RELATED APPLICATIONS

This application claims the benefit of the priority of U.S. provisional application No. 63/351,757, filed June 13, 2022, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to novel polymers with antimicrobially-active moieties covalently incorporated into their molecular structures.

BACKGROUND

Antimicrobials are chemical compounds that inhibit the growth or development of microbial organisms through a variety of mechanisms that depend upon factors including mode of action, composition, degree of activity, and application. The use of the antimicrobial compounds leads to either death or arrested growth of the targeted microorganisms. Since their discovery in the early 1900s, antimicrobial agents have transformed the prevention and treatment of infectious diseases. They are currently employed across a very broad spectrum of applications.

In recent years, the prevalence of nosocomial infections, also known as “healthcare-associated infections” or “HAIs,” has had serious implications for both patients and healthcare workers. In general, nosocomial infections are more serious and dangerous than external, community-acquired infections because the pathogens in hospitals are more virulent and resistant to typical antibiotics. Nosocomial infections are responsible for about 20,000-100,000 deaths in the United States per year. About 5% to 10% of American hospital patients (about 2 million per year) develop a clinically significant nosocomial infection. These hospital-acquired infections (HAIs) are usually related to a procedure or treatment used to diagnose or treat the patient's illness or injury.

Antiseptics and disinfectants are extensively used in hospital and other health care settings for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and help in the prevention of nosocomial infections. In recent years, mounting concerns over the potential for microbial contamination and infection risks has increased the use of antimicrobial products that contain chemical biocides. In general, biocides have a broader spectrum of activity than antibiotics, and, while antibiotics tend to have specific intracellular targets, biocides may have multiple targets. Nonetheless, some conventional biocides typically either need to be ingested by the pathogen or be leached from a contact surface to be effective against microbes.

Ideally, antimicrobial agents should have proven history of use and display broad spectrum activity against various microorganisms without adversely affecting patients' health. The antimicrobial material, or other materials containing the antimicrobial agent, should be applicable to a configured medical or other health care product surface by commercially-viable manufacturing methods such as molding, extrusion, and all other thermoplastic methods of conversion or solvent-based processing, water-borne systems, and 100%-solids (crosslinkable) liquid. In addition, the antimicrobial additive should not interfere with physiochemical and mechanical properties of the treated material and must be compatible with existing formulations and manufacturing processes. Further, the integration of new antimicrobial properties in products should be economical. Bacterial infection is one of the common complications related to the use of medical devices. Advances in medical devices such as catheters, vascular access devices, peripheral lines, intravenous (IV) sites, drains, gastric feeding tubes, trachea tubes, stents, guidewires, pacemakers, and other implantable devices have enormously benefited the diagnostic and therapeutic practices in medical care. Unfortunately, however, bacterial infections are becoming one of the most serious complications related to the use of indwelling medical devices. Resistant strains continue to emerge, and more antibiotics are prescribed to treat infection caused by artificial implants.

Beyond the healthcare field, antimicrobial resistance is a massive and growing threat to human health worldwide. Portions of this problem have been attributed to the over-prescription and use of antibiotic drugs. Routine doses of antibiotics in feed are standard practice in the livestock industry, and up to half of all antibiotics prescribed to people are not needed or are not optimally effective as prescribed. Outbreaks of antibioticresistant infections typically emerge from factory farms, where healthy animals are routinely fed antibiotics to compensate for dangerous conditions, or from healthcare facilities, where antibiotic-resistant bacteria are a major risk. The widespread use of antiseptic and disinfectant products has prompted concerns about the development of microbial resistance, in particular cross-resistance to antibiotics.

It is widely recognized that warmer temperatures promote bacterial growth. A number of bacteria, like Staphylococcus aureus, thrive in temperatures between 4.5°C and 60°C (40° and 140° F) - a range often referred to as the “danger zone”. As temperatures around the world continue to rise due to climate change, bacteria are expected to reproduce at a faster rate, increasing the opportunity for mutation and transmission. Researchers estimate that a 10°C increase in average minimum temperatures across the U.S. could result in a 2.2% increase in Staphylococcus aureus antibiotic resistance. Climate change is also leading to increased environmental events that can promote growth and mutation of dangerous pathogens. For example, a massive seaweed die-off in the Atlantic Ocean has been found to support growth of Vibrio vulnificus bacteria, which can lead to necrotizing fasciitis.

Recent forecasts indicate that without the development of novel antimicrobial therapies, premature deaths resulting from infection by antibiotic-resistant pathogens could exceed ten million annually by 2050, overtaking cancer death projections. Unfortunately, little progress has been made in recent years in the development of new classes of antibiotics, and antibiotic-resistant strains of bacteria continue to emerge worldwide at an alarming rate. The mechanisms for antimicrobial resistance include inhibition of drug uptake, modification of the drug target, inactivation of the drug, and enhanced drug efflux. There is a critical need for broad-spectrum antibiotics that act independently of molecular pathways that frequently give rise to antimicrobial resistance. Several membrane-disrupting antimicrobial compounds have been developed, but their translation has been hampered by either complex synthesis, poor scalability, toxicity by hemolytic activity, or poor stability. The development of novel classes of safe, highly stable, and broadly-effective antibiotics is therefore an important goal in mitigating the burden of antibiotic resistance globally.

SUMMARY

To address the existing need for improved antimicrobials, disclosed herein are polyacrylamide-derived copolymers for use as broad-spectrum antibiotics. As a class of compounds, polyacrylamides are inexpensive to synthesize at scale and exhibit remarkable chemical stability, facilitating global access without the need for cold chain storage and distribution. The inventive copolymers exhibit stand-alone antimicrobial properties when the polymer is intact - they are not merely carriers of, or delivery vehicles that release, traditional antibiotics. As such, these copolymers are highly versatile with a wide range of antimicrobial applications in medicine, industry, agriculture, and much more. Without intending to be bound by theory, the library of copolymers disclosed herein is believed to function by way of a membrane disruption mechanism, without relying on specific protein transporters or pathways. There is some suggestion that an additional mechanism of cytosolic precipitation may be involved. Each copolymer involves various ratios of three distinct acrylamide-based monomers, including: (1) a cationic monomer to attract the polymer selectively to bacterial surfaces; (2) a hydrophilic carrier monomer to impart water solubility; and (3) a hydrophobic dopant monomer. Nonlimiting examples of cationic monomers include (3- acrylamidopropyl)trimethylammonium chloride (TMA), ((2- acrylamidoethyl)amino)(amino)methaniminium, and 3-(2-acrylamidoethyl)-l-methyl- lH-(3-methoxypropyl)acrylamide. Non-limiting examples of hydrophilic carrier monomers include 4-acryloylmorpholine (MORPH or Mo) and N-(3- methoxypropyl)acrylamide (MP AM or Mep). Non-limiting examples of hydrophobic dopant monomers include N-phenyl acrylamide (PHE or Phe), N-hexyl acrylamide, N- isopropyl acrylamide (NIP or Ni), and N-dodecylacrylamide (Do). Each polymer has a molecular weight below 30kDa. Evaluation of the antimicrobial activity of several of these polymers has revealed broad antimicrobial activity against gram-positive and gramnegative bacteria. They also exhibit selectivity for killing bacteria (gram-positive and gram-negative) over lysing red blood cells. Several demonstrate an HC50 (the concentration at which 50% of red blood cells are lysed) over twenty times greater than the MIC (minimum inhibitory concentration to prevent bacterial growth).

In one aspect, the inventive polyacrylamide-based copolymer includes a cationic monomer configured for attraction to a bacterial surface; a hydrophilic carrier monomer; and a hydrophobic dopant monomer; wherein the copolymer has an anti-microbial effect. In some embodiments, the hydrophilic carrier may be a water-soluble carrier monomer selected from the group consisting of 4-acryloylmorpholine (Mo) and N-(3- methoxypropyl)acrylamide (Mep); and the hydrophobic dopant is a functional dopant monomer selected from the group consisting of N-phenyl acrylamide (Phe), N- hexyl acrylamide, N-isopropylacrylamide (Ni), and N-dodecylacrylamide (Do), N- butyl acrylamide (Bam), N-(butoxymethyl)methacrylamide (Bmam), N- tertbutyl acrylamide (Tmb), N-octylacrylamide (Oct), and oleyl acrylamide (01am). The water-soluble carrier may have a weight percent of about 4% to about 10%, and the functional dopant monomer may have a weight percent of about 10% to about 31%. In some embodiments, the water-soluble carrier is Mep The Mep may have a weight percent of about 4%, and the copolymer has a low degree of polymerization (DP) and a minimum inhibitory concentration MIC) of from about 32 to about 96 pg/mL against Staphylococcus aureus. The functional dopant monomer may be selected from Do, Ni, and Phe and the MIC is about 32 pg/mL against Staphylococcus aureus. In other embodiments, the Mep may have a weight percent of about 10%, where the functional dopant monomer is Do, Tmb, Oct, Bmam, and 01am. The MIC may be from about 32 to about 512 pg/mL against Staphylococcus aureus and/or about 64 to about 192 pg/mL against Escherichia coli.

In other embodiments, the water-soluble carrier may be Mo. In some embodiments, the Mo may have a weight percent of about 4%, the copolymer has a low degree of polymerization (DP) and a minimum inhibitory concentration MIC) of about 32 pg/mL against Staphylococcus aureus. The functional dopant monomer may be selected from Do, Ni, and Phe. In other embodiments, the Mo may have a weight percent of about 10%, the functional dopant monomer is selected from Phe, Ni, and Do, and the MIC is from about 32 to about 192 pg/mL against Staphylococcus aureus.

In some embodiments, the cationic monomer is selected from the group consisting of (3-acrylamidopropyl)trimethylammonium chloride (Tma), ((2- acrylamidoethyl)amino)(amino)methaniminium (Api), and 3-(2-acrylamidoethyl)-l- methyl-lH-(3-methoxypropyl)acrylamide (Aeg). The copolymer may have a molecular weight below 30 kDa.

In some embodiments, a composition comprising the copolymer may be incorporated into a polymer used for fabrication of a molded product or fabric. In other embodiments, a composition comprising the copolymer may be incorporated into a film or coating applied to an object.

In another aspect, a polyacrylamide-based copolymer includes a cationic monomer selected from the group consisting of (3-acrylamidopropyl)trimethylammonium chloride (Tma), ((2-acrylamidoethyl)amino)(amino)methaniminium (Api), and 3-(2- acrylamidoethyl)-l-methyl-lH-(3-methoxypropyl)acrylamide (Aeg); a water-soluble carrier monomer selected from the group consisting of 4-acryloylmorpholine (Mo) and N- (3-methoxypropyl)acrylamide (Mep)hydrophilic carrier monomer; and a functional dopant monomer selected from the group consisting of N-phenyl acrylamide (Phe), N- hexyl acrylamide, N-isopropylacrylamide (Ni), and N-dodecylacrylamide (Do), N- butyl acrylamide (Bam), N-(butoxymethyl)methacrylamide (Bmam), N- tertbutyl acrylamide (Tmb), N-octyl acrylamide (Oct), and oleyl acrylamide (01am) hydrophobic dopant monomer; wherein the copolymer has an anti-microbial effect. The water-soluble carrier may have a weight percent of about 4% to about 10%, and the functional dopant monomer has a weight percent of about 10% to about 31%. In some embodiments, the water-soluble carrier is Mep. The Mep may have a weight percent of about 4%, the copolymer has a low degree of polymerization (DP) and a minimum inhibitory concentration MIC) of from about 32 to about 96 pg/mL against Staphylococcus aureus. The functional dopant monomer may be selected from Do, Ni, and Phe and the MIC is about 32 pg/mL against Staphylococcus aureus. In other embodiments, the Mep may have a weight percent of about 10%, where the functional dopant monomer is Do, Tmb, Oct, Bmam, and 01am. The MIC may be from about 32 to about 512 pg/mL against Staphylococcus aureus and/or about 64 to about 192 pg/mL against Escherichia coli.

In other embodiments, the water-soluble carrier may be Mo. In some embodiments, the Mo may have a weight percent of about 4%, the copolymer has a low degree of polymerization (DP) and a minimum inhibitory concentration MIC) of about 32 pg/mL against Staphylococcus aureus. The functional dopant monomer may be selected from Do, Ni, and Phe. In other embodiments, the Mo may have a weight percent of about 10%, the functional dopant monomer is selected from Phe, Ni, and Do, and the MIC is from about 32 to about 192 pg/mL against Staphylococcus aureus. The copolymer may have a molecular weight below 30 kDa.

In some embodiments, a composition comprising the copolymer may be incorporated into a polymer used for fabrication of a molded product or fabric. In other embodiments, a composition comprising the copolymer may be incorporated into a film or coating applied to an object.

Copolymers from the library can be incorporated into articles such as medical devices urinary catheters, percutaneous catheters, central venous catheters, vascular access devices, peripheral lines, IV sites, drug delivery catheters, drains, gastric feeding tubes, trach tube, contact lenses, orthopedic implants, neuro-stimulation leads, pacemaker leads, blood bags, wound care products, a personal protection devices, birth control devices, and more. Such polymers may likewise be made into articles such as surface coatings for a wide range of objects ranging from hospital equipment to product packaging to marine ships, which come into contacting with microorganisms and which benefit from the control of bacterial adhesion and bio-film formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of antimicrobial polymer composition, indicating monomer classes and examples; FIG. IB is a diagram of possible antibacterial mechanism of action through membrane-disruption; FIG. 1C shows one example a copolymer within the inventive library, and FIG. ID provides results of characterization of the example copolymer of FIG. 1C by ^X H NMR.

FIGs. 2A and 2B provide results of testing for antibacterial activity of 18 sample copolymers against E. coli (a gram -negative bacterial species) (FIG. 2A) and S. aureus (a gram-positive bacterial species) (FIG. 2B). This figure indicates that several polymers have broad antimicrobial activity and approach clinically-relevant MICs for S. aureus.

FIGs. 3A and 3B provide results of testing for antibacterial activity of 18 sample copolymers against. E. coli (a gram -negative bacterial species) (FIG. 3A) and S. aureus (a gram-positive bacterial species) (FIG. 3B). This figure indicates that several polymers have broad antimicrobial activity and approach clinically-relevant MICs.

FIG. 4 is a plot of measured hemolytic activity for twenty-four sample polymers within the inventive library.

DETAILED DESCRIPTION OF EMBODIMENTS

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present application; other, suitable methods and materials known in the art in some aspects of this disclosure are also used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entireties. In case of conflict, the present specification, including definitions, will control. When trade names are used herein, the trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.

As used herein, the terms "antimicrobial agent" or "antimicrobial agents" refer to chemicals or other substances that either kill or slow the growth of microbes. Among the antimicrobial agents in use today are antibacterial agents (which kill bacteria), antiviral agents (which kill viruses), antifungal agents (which kill fungi), and antiparasitic drugs (which kill parasites). The two main classes of antimicrobial agents are "antibiotics" and surface disinfectants, otherwise known as "biocides." Biocides and antibiotics are both antimicrobial agents.

The term "antibiotics" refer to a synthetic or naturally-derived organic chemical substance, used most often at low concentrations, in the treatment of infectious diseases of man, animals, and plants, which prevents or inhibits the growth of microorganisms. Examples of antibiotics include therapeutic drugs, like penicillin, while biocides are disinfectants or antiseptics like iodine. Antibiotics typically have a single target and a very specific mode of action, thus interacting with either receptors in the cellular membrane, or the metabolic or nucleic functions of the cell, causing inhibition of enzymatic or metabolic processes, whereas biocides have multiple targets and modes of action, which may include physical disruption and permanent damage to the outer cell membrane of a bacterial microbe.

As used herein, the term "containing" refers to a product generated according to any method of incorporating an antimicrobial agent into a desired item. This can include melt addition of the active agent to a polymer melt during extrusion and spinning of fibers and manufacturing of nonwoven materials used in making products; topical application methods that may or may not impart "sidedness" to the fabrics used in constructing the finished products; and other non-standard methods such as plasma treatment, electrostatic attachment, radiation surface graft copolymerizations using for example UV, gamma rays and electron-beam radiation sources, or the use of chemical initiation to produce graft copolymerized surfaces having anti-microbial activity, etc.

As used herein, the phrase "broad spectrum of microorganisms," is defined to include at a minimum Gram positive and Gram negative bacteria, including resistant strains thereof, for example methicillan-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) strains. Preferably, it is defined to include all bacteria (Gram +, Gram - and acid fast strains) and yeasts such as Candida albicans. Most preferably, it is defined to include all bacteria (Gram +, Gram -, and acid fast), yeasts, and both envelope and naked viruses such as human influenza, rhinovirus, poliovirus, adenovirus, hepatitis, HIV, herpes simplex, SARS, and avian flu. The term “polymer” as used herein refers to a substance or material consisting of repeating monomer subunits.

An “acrylamide monomer,” as used herein, refers to a monomer species that O possesses an acrylamide functional group . The term “acrylamide monomer” includes not only monomeric acrylamide, but derivatives of monomeric acrylamide. Examples of acrylamide monomers include, but are not limited to, acrylamide (AM), N- (3-methoxypropoyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), N- [tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA or Tma), N- isopropyl acrylamide (NIP or Ni), N-tert-butylacrylamide (TBA or Tmb), and N- phenyl acrylamide (PHE), ((2-acrylamidoethyl)amino)(amino)methaniminium, 3-(2- acrylamidoethyl)-l-methyl-lH-(3-methoxypropyl)acrylamide, N-hexyl acrylamide, and N-dodecylacrylamide (Do).

The term “polyacrylamide-based copolymer” refers to polymers that are formed from the polymerization of two or more monomer species, in which at least one of the monomer species possesses an acrylamide functional group (acrylamide monomer) and the monomers are structurally different. In some embodiments, the polyacrylamide-based copolymer is formed from the polymerization of two structurally different acrylamide monomers (two structurally different monomers that each possess an acrylamide functional group). The resulting copolymer can be an alternating copolymer wherein the monomer species are connected in an alternating fashion; a random copolymer, wherein the monomer species are connected to each other within a polymer chain without a defined pattern; a block copolymer, wherein polymeric blocks of one monomer species are connected to polymeric blocks made up of another monomer species; and graft copolymer, wherein the main polymer chain consists of one monomer species, and polymeric blocks of another monomer species are connected to the main polymer chain as side branches. In some embodiments, the polyacrylamide-based copolymers of the present disclosure are formed from the polymerization of a water-soluble carrier monomer and a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymers of the present disclosure are random copolymers. As defined herein, the term “water-soluble carrier monomer” or “hydrophilic carrier monomer” refers to an acrylamide monomer species that is the water-soluble species within the polyacrylamide-based copolymer. In some embodiments, the water- soluble carrier monomer is the predominant species within the polyacrylamide-based copolymer. In some embodiments, the water-soluble carrier monomer imparts aqueous solubility to the copolymer. The water-soluble carrier monomer within the polyacrylamide-based copolymer may also act to provide an inert barrier at the interface of an aqueous formulation to prevent protein-protein interactions. In some embodiments, the interface is an air-water interface. The interface may be an enclosure-water interface, including, but not limited to, a glass-water interface, a rubber-water interface, a plasticwater interface, or a metal-water interface. The interface may also be an oil-water interface. In some embodiments, the interface is an interface between a liquid and tubing. In some embodiments, the interface is an interface between a liquid and a catheter. In some embodiments, the enclosure-water interface is in a pump system. In some embodiments, the enclosure-water interface is in a closed-loop system. In some embodiments, the water- soluble carrier monomer is nonionic. Examples of water-soluble carrier monomers include, but are not limited to, acrylamide (AM), N-(3-methoxypropoyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), and N- hydroxyethyl acrylamide (HEAM).

The term “functional dopant monomer,” as used herein, refers to an acrylamide monomer species that has physicochemical properties (e.g., hydrophobicity, charge) different from those of the water-soluble carrier monomer. In some embodiments, the functional dopant monomer within the polyacrylamide-based copolymer promotes association of the polymers to an interface; such interfaces can include, but are not limited to, polymer-air-water interface interactions, polymer-protein interactions, polymerpeptide interactions, polymer-micelle interactions, polymer-liposome interactions, and polymer-lipid nanoparticle interactions. The functional dopant monomer can act as a stabilizing moiety to facilitate interactions with biomolecules, for example, proteins, peptides, antibodies, antibody-drug conjugates, nucleic acids, lipid particles, and combinations thereof (e.g., to prevent aggregation of the biomolecules). The functional dopant monomers can be further classified into hydrogen-bonding, ionic, hydrophobic, and aromatic monomers based on their chemical composition. The functional dopant monomers may be copolymerized at a lower weight percentages as compared to the water- soluble carrier monomers.

The term “polymerization” refers to the process in which monomer molecules undergo a chemical reaction to form polymeric chains or three-dimensional networks. Different types of polymerization reactions are known in the art, for example, addition (chain-reaction) polymerization, condensation polymerization, ring-opening polymerization, free radical polymerization, controlled radical polymerization, atom transfer radical polymerization (ATRP), single-electron transfer living radical polymerization (SET-LRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, nitroxide-mediated polymerization (NMP), and emulsion polymerization. In some embodiments, the copolymers of the present disclosure are prepared using RAFT polymerization.

The term “degree of polymerization” (“DP”) refers to the number of monomer units in a polymer. It is calculated by dividing the average molecular weight of a polymer sample by the molecular weight of the monomers. As defined herein, the average molecular weight of a polymer can be represented by the number-averaged molecular weight (Mn), the weight-average molecular weight (M _w ), the Z-average molecular weight (M _z ) or the molecular weight at the peak maxima of the molecular weight distribution curve (M _p ). The average molecular weight of a polymer can be determined by a variety of analytical characterization techniques known to those skilled in the art, for example, gel permeation chromatography (GPC), static light scattering (SLS) analysis, multi-angle laser light scattering (MALLS) analysis, nuclear magnetic resonance spectroscopy (NMR), intrinsic viscometry (IV), melt flow index (MFI), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and combinations thereof. Degree of polymerization can also be determined experimentally using suitable analytical methods known in the art, such as nuclear magnetic spectroscopy (NMR), Fourier Transform infrared spectroscopy (FT-IR) and Raman spectroscopy.

The term “amphiphilic” refers to chemical substances that possess both hydrophilic (water-loving, polar) and lipophilic (fat-loving, nonpolar) properties. Examples of common amphiphilic compounds include detergents, soaps, surfactants, lipoproteins, and phospholipids. In some embodiments, the amphiphilic substance is a charged species. In some embodiments, the amphiphilic substance is a neutral species. “Biologic molecule,” as used herein, refers to molecules such as proteins, nucleic acids, polysaccharides, and lipids.

Polyacrylamide-based copolymers

Provided in the present disclosure are polyacrylamide-based copolymers. In some embodiments, the polyacrylamide-based copolymers contain a water-soluble (hydrophilic) carrier monomer and a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer is amphiphilic.

In some embodiments, the polyacrylamide-based copolymer comprises a non-ionic water-soluble acrylamide monomer and a functional acrylamide dopant monomer selected from the group consisting of a hydrophobic functional acrylamide dopant monomer, an aromatic functional acrylamide dopant monomer, a hydrogen-bonding functional acrylamide dopant monomer, and an ionic functional acrylamide dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises a non-ionic water- soluble acrylamide monomer and the functional acrylamide dopant monomer is a hydrophobic functional acrylamide dopant monomer. In some embodiments, the copolymer comprises a non-ionic water-soluble acrylamide monomer and the functional acrylamide dopant monomer is an aromatic functional acrylamide dopant monomer. In some embodiments, the copolymer comprises a non-ionic water-soluble acrylamide monomer and the functional acrylamide dopant monomer is a hydrogen-bonding functional acrylamide dopant monomer. In some embodiments, the copolymer comprises a non-ionic water-soluble acrylamide monomer and the functional acrylamide dopant monomer is an ionic functional acrylamide dopant monomer.

The polyacrylamide-based copolymers of the present disclosure contain a water- soluble carrier monomer. In some embodiments, the water-soluble carrier monomer is non-ionic. The water-soluble carrier monomer may be selected from the group consisting of 7V-(3-methoxypropoyl)acrylamide (MP AM or Mep), 4-acryloylmorpholine (MORPH or Mo), 7V,7V-dimethylacrylamide (DMA), A-hydroxyethyl acrylamide (HEAM), and acrylamide (AM), or combinations thereof. In some embodiments, the water-soluble carrier monomer is selected from the group consisting of MP AM (Mep) and MORPH (Mo).

The polyacrylamide-based copolymers of the present disclosure also contain a functional dopant monomer. The functional dopant monomer may be selected from the group consisting of a hydrophobic functional acrylamide dopant monomer, an aromatic functional acrylamide dopant monomer, a hydrogen-bonding functional acrylamide dopant monomer, and an ionic functional acrylamide dopant monomer, or mixtures thereof.

In some embodiments, the functional acrylamide dopant monomer is a hydrophobic functional acrylamide dopant monomer. In some embodiments, the hydrophobic functional acrylamide dopant monomer is A-isopropylacrylamide (NIP orNi) or -tert-butyl acrylamide (TBA or Tmb). In some embodiments, the hydrophobic functional acrylamide dopant monomer is /'/-isopropylacrylamide (NIP or Ni). In some embodiments, the hydrophobic functional acrylamide dopant monomer is N-tert- butyl acrylamide (TBA or Tmb). In some embodiments, the functional acrylamide dopant monomer is an aromatic functional acrylamide dopant monomer. In some embodiments, the aromatic functional acrylamide dopant monomer is A-phenylacrylamide (Phe). In some embodiments, the aromatic functional acrylamide dopant monomer is N- hexyl acrylamide. In some embodiments, the aromatic functional acrylamide dopant monomer is N-dodecylacrylamide (Do). In some embodiments, the aromatic functional acrylamide dopant monomer is N-butyl acrylamide (Bam). In some embodiments, the aromatic functional acrylamide dopant monomer is N-(butoxymethyl)methacrylamide (Bmam). In some embodiments, the aromatic functional acrylamide dopant monomer is N-octylacrylamide (Oct). In some embodiments, the aromatic functional acrylamide dopant monomer is (Z)-N-(octadec-9-en-l-yl)acrylamide or oleyl acrylamide (01am).

In some embodiments, the polyacrylamide-based copolymer comprises a water- soluble carrier monomer selected from the group consisting of N-(3- methoxypropoyl)acrylamide (MP AM or Mep), 4-acryloylmorpholine (MORPH or Mo), N,N-dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), and acrylamide (AM); and a functional dopant monomer selected from the group consisting of N- phenyl acrylamide (Phe), N-hexylacrylamide, N-isopropylacrylamide (Ni), and N- dodecyl acrylamide (Do), N-butyl acrylamide (Bam), N-(butoxymethyl)methacrylamide (Bmam), N- tert-butyl acrylamide (Tmb), N-octylacrylamide (Oct), and (Z)-N-(octadec-9- en-l-yl)acrylamide or oleyl acrylamide (01am).

In some embodiments, the weight percentage (wt%) of hydrophilic carrier monomer is greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, or greater than 10%. In some embodiments, the weight percentage of hydrophilic carrier monomer is within a range of 3% to 12%, or within a range of 4% to 10%. In some embodiments the weight percentage of hydrophilic carrier monomer is about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

In some embodiments, the weight percentage (wt%) of hydrophobic dopant monomer is within a range of 10% to 40%, within a range of 11% to 35%, or within a range of 10% to 31%. In some embodiments, the weight percentage (wt%) of hydrophobic dopant monomer is greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. In some embodiments, the weight percentage (wt%) of hydrophobic dopant monomer is about 10%, about 13%, about 15%, about 20%, about 25%, about 30%, or about 31%.

Compositions containing a polyacrylamide-based copolymer.

Also provided are compositions containing the polyacrylamide-based copolymers described in the present disclosure. In some embodiments, the composition comprises a polyacrylamide-based copolymer of the present disclosure and a pharmaceutically acceptable excipient.

In addition to refrigerated transport in the early stages of the cold chain, maintaining proper transport and storage conditions during local distribution and once in patients’ hands presents a challenge in many parts of the world. As described herein, the addition of the polyacrylamide-based copolymers can preserve protein formulation integrity during even severe cold chain interruptions. In some embodiments, this enables a reduction in cold chain requirements for protein transportation and storage that are difficult to maintain in under-resourced environments. As disclosed herein, the polyacrylamide-based copolymers as formulation additives can improve cold chain resilience, thereby expanding global access to critical drugs and vaccines.

The present disclosure describes the synthesis and characterization of a library of copolymers comprising three distinct acrylamide-based monomers at a variety of weight ratios, molecular weights, and degree of polymerization. In general, the RAFT (reversible- addition-fragmentation chain transfer) polymerization methods employed for synthesis of the copolymers are described in U.S. Patent No. 6,291,620, to Moad, et al., which is incorporated herein by reference. The copolymers of the present disclosure exhibit antimicrobial activity against both gram-positive and gram-negative bacteria.

Each copolymer in the library is synthesized using varied ratios of acrylamide- based monomers selected from three distinct classes. FIG. 1A provides a simple diagram of an unnamed polymer synthesized based on monomers from members of the three classes, examples of which are shown in their respective groups. Class 1 : a cationic monomer, to attract the polymer selectively to bacterial surfaces; Class 2: a hydrophilic carrier monomer, to impart water solubility; and Class 3 : a hydrophobic dopant monomer, which may help permeabilize the bacterial membrane. The hypothesized membrane disruption approach is diagrammatically illustrated in FIG. IB. As previously noted, this is only one possible mechanism of action. Additional mechanisms, including cytosolic precipitation and others, may be involved.

The first class of monomers, cationic monomers, includes the following nonlimiting examples:

(3-acrylamidopropyl)trimethylammonium chloride (TMA or Tma), ((2-acrylamidoethyl)amino)(amino)methaniminium (Api), and

3-(2-acrylamidoethyl)-l-methyl-lH-(3-methoxypropyl)acryla mide (Aeg).

The second class of monomers, hydrophilic (water-soluble) carrier monomers, include the following non-limiting examples:

4-acryloylmorpholine (MORPH or Mo),

N-(3-methoxypropyl)acrylamide (MP AM or Mep) N,N-dimethylacrylamide (DMA or DMAM), and N-hydroxyethyl acrylamide (HEAM).

The third class of monomers, hydrophobic dopant monomers, include the nonlimiting examples of:

N-phenyl acrylamide (PEE or Phe),

N -hexyl aery 1 ami de

N-isopropyl acrylamide (NIP or Ni),

N-dodecylacrylamide (DAM or Do),

N-butylacrylamide (NTB or Bam),

N-(butoxymethyl)methacrylamide (Bmam),

N- tert-butyl acrylamide (TBA or Tmb),

N-octylacrylamide (Oct),

(Z)-N-(octadec-9-en-l-yl)acrylamide or oleyl acrylamide (01am).

Each polymer has a molecular weight up of to 30kDa. Library Design and Synthesis

An exemplary synthesis for one of the novel copolymers is illustrated in FIG. 1C based on monomers Tma, Mo, and Ni. As noted above, RAFT polymerization processes as art known in the art were used. Briefly, the monomers were dissolved in a solvent (dimethylformamide (DMF)) and added with an initiator (in this case, AIBN (azobisisobutyronitrile)) to a reactor vessel with water. Note that the water was degassed in N2 prior to addition of the monomers and initiator. The solution was heated under nitrogen to 65°C and maintained at temperature overnight. The resulting copolymer was analyzed via NMR, the results of which are shown in FIG. ID. Synthesis of the polymers within the library resulted in high conversion and high yield. The naming convention for the polymers included the degree of polymerization, the hydrophobic dopant monomer with its weight percent, and the hydrophilic carrier monomer with its weight percent, i.e., “DP-(Hydrophobic Monomer) _w t%(Hydrophilic Monomer)wt%.” High (“H”) DP was 115, while low DP was 70. Table 1 below lists the various polymers that were synthesized for evaluation and their respective yield percentages.

TABLE 1 Minimum inhibitory concentration (MIC) is the lowest concentration of an antibacterial agent expressed in mg/L (pg/mL) which, under strictly controlled in vitro conditions, completely prevents visible growth of the test strain of an organism. Antibacterial activity of 18 sample copolymers (Nos. 1-18 in Table 1) against Escherichia coli (a gram-negative bacterial species) and Staphylococcus aureus (a gram-positive bacterial species) was evaluated using standard MIC methods. FIGs. 2A and 2B show the results for A. coli (ATCC® 25922) and S. aureus (ATCC® 29213), respectively, where the vertical axis corresponds to the hydrophobic monomer (Ni, Phe, or Do), and the horizontal axis represents the hydrophilic monomer along with the DP. The relative degree of hydrophobicity is indicated along the right axis of each graph. Regions within the dashed lines had a high cationic density. The white regions within the plots indicate that no antibacterial activity was seen for the specified polymer. The MIC scale is provided at the bottom of the figures. This figure indicates that several of the polymers exhibited broad antimicrobial activity and approach clinically-relevant MICs for S. aureus. Table 2 summarizes the MIC test results shown in FIGs. 2A and 2B, represents the results for E. coli and “MIC + refers to S. aureus.

TABLE 2

FIGs. 3A and 3B provide test results for antibacterial activity of eight sample polymers in which the Class 2 monomer is Mep (wt%10) with a low DP. These polymers correspond to polymer numbers 2, 4, 6, and 19-23 in Table 1. Table 3 summarizes the MIC test results shown in FIGs. 3A and 3B, where “MIC represents the results for E. coll and “MIC + refers to S. aureus. These results indicate that several polymers have broad antimicrobial activity that approach clinically-relevant MICs.

TABLE 3

Beyond the antimicrobial activity, the toxicity towards mammalian cells is a key factor for the clinical applicability of antimicrobial polymers. Contrary to the microbial membrane, the nature of mammalian cell membrane is zwitterionic, and thus the cationic polymers preferably interact with the microbial membrane over the mammalian membrane. Nonetheless, the toxicity toward host mammalian cells is not always as low as desired, in particular in polymers with high hydrophobic content. The toxicity of polymers is typically evaluated by the hemolytic activity towards mammalian red blood cells (RBCs). This is a more susceptible situation in comparison with real blood, as the plasma proteins adsorbed to the membrane provide a protective layer that prevents contact with other compounds.

Using established procedures, hemotoxicity of synthesized polymers was evaluated on red blood cells (RBCs) from fresh human blood obtained from healthy donors The HCso value, the value at which 50% of RBCs are lysed upon exposure to the polymer within an incubation period of one hour was determined for 24 sample polymers. The results are plotted in FIG. 4 for the listed polymers. Except for L-D013M04 and L- TmbsiMepio, all polymers sampled exhibited remarkably low hemolytic activity, showing HC50 > 2000 pg/mL, demonstrating selectivity for bacterial cells over red blood cells.

Uses of antimicrobial polymers

The present disclosure relates to various applications of the antimicrobial polymer compositions and the polymer structures formed therefrom. The polymer composition can be incorporated into any of a variety of products for which prolonged protection from microbial infection and/or pathogenesis may be desirable. Copolymers from the library can be incorporated into articles such as medical devices urinary catheters, percutaneous catheters, central venous catheters, vascular access devices, peripheral lines, IV sites, drug delivery catheters, drains, gastric feeding tubes, trach tube, contact lenses, orthopedic implants, neuro-stimulation leads, pacemaker leads, blood bags, wound care products, a personal protection devices, birth control devices, and more. Such polymers may likewise be made into articles such as surface coatings for a wide range of objects ranging from hospital equipment to product packaging to marine ships, which come into contacting with microorganisms and which benefit from the control of bacterial adhesion and bio-film formation. Some exemplary products are described below.

The novel polymer composition may be used in the preparation of products intended for high-contact with a user. A high-contact product may be any product that is handled, e.g., touched, by a user or otherwise comes into contact with the user during conventional use. The polymer compositions may be utilized for high-contact products used in any setting.

In some embodiments, a disclosed polymer composition alone may be used to fabricate a high-contact product, i.e., a high-contact product may be entirely composed of a polymer composition. In some embodiments, a disclosed polymer composition is a component of the high contact product. For example, the polymer composition may form a layer, e.g., a surface coating, on the high-contact product.

As discussed above, the polymer compositions described herein demonstrate antimicrobial properties, and these properties may be surprisingly enhanced by certain characteristics of the polymer composition. For example, the use of a hydrophilic and/or hygroscopic polymer may increase the antimicrobial activity of the polymer composition. Moisture, e.g., moisture present on the skin, in sweat, or in saliva, typically facilitates pathogen transmission, and a hydrophilic and/or hygroscopic polymer composition may draw in pathogen-containing moisture. Thus, the inventive polymer compositions may be especially useful for high-contact products that come into contact with moisture during typical use. In particular, the moisture may be attracted to the composition, e.g., on a surface of the high-contact product, and the composition may then kill microbes contained therein. Thus, the disclosed polymer compositions may be used in forming (in whole or in part) high-contact products that greatly reduce transmission of a virus. For example, the polymer compositions may be especially useful for medical devices, e.g., catheters, masks, and air filters, or medical drapes, blankets, dressing, etc.

The following non-limiting examples are illustrative of a high-contact product. In some cases, the high-contact product may be a piece or portion of furniture, e.g., for use in an academic, business, or medical setting. In some cases, the high-contact product may be a piece or portion of a consumer product, e.g., consumer electronics. For example, the polymer composition may be used in the preparation of a housing or case for a mobile device, a component of computer, e.g., a housing, a display, a keyboard, or a mouse of a desktop computer or a laptop computer, a component of a kitchen or culinary item (e.g., a refrigerator, oven, stove, range, microwave oven, cookware, or cooking utensil), or a component of a personal hygiene product e.g., a toothbrush (or the fibers/bristles thereof), hair brush, comb, toilet seat, razor, or an air filter. In some applications, the high-contact product may be a piece or portion of medical equipment or tools. For example, the polymer composition may be used in the preparation of monitor equipment, e.g., a blood pressure monitor or an ultrasound probe, radiology equipment e.g., a portion of an MRI machine or a CT machine, a ventilator, or a patient transfer sheet.

In some cases, the high-contact product may be a piece or portion of a textile product. For example, the polymer composition may be used in the preparation of clothing, medical gowns, medical masks, medical drapes, patient transfer sheets, curtains, bedding, e.g., bedsheets and pillows, towels, suitcases, shoes, and much more.

In some cases, the high-contact product may be a molded article or a fabric-based product. For example, the novel polymer compositions may be used in the preparation of packaging, e.g., disposable or reusable food and/or liquid packaging, automotive parts or components, mechanical parts, toys, musical instruments, furniture, storage containers. Examples of fabric-based products include garments, athletic gear, wound dressings, tarps, and more.

Methods of making a high-contact product are not particularly limited, and conventional methods may be used. In some embodiments, for example, a hot melt polymerization, e.g., as discussed above with respect to fibers and nonwoven polymer structures, may be used to prepare the polymer composition, which may then be extruded and/or formed into the high-contact product.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.