Magnetic Glycol Chitin-Based Hydrogel Nanocomposite for Combined

Article Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/aidcbc

Magnetic Glycol Chitin-Based Hydrogel Nanocomposite for Combined Thermal and D‑Amino-Acid-Assisted Biofilm Disruption Eric C. Abenojar,†,# Sameera Wickramasinghe,†,# Minseon Ju,† Sarika Uppaluri,† Alison Klika,‡ Jaiben George,‡ Wael Barsoum,‡ Salvatore J. Frangiamore,§ Carlos A. Higuera-Rueda,*,‡ and Anna Cristina S. Samia*,† †

Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, United States Department of Orthopaedic Surgery, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, United States § Summa Health Orthopaedics and Sports Medicine, 1 Park West Boulevard, Akron, Ohio 44320, United States ‡

S Supporting Information *

ABSTRACT: Bacterial biofilms are highly antibiotic resistant microbial cell associations that lead to chronic infections. Unlike free-floating planktonic bacterial cells, the biofilms are encapsulated in a hardly penetrable extracellular polymeric matrix and, thus, demand innovative approaches for treatment. Recent advancements on the development of gel-nanocomposite systems with tailored therapeutic properties provide promising routes to develop novel antimicrobial agents that can be designed to disrupt and completely eradicate preformed biofilms. In our study, we developed a unique thermoresponsive magnetic glycol chitin-based nanocomposite containing D-amino acids and iron oxide nanoparticles, which can be delivered and undergoes transformation from a solution to a gel state at physiological temperature for sustained release of Damino acids and magnetic field actuated thermal treatment of targeted infection sites. The D-amino acids in the hydrogel nanocomposite have been previously reported to inhibit biofilm formation and also disrupt existing biofilms. In addition, loading the hydrogel nanocomposite with magnetic nanoparticles allows for combination thermal treatment following magnetic field (magnetic hyperthermia) stimulation. Using this novel two-step approach to utilize an externally actuated gel-nanocomposite system for thermal treatment, following initial disruption with D-amino acids, we were able to demonstrate in vitro the total eradication of Staphylococcus aureus biofilms, which were resistant to conventional antibiotics and were not completely eradicated by separate D-amino acid or magnetic hyperthermia treatments. KEYWORDS: hydrogel nanocomposite, iron oxide nanoparticles, D-amino acids, magnetic hyperthermia, Staphylococcus aureus, biofilm

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treatment of medical device related infections remains a huge challenge in the clinical practice setting, mainly because of the extreme difficulty in the disruption and eradication of bacterial biofilm growth, which is resistant to antibiotics, as well as mechanical treatment approaches.11−13 A biofilm is an association of microorganisms that attaches on biological or nonbiological surfaces, which is encased in a matrix of self-produced extracellular polymeric substances (EPS) that acts as a protective barrier and provides structural stability to the biofilm.3,14 Unlike planktonic bacterial cells, which are susceptible to antibiotics and can be eliminated by antibodies and phagocytes, bacterial cells embedded inside biofilms show increased immunity and resistance to antimicrobials used in the treatment of infectious diseases. The detailed process by which biofilms becomes inherently resistant to antimicrobial agents is not well understood but two

ntimicrobial resistance is a global health crisis that complicates the prevention and treatment of diseases caused by pathogenic microbes.1 People infected with antibiotic resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus, have significantly higher morbidity and mortality rates (∼64%) than those exposed to nonresistant bacterial cell lines.1,2 Moreover, the formation of bacterial biofilms protects the pathogen from the host’s immune response and antibiotic treatment, causing chronic infections that form the basis of more than 80% of persistent microbial infections in the human body.3,4 These reports include nondevice-related infections, such as osteomyelitis, chronic wounds,4 and medical-device-related infections, involving orthopedic prostheses, pacemakers, and catheters.5,6 Different types of bacterial strains, such as Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus epidermidis (S. epidermidis) can easily deposit and form biofilms on implant material components leading to disease complications and untimely implant failures.7−10 As such, the © XXXX American Chemical Society

Received: March 21, 2018

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DOI: 10.1021/acsinfecdis.8b00076 ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

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mechanisms have been proposed.15−17 The first mechanism involves the inability of antimicrobial agents to penetrate the biofilm completely due to the presence of the EPS, which retards the diffusion of antibiotics, rendering the drug to be less effective.15,16 The second resistance mechanism involves the fact that bacterial cells in biofilms are nutrient deficient and exist in a starved or slow growth state.16,17 These kinds of bacterial cells (slow growing/nongrowing) are not susceptible to antimicrobial agents and serve as important targets for innovative nanoparticle-based antimicrobial therapeutic approaches.18−20 Recently, the use of magnetic hyperthermia for antibacterial applications has been explored, and it has been demonstrated to be a promising approach to inactivate bacteria both as planktonic cells and as associated biofilms.21−28 Magnetic hyperthermia using biocompatible iron oxide nanoparticles that generate heat when exposed to an alternating current (AC) magnetic field has been reported to enhance the antibiotic efficacy of drugs, as well as stimulate biofilm dispersal.21,22,24−29 Moreover, magnetic hyperthermia has also been demonstrated to be much more effective than direct heating toward the destruction of the bacterial cell membrane integrity, as well as promoting modifications on the bacterial cell surface and biofilm structure that can eventually lead to successful eradication.23,24 The thermal disruption of bacterial biofilms using magnetic hyperthermia is attractive because it does not rely on the use of antibiotics and could treat antibiotic-resistant bacterial strains. In addition, magnetic hyperthermia can be exploited to enable localized heating for targeted biofilm eradication.30 On the other hand, amino acids have been reported to exhibit biofilm disruption activity by interfering with the metabolic pathway of the bacteria.31−34 In particular, various Damino acids (D-AAs) have shown biofilm inhibition and disruption activities against several bacterial strains such as S. aureus and P. aeruginosa.35,36 The investigated D-AAs were found to disrupt bacterial biofilms by integrating into the cell wall of the bacteria cells.35 A combination of D-AAs has also shown better biofilm dispersal activity than their individual DAA counterparts toward S. aureus.37 In addition, mixtures of DAAs in combination with common antibiotics have been reported to demonstrate enhanced biofilm dispersal activities.38 Likewise, D-AAs have been shown to inhibit biofilm formation but did not affect new bone formation at high D-AA mixture concentrations (200 mM).39 A unique advantage of utilizing DAAs in the treatment of microbial infections in humans is that these isomers are not involved in normal biological processes. Nevertheless, with recent advancements in analytical methods, researchers were able to detect D-AAs in the human body as a result of consuming processed foods.40,41 Moreover, two enzymes were identified to be involved in the metabolism of 40,41 D-AAs; amino acid racemase and D-amino acid oxidase. However, the enzymatic hydrolysis of proteins containing DAAs, as well as the activity of mammalian D-amino acid oxidase toward many D-AAs, are demonstrated to be inefficient, thus, majority of consumed D-AAs are excreted from the body.41 With the identification of promising antimicrobial agents, another important consideration is the formulation by which the components will be introduced to the infected sites. To keep the treatment solution at the infected site, the use of biocompatible hydrogels, such as glycol chitin, as a suitable medium for drug delivery has been previously explored.42 Glycol chitin is a water-soluble thermoresponsive polymer

derived from glycol chitosan, which can be tuned to change from the sol to the gel phase at different temperatures by controlling the degree of N-acetylation and concentration of the polymer in the delivery solvent.42 Polymers derived from natural polysaccharides such as chitosan are ideal for biomedical applications because of their low toxicity, biocompatibility, and biodegradability.43,44 Gelation occurs for chitosan-based hydrogels via hydrophobic and hydrogen bonding molecular interactions between the polymer chains, and because formation of a network by this gel is based on these weak interactions, the gelation process is reversible.44 In this study, we combine the unique attributes of a magnetic glycol chitin-based, D-AA loaded hydrogel (MagDAA gel) nanocomposite to disrupt and effectively eradicate preformed bacterial biofilms of S. aureus. We report on the synthesis and optimization of the physical, chemical, and antibacterial properties of a novel magnetic hydrogel system made of iron oxide nanoparticles and a glycol chitin-based hydrogel loaded with a mixture of D-AAs (D-tyrosine, D-tryptophan, and Dphenylalanine) as a new treatment approach toward biofilm disruption that takes advantage of the biofilm dispersal activity of D-AAs and the magnetic hyperthermia effect of the magnetic nanoparticles. In our study, we could effectively demonstrate, for the first time, that a two-step treatment approach that utilizes an externally actuated gel-nanocomposite system for thermal treatment, following initial disruption with D-AAs, is effective in the disruption and total eradication of S. aureus biofilms, which were resistant to conventional antibiotics and were not completely eradicated by separate D-AA or magnetic hyperthermia treatments.

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RESULTS AND DISCUSSION Synthesis and Characterization of Magnetic Nanoparticles (MNPs). The use of magnetic hyperthermia for heat treatment-aided biofilm disruption involving iron oxide nanoparticles, which release localized heat upon excitation with an AC magnetic field, was achieved by first optimizing the heating efficiency of the magnetic nanoparticles (MNPs). To accomplish this task, magnetic nanoparticles with spherical (sMNPs) and cubic (c-MNPs) shapes with similar volumes were synthesized by adapting previously reported thermal decomposition synthetic methods with slight modifications.45−47 Shown in Figure 1A and 1B are the transmission electron microscope (TEM) images of the synthesized s-MNPs that have an average diameter of 30 nm and the prepared c-MNPs with an average size of 25 nm, respectively; wider view TEM images of the MNPs are presented in Figure S1. The powder Xray diffraction (PXRD) patterns obtained from the samples show that both types of MNPs have the magnetite (Fe3O4) crystallographic phase (Figure 1C and 1D). Moreover, results from dynamic light scattering (DLS) measurements showed that the as-prepared oleic acid coated-MNPs did not significantly change in size (based on the evaluated hydrodynamic diameters) after conversion to their water-soluble forms using a silane-based ligand exchange process46,48 (Figure 1E and 1F). The magnetic hyperthermia performance of the synthesized MNPs was evaluated by measuring the change in temperature under AC magnetic field excitation at different sample concentrations (250−750 μg Fe/mL) and under varying AC field amplitudes (H from 1−5 kA/m) at a constant excitation frequency ( f) of 380 kHz. In our study, the values of H and f (H × f ≤ 1.90 × 109 A m−1 s−1) were judiciously selected to B



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Figure 2. (A) Specific absorption rate (SAR) values evaluated for the s-MNPs and c-MNPs, respectively, measured at different concentrations of Fe (250−750 μg Fe/mL) and variable AC magnetic field amplitudes (1−5 kA/m) excitation at a fixed frequency of 380 kHz. (B) Field-dependent magnetization of the s-MNP and c-MNP samples measured from −1 to 1 T at 300 K using a vibrating sample magnetometer (VSM).

Figure 1. TEM images of (A) spherical magnetic nanoparticles (sMNPs) and (B) cubic magnetic nanoparticles (c-MNPs). The corresponding PXRD patterns of the (C) s-MNP and (D) c-MNP samples with reference to the magnetite, Fe3O4, iron oxide crystallographic phase. Hydrodynamic sizes of the (E) s-MNP and (F) c-MNP samples measured using DLS, respectively.

avoid possible side effects, such as damage to noninfected cells, because of excess heat production by MNPs at much higher magnetic fields and excitation frequencies; this value is within the range of the acceptable threshold limit for clinical magnetic hyperthermia that avoids any adverse effects to healthy cells.49−51 Moreover, the tolerable magnetic field strengths for thermotherapy range from 3.8 to 13.5 kA/m.52 The heating efficiency was assessed by estimating the specific absorption rate (SAR) based on the obtained temperature curves during AC field exposure. Comparison of the magnetic hyperthermia performance of the differently shaped MNPs showed that the cubic sample (c-MNPs) has higher heating efficiency (larger SAR values across the concentration range) than its spherical counterpart (s-MNPs) (Figure 2A). This result can be both attributed to the enhanced shape anisotropy in the cubic shaped MNPs53 and its higher saturation magnetization (Figure 2B that was evaluated using vibrating sample magnetometry, VSM), which together lead toward enhanced magnetic hyperthermia performance.47 Since the cMNPs at a concentration of 750 μg Fe/mL demonstrated the best heating efficiency, this cubic MNP formulation was used in the succeeding preparation of the magnetic hydrogels for biofilm disruption. Moreover, to prepare for the in vitro cell studies, the biocompatibility of the water-soluble MNPs was also assessed through a PrestoBlue cell viability assay using HeLa cells. The results of the cell toxicity assay show that both s-MNPs and c-MNPs samples are nontoxic in the concentration range of 250−750 μg Fe/mL for both 2 and 24 h cell exposure times (Figure 3), and can thus be used in the following cell experiments.

Figure 3. Time-dependent HeLa cell cytotoxicity assay results for the s-MNPs and c-MNPs samples in the concentration range from 250 to 750 μg Fe/mL at 2 and 24 h cell exposure times using saline (0.9% NaCl) as positive control.

Preparation of Thermoresponsive Magnetic Glycol Chitin Hydrogel. We prepared a thermoresponsive magnetic glycol chitin based hydrogel that is nontoxic and has a gelation temperature that can be tuned by changing the concentration of glycol chitin solution loading. The glycol chitin polymer was prepared via the controlled N-acetylation of the glycol chitosan precursor (Figure 4A). The successful conversion of the glycol chitosan precursor to the desired glycol chitin derivative can be confirmed from the obtained 1H NMR spectra from the two molecular forms, which show the loss of the amine group signal following successful conversion to glycol chitin (Figure 4B). FT-IR spectroscopy also confirms the formation of the glycol C



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Figure 4. (A) Synthetic route for the preparation of the thermoresponsive glycol chitin-based hydrogel from the selective N-acetylation of glycol chitosan. (B) 1H NMR spectra representing the successful conversion of glycol chitosan to glycol chitin as indicated by the absence of the amine (−NH2) peak in the spectrum for glycol chitin. (C) FT-IR spectra showing the conversion of the precursor glycol chitosan to glycol chitin as revealed by the loss of the amine peak (−NH2) and the emergence of the more pronounced carbonyl (-C = O) and amide (−CONH2) peaks in glycol chitin derivative.

Figure 5. (A) Rheological measurements of different glycol chitin mixtures in 0.9% NaCl at varying glycol chitin loadings (1−5% wt.) showing the concentration dependence of the viscosity over a temperature range of 5−75 °C. (B) Photograph of the synthesized magnetic D-amino acid loaded hydrogel (MagDAA gel containing 5% wt. glycol chitin in 0.9% NaCl) showing its sol−gel transition at the physiological temperature of 37 °C. (C) Schematic diagram of the magnetic hyperthermia measurement setup used in the study, wherein the sample to be tested is placed in an insulator in the middle of water cooled AC magnetic field coil, and the temperature is measured using a temperature sensitive fiber optic probe. (D) Magnetic hyperthermia heating profiles of the synthesized c-MNPs dispersed in 0.9% NaCl and suspended in the MagDAA gel matrix (5% wt. glycol chitin solution and 750 μg Fe/mL of c-MNPs) measured at an excitation AC field amplitude (H) of 5 kA/m and a frequency (f) of 380 kHz.

chitin derivative, which is reflected by the loss of the amine peak and the appearance of stronger peaks for the carbonyl and amide groups (Figure 4C). After verifying the successful conversion of glycol chitosan to the glycol chitin derivative, the changes in viscosity as a function of glycol chitin mass loading across a temperature range of 5−75 °C were evaluated using a Thermo Scientific HAAKE MARS III Rheometer. The rheological measurement

data obtained for different glycol chitin mixtures in 0.9% NaCl at varying glycol chitin loadings (1−5% wt.) are shown in Figure 5A. The 5% wt./vol glycol chitin solution showed an abrupt rise in viscosity at around the physiological temperature of 37 °C, which signifies the onset of the sol to gel phase transition. In contrast, lower mass loadings (1 and 3% wt./vol glycol chitin solutions) barely showed changes in viscosity across the temperature range indicating that the gelation D



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Figure 6. (A) Concentration dependent biofilm disruption effects of vancomycin, and the corresponding (B) crystal violet stained samples of the remaining biofilms after treatment with different amounts of vancomycin for 24 h. (C) Concentration dependent biofilm disruption effects of Damino acid mixtures (D-trp, D-tyr, and D-phe) and the corresponding (D) crystal violet stained samples of the remaining biofilm after treatment with different amounts of the D-amino acid mixtures for 24 h. The biofilms were grown from bacterial cultures of S. aureus (2 × 105 cells/well) in modified tryptic soy broth (3% NaCl, 0.5% glucose) for 24 h at 37 °C. An asterisk (*) indicates that the difference in means of the treatments are statistically significant at p < 0.05, while NS indicates that the difference in means of the treatments are not statistically significant at p > 0.05.

not disrupt preformed biofilms. In addition to the above concentrations of vancomycin, we further explored the biofilm disruption property of vancomycin at a much higher concentration (200 mM), as well as the dispersal properties of other clinically used antibacterial reagents, such as bacitracin,58,59 NeutroPhase,60 and Hibiclens,61 after a 24 h incubation period (Figure S2). Even though vancomycin (200 mM) showed promising results within 24 h, the in vitro cytotoxicity assay proved that 200 mM of vancomycin is extremely toxic to mammalian cells to be of use in the clinical setting (Figure S3). On the other hand, previous reports on D-AAs have shown their potential to interfere with the metabolic pathway of bacterial cells leading to significant biofilm disruption activity.31 Various D-AAs [D-tyrosine (D-tyr), D-methionine (D-met), Dtryptophan (D-trp), D-phenylalanine (D-phe), and D-leucine (Dleu)] have been reported to show biofilm inhibition and disruption activities against several bacterial strains such as S. aureus and P. aeruginosa. Considering the structural properties of the D-AAs, we hypothesized that having aromatic rings on the D-AAs would be structurally advantageous to promote interactions with the protective extracellular polymeric matrix of the biofilms, and as such, we investigated the effects of individual D-AAs on bacterial biofilm disruption (Figure S4). The results showed that none of the individual D-AAs were able to implement the complete eradication of the biofilms at their highest possible water-soluble concentrations. In this regard, we evaluated the biofilm dispersal activity of a mixture of three aromatic D-AAs (D-trp, D-tyr, and D-phe) at concentrations close to the sum of their maximum individual solubilities (100− 200 mM) (Figures 6C and 6D). Increasing biofilm disruption activity was observed with increasing amino acid concentration, and total biofilm disruption was observed at a concentration of 200 mM. To test for biocompatibility, HeLa cell toxicity assays

process was not achieved at these low glycol chitin polymer concentrations at the specified temperature range (Figure 5A). The concentration and temperature dependent sol−gel transition at 37 °C was further confirmed through the tube inversion method where sample gelation was characterized by absence of flow for 30 s (Figure 5B). Using the 5% wt. glycol chitin solution (with sol−gel transition at 37 °C) and 750 μg Fe/mL c-MNPs (MNP with highest heating efficiency) as sample matrix, a magnetic glycol chitin-based, D-AA loaded hydrogel (MagDAA gel) nanocomposite was prepared. The magnetic hyperthermia property of the resulting MagDAA gel nanocomposite was evaluated and compared to the water dispersed c-MNPs (Figure 5C and 5D). It can be observed that a lower increase in temperature was achieved in the hydrogel sample (5 °C) compared to when the MNPs are in solution (25 °C). The decrease in heating efficiency of the c-MNPs in the gel matrix can be attributed to the viscous nature of the hydrogel whereby Brownian rotation is restricted leading to lower heating performance24,53 (Figure 5D). Biofilm Disruption Using Magnetic Glycol ChitinBased, D-Amino-Acid-Loaded Hydrogel (MagDAA gel) Nanocomposite. S. aureus is one of the major causes of hospital- and community-based acquired infections resulting in the formation of methicillin-resistant S. aureus (MRSA) strains that is more difficult to treat using antibiotics that are currently available.54 In our study, one of the most widely used antibiotics in the clinical setting, vancomycin, was evaluated for its biofilm dispersal activity against S. aureus at relevant clinical concentrations (16 mg/kg or ppm every 12 h for vancomycin), as well as concentrations that are lower and higher than the clinical dosage to establish if there is a concentration effect.55−57 Figures 6A and 6B show that vancomycin at concentrations ranging from 2 to 256 ppm do E



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were performed on the D-AA and antibiotic treatments. Results showed that the D-AA mixture at a concentration of 200 mM was toxic to HeLa cells at 24 h incubation period (Figure 7).

Figure 7. Time-dependent HeLa cell cytotoxicity assay results for the c-MNPs, hydrogel (5% wt. glycol chitin in 0.9% NaCl), D-amino acid mixture (D-AA with D-tyr: D-trp: D-phe in 1:22:57 molar ratio), and MagDAA gel at 2 and 24 h cell exposure time periods; NS indicates that the difference in means of the treatments are not statistically significant at p > 0.05.

Figure 8. Biofilm disruption activity comparison of the different treatments used in this study and the corresponding crystal violet stained samples of the remaining biofilms after the different treatments. The use of the MagDAA gel, which involves initial treatment for 2 h followed by additional AC magnetic field (AMF) treatment for 10 min at AC field amplitude of 5 kA/m and a frequency of 380 kHz, showed complete biofilm disruption. NS indicates that the difference in means of the treatments are not statistically significant at p > 0.05.

From this result, we decided to perform a time dependent biofilm disruption assay using 200 mM D-AAs (15 min to 24 h) to find a viable incubation time point that shows significant biofilm disruption, as well as nontoxicity to HeLa cells (Figures 7 and S5). Figure S5 shows that biofilm disruption starts to plateau after 2 h with ∼85% disruption activity. In addition, the 2 h incubation of HeLa cells with the 200 mM D-AA mixture demonstrates that it is nontoxic at this incubation time point (Figure 7). In contrast, at a similar vancomycin concentration of 200 mM, the antibiotic showed extreme cell toxicity for both at 2 and 24 h incubation periods (Figure S3). Considering the results of the biofilm disruption and cell toxicity assays, we then formulated our MagDAA gel with 200 mM loading of D-AAs consisting of 54 mM D-typ, 2.5 mM Dtyr, and 143.5 mM D-phe. A combination of 2 h D-AA biofilm disruption and 10 min application of an alternating magnetic field (AMF-induced hyperthermia at H = 5 kA/m; f = 380 kHz) showed complete biofilm disruption with the appropriate positive (saline treatment) and negative (no bacterial cell treatment and no biofilm formation) controls (Figure 8). In contrast, individual treatments with D-AA or magnetic hyperthermia have not led to complete biofilm eradication (Figure 8). The observed incomplete biofilm eradication by magnetic hyperthermia can be attributed to the selected low field (H) and frequency ( f) values that were adopted to avoid damage to healthy cells.49,51,62 On the other hand, hydrophobic D-AAs have been known to permeabilize bacterial lipid cell membrane causing cell wall disruption, but not complete biofilm damage.40,63 Therefore, in our study, we have adapted an adjuvant treatment approach using a combination of D-AA pretreatment (for initial disruption of the EPS and microbial cell associations) followed by magnetic thermal therapy, to completely destroy remaining bacterial biofilms. In order to investigate the effects of the D-AA and combined D-AA + AMFinduced hyperthermia treatment on the biofilm disruption, we have performed cryo-scanning electron microscopy (cryoSEM) analyses on treated and untreated preformed biofilms (Figure S6). Our cryo-SEM studies demonstrate the disruptive effects of the D-AAs on the EPS of the biofilms and the complete eradication of the biofilms upon additional AMFinduced hyperthermia treatment (Figure S6).

The magnetic hyperthermia effect of the c-MNPs in the MagDAA gel served two purposes; enhancement of the cumulative release of the D-AAs in the gel, which was estimated by monitoring the absorbance of the released D-AA mixture at 280 nm (Figure S7), and enabling the complete disruption of the biofilm after pretreatment with the D-AA mixture (Figure 8). Moreover, cell toxicity assay showed that 2 h exposure of HeLa cells to the MagDAA gel is nontoxic to the cells (Figure 7). We have also investigated the effect of using lower concentrations of D-AAs and found that only the 200 mM concentration combined with AMF treatment leads to complete biofilm eradication (Figure S8). While our in vitro HeLa cell toxicity studies indicate nontoxicity of the 200 mM DAA mixture at a 2-h incubation period, the actual release of the D-AAs from the gel matrix is only up to a total of ∼70% or 140 mM for both the 24 h incubation time point or the combined 2 h + AMF treatment approach (Figure S7). From the recent in vivo studies conducted by Harmata et al.39 using a sheep animal model, they have demonstrated that D-AA mixture concentrations are nontoxic in vivo from 0 to 200 mM for a 24 h incubation period and also do not inhibit new bone formation. Our treatment approach is within the range of the reported in vivo concentration tolerance for D-AA with minimal side effects. To further investigate whether the biofilms have been completely eradicated or there are remaining viable bacterial cells that could reattach to the surface and seed new biofilm formation, we have performed a microbial cell viability assay that provides a method to determine the number of viable bacterial cells based on the quantitation of the ATP present. This assay involves adding a commercial reagent (BacTiter-Glo Reagent, Promega) directly to the bacterial cells released into the test medium and measuring luminescence to evaluate if the cells that have been freed from the biofilms following D-AA or combined D-AA + AMF treatments are viable or not. Shown in Figure 9A is the result of our microbial cell viability assay that demonstrates that the bacterial cells exposed to the combined D-AA and AMF-induced hyperthermia treatment are not viable, which would prevent them from reattaching to the surface to F



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nanocomposite was nontoxic toward HeLa cells when exposed for 2 h, in contrast to similar concentrations of antibiotics used in the clinical setting (i.e., vancomycin). Moreover, separate DAA or magnetic hyperthermia treatments did not result in complete biofilm eradication. Using our fabricated MagDAA gel nanocomposite system, we were able to develop a unique twostep biofilm treatment approach that utilizes an externally magnetic field actuated gel-nanocomposite for thermal treatment, following initial disruption with D-AAs. This innovative nanocomposite-enabled treatment approach has great potential to treat chronic infections linked to bacterial biofilm growth, which have been associated with recurring external wounds of bedridden patients and patients with chronic diabetic foot ulcers. Moreover, this platform technology could also be adapted in the treatment of biofilm related infections associated with plastic based implants involving an open surgery treatment approach.

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EXPERIMENTAL SECTION Materials and Reagents. The following chemicals were purchased from Sigma-Aldrich and used as received: D-tyrosine (99%), D-tryptophan (≥98.0%), D-phenylalanine (≥98%), Dmethionine (≥98%), vancomycin hydrochloride, acetic acid (glacial), methanol (≥99.8%), ethanol (70%), toluene (99.9%), hexane (≥98.5%), acetic anhydride (≥99%), acetone (≥99.5%), glycol chitosan (≥50%), iron(III) chloride hexahydrate (98%), iron(III) acetylacetonate (97%), oleic acid (90%), 4-biphenylcarboxylic acid (95%), benzyl ether (98%), 1octadecene (90%), triethylamine (≥99%), and trimethylamine N-oxide (98%). Sodium hydroxide (99%), ammonium hydroxide (14.8 N), 1-butanol (≥99.4%), crystal violet, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (10 000 U/mL), trypsinEDTA (0.25%) phenol red, phosphate buffered saline (PBS) solution, sodium chloride solution (0.9%, w/v), LB broth powder (50% tryptone, 25% yeast extract, 25% sodium chloride), LB agar powder (25% tryptone, 12.5% yeast extract, 25% sodium chloride and 37.5% agar) and PrestoBlue assay kit were purchased from Fisher Scientific. Sodium oleate (>97%) was obtained from TCI America. The ligand, 3-(triethoxysilyl)propyl succinic anhydride was purchased from Gelest, Inc. S. aureus (ATCC 10832) and HeLa cells (ATCC CCL-2) were purchased from the American Type Culture Collection. Hibiclens (Mö l nlycke Health Care Inc.), Neutrophase (NovoBay Pharmaceuticals, Inc.), and bacitracin (Sandoz) were obtained from Dr. Higuera-Rueda. BacTiter-Glo microbial cell viability assay was purchased from Promega. Methods. Synthesis of Spherical Magnetic Nanoparticles (s-MNPs). s-MNPs were prepared via a two-step synthetic approach: the first step involved the thermal decomposition of an iron oleate complex to form wüstite (FeO) nanoparticles, which was then subsequently converted to the magnetite (Fe3O4) phase using a mild oxidation step.45 The iron oleate precursor was prepared by dissolving iron(III) chloride hexahydrate (FeCl3·6H2O, 40 mmol) and sodium oleate (120 mmol) in a flask containing deionized (DI) water (60 mL), ethanol (80 mL), and hexane (140 mL). The mixture was then heated at reflux for 4 h. After reflux, the organic layer containing the iron oleate precursor was separated from the aqueous layer and washed three times with warm water to remove salt byproducts and excess reagents. The iron oleate mixture was then dried under vacuum for 72 h and stored for further use.

Figure 9. (A) Microbial cell viability assay involving luminescence measurement based on the ATP quantification in viable bacterial cells following the addition of the BacTiter-Glo reagent on the cell culture medium after different biofilm treatment conditions; the positive control represents the same number of planktonic S. aureus cells (105) that was used in the formation of the biofilms, and the negative control is the cell culture medium in the absence of any bacterial cells. (B) Colony-forming assay involving the regrow of any remaining adherent bacterial cells following biofilm treatments; the positive control is the untreated biofilm and no biofilm was grown for the negative control. NS indicates that difference of means are not statistically significant at p > 0.05.

form new biofilms. On the other hand, to investigate whether there are adherent bacterial cells remaining that could regrow the biofilms, we performed a set of experiments to test the survival of remaining adherent bacterial cells using the colonyforming assay approach. Our results demonstrate that no new colonies can be formed from the biofilms subjected to the combined D-AA and AMF-induced hyperthermia treatments, while some colonies formed in the biofilms treated with only DAA (Figure 9B). These results demonstrate the potential viability of the two-step biofilm treatment approach, which utilizes a magnetically actuated gel nanocomposite system for thermal treatment that follows an initial disruption treatment with D-AAs, for complete biofilm eradication in the clinical setting.

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CONCLUSIONS We have successfully prepared a novel thermoresponsive magnetic hydrogel nanocomposite (MagDAA gel) using a glycol-chitin based hydrogel for combined thermal and D-AA assisted S. aureus biofilm disruption in vitro.. The prepared MagDAA gel contains 200 mM D-AA mixture of D-tyr, D-trp, and D -phe and 750 μg Fe/mL of cubic shaped Fe 3O4 nanoparticles in a 5% glycol chitin solution matrix in 0.9% NaCl. Complete eradication of the preformed biofilms was achieved by exposing the sample for 2 h with the MagDAA gel, followed by the application of an applied magnetic field (hyperthermia) for 10 min at a low amplitude (H) of 5 kA/m and a frequency ( f) of 380 kHz. The prepared hydrogel G



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temperature probe (Neoptix T1) and was recorded every 5 s. Prior to turning the magnetic field on, the sample temperature was recorded for 30 s to obtain a stable baseline for the calculation of the SAR values. The SAR was calculated from the initial slope over the first 30 s of the heating curve using the equation

To synthesize 30 nm s-MNPs, iron oleate (3.6 g) and oleic acid (15 mL) were vigorously stirred under argon (Ar) atmosphere. The solution was then heated to 380 °C (3 °C/ min) and refluxed for an hour. The reaction mixture was then cooled to ambient temperature and FeO nanoparticles were precipitated via the addition of 1:1 toluene: ethanol solvent mixture (35 mL) and centrifuged at 7000 rpm for 20 min. FeO nanoparticles were converted to Fe3O4 using trimethylamine N-oxide [(CH3)3NO] as an oxidizing agent. Briefly, (CH3)3NO (0.1 mmol) was added to FeO nanoparticles (100 mg), oleic acid (0.5 mL), and 1-octadecene (20 mL). The reaction mixture was heated to 130 °C (10 °C/min) for 2 h and the temperature was further raised to 280 °C (10 °C/min) and held at that temperature for 1 h. The nanoparticles were then cooled down to ambient temperature and transferred to a 50 mL centrifuge tube. A 30 mL solvent mixture of 1:1 toluene: ethanol was added to the reaction mixture and centrifuged at 7000 rpm for 20 min. The s-MNP precipitate collected was dissolved in 10 mL of toluene, degassed with Ar, and stored for further use. Synthesis of Cubic Magnetic Nanoparticles (c-MNPs). cMNPs were synthesized using a thermal decomposition approach.47 Iron(III) acetylacetonate (0.5 mmol), 4-biphenylcarboxylic acid (0.5 mmol), oleic acid (1.90 mmol), and benzyl ether (52.61 mmol) were placed in a 50 mL three-neck roundbottom flask. The mixture was heated for 30 min at 70 °C under an Ar atmosphere, then again for 90 min at 300 °C. After the reaction mixture has cooled down to 60 °C, ethanol was added and the nanoparticles were centrifuged, isolated, and redispersed in toluene. Characterization of Magnetic Nanoparticles (MNPs). The MNP size and shape were evaluated by transmission electron microscopy (TEM). TEM samples were prepared by placing 5 μL of a dilute suspension of the MNPs on a 400 mesh Formvarcoated copper grid and allowing the solvent to evaporate slowly at room temperature. TEM images were obtained with a FEI Tecnai G2 Spirit BioTWIN transmission electron microscope operated at 120 kV. The mean particle size and size distribution were evaluated by measuring at least 200 nanoparticles for each sample. The crystal structure of the samples was analyzed by powder X-ray diffractometry (PXRD) performed in a Rigaku MiniFlex powder X-ray diffractometer using Cu Kα radiation (λ = 0.154 nm). For the XRD analysis, the diffraction patterns were collected within a 2θ range of 25−75°. The average hydrodynamic radii of the different oxidized MNP samples were measured by dynamic light scattering (DLS) on a ZetaPALS particle size analyzer (Brookhaven) at a scattering angle of 90°. The total Fe concentration in each sample was measured using a fast sequential atomic absorption spectrophotometer (AAS) Varian 220FS AA. For the elemental Fe analysis, the samples were digested in concentrated hydrochloric acid overnight to completely dissolve the MNPs. The magnetic hyperthermia measurements were performed using a MSI Automation bench mount magnetic induction heating system. The MNPs samples were exposed to an alternating current (AC) magnetic field excitation with variable magnetic field amplitude (H) of 1−5 kA/m at a fixed frequency (f) of 380 kHz. All samples were measured inside insulated NMR glass tubes with an internal diameter of 7.5 mm, at different iron concentrations (250−750 μg/mL). To evaluate the temperature profiles of the samples upon excitation with a continuous alternating magnetic field, the change in temperature of the samples was monitored with a fiber optic

SAR =

CVs dT m dt

where dT/dt is the initial slope of the heating curve, C is the volumetric specific heat capacity of the solvent, Vs is the volume of the sample, and m is the mass of magnetic material (i.e., g of Fe) in the sample. The reported average SAR values were calculated from three repeat measurements. The field-dependent magnetization data of the MNPs were measured using a Lakeshore 7307 vibrating sample magnetometer (VSM) from −1 to 1 T at 300 K. Synthesis of Glycol Chitin Hydrogel. A thermoresponsive hydrogel based on glycol chitosan was prepared according to a previously reported procedure.42 Briefly, glycol chitosan (0.25 g) was dissolved in a 50:50 mixture of water to methanol (50 mL), followed by the slow addition of acetic anhydride (0.83 mL). The resulting reaction mixture was then left to stir for 48 h. The product was then precipitated with acetone and centrifuged at 17000 rpm for 10 min at 4 °C. The isolated glycol chitin was then redissolved in deionized water and incubated in 1 M NaOH solution for 12 h to remove undesired reaction byproducts. The sample was then dialyzed in a dialysis membrane (molecular weight cut off = 2000 Da) for 3 days. The purified product was then reprecipitated with acetone and centrifuged at 4 °C to isolate the glycol chitin. Following lyophilization, the product was collected and stored for further use. The successful conversion of glycol chitosan to glycol chitin was confirmed via 1H NMR spectroscopy using a 500 MHz Bruker Ascend Avance III HD. Moreover, FT-IR spectroscopy measurements were performed using a Thermo Scientific Nexus 870 ATR-FTIR spectrometer in the spectral range of 600−4000 cm−1. Rheological studies on glycol chitin hydrogels prepared by mixing different mass loadings of glycol chitin in 0.9% NaCl were performed by measuring temperature dependent changes in viscosity of the hydrogels using a Thermo Scientific HAAKE MARS III Rheometer. The viscosities of the hydrogels were measured in the temperature range of 5−75 °C at a heating rate of 0.0575 °C/s and a shear rate of 0.1/s. Preparation of Magnetic Hydrogels Using Water-Soluble c-MNPs. Magnetic hydrogels were prepared by mixing watersoluble c-MNPs with the glycol chitin-based hydrogels at the appropriate loading concentrations. Water-soluble c-MNPs were prepared by modifying the surface of the as-prepared oleic acid coated nanoparticles with a carboxyl-terminated silane ligand using a previously reported ligand exchange process.46,48 A solution containing NH4OH in 1-butanol (1 M), triethylamine (1.4 mL), deionized water (0.5 mL), and 3(triethoxysilyl)propyl succinic anhydride (100 μL) was vortexed for 5 min. The c-MNPs in toluene (25 mg/mL) were added to the previous solution and vortexed for another 5 min. The sample was allowed to sit for at least 1 h after which the c-MNPs transferred from the organic to the water phase. The resulting mixture was then centrifuged at 8500 rpm for 30 min. The supernatant was removed and the c-MNP sample was redispersed in deionized water. H



Article

Biofilm Formation and Dispersal Assays. S. aureus were cultured overnight in agar plates or in lysogeny broth (LB) with agitation (200 rpm) at 37 °C. Biofilm formation was evaluated under static conditions using 12-well plates (Falcon, USA). Bacterial cultures made overnight were diluted to an OD595 of 0.1 and further diluted 100× (∼105 CFU/mL) in modified tryptic soy broth (3% NaCl, 0.5% glucose) and each well was filled with 2 mL of the diluted bacterial solution and incubated at 37 °C for 24 h. Biofilm dispersal activities were evaluated by removing the culture media from the biofilms after 24 h and replacing it with saline solution (0.9% NaCl) containing either individual or a combination of amino acids at the specified concentrations (pH 7.4), or the other antimicrobial agents used in the clinic (vancomycin, bacitracin, NeutroPhase, Hibiclens). After exposure to the different treatment methods, the plates were gently washed with phosphate buffered saline (PBS, 1×) thrice and then stained with 500 μL of 0.1% (w/v) crystal violet for 30 min. The wells were then washed with PBS and the crystal violet stain was solubilized with 30% acetic acid. The solution was diluted 20 times and biofilm biomass was estimated by measuring the crystal violet stained biofilm remnants, which absorbs at 595 nm. Magnetic Hyperthermia-Aided Biofilm Dispersal Assays. For samples treated with magnetic hyperthermia, the biofilms were grown on individual 35 × 10 mm polystyrene cell culture plates. Magnetic nanoparticles or MagDAA gel were incorporated in the treatment solutions. Each cell culture plate was placed in the middle of a water-cooled coil and exposed to an alternating current (AC) magnetic field with amplitude (H) of 5 kA/m and a frequency (f) of 380 kHz. All assays were repeated in triplicates. Cryo-Scanning Electron Microscopy (SEM) Analyses of Biofilms. S. aureus biofilm samples for cryo-SEM analyses were grown on Formvar-coated 400 mesh copper grids, following the procedure previously described. The preformed biofilms were then subjected to the different treatments and fixed using icecold methanol for 20 min prior to analysis on a Phenom ProX SEM operated at 5 kV. The treated and untreated biofilms were placed on the SEM sample holder using conductive carbon tape and a temperature controlled sample holder that was set and maintained at −25 °C. Cell Viability Assays. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2 in a humidified atmosphere. The cells were seeded to 100% confluence in clear 96-well tissue culture plates (Costar, Corning, NY, USA) for 24 h. The cells were then exposed to the different treatments in media and incubated for the specified time. After treatment, the cells were washed in sterile phosphate buffered saline and incubated in 100 μL/well of DMEM (10% FBS, 1% penicillin/streptomycin). Cell viability was evaluated using the PrestoBlue assay (Invitrogen, USA), following the manufacturer’s instructions. Briefly, 10 μL of PrestoBlue assay reagent was directly added to each well and the plates were incubated at the cell culture incubator (37 °C and 5% CO2) for 2 h. The absorbance at 570 and 600 nm were then measured using a SpectraMax i3 microplate reader (Molecular Device Inc., Sunnyvale, CA). All assays were performed in triplicates. Luminescent Assay for Microbial Cell Viability. Bacterial cell cultures of S. aureus (2 × 105 cells/well) in modified tryptic soy broth (3% NaCl, 0.5% glucose) were added to 12-well plates and incubated for 24 h at 37 °C. After incubation, the cell

culture medium was removed and 2 mL of MagDAA gel was added to the appropriate samples and the sample was further incubated for 2 h (n = 6) and then treated with 10 min of AMF-induced hyperthermia. After treatment, the supernatant was collected (including loosely bound cells) from the individual wells and then centrifuged at 13000 rpm for 1 min. The bacterial cells were then dispersed in 1 mL of PBS and 100 μL of the bacterial cell sample was plated in an opaque white 96-well plate. 100 μL of the BacTiter-Glo reagent was added to each well and the sample was mixed thoroughly and incubated for 5 min before luminescence measurements were performed. All measurements used to quantify luciferase activity were performed using a luminescent multiplate reader (MPL2 and Orion Microplate Luminometer, Berthold Detection Systems, Pforzheim, Germany). To eliminate the interference of the luciferase reaction with the solvent, additional negative control containing only the cell culture medium without bacterial cells was included in the measurement. Colony-Forming Assay to Evaluate the Survival of Remaining Adherent Microbial Cells. The S. aureus bacterial biofilms treated with MagDAA gel for 2 h or subjected to combined MagDAA gel+ AMF treatments were individually scraped using sterile cotton swabs, and the swabs were separately incubated in 10 μL deionized water with 990 μL modified LB broth. A 20 μL aliquot of the incubated cell culture medium from each of the incubated swabs were then evenly dispersed into preprepared LB agar gel plates, and the LB agar gel plates were then incubated at 37 °C for 24 h to check the regrowth of bacterial colonies following treatments. Statistical Analyses. Statistical analyses were performed using one-way ANOVA with a Tukey test to determine statistical difference between the groups (p < 0.05).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.8b00076. Wide view TEM images of MNPs; biofilm disruption and in vitro cytotoxicity assays of vancomycin, bacitracin, NeutroPhase, and Hibiclens at 24 h; biofilm disruption activities of individual D-amino acids; time-dependent biofilm disruption activity of the 200 mM of D-amino acid mixture (D-trp, D-tyr, and D-phe); the cumulative release of D-amino acids with and without of magnetic field excitation; cryo-SEM images of treated biofilms; and biofilm disruption with lower D-AA concentrations with and without AMF excitation (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (A.C.S.S.). *E-mail: [email protected] (C.A.H.-R.). ORCID

Anna Cristina S. Samia: 0000-0002-8871-3851 Author Contributions #

E.C.A. and S.W. equally contributed to this work.

Notes

The authors declare no competing financial interest. I



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Article

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ACKNOWLEDGMENTS A.C.S.S., E.C.A., S.W., and M.J. were supported by a NSFCAREER Grant (DMR-1253358) from the Solid State and Materials Chemistry Program, and the Cleveland Clinic Foundation. We would also like to thank Prof. Yumi Ijiri from the Department of Physics and Astronomy at Oberlin College for access to the vibrating sample magnetometer that was purchased through funds from NSF (DUE-9950606).

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