-type, genetically engineered, or modified organisms. In some embodiments, the formulations and methods disclosed herein are for topical use or for the treatment of surfaces such as the oral mucosa.

[0168] In some embodiments, the scaffold and / or composition thereof may be administered, for example, by direct injection or application to the damaged tissue. Suitable routes also include injection or application to a site adjacent to the damaged tissue. Administration may include parenteral administration (e.g., intravenous, intramuscular, or intraperitoneal injection), subcutaneous administration, administration into a vascular gap, and / or administration into an arthritis (e.g., intra-articular injection). Additional routes of administration include intranasal, topical, vaginal, rectal, intrathecal, intra-arterial, and intraocular routes. In some embodiments, the scaffold and composition disclosed herein may be applied to the treatment site as a gel. In some embodiments, the scaffold and composition may be applied as a liquid.

[0169] In some embodiments, liquid preparations for oral or topical administration can take the form of, for example, a solution, syrup, or suspension, or can be presented as a dry product for composition with water or another suitable vehicle before use. Such liquid preparations can be prepared by conventional techniques using pharma- ceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid). The preparations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents as necessary. Preparations for oral administration can be suitably formulated to allow controlled release of active compounds. For buccal administration, the composition can take the form of tablets or lozenges formulated in a conventional manner.

[0170] In some embodiments, the disclosed compounds can also be formulated as a preparation for implantation or injection.Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in acceptable oil) or ion exchange resin, or as poorly soluble derivatives (for example, as poorly soluble salts).The compounds can also be formulated into rectal compositions (for example, suppositories or retention enemas that contain conventional suppository bases such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.

[0171] Pharmaceutical formulations suitable for administration as an aerosol by inhalation are also provided. In some embodiments, the polymeric structures described herein are formulated in solution and / or aerosol form. In some embodiments, these formulations include solutions or suspensions of the polymers described herein. In some embodiments, the desired formulations can be placed in a small chamber and nebulized. Nebulization can be achieved by compressed air to form multiple liquid droplets or solid particles containing the NO-releasing hyperbranched polyamidomines, or by ultrasonic energy. For example, the NO-releasing hyperbranched polyamidomines of the present disclosure can be administered via inhalation to treat bacterial infections associated with cystic fibrosis. Cystic fibrosis-associated bacterial infections include, but are not limited to, Stenotrophomonis, M. avium intracellulare, and M. abscessus, Burkholderia cepacia, and P. aeruginosa infections. The subject matter described herein is directed to the following embodiments: 1. A NO-releasing carboxymethylcellulose-derived polymeric compound comprising a unit structure of formula I: [ka] Formula I During the ceremony, R1, R2, and R3 are independently -OH, CH2OH, OCH2C(O)OH, CH2OCH2C(O)OH, C(O)O((CH2) a O) b H, C(O)O((CH2) a O) b (CH2) c H, -C(O)-O-(C1-5 alkyl), C(O)NH((CH2) d N.H. e H, C(O)NH((CH2) d N.H. e (CH2) f H, CH2C(O)NH((CH2) d N.H. e H, CH2C(O)NH((CH2) dN.H. e (CH2) f H, C(O)X 1 ((CH2) g X 2 ) h (CH2) i H, CH2C(O)X 1 ((CH2) g X 2 ) h (CH2) i H, C(O)X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k (CH2) l H, O((CH2) a O) b H, O((CH2) a O) b (CH2) c H, -O-(C1-5 alkyl), NH((CH2) d N.H. e H, NH((CH2) d N.H. e (CH2) f H, X 1 ((CH2) g X 2 ) h (CH2) i H, X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k (CH2) l H, CH2C(O)X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l H, Each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X1 , X 2 , and X 3 each instance of is independently selected from: -O-, -S-, -NH-, C(O)NH-; X 1 , X 2 , and X 3 At least one of is represented by one of the following: [ka] ; and wherein the compound has a viscosity of about 10 mPa·s or greater at a concentration of 5% w / w in water at 20° C.

[0172] 2. The compound according to embodiment 1, wherein formula I has the stereochemical configuration shown in formula I': [ka] Formula I'.

[0173] 3. The compound according to embodiment 1 or 2, wherein X 1 , X 2 , and X 3 At least one of the following is represented by: [ka]

[0174] 4. The compound according to any one of embodiments 1 to 3, wherein R 1 is -CH2C(O)-X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l It's H.

[0175] 5. The compound according to any one of embodiments 1-4, wherein R2 and R3 are -OH.

[0176] 6. A compound according to any one of embodiments 1-5, wherein one or more of R1, R2, and R3 are independently selected from the group consisting of: [ka]

[0177] 7. The compound according to any one of embodiments 1-6, wherein the compound has a viscosity of about 20 mPa·s or greater at 20° C. at a concentration of 20% w / w in water.

[0178] 8. The compound according to any one of embodiments 1-7, wherein the compound is soluble in water at a concentration of 50 mg / ml.

[0179] 9. The compound according to any one of embodiments 1-8, wherein the compound has a total releasable NO pool in the range of 0.1-1.0 μmol NO / mg of compound.

[0180] 10. A compound according to any one of embodiments 1-9, wherein the compound has a NO half-life in the range of 0.1 to 24 hours.

[0181] 11. The compound according to any one of embodiments 1-8, wherein the compound has a total duration of NO release in the range of 1-60 hours.

[0182] 12. The compound according to any one of embodiments 1-8, wherein the total NO release after 4 hours is in the range of 0.1-1.0 μmol NO / mg of compound.

[0183] 13. A compound according to any one of embodiments 1 to 12, wherein more than 15% of the repeat units in the compound are monomers of formula I.

[0184] 14. The compound according to any one of embodiments 1-13, wherein the compound has a molecular weight in the range of about 90 kDa to about 700 kDa.

[0185] 15. The compound according to any one of embodiments 1-14, wherein the compound comprises two or more different covalently modified monomers of formula I.

[0186] 16. A NO-releasing hyaluronic acid-derived polymeric compound comprising a unit structure of formula II: [ka] Formula II During the ceremony, R1, R2, R3, R4, R5, and R6 are independently -OH, -NH2, -CH2OH, -C(O)OH, -NHC(O)-CH3, -O-((CH2) a O) b -H, -O-((CH2) a O) b -(CH2) c H, -O-(C1-5 alkyl), -NH-((CH2) d N.H. e -H, -NH-((CH2) d N.H. e -(CH2) f H, -X 1 -((CH2) g X 2 ) h -H, -X 1 -((CH2) g X 2 ) h -(CH2) i H, -CH2C(O)-X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l H, and -X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l H; Each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X 1 , X 2 , and X 3 each instance of is independently selected from: -O-, -S-, -NH-, C(O)NH-; X 1 , X 2 , and X 3 At least one of is represented by one of the following: [ka] ; and wherein the compound has a viscosity of about 10 mPa·s or greater at a concentration of 5% w / w in water at 20° C.

[0187] 17. The compound according to embodiment 16, wherein formula II has the stereochemical configuration shown in formula II'. [ka] Formula II'

[0188] 18. The compound according to embodiment 16 or 17, wherein X 1 , X 2 , and X 3 At least one of is represented by one of the following: [ka]

[0189] 19. The compound according to any one of embodiments 16 to 18, wherein R 1 is -CH2C(O)-X 1 -((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l It's H.

[0190] 20. The compound according to any one of embodiments 16-19, wherein R2 and R3 are -OH.

[0191] 21. A compound according to any one of embodiments 16-20, wherein one or more of R1, R2, R3, R4, R5, R6 are independently selected from the group consisting of: [ka]

[0192] 22. The compound according to any one of embodiments 16-21, wherein the compound has a viscosity of about 20 mPa·s or more at 20° C. at a concentration of 20% w / w in water.

[0193] 23. The compound according to any one of embodiments 16-22, wherein the compound is soluble in water at a concentration of 50 mg / ml.

[0194] 24. The compound according to any one of embodiments 16-23, wherein the compound has a total releasable NO pool in the range of 0.1-1.0 μmol NO / mg of compound.

[0195] 25. The compound according to any one of embodiments 16-24, wherein the compound has a NO half-life in the range of 0.1 to 24 hours.

[0196] 26. The compound according to any one of embodiments 16-24, wherein the compound has a total duration of NO release in the range of 1 to 60 hours.

[0197] 27. The compound according to any one of embodiments 16-24, wherein the total NO release after 4 hours is in the range of 0.1-1.0 μmol NO / mg of compound.

[0198] 28. The compound according to any one of embodiments 1-13, wherein the compound has a molecular weight in the range of about 6 kDa to about 90 kDa.

[0199] 29. A viscosity enhancer comprising: a scaffold comprising a polymer having structural units along the chain of the polymer, wherein one or more of the structural units are functionalized with one or more instances of each of R1, R2, and R3; During the ceremony, R1, R2, and R3 are independently -OH, NH2, OCH3, C(O)OH, CH2OH, CH2OCH3, CH2OCH2CH2OH, OCH2C(O)OH, CH2OCH2C(O)OH, CH2C(O)OH, NHC(O)CH3, C(O)O((CH2) a O) b H, C(O)O((CH2) a O) b (CH2) c H, -C(O)O(C1-5 alkyl), C(O)NH((CH2) d N.H. e H, C(O)NH((CH2) d N.H. e (CH2) f H, CH2C(O)NH((CH2) d N.H. e H, CH2C(O)NH((CH2) d N.H. e (CH2) f H, C(O)X 1 ((CH2) g X 2 ) h (CH2) i H, C(O)X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k (CH2) l H, O((CH2) a O) b H, O((CH2) a O) b (CH2) c H, -O-(C1-5 alkyl), NH((CH2) d N.H. e H, NH((CH2) d N.H. e (CH2)f H, X 1 ((CH2) g X 2 ) h (CH2) i H, CH2C(O)X 1 ((CH2) g X 2 ) h (CH2) i H, X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k (CH2) l H, CH2C(O)X 1 ((CH2) g X 2 ) h ((CH2) j X 3 ) k -(CH2) l H; Each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X 1 , X 2 , and X 3 each instance of is independently selected from: -O-, -S-, -NH-, C(O)NH-; and X 1 , X 2 , and X 3 At least one instance of is represented by one of the following: [ka] ; and The scaffold has a viscosity of approximately 10 mPa·s or greater at 20°C at a concentration of 5% w / w.

[0200] 30. The viscosity-inducing agent of embodiment 29, wherein the scaffold has a gel hardness of about 1.0 mN or greater at a concentration of 5% w / w.

[0201] 31. The viscosity inducing agent according to embodiment 29, wherein the polymer is a biopolymer.

[0202] 32. The viscosity inducing agent of embodiment 29, wherein the polymer is a polysaccharide.

[0203] 33. The viscosity inducing agent according to embodiment 32, wherein one or more structural units are represented by formula I. [ka] Formula I

[0204] 34. The viscosity inducing agent of embodiment 33, wherein the structure of formula I represents a saccharide unit of a carboxymethylcellulose polymer.

[0205] 35. The viscosity-inducing agent of embodiment 33, wherein the structure of formula I represents a saccharide unit of a hyaluronic acid polymer.

[0206] 36. The viscosity inducing agent of embodiment 33, wherein the structure of formula I represents a saccharide unit of a hydroxyethyl cellulose polymer.

[0207] 37. The viscosity inducing agent of embodiment 33, wherein the structure of formula I represents a saccharide unit of a methylcellulose polymer.

[0208] 38. The viscosity-inducing agent of embodiment 33, wherein the structure of formula I represents a saccharide unit of an alginate polymer.

[0209] 39. The viscosity inducing agent according to embodiment 33, wherein the structure of formula I represents a saccharide unit of a cyclodextrin ring structure.

[0210] 40. The viscosity inducing agent of embodiment 29, wherein the polymer comprises a polyaminoglycoside.

[0211] 41. The viscosity inducing agent according to embodiment 40, wherein the polyaminoglycoside is a hyperbranched polyaminoglycoside comprising a first aminoglycoside of formula III: [ka] Formula III; In the formula, G 1 is selected from the group consisting of: [ka] In the formula, G 2 is selected from the group consisting of: [ka] In the formula, R 1 each instance of is independently selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted polyamino having 1-6 repeat units with intervening C1-C6 alkyl groups, optionally substituted polyether having 1-6 repeat units with intervening C1-C6 alkyl groups, or represents a covalent bond to a linking unit; In the formula, X a each instance is independently selected from: -H, -OH, and C1-C6 alkyl; In the formula, R 1 At least one example of the formula (I) represents a covalent bond to one or more linking units selected from the following: [ka] During the ceremony, [ka] indicates attachment to the first aminoglycoside; In the formula, W 1 each instance of, when present, is independently selected from one or more additional aminoglycosides, or one or more end-capping substituents, and at least one linking unit provides a covalent bridge from a first aminoglycoside to a second aminoglycoside; In the formula, R aeach instance of is independently selected from the group consisting of an optionally substituted C1-C6 alkyl, an optionally substituted polyamino having 1-6 repeat units (having C1-C6 alkyl), or an optionally substituted polyether having 1-6 repeat units (having C1-C6 alkyl); and wherein the one or more end-capping substituents, if present, are independently -X 1 -((CH2) h X 2 ) i -(CH2) j It has the formula H.

[0212] 42. The viscosity inducing agent according to embodiment 41, further comprising an end group selected from the group consisting of: [ka] In the formula, R 5 Each example is H or -N + (=NO-)O-.

[0213] 43. The viscosity inducing agent according to embodiment 41 or 42, further comprising an end group selected from the group consisting of: [ka]

[0214] 44. A viscosity-inducing agent according to any one of embodiments 29 to 41, wherein one or more of R1, R2, and R3 are independently selected from the group consisting of: [ka]

[0215] 45. A viscosity-inducing agent according to any one of embodiments 29 to 41, comprising X 1 , X 2 , and X 3 At least one example of is represented by the following structure: [ka]

[0216] 46. ​​A method for delivering nitric oxide to a subject in need of treatment, comprising: The method includes administering to a subject an effective amount of a compound or viscosity inducing agent according to any one of embodiments 1-45.

[0217] 47. The method of embodiment 46, wherein the effective amount of the compound or viscosity-inducing agent is a hydrogel.

[0218] 48. The method of embodiment 46 or 47, wherein the subject has a wound and the compound or viscosity-inducing agent is administered to aid in wound healing.

[0219] 49. The method of embodiment 46 or 47, wherein the subject is in need of tissue replacement and the compound or viscosity-inducing agent is administered as a tissue scaffold.

[0220] 50. A method of treating a disease state, comprising: The method includes administering an effective amount of a compound or viscosity-inducing agent according to any one of embodiments 1 to 45 to a subject in need thereof, wherein the disease state is selected from the group consisting of cancer, cardiovascular disease, microbial infection, platelet aggregation and adhesion caused by exposure of blood to medical devices, pathological conditions resulting from abnormal cell proliferation, transplant rejection, autoimmune disease, inflammation, vascular disease, scar tissue, wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunction, and sexually transmitted diseases.

[0221] 51. A pharmaceutical preparation comprising: A compound or viscosity inducing agent according to any one of embodiments 1 to 45, and A pharmaceutical formulation comprising a pharma- ceutically acceptable excipient.

[0222] 52. A method for reducing or preventing microbial load on a surface, comprising: 46. ​​A method for treating a surface contaminated with a plurality of microorganisms comprising applying a compound or viscosity inducer according to any one of embodiments 1 to 45 to a surface contaminated with a plurality of microorganisms, The compound or viscosity inducing agent according to any one of embodiments 1 to 45 produces nitric oxide and induces oxidative and / or nitrosative damage to microbial DNA and membrane structures, thereby preventing or reducing microbial load, wherein the multiple microorganisms include two or more of gram-positive bacteria, gram-negative bacteria, fungi, yeast, and viruses.

[0223] 53. The method of embodiment 52, wherein the surface is an organic surface.

[0224] 54. The method of embodiment 52 or 53, wherein the surface is human skin.

[0225] 55. The method of embodiment 52 or 53, wherein the surface is a wound surface.

[0226] 56. The method according to embodiment 54 or 55, wherein said application does not induce skin irritation.

[0227] 57. The method of embodiment 52 or 53, wherein the surface is animal skin.

[0228] 58. The method according to embodiment 57, wherein said application does not induce skin irritation.

[0229] 59. The method of embodiment 52, wherein the surface is an inorganic surface.

[0230] 60. The method of embodiment 59, wherein the inorganic surface is an external or internal surface of a medical device.

[0231] 61. The method of embodiment 60, wherein application of the compound produces an antimicrobial coating on the exterior or interior surface of the medical device.

[0232] 62. The method of embodiment 60 or 61, wherein the medical device comprises an endoscope.

[0233] 63. The method of any one of embodiments 52-62, wherein the microbial load comprises drug-resistant bacteria.

[0234] 64. The method according to any one of embodiments 52-63, wherein the microbial load comprises microorganisms associated with the presence of one or more of human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, hepatitis, coxsackie virus, shingles, measles, mumps, rubella, rabies, pneumonia, hemorrhagic viral fever, H1N1, prions, parasites, fungi, molds, Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, Group A Streptococcus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, Carbapenem-Resistant Enterobacteriaceae, Methicillin-Resistant Staphylococcus aureus, and Burkholderia cepacia.

[0235] 65. The method of any one of embodiments 52-63, wherein the microbial load comprises Methicillin-resistant Staphylococcus aureus.

[0236] 66. The method of any one of embodiments 52-63, wherein the microbial load comprises carbapenem-resistant Enterobacteriaceae.

[0237] 67. The method of any one of embodiments 52-63, wherein the microbial load comprises Staphylococcus aureus.

[0238] 68. The method of any one of embodiments 52-63, wherein the microbial load comprises Pseudomonas aeruginosa.

[0239] 69. The method of any one of embodiments 52 to 63, wherein the microbial load comprises Burkholderia cepacia.

[0240] 70. A method for producing any one of the compounds or viscosity inducing agents according to any one of embodiments 1 to 45, comprising: Selecting a polymer; and functionalizing the polymer with an NO-binding moiety.

[0241] 71. The method of embodiment 70, wherein the polymer is a biopolymer.

[0242] 72. The method of embodiment 70 or 71, further comprising exposing the compound or viscosity-inducing agent to NO to provide an NO-donor compound or viscosity-inducing agent.

[0243] 73. Use of a compound or viscosity inducing agent according to any one of embodiments 1 to 45 for the delivery of nitric oxide to a subject in need of treatment of a disease, tissue damage, or reduction of microbial load.

[0244] 74. Use of a compound or viscosity-inducing agent according to any one of embodiments 1 to 45 for the manufacture of a medicament configured to deliver nitric oxide to a subject in need of treatment of a disease, tissue damage, or reduction of microbial load.

[0245] In some embodiments, the polymers disclosed in U.S. Patent Application No. 62 / 441,742, U.S. Patent Application No. 62 / 483,505, International Patent Application No. PCT / IB2018 / 050051, U.S. Patent Application No. 62 / 447,564, International Patent Application No. PCT / IB2018 / 052144, U.S. Patent Application No. 14 / 421525, U.S. Patent Application No. 62 / 639,119, and U.S. Patent Application No. 62 / 737,603 are used. Each of these applications and publications is incorporated by reference in its entirety for all purposes.

[0246] When an amount, concentration, or other value or parameter is given as a range, a preferred range, or a list of an upper preferred value and a lower preferred value, this should be understood as specifically disclosing all ranges formed from any pair of any upper range or preferred value and any lower range or preferred value, regardless of whether the ranges are separately disclosed. When a range of numerical values ​​is recited herein, unless otherwise stated, the range is intended to include its endpoints, and all integers and fractions within the range. The scope of the present invention is not intended to be limited to the specific values ​​recited in defining the range.

[0247] The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. EXAMPLES

[0248] Exemplary synthesis methods: Example 1: Hyaluronic Acid Scaffolds The following examples relate to the synthesis of N-diazeniumdiolate functionalized nitric oxide (NO) releasing hyaluronic acid with tunable NO storage and release kinetics. This embodiment has the following features, advantages, and / or uses: The water solubility and biocompatibility of these scaffolds are high at high (~90 kDa) and low (~6 kDa) molecular weights of the NO releasing scaffolds.

[0249] In some embodiments, these scaffolds may be useful for treating chronic wounds with NO-releasing hyaluronic acid derivatives used for antimicrobial therapy and cell proliferation. In some embodiments, these scaffolds may also be used as treatments for cystic fibrosis with NO-releasing agents acting as antimicrobial agents. ≡

[0250] Composition Details Materials. Low molecular weight (80-110 kDa) and very low molecular weight (<6 kDa) hyaluronic acid (HA) were obtained from Lotion Crafter (Eastsound, WA). Bis(3-aminopropyl)amine (DPTA), diethylenetriamine (DETA), N-propyl-1, 3-propanediamine (PAPA), N-(2-hydroxyethyl)ethylenediamine (HEDA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), neomycin sulfate, phenazine methosulfate (PMS), and hyaluronidase (from bovine testis, type IS) were purchased from MilliporeSigma (St. Louis, MO). 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) was purchased from BioVision (Milpitas, CA). General laboratory salts and solvents were purchased from Fisher Scientific (Fair Lawn, NJ). All reagents were used as received without further purification unless otherwise specified. Tryptic soy broth (TSB) and tryptic soy agar (TSA) were obtained from Becton, Dickinson and Company (Franklin Lakes, NJ). Trypsin, penicillin-streptomycin (PS), Dulbecco's modified Eagle's medium (DMEM), and L929 mouse fibroblast cells (ATCC CCL1) were purchased from UNC Tissue Culture Facility (Chapel Hill, NC). Pseudomonas aeruginosa ( P. aeruginosa ; ATCC #47085), Escherichia coli ( E. coli ; ATCC #43888), Staphylococcus aureus ( S. aureus ; ATCC #29213), Enterococcus faecalis ( E. faecalis ; ATCC #29212), multidrug-resistant P. aeruginosa ( ATCC #BAA-2110 ), and methicillin-resistant Staphylococcus aureus (MRSA; ATCC #33591) were obtained from the American Type Tissue Culture Collection (Manassas, VA).Argon (Ar), carbon dioxide (CO2), nitrogen (N2), oxygen (O2), nitric oxide (NO) calibration (25.87 ppm balance N2), and pure NO (99.5%) gas cylinders were purchased from Airgas National Welders (Raleigh, NC). Distilled water was purified to a resistivity of 18.2 MΩ·cm and total organic content of 6 ppb or less using a Millipore Milli-Q UV Gradient A10 system (Bedford, MA).

[0251] Synthesis of alkylamine-modified hyaluronic acid (HAMW-alkylamine). Hyaluronic acid (90 kDa or 6 kDa) material was modified with either N-propyl-1,3-propanediamine (PAPA), N-(2-hydroxyethyl)ethylenediamine (HEDA), bis(3-aminopropyl)amine (DPTA), or diethylenetriamine (DETA). (Scheme 1a) Briefly, HA (1 g) was dissolved in 40 mL (6 kDa HA) or 100 mL (90 kDa HA) of distilled water. A 4:1 molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was added relative to the carboxylic acid moieties on the HA scaffold, and the solution was titrated to pH 3.0 using 0.5 M HCl. After a 20 min activation period at room temperature, PAPA, DPTA, or DETA in an 8:1 molar ratio, or HEDA in a 4:1 molar ratio, were added dropwise to the reaction solution. The solution was stirred at room temperature for 48 h. The amine-modified HA was precipitated in ethanol, collected by centrifugation, washed twice with ethanol, and dried in vacuum to give a white solid for each modification.

[0252] Characterization of alkylamine-modified hyaluronic acid. Elemental (carbon, hydrogen, and nitrogen; CHN) analysis was performed using a Perkin-Elmer elemental analyzer series 2400 instrument (Waltham, MA) (Table 1). Gel permeation chromatography (GPC) measurements were performed in 0.1 M phosphate buffer (pH 7.4) containing 0.1 M sodium nitrate and 0.02 wt% sodium azide using an aqueous GPC system equipped with a Waters 2414 refractive index detector (Milford, MA) coupled to a Wyatt miniDawn TREOS multi-angle light scattering detector (Santa Barbara, CA). The presence of unreacted starting materials was assessed using high-performance liquid chromatography (HPLC; Agilent Technologies 1260 Infinity II LC system; Santa Clara, CA) equipped with a diode array detector (DAD) and an evaporative light scattering detector (ELSD). 0.1 mg mL- 1 Aliquots (20 μL) of HA, EDC, or NHS samples were loaded onto a Synergi 4 μm Hydro-RP column (250 × 4.6 mm, Phenomenex, Torrance, CA) and eluted in 1 mL min- 1 The analysis was performed using a mobile phase consisting of 80:20 acetonitrile:water at a flow rate of 100 Hz. Elution was monitored by ELSD. 1 Monitored via H 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (600 MHz) spectrometer (Billerica, MA).

[0253] Representative examples of HA and alkylamine-modified HA derivatives 1 H and 13 C NMR included the following peaks:

[0254] HA90 and HA6: 1<h2 style=";text-align:left;direction:ltr">H NMR(600MHz,D2O,δ)2.00(NHC(O)CH3),3.50(CHCH2OH),3.60-4.50(OCHCH(OH)CH(OH)),(OCCHCH(OH)CH(OH),4.50-4.60(NHCOCH),5.30(OCH(CHOH)O),6.30(NHCHCH(OH)).<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR(600MHz,D2O,δ)24.0(NHC(O)CH3),65.0(CHCH2OH),70.0-81.0(OCHCH(OH)CH(OH)CH(OH)CH(O)),95.0(NHCHCH(OH)),110.0(OCCHCH(OH)),175.0(CHC(O)OH).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0255] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> HA90-DETAおよびHA6-DETA:<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> H NMR(600MHz,D2O,δ)2.60-3.30(CH2CH2NHCH2CH2NH2),2.00(NHC(O)CH3),3.50(CHCH2OH),3.60-4.50( OCHCH(OH)CH(OH)),(OCHCH(OH)CH(OH),4.50-4.60(NHCOCH),5.30(OCH(CHOH)O),6.10(NHCHCH(OH)).<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR (600MHz, D2O, δ) 24.0(NHC(O)CH3), 40.0-49.0(C(O)NHCH2CH2NHCH2CH2NH2), 65.0(CHCH2OH), 70.0-81.0(OCHCH(OH)CH(OH)CH(OH)CH(OH)CH(O)), 95.0(NHCHCH(OH)), 110.0(OCHCH(OH)), 170.0(CHC(O)NH), 175.0(CHC(O)OH).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0256] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> HA90-DPTAおよびHA6-DPTA:<h2 style=";text-align:left;direction:ltr"> 1<h2 style=";text-align:left;direction:ltr">H NMR (600MHz, D2O, δ) 1.70-1.80(CH2CH2CH2NHCH2CH2CH2NH2), 2.00(NHC(O)CH3), 2.50-2.40(CH2CH2CH2NHCH2CH2CH2NH2), 2.70-3.20(CH2CH2CH2NHCH2CH2CH2NH2), 3.50(CHCH2OH), 3.60-4.50(OCHCH(OH)CH(OH)),(OCHCH(OH)CH(OH), 4.50-4.60(NHCOCH), 5.30(OCH(CHOH)O), 6.10(NHCHCH(OH)).<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR (600MHz, D2O, δ) 24.0(NHC(O)CH3), 32.0NHCH2CH2CH2NH2), 38.0(C(O)NHCH2CH2CH2NH), 39.0(C(O)NHCH2CH2CH2NH), 46.0(C(O)NHCH2CH2CH2NH,NHCH2CH2CH2NH2), 65.0(CHCH2OH), 70.0-81.0(OCHCH(OH)CH(OH)CH(OH)CH(OH)CH(O)), 95.0(NHCHCH(OH)), 110.0(OCHCH(OH)), 170.0(CHC(O)NH), 175.0(CHC(O)OH).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0257] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> HA90-PAPAおよびHA6-PAPA:<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> H NMR (600MHz, D2O, δ) 0.80-0.90(NHCH2CH2CH3), 1.40-1.50(NHCH2CH2CH3), 1.70-1.80(CH2CH2CH2NHCH2CH2CH3), 2.00(NHC(O)CH3), 2.50-3.20(CH2CH2CH2NHCH2CH2CH3), 3.50(CHCH2OH), 3.60-4.50(OCHCH(OH)CH(OH)),(OCHCH(OH)CH(OH), 4.50-4.60(NHCOCH), 5.30(OCH(CHOH)O), 6.10(NHCHCH(OH)).<h2 style=";text-align:left;direction:ltr"> 13C NMR(600MHz,D2O,δ)11.0(NHCH2CH2CH3),23.0(NHCH2CH2CH3),24.0(NHC(O)CH3),24.0-30.0(C(O)NHCH2CH2CH2NH,NHCH2CH2CH3),46.0-52.0(C(O)NHCH2CH2CH 2NH,NHCH2CH2CH3),65.0(CHCH2OH),70.0-81.0(OCHCH(OH)CH(OH)CH(OH)CH(O)),95.0(NHCHCH(OH)),110.0(OCHCH(OH)),170.0(CHC(O)NH),175.0(CHC(O)OH).

[0258] HA90-HEDA and HA6-HEDA: 1 H NMR(600MHz,D2O,δ)1.75(C(O)NHCH2CH2CH2NH),2.50-3.20(C(O)NHCH2CH2CH2NH),2.70-3.50(NHCH2CH2OH),2.00(NHC(O)CH3),3 .50(CHCH2OH),3.60-4.50(OCHCH(OH)CH(OH)),(OCHCH(OH)CH(OH),4.50-4.60(NHCOCH),5.30(OCH(CHOH)O),6.10(NHCHCH(OH)). 13 C NMR(600MHz,D2O,δ)24.0(NHC(O)CH3),29.0(C(O)NHCH2CH2CH2NH),38.0(C(O)NHCH2CH2CH2NH),46.0(C(O)NHCH2CH2CH2NH),52.0(NHCH2CH2OH),61.0(N HCH2CH2OH),65.0(CHCH2OH),70.0-81.0(OCHCH(OH)CH(OH)CH(OH)CH(O)),95.0(NHCHCH(OH)),110.0(OCHCH(OH)),170.0(CHC(O)NH),175.0(CHC(O)OH).

[0259] The purity analysis of the amine-functionalized hyaluronic acid derivatives is depicted in Figure 2. Representative results of unmodified hyaluronic acid 1 H NMR spectrum and 13 The C NMR spectrum is shown in Figure 3. Representative C NMR spectra of secondary amine-functionalized hyaluronic acid1 The H NMR spectrum is shown in Figure 4. Representative spectra of secondary amine functionalized hyaluronic acid. 13 C NMR spectra are set forth in FIG. 5 and are representative of unmodified and amine-modified hyaluronic acid. 13 A comparison of C NMR is also given. The presence of amide peaks and increased nitrogen content confirms the alkylamine modification.

[0260] Synthesis of NO-releasing hyaluronic acid. Alkylamine-modified HA (45 mg) was dissolved in 7:3 methanol:water (3 mL) with sodium methoxide (75 μL, 5.4 mM in methanol) in a 1-dram glass vial. The open vial was placed in a stainless steel reaction vessel and stirred continuously via magnetic stirring. The vessel was purged three times with argon (10 s, 7 atm) followed by three more long purges (10 min, 7 atm) to remove excess oxygen. The vessel was then pressurized to 20 atm with NO gas (Scheme 1b). After 3 days, the same argon purging protocol was followed to remove unreacted NO. The resulting NO-releasing HA was then precipitated in ethanol, collected by centrifugation, dried in vacuum, and stored at -20 °C as a white / yellow powder in a vacuum-sealed bag. Representative UV-Vis spectra of secondary amine-functionalized and NO-releasing hyaluronic acid are listed in Figure 6. [ka] Scheme 1. (A) Modification of hyaluronic acid with secondary amines to synthesize amine-functionalized hyaluronic acid. (B) Formation of N-diazeniumdiolate on secondary amine-modified hyaluronic acid. [Table 1] a Errors represent the standard deviation of n ≥ 3 separate composites.

[0261] Characterization of NO storage and release. To confirm the presence of N-diazeniumdiolate functional groups, absorbance measurements were performed in 50 mM sodium hydroxide (NaOH) using Molecular Devices SpectraMax M2 (San Jose, CA). Real-time nitric oxide release was assessed using a Sievers 280i Nitric Oxide Analyzer (NOA; Boulder, CO). Prior to use, samples were analyzed to confirm the stability of the stored material. The NOA was calibrated with air passed through a NO zero filter (0 ppm NO) and 25.87 ppm NO calibration gas (balance N2) prior to analysis. In a typical experiment, NO-releasing HA (approximately 1 mg) was dissolved in 30 mL of deoxygenated PBS (10 mM, pH 7.4, 37 °C). The solution was diluted in 200 mL min- 1 The instrument was purged with nitrogen gas at a flow rate of 10 ppb NO mg-1, and the liberated NO was transferred to the instrument. The analysis was terminated when the NO level was below the detection limit of the instrument (10 ppb NO mg-1). 1 An analysis of the nitric oxide releasing properties of the NO-releasing hyaluronic acid is set forth in Table 2 and FIG. 7. An analysis of the control NO-releasing properties of unmodified hyaluronic acid and primary amine-modified hyaluronic acid is set forth in Table 3. [Table 2] a Errors represent the standard deviation of n ≥ 3 separate composites. b Total NO released over the entire period. c Maximum instantaneous NO concentration. d Half-life of NO release. e Duration of NO release. f Total NO released over a 4-hour period. [Table 3] a Errors represent the standard deviation of n ≥ 3 separate composites. b It is measured via chemiluminescence in phosphate buffered saline (10 mM, pH 7.4, 37°C).

[0262] Enzymatic degradation of alkylamine-modified HA and NO-released HA. Enzymatic degradation of hyaluronic acid was performed using a procedure adapted from Turner et al. Briefly, 50 mg of HA90, amine-modified HA90, or NO-released HA90 was dissolved in 5 mL of pH 5.0 buffer containing 0.15 M NaCl, 0.1 M CH3COONa, and 1 mM Na2EDTA at 37°C for 30 min with magnetic stirring. Hyaluronidase (2.5 mg) was dissolved in 1 mL of the same buffer and added directly to the HA solution. The mixture was incubated at 37°C for 30 min with vigorous stirring. After digestion, the reaction was terminated by placing the vial in a boiling water bath for 10 min, and then the solution was cooled to room temperature. Insoluble enzyme fragments were removed via centrifugation (7500 rpm, 15 min). The supernatant was filtered and analyzed via GPC-MALS, measurements were performed in 0.1 M phosphate buffer (pH 7.4) containing 0.1 M sodium nitrate and 0.02 wt % sodium azide.

[0263] Exemplary synthesis methods: Example 2: Carboxymethylcellulose scaffolds These examples relate to the synthesis of N-diazeniumdiolate functionalized nitric oxide (NO) releasing carboxymethylcellulose with tunable NO storage and release kinetics. This embodiment has the following features, advantages, and / or uses: The water solubility and biocompatibility of this scaffold is high with the high molecular weight of the NO releasing scaffold.

[0264] In some embodiments, the scaffold may be useful in the treatment of bacterial infections.

[0265] Periodontal disease encompasses a class of inflammatory infections of the gums and surrounding tissues that affect a large proportion of the population. Disease progression is caused by changes in the microbial composition of healthy dental plaque biofilm, resulting in an excess of gram-negative bacteria. Gram-negative bacteria such as Porphyromonas gingivalis induce inflammation in dental tissues (e.g., gums, periodontal ligament, and alveolar bone tissue), resulting in the development of periodontal pockets where these bacteria can continue to grow. If left untreated, chronic periodontitis eventually leads to the degradation of oral tissues, including tooth and bone resorption. Furthermore, studies have shown an association between periodontitis and other systemic inflammatory conditions (including cardiovascular disease, coronary heart disease, and adverse pregnancy outcomes) due to the spread of pathogenic gram-negative oral bacteria to the bloodstream and other areas of the body.

[0266] The primary treatment for periodontal disease is scaling and root planing (SRP), a process that physically scrapes plaque off of the tooth surface. While this is effective in removing the majority of plaque, it does not directly kill pathogenic bacteria or prevent recolonization of the tooth surface. For this reason, patients with severe chronic periodontitis often undergo SRP along with adjunctive antimicrobial therapy. Antimicrobial implants incorporating agents such as chlorhexidine, one of the most commonly prescribed oral antimicrobial agents, have been used for such purposes but face a number of issues that affect their effectiveness. Undesirable side effects of chlorhexidine, including discoloration of teeth and gums, altered taste, and toxicity to healthy cells, may inhibit its usefulness and hinder patient compliance. Furthermore, localized drug delivery to the periodontal pockets can be challenging due to the flow of gingival crevicular fluid (GCF), a constant stream of fluid from the periodontal pockets into the oral cavity. As a result, there is a need for both potent antimicrobial agents with limited side effects and materials that can deliver antimicrobial agents to the periodontal pockets to effectively eradicate periodontal pathogens.

[0267] Nitric oxide (NO) is an endogenous antimicrobial agent that plays a key role in mammalian immune responses. It exhibits broad-spectrum antimicrobial activity due to its multiple killing mechanisms, including the formation of reactive by-products that can exert both nitrosative and oxidative stress on bacteria. As a result, NO is less likely to promote bacterial resistance than many traditional antimicrobial agents. Challenges in effective local delivery of gaseous NO have necessitated the development of NO donors that can store and release nitric oxide. Among the numerous NO donors that have been developed, N-diazeniumdiolates are attractive options for dental treatments due to their proton-mediated release of NO in aqueous environments. Depending on the release kinetics that depend on the external conditions (i.e., pH and temperature), and the chemical structure of the NO donor itself, a variety of kinetic profiles can be achieved. Furthermore, the ability to modify secondary amine-containing structures to impart NO release is advantageous, as the resulting materials can act as both delivery scaffolds and therapeutic agents. Thus, structures can be designed with desired properties to localize the delivery of NO to the periodontal pocket.

[0268] Carboxymethylcellulose (CMC), a water-soluble synthetic derivative of cellulose, is an attractive scaffold for use in periodontal pockets due to its biocompatibility, adhesive properties, and high solution viscosity. It is widely used as a thickening and stabilizing additive in many industries and has also found utility in dental applications. Thus, CMC can enhance retention in the periodontal pocket, while its water solubility allows for natural clearance over time. Furthermore, CMC can be made with a wide range of molecular weights and degrees of substitution, allowing additional tailorability for its intended end use. The presence of carboxylic acid moieties allows for effective modification of the polymer in aqueous solution under mild reaction conditions, which is important in imparting antibacterial properties to the scaffold.

[0269] To form the N-diazeniumdiolate NO donor on the CMC polymer backbone, it must be chemically modified with secondary amine moieties. Carbodiimide crosslinking reactions using EDC and NHS are effective for the aqueous modification of carboxylic acid-containing polysaccharides as previously described, allowing for the modification of CMC with four alkylamines (Scheme 2A). After secondary amine modification, the polymer can be exposed to high pressure gaseous NO under basic conditions to form the N-diazeniumdiolate (Scheme 2B). Scheme 2. (A) Modification of CMC with alkylamines and (B) N-diazeniumdiolate formation on CMC amines. [ka]

[0270] Synthesis details: Materials and Methods - Carboxymethylcellulose (CMC, Mw 90 kDa, DS = 0.7), diethylenetriamine (DETA), bis(3-aminopropyl)amine (DPTA), N-(2-hydroxyethyl)ethylenediamine (HEDA), N-propyl-1,3-propanediamine (PAPA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from MilliporeSigma (St. Louis, MO). Aggregatibacter actinomycetemcomitans (ATCC #43717) was purchased from the American Type Culture Collection (Manassas, VA). Porphyromonas gingivalis strain A7436 was provided by the University of North Carolina School of Dentistry, Chapel Hill, NC, USA. Brain heart infusion (BHI) broth and agar, CDC anaerobic 5% sheep blood agar by volume, and GasPak EZ campy sachets were purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Wilkins-Chalgren (WC) broth was purchased from Thermo Fisher Scientific (Waltham, MA). Human gingival fibroblasts (HGF-1) and FibroLife S2 medium were purchased from Lifeline Cell Technology LLC (Frederick, MD). Pure nitric oxide (99.5%), argon, nitrogen, nitric oxide calibration gas (in 25.87 ppm nitrogen), and anaerobic gas mixture (10% hydrogen, 5% carbon dioxide, balance nitrogen) were purchased from Airgas (Durham, NC). MTS reagents were purchased from BioVision (Milpitas, CA) and phenazine methosulfate (PMS) from MilliporeSigma. General laboratory salts and solvents were purchased from Thermo Fisher Scientific (Waltham, MA).

[0271] Synthesis of amine-modified carboxymethylcellulose. First, a solution of CMC was prepared by dissolving 100 mg of 90 kDa CMC in 10 mL of pH 6.5 phosphate buffered saline (10 mM PBS6.5). After the solid was completely dissolved with magnetic stirring, a 5:1 molar excess of NHS relative to the number of carboxylates on the CMC was added, followed by an equal molar excess of EDC. The mixture was stirred for 20 min, after which the respective alkylamine was added as a bolus at a 1:1 molar ratio to EDC. The solution was stirred overnight (16 h), then transferred to a dialysis tube (MWCO 10 kDa) and dialyzed against deionized water for 3 days with frequent water changes. The final solution was transferred to a plastic tube, frozen at -80 °C, and lyophilized on a Labconco Freezone -50 °C freeze dryer (Labconco, Kansas City, MO) to obtain a fibrous white solid.

[0272] The formation of amine bonds was qualitatively confirmed using a PerkinElmer 100 FTIR (PerkinElmer, Waltham, MA), and the modification efficiency was quantified using a PerkinElmer Series II CHNS / O Analyzer (PerkinElmer, Waltham, MA). Molecular weights were determined using a Breeze 2 size-exclusion chromatography instrument (Waters, Milford, MA) coupled to a 2414 refractive index detector (Waters, Milford, MA) and a miniDawn TREOS multi-angle light scattering detector (Wyatt Technology, Santa Barbara, CA). Briefly, a 0.1 wt% CMC solution was prepared in a mobile phase buffer consisting of 0.1 M acetate (pH 4.6), with 0.1 M NaNO3 and 0.02 wt% NaN3. A 50 μL aliquot of each sample was injected and run through two columns (2x Shodex OHpak LB804, Showa Denko America, New York, NY) in series at a flow rate of 0.6 mL min−1.

[0273] Synthesis of N-diazeniumdiolate modified carboxymethylcellulose. A solution of amine modified CMC was prepared at 2 wt% in 300 mM NaOH. The solution was placed in a Parr hydrogenation reactor and purged six times with argon (three short purges followed by three 10 min purges), then pressurized with 10 bar NO gas for three days. The reaction chamber was then purged again six times with argon, and the N-diazeniumdiolate modified CMC was precipitated in cold ethanol and dried under reduced pressure. All NO release samples were stored at -20°C before use.

[0274] Nitric oxide release from CMC scaffolds was characterized using a Zysense 280i Nitric Oxide Analyzer (NOA, Zysense, Frederick, CO). In a typical experiment, 1 mg of NO release sample was immersed in 25 mL of deoxygenated PBS (10 mM, pH 7.4, 37 °C). Nitrogen gas was flowed through the solution at 200 mL min-1, and NO released from the scaffolds was transferred to the NOA. Release was measured until NO levels were below 10 ppb NO per mg scaffold.

[0275] Analysis of the synthesized CMC scaffolds: Four different amine-modified CMCs were prepared by aqueous carbodiimide crosslinking reactions between 90 kDa CMC and small molecule amines. Reactions of CMC with diethylenetriamine (DETA), bis(3-aminopropyl)amine (DPTA), N-(2-hydroxyethyl)ethylenediamine (HEDA), and N-propyl-1,3-propanediamine (PAPA) produced CMC-DETA, CMC-DPTA, CMC-HEDA, and CMC-PAPA, respectively.

[0276] To qualitatively determine successful amine conjugation, the CMC-amine was analyzed using FTIR spectroscopy (Figure 8). Unmodified CMC exhibited a 1590 cm- 1 When modified with each amine sample, the singular carbonyl stretching signal appears at 1560 cm-, characteristic of NH bending and C=O stretching, respectively. 1and 1630 cm- 1 The peaks are overtaken by two new peaks that appear at 1000 nm. While this FTIR analysis supports that an amide bond was formed and therefore the amine was successfully conjugated to the CMC, elemental analysis was necessary to quantify the extent of modification. Determining the percent nitrogen within each sample using a CHNS / O elemental analyzer allowed for an estimation of the carboxylate modification of the CMC backbone in addition to the primary amine content, as shown in Table 4 (only DETA and DPTA, as HEDA and PAPA lack an additional primary amine after modification). Notably, the modification efficiency remained relatively consistent despite the differences in amine identity. [Table 4]

[0277] Next, the molecular weight (M w ) determined by SEC-MALS and the theoretical M calculated based on elemental analysis. w compared with (Table 5). [Table 5]

[0278] M w A clear increase in M ​​was observed for all CMC-amines compared to the unmodified biopolymer. For CMC-DETA, the experimentally determined M w is much higher than the theoretical value, likely indicating some crosslinking due to the presence of two reactive amine end groups. However, a similar increase was not observed for DPTA, which also has two amine end groups, likely the result of higher steric hindrance resulting from its larger molar mass. M for CMC-HEDA wThe increase in was attributed to stronger hydrogen bonds originating from the hydroxyl end groups. Surprisingly, the dispersibility of the samples decreased after modification, possibly as a result of sample interactions resulting from the introduction of cationic charges into the polymer backbone. Nitric oxide releasing capabilities were imparted to CMC-amines via the formation of N-diazeniumdiolates. These modifications were achieved by reacting the scaffolds with pressurized (10 bar) NO gas in basic aqueous solution for 3 days. Successful N-diazeniumdiolate formation was confirmed using UV-vis spectroscopy to demonstrate the presence of a characteristic absorbance peak at 250 nm (Figure 21). Furthermore, NO release characteristics were determined in real time using a chemiluminescence-based nitric oxide analyzer. Characterization of the full range of NO release under physiological conditions (37°C, pH 7.4) as shown in Table 6 indicates the range of kinetics achievable for CMC-amines as a function of the chemical structure of the amine modification. CMC-DETA / NO has the longest half-life (approximately 3 hours) as a result of primary amine stabilization of the diazeniumdiolates derived from intramolecular ring formation. CMC-DPTA / NO has similar primary amine interactions to the diazeniumdiolates, but due to its longer chain length, it has less good intramolecular stabilization and therefore a shorter NO release half-life. Interestingly, CMC-HEDA / NO has a similar half-life to CMC-DPTA / NO despite lacking a primary amine. This is due to both stronger hydrogen bonding between the primary hydroxyl groups and the diazeniumdiolates compared to the primary amines, and HEDA having an alkyl chain length more similar to DETA. Finally, CMC-PAPA / NO showed the most rapid NO release of the four systems because its alkyl end groups have very few interactions with the NO releasing moiety. [Table 6] a Total amount of NO released from the scaffold. b Half-life of NO release. c 10ppb mg- 1 Duration of NO release before reaching

[0279] Example usage: Example 3A - Hyaluronic Acid Scaffolds The following examples relate to the use of N-diazeniumdiolate functionalized nitric oxide (NO) releasing hyaluronic acid as an antimicrobial agent, for example in wound healing. The scaffold produced in Example 1 is used in the following experiments against various bacterial cultures:

[0280] Planktonic bacterial killing assay. Bacterial cultures of P. aeruginosa, E. coli, E. faecalis, S. aureus, multidrug-resistant (MDR) P. aeruginosa, and methicillin-resistant S. aureus were grown from frozen (-80 °C) stocks overnight at 37 °C in TSB (3 mL). Aliquots (1 mL) of the overnight solutions were diluted with 10 mL of fresh TSB (50 mL) and diluted with 10 mL of ethanol. 8 CFU mL- 1 and then re-cultured at a concentration of 10 in PBS (10 mM, pH 7.4). 6 CFU mL- 1 Weighed samples of control (non-NO releasing) HA, NO releasing HA, or neomycin sulfate were dissolved in PBS and titrated with 1M HCl to adjust the pH to 7.4. Samples were added to a 96-well polystyrene plate and serially diluted in PBS so that each well contained 100 μL of control HA, NO releasing HA, or neomycin. 6 CFU mL- 1 Add bacterial solution containing (100 µL, 1 vol.% TSB-supplemented PBS) to each well, 0.25–32 mg mL 1 Final HA concentrations ranging from 0.5 to 1024 μg mL- 1The 96-well plates were then incubated at 37°C for 4 hours with gentle shaking. Untreated bacterial solutions were included in each experiment to ensure bacterial viability over the consecutive 4 hours. After the 4-hour exposure, the bacterial solutions were serially diluted (10-, 100-, and 1000-fold dilutions) and spiral-plated onto TSA plates using an Edjet Spiral Plater (IUL, Farmingdale, NY) and incubated overnight at 37°C. Bacterial viability after treatment with HA or neomycin was determined using a Flash-and-Go Colony Counter (IUL, Farmingdale, NY). After the 4-hour exposure period (MBC 4時間 ) was defined as the minimum concentration required to achieve a 3 log reduction (≥99.9% reduction) in bacterial viability compared to untreated bacteria (i.e., reducing bacterial numbers by 10 6 From 10 3 CFU mL- 1 The detection limit of this counting method is 2.5 × 10 3 CFU mL- 1 The NO dose required for bactericidal activity was calculated using the NO-releasing HA sample (mg mL- 1 ) MBC 4時間 PBS (pH 7.4; μmol NO mg- 1 The minimum bactericidal concentration (MBC) of NO-releasing hyaluronic acid against various bacteria was calculated by multiplying the total NO released in the hyaluronic acid (HA) by the total NO released in the hyaluronic acid (HA). 4時間 ) are listed in Tables 7 and 9. DPTA-modified HA was ≤2 mg mL- 1 eradicates all bacterial strains. The doses of NO or neomycin required to elicit a 3-log reduction in bacterial viability after 4 h treatment are listed in Tables 8 and 10. The antibacterial efficacy of the active ingredient NO-releasing hyaluronic acid against various bacteria is listed in Figures 10 and 13-15. NO, at low concentrations, is bactericidal against antibiotic-resistant bacteria. All data presented are from separate experiments with n ≥ 3. [Table 7] a MBC determined from experiments with n ≥ 3 4時間 . [Table 8] a MBC 4時間 and NO dose derived from total NO released over a 4-h exposure time in PBS (10 mM, pH 7.4, 37 °C). b MBC 4h However, the NO dose could not be determined because it exceeded the highest HA concentration evaluated. [Table 9] a MBC determined from experiments with n ≥ 3 4時間 . [Table 10] a Bactericidal neomycin dose determined from n ≥ 3 experiments.

[0281] Time-based planktonic bacterial killing assay. For a 4-hour planktonic bacterial killing assay, 6 CFU mL- 1 A bacterial solution containing P. aeruginosa and S. aureus was prepared as described. A weighed sample of 6 kDa NO-releasing HA was added at 4 mg mL- 1 or 32 mg mL- 1 (for P. aeruginosa and S. aureus treatment, respectively) were dissolved in PBS and titrated with 1 M HCl to adjust the pH to 7.4. 6 CFU mL- 1 An equivalent volume of the bacterial solution was added to each vial to give a final concentration of NO-releasing HA of 2 mg mL- 1 or 16 mg mL- 1Untreated bacterial solution was included to ensure viability over the 4-hour exposure period. The bacterial solution was incubated at 37°C with gentle shaking. At the designated time points (i.e., 0, 0.5, 1, 1.5, 2, 3, and 4 hours), 100 μL aliquots of bacterial solution were removed, serially diluted (10- and 100-fold dilutions), plated onto TSA plates using an Eddy-Jet Spiral Plater, and incubated overnight at 37°C. Bacterial viability at each time point was determined using a flash-and-go colony counter. The results of the time-based bactericidal assay of NO-releasing HA derivatives against bacteria are described in Figure 9. HA-DPTA / NO eradicates bacteria rapidly and at low concentrations.

[0282] Biofilm eradication assay. Bacterial cultures of P. aeruginosa and MDR-P. aeruginosa were grown from frozen (-80 °C) stocks in TSB (3 mL) overnight at 37 °C and then diluted in fresh TSB for 10 min. 8 CFU mL- 1 The mixture was then re-cultured to a concentration of 10. 8 An aliquot of the solution (18 μL) was added to 1800 μL of fresh TSB in a 24-well polystyrene plate and incubated at 37 °C for 72 h with gentle shaking. Nitric oxide-releasing DPTA-modified HA, or neomycin was dissolved in PBS (750 μL, pH 7.4, 10 mM) in a 1-dram vial and adjusted to pH 7.4 with 1 M HCl. Biofilms (250 μL) were washed with PBS (pH 7.4, 10 mM) and added to the 1-dram vial. 4–32 mg mL- 1 of NO release from DPTA-modified HA or 30-240 μg mL- 1Treatment with neomycin sulfate was performed for 24 h at 37°C with gentle shaking. Untreated biofilms were included in each experiment to ensure biofilm viability over a continuous 24 h period. After treatment, biofilms (100 μL) were diluted 10-fold and dispersed by pipetting and vortexing. Biofilm solutions were further diluted (1,000-fold and 100,000-fold) and plated onto TSA plates using an Eddy-Jet Spiral Plater and incubated overnight at 37°C. Biofilm viability after treatment with HA or neomycin was determined using a flash-and-go colony counter. After a 24-h exposure period (MBEC 24時間 ) was defined as the minimum concentration required to achieve a 5 log reduction (≥99.999% reduction) in bacterial viability compared to untreated bacteria (i.e., reducing bacterial numbers by 10 8 From 10 3 CFU mL- 1 The NO dose required for biofilm eradication was determined by the NO-releasing HA sample (mg mL- 1 )MBEC 24時間 PBS (μmol NO mg- 1 The results of biofilm viability after 24 h treatment of pre-existing bacterial biofilms with neomycin sulfate or nitric oxide releasing DPTA modified HA are shown in Figures 11, 16, and 17. All data presented are from separate experiments with n ≥ 3. Low molecular weight HA > high molecular weight HA NO > neomycin.

[0283] In vitro cytotoxicity assay. L929 mouse fibroblast cells were grown in DMEM supplemented with 10% FBS (volume) and 1% penicillin-streptomycin (weight). Cells were incubated at 37°C under humidified conditions and 5% CO (volume). After reaching 80% confluency, cells were cultured at 1 × 10 4 Cell Well- 1After incubation at 37°C for 24 h, the supernatant was aspirated and diluted with 0.25–32 mg mL- 1 The cultures were then replaced with 100 μL of either control or NO-releasing HA in fresh growth medium with HA concentrations ranging from 0.1 to 0.2 μL. The cultures were then incubated at 37°C for 24 h. After exposure, the supernatant was aspirated and the wells were washed twice with PBS. A 100 μL mixture of DMEM / MTS / PMS (105 / 20 / 1, v / v / v) was added to each well and incubated at 37°C for 90 min. The absorbance of the solution in each well was measured at 490 nm using a Molecular Devices Spectramax M2 (San Jose, CA). A blank mixture of DMEM / MTS / PMS and untreated cells were used as blank and control, respectively. Cell viability for each sample was calculated as follows:

number

[0284] Dose-response curves were plotted using GraphPad Prism 8 software (San Diego, CA). IC 50 A nonlinear regression (normalized response with variable slope) analysis was performed to determine IC values ​​following treatment with unmodified, amine-modified, and NO-releasing HA. 50 The results and viability results of L929 mouse fibroblast cells are listed in Figures 12, 18, and 19. Cytotoxicity was strongly influenced by the NO payload, with NO-releasing HEDA-modified HA being the least toxic to cells. All data presented are from n ≥ 3 separate experiments.

[0285] Example 3B - Carboxymethylcellulose scaffolds The following examples relate to the use of NO releasing carboxymethylcellulose (CMC). The scaffolds produced in Example 2 are used in the following experiments on various bacterial cultures:

[0286] 2-hour minimum bactericidal concentration (MBC) 2時間 Porphyromonas gingivalis was reinoculated overnight from a frozen stock of WC anaerobic broth in an anaerobic chamber (Koy Laboratory Products, Grayslake, MI). A 300 μL aliquot of bacteria was transferred to fresh broth and incubated for 10 min at 4°C. 8 Aggregatibacter actinomycetemcomitans was similarly prepared using brain heart infusion (BHI) broth under microaerobic conditions using a GasPak EZ Campy vessel system. The optical density at 600 nm (OD 600 The bacterial concentration was confirmed by measuring the number of bacteria in the culture medium. Bacteria were cultured at 10 6 cfu / mL and exposed to control (i.e., non-NO releasing) and NO releasing substances under aerobic conditions for 2 h at 37 °C. Bacteriostatic conditions were confirmed for both bacteria when not exposed to substances. After exposure, samples were diluted 10-1000-fold in PBS7.4 and plated on the corresponding agar plates using an IUL Instruments Edjit 2 Spiral Plater (Neutec Group, Farmingdale, NY). Porphyromonas gingivalis on CDC anaerobic 5% sheep blood agar by volume was incubated for 3 days in an anaerobic chamber, and Aggregatibacter actinomycetemcomitans on brain heart infusion agar (BHA) was incubated for 3 days under microaerobic conditions. After incubation, bacterial concentrations were determined using the plate count method using an IUL Instruments Flash and Go (Neutec Group, Farmingdale, NY).

[0287] The antibacterial efficacy of NO-releasing CMC amines was evaluated against planktonic forms of two prominent periodontopathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Both of these Gram-negative bacteria play important roles in the proliferation of periodontal disease, with Porphyromonas gingivalis acting as a keystone pathogen in the development of chronic periodontitis, and Aggregatibacter actinomycetemcomitans causing a more localized and aggressive form of periodontitis. The antibacterial efficacy presented in Table 11 was measured under static conditions (MBC 2時間 The results were evaluated by determining the minimum bactericidal concentration (i.e., a 3 log reduction in bacterial viability) of each polymer upon a 2-hour exposure to 1000 sucrose.

[0288] With the exception of CMC-DETA / NO, the NO-releasing scaffolds had similar killing efficacy against both pathogens. Notably, the control (i.e., non-NO-releasing) scaffolds showed minimal bactericidal activity, supporting that NO was indeed acting as a bactericide. The slower NO-release half-life of CMC-DETA / NO resulted in a greater reduction in total NO at the 2-h exposure length and therefore required a higher scaffold concentration to elicit bactericidal activity. While both of the periodontal pathogens of interest are Gram-negative, the A7436 strain of Porphyromonas gingivalis, which possesses a capsule that may reduce NO permeability, showed slightly higher MBCs for three of the polymers tested compared to Aggregatibacter actinomycetemcomitans. 2時間 results. [Table 11]

[0289] Cytotoxicity of modified CMCs towards human gingival fibroblasts. HGF-1 cells were cultured in FibroLife S2 medium supplemented with 1% penicillin and streptomycin and incubated at 37°C in humidified 5% CO2 (volume). Upon reaching approximately 80% confluency, cells were trypsinized and plated at 104 Cells were plated at a density of 1000 cells / well. After 24 h of incubation in the 96-well plate, the medium was aspirated and replaced with 100 μL of medium containing control and NO-releasing CMCs. After 24 h of exposure at 37 °C, the medium from each well was aspirated, the cells were washed with PBS, and 100 μL of MTS solution (composed of medium / MTS / PMS at 105 / 20 / 1 v / v / v) was added. After incubating the plate for 2 h, the absorbance of the medium in each well was measured at 490 nm using a Spectramax M2 UV-vis spectrophotometer (Molecular Devices, San Jose, CA). The control (untreated cells) and blank (MTS solution) were used to determine relative cell viability using the following formula:

number

[0290] The toxicity of CMC scaffolds to mammalian cells was evaluated in Figure 20 by exposing human gingival fibroblasts (HGF) to both NO-releasing and non-NO-releasing substances for 24 hours. Metabolic activity was determined at the end point using the MTS assay, with untreated cells showing 100% viability, and correlated with cell viability. For amine-modified CMC, only CMC-DPTA resulted in a significant decrease in HGF viability, decreasing to about 80% at 8 mg / mL and about 30% at 16 mg / mL. More pronounced differences were observed for NO-releasing CMC polymers. Up to 4 mg / mL, CMC-DETA / NO resulted in consistently higher apparent cell viability than baseline, either as a result of the proliferative effects of NO or increased cell metabolism. Above 4 mg / mL, cell viability decreased for all substances, likely in response to increasing NO doses resulting in oxidative and nitrosative stress to fibroblasts. Notably, high cell viability is maintained for 24 h at MBC 2 h for all scaffolds except CMC-DETA / NO.

[0291] This indicates the efficacy of NO-releasing carboxymethylcellulose as an antibacterial agent against two prominent periodontal pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. In vitro results showed that faster NO release kinetics was associated with a lower MBC. 2時間 showed that all scaffolds except CMC-DETA / NO were capable of achieving a 3-log reduction of bacteria at concentrations that were not significantly cytotoxic to HGF-1 cells.

[0292] Example 4 Prophetic Example A solution of CMC-DETA as disclosed in Example 2 is prepared in PBS at a concentration of 10 mg / ml. The solution is injected via a syringe into the dehiscence of a knife wound of an adult male patient. Upon reaching the inner surface of the wound, the viscous solution forms a firm gel. The gel is covered with a bandage. As a control, another patient with a similar wound is treated by cleaning the wound, applying a bacitracin smear, and covering it with a bandage. The experimental and control antimicrobial formulations are reapplied and replaced with a new bandage every day.

[0293] After three days, the experimental patient shows no signs of infection, while the control patient has redness around the wound, indicating bacterial invasion.

[0294] After one week, the wounds in the experimental patients had healed, leaving only scars. The wounds in the control patients took 17 days to heal.

[0295] Example 4 Prophetic Example A solution of HA6-DPTA / NO as disclosed in Example 1 is prepared in PBS at a concentration of 5 mg / ml. A solution of CMC-DETA as disclosed in Example 2 is prepared in PBS at a concentration of 10 mg / ml. The two solutions are mixed to form a firm gel. This gel is applied to a diabetic foot sore infected with MRSA and previously treated with antibiotics. The gel is covered with a bandage. The gel is reapplied daily with a fresh bandage.

[0296] After six days, the patient shows no signs of infection. After three weeks, the patient's wounds are healed.

[0297] It is contemplated that various combinations or subcombinations of the specific features and aspects of the above-described embodiments may be made and still be within the scope of one or more of the inventions. Moreover, the disclosure herein of any particular feature, aspect, method, property, attribute, quality, attribute, element, or the like in connection with an embodiment may be used in all other embodiments described herein. Thus, it should be understood that the various features and aspects of the embodiments of the present disclosure can be combined with or substituted for one another to form various modes of the invention of the present disclosure. Thus, it is intended that the scope of the invention disclosed herein should not be limited by the specific disclosed embodiments described above. Moreover, the present invention is susceptible to various modifications and alternative forms, specific examples of which have been shown in the drawings and are described in detail herein. However, the present invention should not be limited to the specific forms or methods disclosed, but rather should be understood to cover all modifications, equivalents, and alternatives within the spirit and scope of the various embodiments described and the appended claims. Any method disclosed herein need not be performed in the order recited. The methods disclosed herein include specific actions taken by a practitioner, but may also include, explicitly or implicitly, any third-party instructions for those actions. For example, an action such as "administering an NO donor composition" includes "instructing administration of an NO donor composition." Furthermore, when features or aspects of the present disclosure are described in terms of Markush groups, those skilled in the art will recognize that the present disclosure is also described in terms of any individual members or subgroups of members of the Markush group.

[0298] Ranges disclosed herein also encompass any and all overlaps, subranges, and combinations thereof. Phrases such as "up to," "at least," "greater than," "less than," "between," and the like, include the recited numbers. Numbers preceded by terms such as "about" or "approximately" include the recited numbers. For example, "about 10 one millipascal-second" includes "10 one millipascal-second."

Claims

1. 1. A NO-releasing hyaluronic acid derived polymeric compound comprising a unit structure of formula II: 【Chemistry 1】 Formula II During the ceremony, R ２ , R ３ , R ４ , R ５ , and R ６ are independently -OH, -NH ２ , -CH ２ OH, -C(O)OH, -NHC(O)-CH ３ , -O-((CH ２ ) ａ O) ｂ -H, -O-((CH ２ ) ａ O) ｂ - (CH ２ ) ｃ H, —O—(C １ - ５ alkyl), -NH-((CH ２ ) ｄ N.H. ｅ -H, -NH-((CH ２ ) ｄ N.H. ｅ - (CH ２ ) ｆ H, -X １ - ((CH ２ ) ｇ X ２ ) ｈ -H, -X １ - ((CH ２ ) ｇ X ２ ) ｈ - (CH ２ ) ｉ H, -CH ２ C(O)-X １ - ((CH ２ ) ｇ X ２ ) ｈ ((CH ２ ) ｊ X ３ ) ｋ - (CH ２ ) ｌ H, and -X １ - ((CH ２ ) ｇ X ２ ) ｈ ((CH ２ ) ｊ X ３ ) ｋ - (CH ２ ) ｌ H; R １ is -C(O)NH-((CH ２ ) ｇ X ２ ) ｈ -H, -C(O)NH-((CH ２ ) ｇ X ２ ) ｈ - (CH ２ ) ｉ H or -C(O)NH-((CH ２ ) ｇ X ２ ) ｈ ((CH ２ ) ｊ X ３ ) ｋ - (CH ２ ) ｌ H; a, b, c, d, e, f, g, h, i, j, k, and l are each independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X １ , X ２ , and X ３ each is independently selected from -O-, -S-, -NH-, C(O)NH-; Here, R １ In at least one of X, h and k must not both be 0; ２ , and X ３ is an NO-donor moiety represented by one of the following: 【Chemistry 2】 ;and wherein the compound is a NO-releasing hyaluronic acid-derived polymer compound having a viscosity of 10 mPa·s or greater at 20° C. at a concentration of 5% w / w in water.

2. 2. The compound of claim 1, wherein formula II has the stereochemical configuration shown in formula II'. 【Chemistry 3】 Formula II'

3. X ２ , and X ３ 3. The compound of claim 1 or 2, wherein at least one of is an NO donor moiety represented by one of the following: 【Chemistry 4】

4. R ２ and R ３ The compound according to any one of claims 1 to 3, wherein is -OH.

5. R ２ , R ３ , R ４ , R ５ , and R ６ The compound of any one of claims 1 to 4, wherein one or more of: 【Chemistry 5】

6. The compound according to any one of claims 1 to 5, wherein the compound has a molecular weight in the range of 6 kDa to 90 kDa.

7. 7. The compound according to any one of claims 1 to 6, wherein the compound has a viscosity of at least 20 mPa·s at a concentration of 20% w / w in water at 20°C.

8. The compound according to any one of claims 1 to 7, wherein the compound is soluble in water at a concentration of 50 mg / ml.

9. 9. The compound of any of claims 1 to 8, wherein the compound has a total releasable NO pool in the range of 0.1 to 1.0 μmol NO / mg of compound.

10. 10. The compound of any of claims 1 to 9, wherein the compound has a NO half-life in the range of 0.1 to 24 hours.

11. 11. The compound according to any of claims 8 to 10, wherein the compound has a total duration of NO release in the range of 1 to 60 hours.

12. The compound according to any one of claims 8 to 10, wherein the total NO release after 4 hours is in the range of 0.1 to 1.0 μmol of NO / mg of compound.

13. 13. Use of a compound of any of claims 1 to 12 in the manufacture of a medicament for delivering nitric oxide to a subject in need of treatment.

14. 14. The use according to claim 13, wherein the compound is a hydrogel.

15. 13. Use of a compound of any of claims 1 to 12 in the manufacture of a medicament for treating a disease condition, comprising The use, wherein the disease state is selected from the group consisting of cancer, cardiovascular disease, microbial infection, platelet aggregation and adhesion caused by exposure of blood to medical devices, pathological conditions resulting from abnormal cell proliferation, transplant rejection, autoimmune diseases, inflammation, vascular diseases, scar tissue, wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunction, and sexually transmitted diseases.

16. 1. A pharmaceutical formulation comprising: A compound according to any one of claims 1 to 12, and A pharmaceutical formulation comprising a pharma- ceutically acceptable excipient.

17. Use of a compound according to any one of claims 1 to 12 for reducing or preventing the microbial load on inorganic surfaces, comprising The method comprises applying a compound according to any one of claims 1 to 12 to a surface contaminated with a plurality of microorganisms, wherein the compound produces nitric oxide and induces oxidative and / or nitrosative damage to microbial DNA and membrane structures, thereby preventing or reducing microbial burden, and the plurality of microorganisms includes two or more of gram-positive bacteria, gram-negative bacteria, fungi, yeast, and viruses.

18. 18. The use according to claim 17, wherein the inorganic surface is an external or internal surface of a medical device.

19. The use of claim 18 , wherein the medical device comprises an endoscope.

20. 20. The use according to any of claims 17 to 19, wherein the microbial load comprises microorganisms associated with the presence of one or more of the following: human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza virus, hepatitis virus, coxsackie virus, varicella zoster virus, measles virus, mumps virus, rubella virus, rabies virus, pneumonia virus, viruses causing hemorrhagic viral fever, influenza A virus subtype H1N1, parasites, fungi, molds, Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, group A streptococcus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, carbapenem-resistant Enterobacteriaceae, Methicillin-resistant Staphylococcus aureus, and Burkholderia cepacia.

21. 21. The use of claim 20, wherein the microbial load comprises Methicillin-resistant Staphylococcus aureus, Carbapenem-resistant Enterobacteriaceae, Staphylococcus aureus, Pseudomonas aeruginosa or Burkholderia cepacia.

22. 13. Use of a compound according to any one of claims 1 to 12 for the manufacture of a medicament adapted to deliver nitric oxide to a subject in need of treatment of disease, tissue damage or reduction of microbial load.

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