Article pubs.acs.org/Langmuir
Magnetic Surfactants and Polymers with Gadolinium Counterions for Protein Separations Paul Brown,† Lev Bromberg,† M. Isabel Rial-Hermida,‡ Matthew Wasbrough,# T. Alan Hatton,*,† and Carmen Alvarez-Lorenzo*,‡ †
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidade de Santiago de Compostela, 15782-Santiago de Compostela, Spain # NIST Centre for Neutron Research, National Institute of Standards and Technology, 100 Bureau Drive, MS 6100, Gaithersburg, Maryland 20899-6100, United States ‡
S Supporting Information *
ABSTRACT: New magnetic surfactants, (cationic hexadecyltrimethlyammonium bromotrichlorogadolinate (CTAG), decyltrimethylammonium bromotrichlorogadolinate (DTAG), and a magnetic polymer (poly(3-acrylamidopropyl)trimethylammonium tetrachlorogadolinate (APTAG)) have been synthesized by the simple mixing of the corresponding surfactants and polymer with gadolinium metal ions. A magnetic anionic surfactant, gadolinium tri(1,4-bis(2-ethylhexoxy)-1,4dioxobutane-2-sulfonate) (Gd(AOT)3), was synthesized via metathesis. Both routes enable facile preparation of magnetically responsive magnetic polymers and surfactants without the need to rely on nanocomposites or organic frameworks with polyradicals. Electrical conductivity, surface tensiometry, SQUID magnetometry, and smallangle neutron scattering (SANS) demonstrate surface activity and selfaggregation behavior of the magnetic surfactants similar to their magnetically inert parent analogues but with added magnetic properties. The binding of the magnetic surfactants to proteins enables efficient separations under low-strength (0.33 T) magnetic fields in a new, nanoparticle-free approach to magnetophoretic protein separations and extractions. Importantly, the toxicity of the magnetic surfactants and polymers is, in some cases, lower than that of their halide analogues.
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INTRODUCTION Surfactants play a key role in biotechnology by facilitating separation, isolation, manipulation, and solubilization of membrane proteins and peptides, lowering nonspecific adsorption of biomaterials to surfaces, and inducing protein denaturation for electrophoretic analysis.1 Careful selection of surfactants is vital for control of surfactant-protein binding, encapsulation, and release. With this in mind, many groups are developing novel surfactants that have improved biocompatibility2 and are responsive to external stimuli,3 or that are also ionic liquids and may aid in extractions.4 Recently, surfactants responsive to a magnetic field have been developed that offer unique physicochemical properties5,6 and allow for novel approaches to protein adsorption, separation, and purification. Surfactants that contain multivalent d- or f-block metal ions either as a counterion or as an integral part of the surfactant’s polar headgroup have been reported.7 Their aqueous phase behavior is similar to that of conventional surfactants, and selfassembly provides a simple means of preparing well-defined aggregates of metal complexes.8 In particular, reversed micelles have been employed to control the photophysical properties of © 2016 American Chemical Society
lanthanide ions in solution, which have found application as luminescent probes in the investigation of metal-binding sites in biological materials.9 Metallolipids have proven suitable for magnetic resonance imaging.9 Surprisingly, until recently, experiments related to magnetic surfactants had largely been limited to spin-orientation and relaxation studies for application as paramagnetic contrast agents.10 This was due to the assumption that the metal centers remained isolated and lacked long-range interactions to be magnetically active.11 However, it has now been demonstrated that a magnetic response of magnetic surfactants is indeed observable, leading to surfactants that can be controlled by the switching of an external magnetic field “on” and “off”.5,6 As such, the magnetic surfactants have allowed magnetic micro- and nanoemulsions to be generated with potential applications already demonstrated ranging from environmental cleanup to targeted drug delivery.12 Cationic magnetic surfactants (similar to those Received: November 15, 2015 Revised: December 27, 2015 Published: January 3, 2016 699
DOI: 10.1021/acs.langmuir.5b04146 Langmuir 2016, 32, 699−705
Article
Langmuir presented in this paper) have also been shown to complex with DNA, and with proteins such as myoglobin (low-spin diamagnetic) and green fluorescent protein (GFP), without significant alteration of the DNA or protein tertiary structure.13,14 It is assumed that the change from nonmagnetic halide to magnetic chlorometalate anions does not modify the mechanism of binding of magnetic surfactants and polymers to proteins. Now, however, the protein−surfactant complexes have been rendered magnetic and can be manipulated and concentrated in a low strength magnetic field (gradient ∼36 mT mm−2) even at μM levels of surfactant.13 It has been suggested that the response arises from a combination of undissociated surfactant binding to the surface and dissociated surfactants that retain their counterion in a diffuse layer near the protein−surfactant interface, and that by movement of the counterions in the magnetic field the protein−surfactant complex must be “dragged” with them osmotically.15 A monolayer of magnetic surfactant may lead to superparamagnetic behavior of the complex due to a lack of bulk anisotropy and therefore an enhanced magnetic response16 or even the formation of ferromagnetic domains.17 These studies suggested potential use of the magnetic surfactants in separations and purification.13 Adsorptive, extractive, and chromatographic processes have proved to be the most powerful separation procedures in terms of both selectivity and recovery.18−20 However, standard column systems are usually performed in packed beds that cannot handle samples containing large amounts of particulate material such as cell debris, prohibiting use in the early stages of the purification process. The removal of cell disintegrate and nucleic acids often involves aqueous two-phase partitioning systems in combination with partial protein purification.21 Magnetic separation processes can be advantageous in clarified fermentation broths and other cellfree media where high throughput and larger scales are required.22 Magnetic separation occurs when magnetic carriers with a certain binding affinity toward target compounds are dispersed in a fluid that flows through magnetic fields. Once the target is captured by the magnetic carrier, the resulting complex can be separated from the fluid by means of a magnet. To date, all magnetic field-based approaches toward protein separation have relied on using dispersions of magnetic nanoparticles.22,23 However, nanoparticles are bioactive, and the protein−particle surface interactions induce conformational changes in the adsorbed protein molecules, which in turn, may influence cellular uptake and inflammation as well as accumulation, degradation, and clearance of the nanoparticles.24−26 The synthesis of ultrafine particles can be challenging, and interactions between particle surfaces and the associated biomolecules can often disrupt the native conformation and subsequent functioning of the biomolecules.27 The advantages of employing magnetic surfactants over nanoparticles include their facile fabrication (one-step synthesis), fast and effective binding, which can be readily tuned via alteration of the surfactant aliphatic groups and their counterions, and their good dispersibility and stability in solutions.28 The present work introduces three new gadolinium-based magnetosurfactants (cationic and anionic) and a structurally similar cationic homopolymer, poly(3-acrylamidopropyl)trimethylammonium chloride (APTAC, Figure 1), as a new family of magnetic nanocarriers with the potential for aiding protein separation and purification. Biocompatibility of the
Figure 1. Surfactants and polymer studied. (a) CTAG; (b) poly(APTAC); (c) DTAG; (d) AOT.
magnetic surfactants and polymers, an important consideration in biotechnology applications, is also addressed.
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EXPERIMENTAL SECTION
Materials and Methods. Decyltrimethylammonium bromide (DTAB, ≥ 98%), hexadecyltrimethylammonium bromide (CTAB, ≥ 99%), (3-acrylamidopropyl)trimethylammonium chloride (APTAC) solution (75 wt % in H2O), gadolinium chloride hexahydrate (99.9%), ethyl-α-bromoisobutrate (98%), copper(I) bromide (≥99%), 2,2′bipyridine (bipy, ≥ 99%), N,N,N′,N′-tetramethylethylenediamine (TEMED), ammonium persulfate (PSA), sodium dodecyl sulfate (SDS, 99%), sodium bis(2-ethylhexyl) sulfosuccinate (AOT, 99%), EDTA (99.5% tetrasodium salt hydrate), and phosphate buffer saline 10x pH = 7.4 (PBS) were all purchased from Sigma-Aldrich Chemical Co. and used without further purification. Cell Proliferation Kit I (MTT) was purchased from Roche, Germany. Heat-inactivated fetal bovine Serum (FBS) and penicillin/streptomycin were from Thermo Scientific (UK); acrylamide from NZYTech (Portugal); and fertilized broiler chicken eggs from Avirojo (Spain). Synthesis of Polymer Precursor. APTAC polymerization was carried out via atom transfer radical polymerization (ATRP). In brief, 8.3 g (75 wt % in H2O) of APTAC (30.0 mmol, 30 equiv) and 0.147 mL of ethyl-α-bromoisobutyrate (0.195g, 1.0 mmol) were solubilized in 6.2 mL of a water:methanol (1:3 v/v) mixture. After deoxygenation of the solution via five freeze−thaw cycles (using N2), Cu(I)Br catalyst (0.143 g, 1.0 mmol, 1 equiv) and 2,2′-bipyridine (0.390g, 2.5 mmol, 2.5 equiv) were added against nitrogen purge, at which point the reaction mixture turned dark brown and extremely viscous, indicating the onset of polymerization. After 15 min, an additional 50 mL of degassed methanol was added to the flask, and the solution was stirred for 24 h. 1H NMR (300 MHz, methanol-d4 23 °C, Supporting Information, Figure S1) indicated that polymerization was complete (disappearance of vinyl signals in the δ 5.6−6.3 ppm range). The final solution was filtered through a silica column to remove the spent ATRP catalyst and dried under vacuum to remove the solvent. The resulting polymer was readily soluble in water, but was too viscous to pass through an aqueous column. Conversion of the polymer chloride salt to a [FeCl4]− complex by addition of 1 equiv of FeCl3 enabled polymer characterization in dimethylformamide (DMF). 1H NMR spectra of the polymer solution in D2O did not reveal the presence of residual unreacted monomer. Polymer characterization by size exclusion chromatography (SEC, Supporting Information Figure S2) was carried out using an Agilent 1260 Infinity HPLC system equipped with an isocratic pump, a refractive index detector and PLgel 5 μm MIXED-D Columns. DMF containing 0.1 mol % LiBr was used as an eluent at 50 °C and at a nominal flow rate of 1 mL/min. Linear polystyrene standards were used for calibration. PolyAPTAC weightaverage molecular weight and polydispersity were estimated to be 1.9 kDa and 1.02, respectively. Synthesis of Cationic Magnetic Surfactants and Polymer. Cationic gadolinium-based compounds were synthesized according to the literature methods.13 In brief, 1 equiv of GdCl3.6H2O was added to a methanolic solution of 1 equiv of either DTAB, CTAB, or 700
DOI: 10.1021/acs.langmuir.5b04146 Langmuir 2016, 32, 699−705
Article
Langmuir
°C, 5% CO2, and 90% relative humidity (RH). Surfactants were tested in triplicate at 1, 0.1, 0.01, 0.001 and 0.0001% in Milli-Q water, and 100 μL of these solutions were added to each well. Negative controls were carried out by processing the cells under the same conditions with the addition of 100 μL Milli-Q water. The plates were incubated for 24 and 48 h and the cell proliferation was quantified following Cell Proliferation Kit I (MTT) instructions. HET-CAM Assay. ICCVAM-recommended hen’s test-chorioallantoic membrane test (HET-CAM) method protocol was followed.32 Fertilized broiler chicken eggs were incubated at 37 °C and 60% RH and periodically rotated during 8 days to avoid sticking of the embryo to one side of the shell. The upper part of the eggshell was removed with a rotatory saw (Dremel 300, Breda, The Netherlands), the inner membrane was wetted with NaCl 0.9%, and the eggs were placed again inside the incubator for 30 min. Then, the inner membrane was removed in order to expose the chorioallantoic membrane. One hundred microliters of 0.1% surfactant in PBS solution was placed in contact with the membrane, and hemorrhage (H), lysis (L), and coagulation (C) were monitored over 5 min. Negative (NaCl, 0.9%) and positive (NaOH, 0.1 M) controls were tested analogously. Irritation scores (IS) were calculated as follows:
poly(APTAC), and the mixture was stirred overnight at room temperature before dehydration under vacuum at 80 °C overnight. Synthesis of Anionic Magnetic Surfactant. Gd(AOT)3 was prepared by liquid−liquid ion exchange. Gadolinium chloride was dissolved in an ethanol:H2O (75:25 v/v) mixture. Three molar equivalents of NaAOT were then added, and the solution was stirred for 5 h. Excess solvent was removed under reduced pressure followed by drying of the resultant metallosurfactant under vacuum at 80 °C for 2 days. The surfactant was then resolubilized in a minimum amount of dry dicholoromethane, and the insoluble salt was filtered off. The surfactant was separated by repeated centrifugation. The pure surfactant was then dried under vacuum at 80 °C for 2 days. Analyses showed no chlorine or sodium (
