Adsorbed Polyzwitterion Copolymer Layers Designed for Protein

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Adsorbed Polyzwitterion Copolymer Layers Designed for Protein Repellency and Interfacial Retention S. Kalasin, R. A. Letteri, T. Emrick, and M. M. Santore* Department of Polymer Science and Engineering, University of Massachusetts, 120 Governors Drive, Amherst, Massachusetts 01003, United States S Supporting Information *

ABSTRACT: Poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC), when end-tethered to surfaces by the adsorption of copolymeric cationic segments, forms adsorbed layers that substantially reduce protein adsorption. This study examined variations in the molecular architecture of copolymers containing cationic poly(trimethylammonium ethyl methacrylate (pTMAEMA) anchor blocks that adsorbed strongly to negative surfaces. With appropriate copolymer design, the pTMAEMA blocks were shielded, by pMPC tethers, from solution-phase proteins. The most protein-resistant copolymer layers, eliminating fibrinogen and lysozyme adsorption within detectible limits of 0.01 mg/m2, had metrics (the amount of pMPC at the surface and the reduced tether footprint) consistent with the formation of an interfacial polymer brush. The p(TMAEMA-b-MPC) copolymer layers substantially outperformed the protein resistance of surface-polymerized pMPC layers when compared on a per-polyzwitterion-mass basis or on the basis of the scaled tether area. Additionally, p(TMAEMA-b-MPC) copolymer layers offered advantages over the much-studied cationically anchored poly(ethylene glycol) (PEG) graft copolymer system, which forms PEG brushes by the adsorption of a poly L-lysine (PLL) backbone. Although the optimized p(TMAEMA-b-MPC) and PLL-PEG copolymers were similarly fibrinogen-resistant, the cationic protein lysozyme was repelled by pMPC but adhered to the PEG brush via PEG−lysozyme attractions. Additionally, the adsorbed p(TMAEMA-b-MPC) copolymers were not displaced by poly L-lysine homopolymers, which completely displaced the PLL-PEG copolymer to expose a protein-adhesive surface. Thus, the p(TMAEMA-b-MPC) copolymer system comprises a scalable means to produce protein-repellent surfaces, free of the complexities of surface-initiated polymerization and with the advantages of polyzwitterions.

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substrate, meeting antifouling1,6,13−15 and lubricity targets.16−19 The term “brush” is often applied to the topology of endtethered chains but is rigorous only when high grafting densities and good solvent conditions substantially stretch chains normal to the interface.20,21 Others use the term brush more loosely, when the parabolic segment density profile is not established.22−24 End-tethered PEG layers can be produced by the adsorption of copolymers and surfactants that employ hydrophobic25 or cationic anchoring blocks,26,27 whereas the surface-initiated polymerization of PEGylated acrylates generates end-tethered layers with excellent protein resistance.28−30 Catechol-terminated PEG, covalently attached to TiO2, approaches the protein

INTRODUCTION

The demand for materials with aqueous lubricity and negligible cell and protein adhesion is a major driver in coatings research, with poly(ethylene glycol) (PEG) and polyzwitterions of great longstanding interest. Water-swollen coatings of PEG or polyzwitterions repel cells and biomolecules, and their strong performance is attributed to their neutrality, hydrophilicity, substantial hydration, and a relative lack of hydrogen bonding with biomolecules.4−7 Although PEG- and polyzwitterion-containing hydrogels often form the basis of thick (micrometer-scale) coatings,8−12 added cross-linkers and other components, chemically different from the PEG or polyzwitterion chains, can attract biomolecules. Avoiding the complexities from cross-linking chemical groups, nanometer-thick end-tethered layers of PEG or polyzwitterion chains, if properly designed, can shield approaching biomolecules from attractions to an underlying © XXXX American Chemical Society

Received: September 27, 2017 Revised: October 31, 2017

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protein adsorption by 75−85%.41 Though this reduction still enabled protein adsorption levels shown in other studies to facilitate nonspecific adhesion43 and possible bacterial adhesion,44 the results were sufficiently encouraging to motivate the current study that varied the copolymer architecture to produce interfaces with much greater protein resistance and to rival and exceed the performance of the PEG-containing interfaces. In this study, we compared four p(TMAEMA-b-MPC) copolymers with varied polycation-to-polyzwitterion ratios and overall molecular weights. These compositional variations translated to differences in adsorbed layer features, the ability to prevent protein adsorption, and resistance to displacement by cationic challengers. Fibrinogen was employed as a test protein because it is often used as a benchmark for blood serum36 and adsorbing more aggressively than albumin45 or serum proteins.15,46 Lysozyme was selected as an additional protein challenger because, unlike fibrinogen and most serum proteins, it carries a net positive charge.36 Indeed, PEG layers and brushes having excellent fibrinogen repellence can adhere lysozyme3,47 as a result of the weak attractions between the protein and PEG.48 We demonstrate vanishingly low adsorption of fibrinogen and lysozyme on the best of the p(TMAEMA-b-MPC) layers and explore how architectural variations influence the adsorbed layer and, in turn, protein adsorption. We additionally benchmark our adsorbed pMPC layers against surfacepolymerized pMPC brushes in the literature. This revealed that the adsorbed p(TMAEMA-b-MPC) layers have lower protein adsorption than the chemically attached pMPC when compared in terms of specific surface metrics. We also compared the robustness and protein resistance of the p(TMAEMA-b-MPC) layers to PEO brushes based on wellstudied PLL-PEG copolymers.15,46 A greater resistance was observed for the p(TMAEMA-b-MPC) layers to challenge and displacement by a range of polycations, including pTMAEMA homopolymer challengers. Adsorbed copolymer brushes are typically susceptible to displacement by a homopolymer having the same chemistry as the anchor block of the copolymer.3 The p(TMAEMA-b-MPC) layers reported here therefore comprise easily handled coatings that can be applied in a scalable fashion in a relatively rapid adsorption step to achieve nearly perfect protein resistance and superb retention against solution-phase molecular challengers, making them attractive alternatives to PEG-based coatings.

repellence of adsorbed PLL (poly L-lysine)-PEG copolymers but is not susceptible to the displacement that can compromise adsorbed PLL-PEG layers.14,28,31 Some polyzwitterions offer a potential advantage over PEG because of their insensitivity to ions,5,32−35 motivating their use in end-tethered layers. Here the state of the art is dominated by surface-initiated polymerization (grafting-from) of polyzwitterions such as poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC).1,2,36 The resulting protein-resistant layer can be displaced only if the substrate is compromised; however, the surface polymerization is not currently scalable. The simpler approach of adsorption, or grafting-to, is novel, with a few reports of adsorbed polyzwitterion-containing copolymers reducing but not eliminating protein adhesion.37,38 Between the two strategies of grafting-from and grafting-to lies the innovative approach of a particular surface-initiated polymerization, with polyzwitterion chains grown from a polycationic macroinitiator that has been preadsorbed electrostatically on an oppositely charged surface. This method produced endtethered pMPC layers of high aqueous lubricity, substantially reducing sliding friction at loads exceeding those in physiological systems.39 We describe the protein-repellant character of adsorbed p(TMAEMA-b-MPC) copolymers, the grafting-to approach, containing cationic poly(trimethylammonium ethyl methacrylate)(pTMAEMA)anchoring blocks. The cationic pTMAEMA blocks promote physisorption on negatively charged surfaces and, with appropriate molecular architecture, may produce pMPC end-tethered layers or brushes, as shown schematically in Figure 1A. Our choice of a cationic anchoring

Figure 1. Schematic of (A) a polymer brush showing blobs in the semidilute brush model, (B) adsorbed copolymers in the mushroom regime, (C) a copolymer brush with high-molecular-weight protruding anchor blocks, and (D) the possible mechanism of limited pTMAEMA incorporation into the p(TMAEMA-b-MPC) copolymer brush. In the latter, the homopolymer polycation is compositionally identical to the anchor block but is highlighted for clarity.

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EXPERIMENTS

Diblock Copolymer Synthesis. Diblock copolymers composed of poly(trimethylammonium ethyl methacrylate) cationic blocks and polyMPC zwitterionic blocks were synthesized by reversible additionfragmentation chain transfer (RAFT) polymerization of MPC from a poly(TMAEMA) macromolecular chain transfer agent (macroCTA), as shown in Figure 2.49 RAFT polymerization of 1 was conducted in water with a 5:1 ratio of CTA 2 to radical initiator 4-azobisvaleric acid (ACVA, 3). Cationic macroCTAs 4a−c were synthesized with average degrees of polymerization of 20, 40, and 100 by varying [M]/[CTA] from 25 to 150 and limiting the reaction time to 2−4 h to ensure that functional chain ends were retained. The macroCTAs were isolated by precipitation into acetone and then used as starting materials for the polymerization of MPC 5 in water, targeting [MPC]/[TMAEMA] ratios of 2, 6, and 8. The polymerization of MPC proceeded to >90% conversion, and dialysis in water followed by lyophilization afforded cationic−zwitterionic diblock copolymers 6a−d. The compositions and molecular weights of the macroCTAs and corresponding diblock copolymers were characterized by 1H NMR spectroscopy and size exclusion chromatography (SEC) with elution in trifluoroethanol, and

block is motivated by the ubiquitous negative charge on surfaces and the potential for hydrophobic anchoring blocks (useful for hydrophobic surfaces) to micellize in solution, complicating the formation of a uniform adsorbed brush.40 Nearly monodisperse pMPC-containing diblock copolymers, similar to the polycation-pMPC copolymers synthesized here via reversible addition-fragmentation chain transfer (RAFT) polymerization, were first synthesized by atom-transfer radical polymerization (ATRP).41 The particular class of p(TMAEMAb-MPC) copolymers in this article was previously synthesized, also by ATRP.42 A pMPC copolymer with a different acrylic anchoring polycation was shown, in spin-cast films, to reduce B

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Figure 2. (a) Synthesis of pTMAEMA-b-MPC. (b) SEC trace of cationic macroCTA 4a and diblock 6b, eluting in trifluoroethanol with 0.02 M sodium trifluoroacetate. (c) 1H NMR spectrum of diblock 6a in D2O.

Table 1. Polymerization Data for the Cationic MacroCTAs and Zwitterion-Containing Diblock Copolymers polymera

[M]/[CTA]

conv (%a)

Mn,theo (g/mol)b

Mn,NMR (g/mol)a

Mn (g/mol)c

Đc

polyTMAEMA20, 4a polyTMAEMA40, 4b polyTMAEMA100, 4c polyTMAEMA20-b-MPC105, 6a polyTMAEMA20-b-MPC134, 6b polyTMAEMA40-b-MPC308, 6c polyTMAEMA100-b-MPC177, 6d

25 100 150 120 160 320 200

60 46 50 93 97 93 >99

3100 9600 15 600 37 100 50 000 93 300 79 800

4200 8300 20 700 35 200 43 800 99 200 73 100

15 500 17 500 35 800 54 000 71 500 124 800 71 500

1.05 1.11 1.05 1.08 1.07 1.10 1.12

a Determined by 1H NMR spectroscopy in D2O. bMn,theo = ([M]/[CTA])(conv/100) × repeat unit molecular weight including Cl counterions, rounded to the nearest 100 g/mol. cEstimated relative to poly(methyl methacrylate) standards by SEC elution in TFE with 0.02 M sodium trifluoroacetate.

the results are given in Table 1. NMR spectroscopy showed the compositions of the block copolymers to correlate well with monomer feed ratios, and SEC analysis confirmed the successful chain extension of the macroCTA to afford well-defined diblock copolymers with polydispersity indices of less than 1.15 (Figures 2 and S4). A zwitterionic p(MPC) homopolymer was synthesized as described previously for use as a control sample.50 Cationic polymers pTMAEMA20, pTMAEMA40, and pTMAEMA100 in addition to their use as macroCTAs, were also used as control samples and as cationic challengers. Details are provided in the Supporting Information. Synthesis of PLL-PEG Copolymers. PLL-PEG graft copolymers were synthesized, as described previously,3,51 by attaching PEG sodium valeric acid (5000 molecular weight from Laysan Bio Inc.) to poly Llysine hydrobromide (PLL, nominal molecular weight of 20 000, Sigma Aldrich). The same PLL was employed, alone, as one of the cationic challengers. The reaction was carried out for 6 h in pH 9.1 sodium borate buffer. The products were dialyzed, for 24 h each time, against pH 7.4 phosphate buffer and then against DI water prior to characterization and freeze-drying. The ratio of PLL to PEG in the reaction mixture was adjusted so that the functionalization of the PLL amines was ∼35%, as determined by 1H NMR spectroscopy.3,51 Substrates. The silica surfaces of acid-etched microscope slides (Fisher Finest) were employed as the adsorption substrates. Soaking overnight in concentrated sulfuric acid, followed by rinsing with copious deionized water, produced silica surfaces, which were used immediately. Protein, Polymer, and Buffer Solutions. Studies were conducted in pH 7.4 phosphate buffer (0.002 M KH2PO4 and 0.008 M Na2HPO4), unless otherwise indicated. More dilute buffer was achieved by the addition of deionized water, and NaCl was added to increase the ionic strength. Protein and polymer solutions were prepared in buffers of interest. Bovine fibrinogen (F8630-1G, fraction

I, type 1S) and hen egg white lysozyme (L6876) were purchased from Sigma Aldrich and used as received. Adsorption and Challenge. Adsorbed polymer layers were produced and studied in a laminar slit flow chamber (1 mm × 10 mm × 50 mm) in which the microscope slide comprised one wall. After the chamber was assembled with the microscope slide, pH 7.4 phosphate buffer flowed continuously through the chamber and the chamber was aligned in the near-Brewster reflectometer to establish a baseline signal. Then, a solution of test copolymer or homopolymer control in the same buffer flowed over the surface as the reflectometry signal was recorded. Adsorbed layers were typically established within 10 min, after which the same buffer was reintroduced. Subsequently, various challenge studies were performed. These included immediate exposure to fibrinogen or lysozyme solutions and/or changes in ionic strength. After a pTMAEMA or PLL challenge was performed, buffer was reinjected and fibrinogen solution was introduced. This allowed the assessment of the impact of the challenger on the layer integrity, first by any immediate changes in adsorbed mass and second by changes in the resistance to fibrinogen. All studies employed a wall shear rate of 5 s−1. Near-Brewster Reflectometry. The interfacial mass was tracked in real time during adsorption and challenge studies using a custombuilt near-Brewster reflectometry instrument. Similar to ellipsometry, this method assesses interfacial mass through changes in the intensity of a parallel-polarized HeNe laser beam reflected inside the adsorption substrate under the Brewster condition (where the beam is completely transmitted rather than reflected.) The weak back-reflected beam allows for quantification of the interfacial mass, which is calculated from estimates of the layer thickness and refractive index. Although these two properties evolve and carry error, their product, the interfacial mass, is determined to much higher precision, which is also true of ellipsometry. A detailed description of this method is available in the literature.52 C

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from flowing 100 ppm pH 7.4 solution. After buffer rinses in which each was well-retained, flowing fibrinogen (100 ppm in pH 7.4 buffer) was introduced. The protein mostly failed to adsorb. Indeed, on the best-performing copolymer layer there was no measurable fibrinogen (less than 0.01 mg/m2), on the second surface there was 0.01 mg/m2, and on the third there was 0.2 mg/m2 adsorbed fibrinogen. Table 2 summarizes the full set of protein challenge studies for adsorbed layers of the four p(MPC-b-TMAEMA) copolymers, alongside the adsorbed layer properties and control studies. Except for the column for the calculation of blobs in the semidilute brush model,56 all entries were either directly measured or calculated directly from the observations. Calculations are detailed in the Supporting Information. All of the adsorbed p(TMAEMA-b-MPC) copolymers in Table 2 substantially reduced fibrinogen and lysozyme adsorption compared to that documented on silica54,55 and on the adsorbed pMPC or pTMAEMA homopolymers, which are the controls in Table 2. Moreover, with the best copolymer molecular architecture, p(TMAEMA20-b-MPC105), the adsorption of both fibrinogen and lysozyme was reduced to below a detectable limit of 0.01 mg/m2. Table 2 shows that copolymers producing higher pMPC content in the adsorbed layer more effectively repel proteins. Effect of Ionic Strength. Although the pMPC chain conformation is generally insensitive to ionic strength,5,32,33,35 ionic strength is potentially important when pMPC is tethered to a substrate electrostatically via polycationic anchors. It is therefore significant, in Figure 4, that the adsorbed copolymer layers are robust and remain fibrinogen-repellent over the range of ionic strengths at fixed pH 7. Figure 4 illustrates that only when the ionic strength is increased to above 0.25 M is the adsorbed copolymer removed slightly from the surface, but enough copolymer remains to prevent measurable fibrinogen adsorption. Grafting to and from: Adsorption versus Surface Polymerization. Protein-resistant pMPC brushes, polymerized from (grafting from) a substrate, have been well-studied and comprise an important benchmark for the current (grafting to) adsorbed copolymer pMPC brushes. Figure 5 compares the protein resistance of the current adsorbed copolymer layers to that of the surface-polymerized pMPC layers reported by Feng.1,2 In that work, the surface grafting density and degree of polymerization were systematically varied from 0.1 to 0.4 chains/nm2 and 5 to 200,

RESULTS Protein-Repellent pMPC Copolymer Coatings. The necessity of anchoring pMPC chains to achieve protein repellency is established in the reflectometry trace of Figure 3A. In this experiment, a modest homopolymer pMPC layer

Figure 3. (A) Adsorption of pMPC105, retention in flowing buffer, and its failure to prevent fibrinogen adsorption. (B) Adsorption of pMPCb-pTMAEMA copolymers on silica, followed by the reintroduction of phosphate buffer and then fibrinogen. A slight shift in the time axis between runs enables better viewing.

(0.41 mg/m2) adsorbed to a silica flat from flowing buffered pH 7.4 solution and was well retained in flowing buffer. The surface, however, subsequently adsorbed a substantial amount of fibrinogen (2.4 mg/m2 here compared to 4−6 mg/m2 on non-PEG surfaces),53−55 and the pMPC chains may have been displaced in the process. In contrast, in Figure 3B three different p(MPC-bTMAEMA) copolymers adsorbed in separate runs on silica

Table 2. Properties of Copolymer Layers and Homopolymer Layer Controls, Including the Protein Challenge formula

adsorbed amount, mg/m2 (pMPC, pTMAEMA)

fibrinogen adsorbed, mg/m2a

lysozyme adsorbed, mg/m2a

tether area, nm2

average distance between anchored pMPC chain ends, nm

normalized tether areac

blobs per tetherb

pTMAEMA20-b-MPC105 pTMAEMA20-b-MPC134 pTMAEMA40-b-MPC308 pTMAEMA100-b-MPC177 pMPC105 pTMAEMA20 pTMAEMA40 pTMAEMA100

1.25 (1.10, 0.15) 1.23 (1.11, 0.12) 1.1 (1.01, 0.09) 0.75 (0.54, 0.21) 0.41 0.30 0.36 0.52

0 0.01 0.2 2.7 2.4 6.0 6.1 6.7

0 0 0 0.1 0.15 2.0 2.2 1.9

46.7 59.1 150 172

6.8 7.7 12.2 12.6

4.4 4.3 3.5 2.0

5.6 5.9 6.4 3.4

a Fibrinogen and lysozyme adsorption from 100 ppm in 0.01 M pH 7.4 phosphate buffer. Protein exposure was at least 20 min; however, if the adsorption continued for longer times, we ran up to 45 min until the signal appeared to stabilize. bCalculated according to the semidilute brush model in the Supporting Information. cArea of pMPC tether normalized on the projected area for a free coil57 having the same pMPC molecular weight, with examples given in the Supporting Information.

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Figure 4. Amount of p(TMAMEA20-b-MPC105) retained on a silica flat after rinsing in buffer for 20 min at the ionic strength indicated (blue) and the amount of fibrinogen adsorbed after subsequent exposure to 100 ppm fibrinogen (red). All layers were adsorbed from a 26 mM solution with an initial coverage of 1.25 mg/m2. After being rinsed in 26 mM buffer, the test buffer was introduced, followed by the fibrinogen challenge at the test ionic strength. With no detectible evolution in the signal at these salt and fibrinogen exposure times, we chose not to run longer.

respectively. In the current copolymer adsorption study, the degree of tether polymerization (105−308 for pMPC) is set by copolymer chemistry, but the tether density is limited by the copolymer adsorption (producing 0.006−0.022 tethers/nm2). Notably, copolymer adsorption produces much lower grafting densities compared to surface polymerization. As a result of differences in tether molecular weight and surface density, a direct comparison of adsorbed vs surface-polymerized layers in terms tether length or grafting density is not possible. In studies of PEG-tethered surfaces, however, Dalsin reported a collapse of most protein adsorption data, along with varying tether length and graft density, using the total PEG in the layer or a normalized tether spacing as the independent variable.31 Accordingly, Figure 5 compares fibrinogen adsorption, with lysozyzme adsorption in the inset, on the current copolymer layers (hollow circles) to data from Feng et al. (solid symbols). In part A, data are summarized as a function of the total pMPC in the layer and, in part B, as a function of a normalized tether footprint (normalized on the projected area that the pMPC tether would form were it a free chain in solution). The curves demonstrate the excellent performance of the p(TMAEMA-bMPC) copolymers in their resistance to fibrinogen and lysozyme on a time scale of 20−45 min, with experiments run sufficiently long to produce a flat reflectometer signal with no detectible change over 10 min. Though copolymer adsorption produces layers containing far less pMPC than does surface polymerization, protein adsorption on the adsorbed copolymers is miniscule. In fact, the adsorbed copolymers exhibit greater protein resistance when compared to the surface-initiated pMPC in terms of the total pMPC on the interface or the reduced pMPC footprint. It is interesting that the adsorbed PEG layers from our prior work3 and included in Figure 5 as the hollow squares have excellent fibrinogen resistance similar to that of the pMPC copolymers and better than the surface-initiated pMPC layers. The adsorption of lysozyme is a weakness of PEG brushes.

Figure 5. Comparison of fibrinogen adsorption (main figures) and lysozyme adsorption (insets) in the current work (hollow circles) against data for surface-initiated pMPC tethered layers from Feng et al.,1,2 (solid points) and with PLL-PEG brushes from Gon et al.3 (hollow squares). (A) Data are summarized as a function of the pMPC mass in each layer. (B) Data are summarized as a function of the reduced tether footprint. Within the data sets from Feng et al, there are systematic variations in the chain length for different nominal grafting densities: triangles (0.1 chains /nm2), squares (0.14 chains/ nm2), diamonds (0.29 chains/ nm2), and circles (0.39 chains/nm2). The blue curve is drawn through the current p(TMAEMA-b-MPC) data, and the gray curve is drawn through the si-ATRP pMPC data of Brash to guide the eye.

Retention of Copolymers during Macromolecular Challenge. In our system, the pTMAEMA block of the copolymer is electrostatically driven to the surface. To the extent that the pMPC chains are tethered by the pTMAEMA, the polycation is charged an entropic penalty for holding the tether, equal to kT for each blob enumerated in Table 2. In a competitive situation, a cationic homopolymer matching the anchoring block would be preferred on the substrate as it does not pay this entropic penalty. It would therefore be expected to displace the copolymer. The potential for desorption and displacement of adsorbed copolymers motivates the chemical attachment of pMPC tethers, for instance, by surface-initiated polymerization, and also motivates the polycation challenge studies presented here. We investigated the extent to which p(TMAEMA20-bMPC105) could be displaced by a homopolymer comprising its own anchoring block (pTMAMEA20), as well as other polycations. These experiments were benchmarked against PLL-PEG from our previous studies. For the latter, additional challenge experiments were conducted to provide a direct comparison. E

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layers are substantially more stable to polycation challenge by their respective anchoring polycation blocks. Because the anchoring blocks (pTMAEMA versus PLL) in the polyzwitterion and PEG copolymer systems are different, we further probed the potential for polycation displacement of both copolymers. Figure 7 summarizes the results of six sets of

Figure 6A illustrates a pair of polycation challenge experiments on two identical p(TMAEMA20-b-MPC105) layers. After

Figure 6. Polycation challenge experiments. (A) Adsorbed pTMAEMA20-pMPC105 challenged by pTMAEMA20 for 10 and 60 min and then exposed to fibrinogen. (B) Adsorption of PLL-PEG (shown to be albumin-resistant) and then a challenge by PLL and exposure to albumin again. The light plot is the direct adsorption of PLL followed by albumin, from Gon et al.3

the layers were adsorbed and exposed to flowing buffer, a 100 ppm solution of pTMAEMA20 in the same buffer flowed over the surface for 10 min in one run and for 60 min in the other. The small resulting increases in signal are magnified and superposed for the two runs in the inset. After this, the layers were exposed to fibrinogen to assess how exposure to polycations may have compromised protein repellence. Because fibrinogen adsorbs to small asperities in protein-resistant brushes,51 any fibrinogen adsorption represents a measure of the extent to which a layer was compromised. Indeed, there was about 0.35−0.40 mg/m2 of fibrinogen (superposed in the second inset), which adsorbs after the pTMAEMA challenge, increasing only slightly with the pTMAEMA exposure time. Figure 6A suggests that a small amount of pTMAEMA penetrates the pMPC layer and is retained; however, there is no evidence for copolymer displacement. This behavior is in marked contrast to the instability of PLL-PEG layers when challenged by their anchoring polycation, a homopolymer of PLL of the same molecular weight used to synthesize the PLLPEG copolymer. In Figure 6B, reproduced from Gon et al.,3 a PLL-PEG layer is completely displaced within minutes of exposure to a PLL solution, producing an ultimate interfacial coverage matching that of direct PLL homopolymer adsorption to the bare silica substrate. The subsequent albumin adsorption onto the surface initially holding the PLL-PEG layer matches the albumin adsorption on a PLL homopolymer layer, which is further evidence for the nearly complete replacement of the PLL-PEG copolymer by a layer of PLL. Figure 6A,B shows that, compared to PLL-PEG layers, the p(TMAEMA20-b-MPC105)

Figure 7. Summary of cationic challenge studies on layers of (A) p(TMAEMA20-b-MPC105) and (B) PLL-PEG. For each, there are three challengers: pTMAEMA20, pTMAEMA100, and PLL. For each challenge study, the blue bars show the mass change during the cationic challenge and the red bars show the subsequent fibrinogen uptake. Two challenge times are included for each challenger, as was the case in Figure 6. The PLL challenge of PLL-PEG, taken from the literature,3 employs an albumin probe after the PLL challenge.

challenge studies for three different cationic challengers (pTMAEMA20, pTMAEMA100, and PLL) and two copolymer layers (p(TMAEMA20-b-MPC105) and PLL-PEG). The runs summarized in Figure 7 resemble those in Figure 6A, with the fibrinogen challenge run 20−40 min, each sufficiently long to produce a flat signal with no detectible changes in 10 min. The superior performance of the p(TMAEMA20-b-MPC105) layers relative to the PLL-PEG layers is clear. In general, we found that pTMAEMA does not displace either p(TMAEMA20-bMPC105) or PLL-PEG and that increasing the chain length of the pTMAEMA to increase its surface affinity did not lead to copolymer displacement. Notably, copolymer displacement and adsorption of pTMAEMA would produce much greater extents of fibrinogen adsorption than was observed, approaching 6 mg/ m2 in Table 2. An important distinction between the two copolymer layers is the complete resistance of the p(TMAEMA 20 -b-MPC 105 ) layer to the same PLL that completely displaced the PLL-PEG layer. Challenge by 20 kDa PLL had no apparent impact on the adsorbed F

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Langmuir p(TMAEMA20-b-MPC105). The fibrinogen resistance matches that of newly adsorbed copolymer layers.

p(TMAEMA40-b-MPC308) has a similar molecular proportion to p(TMAEMA20-b-MPC134) while having double the molecular weight of each block. We suggest that, as a result of the higher pTMAEMA molecular weight in pTMAEMA40-bMPC308, its adsorbed layer may contain cationic loops or tails (Figure 1C) due to a lack of full equilibration. This was seen in PLL-PEG graft copolymers with high PLL segments, despite seemingly sufficient PEG content.58 Table 2 includes data for brush-forming PLL-PEG layers from our prior work.3 Here a graft-copolymer-based brush, containing 5 kDa PEG chains, is fibrinogen-resistant within detection limits. This adsorbed layer possesses an adsorbed PEG mass, a number of blobs, and a footprint ratio (free solution to tether) that approach those of the pMPC copolymer brushes. The same PEG brush, however, absorbs substantial amounts of lysozyme as a result of protein interactions with PEG itself.3,48 The attractions between PEG and positively charged proteins comprise an important distinction between PEG and pMPC brushes. Impact of Polycation Challenge. Retaining electrostatically anchored copolymer layers in the face of variations in ionic strength, exposure to cationic and anionic proteins, and the challenge by cationic polymers is of practical and fundamental importance. Key features of the pTMAEMA challenge are (1) a small amount of pTMAEMA incorporated, (2) a very weak dependence (if any) on pTMAEMA exposure time or molecular weight, and (3) modest subsequent fibrinogen adsorption, roughly proportional to the increase in mass upon pTMAEMA exposure. That the fibrinogen adsorption is minimal suggests that the pMPC and PEG layers remain intact. Notably, much greater fibrinogen adsorption occurred on homopolymer pTMAEMA layers and on p(TMAEMA100-bMPC177) layers in Table 2. pTMAMEA may be retained in small amounts in challenge studies if chains successfully anchor to an available area of the substrate, as shown in Figure 1D. A similar accumulation of PLL homopolymers to bare regions of silica at the base of adsorbed PLL-PEG layers was previously demonstrated.3 However, it is a puzzle as to why p(TMAEMAb-MPC) or PLL-PEG is not displaced by the pTMAEMA homopolymer. In the case of the PLL challenge of an adsorbed PLL-PEG layer, the PLL rapidly and completely displaced the initially adsorbed PLL-PEG copolymers. In contrast, pTMAEMA appears to penetrate both pMPC and PEG layers without displacing them. Also interesting is the marked difference between the PLL challenge of the p(TMAMEA-b-MPC) and PLL-PEG layers. The polyzwitterion is completely unaffected by exposure to PLL, apparently repelling this cationic random-coil polypeptide, while PLL rapidly penetrates the PLL-PEG layer. We ascribe these results to differences in the hydration of the polyzwitterions versus PEG7 and the relative solution-phase interactions between PLL and pMPC versus PLL and PEG. This polymer-specific effect may be similar to the different interactions of lysozyme, a substantially cationic protein, with pMPC and PEG. Although we know of no solution-phase studies of the virial coefficiencts of lysozyme and pMPC, solution-phase attractions have been established for lysozyme and PEG.48 Overall, the interactions of pMPC brush layers with cationic displacers and cationic proteins are generally repulsive but polycation-specific, with our study suggesting that pTMAEMA

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DISCUSSION Arguments for Brushlike pMPC Layers. The surface layer structure and its complex dependence on copolymer architecture are critical to rationally designed protein-repellant coatings. Table 2 suggests that one criterion for protein repellency is that the pMPC layer content exceeds 1 mg/m2. The pMPC chains must, however, be irreversibly tethered (on the time scales of interest) to the substrate. Thus the tethering block of the copolymer must bind with appropriate strength, but if it is too large, it will not be sufficiently shielded from species in solution. We find that about 20 pTMAEMA units are sufficient to anchor an appropriately high MW pMPC tether (in the range of 100−140 monomers) to eliminate detectible protein adsorption. Keeping the proportion of polyzwitterion and polycation groups constant but doubling the molecular weight produces interfaces that are more protein-adhesive. Higher molecular weight tethers with proportionally less pMPC allow even greater protein adsorption. Treating tethered layers as solvated brushes, when appropriate, is a useful exercise because it allows a comparison of different brush systems and enables estimations of layer thickness and repulsive forces. In adsorbed copolymer brushes, the anchor blocks hold the ends of the tethers to the surface. The tethers themselves are well-solvated and stretch osmotically into solution.20,21,25 To avoid protein adsorption, the anchor blocks must not be exposed to the solution in either a mushroom-like layer (Figure 1B) or in loops (Figure 1C). Several features are characteristic of brushlike layers. First, the adsorbed mass of the anchor block in a copolymer brush is substantially less than in an equivalent homopolymer layer (of the anchor block homopolymer alone). Second, in brushes, tethers are crowded at the interface so that the tether footprint is considerably smaller than its free end-to-end distance squared.20,21 When the tethers are described as a semidilute solution,56 each correlation blob in the brush contributes kT worth of osmotic energy to repel approaching proteins. The number of blobs in a tether is equal to the tether stretching energy and is summarized in Table 2 (with details in the Supporting Information). The data in Table 2 suggests that the most protein-resistant copolymer layers, p(TMAEMA 20 -b-MPC 105 ) and p(TMAEMA 20-b-MPC 134 ), conform quantitatively to the description of a brushlike layer. Both exhibit the first signature of brush formation, that the adsorbed amount of anchoring blocks in the brush (0.12−0.15 mg/m2) is considerably less than the homopolymer saturation coverage of the same anchoring block alone (0.3 mg/m2 for the pTMAEMA20 homopolymer). This criterion is not upheld for the other two copolymers. Next, the ratio of the projected area of the pMPC block in free solution exceeds the tether footprint for layers of adsorbed p(TMAEMA20-b-MPC105) and p(TMAEMA20-bMPC134). This ratio is 4.3−4.4 for p(TMAEMA20-b-MPC105) and p(TMAEMA20-b-MPC134) but drops to 2.0 for p(TMAEMA100-b-MPC177). Finally, p(TMAEMA20-b-MPC105) and p(TMAEMA20-b-MPC134) adsorb to form a substantial number of effective blobs per tether, also consistent with a brush. p(TMAEMA40-b-MPC308) is an exception. This copolymer adsorbs to form a higher calculated number of blobs in Table 2 but still adsorbs fibrinogen. It is worth noting that G

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Langmuir

less, was needed in the copolymer layers than in the chemically anchored layers. Also, compared to surfaces containing tethered PEG chains anchored by cationic copolymer blocks, p(TMAEMA-b-MPC) exhibited two main advantages: (1) the optimal copolymer layers were completely nonadhesive to lysozyme, a model cationic protein that is attracted in solution to PEG and adsorbs to layers of tethered PEG (especially at higher molecular weight PEG), and (2) the p(MPC-bTMAEMA) layers were unaffected by poly L-lysine, a challenger that rapidly and completely displaces PEG-PLL chains to produce cationic protein-adsorbing surfaces. Overall, the adsorption of p(MPC-b-TMAEMA) chains on negative surfaces from aqueous solution is a versatile, environmentally friendly, scalable approach to producing protein-repellent biocompatible surfaces that exploits both the unique properties of the pMPC polyzwitterion and the advantages of strong electrostatic attachment to surfaces of negative charge.

can penetrate a pMPC brush slightly but to a greater extent than does PLL. Prospects. pMPC coatings have proven useful in preventing protein adsorption but have generally been produced by surface polymerization. This is a multistep process involving the surface anchoring of initiator groups and subsequent polymerization, prohibiting the large-scale production of coatings. The simpler, scalable approach of adsorbing pMPC copolymers from aqueous solution to generate a brushlike pMPC layer has not generally been undertaken (though pMPC copolymers are sometimes employed in electrodeposited coatings and form the basis for bulk materials with reduced but still significant protein adsorption).37,38 With the appropriate choice of pMPCcontaining copolymer architecture to produce relatively dense pMPC tethered layers, fibrinogen and lysozyme adsorption can be avoided within detectible limits of 0.01 mg/m2. This lack of fibrinogen adsorption bodes well for the general elimination of serum protein adsorption based on correlations published by Brash.1 Additionally, the lysozyme repellence of the pMPC brushes is a substantial improvement over that of proteinresistant PEG layers. Worth noting is that anchoring by polycationic copolymer blocks generally facilitates adsorption to negative surfaces, with the particular copolymer architectures optimized here for silica. The use of cationic, rather than hydrophobic, anchors enables the formation of polymer brushes without complications from micelles and surface clusters,40 with reduced protein repellence. The disadvantage of adsorbed copolymer-based brushes is their potential displacement by species from solution. In this regard, the particular p(TMAEMA-b-MPC) copolymers perform exceptionally well, withstanding elevated ionic strength and challenge by cationic polymers and proteins. p(TMAEMAb-MPC) qualitatively and substantially outperformed an optimized PLL-PEG copolymer, a prominent member of the much-studied library of PEG−polycation copolymers. Most notably, the PLL-PEG copolymers were completely displaced by the PLL homopolymer, which had no effect on the p(TMAEMA-b-MPC) layers.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b03391. Experimental details and explanations and example calculations for Table 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] ORCID

T. Emrick: 0000-0003-0460-1797 M. M. Santore: 0000-0003-3689-5064 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF-CBET-1264855 (M.M.S.) and NSF-CBET-1403742 (T.E.).

SUMMARY We demonstrated that p(TMAEMA-b-MPC) copolymers adsorb to negatively charged silica surfaces to produce nanometer-thick layers. Although all of the copolymer layers reduced protein adsorption, the most protein-repellent layers (with protein adsorption of 0.01 mg/m2 or less) possessed metrics that were quantitatively consistent with pMPC brush formation. The best copolymer architectures corresponded to about 20 pTMAEMA units with pMPC chains with a degree of polymerization in the range of 100−140. Doubling the molecular weights of each block together or increasing the pTMAEMA length partially compromised protein resistance. The adsorbed copolymers were well-retained upon exposure to elevated ionic strengths up to the maximum of 1 M tested and were not compromised in their protein resistance. The adsorbed copolymers were not displaced by cationic challengers including cationic protein lysozyme, random-coil cationic polypeptide poly L-lysine, and pTMAEMA homopolymers. Relative to chemically anchored pMPC chains reported in the literature, the current adsorbed copolymers achieved comparable protein resistance overall and far superior protein resistance when compared at the same nominal amounts of pMPC and normalized pMPC tether footprints. Specifically, a smaller amount of tethered pMPC, on the order of one-fifth or

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