Nitric oxide-releasing hyperbranched compounds as antibacterial scaffolds and methods pertaining thereto

Abstract

To provide nitric oxide-releasing hyperbranched structures having units that store and / or release nitric oxide in a controlled manner.SOLUTION: In some embodiments, a hyperbranched structures are covalently modified to store and release nitric oxide. Some embodiments pertain to methods of making and also to the use of hyperbranched structures. The covalently modified hyperbranched structures may be tailored to release nitric oxide in a controlled manner and are useful for eradication of both gram positive and gram negative bacteria as well as other microbes.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 586,404, filed on November 15, 2017, and U.S. Provisional Patent Application No. 62 / 737,603, filed on September 27, 2018, the entire contents of which are incorporated herein by reference.

[0002] Government Support This invention was made with government support under grant DE025207 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The subject matter of this disclosure generally relates to nitric - oxide - releasing hyperbranched structures having units that store and / or release nitric oxide in a controlled manner. Its synthetic method as an antibacterial agent and its use in that method are further disclosed.

Background Art

[0004] Bacterial infections pose a major challenge to human health in both community and hospital settings. A biofilm is a cooperative community 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, as disclosed herein, can play an important role, for example, as a therapeutic agent in the treatment or amelioration of pathophysiology. NO as a therapeutic agent has not been fully utilized heretofore, at least in part due to the limited NO payload of therapeutic compositions, a NO release rate that is more rapid than desired, and a lack of targeted NO delivery. Provided herein are methods of treating various pathophysiologies using hyperbranched NO-releasing constructs, methods of making such constructs, and such constructs that utilize improved NO release characteristics to take advantage of the rich potential of NO-releasing pharmacological compounds. In particular, compounds that are highly effective as antibacterial agents are provided herein.

[0006] For example, in some embodiments, one or more hyperbranched compounds that release NO and exhibit potent antibacterial characteristics are provided. In some embodiments, the hyperbranched compound is a functionalized hyperbranched poly(amidoamine) (PAMAM). In some embodiments, the hyperbranched poly(amidoamine) is provided in a single step that mixes acrylate and amine-containing starting materials to provide a structure that includes amide bonds (e.g., by reaction of an amine starting material with an ester or carboxylate of acrylate), secondary amine bonds (e.g., by reaction of a starting material amine with acrylate via Michael addition), and / or tertiary amine bonds (e.g., by reaction of a starting material amine with multiple acrylates via Michael addition). In some embodiments, the hyperbranched compound is a functionalized hyperbranched hydroxyl-terminated poly(amidoamine).

[0007] In some embodiments, at least one secondary amine of the hyperbranched compound includes a NO donor. In some embodiments, at least one secondary amine of the hyperbranched compound includes a NO donor. In some embodiments, the hyperbranched construct includes an amine-containing group covalently bonded to a NO donor. In some embodiments, the NO donor of the hyperbranched construct generates NO and induces damage to the membranes and / or DNA of microorganisms, thereby reducing the number of viable microorganisms.

[0008] Some embodiments relate to methods for reducing microbial contamination. In some embodiments of the method, the method comprises contacting a surface contaminated with a plurality of microorganisms with a compound comprising a NO-releasing hyperbranched compound. In some embodiments of the method, the NO donor generates NO and induces damage to the membranes and / or DNA of the microorganisms, thereby reducing the number of viable 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 the oral cavity or surrounding tissues (e.g., lips, nostrils, teeth, gums, etc.). In some embodiments, the surface comprises oral mucosa. Advantageously, in some embodiments of the method, the application step does not induce skin or tissue irritation. In some embodiments, the plurality of microorganisms includes one or more viruses, Gram-positive bacteria, Gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof.

[0009] In some embodiments, the hyperbranched NO-donating compound is hyperbranched PAMAM. In some embodiments, the hyperbranched NO-donating compound comprises one or more linking groups. In some embodiments, the linking group comprises any one or more of Formulas A, B, C, or D.

Chemical formula

[0010] In some embodiments,

Chemical formula

Chemical formula

[0011] In some embodiments, the hyperbranched compound does not contain aminoglycoside or glycoside units.

[0012] In some embodiments, the hyperbranched NO-donating compound comprises at least one example of the following structure,

Chemical formula

[0013] In some embodiments, the linking group comprises Formula A. In some embodiments, the linking group comprises Formula B, wherein R 1 is as disclosed elsewhere herein and / or -R b N(R c )R d -(N(R c )) n -R b -, n is as disclosed elsewhere herein and / or 1, R d is as disclosed elsewhere herein and / or -CH 2 -, R b is as disclosed elsewhere herein and / or a single bond, and each R c is as disclosed elsewhere herein and / or H.

[0014] In some embodiments, the linking group of Formula B is represented by the following structure.

Chemical formula

[0015] In some embodiments, each instance of R a is a NO-donating moiety or -H.

[0016] In some embodiments, the linking group of formula A is represented by the following structure. [Chemical formula]

[0017] In some embodiments, the hyperbranched NO-donating compound further comprises a terminal group selected from the group consisting of [Chemical formula] wherein each instance of R 5 is H or -N + (=N-O - )O - .

[0018] In some embodiments, the hyperbranched NO-donating compound further comprises a terminal group selected from the group consisting of [Chemical formula]

[0019] In some embodiments, the hyperbranched NO-donating compound further comprises one or more of the following groups. [Chemical formula]

[0020] In some embodiments, the hyperbranched structure lacks a dendritic core having any symmetric dendrons.

[0021] In some embodiments, each instance of any substitution is selected from C 1 -C 6 alkyl or -OH.

[0022] Some embodiments relate to hyperbranched nitric oxide (NO)-donating compounds and include a linking group of formula A or formula B.

Chemical formula

[0023] In some embodiments,

Chemical formula

[0024] In some embodiments, X 1 and X 2 are as disclosed elsewhere herein and / or independently are selected from the group consisting of -NH-, -N(R a ), -O-, and -S-.

[0025] In some embodiments, each instance of R a is as disclosed elsewhere herein and / or is selected from an NO-donating moiety or -H.

[0026] In some embodiments, R 1 is as disclosed elsewhere herein and / or independently is -N(R c )R d -N(R c )-, -R b (OR d -) n O-R b -, and C 1 -C 6 alkyl selected from the group consisting of.

[0027] In some embodiments, each instance of R c is as disclosed elsewhere herein and / or independently is an NO-donating moiety or -H, optionally substituted C1 -C 6 It is selected from an alkyl group or an optionally substituted polyether having 1 to 6 repeating units.

[0028] In some embodiments, R b is as disclosed elsewhere herein and / or is a single bond or an optionally substituted C 1 -C 6 alkylene group.

[0029] In some embodiments, R d is as disclosed elsewhere herein and / or is -CH 2 - or -CH 2 -CH 2 -.

[0030] In some embodiments, n is an integer selected from 0, 1, 2, 3, 4, 5, or 6 as disclosed elsewhere herein and / or.

[0031] In some embodiments, the hyperbranched compound contains at least one of the following NO-donating moieties.

Chemical formula

[0032] In some embodiments, the hyperbranched NO-donating compound further contains at least one example of the following structures.

Chemical formula

[0033] In some embodiments, R 4 is -N + (=N-O - )O - .

[0034] In some embodiments, the hyperbranched NO-donating compound contains formula A.

[0035] In some embodiments, the hyperbranched NO-donating compound comprises Formula B, wherein R 1 is as disclosed elsewhere herein and / or -N(R c )R d -N(R c )-, where R d is as disclosed elsewhere herein and / or -CH 2 -, and each R c is as disclosed elsewhere herein and / or H.

[0036] In some embodiments, the hyperbranched NO-donating compound further comprises a linking group represented by the following structure.

Chemical formula

[0037] In some embodiments, each example of R a is an NO-donating moiety or -H.

[0038] In some embodiments, the linking group comprises Formula A and is represented by the following structure.

Chemical formula

[0039] In some embodiments, the hyperbranched NO-donating compound further comprises a terminal group selected from the group consisting of the following.

Chemical formula

[0040] In some embodiments, each example of R 5 is H or -N + (=N-O - )O - .

[0041] In some embodiments, the hyperbranched NO-donating compound further comprises a terminal group selected from the group consisting of the following.

Chemical formula

[0042] In some embodiments, the hyperbranched NO-donating compound further comprises one or more of the following groups.

Chemical formula

[0043] In some embodiments, the hyperbranched NO-donating compound lacks a dendritic core having any symmetric dendrons.

[0044] In some embodiments, the hyperbranched NO-donating compound does not contain aminoglycoside or glycoside units.

[0045] In some embodiments, each instance of any substitution is selected from C 1 -C 6 alkyl or -OH, or is as disclosed elsewhere in this specification.

[0046] In some embodiments, the hyperbranched NO-donating compound comprises dendritic units, linear units, and / or terminal units, wherein the dendritic units contain tertiary amines, the linear units contain secondary amines, and the terminal units contain primary amines.

[0047] In some embodiments, the hyperbranched NO-donating compound N-diazanium dioate exhibits an intramolecular hydrogen bond with the primary amine of the terminal unit. In some embodiments, a portion of the amine modified by the N-diazanium dioate moiety is a secondary amine.

[0048] In some embodiments, the hyperbranched NO-donating compound contains at least about 2 wt% NO. In some embodiments, the hyperbranched NO-donating compound contains at least about 1 μmol NO per mg of the compound, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37 °C). In some embodiments, the hyperbranched NO-donating compound contains at least about 2 μmol NO per mg of the compound, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37 °C). In some embodiments, the hyperbranched NO-donating compound contains more than about 2 μmol NO per mg of the compound, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37 °C).

[0049] In some embodiments, the hyperbranched NO-donating compound contains a hydroxy moiety. In some embodiments, the hydroxy moiety is linked to the amine moiety via an alkyl moiety, whereby the amine moiety becomes a secondary amine.

[0050] In some embodiments, the hyperbranched compound has a weight-average molecular weight (MW) of about 2×10 3 gmol -1 ~ about 15×10 3 gmol -1 as determined by size-exclusion chromatography using a multi-angle light scattering (SEC-MALS) detector. In some embodiments, the hyperbranched compound has a weight-average molecular weight (MW) of about 3×10 3 gmol -1 ~ about 10×10 3 gmol -1 as determined by size-exclusion chromatography using a multi-angle light scattering (SEC-MALS) detector. In some embodiments, the hyperbranched compound has a weight-average molecular weight (MW) of about 3×10 3 gmol -1 ~ about 6×10 3 gmol -1 as determined by size-exclusion chromatography using a multi-angle light scattering (SEC-MALS) detector.

[0051] Some embodiments relate to methods for preparing the hyperbranched NO-donating compounds disclosed herein. In some embodiments, an acrylate is contacted with a nucleophile to form a hyperbranched compound.

[0052] In some embodiments, the acrylate is a monoacrylate, diacrylate, triacrylate, or tetraacrylate. In some embodiments, the nucleophile is a bifunctional, trifunctional, or tetrafunctional molecule. In some embodiments, the nucleophile comprises H-R h N(R e )R f -(N(R e )) n -(R g O-) m R h -H, wherein each instance of R e is independently -H, an optionally substituted C 1 -C 6 alkyl group, or an optionally substituted polyether having 1 to 6 repeating units, and / or is optionally substituted as disclosed elsewhere herein. In some embodiments, each instance of R h is a single bond or an optionally substituted C 1 -C 6 alkylene group. In some embodiments, R f and R g are independently an optionally substituted C 1 -C 6 alkylene group. In some embodiments, the nucleophile is H 2 N-((CH 2 ) a NH) b -H, H 2 N-((CH 2 ) a NH) b -(CH 2 ) c H, H 2 N-((CH 2 ) a X 5 ) b -(CH2 ) c H, and HX 5 -((CH 2 ) a X 6 ) b ((CH 2 ) c X 7 ) d -(CH 2 ) e including one or more of H. In some embodiments, each instance of a, b, c, d, or e is independently selected from the integers 0 to 10. In some embodiments, X 5 , X 6 , and X 7 each instance of is independently selected from O, S, or NH.

[0053] In some embodiments, the nucleophile is H 2 NCH 2 CH 2 NHCH 2 CH 2 NH 2 , H 2 NCH 2 CH 2 NHCH 2 CH 2 OH, and

Chemical formula

[0054] In some embodiments, the acrylate includes one or more of the following structures.

Chemical formula

[0055] In some embodiments, R 1 , R 2 , and R 3 are, independently, -R b N(R c )R d -(N(R c )) n -R b -, -R b (OR d -) n O-R b -, and C 1 -C 6 alkyl selected from the group consisting of. In some embodiments, each instance of R c is, independently, an NO-donating moiety, -H, an optionally substituted C 1 -C 6 alkyl group, or an optionally substituted polyether having 1 to 6 repeating units. In some embodiments, R b is a single bond or an optionally substituted C 1 -C 6 alkylene group. In some embodiments, R d is an optionally substituted C 1 -C 6 alkylene group. In some embodiments, n is an integer selected from 0, 1, 2, 3, 4, 5, or 6. In some embodiments, when R 2 is present, at least one instance of R d contains a -C(O)-CH=CH 2 group. In some embodiments, when R 3 is present, at least one instance of the carbon of R d contains a -C(O)-CH=CH 2 group. In some embodiments, the acrylate is N,N'-methylenebis(acrylamide).

[0056] In some embodiments, a hyperbranched compound is exposed to a NO source to provide a hyperbranched NO-donating compound. In some embodiments, the NO exposure step is carried out under alkaline conditions.

[0057] In some embodiments, a nucleophile-to-acrylate molar ratio of about 2:1 to about 5:1 is used. In some embodiments, an amine-to-acrylate molar ratio of about 3:1 to about 4:1 is used.

[0058] Some embodiments relate to a method of reducing microbial contamination. In some embodiments, a surface contaminated with a plurality of microorganisms is contacted with a hyperbranched NO-donating compound disclosed herein. In some embodiments, the NO donor generates nitric oxide and induces damage to the membranes and / or DNA of the microorganisms, thereby reducing the number of viable microorganisms.

[0059] In some embodiments, the plurality of microorganisms includes one or more of viruses, Gram-positive bacteria, Gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof.

[0060] In some embodiments, the surface is an organic surface. In some embodiments, the surface is human skin or animal skin. In some embodiments, the surface is in the oral cavity. In some embodiments, the application does not induce skin irritation.

[0061] In some embodiments, the surface is an inorganic surface. In some embodiments, the inorganic surface is the outer or inner surface of a medical device. In some embodiments, the device is a dental device.

[0062] In some embodiments, the microbial load includes drug-resistant bacteria. In some embodiments, the microbial load includes one or more oral pathogens. In some embodiments, the microbial load includes one or more of P. aeruginosa, S. aureus, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans.

[0063] Some embodiments relate to methods of treating and / or preventing dental caries. In some embodiments, the surface of a patient's mouth contaminated with one or more oral pathogens is contacted with a hyperbranched NO-donating compound disclosed elsewhere herein. In some embodiments, the hyperbranched NO-donating compound generates nitric oxide and induces damage to the membranes and / or DNA of the pathogens, thereby reducing the number of viable pathogens. In some embodiments, the microbial load includes one or more of P. aeruginosa, S. aureus, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans.

[0064] Some embodiments relate to the use of a hyperbranched NO-donating compound disclosed elsewhere herein in the preparation of a medicament for reducing microbial contamination. In some embodiments, the hyperbranched NO-donating compound generates nitric oxide and induces damage to the membranes and / or DNA of the microorganisms, thereby reducing the number of viable microorganisms. In some embodiments, the compound is formulated to treat a plurality of microorganisms including one or more of viruses, Gram-positive bacteria, Gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof.

[0065] In some embodiments, the compound is formulated to be delivered to an organic surface. In some embodiments, the compound is formulated to be delivered to human skin or animal skin. In some embodiments, the surface is in the oral cavity. In some embodiments, the compound is formulated to be delivered to an inorganic surface. In some embodiments, the surface is the outer or inner surface of a medical device. In some embodiments, the device is a dental device.

[0066] Some embodiments relate to a polyamideamine PAMAM composition comprising a hyperbranched copolymer of an amine and an acrylate, wherein at least a portion of the amine is modified with an N-diazeniumdiolate moiety. In some embodiments, the hyperbranched copolymer comprises dendritic units, linear units, and terminal units, the dendritic units comprising tertiary amines, the linear units comprising secondary amines, and the terminal units comprising primary amines.

[0067] In some embodiments, the N-diazeniumdiolate exhibits an intramolecular hydrogen bond with the primary amine of the terminal unit. In some embodiments, the portion of the amine modified with the N-diazeniumdiolate moiety is a secondary amine.

[0068] In some embodiments, the amine has the structure: H 2 N-A 1 -(NH)-A 2 -NH 2 and is derived from a polyfunctional amine monomer. In some embodiments, A 1 and A 2 are independently selected from an alkyl moiety or hydrogen. In some embodiments, the acrylate is derived from the following monomers,

Chemical formula

[0069] In some embodiments, the composition comprises at least about 2% by weight of NO. In some embodiments, the composition comprises at least about 1 μmol of NO per mg of copolymer, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37°C). In some embodiments, the composition comprises at least about 2 μmol of NO per mg of copolymer, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37°C). In some embodiments, the composition comprises more than about 2 μmol of NO per mg of copolymer, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37°C).

[0070] In some embodiments, the copolymer further comprises a hydroxy moiety. In some embodiments, the hydroxy moiety is linked to the amine moiety via an alkyl moiety, whereby the amine moiety is a secondary amine.

[0071] In some embodiments, the molar ratio of amine to acrylate is from about 2:1 to about 5:1. In some embodiments, the molar ratio of amine to acrylate is from about 3:1 to about 4:1.

[0072] In some embodiments, the hyperbranched copolymer has a weight average molecular weight (MW) of about 2×10 3 g / mol -1 ~ about 15×10 3 g / mol -1 as determined by size exclusion chromatography using a multi-angle light scattering (SEC-MALS) detector. In some embodiments, the hyperbranched copolymer has a weight average molecular weight (MW) of about 3×10 3 g / mol -1 ~ about 10×10 3 g / mol -1has a weight average molecular weight (MW). In some embodiments, the hyperbranched copolymer has a weight average molecular weight (MW) of about 3×10 3 g / mol -1 to about 6×10 3 g / mol -1 as determined by size exclusion chromatography using a multi-angle light scattering (SEC-MALS) detector.

[0073] In some embodiments, the hyperbranched copolymer of the polyfunctional amine and acrylate is soluble in water at levels greater than about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 50 mg / mL, or about 100 mg / mL. In some embodiments, the acrylate is derived from monomers selected from salts, esters, and conjugate bases of acrylic acid and its derivatives. In some embodiments, the acrylate is derived from the monomer methacrylate. In some embodiments, the acrylate is selected from the group consisting of methyl acrylate, ethyl acrylate, methyl methacrylate, acrylamide, ethyl methacrylate, 2-chloroethyl vinyl ether, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, butyl acrylate, butyl methacrylate, N-(2-hydroxypropyl) methacrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, N-(3-BOC-aminopropyl) methacrylamide, 2-aminoethyl methacrylate hydrochloride, 2-(tert-butylamino) ethyl methacrylate, n-isopropylacrylamide, 2-methoxyethyl acrylate, n-ethyl methacrylamide, n-vinylacetamide, 2-N-morpholinoethyl acrylate, methacryloyl-L-lysine, 2-(methylamino) ethyl acrylate, and 2-(methylamino) ethyl methacrylate. In some embodiments, the acrylate is derived from a diacrylate. For example, the diacrylate is selected from the group consisting of ethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, tricyclodecane dimethanol diacrylate, N-acryloxysuccinimide, N-(2-hydroxypropyl) methacrylamide, bis[2-(methacryloyloxy)ethyl] phosphate, diacrylamide, and N,N'-methylenebisacrylamide.

[0074] In some embodiments, the amine is derived from diethylenetriamine. In some embodiments, the amine is derived from a monomer selected from the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, polyetheramine, and bis(hexamethylene)triamine. In some embodiments, upon release of NO, the hyperbranched copolymer 1 H NMR: (400 MHz, CD 3 OD, δ): 2.22 - 2.90 (COCH 2 , NHCH 2 , and NH 2 CH 2 ), 3.15 - 3.58 (CONCH 2 ), 3.60 (CH 3 O). 13 C (600 MHz, CD 3 OD, δ): 30 - 60 (CH 2 and CH 3 ), 170 - 175 (C=O). FTIR (cm -1 ): 3308 (NH 2 ), 2957 (CH 2 ), 2848 (CH 2 ), 1647 (C=O), and 1556 (NH), showing characteristics consistent therewith.

[0075] In some embodiments, the hyperbranched PAMAM copolymer has a polydispersity index (PDI) greater than about 1.1. In some embodiments, the hyperbranched copolymer has a polydispersity index (PDI) of 1.1 to 2. In some embodiments, the hyperbranched copolymer has a polydispersity index (PDI) of 1.5 to 1.9.

[0076] Some embodiments relate to PAMAM hyperbranched copolymers comprising one or more dendritic units containing one or more tertiary amines, a plurality of terminal units containing a plurality of primary amines, and a plurality of linear units containing a plurality of secondary amines, wherein at least a portion of the plurality of secondary amines is bonded to a plurality of N-diazinium dioate moieties. In some embodiments, at least a portion of the dendritic units have the structure: [Chemical formula] comprising, at least a part of the plurality of linear units having the structure: [Chemical formula] comprising a group selected from, or a combination of these.

[0077] In some embodiments, at least a part of the plurality of N-diazinium dioleate moieties is stabilized by a hydrogen bond between the oxygen of the N-diazinium dioleate moiety and at least one hydrogen of the plurality of primary amines.

[0078] Some embodiments relate to a method of delivering nitric oxide to a subject. In some embodiments, an effective amount of a hyperbranched compound or polyamidoamine composition disclosed herein is administered to the subject.

[0079] Some embodiments relate to a method of treating a disease state. In some embodiments, an effective amount of a hyperbranched compound or polyamidoamine composition disclosed herein is administered to a subject in need of treatment. In some embodiments, the disease state is selected from the group consisting of gingivitis, cancer, cardiovascular disease, microbial infections, platelet aggregation and platelet adhesion caused by blood exposure to medical devices, pathological conditions resulting from abnormal cell growth, transplant rejection, autoimmune diseases, inflammation, vascular diseases, scar tissue, wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunction, and sexually transmitted infections.

[0080] Some embodiments relate to a method of manufacturing a polyamideamine composition. In some embodiments, a polyfunctional amine (e.g., having 2, 3, 4, 5, or more amines) is mixed with an acrylate monomer in a suitable solvent to form a reaction mixture. In some embodiments, the reaction mixture is mixed for a time sufficient for a significant proportion of the polyfunctional amine to react with the acrylate monomer to form a hyperbranched copolymer. In some embodiments, the reaction mixture is heated to complete the polymerization and remove unreacted monomers to form a basic polyamideamine composition. In some embodiments, the hyperbranched structure is mixed with gaseous NO under basic conditions and at high pressure for a time sufficient to obtain an N-diazinium dioate moiety in the polyamideamine composition. The mixing time is longer than about 6 hours, about 6 hours to about 2 weeks, about 6 hours to 1 week, about 12 hours to 5 days, or about 1 day to about 3 days.

[0081] In some embodiments, heating the reaction mixture to complete the polymerization and remove unreacted monomers includes heating under subatmospheric pressure. In some embodiments, heating the reaction mixture to complete the polymerization and remove unreacted monomers includes heating to a first temperature of about 50°C to about 70°C for a first time of about 30 minutes to about 2 hours, heating to a second temperature of about 90°C to about 110°C for a second time of about 30 minutes to about 4 hours, and heating to a third temperature of about 120°C to about 150°C for a third time of about 30 minutes to about 4 hours. In some embodiments, heating the reaction mixture to complete the polymerization and remove unreacted monomers includes heating to a first temperature of about 60°C for a first time of about 1 hour, heating to a second temperature of about 100°C for a second time of about 2 hours, and heating to a third temperature of about 140°C for a third time of about 2 hours.

[0082] In some embodiments, the polyfunctional amine is ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, a polyetheramine (e.g., selected from the group consisting of Jeffamine, the polyetheramine being an amine having an average molecular weight of about 1700 based on PTMEG [poly(tetramethylene ether glycol)], a diamine having a molecular weight of about 1000 based on a PTMEG [poly(tetramethylene ether glycol)] / PPG (polypropylene glycol) copolymer), and bis(hexamethylene)triamine).

[0083] In some embodiments, the polyetheramine is JEFFAMINE®. In some embodiments, JEFFAMINE® is selected from M-600, M-2005, M-1000, M-2070, D-230, D-400, D-2000, D-4000, ED-600 amine, ED-900 amine, ED-2003 amine, EDR-148 amine, EDR-176 amine, T-403 amine, T-3000 amine, T-5000 amine, THF-100 amine, THF-170 amine, XTJ568, XTA801, RFD-270, and XTJ-616. In some embodiments, the polyetheramine comprises one of the following structures,

Chemical formula

[0084] In some embodiments, the acrylate is selected from the group consisting of methyl acrylate, ethyl acrylate, methyl methacrylate, acrylamide, ethyl methacrylate, 2-chloroethyl vinyl ether, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, butyl acrylate, butyl methacrylate, N-(2-hydroxypropyl) methacrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, N-(3-BOC-aminopropyl) methacrylamide, 2-aminoethyl methacrylate hydrochloride, 2-(tert-butylamino)ethyl methacrylate, n-isopropylacrylamide, 2-methoxyethyl acrylate, n-ethylmethacrylamide, n-vinylacetamide, 2-N-morpholinoethyl acrylate, methacryloyl-L-lysine, 2-(methylamino)ethyl acrylate, and 2-(methylamino)ethyl methacrylate.

[0085] In some embodiments, the solvent is an alcohol or a mixture of alcohols. In some embodiments, the polyamidoamine compound is modified at the hydroxyl moiety by mixing the polyamidoamine composition with a hydroxy-containing compound or a hydroxyl-forming compound (e.g., an epoxide). In some embodiments, the hydroxy-containing compound is an epoxide. In some embodiments, the epoxide is a polypropylene oxide. In some embodiments, the polyfunctional amine is combined with the acrylate monomer at a molar ratio of amine to acrylate of about 2:1 to about 5:1. In some embodiments, the polyfunctional amine is combined with the acrylate monomer at a molar ratio of amine to acrylate of about 3:1 to about 4:1.

[0086] In some embodiments, a hyperbranched compound or polyamidoamine composition, as disclosed elsewhere herein, is formed. In some embodiments, the hyperbranched compound has an NO storage capacity of about 0.4 μmol or more of NO per 1 mg of the hyperbranched compound. In some embodiments, the hyperbranched compound provides a bacterial reduction of about 90% or more (e.g., 90%, 95%, 97%, 98%, 99%, or 100%) of bacterial viability against one or more of P. aeruginosa, S. aureus, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans. In some embodiments, such reduction is achieved at a concentration of the hyperbranched compound of 2 mg / mL or less.

Brief Description of the Drawings

[0087]

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Mode for Carrying Out the Invention

[0088] Some embodiments disclosed herein provide for the synthesis and characterization of hyperbranched structures by N-diazaniumdiolate NO donor modification. In some embodiments, the hyperbranched structure is a hyperbranched PAMAM (h-PAMAM) polymer. In some embodiments, the h-PAMAM is partially or substantially hydroxylated (e.g., using an epoxide) to provide additional secondary amines that can act as NO donors. In some embodiments, the hyperbranched PAMAM scaffolds, with or without hydroxyl (e.g., propylene oxide (PO)) modification, can both release NO. In some embodiments, for example, the compound (h-PAMAM-PO-2 / NO described below) enables an efficient reduction in the viability and / or eradication of microorganisms (e.g., selected oral pathogens) and mammalian cells (e.g., to human gingival fibroblasts) having low toxicity and / or minimal toxicity natural tissues. In some embodiments, despite structural defects (substantially absent in dendrimers and present in hyperbranched structures), the disclosed hyperbranched structures are antibacterial. In some embodiments, the hyperbranched structures disclosed herein benefit from structural defects compared to pure dendrimer compounds. In some embodiments, the antibacterial activity of the hyperbranched structures (e.g., h-PAMAM-PO-2 / NO) is comparable to and / or improved compared to dendrimers (e.g., G3-PAMAM-PO / NO). In this regard, the disclosed hyperbranched structures (e.g., h-PAMAM-PO-2 / NO) are potentially scalable therapeutic methods. In some embodiments, the hyperbranched structures disclosed herein are neither dendrimers nor dendrons and lack one or more properties of dendrimers and / or dendrons.

[0089] The subject matter of the present disclosure will now be described more fully hereinafter. However, many modifications and other embodiments of the subject matter of the present disclosure described herein will come to mind to those of ordinary skill in the art to which the present disclosure pertains, having the benefit of the teachings presented in the foregoing description. Accordingly, the subject matter of the present disclosure should not be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein includes all alternatives, modifications, and equivalents. If 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, usage of terms, described techniques, etc., this application prevails. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0090] As used herein, "and / or" refers to any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in terms of alternatives ("or"), and includes these.

[0091] As used herein, "a", "an", or "the" can mean one or more than one. For example, "a" NO-releasing moiety can mean a single or multiple ones.

[0092] As used herein, the term "about", when referring to a measurable value such as an amount, dosage, time, temperature, bactericidal efficacy, etc. of a compound or agent of the present subject matter, means an inclusion of a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0093] As used herein, the term "dendrimer" refers to a molecule having repeating branched moieties that emanate from a core and radiate outwardly from the dendron. Dendrimers are characterized by their complete or nearly complete symmetry. Dendrimers are typically symmetric or substantially symmetric around the core. Dendrimers are characterized by their stepwise synthesis with repeating structural units.

[0094] As used herein, the term "dendron" refers to a molecule having repeating branched moieties that emanate from a focal point and radiate outwardly. Dendrons are characterized by their complete or nearly complete repeating units. Dendrons are characterized by their stepwise synthesis with repeating structural units that emanate from the focal point.

[0095] As used herein, the term "hyperbranched" refers to a branched compound or structure that has no dendrimers symmetrically or (e.g., having symmetric or substantially symmetric dendrons) around the acorn and lacks dendrons. As used herein, hyperbranched is dendritic, although a "functionalized hyperbranched" structure may or may not have a nitric oxide donor moiety attached. Hyperbranched structures can be generated by a one-pot synthesis.

[0096] As used herein, the terms "therapeutically effective amount" or "effective amount" refer to an amount of an enumerated compound that confers a modulating effect, which may be a beneficial effect on a subject afflicted with a disorder, disease, or illness, including, for example, improvement of the subject's condition (e.g., of one or more symptoms), delay or reduction in the progression of a condition, prevention or delay in the onset of a disorder, and / or changes in clinical parameters, disease, or illness. For example, an effective amount can refer to an amount of a composition, compound, or agent that improves the subject's condition by at least 5%, for example, 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 condition can be a reduction in infection. In some embodiments, the improvement can be a reduction in the bacterial load (e.g., bioburden) on the surface or within the subject. The actual dosage level of the active ingredient in the active compositions of the subject matter of the present disclosure can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired response for a particular subject and / or use. The selected dosage level will depend on a variety of 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 health status and prior medical history of the subject being treated. In some embodiments, a minimum dose is administered and the dose is titrated up to the minimum effective amount in the absence of dose-limiting toxicity. Determination and adjustment of effective amounts, and evaluation of when and how such adjustments are made, are contemplated herein.

[0097] "Treatment" or "treating" or "treat" refers to any kind of action that confers a regulatory effect, which can be a beneficial effect on a subject suffering from a disorder, disease, or illness, including, for example, improvement of the condition of the subject (e.g., of one or more symptoms), delay or reduction in the progression of the condition, and / or change in clinical parameters, disease, or illness, cure of the disease, etc.

[0098] The terms "disrupting" and "eradicating" refer to the ability of the hyperbranched structures of the present disclosure to combat biofilms. A biofilm may be partially eradicated or disrupted, which means that the cells no longer bind to each other or to the surface. A biofilm may be completely eradicated, which means that the biofilm is no longer a substantially interconnected, aggregated, or continuous network of cells.

[0099] The term "nitric oxide donor" or "NO donor" refers to species and / or molecules that donate, release, and / or directly or indirectly transfer nitric oxide species such that the biological activity of the nitric oxide species is expressed at the intended site of action, and / or stimulate the endogenous production of nitric oxide in vivo, and / or increase the endogenous levels of nitric oxide in vivo.

[0100] The term "nitric oxide release" or "nitric oxide donation" refers to species that donate, release, and / or directly or indirectly transfer any one (or more than one) of the three redox forms of nitric oxide (NO+, NO−, NO (e.g., ·NO)), and / or a method of donating, releasing, and / or directly or indirectly transferring any one (or more than one) 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.

[0101] As used herein, the term "microbial infection" refers to bacterial, fungal, viral, yeast infections, and other microorganisms, as well as combinations thereof.

[0102] The "patient" or "subject" to be treated, as disclosed herein, is in some embodiments a human patient, but it should be understood that the principles of the subject matter of the present disclosure are effective for all vertebrate species, including mammals, for which the subject matter of the present disclosure is intended to be included within the terms "subject" and "patient". Suitable subjects are generally mammalian subjects. The subject matter described herein has found use in research as well as in 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, etc. Human subjects include neonatal, infant, juvenile, adult, and geriatric subjects. A subject is a subject "in need of" the methods disclosed herein who may be experiencing a disease state and / or is expected to experience a disease state, and the methods and compositions of the invention are used for therapeutic and / or prophylactic treatment.

[0103] For the general chemical formulas provided herein, when a substituent is not indicated, one of ordinary skill in the art will understand that the substituent is hydrogen. Bonds that are shown but not connected to an atom indicate that the position of such a substituent is variable. Zigzag lines, wavy lines, two wavy lines drawn through or at the end of a bond indicate that some additional structure is attached at that position. For a number of additional monomers that are disclosed herein but not explicitly shown in the structure, even if elemental analysis cannot indicate that such differences can be expected, it will be understood by one of ordinary skill in the polymer art that adding these monomers can potentially change the physical properties of the resulting polymeric material. Such physical properties include solubility, charge, stability, crosslinking, secondary and tertiary structure, and the like. Further, when stereochemistry is not indicated for a compound having one or more chiral centers, all enantiomers and diastereomers are included. Similarly, for the listing of aliphatic or alkyl groups, all of their structural isomers are also included. Unless otherwise specified, in the general formulas provided herein, A 1 ~A n as shown, and the groups referred to herein as alkyl groups are independently selected from alkyl groups or aliphatic groups, particularly alkyls having 20 or fewer carbon atoms, and more typically from lower alkyls having 10 or fewer atoms such as methyl, ethyl, propyl, isopropyl, and butyl. Alkyl may be optionally substituted (e.g., as disclosed elsewhere herein, it may or may not be substituted). Alkyl may be a substituted alkyl group such as a halogenated alkyl (e.g., -CX 3 wherein X is a halide and combinations thereof in the chain or attached thereto), an alcohol (i.e., an aliphatic or alkyl hydroxyl, particularly a lower alkyl hydroxyl), or other similarly substituted moieties, e.g., an amino-, amino acid-, aryl-, alkylaryl-, alkyl ester-, ether-, keto-, nitro-, sulfhydryl-, sulfonyl-, sulfoxide-modified-alkyl group.

[0104] The terms "amino" and "amine" refer to nitrogen-containing groups such as NR 3 , NH 3 , NHR 2 , and NHR 2 R, etc., where R can be as described elsewhere in this specification. Thus, "amino" as used herein can refer to a primary amine, a secondary amine, or a tertiary amine. In some embodiments, one R of the amino group can be a diazeniumdiolate (i.e., NONO).

[0105] Whenever a group is described as "optionally substituted", the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when substituted, when a group is described as "unsubstituted or substituted" (or "substituted" or "unsubstituted"), the substituent(s) can be selected from one or more of the indicated substituents. When no substituents are indicated, the indicated "optionally substituted" or "substituted" group, when unsubstituted, can be 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-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, amino, mono-substituted amine group, di-substituted amine group, mono-substituted amine(alkyl), di-substituted amine(alkyl), diamino group, polyamino, diether group, and polyether - individually, independently, and can be substituted with one or more groups(s) selected therefrom.

[0106] As used herein, "C a ~C b」(where "a" and "b" are integers) refers to the number of carbon atoms in the group. The indicated group can contain carbon atoms and can include from "a" to "b". Thus, for example, "C 1 ~C 4 alkyl" or "C 1 ~C 4 alkyl" groups refer to all alkyl groups having from 1 to 4 carbon atoms, i.e., CH 3 -, CH 3 CH 2 -, CH 3 CH 2 CH 2 -, (CH 3 ) 2 CH-, CH 3 CH 2 CH 2 CH 2 -, CH 3 CH 2 CH(CH 3 )-, and (CH 3 ) 3 C-. When "a" and "b" are not specified, the broadest scope described by these definitions is assumed.

[0107] When two "R" groups are described as being "together", the R groups and the atoms to which they are attached can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle. For example, but not limited to, when the R a R b groups of the NR a and R b are shown as being "together", it means that they are covalently bonded to each other to form a ring.

Chemical Structure

[0108] 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, etc. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, etc. An alkyl group may have 1 to 30 carbon atoms (whenever it appears herein, a numerical range such as "1 to 30" refers to each integer within the given range; for example, "1 to 30 carbon atoms" means that the 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 occurrences of the term "alkyl" when no numerical range is specified). The alkyl group may also be a medium-sized alkyl having 1 to 12 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may be substituted or unsubstituted. By way of example only, "C 1 ~C 5 alkyl" indicates that there are 1 to 5 carbon atoms in the alkyl chain, 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 not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl.

[0109] As used herein, the term "alkylene" refers to a divalent, fully saturated, straight-chain aliphatic hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene. The alkylene group is

Chemical formula

[0110] As used herein, the term "alkenyl" refers to a monovalent straight-chain or branched-chain radical containing 2 to 20 carbon atoms and containing one or more carbon-carbon double bonds, including but not limited to 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc. The alkenyl group can be unsubstituted or substituted.

[0111] As used herein, the term "alkynyl" refers to a monovalent straight-chain or branched-chain radical containing 2 to 20 carbon atoms and containing one or more carbon-carbon triple bonds, including but not limited to 1-propynyl, 1-butynyl, 2-butynyl, etc. The alkynyl group can be unsubstituted or substituted.

[0112] As used herein, "cycloalkyl" refers to a fully saturated (containing no double or triple bonds) monocyclic or polycyclic (such as bicyclic) hydrocarbon ring system. When composed of two or more rings, these rings may be linked in a fused, bridged, or spiro-bonded form. As used herein, the term "fused" refers to two rings sharing two atoms and one bond. As used herein, the term "bridged cycloalkyl" refers to a compound in which the cycloalkyl contains a bond of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings sharing one atom, and the two rings are not linked by a bridge. The cycloalkyl group can contain 3 to 30 atoms in the ring(s), 3 to 20 atoms in the ring(s), 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s), or 3 to 6 atoms in the ring(s). The cycloalkyl group can be unsubstituted or substituted. Examples of monocycloalkyl groups include, but are not 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.

[0113] 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, provided that when there is more than one, the double bonds cannot form a completely delocalized π - electron system throughout all the rings (otherwise, the group could be "aryl" as defined herein). The cycloalkenyl group can contain 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s), or 3 to 6 atoms in the ring(s). When composed of two or more rings, these rings may be linked in a fused, bridged, or spiro - bonded form. The cycloalkenyl group can be unsubstituted or substituted.

[0114] 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 π - electron system throughout all the rings. The number of carbon atoms in the aryl group can vary. For example, the aryl group can be C 6 -C 14 aryl group, C 6 -C 10 aryl group, or C 6It can be an 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(s) of the heteroaryl group can vary. For example, the heteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10 atoms in the ring(s), or 5 to 6 atoms in the ring(s), such as 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. Further, the term "heteroaryl" includes fused ring systems in which two rings, such as 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, benzisoxazole, 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.

[0115] As used herein, "heterocyclyl" or "heteroalicyclic" refers to monocyclic, bicyclic, and tricyclic ring systems of 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, up to 18-membered rings in which 1 to 5 carbon atoms are joined with heteroatoms to form the ring system. The heterocyclic ring can optionally contain one or more unsaturated bonds positioned as such, but a completely delocalized π-electron system does not occur throughout all the rings. The heteroatom(s) is / are elements other than carbon, including but not limited to oxygen, sulfur, and nitrogen. The heterocyclic ring can further contain one or more carbonyl or thiocarbonyl functional groups such that the definition includes oxo- and thio-based systems such as lactam, lactone, cyclic imide, cyclic thioimide, and cyclic carbamate. When composed of two or more rings, these rings may be linked in a fused, bridged, or spiro-bonded form. As used herein, the term "fused" refers to two rings sharing two atoms and one bond. As used herein, the term "bridged heterocyclyl" or "bridged heteroalicyclic" refers to a compound in which a heterocyclyl or heteroalicyclic contains a bond of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings sharing one atom, and the two rings are not linked by a bridge. Heterocyclyl and heteroalicyclic groups can contain 3 to 30 atoms in the ring(s), 3 to 20 atoms in the ring(s), 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s), or 3 to 6 atoms in the ring(s). For example, 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, 2 carbon atoms and 3 heteroatoms, 1 carbon atom and 4 heteroatoms, 3 carbon atoms and 1 heteroatom, or 2 carbon atoms and 1 heteroatom. Additionally, any nitrogen in the heteroalicyclic can be quaternized. The heterocyclyl or heteroalicyclic group can be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroalicyclic" 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-oxathiin, 1,3-oxathiolane, 1,3-dithiol, 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, 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, thiomorpholine, thiomorpholine sulfoxide, thiomorpholine sulfone, and their benzofused analogs (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.

[0116] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group connected as a substituent via a lower alkylene group. The lower alkylene and aryl groups of aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0117] As used herein, "cycloalkyl(alkyl)" refers to a cycloalkyl group connected via a lower alkylene group as a substituent. The lower alkylene and cycloalkyl groups of cycloalkyl(alkyl) may be substituted or unsubstituted.

[0118] As used herein, the terms "heteroalkyl" and "heteroaryl(alkyl)" refer to a heteroaryl group connected via a lower alkylene group as a substituent. The lower alkylene and heteroaryl groups of heteroalkyl may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and their benzo-fused analogs.

[0119] "Heteroaracyclic(alkyl)" and "heterocyclic(alkyl)" refer to a heterocyclic or heteroalicyclic group connected via a lower alkylene group as a substituent. The lower alkylene and heterocyclic of (heteroaracyclic)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-thiazinan-4-yl(methyl).

[0120] As used herein, the term "hydroxy" refers to an -OH group.

[0121] As used herein, "alkoxy" refers to the formula -OR, where R can be 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 alkoxys is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, phenoxy, and benzyloxy. Alkoxy may be substituted or unsubstituted.

[0122] As used herein, "acyl" refers to hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), and heterocyclyl(alkyl) connected via a carbonyl group as a substituent. Examples include formyl, acetyl, propanoyl, benzoyl, and acrylyl. Acyl may be substituted or unsubstituted.

[0123] As used herein, the "cyano" group refers to the "-CN" group.

[0124] As used herein, the term "halogen atom" or "halogen" means any one of the radioactively stable atoms in Group 7 of the periodic table of the elements, such as fluorine, chlorine, bromine, and iodine.

[0125] The "thiocarbonyl" group refers to the "-C(=S)R" group, where R can be the same as defined for O-carbonyl. Thiocarbonyl may be substituted or unsubstituted. The "O-carbamyl" group refers to the "-OC(=O)N(R A R B )" group, where R A and R Bindependently can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). O-carbamyl may be substituted or unsubstituted.

[0126] The "N-carbamyl" group refers to the "ROC(=O)N(R A )-" group, where R and R A independently can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-carbamyl may be substituted or unsubstituted.

[0127] The "O-thiocarbamyl" group refers to the "-OC(=S)-N(R A R B )" group, where R A and R B independently can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). O-thiocarbamyl may be substituted or unsubstituted.

[0128] The "N-thiocarbamyl" group refers to the "ROC(=S)N(R A )-" group, where R and R A independently can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-thiocarbamyl may be substituted or unsubstituted.

[0129] The "C-amide" group refers to the "-C(=O)N(R A R B )" group, where R A and R B are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The C-amide may be substituted or unsubstituted.

[0130] The "N-amide" group refers to the "RC(=O)N(R A )-" group, where R and R A are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The N-amide may be substituted or unsubstituted.

[0131] The "S-sulfonamide" group refers to the "-SO 2 N(R A R B )" group, where R A and R B are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The S-sulfonamide may be substituted or unsubstituted.

[0132] The "N-sulfonamide" group refers to the "RSO 2 N(R A )-" group, where R and R Aindependently can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The N-sulfonamide may be substituted or unsubstituted.

[0133] The "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 may be substituted or unsubstituted.

[0134] The terms "ester" and "C-carboxy" refer to a "-C(=O)OR" group, where R can be the same as defined for O-carboxy. Ester and C-carboxy may be substituted or unsubstituted.

[0135] The "nitro" group refers to a "-NO 2 " group.

[0136] The "sulfenyl" group refers to a "-SR" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). Sulfenyl may be substituted or unsubstituted.

[0137] The "sulfinyl" group refers to a "-S(=O)-R" group, where R can be the same as defined for sulfenyl. Sulfinyl may be substituted or unsubstituted.

[0138] The "sulfonyl" group refers to "SO 2The term "R" group, where R can be the same as defined for sulfenyl. The sulfonyl group may be substituted or unsubstituted.

[0139] As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen (e.g., mono-haloalkyl, di-haloalkyl, tri-haloalkyl, and polyhaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl, and pentafluoroethyl. The haloalkyl group may be substituted or unsubstituted.

[0140] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more of the hydrogen atoms are replaced by halogen (e.g., mono-haloalkoxy, di-haloalkoxy, and tri-haloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy, and 2-fluoroisobutoxy. The haloalkoxy group may be substituted or unsubstituted.

[0141] As used herein, the terms "amino" and "substituted amino" refer to the -NH 2 group.

[0142] The "monosubstituted amine" group refers to the "-NHR A " group, where R A can be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). R A may be substituted or unsubstituted. Monosubstituted amine groups include, for example, mono-alkylamine groups, mono-C 1 -C 6An alkylamine group, a mono-aryl amine group, a mono-C 6 -C 10 An arylamine group and the like may be included. Examples of the mono-substituted amine group include, but are not limited to, -NH(methyl), -NH(phenyl), and the like.

[0143] The "di-substituted amine" group refers to the "-NR A R B " group, wherein R A and R B are independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl) as defined herein. R A and R B may be independently substituted or unsubstituted. Examples of the di-substituted amine group include, for example, a di-alkylamine group, a di-C 1 -C 6 alkylamine group, a di-arylamine group, a di-C 6 -C 10 arylamine group and the like may be included. Examples of the di-substituted amine group include, but are not limited to, -N(methyl) 2 , -N(phenyl)(methyl), -N(ethyl)(methyl), and the like.

[0144] As used herein, the "mono-substituted amine(alkyl)" group refers to a mono-substituted amine provided herein connected via a lower alkylene group as a substituent. The mono-substituted amine(alkyl) may be substituted or unsubstituted. Examples of the mono-substituted amine(alkyl) group include, for example, a mono-alkylamine(alkyl) group, a mono-C 1 -C 6 alkylamine(C 1 -C 6 alkyl) group, a mono-arylamine(alkyl group), a mono-C 6 -C 10 arylamine(C 1 -C 6An alkyl) group etc. may be included. Examples of the mono-substituted amine(alkyl) group include -CH 2 NH(methyl), -CH 2 NH(phenyl), -CH 2 CH 2 NH(methyl), -CH 2 CH 2 NH(phenyl) etc. are included, but are not limited thereto.

[0145] As used herein, the "di-substituted amine(alkyl)" group refers to a di-substituted amine provided herein connected via a lower alkylene group as a substituent. The di-substituted amine(alkyl) may be substituted or unsubstituted. Examples of the di-substituted amine(alkyl) group include, for example, a dialkylamine(alkyl) group, a di-C 1 -C 6 alkylamine(C 1 -C 6 alkyl) group, a di-arylamine(alkyl) group, a di-C 6 -C 10 arylamine(C 1 -C 6 alkyl) group etc. may be included. Examples of the di-substituted amine(alkyl) group include -CH 2 N(methyl) 2 、-CH 2 N(phenyl)(methyl), -CH 2 N(ethyl)(methyl), -CH 2 CH 2 N(methyl) 2 、-CH 2 CH 2 N(phenyl)(methyl), -NCH 2 CH 2 (ethyl)(methyl) etc. are included, but are not limited thereto.

[0146] As used herein, the term "diamino-" represents the "-N(R A )R B -N(R C )(R D )" group, wherein R A , R C , and R Dis, independently, as defined herein, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), and R B connects two "N" groups and may be a substituted or unsubstituted alkylene group (independent of R A , R C , and R D ). R A , R B , R C , and R D may be further substituted or unsubstituted.

[0147] As used herein, the term "polyamino" refers to "-(N(R A )R B )-) n -N(R C )(R D )". By way of example, the term "polyamino" may include -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 described elsewhere herein. This example has only four repeating units, but the term "polyamino" may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units. R A , R C , and R D are, independently, as defined herein, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), and in the formula, R B connects two "N" groups and (R A , R C , and R D(independently of each other) may be a substituted or unsubstituted alkylene group. R A , R C , and R D may each independently be further substituted or unsubstituted. When described herein, polyamino includes an amine group with an intervening alkyl group (alkyl being as defined elsewhere in this specification).

[0148] As used herein, the term "diether-" refers to a "-OR B O-R A " group, where R A may each 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 the two "O" groups and may be a substituted or unsubstituted alkylene group. R A may each independently be further substituted or unsubstituted.

[0149] As used herein, the term "polyether" refers to a repeating -(OR B -) n OR A group. By way of example, the term polyether may include -Oalkyl-Oalkyl-Oalkyl-Oalkyl-OR A . In some embodiments, the alkyl of the polyether is as described elsewhere in this specification. This example has only 4 repeating units, but the term "polyether" may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units. 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. R Bcan be a substituted or unsubstituted alkylene group. R A are each independently further optionally substituted or unsubstituted. When described herein, a polyether contains an ether group with an intervening alkyl group (alkyl is as defined elsewhere herein and can be optionally substituted).

[0150] When the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may contain one or more of the same or different halogens. As another example, "C 1 -C 3 alkoxyphenyl" may contain one or more of the same or different alkoxy groups containing 1, 2, or 3 atoms.

[0151] As used herein, a radical refers to a species having a single unpaired electron such that the species containing the radical can form a covalent bond with 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".

[0152] When a range of integers is given, the range includes any number within the range and the numbers defining the ends of the range. For example, when the term "integers 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.

[0153] Also, as used herein, "and / or" refers to any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or"), and encompasses these.

[0154] Furthermore, as used herein when referring to measurable values such as the amount, dosage, time, temperature, etc. of a compound or agent of the present invention, the term "about" is intended 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 variations) is to be given its ordinary meaning and is meant that the recited composition or method can include additional components so long as those additional components do not substantially alter the composition or method. The term "consisting of" (and grammatical variations) is to be given its ordinary meaning and is meant that the recited composition or method is closed to additional components. The term "comprising" (and grammatical variations) is to be given its ordinary meaning and is also meant that the recited composition or method is open to including additional components.

[0155] Nitric oxide (NO) is an endogenously produced diatomic free radical that mediates angiogenesis. It has a relatively short biological half-life (seconds) and is inherently reactive. As disclosed herein, the synthesis of scaffolds that enable controlled storage and release of NO is important for exploiting the role of NO in physiology and for developing NO-based therapies. In addition to the effects of NO disclosed above, NO is also a potent antibacterial agent that acts on bacteria via nitrosation and oxidative stress. NO is a broad-spectrum antibacterial agent and in some embodiments, the scaffold that delivers NO can eradicate both bacteria and biofilms by the formation of reactive NO by-products (e.g., peroxynitrous acid and dinitrogen trioxide) that primarily cause oxidative and nitrosative damage to microbial DNA and / or membrane structures. Advantageously, the broad range of mechanisms by which NO exerts its antibacterial effect reduces the risk that bacteria will develop resistance. Thus, NO-releasing substances can be good targets for combating bacterial infections. The antibacterial efficacy of NO-releasing substances can depend on both the NO payload and the associated release reaction rate.

[0156] Nitric oxide, an endogenously produced diatomic free radical, is associated with many biological processes, including platelet aggregation and adhesion, vasodilation, wound repair, immune response, and carcinogenesis. Deficiency of NO can lead to some degree of dysfunction of NO-related physiological systems. Exogenous NO delivery can be an effective strategy for the resolution of biomedical therapies ranging from cardiovascular diseases to antibacterial and anticancer therapies. However, the difficulty in regulating gaseous NO as a therapeutic agent justifies the use of synthetic NO donors (e.g., N-diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates) to control NO delivery. N-diazeniumdiolates (NONOates) can be useful as NO donors due to their good stability and their ability for proton-induced NO delivery under physiological conditions. In some examples, high total NO is an important parameter for effectively evaluating the storage capacity of a good scaffold. In addition, a high density of secondary amine groups accepts specific donors with a high NO storage capacity. However, too fast NO release and high NO storage amounts can result in undesired toxicity to mammalian cells. Thus, there are challenges in the preparation of biocompatible NO-releasing materials with high NO storage amounts and low cytotoxicity, and such challenges are addressed, among other things, according to some embodiments disclosed herein. Some embodiments herein have one or more of the advantages of chemical compositions resulting from efficient and unique synthetic routes and hyperbranched architectures. A controllable amount of secondary amines and diverse external end groups (e.g., hydroxyl, methyl, hydroxymethyl, and primary amines) can be provided. The NO storage and NO release reaction rates of the generated nitric oxide-releasing scaffolds can be adjusted for specific applications. In some embodiments, this adjustment is achieved by changing the type and / or number of functionalized monomers of the formulas disclosed herein. In some embodiments, for example, further functionalization of the amines in the generated nitric oxide-releasing scaffolds by compounds having different compositions further enables control of the NO release reaction rate.In some embodiments, the secondary amine group directly affects the stability of the N-diazeniumdiolate (or other NO carrier group) and enables control over both the NO storage and release reaction rates.

[0157] Nitric oxide, an endogenously produced diatomic free radical, not only plays a fundamental role in several important biological processes but also exhibits emerging functions as an antibacterial or anticancer agent. Controlled exogenous NO delivery can be achieved using various NO donors (e.g., N-diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates). Delivery agents are beneficial because gaseous NO is difficult to control. N-bonded diazeniumdiolates (NONOates) are particularly attractive because they spontaneously undergo proton-induced dissociation under physiological conditions to regenerate the NO radical, making them highly stable and easy to store. There has been progress in the discovery of biocompatible N-diazeniumdiolate-modified scaffolds, including linear and dendritic polymers, silicone nanoparticles, chitosan, liposomes, and organometallic frameworks.

[0158] Among hyperbranched materials, dendrimers, which are a family of globular polymers with a theoretically perfect branched architecture and a well-defined molecular weight, are attractive due to the high density of external functional groups that are available for further modification and NO loading. The NO-releasing dendrimers and hyperbranched structures disclosed herein are characterized by a large NO payload and antibacterial activity against a wide range of pathogenic bacteria, including Pseudomonas aureginosa and Staphylococcus aureus. NO-releasing first-generation (G1)-polyamidoamine (PAMAM) dendrimers can be effective against certain periodontal pathogens (e.g., P. gingivalis and A. actinomycetemcomitans). Unfortunately, these dendrimers can show a lack of bactericidal action against cariogenic bacteria (e.g., S. mutans and S. sanguinis). These PAMAM dendrimers can be modified with long alkyl chains to promote antibacterial activity against cariogenic bacteria by both membrane disruption and NO-related stress, but the use of these dual-action dendrimers results in toxicity to human gingival fibroblasts and limits their clinical application. Higher-generation NO-releasing PAMAM dendrimers can promote enhanced antibacterial activity against pathogens without compromising the viability of mammalian cells. Unfortunately, the synthesis of higher-generation PAMAM dendrimers is difficult because it is both time- and labor-intensive due to the multi-step purification. These overall challenges limit the scale-up and potential clinical use of these agents. Repeating the dendrimer growth or purification steps and modifying the secondary amines requires tedious work, undesirable expenses, and high synthetic skills, further hindering commercial exploitation. Furthermore, the residual primary amine groups used to synthesize the secondary amines essential for the preservation of N-diazeniumdiolate also cause unexpected cytotoxicity. One effective strategy for reducing cytotoxicity is to functionalize the dendrimer with a PEGylated outer surface, but this strategy has several drawbacks, including both a cumbersome modification route and a decrease in the NO-preserving ability.Some embodiments disclosed herein solve these or other problems, and in some embodiments, provide a low-cost and biocompatible polymer scaffold for exogenous NO delivery.

[0159] In some embodiments, hyperbranched polymers (HBPs) are a category of dendrimers but have an irregular three-dimensional highly branched architecture. Distinguished from dendrimers, in some embodiments, HBPs consist of dendritic units, linear units, and terminal units randomly dispersed in the polymer backbone. In some embodiments, HBPs can be advantageously synthesized using "one-pot" polymerization, significantly reducing the polymerization steps and time or material consumption. Benefiting from unique physical and chemical properties and facile polymerization, HBPs can be attractive in various fields such as the biomedical field.

[0160] In some embodiments, the hyperbranched polymer can be polymerized by Michael addition polycondensation between a polyfunctional amine (e.g., diamine, triamine, tetraamine, etc.) monomer for providing a promising biocompatible material for biomedical clinical applications and a diacrylamide / diacrylate / divinyl sulfone monomer. For example, cationic hyperbranched poly(amidoamine) (HBPAA). In some embodiments, HBPs such as HBPAA can be used as a biocompatible scaffold to deliver N-diazeniumdiolate NO donors due to the presence of a large number of secondary amines in the polymer backbone. Some embodiments disclosed herein relate to NO-donating hyperbranched polymer structures. In some embodiments, these hyperbranched polymer structures are low-cost and effective "one-pot" polymerizations. In some embodiments, the synthetic sequence disclosed herein can be carried out to obtain a hydroxyl-terminated hyperbranched polymer structure (e.g., hyperbranched poly(methylenebisacrylamide-hydroxyethyl ethylenediamine) (HBPMH)). In some embodiments, the obtained hyperbranched polymer structure rich in secondary amines can be reacted with NO gas to form N-diazeniumdiolate in the polymer backbone. In some embodiments, the hyperbranched polymer structure has antibacterial properties evaluated against Gram-negative bacteria (e.g., Pseudomonas aeruginosa, etc.) and / or Gram-positive bacteria (e.g., Staphylococcus aureus, etc.). In some embodiments, the hyperbranched structure has low cytotoxicity against mammalian cells (e.g., L929 mouse fibroblasts, etc.) in vitro and in vivo.

[0161] Dental caries (e.g., tooth decay) affects 60 - 70% of school-aged children and most adults in most developed countries. Worldwide, 11% of the total population suffers from severe periodontitis, which contributes to tooth loss and systemic diseases such as coronary artery, cardiovascular, stroke, and adverse pregnancy outcomes. Among the over 700 microorganisms in the oral cavity, cariogenic bacteria (e.g., Streptococcus mutans, Actinomyces viscosus) and periodontal pathogens (e.g., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans) play a major role in the onset and progression of oral diseases. Oral diseases are one of the most common health problems faced by humans. Gram-positive cariogenic (e.g., Streptococcus mutans, Actinomyces viscosus) and Gram-negative periodontal (e.g., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans) bacteria are the main exacerbating factors associated with the development and progression of dental caries and periodontal disease, respectively. Unfortunately, current treatments to combat these pathogens are associated with undesirable side effects. For example, systemic use of antibiotics can cause gastrointestinal disorders and promote bacterial resistance. The common oral disinfectant chlorhexidine can change taste, stain teeth and tongue, and irritate the buccal mucosa. Vehicles that deliver polymeric NO (e.g., silica nanoparticles, gold, etc.) kill Gram-negative periodontal pathogens. However, these substances have not been demonstrated to kill Gram-positive cariogenic bacteria at a safe concentration (e.g., a concentration that is bactericidal but non-toxic to mammalian cells). Similar to those nanomaterials, the lack of biodegradability and potential cytotoxicity of silica nanoparticles also hinder their future for biomedical applications. Current research also focuses on using nanomaterials containing silver, gold, zinc, and copper as alternatives to conventional antibiotics that are troubled by promoting bacterial resistance. However, these nanomaterials can accumulate in the body, cause cumulative toxicity, and limit their future for specific applications.To maintain a healthy oral cavity, it is important to develop oral therapeutic agents that can kill the bacteria that cause those diseases. The hyperbranched structures (e.g., HBPs that carry NO) disclosed herein solve one or more of these problems or other problems. As disclosed herein, hyperbranched structures can be advantageously easily synthesized, obtain a unique three-dimensional dendritic shape, and have low cytotoxicity.

[0162] As disclosed herein, hyperbranched polymers are an important subclass of compounds. In some embodiments, hyperbranched polymers have properties similar to dendrimers, but are advantageously easily prepared in bulk by a one-pot reaction with minimal purification. Compared to their substantially structurally defect-free dendrimer counterparts (including the same dendrons derived from a common core of the dendrimer), hyperbranched polymers have an irregular structure. However, as disclosed herein, in some embodiments, hyperbranched polymers still retain a high density of external functional groups.

[0163] Despite having a spherical structure, dendrimers and hyperbranched polymers are clearly different in their structures. For example, dendrimers have two types of structural units, namely, terminal units on the surface and dendritic units inside the spherical structure. Except for defects, dendrimers have a consistent and distinct structure. In contrast to this distinct structure, hyperbranched polymers have three types of structural units that are not consistently oriented across a given population, namely, dendritic units, linear units, and terminal units. The terminal units of the hyperbranched structure are at the ends, but the dendritic units and linear units are distributed throughout the polymer framework, resulting in an irregular structure. The difference in structure is related to different formation mechanisms and may further be related to their different synthetic approaches. Dendrimers are synthesized using stepwise iteration, most commonly by a branching process starting from a multifunctional core. In some embodiments, hyperbranched polymers can be synthesized in one step by a polymerization reaction. For example, in some embodiments, hyperbranched polyamidoamine (h-PAMAM) can be synthesized with a unit structure and molecular weight similar to that of G3-PAMAM dendrimers. In some embodiments, the hyperbranched structure induces low toxicity to mammalian cells.

[0164] In some embodiments, a polyamidoamine composition comprising a hyperbranched copolymer of an amine and an acrylate is provided. In some embodiments, at least a portion of the amine is modified with an N-diazinium dioleate moiety. In some embodiments, the hyperbranched copolymer comprises dendritic units, linear units, and terminal units, wherein the dendritic units comprise tertiary amines, the linear units comprise secondary amines, and the terminal units comprise primary amines. In one embodiment, the N-diazinium dioleate moiety can be attached via a secondary amine and exhibits intramolecular hydrogen bonding with the primary amine of the terminal unit. In one embodiment, the amine has the structure: H 2 N-A 1 -(NH)-A 2 -NH 2 and is derived from a polyfunctional amine monomer having, wherein A 1 and A 2is, independently, a selected alkyl moiety. In a further embodiment, the acrylate is derived from the following monomers, [Chemical formula] wherein A 3 and A 4 are, independently, a selected alkyl moiety or hydrogen.

[0165] In a further embodiment, the polyamidoamine composition is further modified to include a hydroxy moiety. The hydroxy moiety is linked to the amine moiety via an alkyl moiety, whereby the amine moiety is a secondary amine. In some embodiments, the polyamidoamine composition can include a molar ratio of amine to acrylate of from about 2:1 to about 5:1. In some embodiments, the molar ratio of amine to acrylate is from about 3:1 to about 4:1. In some embodiments, ratios between those recited are used.

[0166] In some embodiments, the acrylate residues in the hyperbranched copolymer are derived from monomers selected from salts, esters, and conjugate bases of acrylic acid and its derivatives. In some embodiments, the acrylate is derived from the monomer methacrylate. In some embodiments, the acrylate is methyl acrylate, ethyl acrylate, methyl methacrylate, acrylamide, ethyl methacrylate, 2-chloroethyl vinyl ether, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, butyl acrylate, butyl methacrylate, N-(2-hydroxypropyl) methacrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, N-(3-BOC-aminopropyl) methacrylamide, 2-aminoethyl methacrylate hydrochloride, 2-(tert-butylamino)ethyl methacrylate, n-isopropylacrylamide, 2-methoxyethyl acrylate, n-ethyl methacrylamide, n-vinylacetamide, 2-N-morpholinoethyl acrylate, methacryloyl-L-lysine, 2-(methylamino)ethyl acrylate, and 2-(methylamino)ethyl methacrylate, or is derived from one or more selected monomers thereof. In another embodiment, the acrylate is derived from a diacrylate. For example, the diacrylate can be ethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, tricyclodecane dimethanol diacrylate, N-acryloxysuccinimide, N-(2-hydroxypropyl) methacrylamide, bis[2-(methacryloyloxy)ethyl] phosphate, diacrylamide, and N,N'-methylenebisacrylamide.

[0167] In some embodiments, the amine residue is derived from (e.g., synthesized using) diethylenetriamine. In some embodiments, the amine residue is derived from (e.g., synthesized using) one or more of ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, polyetheramine, and bis(hexamethylene)triamine.

[0168] In some embodiments, the polyamidoamine hyperbranched copolymer comprises one or more dendritic units containing a tertiary amine, a plurality of terminal units containing a primary amine, and a plurality of linear units containing a secondary amine. The N-diazeniumdiolate moiety can be reversibly bound to the secondary amine present in the linear unit. In one embodiment, the polyamidoamine hyperbranched copolymer has a dendritic unit comprising the following structure,

Chemical formula

Chemical formula

[0169] In some embodiments, the hyperbranched polyamidoamine (h-PAMAM) is synthesized by the polymerization of diethylenetriamine (DETA) and methyl acrylate (MA). In some embodiments, the hyperbranched structure is synthesized by adding any of the nucleophilic amine compounds disclosed elsewhere herein together with one or more acrylates.

[0170] In some embodiments, size exclusion chromatography (SEC) can be used to measure the molecular weight of the hyperbranched structures disclosed herein. In some embodiments, a multi-angle light scattering (SEC-MALS) detector is used. In some embodiments, the weight average molecular weight (M w ) of the hyperbranched structures disclosed herein is about 2,500 g / mol, 5,000 g / mol, 6,000 g / mol, 7,000 g / mol, 8,000 g / mol, 10,000 g / mol, 15,000 g / mol or more, or ranges including and / or spanning the aforementioned values. In some embodiments, the M w of the hyperbranched structures disclosed herein is about 6.39×10 3 g / mol or more. In some embodiments, the number average molecular weight (M n ) of the hyperbranched structures disclosed herein is about 2,500 g / mol, 5,000 g / mol, 6,000 g / mol, 7,000 g / mol, 8,000 g / mol, 10,000 g / mol, 15,000 g / mol or more, or ranges including and / or spanning the aforementioned values. In some embodiments, the M n of the hyperbranched structures disclosed herein is about 6.39×10 3 g / mol or more. The size of the hyperbranched copolymer can be described as a weight average molecular weight (MW) of about 2×10 3 gmol -1 to about 15×10 3 gmol -1 , as determined by size exclusion chromatography using a multi-angle light scattering (SEC-MALS) detector. At such sizes, such compounds can be referred to as oligomers. In other embodiments, the size of the hyperbranched copolymer can be described as a weight average molecular weight (MW) of about 3×10 3 gmol -1 to about 10×10 3 gmol -1 . In another embodiment, the size is about 3×10 3 gmol -1 to about 6×10 3 gmol -1It can be. The hyperbranched copolymer can include a population of molecules having different shapes and sizes. For example, the hyperbranched copolymer can have a polydispersity index (PDI) greater than about 1.1. In another embodiment, the hyperbranched copolymer has a PDI of 1.1 to 2. In another embodiment, the PDI can be 1.5 to 1.9.

[0171] In some embodiments, the hyperbranched 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 by the number average molecular weight. In some embodiments, the hyperbranched structure has a PDI of about 1.05, 1.1, 1.2, 1.3, 1.5, 1.7, 1.8, 1.9, 2.0, or greater than or equal to a range including and / or encompassing the values described above. In some embodiments, the hyperbranched structure has a PDI greater than about 1.89. In some embodiments, the hyperbranched structure has a polydispersity (PDI) greater than that of the dendrimer. In some embodiments, as disclosed elsewhere herein, the third generation PAMAM (G3 - PAMAM; theoretical molecular weight 6909 gmol -1 ) dendrimer was also adjusted to investigate the effect of the architecture on the properties of the PAMAM scaffold, which is independent of the molecular weight. The measured M of the G3 - PAMAM dendrimer w was 7.19×10 3 gmol -1 and the PDI was 1.04.

[0172] In some embodiments, compared to dendrimers, the hyperbranched structures (e.g., polymers) disclosed herein with approximately the same molecular weight contain fewer end groups (e.g., primary amines). In some embodiments, per 1000 g / mol of the molecular weight of the hyperbranched molecule, the number of end groups on the hyperbranched molecule is about 0.5, 1, 2, 3, 5 or less, or ranges including and / or extending between the above values. In some embodiments, compared to the G3-PAMAM dendrimer (i.e., 32 primary amines per scaffold of about 7,200 g / mol), the h-PAMAM polymer contains fewer primary amine groups (about 8 primary amines per scaffold of about 6,400 g / mol). This result is based on the difference in the structures of the two materials. As shown in FIG. 1, the h-PAMAM polymer consists of dendritic units, linear units, and terminal units. In some embodiments, the linear units induce structural defects in the polymer structure. In some embodiments, the incorporation of the linear units decreases the number of external primary amines while increasing the secondary amines along the polymer backbone. In contrast, dendrimers (e.g., G3-PAMAM dendrimers) should theoretically be structurally perfect and consist only of dendritic units and terminal units. Thus, the G3-PAMAM dendrimer lacks the linear units containing secondary amines that are most available for the attachment of the N-diazeniumdiolate moiety in the examples shown herein. In some embodiments, the secondary amine functional group is necessary to form a stable N-diazeniumdiolate nitric oxide (NO) donor. In contrast to PAMAM dendrimers that require subsequent reactions to generate secondary amines prior to NO loading, the h-PAMAM polymer can react directly with NO gas to form N-diazeniumdiolate because secondary amines are present in the linear units along the polymer backbone.

[0173] In some embodiments, the NO payload can be further enhanced on the hyperbranched structure by further modification of the terminal primary amine units. In some embodiments, the primary amines of the h-PAMAM polymer (for an exemplary h-PAMAM polymer, there are about 8 primary amines per generation) were modified with 1 molar equivalent of a hydroxy-containing compound. One exemplary compound used was propylene oxide (PO), which is an epoxide that, following a ring-opening reaction, yields a hydroxy functional group and results in the compound described herein as h-PAMAM-PO-1. In a comparative study, the h-PAMAM polymer and the G3-PAMAM dendrimer were modified with 1 molar equivalent of PO relative to the primary amines of the G3-PAMAM dendrimer (32 primary amines per generation) to afford h-PAMAM-PO-2 and G3-PAMAM-PO, respectively. In this way, the effect of external modification on the NO release properties (e.g., payload and release reaction rate) of the h-PAMAM polymer could be manipulated.

[0174] Some embodiments relate to a method of delivering nitric oxide to a subject, comprising administering to the subject an effective amount of a hyperbranched structure. In some embodiments, a method of treating a disease state, comprising administering to a subject in need of treatment an effective amount of a hyperbranched structure, wherein the disease state is selected from the group consisting of gingivitis, cancer, cardiovascular disease, microbial infection, platelet aggregation and platelet adhesion caused by exposure of blood to a medical device, 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 infections, is disclosed.

[0175] In some embodiments, the hyperbranched structures disclosed herein are used in methods of treating a patient and / or killing bacteria (e.g., as an antibacterial agent). Also provided herein are methods for delivering nitric oxide to a subject, including administering to the subject an effective amount of any of the functionalized hyperbranched structures disclosed herein. Methods of treating a disease state are also provided herein, and in some embodiments, the method includes administering to a subject in need of treatment an effective amount of any of the functionalized hyperbranched structures disclosed herein, wherein the disease state is selected from the group consisting of cancer, cardiovascular disease, microbial infections; platelet aggregation and platelet adhesion caused by exposure of blood to a medical device; pathological conditions resulting from abnormal cell growth; transplant rejection, autoimmune diseases, inflammation, vascular diseases; scar tissue; wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunction, and sexually transmitted infections. In some embodiments, the disease state is a microbial infection. In some embodiments, the disease state is dental caries or another disease of the mouth (such as gingivitis, periodontitis, etc.).

[0176] In some embodiments, provided herein is a method for reducing microbial load on a surface, comprising applying a compound to a surface contaminated with a plurality of microorganisms, the compound comprising a nitric oxide (NO)-releasing hyperbranched structure, the functionalized hyperbranched structure comprising an NO donor, the NO donor generating NO and inducing oxidative and / or nitrosative damage to microbial DNA and membrane structures, thereby reducing the microbial load, the plurality of microorganisms comprising 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 a human skin or mucosal surface. In some embodiments, application of the compound does not induce skin irritation or mucosal irritation. In some embodiments, the surface is an animal skin. In some embodiments, the surface is in or around the oral cavity of a human or animal. In some embodiments, application of the compound does not induce skin irritation or irritation of the mouth or surrounding tissues. In some embodiments, the surface is a human airway tissue. In some embodiments, application of the compound (e.g., inhalation) does not induce irritation of airway epithelial cells. In some embodiments, the surface is an inorganic surface. In some embodiments, the inorganic surface is an outer or inner surface of a medical device. In some embodiments, the medical device is a dental tool. In some embodiments, application of the compound generates an antibacterial coating on the outer or inner surface of the medical device. In some embodiments, the medical device comprises an endoscope, a dental drill or other dental device, a dental implant, or a dental fixture.

[0177] In some embodiments, the microbial load that is to be reduced and / or removed includes drug-resistant bacteria. In some embodiments, the drug-resistant bacteria include carbapenem-resistant Enterobacteriaceae. In some embodiments, the drug-resistant bacteria include methicillin-resistant Staphylococcus aureus. In some embodiments, the microorganisms include human immunodeficiency virus, herpes simplex virus, papillomavirus, parainfluenza virus, influenza, hepatitis, coxsackievirus, herpes zoster, measles, mumps, rubella, rabies, pneumonia, hemorrhagic viral fevers, H1N1, etc.), prions, parasites, fungi, molds, yeasts, and, in particular, Candida albicans, Aspergillus niger, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus (S. aureus), Group A streptococci, S. pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans, and various drug-resistant bacteria, including bacteria (both gram-positive and gram-negative). The terms microorganism and microbe shall be used interchangeably. The microorganisms can 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 oral mucosa.

[0178] In some embodiments, treating and / or preventing microbial infection and / or growth includes contacting a surface (e.g., the mouth, which is contaminated with or susceptible to the effects of microbes) with a compound comprising a nitric oxide (NO)-releasing hyperbranched structure, the functionalized hyperbranched structure comprising an NO donor, the NO donor generating NO and inducing damage to the membranes and / or DNA of microbes, thereby reducing the number of viable microbes and treating and / or preventing infection or infestation, the plurality of microbes including one or more of viruses, gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof.

[0179] In some embodiments, the methods and uses are formulated for administration via topical routes, oral administration, oral-topical (e.g., oral rinses, mouthwashes, liquids, solids, gels, pastes, etc.), irrigation (e.g., dental irrigation), injection, spraying, solid depot, ingestion, or inhalation, using the compounds disclosed herein. In some embodiments, a strip or other substrate is used for application of the formulation. The strip is made of a polymer, including but not limited to, polyethylene in some embodiments. In some embodiments, the route is topical and the methods and uses of the NO-releasing hyperbranched structure are for the treatment of oral pathogens (e.g., one or more of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Actinomyces viscosus). In some embodiments, the NO-releasing hyperbranched structure does not substantially damage human cells, including gingival fibroblasts, oral mucosal epithelium, or other cells in or around the mouth.

[0180] In some embodiments, the method includes administering a composition comprising the hyperbranched structure disclosed herein. In some embodiments, the composition used in the methods of the present disclosure can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and can include formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, prior to use.

[0181] By way of example, the hyperbranched copolymer can be water-soluble and can be soluble in water, for example, at levels greater than about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 50 mg / mL, or about 100 mg / mL.

[0182] In some embodiments, the therapeutic composition can be provided in unit dose or multi-dose containers, for example, sealed ampules and vials, and stored under frozen or freeze-dried (lyophilized) conditions that require only the addition of a sterile liquid carrier immediately prior to use.

[0183] In some embodiments, for oral administration, the composition can take the form of tablets or capsules prepared by conventional techniques using pharmaceutically acceptable excipients such as, for example, binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (such as lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (such as magnesium stearate, talc, or silica), disintegrants (such as potato starch or sodium starch glycolate), or wetting agents (such as sodium lauryl sulfate). Tablets can be coated by methods known in the art. For example, the therapeutic agent can be formulated in combination with hydrochlorothiazide as a pH-stabilized core having an enteric coating or a delayed release coating that protects the therapeutic agent until it reaches the target organ.

[0184] In some embodiments, liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or can be presented as dry products for constitution with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional techniques using pharmaceutically 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 can also optionally contain buffering salts, flavoring agents, coloring agents, and sweetening agents. Preparations for oral administration can be suitably formulated to provide slow release of the active compound. For buccal administration, the composition can take the form of tablets or lozenges formulated in a conventional manner.

[0185] In some embodiments, the disclosed compounds can also be formulated as preparations for implantation or injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or insoluble derivatives (e.g., as an insoluble salt). The compounds can also be formulated in rectal compositions (e.g., suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.

[0186] Also provided are pharmaceutical formulations suitable for administration as an aerosol by inhalation. In some embodiments, the hyperbranched structures described herein are formulated in the form of a solution and / or an aerosol. In some embodiments, these formulations comprise a solution or suspension of a NO-releasing hyperbranched structure (e.g., polyamidomine) described herein. In some embodiments, the desired formulation can be placed in a small chamber and sprayed. Atomization can be achieved by compressed air or by ultrasonic energy to form a plurality of droplets or solid particles containing the NO-releasing hyperbranched polyamidomine. For example, the NO-releasing hyperbranched polyamidomine of the present disclosure can be administered via inhalation to treat bacterial infections associated with cystic fibrosis. Bacterial infections associated with cystic fibrosis include, but are not limited to, stenotrophomonis, mybacterium avium intracellulaire, and m. abcessus, burkhoderia cepacia, and Pseudomonas aeruginosa (P. aeruginosa) infections.

[0187] In some embodiments, the hyperbranched structures disclosed herein consist of dendritic units, linear units, and terminal units along and / or within the chain length or arms of the hyperbranched structure (as shown in FIG. 1). In some embodiments, the linear units and / or chains along the hyperbranched structure provide secondary amines as potential reactive sites for the addition of NO donor moieties. N-diazeniumdiolate NO donors.

[0188] In some embodiments, the NO-donating hyperbranched structure, as shown in FIG. 1, includes, for example, NO-donating substituents that decorate the hyperbranched structure along the chain length or arms within the hyperbranched structure.

[0189] In a further embodiment, the present disclosure describes a method for manufacturing a polyamideamine composition, comprising combining a polyfunctional amine with an acrylate monomer (e.g., in a suitable solvent to form a reaction mixture), mixing the reaction mixture for a time sufficient for a significant proportion of the polyfunctional amine to react with the acrylate monomer to form a hyperbranched copolymer, heating the reaction mixture to complete polymerization and remove unreacted monomers to form a basic polyamideamine composition, and mixing the basic polyamideamine composition with gaseous NO at high pressure for a time sufficient to obtain an N-diazinium dioleate moiety in the polyamideamine composition under basic conditions. In some embodiments, the time sufficient for a significant proportion of the polyfunctional amine to react is about 6 hours, 12 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks or more, or ranges including and / or extending between the above values.

[0190] In some embodiments, heating the reactants to complete polymerization and remove unreacted monomers comprises heating under subatmospheric pressure. In another embodiment, heating the reaction mixture to complete polymerization and remove unreacted monomers comprises heating to a first temperature of about 50 °C to about 70 °C for a first time of about 30 minutes to about 2 hours, heating to a second temperature of about 90 °C to about 110 °C for a second time of about 30 minutes to about 4 hours, and heating to a third temperature of about 120 °C to about 150 °C for a third time of about 30 minutes to about 4 hours. In yet another embodiment, heating the reaction mixture to complete polymerization and remove unreacted monomers comprises heating to a first temperature of about 60 °C for a first time of about 1 hour, heating to a second temperature of about 100 °C for a second time of about 2 hours, and heating to a third temperature of about 140 °C for a third time of about 2 hours.

[0191] In some embodiments, the electrophilic polymerizing agent is used to prepare a hyperbranched structure. In some embodiments, the electrophilic agent comprises one or more Michael acceptors (e.g., α,β-unsaturated carbonyl compounds, enolates, etc.) that act as electrophilic agents. In some embodiments, the polymerizing agent comprises one or more acrylate functional groups. In some embodiments, the Michael acceptor is acrylate. In some embodiments, the polymerizing agent is a diacrylate (e.g., N,N'-methylenebis(acrylamide), ethylene glycol diacrylate, propanediol diacrylate, butanediol diacrylate, etc.), a triacrylate (e.g., trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol triacrylate, glycerol propoxylate (1PO / OH) triacrylate, trimethylolpropane propoxylate triacrylate), etc.), a tetraacrylate, or another acrylate having a plurality of acrylate groups (e.g., 5, 6, 7, or more).

[0192] In some embodiments, after polymerization with the nucleophilic unit, one or more of the hyperbranched structures may contain unreacted end groups. In some embodiments, the end groups are end-capped using an epoxide that opens the ring to provide a hydroxyl. In some embodiments, the end-capping agent is one or more of ethylene oxide, glycidol, propylene oxide, ethyl-2,3-epoxypropionate, methyl 2-methylglycidate, ethyl glycidyl ether, etc. In some embodiments, the resulting end groups comprise an optionally substituted mono-alkylamine group. In some embodiments, the optionally substituted mono-alkylamine group is -NH(C 1 -C 6 alkyl), wherein C 1 -C 6 alkyl is substituted anywhere along the alkyl chain with at least one -OH (resulting from the ring opening of the epoxide ring), C 1 -C 6Optionally substituted with alkyl or polyether. In some embodiments, their end groups can be end-capped by further reacting the hyperbranched structure with an amine end-capping agent. In some embodiments, the end-capping agent is H 2 N-((CH 2 ) a NH) b -H, H 2 N-((CH 2 ) a NH) b -(CH 2 ) c H, H 2 N-((CH 2 ) a X a ) b -(CH 2 ) c H, HX a -((CH 2 ) a X b ) b ((CH 2 ) c X c ) d (CH 2 ) e H, -((CH 2 ) a NH) b -, -((CH 2 ) a NH) b -(CH 2 ) c X a , -((CH 2 ) a X a ) b -(CH 2 ) c X b , and -((CH 2 ) a X a ) b ((CH 2 ) c X b ) d -(CH 2 ) e -X cincluding one or more of, each example of a, b, c, d, or e is independently selected from integers from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, X a X b and X c each example is independently selected from O, S, or NH. In some embodiments, the end-capping agent is H 2 NCH 2 CH 2 NH 2 and H 2 NCH 2 CH 2 OH, or one or more thereof. In some embodiments, the end-capping agent is -NH-((CH 2 ) a NH) b -H, -NH-((CH 2 ) a NH) b -(CH 2 ) c H, -NH-((CH 2 ) a X a ) b -(CH 2 ) c H, ((CH 2 ) a X b ) b ((CH 2 ) c X c ) d -(CH 2 ) e H, -((CH 2 ) a NH) b -, -((CH 2 ) a NH) b -(CH 2 ) c X a -, -((CH 2 ) a X b ) b -(CH 2 ) c X b and -((CH 2 ) aX a ) b ((CH 2 ) c X b ) d -(CH 2 ) e -X c results in a substituent selected from one or more of the following. In some embodiments, the end-capping agent is -NHCH 2 CH 2 NH 2 and -NHCH 2 CH 2 OH.

[0193] In some embodiments, the NO donor comprises any one of the following nitric oxide-releasing moieties,

Chemical formula

[0194] wherein

Chemical formula

[0195] In some embodiments, the reaction of the hyperbranched structure with NO is carried out under basic or alkaline conditions. In some embodiments, the alkaline conditions include those having a pH value of at least about 7.5, 8.0, 9.0, 10.0, 12.0 or less, or a range including and / or extending between the above values.

[0196] In some embodiments, the methods disclosed herein provide NO-releasing hyperbranched structures having a NO storage capacity (NO in μmol per mg of hyperbranched structure) of about 0.25, 0.4, 0.5, 1.0, 1.5, 2.0, 3.0 or more, or ranges including and / or encompassing the foregoing values. In some embodiments, within 2 hours of being added to PBS buffer as described in the examples, the NO-releasing hyperbranched structures release about 25%, 50%, 75%, 85%, 90%, 95%, 100%, or ranges including and / or encompassing the foregoing values of their total weight % of bound NO. In some embodiments, NO release in the use for reducing or eliminating biofilms occurs in similar amounts, e.g., about 20-25%, about 30-50%, about 60-75%, at least 80%, at least 85%, at least 90%, at least 95%, and ranges including and / or encompassing the foregoing values of the total weight % of bound NO.

[0197] In some embodiments, NO release can occur over a time of about 0.01 hour, 0.1 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, or 60 hours. In some embodiments, NO release occurs within about 0.01 hour, 0.1 hour, 0.25 hour, 0.5 hour, 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 ranges including and / or encompassing the foregoing values. In some embodiments, no nitrosoamine is present during NO release. As used herein, the phrase "no nitrosoamine is present" refers to levels of nitrosoamine that are not detectable as measured by ultraviolet-visible spectroscopy (or by other approved methods in the art).

[0198] In some embodiments, the disclosed functionalized NO-releasing hyperbranched structures have antibacterial activity. In some embodiments, the disclosed functionalized NO-releasing hyperbranched structures provide at least 90% bacterial reduction against one or more of P. aeruginosa, S. aureus, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans 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 less, or in a range including and / or extending between the aforementioned values, in a bacterial viability assay conducted under static conditions over 2 hours. In some embodiments, the disclosed functionalized NO-releasing hyperbranched structures provide at least 99% bacterial reduction 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 less, or in a range including and / or extending between the aforementioned values, in a bacterial viability assay conducted under static conditions over 2 hours. In some embodiments, the disclosed functionalized NO-releasing hyperbranched structures provide at least 99% bacterial reduction 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 less, or in a range including and / or extending between the aforementioned values, in a bacterial viability assay conducted under static conditions over 2 hours. In some embodiments, the bacterial reduction is greater than 95%, greater than 98%, or greater than 99%.

[0199] Some embodiments relate to a method of killing bacteria and / or microorganisms by applying a NO-donating hyperbranched structure to the bacteria and / or microorganisms. In some embodiments, the bacteria are oral bacteria. In some embodiments, the disclosed compounds can be used in a method of preventing dental caries.

[0200] In some embodiments, the hyperbranched structures disclosed herein lack a core (e.g., derived from a dendron). In some embodiments, the hyperbranched structures disclosed herein lack an ethylenediamine core. In some embodiments, the hyperbranched structures disclosed herein lack an ethylenediamine core from which one or more dendrons protrude. In some embodiments, the hyperbranched structures disclosed herein lack complete, nearly complete symmetry, lack a plane or axis of symmetry, and / or lack symmetry. In some embodiments, the hyperbranched structures disclosed herein lack repeating structural units generated by stepwise synthesis.

[0201] The subject matter described herein relates to the following embodiments: 1. A hyperbranched nitric oxide (NO)-donating compound comprising a linking group comprising any one or more of Formulas A, B, C, or D, wherein

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Examples

[0202] The NO release properties of h-PAMAM (i.e., payload and release reaction rate) were evaluated as a function of chemical modification. The potential of this scaffold as an oral therapeutic was evaluated in terms of antibacterial activity against common oral pathogens and toxicity to human gingival fibroblasts. It should be understood that studying the antibacterial activity of Gram-negative and Gram-positive bacteria in the context of oral hygiene reveals the use of these compounds in a broader category of applications. Finally, the properties of the h-PAMAM derivatives were compared to the G3-PAMAM counterparts with respect to therapeutic potential.

[0203] Example 1: Synthesis of Hyperbranched Polyamidoamine Hyperbranched polyamidoamine (h-PAMAM) was prepared using a one-pot reaction to the 3rd generation PAMAM (G3-PAMAM) dendrimer and similar molecular weights, followed by functionalization with N-diazeniumdiolate nitric oxide (NO) donor.

[0204] Materials and Methods Ethylenediamine (EDA), diethylenetriamine (DETA), methyl acrylate (MA), propylene oxide (PO), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Sigma-Aldrich (St. Louis, MO). Streptococcus mutans (ATCC number 25715), Actinomyces viscosus (ATCC number 15987), and Aggregatibacter actinomycetemcomitans (ATCC number 43717) were purchased from the American Type Culture Collection (Manassas, VA, USA). Porphyromonas gingivalis strain A7436 was provided by the UNC School of Dentistry, Chapel Hill, NC. CDC anaerobic 5 vol% sheep blood agar, brain heart infusion (BHI) broth and agar, and GasPak (trademark) EZ Campy container system sachets were purchased from Becton, Dickinson, and Company (Franklin Lakes, NJ). Wilkins-Chalgren (W-C) broth was purchased from Acumeida Neogen Corporation (Lansing, MI). Human gingival fibroblast cell lines and FibroLife fibroblast serum-free medium were purchased from Lifeline Cell Technology LLC (Frederick, MD). Pure nitric oxide (99.5%), argon, nitrogen, and nitric oxide calibration (25.87 ppm in nitrogen) were purchased from Airgas (Durham, NC). Common laboratory salts and solvents were purchased from Fisher Scientific (Pittsburgh, PA). Water was purified to a final resistivity of 18.2 MΩcm and a total organic content of 10 ppb or less using a Millipore Milli-Q UV Gradient A10 system (Bethlehem, PA).The third-generation polyamidoamine (G3-PAMAM) dendrimer was prepared by repeating the alkylation and amidation reactions using the MA / EDA monomer from the EDA core.

[0205] Proton nuclear magnetic resonance ( 1 1H NMR) spectra were recorded on a 400 MHz Bruker spectrometer. Carbon nuclear magnetic resonance (13C NMR) spectra were collected on a 600 MHz Bruker instrument. Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) was used to determine the molecular weight and polydispersity of the polymers. The eluent (PBS, 0.01% azide, pH 7.4) was passed through a miniDawn TREOS multi-angle light scattering detector (Wyatt Technology; Santa Barbara, CA) connected to a Waters 2414 refractive index detector (Waters Chromatography, Milford, MA).

[0206] Synthesis of hyperbranched polyamidoamine (h-PAMAM)

Chemical formula

[0207] The hyperbranched polyamidoamine (H-PAMAM) polymer was synthesized by the polymerization of diethylenetriamine and methyl acrylate. Briefly, DETA (6.8 mL; 0.06 mol) and MA (6.8 mL; 0.072 mol) were mixed with methanol (10 mL) and stirred for 2 days. The reaction mixture was then heated under a rotary evaporator at 60 °C for 1 hour, 100 °C for 2 hours, 120 °C for 2 hours, and 140 °C for 2 hours to complete the polymerization and remove unreacted monomers. The product (i.e., h-PAMAM) was a yellow oil. h-PAMAM was redissolved in methanol at 100 mg mL -1 and stored in a freezer until future use. h-PAMAM was 1 characterized by 1H NMR, 13 13C NMR, and FTIR at the following peaks.1 H NMR: (400 MHz, CD 3 OD, δ): 2.22 - 2.90 (COCH 2 , NHCH 2 , and NH 2 CH 2 ), 3.15 - 3.58 (CONCH 2 ), 3.60 (CH 3 O). 13 C (600 MHz, CD 3 OD, δ): 30 - 60 (CH 2 and CH 3 ), 170 - 175 (C=O). FTIR (cm -1 ): 3308 (NH 2 ), 2957 (CH 2 ), 2848 (CH 2 ), 1647 (C=O), and 1556 (NH).

[0208] Characteristic Evaluation The free primary amine content of h-PAMAM was measured using the ninhydrin assay. Briefly, a 2 wt% ninhydrin stock solution was freshly prepared before use by dissolving 0.2 g of ninhydrin in a mixture of 7.5 mL of DMSO and 2.5 mL of 0.2 M sodium acetate buffer (pH 5.4). In the experiment, 2 mg of h-PAMAM was dissolved in 1 mL of sodium acetate buffer and mixed with 0.5 mL of the ninhydrin stock solution. This solution was heated at 100 °C for 5 minutes, cooled to room temperature, and diluted 10-fold with ethanol. The absorbance was measured at 570 nm using an ultraviolet-visible Lambda 40 spectrophotometer (PerkinElmer; Waltham, MA) and compared with a similarly prepared G3-PAMAM standard solution. Diethylamine was also examined by the ninhydrin assay to investigate the potential influence of secondary amine functional groups. The results indicate that the iminium salt, which is a product from the reaction between the secondary amine and the ninhydrin reagent, has negligible absorbance at 570 nm, suggesting that the influence from the secondary amines of h-PAMAM is minimal.

[0209] Size exclusion chromatography using a multi-angle light scattering (SEC-MALS) detector showed that the weight-average molecular weight (MW) of the h-PAMAM polymer was 6.39×10 3 g / mol -1 and the PDI was 1.89. The third-generation PAMAM (G3-PAMAM; theoretical molecular weight 6909 g / mol -1 ) dendrimer was also prepared to investigate the effect of the architecture on the properties of the PAMAM scaffold, which is independent of the molecular weight. The measured MW of the G3-PAMAM dendrimer was 7.19×10 3 g / mol -1 and the PDI was 1.04. The h-PAMAM polymer contained fewer primary amine groups (about 8 primary amines per scaffold) compared to the G3-PAMAM dendrimer (i.e., 32 primary amines per scaffold). As shown in Figure 1, the h-PAMAM polymer consists of dendritic units, linear units, and terminal units. The linear units induce structural defects and decrease the number of external primary amines while increasing the secondary amines along the polymer backbone. In contrast, the structurally perfect G3-PAMAM dendrimer (or substantially defect-free or substantially perfect) consists of dendritic units and terminal units attached to the core.

[0210] As shown in Figure 2(b), 1 1H NMR showed the appearance of a broad peak at 2.22 - 2.90 ppm accompanied by the disappearance of peaks at 5.80 ppm to 6.60 ppm (not shown), indicating the consumption of vinyl groups from methyl acrylate during the reaction. Figure 2(e) shows the 13 13C NMR of the hyperbranched structure, providing evidence for the formation of amide bonds (170 - 180 ppm) and -CH 2 CH 2 - bonds (30 - 60 ppm) and confirming the disappearance of vinyl groups (120 - 140 ppm; not shown). As shown in Figure 2(f), the FTIR spectrum showed strong peaks at 1647 cm -1 and 1556 cm -1 assigned to amide bonds, further confirming the success of the synthesis of h-PAMAM.

[0211] Example 2: Functionalization of h-PAMAM with Propylene Oxide Synthesis of PAMAM Scaffolds by Propylene Oxide Modification

Chemical Structure

[0212] As disclosed above, secondary amine-modified PAMAM scaffolds (including h-PAMAM and G3-PAMAM) were synthesized. To enhance the NO payload, the primary amines of the h-PAMAM polymer (about 8 primary amines per scaffold) were modified with 1 molar equivalent of propylene oxide (PO) via a ring-opening reaction to obtain h-PAMAM-PO-1. In a comparative study, the h-PAMAM polymer and G3-PAMAM dendrimer were modified with 1 molar equivalent of PO with respect to the primary amines of the G3-PAMAM dendrimer (32 primary amines per scaffold) to obtain h-PAMAM-PO-2 and G3-PAMAM-PO, respectively. In this way, the effect of external modification on the NO release properties (e.g., payload and release reaction rate) of the h-PAMAM polymer could also be studied. To provide additional secondary amines, 300 mg of h-PAMAM was reacted with 24 μL of propylene oxide (PO) (i.e., 1 equivalent with respect to the molar amount of the primary amines of h-PAMAM) to obtain h-PAMAM-PO-1. To achieve comparable NO release properties, 300 mg of h-PAMAM or G3-PAMAM was reacted with 97 μL of PO (i.e., 1 equivalent with respect to the molar amount of the primary amines of G3-PAMAM) to obtain h-PAMAM-PO-2 or G3-PAMAM-PO. The reagents were mixed in 6 mL of methanol and stirred for 3 days. Unreacted PO and the solvent were removed under reduced pressure. The PO modification was 1 confirmed using 1H NMR spectroscopy. The PO modification was 1 confirmed using 1H NMR spectroscopy. As shown in Figure 2(c), the appearance of a distinct peak at 3.82 ppm was assigned to the proton adjacent to the hydroxyl group (R 2 CHOH) of the product.

[0213] The calculations are based on 2.5 active amino groups per monomer, and 1 using the proton integration ratio from 3.82 ppm to 2.2 - 3.60 ppm from 1H NMR, the conversion of total amino groups (both primary and secondary amines) was estimated to be approximately 11% and approximately 49% for h-PAMAM-PO-1 and h-PAMAM-PO-2, respectively. The resulting PAMAM scaffolds modified with PO 1 1H NMR data consisted of the following peaks: h-PAMAM-PO-1 or h-PAMAM-PO-2 (400 MHz, D 2 2O, δ): 1.05 (NHCH 2 CH(OH)CH 3 ), 2.22 - 2.90 (COCH 2 , NHCH 2 , and NH 2 CH 2 ), 3.15 - 3.58 (CONHCH 2 ), 3.60 (CH 3 2O) and 3.82 (NHCH 2 CH(OH)CH 3 2). G3-PAMAM-PO consisted of the following peaks (400 MHz, D 2 2O, δ): 1.05 (NHCH 2 CH(OH)CH 3 2), 2.37 (CH 2 2N(CH 2 2CH 2 2CO) 2 2), 2.38 - 2.78 (NCH 2 2, NHCH 2 2), 3.08 - 3.28 (CONHCH 2 2CH 2 2) and 3.82 (NHCH 2 CH(OH)CH 3 2). 1 According to the 1H NMR data, the conversion efficiencies of h-PAMAM-PO-1, h-PAMAM-PO-2, and G3-PAMAM-PO were estimated to be 11, 49, and 63%, respectively.

[0214] Example 3: Functionalization of h-PAMAM with Nitric Oxide Synthesis of Hyperbranched Scaffolds by Modification with N-Diazeniumdiolate Nitric Oxide Donors

Chemical Structure

[0215] Scaffolds containing secondary amines were exposed to high-pressure gaseous NO under basic conditions to obtain NO-releasing PAMAMs, designated as h-PAMAM / NO, h-PAMAM-PO-1 / NO, h-PAMAM-PO-2 / NO, and G3-PAMAM-PO / NO. Specifically, 50 mg of the scaffold (h-PAMAM, h-PAMAM-PO-1, h-PAMAM-PO-2, or G3-PAMAM-PO) was mixed with 50 μL of NaOMe in 1 mL of anhydrous methanol (5.4 M in MeOH, approximately 1.2 equimolar amount compared to the primary amines of G3-PAMAM). This solution was placed in a Parr hydrogenation reactor and continuously stirred. The reactor was purged six times with argon to remove oxygen and then pressurized to 10 atm with NO gas for 3 days to obtain the NO donor-modified system (i.e., h-PAMAM / NO, h-PAMAM-PO-1 / NO, h-PAMAM-PO-2 / NO, or G3-PAMAM-PO / NO). The reactor was then purged with argon to remove unreacted NO, and the solvent was removed under reduced pressure. Under high pressure (10 atm) and high pH (basic conditions) (e.g., in some embodiments, about 8, 9, 10, 11, 12, 13, 14 or greater, or a range of pH values encompassing and / or including the aforementioned values), the reaction of the PAMAM scaffold with NO gas yielded NO-releasing PAMAMs, designated as h-PAMAM / NO, h-PAMAM-PO-1 / NO, h-PAMAM-PO-2 / NO, and G3-PAMAM-PO / NO. The formation of the N-diazeniumdiolate NO donor was confirmed by the appearance of a characteristic ultraviolet-visible peak at approximately 250 nm (see Figures 3a and 3b). The FT-IR spectrum also showed O-N-N-O deformation (1350 - 1370 cm-1) and N-N stretching (1230 - 1250 cm-1) vibrations.

[0216] The NO-releasing material was redissolved in anhydrous MeOH at 50 mg mL -1 and stored at -20 °C until use later.

[0217] Characterization of nitric oxide release A wide range of NO storage capacities (about 1 - 2.50 μmol mg-1) and NO release reaction rates (T 1 / 2 about 30 - 80 minutes) were achieved by varying the degree of propylene oxide (PO) modification. Real-time NO release data in PBS (10 mM, pH 7.4, 37 °C) were measured. The nitric oxide-releasing PAMAM scaffold (1 mg) in 20 μL of MeOH was added to 10 mM phosphate-buffered saline (30 mL, pH 7.4) deoxygenated at 37 °C. Nitrogen was bubbled through this solution at a flow rate of 70 mL min -1 to carry the liberated NO to a Sievers chemiluminescence nitric oxide analyzer (Boulder, CO). An additional nitrogen stream was supplied to the flask to match the collection rate of the instrument (200 mL min -1 ). The real-time NO release profile was recorded until the observed NO level decreased to scaffolds less than 10 ppb mg -1 .

[0218] As shown in Table 1, h-PAMAM-PO-1 / NO was stored with the maximum amount of NO (about 2.50 μmol mg -1 ), followed by h-PAMAM / NO (about 2.16 μmol mg -1 ). This resulted in a greater number of secondary amine groups in h-PAMAM-PO-1 via PO modification. In fact, during the formation of h-PAMAM-PO-1, about half of the primary amines of h-PAMAM were converted to secondary amines. 1As demonstrated by the \(^1H\) NMR spectrum, the additional PO modification of h-PAMAM (i.e., h-PAMAM-PO-2) began to consume the secondary amines along the polymer backbone. Larger PO conversion efficiencies (11 and 49% for h-PAMAM-PO-1 and h-PAMAM-PO-2, respectively) actually resulted in lower total NO (about 1.33 μmol mg -1 ) for h-PAMAM-PO-2 / NO. The degree of PO modification also affected the NO release reaction rate, and faster NO release correlated with the degree of PO modification (see Figure 4). For example, h-PAMAM-PO-2 / NO (49% PO modification) released NO the fastest (t 1 / 2 ~30 min), while h-PAMAM-NO (0% PO modification) showed the longest-term NO release (t 1 / 2 ~80 min). The N-diazeniumdiolate anion is stabilized by the adjacent cationic amine via intramolecular hydrogen bonding (Figure 1), and these amino PO modifications are thought to reduce such stabilization and result in more rapid NO release (i.e., a lower half-life).

Table 1

[0219] The NO release characteristics of h-PAMAM-PO-2 / NO and G3-PAMAM / NO were nearly identical (see Figure 4 showing the cumulative NO release from (a) h-PAMAM / NO, (b) h-PAMAM-PO-1 / NO, (c) h-PAMAM-PO-2 / NO, (d) G3-PAMAM-PO / NO in PBS (10 mM, pH 7.4, 37 °C)). In addition, the total 2-hour NO release corresponding to the dose of NO delivered during a 2-hour sterilization assay was also equivalent, enabling a direct examination of the PAMAM polymer structure (i.e., dendrimer vs. hyperbranched) with respect to bactericidal action (Table 1).

[0220] In an exemplary embodiment, the polyamine amine composition comprises at least about 2 wt% NO. In one embodiment, the polyamine amine composition comprises at least about 1 μmol of NO per mg of copolymer, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37 °C). In another embodiment, the composition comprises at least about 2 μmol of NO per mg of copolymer, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37 °C). In another embodiment, the composition comprises more than about 2 μmol of NO per mg of copolymer, as measured by real-time NO release in PBS (10 mM, pH 7.4, 37 °C).

[0221] Example 4: Suspension Bactericidal Assay in h-PAMAM Structure Suspension Bactericidal Assay The bactericidal activity of the PAMAM scaffolds was evaluated against Gram-negative periodontal pathogens (P. gingivalis and A. actinomycetemcomitans) and Gram-positive cariogenic bacteria (S. mutans and A. viscosus). The assay was performed under static conditions for 2 hours. The minimum bactericidal concentration (MBC, mg mL -1 ) was used to determine and compare the antibacterial efficacy of the materials against the bacteria. The bactericidal NO dose was derived by multiplying the amount of NO delivered over a 2-hour exposure time, as measured by NOA in PBS, by the corresponding MBC value (i.e., t[NO] 2時間 × 30 g mol -1 ).

[0222] Planktonic bacteria (i.e., P. gingivalis, A. actinomycetemcomitans, S. mutans, and A. viscosus) were stored at -80 °C in 15% (v / v) glycerol PBS. To perform the bactericidal assay, this frozen stock was incubated overnight at 37 °C in BHI broth (W-C anaerobic broth for P. gingivalis). An aliquot of 500 μL of this solution was added to fresh broth and incubated at 37 °C until the bacterial concentration reached 1 × 10 8 colony-forming units per milliliter (CFU mL -1 ). P. gingivalis was cultured anaerobically in an atmosphere of 5% (v / v) CO 2 , 10% (v / v) H 2 , and 85% N 2 . A. actinomycetemcomitans and A. viscosus were cultured microaerobically in a GasPak EZ Campy Container System (Becton, Dickinson and Company; Franklin Lakes, NJ) under a microaerophilic environment (6 - 16% (v / v) O 2 and 2 - 10% (v / v) CO 2 ). S. mutans was cultured aerobically. Prior to the 2-hour planktonic bactericidal assay, the bacteria were adjusted to 10 6 CFU mL -1It was diluted. Then, the NO-releasing or corresponding control material was introduced at 37 °C. Notably, the addition of broth had little effect on the total NO release (i.e., the total NO release was 5% or less lower in 1% volume broth-added PBS). After 2 hours, the bacterial suspension was diluted 10-fold and 100-fold and spiral plated using BHI agar (CDC anaerobic agar for P. gingivalis). To quantify the antibacterial capacity of the material against planktonic bacteria, the minimum bactericidal concentration (i.e., the minimum concentration of the material required to achieve a three-log reduction in viability after 2 hours) was determined by counting the colonies formed on the agar plates. Notably, the detection limit of the plate counting method was 2.5×10 3 CFU mL -1 was.

[0223] The h-PAMAM scaffolds exhibited some bactericidal properties as a result of the high density of primary and secondary amines. As shown in Table 2, the gram-negative pathogens tested were more sensitive to h-PAMAM treatment than the gram-positive pathogens, as demonstrated by the lower MBC. This behavior may be due to the thicker peptidoglycan layer of gram-positive bacteria, which can limit scaffold association and reduce membrane degradation. Next, the antibacterial effect of h-PAMAM was evaluated as a function of PO modification. A decrease in antibacterial effect was observed in h-PAMAM-PO-1 compared to h-PAMAM, which is thought to be the result of the conversion of some (about 50%) of the external primary amines to less potent secondary amines via PO modification. The non-ionic hydroxyl groups from PO may also protect the cationic amines and thus inhibit interaction with the bacterial membrane. Extensive PO modification of h-PAMAM, as in the case of h-PAMAM-PO-2, further reduced the antibacterial effect based on more consumption of the shielding effect of the cationic amines and / or hydroxyl groups. In this study, the observed decrease in antibacterial effect was most prominent against gram-positive bacteria. For example, the use of h-PAMAM-PO-2 was 16 mg mL -1However, it did not induce antibacterial activity against S. mutans (defined by a minimum 3-log reduction in viability). Modifying PAMAM dendrimers with neutral functional groups such as polyethylene glycol (MW = 685 g mol -1 ) is thought to reduce the eradication efficacy against Gram-positive S. aureus and have little effect on Gram-negative P. aeruginosa (i.e., loss of titer).

[0224] Nitric oxide release contributed to the bactericidal action of h-PAMAM-PO-2 (Table 2). For example, the concentration of h-PAMAM-PO-2 / NO required to eradicate S. mutans was less than 25% of the concentration of h-PAMAM-PO-2. Both h-PAMAM / NO and h-PAMAM-PO-1 / NO showed lower antibacterial activity compared to the control. The charge (negative) of the N-diazene dioate functional group decreased the ability to bind to bacteria and reduced the amine-directed contact killing observed with h-PAMAM and h-PAMAM-PO-1. This result is supported by the extended NO release rate of h-PAMAM / NO and h-PAMAM-PO-1 / NO (T 1 / 2 ~60 - 80 minutes) compared to h-PAMAM-PO-2 / NO (t 1 / 2 ~30 minutes). The increased NO release half-life indicates the sustained effect of the negatively charged N-diazene dioate in h-PAMAM / NO and h-PAMAM-PO-1 / NO throughout the bactericidal assay. Similar doses of NO-releasing scaffolds are required to eradicate oral pathogens regardless of the degree of PO modification, suggesting similar effectiveness of NO delivery. The Gram-positive bacteria evaluated here were slightly more resistant to NO treatment compared to Gram-negative bacteria.

Table 2

[0225] The antibacterial activities of the h-PAMAM and G3-PAMAM systems were compared to elucidate the effect of the PAMAM structure on the bactericidal properties. Equivalent antibacterial activities were achieved when both were modified with PO against both the NO-releasing scaffolds and control scaffolds and controls (Table 2). Considering the same NO release payload and kinetics between h-PAMAM-PO-2 / NO and G3-PAMAM-PO / NO, this equivalent antibacterial activity suggests equivalent polymer-bacteria associations. Notably, the MBC values determined for both the NO-releasing hyperbranched PAMAM and G3-PAMAM were lower than those of the smaller PAMAM dendrimer polymers (i.e., G1-PAMAM-PO / NO), especially in the case of the cariogenic bacterium (S. mutans). This result indicates that increasing size can enhance the killing of oral pathogens, consistent with data for other planktonic pathogens. Overall, h-PAMAM-PO-2 / NO is equivalent to G3-PAMAM-PO / NO with respect to bactericidal efficacy but is available at a much lower synthesis cost.

[0226] As described above, the therapeutic potential of these materials was evaluated by studying their antibacterial activities and toxicity against common oral pathogens and human gingival fibroblasts, respectively. These results indicate that a combination of NO release and PO modification results in h-PAMAM materials with effective bactericidal action without inducing unnecessary cytotoxicity. Importantly, the NO-releasing PO-modified h-PAMAM polymers exhibited biological properties (i.e., antibacterial action and cytotoxicity) comparable to those of defect-free G3-PAMAM dendrimers, but with a much lower synthetic burden. These results also confirm the importance of size in killing oral pathogens. Overall, h-PAMAM-PO-2 / NO is equivalent to G3-PAMAM-PO / NO with respect to bactericidal efficacy but is available at a much lower synthesis cost.

[0227] Confocal microscopy Rhodamine B isothiocyanate (RITC)-modified h-PAMAM-PO-2 and G3-PAMAM-PO materials were prepared to facilitate the visualization of polymer scaffolds containing bacteria (S. mutans) via confocal fluorescence microscopy. Rhodamine B isothiocyanate (RITC)-labeled h-PAMAM-PO-2 and G3-PAMAM-PO were synthesized. S. mutans was cultured as described above and diluted to 10 7 CFU / mL -1 . This bacterial solution (3 mL) was incubated in a glass-bottom confocal dish at 37 °C for 30 min. Fluorescence images of RITC-modified PAMAM-PO were obtained using a Zeiss 510 Meta inverted laser scanning confocal microscope (Carl Zeiss; Thornwood, NY) equipped with a 543 nm HeNe excitation laser (1.0 mW, 25.0% intensity) and a BP 560-615 nm filter. Both bright-field and fluorescence images were collected using a 40× objective, N.A. 1.2 C-apochromat water immersion lens. RITC-labeled PAMAM-PO was added to the bacterial solution to achieve a final concentration of 100 μg / mL -1 . Images were collected every 10 min to visualize the association of PAMAM-PO with bacteria.

[0228] Accumulation of nearly identical fluorescence signals was observed at each time point (Figs. 5(a)–(b)), confirming a very important killing mechanism. These dendrimer scaffolds (e.g., G1-PAMAM-PO) can serve as NO-releasing agents superior to other polymer scaffolds (e.g., silica) because of their improved association and bactericidal efficacy. The studies herein confirm equivalent behavior between h-PAMAM-PO-2 and G3-PAMAM-PO despite the characterization of the imperfect structure of hyperbranched polymers.

[0229] In Vitro Cytotoxicity The toxicity of PAMAM scaffolds to human gingival fibroblasts (HGF-1) was evaluated after 2 h and 24 h exposure times. Human gingival fibroblasts (HGF-1) were grown in FibroLife fibroblast serum-free medium under humidified conditions at 37 °C with 5% CO 2Incubated therein. Cells were trypsinized at 80% confluence and seeded at a density of approximately 10 4 cells / well in tissue culture-treated polystyrene 96-well plates. The plates were incubated at 37 °C for an additional 24 h. The supernatant was aspirated and replaced with 100 μL of fresh growth medium containing various concentrations of PAMAM scaffolds. After two incubation time points at 37 °C (i.e., 2 h and 24 h), the supernatant was aspirated and the cells were washed with DPBS. A 100 μL solution of medium / MTS / PMS (105 / 20 / 1, volume / volume / volume) solution was added to each well and incubated at 37 °C for 3 h. The absorbance of the colored supernatant was quantified at 490 nm using a Thermoscientific Multiskan EX plate reader (Waltham, MA). Measurements of untreated cells (control) and medium / MTS / PMS mixture (blank) were also collected. Results were expressed as a percentage of relative cell viability as follows. % Cell viability = [(Abs 490 - Abs ブランク ) / (Abs 対照 - Abs ブランク )] × 100% (Equation 1)

[0230] % Cell viability vs. concentration (mg mL -1 ) was plotted to construct the killing curves of NO-releasing and control PAMAM scaffolds.

[0231] The 2 h incubation period was chosen to correspond to the exposure time for the bactericidal assay. As shown in FIGS. 6(a) and 6(b), h-PAMAM was demonstrated to be the most toxic to HGF-1, probably due to disruption of the cationic amines in the cell membrane. The toxicity of h-PAMAM was reduced by a decrease in amine content and nonionic hydroxyl group shielding. The addition of NO further alleviated the toxicity of h-PAMAM and h-PAMAM-PO-1 (FIG. 6b), which is the effect of the negatively charged N-diazene dioate functional group that suppresses the interaction of HGF-1.

[0232] During the 24-hour incubation period (see Figures 7(a) and 7(b)), h-PAMAM and h-PAMAM-PO-1 showed significant toxicity to HGF-1 even at low concentrations (i.e., 0.1 mg mL -1 ), compared to h-PAMAM-PO-2 (i.e., 4 mg mL -1 ) with a survival rate of over 80%). These results suggest that partial PO modification of the external primary amine (i.e., h-PAMAM-PO-1) is insufficient to mitigate polymer toxicity over a long period. The addition of NO release to the scaffold via NO donor modification (i.e., h-PAMAM, h-PAMAM-PO-1, and h-PAMAM-PO-2) resulted in a decrease in the survival rate of HGF-1 cells at high concentrations (>0.5 mg mL -1 ), indicating that high doses of NO are toxic to mammalian cells. See Worley et al. In contrast, low-concentration (0.1 mg mL -1 ) NO-releasing h-PAMAM polymers are less toxic than the control, suggesting that low levels of NO are acceptable and may even promote cell growth. The h-PAMAM-PO-2 / NO polymer caused some toxicity to HGF-1 cells at 4 mg mL -1 (the most effective bactericidal concentration), representing an advantage of h-PAMAM-PO-2 / NO over NO-releasing polymer scaffolds (silica nanoparticles and G1-PAMAM dendrimer).

[0233] Finally, cytotoxicity was compared between h-PAMAM polymers and G3-PAMAM dendrimers using HGF-1 cells. Although lower toxicity was observed for unmodified G3-PAMAM dendrimers compared to h-PAMAM polymers at concentrations below 2 mg mL -1 , the G3-PAMAM dendrimer showed toxicity at 0.5 mg mL -1However, significant toxicity was still observed. When modified with PO, comparable toxicity was achieved with h-PAMAM polymer and G3-PAMAM dendrimer (i.e., h-PAMAM-PO-2 and G3-PAMAM-PO systems, respectively). Collectively, these data indicate the potential of h-PAMAM-PO-2 / NO as a safe antibacterial agent for oral antibacterial therapy.

[0234] As described above, the synthesis and characterization of h-PAMAM polymers by N-diazeniumdiolate NO donor modification are provided. In some embodiments, the NO release characteristics of h-PAMAM (i.e., payload and release reaction rate) were evaluated as a function of chemical modification. The potential of this scaffold as an oral therapeutic was evaluated from the perspective of antibacterial activity against common oral pathogens and toxicity to human gingival fibroblasts. Finally, the characteristics of the h-PAMAM structure were compared to the G3-PAMAM counterpart with respect to therapeutic potential, and favorable results were obtained. The combination of NO release ability and propylene oxide (PO) modification of the hyperbranched PAMAM scaffold (h-PAMAM-PO-2 / NO) was able to effectively eradicate oral pathogens with low / minimal toxicity to human gingival fibroblasts. Despite structural deficiencies, the antibacterial activity of h-PAMAM-PO-2 / NO was comparable to that of structurally complete G3-PAMAM-PO / NO. In this regard, h-PAMAM-PO-2 / NO shows potential as a therapy. In some embodiments, the antibacterial action of h-PAMAM-PO-2 / NO can be used against clinically relevant ex vivo multispecies dental biofilms. In some embodiments, the disclosed NO-releasing hyperbranched polymers can be used for oral care applications. In some embodiments, the disclosed NO-releasing hyperbranched polymers can be used for oral care applications in the oral microbiota.

[0235] Example 5: Synthesis of Hydroxyl-Terminated Hyperbranched Polymer Materials and Methods N,N'-Methylenebisacrylamide (MBA), N-(2-hydroxyethyl)ethylenediamine (HEDA), fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), phenazine methosulfate (PMS), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), trypsin, Dulbecco's phosphate-buffered saline (DPBS), and penicillin streptomycin (PS) were purchased from Sigma-Aldrich and used without further purification. Sodium methoxide (5.4 M solution in methanol) was purchased from Acros Organics. Nitric oxide (NO) gas (99.5%) was purchased from Praxair. Distilled water was purified to a final resistivity of 18.2 MΩCM and a total organic content of 6 ppb or less using a Millipore Milli-Q UV Gradient A-10 system. Pseudomonas aeruginosa (P. aeruginosa; ATCC number 19143) and Staphylococcus aureus (S. aureus; ATCC number 29213) were purchased from the American Type Culture Collection. Tryptic soy agar (TSA) and tryptic soy broth (TSB) were purchased from Becton, Dickinson, and Company. L929 mouse fibroblasts (ATCC number CCL-1) were obtained from the University of North Carolina Tissue Culture Facility. All other materials were obtained from commercial sources and used without further purification.

[0236] Nuclear magnetic resonance ( 1 1H NMR) spectra were recorded on a Bruker (400 MHz) spectrometer. Quantitative 13 13C nuclear magnetic resonance ( 13The 13C NMR spectra and 2D NMR techniques were recorded on a 600 MHz Bruker instrument. The ultraviolet-visible absorption spectra were measured on a PerkinElmer Lambda 40 spectrophotometer. Gel permeation chromatography measurements were performed on an aqueous GPC multi-angle light scattering system equipped with a Waters 2414 refractive index detector (Milford, MA) connected to a Wyatt miniDawn TREOS multi-angle light scattering detector (Santa Barbara, CA).

[0237] Synthesis of Hydroxyl-Terminated Hyperbranched Polymer (HBPMH) [Chemical formula]

[0238] The Michael addition polycondensation procedure was as follows: MBA (1.540 g, 10 mmol) was added to a mixture of HEDA (1.041 g, 10 mmol) in 15 ml of deionized water, and then reacted at 75 °C for 72 hours with vigorous stirring. The resulting solution was precipitated with cold acetone and centrifuged to obtain a white solid product. The product was dried under vacuum at room temperature for 3 days. The final product was obtained as a white solid powder (1.212 g, yield 47.0%). Hyperbranched poly(methylenebisacrylamide-hydroxyethylethylenediamine) (HBPMH) was synthesized at 75 °C for 3 days at an equal feed molar ratio by Michael addition polycondensation between the A 2 -monomer of N,N'-methylenebisacrylamide and the trifunctional amine monomer of N-(2-hydroxyethyl)ethylenediamine. The polymerization process was first monitored by 1 1H NMR spectra (Figure 8). Two peaks at approximately 5.70 ppm and 6.10 ppm assigned to the double bonds of the MBA monomer gradually decreased during the polymerization procedure. The conversion efficiency of the vinyl group (CR V ) can be calculated according to the formula CR V %=(1 - S 5.60~6.20 / 3S 4.40~4.65 ) * 100%, where S 5.60~6.20is the integration of the proton signal (5.60 - 6.20 ppm) assigned to the double bond, S 4.40~4.65 was the integration of the proton signal (4.40 - 4.65 ppm) assigned to the methylene group between two amide groups. CE V was 31.6% in 10 minutes, 74.1% in 2 hours, and 97.2% in 6 hours. The results indicated that the Michael addition polymerization proceeded very rapidly at the initial stage. Two new proton signals in the ranges of 2.26 - 2.35 ppm and 2.41 - 2.47 ppm were also observed after 10 minutes of reaction, and these were assigned to the newly formed methylene groups adjacent to the amide groups of intermediate I and intermediate II. The integration area of the signal (2.26 - 2.35 ppm) was larger than that of the signal (2.41 - 2.47 ppm), indicating that the primary amine had higher reactivity than the secondary amine. After 12 hours, the proton signal of the vinyl group almost disappeared. All sharp peaks changed to broad peaks, indicating the occurrence of polymerization. Similar time - dependent NMR traces have also been reported in the synthesis of primary - amine - terminated hyperbranched poly(amidoamine). Notably, there were residual double bonds (less than 0.2%) in the final polymer architecture.

[0239] Generally, hyperbranched polymers can consist of dendritic units, linear units, and terminal units, as shown in Figure 9. Six possible structural segments of the newly synthesized HBPMH are shown in Figure 9, D, L a , L b , T a , and T b represent the fractions of dendritic units (D), linear units (L a , L b ), and terminal units (T a , T b ), respectively. 1 H - 13 C HSQC, 1 H - 1 H COSY, 1 H NMR, quantitative 13 C NMR spectra were run to carefully confirm the 3D topological structure of HBPMH (Figure 10a - d).13 In the 13C NMR spectrum (Figure 10b), no signal was observed in the range of 37 - 42 ppm, indicating that the terminal unit T having a primary amine in the polymer backbone of HBPMH was not found. However, due to the complex structure of HBPMH, c it was difficult to determine the branching degree (DB) based on 1H NMR or quantitative 1 13C NMR. The molecular weight and polydispersity index (PDI) of HBPMH were characterized by gel permeation chromatography (GPC). The weight-average molecular weight (Mw) of HBPMH was 6,368 g / mol, and the PDI was approximately 1.78. The number-average degree of polymerization (DPn) was calculated by dividing the Mw of HBPMH by the total molecular weight of the two monomers (258 g / mol), and it was 24.7. Each group in Figure 9 was confirmed by NMR, except for the terminal unit Tc. 13 Synthesis of Hyperbranched Scaffolds by N-Diazeniumdiolate Nitric Oxide Donor Modification

[0240] N-Diazeniumdiolate Nitric Oxide Donor Modification The HBPMH resulting from the abundance of secondary amines was reacted with NO gas (10 bar) under strongly alkaline conditions to obtain an N-diazene dioate NO donor (Figs. 11(a - d)). To synthesize N-diazene dioate-functionalized HBPMH, secondary amine-containing HBPMH was added to 2.0 mL of anhydrous methanol (MeOH). In the following step, 1 equivalent of sodium methoxide in methanol (relative to the molar amount of secondary amines in the polymer backbone) was added to the mixture, followed by vortexing to obtain a homogeneous solution. The mixture was placed in a stainless-steel reactor with strong magnetic stirring. The vessel was rapidly purged three times with argon to a pressure of 7 bar, followed by three long argon purge cycles (10 minutes) to remove residual oxygen from the solution. The vessel was then pressurized with 10 bar of NO gas, and this pressure was maintained for 3 days. The solution was purged three times briefly with argon, followed by a purge three times as long (10 minutes) to remove unreacted NO gas. The solution was precipitated once with 15 mL of acetone and then centrifuged to remove the solvent. The final product was dried in a drying oven under vacuum at room temperature for 2 hours. The resulting N-diazene dioate-functionalized HBPMH was parafilm-sealed and stored at -20 °C for future use.

[0241] The obtained HBPMH modified with N-diazene dioate (HBPMH / NO) was characterized by ultraviolet-visible spectrum, 1 1H NMR spectrum and FTIR spectrum. Compared with the ultraviolet-visible spectrum of HBPMH, a strong and typical absorption peak (at approximately 254 nm) assigned to the N-diazene dioate structure appeared in the ultraviolet-visible spectrum (Fig. 11b), indicating the formation of HBPMH / NO. In addition, a broad absorption peak in the range of approximately 330 - 360 nm assigned to carcinogenic N-nitrosoamine species was not observed, indicating that N-nitrosoamine was not formed in the final NO donor of HBPMH / NO. The formation of HBPMH / NO also 1Characterized by the ¹H NMR spectrum (Figure 11c). By diazeniumdioation, the proton signal in the range of 2.50 - 2.90 ppm corresponding to the bonding of the methylene group to the secondary amine is changed to a high chemical shift in the range of 2.95 - 3.20 ppm due to the electron-withdrawing effect of the newly formed diazeniumdioate group. Peak 1 (3.10 - 3.20 ppm) corresponded to N-diazeniumdioate linked to the secondary amine close to the hydroxyl group (Figure 12a and Figure 12b), while peak 2 (2.95 - 3.10 ppm) represented N-diazeniumdioate far from the hydroxyl group (Figure 12c and Figure 12d). The conversion efficiency (CE SA ) of the secondary amine to N-diazeniumdioate can be calculated by the formula CE SA %=(S 1,2 / 4) / (S 4.50 / 2)×100%, where S 4.50 and S 1,2 represent the integration of the peak at about 4.50 ppm and the total integration of peak 1 and peak 2, respectively. The resulting CE SA was 27.3%. The FTIR spectrum (Figure 11d) provided further evidence for the formation of HBPMH / NO. Compared with non-functionalized HBPMH, the FTIR band at 1361 cm -1 was enhanced, and a new FTIR band at 945 cm -1 , which is assigned to the characteristic peaks for the O-N-N-O asymmetric stretch and in-plane N-N symmetric stretch, appeared in the FTIR spectrum. Both different FTIR bands revealed the successful introduction of N-diazeniumdioate into the HBPMH backbone. The formation of HBPMH / NO was also demonstrated using GPC (Figure 13). The Mw of HBPMH / NO increased up to 6,973 g / mol by diazeniumdioation, but there was no obvious change in the PDI (PDI = 1.69). Compared with the Mw of HBPMH, the significant increase in molecular weight (569 g / mol) indicated that the N-diazeniumdioate NO donor segment (-N 2 O 2 Na) was successfully introduced into the polymer backbone of HBPMH.

[0242] Characteristics evaluation of nitric oxide release Real-time NO release was monitored by using a Sievers NOA 280i chemluminescence NO analyzer (NOA, Boulder, CO). Prior to analysis, the NO analyzer was calibrated using air that had passed through a NO zero filter (0 ppm of NO) and a 25.87 ppm NO standard gas. In a typical measurement, 1 mg of N-diazene dioate-functionalized HBPMH was added to a sample container containing 30 mL of deoxygenated PBS (pH 7.4, 37 °C) to initiate NO release. The container was purged with nitrogen at a flow rate of 80 mL / min to transport the liberated NO gas to the NOA analyzer. An additional nitrogen stream was supplied to the container to match the collection rate of the instrument (200 mL / min). The NO analysis was terminated when the NO level dropped below 10 ppb of NO / mg (HBPMH functionalized with N-diazene dioate).

[0243] The N-diazeniumdiolate NO donor proceeds through the process of pH-triggered NO release. Figure 14(a) shows the proposed dissociation procedure of the hyperbranched polymer scaffold by the new N-diazeniumdiolate modification. When HBPMH / NO is immersed in physiological conditions (i.e., 37 °C, pH 7.4), 1 mole of N-diazeniumdiolate reacts with 1 mole of proton to regenerate 1 mole of the parent secondary amine compound and 2 moles of NO radicals. A chemiluminescence-based nitric oxide analyzer (NOA) was used to detect NO radicals in real time, and the NO release total storage and dissociation reaction rate of water-soluble HBPMH / NO in PBS buffer (pH 7.43) at physiological temperature (37 °C) were investigated. The calculated NO release parameters (e.g., total NO storage, half-life of NO release, maximum flux, time to maximum flux, and conversion rate) are shown in Table 3. Representative real-time NO release profiles of HBPMH / NO are shown in Figures 14b and 14c. Generally, HBPMH / NO showed a high NO storage capacity (i.e., total NO of about 2.01 μmol / mg) and a fast NO release reaction rate (i.e., half-life of NO release of about 20 minutes). The rapid NO release reaction rate was caused by the water absorption ability of the hydroxyl-terminated HBPMH, which is faster than the water absorption ability of the dendrimer by alkyl (alky) chain modification. Further calculations showed that the conversion efficiency of the secondary amine of HBPMH to N-diazeniumdiolate was 25.9%, which was in good agreement with the NMR data. This can occur due to steric hindrance by the dense topological architecture of the hyperbranched polymer and repulsive interactions between the negatively charged N-diazeniumdiolate.

Table 3

[0244] Example 6: Suspension Bactericidal Assay in the HBPMH Hyperbranched Structure It was tested whether NO is an effective antibacterial agent for removing planktonic bacteria. The antibacterial ability of hydroxyl-terminated HBPMH / NO was evaluated against model pathogens of Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus associated with severe diseases (e.g., wounds). The planktonic bacteria viability assay was performed under static conditions, and the minimum bactericidal concentration (MBC 4時間 ) required to reduce the bacterial survival rate by three logarithms (i.e., kill 99.9%) over 4 hours, a common parameter for quantifying antibacterial activity, was determined. The amount of NO delivered by the NO-releasing HBPMH / NO during this period was calculated to quantitatively evaluate the NO dosage required for bactericidal activity.

[0245] Colonies of P. aeruginosa or S. aureus were cultured in 3 mL of TSB at 37 °C overnight (about 16 hours). A 1000 μL aliquot of the resulting suspension was added to 15 mL of fresh TSB and incubated at 37 °C for an additional 2 hours to achieve a concentration of 10 8 colony-forming units per milliliter (CFU / mL, confirmed by OD600). The bacteria were collected by centrifugation, resuspended in sterile PBS, and diluted to 10 6 CFU / mL. The antibacterial efficacy of both non-NO-releasing and NO-releasing HBPMH against planktonic bacteria was evaluated under static conditions at 37 °C for 4 hours. A blank (untreated cells) was incubated in each experiment, and it was confirmed that the bacteria were viable at 10 6 CFU / mL over the 4-hour assay. 100 μL aliquots of the blank, control, or NO-releasing HBPMH-treated bacterial suspension were serially diluted 10-fold with sterile H 2 O and plated onto TSA plates using an Eddy Jet spiral plater (IUL; Farmingdale, NY), followed by incubation at 37 °C overnight. Bacterial survival was evaluated by counting all colonies on the TSA plates using a Flash & Go colony counter (IUL; Farmingdale, NY). The minimum bactericidal concentration (MBC4時間 ) resulted in a three-log reduction in bacterial viability compared to the blank and was expressed as the minimum concentration of NO-releasing HBPMH over a 4-hour exposure. Notably, the detection limit of this selected plate count method was 2.5×10 3 CFU / mL. Both the MBC and the required NO dosage are shown in Table 4.

[0246] The antibacterial activities of both the control hydroxyl-terminated HBPMH and NO-releasing HBPMH / NO against planktonic bacteria were performed to evaluate the function of nitric oxide. HBPMH showed bactericidal efficacy against P. aeruginosa, and its MBC 4時間 value was 500 μg / mL, while HBPMH did not show a significant effect on irradiation against S. aureus at concentrations below 16000 μg / mL (Figures 15a and 15b). The MBC of the primary amine-terminated G4 PAMAM dendrimer against P. aeruginosa and S. aureus 4時間 values were 30 μg / mL and 1000 μg / mL, respectively (not shown). Without being restricted to a specific mechanism, the increased MBC 4時間 value for HBPMH indicated the low cytotoxicity of hydroxyl-terminated HBPMH due to PEGylation protection. However, NO-releasing HBPMH / NO showed excellent bactericidal efficacy against both P. aeruginosa and S. aureus, and their MBC 4時間 values were 50 μg / mL and 1000 μg / mL, respectively. The significantly decreased MBC 4時間 value indicated that NO acts as a very important antibacterial agent, especially for S. aureus. Further examination of the MBC 4時間 value and the required NO dosage showed that HBPMH / NO was a more effective reduction against P. aeruginosa. The increased antibacterial activity against P. aeruginosa was due to the rapid association between HBPMH / NO and the thin peptidoglycan layer of the Gram-negative bacterial membrane and the resulting high-efficiency NO delivery.

Table 4

[0247] In vitro cytotoxicity Despite effective antibacterial activity, the usefulness of new antibacterial materials is also related to their toxicity to mammalian cells. Through the delicate design of hydroxyl-terminated HBPMH, the cytotoxicity was effectively reduced.

[0248] L929 mouse fibroblasts were cultured in DMEM supplemented with 10% v / v fetal bovine serum (FBS) and 1 wt% penicillin / streptomycin and incubated at 37 °C with 5% v / v CO 2 in a humidified atmosphere. After reaching confluence (80%), the cells were trypsinized and seeded at a density of 1×10 4 cells / mL in tissue culture-treated polystyrene 96-well plates and incubated at 37 °C for 24 h. The supernatant was then aspirated and replaced with 100 μL of fresh growth medium containing various concentrations of both non-NO-releasing and NO-releasing HBPMH in each well. Over a 4-h incubation at 37 °C, the supernatant was aspirated and 100 μL of a mixture of DMEM / MTS / PMS (105 / 20 / 1, volume / volume / volume) was added to each well. The absorbance of the colored solution obtained over a 3-h incubation was quantified at 490 nm by using a Thermo Scientific Multiskan EX plate reader (Waltham, MA). Mixtures of DMEM / MTS / PMS and untreated cells were used as blank and control, respectively. The cell viability was calculated according to the following formula:

Chemical formula

[0249] The cytotoxicity of both the control and NO-releasing HBPMH / NO was evaluated against mouse fibroblasts at various concentrations from 0 to 16,000 μg / ml. The normalized cell viability of both the control and NO-releasing HBPMH / NO over a 4-hour incubation is shown in FIGS. 16(a)–(b). Hydroxyl-terminated HBPMH showed non-toxic properties (cell viability > 50%) against mouse fibroblasts at concentrations below 8000 μg / mL (FIGS. 16a and 16b). By diazeniumdiolation, the cell viability of more than 50% of NO-releasing HBPMH / NO decreased to 1000 μg / mL because higher NO concentrations may cause cell death due to nitrosation stress or oxidative stress. It was found that both the control and NO-releasing HBPMH / NO were non-toxic to mouse fibroblasts at the MBC 4時間 value. In summary, the bactericidal activity against planktonic bacteria and the low cytotoxicity of NO-releasing HBPMH / NO suggest that the new NO-releasing HBPMH / NO can be utilized as an ideal antibacterial agent for wound healing or cystic fibrosis applications.

[0250] Biocompatible hydroxyl-terminated HBPMH was successfully synthesized by Michael addition polycondensation. N-Diazeniumdiolate-functionalized HBPMH with high total NO and fast NO kinetics was also reported in this study. The usefulness of NO-releasing hydroxyl-terminated HBPMH as a new antibacterial agent was demonstrated by antibacterial and cytotoxicity assays. The hydroxyl-terminated HBPMH and other hyperbranched structures disclosed herein are thought to be useful as biocompatible scaffolds for other biomedical applications (e.g., gene delivery or drug delivery). HBPMH functionalized with N-diazeniumdiolate can function as an effective NO-releasing material for targeted applications.

[0251] In summary, biocompatible hydroxyl-terminated hyperbranched poly(methylenebisacrylamide-hydroxyethyl ethylenediamine) (HBPMH) was successfully synthesized by green, low-cost, and efficient Michael addition polycondensation. The resulting secondary amine-containing HBPMH was reacted with nitric oxide (NO) gas under alkaline conditions to obtain N-diazinium dioate-functionalized HBPMH (HBPMH / NO). The total NO and half-life of HBPMH / NO were approximately 2.01 μmol / mg and approximately 20 minutes, respectively. Using Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus, the antibacterial activity of HBPMH / NO was evaluated, and the minimum bactericidal concentrations against 4-hour incubation were 50 μg / mL and 1000 μg / mL. Furthermore, both the control HBPMH and NO-releasing HBPMH / NO showed non-toxic properties to mammalian L929 mouse fibroblasts in vitro.

[0252] Example 7: Synthesis of Hyperbranched Poly(methylenebisacrylamide-aminoethylpiperazine) Synthesis of Hyperbranched Polymer (HBPMA)

Chemical formula

[0253] Hyperbranched poly(methylenebisacrylamide-hydroxyethyl ethylenediamine) (HBPMA) was synthesized at 60 °C for 3 days at an equal feed molar ratio by Michael addition polycondensation between the A 2 -monomer of N,N'-methylenebisacrylamide (MBA) and the trifunctional amine monomer of 1-(2-aminoethyl)piperazine (AP). Some possible structural units of HBPMA are shown in Figure 17. 1 1H NMR and quantitative 13 13C NMR spectra were used to characterize the resulting hyperbranched polymer (Figures 18(a) and (b)). These results were the same as those reported in the literature. Quantitative 13Based on the 13C NMR spectrum, the degree of branching (DB) of HBPMA can be calculated according to the formula DB = (D + T) / (D + L + T), where D, T, and L represent the ratios of branched units, terminal units, and linear units, respectively. The calculated DB was 40.0%.

[0254] Synthesis of Hyperbranched Scaffolds Modified with N-Diazeniumdiolate Nitric Oxide Donors Synthetic HBPMA rich in secondary amines was reacted with NO gas (10 bar) under strongly alkaline conditions to obtain an N-diazeniumdiolate NO donor (Figure 19(a)). The resulting HBPMA modified with N-diazeniumdiolate (HBPMA / NO) 1 was characterized by 1H NMR spectra and ultraviolet-visible spectra. The formation of HBPMA / NO was first 1 characterized by 1H NMR spectra (Figure 19(b)). Due to diazeniumdiolation, the proton signal in the range of 2.50 - 2.85 ppm corresponding to the binding of methylene groups to secondary amines changes to a higher chemical shift in the range of 2.95 - 3.18 ppm due to the electron-withdrawing effect of the newly formed diazeniumdiolate group. The ultraviolet-visible spectrum (Figure 19(c)) provided further evidence for the formation of HBPMH / NO. Compared with the ultraviolet-visible spectrum of HBPMH, a strong and typical absorption peak (at approximately 256 nm) assigned to the N-diazeniumdiolate structure appeared in the ultraviolet-visible spectrum (Figure 19(c)), indicating the formation of HBPMH / NO. In addition, a broad absorption peak in the range of approximately 330 - 360 nm assigned to carcinogenic N-nitrosoamine species was not observed, indicating that N-nitrosoamine was not formed in the final NO donor of HBPMH / NO.

[0255] Characterization of Nitric Oxide Release Properties Figure 20(a) shows the proposed dissociation procedure of HBPMA by N-diazanium dioate modification. In the dissociation of HBPMA / NO under physiological conditions (i.e., 37 °C, pH 7.4), 1 mol of N-diazanium dioate can react with 1 mol of proton to regenerate 1 mol of the parent secondary amine compound and 2 mol of NO radicals. A chemiluminescence-based nitric oxide analyzer (NOA) was used to detect NO radicals in real time, and the total NO release storage and dissociation reaction rate of water-soluble HBPMH / NO in PBS buffer (pH 7.43) at physiological temperature (37 °C) were investigated. The calculated NO release parameters (e.g., total NO storage, half-life of NO release, maximum flux, time to maximum flux, and conversion rate) are shown in Table 5. Representative real-time NO release profiles of HBPMH / NO are shown in Figures 20(b) and 20(c). In general, HBPMH / NO showed a higher NO storage capacity (i.e., total NO of about 2.08 μmol / mg) and a similar NO release reaction rate (i.e., half-life of NO release of about 60 minutes) compared to the alkyl-modified G3 PAMAM dendrimer (i.e., total NO of about 1.0 μmol / mg and half-life of NO release of about 60 minutes). The conversion efficiency of the secondary amine of HBPMA to N-diazanium dioate was 29.4%. The conversion efficiency may occur due to steric hindrance by the high-density topological architecture of the hyperbranched polymer and repulsive interaction between the negatively charged N-diazanium dioate.

Table 5

[0256] Conclusion In this specification, a synthetic protocol for preparing NO-releasing hyperbranched structures capable of a wide range of NO storage and release reaction rates was provided. The advantageous NO payload, release reaction rate, bactericidal effect, and cytotoxicity suggest that these scaffolds can be used for many therapeutic applications beyond oral hygiene.

Claims

1. 1. A hyperbranched nitric oxide (NO) donor compound comprising: comprising a linking group comprising any one or more of formulae A, B, C, or D; 【Chemistry 1】 X １ , X ２ , X ３ , and X ４ are independently -NH-, -N(R ａ )-, -O-, and -S-; R ａ Each example is selected from the group consisting of an NO donor moiety, —H, an optionally substituted C １ -C ６ is selected from an alkyl group, or an optionally substituted polyether having 1 to 6 repeat units; R １ , R ２ , and R ３ is independently -R ｂ N (R ｃ ) R ｄ -(N(R ｃ )) ｎ -R ｂ -, -R ｂ (OR ｄ -) ｎ O-R ｂ - and C １ -C ６ is selected from the group consisting of alkyl, R ｃ Each instance of is independently an NO donating moiety, —H, an optionally substituted C １ -C ６ is selected from an alkyl group, or an optionally substituted polyether having 1 to 6 repeat units; R ｂ is a single bond or an optionally substituted C １ -C ６ is an alkylene group, R ｄ is optionally substituted C １ -C ６ is an alkylene group, n is an integer selected from 0, 1, 2, 3, 4, 5, or 6; R ２ If R ｄ At least one example of the group is —C(O)—(CH ２ ) ２ -X ３ - group, R ３ If R ｄ At least one example of the group is —C(O)—(CH ２ ) ２ -X ３ - group, R ｄ At least one example of the group is —C(O)—(CH ２ ) ２ -X ４ - group, The hyperbranched compound comprises at least one of the following NO-donating moieties: 【Chemistry 2】 and A hyperbranched NO-donor compound, wherein said hyperbranched compound does not contain aminoglycoside or glycoside units.

2. Further examples include at least one of the following structures: 【Chemistry 3】 In the formula, R ４ But -N ＋ (=N-O － ) O － 2. The hyperbranched NO donor compound of claim 1, wherein

3. 3. The hyperbranched NO-donor compound of claim 2, wherein the linking group comprises Formula A.

4. The linking group comprises formula B, wherein R １ But, -R ｂ N (R ｃ ) R ｄ -(N(R ｃ )) ｎ -R ｂ - and n is 1, R ｄ But -CH ２ - and R ｂ is a single bond, Each R ｃ The hyperbranched NO donor compound of claim 1 , wherein

5. 5. The hyperbranched NO-donor compound of claim 4, wherein the linking group of formula B is represented by the following structure: 【Chemistry 4】 。

6. R ａ 6. The hyperbranched NO-donor compound of claim 5, wherein each instance of is said NO-donor moiety or -H.

7. 7. The hyperbranched NO-donor compound of claim 6, wherein the linking group of formula A is represented by the following structure: 【Chemistry 5】

8. Further comprising an end group selected from the group consisting of: 【Chemistry 6】 In the formula, R ５ Each instance of is H or -N ＋ (=N-O － ) O － 2. The hyperbranched NO donor compound of claim 1, wherein

9. 2. The hyperbranched NO-donor compound of claim 1, further comprising a terminal group selected from the group consisting of: 【Chemistry 7】

10. 2. The hyperbranched structure of claim 1 further comprising one or more of the following groups: 【Chemistry 8】

11. The hyperbranched structure of claim 1 , wherein the hyperbranched structure lacks a dendritic core having any symmetric dendrons.

12. Each instance of any substitution is C １ -C ６ 2. The hyperbranched compound of claim 1, wherein the group is selected from alkyl or -OH.

13. 1. A hyperbranched nitric oxide (NO) donor compound comprising: comprising a linking group of formula A or formula B, 【Chemistry 9】 X １ and X ２ are independently -NH-, -N(R ａ )-, -O-, and -S-; R ａ each instance of is selected from an NO donating moiety or -H; R １ is independently -N(R ｃ ) R ｄ -N(R ｃ ) -, -R ｂ (OR ｄ -) ｎ O-R ｂ - and C １ -C ６ is selected from the group consisting of alkyl, R ｃ Each instance of is independently an NO donating moiety or -H, optionally substituted C １ -C ６ is selected from an alkyl group, or an optionally substituted polyether having 1 to 6 repeat units; R ｂ is a single bond or an optionally substituted C １ -C ６ is an alkylene group, R ｄ But -CH ２ - or -CH ２ -CH ２ - and n is an integer selected from 0, 1, 2, 3, 4, 5, or 6; A hyperbranched (NO) donor compound, wherein the hyperbranched compound comprises at least one of the following NO donor moieties: 【Chemistry 10】

14. Further examples include at least one of the following structures: 【Chemistry 11】 In the formula, R ４ But -N ＋ (=N-O － ) O － 14. The hyperbranched NO donor compound of claim 13, wherein

15. 14. The hyperbranched NO-donor compound of claim 13 comprising formula A.

16. Formula B, wherein R １ But -N(R ｃ ) R ｄ -N(R ｃ ) - and R ｄ But -CH ２ - and Each R ｃ The hyperbranched NO-donor compound of claim 13, wherein is H.

17. 14. The hyperbranched NO donor compound of claim 13, comprising a linking group represented by the structure: 【Chemistry 12】

18. R ａ 20. The hyperbranched NO-donor compound of claim 17, wherein each instance of is said NO-donor moiety or -H.

19. 20. The hyperbranched NO-donor compound of claim 18, wherein the linking group of formula A is represented by the following structure: 【Chemistry 13】

20. Further comprising an end group selected from the group consisting of: 【Chemistry 14】 In the formula, R ５ Each instance of is H or -N ＋ (=N-O － ) O － 14. The hyperbranched NO donor compound of claim 13, wherein

21. 14. The hyperbranched NO donor compound of claim 13, further comprising a terminal group selected from the group consisting of: 【Chemistry 15】

22. 14. The hyperbranched NO donor compound of claim 13 further comprising one or more of the following groups: 【Chemistry 16】

23. 14. The hyperbranched NO-donor compound of claim 13, wherein said hyperbranched structure is devoid of a dendritic core having any symmetrical dendrons.

24. 14. The hyperbranched NO-donor compound of claim 13, wherein the hyperbranched compound does not contain aminoglycoside or glycoside units.

25. Each instance of any substitution is C １ -C ６ 14. The hyperbranched NO-donor compound of claim 13, selected from alkyl or -OH.

26. 2. The hyperbranched NO donor compound of claim 1, wherein the hyperbranched compound comprises a dendritic unit, a linear unit, and a terminal unit, the dendritic unit comprises a tertiary amine, the linear unit comprises a secondary amine, and the terminal unit comprises a primary amine.

27. 2. The hyperbranched NO-donor compound of claim 1, wherein the N-diazeniumdiolate exhibits intramolecular hydrogen bonding with the primary amine of the terminal unit.

28. 2. The hyperbranched NO-donor compound of claim 1, wherein the amine moiety modified by the N-diazeniumdiolate moiety is a secondary amine.

29. 2. The hyperbranched NO-donor compound of claim 1, wherein the compound contains at least about 2% NO by weight.

30. 2. The hyperbranched NO-donor compound of claim 1, wherein the composition comprises at least about 1 μmol of NO per mg of the compound as measured by real-time NO release in PBS (10 mM, pH 7.4, 37° C.).

31. 2. The hyperbranched NO-donor compound of claim 1, wherein the compound contains at least about 2 μmol of NO per mg of the compound as measured by real-time NO release in PBS (10 mM, pH 7.4, 37° C.).

32. 2. The hyperbranched NO-donor compound of claim 1, wherein the composition comprises greater than about 2 μmol of NO per mg of the copolymer as measured by real-time NO release in PBS (10 mM, pH 7.4, 37° C.).

33. The hyperbranched NO-donor compound of claim 1 , wherein the compound comprises a hydroxy moiety.

34. 34. The hyperbranched NO-donor compound of claim 33, wherein said hydroxy moiety is linked to an amine moiety via an alkyl moiety, such that said amine moiety is a secondary amine.

35. The hyperbranched compound has a molecular weight of about 2×10 as determined by size exclusion chromatography with multi-angle light scattering (SEC-MALS) detection. ３ gmol －１ ~Approx. 15×10 ３ gmol －１ 2. The hyperbranched NO donor compound of claim 1 having a weight average molecular weight (MW) of:

36. The hyperbranched compound has a molecular weight of about 3×10 as determined by size exclusion chromatography with multi-angle light scattering (SEC-MALS) detection. ３ gmol －１ ~About 10×10 ３ gmol －１ 2. The hyperbranched NO donor compound of claim 1 having a weight average molecular weight (MW) of:

37. The hyperbranched compound has a molecular weight of about 3×10 as determined by size exclusion chromatography with multi-angle light scattering (SEC-MALS) detection. ３ gmol －１ ~Approx. 6×10 ３ gmol －１ 2. The hyperbranched NO donor compound of claim 1 having a weight average molecular weight (MW) of:

38. 10. A method for preparing the hyperbranched NO-donor compound of claim 1, comprising: contacting an acrylate with a nucleophile to form a hyperbranched compound.

39. 39. The method of claim 38, wherein the acrylate is a monoacrylate, diacrylate, triacrylate, or tetraacrylate.

40. 39. The method of claim 38, wherein the nucleophile is a difunctional, trifunctional, or tetrafunctional molecule.

41. The nucleophile is H-R ｈ N (R ｅ ) R ｆ -(N(R ｅ )) ｎ - (R ｇ O-) ｍ R ｈ Contains -H, In the formula, R ｅ Each instance of is independently -H, optionally substituted C １ -C ６ an alkyl group or an optionally substituted polyether having 1 to 6 repeat units; R ｈ Each instance of １ -C ６ is an alkylene group, R ｆ and R ｇ independently represents an optionally substituted C １ -C ６ The method of claim 38, wherein the alkylene group is an alkylene group.

42. The nucleophile is H ２ N-((CH ２ ) ａ N.H. ｂ -H, H ２ N-((CH ２ ) ａ N.H. ｂ - (CH ２ ) ｃ H, H ２ N-((CH ２ ) ａ X ５ ) ｂ - (CH ２ ) ｃ H, and HX ５ - ((CH ２ ) ａ X ６ ) ｂ ((CH ２ ) ｃ X ７ ) ｄ - (CH ２ ) ｅ H, each instance of a, b, c, d, or e is independently selected from an integer from 0 to 10; X ５ , X ６ , and X ７ 39. The method of claim 38, wherein each instance of is independently selected from O, S, or NH.

43. The nucleophile is H ２ NCH ２ CH ２ N.H.C.H. ２ CH ２ N.H. ２ , H ２ NCH ２ CH ２ N.H.C.H. ２ CH ２ OH, and 【Chemistry 17】 40. The method of claim 38, comprising one or more of:

44. 39. The method of claim 38, wherein the acrylate comprises one or more of N,N'-methylenebis(acrylamide), ethylene glycol diacrylate, propanediol diacrylate, butanediol diacrylate, trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol triacrylate, glycerol propoxylate (1PO / OH) triacrylate, or trimethylolpropane propoxylate triacrylate.

45. The acrylate comprises one or more of the following structures: 【Chemistry 18】 In the formula, R １ , R ２ , and R ３ is independently -R ｂ N (R ｃ ) R ｄ -(N(R ｃ )) ｎ -R ｂ -, -R ｂ (OR ｄ -) ｎ O-R ｂ - and C １ -C ６ is selected from the group consisting of alkyl, R ｃ Each instance of is independently an NO donating moiety, —H, an optionally substituted C １ -C ６ is selected from an alkyl group, or an optionally substituted polyether having 1 to 6 repeat units; R ｂ is a single bond or an optionally substituted C １ -C ６ is an alkylene group, R ｄ is optionally substituted C １ -C ６ is an alkylene group, n is an integer selected from 0, 1, 2, 3, 4, 5, or 6; R ２ If R ｄ At least one example of the -C(O)-CH=CH ２ group, R ３ If R ｄ At least one example of the carbon atom is the -C(O)-CH=CH ２ The method of claim 38, comprising a group.

46. The method of claim 45, wherein the acrylate is N,N'-methylenebis(acrylamide).

47. 40. The method of claim 38, further comprising exposing the hyperbranched compound to a source of NO to provide the hyperbranched NO-donor compound.

48. 48. The method of claim 47, wherein the NO exposure step is carried out under alkaline conditions.

49. 39. The method of claim 38, wherein the molar ratio of nucleophile to acrylate is from about 2:1 to about 5:

1.

50. 39. The method of claim 38, wherein the molar ratio of amine to acrylate is from about 3:1 to about 4:

1.

51. 1. A method for reducing microbial contamination, comprising: contacting a surface contaminated with a plurality of microorganisms with a compound comprising the hyperbranched NO-donor compound of claim 1; The method, wherein said nitric oxide donor produces nitric oxide and induces damage to the membrane and / or DNA of said microorganisms, thereby reducing the number of viable microorganisms.

52. 52. The method of claim 51, wherein 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.

53. 52. The method of claim 51 , wherein the surface is an organic surface.

54. 52. The method of claim 51, wherein the surface is human skin or animal skin.

55. 52. The method of claim 51, wherein the surface is within the mouth.

56. 52. The method of claim 51, wherein said application does not induce skin irritation.

57. 52. The method of claim 51 , wherein the surface is an inorganic surface.

58. 58. The method of claim 57, wherein the inorganic surface is an exterior or interior surface of a medical device.

59. 60. The method of claim 58, wherein the appliance application is a dental appliance.

60. 52. The method of claim 51, wherein the microbial burden comprises drug-resistant bacteria.

61. 52. The method of claim 51, wherein the microbial load comprises one or more oral pathogens.

62. 52. The method of claim 51, wherein the microbial load comprises one or more of P. aeruginosa, S. aureus, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans.

63. 1. A method for treating and / or preventing dental caries, comprising: comprising contacting a surface of a patient's mouth contaminated with one or more oral pathogens with a compound of claim 1; The method wherein said nitric oxide donor produces nitric oxide and induces damage to the membrane and / or DNA of said pathogen, thereby reducing the number of viable pathogens.

64. 64. The method of claim 63, wherein the microbial load comprises one or more of P. aeruginosa, S. aureus, P. gingivalis, A. actinomycetemcomitans, A. viscosus, and / or S. mutans.

65. 2. Use of a compound according to claim 1 in the preparation of a medicament for reducing microbial contamination, said compound comprising: Contains nitric oxide-releasing hyperbranched polyaminoglycosides, The nitric oxide donor produces nitric oxide and induces damage to the membrane and / or DNA of the microorganisms, thereby reducing the number of viable microorganisms.

66. 66. The use of claim 65, wherein the compound is formulated to treat multiple microorganisms including one or more of viruses, gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria, molds, yeasts, fungi, and combinations thereof.

67. 66. The use of claim 65, wherein the compound is formulated for delivery to an organic surface.

68. 66. The use of claim 65, wherein the compound is formulated for delivery to human or animal skin.

69. 69. The use of claim 68, wherein the surface is in the mouth.

70. 66. The use of claim 65, wherein the compound is formulated for delivery to an inorganic surface.

71. 71. The use according to claim 70, wherein the surface is an exterior or interior surface of a medical device.

72. 72. The use of claim 71, wherein the device is a dental device.

73. A polyamidoamine composition comprising a hyperbranched copolymer of an amine and an acrylate, at least a portion of the amines being modified with N-diazeniumdiolate moieties.

74. 74. The polyamidoamine composition of claim 73, wherein the hyperbranched copolymer comprises dendritic units, linear units, and terminal units, the dendritic units comprise a tertiary amine, the linear units comprise a secondary amine, and the terminal units comprise a primary amine.

75. 74. The polyamidoamine composition of claim 73, wherein the N-diazeniumdiolate exhibits intramolecular hydrogen bonding with the primary amine of the terminal unit.

76. 74. The polyamidoamine composition of claim 73, wherein the portion of the amine modified by the N-diazeniumdiolate moiety is a secondary amine.

77. The amine is derived from a multifunctional amine monomer having the structure: H ２ P. １ -(NH)-A ２ -NH ２ In the formula, A １ and A ２ is independently selected from an alkyl moiety or hydrogen.

78. The acrylate is derived from the following monomers: 【Chemistry 19】 In the formula, A ３ and A ４ is independently selected from an alkyl moiety or hydrogen.

79. 74. The polyamidoamine composition of claim 73, wherein the composition comprises at least about 2 wt% NO.

80. 74. The polyamidoamine composition of claim 73, wherein the composition comprises at least about 1 μmol of NO per mg of the copolymer as measured by real time NO release in PBS (10 mM, pH 7.4, 37° C.).

81. 74. The polyamidoamine composition of claim 73, wherein the composition comprises at least about 2 μmol NO / mg of copolymer as measured by real time NO release in PBS (10 mM, pH 7.4, 37° C.).

82. 74. The polyamidoamine composition of claim 73, wherein the composition comprises greater than about 2 μmol NO / mg of copolymer as measured by real time NO release in PBS (10 mM, pH 7.4, 37° C.).

83. 74. The polyamidoamine composition of claim 73, wherein the copolymer further comprises a hydroxy moiety.

84. 84. The polyamidoamine composition of claim 83, wherein the hydroxy moiety is linked to an amine moiety through an alkyl moiety, thereby making the amine moiety a secondary amine.

85. 74. The polyamidoamine composition of claim 73, wherein the molar ratio of amine to acrylate is from about 2:1 to about 5:

1.

86. 74. The polyamidoamine composition of claim 73, wherein the molar ratio of amine to acrylate is from about 3:1 to about 4:

1.

87. The hyperbranched copolymer has a molecular weight of about 2×10 as determined by size exclusion chromatography with multi-angle light scattering (SEC-MALS) detection. ３ gmol －１ ~Approx. 15×10 ３ gmol －１ 74. The polyamidoamine composition of claim 73 having a weight average molecular weight (MW) of

88. The hyperbranched copolymer has a molecular weight of about 3×10 as determined by size exclusion chromatography with multi-angle light scattering (SEC-MALS) detection. ３ gmol －１ ~About 10×10 ３ gmol －１ 74. The polyamidoamine composition of claim 73 having a weight average molecular weight (MW) of

89. The hyperbranched copolymer has a molecular weight of about 3×10 as determined by size exclusion chromatography with multi-angle light scattering (SEC-MALS) detection. ３ gmol －１ ~Approx. 6×10 ３ gmol －１ 74. The polyamidoamine composition of claim 73 having a weight average molecular weight (MW) of

90. 74. The polyamidoamine composition of claim 73, wherein the hyperbranched copolymer of multifunctional amine and acrylate is soluble in water at a level of about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 50 mg / mL, or greater than about 100 mg / mL.

91. 74. The polyamidoamine composition of claim 73, wherein the acrylate is derived from a monomer selected from salts, esters, and conjugate bases of acrylic acid and its derivatives.

92. 74. The polyamidoamine composition of claim 73, wherein the acrylate is derived from a monomeric methacrylate.

93. 74. The polyamidoamine composition of claim 73 wherein the acrylate is derived from a monomer selected from the group consisting of methyl acrylate, ethyl acrylate, methyl methacrylate, acrylamide, ethyl methacrylate, 2-chloroethyl vinyl ether, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, butyl acrylate, butyl methacrylate, N-(2-hydroxypropyl)methacrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, N-(3-BOC-aminopropyl)methacrylamide, 2-aminoethyl methacrylate hydrochloride, 2-(tert-butylamino)ethyl methacrylate, n-iso-propylacrylamide, 2-methoxyethyl acrylate, n-ethyl methacrylamide, n-vinyl acetamide, 2-N-morpholinoethyl acrylate, methacryloyl-L-lysine, 2-(methylamino)ethyl acrylate, and 2-(methylamino)ethyl methacrylate.

94. 74. The polyamidoamine composition of claim 73, wherein the acrylate is derived from a diacrylate.

95. 95. The polyamidoamine composition of claim 94, wherein the diacrylate is selected from the group consisting of ethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, tricyclodecane dimethanol diacrylate, N-acryloxysuccinimide, N-(2-hydroxypropyl)methacrylamide, bis[2-(methacryloyloxy)ethyl]phosphate, diacrylamide, and N,N'-methylenebisacrylamide.

96. 74. The polyamidoamine composition of claim 73, wherein the amine is derived from diethylenetriamine.

97. 74. The polyamidoamine composition of claim 73, wherein the amine is derived from a monomer selected from the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, polyetheramines (e.g., JEFFAMINE®), and bis(hexamethylene)triamine.

98. The hyperbranched copolymer, upon release of NO, １ H NMR: (400MHz, CD ３ OD, δ): 2.22-2.90 (COCH ２ , N.H.H. ２ , and N.H. ２ CH ２ ), 3.15-3.58 (CONCH ２ ), 3.60 (CH ３ O). １３ C (600MHz, CD ３ OD, δ): 30-60(CH ２ and C.H. ３ ), 170-175 (C=O). FTIR (cm －１ ): 3308 (NH ２ ), 2957 (CH ２ ), 2848 (CH ２ 74. The polyamidoamine composition of claim 73, exhibiting properties consistent with: 1,647 (C=O), 1647 (C=O), and 1556 (NH).

99. 74. The polyamidoamine composition of claim 73, wherein the hyperbranched copolymer has a polydispersity index (PDI) greater than about 1.

1.

100. 74. The polyamidoamine composition of claim 73, wherein the hyperbranched copolymer has a polydispersity index (PDI) of 1.1 to 2.

101. 74. The polyamidoamine composition of claim 73, wherein the hyperbranched copolymer has a polydispersity index (PDI) of 1.5 to 1.

9.

102. 1. A polyamidoamine hyperbranched copolymer comprising one or more dendritic units comprising one or more tertiary amines, a plurality of terminal units comprising a plurality of primary amines, and a plurality of linear units comprising a plurality of secondary amines, at least a portion of the plurality of secondary amines being bonded to a plurality of N-diazeniumdiolate moieties.

103. At least a portion of the dendritic units comprise the following structure: 【Chemistry 20】 At least a portion of the plurality of linear units has the structure: 【Chemistry 21】 or a combination thereof.

104. 103. The polyamidoamine hyperbranched copolymer of claim 102, wherein at least a portion of the plurality of N-diazeniumdiolate moieties are stabilized by hydrogen bonding between an oxygen of the N-diazeniumdiolate moiety and a hydrogen of at least one of the plurality of primary amines.

105. 1. A method for delivering nitric oxide to a subject, comprising:

74. A method comprising administering to the subject an effective amount of the hyperbranched compound or polyamidoamine composition of claim 73.

106. 1. A method of treating a disease state, comprising:

13. A method comprising administering an effective amount of the hyperbranched compound or polyamidoamine composition of claim 1 to a subject in need of treatment, wherein the disease state is selected from the group consisting of gingivitis, 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.

107. 1. A method for making a polyamidoamine composition comprising the steps of:

1. A method comprising: combining a multifunctional amine with an acrylate monomer to form a reaction mixture in a suitable solvent; mixing the reaction mixture for a sufficient time to cause a substantial proportion of the multifunctional amine to react with the acrylate monomer to form a hyperbranched copolymer; heating the reaction mixture to complete polymerization and remove unreacted monomer to form a basic polyamidoamine composition; and mixing the basic polyamidoamine composition with gaseous NO at elevated pressure under basic conditions for a sufficient time to provide N-diazeniumdiolate moieties in the polyamidoamine composition.

108. 108. The method of claim 107, wherein the time sufficient for a substantial proportion of the multifunctional amine to react is greater than about 6 hours, from about 6 hours to about 2 weeks, from about 6 hours to 1 week, from about 12 hours to 5 days, or from about 1 day to about 3 days.

109. 108. The method of claim 107, wherein said heating of the reaction mixture to complete polymerization and remove unreacted monomer comprises heating at subatmospheric pressure.

110. 108. The method of claim 107, wherein said heating of the reaction mixture to complete polymerization and remove unreacted monomer comprises heating to a first temperature of about 50° C. to about 70° C. for a first period of about 30 minutes to about 2 hours, heating to a second temperature of about 90° C. to about 110° C. for a second period of about 30 minutes to about 4 hours, and heating to a third temperature of about 120° C. to about 150° C. for a third period of about 30 minutes to about 4 hours.

111. 108. The method of claim 107, wherein said heating of the reaction mixture to complete polymerization and remove unreacted monomer comprises heating to a first temperature of about 60° C. to about 60° C. for a first time period of about 1 hour, heating to a second temperature of about 100° C. for a second time period of about 2 hours, and heating to a third temperature of about 140° C. for a third time period of about 2 hours.

112. 108. The method of claim 107, wherein the polyfunctional amine is selected from the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, polyetheramines, and bis(hexamethylene)triamine.

113. 113. The method of claim 112, wherein the polyetheramine is JEFFAMINE®.

114. 114. The method of claim 113, wherein the JEFFAMINE® is selected from the group consisting of M-600, M-2005, M-1000, M-2070, D-230, D-400, D-2000, D-4000, ED-600 amine, ED-900 amine, ED-2003 amine, EDR-148 amine, EDR-176 amine, T-403 amine, T-3000 amine, T-5000 amine, THF-100 amine, THF-170 amine, XTJ568, XTA801, RFD-270, and XTJ-616.

115. The polyetheramine comprises one of the following structures: 【Chemical 22】 wherein g, h, i, and j are independently integers from 1 to 100; X １ is as defined elsewhere herein or optionally substituted C １－６ 114. The method of claim 112 or 113, wherein said alkyl is

116. 108. The method of claim 107, wherein the acrylate is selected from the group consisting of methyl acrylate, ethyl acrylate, methyl methacrylate, acrylamide, ethyl methacrylate, 2-chloroethyl vinyl ether, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, butyl acrylate, butyl methacrylate, N-(2-hydroxypropyl)methacrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, N-(3-BOC-aminopropyl)methacrylamide, 2-aminoethyl methacrylate hydrochloride, 2-(tert-butylamino)ethyl methacrylate, n-iso-propylacrylamide, 2-methoxyethyl acrylate, n-ethyl methacrylamide, n-vinyl acetamide, 2-N-morpholinoethyl acrylate, methacryloyl-L-lysine, 2-(methylamino)ethyl acrylate, and 2-(methylamino)ethyl methacrylate.

117. 108. The method of claim 107, wherein the suitable solvent is an alcohol or a mixture of alcohols.

118. 108. The method of claim 107, further comprising modifying the polyamidoamine composition with hydroxyl moieties by mixing the polyamidoamine composition with a hydroxy-containing compound.

119. 119. The method of claim 118, wherein the hydroxy-containing compound is an epoxide.

120. 119. The method of claim 118, wherein the epoxide is a propenoic oxide.

121. 108. The method of claim 107, wherein the combination of the multifunctional amine and the acrylate monomer comprises a molar ratio of amine to acrylate of from about 2:1 to about 5:

1.

122. 108. The method of claim 107, wherein the combination of the multifunctional amine and the acrylate monomer comprises a molar ratio of amine to acrylate of about 3:1 to about 4:

1.

123. 108. The method of claim 107, forming a hyperbranched compound or a polyamidoamine composition.

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