Deep Eutectic Solvent-Assisted Synthesis of Biodegradable

Article pubs.acs.org/Langmuir

Deep Eutectic Solvent-Assisted Synthesis of Biodegradable Polyesters with Antibacterial Properties Sara García-Argüelles,† M. Concepción Serrano,*,† María C. Gutiérrez,*,† M. Luisa Ferrer,† Luis Yuste,‡ Fernando Rojo,‡ and Francisco del Monte† †

Instituto de Ciencia de Materiales de Madrid (ICMM), Consejo Superior de Investigaciones Científicas (CSIC), C/Sor Juana Inés de la Cruz 3, 28049-Madrid, Spain ‡ Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), C/Darwin 3, 28049-Madrid, Spain S Supporting Information *

ABSTRACT: Bacterial infection related to the implantation of medical devices represents a serious clinical complication, with dramatic consequences for many patients. In past decades, numerous attempts have been made to develop materials with antibacterial and/or antifouling properties by the incorporation of antibiotic and/or antiseptic compounds. In this context, deep eutectic solvents (DESs) are acquiring increasing interest not only as efficient carriers of active principle ingredients (APIs) but also as assistant platforms for the synthesis of a wide repertoire of polymer-related materials. Herein, we have successfully prepared biodegradable poly(octanediol-co-citrate) polyesters with acquired antibacterial properties by the DES-assisted incorporation of quaternary ammonium or phosphonium salts into the polymer network. In the resulting polymers, the presence of these salts (i.e., choline chloride, tetraethylammonium bromide, hexadecyltrimethylammonium bromide, and methyltriphenylphosphonium bromide) inhibits bacterial growth in the early postimplantation steps, as tested in cultures of Escherichia coli on solid agar plates. Later, positive polymer cytocompatibility is expected to support cell colonization, as anticipated from in vitro preliminary studies with L929 fibroblasts. Finally, the attractive elastic properties of these polyesters permit matching those of soft tissues such as skin. For all of these reasons, we envisage the utility of some of these antibacterial, biocompatible, and biodegradable polyesters as potential candidates for the preparation of antimicrobial wound dressings. These results further emphasize the enormous versatility of DES-assisted synthesis for the incorporation, in the synthesis step, of a wide palette of APIs into polymeric networks suitable for biomedical applications.

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INTRODUCTION Despite the enormous progress that surgery and aseptic practices have experienced in the last few decades, infections associated with the implantation of medical devices still represent one of the most serious and dramatic complications in daily clinical practice.1 Orthopedic devices, prosthetic heart valves, vascular and urinary catheters, intrauterine devices, and oral implants are all critical targets for an important number of pathogens.2,3 Dramatically, most of these microorganisms tend to form organized communities (i.e., biofilms) that are embedded into autosecreted matrices of extracellular polymeric substances.3 In these biofilms, bacteria individuals usually display altered phenotypes and acquire extensive antibiotic resistance, thus extensively complicating the achievement of an effective treatment.4 Initial strategies to combat biofilm © 2013 American Chemical Society

formation include the prevention of both device contamination and microbial attachment to the device, among others.3 With this purpose, numerous attempts have been made to develop antifouling coatings5 as well as bactericidal materials by the incorporation of antibiotics, antiseptics, or silver atoms.3,4,6 For instance, silver-poly(amidoamine) dendrimer nanocomposites have shown promise in combating human pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.7 In a different approach, cationic betaine ester molecules were introduced into poly(N-isopropylacrylamide)-based triblock hydrogels to prepare thermoresponsive antimicrobial Received: April 12, 2013 Revised: June 6, 2013 Published: June 28, 2013 9525

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wound dressings.8 Polymers have been also explored as tunable material platforms able to display antimicrobial activity per se (e.g., those containing quaternary nitrogen atoms, such as imidazole derivatives, or phospho- and sulfoderivatives) or after the incorporation of organic or inorganic bactericidal molecules through covalent chemical modification, coupling, addition, or blending.9 In this sense, quaternary ammonium compounds (QACs), whose antiseptic and disinfectant activities have been known for decades,10 have been successfully incorporated into different types of materials to confer to them effective antibacterial properties for biomedical applications.11,12 Interestingly, their safe and effective use for the fabrication of dental resins has been widely explored to prevent the development of caries and aid in pulp care.13 QACs are membrane-active agents that target cytoplasmic membrane in bacteria by causing the loss of its structural organization and integrity.10 Deep eutectic solvents (DESs) are a new type of ionic liquid (IL) obtained by the simple mixture of two or three components, generally a quaternary ammonium salt and a hydrogen bond donor, which are capable of forming a eutectic mixture.14 The charge delocalization occurring through hydrogen bond formation between the halide anion and the hydrogen donor moiety is responsible for the decrease in the freezing point of the mixture relative to the melting points of its individual components. In combining all of the interesting features characteristic of ILs (e.g., high viscosity, thermal and chemical stability, negligible volatility, and the ability to dissolve a wide spectrum of solutes), DESs offer certain advantages versus ILs and can replace them in many applications. For instance, DESs can be prepared in a cheaper (from common reagents) and easier way than ILs, with an almost unlimited range of compositions and with no need for postsynthesis purification (depending on the degree of purity of the resulting DES and the purity of its individual components), making their large-scale use feasible.15 As a consequence of all of these remarkable properties, DESs are currently attracting significant attention as alternative and greener media for organic synthesis and biotransformations, being even more attractive than ILs because some of them have been proven to be biodegradable and compatible with enzymes.15 As ILs,16−18 DESs have proven key roles in the synthesis of a wide repertoire of polymerrelated materials19−21 as well as being efficient carriers of active principle ingredients (APIs).15,22−24 Unfortunately, although ILs have already shown promise as unique stabilizing platforms for antibiotic loading and release,25 DESs have been rarely explored to date as antimicrobial carriers.26 Biodegradable elastomers have demonstrated remarkable versatility as polymeric networks for regenerative medicine as a result of their tunable degradation profiles, their ability to incorporate attractive functionalities, and their useful mechanical properties to fulfill soft tissue compliance, among others.27,28 This type of material has also shown promise for controlled release29 because drug delivery has been pursued as an advanced property in the design of smart materials for biomedical applications.30 In this context, several attempts have been made to achieve antibiotic release from this type of polymer. For instance, Kushwaha et al. reported the preparation of viscoelastic polymer blends composed of poly(vinyl alcohol) and gum arabica for the sustained release of diverse antimicrobial drugs.31 Polyhydroxyalkanoates have been also explored as effective carriers of antibiotics (e.g., cefoperazone, gentamicin, and tetracycline) for the treatment of severe infections.32 However, most of the approaches used to achieve

drug loading into polymers involves solvent evaporation techniques,33 thus limiting the amount of drug that can be incorporated and requiring the use of organic solvents that are usually highly toxic to cells. Other synthesis approaches not involving polymer curing at high temperatures, such as UV curing, have also been investigated for the incorporation of antibiotics into elastomeric matrices.34 In this context, our group has recently reported on the preparation of lidocainereleasing poly(diol-co-citrate) (PDC) elastomers by the use of a DES-assisted synthesis.23 PDC elastomers were first described by Ameer and co-workers in 200435,36 and have already been explored as gene-delivery systems and shape-memory polymers for temperature-controlled drug delivery,37,38 among others. In these materials, the original scheme for the thermal condensation of 1,8-octanediol and citric acid is implemented by the use of a DES composed of 1,8-octanediol and lidocaine that serves as a reaction medium and simultaneously provides one of the polymer precursors (i.e., 1,8-octanediol) and the API (i.e., lidocaine). By means of this simple approach, we achieved a significant decrease in the synthesis temperature that allowed the preservation of the API integrity. Moreover, high compound loadings into the polymer and a homogeneous incorporation of the active compounds into the entire network were attained without the use of any additional organic solvents. Because both quaternary ammonium and phosphonium salts have already demonstrated antimicrobial activity when incorporated into polymers,39,40 herein we have explored the formation of new octanediol/API-based DESs in an attempt to assist a mild one-pot synthesis of PDC elastomers with conferred antibacterial properties and potential utility as bactericidal wound dressings. In particular, we have selected the following quaternary ammonium salts as APIs: choline chloride (abbreviated as C), tetraethylammonium bromide (T), and hexadecyltrimethylammonium bromide (H), frequently abbreviated in the literature as CTAB. Additionally, we have explored the incorporation of a phosphonium salt: methyltriphenylphosphonium bromide (M). The resulting biodegradable polymers, used either as bulk materials or as coatings, are expected to display dual behavior. Specifically, the presence of the quaternary ammonium and phosphonium salts will allow the inhibition of bacterial growth at an early stage, whereas the positive polymer cytocompatibility will support later cell colonization, consequently benefitting tissue regeneration in the implanted area.

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EXPERIMENTAL SECTION

Chemicals and Bacterial Strain. All chemicals were purchased from Sigma-Aldrich and used as received. The bacterial strain used was Escherichia coli TG1, which was grown on a complete LB medium.41 Cell culture media and supplements were purchased from Lonza and used following the manufacturer’s instructions. DES Preparation and Poly(octanediol-co-citrate) (POC) Polymer Synthesis. To achieve DES formation, a mixture of 1,8octanediol with either choline chloride (DES-C), tetraethylammonium bromide (DES-T), hexadecyltrimethylammonium bromide (DES-H), or methyltriphenylphosphonium bromide (DES-M) in a 3:1 molar ratio (typically 9 mmol:3 mmol) was held at 90 °C for 24 h for the complete formation of homogeneous transparent liquids, except for DES-H that required 48 h. DES-M was also prepared in a molar ratio of 3:0.75. On the preheated DES at 90 °C and under stirring, DESassisted synthesis of the POC prepolymer was initiated upon addition of citric acid (1:1 molar ratio with respect to 1,8-octanediol). The resulting prepolymer solutions (90 °C, 6 h) were aged at 80 °C for 10 days to obtain the cross-linked polymers (i.e., POC-C, POC-T, POCH, and POC-M). The polymer that resulted from using DES-M in a 9526

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Scheme 1. Summary of the DES-Assisted Synthesis of Poly(octanediol-co-citrate) Polyestersa

a

(A) Preparation of the different DESs and physical appearance of the components before and after DES formation. (B) DES-assisted synthesis of POC elastomers containing antibacterial compounds. 1,8-Octanediol molecules are schematized as black curled lines; antibacterial compounds, as blue stars; and citric acid, as red trident figures. (C) Diagram of the synthesis of standard POC. t is the curing temperature. Assays in Solid Agar Plates. Petri plates containing solid LB-agar (LB with 1.5% agar) were overlaid with 3 mL of melted soft LB-agar (LB with 0.6% agar) to which about 108 E. coli cells had been added. After the solidification of the soft agar, nonconditioned and conditioned polymer discs (0.6 cm in diameter, thickness ca. 0.7 ± 0.2 mm) were placed on top of the agar. Cells were allowed to grow for 24 h at 37 °C. The antibacterial properties of the polymer discs were then measured as the extension of the growth-inhibition zone around discs, normalized by the grams of polymer added. Polymer disc conditioning was performed by incubating the samples in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C and a CO2 (5%) atmosphere for 7 days. Mammalian Cell Cytocompatibility Studies. Polymers discs (0.6 cm in diameter, thickness ca. 0.7 ± 0.2 mm) were first sterilized under UV radiation for 20 min per side and then preconditioned in culture medium for 7 days to eliminate any nonreacted acidic residues from the synthesis. Polymer samples were then placed in a 24-well plate, and murine L929 fibroblasts were seeded on the polymers at a density of 3 × 104 cells per disc. To further evaluate the capacity of cells to grow in the vicinity of the polymer discs, 1.5 × 104 cells per well were seeded around the samples. DMEM supplemented with fetal bovine serum (10%), streptomycin (100 UI mL−1), penicillin (100 UI mL−1), and L-glutamine (1 mM) were used as culture medium. Cultures were maintained at 37 °C in a sterile incubator under a CO2 (5%) atmosphere for up to 96 h. Polystyrene from standard cell culture plates (tissue culture plastic) was used as a control surface. Cell cultures were followed over time by using an Axiovert CFL-40 optical microscope with a coupled Axiocam ICC-1 digital camera (Zeiss). Statistics. Values were expressed as mean ± standard deviation. Statistical analysis was performed using Statistical Package for the Social Sciences software (SPSS), version 17.0. Comparisons among groups were made by analysis of variance (ANOVA), followed by Scheffé post hoc test as the variance homogeneity criteria among groups were satisfied (Levene test). In all statistical evaluations, p < 0.05 was considered to be statistically significant.

molar ratio of 3:0.75 was named POC-M075. In all syntheses, the temperature was controlled by using a thermocouple with an accuracy of ±5 °C. Conventional POC (in the absence of DES) was also synthesized as previously described.35,36 Polymer Characterization. 1H NMR spectra (500 MHz) were recorded using a Bruker DRX-500 spectrometer. DES samples were placed in capillary tubes and analyzed in DMSO-d6 as an external reference (δ 2.5) at 90 °C, below the melting points of the quaternary ammonium and phosphonium salts. Prepolymer samples (typically 10 mg) were dissolved in DMSO-d6, placed in RMN tubes, and analyzed at room temperature. DSC studies were performed using a DSC Q100 calorimeter. Briefly, samples were exposed to an initial cooling segment (at 5 °C min−1) from room temperature to −90 °C, followed by an isothermal segment at −90 °C that was held for 10 min, ramped from −90 to 100 °C (at 5 °C min−1), and finally cooled (at 5 °C min−1) from 100 to −90 °C. This cycle was repeated twice, and the behavior of the second cycle was used to determine the thermal transitions of the samples. Finally, the degree of postpolymerization was evaluated by calculating the molecular weight between cross-links (Mc) by swelling studies in DMSO.42 The complex Young’s modulus (viscoelastic behavior) of samples soaked in distilled water at 37 °C for 4 h was measured at 1 Hz in a triple-point bending configuration by using a DMA 7e dynamic mechanic analyzer (Perkin-Elmer). The force used in the experiment was chosen so as not to produce strains larger than 0.3% in order to maintain the mechanical response within the linear range. The dimensions of the samples were 12 × 2.5 × 3 mm3 parallelepipeds. Data were collected in triplicate. The error in the measurement was estimated to be around 20% (intrinsic to the measurement technique), which allowed comparison between the outgoing data. The storage viscosity was also reported for each polymer sample. Bactericidal Studies. Determination of the Minimal Inhibitory Concentration (MIC). Sterile test tubes containing 1 mL of LB medium were inoculated with about 5 × 106 E. coli cells obtained from an exponentially growing culture in the same medium. After the addition of the antibacterial compound at different concentrations, the tubes were incubated at 37 °C under vigorous shaking and bacterial growth was monitored at 15 h. 9527

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Table 1. 1H NMR Spectroscopy Data of DESs Used for the Preparation of Biodegradable Polyesters

RESULTS AND DISCUSSION DESs were initially prepared by mixing 1,8-octanediol with either C, T, H, or M compounds in their solid state in a molar

octanediol DES-C

DES-T

DES-H

H

−(CH2)4−

0.9 (24H)

3.5 (2H)

−CH2−CH2− OH −CH2−OH −OH −(CH2)4− −CH2−CH2− OH −CH2−OH −OH −(CH2)4−

1.1 (12H)

N+−CH2−CH2− OH N+−CH2−CH2− OH (CH3)3−N+ N+−(CH2−CH3)4 N+−(CH2−CH3)4

0.8 (12H) 2.9 (8H)

−CH2−CH3

0.4 (3H)

−(CH2)13−

0.9* (26H) 1.3 (2H)

−CH2−CH2− OH −CH2−OH

Figure 1. DSC spectra of the different DESs obtained after mixing 1,8octanediol with choline chloride (DES-C, blue line), tetraethylammonium bromide (DES-T, red), hexadecyltrimethylammonium bromide (DES-H, green), and methyltriphenylphosphonium bromide (DES-M, orange). Results from the second heat scan are shown. The spectrum from pure 1,8-octanediol is also shown for comparison (black).

DESM075

ratio of 3:1, respectively, and thermally treated at 90 °C. Only in the case of compound M, an additional DES was prepared in a molar ratio 3:0.75. During DES formation, the mixtures were frequently homogenized by vortex mixing. After 24 h, a homogeneous transparent liquid was formed for the mixture with compounds C (DES-C), T (DES-T), and M (DES-M). However, DES containing compound H required 48 h to form (DES-H). Scheme 1 (panel A) summarizes the preparation of the different DESs from their respective solid components. From DSC scans, the melting points (Tm) for DES-C, DES-T, DES-H, and DES-M were identified at 52, 47, 54, and 55 °C, respectively, which were significantly below those of any of its individual components (i.e., 61 °C for 1,8-octanediol, 302−305

DES-M

ammonium or phosphonium salt

δ (ppm)

H

3.1 (12H) 4.1 (6H) 0.8 (24H) 1.0 (12H) 3.0 (12H) 3.8 (6H) 0.8* (24H) 1.1 (12H)

−OH

3.7 (6H)

−(CH2)4− −CH2−CH2− OH −CH2−OH −OH −(CH2)4− −CH2−CH2− OH −CH2−OH −OH

0.6 (24H) 0.8 (12H)

−(CH2)13−CH2− CH3 −N+−(CH3)3 −N+−CH2− (CH2)13− (Ph)3−P+−CH3 (Ph)3−P+−CH3

2.8 (12H) 3.7 (6H) 0.5 (24H) 0.7 (12H)

(Ph)3−P+−CH3 (Ph)3−P+−CH3

3.1 (12H)

δ (ppm)

3.2 (2H) 2.9 (9H)

2.8 (9H) 3.1 (2H) 2.4 (2.3H) 7.0−7.1 (11.3H)

2.4 (3H) 7.0−7.1 (15H)

2.8 (12H) 3.7 (6H)

Chemical shifts marked with * as superscript may be interchanged.

°C for compound C, 285 °C for T, 248−251 °C for H, and 230−234 °C for M) (Figure 1). Hydrogen bonding between the halide anion of quaternary nitrogen or phosphonium salts

Figure 2. 1H NMR spectra of DES-C, DES-T, DES-H, and DES-M. 9528

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Figure 3. 1H NMR spectra of POC-C, POC-T, POC-H, and POC-M prepolymers.

and the hydroxyl groups in 1,8-octanediol (as the hydrogen donor moiety) is the most plausible explanation for the stabilization of the different DESs formed, as previously described for other DESs.23,43 In the DES-T scan, however, another two minority peaks could be observed at 15 and 23 °C and were likely attributed to other minor eutectic compositions. Similarly, DES-M showed a minority peak at 50 °C, and DES-H also displayed another thermal transition at 72 °C. This situation is analogous to that described for DESs made of 1,8octanediol and lidocaine23 or mixtures of ibuprofen and thymol,44 with molar ratios on either side of the eutectic composition. Chemical shifts of the different peaks identified in the 1H NMR spectra (500 MHz, DMSO-d6) of DES-C, DES-T, DES-H, DES-M075, and DES-M were found to be in accordance with the theoretical predictions for all components (Figures 2 and 1-SI and Table 1). The molar ratio of the

Table 2. Physical Properties of DES-Based Elastomers: Molecular Weight between Cross-Links (Mc), Storage Viscosity, and Young’s Modulusa polymer POC POC-C POC-T POC-H POCM075 POC-M a

storage viscosity (MPa s)

Mc (g mol−1) 5094 11 264 10 384 9420 14 970

± ± ± ± ±

785 897b 547b 222b 55b,c,d,e,g

0.10 0.07 0.06 0.04 0.03

22 750 ± 835b,c,d,e,f

± ± ± ± ±

0.03 0.01 0.01 0.02 0.02b

0.03 ± 0.02b

Young’s modulus (MPa) 1.40 1.06 0.83 0.30 0.27

± ± ± ± ±

0.10 0.10b 0.14b 0.01b,c,d 0.04b,c,d

0.27 ± 0.02b,c,d

b

Statistical significance: p < 0.05. Significant differences with respect to POC. cSignificant differences with respect to POC-C. dSignificant differences with respect to POC-T. eSignificant differences with respect to POC-H. fSignificant differences with respect to POC-M075. g Significant differences with respect to POC-M.

Figure 4. Antibacterial activity of choline chloride (C), tetraethylammonium bromide (T), hexadecyltrimethylammonium bromide (H), and methyltriphenylphosphonium bromide (M) salts against E. coli. Arrows indicate the estimated MIC for each compound. Please notice that concentrations in the left panel are expressed as M (C and T) or mM (H and M). 9529

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Figure 5. Antibacterial activity of POC-C, POC-T, POC-H, POC-M075, and POC-M against E. coli. Polymer inhibition of bacterial growth was measured as the diameter of the zone of inhibition around polymer discs normalized by the grams of polymer (representative images displayed as insets). POC was used as a negative control to test the inherent antibacterial properties of the polymer surface. Statistical significance: p < 0.05. There are significant differences with respect to (a) POC, (b) POC-C, (c) POC-T, (d) POC-H, (e) POC-M075, and (f) POC-M.

tested, all of the synthesized polyesters exhibited typical elastic behavior in the same range as that obtained for standard POC. Although the incorporation of these bactericidal molecules into the polymer networks resulted in a significant reduction of the original Young’s modulus of POC, which is more notorious in the case of H and M compounds (Table 2), the low elastic modulus of these polyesters still makes them suitable for applications in soft tissues such as skin.45 Compounds C and T are QACs, whose antimicrobial properties depend on the nature of the organic groups attached to the nitrogen, the number of nitrogen atoms present, and the counterion.46 Compound H has also been known for its potent antibacterial activity.47 In this sense, some reports have already demonstrated its antimicrobial activity against E. coli and S. aureus when used as a thin coating of poly(ethylene terephthalate) films along with polyethylenimine and poly(acrylic acid).48 Similarly, phosphonium salts have also been explored as cationic biocides when immobilized on the surface of polymeric materials such as polypropylene,49 incorporated into thermosensitive copolymers with N-isopropylacrylamide,50 or grafted on gel-type styrene-divinylbenzene copolymers.51 In this sense, their mechanism of action is similar to that described for QACs (i.e., killing bacteria by damaging cell walls and membranes).9 Before evaluating the antibacterial properties of the different biodegradable polyesters resulting from the incorporation of these compounds, we first determined the MIC (lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation) of the four molecules used by directly exposing E. coli cultures to the different salts in solution (Figure 4). Concentrations ranging from 0 to 1.25 M were tested according to the antibacterial activities previously reported for this type of compound.52 Under these conditions, all four molecules demonstrated antibacterial properties. In particular, compounds C and T evidenced the highest MICs (0.75 and 1.0 M, respectively), and H and M showed MICs on the order of