Article pubs.acs.org/Langmuir
Antimicrobial Particles from Emulsion Polymerization of Methyl Methacrylate in the Presence of Quaternary Ammonium Surfactants Alliny F. Naves, Renata R. Palombo, Letícia D. M. Carrasco, and Ana M. Carmona-Ribeiro* Biocolloids Laboratory, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, CEP 05513-970, São Paulo SP, Brazil S Supporting Information *
ABSTRACT: The purpose of this Article is to characterize polymeric particles of poly(methylmethacrylate) (PMMA) synthesized in the presence of one of two different quaternary ammonium surfactants (QACs): cetyltrimethylammonium bromide (CTAB) or dioctadecyldimethylammonium bromide (DODAB). The methods used are dynamic light scattering for sizing, polydispersity and zeta potential analysis, scanning electron microscopy (SEM) for morphology visualization, and plating plus colony-forming unities (CFU) counting for the determination of antimicrobial activity. The results point out the high QAC concentration required to obtain cationic and bioactive antimicrobial particles with good colloidal stability and a permanent load of the polymeric network with QACs. Over a range of micromolar QAC concentrations, there is remarkable antimicrobial activity of PMMA/CTAB or PMMA/DODAB particles, which is much higher than those determined for the QACs by themselves. Loading the biocompatible polyacrylate particles with QACs is a facile, fast, low-cost approach to obtaining highly efficient antimicrobial nanoparticles.
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separation during polymerization.9−11 Microemulsions of styrene stabilized by cetyl trimethyl ammonium chloride (CTAC), dodecyl trimethyl ammonium bromide (DTAB), or mixtures of other emulsifiers led to stable polystyrene latex particles with diameters of between 20 and 120 nm.12,13 The available sites for polymerization are the micelles and the monomer droplets of the microemulsion.14 Slightly hydrophilic monomers such as MMA result in smaller droplets because MMA at the interface of polymerized particles also acts as a cosurfactant.13 The anionic free radicals generated by KPS can react with MMA in the micelles and with MMA inside the droplets in the aqueous phase, yielding oligoradicals. As the chain length of the oligoradicals increases, they can diffuse into the micelle interior where the monomers are, to proceed with the polymerization. Larger polymerized particles are produced when there are fewer micelles and many monomer droplets.3 In summary, although the emulsion and miniemulsion polymerization processes using different QACs have been described, a systematic study of the antimicrobial activity of the resulting dispersions is not yet available. In our group, we have previously combined PMMA and QACs in spin-coated films, describing for the first time the good compatibility between dioctadecyl dimethyl ammonium bromide (DODAB) and PMMA leading to good antimicrobial activity against bacteria upon contact.15 The dependence of the antimicrobial activity on the QAC structure was also evaluated from combinations of
INTRODUCTION Much work has been devoted to the preparation of antimicrobial macro-, micro-, and nanostructures of biomedical importance based on the covalent attachment or grafting of the quaternary ammonium moiety to the structures.1,2 However, polymeric nanoparticles in aqueous dispersions have often been obtained via emulsion or microemulsion polymerization using potassium persulfate (KPS) as an initiator and ternary mixtures of water, monomer, and quaternary ammonium compounds (QACs).3 The emulsion polymerization process occurs in two steps: nucleation and growth of the particles. When the monomer has a high affinity for the micelle interior, the nucleation is driven by the micelles, where the monomers solubilize. When the monomer is more polar and has some affinity for the continuous aqueous phase, the initiation of polymerization also takes place in emulsified monomer droplets and the nucleation becomes homogeneous and coagulative.4−7 The initiation of polymerization in emulsified monomer droplets with a 100−400 nm mean diameter characterizes the process of miniemulsion polymerization. These droplets compete with the micelles for the free radicals, which allow polymerization to start.8 There are also polymerization processes in oil in water emulsions involving four components: an ionic emulsifier or detergent such as cetyltrimethylammonium bromide (CTAB), the monomer (styrene), water, and a cosurfactant or codetergent such as pentanol or hexanol.9 However, attempts to polymerize methyl acrylate (MA) or methyl methacrylate (MMA) in large amounts of monomer (above 1.9 wt %) in oil-in-water (o/w) microemulsions were not successful because of phase © XXXX American Chemical Society
Received: April 22, 2013 Revised: June 19, 2013
A
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PMMA and DODAB, CTAB, or tetrapropyl bromide (TPAB) for spin-coated films.16 Whereas DODAB remains associated with PMMA films and kills bacteria upon contact, CTAB leaks out of the films and kills bacteria in the bulk solution.16 Tetrapropyl bromide (TPAB) does not kill bacteria because of its inappropriate hydrophobic−hydrophilic balance.16 Here we report the preparation, characterization, and antibacterial activity of PMMA/QAC particles prepared by emulsion polymerization over a range of high QAC concentrations. We show that there is remarkable antimicrobial activity with these synthesized PMMA/CTAB or PMMA/DODAB particles over micromolar ranges, with a much higher bactericidal efficacy compared to that observed for the surfactants, CTAB and DODAB, alone. Both PMMA/CTAB and PMMA/DODAB dispersions displayed a bactericidal effect that was at least 10 times greater than that of the surfactants by themselves against P. aeruginosa. Regarding the activity against S. aureus, PMMA/ QACs particles also exhibited a higher antimicrobial effect than did the corresponding surfactant, with PMMA/CTAB particle bactericide present in doses almost 10 times lower than the doses necessary for the bactericidal effect of CTAB alone, and for PMMA/DODAB dispersions, there is 98% cell death whereas DODAB bilayer fragments achieved at most 85% cell death.
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Table 1. Physical Properties of PMMA/QAC Particles Synthesized by Emulsion Polymerization Just after Their Exhaustive Dialysis system A1 A2 A3 A4 B1 B2 B3 B4
QAC (mM)
MMA (M)
Dz (nm)
ξ (mV)
polydispersity
CTAB 0.5 CTAB 2.0 CTAB 4.0 CTAB 4.0 DODAB 0.5 DODAB 2.0 DODAB 10 DODAB 10
0.56 0.56 0.56 1.32 0.56
419 ± 9 395 ± 5 84 ± 1 112 ± 1 707 ± 11
−44 ± 2 −38 ± 1 +25 ± 1 +44 ± 4 −41 ± 1
0.309 0.262 0.019 0.059 0.236
0.56
1260 ± 43
−10 ± 1
0.370
0.56
113 ± 1
+46 ± 3
0.278
1.32
140 ± 1
+48 ± 2
0.280
equation ζ = μη/ε, where η and ε are the viscosity and the dielectric constant of the medium, respectively. Samples were prepared by diluting 50−100 μL of PMMA dispersions in 10 mL of Milli-Q water, and the measurements were performed after thermal equilibrium at 25 ± 1 °C. Determination of PMMA Molecular Weight and Particle Morphology in PMMA/QAC Dispersions. For these analyses, the PMMA/QAC dispersions were lyophilized in order to obtain dry particles. The conversion of monomer into polymer and the solid contents were determined by gravimetric measurements. The degree of polymerization was determined from the mean molecular weight divided by the monomer molecular weight (100.12 g/mol). The mean molecular weight was obtained from size-exclusion chromatography. Size-exclusion chromatography (SEC) was applied to determine M̅ n, M̅ w, and the polydispersity index (PDI) using a Shimadzu HPLC/SEC class-VP equipped with two Viscogel columns (I-MBMMW 3078 and I-Oligo 3078, 30 cm × 8 mm each, Viscotek) with exclusion limits of 20 and 10 KPS, respectively, connected to a Shimadzu RID 10A refractive index differential detector. The solvent was chloroform with a flow rate of 1 mL/min. The SEC system was calibrated using seven polystyrene standards of low PDI (Aldrich/Waters; M̅ w = 820, 2460, 5120, 13 200, 29 300, 47 500, and 216 000 g/mol), and toluene was employed to identify the exclusion limit (conventional calibration). Samples were solubilized previously in chloroform (10−15 mg/mL) 24 h before the SEC analyses. Elemental analyses were performed with Perkin-Elmer CHN 200 equipment, allowing the quantitative determination of carbon, hydrogen, and nitrogen in the lyophilized dispersions. Scanning electron microscopy (SEM) was performed with Jeol JSM-7401F equipment. The lyophilized dispersions were covered with a thin layer of gold before SEM analyses. Organisms and Culture Conditions. Pseudomonas aeruginosa ATCC (American Type Culture Collection) 27853 and Staphylococcus aureus ATCC 25923 were reactivated, each one separately, for 3 h at 37 °C under stirring in 3 mL of tryptic soy broth TSB (Merck KGaA, Darmstadt, Germany). Thereafter, bacteria were spread on plates of Mueller-Hinton agar MHA (Hi-Media Laboratories Pvt, India) and incubated for 18−24 h at 37 °C. Aliquots of each species were taken from the plates and incubated in 10 mL of TSB (160 rpm, 37 °C, 2 to 3 h), allowing both species to reach the exponential phase of growth. Each culture was pelleted and separated from its nutritive medium by centrifugation (8000 rpm, 15 min). The supernatant was replaced with a 0.264 M D-glucose solution before resuspending the bacteria pellets, and the centrifugation/resuspension procedure was repeated twice before using both bacteria to evaluate the antimicrobial activity of PMMA/QAC particles. Because cationic molecules are inactivated by the culture medium that contains a relatively high ionic strength and also negatively charged molecules such as amino acids and polysaccharides, the interaction between particles and bacteria was carried out in an isotonic 0.264 M D-glucose solution. The turbidity of bacteria suspensions at 625 nm was adjusted according to tube 0.5
EXPERIMENTAL SECTION
Chemicals. Methyl methacrylate (MMA, Sigma-Aldrich), hexadecyl trimethyl ammonium bromide (CTAB, Sigma), dioctadecyl dimethyl ammonium bromide (DODAB, Sigma), and potassium persulfate (KPS, K2S2O8, Sigma) were used without further purification. The syntheses were performed using Milli-Q water. The dispersions obtained by emulsion polymerization were purified by dialysis using cellulose acetate membranes (Sigma-Aldrich) with a molecular weight cutoff, MWCO, in the range of 12 400 g/mol. Preparation of Dispersions of Cationic Amphiphiles. DODAB powder was weighted and added to a solution of 1 mM NaCl (pH 6.3) to a final concentration of 2 mM before dispersion by ultrasound with a macroprobe as described previously.17 DODAB or NaCl concentrations were determined by halides microtitration18 or by using a spectrophotometer to measure the absorbance decrease that occurs as a result of the cosolubilization of the orange-G/DODAB complex in Brij micelles.19 CTAB was added to a 1 mM NaCl (pH 6.3) solution to obtain the final desired concentration. Preparation of PMMA/QAC Dispersions by Emulsion Polymerization. Polymerization reactions were carried out inside an external oil bath at 85 °C under reflux for 2 h using 50 mL of aqueous dispersions of CTAB or DODAB at the concentrations described in Table 1. A slight flow of N2 was applied for 30 min before adding radical initiator KPS (0.018 g, 1.33 mmol/L) and maintained for 30 min after its addition. Monomer MMA was added dropwise 3 min after adding the initiator to obtain the final concentrations presented in Table 1. After 2 h, the reactions were removed from the oil bath and allowed to reach room temperature. The dispersions thus obtained were purified by dialysis against Milli-Q water for 3 days or up to pH 6.5−7.0 with water changes every 12 h. Particle characterization took place after the exhaustive dialysis step. Particle Sizing and Zeta-Potential Analysis of PMMA/QAC Dispersions. Size distributions, zeta-average diameters (Dz), and zeta potentials (ζ potentials) were obtained by dynamic light scattering (DLS) using a zeta plus−zeta potential analyzer (Brookhaven Instruments Corporation, Holtsville, NY) equipped with a 677 nm laser with measurements at 90°. The polydispersity of the dispersions was determined by dynamic light scattering (DLS) following welldefined mathematic equations.20 Mean hydrodynamic diameters (mean Dz) were obtained from the log-normal distribution of the light-scattering intensity curve against the diameter. ζ potentials were determined from the electrophoretic mobility μ and Smoluckowski B
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Table 2. Physical Properties of Positively Charged PMMA/QAC Particles Synthesized at High QAC Concentrations as Formulations A3, A4, B3, and B4, Dialyzed and Selected for Determining Antimicrobial Activity [QAC]
[MMA] (M)
4 mM CTAB 4 mM CTAB 10 mM DODAB 10 mM DODAB
0.56 1.32 0.56 1.32
Dz (nm) 84 112 113 140
± ± ± ±
1 1 1 1
P 0.019 0.059 0.278 0.280
ξ (mV)
solid content (mg/mL)
conversion rate (%)
± ± ± ±
47 110 51 35
84 83 91 27
25 44 46 48
1 4 3 2
Np/mL−1 1.3 1.3 2.0 6.6
× × × ×
1014 1014 1013 1012
Table 3. Pondered Molecular Weight (Mw), Polydispersity Index PDI = Mw/Mn, Degree of Polymerization or Number of Monomer Residues per Polymer Chain (DP), Elemental Analyses for Carbon, Hydrogen, and Nitrogen, and Estimated Final QAC Concentrations in Formulations A3, A4, B3, and B4 after Exhaustive Dialysis dispersion
Mw (g/mol)
Mw/ Mn
DP
A3 A4 B3 B4
60 600 73 620 115 320 83 620
1.22 1.18 1.10 1.09
606 736 1153 836
%C 59.37 59.47 60.68 60.32
using the McFarland scale (final cell concentration of 1.5 × 108 CFU/ mL) in order to proceed to bacterial interactions with the PMMA/ QAC particles for cell viability determination. Cell Viability Assays. Interactions between bacteria and dispersions proceeded for 1 h using 0.9 mL of a bacteria suspension and 0.1 mL of different dilutions of each dispersion. After 1 h of interaction, 0.1 mL of the diluted mixture was diluted up to 1000- or 10 000-fold to be spread on agar plates in triplicate. Next, the plates were incubated (24 h/37 °C), and thereafter CFU counting was performed. Cell viability (%) was presented as a mean value ± mean standard deviation. Positive control was performed by interacting 0.9 mL of a standard bacteria suspension and 0.1 mL of a 0.264 M Dglucose solution and then spreading 0.1 mL of this mixture previously diluted 1:20 000.
± ± ± ±
%H 0.04 0.19 0.04 0.07
7.92 8.11 8.68 8.44
± ± ± ±
0.14 0.01 0.01 0.02
%N
[QAC] (mM)
± ± ± ±
0.83 0.78 1.38 0.46
0.05 0.02 0.23 0.12
0.03 0.01 0.01 0.01
Table 1. The zeta-average diameter, polydispersity, and ζ potential for the dispersions from dynamic light scattering were determined after exhaustive dialysis of the dispersions. Some dispersions synthesized in the largest concentrations of CTAB or DODAB presented good colloidal stability and high, positive ζ-potential values, representing good candidates for testing the antimicrobial activity. Over a low range of QAC concentrations, such as those in formulations A1 to A2 with CTAB or B1 to B2 with DODAB, the negative residual charges due to the KPS were not completely neutralized by the QACs employed during the syntheses. The colloidal stability of the particles depends on the negative balance between the negative charges from the initiator and the positive charges from the QACs. In the case of formulations A3, A4, B3, and B4, the QAC concentration was enough to give stability to the positively charged dispersions obtained from the prevalence of positive charges on the particle surfaces. Given their high positive ζ potential and the small particle size (Dz ≤ 140 nm), formulations A3, A4, B3, and B4, as quoted in Table 1, were selected for additional characterization. Determinations of solid contents, the conversion rate of monomer to polymer, the particle number density (Np), the average molecular weight M̅ w (by size-exclusion chromatography, SEC), polydispersity index (PDI), morphology of lyophilized particles from SEM, and antimicrobial activity against bacteria from plating and CFU counting were carried out. Table 2 shows data on the particle number density (Np) calculated from the mass of one particle, mp, taken as its volume (4/3πrp3) times the PMMA density (1.15 × 103 g/dm3). The particle radius, rp, was taken to be Dz/2, and Dz was determined by means of dynamic light scattering. Because the solid content for each formulation was obtained by gravimetry in mg/mL and mp is known, the particle number density (the number of particles in 1 mL of dispersion or Np) could be calculated, and the results are presented in Table 2. As products of the polymerization, chains with different lengths are formed so that there will be a distribution of molecular weights. Two molecular weights can be determined from the distributions: the average molecular weight (M̅ n), that is, the total weight of polymer divided by the total number of polymer molecules (n), and the pondered molecular weight (M̅ w), which corresponds to the pondered sum of molecular weights divided by the total weight. The polydispersity index (PDI) corresponds to the ratio between M̅ w and M̅ n (PDI =
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RESULTS AND DISCUSSION Emulsion polymerizations of MMA in the presence of CTAB or DODAB were carried out with KPS as the inorganic initiator under N2 flow in order to prevent precocious termination of the free radicals required for the MMA addition reaction and the synthesis of the PMMA particles. Table 1 shows the main physical properties of the dispersions as a function of different feed concentrations of the MMA monomer or the cationic amphiphile (CTAB or DODAB). Formulations A3, A4, B3, and B4 yielded cationic dispersions with high colloidal stability at high concentrations of CTAB or DODAB. In general, for syntheses carried out in the presence of the QACs, the zetaaverage diameter for the particles in the dispersions varied between 84 and 1320 nm whereas the ζ-potential values varied between −43 and +51 mV. In all cases, the particle size increases when the concentration of monomer is raised from 0.56 to 1.32 mol/L at a given CTAB or DODAB concentration. The stable dispersions have the smallest sizes: 84 and 113 nm for the zeta-average diameter using CTAB and DODAB, respectively. Sizes varied from 84 to 419 nm for the zetaaverage diameter for formulations with CTAB and from 113 to 1260 nm for formulations with DODAB (Table 1). Potassium persulfate (used at 1.33 mM) is a divalent salt (K2S2O8), as are the sulfate residues left on the particles. CTAB and DODAB are monovalent salts. Possibly, [QAC] < 2.66 mM is not large enough to neutralize the sulfate residues attached to the particles. Above 2.66 mM QAC, the particles are positively charged, as expected and required for the bactericidal activity (Table 1). The effects of QAC and MMA concentration on the physical properties of the particles are also evidenced by the data in C
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Figure 1. Size distributions for cationic PMMA/QAC particles in formulations A3 (A, E), A4 (B, F), B3 (C, G), and B4 (D, H) just after synthesis and dialysis (A−D) and 1 year after synthesis and a second exhaustive dialysis step (E−H).
M̅ w/M̅ n). The higher this index, the broader the length distribution of the polymer chains. It is important to remember that this PDI value is not related to the particle polydispersity determined from the particle size distribution obtained by DLS. Table 3 shows M̅ w, PDI values, the degree of polymerization DP (or mean number of monomer residues per polymer chain), and the elemental analyses for the four selected PMMA/QAC dispersions. From the %N in the particles, mean particle volume, and PMMA density, the millimolar QAC concentration after dialysis was calculated and is displayed in the last column of Table 3. Syntheses of PMMA particles in the presence of DODAB yielded chains with higher Mw and lower PDI compared to syntheses in the presence of CTAB (Table 3). Furthermore, the degree of polymerization or the mean number of monomer residues per polymer chain is larger in the presence of DODAB than in the presence of CTAB. Thus, the nucleation provided by the DODAB bilayer fragments allow the formation of longer polymeric chains with larger Mw than the nucleation provided by CTAB micelles. The CTAB micelles and the MMA droplets
provide two different hydrophobic microenvironments in terms of volume for MMA polymerization. The DODAB bilayer fragments provide a microcompartment that is more similar in volume to the volume of MMA microdroplets in the dispersion. This would yield a lower polydispersity index for the length of the obtained polymer chains in the DODAB bilayer fragments and the MMA microdroplets microenvironment. Another curious result in Table 3 refers to the elemental analysis for the polymeric particles. The nitrogen content in the PMMA/ DODAB particles is always higher than that determined for PMMA/CTAB particles. This finding suggests a higher affinity between DODAB and PMMA than between CTAB and PMMA because there is a larger amount of DODAB in the polymeric matrix when compared to the amount of CTAB. In fact, previous results in the literature related to the presence of DODAB and CTAB at the film/water interface of hybrid PMMA/QACs films showed that CTAB tends to move through the polymeric matrix, leaking into the water, whereas DODAB has a higher affinity for PMMA and remains in the polymeric matrix as well as at the polymer−water interface.16 D
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Table 4. Mean Diameter (Dz), Polydispersity (P), Zeta Potential (ζ), and Elemental Analysis (%C, %H, and %N) for PMMA/ QAC Dispersions 1 Year after Their Synthesis and Two Exhaustive Dialysis Steps dispersion (1 year later) A3 A4 B3 B4
Dz (nm) 82 111 160 136
± ± ± ±
1 1 4 1
ζ (mV)
P 0.019 0.060 0.359 0.308
± ± ± ±
0.007 0.008 0.004 0.003
41 51 42 44
Figure 1 presents the size distributions and ζ potentials for particles in formulations A3, A4, B3, and B4 just after synthesis and dialysis (Figure 1A−D, respectively) and for the same dispersions 1 year after synthesis and a second exhaustive dialysis step (Figure 1E−H, respectively). From the intensity of scattered light as a function of the particle diameter for sample A3 (Figure 1A), it is possible to observe the occurrence of a very narrow size distribution and a positive ζ potential, as should be expected at the high concentration of CTAB employed (4 mM), which is well above the CTAB critical micelle concentration of 1 mM. This dispersion shows a polydispersity that is remarkably low and equal to 0.019 (Table 1), which is consistent with the very narrow size distribution for the particles, even 1 year after the particle synthesis (Figure 1E). In formulation A4 (Figure 1 B), by feeding the reaction with twice the amount of MMA and keeping the same concentration of CTAB (4 mM, Table 1), the small positively charged particles still exhibit a low polydispersity and a high colloidal stability 1 year after synthesis (Figure 1F). For dispersions prepared with MMA and cationic bilayer fragments of DODAB, size and ζ potential characterization showed positively charged polymeric particles that were larger than the particles synthesized in the presence of CTAB (Table 1). Formulation B3 in the presence of 10 mM DODAB employing 0.56 M MMA yielded a particle dispersion with a mean zeta-average diameter of 113 nm and a ζ potential of 46 mV (Figure 1 C), which increased in size and polydispersity 1 year thereafter (Figure 1 G). Doubling the amount of MMA used in the particle synthesis in the presence of 10 mM DODAB yielded positively charged particles with a higher Dz (140 ± 1 nm) and polydispersity (0.280) ,which display a slight increase in polydispersity 1 year thereafter (Figure 1D,H and Tables 2 and 4). The effect of aging on the physical properties of A3, A4, B3, and B4 dispersions (1 year after synthesis) is shown in Table 4. PMMA/CTAB formulations A3 and A4 were stored on the laboratory bench for 1 year at room temperature, and no precipitation/aggregation, flocculation, or apparent growth of microorganisms was observed. The physical properties were the same for A3 and A4 1 year after synthesis and after a second exhaustive dialysis step (Tables 2 and 4). PMMA/DODAB dispersions B3 and B4 stored under the same conditions exhibited some precipitation; however, the size, the ζ potential, and polydispersity measurements revealed the occurrence of slightly larger particles with larger polydispersities and no changes in the ζ potential that were still able to redisperse by stirring 1 year after synthesis (Table 4). Furthermore, plating aliquots of the dispersions on agar showed the total absence of viable microorganisms 1 year after the particle synthesis. For PMMA/CTAB particles, no changes in Dz and polydispersity took place 1 year after synthesis and after two exhaustive dialysis steps, whereas for PMMA/DODAB particles there was an increase in size for B3 and an increase in polydispersity for B3 and B4 but no change in the zeta potentials. A possible
± ± ± ±
2 3 1 1
%C 59.06 59.46 60.57 60.02
± ± ± ±
%H 0.26 0.10 0.03 0.10
8.11 8.08 8.53 8.30
± ± ± ±
0.20 0.09 0.37 0.02
%N 0.10 0.16 0.31 0.26
± ± ± ±
0.05 0.01 0.03 0.09
explanation for the changes obtained with the largest PMMA/ DODAB particles would be aggregation at the secondary minimum of the interaction energy curve as a function of interparticle distance. Aggregation is reversible upon shaking or heating because the minimum is shallow and involves a few kT of energy. The aggregation of PMMA/DODAB particles 1 year after their synthesis and dialysis also explains the increase in polydispersity. For the PMMA/QAC particles used in this work, the exhaustive dialysis step done after particle synthesis and prior to plating removed most loosely bound QACs. The % N 1 year after synthesis and after a second exhaustive dialysis step, in Table 4, either remained unchanged within the limits of the experimental error or slightly increased when compared to %N in Table 3 for data just after synthesis and the first dialysis. This shows that the QACs remaining in the particles after dialysis are tightly bound and are not released. The slight increase in %N after the second dialysis step might be due to the MMA monomer remaining in the particles after the first dialysis; however, the monomer could be completely removed from the particles after the second exhaustive dialysis step. QAC leaching did not take place after the first dialysis step because %N did not decrease after the second dialysis step. SEM images obtained for lyophilized particles just after their synthesis and dialysis revealed the morphology for particles in formulations A3, A4, B3, and B4 (Figure 2). There is good agreement between the data from dynamic light scattering and the sizes seen from the micrographs in Figure 2. The very low polydispersity for A3 and A4 (0.019 and 0.059, respectively) is consistent with images in Figure 2a,b where all particles have similar sizes. In the case of particles synthesized in the presence of DODAB bilayer fragments, the individual
Figure 2. Micrographs of PMMA/CTAB dispersions (a) A3 and (b) A4 and PMMA/DODAB dispersions (c) B3 and (d) B4, obtained by scanning electron microscopy (SEM). E
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Figure 3. Bacterial cell viability (%) as a function of the final concentrations of CTAB alone (△) or [CTAB] in PMMA/CTAB dispersions A3 (□) and A4 (○) after the first dialysis step. There was an interaction time of 1 h between dispersions and S. aureus (A) and P. aeruginosa (B), at final cell concentrations of (4−6) × 106 and 6 × 106 CFU/mL, respectively.
Figure 4. Bacterial cell viability (%) as a function of DODAB concentration alone as bilayer fragments (△) or in PMMA/DODAB dispersions B3 (□) and B4 (○) against S. aureus (A) or P. aeruginosa (B) at final cell concentrations of 1.6 × 108 and (1.5 −3.0) × 108 CFU/mL, respectively. The interaction time between bacteria and dispersions was 1 h.
The microbicidal activity for formulations B3 and B4 against S. aureus and P. aeruginosa is presented in Figure 4, where we observe a much higher bactericidal efficacy of PMMA/DODAB particles compared that observed for DODAB bilayer fragments alone. Above 0.05 mM DODAB in the PMMA/DODAB particles, the bacterial viability increased (Figure 4A). With increasing concentration of PMMA/DODAB particles interacting for 1 h with bacteria, there is a sigmoidal decrease in the cell viability of bacteria. For S. aureus, from 20 to 40 μM DODAB in PMMA/DODAB particles (B3 and B4), there is 98% cell death, whereas DODAB bilayer fragments achieved at most 85% cell death. Against P. aeruginosa, 100% cell death was achieved by PMMA/DODAB particles at 1 (B3), 0.4 (B4), and 7 μM DODAB (DODAB bilayer fragments). Against both bacteria, there is superior performance for DODAB immobilized in the polymeric particles in comparison to the activity of DODAB bilayer fragments only. The mechanism of antimicrobial activity for the particles may be explained only by the direct contact between the particles and the bacteria because the QACs are not released after the exhaustive dialysis of the dispersions (Tables 3 and 4). The almost complete activity for PMMA/DODAB particles against S. aureus over a range of intermediate concentrations (Figure 4) can be understood from the competitive behavior of these particles toward adsorption
morphologies resemble coalescent discs and the size heterogeneity is accordingly higher (Figure 2c,d). The tests for formulations A3 and A4 against Gram-positive microorganism Staphylococcus aureus (Figure 3A) and Gramnegative Pseudomonas aeruginosa (Figure 3B) revealed their strong microbicidal activity, which was superior to that exhibited by CTAB alone (triangles). One should notice that the CTAB concentrations in the particles are calculated after the first dialysis from the %N in Table 3. It is possible that after the second dialysis step and complete leaching of the MMA monomer even higher antimicrobial activity should be obtained for the cationic particles because the methacrylic acid reduces the positive charge density on particles. With increasing concentration of PMMA/CTAB particles interacting for 1 h with bacteria, there is a sigmoidal decrease in the cell viability of bacteria. From 4 μM CTAB in PMMA/ CTAB particles, there is a complete loss of cell viability for S. aureus and for P. aeruginosa. For CTAB alone, 30 and 100 μM CTAB are required to kill S. aureus and P. aeruginosa completely, respectively. Free surfactant molecules tend to self-assemble instead of assembling into the bacterial cell. QAC immobilization in the particles prevents the surfactant selfassembly and increases its availability to the bacterial cells. F
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on the bacterial cells and particle/particle aggregation, which is favored by increasing the particles concentration. Furthermore, this behavior was observed only for the S. aureus coccus, which has a lower total surface area available for adsorption in comparison to that of P. aeruginosa rods at the same concentration of cells. Another possible explanation for the nonmonotonic behavior of antimicrobial activity against S. aureus as a function of DODAB concentration in the PMMA/ DODAB particles might be the occurrence of some MMA monomers even after the first dialysis step so that the particles would display a lower positive zeta potential over a range of high particle concentration resulting from the presence of residual MMA after the first dialysis. This would represent an increase in ionic strength that is able to reduce the positive zeta potential of the particles, thereby reducing their antimicrobial activity. Because high-quality measurements in the Zeta Plus apparatus can be obtained only for highly dilute particle samples, it is not possible to prove this by performing a zetapotential measurement at high particle concentration. However, the second dialysis step has shown an increase in %N in the particle composition, which would be in agreement with the leaching of the residual MMA after the first dialysis step. Again, an even more pronounced antimicrobial effect should be expected for the cationic PMMA/DODAB particles after the second dialysis step. The use of PMMA/CTAB or PMMA/DODAB cationic particles at low concentrations of CTAB and DODAB, respectively, represents substantial progress when compared to QAC applications in vivo in isolated form because the living organisms have phagocytosis mechanisms specially driven to pathogenic bacteria and foreign particles. Phagocytosis eliminates particles and other foreign bodies in such a way that in vivo the cationic particles and the bacteria will colocalize inside the macrophages, which are the phagocytic cells.21,22 Although PMMA is known to be biocompatible, after the loading of QACs future experiments should check the biocompatibility of the modified/loaded PMMA particles, aiming at the many uses in biomedicine, the production of new materials with antimicrobial properties, the production of polymeric films for storing and/or preserving food, and even the production of prosthesis or regenerative devices to be used for bone, teeth, and other tissue replacement, which would also be important in biomedical areas or in the production of polymeric hospital materials such as catheters, probes, and wound dressings. Thus, the present work uses the advantages of quaternary ammonium cationic amphiphiles such as CTAB and DODAB to minimize their concentrations and eventually promote their biocompatibility through their incorporation into polyacrylate polymeric matrixes, taking advantage of PMMA biocompatibility and inexpensive character. The possible applications of particle synthesis in the presence of quaternary ammonium compounds are not restricted to the use of CTAB or DODAB. Many other cationic antimicrobial agents have recently been described, such as the gemini surfactants,23−25 conjugated polyelectrolytes,26 polynorbornenes,27 polydiallydimethylammonium chloride derivatives,28,29 and lipopeptides,30 with all representing good candidates for incorporation into PMMA nanoparticles during their synthesis. The incorporation of antimicrobial surfactants, lipids, and polymers in PMMA particles during their synthesis by emulsion polymerization would represent a simple and inexpensive way of obtaining biocompatible antimicrobial materials.
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ASSOCIATED CONTENT
S Supporting Information *
Calculation of QAC concentration on particles after particle synthesis and the first dialysis step. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from Conselho Nacional de Desenvolvimento Cientı ́fico e Tecnológico (CNPq) for A.M.C.-R. and for R.R.P. (trainee technician fellowship) are gratefully acknowledged. The Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo is also acknowledged for the research grant number 2011/00046-5 to A.M.C.-R. and the PhD fellowship for L.D.M.C. (grant number 2012/24534-1).
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