Hydrogel compositions comprising synergistic antimicrobial peptides (AMPS) and methods of making same

Abstract

The present disclosure relates to antibacterial hydrogel compositions comprising a synergistic combination of antimicrobial peptides, and methods and kits for preparing the same.

Description

[0001] TITLE OF THE INVENTION

[0002] Hydrogel Compositions Comprising Synergistic Antimicrobial Peptides (AMPs) and Methods of Making Same

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 598.782. filed November 14, 2023, which is incorporated herein by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] The XML file named "370431 - 1053 WO I - Sequence Listing. xml" created October 7, 2024, comprising 7.28 Kbytes, is hereby incorporated by reference in its entirety.

[0007] BACKGROUND

[0008] Prosthetic implants and indwelling medical devices are used to replace, reinforce, or support tissues and organs, and their use has increased drastically over the past 50 years. While medical implants have significantly improved the quality of life and prolonged the lives of many patients, implants are at risk for microbial contamination. In the United States, about 2 million hospital-acquired infections (HAIs) occur yearly, and 50-70% of such infections result from indwelling medical devices. Such devices can become contaminated during the surgical procedure, or from bacterial colonization of the skin or other body sites of the patient or healthcare workers. As there are currently no specific markers of device-related infections, detection is typically delayed and eventual replacement of the implant is required, involving additional costly and high-risk surgeries.

[0009] Approximately 80% of all microbial infections in the human body involve formation of multicellular communities called biofilms. Bacterial biofilms formed on the surface of medical devices make the infection inherently more difficult to treat. Bacteria in a biofilm secrete an extracellular polymeric substance (EPS), which is made of polysaccharides, proteins, and extracellular DNA. Since bacteria are encased in a protective matrix, biofilms are extremely difficult to eliminate and exhibit decreased susceptibility to antibiotics. EPS comprises up to 85% of the volume of the biofilm and modulates the distribution of nutrients and oxygen throughout. This results in a decreased growth rate, which renders bacteria less susceptible to antibiotics. As biofilms mature, essential nutrients become limited and toxic byproducts accumulate, causing the surface layer of bacteria to dissociate and migrate to form new biofilms, which can cause recurrent infections in patients. Given the difficulty in detecting and eradicating biofilm-based infections, the best treatment strategy might be to prevent biofilms from forming in the first place.

[0010] There is thus a need in the art for hydrogel compositions comprising synergistic antimicrobial peptides and methods for the preparation thereof. The present disclosure addresses this need.

[0011] BRIEF SUMMARY

[0012] In one aspect, the disclosure provides a method for preparing an antibacterial hydrogel composition. In certain embodiments, the method comprises performing at least one bacterial growth inhibition assay using at least two antimicrobial peptides (AMPs) against at least one bacterial species. In certain embodiments, the method comprises identifying at least one combination of AMPs with synergistic bacterial growth inhibition activity. In certain embodiments, each combination of AMPs comprises at least a first AMP and a second AMP which have a synergistic molar ratio. In certain embodiments, the method comprises preparing a hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide or disaccharide monomers. In certain embodiments, a plurality' of the monomers thereof are independently covalently conjugated to an AMP present in the combination of AMPs with synergistic bacterial growth inhibition activity. In certain embodiments, the monomers are conjugated such that the hydrogel composition has a molar ratio of AMPs present in the at least one combination which is about equivalent to the synergistic molar ratio.

[0013] In another aspect, the disclosure provides a hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide or disaccharide monomers. In certain embodiments, a plurality of the monomers thereof are independently covalently conjugated to a first AMP or a second AMP. In certain embodiments, the first AMP comprises indolicidin (SEQ ID NO:2), or a modified derivative thereof. In certain embodiments, the second AMP comprises PIO (SEQ ID NO:3), or a modified derivative thereof. In certain embodiments, the first AMP and second AMP have a molar ratio in the hydrogel composition of about 1 :4.

[0014] In another aspect, the disclosure provides a kit for preparing hydrogel compositions comprising a synergistic combination of antimicrobial peptides (AMPs). In certain embodiments, the kit comprises at least two AMPs. In certain embodiments, the kit comprises functionalized derivatives of each of the at least two AMPs. In certain embodiments, the kit comprises at least one functionalized polysaccharide. In certain embodiments, the kit comprises eeagents suitable for covalent conjugation of the functionalized antimicrobial peptides and the at least one functionalized polysaccharide. In certain embodiments, the kit comprises instructional materials for identifying a bacterial species, identifying a synergistic combination of AMPs, and preparing a hydrogel composition comprising the synergistic combination of AMPs.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0017] FIG. 1 provides a schematic of an exemplary process for the identification and preparation of hydrogels comprising synergistic antimicrobial peptide (AMP) compositions including a checkerboard AMP screening (left), AMP hydrogel synthesis (center); and inoculation and viability analysis (right). Diagram of 96-well plate array used for high throughput screening of AMPs at different concentrations to determine the synergistic concentrations including representative inhibitory (ICioo), additive, and synergistic combinations for MRSA and MSSA. Chemical components used to synthesize AMP hydrogels. MRSA inoculation, incubation, staining, and imaging process diagram.

[0018] FIGs. 2A-2F depict combinatorial effects of soluble AMPs on methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains. Representative checkerboard assay of fractional inhibitory concentrations (FIC) for each AMP pair for MSSA (FIGs. 2A-2C) and MRSA (FIGs. 2D-2F) strains. FICs were calculated for each drug (concentration / MIC) and added together for all wells where no growth was observed. The red colored boxes indicate wells with no bacterial growth (ODeoo <0. 1) and green colored boxes indicate bacterial grow th. The box in the bottom right comer of each plate contains no drug and serves as a growth control. Yellow shaded boxes indicate additive interactions (FICI between 0.5- 1.0) and blue boxes indicate synergistic interactions (FICI < 0.5).

[0019] FIGs. 3A-3F depict examination of MRSA biofilm formation on different AMP- loaded hydrogels at different MICs. Qualitative images of representative AMP hydrogels at 0.5x, lx, and 2x MIC for tethered DD13-RIP (FIG. 3A), indolicidin (FIG. 3B), and PIO (FIG. 3C). Scale bars: 1 mm. Quantitative analysis of the cell viabilities for DD13-RIP (FIG. 3D), indolicidin (FIG. 3E), and PIO (FIG. 3F) AMPs. All the groups were normalized to the results of a control hydrogel with no AMP. The three asterisks indicate a significant decrease (p-value <0.001) in viability compared the control hydrogels with no AMP (Ox).

[0020] FIGs. 4A-4B depict synergistic effects of PIO and indolicidin on MRSA viability when seeded on different AMP-loaded hydrogels. Qualitative analysis showing representative hydrogels loaded with different multiples of the synergistic concentrations for indolicidin and PIO (3.125 pM and 12.5 pM, respectively) and stained for live (green) and dead (red) cells (FIG. 4A). Scale bar: 1 mm. Quantitative analysis of synergistic AMP hydrogel on biofilm viability (FIG. 4B). The three asterisks indicate a significant decrease (p-value <0.001) in viability compared the control hydrogels with no AMP (Ox).

[0021] FIGs. 5A-5B depict mammalian mesenchymal stem cell (MSC) viability on AMP- loaded hydrogels. Live / dead stain of MSCs seeded on AMP hydrogels at 2x MIC for each group (FIG. 5A). Scale bar: 1 mm. Quantification of the viability of mammalian cells seeded on top of each AMP hydrogel (FIG. 5B). There were no significant differences in viability observed. At most, there was one cell suspected to be dead for every 100 live cells counted.

[0022] FIG. 6 provides a bar graph showing elastic modulus of AMP hydrogels. 8 mm diameter. 1.5-2.0 mm gels were formed using 5% wt. HANor, 8 mM DTT, and 2 mM RGD or the AMPs at 2x MIC (n=6 per group). All groups were compared using an ANOVA test, each test group was then compared directly to the control group, and the Dunnett correction for multiple comparisons was applied. The stiffness was significantly higher for the P10 (p = 0.0357) and DDis-RIP (p = 0.0084) groups. This increase in stiffness corresponds to peptides with the longest amino acid chains and molecular weights. There are likely intramolecular forces or protein entanglements causing this trend.

[0023] FIGs. 7A-7F depict optical density' readings used to calculate combinatorial effects of soluble AMPs on MSSA and MRSA in FIGs. 2A-2F. The MIC for each individual peptide is highlighted in grey and corresponds to the lowest concentration which results in no growth (bold box). The red colored boxes indicate wells with no bacterial growth (ODgoo < 0.1) and green colored boxes indicate bacterial growth. The box in the bottom right comer contains no drug and sen es as a growth control, yellow shaded boxes indicate additive interactions and blue boxes indicate synergistic interactions based on FICI calculations.

[0024] FIG. 8 depicts a ’H-NMR spectrum of the HANor macromer.

[0025] FIGs. 9A-9J: investigation of MRSA cells interacting with AMP -hydrogels. FIGs. 9A-9E: representative high-resolution (60x magnification) images of hydrogels tethered with no AMP (FIG. 9A), DD13RIP (FIG. 9B), indolicidin (FIG. 9C), P10 at their soluble MIC (FIG. 9D), and indolicidin and P10 at their synergistic soluble MIC (FIG. 9E). Scale bars: 100 pm. FIGs. 9F-9J: quantitative spatial viability7analysis of MRSA as a function of hydrogel depth of hydrogels tethered with no AMP (FIG. 9F), DD13RIP (FIG. 9G). indolicidin (FIG. 9H), PIO at their soluble MIC (FIG. 91). and indolicidin and PIO at their synergistic soluble MIC (FIG. 9J).

[0026] FIGs. 10A-10B: schematic representations depicting preparation norbomene hyaluronic acid hydrogels amenable to peptide-functionalization with thiolated peptides (FIG. 10A) and methacrylated hyaluronic acid hydrogels amenable to peptide-functionalization with thiolated peptides (FIG. 10B).

[0027] FIGs. 1 1 A-l IF: functionalization of methacrylated hyaluronic acid hydrogels with adhesive peptide (z. e. thio-RGD). FIGs. 11A-11C: images depicting soft (FIG. 11 A), stiff (FIG. 11B), or in situ stiffened methacrylated hyaluronic hydrogels functionalized with an adhesive peptide (thio-RGD). FIGs. 1 ID- 1 IF: bar graphs depicting area (FIG. 1 ID), aspect ratio (FIG. HE), and circularity (FIG. 1 IF) of soft, stiff, and in situ stiffened methacrylated hyaluronic hydrogels functionalized with an adhesive peptide (thio-RGD).

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0030] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of ‘'about 0.1% to about 5%” or '‘about 0. 1% to 5%” should be interpreted to include notjust about 0.1% to about 5%, but also the individual values (e.g, 1%, 2%, 3%, and 4%) and the sub-ranges (e.g, 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y. or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0031] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as '‘A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0032] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0033] Description

[0034] Staphylococcus aureus is an opportunistic pathogen that lives on surfaces (such as the skin) and can cause serious infections once inside the body. While antibiotics effectively kill bacteria, there are a growing number of infections with antibiotic-resistant strains. Antimicrobial peptides (AMPs) are part of the innate immune system and can eliminate pathogens including bacteria, fungi, and viruses, and are a promising alternative to antibiotics. Although studies have reported that AMP-functionalized hydrogels can prevent bacterial adhesion and biofilm formation, these materials generally consist essentially of one AMP at an arbitrary concentration, and AMP dosing and the combined effects of multiple AMPs are not well understood in the context of functionalized hydrogels.

[0035] In one aspect, the present disclosure describes the synthesis and evaluation of hydrogel compositions comprising synergistic combinations of AMPs. Exemplary AMPs with different antibacterial properties were synthesized and the soluble minimum inhibitory concentrations (MICs) of each AMP against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) were determined. Unexpectedly, it was found that hydrogels with immobilized AMPs at their MIC (DD13RIP 27.5 pM; indolicidin 43.8 pM; PIO 120 pM) were effective in preventing MRSA adhesion and biofilm formation. Checkerboard AMP screens identified synergy between indolicidin (3.1 pM) and PIO (12.5 pM) based on soluble fractional inhibitory concentration indices (FICIs) against MRSA, and hydrogels formed with these AMPs at half of their synergistic concentrations (total peptide concentration, 7.8 pM) were highly effective in killing MRSA. Mammalian cells cultured atop these hydrogels displayed high viabili ty, demonstrating that the AMP hydrogels developed are biocompatible and selectively eradicate bacteria based on soluble checkerboard screening data.

[0036] Definitions

[0037] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%. within 5%. or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0038] The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=C=CH2, -CH=CH(CH.3), -CH=C(CHs)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl. pentadienyl, and hexadienyl among others.

[0039] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, penty loxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxy ethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0040] The term “alkynyl” as used herein refers to straight and branched chain alky l groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms. 2 to about 20 carbon atoms, or from 2 to 12 carbons or. in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - CACH. -CAC1CH3). -C =C(CH2CH3). -CH2OCH, -CHiCACTCH,). and -CH2C^C(CH2CH3) among others.

[0041] The term “alkyl” as used herein refers to straight chain and branched alkyd groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or. in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyd groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-buty l, sec-butyl, t-butyl, neopenty l, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0042] The term “alkylene” or “alkylenyl” as used herein refers to a bivalent saturated aliphatic radical (e.g, -CEE-, -CH2CH2-, and -CH2CH2CEI2-, inter alia). In certain embodiments, the term may be regarded as a moiety' derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from the same (e.g., - CH2-) different (e.g, -CH2CH2-) carbon atoms.

[0043] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g, the formula Nigro up )3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like: and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

[0044] The terms “antibacterial” or “antimicrobial” are used interchangeably herein to refer to a substance or composition capable of killing or inhibiting the growth of microbes, prevents the development of microbes, and / or inhibits the pathogenic action of microbes such as viruses, fungi, and bacteria.

[0045] The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl. heptalenyl. biphenyl, indacenyl. fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl. biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, ar l groups contain about 6 to about 14 carbons in the ring portions of the groups. Ary l groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof. Use of the term “aryl” in combination with another term (e.g, iodide, chloride, boronic acid, and magnesium halide, inter alia), indicates that the and group is substituted at one or more positions with the substituent defined by the term used in the combination. For example, an aryl chloride indicates that the ary l is substituted with at least one chloride.

[0046] The term “assay” as used herein refers to a system or experiment, wherein the effect of potential of one or more substances (e.g. antimicrobial peptide) on living organisms (e.g, bacteria) was tested.

[0047] The term “checkerboard assay” as used herein refers to a laboratory' technique used to assess the interactions between two or more substances (e.g, antimicrobial peptides or AMPs) to determine if their combined effect (e.g, bacterial growth inhibition) is greater than (z.e., synergistic or additive), equal to, or less than (z. e. , antagonistic) the sum of their individual effects. In certain non-limiting embodiments, the checkerboard assay can be used to determine the synergistic effect of two or more AMPs (e.g. , two-dimensional, three- dimensional, or four-dimensional checkerboard). Generally, checkerboard assays according to the methods described herein comprise evaluation of antimicrobial activity of each AMP individually. In non-limiting embodiments, the checkerboard assay comprises a two- dimensional (2D) checkerboard assay. In such embodiments, bacterial strains of interest are grown in media having varied concentrations of each of the two antimicrobial peptides, and their growth is measured. In certain embodiments, the varied concentration combinations are achieved by independent serial dilution of samples comprising each respective antimicrobial peptide in opposing directions (e.g., antimicrobial peptide 1 serially diluted horizontally and antimicrobial peptide 2 serially diluted vertically). In certain embodiments, an output (e.g.. bacterial growth) is measured.

[0048] The term “click chemistry” refers to chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together (see H. C. Kolb, M. G. Finn and K. B. Sharpless (2001). Click Chemistry': Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 40 (11): 2004-2021). Click chemistry does not refer to a specific reaction, but to a concept including, but not limited to, reactions that mimic reactions found in nature. Click chemistry reactions can be modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, exhibit a large thermodynamic driving force to favor a reaction with a single reaction product, and / or can be carried out under physiological conditions. In certain embodiments, a click chemistry reaction exhibits high atom economy, can be carried out under simple reaction conditions, use readily available starting materials and reagents, uses no toxic solvents or uses a solvent that is benign or easily removed (preferably water), and / or provides simple product isolation by non-chromatographic methods (crystallization or distillation). In certain embodiments, the click chemistry reaction is a thiol-ene reaction. In certain embodiments, the click chemistry reaction is a [2+3] dipolar cycloaddition. In certain embodiments, the click chemistry reaction is a Diels-Alder cycloaddition.

[0049] The term "‘click chemistry handle” refers to a reactant, or a reactive group, that can partake in a click chemistry reaction. Exemplary' click chemistry handles are demonstrated in U.S. Patent Publication No. US2013 / 0266512, which is incorporated by reference herein. For example, a strained alkyne, e.g., a cyclooctyne, is a click chemistry handle, since it can partake in a strain-promoted cycloaddition. In general, click chemistry reactions require at least two molecules comprising click chemistry handles that can react with each other. Such click chemi sin handle pairs (e.g., complementary pairs) that are reactive with each other are sometimes referred to herein as partner click chemistry handles or complementary click chemistry pair. For example, an azide is a partner click chemistry handle to a cyclooctyne or any other alkyne. Exemplary click chemistry handles suitable for use according to some embodiments are described herein. In certain embodiments, the click chemistry partners are a thiol and an alkene. In certain embodiments, the click chemistry partners are a conjugated diene and an optionally substituted alkene. In other embodiments, the click chemistrypartners are optionally substituted tetrazine (Tz) and optionally substituted trans-cyclooctene (TCO). Tz and TCO react with each other in a reverse-electron demand Diels-Alder cycloaddition reaction (see e.g., Blackman et al., “The Tetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demand Diels-Alder Reactivity .'’ J. Am. Chem. Soc. 2008; 130, 13518-13519). In other embodiments, the click chemistry partners are an optionally substituted alkyne and an optionally substituted azide. For example, a difluorinated cyclooctyne, a dibenzocyclooctyne, a biarylazacyclooctynone, or a cyclopropyl-fused bicyclononyne can be paired with an azide as a click chemistry pair. In other embodiments, the click chemistry partners are reactive dienes and suitable tetrazine dienophiles. For example. TCO. norbomene, or biscyclononene can be paired with a suitable tetrazine dienophile as a click chemistry- pair. In yet other embodiments, tetrazoles can act as latent sources of nitrile imines, which can pair with unactivated alkenes in the presence of ultraviolet light to create a click chemistry pair, termed a “photo-click” chemistry- pair. The click chemistry pair may also be a cysteine and a mal eimide. For example, the cysteine from a peptide (e.g., GCGGG) may be reacted with a maleimide that is associated with a chelating agent (e.g., NOTA). For two molecules to be conjugated via click chemistry', the click chemistry handles of the molecules have to be reactive with each other, for example, in that the reactive moiety7of one of the click chemistry7handles can react with the reactive moiety7of the second click chemistry7handle to form a covalent bond. Such reactive pairs of click chemistry7handles are well known to those of skill in the art.

[0050] The term “conjugated” as used herein refers to a covalent interaction between two entities, e.g.. molecules, compounds, or a combination thereof, which may comprise one or more linkers.

[0051] The term “contacting” as used herein refers to bringing at least two different compounds and / or compositions in physical proximity7as to allow physical and / or chemical interaction of said compounds. A non-limiting example of facilitating contact include immersion.

[0052] The term “crosslinked” as used herein, in the context of functionalized polysaccharide polymers, refers to the process of forming covalent bonds between either: (1) two reactive moieties present on the same polysaccharide polymer (e.g., dithiol linker reacting with a first alkene of a first monomer and second monomer of the same polysaccharide, thereby forming a macrocycle); or two reactive moieties present on different polysaccharide polymers (e.g., dithiol linker reacting with a first alkene of a first polysaccharide and a first alkene of a second polysaccharide, thereby linking two separate polysaccharides).

[0053] The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5. 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bomyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2.2-, 2,3-. 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbomyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.

[0054] The term “disaccharide” as used herein refers to a saccharide comprising two monosaccharide units, which may be identical or different. Examples of disaccharides comprise hyaluronic acid (N-Acetyl-D-glucosamine and D-glucoronic acid), lactose (Gal- bl,4-Glc), lacto-N-biose (Gal-bl.3-GlcNAc). N- acetyllactosamine (Gal-bl,4-GlcNAc), LacDiNAc (GalNAc-bl,4-GlcNAc), N-acetylgalactosaminylglucose (GalNAc-bl,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and fucopyranosyl- (l-4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc).

[0055] The term “fractional inhibitory concentration index” (FICI) refers to a value that compares the ratio of inhibitory concentration of a combination with that of individual agents separately. The formula used to calculate FICI is described elsewhere herein. The FIC Index (FICI) value is used to determine whether paired combinations of antimicrobial agents can exert inhibitory' effects against a tested organism that are more or less than the sum of their effects alone. In theory, FICK1.0 indicates synergy and FICI>1.0) indicates antagonism. However, a more conservative calculation has been recommended such that FICI data may be interpreted as “synergy ” for a FICI<0.5, “antagonism” for a FICI>4.0 and “no interaction” interaction for an FICI>0.5-4.0. (Odds, F. C., Synergy, antagonism, and what the checkerboard puts between them, Journal of Antimicrobial Chemotherapy (2003) 52, 1. As used herein, the FICI is interpreted as follows: FICI < 0.5 = synergy; FICI > 0.5 - < 1 = additive effect; FICI > 1 - < 4 = indifference; and FICI > 4 = antagonism.

[0056] The term “functionalized” as used herein, in the context of monomers and / or polymers, refers to a monomer or polymer comprising one or more reactive functional groups suitable for covalent modification upon contact with a suitable compound or composition under suitable reaction conditions. Non-limiting examples of “functionalized” polymers include hyaluronic acid amidated with 5-norbomene-2 -methylamine, wherein the resultant norbomene may react with any of a number of alkene reactive groups, including but not limited to thiols, under conditions including but not limited to irradiation.

[0057] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0058] The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, polyhalo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1 -dichloroethyl, 1,2-dichloroethyl, l,3-dibromo-3,3- difluoropropyl. perfluorobutyl, and the like.

[0059] The term “heteroalkylene” or “heteroalkylenyl” as used herein refers to a bivalent saturated aliphatic radical comprising one or more heteroatoms, wherein the heteroatom may be internal or position at a terminus of the bivalent radical (e.g.. -OCH2-, -SCH2-, -NHCH2-, - CH2OCH2-, -CH2CH2CH2O-, and -SCH2CH2CH2S-, inter alia). In certain embodiments, the term may be regarded as a moiety derived from an alkene by opening of the double bond or from a heteroalkane by removal of two hydrogen atoms from different carbon and / or heteroatoms (e.g. , -CH2OCH2- and -OCH2-).

[0060] The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to. N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl. pyrazolyl, triazolyl, tetrazolyl. oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl. indolyl. azaindolyl. indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroary l groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

[0061] Additional examples of ary l and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl. N- hydroxytriazolyl, N-hydroxyimidazolyL anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2 -thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydry 1, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl). triazolyl (1,2.3-triazol-l-yl, l,2,3-triazol-2-yl 1.2,3-triazol-4-yl. l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2 -thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5 -pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1 -isoquinolyl, 3-isoquinolyl, 4-isoquinolyL 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl. 4-benzo[b] furanyl. 5-benzo[b]furanyl. 6-benzo[b] furanyl. 7- benzofb] furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3- dihydro-benzo[b] furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl),

[0062] 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b] furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl). 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl). 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl. 2-indolyl,

[0063] 3-indolyl, 4-indolyl, 5-indolyl. 6-indolyl, 7-indolyl), indazole (1-indazolyl. 3-indazolyl,

[0064] 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5 -benzothiazolyl, 6-benzothiazolyl,

[0065] 7-benzothiazolyl), carbazolyl (1-carbazolyL 2-carbazolyl. 3-carbazolyl. 4-carbazolyl), 5H-dibenz[b,f|azepine (5H-dibenz[b,f| azepin- 1-yl, 5H-dibenz[b,f|azepine-2-yl, 5H-dibenz[b,f|azepine-3-yl, 5H-dibenz[b,f|azepine-4-yl, 5H-dibenz[b,f|azepine-5-yl),

[0066] 10.1 l-dihydro-5H-dibenz[b,f| azepine (10.1 l-dihydro-5H-dibenz[b.f]azepine-l-yl,

[0067] 10.1 l-dihydro-5H-dibenz[b.f|azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f|azepine-3-yl.

[0068] 10.1 l-dihydro-5H-dibenz[b,f|azepine-4-yl, 10,1 l -dihydro-5H-dibenz[b,f|azepine-5-yl), and the like.

[0069] The term “hydrogel” as used herein refers to a crosslinked polymeric material which is not water-soluble and can contains at least 10% by weight of water within its polymer matrix when fully hydrated.

[0070] The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3are all the same, where X1, X2, and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations.

[0071] The term “linker” as used herein refers to a chemically inert region between two linked molecules.

[0072] The term “minimum inhibitory7concentration” or “MIC” as used herein refers to the lowest concentration of the corresponding antimicrobial agent required to prevent or reduce microbial growth. In certain embodiments, microbial growth is assessed by measurement of optical density at 600 nm (ODeoo), wherein ODeoo < 0.1 means no growth..

[0073] The term “monomer” as used herein refers to a molecular compound that takes its usual definition in the art and thus can chemically bond to other monomers to form a polymer. Non-limiting exemplary monomers of polysaccharides include monosaccharides and disaccharides.

[0074] The term “monosaccharide” as used herein refers to a sugar having a five- or sixmembered carbon backbone (e.g., pentose or hexose). Examples of monosaccharides include, but are not limited to, glucose (Glc), galactose (Gal), mannose (Man), glucuronic acid (GlcA), and iduronic acid (IdoA). Monosaccharides also include hexoses substituted with hydroxy groups, oxo groups, amino groups, acetamido groups, and other functional groups. “Deoxy” monosaccharides refer to monosaccharides having carbon atoms one or more carbon atoms in the hexose backbone having only hydrogen substituents. Monosaccharides also include, but are not limited to, glucosamine (2-amino-2-deoxy -glucose; GlcN), N- acetylglucosamine (2-acetamido-2-deoxy-glucose; GlcNAc). galactosamine (2-amino-2- deoxy-galactose; GalN;), N-acetylgalactosamine (2-acetamido-2-deoxy -galactose; GalNAc), mannosamine (2-amino-2-deoxy-mannose; ManN), and N-acetylmannosaminc (2-acetamido- 2-deoxy-mannose; ManNAc.

[0075] The term “multiwell plate” as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to any plate-shaped device configured to permanently or temporarily store a small amount of a fluid. A small amount in this respect is to be understood as a quantity of fluid in the range of nL to mL such as 10 nL to 100 mL, preferably 0.1 pL to 10 mL and even more preferably 0. 1 pL to 5 mL. Basically, the design of the multiwell plate may depend on the respective application of the laboratory instrument. The multiwell plate may be designed as a device for storing a single fluid sample or a plurality’ of fluid samples. Similarly, the geometry of a respective storage area of the multiwell plate may depend on the respective application of the multiwell plate. The storage area may be designed as a well, channel, depression, recess or the like. For example, the multiwell plate may comprise a plurality of wells. The term “multiwell plate” as used herein may refers to a flat plate with multiple “wells” used as small test tubes. Such a multiwell plate is also known as microtiter plate. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories. A multiwell plate typically has 6, 24. 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix. Some microplates have even been manufactured with 3456 or 9600 wells, and an “array tape” product has been developed that provides a continuous strip of microplates embossed on a flexible plastic tape. Each well of a microplate typically holds somewhere between tens of picolitres to several mL of liquid. Wells can be either circular or square.

[0076] The term “nosocomial infection” as used herein refers to an infection arising from a medical procedure performed in a hospital, or in or outside a hospital. Non-limiting, exemplary nosocomial infections include sepsis (e.g, bloodstream infection), surgical site infection, or pneumonia.

[0077] The term “polymer” may include, according to some embodiments, any molecule comprising repeating structural units connected to each other, typically, by covalent chemical bonds. The term “polymer” may include, according to some embodiments, a homopolymer (which is a polymer derived from one monomer species), a copolymer (which is a polymer derived from two (or more) monomeric species) or a combination thereof. A polymer, as referred to herein, may include a mixture of polymers. A polymer, as referred to herein, may include linear and / branched polymers which consist of a single main chain with one or more polymeric side chains.

[0078] The term “polypeptide” as used herein refers to a polymer of amino acids residues of at least eight or more amino acids bonded via covalent peptide bonds. The polypeptide can be linear or branched and may comprise naturally occurring and / or amino acid analogs.

[0079] The term “polysaccharide” as used herein refers to polymeric carbohydrate structures, formed of repeating units (e.g., mono- or di-saccharides) joined together by glycosidic bonds. These structures may be linear, but may also contain various degrees of branching. The polysaccharide may be a homo- or heteropolysaccharide. The polysaccharide may contain non-carbohydrate units. Examples of polysaccharides include, but are not limited to, hyaluronic acid, amylose, amylopectin, pectins, glycogen, agar, alginate, carrageenans, chitin, beta-glucans, dextrins, carboxymethylcellulose, carboxyethylcellulose, hydroxypropylcellulose, methylcellulose and combinations thereof. One exemplary combination of the afore-mentioned polysaccharides is starch which consists of amylopectin and amylose. Usually polysaccharides comprise more than 100 monosaccharide or disaccharide units, such as 200 to 25,000. The molecular weight of the polysaccharides can be greater than 20,000. for example 20,000 to 1.000,000 g / mol or even higher.

[0080] The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxygroups. alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, 0C(0)N(R)2, CN, NO, NO2, ONO2, azido, CFs, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R. C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR. OC(O)R. C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)O- 2N(R)C(0)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(0)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example. R can be hydrogen, (Ci- C100) hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

[0081] The term “synergy” or “synergistic” as used herein refers to the interaction of discrete antimicrobial agents in which the total antimicrobial effect is greater than the sum of the effects of the antimicrobial agent alone. Conversely, the term “antagonism” is intended to refer to an interaction of antimicrobial agents in which the total antimicrobial effect is less than the sum of the effects of the antimicrobial agents alone.

[0082] Methods

[0083] In one aspect, the present disclosure provides a method for preparing an antibacterial hydrogel composition.

[0084] In certain embodiments, the method comprises performing at least one bacterial growth inhibition assay using at least two antimicrobial peptides (AMPs) against at least one bacterial species. In certain embodiments, the method comprises identifying at least one combination of AMPs with synergistic bacterial growth inhibition activity. In certain embodiments, each combination of AMPs comprises at least a first AMP and a second AMP which have a synergistic molar ratio. In certain embodiments, the combination further comprises a third AMP. In certain embodiments, the combination further comprises a fourth AMP. In certain embodiments, the combination further comprises a fifth AMP.

[0085] In certain embodiments, the method comprises preparing a hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide and / or disaccharide monomers.

[0086] In certain embodiments, a plurality of the monomers thereof are independently covalently conjugated to an AMP present in the combination of AMPs with synergistic bacterial growth inhibition activity. In certain embodiments, a plurality of the monomers are crosslinked with monomers of the same or another polysaccharide.

[0087] In certain embodiments, the monomers are conjugated such that the hydrogel composition has a molar ratio of AMPs present in the at least one combination which is about equivalent to the synergistic molar ratio.

[0088] In certain embodiments, the bacterial growth inhibition assay comprises a two- dimensional (2D) checkerboard assay. In certain embodiments, the bacterial growth inhibition assay comprises a three-dimensional (3D) checkerboard assay. In certain embodiments, the bacterial growth inhibition assay comprises a four-dimensional (4D) checkerboard assay. In certain embodiments, the bacterial growth inhibition assay comprises a five-dimensional (5D) checkerboard assay.

[0089] In certain embodiments, the bacterial growth inhibition assay comprises adding a solution comprising the first AMP to a first column of a multiwell assay plate and performing a horizontal serial dilution thereof.

[0090] In certain embodiments, the bacterial growth inhibition assay comprises adding a solution comprising the second AMP to a first row' of the multi well assay plate and performing a vertical serial dilution thereof. In certain embodiments, the bacterial growth inhibition assay comprises adding a medium comprising the at least one bacterial species to each well of the multiwell assay plate. In certain embodiments, the bacterial growth inhibition assay comprises incubating the multiwell assay plate. In certain embodiments, the bacterial growth inhibition assay comprises measuring growth of the at least one bacterial species in each well of the multiwell assay plate. In certain embodiments, measurement of growth is performed by absorbance measurements. In certain embodiments, the multi well plate is a 96-well plate (e.g., 2D checkerboard). In certain embodiments, higher dimension checkboards assays can be used according to methods known to those of ordinary skill in the art to permit use of more than tw o AMPs (e.g., multiple 96-well plates).

[0091] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial grow th inhibition activity comprises independently calculating a minimum inhibitory concentration of each AMP alone (e.g., first AMP. second AMP, third AMP, fourth AMP, and fifth AMP) against a bacterial species (e.g, AMP MIC-1, AMP MIC- 2, AMP MIC-3, AMP MIC-4, and AMP MIC-5)

[0092] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial growth inhibition activity comprises calculating a minimum inhibitory' concentration (MIC) for each bacterial growth inhibition assay comprising at least the first AMP and second AMP (MIC-1 / 2).

[0093] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial growth inhibition activity comprises calculating a fractional inhibitory' concentration index (FICI) using the MIC for each bacterial growth inhibition assay and an independent MIC value for the first AMP (MIC AMP-1) and second AMP (MIC AMP-2) according to Eq. 1 : wherein each combination of first AMP and second AMP having a FICI value of < 0.5 has synergistic bacterial growth inhibition activity.

[0094] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial growth inhibition activity comprises calculating a minimum inhibitory concentration (MIC) for each bacterial growth inhibition assay comprising the first AMP, second AMP, and third AMP (MIC-1-3).

[0095] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial growth inhibition activity comprises calculating a fractional inhibitory concentration index (FICI) using the MIC for each bacterial growth inhibition assay and an independent MIC value for the first AMP (MIC AMP-1), second AMP (MIC AMP -2), and third AMP (MIC AMP-3) according to Eq. 2:

[0096] „T„TMIC-1-3 , MIC-1-3 , MIC-1-3

[0097] FICI — - - - (Eq. 2).

[0098] MIC AMP-1 MIC AMP-2 MIC AMP-3Mwherein each combination of first AMP. second AMP, and third AMP having a FICI value of < 0.8 has svnergistic bacterial growth inhibition activity. In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial growth inhibition activity comprises calculating a minimum inhibitory’ concentration (MIC) for each bacterial growth inhibition assay comprising the first AMP, second AMP, third AMP, and fourth AMP (MIC-1-4).

[0099] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial grow th inhibition activity comprises calculating a fractional inhibitory’ concentration index (FICI) using the MIC for each bacterial growth inhibition assay and an independent MIC value for the first AMP (MIC AMP-1), second AMP (MIC AMP -2), third AMP (MIC AMP-3), and fourth AMP (MIC AMP-4) according to Eq. 3:

[0100] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial growth inhibition activity comprises calculating a minimum inhibitory’ concentration (MIC) for each bacterial growth inhibition assay comprising the first AMP, second AMP, third AMP, fourth AMP, and fifth AMP (MIC- 1-5).

[0101] In certain embodiments, the identifying the at least one combination of AMPs with synergistic bacterial grow th inhibition activity comprises calculating a fractional inhibitory’ concentration index (FICI) using the MIC for each bacterial growth inhibition assay and an independent MIC value for the first AMP (MIC AMP-1), second AMP (MIC AMP -2), third AMP (MIC AMP-3), fourth AMP (MIC AMP-4), and fifth AMP (MIC AMP-5) according to

[0102] (Eq. 4).

[0103] In certain embodiments, the preparing the hydrogel composition comprises contacting at least one functionalized polysaccharide, at least one functionalized first AMP, and at least one functionalized second AMP under conditions suitable for covalent bond formation. In certain embodiments, the preparing the hydrogel composition comprises contacting at least one functionalized polysaccharide, functionalized first AMP, and functionalized second AMP under conditions suitable for covalent bond formation. In certain embodiments, the preparing the hydrogel composition comprises contacting at least one functionalized polysaccharide, functionalized first AMP, functionalized second AMP, and functionalized third AMP under conditions suitable for covalent bond formation. In certain embodiments, the preparing the hydrogel composition comprises contacting at least one functionalized polysaccharide. functionalized first AMP, functionalized second AMP, functionalized third AMP, and functionalized fourth AMP under conditions suitable for covalent bond formation. In certain embodiments, the preparing the hydrogel composition comprises contacting at least one functionalized polysaccharide, functionalized first AMP, functionalized second AMP, functionalized third AMP, functionalized fourth AMP, and functionalized fifth AMP under conditions suitable for covalent bond formation.

[0104] In certain embodiments, the functional groups of the at least one polysaccharide and functionalized first AMP comprise a complementary click chemistry pair. In certain embodiments, the functional groups of the at least one polysaccharide and functionalized second AMP comprise a complementary click chemi stry pair. In certain embodiments, the functional groups of the at least one polysaccharide and functionalized third AMP comprise a complementary click chemistry pair. In certain embodiments, the functional groups of the at least one polysaccharide and functionalized fourth AMP comprise a complementary click chemistry pair. In certain embodiments, the functional groups of the at least one polysaccharide and functionalized fifth AMP comprise a complementary click chemistry pair.

[0105] In certain embodiments, the complementary click chemistry pair comprises an alkene or alkyne and an azide ([3+2] cycloaddition). In certain embodiments, the complementary click chemistry' pair comprises an alkyne and a nitrone (strain-promoted alkyne-nitrone cycloaddition). In certain embodiments, the complementary click chemistry pair comprises a thiol and an alkene (thiol-ene reaction). In certain embodiments, the complementary click chemistry pair comprises an enol, alcohol, thiol, or amine and an a,P-unsaturated carbonyl ([l,4]-conjugate addition). In certain embodiments, the complementary' click chemistry' pair comprises an alkene and a diene (Diels-Alder [4+2] cycloaddition). In certain embodiments, the complementary click chemistry pair comprises an isonitrile and a tetrazine ([4+1] cycloaddition). In certain embodiments, the complementary click chemistry pair comprises an epoxide or aziridine and an amine or hydroxyl (nucleophilic substitution).

[0106] In certain embodiments, the functionalized polysaccharide is a substituted hyaluronic acid. In certain embodiments, the functionalized polysaccharide comprises a norbomene- substituted hyaluronic acid. In certain embodiments, the functionalized polysacchande comprises a methacrylate-substituted hyaluronic acid.

[0107] In certain embodiments, the functionalized first AMP comprises the first AMP further comprising N-terminal functionalization with SEQ ID NO:4. In certain embodiments, the functionalized second AMP comprises the first AMP further comprising N-terminal functionalization with SEQ ID NO:4. In certain embodiments, the functionalized third AMP comprises the first AMP further comprising N-terminal functionalization with SEQ ID NO:4. In certain embodiments, the functionalized fourth AMP comprises the first AMP further comprising N-terminal functionalization with SEQ ID NO:4. In certain embodiments, the functionalized fifth AMP comprises the first AMP further comprising N-terminal functionalization with SEQ ID NO:4.

[0108] In certain embodiments, the functionalized polysaccharide is a norbomene-substituted hyaluronic acid. In certain embodiments, conditions suitable for covalent bond formation comprise further contacting the at least one functionalized polysaccharide, the at least one functionalized first AMP, and the at least one functionalized second AMP with a cross-linker and photoinitiator to provide a first mixture. In certain embodiments, conditions suitable for covalent bond formation comprise further contacting the at least one functionalized polysaccharide, the functionalized first AMP, and the functionalized second AMP with a cross-linker and photoinitiator to provide a first mixture. In certain embodiments, conditions suitable for covalent bond formation comprise further contacting the at least one functionalized polysaccharide, the functionalized first AMP, the functionalized second AMP, and the functionalized third AMP with a cross-linker and photoinitiator to provide a first mixture. In certain embodiments, conditions suitable for covalent bond formation comprise further contacting the at least one functionalized polysaccharide, the functionalized first AMP, the functionalized second AMP, the functionalized third AMP, and the functionalized fourth AMP with a cross-linker and photoinitiator to provide a first mixture. In certain embodiments, conditions suitable for covalent bond formation comprise further contacting the at least one functionalized polysaccharide, the functionalized first AMP, the functionalized second AMP, the functionalized third AMP, the functionalized fourth AMP, and the functionalized fifth AMP with a cross-linker and photoinitiator to provide a first mixture.

[0109] In certain embodiments, conditions suitable for covalent bond formation further comprise irradiating the first mixture. In certain embodiments, the irradiation comprises exposure to ultraviolet light.

[0110] In certain embodiments, the cross-linker is dithiothreitol. In certain embodiments, the photoinitiator is 2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959). In certain embodiments, each contacting occurs in a suitable solvent. In certain embodiments, the suitable solvent is a phosphate buffered saline (PBS) solution.

[0111] In certain embodiments, the functionalized polysaccharide is a methacrylatesubstituted hyaluronic acid. In certain embodiments, the conditions suitable for covalent bond formation comprise aqueous conditions. In certain embodiments, conditions suitable for covalent bond formation comprise a pH of about 8 to about 10. In certain embodiments, conditions suitable for covalent bond formation comprise a pH of about 9. In certain embodiments, conditions suitable for covalent bond formation do not comprise or require exposure to light.

[0112] In certain embodiments, the contacting further comprises further contacting the at least one functionalized polysaccharide, the at least one functionalized first AMP, and the at least one functionalized second AMP with a cross-linker. In certain embodiments, the crosslinker is dithiothreitol. In certain embodiments, the contacting occurs at a pH of about 8 to about 10. In certain embodiments, the contacting occurs at a pH of about 9.

[0113] In certain embodiments, the at least one bacterial species is a pathogen associated with nosocomial infection. In certain embodiments, at least one bacterial species is selected based on a clinical isolate from a hospital or healthcare setting.

[0114] In certain embodiments, the bacterial species is at least one selected form the group consisting of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae. Enterococcus faecalis, Enterococcus faecium. Pseudomonas aeruginosa, Acinetobacter baumannii,Clostridioides difficile, Methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Haemophilus influenzae, Clostridioides perfr ingens, Enterobacter cloacae, Salmonella spp. , Serratia marcescens. Citrobacter freundii. Neisseria meningitidis, Campylobacter spp., Streptococcus pyogenes, Streptococcus agalactiae, Enterobacter aerogenes, Mycobacterium tuberculosis, Listeria monocytogenes, Stenotrophomonas maltophilia, Morganella morganii, Clostridioides septicum, Group B Streptococcus (Streptococcus agalactiae), Coagulase-negative Staphylococci (e.g., Staphylococcus epidermidis). Clostridioides botulinum, Burkholderia cepacia, Clostridioides tetani, Clostridioides difficile (specifically associated with antibiotic- associated diarrhea), Clostridioides perfringens, Burkholderia pseudomallei, Proteus vulgaris, Enterobacter sakazakii (Cronobacter spp ), Proteus penneri, Mycobacterium avium complex, Proteus rettgeri, Acinetobacter calcoaceticus , Aeromonas hydrophila, Elizabethkingia anophelis. Clostridium ramosum. Corynebacterium jeikeium, Bacteroides fragilis, Klebsiella terrigena, Haemophilus parainfluenzae, and Mycobacterium chelonae.

[0115] In certain embodiments, the bacterial species is methicillin resistant S. aureus (MRSA). In certain embodiments, the bacterial species is methicillin sensitive S. aureus (MSSA).

[0116] In certain embodiments, each AMP (z. e. , first AMP, second AMP, third AMP, fourth AMP, and fifth AMP, if present) is independently selected from the group consisting of DD13RIP, indolicidin, PIO, Alvinellacin, Ascaphin, Aurein. Apidaecin, Bac8c, Bacitracin, Bominin, Buforin, Buforin II, Camocyclin, Cathelicidin, Cecropin, Cecropin A (l-7)-Melittin A (2-9), CLP-19, Citropin, CPC 2019, Cystatin C, Defensin, Dermaseptin, Dermicidin, Dermcidin, Drosocin, Epidermin, Epinecidin, Esculentin, Falchin, Fallaxin, Gaegurin, HBD- 2, Hepcidin, Histatins, Hongkongin, Human Beta-Defensin 3 (hBD-3), Iseganan. Jelleines, Lactoferrin, Lachesin, Lysostaphin, Maculatin, Magainin. Maximin, Melittin, MccJ25. Microcin Pl, Muscin, Mucroporin, Myxinidin, Nisin, Novicidin, NZ21 14, NVB302, Omiganan, Onocin, Otosenin, Ovotransferrin, Palustrin, Pardaxin, Phylloseptin, Phylloxin, Platelet antimicrobial proteins, Platicin, Pleurocidin, Plectasin, Plectasin Analogs, Polybia- MP1. Polyphemusin. Protegrin, Pseudomycoicidin, Psacotheasin. Pseudin, Pyrrhocoricin, Ranalexin, Ranatuerin, Retapamulin, RNAIII inhibiting peptide, Robustin, Rugosin, Sacovirin, Saposin, TAS-101, Tachyplesin, Temporin, Thermonuclease, and Virginiamycin Ml, or a hybrid peptide comprising a combination thereof. In certain embodiments, the hybrid peptide comprises Cecropin (l-7)-Melittin A (2-9), wherein the hybrid peptide comprises two cecropin peptides and a melittin peptide.

[0117] In certain embodiments, the first AMP and second AMP are different. In certain embodiments, each of the first AMP, second AMP, and third AMP are different. In certain embodiments, the first AMP, second AMP, third AMP, and fourth AMP are different. In certain embodiments, the first AMP. second AMP, third AMP, fourth AMP, and fifth AMP are different.

[0118] Hydrogel Compositions

[0119] In another aspect, the present disclosure provides a hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide or disaccharide monomers. In certain embodiments, a plurality of the monomers thereof are independently covalently conjugated to a first AMP or a second AMP. In certain embodiments, the first AMP comprises indolicidin (SEQ ID NO:2), or a modified derivative thereof. In certain embodiments, the second AMP comprises PIO (SEQ ID NO: 3), or a modified derivative thereof.

[0120] In certain embodiments, the first AMP and second AMP have a molar ratio in the hydrogel composition of about 1:4.

[0121] In certain embodiments, the first AMP and second AMP have a concentration in the hydrogel composition of about 0.31 pM and about 1.25 pM, respectively. In certain embodiments, the first AMP and second AMP have a concentration in the hydrogel composition of about 0.78 pM and about 3.13 pM, respectively. In certain embodiments, the first AMP and second AMP have a concentration in the hydrogel composition of about 1.56 pM and about 6.25 pM, respectively. In certain embodiments, the first AMP and second AMP have a concentration in the hydrogel composition of about 3.125 pM and about 12.50 pM, respectively. In certain embodiments, the first AMP and second AMP have a concentration in the hydrogel composition of about 6.25 pM and about 25.00 pM, respectively.

[0122] In certain embodiments, the polysaccharide comprises hyaluronic acid, or a modified derivative thereof.

[0123] In certain embodiments, wherein the first AMP and second AMP are covalently conjugated to the polysaccharide via a linker. In certain embodiments, the linker comprises a moiety formed by a click reaction.

[0124] In certain embodiments, the compositions comprises a crosslinked product of: (a) at least one polysaccharide compound of Formula (I): wherein: each occurrence of Y is independently selected from the group consisting of O and NRle; each occurrence of Rla, Rlb, Rlc, Rld, Rle, and Rlfis independently selected from the group consisting of R2, H, optionally substituted Ci-Ce alkyd, optionally substituted Cs-Cs cycloalky l, optionally substituted C2-C8 heterocycloalkyl, optionally substituted Ce-Cio aryl, optionally substituted C2-C10 heteroaryd. C(=O)RA, C(=O)ORA, C(=O)N(RA)(RB), S(=O)RA, S(=O)2RA, and S(=O)2N(RA)(RB), wherein, in each monomeric unit of the compound of formula (I), no more than one of Rla, Rlb, Rlc, Rld, Rle, and Rlfis R2; each occurrence of L1is independently selected from the group consisting of a bond, optionally substituted C2-C6 alkydenyl, optionally substituted C2-C6 heteroalkylenyl, - C(=O)N(RA)-, and -C(=O)O-; each occurrence of R2is independently an alkenyl moiety: R3aand R3bare each independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl. C(=O)(optionally substituted Ci-Ce alkyl), and a monosaccharide or a modified derivative thereof; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, optionally substituted Cs-Cx cycloalkyd, optionally- substituted C2-C8 heterocycloalkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl; m is an integer between 2 and 25,000;

[0125] (b) a first polypeptide comprising SEQ ID NO:2, wherein the first polypeptide further comprises an N-terminal polypeptide of one to ten amino acids comprising at least one cysteine;

[0126] (c) a second polypeptide comprising SEQ ID NO:3, wherein the second polypeptide further comprises an N-terminal polypeptide of one to ten amino acids comprising at least one cysteine; and

[0127] (d) a crosslinker selected from the group consisting of -S-(optionally substituted C2-C8 alkylenyl)-S- and -S-(optionally substituted C2-C8 heteroalkylenyl)-S-.

[0128] In certain embodiments, the compound of formula (I) is a compound of formula (la):

[0129] In certain embodiments, Rlais H. In certain embodiments, Rlbis H. In certain embodiments, Rlcis H. In certain embodiments, Rldis H. In certain embodiments, Rleis H.

[0130] In certain embodiments, Rlais C(=O)OCH3. In certain embodiments, Rlbis C(=O)OCH3. In certain embodiments, Rlcis C(=O)OCH3. In certain embodiments, Rldis C(=O)OCH3. In certain embodiments, Rleis C(=O)OCH3.

[0131] In certain embodiments, Y is NRle.

[0132] In certain embodiments, L1is -C(=O)NH-. In certain embodiments, L1is -C(=O)O-.

[0133] In certain embodiments, R2is in certain embodiments, R2is certain embodiments, R2is H.

[0134] In certain embodiments, R3ais H. In certain embodiments, R31’ is H. In certain embodiments, R'ais CH3. In certain embodiments, R?bis CH3. In certain embodiments, R?ais ,

[0135] In certain embodiments, each monomeric unit of the compound of formula (la) is independently selected from the group consisting of:

[0136] In certain embodiments, the compound of formula (I) is a compound of formula (lb):

[0137] In certain embodiments, Rlais H. In certain embodiments, Rlbis H. In certain embodiments, Rldis H. In certain embodiments, Rlfis H. In certain embodiments, Y is NRle.

[0138] In certain embodiments, Rleis C(=O)CH3.

[0139] In certain embodiments, L1is -C(=O)NH-. In certain embodiments, L1is -C(=O)O-.

[0140] O

[0141] In certain embodiments, R2is1. In certain embodiments, R2is H.

[0142] In certain embodiments, each monomeric unit of the compound of formula (lb) is independently selected from the group consisting of:

[0143] In certain embodiments, the first polypeptide is SEQ ID NO:6. In certain embodiments, the second polypeptide is SEQ ID NO:7.

[0144] In certain embodiments, the crosslinker is dithiothreitol.

[0145] Kits

[0146] In another aspect, the present disclosure provides a kit for preparing hydrogel compositions comprising a synergistic combination of antimicrobial peptides (AMPs). In certain embodiments, the kit comprises at least two AMPs. In certain embodiments, the kit comprises functionalized derivatives of each of the at least two AMPs. In certain embodiments, the kit comprises at least one functionalized polysaccharide. In certain embodiments, the kit comprises reagents suitable for covalent conjugation of the functionalized antimicrobial peptides and the at least one functionalized polysaccharide. In certain embodiments, the kit comprises instructional materials for identifying a bacterial species, identifying a synergistic combination of AMPs, and preparing a hydrogel composition comprising the synergistic combination of AMPs. In certain embodiments, the functionalization of the at least one polysaccharide and each of the functionalized AMPs are selected such that functional groups thereof comprise a complementary' click chemistry' pair.

[0147] EXAMPLES

[0148] Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.

[0149] Materials and Methods

[0150] Peptide Synthesis

[0151] The peptides DD13RIP (ALWKTLLKKVLKAYSPWTNF) (SEQ ID NOT), indolicidin (ILPWKWPWWPWRR) (SEQ ID NO:2), and PIO (LAREYKKIVEKLKRWLRQVLRTLR) (SEQ ID NOT) were prepared using a solrd-state peptide synthesizer (Liberty Blue, CEM). The sequence GCGGG (SEQ ID NO:4) was added to the N-terminus of these peptides to provide a thiol group (in cysteine, C) for covalent binding to norbomene groups in the hydrogel network. Peptides were cleaved from the resin (ProTide Rink, CEM) using a solution of 92.5% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% 2, 2-(Ethylenedioxy)di ethanethiol for 3 hours. The peptides were precipitated three times in cold diethyl ether and lyophilized. Peptide synthesis was confirmed using MALDI- TOF (matrix-assisted laser desorption / ionization-time of flight) spectroscopy (Table 1).

[0152] Table 1. MALDI-TOF characterization of exemplary thiolated AMPs

[0153] Bacterial strains, media, and growth conditions

[0154] Deidentified patient isolates of MSSA and MRSA strains were obtained and utilized herein. Bacterial strains were grown overnight at 37 °C, with aeration in lysogeny broth (LB) or Mueller-Hinton broth (MHB), when appropriate. The optical density at 600 nm (ODeoo) was determined and cells were diluted to a starting ODeoo to 0.05 in MHB for MIC determination and checkerboard assays, and to 0.1 in Trypticase-soy broth (TSB) for biofilm growth. For biofilm growth, 1 rnL of the diluted bacterial culture was added to each well of a 24-well plate containing a hydrogel mounted on a glass coverslip, incubated for 2 days at 37 °C, and samples were then stained and prepared for confocal microscopy.

[0155] Determination of the minimum inhibitory concentration (MIC)

[0156] MIC determination w as performed by broth microdilution according to standard protocols. Briefly, each soluble AMP was added to the first column of a 96-well plate and serially diluted 2-fold, ten times. The starting concentrations for the AMPs were as follows: 100 pM for DD13RIP and 50 pM for indolicidin and P 10. These values were selected using previously determined MICs as a guide, where the starting value w as at least 1 -fold higher to allows for differences to be observed. Following dilutions, 50 pL of diluted MSSA or MRSA cells were added. Plates were incubated overnight at 37 °C, without shaking. The following morning, the ODeoo was read in a Synergy plate reader (Biotek). The MIC was determined as the lowest concentration of AMP where no bacterial growth w as observed (ODeoo < 0. 1). Checkerboard assays

[0157] Checkerboard assays were carried out as described previously in the literature. Briefly, each pairwise combination of AMPs was tested against MSSA and MRSA strains. The first AMP was added to the first column of the 96-well plate and diluted horizontally as described for MIC determination. The second AMP was added to the first row and serially diluted 2-fold vertically (FIG. 1). 50 pL of cells were added to the plate and analyzed as described above. To determine potential additive or synergistic effects, the fractional inhibitory concentration index (FICI) was calculated using the following formula:

[0158] In this formula. MIC AA+B and MIC BA+B are the MICs of the AMPs in combination and MIC A and MIC B are the MICs of each AMP alone. If the FICI is above 4.0, the AMPs at those concentrations were antagonistic. If the FICI was between 1 and 4, the AMPs in combination had no effect. Additive effects are defined by the combination of AMPs producing a larger effect than either alone and are present when the FICI is between 0.5 and 1. Synergistic effects, as defined by the combination of AMPs producing a significantly larger effect than either alone are denoted by FICI values less than 0.5.

[0159] The description provided herein relates to two-dimensional (2D) checkerboard assays. The method described herein are not limited to the application of 2D checkerboard assays. The methods described herein may utilize higher order checkerboard assays, for example 3D checkerboard assays, wherein the same techniques are applied using a third AMP and additional 96-well plates.

[0160] HANor macromer synthesis

[0161] Hyaluronic acid (HA) modified with norbomene (HANor) was synthesized as described previously. Briefly, sodium hyaluronate (NaHA, 2% w / v) was mixed with Dowex resin in distilled w ater for tw o hours at room temperature. The resin was then vacuum filtered, and tetrabutylammonium hydroxide (TBA-OH, ~2.4 mmol per gram of NaHA) added to form HA tetrabutylammonium salt (HA-TBA). The resulting HA-TBA solution was then frozen and lyophilized. Carboxyl groups in HA-TBA were then modified with norbomenes via amidation with 5-norbomene-2-methylamine in an anhydrous solution of dimethyl sulfoxide (DMSO, 2% w / v) and benzotriazole-l-yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate (BOP) under nitrogen for two hours at room temperature. The reaction was quenched with ice cold water, dialyzed (SpectraPor, 6-8 kDa molecular weight cutoff), frozen, and lyophilized. The synthesized HANor macromer had -45% of its repeat units functionalized with norbomene. as confirmed with NMR spectroscopy (FIG. 8). Norbomene functionalized percent was determined from the integration of the norbomene vinyl groups (3=5.9-6.36) relative to the 2.0 ppm peak attributed to the protons of the methyl group of hyaluronic acid.

[0162] AMP Hydrogel Synthesis

[0163] HANor macromers (5 wt%) were mixed in phosphate buffered saline (PBS) with dithiol crosslinkers (dithiothreitol, DTT at 8 mM concentration), photoinitiator (Irgacure 2959, 0.05 wt%), and appropriate thiolated AMP concentrations, and injected into cylindrical molds (3 mm diameter, 0.8 mm height) (FIG. 1). The hydrogels were photopolymerized by irradiating with ultraviolet light (10 minutes, 10 mW / cm2) and washed twice with PBS. Compression testing showed that the stiffness of the formed hydrogels ranges from 27.9 to 36.2 kPa, with P10 and DD13-RIP hydrogels being slightly stiffer than the other peptide- loaded hydrogel groups (FIG. 6). Using soluble AMP screening data, hydrogels containing 0.5x. l.Ox. and 2. Ox DD13RIP, indolicidin and P10 MICs against MRSA (9 groups) were synthesized. A synergistic combination consisting of indolicidin and P10 was identified against MRSA and hydrogels were formed with both AMPs at O. lx, 0.25x, 0.5x, lx, and 2x their synergistic concentrations. AMP-free hydrogels were also formed as peptide-free controls. For MSC viability studies, hydrogels were formed with 2 mM thiolated RGD peptide for adhesion and DD13RIP, indolicidin, P10, or synergistic two- AMP combination at double their MIC or synergistic concentrations to evaluate AMP effects on mammalian cell adhesion and viability.

[0164] Biofilm viability

[0165] Biofilms w ere washed three times with sterile w ater to remove unattached cells. The live / dead biofilm viability assay (FilmTracer, Invitrogen) was performed according to the manufacturer’s instructions. 200 pL of staining solution was added to each well and plates were incubated for 20 minutes in the dark at room temperature. Following incubation, wells w ere w ashed once with 1 mL of deionized sterile water to remove excess stain. The hydrogel assembly w as then placed upside dow n on a thin glass coverslip and imaged using an inverted confocal microscope (Nikon AIR) within two hours of staining (FIG. 1). For each hydrogel, a 5x5 mm2tile scan image with a z-depth of 250 pm (step size of 10 pm) was acquired, and the green and red channel acquisition parameters were kept constant throughout all experiments. The images were analyzed using ImageJ software by extracting 100 x 100 pm2square image stacks from each hydrogel image stack (n=4 per hydrogel), identifying focal plane image(s) within the z-stack, and by applying an Otsu threshold to create binary images from the green (live) and read (dead) channels. Twelve cross sections taken from the three gels of each sample group were analyzed in this manner. Viability (% viable) was then quantified using the following equation:

[0166] Red % Area \

[0167] % viable = - x 100 Green % Area /

[0168] The % viable values w ere then normalized to the average viability from the control samples with no peptide such that 100% represents a typical bacterial death rate on these hydrogels over the course of the experiment:

[0169] Sample % viable

[0170] Normalized viablity = - - - — x 100

[0171] Control % viable

[0172] Mammalian cell viability

[0173] Human bone-marrow derived mesenchymal stem cells (MSCs, Lonza, passage 4) were seeded on RGD-functionalized AMP -hydrogels at a density of 20,000 cells / cm2. MSCs were incubated at 37 °C, 5% CO2 for two days in grow th medium (a-minimum essential medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin). Three hydrogels were seeded for each AMP group. After two days, a Mammalian cell LIVE / DEAD Viabilify / Cytotoxicity Kit (Invitrogen) was constituted in growth medium per the manufacturer’s instructions. Samples were exposed to the staining solution for 30 minutes, then the constituted live / dead solution was replaced with grow th medium, and samples were imaged within 2 hours. Live and dead cells were quantified by counting green and red individual cells, respectively. Live cells were quantified by hand by selecting four 1 x 1 mm2image sections for each hydrogel group (n=l 2 images analyzed per group). Viability was calculated by dividing the number of living cells by total cell count (living plus dead cells).

[0174] Statistical Analysis

[0175] All experiments were carried out at least three independent times. Data was analyzed by one-way analysis of variance (ANOVA) and tested versus control hydrogels using Dunnetfs multiple comparisons test. Bar graphs represent the mean, and the error bars represent ± standard deviation. Differences among groups are stated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

[0176] Example 1: Determination of minimum inhibitory concentrations (MICs) of certain exemplary soluble antimicrobial peptides (AMPs) against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)

[0177] In this study, DDBRIP, indolicidin, and P10 were chosen as candidate AMPs due to their different mechanisms of action and anti-biofilm properties. The MIC is a commonly used laboratory measurement to determine the in vitro activity of an antimicrobial agent against bacteria. A lower MIC typically indicates that less of the agent is required to inhibit growth of the organism, thus implying that the lower the MICs, the more potent the agent. The MICs for MSSA were 13.5 pM. 18.8 pM, and 87.5 pM for DD13RIP, indolicidin, and P10, respectively (Table 2). The MICs for MRSA were over 2-fold higher for DDBRIP, 2.5- fold higher for indolicidin, and 1.4-fold higher for P10 in comparison to MSSA (Table 2).

[0178] Table 2. Average minimum inhibitory concentration of AMP against S. aureus strains (n=3)

[0179] The MIC values observed for the clinical isolates were higher than those previously reported in the literature, which is likely due to strain differences. It is well known that MRSA infections are more difficult to treat than MSSA infections due to increased drug resistance, therefore limiting treatment options for MRSA infections. The data provided herein demonstrates that MRSA is also more tolerant of AMPs than MSSA, based upon comparison of MIC values. It is important to note that no clinical breakpoint data is available for AMPs, so susceptibility cannot be determined. A possible explanation of increased MICs in MRSA strains is that there is some level of cross-protection provided by one or more drugresistance mechanisms, with a possible candidate being multidrug efflux pumps. The data provided herein demonstrates that drug resistance status influences the efficacy of AMPs, highlighting the need to identify optimal effective concentrations tailored to specific bacterial strains.

[0180] Example 2: Checkboard arrays identify additive and synergistic AMP combinations against S. aureus isolates Antibiotics used in combination allow for increased potency at lower bactericidal concentrations. Combinatorial effects are determined in vitro using the standard checkerboard array, where two antimicrobial agents are serially diluted in different directions in a 96-well plate to assess the effectiveness of antibiotic cocktails. Often, when drugs are tested together, the effect is similar to that of one of the drugs alone. An additive effect is present when the two agents have a larger effect than either alone, as if adding their efficacy together. Synergistic effects occur when their combined effect is significantly larger than either alone. Combination therapy offers an effective approach to mitigate potential cytotoxicity, adverse reactions, and the emergence of bacterial resistance to each antimicrobial peptide. Thus, checkerboard arrays were used to examine pairwise combinations of DD13RIP, indolicidin, and PIO AMPs for potential additive and synergistic effects against MSSA (FIGs. 2A-2C) and MRSA strains (FIGs. 2D-2F).

[0181] For MSSA, the combination of indolicidin and PIO yielded multiple combinatorial effects, including eight additive combinations and five synergistic combinations (FIG. 2A). The concentrations of the AMPs in the synergistic combinations are significantly lower (FIG. 7 A) than the MIC of individual AMPs (Table 2). For DD13-RIP and PIO, seven additive combinations were identified, but no synergistic ones (FIG. 2B and FIG. 7B). Surprisingly, no combinatorial effects were identified for indolicidin and DD13-RIP (FIG. 2C and FIG. 7C). For MRSA, seven additive combinations were identified between indolicidin and PIO AMPs (FIG. 2D). Only one combination yielded a synergistic effect, which was at 12.5 pM PIO and 3.125 pM indolicidin. Nine additive combinations were observed using DD13-RIP in combination with either PIO or indolicidin (FIGs. 2E-2F). Interestingly, the combination of DD13-RIP and indolicidin showed additive effects against MRSA but not MSSA, demonstrating that combinatorial effects of AMPs are strain specific. Due to clinical challenges in treating MRSA infections and the observation that higher soluble AMP concentrations are needed to prevent MRSA growth in vitro, soluble AMP screening data were used to create custom bactericidal AMP hydrogels against MRSA.

[0182] Example 3: Single AMP-loaded hydrogels at the MIC retain antimicrobial properties against MRSA

[0183] While antimicrobial peptides offer a viable replacement for antibiotics, in their soluble form, the clearance rate of fluid surrounding a w ound or implant has limited their use for biomedical applications. The immobilization of AMPs to a biocompatible substrate can mitigate this dilution effect and expand the lifetime of AMPs. Towards this, the transferability of the soluble peptide concentrations determined in broth into AMP hydrogels was evaluated by tethering thiolated AMPs at concentrations equivalent to 0.5x, lx, and 2x the MICs to HANor hydrogels using thiol-ene click chemistry reactions. MRSA was incubated with AMP -functionalized hydrogels (3 mm diameter, 0.5 mm thickness) for 2 days under biofilm-forming conditions. Next, non-adherent cells were removed, and hydrogels stained with a mixture of Syto-9 and propidium iodine (live-dead) for imaging by confocal microscopy (FIGs. 3A-3C).

[0184] For DDis-RIP, all the AMP concentrations tested showed antimicrobial properties, evidenced by a visibly high red fluorescence, indicating dead cells (FIG. 3A). The percent of viable bacteria cells was calculated to be 21%, 5%, and 13% relative to AMP-free control gels at 0.5x, lx and 2x MIC respectively (FIG. 3D). The DDis-RIP peptide MIC from the checkerboard arrays was the lowest at 27.5 pM and these results suggest that even at 13.8 pM, this peptide can greatly reduce the bioburden of the hydrogels. If total peptide concentration is a concern for any application, DD13-RIP laden hydrogels may be an appropriate option.

[0185] At the MIC, indohcidin hydrogels had patches of dead cells, but many live bacterial cells remained, while at 0.5x MIC there w as a minimal killing effect (FIG. 3B). At 2x MIC, there was a drastic decrease in viability. Quantification of these findings show ed a 55% and 88% drop in viability for indolicidin hydrogels formed with lx and 2x soluble MICs. respectively, relative to AMP-free control hydrogels (FIG. 3E). These results highlight the ability of indolicidin to reduce bacterial burden within these hydrogels. As a potential clinical application, there would be a requirement for a substantial reduction in viability, so it w ould be advisable to employ double the MIC concentration or higher. The 2x MIC corresponds to 88 pM of indohcidin, a concentration that is well-tolerated by human cells, as validated by a Mammalian cell live-dead assay discussed below.

[0186] Starting at the MIC, the PIO AMP hydrogel demonstrated a pronounced decrease in the number of viable cells (FIG. 3C). The normalized viability in P 10-functionalized hydrogels was reduced from 100% in the 0.5x MIC to just 1.6% in the lx MIC hydrogels, and this bactericidal effect was retained for PIO hydrogels formed at 2x MIC (FIGs. FIG. 3F). Taken together, these findings demonstrate that these three AMPs, when tethered to a hydrogel surface, retain excellent antimicrobial activity, and can prevent MRSA biofilms at or below their individual MICs. In addition, our results demonstrate that in vitro MIC screening with soluble AMPs can be used to create effective, antimicrobial AMP-loaded hydrogels. Example 4: Combinatorial AMP hydrogels are effective against MRSA

[0187] The use of AMPs at doses which display a synergistic effect has the potential to reduce the risk of antimicrobial resistance development, and by tethering these peptides to hydrogels the dosage can be held consistent in the targeted area without the need to overcompensate for clearance. Towards this, the coupling of multiple AMPs to HANor hydrogels at the concentrations that yielded a synergistic effect against MRSA based on checkerboard array screening data was performed. From these experiments, the combination of 3. 125 pM indolicidin and 12.5 pM PIO yielded a synergistic effect against MRSA and an additive effect against MSSA. Multiples of these concentrations were tested, which represented a proportional change of 0. lx. 0.25x, 0.5x, lx and 2x the synergistic MIC (FIGs. 4A-4B). At low PIO and indolicidin concentrations representing O. lx and 0.25x the synergistic MIC, the multi-AMP hydrogels formed had no effect on MRSA viability (FIG. 4A). At PIO and indolicidin AMP concentrations above 0.5x the synergistic MIC, hydrogels completely killed the bacteria (FIG. 4A). This can be seen visually in FIG. 4A, which shows the switch from entirely green (live) to entirely red (dead) starting at the 0.5x MIC. and quantitatively in FIG. 4B, which shows a statistically significant decrease in viability' compared to the AMP-free control group. This observed effect at 0.5x MIC is reflective of a concentration of only 1.56 pM and 6.25 pM for indolicidin and PIO, respectively.

[0188] Further, it was observed that the MIC of indolicidin was 43.8 pM and PIO was 120 pM when used alone against MRSA (Table 2). When used in combination, they were effective at ~28- and ~20-fold lower concentrations, respectively. AMPs used at high concentrations tend to be cytotoxic. As seen here, the combination of AMPs allows for the use of the antimicrobial agent at lower effective concentrations, which could reduce negative side effects. In addition, using two different AMPs with different mechanisms of actions reduces the risk of developing resistance. These findings highlight the value of the high throughput assay described herein in identify ing AMP combinations that minimize the total concentration needed to kill a specific pathogen and prevent biofilm formation for use in AMP-loaded hydrogels.

[0189] Example 5: Single AMP-loaded hydrogels at the MIC and Combinatorial AMP hydrogels reduce bacterial bioburden on and within hydrogels

[0190] In view of the findings that single- AMP and dual-AMP hydrogels at their soluble MIC exhibited excellent antimicrobial activity’, efforts were directed to evaluating bacteria- AMP hydrogel interactions on a finer scale. Towards this end, hydrogels without AMPs or functionalized with DD13RIP. indolicidin, PIO. or an exemplary synergistic combination thereof (PIO and indolicidin) at their soluble MIC were formed. MRS A were cultured atop the hydrogels as described previously; two days later, the samples were stained for Syto-9 (live) and propidium iodide (dead), and high-resolution z-stacks (60* magnification, surface- imaged for every 1 pm of the hydrogels for 5 pm) were acquired to evaluate 3D viability and extent of bacteria infiltration into the hydrogels.

[0191] On AMP-free hydrogels, live bacteria covered about 46% of the hydrogel surface (FIG. 9A and FIG. 9F). In contrast, AMP-functionalized hydrogels all showed fewer live cells at the hydrogel surface. DD13RIP-functionalized hydrogels (27.5 pM) showed a lower total bacterial load on the surface compared to the no-AMP group with 7% covered by dead cells and 2% covered with live bacteria (FIG. 9B and FIG. 9G). However, the total surface coverage of bacteria on indolicidin-functionalized hydrogels (43.8 pM) was highest among other AMPs (37%), and the majority of these cells (70% of surface bacteria) were dead (FIG. 9C and FIG. 9H). PlO-functionalized hydrogels (120 pM) were best among single-AMP hydrogels at preventing initial bacterial adhesion at the surface, and less than 1% of the cells bound to the hydrogel surface had living MRS A (FIG. 9D and FIG. 9 J). PIO prevents bacterial biofilm formation by disrupting the bacterial cell membrane, which results in the near complete elimination of bacteria on the hydrogel surface at the MIC. An exemplary synergistic hydrogel of the disclosure (z.e., 3. 1 pM indolicidin and 12.5 pM PIO) showed essentially no live bacterial cells at the surface, and just 2% of the surface contained cells, almost all of which were dead (FIG. 9E and FIG. 9J). These findings suggest that these dualAMP hydrogels leverage the bactericidal effects of indolicidin-functionalized hydrogels and the anti-biofilm effects of PlO-functionalized hydrogels, allowing for maximal antibacterial properties at a significantly lower total peptide concentration.

[0192] At a 5 pm depth inside AMP-free hydrogels, about 6% of the area was covered with living MRSA cells. While it is unlikely that the bacteria can immediately infiltrate the hydrogel, Gram-positive bacteria such as MRSA can secrete hyaluronidases and degrade hyaluronic acid, allowing them to burrow into hyaluronic acid hydrogels over time. None of the AMP -loaded hydrogels showed a greater than 1 % area of live cells within the gel at 5 pm depth. The AMP-loaded hydrogels appear to prevent the process of bacteria making their way below the hydrogel surface either by killing the bacteria on the surface or preventing them from collecting on the surface at all. Overall, the antimicrobial properties of all these peptides appear to function for improving the natural antimicrobial properties of hyaluronic acid and minimize the bacterial burden at the surface and within the hydrogel.

[0193] Example 6: Mammalian cells are viable on single and combinatorial AMP hydrogels

[0194] Towards establishing whether these AMP-loaded hydrogels could be used in practical biomedical applications, the effects of these compositions on mammalian cell viability was assessed. Mesenchymal stem cells (MSCs) were seeded onto RGD-functionalized (2 mM, to allow for MSC adhesion) AMP hydrogels at the highest concentration, 2x MIC for each AMP as well as the synergistic concentrations for PIO and indolicidin. All AMP hydrogels promoted MSC cell attachment, growth, and spreading at these concentrations, as evidenced by the coverage, and spread of live (green) MSCs on each hydrogel group (FIG. 5 A). For every 100 cells counted across each AMP group there was at most one cell which displayed a nuclear red stain, resulting in viability at or above 99% (FIG. 5B).

[0195] This data demonstrates that even at double the concentration required for antimicrobial properties, the tethered AMPs do not display significant cytotoxic effects. Thus, using the high throughput screening methodology described herein. AMP-loaded hydrogels could be created for infection prevention, providing an effective alternative to antibiotics and minimizing the potential for resistance development, while allowing for the grow th of healthy mammalian cells.

[0196] Sequence Listing

[0197] DD13RIP (SEQ ID NO : 1 )

[0198] AL WKT L L KKVL KAY S P WT N F

[0199] Indolicidin (SEQ ID NO : 2 )

[0200] LLPWKWPWWPWRR

[0201] PIO (SEQ ID NO : 3)

[0202] LAREYKKIVEKLKRWL RQVL RT L R

[0203] Thio-Linker (SEQ ID NO : 4 )

[0204] GCGGG

[0205] Thio-DDi3RIP (SEQ ID NO : 5)

[0206] GC GGG AL WKT L L KKVL KAY S P WT N F Thio-Indolicidin (SEQ ID NO : 6)

[0207] GCGGGILPWKWPWWPWRR

[0208] Thio-PIO (SEQ ID NO : 7 )

[0209] GC G G G L ARE Y KK I VE KL KRWL RQVL RT L R

[0210] Enumerated Embodiments

[0211] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0212] Embodiment 1 provides a method for preparing an antibacterial hydrogel composition, the method comprising:

[0213] (a) performing at least one bacterial growth inhibition assay using at least two antimicrobial peptides (AMPs) against at least one bacterial species;

[0214] (b) identifying at least one combination of AMPs with synergistic bacterial growth inhibition activity, wherein each combination of AMPs comprises at least a first AMP and a second AMP which have a synergistic molar ratio;

[0215] (c) preparing a hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide or disaccharide monomers, wherein:

[0216] (i) a plurality of the monomers thereof are independently covalently conjugated to an AMP present in the combination of AMPs with synergistic bacterial growth inhibition activity, and

[0217] (ii) the monomers are conjugated such that the hydrogel composition has a molar ratio of AMPs present in the at least one combination which is about equivalent to the synergistic molar ratio.

[0218] Embodiment 2 provides the method of Embodiment 1, wherein the bacterial growth inhibition assay comprises a two-dimensional (2D) checkerboard assay.

[0219] Embodiment 3 provides the method of Embodiment 2, wherein the bacterial growth inhibition assay comprises:

[0220] (a) adding a solution comprising the first AMP to a first column of a multiw ell assay plate and performing a horizontal serial dilution thereof;

[0221] (b) adding a solution comprising the second AMP to a first row of the multiwell assay plate and performing a vertical serial dilution thereof;

[0222] (c) adding a medium comprising the at least one bacterial species to each well of the multiwell assay plate;

[0223] (d) incubating the multiwell assay plate; and

[0224] (e) measuring growth of the at least one bacterial species in each well of the multiwell assay plate.

[0225] Embodiment 4 provides the method of Embodiment 2 or 3, wherein the identifying the at least one combination of AMPs with synergistic bacterial grow th inhibition activity comprises:

[0226] (a) calculating a minimum inhibitor}' concentration (MIC) for each bacterial growth inhibition assay comprising at least the first AMP and second AMP (MIC- 1 / 2);

[0227] (b) calculating a fractional inhibitory' concentration index (FICI) using the MIC for each bacterial growth inhibition assay and an independent MIC value for the first AMP (MIC AMP-1) and second AMP (MIC AMP-2) according to Eq. 1: w herein each combination of first AMP and second AMP having a FICI value of < 0.5 has synergistic bacterial growth inhibition activity'.

[0228] Embodiment 5 provides the method of any one of Embodiments 1-4, wherein the preparing the hydrogel composition comprises contacting at least one functionalized polysaccharide, at least one functionalized first AMP, and at least one functionalized second AMP under conditions suitable for covalent bond formation.

[0229] Embodiment 6 provides the method of Embodiment 5, wherein functional groups of the at least one polysaccharide, at least one functionalized first AMP. and at least one functionalized second AMP comprise a complementary click chemistry pair.

[0230] Embodiment 7 provides the method of Embodiment 6, w herein the complementary click chemistry' pair is selected from the group consisting of a thiol and alkene (thiol-ene reaction), an alkene or alkyne and azide ([3+2] cycloaddition), an alky ne and nitrone (strain-promoted alkyne-nitrone cycloaddition), an enol, alcohol, thiol, or amine and a.[3-unsaturated carbonyl ([1,4] -conjugate addition), an alkene and diene (Diels-Alder [4+2] cycloaddition), an isonitrile and tetrazine ([4+1] cycloaddition), and an epoxide or aziridine and amine or hydroxyl (nucleophilic substitution). Embodiment 8 provides the method of Embodiment 7, wherein the complementary click chemistry pair is a thiol and an alkene (thiol-ene reaction) or a thiol and an a.p- unsaturated carbonyl ([l,4]-conjugate addition).

[0231] Embodiment 9 provides the method of any one of Embodiments 6-8, wherein the functionalized polysaccharide is selected from the group consisting of a norbomene- substituted hyaluronic acid and a methacrylate-substituted hyaluronic acid. Embodiment 10 provides the method of any one of Embodiments 6-9, wherein at least one of the following applies:

[0232] (a) the functionalized first AMP comprises the first AMP further comprising N- terminal functionalization with SEQ ID NO:4; and

[0233] (b) the functionalized second AMP comprises the second AMP further comprising N-terminal functionalization with SEQ ID NO:4.

[0234] Embodiment 11 provides the method of any one of Embodiments 8-10, wherein the functionalized polysaccharide is a norbomene-substituted hyaluronic acid and conditions suitable for covalent bond formation comprise:

[0235] (a) further contacting the at least one functionalized polysacchande, the at least one functionalized first AMP, and the at least one functionalized second AMP with a cross-linker and photoinitiator to provide a first mixture; and

[0236] (b) irradiating the first mixture, optionally wherein the irradiation comprises exposure to ultraviolet light.

[0237] Embodiment 12 provides the method of Embodiment 11, wherein at least one of the following applies:

[0238] (a) the cross-linker is dithiothreitol;

[0239] (b) the photoinitiator is 2-hydroxy-4’-(2-hydroxyelhoxy)-2 -methylpropiophenone (Irgacure 2959); and

[0240] (c) each contacting occurs in a suitable solvent, optionally wherein the suitable solvent is a phosphate buffered saline (PBS) solution.

[0241] Embodiment 13 provides the method of any one of Embodiments 8-10, wherein the functionalized polysaccharide is a methacrylate-substituted hyaluronic acid and conditions suitable for covalent bond formation comprise aqueous conditions, optionally wherein the conditions suitable for covalent bond formation comprise a pH of about 8 to about 10, optionally wherein the conditions suitable for covalent bond formation comprise a pH of about 9.

[0242] Embodiment 14 provides the method of any one of Embodiments 8-10 and 13, wherein the functionalized polysaccharide is a methacrylate-substituted hyaluronic acid and the contacting further comprises further contacting the at least one functionalized polysaccharide, the at least one functionalized first AMP, and the at least one functionalized second AMP with a cross-linker, optionally wherein at least one of the following applies:

[0243] (a) the cross-linker is dithiothreitol; and

[0244] (b) the contacting occurs at a pH of about 8 to about 10. optionally wherein the contacting occurs at a pH of about 9.

[0245] Embodiment 15 provides the method of any one of Embodiments 1-14, wherein the at least one bacterial species is a pathogen associated with a nosocomial infection.

[0246] Embodiment 16 provides the method of any one of Embodiments 1-15, wherein the at least one bacterial species is selected based on a clinical isolate from a hospital or healthcare setting.

[0247] Embodiment 17 provides the method of any one of Embodiments 1-16, wherein the bacterial species is at least one selected form the group consisting of Staphylococcus aureus, Staphylococcus epidermis. Escherichia coll. Klebsiella pneumoniae, Enterococcus faecalis, C lostridioides difficile, Enterococcus faecium, and Pseudomonas aeruginosa, optionally wherein the S. aureus is methicillin resistant (MRSA) or methicillin sensitive (MSSA).

[0248] Embodiment 18 provides the method of any one of Embodiments 1-17, wherein each AMP is independently selected from the group consisting of DD13RIP, indolicidin, PIO, Alvinellacin. Ascaphin, Aurein, Apidaecin, Bac8c, Bacitracin, Bominin, Buforin, Buforin II, Camocyclin, Cathelicidin, Cecropin, Cecropin A (l -7)-Melittin A (2-9), CLP-19, Citropin, CPC 2019, Cystatin C, Defensin, Dermaseptin, Dermi cidin, Dermcidin, Drosocin, Epidermin, Epineci din. Esculentin, Falchin, Fallaxin, Gaegurin. HBD-2, Hepcidin, Histatins. Hongkongin. Human Beta-Defensin 3 (hBD-3), Iseganan, Jelleines, Lactoferrin, Lachesin. Lysostaphin, Maculatin, Magainin, Maximin, Mehttin, MccJ25, Microcin Pl, Muscin, Mucroporin, Myxinidin, Nisin, Novicidin, NZ2114, NVB302, Omiganan, Onocin, Otosenin, Ovotransferrin, Palustrin, Pardaxin, Phylloseptin, Phylloxin, Platelet antimicrobial proteins, Platicin, Pleurocidin. Plectasin, Plectasin Analogs, Polybia-MPl, Polyphemusin, Protegrin, Pseudomycoicidin. Psacotheasin, Pseudin, Pyrrhocoricin. Ranalexin, Ranatuerin. Retapamulin, RNAIII inhibiting peptide, Robustin, Rugosin, Sacovirin, Saposin, TAS-101, Tachyplesin, Temporin, Thermonuclease, and Virginiamycin Ml, or a hybrid peptide comprising a combination thereof.

[0249] Embodiment 19 provides a hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide or disaccharide monomers, wherein: (a) a plurality of the monomers thereof are independently covalently conjugated to a first AMP or a second AMP;

[0250] (b) the first AMP comprises indolicidin (SEQ ID NO:2), or a modified derivative thereof;

[0251] (c) the second AMP comprises PIO (SEQ ID NO:3), or a modified derivative thereof; and

[0252] (d) the first AMP and second AMP have a molar ratio in the hydrogel composition of about 1 :4.

[0253] Embodiment 20 provides the hydrogel composition of Embodiment 19, wherein the first AMP and second AMP have a concentration in the hydrogel composition selected from the group consisting of about 0.31 pM and about 1.25 pM, about 0.78 pM and about 3. 13 pM, about 1.56 pM and about 6.25 pM, about 3.125 pM and about 12.50 pM, and about 6.25 pM and about 25.00 pM, respectively.

[0254] Embodiment 21 provides the hydrogel composition of Embodiment 19 or 20, wherein the polysaccharide comprises hyaluronic acid, or a modified derivative thereof.

[0255] Embodiment 22 provides the hydrogel composition of any one of Embodiments 19- 21, wherein the first AMP and second AMP are covalently conjugated to the polysaccharide via a linker.

[0256] Embodiment 23 provides the hydrogel composition of Embodiment 22, wherein the linker comprises a moiety formed by a click reaction.

[0257] Embodiment 24 provides the hydrogel composition of any one of Embodiments 19- 23, wherein the composition comprises a crosslinked product of:

[0258] (a) at least one polysaccharide compound of Formula (I): wherein: each occurrence of Y is independently selected from the group consisting of O and NR1e; each occurrence of Rla, Rlb, Rlc, Rld, Rle, and Rlfis independently selected from the group consisting of R2, H, optionally substituted Ci-Ce alkyl, optionally substituted Cfi-Cs cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted Ce-Cio aryl, optionally substituted C2-C10 heteroaryl, C(=O)RA, C(=O)ORA, C(=O)N(RA)(RB), S(=O)RA, S(=O)2RA, and S(=O)2N(RA)(RB), wherein, in each monomeric unit of the compound of formula (I), no more than one of Rla, Rlb, Rlc, Rld, Rle, and Rlfis R2;each occurrence of L1is independently selected from the group consisting of a bond, optionally substituted C2-Ce alkylenyl, optionally substituted C2- Co heteroalkylenyl, -C(=O)N(RA)-, and -C(=O)O-; each occurrence of R2is independently an alkenyl moiety or H;

[0259] R3aand R3bare each independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, C(=O)(optionally substituted Ci-Ce alkyl), and a monosaccharide or a modified derivative thereof; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, optionally substituted C?-Cs cycloalkyl, optionally substituted C2-Cs heterocycloalkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-Cio heteroaryl; and m is an integer between 2 and 25,000;

[0260] (b) a first polypeptide comprising SEQ ID NO:2, wherein the first polypeptide further comprises an N-terminal polypeptide of one to ten amino acids comprising at least one cysteine;

[0261] (c) a second polypeptide comprising SEQ ID NO:3, wherein the second polypeptide further comprises an N-terminal polypeptide of one to ten amino acids comprising at least one cysteine; and

[0262] (d) a crosslinker selected from the group consisting of -S-(optionally substituted C2-Cs alkylenyl)-S- and -S-(optionally substituted C2-Cs heteroalkylenyl)-S-.

[0263] Embodiment 25 provides the hydrogel composition of any one of Embodiments 19- Embodiment 26 provides the hydrogel composition of Embodiment 25, wherein the compound of formula (I) is the compound of formula (la), and at least one of the following applies:

[0264] (a) Rla, Rlb, Rlcand Rldare each independently H;

[0265] (b) Y is NRle;

[0266] Embodiment 27 provides the hydrogel composition of Embodiment 25 or 26. wherein each monomeric unit of the compound of formula (la) is independently selected from the group consisting of:

[0267] Embodiment 28 provides the hydrogel composition of Embodiment 25. wherein the compound of formula (I) is the compound of formula (lb), and at least one of the following applies:

[0268] (a) Rla, Rlb, Rldand Rlfare each independently H;

[0269] (b) Y is NRle;

[0270] 0

[0271] (e) R2is -V ' or H.

[0272] Embodiment 29 provides the hydrogel composition of Embodiment 25 or 28, wherein each monomeric unit of the compound of formula (lb) is independently selected from the group consisting of:

[0273] Embodiment 30 provides the hydrogel composition of any one of Embodiments 24-

[0274] 29, wherein at least one of the following applies:

[0275] (a) the first polypeptide is SEQ ID NO:6; and

[0276] (b) the second polypeptide is SEQ ID NO:7.

[0277] Embodiment 31 provides the hydrogel composition of any one of Embodiments 24-

[0278] 30, wherein the crosslinker is dithiothreitol.

[0279] Embodiment 32 provides a kit for preparing hydrogel compositions comprising a synergistic combination of antimicrobial peptides (AMPs), the kit comprising:

[0280] (a) at least two AMPs;

[0281] (b) functionalized derivatives of each of the at least two AMPs;

[0282] (c) at least one functionalized polysaccharide;

[0283] (d) reagents suitable for covalent conjugation of the functionalized antimicrobial peptides and the at least one functionalized polysaccharide; and

[0284] (e) instructional materials for identifying a bacterial species, identifying a synergistic combination of AMPs, and preparing a hydrogel composition comprising the synergistic combination of AMPs.

[0285] Embodiment 33 provides the kit of Embodiment 32. wherein the functionalization of (b) and (c) are selected such that functional groups thereof comprise a complementary click chemistry pair.

[0286] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.

Claims

CLAIMSWhat is claimed is:

1. A method for preparing an antibacterial hydrogel composition, the method comprising:(a) performing at least one bacterial growth inhibition assay using at least two antimicrobial peptides (AMPs) against at least one bacterial species;(b) identifying at least one combination of AMPs with synergistic bacterial grow th inhibition activity, wherein each combination of AMPs comprises at least a first AMP and a second AMP which have a synergistic molar ratio;(c) preparing a hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide or disaccharide monomers, wherein:(i) a plurality of the monomers thereof are independently covalently conjugated to an AMP present in the combination of AMPs with synergistic bacterial growth inhibition activity, and(ii) the monomers are conjugated such that the hydrogel composition has a molar ratio of AMPs present in the at least one combination which is about equivalent to the synergistic molar ratio.

2. The method of claim 1, wherein the bacterial grow th inhibition assay comprises a two-dimensional (2D) checkerboard assay.

3. The method of claim 2, wherein the bacterial growth inhibition assay comprises:(a) adding a solution comprising the first AMP to a first column of a multiwell assay plate and performing a horizontal serial dilution thereof;(b) adding a solution comprising the second AMP to a first row of the multiwell assay plate and performing a vertical serial dilution thereof;(c) adding a medium comprising the at least one bacterial species to each w ell of the multi w ell assay plate;(d) incubating the multiw-ell assay plate; and(e) measuring growth of the at least one bacterial species in each well of the multiwell assay plate.

4. The method of claim 2 or 3, wherein the identifying the at least one combination of AMPs with synergistic bacterial growth inhibition activity comprises:(a) calculating a minimum inhibitory concentration (MIC) for each bacterial grow th inhibition assay comprising at least the first AMP and second AMP (MIC-1 / 2);(b) calculating a fractional inhibitory concentration index (FICI) using the MIC for each bacterial growth inhibition assay and an independent MIC value for the first AMP (MIC AMP-1) and second AMP (MIC AMP-2) according to Eq. 1:wherein each combination of first AMP and second AMP having a FICI value of < 0.5 has synergistic bacterial growth inhibition activity.

5. The method of any one of claims 1-4, wherein the preparing the hydrogel composition comprises contacting at least one functionalized polysaccharide, at least one functionalized first AMP. and at least one functionalized second AMP under conditions suitable for covalent bond formation.

6. The method of claim 5, wherein functional groups of the at least one polysaccharide, at least one functionalized first AMP, and at least one functionalized second AMP comprise a complementary' click chemistry pair.

7. The method of claim 6, wherein the complementary click chemistry pair is selected from the group consisting of a thiol and alkene (thiol-ene reaction), an alkene or alkyne and azide ([3+2] cycloaddition), an alkyne and nitrone (strain-promoted alkyne-nitrone cycloaddition), an enol, alcohol, thiol, or amine and a,P-unsaturated carbonyl ([1,4]- conjugate addition), an alkene and diene (Diels-Alder [4+2] cycloaddition), an isonitrile and tetrazine ([4+1] cycloaddition), and an epoxide or aziridine and amine or hydroxyl (nucleophilic substitution).

8. The method of claim 7, w herein the complementary' click chemistry' pair is a thiol and an alkene (thiol-ene reaction) or a thiol and an a,|3-unsaturated carbonyl ([l,4]-conjugateaddition).

9. The method of any one of claims 6-8, wherein the functionalized polysaccharide is selected from the group consisting of a norbomene-substituted hyaluronic acid and a methacrylate-substituted hyaluronic acid.

10. The method of any one of claims 6-9, wherein at least one of the following applies:(a) the functionalized first AMP comprises the first AMP further comprising N- terminal functionalization with SEQ ID NO:4; and(b) the functionalized second AMP comprises the second AMP further comprising N-terminal functionalization with SEQ ID NO:4.

11. The method of any one of claims 8-10, wherein the functionalized polysaccharide is a norbomene-substituted hyaluronic acid and conditions suitable for covalent bond formation comprise:(a) further contacting the at least one functionalized polysacchande, the at least one functionalized first AMP, and the at least one functionalized second AMP with a cross-linker and photoinitiator to provide a first mixture; and(b) irradiating the first mixture, optionally wherein the irradiation comprises exposure to ultraviolet light.

12. The method of claim 11, wherein at least one of the following applies:(a) the cross-linker is dithiothreitol;(b) the photoinitiator is 2-hydroxy-4’-(2-hydroxyelhoxy)-2 -methylpropiophenone (Irgacure 2959); and(c) each contacting occurs in a suitable solvent, optionally wherein the suitable solvent is a phosphate buffered saline (PBS) solution.

13. The method of any one of claims 8-10, wherein the functionalized polysaccharide is a methacrylate-substituted hyaluronic acid and conditions suitable for covalent bond formation comprise aqueous conditions, optionally wherein the conditions suitable for covalent bond formation comprise a pH of about 8 to about 10, optionally wherein the conditions suitable for covalent bond formation comprise a pH of about 9.

14. The method of any one of claims 8-10 and 13, wherein the functionalized polysaccharide is a methacrylate-substituted hyaluronic acid and the contacting further comprises further contacting the at least one functionalized polysaccharide, the at least one functionalized first AMP, and the at least one functionalized second AMP with a cross-linker, optionally wherein at least one of the following applies:(a) the cross-linker is dithiothreitol; and(b) the contacting occurs at a pH of about 8 to about 10. optionally wherein the contacting occurs at a pH of about 9.

15. The method of any one of claims 1-14, wherein the at least one bacterial species is a pathogen associated with a nosocomial infection.

16. The method of any one of claims 1-15, wherein the at least one bacterial species is selected based on a clinical isolate from a hospital or healthcare setting.

17. The method of any one of claims 1-16, wherein the bacterial species is at least one selected form the group consisting of Staphylococcus aureus, Staphylococcus epidermis, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Clostridioides difficile, Enterococcus f aecium, and Pseudomonas aeruginosa, optionally wherein the S. aureus is methicillin resistant (MRSA) or methicillin sensitive (MSSA).

18. The method of any one of claims 1-17, wherein each AMP is independently selected from the group consisting of DD13RIP, indolicidin, PIO, Alvinellacin, Ascaphin, Aurein, Apidaecin, Bac8c, Bacitracin, Bominin, Buforin, Buforin II, Camocyclin, Cathelicidin, Cecropin, Cecropin A (l-7)-Melittin A (2-9), CLP-19, Citropin, CPC 2019, Cystatin C, Defensin, Dermaseptin, Dermicidin, Dermci din, Drosocin, Epidermin, Epineci din, Esculentin, Falchin, Fallaxin, Gaegurin, HBD-2, Hepcidin, Histatins, Hongkongin, Human Beta-Defensin 3 (hBD-3), Iseganan, Jelleines. Lactoferrin, Lachesin, Lysostaphin, Maculatin, Magainin, Maximin. Melittin, MccJ25, Mi crocin Pl, Muscin. Mucroporin. Myxinidin. Nisin, Novicidin, NZ2114, NVB302, Omiganan, Onocin, Otosenin, Ovotransferrin, Palustrin, Pardaxin, Phylloseptin, Phylloxin, Platelet antimicrobial proteins, Platicin, Pleurocidin, Plectasin, Plectasin Analogs, Polybia-MPl, Polyphemusin, Protegrin, Pseudomycoicidin, Psacotheasin, Pseudin. Pyrrhocoricin, Ranalexin, Ranatuerin. Retapamulin, RNAIII inhibiting peptide, Robustin, Rugosin, Sacovirin, Saposin, TAS-101, Tachyplesin, Temporin,Thermonuclease, and Virginiamycin Ml, or a hybrid peptide comprising a combination thereof.

19. A hydrogel composition comprising at least one optionally crosslinked polysaccharide comprising monosaccharide or disaccharide monomers, wherein:(a) a plurality of the monomers thereof are independently covalently conjugated to a first AMP or a second AMP;(b) the first AMP comprises indolicidin (SEQ ID NO:2), or a modified derivative thereof;(c) the second AMP comprises PIO (SEQ ID NO:3), or a modified derivative thereof; and(d) the first AMP and second AMP have a molar ratio in the hydrogel composition of about 1:4.

20. The hydrogel composition of claim 19, wherein the first AMP and second AMP have a concentration in the hydrogel composition selected from the group consisting of about 0.31 pM and about 1.25 pM, about 0.78 pM and about 3.13 pM, about 1.56 pM and about 6.25 pM, about 3.125 pM and about 12.50 pM, and about 6.25 pM and about 25.00 pM, respectively.21 . The hydrogel composition of claim 19 or 20, wherein the polysaccharide comprises hyaluronic acid, or a modified derivative thereof.

22. The hydrogel composition of any one of claims 19-21, wherein the first AMP and second AMP are covalently conjugated to the polysaccharide via a linker.

23. The hydrogel composition of claim 22, wherein the linker comprises a moiety formed by a click reaction.

24. The hydrogel composition of any one of claims 19-23, wherein the composition comprises a crosslinked product of:(a) at least one polysaccharide compound of Formula (I):wherein: each occurrence of Y is independently selected from the group consisting of O and NRle; each occurrence of R1a, R1b, Rlc, R1d, Rle, and R1fis independently selected from the group consisting of R2, H, optionally substituted Ci-Ce alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C6-C10 aryl, optionally substituted C2-C10 heteroaryl, C(=O)RA, C(=O)ORA, C(=O)N(RA)(RB), S(=O)RA, S(=O)2RA, and S(=O)2N(RA)(RB), wherein, in each monomeric unit of the compound of formula (I), no more than one of Rla, Rlb, Rlc, Rld, Rle, and Rlfis R2;each occurrence of L1is independently selected from the group consisting of a bond, optionally substituted C2-C6 alkylenyl, optionally substituted C2- Ce heteroalkylenyl, -C(=O)N(RA)-, and -C(=O)O-; each occurrence of R2is independently an alkenyl moiety or H;R3aand R3bare each independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, C(=O)(optionally substituted Ci-Ce alkyl), and a monosaccharide or a modified derivative thereof; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-C10 heteroaryl; and m is an integer between 2 and 25,000;(b) a first polypeptide comprising SEQ ID NO:2, wherein the first polypeptide further comprises an N-terminal polypeptide of one to ten amino acids comprising at least one cysteine;(c) a second polypeptide comprising SEQ ID NO:3, wherein the second polypeptide further comprises an N-terminal polypeptide of one to ten amino acids comprising at least one cysteine; and(d) a crosslinker selected from the group consisting of -S-(optionally substituted C2-C8 alkylenyl)-S- and -S-(optionally substituted C2-C8 heteroalkylenyl)-S-.

25. The hydrogel composition of any one of claims 19-24, wherein the compound of formula (I) is selected from the group consisting of:

26. The hydrogel composition of claim 25, wherein the compound of formula (I) is the compound of formula (la), and at least one of the following applies:(a) Rla, Rlb, Rlcand Rldare each independently H;(b) Y is NR1C;27. The hydrogel composition of claim 25 or 26, wherein each monomeric unit of the compound of formula (la) is independently selected from the group consisting of:

28. The hydrogel composition of claim 25, wherein the compound of formula (I) is the compound of formula (lb), and at least one of the following applies:(a) Rla, Rlb, Rldand Rlfare each independently H;(b) Y is NRle;(c) Rleis C(=O)CH3;(d) L1is -C(=O)NH- or -C(=O)O-; and29. The hydrogel composition of claim 25 or 28, wherein each monomeric unit of the compound of formula (lb) is independently selected from the group consisting of:

30. The hydrogel composition of any one of claims 24-29, wherein at least one of the following applies:(a) the first polypeptide is SEQ ID NO:6; and(b) the second polypeptide is SEQ ID NO:7.

31. The hydrogel composition of any one of claims 24-30, wherein the crosslinker is dithiothreitol.

32. A kit for preparing hydrogel compositions comprising a synergistic combination of antimicrobial peptides (AMPs), the kit comprising:(a) at least two AMPs;(b) functionalized derivatives of each of the at least two AMPs;(c) at least one functionalized polysaccharide;(d) reagents suitable for covalent conjugation of the functionalized antimicrobial peptides and the at least one functionalized polysaccharide; and(e) instructional materials for identifying a bacterial species, identifying a synergistic combination of AMPs, and preparing a hydrogel composition comprising the synergistic combination of AMPs.

33. The kit of claim 32, wherein the functionalization of (b) and (c) are selected such that functional groups thereof comprise a complementary click chemistry pair.

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