Solvent Tunable Self-Assembly of Amphiphilic Rod–Coil Block

Article pubs.acs.org/Macromolecules

Solvent Tunable Self-Assembly of Amphiphilic Rod−Coil Block Copolymers with Chiral, Helical Polycarbodiimide Segments: Polymeric Nanostructures with Variable Shapes and Sizes James F. Reuther,†,‡ Dumindika A. Siriwardane,† Raymond Campos,†,§ and Bruce M. Novak*,† †

Department of Chemistry and Alan G. MacDiarmid NanoTech Institute, The University of Texas at Dallas, Richardson, Texas 75080, United States ‡ Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States § The Air Force Research Laboratory, ERC, Inc., Edwards AFB, California 93524-7680, United States S Supporting Information *

ABSTRACT: The nanoscale self-assembly of four amphiphilic rod−coil di- and triblock copolymers with chiral, rodlike poly(N-1-phenethyl-N′-methylcarbodiimide) (PPMC) segments and random coil, hydrophilic PEG blocks has been investigated using dynamic light scattering (DLS) and tappingmode atomic force microscopy (AFM). This self-assembly proved to be highly tunable simply upon altering the concentration and chemical structure of the hydrophilic selective solvent and/or blending the copolymers with polycarbodiimide homopolymer. When spin-coated from dilute (c = 0.5 mg/mL) THF/H2O solutions, these interesting polymers adopted either simple spherical micelles or spherical polymersomes depending on the relative amount of H2O used for dissolution. Switching selective solvent from H2O to MeOH induced changes in aggregation behavior, as evidenced by DLS and AFM, with interesting nanoworm and nanomaggot micelle assemblies observed when spin-coated from dilute THF/MeOH solutions. Blending high-MW PPMC homopolymer with the block copolymers and spin-coating from dilute THF/25 vol % MeOH solutions resulted in the formation of long, interconnected nanofibers with several different observed tangling pathways including parallel packing, perpendicular wrapping, and helical twisting of nanofibers. Additionally, a large number of toroid nanostructures were also identified by AFM when spin-coated from these conditions. Finally, spin-coating copolymer/homopolymer blends from THF/25 vol % EtOH induced the nanoscale formation of long, bundled superhelical nanofibers with defined helical structures depending on the homopolymer−copolymer chiral pairing (i.e., (R)-(R) pairing formed P superhelical nanofibers and M superhelix for (S)-(S) pairing). The highly tunable nature of these polymeric nanostructures offers new opportunities for the formation of nanoparticles with variable shapes and sizes simply upon altering the solvent combinations opening up new applications as biological mimics and drug delivery agents.

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features and patterns with unprecedented control.1,2 Amphiphilic block copolymers, consisting of covalently bound hydrophobic and hydrophilic segments, have been an intense area of interest due to their wide applicability as biological mimics, drug delivery agents, soft templates for nanoparticle synthesis, and tunable nanoscale patterning.3−7 The selfassembly of amphiphilic coil−coil diblock copolymers (CCPs) has proven to be somewhat limited with such copolymers adopting only spherical/cylindrical micelles and vesicles/polymersomes in solution.8−10 The self-assembly of amphiphilic dendrimers has also been studied in great detail with several reports describing their aggregation into uniform dendrimersomes for applications as drug nanocarriers.11−13

INTRODUCTION In nature, the self-assembly of biological small molecules and macromolecules is one of the fundamental driving forces for the formation of life. Such natural examples include phospholipids (e.g., cell membranes), proteins/peptides (e.g., tertiary and quaternary structures of enzymes), and nucleic acids (e.g., double-helical assembly of DNA and RNA). Understanding the intricacies of natural assemblies is of the utmost importance and has led to the development of a plethora of synthetic analogues with the capabilities of mimicking biological systems in a variety of ways. Covalently attaching macromolecular systems with diverse chemical characteristics and properties can result in the formation of ordered nanostructures arising from the microphase immiscibility of each chemically distinct segment. Recent advances in directed block copolymer self-assembly have provided new, exciting methods for tuning nanometer-size © 2015 American Chemical Society

Received: July 14, 2015 Revised: August 25, 2015 Published: September 17, 2015 6890

DOI: 10.1021/acs.macromol.5b01564 Macromolecules 2015, 48, 6890−6899

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Figure 1. Amphiphilic RCPs with chiral, helical PPMC blocks and hydrophilic, random coil PEG blocks covalently attached via CuAAC “click” reaction.

Figure 2. 3D height AFM micrographs (scan size = 3.0 × 3.0 μm) of RCP-1 (a), -2 (b), and -3 (c) spin-coated from H2O/10 vol % THF and RCP-4 (d) spin-coated from THF/10 vol % H2O (c = 0.5 mg/mL) displaying spherical micelle assemblies. Hypothetical micellar structures formed by the diblock (e) and triblock (f) RCPs are also shown with the hydrophobic PPMC blocks occupying the core.

incorporated into amphiphilic RCPs. Polycarbodiimides, the class of helical polymers discussed herein, are synthesized via “living” reversible coordination−insertion polymerizations mediated by various transition metal initiators such as Ti(IV), Cu(I/II), and Ni(II).49−51 The steric repulsion of adjacent pendant groups causes these polymers to adopt a stable helical conformation in solution and the solid state. Furthermore, the partial conjugation of the nitrogen-rich, repeating amidine backbone causes these polymers to act as rigid rods in solution with persistence lengths (P) of ca. 42 ± 8 nm in the case of poly(N-1-phenethyl-N′-methylcarbodiimide) (PPMC) when incorporating enantiopure 1-phenethyl side groups.52 Recently, we reported the facile synthesis of a novel class of helical-b-random coil block copolymers with chiral, helical PPMC segments using copper-catalyzed alkyne−azide [3 + 2] cycloaddition (CuAAC) “click” reactions of functional end groups.53 Some of the RCPs synthesized in this report included amphiphilic PPMC−PEG block copolymers displayed in Figure 1. These copolymers include two diblock copolymers with varying PEG molecular weights (R-PPMC202-b-PEG225 = RCP1; R-PPMC202-b-PEG455 = RCP-2) and one triblock copolymer (S-PPMC146-b-PEG455-b-S-PPMC146 = RCP-3) with the random coil, hydrophilic PEG segments as the center block. Additionally, one newly synthesized triblock copolymer was included in this study with shorter PEG center blocks incorporated (Mn = 49.5 kDa, Đ = 1.30; R-PPMC123-bPEG225-b-R-PPMC123 = RCP-4). By VCD, we have shown that incorporating the (R)- or (S)-1-phenethyl side chain into

Block copolymers containing poly(ferrocenyldimethylsilane) (PFS) blocks adopt rigid, patchy, or branched cylindrical micelles with controlled lengths and thickness as demonstrated in several recent reports.14−16 The incorporation of rigid-rod segments into amphiphilic block copolymer systems has drawn significant attention due to the wide assortment of supramolecular structures adopted in solution and the solid state.17−20 The assembly of rod−coil block copolymers (RCPs) is driven by a combination of microphase immiscibility of the different blocks and the selfassociation behaviors of the anisotropic, rodlike block. A variety of structurally diverse, oligomeric/polymeric rod blocks have been incorporated into RCPs including π-conjugated and/or helical segments. Different supramolecular assemblies can be fabricated depending on the structure and characteristics of the RCP including spherical micelles,21−25 vesicles,21,23 superhelices,26,27 wormlike micelles,21 ribbons,28,29 microcapsules,30,31 tubules,32 and nanofibers.33,34 Typically, however, a single copolymer system adopts no more than two to four different nanostructures depending on the specific sample preparation conditions. The ability to form synthetic helical macromolecules via helix sense selective polymerization techniques has expanded tremendously in the past two decades owing to their potential in applications such as chiral stationary phases, asymmetric catalyst supports, and chiral sensors.35−41 With the exception of polypeptides32,42−46 and poly(n-hexyl isocyanate)23,25,47,48 (PHIC), synthetic helical high-MW polymers have yet to be 6891

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Figure 3. Height (a, c) and phase (b, d) AFM micrographs (scan size = 5.0 × 5.0 μm; inset size = 1.0 × 1.0 μm) of RCP-1 (a, b) and RCP-2 (c, d) spin-coated from THF/10 vol % H2O (c = 0.5 mg/mL) showing large polymersome assemblies. A proposed polymersome structure (e) based on measured height and phase step functions ((f); AFM trace shown in (d) with blue line), which show distinct phase changes between the edge and center of aggregates.

PPMC results in the preferential formation of the P or M helical polymer backbone, respectively.50 The amphiphilic character of this new class of RCPs should result in the formation of interesting supramolecular assemblies when dissolved in or cast from selective solvents for PEG such as H2O, MeOH, and EtOH. Herein, we report the highly tunable nanoscale self-assembly of these novel RCPs, studied by tapping-mode atomic force microscopy (AFM) and dynamic light scattering (DLS).

adopted similar spherical micelle aggregates to that observed when spin-coating RCP-1−3 from H2O/10 vol % THF. By DLS, two distinct size distributions of equal intensity are observed for both solvent combinations with one small distribution corresponding to Dh = 16.2 ± 4.6 nm, believed to be solubilized copolymer, and one distribution corresponding to Dh = 198 ± 65.2 nm (Figure S7). The DLS calculated hydrodynamic diameters of all four RCPs were compared to the solid state diameters calculated using AFM statistical analysis. In our previous report, we calculated the unperturbed end-to end distance of RCP-1, -2, and -3 to be 49, 78, and 82 nm, respectively.53 Using the same method, the overall dimensions of RCP-4 were calculated to be 55 nm. The measured diameter of these spherical aggregates by AFM statistical analysis (ca. 96 ± 16, 148 ± 25, 149 ± 17, and 172 ± 14 nm, respectively) matches closely to calculated unperturbed end-to-end distance of RCP-1 and -2 multiplied by two further eluding to the proposed micellar structure. One can envision the only possible way for RCP-3 and -4 to assemble into micelles would be for the PEG chains to fold in half to allow for both hydrophobic PPMC blocks to occupy the core of the micelle. This should essentially cut the end-to-end distance of the copolymer in half (ca. 41 and 28 nm for RCP-3 and -4, respectively) when considering the micelle size; however, the measured micelle radius by AFM and DLS was substantially larger in both cases. This could be due to encapsulation of some copolymers within these micelle assemblies, depicted in Figure 2f, due to the higher degree of difficulty for these copolymers to adopt the micellar structure when incorporating the hydrophilic PEG block as the center block. For RCP-4, the deviation between calculated and AFM measured micelle diameter was substantially larger than RCP-3 possibly due to the highest degree of copolymer encapsulation. This is hypothesized to be due to RCP-4 containing the lowest overall PEG content of all RCPs (ca. f rod = 0.52 for RCP-4 compared to f rod = 0.47, 0.31, and 0.39 for RCP-1−3, respectively).53 RCP-3 was also previously shown to adopt large polymersome aggregates by DLS and polarizing optical microscopy (POM) that increase diameter in a linear fashion upon

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RESULTS AND DISCUSSION Self-Assembly of RCPs Cast from Dilute THF/H2O Solutions. To study the aggregation behavior of all synthesized copolymers, the same sample preparation procedure was followed in making the specific solutions studied by DLS and spin-coated for AFM. Each of the copolymers was first dissolved in THF (c = 5.0 mg/mL) and injected into specific amounts of H2O, MeOH, or EtOH to achieve a final c = 0.50 mg/mL and the specific solvent combinations reported herein. The solutions were then allowed to incubate at room temperature under open air for 8−10 h to allow for full formation of each aggregated nanoparticle. At this point the solutions were analyzed by DLS to observe the presence of aggregates in solution. Following DLS analysis, each solution was spin-coated onto silicon wafers, and the polymeric aggregates were imaged using AFM to provide quantitative 3D topographical information on each nanoparticle set. In dilute H2O/10 vol % THF solutions, the first three copolymers display a single size distribution by DLS size analysis corresponding to hydrodynamic diameters (Dh) of 146 ± 38.8, 242 ± 46.2, and 216 ± 49.8 nm for RCP-1, -2, and -3, respectively. These same solutions were then spin-coated and imaged AFM revealing spherical aggregates hypothesized to be micelle assemblies (Figure 2). RCP-4 showed no solubility at this specific solvent combination due to the higher relative amount of hydrophobic PPMC in the system, when compared to RCP-1−3. When spin-coated from THF/10 vol % H2O (Figure 2d; the maximum amount of water prior to precipitation), RCP-4 6892

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Macromolecules increasing the concentration of THF solutions from 5.0 to 60.0 mg/mL.53 When altering the water content to THF/10 vol % H2O for RCP-1−3, significant increases in aggregate diameters were noted by DLS suggesting changes in aggregation behavior. These diameters (ca. 656 ± 127, 410 ± 88.8, and 629 ± 97.3 nm for RCP-1−3, respectively) are significantly larger than the unperturbed end-to-end distance of each copolymer. To identify the specific aggregation behavior, these solutions were again spin-coated onto silicon wafers and imaged via AFM. This analysis revealed large spherical aggregates for all RCPs with substantially larger diameters than seen previously (Figure 3). Additional AFM images of RCP-1 and -2 can be found in Figures S12 and S13, respectively. The AFM micrographs of RCP-3 spin-coated from THF/10 vol % H2O can be found in Figure S14. Interestingly, the phase AFM images exhibited distinct phase differences between the edge and center of the aggregates in all cases. This phenomenon is theorized to be attributed to the hollow nature of these aggregates leading to the proposed polymersome nanostructure depicted (Figure 3e). The measured diameters of the individualized aggregates by AFM statistical analysis (ca. 294 ± 22, 289 ± 19, and 299 ± 19 nm for RCP-1−3, respectively) again are significantly smaller than the observed Dh by DLS. This could be attributed to the self-association behaviors of the polymersome aggregates, which is clearly observed in Figure 3, to form larger, higherorder compound vesicles. This would inflate the overall average Dh and increase the standard deviations significantly. The observed, individualized polymersome aggregates by AFM, however, are very uniform in size for all three copolymers. Using the AFM phase step function (Figure 3e), the diameter of the polymersome bilayer walls for RCP-1−3 were measured (ca. 76 ± 8, 86 ± 11, and 105 ± 10 nm, respectively) to correlate with the calculated unperturbed end-to-end distance. The average bilayer thickness of RCP-1 and -2 matches closely with the calculated diameter for the bilayer structure depicted in Figure 3f (ca. 68 and 106 nm). For RCP-3, however, the bilayer diameter is substantially larger than the estimated value calculated (ca. 59 nm for a similar nanostructure depicted in Figure 3e) which leads to the different bilayer structure proposed in Figure S14e. This consists of two rows of RCP-3 aggregating with PPMC blocks occupying the core of the polymersome walls with a diameter of ca. 82 nm matching closer to the measured bilayer walls provided by AFM statistical analysis. Self-Assembly of RCPs Cast from Dilute THF/MeOH Solutions. Switching the selective solvent from H2O to MeOH induced significant size changes observed via DLS. These solutions were prepared in the same manner as previously stated by dissolving the RCPs in THF and injecting the solutions into specific amounts of MeOH to achieve the final c = 0.5 mg/mL. Each RCP displays the same trend by DLS with two size distributions observed in both THF/10 vol % MeOH and MeOH/10 vol % THF solutions (see Supporting Information). RCP-1 was first spin-coated from THF/10 vol % MeOH solutions onto silicon wafers and imaged using AFM revealing interesting nanomaggot micelle assemblies (Figure 4; also in Figure S15). These unique aggregates possess an oblong shape with substantially larger lengths (ca. 247 ± 42 nm) than widths (ca. 109 ± 13 nm) measured by AFM statistical analysis. The widths of these aggregates closely mimic the measured average diameter of spherical micelles observed when RCP-1 is spin-

Figure 4. Height (a) and phase (b) AFM micrographs (scan size = 3.0 × 3.0 μm; inset size = 500 × 500 nm) of RCP-1 spin-coated from THF/10 vol % MeOH (c = 0.5 mg/mL) showing nanomaggot micelle assemblies. Also shown are the height step functions (c) used for statistical analysis (d) to measure the width and length of the aggregates.

coated from H2O/10 vol % THF solutions. These sizes do not match up closely with two, equally distributed sizes observed by DLS in THF/10 vol % MeOH (ca. Dh = 21.7 ± 5.0 and 273 ± 71.7 nm), but they do agree closely with the observed sizes of RCP-1 in MeOH/10 vol % THF (ca. Dh = 111 ± 45.7 and 296 ± 102 nm), suggesting that the aggregation behavior in solution may follow a slightly different trend. To confirm or refute this, a follow-up study employing cryo-TEM to image solution aggregates will be conducted. The AFM micrographs of RCP-1 spin-coated from dilute MeOH/10 vol % THF solutions revealed another interesting set of aggregates and provided possible evidence for the formation of these unique assemblies (Figure 5). In these images, we visualized possible transient species with a large 6893

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Figure 5. Height (a) and phase (b, c) AFM micrograph of RCP-1 (a, b: scan size 4 × 4 μm; c: scan size = 1.5 × 1.5 μm) spin-coated from MeOH/ 10 vol % THF displaying mixtures of spherical and nanoworm micelles hypothesized to be formed via the self-association of spherical micelles.

Figure 6. Height (a) and phase (b) AFM micrographs (scan size = 5.0 × 5.0 μm; inset size = 1.0 × 1.0 μm) of RCP-2 spin-coated from MeOH/10 vol % THF solutions (c = 0.5 mg/mL) displaying long, nanoworm micelle aggregates. Also shown is the height step function (c) across the length of one aggregate showing the apparent undulation leading to the hypothesized structure depicted (d).

size distribution corresponding to Dh = 18.7 ± 3.7 nm was also observed, suggesting that at this concentration only some of the copolymer chains aggregate in solution whereas some chains are well solubilized. RCP-2 dissolved in dilute MeOH/10 vol % THF solutions displayed two size distributions with Dh = 317 ± 41 and 974 ± 162 nm via DLS. When spin-coated from MeOH/10 vol % THF solutions, RCP-2 aggregates into long, nanoworm micelles exclusively as evidenced by AFM (Figure 6; also in Figure S17). The average extended length of the nanoworm aggregates measured by AFM is 842 ± 508 nm ranging from 275 nm to 2.48 μm long. The widths (ca. 130 ± 20 nm) match closely with the diameters of the spherical micelles observed for RCP-2 previously. Additionally, the apparent undulation across the lengths of these aggregates, best shown in the step function in Figure 6c, similarly display diameters closely relating to the diameters of the spherical micelles of RCP-2 when spin-coated from H2O/10 vol % THF and THF/10 vol % MeOH. These results further support the hypothesized structure shown in Figure 6d where the long, wormlike assemblies are formed upon association of spherical micelles in solution/solid state. RCP-3 spin-coated from THF/MeOH solutions displayed slightly different morphologies by AFM. When spin-coated

number of simple, spherical micelles present throughout. Intermittent with the spherical micelles are several much larger nanoworm micelles (best visualized in Figure 5c). The diameters of the spherical micelles by AFM statistical analysis revealed diameters of ca. 87 ± 19 nm, which is slightly smaller than the smaller size distribution observed via DLS in MeOH/ 10 vol % THF solutions. In the zoomed AFM image (Figure 5c), the transient formation of the largest observed nanoworm micelle can be seen where it appears as if the self-association of spherical micelles causes the formation of the long nanoworm micelle aggregates. Similar formation of nanoworm micelles have been reported previously for amphiphilic block copolymers.54,55 The longest wormlike micelle displayed a fully extended length greater than 2.09 μm with a range of widths from 42−130 nm depending on the spot measured. AFM analysis of RCP-2 spin-coated from dilute THF/10 vol % MeOH solutions exhibited simple, spherical micelle assemblies with comparable diameters (ca. 138 ± 11 nm) to the micelle aggregates observed when spin-coated from H2O/ 10 vol % THF solutions (Figure S16). These diameters are also slightly smaller with the larger observed size distribution via DLS of Dh = 211 ± 42.8 nm following the trend observed throughout this study. Additionally, a smaller, equally intense 6894

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Macromolecules from THF/10 vol % MeOH, we observed shorter nanomaggot micelles, similar to RCP-1 spin-coated from THF/10 vol % MeOH, intermittent with large amounts of spherical micelles (Figure S18). Spin-coating RCP-3 from MeOH/10 vol % THF solutions, however, portrayed in interesting phenomenon. Toward the center of the silicon wafer, long nanoworm micelles were again observed although less spread out than observed for RCP-2. Toward the edge of the wafer, the more concentrated portion when spin-coating, large bundles of wormlike micelles were observed, suggesting that these aggregates prefer to self-associate upon solvent evaporation (Figure S19) possibly due to unfavorable interactions between the polymer and silicon substrate. Furthermore, RCP-4 spin-coated from THF/10 vol % MeOH displayed small spherical micelles with a measured diameter of 104 ± 13 nm (Figure S20). Cast from THF/25 vol % MeOH, the AFM micrographs of RCP-4 display mixtures of the spherical micelles intermittent with shorter nanoworm micelles (Figure S21). Again, like in the case of THF/H2O solutions, RCP-4 completely precipitated from solution at concentrations of MeOH greater than 25 vol %. For all AFM micrographs of RCP-3 and RCP-4 spin-coated from THF/ MeOH solutions, see the Supporting Information. Self-Assembly of RCP/PPMC Homopolymer Blends Cast from THF/MeOH. In certain cases, blending high-MW, hydrophobic rod homopolymer with the amphiphilic RCPs has been shown to induce significant alterations to the observed morphologies.26,27,34 To apply this to our system, a high MW (R)-PPMC774 (n = 774, Mn = 124 kDa, Đ = 1.60) was synthesized and blended with each of the RCPs at different proportions (25 and 50 wt % with respect to the homopolymer) in different solvent combinations. To prepare each solution, the copolymers and homopolymer were first mixed at c = 5.0 mg/mL in THF and injected into the corresponding amount of THF/H2O, THF/MeOH, or THF/ EtOH solutions so that, again, the final c = 0.5 mg/mL. THF/ H2O solutions were first attempted, but with the exception of RCP-2, the RCPs could not effectively encapsulate the hydrophobic PPMC homopolymers causing the polymer to precipitate with as little as 10 vol % water introduced. For RCP2, we imaged the aggregates formed when the 15 wt % PPMC774 blend was spin-coated from THF/10 vol % H2O by SEM revealing large microcapsules with the homopolymer hypothesized to be encapsulated inside (Figure S22). Any more homopolymer/H2O also caused the polymer to precipitate. This is believed to be due to the difficulties in encapsulating such high-MW rigid-rod polymer into the spherical structures observed for all RCPs cast from THF/H2O. We then switched our focus to blended THF/MeOH solutions to see if different aggregation behaviors would be observed, as was the case for the RCPs absent of homopolymer. Casting the 25 and 50 wt % blended systems resulted in the formation of bundled, interconnected nanofiber networks with the high-MW homopolymer effectively encapsulated within (Figure 7 displays 50 wt % blend with RCP-2 and -3; for all other nanofiber AFMs see Supporting Information). The height AFM micrographs can to go with the phase images in Figure 7 can be found Figures S27 and S28. Also present in some of the AFM images, intermittent with the long nanofibers, were smaller toroidal nanostructures which can be visualized in Figures 7b,d. RCP-1 and -2 have previously been shown to adopt nanofibullar morphologies when cast from THF solutions and annealed above the melting temperature at 65

Figure 7. Phase AFM micrographs (a, c: scan size = 10 × 10 μm; b, d: scan size = 2.0 × 2.0 μm) of RCP-2 (a, b) and RCP-3 (c, d) blended with 50 wt % PPMC774 homopolymer and spin-coated from THF/25 vol % MeOH displaying interconnected, bundled nanofibers.

°C for 24 h.53 Furthermore, hydrophobic polycarbodiimide homopolymers and graft copolymers can adopt nanofibullar and toroidal morphologies when subjected to specific conditions.53,56 The spin-cast 25 wt % blended solutions displayed mostly individualized nanofibers by AFM without significant entanglements or higher-order nanofiber association present (see Supporting Information). For the 50 wt % blended systems, however, the AFM micrographs displayed significant nanofiber entanglements throughout a continuous fibrous network. For RCP-2, several different tangling pathways can be easily visualized by AFM including parallel packing, perpendicular wrapping, and helical twisting of nanofibers (all shown distinctly in Figure 7b). RCP-3, however, only displayed parallel bundles of nanofibers with a large number of branching points observed by AFM. Interestingly, altering the RCP or relative amount of homopolymer did not alter the thickness of the nanofibers with average diameters of 167−171 nm for all nanofibers analyzed. For RCP-4, blending 50 wt % PPMC774 induced the formation of toroid-like aggregates (Figure 8) intermittent with the long, tangled nanofibers similar to the ones observed for RCP-1−3 by AFM. These copolymer blends also showed some toroid nanostructures scattered throughout but not with the same frequency as observed for RCP-4. Typically, these toroidal nanostructures have only observed in the self-assembly of complex triblock copolymers57 and unnatural oligopeptides.58,59 Because of their relatively small average diameters (ca. 222 ± 39 nm for RCP-4 by AFM statistical analysis) and overall curvature, we hypothesize that these toroid assemblies do not contain the high-MW, rodlike PPMC774 encapsulated unlike the long interconnected nanofibers (see Figure 8d). Self-Assembly of RCP/PPMC Homopolymer Blends Cast from THF/EtOH. The superhelical motif has been identified in self-assembled peptide,60 peptide-doped polyaniline,61 carbon nanotube,62 and chiral supramolecular polymer63 nanostructures, to name a few. So far, we have observed six different morphologies adopted by these novel RCPs which can 6895

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Figure 8. Height (a, b) and phase (c) AFM micrographs (a: scan size = 10 × 10 μm; b, c: scan size = 3.0 × 3.0 μm) of RCP-4 blended with 50 wt % PPMC774 and spin-coated from THF/25 vol % MeOH displaying mixtures of tangled nanofiber and toroidal nanostructures with the hypothesized structures depicted (d).

Figure 9. Height (a, b, d, e) and phase (c, f) AFM micrographs (a, d: scan size = 3.0 × 3.0 μm; b, c, e, f: scan size = 1.0 × 1.0 μm) of RCP-1 (a, b, c) and RCP-2 (d, e, f) blended with 50 wt % (R)-PPMC774 homopolymer and spin-coated from THF/25 vol % EtOH revealing interconnected, P superhelical nanofibers assemblies.

seems to template the formation of specific superhelical handedness since both RCP-1 and -2 possess (R)-1-phenethyl side chains on the PPMC blocks. These superhelical nanofibers appeared to be highly tangled, much like the straight nanofibers observed when the RCP/homopolymer blends were spincoated from THF/25 vol % MeOH. The average diameters of the nanofibers formed from RCP-1 and -2 were measured by AFM statistical analysis to be 131 ± 23 and 146 ± 32 nm, respectively. Also, the average helical pitch for these aggregates was measured for RCP-1 and -2 to be 58 ± 9 and 75 ± 16 nm, respectively. Additionally, the chiral pairing with the respective homopolymer in the blend greatly affects the nanostructures observed. When the chiral pairing was mismatched, i.e., (R)-(S) RCPhomopolymer blends for RCP-1 and -2, we observed what appeared to be frustrated nanofiber assemblies in which defined

be selectively tuned simply by altering the solvent combinations employed for spin-coating or blending high-MW PPMC homopolymer. Because of the chiral, helical nature of the PPMC blocks, we hypothesized that these copolymers may also self-assemble into ordered superhelical nanofibers similar to peptide-b-PEG block copolymer blends reported by Cai et al.26,27 This was confirmed with RCP-1 and -2 adopting interconnected, right-handed P superhelical nanofiber assemblies when blended with 50 wt % (R)-PPMC774 and spin-coated from THF/25 vol % EtOH (Figure 9; additional images of RCP-1 and 2 superhelices can be found in Figures S29 and S30). Using VCD spectroscopy, we have previously assigned righthanded, P helical backbone rotation to (R)-PPMC and vice versa (M helix) when incorporating (S)-1-phenethyl side chains.50 The helical rotation of the polymer backbone also 6896

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Figure 10. Height (a, b) and phase (c) AFM micrographs (a: scan size = 5 × 5 μm; b, c: scan size = 2.0 × 2.0 μm) of RCP-3 blended with 50 wt % (S)-PPMC133 and spin-coated from dilute THF/25 vol % EtOH displaying bundled, left-handed M superhelical nanofibers.

Table 1. Summarized Nanostructure Morphologies Adopted by Each Copolymer Spin-Coated from Different Solvent Combinations spin-coating conditions RCP

H2O/10 vol % THFa

(R)-1

spherical micelles

polymersomes

(R)-2

spherical micelles

(S)-3 (R)-4

THF/10 vol % H2Oa

MeOH/10 vol % THFa

THF/10 vol % MeOHa nanomaggot micelles

polymersomes

spherical and nanoworm micelles nanoworm micelles

spherical micelles

polymersomes

bundled nanoworm micelles

N/Ac

spherical micelles

N/Ac

spherical and nanomaggot micelles Spherical micelles

spherical micelles

THF/25 vol % MeOH blendb bundled nanofibers toriods bundled nanofibers toroids bundled nanofibers toriods bundled nanofibers toroids

and and and and

THF/25 vol % EtOH blendb P superhelical nanofibers P superhelical nanofibers M superhelical nanofibers P superhelical nanofibers

a Spin-coated from dilute 0.5 mg/mL solutions. bBlended with 50 wt % PPMC homopolymer (containing the same chiral side chain as the corresponding RCP) at final total c = 0.5 mg/mL and spin-coated. cShowed no solubility.

defined, tunable structures. All of these potential applications are currently being explored. All four synthesized RCPs follow very similar, solvent-tunable trends, and to better portray the multitude of self-assembled nanostructures adopted by each copolymer, these results are summarized and tabulated in Table 1.

right- or left-handed helices could not be distinguished (Figures S33a and S33b). When blended with (R)-PPMC774 and spincoated from THF/25 vol % EtOH, RCP-3 displayed interconnected nanofibers with some distinguishable righthanded superhelices by AFM analysis. Furthermore, the hydrophobic homopolymers were encapsulated into large sheets from which both long, straight nanofibers and superhelical nanofibers emanate (Figure S34b). This is believed to be attributed to the less favorable encapsulation of the highMW (R)-PPMC774 homopolymer due chiral mismatching of the (S) 1-phenethyl side chain in RCP-3. When RCP-3 was blended with (S)-PPMC homopolymer (n = 133, Mn = 21.2 kDa) and spin-coated from THF/25 vol % EtOH, however, the formation of left-handed M superhelical nanofibers was confirmed by AFM (Figure 10; also in Figure S31), allowing for the selective formation of superhelical nanostructures with defined chirality simply by attaching different enantiopure side chains. These nanofibers possess smaller diameters (ca. 123 ± 20 nm) and helical pitch (ca. 48 ± 5 nm) when compared to the previous examples believed to be consequence of the lower MW of the (S)-PPMC 133 homopolymer in the blend. RCP-4 was also found to adopt P superhelical nanofibers when blended with (R)-PPMC774, similar to RCP-1 and RCP-2 (Figure S32). This novel block copolymer system has proven to be one of the most versatile reported to date with significant promise in applications such as nanoscale lithography, biomimetics, and drug delivery. Because of the relatively low degradation temperatures (ca. Td = 174 °C)50 needed to revert the PPMC backbone back to starting carbodiimide monomer, another potentially fruitful application for these copolymers includes using them as degradable, soft polymeric templates for preparation of organic/inorganic nanomaterials with well-

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CONCLUSION The self-assembly of helical-b-random coil RCPs with chiral PPMC and hydrophilic PEG segments was controlled by augmenting the processing solvent composition and/or blending with hydrophobic homopolymer. Up to seven different structures were observed using these two simple approaches to control self-assembly introducing a potentially useful family of block copolymer in the field of tunable polymeric nanostructure assemblies. Spherical micelle assemblies or polymersomes were observed for all copolymers spincoated from THF/10 vol % H2O. Switching the selective solvent from 10 vol % H2O to 10 vol % MeOH in THF solutions produced nanomaggot assemblies for RCP-1 and -3. Methanol solutions of RCP-1−3 with 10 vol % THF produced long nanoworm micelles. Blending all copolymers with 50 wt % high-MW PPMC homopolymer and spin-coating from THF/ 25 vol % MeOH solutions induced the formation of long, interconnected nanofibers with several different tangling pathways constructing a continuous nanofiber network. A large number of toroidal nanostructures were also observed intermittent with the long, bundled nanofibers, with the highest frequency observed for RCP-4. Finally, switching the selective solvent from MeOH to EtOH in the blended solutions templated the formation of bundled superhelical nanofibers with defined helicity depending on the chiral pairing of RCP to 6897

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homopolymer (R-R formed P superhelical nanofibers and vice versa for S-S).

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b01564. Additional AFM analysis of all copolymers and synthesis/characterization of RCP-4 (PDF)

EXPERIMENTAL SECTION

Materials. The synthesis and characterization of RCP-1−3 have been previously reported.53 The NMR, FTIR, and GPC characterization of RCP-4 can be found in the Supporting Information. All solvents used for AFM sample preparation and DLS analysis were purchased from Fisher Scientific and used as received. The silicon wafers used as thin-film substrates were purchased from Wafer World and used as received. Equipment. AFM was employed to investigate the aggregation behavior of the coupled, amphiphilic block copolymers using a Nanoscope IV-Multimode Veeco instrument equipped with an E-type vertical engage scanner. All images were recorded with resonant frequencies of 320 kHz and lateral scan frequencies of 0.996 Hz using silicon TESP, V-shaped cantilever probes with a nominal spring constant of 42 N/m. Scan areas ranged from 500 × 500 nm to 10 × 10 μm. DLS measurements were conducted on Malvern Zetasizer particle sizer Nano ZS model equipped with He−Ne laser source (633 nm; Max 4 mW) to measure the hydrodynamic aggregate size in solution for comparison. DLS Analysis of Copolymers. All copolymers were first dissolved in stock solutions of THF (c = 5.0 mg/mL) in a clean Eppendorf tube, and a small amount (typically 100 μL) was injected into the corresponding amount of THF/H2O or THF/MeOH to achieve the reported solvent combination and a final c = 0.5 mg/mL. Then, ∼1.0 mL of the solution was filtered through 0.45 μm PTFE syringe filter into a clean quartz cuvette, and the DLS measurement was performed at 25 °C. The DLS plots of hydrodynamic diameter vs intensity and delay time vs correlation coefficient for RCP-1−3 in H2O/10 vol % THF, THF/10 vol % H2O, MeOH/10 vol % THF, and THF/10 vol % MeOH solutions are displayed in the Supporting Information. Because of poor solubility, DLS of RCP-4 was not studied in H2O/10 vol % THF and MeOH/10 vol % THF. Also included in the Supporting Information are the corresponding intensity-average sizes and standard deviations for each solution. Each of the THF/MeOH solutions displayed two distinct size distributions denoted Dh(s) for the smaller size distribution and Dh(L) for the larger size distribution. All DLS values presented in this article are intensity-average size values due to the presence of bimodal size distributions in many cases which has been shown to skew cumulant analysis of the polymeric aggregate, according to previous reports.64 AFM Imaging of Copolymers. The self-assembly behaviors of all PPMC−PEG copolymers depend greatly on the specific sample preparation procedures employed. All TMAFM samples reported were prepared by passing dilute copolymer solutions through 0.45 μm PTFE syringe filters and spin-coating onto silicon wafers (Wafer World) at 1000 rpm for 30 s to allow for the uniform distribution of the polymer aggregates across the entire silicon substrate. Specific aggregation behaviors in mixed-solvent systems were prepared by first dissolving the copolymers in THF stock solutions (5.0 mg/mL) and injecting specific amounts (typically 100 μL) into corresponding solvent mixtures of THF/H2O or THF/MeOH so that the final solvent combinations are those outlined in the discussion (final c = 0.5 mg/mL). Each solution was then spin-coated onto silicon wafers as before and dried/stored under vacuum in a desiccator prior to imaging. For blended systems, the stock solutions of copolymer and homopolymer (both at c = 5.0 mg/mL) were premixed (50:50 by weight) and injected into the corresponding solvent mixture to achieve the final c = 0.5 mg/mL and solvent proportions of THF/25 vol % MeOH and THF/25 vol % EtOH. All additional AFM images referred to in the main text can be found in the Supporting Information.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (B.M.N.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding for this work was provided by the Faculty start-up fund from the University of Texas at Dallas (UTD) and the Endowed Chair for Excellence at UTD. We gratefully acknowledge the NSF-MRI grant (CHE-1126177) used to purchase the Bruker AVANCE III 500 NMR instrument.

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