Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
Design Principles for Triggerable Polymeric Amphiphiles with Mesogenic Side Chains for Multiscale Responses with Liquid Crystals Lisa Adamiak,‡ Joel Pendery,† Jiawei Sun,† Kazuki Iwabata,† Nathan C. Gianneschi,*,‡,§ and Nicholas L. Abbott*,† †
Department of Chemical and Biological Engineering, University of WisconsinMadison, Madison, Wisconsin 53706, United States Department of Chemistry and Biochemistry, University of CaliforniaSan Diego, 9500 Gilman Drive, La Jolla, California 92093-0303, United States § Department of Chemistry, Department of Materials Science & Engineering, and Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60091-3113, United States ‡
S Supporting Information *
ABSTRACT: Interfacial assemblies formed by polymeric amphiphiles at aqueous interfaces of thermotropic liquid crystals (LCs) can trigger multiscale changes in the organization of the LCs in response to recognition events. However, we have a limited understanding of the rules governing the rational design of LC-integrated polymeric amphiphiles. Herein, we report the synthesis of families of amphiphilic polymers that differ in (i) side-chain molecular structure, (ii) polymer architecture, and (iii) copolymer composition. We used this library in experiments to establish structure−property relationships relevant to the design of multifunctional polymers that can amplify and transduce biomolecular recognition events into optically detectable, macroscopic ordering transitions in LCs. We then utilized these structure−property relationships to guide the design of a peptide−polymer amphiphile (PPA) that assembles at the interface of LC droplets. Enzymatic cleavage of PPA-coated LC droplets by thermolysin directly triggered a change in the internal ordering of the LC within the droplets and the scattering of light from the droplets. The results of our study provide important guidance to future designs of triggerable LC systems.
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INTRODUCTION The orientations assumed by liquid crystals (LCs) at the surfaces of solids have been extensively explored because of their utility in electrooptical devices, such as LC displays.1,2 These studies have established that the chemical functionality of molecules and their ordering over a range of length scales (angstroms to micrometers) can impact the orientations of LCs.1−4 More recently, it has been revealed that molecular assemblies formed at interfaces between LCs and immiscible aqueous phases can also influence the orientations of LCs.5,6 These interfaces are particularly promising for the design of LC systems that respond to biomolecular interactions because biological stimuli (e.g., targeted proteins) can be transported to the LC interface through the aqueous phase.5−7 The LC systems are passive (do not require power to amplify and transduce the arrival of the stimulus) and inexpensive (they can be a LC droplet in water), making them potentially suitable as sensors, including low-cost diagnostics for use outside central facilities.7,8 In addition, molecules assembled at LC−aqueous interfaces are mobile, and thus changes in the lateral organization of biological species at the LC interface can be transduced.9 While LC−aqueous interfaces offer the basis of a © XXXX American Chemical Society
broadly useful class of responsive materials, an incomplete understanding exists of the designs of molecules that will assemble at the LC−aqueous interface to trigger changes in the ordering of the LC in response to a stimulus. To address the above-described gap in knowledge, in this paper, we report structure−property relationships governing polymeric assemblies formed at liquid crystal (LC)−aqueous interfaces with the goal of propagating molecular-level surface recognition events throughout the LC. The study is motivated by several past studies that have reported the use of interfacial assemblies formed by polymeric amphiphiles to create stimuliresponsive LCs.10−17 A change in molecular-level organization of the polymeric amphiphiles at the interface leads to a change in ordering of the LC that propagates tens of micrometers from the interface and thus can be observed optically.5,18 These past studies establish the feasibility of using copolymers to trigger changes in the ordering of LCs in response to pH,10−13,17 the presence of polyelectrolytes of opposite charge,15 or enzymatic Received: October 4, 2017 Revised: January 12, 2018
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DOI: 10.1021/acs.macromol.7b02140 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. (A) Library of homopolymers with distinct side-chain groups, R. (B) Percentage of radial droplets triggered by 113, 215, 321, and 415 as a function of homopolymer concentration. Error bars represent the standard deviation of at least four experiments. (C−E) The percentage of radial droplets triggered by homopolymers at varying concentrations, with different linker functional group (ether or amide), linker length, and the presence or absence of a nitrile functional group. Error bars represent the standard deviation of three experiments.
Table 1. Characterization of Polymers Used in This Study polymer type homopolymers
block copolymers
random copolymers
identifiera
m block “R”b
113 215 321 415 520 616 716 816 916 1019 117-b-PEG29 130-b-PEG26 126-b-PEG16 147-b-PEG15 112-ran-PEG20 114-ran-PEG17 126-ran-PEG19 133-ran-PEG17 PPA
1 2 3 4 5 6 7 8 9 10 1 1 1 1 1 1 1 1 1
n block
DPmc
PEG-12 PEG-12 PEG-12 PEG-12 PEG-12 PEG-12 PEG-12 PEG-12 peptide 1
13 15 21 15 20 16 16 16 16 19 17 30 26 47 12 14 26 33 22
DPnc
Mn (g/mol)c
Đc
m (mol %)
29 26 16 15 20 17 19 17 5
6195 7342 9426 4726 7142 8139 8077 9746 7880 8011 27680 32350 22970 32170 19290 18650 25240 27090 15800
1.030 1.050 1.010 1.437 1.024 1.017 1.017 1.021 1.035 1.063 1.018 1.022 1.011 1.016 1.024 1.009 1.019 1.019 1.015
100 100 100 100 100 100 100 100 100 100 37 54 62 76 38 45 58 66 81
a
Subscript indicates degree of polymerization. bR groups are shown structurally in Figure 1, with monomers described in the Supporting Information. cDegree of polymerization (DP) for each block as indicated, m and n, determined by SEC-MALS, as is Mn and dispersity (Đ).
cleavage.16 However, the diversity of hydrophobic and hydrophilic monomer structures used, as well as the copolymer compositions and architectures deployed, prevents identification of broadly applicable design rules for this promising class of stimuli-responsive materials. In this work, design rules for triggerable polymeric amphiphiles to be used with LCs were sought by generating a family of copolymers that differed systematically in their composition and architecture. First, we studied the effect of the
hydrophobic side-group structure on the anchoring of LCs at the nematic LC/aqueous interface utilizing homopolymers prepared via ring-opening metathesis polymerization (ROMP).19−27 From this initial library of structures, we identified an optimal mesogen-like side chain that can efficiently cause perpendicular (homeotropic28) anchoring of LCs and can be readily incorporated into responsive copolymer structures. Second, we incorporated the optimal hydrophobic side chain into block and random copolymers to study how B
DOI: 10.1021/acs.macromol.7b02140 Macromolecules XXXX, XXX, XXX−XXX
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Scheme 1. Representative Scatter Plots of (A) Bipolar and (B) Radial Nematic LC Droplets (Droplets Are Depicted in the Insets)
BioSciences). A detailed description of the use of flow cytometer to characterize the configurations of LC droplets can be found in a previous publication.22 Optical microscopy measurements were performed on an inverted optical microscope with crossed polarizers and in bright field with a 60× objective and a 100× oil-immersion objective. To perform Langmuir isotherm studies, polymer solutions in HPLC grade chloroform were prepared at a concentration of 0.5 mg/mL in glass vials with PTFE tops. The Langmuir trough (Nima 602A film balance (Coventry, England)) was cleaned with HPLC grade chloroform while the barriers were cleaned with ethanol and dried with air from “AirIt” dry air canisters. Water with a resistivity of 18.2 MΩ cm (Milli-Q, MilliPore; Bedford, MA) was introduced into the trough, and a Wilhelmy plate (Biolin Scientific) was attached to the force sensor, submerged into the subphase, and left to equilibrate for at least 20 min. Before the addition of the polymer solution, the pressure sensor was zeroed, and the barriers were closed to ensure that the surface was clean. If the measured surface pressure was >0.5 mN/m, the surface was cleaned using an aspirator. Once the surface was cleaned, a 100 μL glass syringe (Hamilton) was cleaned with HPLC grade chloroform and dried under a stream of nitrogen. The polymer solution was added in a dropwise fashion using the syringe, across the surface of the subphase, and then left to equilibrate for a minimum of 30 min. Finally, the barrier speed was set to +35 cm2/min (compression), and an isotherm was recorded. Immediately after completion of the isotherm, the barrier speed was set to −35 cm2/min (expansion), and another isotherm was recorded. To cleave the peptide in the peptide polymer amphiphiles (PPAs), we aliquoted 400 μL of LC emulsion into a tube and added 2.0 μL of 100 mM CaCl2 to reach a concentration of 0.5 mM CaCl2. We used the emulsion immediately after its preparation without equilibration for 60 min. Thermolysin (Promega; Madison, WI) was aliquoted into 2 mL vials and stored in the freezer at −4 °C until needed. The aliquoted thermolysin was dispersed in a 0.5 mM CaCl2 in PBS as needed and used within 2 weeks of reconstitution. To inactivate thermolysin for use as a control, the aliquoted thermolysin was dispersed in a 10 mM EDTA in PBS and then incubated at 65 °C for 3 h. Reconstituted thermolysin or inactive thermolysin was added to the tube containing the uncleaved PPA at a concentration of 125 nM and incubated at room temperature for half an hour.
polymer architecture influences LC anchoring at aqueous interfaces, leading to the finding that polymer architecture exhibits pronounced effects on the LC organization. We also explored the effect of composition of the copolymers on LC ordering, namely the hydrophobic:hydrophilic block ratio, to identify copolymer compositions that can trigger changes in the ordering of the LCs. Finally, we tested the utility of these structure−property relationships by designing polymeric amphiphiles that are targeted by enzymes and can be used to trigger multiscale responses in aqueous dispersions of LC droplets. Other designs and linker structures proved less effective, including use of amide functional groups and terminal nitrile groups. Furthermore, random copolymer architectures of biphenyl side chains and hydrophilic oligoethylene glycol groups were found to be preferred over block copolymers, allowing even low mole fractions of hydrophobic side chains to cause homeotropic anchoring and triggering LC responses when incorporating a targeted, responsive functionality.
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EXPERIMENTAL SECTION
Detailed information is provided in Supporting Information regarding the use of ROMP to synthesize the polymers shown in Figure 1 and Table 1 and the methods used to characterize the polymeric products. To prepare LC droplets decorated with the polymers shown in Table 1, we dissolved the polymers (10% v/v) in HPLC grade chloroform (Fisher Scientific). The polymer solutions were added to 2 mL glass vials with PTFE caps to which the LC 4′-pentyl-4cyanobiphenyl (5CB, EMD Sciences; New York, NY, and HCCP, China) was also added. All polymer concentrations noted below are defined with respect to the volume of 5CB added to each vial (0.5 μL). After addition of the 5CB, the chloroform was removed with a nitrogen stream, and then the vial was placed in a vacuum chamber for ∼60 min. After evaporation of the solvent, we added 1000 μL of 10 mM phosphate buffered saline (PBS Sigma-Aldrich; Milwaukee, WI) containing 10 μM SDS to each tube and capped them. We emulsified the aqueous dispersion of polymer and 5CB in each tube by sonication in a FS20 bath sonicator (Fisher Scientific) until the emulsion appeared opaque, indicating the formation of microdroplets. We subsequently vortex mixed (Fisher Scientific Digital Vortex Mixer) each tube for 15 s. The emulsions were allowed to equilibrate for at least 60 min before use. We prepared control emulsions to determine the percentage of radial droplets (see Supporting Information). Positive control emulsions (emulsions with droplets in radial configuration) were prepared using 1 mM SDS. Negative control emulsions (emulsions with droplets in bipolar configuration) were prepared using 10 μM SDS. The LC emulsions were characterized by using the light scattering mode of an Accuri C6 flow cytometer (BD, New Jersey) and a BD FACSCalibur flow cytometer (BD
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RESULTS AND DISCUSSION Influence of Side-Chain Structure on LC Ordering. We prepared LC−aqueous interfaces using microdroplets of nematic 5CB dispersed in PBS, as described in the Experimental Section. The assembly of polymeric amphiphiles at the LC−aqueous interface is amplified through changes in the orientational anchoring of the LC at the droplet interface and thus the overall configuration of LC within the droplet.5,18 The anchoring of LC at the undecorated LC/aqueous interface C
DOI: 10.1021/acs.macromol.7b02140 Macromolecules XXXX, XXX, XXX−XXX
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compared to an amide in 3. To study these differences, we synthesized homopolymers 520 and 616 (Figure 1 and Table 1). For the mesogen side chain structure in polymers 113 and 520, we observed that the length of the alkyl linker affects the percentage of radial droplets generated. Specifically, 113, consisting of the longer alkyl linker (C10), was able to generate a higher percentage of radial droplets than 520, which has a shorter alkyl linker (C5) (Figure 1C). This is consistent with other reports, where an alkyl length dependence of the anchoring of LC at the LC/aqueous interface was observed.23 This suggests that the mesogenic side chain can fail to generate radial droplet configurations if the alkyl linker is too short.16 Homopolymers 321 and 616 generated very few radial droplets (Figure 1C). These amide-linked systems perform poorly when compared to homopolymers 113 and 520, which possess ether-functionalized mesogen side chains. The etherlinked systems prove far more effective at generating radial droplets. This striking difference may be due to the large lateral dipole moment of the amide, perpendicular to the longitudinal axis of the side chain, promoting an orientation of the side chain that is conducive to tangential rather than homeotropic anchoring of the LC. More broadly, the low radial LC droplet population observed for homopolymers 321 and 616 suggests that the amide is dominating the interactions of the polymer side chain with the LC. This finding also illustrates the high sensitivity of LC ordering to the structure of the hydrophobic side chain. Inspection of the data revealed a small difference in the percentage of LC droplets that assume a radial configuration in the presence of 113 and 215, which differ by a single carbon in the aliphatic linker. To systematically investigate the influence of side-chain linker length, we synthesized homopolymers 716 and 816 to correlate the strength of the homeotropic anchoring to the number of carbons in the aliphatic linker. To this end, polymers 716, 113, 215, and 816 have alkyl chain lengths of C9, C10, C11, and C12, respectively. At a polymer concentration of 0.3 mM, we observed a modest difference in the ability of the homopolymers to generate radial droplets as a function of the alkyl linker length (Figure 1D). The homopolymer with the C10 linker (113) generated a greater percentage of LC droplets in the radial configuration as compared to homopolymers with aliphatic linkers of C9, C11, or C12. This result suggests that polymers with an aliphatic chain length of C10 are optimal for triggering radial configurations of 5CB droplets. Motivated by observations reported by Ma16 and Lee and Khan,13,17 we compared designs of side chains with biphenyl versus nitrile−biphenyl groups. We synthesized homopolymers 916 and 1019, which contain a terminal nitrile group, and compared them to homopolymers 113 and 215. Interestingly, we observed that side chains lacking a nitrile functional group are able to generate a higher percentage of radial droplets than side chains with the nitrile group (Figure 1E). This is surprising, as we anticipated that the presence of a nitrile functional group in the hydrophobic side chain would enhance the interactions between the polymer mesogens and the 5CB molecules due to dipole−dipole interactions of their respective polar nitrile groups. On the basis of the above-described studies using homopolymer systems, we reasoned that amphiphilic copolymers should incorporate a biphenyl that is coupled to the polymer backbone through a C10 aliphatic linker to optimally achieve homeotropic anchoring of LCs. The use of an amide moiety in the linker inhibited homeotropic anchoring of 5CB
is tangential (planar) and minimization of the elastic energy of the LC constrained by tangential surface anchoring causes LC droplets to adopt bipolar droplet configurations with two diametrically opposite defects called boojums (Scheme 1).28,33,34 A change in anchoring of the LC to the homeotropic orientation transforms the LC droplet into a radial configuration.5,18 These configurations can be readily characterized by using the light scattering mode of a flow cytometer because the bipolar and radial droplet configurations are cylindrically and spherically symmetric, respectively (Scheme 1).35 For bipolar droplets, we observe a broad scatter plot because of the orientation-dependent scattering of light from the LC droplets. In contrast, for radial droplets, we observe a narrow “S”-shaped scatter plot because of the spherical symmetry of the droplet wherein scattering of light is invariant with droplet rotation.35 In the studies described below, we use flow cytometry and optical microscopy of LC droplets to screen for and to establish structure−property relationships for several families of homopolymers and amphiphilic copolymers. To provide insight into side-chain designs that cause homeotropic anchoring of LCs, we first tested homopolymers of types 1−10 (Figure 1 and Table 1). The homopolymers (synthesized using ROMP, see Supporting Information) possessed a range of side-chain structures consisting of hydrophobic anchoring groups linked to the polymer backbone and were dispersed into the LC prior to emulsification of the LC in phosphate buffered saline (PBS). Homopolymers 113, 215, 321, and 415 were synthesized targeting similar degrees of polymerization (DP ∼ 20) but varying side-chain linker length and conjugation chemistry from ethers to amides (Figure 1). We measured the ability of homopolymers 113, 215, 321, and 415 to generate radial droplets using concentrations of the polymers in the LC that ranged from 0.05 to 1 mM. In general, we observed an increase in the percentage of radial droplets increased with polymer concentration in the LC, consistent with an increase in the density of hydrophobic side-chains at the LC droplet interface (Figure 1B). Droplets decorated with polymer 415, consisting of the aliphatic side chain, required a higher polymer concentration to transform the LC droplets to a radial configuration compared to 113 or 215, which possess the biphenyl side chain. At a polymer concentration of 0.2 mM, a small difference is evident between 113 and 215, which differ by one carbon in their side-chain alkyl linker. We calculated the surface area of the LC droplets in the aqueous dispersion (see Supporting Information). By assuming that all polymer within the LC adsorbs onto the droplet interface, for a polymer concentration of 0.25 mM, we calculated an average surface area per hydrophobic side chain of 113, 215, 321, and 415 to be ∼44 ± 7, 42 ± 1, 23 ± 2, and 55 ± 22 Å2, respectively. These values of surface area per side chain of the homopolymers (∼20−55 Å2) are comparable to the limiting area per molecule of classical single tail surfactants, such as SDS or CnTAB (∼40− 70 Å2), which also cause homeotropic anchoring.36 Interestingly, by employing the aliphatic tail as with homopolymer 415, we were unable to generate radial droplets at surface coverages comparable to those of standard small molecule surfactants (Figure 1B). In addition, we observed that polymer 321 did not generate radial droplets (Figure 1B). Comparing polymers 113 and 321, we note two differences in the structure of the side chains: (i) the length of the alkyl linker is much longer for 1 (C10) than in for 3 (C5) and (ii) the presence of an ether moiety in 1 D
DOI: 10.1021/acs.macromol.7b02140 Macromolecules XXXX, XXX, XXX−XXX
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the LC of 0.2 mM whereas the block copolymer was unable to generate a significant percentage of radial droplets. Even at high polymer concentrations (1 mM), the block copolymer was unable to generate a substantial fraction of LC droplets with radial configurations (
