Article pubs.acs.org/Langmuir
Dual Functionality of Antimicrobial and Antifouling of Poly(N‑hydroxyethylacrylamide)/Salicylate Hydrogels Chao Zhao,† Xiaosi Li,† Lingyan Li,† Gang Cheng,† Xiong Gong,‡ and Jie Zheng*,† †
Department of Chemical and Biomolecular Engineering and ‡Department of Polymer Engineering, The University of Akron, Akron, Ohio 44325, United States ABSTRACT: The emergence and reemergence of microbial infection demand an urgent response to develop effective biomaterials that prevent biofilm formation and associated bacterial infection. In this work, we have synthesized and characterized hybrid poly(N-hydroxyethylacrylamide) (polyHEAA)/salicylate (SA) hydrogels with integrated antifouling and antimicrobial capacities. The antifouling efficacy of polyHEAA hydrogels was examined via exposure to proteins, cells, and bacteria, while the antimicrobial activity of SA-treated polyHEAA hydrogels was investigated against both Gram-negative Escherichia coli RP437 and Gram-positive Staphylococcus epidermidis. The results showed that polyHEAA/SA hydrogels exhibited high surface resistance to protein adsorption, cell adhesion, and bacteria attachment. The polyHEAA hydrogels were also characterized by their water content and state of water, revealing a strong ability to contain and retain high nonfreezable water content. This work demonstrates that the hybrid polyHEAA/SA hydrogels can be engineered to possess both antifouling and antimicrobial properties, which can be used for different in vitro and in vivo applications against bacterial infection.
1. INTRODUCTION Bacterial infection remains one of the most serious complications to human health, and it costs ∼$25 billon dollars per year in the United States alone.1−3 The widespread use of antibiotics has promoted the enormous and growing threat of bacteria that are resistant to almost all available antibiotics due to natural selection.4 Extensive efforts have been conducted toward finding multidrug-resistant antibiotics with low toxicity to the host cell and a broad spectrum against a wide diversity of bacterial pathogens.5 Antimicrobial agents and peptides alone, however, usually suffer from environmental toxicity, short-term antimicrobial activity, and proteolytic instability and degradation.6 To overcome such disadvantages, antimicrobial agents are often physically incorporated into or chemically conjugated with polymers to enhance their antimicrobial efficacy and specificity, reduce cytotoxicity, prolong biostability and biocompatibility, and promote other biomimetic physicochemical properties.7 Moreover, because many antibiotics cannot effectively cross the blood−brain barrier to achieve therapeutic concentrations at infection sites such as brains or cerebrospinal fluid, in parallel to develop new antibiotic agents, it is equally important to develop effective delivery systems that integrate existing antibiotics with novel drug delivery systems to promote their antimicrobial activity.8 About half of bacterial infections are related to medical devices and implants for different biomedical applications. Biomimetic hydrogels have attracted much attention due to their great potential in biomedical applications; e.g., they can be used as loading carriers to deliver different bioactive agents to target organisms, tissues, cells, and areas of the body. Apart © 2013 American Chemical Society
from possessing the general properties of hydrogels such as high water content, high diffusive permeability, and mechanical strength, biomimetic hydrogels also require a bioinert background with specific bioactive functionalities to achieve their desirable activities, because any adsorbed protein, e.g., even 5 ng/cm2 of fibrinogen adsorption, can induce full-scale platelet adhesion and bacterial colonization, leading to the failure of practical applications.9−11 Many hydrophilic polymers (e.g., poly(ethylene glycol)-based derivatives, poly(2-hydroxyethyl methacrylate) (polyHEMA), and poly(hydroxypropyl methacrylate) (polyHPMA)) and zwitterionic polymers (e.g., 2methacryloyloxyethylphosphorylcholine (MPC), poly(sulfobetaine methacrylate) (polySBMA), and poly(carboxybetaine methacrylate) (polyCBMA) have been synthesized as biomimetic hydrogels for various biomedical and industrial applications.2,12−19 Most of these hydrogels, while low fouling (bioinert or stealthy), still cause nonspecific protein adsorption, cell adhesion, and even bacterial accumulation in long-term contact with biological media. PEG- and polyHEMAbased hydrogels are the most commonly used biomimetic hydrogels.20 However, the PEG-based hydrogels are known to oxidatively degrade, which limits their uses required for longterm material stability. The polyHEMA-based hydrogels have relative lower hydration than native tissues, which may induce a local dehydration near native tissues driven by water diffusion through a concentration gradient. Thus, searching for new Received: November 12, 2012 Revised: January 9, 2013 Published: January 14, 2013 1517
dx.doi.org/10.1021/la304511s | Langmuir 2013, 29, 1517−1524
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(HEMA, 97%), 2-(methacryloyloxy)ethyltrimethylammonium (TM, 80 wt % in H2O), poly(ethylene glycol) methacrylate (Me(EG)8OH, average Mn 475), N,N′-methylene-bis-acrylamide (MBAA, 99%), N(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (SBMA, 97%), and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (98%), salicylic acid sodium salt (SA, 99%), phosphate buffered saline (PBS, pH 7.4, 0.15 M, 138 mM NaCl, 2.7 mM KCl), phosphate citrate buffer (pH 5.0), hydrogen peroxide (30 wt % in H2O), sulfuric acid (99.999%), o-phenylenediamine (98%), ethylene glycol (99.5%), ethanol (absolute 200 proof), human plasma fibrinogen (Fg), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Milwaukee, WI). Horseradish peroxidase (HRP) conjugated polyclonal goat antihuman fibrinogen was obtained from USBiological (Swampscott, MA). Water used in these experiments was purified to a minimum resistivity of 18.0 MΩ cm by a Millipore water purification system. Bovine aortic endothelial cells (BAECs) and all cell culture media and reagents were obtained from Gibco (Gaithersburg, MD). Escherichia coli RP437 and Staphylococcus epidermidis were kindly supplied by Dr. Gang Cheng of The University of Akron. A Live/Dead BacLight bacterial viability kit was purchased from Invitrogen (Carlsbad, CA). 2.2. Hydrogel Preparation. Hydrogel samples were prepared by adding “monomer solution” (4 mM monomer, 10 mg of photoinitiator, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) and MBAA as cross-linker to “mixed solvent” (0.375 mL of ethanol, 0.565 mL of ethylene glycol, and 0.565 mL of H2O). The resulting solution was gently sonicated to mix well in an ice bath to prevent premature polymerization. To polymerize, the solution was cast between a pair of glass slides separated by a 1-mm-thick poly(tetrafluoroethylene) (PTFE) spacer. Polymerization was initiated at room temperature with 362 nm UV light for 1 h. The resulting hydrogels were then removed from the slides and soaked in deionized water (or PBS) for 24 h. The water was changed every 3 h to remove unreacted chemicals and excess salt. 2.3. Characterization of Hydrogels. The equilibrium water contents (EWCs, %) of four hydrogels (polyHEAA, poly(Me(EG)8OH), polySBMA, polyHEMA) were assessed by comparing weights of the hydrated and dry samples. First, hydrated hydrogel samples were withdrawn from PBS, and the excess surface water was removed with a Kimwipe. The wet samples were then punched into 8 mm disks (8 mm biopsy punch, Acuderm, USA) and were weighed as mw. The wet samples were dried at 65 °C under vacuum for 72 h and reweighed again as md. The EWCs of the samples were calculated by (mw − md)/mw·100%, and all samples were averaged from three reversible processes. Differential scanning calorimetry (DSC) is a common tool to identify the different states of water (i.e., freezable and nonfreezable water) in a polymer matrix. Hyrdogel samples of 4−6 mg were placed in an aluminum pan after gently wiping off the excess water on its surface, and then the pan was hermetically sealed. An empty aluminum pan was used as the control. The enthalpy change associated with the melting of freezable water per weight of a hydrogel (ΔHf) was measured using a DSC (Q2000, TA Instruments, USA) equipped with a liquid nitrogen cooling system (LNCS) for nonisothermal experiments. Temperature and enthalpy calibration were performed using an indium standard. Sapphire disks were used for heat capacity calibration. During the cooling and heating experiments, the samples were cooled from room temperature to −100 °C and then heated to 40 °C at a rate of 5 °C/min, under the protection of nitrogen gas at a flow rate of 25 mL/min. The weight ratio (Wnonfreezable) of nonfreezable water:polymer in the hydrogel was calculated by Wnonfreezable = wnonfreezable/wpolymer = (EWC − wfreezable)/wpolymer, where wnonfreezable, wfreezable, and wpolymer are the weight percentages of nonfreezable water, freezable water, and polymer in hydrogels, respectively. wfreezable can be experimentally obtained by wfreezable = ΔHf/ΔHw·100%, where ΔHf is the enthalpy change associated with the melting of freezable water per weight of a hydrogel as measured by DSC and ΔHw is the enthalpy change for the melting of bulk water by DSC. For polyHEAA hydrogels, the ratio of the Wnonfreezable value can be converted into the number (Nw) of
hydrogels with maximal antifouling capacity, high biological stability, low cytotoxicity, and high water retention is critical for the development of novel drug delivery systems, tissue engineering scaffolds, and wound healing. To achieve antimicrobial activity of hydrogels, two common approaches have been extensively employed to load and release antimicrobial agents. The first one is to simultaneously load drugs upon hydrogel synthesis. This approach usually has relatively high drug loading, but harsh synthesis conditions can also deactivate drug functions. The other approach is to first synthesize hydrogels, followed by immersion of hydrogels in a drug solution that allows drugs to physically adsorb on or diffuse into the hydrogels. The partition of drugs into the hydrogels is usually driven by specific gel−drug interactions (e.g., electrostatic interactions, hydrogen bonds, hydrophobic interactions, or combined interactions). The amount of loading drugs, while a lower loading initially, can be readily increased as immersion time and drug concentration in solution. Generally, the second approach is more convenient for practical uses. A number of commonly used antibacterial agents such as metal salts,21,22 quaternary ammonium compounds (QACs),23−25 polybiguanides,26 N-halamine,27,28 chitosan,29,30 and triclosan31 have been widely used to incorporate into a polymeric matrix to produce antimicrobial coatings using different coating strategies such as the layer-by-layer deposition,32 spin-coating,33 and solvent-casting.34 These antimicrobial polymers mainly achieve their antimicrobial activity by “release-killing” microorganisms in solution and/or by “contact-killing” microorganisms upon adsorption. However, a major challenge for these antimicrobial agent coated surfaces still remains, e.g., how to remove dead microorganisms from antimicrobial coatings. The dead microorganisms on the surfaces will block active groups, impair antibacterial activity, and trigger the immune response and chronic inflammation.35 Our previous works have demonstrated that poly(Nhydroxyethylacrylamide) (polyHEAA)-based materials of different polymeric architecture forms (brushes, nanoparticles, and nanogels) exhibited a superlow fouling property in different biomedia. For example, the polyHEAA brushes were highly resistant to protein adsorption from undiluted blood serum and plasma (
