Self-Assembly of Catecholic Moiety-Containing Cationic Random

Article pubs.acs.org/JPCB

Self-Assembly of Catecholic Moiety-Containing Cationic Random Acrylic Copolymers Vincenzo Taresco,† Lorenzo Gontrani,*,† Fernanda Crisante,† Iolanda Francolini,† Andrea Martinelli,† Lucio D’Ilario,† Federico Bordi,‡ and Antonella Piozzi*,† †

Department of Chemistry and ‡Department of Physics, Sapienza University of Rome, P. le Aldo Moro 5, 00185, Rome, Italy ABSTRACT: Amphiphilic polyelectrolytes (APEs), exhibiting particular selfassociation properties in aqueous media, can be used in different industrial applications, including drug delivery systems. Their typical core−shell structure (micelle) depends on the balance of interactions between hydrophobic and ionizable monomer units. In this work, the structure of amphiphilic cationic random copolymers, obtained by employing different molar ratios of two acrylic monomers, one bearing in the side chain a tertiary amine (N,N-diethylethylendiamine, DED) and the other one a hydrophobic catecholic group (hydroxytyrosol, HTy), was investigated by atomistic molecular dynamics (MD) simulation, 1H NMR analysis, dynamic light scattering (DLS), and zeta potential measurements. The structures of p(AcDED-co-AcHTy) copolymers were compared with that of the cationic homopolymer (pAcDED). MD simulation showed a chain folding in water solution of all polymer materials consistent with the degree of hydrophobicity of the chain, that increases with the number of aromatic residues. This phenomenon was induced by the interaction between the charged amine groups with water and by the associated attraction between aromatic rings inside the molecule. In addition, the p(AcDED-co-AcHTy) 70/30 copolymer had a marked tendency to self-assemble as shown by the radial distribution function among catechol carbon atoms. Electrical conductivity measurements evidenced a micellar arragment for all of the synthesized copolymers, and specially for p(AcDED-coAcHTy) 70/30, a flower micelle structure seem to be more likely. The stacking interactions among catecholic groups present in the side chain of the copolymers reduced the size and charge density specially for the p(AcDED-co-AcHTy) 70/30 copolymer. Finally, the good antimicrobial activity of all copolymers confirmed the right reached amphiphilic balance. Indeed, a considerable reduction of the minimum inhibitory concentration (from 100 μg/mL to 40 μg/mL for pAcDED and p(AcDED-co-AcHTy) 70/ 30, respectively) was obtained by introducing a hydrophobic group molar fraction of 0.3.

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INTRODUCTION In the last few decades, much interest has been directed at the characterization and synthesis of amphiphilic copolymers because of their scientific and technological relevance. Among amphiphilic copolymers, amphiphilic polyelectrolytes (APEs), constituted from hydrophobic and ionizable monomer units, have received remarkable attention since they exhibit particular self-association properties in aqueous media.1,2 These properties can be exploited in drug delivery systems, specially for the active transport of drugs or biological agents otherwise incompatible with the aqueous media,3,4 and in different industrial applications, namely, cosmetics, paints, inks, coatings, foodstuffs, and personal care items.5−9 APEs when dissolved in aqueous media self-assemble forming various types of core−shell architectures (micelles), the structure of which depends on the balance between hydrophobic interactions among hydrophobes and electrostatic repulsions among the charged groups.10 These interactions are aimed at minimizing the water−hydrophobe contact. The distribution of hydrophilic and hydrophobic monomers in the polymer chain together with the number of hydrophobic groups can greatly influence polymer conformation.11 In © 2015 American Chemical Society

addition, since hydrophobic interactions can establish among hydrophobes belonging either to the same or to different polymer chains, the hydrophobic associations can occur in an intra- or intermolecular fashion. APEs can exist as block or statistical (random) copolymers. Block polymers are known to form multicore spherical micelles with a well-defined core−shell structure in aqueous media, specifically a core of hydrophobic blocks surrounded by flexible hydrophilic blocks, promoted by intermolecular interactions.12,13 On the contrary, random APEs can form unicore or multicore micelles with a less defined structure since interactions among hydrophobic and charged groups can occur both in the same and in different polymer chains.14,15 Then, when hydrophobic groups are arranged in blocks with long hydrophilic sequences, the hydrophobic interactions promote intermolecular association while a random distribution of the hydrophilic and hydrophobic units encourages an intramolecular associative behavior. Received: May 26, 2015 Revised: June 11, 2015 Published: June 15, 2015 8369

DOI: 10.1021/acs.jpcb.5b05022 J. Phys. Chem. B 2015, 119, 8369−8379

Article

The Journal of Physical Chemistry B

associative phenomena of hydrophobic side groups and the antimicrobial activity of copolymers. Indeed, the self-assembly of hydrophobic moieties of amphiphilic polymers can negatively influence their biological properties.41 To find the best hydrophobic/hydrophilic balance, a series of copolymers was prepared by varying the molar ratios of monomers. The structure of polymer aggregates was elucidated by atomistic molecular dynamics (MD) simulation and characterized in terms of the radius of gyration, head-to-tail (or end-to-end) distance, intramolecular and intermolecular radial distribution functions of selected atoms, and spatial distribution function. 1H NMR analysis, dynamic light scattering (DLS), and zeta potential measurements as well as determinations of CMC by electrical conductivity experiments were also carried out on the copolymers to examine the consistency of the results of MD simulation with the experimental data. Finally, the antimicrobial activity of the amphiphilic random copolymers was determined by broth microdilution assay and related to their structure in aqueous medium.

The block micelle structure (in terms of aggregation number, micellar and core sizes, and critical micelle concentration) has been extensively investigated to date.16,17 Block micelles offer many advantages such as colloidal stability with low critical micelle concentration (CMC), predictable narrow size distribution, ability to protect drugs from possible deactivation, and improved pharmacokinetics.18,19 However, sequential controlled radical polymerizations [atom transfer radical polymerization (ATRP), reversible addition−fragmentation chain transfer (RAFT), etc.] or multistep sequential polycondensations are necessary to prepare block APEs. Conversely, random copolymers can be easily obtained by a single step synthesis and using a wide range of monomers.20 The properties of random APEs can be tuned by changing (i) the types of hydrophobic and ionic monomers;21,22 (ii) the content of each monomer;23,24 (iii) the degree of polymerization;25 and (iv) the spacer length between the hydrophobe and polymer backbone. To date, various studies have attempted to provide a general shape for the random polymer micelle structure.26−29 In this regard, Kawata et al.30 proposed a flower micelle model of the minimum loop size. These authors specifically investigated the dependence of hydrophobicity of each monomer unit on the micellar structure. Tominaga et al.31 studied the structural arrangement of a sodium (2-acrylamido)2-methylpropanesulfonate and N-dodecylmethacrylamide p(AMPS/C12) random copolymer by light scattering and atomistic molecular dynamic simulations confirming the predictions of the flower micelle model of the minimum loop size proposed by Kawata. Among APEs, cationic amphiphilic random copolymers are very interesting as a new class of antimicrobial agents since they are able to mimic the amphiphilic structures of antimicrobial helix-forming peptides, such as magainin.32−35 Such copolymers can arrange in separated domains or facially amphiphilic structures, the latter obtained by separation of the cationic side chains and hydrophobic segments, under the influence of the cell membrane interface.36,37 Even if these APEs seem to form vesicles or micelles at high concentration, it was observed that they can also display antimicrobial activity below the critical micelle concentration.38 It was hypothesized that the water-soluble segments shielding the hydrophobic ones lead to the formation of a core−shell structure responsible for the biological activity.39 A suitable balance of hydrophobic content and cationic charges in these polymers is necessary to have high antimicrobial activity. Indeed, it is generally accepted that the copolymer positive charges attach to the negatively charged bacterial membrane while the hydrophobic groups promote membrane permeation and disruption of the pathogen cell with a consequent cell death. Generally, antimicrobial cationic copolymers with proper hydrophilic/hydrophobic content are obtained by the polymerization of an antimicrobial cationic monomer, containing primary or tertiary amines, and a hydrophobic monomer, that most of time does not possess antimicrobial activity. In this work, the structure in water of antimicrobial amphiphilic cationic random copolymers, previously synthesized by copolymerization of two monomers,40 one bearing in the side chain a tertiary amine and the other one a hydrophobic catecholic group, was investigated. The catecholic group-containing monomer was chosen to increase not only the polymer chain hydrophobicity, but also its stiffness in order to study how these features could affect the

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MATERIALS AND METHODS Materials. Sodium metabisulfite (Na2S2O5) and potassium monobasic phosphate (K2HPO4) were purchased from Carlo Erba. Acryloyl chloride 96% and N,N-diethylethylendiamine (DED) were supplied by Fluka. Tyrosol (2-(4-hydroxyphenyl)ethanol, TY), acrylic acid (AA), hydroquinone, potassium persulfate (K2S2O8), p-toluensulfonic acid (pTSOH), sodium dithionite (Na2S2O4), and ferrous sulfate (Fe2SO4) were purchased from Sigma-Aldrich as well as other solvents and reagents. DMSO-d6 100% and D2O were supplied from CIL (Cambridge Isotope Laboratories, Inc.). All chemicals were of analytical grade and used as received. 2-Iodoxybenzoic acid (IBX) was prepared in the laboratory as described in the literature.42,43 Synthesis of AcDED and AcTy Monomers. The synthesis of acrylate monomers based on AcDED was already described.44 Briefly, AcDED was synthesized by reaction between acryloyl chloride (0.038 mol Ac, Fluka) and DED (0.029 mol, Sigma-Aldrich) in dimethylcarbonate (DMC, 75 mL, Sigma-Aldrich) containing K2HPO4 (0.08 mol), used as base.45 The reaction was carried out for 4 h at room temperature. Then, the solution was filtered to remove the inorganic and organic salt and the monomer recovered by solvent evaporation (yield ranging from 85% to 90%). As for the tyrosol-based acrylic monomer (AcTy) synthesis, 0.1 mol of Ty was dissolved in 75 mL of toluene in the presence of 20 mg of hydroquinone and 750 mg of ptoluensulfonic acid (pTSOH). Then, 0.12 mol of acrylic acid was added and the mixture was refluxed for 1.5 h in a flask equipped with Dean−Stark apparatus. At the end of the reaction, the mixture was cooled and treated with sodium bicarbonate (6 g) and water (1.5 mL), until carbon dioxide evolution ended. The mixture was then dried with anhydrous sodium sulfate and filtered. The solvent (toluene) was removed under vacuum. Synthesis of pAcDED Homopolymer and p(AcED-coAcTy) Copolymers. The homopolymer synthesis, previously reported,44 was carried out at 25 °C for 24 h by using a 1 M water solution of AcDED (5 mL) and K2S2O8 (2.8 × 10−4 mmol) plus FeSO4 (2.4 × 10−4mmol) as radical initiators and sodium metabisulfite (Na2S2O5) as chain transfer agent (chain 8370


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The Journal of Physical Chemistry B Scheme 1. Synthesis of p(AcDED-co-AcHTy)

Table 1. Physical and Biological Parameters of the Synthesized Polymers: Number Average Molecular Weight (Mn), Dispersity (D), Critical Micelle Concentration (CMC), Hydrodynamic Diameter, Zeta Potential, and Minimun Inhibitory Concentration (MIC) of Bacterial Growth polymer pAcDED p(AcDED-co-AcHTy) 90/10 p(AcDED-co-AcHTy) 80/20 p(AcDED-co-AcHTy) 70/30

Mn ×103a (g/mol) 69.0 15.0 13.0 8.00

D = Mw/Mn CMC (mM) hydrodynamic diameterb (nm) 1.60 1.55 1.85 1.85

b

500 300 215 90

1.99 1.64 1.59

± ± ± ±

70 25 15 10

zeta Potentialc (mV) MIC (μg/mL) (mM) 20 11 9 6

± ± ± ±

1 1 2 1

100 69 55 40

(0.59) (0.40) (0.31) (0.22)

a

PEO equivalent-molecular weights. bValue not determinable. cThe data, representative of at least six measurements, are reported as arithmetic mean ± standard deviation.

After the reduction reaction, the copolymers containing a AcDED/AcTy ratio higher than 70/30 precipitated.40 Therefore, since the aim of this work was to study the associative properties of antimicrobial water-soluble polymer materials, only the copolymers containing AcDED/AcTy molar ratio until 70/30 were considered. Molecular Dynamics Study. Three copolymers belonging to the p(AcDED-co-AcHTy) class, namely, 70/30, 80/20, and 90/10, were simulated in water at the molar ratio solute:water = 1:100, in compliance with the concentration of the solutions employed in the DLS study (∼3 mg/L). Since the average molecular weight of the synthesized copolymers was in the range 8000−15 000 Da (see Table 1), an average length of about 50 units for each fragment can be expected. We used this size value as the center of a Gaussian distribution of lengths, whose variance was chosen as to span the interval 40−60 and to produce a total of 100 molecules. Such a number was deemed appropriate to investigate the role of both intra- and intermolecular interactions between the organic fragments. The generation procedure, which is described here fully for 70/ 30 copolymer but was employed for the other two systems with the necessary composition modifications, can be summarized as follows: 1. A model decamer of the p(AcDED-co-AcHTy) system was built. Among the 55 possible decamers obtainable combining 7 AcDED and 3 AcHTy fragments, the sequence A-HTy-A-AHTy-A-A-A-HTy-A was selected to minimize possible associ-

transfer agent/monomer molar ratio = 0.5). The resulting polyacrylamide was called pAcDED (pKb = 8.61). As for copolymer synthesis (Scheme 1),40 an aqueous solution of AcDED monomer (in variable quantity according to the chosen ratio between comonomers) was added to an AcTy acetone solution in 1:1 ratio (v/v). The initiator (potassium persulfate) and the chain transfer agent (sodium metabisulfite) were then added. The solution was heated up to 80 °C temperature for 2 h to distill acetone and then kept at room temperature for 6 h. Finally, the solution was dialyzed in water using a membrane of regenerated cellulose (Spectra/por membrane BIOTECH) with a cut off of 3500 Da. Different molar ratios between the monomers AcDED and AcTy were used. The reaction conversion and the ratio of the monomers within the repeat unit of the copolymer were assessed by 1H NMR. The copolymers, named p(AcDED-co-AcTy)s, were soluble in water, dimethyl sulfoxide, and dimethylformamide. The aromatic hydroxylation of copolymers was performed by IBX (in stoichiometric amount to the phenolic fraction) added to an aqueous solution of p(AcDED-co-AcTy) copolymer (200 mg/mL), at 0 °C under magnetic stirring. Later, sodium dithionite (stoichiometric to IBX) was added to the reaction batch and the mixture was kept under stirring until a chromatic variation from orange to white was observed. The solution was dialyzed in water. The resulting copolymers, dried in vacuum oven at 25 °C for 1 day, were named p(AcDED-co-AcHTy) (Scheme 1). 8371


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The Journal of Physical Chemistry B ation effects due to proximity at startup, namely, requiring that the hydroxytyrosol residues (HTy) were dispersed in the chain having at least two amine groups (A) around. 2. Three other equivalent decamers were linked to the first one to obtain a fundamental 40-mer unit. 3. The fragment was extended by adding A and HTy fragments alternatively at the end of the sequence (e.g., the structure 49 units long is A-HTy-A-A-(40-mer)-HTy-A-A-AHTy, containing 34 A and 15 HTy). Methyl groups terminated every monomer sequence. Starting from the SMILES strings,46 hydrogen atoms were added to all amine nitrogen atoms, and a starting three-dimensional structure was obtained using OpenBabel Toolkit molecular builder.47 The generalized amber force field (GAFF) was assigned using Antechamber/Tleap,48 and the necessary number of chloride counteranions was added. Gasteiger-Marsili empirical atomic partial charges were used to model electrostatic interactions.49 The 100 polymer fragments were put into a cubic box of edge 62 Å using the code Packmol,50 that places the molecules in the simulation box at random, using rigid movements (translation and rotation) with a minimum interatomic separation of 2 Å as the only constraint. The system was then soaked with 10 000 TIP3P water molecules (183 730 atoms in total). The integration time step was 2 fs for all calculations and an 8 Å cutoff was used for van der Waals interactions and for the direct-space term of Ewald summation; cubic periodic boundary conditions were applied throughout the simulation. The simulation protocol consisted of the following steps: after the initial energy minimization, the cell was thermalized at 300 K in the NPT ensemble for 4 ns, reaching an equilibrated density of 1.15 g cm−3. A productive NVT trajectory of 2 ns was then obtained at that density, dumping the configuration every 0.2 ps; a total of 10 000 configurations were saved. A typical frame is shown in Figure 1. The following quantities were calculated over the productive trajectory to assess the polymer conformation: (1) radius of gyration, defined as the root-mean-square distance of all the atoms constituting the chain from the center of mass

Figure 1. Simulation cell for 70/30. Polymer: blue; water: red−white; chloride: green.

values obtained at the end of the equilibration phase were 1.12 and 1.08 g cm−3 for 80/20 and 90/10, respectively. Polymer Characterization. NMR Spectroscopy and Elemental Analysis. 1H NMR spectra were performed employing a Varian XL 300 instrument and chloroform-d, D2O, or DMSO-d6 100% CIL as solvent. The monomer progression in the copolymerization was monitored using in situ 1H NMR analysis. Specifically, the disappearance rates of the peaks at 5.9 and 5.6 ppm, relative to double bond of the AcTy and AcDED monomers, respectively, in DMSO-d6 solutions were examined. Dynamic Light Scattering and Zeta Potential Measurements. The sizes of the pAcDED homopolymer and pAcDEDco-pAcHTy copolymer particles were determined by dynamic light scattering analysis (Coulter LS-13320), while the zeta potential was measured by using a NanoZetasizer instrument (Malvern, UK). Both measurements were carried out by employing deionized (DI) water as a solvent and a concentration of the polymer solution of 3 mg/mL. Gel Permeation Chromatography. Molecular weight measurements were carried out at 25 °C using a gel permeation chromatography (GPC) system equipped with a Shimadzu RID-10A differential refractive index detector and Tosoh TSK polyacrylamide gel columns. The calibration curve was obtained by using poly(ethylene oxide) standards. Aqueous sample solutions (1 mg/mL) containing 0.1 M NaCl were injected by an autosampler Hitachi AS-2000 at a flow rate of 0.8 mL/ min−1. Conductivity Measurements. Electrical conductivity measurements, performed by a Crison GLP-32 conductivity meter with a cell constant of 0.1 cm−1, allowed determination of the critical micelle concentration by the variation of specific conductivity of aqueous solutions of the copolymers as a function of their concentration.

N

s2 =

∑i = 1 mi r i2 N

∑i = 1 mi

(2) head-to-tail (or end-to-end) distance between the terminal methyl carbon atoms; (3) intramolecular and intermolecular Radial Distribution Functions (RDFs) of selected atoms; (4) spatial distribution functions (SDFs). MD Simulations were carried out with AMBER12 (PMEMD),51 radial and spatial distribution functions were calculated with Ptraj,51 Gromacs52 tools, and TRAVIS,53 as well as with in-house written codes. Concerning the models of the other three systems (p(AcDED-co-AcHTy) 80/20−p(AcDED-co-AcHTy) 90/10− pAcDED), the fundamental decamer sequence was obtained from that of p(AcDED-co-AcHTy) 70/30 reported above, by mutating the hydroxytyrosol residue (HTy) into the amine one (A) moving from left to right, obtaining A-A-A-A-HTy-A-A-AA-HTy-A for p(AcDED-co-AcHTy) 80/20 and A-A-A-A-A-AA-A-A-HTy-A for p(AcDED-co-AcHTy) 90/10. The generation of the 100 fragments and the calculation protocol adopted was similar to what has already been described; the density 8372


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The Journal of Physical Chemistry B Antibacterial Activity. The antibacterial activity of the watersoluble copolymers was assessed against Staphylococcus epidermidis (ATCC 35984) by broth microdilution assay which allowed determination of their minimum inhibitory concentration (MIC). S. epidermidis was grown in triptic soy agar (TSA) and tryptic soy broth (TSB) at 37 °C. Briefly, 1 mL of polymer solution at various concentrations was added to 1 mL of microorganism solution at a concentration which gave an optical density reading of 0.1−0.2 at 600 nm. Then, a set of dilutions was prepared and inoculated with a 106 colony forming unit (CFU)/mL bacterial concentration (explored concentration range 10−1000 μg/mL). At the highest concentration of pAcDED-co-pAcHTy solutions no precipitate was observed. After 24 h incubation at 37 °C, bacterial growth was determined by measuring the absorbance at 600 nm. Broth containing cells alone was used as control. S. epidermidis was chosen for its implications in medical device-related infections. All the experiments were done in triplicate. Differences were considered significant for P < 0.05.

Figure 3. Head-to-Tail (HT) distance of the polymer fragment calculated over the productive trajectory. Top (green): 90/10; Middle (red): 80/20; Bottom (black): 70/30. The histogram population of each HT value is shown (histogram bin width 0.05 Å).

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RESULTS AND DISCUSSION Molecular Dynamics study. The distribution of radius of gyration and head-to-tail distances values calculated along the trajectory of each system are shown in Figures 2 and 3,

well above the uniform distribution. The comparison among the three polymers (different columns in Figure 4, upper panel) points out, though, that in 70/30 the interaction with water is more relevant for amine nitrogen with respect to catechol oxygen than in the other two systems (taller peak), i.e., oxygen atoms, in spite of their larger number (two OH instead of one NH+) have less “free” interaction surface available for the interaction with water. The interaction propensity between aromatic fragments can be evaluated more precisely through the RDFs between “CA” atom types (i.e., aromatic carbon atoms), which is shown in Figure 4, bottom panel. From the intramolecular radial distribution curves (left panel), the same 70/30 > 80/20 > 90/10 trend can be observed. Notice that only the distances beyond 3.0 Å (i.e., atoms not in the same ring) are plotted. The reverse behavior appears for intermolecular interactions, that show the presence of a peak at around 5 Å, though the relative function is lower than the uniform density (dashed line in the figure, corresponding to 1). Two typical examples of the aromatic−aromatic interactions revealed by the functions of Figure 4 (bottom) are shown in Figure 5 for a 70/30 trajectory frame. In particular, the upper panel shows a π-stacking type contact between two distant catechol rings in residue 45 (intramolecular), while the lower panel shows an analogous interaction between two rings of different residues (intermolecular). Even deeper insight can be gained from the analysis of SDFs, which, unlike monodimensional RDF that give radial occupancies averaged over the entire space, are able to describe the probability density of finding a particle or group of particles in the volume element (voxel) around a center taken as reference.55 In Figure 6, the isodensity plots (contour plots) of the other five HTy oxygen atoms, around the sixth oxygen taken as reference, for the copolymer 70/30 are reported. The threshold value for the contour plot is five times the uniform density for oxygens of different rings (blue, red, orange, and magenta), and 5000 times for the oxygen of the same ring (green), that otherwise would cover all other contributions, belonging to the same structural moiety. It is worth noting that, for instance, the distribution of the “magenta” oxygen has maximum values above and below the ring plane, indicating a stacking interaction among the aromatic rings during the

Figure 2. Radius of gyration calculated over the productive trajectory. The population of each s2 value is shown (histogram bin width 0.05 Å). Top (green): 90/10; Middle (red): 80/20; Bottom (black): 70/30.

respectively. These two quantities, which are related by the equation ⟨r2⟩ = 6⟨s2⟩ between their quadratic mean values for ideal polymers,54 both account for the existence of a sort of chain folding in water solution that follows the general trend p(AcDED-co-AcHTy) 70/30 > p(AcDED-co-AcHTy) 80/20 > p(AcDED-co-AcHTy) 90/10, consistent with the degree of hydrophobicity of the chain, that increases with the number of aromatic residues. Parallel to this, MD simulations show that the attraction between aromatic groups inside the molecule increases the interaction between the charged amine groups with water. Such an issue is clearly pointed out by the comparison of radial distribution functions between aminic nitrogen and water and between hydroxytyrosol oxygen (HTyO)−water of the systems studied (Figure 4). As can be seen, the first hydration shell is rather evident for both atom types in the three systems examined, and has a minimum distance around 2.8 Å, with a maximum of intensity 8373


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Figure 4. Top: Radial distribution function of polymer hydration: amine nitrogen vs water oxygen (black); catechol oxygen vs water oxygen (red). Bottom: Radial distribution function between cathecol carbon atoms. Left: Intramolecular; right: Intermolecular.

simulation. This result, though it should not be considered as fully quantitative, can be invoked to explain the findings of 1H NMR analysis reported below. Polymer Characterization. In Table 1, the PEOequivalent molecular weight and dispersity of water-soluble copolymers, determined by GPC analysis, are reported. The decrease of the number-average molecular weight (Mn) of the p(AcDED-co-AcHTy)s with respect to that of the homopolymer could be explained by the formation of more compact polymer structures with increasing the HTy content. Indeed, as evidenced by MD simulations, the HTy aromatic rings can interact with each other and lead to a decrease of the hydrodynamic volume of copolymers. Given poor solubility of the copolymers in common organic solvents, it was not possible to determine the molecular weight of p(AcDED-co-AcHTy)s without aggregation. 1 H NMR spectra confirmed the quantitative conversion of monomers by the disappearance of peaks relative to acrylic group double bond. Particularly, in situ 1H NMR analysis evidenced a reactivity of AcDED monomer only slightly higher than the AcTy one, suggesting the formation of essentially random copolymers. As an example, in Figure 7a the spectrum of a p(AcDED-co-AcTy) copolymer carried out in DMSO-d6 is reported. For all of p(AcDED-co-AcTy) copolymers, resonances between 6.5 and 6.6 ppm relative to aromatic protons were observed. In the spectral range 4.2−3.1 ppm, the copolymer resonances were covered or modified by water

peak, while in the 1.0−1.8 ppm range, the two broad and very low intensity peaks were attributed to alkyl protons of the polymer backbone. Finally, at 0.9 ppm the methyl hydrogen resonance belonging to AcDED moiety was observed. To evaluate the molar ratio of the co-monomers in the repeat unit, the following equation that takes into account the integral values of resonances at 0.9 ppm and 6.5−6.6 ppm was used: ∫0.9ppm(CH3 − CH 2 − N)

AcDED/AcTy =

3

∫0.9ppm(CH3 − CH 2 − N) 3

+

∫6.5 − 6.6ppm(−Ar − H) 4

The obtained values were in agreement with the monomer molar ratios used for polymerization. Given the poor solubility of the p(AcDED-co-AcHTy)s in DMSO-d6, the1H NMR spectra were carried out employing D2O as a solvent. In aqueous medium, the peaks relative to aromatic protons disappeared (Figure 7b). This was probably due to the supramolecular restructuring of our copolymers involving the stacking interactions among aromatic rings of AcHTy that could lead the aromatic protons to be shielded against magnetic field. These findings are in agreement with those obtained from MD simulation. Proton shielding effects were already observed by Bütün et al. on block copolymers containing two different tertiary amine groups in the side chain of the two employed acrylic monomers.56 The authors hypothesized micelle formation 8374


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Figure 5. Distance between hydroxytyrosol carbon atoms in typical hydrophobic contacts between the aromatic moieties. Top: Intramolecular; Bottom: Intermolecular.

from the disappearance of some 1H NMR signals relative to one of the two blocks containing quaternized tertiary amine groups Therefore, a micellar arragment could also be hyphothesized for our random p(AcDED-co-AcHTy) copolymers. The micelles should consist of an inner core, comprising HTy-based units, and an exterior shell of hydrophilic units swollen by the solvent. Since a flower-like micelle model was proposed for several cationic random copolymers, it can be hypothesized that flower-type spherical micelles are also formed by our random copolymers. The change of specific conductivity of aqueous solutions of the copolymers as a function of the p(AcDED-co-AcHTy) concentration allowed us to determine the CMC values, reported in Table 1. It can be observed that CMC of the amphiphilic copolymers decreases with hydrophobic group increasing. This result confirms that the driving force of the

micellization of the random p(AcDED-co-AcHTy)s is the hydrophobic interaction among the HTy-based units. Hydrodynamic diameter measurements (DLS data), reported in Table 1, showed that the copolymer series possessed a size larger than normal flower micelles reported in the literature.57 Probably, in our case the high polymer concentration used in DLS analysis induced the formation of three-dimensional networks of swollen micellar aggregates (multicore flower-like micelle).58 However, the micellar aggregate size decreased with increasing hydrophobic content. The small size of p(AcDEDco-AcAHTy) 70/30 can be justified by the tendency of this copolymer to self-assemble by intramolecular interactions of HTy units (π-stacking interactions) as evidenced by MD simulation (Figure 4). Zeta potential measurements showed that all synthesized materials were positively charged. The charge density of 8375


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p(AcDED-co-AcHTy)s, lower than the pAcDED one, evidenced the low stability of the copolymers in aqueous medium. Probably, the great decrease of zeta potential from the homopolymer to the 90/10 is due not only to the polymer composition (decrease of the positive charge carrier), but also to the bend of that copolymer to give a more compact structure thanks to hydrophobic interactions among aromatic groups. This occurs more for the 70/30 copolymer, as highlighted by MD simulations. In Table 1 the antimicrobial activity of the homopolymer and copolymers in terms of MIC is also reported. It can be observed as the MIC values decreased with increasing copolymer hydrophobicity and were low with respect to other random acrylic copolymers reported in the literature.59−61 Generally, amphiphilic random copolymers show an enhancement of antimicrobial activity with increase of their hydrophobicity reaching in some cases a constant value at high hydrophobic degree. This behavior is due to favorable interaction of the cationic and hydrophobic groups of the copolymers with bacterial cell membranes. However, high hydrophobicity can

Figure 6. Spatial distribution function (isodensity plot) of hydroxytyrosol oxygen atoms; one of the six oxygen is taken as reference (see text).

Figure 7. Repeat unit and 1H NMR spectra of p(AcDED-co-AcTy) 70/30 in DMSO-d6 (a) and p(AcDED-co-AcHTy) 70/30 in D2O (b). In spectrum (a) the peaks at 2.5 and 3 ppm are related to DMSO-d6 and adsorbed water, respectively. In spectrum (b) the peak at 4.2 ppm is due to D2O. 8376


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*E-mail: [email protected]. Tel/Fax: +39 06 49913692.

also induce self-assembly of polymer chains in aqueous media lowering antimicrobial activity.41 The reduction of antimicrobial efficiency is due to a decrease of polymer chains available for insertion into the hydrophobic zone of the membrane. Also, polymer backbone flexibility can promote intramolecular association of the chains in aqueous solution contributing to decrease in the available hydrophobic groups.41,59 When chain self-assembly occurs, high MIC values (>1000 μg/mL) are generally obtained.41 In our case, the MIC values are low and below the CMC of the copolymers (see Table 1). Therefore, it can be considered that the single chains are the species that interact with the cell membrane and not the aggregates. Although MD simulations have shown intramolecular hydrophobic aggregations (chain folding) for all copolymers, more dominant for p(AcDED-coAcHTy) 70/30, the copolymer self-assembly did not reduce the antibacterial activity differently from what was reported in the literature.41 In our case, the formation of structures with a hydrophobic aromatic core permits exposure of the amine groups to water medium promoting the electrostatic binding with the anionic lipopolysaccharides of the bacterial cell surface. The strong polymer/membrane interactions probably destabilize the copolymer structure making the hydrophobic groups available for their insertion into the membrane. The steric hindrance of the catecholic groups seems to have no significant effect on polymer stiffness, presumably due to the random distribution of the monomers in the copolymer chain. Therefore, thanks to the introduction in a cationic polyacrylate of a 0.3 molar fraction of catecholic groups, it was possible to properly balance the amphiphilic ratio and minimize intermolecular polymer chain aggregations to the benefit of antimicrobial properties. In conclusion, in the design of antimicrobial amphiphilic random copolymers it is necessary to choose both suitable hydrophobic monomers and hydrophilic/hydrophobic ratios to control their self-assembly and antimicrobial properties.

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SUMMARY

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was financially supported by the Italian Ministry of Education, University and Research. L. G. thanks Prof. Ruggero Caminiti (Sapienza University of Rome Chemistry Department) for providing free usage of Narten computing cluster facility.

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REFERENCES

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The structure of catecholic moiety-containing cationic random acrylic copolymers was investigated by MD, 1H NMR analysis, DLS, and zeta potential measurements. To evaluate the influence of hydrophobic/hydrophilic balance on polymer micellar structure, a series of copolymers was prepared by varying the molar ratios of two monomers, one bearing in the side chain a tertiary amine and the other one a hydrophobic catecholic group. Electrical conductivity measurements evidenced a micellar arragment for all of p(AcDED-co-AcHTy) copolymers. The stacking interactions among aromatic rings of HTy promoted the hydrophobic core formation reducing the size and charge density of cationic random copolymers, especially in the p(AcDED-co-AcHTy) 70/30 copolymer where a flower micelle structure could seem more likely. The low MIC values obtained for all of the copolymers confirmed the good amphiphilic balance reached. Particularly, in the case of the p(AcDED-co-AcHTy) 70/30 copolymer, the introduction of 0.3 molar fraction of hydrophobic catecholic groups minimized intermolecular polymer chain aggregations to the benefit of antimicrobial properties.

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Self-Assembly of Catecholic Moiety-Containing Cationic Random

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