Article pubs.acs.org/Biomac
Poly(carboxybetaine methacrylamide)-Modified Nanoparticles: A Model System for Studying the Effect of Chain Chemistry on Film Properties, Adsorbed Protein Conformation, and Clot Formation Kinetics Sinoj Abraham, Alan So, and Larry D. Unsworth* Chemical and Materials Engineering Department, University of Alberta, National Research Council (Canada), National Institute for Nanotechnology, Edmonton, Alberta, Canada ABSTRACT: Nonfouling polymer architectures are considered important to the successful implementation of many biomaterials. It is thought that how these polymers induce conformational changes in proteins upon adsorption may dictate the fate of the device being utilized. Herein, oxidized silicon nanoparticles (SiNP) were modified with various forms of poly(carboxybetaine methacrylamide) (PCBMA) for the express purpose of understanding how polymer chemistry affects film hydration, adsorbed protein conformation, and clot formation kinetics. To this end, carboxybetaine monomers differing in intercharge separating spacer groups were synthesized, and nitroxide-mediated free radical polymerization (NMP) was conducted using alkoxyamine initiators with hydrophobic (TEMPO) and hydrophilic (β-phosphonate) terminal groups. The physical properties (surface composition, thickness, grafting density, etc.) of the resulting polymer− SiNP conjugates were quantified using several techniques, including Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and thermogravimetric analysis (TGA). The effect of spacer group on the surface charge density was determined using zeta potential measurements. Three proteins, viz., lysozyme, bovine α-lactalbumin, and human serum albumin, were used to evaluate the effect film properties (charge, hydration, end-group) have on adsorbed protein conformation, as determined by circular dichroism (CD), fluorescence spectroscopy, and fluorescence quenching techniques. Hemocompatibility of these surfaces was observed by measuring clot formation kinetics using the plasma recalcification time assay. It was found that chain chemistry, as opposed to end-group chemistry, was a major determiner for water structure, adsorbed protein conformation, and clotting kinetics. It is thought that the systematic evaluation of how both chain (internal) and end-group (external) polymer properties affect film hydration, protein conformation, and clot formation will provide valuable insight that can be applied to all engineered surfaces for biomedical applications.
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research has been employed to investigate this effect, especially for colloidal metallic nanoparticles,22−25 as these may have significant application within a physiological environment and are able to be characterized using various spectroscopic techniques. Oxidized silicon nanoparticles (SiNPs) with surface-grafted zwitterionic poly(carboxybetaine methacrylamide)-based polymers (PCBMA), of differing internal and end-group chemistries, were used in our study to understand the effect of internal polymer properties as well as end-group chemistry on film hydration, adsorbed protein conformation, and the rate of clot formation. Specifically, zwitterionic carboxybetaine methacrylate, with varying spacer groups (1 and 5 −CH2− groups) and end-groups (hydrophilic β-phosphonate or hydrophobic 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO)), were synthesized via the quaternization reaction between N-[3-(dimethylamino)propyl]methacrylamide and
INTRODUCTION The therapeutic efficacy of implanted materials largely depends on the resulting host response. 1−3 Nonspecific protein adsorption plays an important role, as it is thought to be the impetus for the initiation of several deleterious host responses (complement activation, inflammation, thrombosis, etc.) that can ultimately cause implant rejection.4−15 For valid reasons, significant research has focused on the amounts of adsorbed protein as well as understanding the composition of adsorbed proteins from complex solutions (i.e., plasma) as a means of identifying the molecular markers that may lead to the initiation of various host responses.16−18 Recently, however, more attention has been given to the effects of material properties on adsorbed protein conformation. For example, it has been found that it is not the adsorbed amount of protein, nor the composition of the adsorbed protein layer only, but also the degree of protein denaturing that can initiate host responses, viz., the activation of platelets. 19 It has been generally recognized that protein molecules undergo conformational changes upon adsorption onto solid surfaces.20,21 Extensive © 2011 American Chemical Society
Received: June 9, 2011 Revised: August 2, 2011 Published: September 5, 2011 3567
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alkylbromo esters.26 NMP techniques were employed for surface-grafted polymerization of these CBMA monomers on the SiNPs. Secondary structures of adsorbed proteins were determined using circular dichroism (CD), whereas fluorescence spectroscopy techniques were used to further determine the extent of denaturing. Clotting kinetics was evaluated using a turbidimetric assay for Ca2+ reconstituted human plasma, in the absence and presence of nanoparticles (i.e., platelet-free technique). It was observed that both internal and end-group properties of the polymer play a role in the film hydration, adsorbed conformation of proteins, and the kinetics of clot formation. Polymer-modified surfaces have been touted for their ability to inhibit nonspecific protein adsorption.3 However, the role that different polymer properties have on protein adsorption is only now being elucidated. Polymer properties like chain density, chain length, extent of hydration, interaction potential with the modified surface, end-group chemistry, etc., are considered to be important parameters for controlling the nonspecific adsorption of protein.16−18,27 In particular, polymer properties at high chain density have been discussed at length, 27 where it has been suggested that the thin polymer film endgroup chemistry and the internal hydration are important to inhibiting protein adsorption.28 Zwitterionic compounds, such as phosphobetaine (phosphorylcholine), sulfobetaine, and carboxybetaine polymers, have garnered considerable interest as nonfouling polymers.29−32 It has also been demonstrated that carboxybetaine polymers inhibit protein adsorption and platelet adhesionsurface-initiated polymerization being achieved via atom transfer radical polymerization (ATRP) strategies.33,34 It has been suggested that the major factor in the ability of these polymers to inhibit protein adsorption is due to their propensity to form strong hydration layers by ionic solvation, in addition to hydrogen bonding.29−32 It is known that the physicochemical properties of zwitterionic carboxybetaine polymers are also highly influenced by the spacer group between its positive quaternary amine and negative carboxyl groups.35−37 However, the systematic evaluation of the effect these properties have on protein adsorption events has yet to be conducted. Although several polymer modification strategies and chemistries have been developed, surface-initiated polymerization regimens allow for particularly precise control over the resultant polymer properties (molecular weight, polydispersity, etc.).38−40 Recently, several methods were reported for grafting polymers to surfaces, and controlled/“living” free radical polymerizations (CRP) presents many advantages over other techniques: self-assembly or traditional polymerization methods.41−46 Nitroxide-mediated polymerization (NMP) is a stable CRP technique that allows surface grafting of polymer chains, while possessing some significant advantages over other techniques.47−49 For example, unlike commonly employed ATRP,33,34,50,51 neither cytotoxic organometallic catalysts nor bromide chain ends are required for polymerization using NMP techniques. Alkoxyamines are the starting compounds for NMP and function by generating equal amounts of initiating radical and propagating nitroxide upon dissociation.52−55 Therefore, surface-initiated NMP can be performed by designing the alkoxyamine initiator with both a tethering group, for polymer growth on specific surfaces, and chain ends of various physicochemical properties. Herein, we discuss a facile strategy for the surface-initiated synthesis of PCBMA by employing the NMP technique, using
initiators with a recently reported β-phosphonate and a common TEMPO moiety at the chain end. This single step polymerization protocol yields a zwitterionic polymer coating along with an assemblage of β-phosphonate groupsa result usually achieved through complex organic reactions. The effect of spacer length and end-group chemistry on surface properties (hydration and charge) was determined. Interaction of water molecules on the polymeric surface was investigated, and the amount of nonfreezing bounded water was quantified. Moreover, the conformational changes of adsorbed lysozyme, bovine α-lactalbumin, and human serum albumin onto SiNPs grafted with PCBMA of different spacer lengths (1 and 5 −CH2− groups) and end-groups (phosphonate or TEMPO) were investigated. Clot formation kinetics, in the presence of modified SiNPs, was also evaluated using a turbidimetric assay. This PCBMA system provided a systematic experimental system for elucidating the effect that fundamental polymer properties have upon film hydration, protein adsorption, conformation, and final plasma compatibility.
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EXPERIMENTAL SECTION
Materials and Methods. Employing a quaternization reaction between N-[3-(dimethylamino)propyl]methacrylamide and methyl bromoacetate or ethyl 6-bromohexanoate, according to our previous report,26 yielded carboxybetaine methacrylamide monomers (CBMA) with intercharge separation groups of one (CBMA-1) and five (CBMA-5) CH2 units, respectively. All initiators and monomers used were prepared using previously published techniques and are briefly outlined below.26 All chemicals were purchased from Sigma-Aldrich and used as received without further purification, unless specifically stated herein. Solvents used were purified by standard purification methods prior to use. All chemical synthesis procedures were carried out under pure nitrogen using standard Schlenk techniques. Chicken egg white lysozyme (lyophilized powder, 14 kDa, pI = 11, protein ≥90%), bovine α-lactalbumin (calcium depleted, type II, 14 kDa, pI = 4.3, protein ≥85%), human serum albumin (lyophilized powder, 66 kDa, pI = 4.7, protein ≥96%), and oxidized silicon nanoparticles (10−20 nm avgerage particle size, 99.8% trace metal basis) were purchased from Sigma-Aldrich (St. Louis, MO). Disodium phosphate and potassium phosphate, used to prepare phosphate buffer, were also purchased from Sigma-Aldrich. Additionally, acrylamide, sodium thiosulfate, and sodium chloride were purchased from Sigma-Aldrich and used for quenching experiments. All dilutions and buffers were prepared with syringe filtered (22 μm) Milli-Q distilled deionized water. Finally, human plasma was obtained from Canadian Blood Research Services (Vancouver, British Columbia, Canada), where all appropriate ethical procedures were strictly adhered to for the collection, release, and use of blood products. Ethics approval for this work was approved by two national bodies (Canadian Blood Services, National Research Council of Canada) and the University of Alberta ethics board. Moreover, all donors were considered to be free of medications or other drugs, and pooled blood was screened (as per standard protocols) by Canadian Blood Services prior to distribution for research. NMR spectroscopy (Varian UNITY 500 NMR, Varian, Inc., Walnut Creek, CA) was used to characterize initiators, monomers, and resulting polymers. The polymer molecular weight (Mn) and polydispersity index (I) were determined using gel permeation chromatography (GPC, Agilent G1311A quaternary pump, G1362A refractive index detector), where a PL-gel (5 μm) mixed-D type column was employed (DMF eluent, 1 mL/min). A 0.4% (w/w) polymer−DMF solution was prepared; 10 μL was used for each analysis. Polystyrene standards (Polysciences) were used for calibration purposes. Dynamic light scattering studies were conducted using the Malvern Zetasizer (Nano ZS) spectrometer, where the mean SiNPs particle size, before and after surface polymerization, was
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determined using a 633 He−Ne laser at a scattering angle of 90°. This same equipment was employed to measure zeta potential values. Thermogravimetric analysis (TGA Q50, TA Instruments, New Castle, DE) was conducted using the ASTM D3850-94 standard. Briefly, under dry nitrogen samples were heated from 25 to 700 °C at a constant heating rate of 10 °C/min. Infrared spectra was measured using a Varian FTS-7000 (Digilab) FTIR spectrometer. Elemental analysis was facilitated using a Costech 4010 elemental analyzer equipped with a multidetector. Modulated differential scanning calorimetery (MDSC, Q1000-TA Instruments) was used to characterize the surface hydration and state of waters within the polymer films formed on the SiNPs. Samples were prepared in sealed aluminum pans, and DSC curves were obtained at a scanning rate of 3 °C/min from −60 to 200 °C. Circular dichroism (Jasco J-810 CD/ORD) was conducted and analysis completed using Jasco Spectrum Analysis software. Fluorescence spectra of adsorbed proteins were recorded with a Varian Cary fluorescence spectrophotometer, using a square quartz cuvette of 1 cm path length. Blood clot formation was analyzed using a BioTek ELx808 plate reader. Synthesis of Alkoxyamine Initiator. Commercially purchased TEMPO radical (1.2 mmol) and brominated trimethoxysilane (BrTMOS, 1 mmol) were reacted in anhydrous toluene (15 mL) in the presence of CuBr (1 mmol) and pentamethyldiethylenetriamine (PMDETA) (1 mmol) with 78% yield. The reaction and work-up conditions are similar to that of the synthesis of β-phosphonato alkoxyamine.10 1H NMR (300 MHz, CHCl3 298 K, δ): 7.84 (1H, NH), 4.45 (1H, CH), 3.48 (9H CH3), 3.22 (2H, CH2), 1.64 (2H, CH2), 1.35 (3H CH3), 1.17 (12H, CH3), 0.56 (2H, CH2) ppm. 13C NMR (600 MHz, CHCl3 298 K, δ): 174.34, 78.56, 52.23, 50.40, 42.06, 37.84, 26.16, 24.58, 17.77, 6.81 ppm. Anal. Calcd for C18H38N2O5Si: C, 55.35; H, 9.81; N, 7. Found: C,55.24; H, 9.56; N, 7.2. Surface Modification of SiNPs. Fused particles, with an average diameter of 16 ± 5 nm (Sigma-Aldrich), were dried overnight at 120 °C under vacuum. Alkoxyamine initiator containing triethoxysilyl functional group (0.5 g, 1.5 mmol) dissolved in toluene was added to a suspension of SiNPs (1 g) in toluene (20 mL). The mixture was stirred for 24 h at 60 °C. The modified SiNPs were isolated by centrifugation (18 000 rpm, 15 min), resuspended in fresh toluene, and centrifuged; this was repeated three times to ensure complete removal of unreacted alkoxyamine. The grafted SiNPs were dried in a vacuum oven at 60 °C for 6 h, and characterized using 29Si NMR. The polymerization of carboxybetaine monomers was conducted via initiator grafted SiNPs. In a typical experiment, monomer (initiator:monomer = 1:50), the alkoxyamine grafted SiNPs (0.4 g), DMF (10 mL), and a known amount of free initiator corresponding to a total monomer to total initiator ratio of 50 (total initiator = surface grafted + free initiator) were introduced in a dried Schlenk flask under nitrogen. As it was previously determined that the homolysis of the C−O bond and, thus, the generation of a nitroxide free radical occurred at ∼110 °C for these initiators, all polymerization reactions were conducted at this temperature.26 Thus, the polymerization mixture was then heated to 110 °C for 5 h. The solution-free polymer was isolated from the grafted SiNPs by eight successive centrifugation/ redispersion cycles in distilled water. After each cycle, the solvent was collected and replaced by fresh water. The SiNP grafted and fresh polymer (precipitated in diethyl ether) were collected separately and dried overnight under vacuum at 60 °C. Molecular weights of the polymer chain were determined using GPC and NMR spectroscopy. Protein Adsorption. A 10 mM phosphate buffer (PB, pH = 7) was prepared and used for all single protein adsorption experiments. Protein−SiNP samples were prepared with final concentrations of 475 μg/mL nanoparticles and 50 μg/mL of protein, which corresponded to a 10:1 protein to particle ratio: being previously reported as a ratio that tends toward the adsorption of a monolayer of lysozyme adsorbed onto SiNPs.56 Given that α-lactalbumin is similar to lysozyme, in shape and size, a similar ratio was employed. However, HSA adsorption involved increasing the nanoparticle concentration to 1.02 mg/mL, so as to provide 1:1 protein to nanoparticle ratio, as discussed elsewhere.25,57,58 Control samples of protein and unmodified SiNPs were prepared with the same final concentrations.
Single protein concentrations were verified by measuring the protein solution absorbance (Agilent 8453 UV−vis spectrophotometer, λabs = 280 nm) and the respective molar extinction coefficients of 36 000, 28 175, and 36 600 L M−1 cm−1 for lysozyme, α-lactalbumin, and HSA. All samples were incubated for 3 h at 37 °C with shaking every 30 min. Changes in protein conformation upon adsorption to virgin and modified SiNPs were characterized using CD techniques, solution temperature being 37 °C and a cuvette path length of 2 mm. Measurements were taken at standard sensitivity (100 mdeg), data pitch of 0.5 nm, and bandwidth of 1 nm, each spectrum representing an average of 10 readings. Baseline correction was accomplished using control samples containing no protein. Native protein spectra were obtained using protein solutions that were prepared for SiNPs adsorption experiments. Instrument ellipticity measurements were converted into mean residue ellipticity using standard equations. 57−61 CD spectra were analyzed using the CDSSTR program via the Dichroweb server, utilizing the best fitting reference sets for the specific spectra.59−61 It is important to note that, opposed to the fluorescence studies outlined below, the CD experiments were conducted looking at the change in conformation of protein upon introduction of the SiNPs to a protein solution. Although samples were also measured using CD on nanoparticles spun down and resuspended after protein adsorption, the CD spectra could not be properly analyzed as the resulting protein concentration could not be accurately determined; that said, similar trends were observed. Tryptophan fluorescence intensity was determined by monitoring the fluorescence over a wavelength range of 300−450 nm, using an excitation wavelength of 295 nm.62,63 All samples were centrifuged (VWR microcentrifuge) at 13 000 rpm for 15 min, and the supernatant was extracted. The remaining SiNPs were washed and resuspended in 10 mM PB in order to ensure that fluorescence signal was only from adsorbed protein. All intensity readings were baseline corrected with corresponding solutions of all components, excluding protein, smoothed, and then normalized. Fluorescence quenching experiments followed previously described protocols, using acrylamide as a collision based quencher. 64 Briefly, a 4 M stock solution of quencher was prepared by dissolving appropriate amounts of solid acrylamide in 10 mM PB. An incremental volume of stock quencher solution was added to the resuspended SiNP−protein samples to vary the quencher concentrations from 0.0 to 0.7 M. The intensity spectrum was first baseline subtracted and then corrected for dilution. Intensities were also corrected for the absorbance of acrylamide at 295 nm using a molar extinction coefficient at 295 nm of 0.23 and the following equation:63 (1) Peak intensities were read directly from the corrected emission spectra and analyzed using a modified Stern−Volmer (Lehr) plot. 62−66 Plasma Clot Analysis. Clot formation was assessed using previously described protocols, viz., the plasma recalcification turbidimetric assay.77 Normal human plasma (100 μL) was incubated with SiNPs in a 96-well microtiter plate. After 5 min of equilibration period, an equal volume of 0.025 M CaCl2 was injected into the wells. Absorbance at 450 nm was measured (BioTek ELx808 plate reader) at 1 min intervals over a 60 min period. All experiments were repeated five times.
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RESULTS AND DISCUSSION SiNP Surface Modification. Alkoxyamines were synthesized via the addition reaction between BrTMOS and β-phosphonylated nitroxide radical,26 or commercially available 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) free radical, as discussed elsewhere.48 This initiator design allowed for its tethering to SiNPs via the trimethoxysilane end-group, while affording for direct control over the end-group chemistry. The latter being possible because the NMP reaction results in monomer insertion below the initiator end-group, thus carrying the initiator end-group to the end of the formed polymer chain. 3569
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In order to evaluate the coupling of the initiator to the SiNPs, both 31P and 29Si NMR were conducted. Briefly, fused SiNPs were incubated in an anhydrous toluene solution of alkoxyamine for 3 h at 50 °C. Free alkoxyamine initiators were removed from the system using a toluene-centrifugation step, as discussed above. The expected 1H−31P split and doublet− doublet peaks obtained using 31P NMR were observed at 24.32 and 27.12 ppm, confirming the presence of β-phosphonate (results not shown). Furthermore, the grafting of both alkoxyamine initiators onto SiNPs was confirmed using 29Si NMR spectroscopy, where the framework Si atoms [Si(4OSi)] and surface silanol groups [Si(3OSi, 1OH)] peaks were centered at −108 and −100 ppm, respectively (results not shown). In addition, the presence of a broad peak at −55 ppm, corresponding to grafted Si atoms [Si(2OSi, 1R, 1OR], was observed, suggesting that both initiators were attached to the SiNPs through multiple siloxane bonds.39 Similar to previous work,26 CBMA monomers were prepared with two different spacer lengths (1 and 5 CH2) separating the positively charged quaternary amine and the negatively charged carboxyl group. Initiator bound SiNPs were then used for polymerizing CBMA monomers using NMP techniques. In order to compare the polymer molecular weight in solution to the polymer formed on the surface of the SiNPs, free initiator was added to the reaction solution. Polymer molecular weight (Mn) was calculated using the NMR peak for the β-phosphonate CH3 group (1.37 ppm) and TEMPO CH3 group (1.18 ppm) as reference with respect to the CH2 polymer backbone proton (1.76 ppm). GPC analysis was also used to determine the polymer molecular weight (Table 1).
data show that the polymerization on the surface of the SiNPs was not diffusion limited. Moreover, dynamic light scattering (DLS) results (Table 2) show that, upon reaction with the initiators, the SiNP hydrodynamic diameter increased by ∼7 and ∼5 nm for β-phosphonate and TEMPO initiators, respectively, supporting the NMR results suggesting that the initiator was successfully coupled to the SiNPs. Finally, thermogravimetric analysis also showed a weight loss of ∼13%, which corresponded to macromolecular decomposition within the temperature ranges of 60−700 °C. Despite any difference in weight loss as a function of initiator type, these data also suggest the presence of a bound initiator at the SiNP surface. Surface Grafting. SiNP hydrodynamic diameters increased substantially upon polymerization with both CBMA-5 and CBMA-1 monomers. Specifically, PCBMA-1-modified SiNPs showed particle diameter increases of ∼63 and ∼51 nm, compared to initiator-grafted SiNPs, for β-phosphonate and TEMPO initiators, respectively. Also, PCBMA-5-modified SiNPs showed particle diameter increases of ∼66 and ∼52 nm, compared to initiator-grafted SiNPs, for βphosphonate and TEMPO initiators, respectively. The hydrodynamic diameter seemed to be related to the end-group. For both PCBMA-1 and -5, there was a decrease of ∼16 nm between the β-phosphonate and TEMPO capped polymer systems. This may suggest that, despite similarities in amount of polymer present per area, TEMPO polymer modified SiNPs have a smaller diameter. It may be that the rather hydrophobic TEMPO end-group is burying itself within the polymer layer, leading to an overall decrease in the polymer film thickness. In particular, a moderate difference of only ∼0.1 μmol/m2 in polymer graft density between PCBMA 5 β-phosphonate and TEMPO end-groups lead to a hydrodynamic radius difference of ∼17 nm. Whereas, a difference of ∼0.2 μmol/m2 in polymer graft density between PCBMA-5 and -1 β-phosphonateterminated systems lead to a hydrodynamic radius difference of only ∼3 nm. Similarly, PCBMA-1 systems with β-phosphonate and TEMPO end-groups, with only a difference of ∼0.08 μmol/m2 in polymer graft density, exhibited a decrease in hydrodynamic radius of ∼15 nm, having similar Mn values. These data suggest that the formation of a polymer shell over the NPs occurs upon polymerization (Scheme 1). The surface graft density (eq 2) of the alkoxyamine initiator was determined using quantitative thermogravimetric analysis (TGA):67
Table 1. Molecular Weights and Polydispersity (PDI) of Solution Free and Surface Bonded PCBMA Polymers, Obtained after 5 h of Polymerization silica grafted polymer sample PCBMA5 PCBMA1 PCBMA5 PCBMA1
(Phospho) (Phospho) (TEMPO) (TEMPO)
free polymer in solution
Mn(GPC) Mw/Mn Mn(GPC) Mn(NMR) Mw/Mn 5450 4800 5700 4300
1.15 1.28 1.07 1.13
5050 4500 5800 4600
5200 4550 5900 4600
1.18 1.30 1.11 1.16
yielda (%) 70 65 74 69
a
Yield percentage of polymerization was calculated from the initial monomer concentration and pure polymer obtained together as grafted and free polymer.
It was observed that the free polymer molecules formed in solution have similar molecular weights, as well as molecular weight distributions, to those grafted to SiNPs (Table 1). These
(2)
Table 2. Thermogravimetric and DLS Analysis of Alkoxyamine and PCBMA-Modified SiNPs a thermogravimetric analysisb sample phosphonate initiator TEMPO initiator SiNP-PCBMA5 (Phospho) SiNP-PCBMA1 (Phospho) SiNP-PCBMA5 (TEMPO) SiNP-PCBMA1 (TEMPO)
weight loss (%) 13.3 12.9 54.4 44.1 44.4 36.8
± ± ± ± ± ±
0.1 0.2 0.2 0.2 0.1 0.1
grafting density (μmol/m2) 2.482 2.337 0.927 0.738 0.827 0.656
± ± ± ± ± ±
0.006 0.007 0.003 0.002 0.007 0.004
grafting yield (%) 10.60 9.7 43.3 45.9 44.7 49.2
± ± ± ± ± ±
0.04 0.1 0.8 0.4 0.6 0.2
hydrodynamic diam (±fwhm) (nm) 23 21 89 86 72 71
± ± ± ± ± ±
3 3 10 9 9 10
a
In general, the data represent average ±1 SD (n = 3), except for the hydrodynamic diameter where the values represent the peak maximum ± the full width at half maximum (fwhm) of the distribution; this was repeated thrice to confirm reproducibility. bThe values for initiators are with respect to the amount of alkoxyamine initiator taken for functionalization, and those for the polymers are with respect to the amount of initiators grafted on the SiNPs. 3570
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Scheme 1. Illustration of SiNPs Grafted with Poly(carboxybetaine methacrylic acid) and the Two Possible End-Groups
where the weight loss (Wloss) corresponds to polymer decomposition that occurs between 60 and 700 °C, the molecular weight (M) being for either the grafted initiator or polymer (Table 1), specific surface area for the SiNPs (Sspec, m2 g−1), and the weight loss (WSiNPs) of the substrate determined before grafting are all used to determine the surface graft density. The grafting yield percentage was calculated using the graft density (eq 3). This value represents the amount of initiator molecules at the interface of the SiNPs:
chains after subtracting the contribution of adsorbed water and bonded initiator. The surface graft density and grafting yield of the polymerization were calculated with respect to the amount of initially grafted initiator on the SiNPs (being [I] in this case). The highest grafting density of 0.927 μmol/m2 was observed for the β-phosphonate-terminated PCBMA-5. At this polymer grafting density, it is evident that only 43 mol % of the surface bonded initiators were involved in the polymerization. This is not unusual, and the limited use of surface bonded initiators is usually attributed to the steric hindrance imposed as initial monomer insertion into growing chains may impede the growth of near neighboring initiator molecules.68 Moreover, the chain density seems to be similar based upon monomer type and relatively insensitive to end-group chemistry. Polymer Chemistry Effect on Zwitterion and Surface Charge. Zwitterionic PCBMA contains carboxylic acid moieties, which facilitates protonation and deprotonation as a function of pH. This ability largely affects the overall charge density of surfaces modified with these zwitterionic moieties. Polymer-grafted SiNPs were immersed into acidic and basic pH buffer solutions and analyzed using FTIR to evaluate the influence of pH on polymer chains. pH-induced protonation and deprotonation of the carboxylic acid moiety were evidenced from the change in carbonyl group stretching peaks in FTIR. At low and high pH conditions, protonation and deprotonation of carboxyl groups occur, and the corresponding CO stretching peak of COOH is visible at 1735 cm−1 and COO− at 1670 cm−1.
(3)
where [I] is the initial concentration of initiator added to the reaction vessel, being the mole of functional alkoxyamine molecules introduced per square meter of SiNPs surface (Table 2). Thus, the grafting yield of the initiators represents the amount of the total initiators that bound to the SiNPs. It was observed that the SiNPs were modified using ∼10% of the total initiators added to the reaction solution. The amount of tethered initiators was observed to be similar at 2.48 and 2.34 μmol/m2 for the β-phosphonate and TEMPO end-groups, respectivelythe TEMPO density being less, perhaps due to the presence of the large piperidine ring sterically hindering the addition of more initiators to the interface. Regardless, this tethered amount is similar to an expected monolayer of ∼2.8 μmol/m2, as estimated using the initiator dimensions. TGA was also used to evaluate the polymer graft density, using the weight loss and molar mass of the grafted polymer 3571
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The C−O corresponding to these two states are also visible at 1310 and 1270 cm−1, respectively. Although similar for all zwitterionic polymers studied, Figure 1 is a representative
Figure 2. Zeta potential of PCBMA-modified SiNPs as a function of solution pH. Isoelectric point of SiNP-PCBMA5 and SiNP-PCBMA1 was determined to be 8.7 and 8.5, respectively. Values represent mean ±1 SD, n > 3; errors bars are present but smaller than point size. Note that lines are to guide the eye only and do not represent modeling results. Figure 1. Representative FTIR spectra of SiNP-PCBMA5 at a pH of 2, 7, and 9. IR absorption peaks of CO of COOH and COO− are visible at 1735 and 1670 cm−1; similarly, C−O peaks are visible at 1310 and 1270 cm−1. This is due to the ionization of carboxylic acid to carboxylate group at high pH values.
adsorbed proteins, the states of water in these hydrated polymeric films were characterized. The amounts of different types of water, viz., total water, free water, freezing bound water, and nonfreezing bound water,70−72 were estimated from the thermogram profile (Figure 3). Using MDSC, a
spectrum of SiNP-PCBMA5 synthesized using the β-phosphonate initiator. Zwitterionic materials contain opposite charges within molecular distance and have permanent dipole moments, which have been shown to depend on intercharge distance.35,54 Thus, due to protonation−deprotonation that can be induced upon carboxylic acid moieties, PCBMA could be overall anionic, neutral, or cationic at specific pH conditions. Zeta potential values, as a function of solution pH, were determined for all systems (Figure 2). It was apparent that the pI for these systems was similar at ∼8.7. For pH < 7 the surface becomes positively charged and the zeta potential decreased with increasing solution pH; the net negative charge at high pH being due to the dissociation of carboxylic acid groups. Among the two polymer systems, PCBMA-5 exhibited the largest variation in surface charge as a function of pH. This might be expected, as PCBMA-5 has the largest intercharge separation distance with highest pKa value of 4.37. It has been reported that the pKa decreases with decreasing spacer lengths and for PCBMA-1 is 2.03.69 Therefore, the anionic headgroup of both PCBMA systems remained deprotonated at the experimental pH conditions, but the chances for electrostatic coupling increases for the systems with smaller spacer groups that separate the cations from anions. Because of this charge separation, the dipole moment increases (6.15 Å in aqueous media), which may affect the overall polymer hydrophilicity. Polymer Chemistry Effect on Water States. In order to better understand how polymer chemistry might affect the resulting film properties and, ultimately, the conformation of
Figure 3. Representative MDSC thermograms of pure water and hydrated SiNP-PCBMA samples. The heating rate is 2.5 °C/min modulated at every 60 s. Experiments were repeated twice for each sample and absorption peaks observed at −48 °C for SiNP-PCBMAs are associated with the cold crystallization of water. 3572
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nonfreezing bound water content is the difference between the actual water content and the freezing water content (Table 3).
characteristic exothermic peak was found for these PCBMAmodified SiNPs at around −48 °Ca peak that becomes evident only in the presence of PCBMA. This peak has, previously, been attributed to the cold crystallization of waters present in the hydrated polymer film,73,74 whereas the relatively large endothermic peak in the range of −8 to 2 °C has been associated with the melting of the ice from free water and freezing bound water. Cold crystallization occurs in systems where nonfreezing and freezing bound waters are present. 71,75 As opposed to crystallization occurring as the system is cooled, cold crystallization of water is the phase transition of ice from amorphous to crystalline. In this case, the amorphous structure has been shown to be related to the freezing bound water in the hydrated polymer.73,74 A broadening of the cold crystallization peak was observed to occur with PCBMA-1 compared to PCBMA-5 systems. It is thought that this was a result of the difference in charge separating spacer group that may lead to a change in the water environment; this change in charge separating spacer group leading to either the reduced flexibility of the formed polymer with increasing side chain length of the PCBMA-5 and/or the change in dipole moment that exists between the quaternary amine and the carboxylic acid moieties of the zwitterion. The actual water content (Wc, %) of the samples was calculated using
Table 3. MDSC-Based Characterization of the Hydrated SiNPs and Calculated Amount of Different Types of Waters in the Polymer Matrixa water content (%) sample SiNP-PCBMA5 (Phospho) SiNP-PCBMA1 (Phospho) SiNP-PCBMA5 (TEMPO) SiNP-PCBMA1 (TEMPO) a
actual Wc
freezing bound Wfb
nonfreezing bound Wnf
unbounded free Wf
34.2 ± 0.5
12.2 ± 0.4
7.08 ± 0.7
15 ± 1
30.7 ± 0.7
8.5 ± 0.4
10.5 ± 0.5
11.6 ± 0.7
32.8 ± 0.7
9.1 ± 0.1
8±1
15 ± 1
28.9 ± 0.4
6.8 ± 0.3
7±2
14 ± 1
The values represent an average ±1 SD, n = 3.
Polymeric systems formed with CBMA-5 can accommodate more water than PCBMA-1 irrespective of their end-groups. This may be attributed to the influence of the electrostatic forces between the positive quaternary ammonium salt and negative carboxylate of the monomers. The delocalization of the charges can be reduced by the large spacer groups in PCBMA-5, which may increase it affinity toward water. Similarly, polymers with hydrophilic β-phosphonate end-groups had more water content that the TEMPO-terminated systems. The influence of end-groups is largely observed in the amount of the bound water (freezing and nonfreezing) and seemed to have little influence upon the unbounded free water present in these systems. The freezing bound water content in these SiNP-PCBMA systems was also substantially influenced by the space between the opposite charges within the zwitterions (Table 3). Polymer Chemistry Effects on Adsorbed Protein Conformation (CD Investigation). CD techniques were employed for analyzing protein secondary structures, providing a unique characteristic spectrum that can be used to identify relative concentrations of α-helices, β-sheets and turns, and unordered structures.62 The CD spectrum for a protein is the representation of the combination of all secondary structures comprising the final protein conformation and can be used to evaluate changes in secondary structures as proteins undergo conformational changes upon adsorption, as judged by comparisons with reference spectra. The CD spectra of all samples were recorded, and the change in ellipticity, compared to the native state, due to adsorption on the SiNPs was monitored. CD spectra were analyzed using the CDSSTR program to determine the secondary structural fractions by utilizing the best fitting references. CDSSTR reported protein α-helix and β-sheet conformations were classified with two components (1 and 2) that correspond to the regular and distorted fractions, by considering the certain number of distorted terminal residues in a given α-helix or β-sheet segment, which was directly obtained from CDSSTR analysis. 61 Secondary structure changes upon adsorption of lysozyme (lys), α-lactalbumin (α-la), and human serum albumin (HSA) to PCBMA modified SiNPs were evaluated using CD techniques. Lys and α-la were employed in this study as they have a similar size and shape but differ in net surface charge. These differences should allow for further understanding of how charge differences may influence adsorption as well as final
(4)
where Wi is the initial mass of the sample after soaked in water overnight and Ws is the mass of dried sample at 80 °C in vacuum until weight become constant. The actual water content on the hydrated SiNPs can be expressed with the following relationship: (5)
where Wnf (%) is the nonfreezing water content, Wfb (%) is the freezing bound water content, and Wf (%) is free water content present in the samples. Using Wc and MDSC obtained enthalpies (ΔHc = cold crystallization of Wfb and ΔHm = fusion of Wfb + Wf), the amount of these waters (nonfreezing, freezing bound, and free water) may be quantified. Water states were determined on the basis of (1) the crystallizing water is composed of only freezing bound water (ΔHc for Wfb) and ΔHm is the sum of the fusion enthalpies of the ices derived from the free and the freezing bound water in the polymer (Wfb + Wf) and (2) the fusion enthalpies of the ice is the fusion enthalpy of ice from bulk pure water, which ranges between 311 and 334 J/g due to different polymorphic forms of ice.72 The experimental enthalpy of the fusion of water and cold crystallization was determined to be within this range at 318 J/g. The free water melting peak and the freezing bound water melting peak are fused and appeared as one broad melting endothermic peak, centered at ∼0 °C. Generally hydrophilic polymers exhibit a separate melting peak for freezing bound water at a temperature lower than 0 °C.71 The polymers with β-phosphonate endgroup exhibit broad and slightly separated thermograms in this region, showing their affinity toward water compared to TEMPO-terminated polymers that exhibit narrow and sharp peaksboth regardless of monomer type. However, while calculating the enthalpy of fusion, this was considered to be a single peak. The melting enthalpy of this peak was less than that of the pure water added to the sample, indicating that some of the bound water does not freeze. Therefore, the 3573
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protein conformation. However, lys and α-la differ in that lys has a large number of disulfide bonds that may inhibit unfolding. HSA is used in this study for multiple reasons. HSA makes up a large fraction of the total proteins found in human plasma57 and is considered to readily undergo conformational changes. HSA also has a similar charge, but drastically different molecular weight, as that of α-la. This should provide insight into the effect of protein size on surface-induced conformational rearrangement. Finally, recent studies have shown that HSA can directly affect platelet activity through the revealing of occult epitopes exposed upon adsorption.19 In comparison with the CD spectra of native lys (Figure 4), it is evident that bare SiNPs induced the most profound variation
Figure 5. Lysozyme secondary structure changes upon interaction with unmodified and PCBMA-modified SiNPs. Data shown represent average ±1 SD, n = 3. *, p < 0.05 (one way ANOVA); **, p < 0.001 (one way ANOVA).
lys. However, these changes in secondary structure due to endgroup effects are generally not as profound as the effect of chain chemistry. CD spectra for the α-la adsorbed onto the modified SiNPs illustrate an opposite trend to that of lys (Figures 6 and 7). Bare
Figure 4. Representative circular dichroism spectra of native lys (a) and lys adsorbed to unmodified (b) and modified SiNPs (c−f). The measurements reflect the collection of 10 scans, repeated thrice, and the changes in the ellipticity while interacting with SiNPs were compared with the native state.
in both shape and intensity of the CD spectrum. Given the difference in charge between lys and the SiNPs in this situation, it may be that this change in secondary structure may be furthered by an electrostatically driven interaction between lys and unmodified SiNPs. Quantified changes in secondary structure for lys adsorption to bare SiNPs are presented in Figure 5. It is evident that helical content decreased from 19 to 3% (p < 0.001, one way ANOVA), whereas β-sheet content increased from 12 to 29% (p < 0.001, one way ANOVA) and unordered structures decreases from 28 to 21% (p < 0.001, one way ANOVA) upon adsorption of lys to unmodified SiNPs. Lys incubated with PCBMA1-modified SiNPs showed statistically significant differences in helical and β-sheet content compared to the native state. However, in general, PCBMA5-modified SiNPs showed minimal changes in secondary structure. This may be due to the greater zeta-potential of the larger spacer group polymer, contributing greater charge repulsion between the polymer and protein that impedes surface-induced spreading of the adsorbed proteins. On average the samples with TEMPO end-groups seem to show comparatively more adsorption-induced changes in adsorbed protein conformation (compared to β-phosphonate-terminated polymers), suggesting that hydrophobic end-group plays a role in denaturing adsorbed
Figure 6. Representative circular dichroism spectra of native αlactalbumin (a) and α-lactalbumin adsorbed to unmodified (b) and modified SiNPs (c−f). The measurements reflect the collection of 10 scans, repeated thrice, and the changes in the ellipticity while interacting with SiNPs were compared with the native state.
SiNPs and α-la are both negatively charged at the pH of 7; hence, it is probable that due to electrostatic repulsion, α-la adsorption yielded only minor changes in secondary structure that were statistically significant. Conversely, the CD spectra of α-la incubated with PCBMA-SiNPs showed striking changes from the native spectrum. It may be that conformational 3574
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Figure 8. Representative circular dichroism spectra of native human serum albumin (HSA) (a) and HSA adsorbed to unmodified (b) and modified SiNPs (c−f). The measurements reflect the collection of 10 scans, repeated thrice, and the changes in the ellipticity while interacting with SiNPs were compared with the native state.
Figure 7. α-Lactalbumin secondary structure changes upon interaction with unmodified and PCBMA-modified SiNPs. Data shown represent average ±1 SD, n = 3. *, p < 0.05 (one way ANOVA); **, p < 0.001 (one way ANOVA).
changes upon adsorption were facilitated by the positive charge of modified polymer samples, leading to an electrostatic attraction toward negatively charged α-la. In comparing spacer lengths, the opposite trend to lys was evident; the single spacer group samples showed less adsorption-induced conformational changes than the longer spacer group. Moreover, the β-phosphonate end-group samples showed greater decreases in ellipticity as compared to TEMPO samples. α-la incubated with unmodified SiNPs show a minimal change in secondary structure compared to that of native protein. But a dramatic change in helical content and in sheet conformation was observed for α-la incubated with modified SiNPs. Several statistically significant relationships were observed in the CDSSTR analysis of molar ellipticity obtained for adsorbed α-la on modified SiNPs, based on one way ANOVA statistical analysis compared with native protein structure. There is dramatic decrease in the α-helix content (p < 0.001) and an increase in the β-sheet content (p < 0.001) (Figure 7). Adsorption and adsorption-induced denaturing of α-la are highly influenced by the end-group chemistry of the surfacegrafted PCBMA. The conformational elements of α-la on PCBMA-1 (Phospho) show prominent variation from the native structure (α-helix decreased by ∼17%, β-sheet increased by ∼17%, p < 0.001), but PCBMA-1 (TEMPO) shows relatively less variation (α-helix decreased by ∼12%, β-sheet increased by ∼7%, p < 0.001). However, α-la adsorbed on PCBMA-5 surfaces undergoes a comparable degree of denaturing to that observed with PCBMA-1 system, indicating that the influence of spacer group and end-groups affects the adsorption of these small negatively charged proteins. The CD spectra of native and adsorbed HSA are illustrated in Figure 8, whereas the quantitive analysis of secondary structure is summarized in Figure 9. HSA shows a minor interaction with bare SINPs, but this interaction is significant compared to that observed with α-la with bare SINPs. Even though both α-la and HSA are negatively charged, HSA has a higher pI value of 4.7. However, this is in contrast to the
Figure 9. HSA secondary structure changes upon interaction with unmodified and PCBMA modified SiNPs. Data shown represent average ±1 SD, n = 3. *, p < 0.05 (one way ANOVA); **, p < 0.001 (one way ANOVA).
apparent change in conformation upon introduction to PCBMmodified surfaces. HSA adsorption trends were very similar to that of α-la, which may be expected based upon their charge similarity. CDSSTR analysis shows that native HSA is primarily helical. Generally for all surface-modified systems, α-helical content of HSA decreased to ∼25% (p < 0.001, one way ANOVA), β-sheet content increased to ∼20% (p < 0.001), and unordered structures increased to ∼5% (p > 0.05) upon adsorption. Unlike α-la adsorption to these surfaces, HSA conformational changes seem to be highly dependent on the effect of spacer group length and not end-group chemistry (see Figure 9). This decreased dependence on end-group in 3575
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A considerable degree of adsorption-induced conformational change was expected for α-la based on the CD results. Native α-la exhibited an emission peak maximum at ∼335 nm. In comparing peak maxima, it is evident that a red shift occurs upon adsorption to PCBMA samples, being most prominent for SiNP-PCBMA5 with TEMPO end-groups (∼338 nm), perhaps indicating that the protein matrix changed such that tryptophan residues are in a more solvated local environment the effect of monomer being considered to be greater than the end-group in altering this protein conformation, from this measurement. The 3D structure of α-la has previously been reported, and it is known that the four tryptophan residues are generally located in the internals of the protein, away from solvent.66 Hence, a red shift would most likely indicate a loosening/unfolding of the protein induced by the adsorption onto various SiNPs. HSA contains a single tryptophan that is located in the physiologically important ligand binding site at amino acid position 214.77,78 Native HSA has an emission peak maximum at ∼336 nm. Upon adsorption of HSA to PCBMA-modified SiNPs, a noticeable shift was observed toward shorter wavelength region (∼328 nm) for both TEMPO-terminated polymers, whereas β-phosphonate-terminated polymers yielded a blue shift to ∼331 nm. HSA generally exhibit a blue shift upon adsorption, which indicates a conformational change that promotes the π−π* excitation.79 Secondary structures of HSA seemed to undergo a greater change for β-phosphonate vs TEMPO end-groups, as indicated from CD results. However, tertiary structural changes seemed slightly less when HSA adsorbed to surfaces with β-phosphonate end-groups than on the TEMPO surfaces. The influence of end-groups toward the adsorption-induced conformational changes of HSA would seem to be the main cause for the difference in λmax. That said, although all adsorbed systems are evidently different than the native state, differences of
