ARTICLE pubs.acs.org/Biomac
Recyclable Antibacterial Magnetic Nanoparticles Grafted with Quaternized Poly(2-(dimethylamino)ethyl methacrylate) Brushes Hongchen Dong,† Jinyu Huang,‡ Richard R. Koepsel,§ Penglin Ye,† Alan J. Russell,*,§ and Krzysztof Matyjaszewski*,† †
Carnegie Mellon University, Chemistry Department, Pittsburgh, Pennsylvania 15213, United States CIBA Vision Corporation, 11460 Johns Creek Parkway, Duluth, Georgia 30097, United States § Department of Surgery and McGowan Institute for Regenerative Medicine, University of Pittsburgh, Suite 300, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, United States ‡
ABSTRACT: Highly efficient recyclable antibacterial magnetite nanoparticles consisting of a magnetic Fe3O4 core with an antibacterial poly(quaternary ammonium) (PQA) coating were prepared in an efficient four-step process. The synthetic pathway included: (1) preparation of Fe3O4 nanoparticles via coprecipitation of Fe2þ/Fe3þ in the presence of an alkaline solution; (2) attachment of an ATRP initiating functionality to the surface of the nanoparticles; (3) surface-initiated atom transfer radical polymerization (ATRP) of 2(dimethylamino)ethyl methacrylate (DMAEMA); and (4) transformation of PDMAEMA brushes to PQA via quaternization with ethyl bromide. The success of the surface functionalization was confirmed by FT-IR, thermal gravimetric analysis (TGA), elemental analysis, and transmission electron microscopy (TEM). The PQA-modified magnetite nanoparticles were dispersed in water and exhibited a response to an external magnetic field, making the nanoparticles easy to remove from water after antibacterial tests. The PQA-modified magnetite nanoparticles retained 100% biocidal efficiency against E. coli (105 to 106 E. coli/mg nanoparticles) during eight exposure/collect/recycle procedures without washing with any solvents or water.
1. INTRODUCTION Magnetic nanoparticles have been extensively studied over the past decade in various medical applications such as magnetic resonance imaging (MRI),1,2 site-specific drug delivery,1,3-5 labeling and sorting of biological species,1,2,6,7 and therapy.1,8,9 The use of superparamagnetic iron oxide nanoparticles as contrast agents in MRI has been approved by the Food and Drug Administration (FDA).10,11 However, because of their large surface area/volume ratio, the nanometer-sized iron oxide cores tend to agglomerate into large clusters and lose the specific properties associated with their initial nanometer dimensions. Therefore, appropriate surface modification and particle stabilization have become critical challenges for extending the utility of magnetic nanoparticles into bioapplications. Surface grafting of polymer brushes is one of the most effective methods to achieve surface modification because the surface properties can be predictably changed by tethering a variety of functional polymers to the target surface, and these permanently attached polymers ensure long-term stability. In general, surface grafting of polymer chains can be achieved via either a “grafting onto”12-15 or a “grafting from”16-20 approach. The “grafting onto” method involves the reaction of reactive functional groups in the polymer with complementary active groups on the surface. In general, because of the steric hindrance effect, the grafting density of “grafted onto” polymer chains is limited. Conversely, the “grafting from” approach, which utilizes active species on the surface to initiate polymerization, usually results in high grafting density, up to r 2011 American Chemical Society
Scheme 1. Synthesis of 2-(2-Bromoisobutyryloxy)ethyl) Phosphonic Acid (BiBEP)
1 chain/nm2. In conjunction with recently developed controlledliving radical polymerization techniques,21-24 especially atom transfer radical polymerization (ATRP),22,23,25-30 the “grafting from” approach provides an effective synthetic route to functional surfaces with designed properties by allowing control over the graft polymer composition and structure.16,18,19,31-34 Antibacterial surfaces have attracted considerable interest, among a variety of applications for functional polymer brushes fabricated via surface-initiated ATRP, because of the increasing demand for hygienic living conditions.35-40 Antibacterial surfaces with adjustable biocidal activity have been prepared from a variety of substrates such as glass slides,17,35,41-44 paper,43 and plastics.37,45-47 The surface charge density of poly(quaternary ammonium) (PQA)-modified surfaces has been shown to be the critical element that determines Received: January 6, 2011 Revised: February 16, 2011 Published: March 08, 2011 1305
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Table 1. PDMAEMA-Modified Magnetite Nanoparticlea entry
[I]0/[M]0/[CuCl]0/[CuCl2]0/[L]0b
1
Mn (GPC)
Mw/Mn (GPC)
organic mass (%) (TGA)
initiators only
organic mass (%) (EA)
10
12
2
1:1570:4.1:0.8:5
10 700
1.6
55
50
3 4
1:1570:4.1:0.8:5 1:1570:8.2:1.7:10
44 700 152 100
1.2 2.1
61 93
58 88
a GPC: gel permeation chromatography; TGA: thermal gravimetric analysis; EA: elemental analysis. b M = DMAEMA; L = HMTETA; [DMAEMA]0 = 5.5 M; solvent: acetone. Polymerization was carried out at 40 °C.
antibacterial efficiency.17 The most effective biocidal surfaces had charge densities greater than (1 to 5) 1015 accessible quaternary ammonium groups/cm2, which can be readily achieved through surface-initiated ATRP. Magnetic nanoparticles have shown multiple benefits in many applications.7 Because of their unique characteristics, such as response to external magnetic force and high surface-to-volume ratio, it is of interest to explore the use of magnetic nanoparticles as carriers of antibacterial polymers. These antibacterial nanomaterials could be highly efficient biocides that could be easily recycled via application of an external magnetic field. Whereas antimicrobial magnetic particles have been made by a “grafting to” method, the material properties and recyclability of these particles was not reported.48 In this Article, the synthesis of antibacterial magnetite nanoparticles and investigation of the biocidal activity of these nanoparticles is reported. Control of coating properties was achieved by surface-initiated ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA), followed by postpolymerization quaternization giving particles that were readily dispersible. The recyclability of PQA-modified magnetite nanoparticles is also discussed.
2. EXPERIMENTAL SECTION 2.1. Materials. All chemicals were purchased from Aldrich and used without further purification unless otherwise stated. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) (Aldrich, 98%) was purified by passing the monomer through a column filled with basic alumina. 2.2. Synthetic Procedures. Synthesis of Magnetite Nanoparticles. We dissolved 1.6 g FeCl3 (0.01 mol) and 0.6 g FeCl2 (0.005 mol) in 10 mL of degassed H2O. The solution of iron chlorides was then slowly added to 100 mL of N2 purged NaOH solution (0.5 M) under sonication. The formation of a black precipitate of magnetite was immediately observed, and the precipitate was then collected by applying a magnetic force. The magnetic nanoparticles were extensively washed with deionized water to remove unreacted materials and impurities (yield 85%). Synthesis of Dimethyl 2-(2-Bromoisobutyryloxy)ethyl) Phosphonate (DMBiBEP). We added 8.9 mL (0.07 mol) of 2-bromoisobutyryl bromide and 10.3 mL (0.07 mol) of triethylamine to 120 mL of methylene chloride; then, 10 g (0.065 mol) of dimethyl(2-hydroxyethyl) phosphonate was slowly added to the reaction mixture at 0 °C. The reaction was held at room temperature overnight; then, the white precipitate was filtered off, and the clear filtrate was extracted with (2 150 mL) saturated sodium bicarbonate in water, followed by (1 100 mL) water. The solution was then dried over anhydrous MgSO4 overnight. The final oil-like product was obtained after filtration and evaporation of the solvent (yield 80%): 1H NMR (300 MHz) in CDCl3 δ 5.3 (s 6H), 4.5 (t 2H), 2.3 (t 2H), 1.9 (s 6H). Preparation of ATRP Initiator, (2-(2-Bromoisobutyryloxy)ethyl) Phosphonic Acid (BiBEP). We introduced 0.5 g of DMBiBEP (1.7 mmol phosphate groups) and 5 mL of methylene chloride to a 10 mL round-
bottomed flask. After complete dissolution of the DMBiBEP, 1.3 mL (8.5 mmol) of bromotrimethylsilane was added in a dropwise manner, and the mixture was stirred at room temperature for 12 h. The solvent and volatile residues were removed then by evaporation, and methanolysis of the silylated intermediate was realized by adding an excess of methanol (5 mL). The mixture was stirred at room temperature for another 12 h, and the solvent was evaporated to provide a quantitative yield of a slightly yellowish oil after washing with ether (yield 92%): 1H NMR (300 MHz) in CDCl3 δ 1.97 (s, 6H, BrC(CH3)2COO), 2.30 (t, 2H, J = 9.7 Hz, CH2CH2COO), 4.47 (t, 2H, J = 6.8 Hz, CH2CH2COO), 10 (broad s, 1H, acid). 13C NMR (300 MHz) in CDCl3 δ 171.8 (CdO), 60.1 (BrC(CH3)2), 55.5 (COOCH2CH2), 30.5 (BrC(CH3)2), 27.2 (COOCH2CH2). Immobilization of BiBEP on the Surface of Magnetite Nanoparticles. A slurry of magnetite nanoparticles (0.25 g) in a solution of BiBEP (0.25 g, 0.9 mmol) in 10 mL of methanol were shaken overnight. The resulting magnetite particles were collected, washed with acetone five times, and dried at room temperature under vacuum (quantitative yield). Synthesis of PDMAEMA Brushes from BiBEP-Modified Magnetite Nanoparticles. A typical “grafting from” polymerization procedure follows (Table 1, entry 3): 50 mg of BiBEP initiator modified nanoparticles (0.02 mmol ATRP initiators), 5.6 mL (33 mmol) of DMAEMA, 28 μL (0.1 mmol) of 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), and 0.5 mL of acetone were added to a 50 mL Schlenk flask. The reaction mixture was purged with N2 for 1 h. Then, CuCl (8.8 mg, 0.08 mmol) and CuCl2 (2.2 mg, 0.016 mmol) were added under N2. The reaction was carried out at 40 °C with mechanical stirring. Samples were taken throughout the polymerization to determine monomer conversion by 1H NMR. Gel permeation chromatography (GPC) of the cleaved PDMAEMA was conducted to determine the molar mass of the tethered polymer. The polymerization was stopped by opening the flask to air when the targeted monomer conversion was reached. The particles were washed with acetone and collected after centrifugation at 4000 rpm for 30 min. This procedure was repeated at least five times, yielding 103 mg PDMAEMA-modified magnetite particles. Cleavage of PDMAEMA Brushes. A 37% HCl (aq) solution (0.5 mL) was added to 2 mL of a solution of the particles in DMF (∼ 10 mg/mL), and the reaction was allowed to stir at room temperature overnight. An ammonium hydroxide solution (28.0-30.0% NH3 basis) was added very slowly to the mixture in ice bath until the pH value was >8. The cleaved polymer was extracted using chloroform and analyzed by GPC. Quaternization of PDMAEMA Brushes. About 200 mg PDMAEMA grafted magnetite nanopartices were added to a mixture of isopropanol (5 mL) and ethyl bromide (5 mL). The quaternization reaction was carried out at room temperature over a 24 h period. The particles were collected by centrifugation at 4000 rpm for 30 min and then washed with deionized water. This purification procedure was repeated at least four times. Determination of the Amount of Quaternary Amines on Magnetite Nanoparticles. The density of quaternary ammonium groups on magnetite nanoparticles was measured as the amount of fluoresceinbound per gram of particles.17 We placed 1 mg of PQA-modified 1306
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Scheme 2. Synthesis of Antibacterial Magnetite Nanoparticles
magnetite particles in a 15 mL test tube containing 5 mL of a 1 wt % solution of fluorescein (sodium salt) in distilled water for 10 min. Then, the tube was placed in a magnetic separation stand at room temperature for 10 min. The nanoparticles were attracted to the wall of the tube via magnetic force. The fluorescein supernatant was decanted, and the particles were extensively washed with distilled water and collected via magnetic force to remove unattached fluorescein molecules. The particles were then mixed with 3 mL of 0.1% solution of cetyltrimethylammonium chloride and shaken for 20 min at 300 rpm on an orbital shaker to desorb the dye. The absorbance of the resultant aqueous solution was measured at 501 nm after adding 10% v/v of 100 mM phosphate (pH 8.0). The amount of fluorescein bound to the PQA brush was calculated using a value of 77 mM-1 cm-1 as the extinction coefficient.17 The number of QA units on the brush was determined by using the fluorescein concentration assuming a 1:1 fluorescein to accessible QA ratio. The solvent accessible density of quaternary ammonium groups on magnetite nanoparticles was 4.8 1019/g particles. Antimicrobial Activity Determination. Antimicrobial testing was performed using a modified ASTM standard: E2149-01 Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions. A colony of E. coli K12 grown on a Luria agar (L-agar) plate was used to inoculate 5 mL of Luria broth in a sterile 50 mL conical tube. The culture was incubated at 37 °C while being shaken at 300 rpm (G24 Environmental Incubator Shaker, New Brunswick Scientific) for 18-20 h. The cells were diluted with Sorensen’s phosphate buffer (pH 6.8, 0.3 mM KH2PO4) to the desired concentration. The actual number of cells used for a given experiment was determined by standard serial dilution. PQA-modified magnetite nanoparticles (1.0 mg) were incubated with 1 mL of cell suspension (105 to 106 E. coli/mL) in a 10 mL conical tube (Falcon) at 37 °C and 300 rpm. Bare magnetite nanoparticles were used as a control. After 50 min of incubation, the tube was placed in a magnetic separation stand at room temperature for 10 min. The nanoparticles were attracted to the wall of the tube via magnetic force. The supernatant was removed from the tube, and 0.5 mL of the supernatant was diluted appropriately and plated on L-agar plates. Each viable bacterium developed into a bacterial colony that was counted. The recycled magnetite nanoparticles were incubated with a fresh sample of 1 mL of cell suspension in the same conical tube at 37 °C and 300 rpm for 50 min. The procedures described above were repeated to determine the antibacterial activity of the PQA-modified magnetite nanoparticles after repeated exposure to E. coli, employing the same procedures, six more times. In each cycle, the magnetic nanoparticles were collected only by exposure to the external magnetic field without washing with any solvents or water. The biocidal efficiency of PQA-modified magnetic nanoparticles was represented by the percentage of bacteria killed by those particles that
Figure 1. FT-IR spectra of magnetite nanoparticles (a) grafted with ATRP initiator BiBEP and (b) grafted with PDMAEMA.
Figure 2. GPC trace of cleaved PDMAEMA chains (Table 1, entry 3). was calculated based on eq 1 where Ncontrol and Nsample correspond to the colonies on the L-agar plates of the control and the sample, respectively, whereas Fcontrol and Fexperiment represent the dilution factor of the control and experiment, respectively. Calculation of the percentage of bacteria killed by PQA-modified magnetite nanoparticles biocidal efficiency ¼
Fcontrol Ncontrol - Fsample Nsample 100 ð1Þ Fcontrol Ncontrol
2.3. Analyses. Conversion of DMAEMA was determined by 1H NMR in CDCl3 on a Bruker AM 300 MHz spectrometer. Molecular weight and molecular weight distribution of PDMAEMA were measured by a GPC system consisting of a Waters 510 HPLC pump, three Waters Ultrastyragel columns (500, 103, and 105 Å), and a Waters 410 DRI detector, with dimethylformamide (DMF) as an eluent (50 °C, flow rate = 1.0 mL/min). Linear poly(methyl methacrylate) standards were used for calibration. Transmission electron microscopy (TEM) pictures were taken on a JEOL JEM-2000 EX II with acceleration voltage 200 kV on THF-cast samples. Elemental analysis (EA) was performed by Midwest Microlab. Thermal characterization of samples was carried out with the aid of Seiko thermogravimetric analysis (TGA)/differential thermal analysis 1307
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Figure 3. TEM images of (A) the initiator-modified and (B,C) PDMAEMA-grafted magnetite particles (Table 1, entry 3).
Figure 5. Images of PQA-modified nanoparticles (Table 1, entry 3) in water (2 mg/mL) (a) prior to and (b) after exposure to a permanent magnet for 10 min.
Figure 4. Stability of the suspensions of initiator, PDMAEMA, and PQA-modified nanoparticles in water and toluene (0.5 wt % of nanoparticles, Table 1, entry 3). 300 instruments (Seiko Instruments) operated at the heating rate of 20 °C/min under N2.
3. RESULTS AND DISCUSSION Synthesis of PDMAEMA Brushes from Magnetite Nanoparticles. The pathway applied to the synthesis of antibacterial
magnetite nanoparticles (Scheme 2) involved the formation of nanoparticles via coprecipitation of Fe2þ/Fe3þ in the presence of an alkaline solution, followed by immobilization of an ATRP initiator onto the surface of the nanoparticles, subsequent grafting from polymerization of DMAEMA via surface-initiated ATRP,49,50 and quaternization of the tethered PDMAEMA with ethyl bromide. Alkaline precipitation has been demonstrated to be a very effective approach for preparing magnetite nanoparticles.51 According to TEM images, the magnetite nanoparticles formed clusters in a range of sizes, from 20 to 100 nm because of the large surface of the nanoparticles. An ATRP initiator, 2-(2bromoisobutyryloxy)ethyl) phosphonic acid, (BiBEP), was then chemically attached to the surface of the magnetite nanoparticles via reaction between the phosphonic acid group and Fe-OH groups inherently present on the surface.52 Phosphonic acid
shows a strong affinity to the surfaces of the iron oxide particles through the formation of Fe-O-P- bonds, which are more stable than the carboxylic acid bonds.53 The presence of the initiator was verified by FT-IR spectroscopy and quantified through both EA and TGA. A characteristic CdO stretch vibration band (1750 cm-1) was clearly observed in the IR spectrum of the modified magnetite nanoparticles (Figure 1a), confirming the presence of the initiator. The amount of initiators present on the modified nanoparticles was 10 wt % based on EA or 12 wt % based on TGA (Table 1). Because of the size variation of the magnetite nanoparticles, it is difficult to estimate the density of initiators on the surface and hence the grafting density of the resulting polymer brushes. Magnetite nanoparticles with attached initiators were used for subsequent surface-initiated ATRP of DMAEMA with CuCl/ HMTETA as a catalyst. As shown in Table 1, a series of PDMAEMA-modified magnetite nanoparticles with varied polymer chain lengths were prepared. To determine the molecular weight of the tethered chains the magnetite core was etched with hydrochloric acid, and the cleaved polymer chains were characterized using GPC. The average molecular weights (Mn) of cleaved polymer chains varied from 10 700 to 152 100. The GPC trace of one sample of cleaved polymer chains (Table 1, entry 3) is shown in Figure 2. In the final step of the synthesis, the PDMAEMA brushes tethered to magnetite surfaces were converted to PQA through quaternization with bromoethane. Fluorescein staining of PQA-modified magnetite nanoparticles (Table 1, entry 3) showed that the solvent-accessible density of quaternary ammonium groups on magnetite nanoparticles was 4.8 1019/g particles. 1308
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Biomacromolecules Scheme 3. Schematic Illustration of Recycling PQA-Modified Magnetite Nanoparticles for Antibacterial Application
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Table 2. Antimicrobial Activity of PQA-Modified Magnetic Nanoparticlesa number of survived E. coli/mL PQA-modified controlb
PQA-modified
magnetite no. 1c magnetite no. 2c
antimicrobial efficiency
1st cycle 2.5 105
0
0
100%
2nd cycle 2.7 105 3rd cycle 3.6 105
0 0
0 0
100% 100%
4th cycle 4.0 105
0
0
100%
5th cycle 6.9 105
0
0
100%
6th cycle 6.1 105
0
0
100%
7th cycle 2.5 105
0
0
100%
8th cycle 2.8 105
0
0
100%
a
Concentration of PQA-modified magnetite particles = 1 mg/mL. b Control: bare magnetite particles (1 mg/mL). c PQA-modified magnetite nos. 1 and 2 are identical (Table 1, entry 3).
The presence of polymer on the surface of the nanoparticles was verified through FT-IR spectroscopy and quantified by TGA. In comparison with the spectrum of the initiator-immobilized magnetite nanoparticle (Figure 1a), a much stronger CdO stretch vibration band at 1750 cm-1 was observed after polymerization (Figure 1b). The increase in the intensity of the absorption band corresponded to the presence of grafted PDMAEMA brushes. More quantitative characterization results were obtained from both TGA and EA. On the basis of TGA, the weight loss of the initiator-immobilized magnetite nanoparticle was 10 wt %, whereas the weight loss of the PDMAEMA-modified magnetite nanoparticles was significantly higher and depended on the molecular weight of the PDMAEMA brushes. The weight loss increased from 55 to 93 wt % when Mn of the grafted PDMAEMA increased from 10 700 to 152 100. The TGA results were in good agreement with EA results. TEM was used to characterize the size distribution of the PDMAEMA-modified magnetite nanoparticles. The size of the particles was in a range of 50 to 150 nm due to the aggregation of magnetite cores before and during the polymerization. Upon close inspection, the polymer layer (light gray area) was observed on the coated magnetite core (dark black area) with average thickness around 20 nm. Properties of Modified Magnetite Nanoparticles. The particles described in Table 1, entry 3 were selected for their narrow size range and consistent coating thickness (Figure 3B,C) and for their ready dispersibility in water and toluene and were used for the functional testing. Initiator, PDMAEMA, and PQAmodified nanoparticles were each mixed with water or toluene to examine their dispersibility and the stability of the dispersion (Figure 4). PDMAEMA-modified nanoparticles were well-dispersed in toluene and H2O, whereas PQA-modified particles only dispersed well in water because of the hydrophilicity of the quaternized chains. The aqueous suspensions of PQA- and PDMAEMA-modified magnetite nanoparticles as well as the toluene suspension of PDMAEMA-modified particles with up to 2 wt % of the nanoparticles remained stable for several months without sedimentation. In contrast, the initiator-modified magnetite nanoparticles precipitated shortly after being dispersed in either solvent. The presence of tethered polymer layers promoted the stability of the magnetite nanoparticles in solvents due to steric effects or electrostatic repulsion of the polymer chains.
PQA-modified magnetite nanoparticles exhibited a typical response to the presence of a permanent magnet. After exposure to an external magnetic field for 20 min, the majority of the nanoparticles were attracted to the source of the magnetic field. However, there was a small portion of magnetite nanoparticles remaining in the supernatant, which could be due to the low mass fraction of magnetite in these hybrid particles. To recycle efficiently the nanoparticles in the antibacterial tests where a short response time (
