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Inherently Antimicrobial Biodegradable Polymers in Tissue Engineering Emma Watson, Alexander M. Tatara, Dimitrios P. Kontoyiannis, and Antonios G. Mikos ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00501 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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ACS Biomaterials Science & Engineering

Inherently Antimicrobial Biodegradable Polymers in Tissue Engineering

Emma Watson1,2, Alexander M. Tatara1,2, Dimitrios P. Kontoyiannis3, and Antonios G. Mikos1,4*

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Department of Bioengineering, Rice University, Houston, TX

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Medical Scientist Training Program, Baylor College of Medicine, Houston, TX

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Department of Infectious Diseases, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX

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Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX

*

Corresponding author

Antonios G. Mikos, PhD

University of Texas MD Anderson Cancer

Department of Bioengineering, MS-142

Center

BioScience Research Collaborative

1515 Holcombe Blvd.

Rice University

Houston, TX 77030

6500 Main Street Houston, TX 77030 e-mail: [email protected]; Tel: (713) 348-5355 Fax: (713) 348-4244

Baylor College of Medicine 1 Baylor Plaza Houston, TX 77004

ACS Paragon Plus Environment


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ABSTRACT Many of the strategies currently being explored in the field of tissue engineering involve the combination of cells and degradable engineered scaffolds for the regeneration of biological tissues. However, infection of the wound or the scaffold itself results in failure of healing. Therefore, a new area of development in the field is the synthesis of polymer-based scaffolds that inherently have the ability to resist microbial infection as degradation occurs and new tissue replaces the scaffold. These scaffolds, defined as inherently antimicrobial biodegradable polymers (IABPs), can be classified based on their monomeric components as follows: 1) traditional antimicrobials (such as beta-lactams, fluoroquinolones, glycopeptides, and aminoglycosides), 2) naturally-derived compounds (such as extracellular matrix components, chitosan, and antimicrobial peptides), and 3) novel synthetic antimicrobials. After validation of chemical synthesis as well as physicochemical characterization of a newly-created IABP, thorough in vitro and in vivo assays must be conducted to ensure antimicrobial efficacy as well as biocompatibility as a tissue-engineered scaffold system. In this review, we will introduce existing IABPs, discuss the current platforms that have been developed for the synthesis of IABPs, and highlight future directions as well as challenges in the field.

Keywords: Antimicrobial, tissue engineering, biodegradable, polymer

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ABBREVIATIONS: AM = acrylamide AMPs = antimicrobial peptides BA = butyl acrylate DADMAC = diallyldimethyl ammonium chloride DCC = dicyclohexylcarbodiimide DMAP = 4-(dimethylamino)pyridine EA = ethyl acrylamide ECM = extracellular matrix EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide IABPs = inherently antimicrobial biodegradable polymers MIC = minimum inhibitory concentration MMA = methylmethacrylate MRSA = methicillin-resistant Staphylococcus aureus MSSA = methicillin-susceptible Staphylococcus aureus NHS = N-hydroxysuccinimide NO = nitric oxide PCL = poly(ε-caprolactone) PEG = poly(ethylene glycol) PEGMEM poly(ethylene glycol) methyl ether methacrylate PLA = poly(lactic acid) PMMA = poly(methylmethacrylate) PN = poly(norbornene) PVA = poly(vinyl alcohol) VRE = vancomycin-resistant enterococci

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INTRODUCTION Infection remains the third-leading cause of death in developed countries and the second-leading cause of death-worldwide.1 The antibiotic era began in the 1930s with the development of penicillin-based therapy and continued to rapidly evolve over several decades, but development of new antimicrobial agents has recently slowed.2 With widespread use, resistance to existing antibiotics is also increasing, with several known organisms demonstrating resistance to multiple commonly used antibiotics.3,4 Therefore, there is a clinical need for the development of new antimicrobial therapies. Cells, scaffolds, and signals form the backbone of tissue engineering.5 Scaffolds can serve many purposes, such as promoting native tissue ingrowth, releasing signaling molecules, and delivering cells to assist in regeneration.5 These scaffolds have been used to deliver a variety of molecules, from growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor6,7 to antibiotics.8,9 Many of these scaffolds are fabricated from polymers which are capable of serving as drug depots for the controlled release of antibiotics. Unlike traditional systemic antibiotic therapy, implantation of polymer systems loaded with antibiotic permits local release of drug to a specific region.6–9 Targeted delivery allows for high tissue concentrations of an antibiotic at the site of infection while minimizing its systemic side effects. This is particularly advantageous for drugs with short half-lives. While antimicrobials can be loaded into polymer matrices without covalent attachment to the polymer chain, the subsequent release of freely-loaded antibiotic is dictated by diffusion of the drug through the polymer matrix, and may precede polymer degradation (in the case of biodegradable systems). After drug is released, the tissue-engineered scaffold may remain and provide a nidus for bacterial colonization and subsequent infection.10 By covalently bonding antibiotics or other antimicrobial agents to polymer systems, the release of drug is dependent on polymer degradation rather than drug diffusion. Through a variety of different chemistries (such as acrylation11 and isocyanation12), therapeutics can be covalently attached to polymer systems via

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hanging pendant chains or incorporated directly into the polymer backbone (Fig. 1). In addition, certain chemistries (such as pH-sensitive imine bonds13 or thrombin-cleavable linkages14) can be incorporated to trigger the release of antibiotics in response to infection-related environmental cues, such as local pH changes or exposure to bacterial enzymes. To combat infection of medical materials and develop new therapeutics for resistant pathogens, there has been recent work in the field in the development of inherently antimicrobial biodegradable polymers (IABPs). In this work, we will review current advances in traditional antibiotics and their covalent incorporation into polymers, naturally-occurring antimicrobial polymers, and synthetic antimicrobial polymers (Fig. 2). Given their prevalence in infection of implanted materials, the majority of the reviewed literature will focus on bacterial infection; however, fungal and viral infection are areas of increased interest for future investigation. TRADITIONAL ANTIBIOTICS The current clinical gold standard for treatment of infectious diseases is antimicrobial therapy. Traditionally, antibiotics are delivered in free form via systemic administration such as oral, intravenous, and subcutaneous. However, interest in localized delivery of antibiotics via polymer-based carriers has grown given the ability to create high local concentrations of antibiotic while mitigating off-target side effects.15,16 In addition, traditional antibiotics have diminished efficacy in the treatment of biofilm-related infections as well as multidrug-resistant pathogens;17,18 therefore, there are opportunities to create new polymer-based forms of traditional antibiotics to better control delivery as well as enhance efficacy. Lastly, by slowly releasing traditional antibiotics as polymer chains degrade, it is possible that the half-life of these agents (Table 1) may be extended.19 In this section, IABPs derived from four classes of commonly used traditional antibiotics (beta-lactams, fluoroquinolones, glycopeptides, and aminoglycosides) will be evaluated and discussed to better understand structure/function relationships and currently available synthetic schemes.

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Beta-Lactams. Named after the lactam ring (a four carbon cyclic amide), the betalactams are a large class of antibiotics that comprise some of the earliest antibiotics, such as penicillin.20 Beta-lactam antibacterial activity is due to inhibition of peptidoglycan crosslinking in the bacterial cell wall.20 Interestingly, certain beta-lactams have been reported to self-assemble into polymeric structures.21 These self-assembling chains polymerize via peptide bond formation, have diminished antimicrobial bioactivity, and may illicit a negative host immune response.22 Therefore, other techniques to create beta-lactam-based polymers are currently being explored. For example, ampicillin pendant groups were attached via ester bond to either poly(lactic acid) (PLA) or poly(ε-caprolactone) (PCL) by N,N’-dicyclohexylcarbodiimide (DCC) and 4(dimethylamino)pyridine (DMAP)-mediated conjugation.23 Under incubation at pH 7.0 and 37oC, 60% and 14% of ampicillin was released from PLA and PCL over 21 days, respectively. The authors speculated that the difference in release rates may be due to dissimilarities in polymer crystallinity and hydrophobicity, as antibiotic release was mediated by hydrolytic ester bond cleavage.23 However, we note that head-to-head testing against ampicillin encapsulated without conjugation with these polymers was not performed within the study, nor was in vitro efficacy against ampicillin-susceptible pathogens. Another technique in which pendant beta-lactam groups were introduced to polymer chains was by synthesis of acrylated beta-lactams via acryloyl chloride.24 Due to the addition of the reactive vinyl group, these acrylated monomers could then be polymerized with other acrylate-containing monomers via free radical polymerization. Acrylated beta-lactam was polymerized with a 7:3 wt/wt combination of butyl acrylate and styrene to form carbon chains with beta-lactam pendant chains conjugated via ester bond. These polymers were further emulsified to form nanoparticle structures and analyzed for bioactivity against methicillinsusceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA), using acrylated antibiotic monomers as control groups. The polymerized nanoparticle structures had greater

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bioactivity (both against MSSA and MRSA) than monomers alone per MIC (monomer MICs ranging from 64-128 µg/mL and polymer nanoparticle MICs ranging from 8-32 µg/mL).24 The mechanism of this relatively enhanced activity against resistant strains was found to be protection from beta-lactamases.25 Further complexity was created by glycosylating the antibiotics, such that the pendant chain contained an ester group followed by glucose followed by the antibiotic.11 The resulting “glyconanobiotic” nanoparticles retained activity against MRSA. Glycosylation, and other methods of adding carbohydrate chains to polymers, may function as cell recognition motifs for targeted delivery, alter drug release kinetics through drug binding affinities, and alter drug vehicle immunogenicity.26 Incorporation of carbohydrates may have future uses within drug delivery. Rather than as pendant chains, polymers with antimicrobial activity have also been synthesized using beta-lactams as units in the polymer backbone. By developing a novel set of beta-lactams, Nylon-3 materials were synthesized by anionic ring-opening polymerization.27 As beta-lactam rings can be considered critical functional groups in many traditional antibiotics as well as fundamental building blocks in synthetic nylons via ring-opening reactions, it can be noted that the distinction between traditional and synthetic IABPs is not always straightforward. A variety of different co-polymers from these beta-lactams were created with different functional groups, including zwitterionic combinations.27 Dependent on the functional group, these new beta-lactam-derived polymers demonstrated efficacy against a gram-negative organism (Escherichia coli), and several different gram-positive organisms (Bacillus subtilis, Enterococcus faecium, and MRSA) as measured by MIC testing (Table 2).27 Interestingly, modifying the Nand C-termini of these polymers has differential effects on antimicrobial properties as well as propensity to cause hemolysis in eukaryotic blood cells.28 Fluoroquinolones. Fluoroquinolones, inhibitors of proteins required for the unwinding of DNA, are effective antibiotics for the treatment of a variety of diseases including pneumonia, genitourinary infections, cellulitis, and endocarditis.29 Given their wide spectrum against both

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gram-positive and gram-negative bacteria, fluoroquinolones are attractive agents for local delivery strategies. Ciprofloxacin in particular has been successfully modified by a number of mechanisms to be covalently incorporated into polymer structures (Fig. 3). For example, using the same “glyonanobiotic” microparticle synthesis technique previously discussed for betalactams, ciprofloxacin was also acrylated, polymerized with styrene and butyl acrylate, and emulsified to form nanoparticles with efficacy against Bacillus anthracis, MRSA, and MSSA per MIC testing (Table 2).11 In head-to-head study, the ciprofloxacin-derived nanoparticles had superior activity against both species compared to the beta-lactam-derived nanoparticles.11 In addition to acrylation, ciprofloxacin-derived monomers have also been synthesized by methacrylation via glycidyl methacrylate.30 These monomers were then polymerized with acrylamide and diallyldimethyl ammonium chloride (DADMAC) in different feed ratios by free radical polymerization. The ciprofloxacin-based monomer was more difficult to incorporate in the polymer than the other two monomers; the authors speculate steric hindrance by the drug may hinder reaction. Nevertheless, polymers exhibited activity against E. coli per MIC testing and this activity was increased with greater incorporation of ciprofloxacin within the polymer, ranging from 4.62-17.61 wt% ciprofloxacin with resulting MIC of 250-15 µg/mL.30 In addition to acrylation and methacrylation, ciprofloxacin has also been acetylated via chloroacetic acid.31 The acetylated ciprofloxacin was then conjugated to PLA as a terminal group and the co-polymer was electrospun into nanofiber sheets. These sheets inhibited the growth of S. aureus and E. coli per colony counting and released ciprofloxacin for at least two days in a statistically significant pH-dependent manner (increased acidity contributing to increased release, most likely though catalysis of ester hydrolysis).31 Specifically, 55% of conjugated ciprofloxacin was released at pH=7.4 and 85% at pH=1. Unfortunately, the authors did not specify the absolute concentrations of ciprofloxacin that were conjugated and measured.31 In addition to ciprofloxacin, other fluoroquinolones have also been explored. Norfloxacin also has successfully been methacrylated (via 3-(acryloyloxy)-2-hydroxypropyl methacrylate and

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N,N-dimethylformamide) with activity against S. aureus and E. coli as assayed by colony counting from liquid culture (7 orders of magnitude reduction in bacteria).32 In addition, norfloxacin was incorporated into the backbone of a PCL co-polymer via urethane linkages mediated by 1,12-diisocyanatododecane.33 Following a similar reaction scheme, norfloxacin was incorporated into the backbone of PCL and PLA oligomers via urethane linkages mediated by 1,6-diisocyanatohexane.34 Norfloxacin release from the polymers under acidic and normal pH conditions at days 7, 14, and 21 was characterized; a greater amount of polymer was released under acidic conditions and from PLA co-polymers compared to PCL co-polymers, although no statistical comparisons were made.34 These results may in part be explained due to differences in PCL and PLA physicochemical properties such as, for instance, variations in crystallinity and hydrophobicity.35,36 Alternative methods to attach fluoroquinolones to PCL and PLA oligomers included DMAP/DCC conjugation (ofloxacin)37 and pyridine-mediated esterification (ciprofloxacin).38 Similar to fluoroquinolones co-polymerized via urethane linkages, greater amounts of antibiotic were released from PLA-based co-polymers compared to PCL-based copolymers. Depending on IABP formulation, 22-29% of loaded ciprofloxacin was released after 35 days of incubation at pH=7 from PLA-based co-polymers compared to 17% from PCL-based co-polymers under the same conditions.38 Unfortunately, in vitro efficacy against bacterial pathogens was not measured in any of these studies.37,38 In the previous examples, release of fluoroquinolone from the polymer was mediated by passive processes (primarily hydrolysis). An exciting direction in antibiotic-polymer conjugation is enzymatically-cleavable linkages. For example, bacteria such as Mycobacterium tuberculosis are known to infect intracellular compartments such as macrophage lysosomes and resist traditional antibiotic therapy and immune responses. To overcome this obstacle, norfloxacin was conjugated to the end of a peptide sequence such that it was cleavable by cathepsin B, a lysosomal protease.39 This peptide sequence was further attached to mannosylated dextran, a carbohydrate sequence recognized by macrophages, to create a hybrid polymer system in

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which norfloxacin is specifically delivered to and released in lysosomes. This polymer demonstrated greater efficacy against Mycobacterium tuberculosis compared to norfloxacin alone as well as efficacy against isoniazid-resistant strains of Mycobacterium bovis in murine models of disease per colony counting from recovered organs (Table 2).39,40 In addition to incorporating naturally-occurring peptide sequences into the IABP backbone, work has also been done using only synthetic sequences to preferentially promote enzymatic cleavage. Specifically, when ciprofloxacin-PCL co-polymer was synthesized by generating urethane linkages with either 1,6-diisocyanatohexane or 1,12-diisocyanatododecane, exposure of the polymer to inflammatory enzymes increased release of ciprofloxacin compared to buffer alone only when synthesized with 1,12-diisocyanatododecane.12,41 The authors hypothesized that the increased carbon chain length between functional units in the backbone may have increased recognition by cleaving enzymes (in this case, cholesterol esterase) by creating structure more similar to natural substrate (long-chain fatty acid esters of cholesterol).12 When exposed to leukocytes, ciprofloxacin was released from these polymers and demonstrated activity against Pseudomonas aeruginosa and E. coli per MIC testing (Table 2).12 Given their broad spectrum of activity, as well as capacity to be chemically modified to function as either a backbone or pendant monomer, the fluoroquinolones are an attractive class of therapeutics for incorporation into biodegradable polymers as IABPs. Glycopeptides. An inhibitor of bacterial cell wall synthesis, vancomycin (the most clinically-relevant glycopeptide) is a first-line agent used to treat many life-threatening grampositive infections.42 In recent years, the emergence of vancomycin-resistant enterococci (VRE) has become a major public health concern. Dimers of vancomycin were shown to have activity against VRE species, including Enterococcus faecalis and Enterococcus faecium,43 generating interest in creating vancomycin-derived polymers. Furthermore, another appealing property of vancomycin for use in biomaterial applications is its ability to retain activity even when permanently fixed to surfaces; there is evidence that suggests vancomycin does not need to be

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free in solution to have antimicrobial efficacy.44 By attaching a norborene ring with an aromatic aldehyde group to vancomycin via reductive amination, a vancomycin-derived polymer was synthesized by ring-opening metathesis polymerization.45 Per MIC testing, the resulting polymer (“Compound 3”) had activity against MRSA (2.3 µg/mL) as well as VRE (2-31 µg/mL) and nonresistant strains of E. faecalis (2 µg/mL) (Table 2).45 Compared to the monomeric form of VRE, these MICs were an 8 to 60-fold increase in potency against VRE, while retaining similar MIC for S. aureus and E. faecalis.45 Without evaluating these polymers in a preclinical animal model, it is unclear if this strategy will result in clinically-relevant bioactivity. While these polymers may not be considered biodegradable, they demonstrate that polyvalent forms of antibiotics may have enhanced activity against strains otherwise resistant to monovalent antibiotics. In one study, vancomycin was acrylated (via substitution of a N-hydroxysuccinimide (NHS) group) at different points along the glycopeptide (either at a primary or secondary amine).46 In addition, at the primary amide, the effect of adding a large (n=80) poly(ethylene glycol) (PEG) spacer between vancomycin and the polymer backbone was studied. These vancomycin acrylates were co-polymerized with bone cement (poly(methylmethacrylate) (PMMA)) and tested against biofilm-producing Staphylococcus epidermidis. The tested groups were as followed: Blank (unloaded) PMMA; PMMA loaded with monomer vancomycin (2.5 wt% and 10 wt%); PMMA with vancomycin pendant chains (10 wt%); PMMA with vancomycin pendant chains with PEG spacer (10 wt%); and PMMA with PEG spacer. Compared to unloaded PMMA, only PMMA with vancomycin as a pendant group with PEG spacer as well as PMMA with PEG spacer and no vancomycin significantly reduced S. epidermidis biofilm mass (~45 mg reduced to ~20 mg biofilm dry mass for both groups).46 Vancomycin co-polymer without PEG spacer performed similarly to free vancomycin encapsulated within bone cement (non-conjugated), but these groups were not statistically different from unloaded bone cement, and there were no differences dependent on specific amine group acrylation. However, the vancomycin co-polymer without PEG spacer had mechanical properties most similar unloaded

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bone cement, compared to vancomycin co-polymer with PEG spacer as well as bone cement loaded with free vancomycin. For example, the yield strength of PMMA loaded with vancomycin monomer (10 wt%), PMMA with vancomycin pendant chains (10 wt%), PMMA with vancomycin pendant chains with PEG spacer (10 wt%) and PMMA with PEG spacer alone (10 wt%) was 84%, 102%, 59%, and 52% that of unloaded PMMA, respectively.46 In sum, specifically for antibiofilm applications, vancomycin-derived monomers required a PEG chain for efficacy, but addition of this bulky modification reduced final polymer mechanical properties. Aminoglycosides. Including antibiotics such as gentamicin, streptomycin, and tobramycin, the aminoglycosides function by inhibiting protein synthesis and are effective against many gram-negative bacteria and some gram-positive organisms.47 Among the first classes of antibiotics developed for clinical application, the aminoglycosides are seeing a resurgence in use due to the emergence of multidrug-resistant bacteria.47 In one study, streptomycin was conjugated to latex microparticles composed of methyl methacrylate, ethyl acrylate, and acrylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).48 As a pendant group, streptomycin was either conjugated directly to the free carboxylic acid groups from the latex or with a 6-aminocaproic acid spacer. In a turbidity assay against E. coli, streptomycin-latex co-polymer microparticles performed similarly to free streptomycin (Table 2).48 However, as the co-polymer was not tested with and without 6-aminocaproic acid, the effects of the spacer on antimicrobial activity remain unclear. In an attempt to create targeted delivery, a Schiff base was created by reacting gentamicin with an aldehyde at the end of a PEG chain to tether the antibiotic via a pH-sensitive imine bond.13 The PEG chain was conjugated to a norbornene group and polymerized via ringopening metathesis polymerization to form self-assembling nanoparticles, with the hydrophobic norbornene backbone in the center and the hydrophilic PEG chains (ending in gentamicin groups) radiating outward. As designed, the nanoparticle release products at 6 and 16 hours of incubation (for an elution time of either 6 or 16 hours total) had statistically significant increased

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activity against S. epidermidis per MIC measurements when the nanoparticles were incubated at lower pH (direct measurement on concentration of release was not measured). Specifically, at pH of 6, 5, and 4, MIC was 22, 11, and 10 µg/mL at 6 hours and 6, 5, and 3 µg/mL at 16 hours, respectively.13 At a pH of 7, the release products of the nanoparticles had no efficacy compared to blank nanoparticles against S. epidermidis as measured by MIC testing. In addition to pH-triggered release, the aminoglycosides have also been explored as enzyme-cleavable pendant groups. Specifically, EDC conjugation was performed to link gentamicin to a thrombin-cleavable peptide sequence, which in turn was attached as pendant groups to a poly(vinyl alcohol) (PVA) hydrogel to be used as a wound dressing.14 A subcutaneous wound was created in rats and exudate was collected from animals with sterile wounds and from wounds infected with P. aeruginosa.14 Gentamicin was only released when exposed to exudate from infected animals. Furthermore, gentamicin-peptide-PVA wound dressings statistically significantly inhibited the in vitro growth of P. aeruginosa compared to PVA alone by nearly 5 orders of magnitude in a colony counting in vitro assay (Table 2).14 The authors further speculated that by using peptide sequences associated with pathogen-specific enzymes (for example, a protease secreted specifically by E. coli), this same technique could function as a platform to create polymers to target specific infections.

Summary of Traditional Antibiotics. From a synthesis standpoint, significant progress has been made in the field regarding the modification of traditional antibiotics for conversion to monomers (as summarized in Table 1). These include modifying antibiotics by acrylation, methacrylation, acetylation, isocyanation, carbodiimide-based conjugation, and azomethination. A single antibiotic, such as ciprofloxacin, can be successfully modified by multiple methods for different purposes (Fig. 3). Traditional antibiotics have been incorporated in the backbone of biodegradable polymers, or more commonly, as pendant groups with degradable linkages such as ester bonds, imine bonds, amide bonds, or enzyme-cleavable bonds. It has been

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demonstrated that the choice of co-polymer,23 the location of the antibiotic in its attachment to the polymer backbone,28 and use of spacers to create distance between the backbone and antibiotic pendant chain46 can contribute to the efficacy of antibiotic-derived IABP systems and are important design considerations. Depending on the chemistry chosen, antibiotic release can be triggered based on environmental signals such as pH changes or exposure to specific enzymes.13,39–41 Lastly, presentation of naturally-occurring molecules such as polysaccharides may promote biointegration or targeting of specific tissues/cell populations vulnerable to infection.11,39,40 From a functional standpoint, it has been demonstrated that antibiotic-derived IABPs may have increased efficacy against resistant pathogens compared to their monovalent counterparts.11,24,40,45 Given the rising prevalence of multidrug resistant organisms, this property of the antibiotic-derived IABPs may be of interest for future study. However, steps taken in the translation of these technologies have been limited. Out of all of the studies in Table 2, only two included analyzing efficacy in an animal model of infection (and both were from the same group).39,40 In order for these strategies to be further developed and eventually brought into clinical practice, more validation in physiologic models of disease must be performed.

NATURALLY-DERIVED ANTIMICROBIAL POLYMERS In addition to providing many of the traditional antibiotics, nature has created other means to dissuade pathogens. For example, plants can produce phenols to inhibit bacterial, viral, and fungal growth49, while mammalian cells produce antimicrobial peptides50. The presentation of these molecules, either in polymerized or degraded form, can alter their potency. In this section, two classes of natural antimicrobial polymers will be discussed: those created entirely in nature and those synthesized from antimicrobial monomers found in nature. These data have been summarized in Table 3.

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Naturally-occurring polymers. Extracellular matrix (ECM) components from multiple tissue types have been shown to have antimicrobial activity. However, these properties arise in response to certain stimuli. For example, ECM consisting of intact porcine small intestine submucosa was shown to have no antimicrobial activity.51 Generally, ECM degrades soon after tissue injury, with complete resorption after 90 days. After degradation in vitro using acetic acid and neutralizing to pH of 7 to remove any possible effects due to decreased pH, porcine small intestine submucosa and urinary bladder ECM were shown to have antibacterial effects against E. coli and S. aureus as measured by decreased optical density of culture medium (Table 3).52 The substances isolated from the acid-digest extract were peptides and proteins of a variety of sizes. Likewise, porcine liver ECM also had antimicrobial properties after digestion in acid.53 After further fractionation via ammonium sulfate precipitation, different fractions were shown to have varying levels of activity, likely due to fractions containing different ratios of antimicrobial peptides and their inhibitors. Like ECM, naturally-produced antimicrobial peptides (AMPs) can have different properties depending on length. Produced by a variety of cell types, AMPs have activity against many different pathogens.54 As these usually cationic proteins target the negatively-charged phospholipid bilayer, development of resistance is much less common than traditional antibiotics.54 AMPs are synthesized from amino acids and can be degraded or further processed into shorter chains. LL-37, an AMP belonging to the cathelicidin family, is produced by skin epithelium.50 While LL-37 has some in vitro efficacy against common pathogens, smaller peptides derived from LL-37 demonstrated improved MIC in comparison against S. aureus, E. coli, C. albicans, and Vaccinia virus, illustrating that specific amino acid sequences as well as length play important roles in AMP activity (Table 3).50 Given their broad-spectrum activity, biomimetic synthetic AMPs are currently being explored and will be discussed elsewhere in this review.

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Chitosan, produced by exposing natural chitin to a strong base, has been shown to possess a variety of benefits when used as a tissue engineering scaffold.55 Not only is it biodegradable and non-toxic, but it also has the additional benefit of selective action against gram-positive and gram-negative bacteria and fungi, with much lower action against mammalian cells.56 In part, this selectivity has been attributed to the cationic charge density of chitosan.56 As chitosan is degradable, it is important to investigate the effects of molecular weight on chitosan activity. Antimicrobial activity of twelve chitosan chains of molecular weight ranging from 1-1671 kDa were compared.57 These solutions were then tested against seven different gram-positive and four different gram-negative bacteria. Higher molecular weight chitosan was shown to be more efficacious at inhibiting bacterial growth by MIC testing (Table 3), with slightly increased action against gram-positive organisms.57 In another study, wherein only E. coli was tested, it was shown that lower molecular weight chitosan had a greater antimicrobial effect (as measured by decreasing optical density) with more pronounced reduction in bacteria number when chitosan was introduced at earlier timepoints during bacteria culture.58 As chitosan is more soluble in acidic solutions, often it can be difficult to discern the effect of chitosan from the effect of acid; however, the activity of chitosan in a solution of pH 7 was found to eliminate nearly all exposed E. coli at chitosan concentrations of 200 ppm and higher.58 Yet another study investigated the use of chitosan and degraded chitosan chains.59 Here, it was found that chitosan of lower molecular weights inhibited bacterial growth to a point. After which, the very low molecular weight chitosan did not inhibit bacterial growth in food or beverage items.59 Due to these conflicting data regarding chitosan chain length and in vitro efficacy in the literature, further investigation may be required to definitively understand the relationship between linear chitosan molecular weight and antimicrobial properties. Another tunable parameter of chitosan that can affect antibacterial activity, in addition to molecular weight, is degree of deacetylation.60,61 One study investigated the antibacterial properties of chitosan membranes using a bactometer and MIC testing of S. aureus and E. coli. 61 While it was found that the MIC

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of S. aureus was lower than that of E. coli, the authors also found that increasing degree of deacetylation further lowered the MIC. It was also noted that the antibacterial properties of chitosan were dependent on pH, with increased activity at lower pH (Table 3). 61 Threedimensional matrices of chitosan also exhibited antimicrobial properties.62 Streptococcus mutans and Actinobacillus actinomycetemcomitans were cultured on chitosan matrices that had been neutralized to pH of 7, either with ethanol or sodium hydroxide. When these matrices were exposed to lysozyme, an enzyme capable of hydrolyzing chitosan, increased antimicrobial activity was seen against both bacteria as determined by optical density and imaging (Table 3)62; however, it was not determined if a decreased pH were responsible for these effects. Other matrix parameters such as pore size did not affect antimicrobial activity, while blending with PCL reduced it.62 The authors believe this is due to the contact-dependent nature of chitosan’s antimicrobial properties. When PCL, a non-antimicrobial polymer, was introduced to the blend, it provided a site where bacteria could adhere without contacting chitosan, allowing it to grow.62 Another class of naturally-occurring antimicrobial molecules are tannins. Tannins are produced by plants and are conventionally classified into two groups: proanthocyanidins and hydrolysable tannins63 (some classification schemes further subcategorize tannins64). Proanthocyanidins are much less susceptible to hydrolysis and will not be discussed. Hydrolysable tannins consist of biodegradable esters of phenolic acids and glucose. Two hydrolysable tannins, tellimagrandin I and corilagin, were shown to restore antibiotic sensitivity when tested against MRSA (Table 3).65–67 The authors speculated that this effect was likely due to binding penicillin-binding proteins or inhibiting ߚ-lactamases.67 In addition to restoring antibiotic sensitivity, hydrolysable tannins are also capable of damaging Helicobacter pylori, a gram-negative rod responsible for gastritis and ulcers. Several hydrolysable tannins tested showed moderate antibacterial activity at MIC of 12.5 µg/mL,68 with tellimagrandin I showing promise even after treatment with acid to recapitulate stomach environment. Simultaneously, a

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gastric cancer cell line MKN-28 showed no reduction in viability, indicating that tannins may be a safe alternative to treating H. pylori infections.68 Naturally-occurring monomers. Just as nature has made use of AMPs within ECM or phenolic acids to make tannins, these compounds can also be used to generate semi-synthetic polymers with antimicrobial properties. Using naturally-occurring AMPs for peptide sequences, a monovalent structure was found to be inactive against E. coli.69 However, incorporating these amino acids into a dendritic structure allowed for the formation of divalent and tetravalent polymers. These divalent and tetravalent structures of the same series of amino acids afforded a large increase of MIC to 292 and 29 µg/mL, respectively.69 It was hypothesized that multivaliancy increases resistance to peptidases.70–72 While higher multivalences increased antimicrobial activity, it also resulted in greater cytotoxicity as assayed by red blood cell hemolysis.69 Salicylic acid, coumaric acid, and other phenolic acids are naturally-occurring compounds with at least two different functional groups, lending themselves to polymerization. Salicylic acid has been shown to inhibit bacterial adhesion and biofilm formation73; however, it has a short, concentration-dependent half-life. Local release of salicylic acid could therefore prove beneficial. To this end, poly(anhydride esters) based on salicylic acid were developed.74 Salicylic acid was coupled to adipoyl chloride to form a diacid, which was then reacted via melt polymerization to form a polyanhydride. To determine efficacy, the amount of biofilm deposition by Salmonella enterica was measured on glass slides coated with these polymers (Table 3). While biofilm formation was inhibited, it was not due to inhibited attachment as hypothesized but was more likely due to damaging bacterial cells.74 A similar synthesis was utilized to create polymers of coumaric acid.75 While high drug loading and sustained release were observed, its antimicrobial efficacy was not measured.75 Phenols act by disrupting cell membranes and coagulating cytoplasm, resulting in cell death.49 With usually only one functional group, they are more easily grafted onto a polymer as

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pendant groups rather than part of the backbone compared to the phenolic acids. Carvacrol, thymol, and eugenol were reacted with ethylenediaminetetraacetic acid dianhydride to form a diacid.76 The diacids were then polymerized in the presence of triphosgene to form a poly(anhydride ester) with pendant phenol groups. The resulting polymers were shown to degrade over 16 days under physiological conditions, to have in vitro activity against both E. coli and S. aureus as assayed by disc diffusion, and to have degradation products that retained antioxidant properties of phenols.76 Similar phenolic polymers can be synthesized using pyromellitic anhydride.77 While the focus of this study was not antimicrobial activity, the resulting biomaterials were non-cytotoxic to mouse fibroblasts.77 Like phenols, benzoic acid is found in many plants and has a single carboxylic acid functional group. As such, it can be attached as a pendant group cleavable via hydrolysis. Vinyl monomers of phenols and benzoic acid derivatives were prepared by reacting the derivatives with acryloyl chloride.78 These monomers were individually tested for antimicrobial activity against S. aureus and P. aeruginosa using the disc diffusion method. The monomers were then polymerized via free radical polymerization using benzoyl peroxide as an initiator. The zones of inhibition of each of the monomers was greater than its respective polymer (Table 3). The reduction in activity could be due to loss of vinyl bonds which may convey mild antimicrobial activities or reduction in diffusion of antimicrobials as hydrolysis must occur before antimicrobials are fully mobile. Benzoic acid derivatives were also successfully attached to polyacrylamide modified with ethylenediamine.79 All polymers synthesized demonstrated activity against two species of bacteria and two species of fungi, with greater inhibition observed in fungal species.79 Summary of naturally-derived antimicrobial polymers. Eukaryotic organisms have evolved to resist bacteria through a variety of mechanisms, including secreted antimicrobial compounds and matrix components. In addition to antimicrobial activity, these compounds may have other effects, such as inhibition of biofilm formation (salicylic acid)74 and induction of

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increased sensitivity to traditional antibiotics (hydrolysable tannins).65 Despite showing great promise in vitro, very little in vivo work has been done, and most studies utilize a single grampositive and/or gram-negative organism. Nevertheless, by studying the structures and mechanisms of these naturally-occurring IABPs, synthetic polymers can be created with these properties in mind.

SYNTHETIC ANTIMICROBIAL POLYMERS Synthetic polymers have been created to mimic various properties of naturally-occurring IABPs. Structural conformation and cationic charge density serve as the basis for many of the synthetic properties developed and tested in vitro. Other physiological antimicrobial responses, such as generation of nitric oxide, have also been incorporated into polymers. In this section, synthetic mimics of AMPs and other biodegradable synthetic polymers are discussed, with further detail given in Table 4. Synthetic mimics of AMPs. The discovery that the α-helices within many AMPs change conformation and interact with bacterial membranes have prompted investigation into synthetic molecules that can mimic this ability to change structural conformation. While α-helix formation upon membrane contact is thought to be critical to AMP function,80 it is unclear if it is absolutely required for the success of synthetic analogues. Using a nylon-3-based polymer backbone, it was demonstrated that random co-polymers featuring lipophilic and cationic side chains were capable of disrupting membrane bilayers without α-helix formation.80 A mixture of beta-lactams was initiated with 4-tert-butylbenzoyl chloride and used to synthesize random copolymers of varying chain lengths, which were then evaluated for MIC against E. coli, S. aureus, Enterococcus faecium, and Bacillus subtilis (Table 4).80 It was shown that increasing chain length increased cytotoxicity against red blood cells without much increase in antimicrobial activity. A chain length of 10-30 units saw favorable antimicrobial activity with very little

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hemolysis.80 The mechanism of action of these nylon-3 polymers was the ability to interact with negatively charged membranes at low concentrations, and the ability to prevent passage of solutes into intermembrane space at high concentrations based on testing done against E. coli and S. aureus (Table 4).81 Further studies of nylon-3 AMP-mimics indicated that conformation of sidechains also affects selectivity and potency of the resulting polymer.82,83 Using similar betalactams with cyclic or non-cyclic side groups, it was shown that cyclic groups tended to have higher MICs and lower degrees of hemolysis.82 This was believed to be due to increased rigidity of the polymer chain.82 These polymers have also been tailored to selectively target fungi without increase in hemolysis, indicating specificity between two eukaryotic cell types.83 Attempts to mimic the α-helical structure using solid-phase peptide synthesis have also been performed.84,85 By closely controlling the peptide sequence, arylamide polymers with hydrophilic and hydrophobic side groups were created.85,84 Small arylamide molecules can fold and have been shown to increase permeability of bacterial membranes.85 Computational models were able to successfully predict the structure of novel arylamide polymers. When evaluated against E. coli and Klebsiella pneumoniae, it was shown that an 8-unit repeating sequence had the highest MIC activity (Table 4).84 However, these resulting peptides were hemolytic,84 and further investigation into different peptide sequences or side chains is needed. Recent advances in synthetic techniques has facilitated the production of new biomimetic AMP analogues. Peptide synthesis generally relies on solid-phase synthesis, which, although recent developments in automation have greatly sped up the process, is still timeconsuming and requires specialized equipment. Synthesis of peptides via ring-opening of Ramino acid N-carboxyanhydrides offers an alternative mechanism of synthesis.86 First, protected lysine and hydrophobic amino acids were reacted with triphosgene to produce a cyclic compound. These cyclic compounds were combined in presence of a nickel initiator. The resulting solution was purified via dialysis and tested against E. coli, P. aeruginosa, Serratia marcescens, S. aureus, and C. albicans, and some of the resulting peptides had lower MICs

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than naturally-produced AMPs (Table 4).86 However, these polymers had significant hemolytic activity that would inhibit their use in vivo without further exploration.86 Other synthetic polymers. Nitric oxide (NO) is produced endogenously by immune cells. At low concentrations, it modulates chemokine production; at high concentrations, it covalently binds proteins and DNA, resulting in cell death.87 As its half-life is 3 seconds, local delivery is necessary for antimicrobial effects.88 Several IABPs have been designed to leverage NO’s antimicrobial activities.88,89 Poly(sulfhydrylated polyesters) were first created through the polycondensation of ethylene glycol and mercaptosuccinic acid.89 These polyesters then underwent S-nitrosation with nitrous acid, resulting in biodegradable polymers capable of releasing NO. Efficacy of these polymers as a coating of intravascular catheters was tested after blending with PMMA. S. aureus and multidrug-resistant P. aeruginosa showed a 98% reduction in viability after 12 hours of incubation (Table 4).89 In another study, glutaric acid and glycerol were reacted to form a crosslinked polyester scaffold.88 After curing, the acid end groups were modified via NHS and EDC hydrochloride followed by either penicillamine or cysteamine to have thiol end groups. This was converted to NO via sodium nitrite. Under physiological conditions, NO was released for 6 days and growth of P. aeruginosa was reduced by 80% by colony counting method.88 N-halamines have been shown to have potent antimicrobial activity via release of chlorine, bromine, or iodine atoms. They are also stable over a wide range of temperatures and can be regenerated upon exposure to chlorine, bromine, or iodine.90 N-halamines can be attached as pendant groups via ester bonds to polymers such as cellulose or surfaces such as glass.91 They can also be polymerized via free-radical polymerization when modified with vinyl groups. N-halamine precursors can be bound to epoxide rings that can be easily grafted onto the surface of cellulose.91 3-Glycidyl-5,5-dimethylhydantoin, a N-halamine precursor, was grafted onto the surface of cellulose using an epoxide ring-opening reaction. These fibers were

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able to disinfect E. coli and S. aureus within minutes after contact as shown by broth dilution assay and were capable of being regenerated multiple times (Table 4).91 Polyammonium salts can also be attached as pendant groups via ester bonds or can undergo free radical polymerization after modification with vinyl bonds. Quaternary ammonium salts are antibacterial while maintaining low cytotoxicity.92 When quaternary ammonium compounds are attached to polymers, increases in antimicrobial activity with decreases in cytotoxicity and tissue irritation were observed.93 Polymers with high charge density act similar to the cationic chitosan chains and AMPs in disrupting cytoplasmic membranes.93 Cationic IABPs can be created using polycarbonates with tertiary amine groups. When the alkyl chain connecting the tertiary amine group to the polycarbonate was varied, different levels of selectivity were observed.93 A four-carbon alkyl chain with degree of polymerization of 20 was very potent against and selective for MRSA and VRE as shown by MIC testing (Table 4).93 The selectivity of cationic antimicrobials can also be tuned by the counter-anion.94 While it is most commonly chlorine, other possibilities include citrate and lactate.94 Trifluoroacetate and benzoate yielded the highest degree of antimicrobial activity (via MIC testing) while also showing low levels of hemolysis.94 One mechanism of grafting monomers that contain tertiary ammonium is reversible addition-fragmentation chain transfer.92 With this strategy, 2(dimethylamino)ethyl methacrylate was grafted onto cellulose fibers and quarternized with alkyl bromide. A variety of alkyl lengths and density of quarternary groups were tested, with the shortest alkyl chain and highest number of quarternized ammoniums having the greatest effect against E. coli.92 A similar strategy can be adopted for phosphonium salts, with activity seen against E. coli and P. aeruginosa (Table 4).95 Summary of synthetic antimicrobial polymers. Through a better understanding of the structure/function relationships observed in naturally-occurring IABPs, synthetic IABPs, have been developed to resist infection. However, despite initial promise with low MICs against grampositive and gram-negative bacteria, several of these polymers demonstrated poor

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biocompatibility (as reflected by a high degree of hemolysis).84,86 Further studies and alterations of polymer sequence should be performed before these compounds are transferred to in vivo models. Nevertheless, synthetic antimicrobial polymers show promise as potent new antibacterial agents.

CONCLUSIONS AND FUTURE DIRECTIONS Much work remains to be performed for the translation of IABPs as tissue-engineered scaffolds for regeneration in the clinical setting. The workflow for the development of novel IABPs can be found in Figure 4. Firstly, the novel polymer should be synthesized and characterized by means such as nuclear magnetic resonance spectroscopy, size exclusion chromatography, differential scanning calorimetry, as well as measurement of the rate of material degradation under physiologic conditions and conditions associated with active infection, such as decreased pH. After synthesis and physicochemical characterization, polymers should subsequently be assayed for antimicrobial activity against a variety of pathogens. As can be seen in Tables 2, 3, and 4, the assays utilized to measure antimicrobial efficacy are varied, including MIC testing, optical density, disc diffusion, and other techniques. While without head-to-head comparisons it is difficult to definitively determine which is the optimal test for every system, MIC testing offers information that can be useful for scaling to different defect sizes when focusing on local infection. In addition, MIC testing is the current clinical laboratory gold standard for the measurement of antimicrobial therapeutic susceptibility.96 Given the familiarity with MIC testing of clinicians and regulatory bodies, its use as a validation method may translate eventual commercialization of IABP technologies. Many current studies utilize a single gram-positive and/or single gram-negative bacterium, but this strategy neglects drug-resistant strains and other organisms such as fungi. In order to fully evaluate the efficacy of an IABP, the polymer, its degradation products, and its monomers should be tested against multiple bacteria, including gram-positive, gram-negative, aerobic, and anaerobic species. In addition to efficacy against

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pathogens, in order to be successfully implanted in a host, IABP cytotoxicity must be evaluated using mammalian cell lines using a concentration larger than the MIC discovered in in vitro testing. Lastly, new IABPs must be validated in an in vivo environment for both biocompatibility and efficacy against infection. In the current body of literature, there are very few IAPB studies in which new polymers were evaluated in an infected in vivo model of disease after characterization and in vitro studies. With a variety of animal models available in the literature, IAPBs should be validated in a clinically-relevant setting.97 Given increases in bacterial resistance to traditional therapies and the advantage of controlled delivery to a local site, IABPs represent a new and exciting class of antimicrobial polymers which may be promising for use as tissue scaffolds to prevent and/or treat infected wounds. In this work, the current state-of-the-art in IABP research has been presented. From conjugating traditional antibiotics to adapting properties of naturally-occurring antimicrobial agents to create biomimetic synthetic polymers, IABPs are a promising alternative to many currently-used treatment options. However, there is a dearth of in vivo studies applying novel IABPs in the literature. For the translation of IABP-based technologies from the laboratory to the bedside, polymer characterization, antimicrobial profile against a variety of pathogens, and in vivo biocompatibility and efficacy must be determined. In light of the current clinical need for new therapies against infection, further investigation of these versatile polymers is warranted.

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ACKNOWLEDGEMENTS This work was supported by the Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-14-2-0004. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick MD 21702-5014 is the awarding and administering acquisition office. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. In addition, further support towards the development of new biomaterials has been provided by the John S. Dunn Foundation and the National Institutes of Health (R01 AR068073). E.W. and A.M.T. would like to thank the Baylor College of Medicine Medical Scientist Training Program, and A.M.T. would like to acknowledge the Barrow Scholars Program. There are no conflicts of interest to report in this work.

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For Table of Contents Use Only

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Figure 1. Antimicrobial subunits can be covalently conjugated to polymers by (A) attachment via pendant groups or (B) direct incorporation into a polymer backbone. Blue circle = antimicrobial subunit, yellow lines = cleavable bonds. Fig. 1



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Figure 2. Classes of inherently antimicrobial biodegradable polymers (IABPs). Fig. 2


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Figure 3. Examples of some of the modifications of ciprofloxacin as performed in the literature. Fig. 3



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Figure 4. Envisioned workflow for validating IABPs. Figure 4


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Table 1. Reported half-lives of a selection of traditional antibiotics.19 Table 1



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Table 2. Summary of traditional antibiotic-derived polymers. Table 2


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Table 3. Summary of naturally-derived antimicrobial polymers. Table 3



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Table 4. Summary of synthetic antimicrobial polymers Table 4


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Table of Contents Graphic. FOR TABLE OF CONTENTS USE ONLY


Inherently Antimicrobial Biodegradable Polymers ... - ACS Publications

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