Temperature-Induced Shape Changing of Thermosensitive Binary

Article pubs.acs.org/Macromolecules

Temperature-Induced Shape Changing of Thermosensitive Binary Heterografted Linear Molecular Brushes between Extended Wormlike and Stable Globular Conformations Daniel M. Henn,† Wenxin Fu,† Shan Mei,‡ Christopher Y. Li,‡ and Bin Zhao*,† †

Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, United States Department of Materials Science and Engineering, Drexel University, Philadelphia, Pennsylvania 19104, United States

‡

S Supporting Information *

ABSTRACT: Inspired by stimuli-triggered unraveling of the von Willebrand factor from a nonsticky globular to a stretched linear shape with exposure of functional groups in blood clotting, this article reports on the synthesis of thermosensitive binary heterografted linear molecular brushes that exhibit temperature-induced shape changing between extended wormlike and collapsed yet stable globular conformations in water. The molecular brushes are composed of two distinct side chain polymers: thermosensitive poly(ethoxydi(ethylene glycol) acrylate) (PDEGEA), which undergoes a lower critical solution temperature (LCST) transition at 9 °C in water, and poly(ethylene oxide) (PEO), which serves as a stabilizer for the collapsed state. A “grafting to” method was developed to construct molecular brushes by clicking alkyne end-functionalized side chain polymers onto an azide-bearing backbone polymer. While a 1.0 mg/g aqueous solution of PDEGEA homografted molecular brushes turned cloudy upon heating from 0 to 22 °C, at the same concentration the aqueous solution of PEO/PDEGEA binary molecular brushes remained clear, indicating the stabilization of the collapsed state against aggregation by PEO side chains. Atomic force microscopy study revealed a stretched, wormlike morphology of brushes at 0 °C and compact, globular nano-objects at 40 °C. The thermally induced shape changing was exploited to regulate the binding of biotin, which was incorporated into the thermosensitive side chains along with a fluorescent resonance energy transfer (FRET) donor, and Rhodamine B (FRET acceptor)-labeled avidin. FRET study showed that when the molecular brushes changed from the globular to the wormlike state, the binding of biotin and avidin occurred and increased significantly with time.

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INTRODUCTION

functional groups upon unraveling, allowing the macromolecules to interact with environment. Molecular brushes or bottlebrushes,4−7 composed of polymer side chains densely grafted to a backbone polymer, are excellent synthetic polymer candidates for mimicking some aspects of the VWF’s behavior. These brush molecules are a unique class of macromolecules, whose shapes and dimensions are determined by backbone and side chain lengths as well as interactions of side chains with themselves and environment, and have attracted tremendous attention due to their interesting behavior and potential applications.4−23 In general, molecular brushes are prepared by three methods: (i) “grafting through”, where macromonomers are directly polymerized into brushes; (ii) “grafting from”, where side chains are grown from the backbone, typically by a “living”/controlled polymerization; and (iii) “grafting to”, where end-functionalized polymers are grafted onto the backbone.4−7 Each of these methods has its

The von Willebrand factor (VWF) is a fascinating glycoprotein in our body, which plays an indispensable role in the cascade of molecular events in the blood clotting process.1−3 This protein is a “complex homopolymer”, where each monomer unit is made up of 4100 amino acids and contains ∼15 different folded domains. The VWF is an interesting stimuli-responsive molecular glue. In its quiescent globular state, it is nonsticky and soluble. When an injury occurs, the mechanical and chemical cues activate the VWF, which undergoes unraveling from a compact globular to an extended shape and exposes a large number of binding sites, enabling VWF to bind rapidly to the injured vessel area. The intriguing behavior of VWF in blood clotting provides an inspiration for design of advanced stimuli-responsive soft materials with unique structures and properties.1 We are particularly interested in designing and synthesizing organic polymers that can exhibit two features of the VWF in blood clotting: (i) on demand stimuli-triggered shape changing from a compact globular to an extended wormlike nanostructure; (ii) exposure of a large number of © XXXX American Chemical Society

Received: January 20, 2017 Revised: February 4, 2017

A

DOI: 10.1021/acs.macromol.7b00150 Macromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Temperature-Induced Shape Changing of Thermosensitive Binary Heterografted Linear Molecular Brushes between Stable Globular and Extended Wormlike Conformations

Scheme 2. Synthesis of Binary Heterografted Linear Molecular Brushes by a “Grafting To” Method Using Cu(I)-Catalyzed Azide−Alkyne Cycloaddition Reaction (CuAAC) and Molecular Structures of Backbone Polymer PTEGN3MA and Side Chain Polymers PEO and PDEGEA

own advantages and disadvantages. In particular, “grafting to” is a straightforward and modular approach, allowing the use of preformed, well-defined backbone and side chain polymers, which is well suited for the synthesis of heterografted molecular brushes composed of two or more different side chain polymers. However, the grafting density tends to be low. This scenario has been changed effectively after the highly efficient click reactions, e.g., copper-catalyzed azide−alkyne cycloaddition (CuAAC), were introduced into polymer chemistry.24−26 It has been shown that the grafting densities of molecular brushes by click “grafting to” methods can be comparable to those from “grafting from”.27−30 When stimuli-responsive polymers, which exhibit abrupt conformational or solubility changes in a solvent in response to external triggers,31,32 are employed as side chains, the shape of molecular brushes can be altered, often reversibly, by external stimuli.4−7,33 There has been increasing interest in stimuliresponsive molecular brushes in recent years.33−51 However, only a limited number of studies focused on triggered shape changing of single molecular brushes in solution. The first thermosensitive example, reported by Schmidt et al.,34 was poly(N-isopropylacrylamide) (PNIPAM) molecular brushes synthesized by a grafting from approach using atom transfer radical polymerization (ATRP). These brushes underwent a shape transition from wormlike to globular conformations upon heating above the LCST of PNIPAM. However, the globular nano-objects were unstable above the LCST; aggregation and eventually precipitation occurred.34 Müller et al. made thermoand pH-responsive poly(N,N-dimethylaminoethyl methacrylate) molecular brushes and observed shape changes induced by environmental variations.43 They also reported the morphological switching of cationic brush molecules using ionic and supramolecular inclusion complexes in dilute conditions;46 the complexation of cationic brushes with sodium dodecyl sulfate (SDS) resulted in collapse of brushes from wormlike to globular. Addition of β-cyclodextrins (β-CD) switched the morphology back to wormlike because of the formation of a SDS/β-CD inclusion complex. The brushes collapsed again into a globular shape when a more competitive inclusion agent was used. However, at a slightly higher concentration (e.g., 1 mg/mL), precipitation occurred immediately when SDS was

added.46 Although the stimuli-induced shape changing makes molecular brushes excellent candidates for mimicking some functions of VWF, the collapsed brushes in solution in the literature were not stabilized after the worm-to-globular transition. Note that Sheiko et al. conducted extensive studies of worm-to-globular transitions of hydrophobic molecular brushes at interface induced by lateral pressure, solvent vapors, etc.5,6,9,52,53 Herein, we report the synthesis of thermosensitive binary heterografted molecular brushes that can undergo switching between extended wormlike and stable globular shapes in water in response to temperature changes (Scheme 1). The binary brushes are composed of two distinct side chain polymers: thermosensitive poly(ethoxydi(ethylene glycol) acrylate) (PDEGEA), which exhibits a lower critical solution temperature (LCST) transition at 9 °C in water,54,55 thus driving the shape transitions of molecular brushes in response to temperature changes, and poly(ethylene oxide) (PEO), which serves as a stabilizer for the collapsed state (Scheme 1). The PDEGEA/PEO brushes were synthesized via a “grafting to” approach using highly efficient CuAAC;24−27 alkyne endfunctionalized side chain polymers were clicked onto an azidebearing backbone polymer (Scheme 2), producing molecular brushes with relatively high grafting densities. The thermally induced shape and size changes of binary brushes between linear wormlike and collapsed yet stable globular single macromolecular nano-objects were studied by visual inspection, dynamic light scattering (DLS), and atomic force microscopy (AFM). We further demonstrated that the temperature-induced shape changing can be exploited to regulate the interaction of brush molecules with their environment using biotin−avidin complexation as an example.

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RESULTS AND DISCUSSION Synthesis of Azide-Functionalized Backbone Polymer PTEGN3MA. To synthesize the targeted binary heterografted molecular brushes that are capable of changing their shapes between wormlike and stable globular in response to temperature changes, a modular “grafting to” method was developed in conjunction with CuAAC click chemistry, which allows for simultaneous, random attachment of two or more B


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Scheme 3. Synthesis of Azide-Functionalized Backbone Polymer PTEGN3MA by Atom Transfer Radical Polymerization (ATRP) and Subsequent Postpolymerization Reactions

different side chain polymers to the backbone and facile tuning of relative molar ratio(s). The azide-functionalized backbone polymer, PTEGN3MA (Scheme 2), was synthesized by ATRP of TEGSiMA, a silyl ether-protected methacrylate monomer, followed by the removal of tert-butyldimethylsilyl ether protective groups, then reaction with 4-toluenesulfonyl chloride (tosyl chloride), and finally substitution with sodium azide to yield PTEGN3MA (Scheme 3). TEGSiMA was designed and synthesized for the following three considerations. (i) ATRP of silyl ether protected methacrylates can be well-controlled, producing well-defined backbone polymer precursors with sufficiently high degrees of polymerization (DPs) and small polydispersity indices.10 A sufficiently long backbone, thus a large aspect ratio, is necessary to achieve shape transitions between wormlike and globular conformations for molecular brushes. (ii) The desired azide functionality can be introduced through a series of nearly quantitative postpolymerization reactions.10,24−26,56 (iii) The incorporation of a tri(ethylene glycol) spacer between backbone and azide moiety should alleviate steric hindrance during the “grafting to” reaction for bulky thermosensitive PDEGEA and result in high grafting densities. We used this synthetic route instead of direct polymerization of a halogen-, tosylate-, or azide-containing methacrylate because it is potentially problematic to synthesize well-defined polymers with both large DPs and narrow polydispersities due to side reactions involving alkyl bromide, tosylate, and azide groups.57,58 TEGSiMA was synthesized by first protecting one hydroxyl group of triethylene glycol with tert-butylchlorodimethylsilane and subsequently reacting with methacryloyl chloride (Scheme S1). The monomer was purified by column chromatography and vacuum distillation, and the molecular structure was verified by 1H and 13C NMR spectroscopy (Figure S1) and high-resolution mass spectrometry analysis. A PTEGSiMA backbone polymer precursor with a DP of 800 was synthesized by ATRP, carried out in anisole at 60 °C using ethyl 2-bromoisobutyrate as initiator and CuBr/CuBr2/ N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) as catalyst and was monitored by 1H NMR spectroscopy and size exclusion chromatography (SEC) analysis. The DP was calculated from the monomer-to-initiator molar ratio and the final monomer conversion determined by 1H NMR analysis. SEC of the purified PTEGSiMA showed that the ATRP of TEGSiMA was well-controlled, yielding a PDI of 1.11 for the final polymer (Figure S2). PTEGSiMA was then transformed into PTEGN3MA through a series of postpolymerization reactions (Scheme 3). The silyl ether protecting group was first removed using HCl in ethanol, followed by tosylation, and substitution with sodium azide to yield PTEGN3MA. Figure 1

Figure 1. 1H NMR spectra of PTEGSiMA, the tosylated backbone intermediate PTEGTsMA, and PTEGN3MA in CDCl3.

shows the 1H NMR spectra of PTEGSiMA, the tosylated intermediate (PTEGTsMA), and PTEGN3MA. The high efficiency of the deprotection and tosylation reactions can be clearly seen from the nearly complete disappearance of the silyl ether peak at 0.05 ppm (−OSi(C(CH3)3)(CH3)2) and the appearance of tosylate peaks at 7.67−7.81 and 7.27−7.38 ppm (aromatic), 4.11 ppm (−CH 2 OTs), and 2.41 ppm (−C6H4CH3). After the reaction of PTEGTsMA with sodium azide, the tosylate peaks almost disappeared, accompanied by the appearance of a peak at 3.34−3.47 ppm (−OCH2CH2N3). By using the integrals of the peaks at 3.98−4.14 ppm (−COOCH2CH2−) and 3.34−3.47 ppm (−OCH2CH2N3), the degree of azide functionalization was calculated to be 90%, which was lower than 100% likely due to a very small amount of incomplete reactions as can be seen from a small silyl ether peak remaining at 0.05 ppm in Figure 1 as well as some unclear side reactions during the postpolymerization modifications. In particular, there appears to be a small second set of tosyl peaks at 7.71−7.77 and 7.08−7.14 ppm (aromatic) and 2.31 ppm (−C6H4CH3) which did not disappear after reaction with sodium azide. SEC analysis of the obtained PTEGN3MA indicated that the peak remained unimodal and narrow (Figure S3). The characterization data for PTEGSiMA and PTEGN3MA are summarized in Table 1. Synthesis of PDEGEA and PEO Side Chain Polymers. Alkyne end-functionalized thermosensitive PDEGEA side chain polymer with a DP of 46 was synthesized by ATRP using an alkyne-containing initiator, propargyl 2-bromoisobutyrate. The polymerization was carried out in anisole at 80 °C using CuCl/ PMDETA as catalyst and stopped when the desired molecular C


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containing PTEGN3MA and alkyne end-functionalized PEO or/and PDEGEA side chain polymers. The reactions were carried out in DMF using CuCl/PMDETA as catalyst at ambient temperature and a feed molar ratio of ∼1:2 for backbone monomer units to side chains. The grafting density, defined as the percentage of backbone repeat units that are grafted with a side chain polymer, was calculated from the SEC chromatogram using the ratio of peak areas from the brushes and the unreacted side chain polymers and the feed molar ratio of backbone monomer units to the side chain polymers. With a series of mixtures of two polystyrene homopolymers with very different molecular weights, we confirmed that the peak area ratio of the two different molecular weight polymers is essentially the same as the mass ratio (Figure S6). For heterografted molecular brushes, we assumed that the molar ratio of side chains in the brushes was the same as in the feed for grafting density calculation. We first prepared PDEGEA and PEO homografted molecular brushes from PTEGN3MA and PDEGEA and PEO-45 or -114, respectively. PDEGEA homografted molecular brushes (HMB-1 in Table 2) were prepared using a molar ratio of 1:1.89 for PTEGN3MA backbone monomer units to PDEGEA. The reaction progress was followed by SEC analysis. Figure 2A shows the SEC data of the final reaction mixture after 48 h, with the SEC trace of PTEGN3MA being included for comparison; a high molecular weight peak appeared with a Mn,SEC of 1 086 000 and PDI of 1.12, indicating the formation of brush molecules. The PDEGEA side chain polymer peak was however broadened, likely because of the coupling reaction59 between PDEGEA side chains. Based on the relative peak areas (40.7% brushes and 59.3% unreacted PDEGEA) and the feed molar ratio, the grafting density was calculated to be 75.9% (see Supporting Information). The excess PDEGEA was removed by fractionation using THF (a good solvent) and hexanes (a poor solvent) as confirmed by SEC analysis (Figure 2A). The 1 H NMR spectrum of HMB-1 is shown in Figure S7. PEO homografted molecular brushes (HMB-2 and -3) were prepared by a procedure similar to that for HMB-1 using PEO45 and PEO-114. A molar ratio of 1:2.01 for the PTEGN3MA backbone monomer units to PEO-45 was used to synthesize HMB-2 (Figure S8A). After the reaction proceeded for 22 h, SEC analysis showed that the mixture contained 45.1% brushes, corresponding to a grafting density of 85.9%. This indicates that almost every azide moiety in PTEGN3MA had reacted with a PEO side chain because the degree of azide functionalization of PTEGN3MA was 90%. The higher grafting density of PEO

Table 1. Characterization Data for PTEGSiMA, PTEGN3MA, and Side Chain Polymers backbone or side chain polymer PTEGSiMA PTEGN3MA PEO-45 PEO-114 PDEGEA P(DEGEA-co-BA-co-NBDA)

Mn,SEC (kDa) a

112.6 313.4b 2.8a 8.7a 8.8a 10.6a

PDI a

1.11 1.09b 1.08a 1.04a 1.17a 1.16a

DP 800 800 45c 114c 46d 50d

a

The number-average molecular weight (Mn,SEC) and polydispersity index (PDI) were determined by SEC relative to polystyrene standards using PL-GPC 20 with THF as solvent. bObtained by SEC relative to polystyrene standards using PL-GPC 50 Plus system with DMF containing 50 mM LiBr as mobile phase. cCalculated from the nominal molecular weight (2 or 5 kDa). dDetermined by end-group analysis from 1H NMR spectra.

weight was achieved. The monomer conversion was kept below 50% in order to minimize any possible side reactions involving the alkyne group.57 SEC analysis showed that the ATRP was well controlled, as reflected by a narrow, monomodal peak with a PDI of 1.17 (Figure S4). The DP of 46 was calculated from the 1H NMR spectrum of the purified polymer using the integrals of the peaks at 4.62 ppm (HCCCH2OOC− of the end group) and 4.07−4.24 ppm (−COOCH2CH2− of DEGEA monomer units) (Figure S5A). Alkyne end-functionalized PEO side chain polymers with DPs of 45 and 114 (PEO-45 and PEO-114, respectively) were synthesized by reacting the −OH end group of commercially available PEO monomethyl ethers with molecular weights of 2 and 5 kDa with 4-pentynoic acid activated by N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride in the presence of DMAP catalyst (Scheme S2).56 Both PEO side chain polymers were purified by washing the dichloromethane reaction mixtures with water and aqueous NaOH, followed by precipitation in diethyl ether. The SEC traces of PEO-45 and PEO-114 are shown in Figure S4. From the 1H NMR spectroscopy analysis, the degree of alkyne functionalization was determined to be essentially quantitative for both PEO-45 (Figure S5B) and PEO-114 using the integral values of the peaks at 4.21 ppm (HCCCH2CH2COOCH2−) and 3.33 ppm (−OCH3 of methyl end group) of the purified polymer. Synthesis of PDEGEA and PEO Homografted and PEO/PDEGEA Binary Heterografted Molecular Brushes. Homografted and binary heterografted molecular brushes were synthesized by using CuAAC “click” reactions between azide-

Table 2. Characterization Data for Homografted and Binary Heterografted Molecular Brushes molecular brushes sample HMB-1 (PDEGEA) HMB-2 (PEO-45) HMB-3 (PEO-114) BMB-4 (PEO/PDEGEA) BMB-5 PEO/P(DEGEA-coBA-co-NBDA)

a

backbone units:PEO:PDEGEA in the feed

Mn,SECb (kDa); PDI

grafting densityc (%)

feed molar ratio of PEO to PDEGEAd

molar ratio of PEO to PDEGEA in brushese

1:0:1.89 1:2.01:0 1:1.81:0 1:1.10:0.723 1:1.15:0.768

1086.0; 1.12 990.5; 1.12 1199.3; 1.15 927.2; 1.11 1226.2; 1.12

75.9 85.9 78.6 74.3 77.5

0:1 1:0 1:0 0.60:0.40 0.60:0.40

0:1 1:0 1:0 0.62:0.38 0.61:0.39

a

HMB stands for homografted molecular brushes, and BMB stands for binary heterografted molecular brushes. bThe Mn,SEC and polydispersity index (PDI) were measured by SEC of the final reaction mixture relative to polystyrene standards using PL-GPC 50 Plus with Agilent Mixed-B columns in DMF containing 50 mM LiBr. cGrafting density was calculated from the ratio of SEC peak areas of molecular brushes and unreacted side chains in the final reaction mixture and the feed molar ratio of backbone monomer units to side chains. dMolar ratios of PEO to PDEGEA in the feed. eMolar ratio of PEO to PDEGEA calculated from the 1H NMR spectrum. D


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Figure 2. SEC traces of (A) PDEGEA HMB-1 homografted molecular brushes before and after removal of unreacted PDEGEA side chains by fractionation and (B) PEO/PDEGEA BMB-4 before and after removal of excess side chains.

Figure 3. (A) Optical photographs of 1.0 mg/g aqueous solutions of PDEGEA homografted HMB-1 and PEO/PDEGEA binary heterografted BMB4 molecular brushes at 0 and 22 °C. (B) Apparent hydrodynamic size (Dh) of HMB-1 in water at a concentration of 0.2 mg/g as a function of temperature, obtained from a DLS study. (C) Plot of Dh of BMB-4 in water at a concentration of 0.2 mg/g versus temperature. (D) Size distribution by intensity from DLS measurements of BMB-4 at 25 °C at concentrations of 0.2 and 1.0 mg/g.

HMB-2 (85.9%), compared with that of PDEGEA HMB-1 (75.9%), likely resulted from the low steric hindrance of PEO45 due to its linear and flexible chain structure. We also prepared PEO brushes HMB-3 using the PTEGN3MA and PEO-114 with a molar ratio of 1:1.81 (Figure S8B and Table 2). After 17 h, the grafting density reached 78.6%, which was lower than that of HMB-2 but slightly higher than HMB-1. PEO/PDEGEA binary heterografted molecular brushes, in which PEO and PDEGEA side chains were randomly distributed along the backbone, were then prepared from PTEGN3MA and a mixture of PEO-45 and PDEGEA. We used a molar ratio of 1:1.82 for PTEGN3MA backbone units to total side chain polymers PEO-45 and PDEGEA, with a molar ratio of 0.60:0.40 for PEO-45 to PDEGEA-46 in the feed, to synthesize heterografted molecular brushes (BMB-4 in Table 2). The feed molar ratio of 0.6:0.4 for PEO-45 to PDEGEA was used to make molecular brushes that were slightly enriched

with PEO so as to provide increased stability to the collapsed state in water. Figure 2B shows the SEC traces of the final reaction mixture at 22 h and BMB-4 after the removal of unreacted side chains by centrifugal filtration and fractionation; the Mn,SEC of the brushes in the mixture was 927 200 Da, and the PDI was 1.11. By using the relative peak areas from SEC analysis and the feed ratio and assuming that the brush composition was the same as the feed, the grafting density of BMB-4 was calculated to be 74.3%. The reaction was stopped by injecting excess propargyl alcohol in an attempt to avoid the slight increase of the front shoulder observed for HMB-1 by capping unreacted azide units, which are known to undergo thermal- and light-induced decomposition into reactive nitrenes capable of resulting in cross-linking reactions.58,60 The mixture was allowed to stir for an additional 1 h before it was opened to air and passed through neutral alumina to remove the catalyst. The unreacted side chains were completely removed by E


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Figure 4. AFM height images of PEO/PDEGEA BMB-4 brush molecules spin coated onto freshly cleaved mica from aqueous solutions at (A) 0 °C with a polymer concentration of 0.1 mg/g and (B) 40 °C with polymer concentration of 0.01 mg/g.

raised to 17 °C. Similar to HMB-1, there was essentially no change in size upon further heating, indicating that BMB-4 underwent unimolecular collapse above the LCST of PDEGEA. Interestingly, the Dh observed for BMB-4 at 1 °C (63 nm) was noticeably smaller than that of HMB-1 (76 nm), while the Dh of BMB-1 at 25 °C (43 nm) was slightly larger than HMB-1 (40 nm). This is likely due to the relatively small size of PEO45 compared with PDEGEA, resulting in a smaller Dh for the wormlike state of BMB-4. Conversely, the slightly larger size of collapsed BMB-4 at 25 °C compared with HMB-1 was due to the hydrated PEO corona of BMB-4. We also conducted a DLS study of a 1.0 mg/g aqueous solution of BMB-4 at 25 °C, which revealed a single size distribution overlapping with that for the 0.2 mg/g BMB-4 solution and a Dh of 43 nm identical to that of BMB-4 at a concentration of 0.2 mg/g (Figure 3D). This further confirmed that the BMB-4 underwent unimolecular collapse at both concentrations of 0.2 mg/g and 1.0 mg/g, likely because of the PEO’s stabilization effect. We then used AFM to characterize the morphological transitions of BMB-4 with the temperature increasing from below to above the LCST of PDEGEA. PEO/PDEGEA BMB-4 brush molecules were spin coated onto freshly cleaved mica from dilute aqueous solutions at 0 °C (0.1 mg/mL) and 40 °C (0.01 mg/mL) and imaged with AFM in tapping mode. The lower polymer concentration of 0.01 mg/g for the 40 °C sample was used in order to avoid possible clustering of collapsed brush molecules during evaporation of water in the sample preparation. For the sample prepared at 0 °C, a wormlike morphology was observed as expected for densely grafted molecular brushes in a good solvent (Figure 4A). Quantitative length analysis yielded an average contour length of 151 ± 29 nm and a typical height of ∼1 nm. Given that the length of a fully extended worm in an all-trans conformation was 203 nm for a DP of 800, the measured average contour length of 151 nm corresponds to the degree of stretching of ∼74%. For the brushes spin coated at 40 °C, globular nanoobjects were observed as anticipated for the collapsed state (Figure 4B). Interestingly, the coiled backbone can be clearly seen in the inset AFM image of Figure 4B. Cross-sectional analysis of the collapsed brushes showed an average diameter of 51 nm and an average height of ∼2.5 nm. More AFM images

repetitive centrifugal filtration in methanol/water (50/50, v/v) using a 50 kDa MWCO dialysis membrane and one round of fractionation using THF and hexanes (Figure 2B). The molar ratio of PEO to PDEGEA side chains in the purified BMB-4 was determined to be 0.62:0.38 from the 1H NMR spectrum (Figure S9) using the integrals of the peaks at 4.08−4.31 ppm (−COOCH2CH2− of DEGEA units) and 3.46−3.72 ppm (−OCH 2 CH 2 − of PEO and −COOCH 2 −CH 2 OCH 2 CH2OCH2CH3 of DEGEA units), which is very close to the feed ratio of 0.60:0.40. SEC analysis, however, still showed the increase of the high molecular weight shoulder (Figure 2B), indicating that the attempt with propargyl alcohol to avoid this was unsuccessful, for which the reason is currently unknown. Temperature-Induced Size and Shape Changes of PEO/PDEGEA Binary Heterografted Molecular Brushes. The thermoresponsive property of PEO/PDEGEA BMB-4 in water was initially studied by visual inspection and DLS with PDEGEA HMB-1 for comparison. Aqueous solutions of HMB1 and BMB-4 in water with a concentration of 1.0 mg/g were prepared by first ultrasonicating in an ice/water bath and then storing in a refrigerator (4 °C) for at least 1 day to ensure complete dissolution. Both solutions were clear at low temperatures. Upon increasing the temperature through the reported LCST of 9 °C for PDEGEA linear homopolymer, the HMB-1 solution turned cloudy (Figure 3A), and eventually the brush molecules precipitated to the bottom of the vial after about 1 day, indicating the aggregation of collapsed brush molecules. In contrast, the BMB-4 solution remained clear at room temperature, suggesting that the collapsed state was stabilized by PEO side chains as designed and expected. DLS was then employed to study the thermoresponsive properties of HMB-1 and BMB-4 in water at a concentration of 0.2 mg/g, at which both solutions stayed transparent at room temperature. At 1 °C, HMB-1 had an apparent hydrodynamic diameter (Dh) of 76 nm (Figure 3B). The Dh decreased upon heating, with the fastest change occurring at ∼8 °C; the apparent size leveled off to ∼40 nm as the temperature was increased to 15 °C, and there was essentially no further change in size above 15 °C, suggesting a unimolecular collapse. BMB-4 showed similar behavior (Figure 3C), with a maximum Dh of 64 nm at 1 °C; the size exhibited the sharpest decrease at ∼10 °C and eventually leveled off to ∼43 nm as the temperature was F


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Scheme 4. Synthesis of Alkyne End-Functionalized Biotin-Containing, Fluorescently Labeled Thermosensitive Side Chain Polymer P(DEGEA-co-BA-co-NBDA)

Figure 5. (A) SEC traces of PEO/P(DEGEA-co-BA-co-NBDA) BMB-5 before and after removal of unreacted side chains by centrifugal filtration. SEC was carried out on PL-GPC 50 Plus using DMF containing 50 mM LiBr as carrier solvent. (B) Plot of apparent hydrodynamic size Dh of BMB5 in water at a concentration of 0.2 mg/g versus temperature, obtained from a DLS study.

(FRET) donor for Rhodamine B,62−65 was used as a probe to investigate the possibility of thermoregulated binding between biotin-containing molecular brushes and Rhodamine B-labeled avidin via fluorescence spectroscopy. The copolymer P(DEGEA-co-BA-co-NBDA) was prepared by ATRP of a mixture of DEGEA, BA, and NBDA in anisole with feed molar ratios of 100:1.1:0.16, respectively. SEC analysis showed that the polymerization was well controlled, yielding an Mn,SEC of 10.6 kDa and a PDI of 1.16 (Figure S12A and Table 1). The DP was 50, determined by end-group analysis from the 1H NMR spectrum (Figure S12B), and the average number of BA monomer units per chain was 0.66. PEO/P(DEGEA-co-BA-co-NBDA) binary heterografted molecular brushes, referred to as BMB-5 in Table 2, were synthesized using PTEGN3MA, PEO-114, and P(DEGEA-coBA-co-NBDA) under similar conditions as for PEO/PDEGEA BMB-4. To reduce the likelihood of interactions between the collapsed P(DEGEA-co-BA-co-NBDA) core with avidin at elevated temperatures, here we used the longer PEO-114 instead of PEO-45. Similar to the synthesis of BMB-4, a molar ratio of 1:1.92 for PTEGN3MA backbone monomer units to total side chain polymers and a molar ratio of 0.60:0.40 for PEO to P(DEGEA-co-BA-co-NBDA) were employed. Figure 5A shows the SEC traces of the final reaction mixture after 44 h and PEO/P(DEGEA-co-BA-co-NBDA) BMB-5 after the removal of unreacted side chains. From the relative peak areas in SEC and the feed molar ratio, the grafting density was

can be found in the Supporting Information (Figures S10 and S11). Regulation of Biotin−Avidin Binding via Temperature-Triggered Shape Changing of Thermosensitive Binary Heterografted Molecular Brushes: Synthesis and Shape Changing Behavior. Having demonstrated the thermally induced shape transitions of PEO/PDEGEA binary brushes between an extended wormlike and a collapsed yet stable globular state, we explored the possibility of utilizing this stimuli-induced shape-changing behavior to control interactions of brush molecules with other molecules in an attempt to mimic the function of the VWF in blood clotting. Our hypothesis is that the functional groups incorporated into the core-forming side chain polymer will have little or no interactions with environment when the brushes are in the collapsed globular state because of the shielding of the stabilizing polymer corona but become accessible when the brushes are in the extended worm state. It is well-known that biotin forms a very strong complex with avidin, a tetrameric protein, and that once formed the complex is essentially irreversible under normal conditions.61 With this in mind, we prepared a thermosensitive PDEGEA side chain polymer incorporated with a small amount of biotin-containing acrylate monomer (D-biotin oxyethyl acrylate, BA) and a fluorescent monomer (4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxodiazole, NBDA), referred to as P(DEGEA-co-BA-co-NBDA) (Scheme 4). NBDA, a fluorescence resonance energy transfer G


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Figure 6. AFM height images of PEO/P(DEGEA-co-BA-co-NBDA) BMB-5 (A) spin coated onto freshly cleaved mica from aqueous solution at 0 °C with a polymer concentration of 0.05 mg/g, (B) spin coated onto freshly cleaved mica from aqueous solution at 40 °C with a polymer concentration of 0.05 mg/g, (C) drop cast onto mica-PS from an aqueous solution at 0 °C with a polymer concentration of 0.05 mg/g, and (D) drop cast onto mica-PS from an aqueous solution at 45 °C with a polymer concentration of 0.01 mg/g.

exhibited a 14% reduction in size over a temperature range of 14 °C compared to a 33% reduction in size over 10 °C for BMB-4. Apparently, this is the result of the use of longer PEO114 side chains in BMB-5, causing the P(DEGEA-co-BA-coNBDA) side chains, whose collapse was responsible for the change in size, to occupy a relatively smaller proportion of volume per brush molecule in both the extended and collapsed states of BMB-5 (52.6 mass % P(DEGEA-co-BA-co-NBDA)) compared with PDEGEA side chains in BMB-4 (68.0 mass % PDEGEA). For AFM study, we initially spin coated dilute aqueous solutions of BMB-5 onto freshly cleaved mica at 0 and 40 °C as for BMB-4. At 0 °C, BMB-5 exhibited an extended wormlike morphology as expected, and the side chains can be clearly seen from Figure 6A and Figure S14, possibly due to the stronger interactions of longer PEO side chains with the hydrophilic mica surface. Image analysis showed that the average contour length was 190 ± 29 nm, with a representative height of ∼0.5 nm. For BMB-5 spin coated onto mica at 40 °C, we found that wormlike and globular morphologies coexisted, with the majority of the brush molecules exhibiting extended wormlike

calculated to be 77.5% (Table 2), assuming the molar ratio of PEO and P(DEGEA-co-BA-co-NBDA) were the same in the brushes as in the feed. This value is similar to that for BMB-4 (74.3%). SEC analysis of the purified BMB-5 indicated that the molecular weight distribution was essentially unchanged, and from 1H NMR analysis (Figure S13), the molar ratio of PEO to P(DEGEA-co-BA-co-NBDA) side chains in BMB-5 was found to be 0.61:0.39, which was very close to the feed ratio of 0.60:0.40. The thermally induced size and shape changing of BMB-5 in water was investigated by DLS and AFM, respectively, as for BMB-4. Figure 5B shows the Dh of BMB-5 as a function of temperature from a DLS study of a 0.2 mg/g aqueous solution. Overall, the DLS results are similar to that for BMB-4, except that the Dh of BMB-5 was generally larger than BMB-4, due to the longer side chain polymer PEO-114. At 1 °C, BMB-5 exhibited a Dh of 79 nm; the size showed a significant decrease at ∼11 °C and leveled off at ∼19 °C with a size of 68 nm. Although still centered around the same temperature, the transition was slightly broader for BMB-5 than BMB-4, and the relative size decrease was also less dramatic for BMB-5, which H


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Figure 7. (A) Fluorescence emission spectra recorded at various time intervals at 40 and 0 °C for an aqueous solution of the mixture containing 0.005 mg/g BMB-5 and 0.025 mg/g avidin. (B) Plot of the ratio of fluorescence emission intensities of the peaks at 570 and 520 nm for the aqueous solution containing a mixture of 0.005 mg/g BMB-5 and 0.025 mg/g avidin as well as solutions containing 0.005 mg/g BMB-5 alone and 0.025 mg/g avidin alone as control samples.

solution and 0.606 g of a 0.050 mg/g avidin solution at 40 °C. Before mixing, both solutions were equilibrated at 40 °C for 2 h to ensure that the BMB-5 molecules were in the globular state. Two additional solutions containing 0.005 mg/g BMB-5 and 0.025 mg/g avidin alone were also prepared and used as controls for comparison. Each solution was prepared and maintained at 40 °C, and fluorescence emission spectra were recorded at various time intervals over a period of 19 h. The temperature of each solution was then lowered to 0 °C, and fluorescence measurements were continued for an additional 29 h. FRET, which occurs when the donor and acceptor molecules are within a distance of 1−10 nm,62−65 between NBD (FRET donor) in the brushes and Rhodamine B (FRET acceptor) in avidin was used to probe the binding behavior between biotincontaining BMB-5 and avidin. Figure 7A shows the fluorescence emission spectra recorded at various time intervals at 40 and 0 °C for the mixture containing 0.005 mg/g BMB-5 and 0.025 mg/g avidin. The corresponding fluorescence emission spectra for the 0.005 mg/ g BMB-5 and the 0.025 mg/g avidin control samples can be found in the Supporting Information (Figure S19). After preparing and maintaining the samples at 40 °C for 19 h, we observed an overall decrease in fluorescence intensity for all three samples over the time. After 19 h, the temperature of the three samples was decreased to 0 °C using an ice/water bath. A gradual decrease in fluorescence intensity was again observed over the time, for which the reason was unclear. However, the peak at 520 nm, from FRET donor NBDA, apparently decreased to a larger extent than the 570 nm peak, from FRET acceptor Rhodamine B, while the peaks for two control samples decreased to a similar degree. To get a better illustration about the interactions between the biotin in BMB-5 and avidin, we plotted the ratio of the fluorescence emission intensities of the peaks at 570 and 520 nm for the sample containing the mixture of BMB-5 and avidin and the two control samples (Figure 7B). For the two control samples, the I570/I520 ratio against temperature was plotted from the two spectra summed together. For the mixture of BMB-5 and avidin at 40 °C, the I570/I520 ratio initially decreased slightly and then leveled off; there was essentially no or very little change in the intensity ratio between 2 and 19 h, with the ratios of 1.23 and 1.21, respectively. The intensity ratio obtained from the two control samples showed a similar trend initially but continued to decrease slightly over the time. After the temperature was lowered to 0 °C, the I570/I520 decreased

conformations (Figure 6B and Figure S15). The predominance of the wormlike morphology observed at 40 °C by AFM is contrary to the size decrease observed by DLS. We believe that this is a result of unfolding of the BMB-5 brush molecules during sample preparation due to a combination of the favorable strong surface interactions and the shear force exerted on the brushes by spin-coating, which was promoted by the high conformational strain on the backbone imposed by long PEO side chains. In fact, for BMB-4 spin-cast onto mica at 40 °C, while globular nano-objects were observed near the center of the substrate, we also saw brush molecules in the wormlike morphology at the edge of the mica where the shearing force was larger during spin coating. To reduce surface interactions of BMB-5 with hydrophilic mica, we hydrophobized the substrate by spin coating a thin layer of polystyrene (PS, 30 kDa, from a 1 wt % solution in CHCl3) onto the freshly cleaved mica surface at 10 000 rpm. Subsequently, we deposited BMB-5 onto the PS-coated mica (mica-PS) surface from dilute aqueous solutions at 0 and 45 °C by drop casting, instead of spin coating, in order to avoid any conformational changes due to the shear force from spin coating. For the sample drop cast on mica-PS at 0 °C, we observed wormlike brush molecules as expected (Figure 6C). The average contour length was 154 ± 28 nm, with a typical height of ∼1 nm. For the brushes drop cast on mica-PS at 45 °C, compact globular nano-objects were seen (Figure 6D), consistent with the DLS results for BMB-5 and the shape change of BMB-4 previously visualized by AFM. The average size of the collapsed brushes was 64 ± 12 nm, with a height of ∼3.5 nm. As a control, we spin coated Milli-Q water onto a mica-PS substrate; AFM showed a clean, flat surface (Figure S16), indicating that the PS film did not interfere with AFM analysis. Additional AFM images can be found in the Supporting Information (Figures S17 and S18). Regulation of Biotin−Avidin Binding via Temperature-Triggered Shape Changing of Thermosensitive Binary Heterografted Molecular Brushes: FRET Study of Binding between Biotin-Containing BMB-5 and Avidin. The binding interactions between biotin-containing PEO/ P(DEGEA-co-BA-co-NBDA) BMB-5 and Rhodamine B-labeled avidin in aqueous solution at different temperatures were studied using fluorescence spectroscopy. We prepared an aqueous solution containing a mixture of BMB-5 with a concentration of 0.005 mg/g and avidin with a concentration of 0.025 mg/g by mixing 0.598 g of a 0.010 mg/g BMB-5 aqueous I


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slightly for the control samples. In contrast, for the BMB-5/ avidin mixture, the I570/I520 ratio jumped upward from 1.21 to 1.36 after just 5 min of incubation time. As the temperature was maintained at 0 °C over a period of 29 h, we saw a continued, gradual increase in I570/I520 for the BMB-5/avidin mixture, reaching 1.92 at 29 h, while the I570/I520 continued decreasing slightly for the control samples over the same time period. These observations suggest the occurrence of FRET in the mixture of BMB-5 and avidin due to the unfolding of BMB-5 molecular brushes from the collapsed globular state to the extended wormlike state, which exposed the P(DEGEA-co-BAco-NBDA) side chains to the environment and enabled the binding between biotin moieties of BMB-5 and avidin. The continued increase in the I570/I520 ratio indicated that more avidin molecules bound to the extended, wormlike brushes with time increasing. Although we cannot rule out the possibility of the occurrence of FRET to some small extent, and thus the binding between BMB-5 brushes and avidin, during the incubation at 40 °C due to the complication by the continued decrease of fluorescence intensity with time, the increase in I570/I520 for the BMB-5/avidin solution relative to the control samples observed upon lowering the temperature to 0 °C demonstrated a significant level of control over the binding between brush molecules and avidin via shape changing.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF through DMR-1607076. C.Y.L. thanks the support from NSF (DMR-1308958).

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CONCLUSIONS Inspired by the intriguing behavior of the VWF in the blood clotting mechanism, we designed and synthesized binary heterografted molecular brushes composed of a thermosensitive side chain polymer and a permanently hydrophilic polymer and demonstrated their thermally induced shape transitions between extended wormlike and collapsed yet stable globular conformations. A click “graft to” method was developed to construct binary molecular brushes from an azide-bearing backbone polymer and alkyne end-functionalized side chain polymers. This modular approach allows for convenient selection of different side chain polymers and easy tuning of relative ratios of different side chain polymers. DLS and AFM studies showed the temperature-induced size and shape changes of the heterografted molecular brushes. As an example of possible applications, the shape changing of thermosensitive binary molecular brushes was used to regulate the binding of biotin-containing brush molecules and avidin. We believe that both the synthetic method and the thermosensitive shape changing binary molecular brushes reported in this article will have great potential in the design, preparation, and applications of stimuli-responsive soft materials.

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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.macromol.7b00150. Experimental section, characterization data for polymers, and additional AFM and fluorescence data (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone 865-974-3399; e-mail [email protected] (B.Z.). ORCID

Bin Zhao: 0000-0001-5505-9390 J


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Temperature-Induced Shape Changing of Thermosensitive Binary

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