ANTIMICROBIAL SHAPE MEMORY POLYMERS

Description

Cross-Reference to Related Application

[0001] This application claims priority to United States Provisional Patent Application No. 62 / 430,620 filed on December 6, 2016 and entitled "Antimicrobial Shape Memory Polymers".Technical Field

[0002] Embodiments of the invention are in the field of shape memory polymer medical devices.Background

[0003] Hemorrhage is the leading cause of potentially preventable death on the battlefield. The current standard of field care is to utilize gauze in combination with tourniquets; however, these treatments are insufficient for up to 80% of wounds. Furthermore, tourniquets only serve as temporary measures against blood loss, as tourniquet use beyond ~4-6 hours is associated with limb damage and loss. An improved hemostat material could enable earlier tourniquet removal before patients can receive treatment at a fixed facility. WO 2004 / 063088 A1 discloses a memory foam with excellent antibacterial activity, deodorant property and far infrared ray radiation. The memory foam is produced by mixing 75-98% by weight of polyurethane, 1-13% by weight of an inorganic porous material and 1-13% by weight of an inorganic antibacterial agent powder, and molding the mixture into the memory foam. CN 104 744 664 A relates to a production method of nano-silver memory foams, wherein nano-silver completely wraps polyurethane molecules by virtue of uniform dispersion. WO 2016 / 149070 A1 teaches a wound dressing comprising a shape memory polymer (SMP) foam, including open cells, having first and second states, and a hydrogel (HG) included within the cells.

[0004] The current standard of care on the battlefield involves the use of a broad spectrum antibiotic regimen in combination with frequent dressing changes to prevent bacterial and fungal infections; however, rising concerns over antibiotic resistance requires the use of alternative treatment methods, and dressing changes are not always feasible during battle. This has been achieved by the subject-matter of the independent claims. Preferred embodiments are defined in the dependent claims.Brief Description of the Drawings

[0005] Features and advantages of embodiments of the present invention will become apparent from the appended claims, the following detailed description of one or more example embodiments, and the corresponding figures. Where considered appropriate, reference labels have been repeated among the figures to indicate corresponding or analogous elements. Figure 1. Schematic representation of cinnamic acid (CA) incorporation into shape memory polymer (SMP) foams. Route A: In an embodiment a pre-polymer was prepared with CA, hydroxypropyl ethylene diamine (HPED), triethanolamine (TEA), and excess hexamethylene diisocyanate (HDI). The pre-polymer was reacted with the remaining CA, HPED, and TEA in the presence of catalysts, surfactants, and blowing agents while heating to form a CA-containing SMP foam. Route B: In an embodiment the carboxylic acid group on CA was esterified with a hydroxyl group on HPED to form HCA. Then, HCA was used as a foaming monomer in place of CA in the same method utilized for Route A to form a CA-containing SMP foam. Figure 2. Spectral confirmation of synthesis of HCA / CA foams. Figure 2(A) Transmission Fourier transform infrared (FTIR) spectra of HCA compared to HPED and CA, Figure 2(B) Nuclear magnetic resonance (NMR) spectra of HCA, Figure 2(C) Attenuate total reflectance (ATR)-FTIR spectra of control, CA, and HCA foams. Figure 3. Phenolic acid-containing foam compositions Figure 4. Pore size and density of SMP foams synthesized with CA and CAOH monomers. Figure 5. Thermal and shape memory properties of CA and CAOH containing SMP foams. Figure 6. E. coli colony forming units after exposure to control SMPs, SMPs with CA or CAOH, and penicillin-streptomycin (antibiotic control). Both CA and CAOH-containing SMPs reduced the size and number of colonies compared to the control SMP, and both reduced the size of colonies compared to the antibiotic control. Figure 7. SMP foam structural properties. Figure 7(A) Density, Figure 7(B) Pore size (axial and transverse foaming directions) and isotropicity (ratio between pore sizes in the axial and transverse foaming directions), and Figure 7(C) Representative scanning electron micrographs in the axial and transverse foaming directions. Scale bar applies to all images. Figure 8. Thermal and shape memory properties of HCA / CA foams. Figure 8(A) Dry and wet glass transition temperatures (Tg), Figure 8(B) Volume recovery profile in 37°C water, Figure 8(C) Time to 100% volume recovery in 37°C water. Figure 9. Representative brightfield images of human dermal fibroblasts after 2 and 72 hr of contact with SMP films. Scale bar applies to all images. Figure 10. Colony forming unit (CFU) density of Figure 10(A) E. coli and Figure 10(B) Staph. Epi. after exposure to unmodified control, CA, HCA, and penicillin-streptomycin (P / S)-soaked control SMP films. *p<0.05 relative to Control. †p<0.05 relative to 10% HCA and 20% HCA, •p<0.05 relative to all other samples. Figure 11. Colony forming unit (CFU) density of (A) E. coli and (B) Staph. Epi. after exposure to CA and HCA SMP films that had been soaked in phosphate buffered saline at 37°C for up to 30 days. Red line- Control CFU density. *p<0.05 relative to Control. •p<0.05 relative to all other time points within formulation, ▪p<0.05 relative to 10 and 20 day samples within formulation, ‡p<0.05 relative to 0 day sample within formulation. Figure 12. Antimicrobial SMP compositions. NCO: Isocyanate; OH: Hydroxyl. Detailed Description

[0006] "An embodiment", "various embodiments" and the like indicate embodiment(s) so described may include particular features, structures, or characteristics, but not every embodiment necessarily includes the particular features, structures, or characteristics. Some embodiments may have some, all, or none of the features described for other embodiments. "First", "second", "third" and the like describe a common object and indicate different instances of like objects are being referred to. Such adjectives do not imply objects so described must be in a given sequence, either temporally, spatially, in ranking, or in any other manner.

[0007] Below various embodiments are addressed. Embodiments are first addressed in the section entitled "OVERVIEW OF AN EMBODIMENT". Embodiments are then further addressed in sections entitled "HIGH LEVEL DESCRIPTION OF EMBODIMENTS" and "MORE DETAILED DESCRIPTION OF EMBODIMENTS."OVERVIEW OF AN EMBODIMENT OR EMBODIMENTS

[0008] There are numerous and growing concerns in the medical community over antibiotic-resistant bacteria that result in infections that are very difficult to treat. To address these concerns, antimicrobials are incorporated into thermoset polyurethane shape memory polymer (SMP) foams, wherein the at least one antimicrobial agent which includes at least one of cinnamic acid, benzoic acid, gentisic acid, 4-hydroxy benzoic acid, p-coumaric acid, vanillic acid, syringic acid, protocatechuic acid, gallic acid, ferulic acid, sinapic acid, caffeic acid, or combinations thereof is chemically bounded to the SMP foam. SMPs are highly valuable materials that may be employed in a range of medical devices or other devices. The presence of antimicrobial agents provides localized infection prevention around SMP-based devices. An embodiment provides that antimicrobial properties are introduced by including small antimicrobial or antimicrobial-producing agents in the polymer synthesis to chemically incorporate antimicrobials into the polymer network.

[0009] Thus, embodiments include SMP-based medical devices with infection resistance. Embodiments include antimicrobial SMPs to be employed in a wide range of medical devices including, but not limited to, endovascular medical devices, wound dressings, hemostat materials, lung puncture sealants, and / or bone grafts. In an embodiment SMP medical devices with incorporated antimicrobials work in conjunction with oral antibiotics to reduce local infection risks in damaged tissue.

[0010] Such embodiments are important for various reasons. For example, these antimicrobial SMP-based medical devices meet large clinical needs in treatment and infection prevention because infections acquired at the point of injury and / or in the hospital environment are a significant source of health care costs and contribute to patient morbidity and mortality rates. By delivering antimicrobial agents directly to the source of wounding or implantation, these infections and their complications are reduced, particularly with the use of broad-spectrum antimicrobials that are effective against drug-resistant bacteria strains. SMP medical devices can be easily delivered into small and / or irregularly-shaped defect sites. Thus, antimicrobial SMPs provide new treatment options with reduced infection risks.

[0011] Such embodiments are novel for various reasons. For example, the incorporation of non-drug-based antimicrobial agents directly into SMPs and the subsequent fabrication of devices with complex architectures are novel. A point of novelty includes incorporating non-drug small antimicrobial molecules (phenolic acids) into a SM polymer foam system to enable delivery in a medical device.

[0012] Further regarding the antimicrobial molecules, specific phenolic acids (PAs) are included. Bees produce plant-derived PAs in their honey, which protect hives against microbes and viruses. PAs exhibit broad antimicrobial properties, and have been shown to be effective against multi-drug resistant organisms (MDROs). For example, antibiotic-resistant bacteria that were obtained from hospitals (Enterobacter aerogenes, Escherichia coli, and Staphylococcus aureus) were susceptible to the PA cinnamic acid. Similarly, two other Pas (ferulic and gallic acids) reduced biofilm activity >70% for four human pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes).

[0013] As an additional advantage, the technique of utilizing antimicrobials as monomers enables fine-tuning of SMP properties using standard variables, such as hydrophobicity and crosslink density. This property expands the potential applications of antimicrobial SMPs, as they can be processed and tuned to match a range of tissue types.

[0014] There are still further advantages with embodiments. Previous work (Landsman, et al. Acta Biomaterialia (2016)) included SMP foams integrated with iodine-containing hydrogels. While these iodine "sponges" also provide an option for antimicrobial SMP system, the antimicrobial properties are fully dependent upon the hydrogel component, with no antimicrobials directly incorporated into the SMP. In contrast, an embodiment utilizes a SMP-only scaffold with varied mechanisms of incorporating antimicrobials.

[0015] Various embodiments of antimicrobial SMP foams are fabricated by a number of routes.

[0016] ROUTE 1: Functionalize an antimicrobial or peroxide-producing monomer with reactive hydroxyl or amine groups to enable their incorporation into the polyurethane. Briefly, the carboxylic acid of a phenolic acid is esterified with a foaming polyol (e.g., N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine (HPED) or triethanolamine) in an appropriate solvent with esterification catalysts (e.g., dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP)). The reaction is filtered and washed to remove byproducts and residual catalysts, and the final product is isolated by rotary evaporation. This method is appropriate for use with any antimicrobial agent that has a carboxylic acid group. The final product is utilized as a polyol in SMP synthesis with other polyols (e.g., HPED and / or TEA) and diisocyanates (e.g. hexamethylene diisocyanate, trimethyl hexamethylene diisocyanate, and / or isophorone diisocyanate).

[0017] ROUTE 2: Direct chemical incorporation into SMP. Briefly, the antimicrobial is reacted with polyols (e.g., HPED and / or TEA) and diisocyanates (e.g., hexamethylene diisocyanate, trimethyl hexamethylene diisocyanate, and / or isophorone diisocyanate) to form a polyurethane SMP.

[0018] ROUTE 3 (not acoording to the invention): Direct physical incorporation into SMP. Briefly, any antimicrobial or peroxide-producing monomer or particle (nano or micro-scale) is mixed with SMP monomers (e.g., polyols (e.g., HPED and / or TEA) and diisocyanates (e.g. hexamethylene diisocyanate, trimethyl hexamethylene diisocyanate, and / or isophorone diisocyanate)) prior to the crosslinking reaction to result in a SMP network with physically incorporated antimicrobials. Thus, the SMP foam is crosslinked around the antimicrobial or peroxide-producing monomer or particle. In an embodiment, the iso or "B" side and "poly" or "A" side of the polymer chain are crosslinked around the antimicrobial or peroxide-producing monomer or particle.

[0019] Generally, a SMP network includes switch units or segments and netpoints or domains. The netpoints determine the permanent shape of the polymer network. In an embodiment the antimicrobial agent is a physically crosslinked netpoint. With the antimicrobial agent the SMP foam comprises a composite and is therefore stronger in response to the inclusion of the antimicrobial agent.

[0020] Any of the above routes are utilized with bulk or porous SMPs. After synthesis, the material is cut, cleaned, processed, and incorporated into a medical device.

[0021] Embodiments include PAs that have been successfully modified with HPED and utilized in SMP foam and bulk film synthesis. The PA-containing SMPs have similar thermal transition temperatures to controls and demonstrate shape memory properties. The PA-containing SMPs demonstrate effective reduction in Escherichia coli growth in comparison to controls.

[0022] Various SMP foams have been discussed. Embodiments include polyurethane SMP foams synthesized by some combination of: (a) Hexamethylene diisocyanate, trimethyl hexamethylene diisocyanate, isophorone diisocyanate, triethanolamine, diethanolamine, butane diol, butyne diol, N,N,N',N' tetrakis (hydroxyl propylene) ethylenediamine, and (b) an antimicrobial agent which is limited to: phenolic acids (cinnamic acid, benzoic acid, gentisic acid, 4-hydroxy benzoic acid, p-coumaric acid, vanillic acid, syringic acid, protocatechuic acid, gallic acid, ferulic acid, sinapic acid, caffeic acid).HIGH LEVEL DESCRIPTION OF EMBODIMENTS

[0023] Antimicrobial agents are incorporated into polyurethane shape memory polymers (SMPs), Figure 1. In some embodiments, incorporated antimicrobials work in conjunction with oral antibiotics to reduce infection risks in implanted SMP-based devices. There are numerous concerns about antibiotic-resistant bacterial strains. To address this issue, antimicrobial phenolic acids (PAs) are utilized in a non-drug approach. Bees produce plant-derived PAs in their honey, which protect hives against microbes and viruses. PAs exhibit broad antimicrobial properties, and have been shown to be effective against multi-drug resistant organisms (MDROs). For example, antibiotic-resistant bacteria that were obtained from hospitals (Enterobacter aerogenes, Escherichia coli, and Staphylococcus aureus) were susceptible to cinnamic acid. A recent review covers the efficacy of a number of PAs, including cinnamic, gentisic, and benzoic acids, against Candida infections (planktonic and biofilms, drug susceptible and drug resistant). Furthermore, ferulic and gallic acids reduced biofilm activity >70% for four human pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes). By taking a single component of honey (e.g., PA) and utilizing it as a foaming monomer, we harness the benefits of PAs while maintaining a synthetic medical device system.

[0024] Study 1.1: Fabricate PA-containing SMP foams. PA (modified and unmodified) antimicrobial efficacy against MDROs is measured to down-select agents prior to foam fabrication. For all agents with acceptable antimicrobial properties, SMP foams are synthesized with varying levels of cinnamic, gentisic, and benzoic acid via (a) prior esterification with foaming polyols to produce a PA-containing polyol (PAOH) and (b) direct incorporation. In an embodiment route (a) requires fewer alterations to foam composition. Prior esterification enhances the antimicrobial properties of PAs. Furthermore, route (a) eliminates / reduces bubble generation from the reaction between carboxylic acids on PAs and isocyanates in the foams to better control foam properties. Route (b) is simpler and less expensive. Thus, in some embodiments foams synthesized via route (b) that meet all success criteria and produce consistent and reliable foams are advantageous.

[0025] Methods: PAOH Synthesis: PAOH is synthesized from hydroxypropyl ethylenediamine (HPED) and select PAs (cinnamic acid, gentisic acid, and benzoic acid) via esterification in chloroform (catalyzed by dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP)) for 3 hours at room temperature, Figure 1. The reaction is filtered and washed to remove byproducts and residual catalysts, and the final product is isolated by rotary evaporation. Successful synthesis of PAOH is confirmed using Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy.

[0026] Results: Cinnamic acid-HPED (CAOH) was successfully synthesized, as indicated by FTIR spectra that show a reduction in hydroxyl groups relative to HPED and formation of ester linkages, Figure 2.

[0027] Foam Synthesis: Because PAs and PAOH derivatives are effective antimicrobials, foams are synthesized with varied amounts of PAOH in combination with HPED and hexamethylene diisocyanate (HDI) as previously described, Figure 1. Briefly, a polyurethane prepolymer is synthesized using an excess of HDI with PAOH and HPED. The remainder of PAOH and HPED are mixed together and added to the prepolymer at a final molar ratio of 1:1 (isocyanates (HDI): hydroxyls (PAOH and HPED)) in some embodiments. Upon addition of the hydroxyl mix to the prepolymer, catalysts, surfactants, water (chemical blowing agent), and Enovate (physical blowing agent) are added, and the components are mixed until homogenous. The resulting foaming mixture is cured at 90°C for 20 minutes in an embodiment. In parallel, PA-containing foams are synthesized via direct incorporation of phenolic acids into the polyurethane backbone (reaction between carboxylic acid on PAs and isocyanates in foam), Figure 3.

[0028] Study 1.2: Characterize PA-containing foam structure; mechanical, thermal, and shape memory properties; and cytocompatibility. After a library of PA-containing foams is synthesized, they are characterized to ensure that desirable foam properties are retained. In particular, embodiments of PA-containing foams have thermal transitions at body temperature in aqueous conditions to enable shape change upon application of a bleed, rapid shape recovery (<2 minutes) to enhance wound filling and clotting speed, and high cytocompatibility as an initial indication of their safety. These properties are achieved with a number of the compositions described herein.

[0029] Methods: Structure: After incorporation of PAOH and PAs into foams, foam pore size and structure are quantified. Thin samples are cut laterally and longitudinally from each foam, sputter coated in gold, and imaged using scanning electron microscopy (SEM). Pore sizes and strut thicknesses are quantified from SEM images using ImageJ software.

[0030] Results: CA and CAOH were successfully incorporated into SMP foams, resulting in low density foams with retained pore sizes of ~1000 µm, Figure 3.

[0031] Thermal Properties: The glass transition temperature (Tg) of control and modified foams are measured using differential scanning calorimetry (DSC) under wet and dry conditions. For dry Tg, a 3-8 mg sample is loaded into an aluminum pan at room temperature, cooled to -40°C using the DSC, and then heated to 120°C. The sample is cooled and heated again, and Tg is recorded from the second cycle as the inflection point of the thermal transition curve. For wet Tg, 3-8 mg foam samples are submerged in reverse osmosis water at 50°C for 5 min to allow full plasticization and then pressed dry with laboratory wipes. Samples are weighed and placed in a vented aluminum pan. The DSC is used to cool the samples to -40°C and heat them to 80°C. The wet Tg (after water plasticization) is determined using the average inflection point of the thermal transition.

[0032] Results: CA and CAOH-containing SMP foams exhibited retained dry thermal properties relative to control, Figure 4. The wet glass transition temperatures were reduced relative to the control, but maintained below body temperature to enable actuation upon implantation.

[0033] Shape Memory Properties: Cylindrical foam samples (2 mm diameter X 1 cm length) are cut, and a 203.20 µm diameter nickel titanium wire is threaded through the length of the sample to serve as a stabilizer. Samples are be crimped to their smallest possible diameter using a stent crimper with heating above the Tg. Initial foam diameter is measured using ImageJ software, and the foams are placed in a water bath at 37°C. Images are taken over 30 minutes, and foam diameter is calculated at each time point using ImageJ ®< . Percent recovery versus the original sample diameter and volume expansion is calculated using the resulting measurements.

[0034] Results: CA and CAOH-containing SMP foams exhibited retained shape memory properties with high volume recovery and volume expansion, Figure 5.

[0035] Study 1.3: Assess antimicrobial properties of PA-containing foams in comparison to clinically available silver-based antimicrobial wound dressings. As an initial indication of the antimicrobial efficacy of PA-containing SMP foams, a series of in vitro studies were conducted with bacteria. Embodiments include formulations with comparable antibacterial properties to those of clinically available antimicrobial oxidized regenerated cellulose hemostats.

[0036] Methods: E. coli (gram-negative) and S. epidermidis (gram-positive) colony forming units (CFUs) are counted after exposure to control foams, PA and PAOH foams, and cellulose hemostats (Surgicel ®< , Ethicon).

[0037] Results: CA and CAOH SMPs greatly inhibit E. coli growth (number and size of colonies) relative to control SMPs, Figure 6.MORE DETAILED DESCRIPTION OF EMBODIMENTS

[0038] SMP foams embodiments addressed herein provide a biomaterial platform with numerous potential benefits for use as hemostatic dressings. Polyurethane SMPs are fabricated as expanded, open porous foams that can be compressed into a temporary, secondary shape. The compressed shape is retained until the foam is exposed to water and heat, upon which it returns to the original expanded shape. Embodiments are designed to actuate in aqueous conditions between 15 and 45°C (~40-70°C in dry conditions), and actuation times are tuned between 30 seconds and 30 minutes. SMP foams demonstrated excellent biocompatibility over 90 and 180 days of implantation in a porcine aneurysm model. Importantly, these foams induce rapid clotting due to their high surface area and thrombogenic material chemistry, in a porcine hind limb vessel, SMP foams promoted arterial hemostasis within 90 seconds of device deployment. SMP foam embodiments have minimal particulate generation, and no undesired downstream clotting has been observed in prior in vivo occlusion studies. Embodiments enable application of a compressed device in a deep, irregularly-shaped bleed site, which rapidly expands upon passive heating to body temperature to space-fill the wound volume and promote hemostasis.

[0039] One of the benefits of SMP system embodiments described herein is tunable material chemistry. To enhance hemostatic device performance, antimicrobial agents are introduced into the polymer network. In an embodiment incorporated antimicrobials work in conjunction with oral antibiotics to reduce infection risks and the need for frequent dressing changes. To address concerns about antibiotic-resistant bacterial strains, antimicrobial phenolic acids were utilized to provide a non-drug approach. Bees produce plant-derived phenolic acids in their honey, which protect hives against microbes and viruses. Phenolic acids exhibit broad antimicrobial properties, and have been shown to be effective against multi-drug resistant organisms (MDROs). For example, antibiotic-resistant bacteria that were obtained from hospitals (Enterobacter aerogenes, Escherichia coli, and Staphylococcus aureus) were susceptible to cinnamic acid. A recent review covers the efficacy of a number of phenolic acids, including cinnamic, gentisic, and benzoic acids, against Candida infections (planktonic and biofilms, drug susceptible and drug resistant).

[0040] Embodiments incorporate native and modified cinnamic acid (CA) into the SMP system, Figure 1. The resulting scaffolds were characterized to ensure that the desirable SMP properties were maintained, including density, pore size and structure, thermal properties, shape recovery profiles, and cytocompatibility. An emphasis was placed on design of a SMP foam hemostat that could be stored in extreme battlefield conditions and actuate quickly once exposed to water in blood at body temperature. Antimicrobial efficacy against Escherichia coli (E.coli) and Staphylococcus epidermidis (Staph. epi.) was characterized after soaking samples for up to 30 days in saline at body temperature to gain an understanding of initial and sustained antimicrobial effects.Results and Discussion

[0041] Synthesis of CA and HCA-containing SMPs: To enable its incorporation without sacrificing polyurethane network crosslink density, cinnamic acid (CA) was modified via esterification with N,N,N',N'-tetrakis(2-hydroxypropyl) ethylenediamine (HPED) to form a CA-containing triol (HCA). Successful synthesis of HCA was confirmed via Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. In the FTIR spectra, a relative decrease in the hydroxyl groups can be observed at ~3350 cm -1< , and ester formation was confirmed via a shift in the carbonyl peak from ~1680 to ~1710 cm -1< , Figure 2A. NMR showed the presence of esterified CA and HPED, with ~90% functionalization, Figure 2B.

[0042] Upon successful synthesis of HCA, SMP foams were prepared with 10, 20, and 30% HCA (mol% of hydroxyl groups, based off of 3 hydroxyl groups per mole of HCA).

[0043] Foams were made with 10, 20 and 30% CA (mol% of hydroxyl groups, based off of 1 hydroxyl group (i.e. carboxylic acid) per mole of CA). The 10% CA foams rose, but 20 and 30% CA, a stable network was not able to form and the foams collapsed. This result was attributed to the termination of the polyurethane network upon reactions between HDI and mono-functional CA. While direct incorporation of CA is faster and simpler, its single functional group is a limitation to its effective use in this SMP system. This result also validates the additional HCA synthesis step, as it allows for more effective incorporation of higher concentrations of CA. The relatively straightforward synthesis method of HCA could be utilized with a range of phenolic acids as claimed or other carboxylic acid-containing functional molecules (not according to the invention), (e.g. drugs, bioactive factors) in some embodiments to impart new properties to the SMP system.

[0044] ATR-FTIR spectra were obtained of CA and HCA containing foams, Figure 2C. There is an additional peak at ~1615 cm -1< in all of the CA and HCA foams that is attributed to the C=C groups in the ring structure. The relative absorbance of this peak increases slightly (relative to the urethane peak at ~1680 cm -1< ) with increased HCA concentration. In the 10% CA spectra, the relative absorbance at the 1615 cm -1< peak is between that of 20 and 30% HCA, indicating a higher incorporation efficiency with the unmodified CA. These spectra confirm successful incorporation of CA and HCA into the SMP network. Both routes are viable for effective synthesis of phenolic acid-containing foams.

[0045] Structural properties: Foam density and pore sizes were assessed to verify the qualitative observations of successful foam blowing with CA and HCA, Figure 3. In general, CA and HCA-containing foams retain the ultralow density that is characteristic of this SMP system, Figure 3A. The 10% HCA foams had a higher density, and there was a trend of decreasing density with increased HCA concentration. These results correspond with pore size measurements, with increasing pore sizes as HCA concentration is increased, Figure 3B. Qualitatively, 10 and 20% HCA foams have less rounded pores, which further correlates with density measurements, Figure 3C. Despite these minor differences, overall, CA and HCA foam pore sizes were comparable to those of the control foam, Figure 3B-C. Improved isotropicity (ratio between pore sizes in the axial and transverse foaming directions) was observed with the CA and HCA foams, indicating that the new monomers aided in constricting the foam rise process to provide more homogeneous pores, Figure 3B. Overall, these results show that CA and HCA can be incorporated into SMP foams with minimal protocol changes to provide structurally similar materials, further indicating the potential for this method to be used effectively with other functional monomers with similar reactive groups.

[0046] Thermal and shape memory properties: For effective field use, SMP foam-based hemostats must retain their compressed geometries at the high temperatures that are reached in desert climates (e.g., up to ~45°C in Iraq) under dry storage conditions and then rapidly expand upon exposure to water in body temperature blood. As an initial indication of these capabilities, SMP foam glass transition temperatures (Tg's) were measured under dry and wet conditions, Figure 8A. The 10% CA foam dry Tg was similar to that of the control, and both were below the required 45°C for field use. CA increases the hydrophobicity of the foams, which generally increases Tg; however, incorporation of the monofunctional group is expected to reduce the overall crosslink density, which had an opposing effect to lower Tg. To increase dry Tg of CA foams to a useful level, overall crosslink density could be increased by utilizing more HPED, or hydrophobicity could be further increased with the introduction of more hydrophobic monomers, such as trimethyl hexamethylene diisocyanate. Incorporation of HCA allowed for crosslink density to be retained while increasing hydrophobicity, resulting in increases in dry Tg with increased HCA content. All HCA-containing foams had dry Tg's at or above 45°C, indicating their potential for use on the battlefield without premature expansion. Additionally, HCA incorporation provides a new tool for tuning SMP foam thermal properties. All CA and HCA foams had wet Tg's below 30°C, allowing for expansion after exposure to body temperature blood, even if the patient is in hypovolemic shock from blood loss and / or subjected to hypothermia.

[0047] To further explore the functional capabilities of CA and HCA SMP foams, their volume expansion profiles were characterized in 37°C water, Figure 8B-C. CA foams had more rapid expansion as compared to controls, which is attributed to their theoretically decreased crosslink density due to the incorporation of a monofunctional monomer. Increasing HCA content resulted in slower volume expansion, correlating with increased Tg measurements, and is attributed to increased foam hydrophobicity and backbone stiffness with the introduction of the ring structure in CA. The increase in expansion rate of 10 and 20% HCA foams relative to the control is likely due to network inconsistencies. Although high average functionalization of HCA was achieved, the synthesized monomer is a mix of HPED with varied numbers of tethered CA molecules. The resulting network inhomogeneity can result in more rapid volume expansion, despite increased hydrophobicity. This effect was outweighed with increased HCA content in the 30% HCA foam, where increased hydrophobicity likely had a larger effect on slowing down expansion than network inconsistencies. To address severe bleeds, SMP-based hemostats should achieve full expansion as quickly as possible. Full expansion of 10 and 20% HCA in less than two minutes in combination with their high dry Tg's is highly promising for their potential use as hemostats on the battlefield. The variations in volume expansion rates with increased HCA content further validates the ability to use HCA incorporation as a tool for tuning SMP foam properties.

[0048] Cytocompatibility: As an initial indication of CA and HCA-containing SMP cytocompatibility, human dermal fibroblasts (HDFs) were indirectly exposed to SMP films, and their morphology, initial attachment, and proliferation were qualitatively assessed, Figure 9. Initial attachment was similar between wells containing SMP films and the positive control, TCPS. HDFs were evenly attached across the well surfaces and well spread. The negative control, BSA-coated TCPS, had lower attachment and rounded cells. At 72 hours, HDFs had elongated and proliferated in all SMP film-containing wells and the TCPS control with some areas of confluence. The BSA negative control wells still had low attachment numbers and lower spreading after 72 hours. These studies indicate that the modifications did not negatively affect the cytocompatibility of the base SMP formulation, which is a benefit for their use as biomaterial scaffolds.

[0049] Antimicrobial properties: To measure antimicrobial efficacy of CA and HCA following incorporation into SMPs, colony forming unit (CFU) density of E. coli (gram negative) and Staph. epi. (gram positive) were measured following exposure to SMP films. There were large reductions in E. coli CFUs after exposure to CA and HCA-containing films in comparison to unmodified control SMP films. CFU density was at or below that of drug-based (penicillin streptomycin, P / S) controls, Figure 10A. CFU reductions relative to unmodified control SMP films were significant for 10% CA and 30% HCA films. The improved performance of CA at lower concentrations is likely due to more effective incorporation, as indicated by an increased C=C peak at 1615 cm-1 in the FTIR spectra (Figure 2C). The increased efficacy of 30% HCA illustrates how antimicrobial properties should increase with increased CA concentration in the films. Similar results were observed with Staph. epi., with significant decreases in CFU density for 10% CA, 20% HCA, and 30% HCA films to levels at or below the drug-based (P / S) control, Figure 10B. There was a trend of decreasing CFU density with increased HCA concentration. Some phenolic acids have shown increased antimicrobial efficacy after esterification. This result has not been observed with CA. Embodiments include incorporation of phenolic acids that benefit from esterification. Overall, these results demonstrate that the antimicrobial properties of CA are retained following incorporation into SMPs with and without prior modification. The comparable efficacy to P / S is highly promising for potential use of CA or HCA-containing SMPs against drug resistant organisms.

[0050] To characterize antimicrobial property retention, CA and HCA containing films (not according to the invention) were soaked in saline solution at 37°C for up to 30 days, and E. coli and Staph. epi. CFU density was measured after exposure to soaked films. All 10% CA films retained significantly lower CFU density compared to the unmodified control SMP for both bacteria types, Figure 11. The formation of a urethane bond between CA and HDI in the SMP network provides a biostable linkage that is not susceptible to hydrolysis. In an embodiment the majority of the CA was retained in the films throughout the soaking period, providing a sustained antimicrobial effect. The 10% HCA films had comparable CFU densities up to 20 days of soaking, with increases for the 30 day samples that approached the control film value (red dashed line) for both bacteria types. Since HCA is incorporated into the SMP network via an ester linkage, hydrolysis likely caused HCA concentration reductions over time. A similar trend was observed with 20% HCA samples, with increases in the CFU density after exposure to the 20 and 30 day soaked films. Increases in E. coli density were lower for 20% HCA at 30 days than those for 10% HCA, indicating a higher retained HCA concentration with increased initial concentration. This trend was further confirmed with 30% HCA films. While CFU density increases were observed between the 0 and 10 day soaked samples, indicating an initial loss of HCA, the CFU density did not dramatically change past 10 days of soaking and never approached that of control films. Thus, even with the hydrolytically-labile linkage, HCA provides a sustained antimicrobial effect in SMPs when incorporated at higher concentrations. These results combined with the favorable thermal and shape memory properties of HCA-containing SMPs indicate their potential for use as antimicrobial hemostats.Experimental Section

[0051] Materials: DC 198, DC 5943, BL-22, T-131, and Enovate ®< were purchased from Evonik ®< (Essen, Germany) and used as received. All other chemicals were purchased from Sigma-Aldrich Inc. (St. Louis, MO) and used as received.

[0052] Phenolic acid monomer synthesis and characterization: N,N,N',N'-tetrakis(2-hydroxypropyl) ethylenediamine (HPED)-cinnamic acid (HCA) was synthesized using an esterification procedure. Cinnamic acid (CA, 1 molar eq.) was added to a round bottom flask and dissolved in chloroform. Then, 4-(dimethylamino) pyridine (DMAP, 0.1 molar eq.) was added to the flask and dissolved. HPED (1 molar eq.) was weighed out in a separate vial, dissolved in chloroform, and added dropwise to the reaction flask. The flask was placed on ice to cool for ~5 minutes. N,N'-dicyclohexyl carbodiimide (DCC, 1.1 molar eq.) was weighed into a separate vial, dissolved in chloroform, and added dropwise to the chilled reaction vessel. The reaction was stirred under nitrogen on ice for 5 minutes and then allowed to proceed at room temperature for 3 hours. After the reaction was complete, the flask was placed at 0°C for 30 minutes to precipitate dicyclohexyl urea, which was then removed via vacuum filtration. The reaction solution was washed twice with 1 molar eq. of 0.1M HCl and then washed with an aqueous saturated sodium bicarbonate solution. The organic phase was dried over magnesium sulfate and filtered. Then, chloroform was removed using rotary evaporation following drying under vacuum overnight. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy were utilized to confirm synthesis of HCA. 1H NMR (CdCl 3 ): 1.05 ppm (m, HOCHCH 3 , 9H), 1.2 ppm (m, -OCHCH 3 , 3H), 2.2-2.6 ppm (m, -CH 2 -, 12H), 3.6 ppm (m, HOCHCH 3 , 3H), 5.1 ppm (m, -OCHCH 3 , 1H), 6.4 ppm (d, - CCH=, 1H), 7.3-7.6 ppm (m, -HC=CHC 6 H 5 , 6H).

[0053] Shape memory polymer (SMP) foam and film (not according to the invention) synthesis: An isocyanate (NCO) pre-polymer was synthesized with appropriate molar ratios of HPED, triethanolamine (TEA), HCA or CA, and hexamethylene diisocyanate (HDI), with a 42 mol% hydroxyl (OH) content. A OH mixture was prepared with the remaining molar equivalents of HPED, TEA, and HCA or CA. Foaming agents (catalysts, surfactants, deionized water, and Enovate) were mixed with the NCO-prepolymer and the OH mixture using a speedmixer (FlackTek, Inc., Landrum, SC) to induce foam blowing.

[0054] The foams were then cured at 50°C for 5-10 minutes and cooled to room temperature before washing via sonication in isopropyl alcohol (IPA) or reverse osmosis (RO) water for 15 minute cycles. The purified foams were lyophilized to dry.

[0055] SMP films (not according to the invention) were synthesized using the same monomer compositions as the foams, but without surfactants, deionized water, or Enovate ®< . Figure 12 shows the SMP compositions that were synthesized and characterized in these studies.

[0056] SMP foam density: SMP foam density (n=3) was quantified on foam blocks cut from the top, middle, and bottom section of the foam, according to ASTM standard D-3574. Foam block masses was measured using a gravimetric scale, and length, width, and height values were measured three times per sample using a digital caliper. Density was calculated as mass divided by volume.

[0057] SMP foam pore size and structure: To assess pore sizes, thin slices (~1 mm, n=3) were cut from each foam composition in the axial (parallel to foam rise) and transverse (perpendicular to foam rise) foaming directions. Samples were mounted to sample holders with carbon black tape and sputter-coated for 60 seconds at 20 mA (Cressington Sputter Coater ®< , Ted Pella, Inc. Redding, CA). Samples were imaged using a Jeol NeoScope JCM-5000 Scanning Electron Microscope (SEM) ®< (Nikon Instruments, Inc., Melviille, NY). A line was drawn through the center of each image, and pore size was measured for 5 randomly selected pores on the line using ImageJ software.

[0058] SMP foam thermal transitions: The glass transition temperature (Tg) was measured under wet and dry conditions (n=5). To measure dry Tg, foam samples (3-8 mg) were cut and stored with desiccant prior to testing. A Q-200 DSC ®< (TA Instruments, Inc., New Castle, DE) was used to obtain the thermogram for each composition using the following program: (1) temperature was decreased to -40°C at 10°C·min-1 and held isothermally for 2 minutes, (2) temperature was increased to 120°C at 10°C·min-1 and held isothermally for 2 minutes, (3) temperature was decreased to -40°C at 10°C·min-1 and held isothermally for 2 minutes, and (4) temperature was increased to 120°C at 10°C·min-1. The dry Tg was recorded from the second heating cycle using the inflection point of the thermal transition curve. TA Instruments ®< software (TA Instruments, Inc., New Castle, DE) was utilized to determine the inflection points.

[0059] For wet Tg measurements, foam samples (3-8 mg) were submerged in reverse osmosis (RO) water at 50°C for 5 minutes to allow full plasticization. The samples were removed from the water, pressed dry with laboratory wipes, weighed, and placed in an aluminum pan with a vented aluminum lid. A Q-200 DSC was used to cool the samples to - 40°C at 10°C·min-1 and hold them isothermally for 2 minutes. The samples were then heated to 80°C at 10°C·min-1. TA Instruments software was used to generate the thermogram and determine the wet Tg using the average inflection point of the thermal transition.

[0060] Various examples at the end of the application address Tg, such as dry Tg and wet Tg. Those terms should be construed in keeping with the immediately preceding two paragraphs (i.e., as used herein dry and wet Tg should be determined using the methods described in the two paragraphs immediately above).

[0061] Volume recovery: Cylindrical foam samples (n=3, diameter = 4 mm, length = 10 mm) were prepared, and a stabilizing 203.20 µm diameter nickel-titanium wire (NDC, Fremont, CA) was threaded through the center of each sample along its length. The foam samples were radially crimped to their smallest possible diameter using an ST 150-42 stent crimper (Machine Solutions, Flagstaff, AZ). Samples were heated to 100°C, held isothermally for 15 minutes, and programmed to the crimped morphology by cooling to room temperature. Initial foam diameter was measured for each sample using Image J ®< software (NIH, Bethesda, MD). The crimped foams were placed in a 37°C water bath, and images were taken every 30 seconds up to 7 minutes. Foam diameter was measured at each time point at 5 evenly spaced locations along the foam length using Image J ®< . Percent volume recovery was calculated using Equation 1. % Volume Recovery = Recovered diameter Original diameter 2 ∗ 100

[0062] Cell interactions: Human dermal fibroblasts (HDFs, Invitrogen, Inc., San Diego, CA) were used to assess cell attachment and spreading. In vitro culture was carried out at 37°C / 5% CO 2 with Medium 105 (Invitrogen) supplemented with low serum growth supplement (Invitrogen) and 1% penicillin-streptomycin (P / S, Gibco). Cells were used at passage 3.

[0063] SMP films (not according to the invention) were cut into 6 mm diameter cylinders and sterilized via incubation in 70% ethanol overnight and subsequent washing in sterile phosphate buffered saline (PBS, 3 washes). As a negative cell attachment control, wells in a 96 well tissue culture polystyrene (TCPS) plate were blocked with a sterile 5% bovine serum albumin (BSA) in PBS. Unmodified TCPS wells served as positive cell attachment controls. HDFs were seeded into wells containing SMP films at 5,000 cells cm -1< . Seeded cells were cultured at 37°C / 5% CO2 for up to 72 hours. Media was changed at 2 and 36 hours. At 2 and 72 hours, brightfield images were obtained to qualitatively assess cell attachment and proliferation. Representative images were obtained using a Nikon Eclipse TE2000-S ®< with 4 field views per specimen and 3 specimens per sample type.

[0064] Antimicrobial properties: To obtain an initial measure of antimicrobial properties, SMP films (not according to the invention) were cut into 6 mm diameter cylinders. To characterize antimicrobial properties over time, samples were incubated in PBS at 37°C for 0, 10, 20, or 30 days. Then, films were sterilized as described in the Cell Interactions Section. Escherichia coli (E. coli) and Staphylococcus epidermidis (Staph. epi.) were grown overnight in 5 ml of lysogeny broth (LB) at 37°C. Subsequently, 500 µl were taken from each overnight culture and grown in 10 ml of fresh LB to optical density (O.D.) 0.6 (i.e. until bacteria had entered log phase growth). O.D. was measured using a Tecan plate reader. Samples were placed into a sterile 96 well plate, and 100 µl of bacteria solution were pipetted onto the surface of each sample. Control SMP films were soaked in P / S overnight to provide a drug-based antimicrobial control. Samples were incubated with bacteria for 1 hour at 37°C and then vortexed to dislodge attached bacteria. Bacterial solutions were diluted by 106 in fresh LB and plated onto LB-agar plates overnight at 37°C. Images were obtained of each specimen plating area. Colony forming unit (CFU) density was measured by counting the number of colonies and dividing by the plating area.

[0065] Statistics: Data are reported as mean ± standard deviation. Student's t-test was used to determine statistical significance, which was accepted at p<0.05.

[0066] Embodiments demonstrate successful incorporation of CA, a honey-based phenolic acid, into SMP foams via two routes. The resulting foams retain the desirable porous structure of the control SMP while providing tunable thermal transitions and shape recovery properties that are ideal for their use in hemostats for bleeding control. Namely, CA-based SMPs were designed with high dry Tg's to enable their storage under extreme battlefield conditions and low wet Tg's to enable their rapid shape recovery upon exposure to blood at body temperature. Furthermore, CA-based SMPs have high cytocompatibility while effectively reducing bacterial growth to levels that are comparable to penicillin / streptomycin-based treatments, even after 30 days of storage in saline at body temperature. Overall, embodiments provide a hemostat device that is easy-to-use, biocompatible, and antimicrobial. An additional benefit to phenolic acids is their antioxidant properties; phenolic acids contain hydrogen donating groups that scavenge free radicals and reduce oxidation. This is ideal for SMP foams that are susceptible to oxidative degradation. In other words, pendent phenolic acids may be used in biodurable implants. For example, occlusive foams (e.g., foams for occulting aneurysms) that are biodurable help prevent recanalization-a problem experienced with hydrogel and coil based aneurysm therapies.

[0067] The foregoing description of the embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

Claims

1. A wound dressing or hemostat device system comprising a thermoset polyurethane shape memory polymer (SMP) foam that includes at least one antimicrobial agent, wherein: the SMP foam is chemically bonded to the at least one antimicrobial agent; the at least one antimicrobial agent includes at least one of cinnamic acid, benzoic acid, gentisic acid, 4-hydroxy benzoic acid, p-coumaric acid, vanillic acid, syringic acid, protocatechuic acid, gallic acid, ferulic acid, sinapic acid, caffeic acid, or combinations thereof; the SMP foam is biocompatible.

2. The system according to claim 1 wherein the at least antimicrobial agent is a pendent group chemically bonded to a polyurethane polymer chain of the SMP foam.

3. The system of claim 2 wherein the pendent group is chemically bonded to the polyurethane polymer chain via an ester.

4. The system according to any of claims 1-3 wherein the SMP foam has a dry glass transition (Tg) above 40 degrees C and a wet Tg below 30 degrees C.

5. The system of claim 1 wherein the SMP foam is physically cross-linked around the at least one antimicrobial agent.

6. The system according to any of claims 1-5 comprising: a thermoset polyurethane second SMP foam that includes a phenolic acid that is a pendent group chemically bonded to a polyurethane polymer chain of the second SMP foam; and a thermoset polyurethane third SMP foam that includes a phenolic acid that is a pendent group chemically bonded to a polyurethane polymer chain of the third SMP foam; wherein (a) the SMP foam comprises a first foam, and (b) the first, second, and third SMP foams are all enclosed in a sealed kit7. A method for preparing the wound dressing or hemostat device system of any of claims 1to 6 comprising: reacting a first polyol portion with the antimicrobial agent to form a first reaction product; reacting a first portion of the first reaction product with a second polyol portion and an isocyanate to form a second reaction product; reacting the second reaction product with a second portion of the first reaction product and a third polyol portion to form a third reaction product; mixing the third reaction product with a blowing agent to form theSMP foam; wherein the SMP foam is a thermoset polyurethane SMP foam.

8. The method according to claim 7 wherein reacting a first polyol portion with the antimicrobial agent to form a first reaction product comprises esterification of the antimicrobial agent; and / or wherein the first reaction product comprises a triol; and / or wherein at least one of the first, second, and third polyol portions includes at least one of triethanolamine (TEA), diethanolamine, butane diol, butyne diol, N,N,N',N' tetrakis (hydroxyl propylene) ethylenediamine (HPED), and combinations thereof; and the isocyanate includes at least one of hexamethylene diisocyanate (TMHDI), isophorone diisocyanate (IPDI), and combinations thereof; and / or wherein the antimicrobial agent is a pendent group chemically bonded to a polyurethane polymer chain of the SMP foam; and / or wherein the pendent group is chemically bonded to the polyurethane polymer chain via an ester; and / or wherein the second reaction product is a prepolymer formed before polymerization of the SMP foam.

9. The method of claim 8 wherein the pendent group is chemically bonded to the polyurethane polymer chain via an ester.

10. A method for preparing the wound dressing or hemostat device system of any of claims 1 to 6 comprising: reacting a first polyol portion with a first antimicrobial agent portion and an isocyanate to form a first reaction product; reacting the first reaction product with a second polyol portion and a second antimicrobial agent portion to form a second reaction product; mixing the second reaction product with a blowing agent to form the SMP foam; wherein the SMP is a thermoset polyurethane SMP foam; wherein the first reaction product is a prepolymer formed before polymerization of the SMP foam.