Chain Length and Grafting Density Dependent Enhancement in the

May 26, 2015 - Determination of the polymer chain length and polydispersity by gel permeation chromatography (GPC) of the hydrolysates is based on the...
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Chain Length and Grafting Density Dependent Enhancement in the Hydrolysis of Ester-Linked Polymer Brushes Kathryn A. Melzak,† Kai Yu,‡ Deng Bo,‡ Jayachandran N. Kizhakkedathu,*,‡,§ and José L. Toca-Herrera*,† †

Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 11, A-1190, Vienna, Austria ‡ Centre for Blood Research and the Department of Pathology and Laboratory Medicine and §Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 S Supporting Information *

ABSTRACT: Poly(N,N-dimethylacrylamide) (PDMA) brushes with different grafting density and chain length were grown from an ester group-containing initiator using surface-initiated polymerization. Hydrolysis of the PDMA chains from the surface was monitored by measuring thickness of the polymer layer by ellipsometry and extension length by atomic force microscopy. It was found that the initial rate of cleavage of one end-tethered PDMA chains was dependent on the grafting density and chain length; the hydrolysis rate was faster for high grafting density brushes and brushes with higher molecular weights. Additionally, the rate of cleavage of polymer chains during a given experiment changed by up to 1 order of magnitude as the reaction progressed, with a distinct transition to a lower rate as the grafting density decreased. Also, polymer chains undergo selective cleavage, with longer chains in a polydisperse brush being preferentially cleaved at one stage of the hydrolysis reaction. We suggest that the enhanced initial hydrolysis rates seen at high grafting densities and high chain lengths are due to mechanical activation of the ester bond connecting the polymer chains to the surface in association with high lateral pressure within the brush. These results have implications for the preparation of polymers brushes, their stability under harsh conditions, and the analysis of polymer brushes from partial hydrolysates.

1. INTRODUCTION Mechanical stress on bonds has been found to enhance reaction rates1 and even to induce bond cleavage.2,3 The stress can be applied through an externally powered force but has also been induced by adsorption to surfaces,2 spreading of drops,3 and the incorporation of bonds into cyclic structures.1 In the experiments described here, we determined the rate of hydrolysis of end-tethered polymers at grafting densities that are associated with high lateral forces within the brush layer.4 At these high grafting densities, the cleavage rate was found to be enhanced by an order of magnitude relative to the rate in less dense brushes, an increase that we propose is due to mechanical stress within the highly crowded hydrated brushes. Polymer brushes have generated significant interest due to their potential biotechnological applications.5−7 These include the development of protein-resistant, cell-resistant, or antimicrobial coatings as well as materials applications such as actuators and sensors.8−11 The poly(N,N-dimethylacrylamide) (PDMA) brushes that were investigated are of interest because they have been shown to reduce protein adsorption and render surfaces biocompatible.9 These are two important applications of polymer brushes that depend on the grafting density and polymer chain length;9−11 reduced protein adsorption and increased biocompatibility are both associated with increased grafting density and increased chain length.9−11 © 2015 American Chemical Society

The results presented here have potential relevance to both the reactivity and the characterization of polymer brushes. The stability of polymer brushes is important for the preservation of their function; it is therefore of interest to understand the manner in which the brushes could degrade under harsh conditions. Conversely, it might be possible to exploit the phenomenon of strain-related instability in order to produce brushes with defined characteristics. Selective hydrolysis of the brushes, which we are suggesting is due to conformationinduced changes in the stability, will affect analysis that is done with polymer chains that have been cleaved off of the surface. Determination of the polymer chain length and polydispersity by gel permeation chromatography (GPC) of the hydrolysates is based on the assumption that the cleavage product is representative of what is on the surface. We demonstrate here that this may not be true and that GPC with an incomplete hydrolysis has the potential to overestimate the chain length.

2. EXPERIMENTAL SECTION 2.1. Materials. N,N-Dimethylacrylamide, sodium methoxide solution (25 wt % in methanol), copper(I) chloride, copper(II) Received: September 19, 2014 Revised: May 22, 2015 Published: May 26, 2015 6463

DOI: 10.1021/acs.langmuir.5b01424 Langmuir 2015, 31, 6463−6470

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modified by 8 μm diameter silica beads (from Microparticles GmbH) that were glued onto the AFM tip. Approach and retraction speeds were kept constant at 1 μm/s. Maximum applied load was 5 nN unless otherwise stated. The AFM chip with the cantilever was cleaned prior to measurements by a 15 s immersion in a basic solution of hydrogen peroxide. The sensitivity of the cantilevers was determined from the constant compliance regime of the force−distance curves recorded on a cleaned glass slide; the spring constant was evaluated by thermal noise analysis before each experiment. The polymer chain length was determined from the tip−sample separation at the point of the last detachment16 as indicated in the force−distance curve. Measurements were made at 100 different points over a 10 × 10 μm square. Repeat sets of measurements were made at different locations and on different days. 2.3. Synthesis of PDMA Brushes: Variation in Initial Grafting Densities, Chain Lengths, and Polydispersity. 2.3.1. PDMA Brush with High Grafting Density on Silicon Substrate (Series A). Copper(II) chloride (CuCl2, 1.35 mg), copper(I) chloride (CuCl, 8 mg), and HMTETA (60 μL) were added successively into a glass tube followed by adding 12 mL of Milli-Q water. The solution was degassed with three freeze−pump−thaw cycles. The solution was then transferred into a glovebox. The catalyst solution (6 mL) was mixed very well before adding 300 μL of DMA. The silicon wafer with deposited surface initiator ((11-(2-bromo-2-methyl)propionyloxy)undecyltrichlorosilane) was immersed in the polymerization mixture. Soluble free initiator, 10 μL of methyl 2-chloropropionate in methanol (stock solution 40 μL in 5 mL of methanol), was added immediately to the reaction mixture. The polymerization was allowed to proceed at RT (22 °C) for 24 h. The substrate was then rinsed thoroughly with methanol and sonicated in methanol for 30 min. This brush sample was labeled as A1. The soluble polymer formed along with the surface grafted polymer was collected by dialysis (molecular weight cutoff: 1000) against water for 1 week. Two other PDMA brush samples labeled as A2 and A3 with a grafting density similar to PDMA brush A1 but with different chain lengths were synthesized by changing the monomer concentration while keeping the other parameters fixed. The PDMA brush A2 was synthesized by adding 100 μL of DMA and 3 μL of methyl 2chloropropionate into the catalyst solution (6 mL). The PDMA brush A3 was prepared by adding 54 μL of DMA and 4.5 μL of methyl 2chloropropionate into the catalyst solution (6 mL). 2.3.2. Variation of Polymer Chain Length, Polydispersity, and Initial Grafting Density. The initial grafting density of polymer brushes produced on silicon substrates was controlled by changing the polymerization parameters, including the ligand and solvent. Briefly, series A with a high grafting density was produced utilizing HMTETA as the ligand; series B with a similar molecular weight but a lower grafting density was produced by changing the ligand to Me6TREN; brush C with an intermediate grafting density and lower polydispersity was produced with Me6TREN as ligand by changing the solvent to DMSO. Details on the synthesis of brush series B and brush C are in the Supporting Information. 2.3.3. Validation of Mn Values Used To Calculate Grafting Density. The Mn values used in the calculations of grafting density were determined from the polymer formed in solution along with the surface grafted polymer from the sacrificial initiator. It was therefore necessary to show that the molecular weight of the polymer produced in solution is similar to that of the grafted polymer. This was done by preparing PDMA brushes on a large silicon wafer (18 cm2), as described in the Supporting Information. The grafted polymer was cleaved with 25 wt % sodium methoxide, producing sufficient polymer for GPC analysis. The data are shown in the Supporting Information Table S1. 2.3.4. Synthesis of PDMA Brush D Grafted on PS Particles. Atom transfer radical polymerization (ATRP) initiator modified polystyrene (PS) particles with diameter size of 669 ± 6 nm were synthesized by a shell growth mechanism utilizing surfactant-free emulsion polymerization of styrene and 2-(methyl 2′-chloropropionato)ethyl acrylate.13 Polymer chains formed on the PS surface have an ester linker similar to that for chains prepared on silicon wafers (series A, B, and C). We

chloride, tris[2-(dimethylamino)ethyl]amine (Me 6 TREN), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), and methyl 2-chloropropionate (97%) were purchased from Sigma-Aldrich (Oakville, ON). N,N-Dimethylacrylamide was purified by distillation under reduced pressure. The synthesis of the ester derivative of the surface ATRP initiator (11-(2-bromo-2-methyl)propionyloxy)undecyltrichlorosilane) and deposition of the surface initiator onto the silicon wafer were done using a procedure similar to that reported in the literature.12 Atom transfer radical polymerization (ATRP) initiatormodified polystyrene (PS) particles (669 nm) were synthesized by a shell growth mechanism utilizing surfactant-free emulsion polymerization of styrene and 2-(methyl 2′-chloropropionato)ethyl acrylate.13 2.2. Characterization. 2.2.1. Gel Permeation Chromatography. Molecular weights and polydispersities of poly(N,N-dimethylacrylamide) (PDMA) were determined using gel permeation chromatography (GPC) on a Waters 2690 separation module fitted with a DAWN EOS multiangle laser light scattering (MALLS) detector from Wyatt Technology Corp. with 18 detectors placed at different angles and a refractive index detector (Optilab DSP from Wyatt Technology Corp.), using aqueous buffer (0.1 M NaNO3) as eluting phase. An Ultrahydrogel linear column with bead size 6−13 μm (elution range 103−5 × 106 Da) and an Ultrahydrogel 120 with bead size 6 μm (elution range 150 to 5 × 103 Da) from Waters were used. The dn/dc value of PDMA in the mobile phase was determined at λ= 620 nm to be 0.15 mL/g and was used for determining molecular weight parameters. The number-average mean-square radius moments were taken as the Rg of the polymer. 2.2.2. Ellipsometry and Grafting Density. The variable-angle spectroscopic ellipsometry (VASE) spectra were collected on an M2000 V spectroscopic ellipsometer (J.A. Woollam Co. Inc., Lincoln, NE) at 55°, 65°, and 75° at wavelengths from 480 to 700 nm with an M-2000 50W quartz tungsten halogen light source. The VASE spectra were then fitted with a multilayer model utilizing WVASE32 analysis software based on the optical properties of a generalized Cauchy layer to obtain the dry thickness of the PDMA layers. The grafting density σ was calculated according to σ = NAhρ/Mn,14 where Mn is the numberaverage molecular weight of sacrificial polymer generated in solution, NA is Avogadro’s number, σ is the density for the dry polymer, and ρ is the mass density, which was assumed to be 1 g/cm3. We determined that the molecular weight of grafted polymer is very similar to that of the polymer formed in solution from the sacrificial initiator (see section 2.3.3) even at a grafting density about 0.7 chains/nm2. It is therefore valid to calculate the grafting density based on the Mn value of sacrificial polymers. The polymer layer thickness in aqueous medium and in ethyl acetate was determined with the Woollam CompleteEASE program (Woollam, NE) using a four-layer model (solvent, polymer, silicon oxide, silicon). The silicon oxide layer was assumed to remain constant at 2.32 nm thick. Fitting the data using small variations in this did not affect the measured values for the PDMA layer. The bulk NaCl solution was treated as a Cauchy medium with nsolvent(λ) = 1.325 + 0.00322/(λ/μm)2.15 Measurements were made in chambers that had been shown to have no significant window effects. The ethyl acetate was treated as a Cauchy medium with n(λ) = 1.349 + 0.00243/(λ/ μm) 2 . These values were obtained by using the Woollam CompleteEASE program to fit data obtained for a calibration wafer of Si having a relatively thick oxide layer. An initial measurement was made with the calibration wafer in air to determine the oxide layer thickness (23.45 nm), which was assumed to remain constant. The only variables remaining for the fitting of the data obtained in the ethyl acetate were therefore the Cauchy coefficients for the ambient medium, i.e., the ethyl acetate. The values thus obtained were used when estimating the thicknesses of the PDMA layers in ethyl acetate. 2.2.3. AFM Measurements. Polymer chain extension lengths were determined using a Nanowizard atomic force microscope (JPK Instruments, Germany). Uncoated SiN cantilevers (MLCT, Veeco Instr., USA) with a pyramidal tip having a nominal radius of 20 nm and a maximum radius of 60 nm and a nominal spring constant of 0.07 N/m were used to acquire the force−distance curves; additional measurements were carried out with the same MLCT cantilevers 6464

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Langmuir Scheme 1. Synthesis of the Poly(N,N-dimethylacrylamide) Brushes on Silicon Wafersa

a

The black line represents the SiOx-coated wafer surface and is shown here with an attached initiator that acts as a grafting point for the brush. On the latex beads, the 2-(methyl-2′-chloropropionato)ethyl acrylate initiator was incorporated into an initiator shell layer as a copolymer of styrene.

Table 1. Polymer Brushes for Reaction Rate Determinationa Rg (nm) Mn (g/mol) PDI initial grafting density σ (chains/nm2) initial hydrolysis rate constant (min−1) later hydrolysis rate constant (min−1) grafting density at transition (chains/nm2) D/2Rg at transition

A1

A2

A3

B1

B2

B3

C

34.2 3.62 × 105 1.4 0.79 1.27 0.024 0.31 0.026

11.4 7 × 104 1.4 0.70 0.080 0.020 0.20 0.098

8.3 4.3 × 104 1.4 0.78 0.050 0.021 0.20 0.13

35.1 3.8 × 105 1.8 0.11 0.41 0.025 0.065 0.055

11.6 7.2 × 104 1.5 0.11 0.023 0.00010 0.039 0.22

8.9 4.5 × 104 1.4 0.13 0.011 0.00082 0.034 0.31

13.6 5.2 × 104 1.1 0.38 0.010 0.00066 0.072 0.14

a

Characterization of PDMA brushes having different grafting density and molecular weight. Series A: high initial grafting density and varied Mn. Series B: low initial grafting density and varied Mn. Series C: intermediate grafting density. Rg is a number-average mean-square radius moments from GPC coupled to a multiangle light scattering system measured in aqueous 0.1 M NaNO3 solution (a good solvent for PDMA), obtained for sacrificial polymer as outlined in section 2.2.1 and in the Results section; the grafting density was calculated from ellipsometric dry thickness as outlined in section 2.2.3; the hydrolysis rates were calculated from the slopes in Figure S1, initial hydrolysis rates were calculated from the first three data points as described in section 3.2, and later hydrolysis rates were calculated from last three data points; grafting density at the transition is determined at the intersection of the two lines for each sample. Using Mw in the calculations of grafting density, as has been suggested elsewhere38 does not have any significant effect on the results presented here (see Supporting Information, Table S2). followed our published protocols for the characterization of ATRP initiator-modified PS particles and PDMA-grafted PS particles.13 ATRP initiator-modified PS particles (45 mg), nonionic surfactant Brij 35 (3.3 mg), and Milli-Q water (1.5 mL) were added successively into a glass tube. The contents of the glass tube were degassed by three freeze−pump−thaw cycles. To help the PS particles disperse in the water homogeneously, the glass tube was subjected to ultrasonication for 15 min before being transferred to the glovebox. In another glass tube, CuCl (8 mg), CuCl2 (0.67 mg), and Me6TREN (72 μL) were added successively followed by the addition of Milli-Q water (5.4 mL). This glass tube was degassed by three freeze−pump−thaw cycles and transferred to the glovebox. DMA (150 μL) was added to the prepared solution (1.35 mL). After the monomer was dissolved completely, the solution was mixed with 1.5 mL of PS particle suspension. Soluble methyl 2-chloropropionate solution (20 μL) (from a stock solution of 40 μL in 5 mL of methanol) was also added along with the substrate to the reaction solution. The suspension was stirred continuously, and the polymerization was allowed to proceed at RT (22 °C) for 24 h. The polymer-grafted PS particles were cleaned by three repeated cycles of centrifugation (10 min at 10 000 rcf) and resuspension in NaHSO3 solution (50 mM) in water to remove adsorbed copper complexes. Finally, the latex suspension was washed with 0.1 M EDTA solution three times and then with water three times to remove any copper complex associated with the grafted surface. 2.4. Hydrolysis of PDMA Brushes from the Silicon Substrate. PDMA brushes grafted on the silicon substrate (dimension 1 cm × 1 cm) were immersed in 1 mL of 25% NaOMe in methanol. At different time intervals, the substrate was taken out and rinsed with methanol thoroughly, sonicated in methanol for 5 min, and then dried under a flow of argon. The thickness of the brush after hydrolysis was then characterized by ellipsometry. 2.5. Hydrolysis of PDMA Brushes from the PS Particles. PS particles grafted with PDMA brushes were initially well dispersed in 3 mL of anhydrous methanol. Sodium hydroxide solution (25 wt %, 0.6 mL) was then added into the suspension. Portions of the suspension were removed at different times and centrifuged at 10 000 rcf. The supernatant solution was then dialyzed against DI water (membrane

cutoff 1000) and analyzed by GPC. The PS particles were then washed with water three times by the centrifugation and redispersion method and analyzed.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PDMA Brushes. For these experiments, PDMA brushes were grown from an ester group-containing initiator using the surface-initiated atom transfer radical polymerization (SI-ATRP) method (Scheme 1). Brushes were prepared on silicon wafers for analysis by ellipsometry and atomic force microscopy. The grafting density σ was calculated according to σ = NAhρ/Mn, where Mn is the number-average molecular weight of polymers generated in solution from sacrificial initiators, NA is Avogadro’s number, and ρ is the density for the dry polymer.14 We found that the molecular weight of the polymers formed in solution from sacrificial initiator was very similar to that of grafted polymer. This was done by modifying a sufficiently large area of silicon wafer that the cleaved polymer could be analyzed directly by GPC for comparison to the values obtained for the polymer formed in solution. These results are summarized in Table S1 of the Supporting Information. Polymer brushes with different properties were synthesized by changing the ligands, the solvent, or the ratios of the components. These different polymerization conditions affect the polymerization kinetics, thus affecting the grafting density and chain length.17−19 The initial grafting density and chain length of the polymer brushes were varied so that the hydrolysis rate could be investigated as a function of chain length at both low and high initial grafting densities and as a function of varying grafting density at an intermediate chain length. Characteristics of the different brushes are given in Table 1. 6465

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Figure 1. Cleavage of PDMA brushes as determined by ellipsometric measurements of the dry brush thickness. Grafting density is determined as described in the text and Supporting Information. Slopes of the initial lines have been determined from the first three data points. Part A shows the effects of varying the polymer chain length at high initial grafting density (0.70−0.79 chains/nm2); the inset shows the first 12 min. Part B shows the effect of varying the chain length at low initial grafting density (0.11−0.13 chains/nm2); the inset shows the first 150 min. Part C shows the effect of varying the initial grafting density for brushes with similar chain lengths.

The plots of ln(grafting density) against time show linear regions, implying that the reaction follows pseudo-first-order kinetics. The distinctive feature of the results shown here is that there are two polymer chain hydrolysis rate regimes, with a higher rate being observed at higher grafting densities. The two linear regions are more clearly differentiated in the cases where the initial rate is lower (samples B2 and B3 in Figure 1B and sample C in Figure 1C). Our results show both the effects of varying the grafting density over the time course of the reaction and the effect of varying the initial grafting density. In addition, we investigated the effect of varying the polymer chain length (Figure 1A,B). The rates calculated from the initial and later parts of the reactions are summarized in Table 1, allowing us to make comparisons between the different samples and different stages of the reactions. The initial rates given in Table 1 have been calculated based on the first three data points because of the importance of the early stages of the hydrolysis for results observed later with latex beads, which are discussed below. The relative increase in the hydrolysis rate at the initial stage of the reaction, when the grafting density is highest, can be estimated from the ratio of the slopes in the initial and later

Changing the grafting density would vary the initial values for D/2Rg (where D is the average separation distance between the nearest neighbor grafting points and Rg is the radius of gyration). The Rg values were determined by GPC in combination with multiangle light scattering, and the average nearest-neighbor separation distance D was estimated according to D = σ−1/2,20,21 where σ is the grafting density (chains/ nm2) (Table 1). 3.2. Hydrolysis of PDMA Brushes: Influence of Grafting Density and Chain Length. PDMA brushes on silicon wafers with different initial grafting density and chain length were subjected to hydrolysis in 25% NaOMe. The ester linkage between the surface and PDMA chain is the cleaving point during the hydrolysis reaction. The PDMA chains are hydrolytically stable, so it is anticipated that they are released without degradation. The brush sample dry thickness was analyzed by ellipsometry at different time points and used to calculate the variation in grafting density during the time course of the hydrolysis reaction. Figures 1A and 1B show the effect of varying the chain length at high and low initial grafting densities, and Figure 1C shows the effect of varying the initial grafting density while maintaining a similar chain length. The base-catalyzed hydrolysis rates were determined from the rate of change of the grafting density as a function of time and are summarized in Table 1. It should be noted here that the hydrolysis rates that are quoted reflect the cleavage of the polymer chains rather than the total cleavage of ester groups on the surface. Unreacted initiator that is present on the surface will also contain ester groups, but cleavage of these groups will not affect the measured ellipsometric thickness.

Table 2. Polymer Brushes on PS Particles; GPC Analysis Showing Selective Hydrolysis hydrolysis time 45 s 2 min 30 min 5 days 6466

Mn of cleaved PDMA (g/mol)

PDI of cleaved PDMA

× × × ×

1.9 2.0 2.2 2.6

2.1 2.3 2.8 1.3

105 105 105 105

Mn of polymer from the shoulder peak (g/mol) 1.2 × 106 3.5 × 106 4.0 × 106

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Figure 2. AFM extension lengths for a polymer series with Mn = 3.8 × 105 and PDI = 1.8, measured in an aqueous environment with an unmodified AFM tip. The grafting density σ in chains/nm2 is shown on each graph.

the increased pressure, as part of the polymer chains, but more work would be required to understand the relationship between the pressure within the brush and the increased ester reactivity. Pseudo-first-order kinetics have been observed previously for base catalyzed hydrolysis of esters in alkylthiol monolayers on gold, when the catalyst is present in sufficient excess and when the ester packing density is sufficiently low.24,25 The hydrolysis rate decreases for alkylthiol layers with higher packing densities due to hindered access of the base to the ester bonds.24,25 For very efficient packing, the kinetics follow a sigmoidal pattern instead of being first order, with an initial slow cleavage rate that has been associated with a reaction that spreads from isolated defect locations.24,25 The cleavage of the PDMA brushes here proceeds differently, with the cleavage rate decreasing at low grafting densities over the time course of the reaction (Figure 1). Cleavage of the PDMA therefore does not seem to be limited by access of the base to the ester bonds, which are situated near the surface; this may be due to the much lower packing densities of the tethered PDMA, when compared to alkylthiol layers. We then turn to the question of the structural differences that occur within a given polymer brush when the grafting density changes, as happens during the hydrolysis reaction. What we are looking for here are differences that could be associated with the change in cleavage rates shown in Figure 1. The tethered polymer chains will change conformation with variations in the grafting density, due to excluded-volume interactions between the chains, and will make a gradual transition away from a brush when the surface coverage becomes low enough that neighboring chains no longer interact with each other.26 The grafting density at the transition points between the high and low rates (see Table 1) is much higher than the densities associated with the transition to a polymer brush, where D/2Rg ≈ 1. At very high surface coverage, the polymer chains can have a highly stretched regime, where the chains are stretched out normal to the substrate.27,28 The onset of this regime has been observed and predicted16 at σπRg2 = 14,

stages of Figure 1. The enhancement factors were found to be 53, 16, and 15 for samples A1, B1, and C, respectively. The effect of varying the initial grafting density can be seen in Figure 1C for polymer brushes with similar Mn values (between 4.3 × 104 and 5.2 × 104): when the grafting density is increased from 0.13 to 0.38 chains/nm2, there is very little effect on the reaction rates; a further increase to 0.78 chains/nm2 leads to an enhancement of factor of 5 on the initial hydrolysis rate. A similar enhancement factor in the initial rate (a factor of 3) is seen for polymer brushes with a greater chain length (Mn 3.6 × 105 and 3.8 × 105), when the grafting density is increased from 0.11 to 0.79 chains/nm2 (see Figure S1). The variations in the initial grafting density investigated here are much smaller than the change that occurs as the hydrolysis reaction progresses. The influence of the polymer chain molecular weight was investigated for two different initial grafting densities. The results are shown in Figures 1A and 1B and are summarized in Table 1. The initial hydrolysis rate decreased by a factor of 25 with a decrease in molecular weight from 3.6 × 105 to 4.3 × 104 at constant grafting density 0.78 chains/nm2 (for series A brush samples). Similarly, a 37-fold decrease was observed in the initial rates for series B samples at a grafting density of about 0.1 chains/nm2 when the molecular weight decreased from 3.8 × 105 to 4.5 × 104. The results clearly demonstrate that the initial hydrolysis rate for the brushes is dependent on both the grafting density and the molecular weight of the polymer chains. Both these factors are known to be related to the lateral pressure within the brush, suggesting that the enhancement of the hydrolysis could be linked to the high lateral pressure that builds up within brushes due to interactions between the chains. The surface pressure scales as π ∼ Nσ5/3, where N is a measure of the chain length and σ is the grafting density.22,23 The initial hydrolysis rate for our brushes was more strongly influenced by molecular weight than by grafting density, implying that the enhancement of the hydrolysis is not directly proportional to the pressure within the brush. The ester linker that is hydrolyzed will be subjected to 6467

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Figure 3. (A) GPC chromatograms of the PDMA hydrolysate from beads, accumulated at 45 s, 2 min, 30 min, and 5 days. All peaks have been normalized. The shoulder to the left of the peak is due to higher molecular weight components. (B) Cumulative weight fraction of polymer cleaved from the bead surface at 30 min and 5 days. At a given Mn (x-axis), the percentage by weight of polymer that is less than the specified Mn value (shown on the y-axis) is higher for polymer cleaved for 5 days than for polymer cleaved in the first 30 min. In other words, the high molecular weight portion for polymer cleaved in the initial stage is larger than for the sample collected after 5 days. Greater amounts of longer chains are hydrolyzed at the early stages of hydrolysis.

where σ is the grafting density. The values calculated from 14/ (πRg2) do not appear to be related to the grafting densities at which the hydrolysis rates change (sample comparison for A1: 14/(πRg2) = 0.0038 while grafting density at transition is 0.31). The onset of the highly stretched state therefore does not seem to be the cause of the pattern observed in Figure 1. In addition to the grafting density, the size of grafted polymer chains (Rg) also affects the degree of lateral interaction within the brush.4 As shown in Table 1, brush B3, with the smallest Rg, has the lowest hydrolysis rates (Table 1). This leads to questions about the hydrolysis within polydisperse brushes and the possibility that the longer chains are cleaved more rapidly than the shorter chains. This was investigated initially using AFM pulling experiments to analyze the polymer chains remaining on the substrate after different hydrolysis reactions (Figure 2). The analysis was done with a PDMA brush similar to sample series B in Table 1 and Figure 1, with Mn and PDI values matching those listed in Table 1. The average extension lengths for samples with grafting densities of 0.08, 0.05, 0.03, and 0.003 chains nm−2 produced after increasing hydrolysis times were respectively 834, 522, 397, and 84 nm. Polymer chain extension lengths measured by AFM can be similar to the polymer chain contour length, if the tip is specific for the chain end.29 In the present case, the interaction with PDMA chains with the AFM tip will be nonspecific, so that the measured extension length will depend on the point of contact between the tethered polymer chain and the tip. We would therefore expect that the extension lengths would be affected by the polymer conformation,30 which was confirmed by measuring the extension lengths in different solvents (Supporting Information, Figure S2). Alternative evidence is therefore required to assess the possibility of preferential cleavage of the longer chains. Variable rates of hydrolysis in polydisperse brushes were investigated using PDMA brushes grown on PS beads (σ = 0.08 chains/nm2; sacrificial polymer Mn = 1.6 × 105 with PDI = 1.4). PDMA chains are attached to PS surface via ester linkages similar to that on silicon wafer and are susceptible to hydrolysis under basic conditions. Cleaved polymer chains from these brushes were collected at different time points and analyzed by GPC. The high specific surface area of the PS beads allowed

quantitative analysis of the cleaved chains. The GPC results illustrated in Figure 3 show the selectivity of the hydrolysis for higher molecular weight components. The relative height of the shoulder to the left of the peak indicates the proportion of higher molecular weight polymer chains that have been cleaved off the surface of the beads. As may be seen in Figure 3A, the shoulder is most prominent at 30 min, indicating that the selectivity for the higher molecular weight chains is greatest for hydrolysis that takes place between 2 and 30 min; the hydrolysate collected within the first 45 s (the amount of polymer collected was smaller in this case) and the first 2 min shows a lower proportion of high molecular weight components. By the end of 5 days, all the shorter chains have been cleaved off, and accumulated hydrolysate no longer shows a preponderance toward the higher molecular weight components. Figure 3B shows the cumulative weight fraction of polymer cleaved from the PS bead surface at 30 min and at 5 days. The cumulative weight fraction of high molecular weight chains is higher for the hydrolysate collected at 30 min than for the hydrolysate collected at 5 days, indicative of selective hydrolysis of longer chains. Greater amounts of longer polymer chains are hydrolyzed in the early stages of the hydrolysis, while the shorter chains are hydrolyzed more slowly. Measurements from an independent experiment on the hydrolysis of PDMA brush from PS beads confirmed these results (Figure S3 and Table S3 in the Supporting Information). If we consider these results, we may see that the hydrolysis is selective for the higher molecular weight components in the early stages of the reaction. We can also see that this effect is not at its most pronounced during the first 2 min, when the grafting density is highest, but rather when the grafting density has been decreased by the initial hydrolysis period. We therefore suggest that the initial high grafting density causes an increased rate of hydrolysis, as seen on the flat surfaces. Our hypothesis is therefore that all molecules initially have sufficient lateral interaction to cause the accelerated hydrolysis; as the reaction progresses, the smaller molecules become less likely to have such interactions, and so the hydrolysis reaction becomes selective for the larger chains. One effect of this is that the 6468

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way in which these forces contribute to the enhanced reactivity of bonds within the brushes is unknown. More work is needed to elucidate this. The AFM force−distance curves show that the polymers interact with the substrate. This is indicated by the characteristic plateaus in some of the force−distance curves obtained at low grafting densities,36 as shown in Figure S5 of the Supporting Information. Adsorption of polymers to surfaces can lead to bond breakage, provided that sufficient interactions are formed.2 The adsorption itself does not seem to be driving the accelerated hydrolysis because the extent of polymer interaction with the substrate would increase at the lower grafting densities,37 where the hydrolysis rate is the lowest. It is however possible that the adsorption in conjunction with the lateral pressure4,35 at higher grafting densities could cause the polymer chains to be subjected to some degree of shear stress.

molecular weight analysis from incomplete hydrolysates may not reflect the values of the tethered polymers. Base-catalyzed hydrolysis of esters is a BAC2 reaction, proceeding through a tetrahedral intermediate with acyl cleavage. Recently, Akbulatov et al.31 found that hydrolysis of esters with straight alkyl chains has relatively low sensitivity to tensile loads because the pulling axis of small molecules with an ester group is nearly orthogonal to the reaction coordinate for the reaction-limiting transition state. The model studies on small molecules containing an ester group suggested that the hydrolysis rate would be decreased at lower forces, through an entropic mechanism, and then increased at forces greater than 500 pN.31 There is also experimental evidence that shear force along the polymer chains can be sufficient to cleave polymers.32 Elongational flow has also been shown to enhance the hydrolysis of DNA33 and of polyacrylamide.34 In these examples, hydrolysis involves breaking of the ester O−PO and the amide N−CO bonds. Tensile stress stretches the chain, facilitating the formation of a transition state and enhancing the chemical process. We will now consider briefly the forces that are acting on the polymer chains within the brush. There will be a force acting normal to the surface, stretching the chains away from their normal solution conformation to the conformation present in the brush. The degree of stretching increases with increase in grafting density. There will also be forces acting laterally within the brush and in the plane of the substrate, related to the surface pressure. We do not have a way to measure the force acting normal to the substrate, but we can estimate the force to stretch the polymer chains using the AFM pulling experiments. The magnitude of the force required to stretch the polymer chains to their full extent with the AFM tip is on the order of 150 pN. The polymer chains in aqueous brushes will be stretched to some extent due to the high grafting density; the AFM pulling experiments do not take this effect into account and do not fully reflect the normal force required to stretch the chains. It may, however, be noted that the results showed little variation with density, polymer chain length, or solvent-induced collapse of the brushes (see Supporting Information, Figure S4). The measured value is well below the 500 pN range at which pulling along the axis of small ester group-containing molecules would be expected to increase the reaction rates.31 We can therefore see that even when the polymer chains are being actively pulled by the AFM tip, we do not expect to apply sufficient force normal to the surface to accelerate the hydrolysis reaction. It may therefore be assumed that the forces acting normal to the surface in the undisturbed brush will not be high enough to affect the hydrolysis rates. Increasing grafting density and molecular weight will also lead to increased lateral pressure within the brush.22,23,26 Forces in the order of 0.5−1.92 nN/nm were reported in the cases of polyelectrolyte brushes grown on gold cantilevers.35 For high molecular weight PDMA brushes similar to those described here the lateral force has been reported to be relatively high (12−74 N/m; increases with grafting density), sufficient to cause buckling of soft substrates, or to stretch the substrates if the brush has been produced on both the top and bottom surfaces.4 If we assume a width equal to twice the Rg for the polymer chains, then we have a force per chain in the 100 nN range (sample calculation: 74 N/m × 2 × 10−9 m = 148 nN). This compressive force is considerably higher than the stretching forces predicted to enhance the hydrolysis of ester groups.31 Because of the absence of experimental evidence, the

4. CONCLUSIONS In summary, we synthesized PDMA brushes with ester-linked polymer chains on substrates. The hydrolysis of polymer brushes from the substrate was accelerated by increasing the chain length and increasing the grafting density. Both of these factors are known to be linked to the lateral pressure within the brush; it therefore appears that the reaction rate is linked to mechanical forces within the brush. For polydisperse brushes, the GPC analysis of hydrolysates indicated that the hydrolysis is selective for longer chains at an early stage of the reaction, possibly due to the accelerated hydrolysis of the longer chains. This effect could potentially be exploited when preparing polymer brushes and should also be considered when analyzing polymers based on hydrolysates.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details, additional results, and calculations. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01424.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (J.N.K.). *E-mail [email protected] (J.L.T.-H.). Author Contributions

K.A.M. and K.Y. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the Canadian Institutes of Health Research (CIHR) and Natural Science and Engineering Research Council (NSERC) of Canada. The authors thank the LMB Macromolecular Hub at the UBC Centre for Blood Research for use of the analytical facilities. The infrastructure facility is supported by Canada Foundation for Innovation (CFI) and Michael Smith Foundation of Health Research (MSFHR). J.N.K. is recipient of a Career Investigator Scholar award from MSFHR.

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REFERENCES

(1) Kucharksi, T. J.; Yang, Q. Z.; Tian, Y.; Boulatov, R. StrainDependent Acceleration of a Paradigmatic SN2 Reaction Accurately

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Article

Langmuir Predicted by the Force Formalism. J. Phys. Chem. Lett. 2010, 1, 2820− 2825. (2) Sheiko, S. S.; Sun, F. C.; Randall, A.; Shirvanyants, D.; Rubinstein, M.; Lee, H.; Matyjaszewski, K. Adsorption-Induced Scission of Carbon−Carbon Bonds. Nature 2006, 440, 191−194. (3) Park, I.; Shirvanyants, D.; Nese, A.; Matyjaszewski, K.; Rubinstein, M.; Sheiko, S. S. Spontaneous and Specific Activation of Chemical Bonds in Macromolecular Fluids. J. Am. Chem. Soc. 2010, 132, 12487−12491. (4) Zou, Y.; Lam, A.; Brooks, D. E.; Phani, S.; Kizhakkedathu, J. N. Bending and Stretching Actuation of Soft Materials through SurfaceInitiated Polymerization. Angew. Chem., Int. Ed. 2011, 50, 5116−5119. (5) Krishnamoorthy, M.; Hakobyan, S.; Ramstedt, M.; Gautrot, J. E. Surface-Initiated Polymer Brushes in the Biomedical Field: Applications in Membrane Science, Biosensing, Cell Culture, Regenerative Medicine and Antibacterial Coatings. Chem. Rev. 2014, 114, 10976− 11026. (6) Ayres, N. Polymer Brushes: Applications in Biomaterials and Nanotechnology. Polym. Chem. 2010, 1, 769−777. (7) Yu, K.; Mei, Y.; Hadjesfandiari, N.; Kizhakkedathu, J. N. Engineering Biomaterials Surfaces to Modulate the Host Response. Colloids Surf., B 2014, 124, 69−79. (8) Senaratne, W.; Andruzzi, L.; Ober, C. K. Self-Assembled Monolayers and Polymer Brushes in Biotechnology: Current Applications and Future Perspectives. Biomacromolecules 2005, 6, 2427−2448. (9) Lai, B. F. L.; Creagh, A. L.; Janzen, J.; Haynes, C. A.; Brooks, D. E.; Kizhakkedathu, J. N. The Induction of Thrombus Generation on Nanostructured Neutral Polymer Brush Surfaces. Biomaterials 2010, 31, 6710−6718. (10) Jiang, H.; Wang, X. B.; Li, C. Y.; Li, J. S.; Xu, F. J.; Mao, C.; Yang, W. T.; Shen, J. Improvement of Hemocompatibility of Polycaprolactone Film Surfaces with Zwitterionic Polymer Brushes. Langmuir 2011, 27, 11575−11581. (11) Feng, W.; Brash, J. L.; Zhu, S. Non-biofouling Materials Prepared by Atom Transfer Radical Polymerization Grafting of 2Methacryloloxyethyl Phosphorylcholine: Separate Effects of Graft Density and Chain Length on Protein Repulsion. Biomaterials 2006, 27, 847−855. (12) Matyjaszewski, K.; Miller, P. J.; Shukla, N.; Immaraporn, B.; Gelman, A.; Luokala, B. B.; Siclovan, T. M.; Kickelbick, G.; Vallant, T.; Hoffmann, H.; Pakula, T. Polymers at Interfaces: Using Atom Transfer Radical Polymerization in the Controlled Growth of Homopolymers and Block Copolymers from Silicon Surfaces in the Absence of Untethered Sacrificial Initiator. Macromolecules 1999, 32, 8716−8724. (13) Kizhakkedathu, J. N.; Brooks, D. E. Synthesis of Poly(N,Ndimethylacrylamide) Brushes from Charged Polymeric Surfaces by Aqueous ATRP: Effect of Surface Initiator Concentration. Macromolecules 2003, 36, 591−598. (14) Luzinov, I.; Julthongpiput, D.; Malz, H.; Pionteck, J.; Tsukruk, V. V. Polystyrene Layers Grafted to Epoxy-Modified Silicon Surfaces. Macromolecules 2000, 33, 1043−1048. (15) Eisele, N. B.; Frey, S.; Piehler, J.; Görlich, D.; Richter, R. P. Ultrathin Nucleoporin Phenylalanine−Glycine Repeat Films and Their Interaction with Nuclear Transport Receptors. EMBO Rep. 2010, 11, 366−372. (16) Pericet-Camara, R.; Papastavrou, G.; Behrens, S. H.; Helm, C. A.; Borkovec, M. Interaction Forces and Molecular Adhesion between Pre-adsorbed Poly(ethylene imine) Layers. J. Colloid Interface Sci. 2006, 296, 496−506. (17) Zou, Y. Q.; Kizhakkedathu, J. N.; Brooks, D. E. Surface Modification of Polyvinyl Chloride Sheets via Growth of Hydrophilic Polymer Brushes. Macromolecules 2009, 42, 3258−3268. (18) Yu, K.; Kizhakkedathu, J. N. Synthesis of Functional Polymer Brushes Containing Carbohydrate Residues in the Pyranose Form and Their Specific and Nonspecific Interactions with Proteins. Biomacromolecules 2010, 11, 3073−3085.

(19) Perrier, S.; Haddleton, D. M. Effect of Water on Copper Mediated Living Radical Polymerization. Macromol. Symp. 2002, 182, 261−272. (20) Zhulina, E. B.; Rubinstein, M. Lubrication by Polyelectrolyte Brushes. Macromolecules 2014, 47, 5825−5838. (21) Brittain, W. J.; Minko, S. A Structural Definition of Polymer Brushes. J. Polym. Sci., Polym. Chem. 2007, 45, 3505−3512. (22) Currie, E. P. K.; Leermakers, F. A. M.; Cohen Stuart, M. A.; Fleer, G. J. Grafted Adsorbing Polymers: Scaling Behavior and Phase Transitions. Macromolecules 1999, 32, 487−498. (23) Birshtein, T. M.; Lakovlev, P. A.; Arnoskov, V. M.; Leermakers, F. A. M.; Zhulina, E. B.; Borisov, O. V. On the Curvature Energy of a Thin Membrane Decorated by Polymer Brushes. Macromolecules 2008, 41, 478−488. (24) Dordi, B.; Schönherr, H.; Vansco, G. J. Reactivity in the Confinement of Self-Assembled Monolayers: Chain Length Effects on the Hydrolysis of N-Hydroxysuccinimide Ester Disulfides on Gold. Langmuir 2003, 19, 5780−5786. (25) Schönherr, H.; Chechik, V.; Stirling, C. J. M.; Vansco, G. J. Monitoring Surface Reactions at an AFM Tip: An Approach To Follow Reaction Kinetics in Self-Assembled Monolayers on the Nanometer Scale. J. Am. Chem. Soc. 2000, 122, 3679−3687. (26) Carignano, M. A.; Szleifer, I. On the Structure and Pressure of Tethered Polymer Layers in Good Solvent. Macromolecules 1995, 28, 3197−3204. (27) Unsworth, L. D.; Tun, Z.; Sheardown, H.; Brash, J. L. Chemisorption of Thiolated Poly(ethylene oxide) to Gold: Surface Chain Densities Measured by Ellipsometry and Neutron Reflectometry. J. Colloid Interface Sci. 2005, 281, 112−121. (28) Zheng, J. X.; Xiong, H.; Chen, W. Y.; Lee, K.; Van Horn, R. M.; Quirk, R. P.; Lotz, E. L.; Shi, A. C.; Cheng, S. Z. D. Onsets of Tethered Chain Overcrowding and Highly Stretched Brush Regime via Crystalline−Amorphous Diblock Copolymers. Macromolecules 2006, 39, 641−650. (29) Yamamoto, S.; Tsujii, Y.; Fukuda, T. Atomic Force Microscopic Study of Stretching a Single Polymer Chain in a Polymer Brush. Macromolecules 2000, 33, 5995−5998. (30) Brotherson, B.; Bottomley, L. A.; Ludovice, P.; Deng, Y. Cationic Polyacrylamide Conformation on Mica Studied by Single Molecule “Pulling” with Scanning Probe Microscopy. Macromolecules 2007, 40, 4561−4567. (31) Akbulatov, S.; Tian, Y.; Kapustin, E.; Boulatov, R. Model Studies of the Kinetics of Ester Hydrolysis under Stretching Force. Angew. Chem., Int. Ed. 2013, 52, 6992−6995. (32) Caruso, M. M.; Davis, D. A.; Shen, Q.; Odom, S. A.; Sottos, N. R.; White, S. R.; Moore, J. S. Mechanically-Induced Chemical Changes in Polymeric Materials. Chem. Rev. 2009, 109, 5755−5798. (33) Adam, R. E.; Zimm, B. H. Shear Degradation of DNA. Nucleic Acids Res. 1977, 4, 1513−1537. (34) Basedow, A. M.; Ebert, K. H.; Hunger, H. Effect of Mechanical Stress on the Reactivity of Polymers: Shear Degradation of Polyacrylamide and Dextran. Makromol. Chem. 1979, 180, 411−427. (35) Kelby, T. S.; Huck, W. T. S. Controlled Bending of Microscale Au−Polyelectrolyte Brush Bilayers. Macromolecules 2010, 43, 5382− 5386. (36) Sonnenberg, L.; Parvole, J.; Kühner, F.; Billon, L.; Gaub, H. E. Choose Sides: Differential Polymer Adhesion. Langmuir 2007, 23, 6660−6666. (37) Goodman, D.; Kizhakkedathu, J. N.; Brooks, D. E. Attractive Bridging Interactions in Dense Polymer Brushes in Good Solvent Measured by Atomic Force Microscopy. Langmuir 2004, 20, 2333− 2340. (38) Matyjaszewski, K.; Dong, H.; Jakubowski, W.; Pietrasik, J.; Kusumo, A. Grafting from Surfaces for “Everyone”: ARGET ATRP in the Presence of Air. Langmuir 2007, 23, 4528−4531.

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