Organometallic–Polypeptide Diblock Copolymers: Synthesis by Diels

Article pubs.acs.org/Macromolecules

Organometallic−Polypeptide Diblock Copolymers: Synthesis by Diels−Alder Coupling and Crystallization-Driven Self-Assembly to Uniform Truncated Elliptical Lamellae Gregory Molev,† Yijie Lu,† Kris Sanghyun Kim,† Ingrid Chab Majdalani,† Gerald Guerin,† Srebri Petrov,† Gilbert Walker,† Ian Manners,*,‡ and Mitchell A. Winnik*,† †

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto M5S 3H6, Ontario, Canada School of Chemistry, University of Bristol, Bristol, United Kingdom BS8 1TS

‡

S Supporting Information *

ABSTRACT: This paper reports a new synthetic strategy for the preparation of polyferrocenylsilane (PFS) block copolymers (BCPs), by conjugation of two independently prepared homopolymers using Diels−Alder cycloaddition. The PFS blocks were synthesized by photocontrolled ring-opening polymerization, yielding polymers with a cyclopentadienyl end group that serves as a diene in the conjugation reaction. In this initial study we focused on the synthesis of organometallic−polypeptide block copolymers PFS-b-PBLG (PBLG = poly(γ-benzyl-L-glutamate) using PBLG polymers with a terminal maleimide attached by one-step postpolymerization modification. Five PFS-b-PBLG copolymers with different degrees of polymerization were synthesized and purified by a series of selective precipitations. The self-assembly of these samples was studied in N,N-dimethylformamide, a solvent selective for PBLG. The BCPs with a PFS block longer than the PBLG block after annealing at 90 °C formed highly uniform platelet micelles with a truncated elliptical shape. Experiments at 75 °C showed that disordered elongated structures formed initially, with fiber-like protrusions from the ends. Over time, the structures became shorter and wider, evolving into uniform truncated elliptical micelles. The process was monitored by X-ray diffraction (XRD) measurements and by 1H NMR spectroscopy. AFM analysis showed that the micelles were flat and that their thickness increased with the overall chain length of the BCP. Selfassembly of these BCPs in the presence of PFS homopolymer led to formation of flower-like mesostructures consisting of stacks of lamellar petals.

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INTRODUCTION Block copolymers (BCPs) have received considerable attention due to their ability to self-assemble into a variety of different morphologies either in bulk1 or in solution.2 One subset of these materials, biopolymers such as polypeptides conjugated with synthetic organic polymers, has been developed significantly in recent years.3 For example, synthetic polymers synthesized by ring-opening metathesis polymerization (ROMP), while grafted with oligopeptides, found applications as polyelectrolytes,4 enzyme substrates,5 or unimers which selfassemble to tubular structures, resembling biological superstructures assembled from proteins.6 Vesicles were prepared from peptidic side-chain polymers synthesized by atom transfer radical polymerization (ATRP).7 Conjugates of antibodies with polymers synthesized by reversible addition−fragmentation chain-transfer (RAFT) polymerization, which are formally organic−polypeptide diblock copolymers, were designed for use in multiplexed immunoassays based on both mass cytometry and fluorescent flow cytometry.8 Additional examples of new organic−polypeptide diblock copolymers © 2014 American Chemical Society

include poly(3-hexylthiophene-b-γ-benzyl-L-glutamate) copolymer (P3HT-b-PBLG), which forms nanoparticles in N,Ndimethylformamide (DMF),9 poly(Z-lysine)-based block copolymers, forming thermoreversible gels in tetrahydrofuran (THF),10 and many others, summarized in a recent review.11 In 2005, our groups reported the synthesis of the first organometallic−polypeptide block copolymer, poly(ferrocenyldimethylsilane-b-γ-benzyl- L -glutamate) (PFS− PBLG),12 and examined its self-assembly in toluene.13 Toluene is a good solvent for PFS but promotes association of PBLG chains. We found that this block copolymer formed welldefined ribbon-like structures that were characterized by transmission electron microscopy (TEM) and by small- and wide-angle X-ray scattering and under certain conditions caused the solutions to gel. To test whether the PFS block played a role in the self-assembly in toluene, we synthesized Received: November 28, 2013 Revised: March 19, 2014 Published: April 3, 2014 2604

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Scheme 1

then coupled by complexation to a ruthenium center to yield a PFS-Ru-PEO diblock copolymer.23 Recently our groups reported a protocol for obtaining PFS-based BCPs by CuAAC, which was the first example of PFS-based BCP preparation by covalent conjugation.24 Using this methodology, we prepared the following PFS-based BCPs: PFS-b-poly(Nisopropylacrylamide), PFS-b-poly(2-(dimethylamino)ethyl methacrylate), PFS-b-poly(methyl methacrylate), PFS-b-poly(dimethylsiloxane) (PDMS), PFS-b-PS, and PFS-b-PEO. In the work described here, we take advantage of the photocontrolled ring-opening polymerization (PROP) of dimethylsila[1]ferrocenophane (Scheme 1).25 PFS chains obtained in this way have a cyclopentadienyl (Cp) end group, which we exploit for conjugation with a second polymer by Diels−Alder cycloaddition. This is an attractive strategy for preparing PFS-based BCPs, as postpolymerization modification of the PFS is unnecessary. Previous reports on coupling of homopolymers by a Diels− Alder reaction may be divided into three major types.26 The first consists of reactions between polymers with an anthracene end group as the diene and a second polymer with a maleimide end group as the dienophile. This reaction usually requires high temperature (≥110 °C) and long reaction times (35−120 h).27 A second approach involves the reaction between polymers capped with a dienophile and polymers with a furan end group as the diene. This reaction works well under mild conditions, but the conjugation product undergoes a retro-Diels−Alder reaction at ca. 90 °C.28 The third method reported by BarnerKowollik and co-workers29 involves a Diels−Alder reaction between polymer capped with a diene and a second polymer with a terminal activated thiocarbonyl as the dienophile. The polymers with the thiocarbonyl end group are prepared by RAFT polymerization using chain transfer agents with an electron-withdrawing group attached to the thiocarbonyl. In this paper we report the synthesis of a series of PFS− PBLG diblock copolymers by Diels−Alder conjugation of two homopolymers: PFS-Cp, synthesized by PROP, and PBLG-Mal (Mal = maleimide), synthesized by one-step postpolymerization modification of PBLG. In addition, we present results on self-assembly of these BCPs in DMF, which is a selective solvent for PBLG. The three BCP samples with a PFS block longer than the PBLG block self-assemble into uniform flat truncated elliptical micelles, with shapes and thicknesses that

corresponding PBLG diblock copolymers with a polystyrene (PS) block and with a poly(ethylene oxide) (PEO) block. These PS−PBLG and PEO−PBLG polymers exhibited similar behavior in toluene.13 At that time, we did not investigate the self-assembly of PFS−PBLG in a solvent selective for PBLG, in which the PFS might drive micelle formation by its crystallization in the micelle core.14 In a more recent publication, we reported the preparation of conjugates of PFS with tetrapeptides and the study of their self-assembly in toluene as well as their solid-state behavior.15 We found that in toluene the PFS−tetrapeptide conjugates formed a cross-linked network of fibers having a PFS corona. The PFS corona was accessible for further chemical oxidation that was demonstrated by the formation of silver nanoparticles on the surface of the fibers. In 2008, we reported the preparation of PFS-b-PZLys (PZLys = poly(ε-benzyloxycarbonyl-L-lysine) copolymers.16 Self-assembly of this BCP at room temperature in a PZLys selective solvent (DMF) alone or in the presence of a common solvent (THF) led to formation of rod-like micelles with a PFS core. In these previous studies,12,15,16 the PFS−polypeptide block copolymers were synthesized by a four-step reaction. In the first step, PFS was synthesized by anionic ring-opening polymerization, and its anionic terminus was quenched with bis-silylprotected 1-amino-3-bromopropane. After deprotecting the amino group with methanol, the amino-end-capped polymer was employed as the initiator for the ring-opening polymerization of the corresponding N-carboxyanhydride. Over the past few years, there has been a growing interest in the synthesis of block copolymers by coupling two homopolymers, synthesized independently, and with appropriate reactive functionality at one end. This strategy relies on a class of reactions like the Cu-catalyzed alkyne/azide cycloaddition (CuAAC) reaction and some other types of reactions (e.g., thiol−ene, Diels−Alder, pyridyl disulfide) that are categorized as robust, efficient, and orthogonal.17 This approach has been used recently for the fabrication of soft materials,18 drug delivery constructs,19 and novel structures20 for applications in biology21 and in semiconductor technology.22 There is an early report in the literature of a PFS block copolymer synthesized in this manner: both PFS and PEO were synthesized with a terpyridine at one end. The polymers were 2605

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A byproduct in this reaction is a PFS dimer, detected as a faster eluting shoulder in gel permeation chromatograms (GPC) of the polymer (marked with an asterisk in Figure S1 of the Supporting Information). The dimer content of the byproduct was determined by fitting Gaussian functions to these GPC traces using Microsoft Excel and calculating the relative fractional area of the dimer peak. After the synthesis, the PFS-Cp polymers were stored as neat solids in screwcapped vials at room temperature. After 3 months, they were analyzed again by GPC. We observed (Figure S1B) that the dimer content had increased. In Table 1, the column labeled “purity” indicates the relative amount of PFS-Cp in the asprepared sample and after storage as a bulk solid for 3 months. Over that time, on the order of 10% of the polymer dimerized. The most obvious pathway for dimerization of PFS-Cp is a Diels−Alder reaction between the terminal Cp rings on two polymer chains, and NMR data support this suggestion (Figure S2). To test whether this is a facile reaction, a solution of PFS25-Cp (10 mg) was heated in 1 mL of 1,2-dichloroethane (DCE), at 75 °C for 24 h. An 1H NMR spectrum of this sample in CD2Cl2 showed preservation of the Cp end group, and the GPC trace was identical to the original trace of PFS25-Cp. This experiment shows that no detectable Diels−Alder reaction between two PFS chains takes place in solution, and this is an important control reaction for the conjugation of PFS-Cp with PBLG-Mal described below. However, we cannot exclude the possibility that some degree of Diels−Alder coupling occurs in the solid state. On the other hand, the Cp group attached to Si is very labile and can act as an anionic or a radical leaving group.30 Therefore, the dimer content might have increased with time due to hydrolysis or oxidation of the Si−Cp bond. Maleimide-End-Capped Poly(γ-benzyl- L -glutamate) (PBLG-Mal). The synthesis of maleimide-functionalized PBLG (PBLG-Mal) is shown in Scheme 2. In the first step, polymerization of γ-benzyl-L-glutamate N-carboxyanhydride was initiated with 2,2-dimethyl-1,3-dioxolane-4-methanamine (DDM) in DMF as described previously.31 In the second step, the amino end group of PBLG was reacted with pentafluorophenyl 6-maleimidohexanoate. DPn values and the degrees of modification with maleimide (see column “purity” in Table 1) were determined by 1H NMR (in DMSO-d6) by end-group analysis. DPn was determined by comparing the integrals of the two methyl signals at 1.2 ppm of the dimethyl-1,3-dioxolane ring with the integral of the benzyl groups at 7−7.5 ppm. The maleimide content was determined from the corresponding integration of the maleimide singlet at 6.8 ppm. The 1H NMR spectra of PBLG30-Mal and PBLG80-Mal are presented in Figure S3, and their GPC traces of are presented in Figure S4. Values of DPn, Mn, the polydispersity index (Đ = Mw/Mn), and the purity (nondimerized PFS-Cp, maleimide content of PBLG-Mal) are collected in Table 1. All of these polymers had narrow polydispersities. For the analysis of GPC traces of PFSCp, we used a universal calibration in conjunction with polystyrene (PS) standards. Previously we reported that PFS molecular weights obtained in this way are in good agreement with molecular weights determined by low-angle laser light scattering.32 Preparation and Purification of Block Copolymers. First, we prepared two block copolymers with different block ratios: PFS40-b-PBLG30 and PFS25-b-PBLG80. Diels−Alder coupling reactions were carried out in 1 mL of 1,2dichloroethane (DCE) in a capped 7 mL glass vial with stirring bar at 75 °C. Samples of PFS-Cp (10 mg) with DPn =

depend on the polymer composition. In the presence of PFS homopolymer the BCPs self-assemble to form flower-like structures consisting of stacks of lamellar petals.

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RESULTS AND DISCUSSION Preparation of the End-Functional Homopolymers. Cyclopentadienyl-End-Functionalized Poly(ferrocenyldimethylsilane) (PFS-Cp). Three samples of PFS with a cyclopentadiene end group (PFS-Cp) were prepared by PROP following the methodology reported in ref 25 (Scheme 1). These are denoted PFS25-Cp, PFS40-Cp, and PFS90-Cp, where the subscripts refer to the target number-average degree of polymerization (DPn). Experimental values of DPn were determined by 1H NMR by comparing the integration of the Cp-Fe end group peak (marked “c” in Figure 1) with the

Figure 1. 1H NMR spectrum of PFS40-Cp in C6D6.

integration of the repeat unit peaks (marked “a” and “b” in Figure 1). These values (Table 1) are very close to the target values (DPn = 24 for PFS25-Cp, DPn = 39 for PFS40-Cp, and DPn = 90 for PFS90-Cp). Table 1. Characterization Data for PFS-Cp and PBLG-Mal polymera PFS25-Cp PFS40-Cp PFS90-Cp PBLG30Mal PBLG80Mal

Mn NMR,b kDa (DPn) 5.8 9.4 22 6.9

(24) (39) (90) (30)

18 (81)

Mn GPCc kDa (DPn)

Đc

purity,d %

5.6 (23) 9.3 (38) 20 (83) 4.5

1.03 1.02 1.02 1.06

94 (79) 95 (88) 94 (90) 99

11

1.07

97

a The values of the DPn have an estimated error ±10% error based on a precision of ±5% in peak integration. bCalculated from 1H NMR spectra by end-group analysis. cCalculated from GPC traces (refractive index detector, THF eluent for PFS-Cp, universal calibration plus PS standards; N-methyl-2-pyrrolidone (NMP) for PBLG-Mal, PMMA standards). For PBLG, calculated Mn values are nominal, and thus DPn(GPC) values were not calculated. dFor PFS-Cp: the percent of nondimerized polymer was calculated by fitting Gaussian functions to GPC traces for the as-synthesized polymer (values in parentheses refer to this quantity measured after storage of the solid polymer for 3 months at room temperature). For PBLG-Mal: the percent of polymer with a maleimide end group, calculated by comparing peak integrals in 1 H NMR spectra corresponding to initiator and maleimide groups.

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Scheme 2

Scheme 3

25 and 40 were reacted with PBLG-Mal with DPn = 80 and 30, respectively, with a 20 mol % excess of PFS-Cp (Scheme 3). After 2 h, an aliquot (0.1 mL) was taken, and after 18 h the DCE was evaporated in vacuum. 1H NMR spectra in CD2Cl2 were recorded, and quantitative PBLG-Mal conversion was detected by disappearance of the maleimide peak at 6.5 ppm (Figure 2A−C). In this reaction, the decrease of the maleimide peak intensity was accompanied by an increase of the intensity of the internal double bond peaks between 5.6 and 6.0 ppm (marked with arrows in Figure 2B). We assign these new peaks to the double bonds of the three isomers of bicycloheptene formed in the coupling reaction (Figure 2, top right corner). Three additional BCPs, PFS40-b-PBLG80, PFS90-b-PBLG30, and PFS90-b-PBLG80 were synthesized by a slightly different protocol (see Experimental Section for details). Characterization data for the BCPs are collected in Table 2. The block copolymers were purified as follows. First, each crude DCE solution was concentrated with a stream of air from an initial volume of 1 mL to a final volume of ca. 0.1 mL. Then the concentrated solution was added via pipet to an Eppendorf vial containing DMF (2 mL) at room temperature. An orange solid precipitated. The DMF solution (also orange) was

separated from the orange solid by centrifugation. GPC analysis of the precipitated orange powder dissolved in C6D6 indicated that it was composed primarily of the unreacted PFS-Cp with the PFS dimer impurity as major peak (cf. Figure S6). The orange solution was dried by DMF evaporation. The remaining solid was dissolved in a minute amount of THF (ca. 0.1 mL) and then added to a methanol/DMF 8:2 mixture (2 mL) at room temperature in another Eppendorf tube. An orange powder precipitated, leaving a transparent colorless methanol/ DMF solution. The residue was separated by centrifugation and dried in vacuum (ca. 10 Torr, 40 °C, 2 h). GPC and NMR analyses indicated that the precipitate included BCP without a detectable amount of the homopolymers. NMR spectra of the precipitated BCPs (cf. Figure 2D for PFS40-b-PBLG30) showed no decomposition upon purification. The final isolated yields were 9.4 mg for PFS40-b-PBLG30 (68%) and 10.3 mg for PFS25b-PBLG80 (32%). Figure 3 compares RI GPC traces of the purified PFS40-bPBLG30 (left peak) with those of the precursor homopolymers PFS40-Cp and PBLG30-Mal. The two peaks on the right were obtained from a mixture of the two polymers employed in the synthesis prior to heating the sample to initiate the reaction 2607

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Figure 2. 1H NMR spectra in CD2Cl2 of PFS40-b-PBLG30. (A) 1H NMR spectrum showing the Mal and Cp end groups of the reaction mixture at t = 0. (B) The portion of the spectrum after 2 h reaction. (C) The portion of the spectrum after 18 h reaction. Peaks assigned to the double bonds of the three isomers of bicycloheptene formed in the coupling reaction are marked with arrows. (D) Full spectrum of the purified polymer. Note that the two methyl groups (i) of the acetonide are not equivalent.

Table 2. Mn and Đ Values of the BCPs Obtained by Diels− Alder Coupling BCPa

Mn NMRb [kDa]

Mn GPCc [kDa]

Đc

PFS25-b-PBLG80 PFS40-b-PBLG30 PFS40-b-PBLG80 PFS90-b-PBLG30 PFS90-b-PBLG80

24 16 27 30 40

18 15 25 25 42

1.14 1.12 1.14 1.19 1.32

was conjugated with PBLG by Diels−Alder coupling. The absence of peaks at 25.8 and 27.5 mL indicates that little or no PFS and PBLG homopolymers were present in the purified diblock copolymer samples. Table 2 summarizes the characteristic of the block copolymers synthesized by Diels−Alder coupling. The righthand column reports block copolymer composition as determined by 1H NMR with that anticipated from coupling of the homopolymer precursors. In traditional block copolymer synthesis, one determines the molecular weight of the first synthesized block and then uses 1H NMR to determine the composition of the block copolymer. The Mn value of the block copolymer is calculated by combining these values. Here we know in advance the Mn values of the two components. The 1H NMR spectra give us a chance to compare the measured values with the anticipated values. With one exception, these values are in good agreement. Differences are found for PFS90-bPBLG30, which we know contained some PBLG homopolymer as an impurity. Self-Assembly Experiments at 23 and 90 °C. Selfassembly experiments were carried out in DMF, a good solvent for PBLG but a nonsolvent for PFS. To test the self-assembly of the BCPs, samples of PFS40-b-PBLG30 and PFS25-b-PBLG80

DPnPBLG in BCP from integration of benzyl CH2 and phenyl C6H5d 87, 29, 87, 48, 80,

95 30 87 49 85

a

The DPn values of each block are from those of the corresponding precursor homopolymers. bCalculated from DPn. cCalculated from GPC (THF) RI traces based on PS standards. dNMR values of the apparent DPn of the PBLG block were calculated by comparing the integration values of the PBLG benzyl protons at 4.9 ppm and the phenyl protons at 7.15 ppm to the integration of the PFS signal at 4.1 ppm (see Figure S7). This calculation assumes that the DPnPFS(NMR) value reported in Table 1 is correct. The PFS90-b-PBLG30 sample contained some PBLG homopolymer as an impurity, and this explains the discrepancy between the values in this column and the expected composition of the block copolymer.

(which we denote t = 0). The main conclusion to be drawn from this figure is that the reaction was successful and that PFS 2608

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Figure 3. GPC (THF, RI detection) traces of a purified sample of PFS40-b-PBLG30 and of the mixture of the two homopolymer reactants used in the synthesis (PFS40-Cp and PBLG30-Mal). The peak of the diblock copolymer is approximately normalized to that of PFS40-Cp in the reaction mixture. The PFS-dimer impurity present in the PFS40-Cp precursor is marked with an asterisk. Figure 4. TEM images of the structures formed at room temperature in DMF solution by (A) PFS25-b-PBLG80 and (B) PFS40-b-PBLG30 and after annealing at 90 °C by (C) PFS25-b-PBLG80 and (D) PFS40-bPBLG30. In (D), our notation for the width at the center (W) and at the termini (W′) of the truncated elliptical structures is indicated on one of the micelles. Additional images are presented in Figures S8 and S9.

(0.2 mg each) were dissolved in DMF (1 mL) at room temperature (ca. 23 °C) in glass vials. Part of the solution was kept at 23 °C. The second part was heated at 90 °C in an oil bath for 30 min, removed from the oil bath, and left to cool at room temperature. Then it was aged at room temperature for 2 days. For TEM analysis, 1 drop of each solution was deposited on a Formvar-carbon-coated TEM grid. The excess solution was removed by touching the edge of the drop with a KimWipe. Then the grid was left to dry for 1 h in a fume hood. For the solution of PFS25-b-PBLG80 prepared and aged at room temperature, clusters of spherical objects were observed on the grid (Figure 4A). In contrast, PFS40-b-PBLG30 at room temperature formed thin, flat elongated structures with fibers protruding from the ends (Figure 4B). Heating the solutions at 90 °C led to different results. PFS25b-PBLG80 yielded smaller objects (10−50 nm) without defined shape and some spherical micelles (Figure 4C). In contrast, for PFS40-b-PBLG30, with a shorter PBLG block relative to PFS, lamellar micelles were seen in the TEM images (Figure 4D). These structures are not strictly rectangular but rather have a truncated elliptical shape, wider at the center, than at the edges. They appear to be remarkably regular in size and shape. Self-assembly experiments at 90 °C using a similar protocol were carried out with the other BCPs. PFS40-b-PBLG80 yielded mostly small objects without defined shape similarly to PFS25-bPBLG80 (Figure S9A,B). However, PFS90-b-PBLG30 and PFS90b-PBLG80 yielded truncated elliptical micelles (Figures 5 and S8). These results show that the micelles with a truncated elliptical shape form preferably from the BCPs, in which the degree of polymerization of PFS block was longer or similar to that of PBLG. Interestingly, on most of the micelles of PFS90-b-PBLG80 areas of darker contrast were observed. These appear to be additional layers of BCP on the larger lamellae. The inset of Figure 5A exhibits a micelle of PFS90-b-PBLG80 with one such thicker layer, on top of which there is another smaller higher contrast region. These can be attributed to secondary and tertiary nucleation, which may be a result of a screw

Figure 5. TEM images of the micelles formed from (A) PFS90-bPBLG80 and (B) PFS90-b-PBLG30 in DMF, annealed at 90 °C. Additional images for each sample are presented in Figure S8.

dislocation36 in the crystalline micelle core, possibly promoted by traces of PFS homopolymer impurity. To test the chemical stability of the diblock copolymer under the self-assembly conditions, a sample of 2 mg of PFS40-bPBLG30 was dissolved in 1 mL of DMF and heated for 30 min at 90 °C. After DMF evaporation the sample was analyzed by NMR and GPC, which showed that the BCP was stable under these conditions. Previously we reported formation of uniform oval micelles in 2-propanol by addition of PFS54-b-PP290 (PP = poly[bis(trifluoroethoxy)phosphazene]) to short rod-like seed micelles of PFS34-b-P2VP272 (P2VP = poly(2-vinylpyridine)).34 Rectangular platelets were formed by self-assembly of PFS114-b2609

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PDMS80 in decane34 and by PFS60-b-PI30 in THF/hexane (3/7, v/v, PI = polyisoprene), but these were not uniform in size.14d In a more recent publication we reported formation of lenticular platelets by self-assembly of PFS74-b-P2VP74 in 2propanol.35 Micelles with a truncated elliptical shape represent a new morphology for PFS block copolymer micelles. It is also remarkable that these structures are uniform in size. The X-ray diffraction (XRD) pattern of a PFS40-b-PBLG30 sample annealed at 90 °C showed a peak at 2θ = 13.9° (Figure S14) that corresponds to a reflection at 6.4 Å. This peak, characteristic of PFS homopolymer samples crystallized from solution,37 can be assigned to the distance between adjacent planes containing planar zigzag PFS chains.38,39 Self-Assembly at 75 °C. To gain further insight about how the truncated elliptical micelles form, we carried out selfassembly experiments with PFS40-b-PBLG30 in DMF (0.25 mg/ mL) at 75 °C, where we could follow the evolution of the structures over time. TEM images from these experiments, with samples taken after 15, 30, 60, and 120 min annealing, are shown in Figure 6. One can observe that there is a gradual

the crystallinity of the core of the PFS40-b-PBLG30 micelles upon annealing at 75 °C in DMF. To test this idea, we examined the X-ray diffraction (XRD) patterns of samples obtained with and without annealing. One sample was dissolved in THF and precipitated with methanol (this sample we denote as “t = 0”). The other samples were suspended in DMF, annealed at 75 °C for 15, 30, 60, and 120 min, then removed from the oil bath, cooled to room temperature, and allowed to age overnight. This was the same protocol used to obtain the micelles shown in the TEM images in Figure 6. Then the samples were concentrated at room temperature (to ca. 0.2 mL) by evaporation of DMF under a stream of air, precipitated in methanol, and dried under vacuum. The results are presented in Figure 7. The sample precipitated from methanol and examined without annealing showed no significant signal. All the annealed samples showed a peak at 2θ = 13.9°, accompanied by a shoulder at 2θ = 13.6°. These peaks did not change in shape or intensity upon further annealing. The absence of a peak in the “t = 0” sample suggests that this sample is amorphous. The broad peak that appeared upon annealing is consistent with an increase in the degree of crystallinity of the sample. No significant changes in the XRD spectra were found in samples annealed for longer times. Thus, the changes in morphology seen in Figure 6 are not explained by differences in the degree of crystallinity of the micelles. We carried out 1H NMR measurements to obtain more information about these micelles. To begin, we ran a 1H NMR spectrum of a micelle solution at 25 °C (spectrum A in Figure S16). For this sample, we took the solution from which the TEM image in Figure 6A was obtained (annealed 15 min at 75 °C and then cooled to room temperature). In the spectrum taken at 25 °C, the peaks associated with the Cp rings of PFS can be identified, but they are weak. From their integrated intensity compared to peaks associated with the benzyl groups of the PBLG, we find that fewer than 10% of the ferrocenes contribute to this signal. The low intensity is consistent with the reduced mobility of the PFS groups in the micelle core. The weak signal itself is likely due to more mobile groups in the amorphous domains of the core near the core−coronal interface.14a Then the NMR tube was heated to 75 °C, and a series of spectra were acquired over 15 min intervals. We anticipated that the polymer in the less crystalline domains of the micelles would dissolve upon heating, leading to an increase in signal from the Cp rings, and that this signal would decrease in intensity as the polymer crystallized to form the more regular structures seen in Figure 6B−D. As seen in Figure S15B, at 75 °C the peaks associated with the ferrocene became sharp and much stronger in intensity. According to the peak integration, approximately 50% of the Cp rings contributed to the 1H NMR signal, implying that about half of the PFS repeat units are mobile. As we continued to monitor the NMR spectrum of this sample at 75 °C, however, the integrated intensity of the PFS ferrocene protons did not change relative to the benzyl protons of the PBLG chains over this time (Figure S17). One explanation for this result is that the polymer that dissolved upon heating to 75 °C remained in solution at this temperature. These polymers would no longer be soluble when the solution was cooled and would grow epitaxially off the edges of the crystals that survived in the annealed solution. Alternatively, the ferrocene peaks that appear at 75 °C are due to mobile repeat units in amorphous domains of the micelle core.14b,43

Figure 6. TEM images of the PFS40-b-PBLG30 micelles formed in DMF (0.25 mg/mL) at 75 °C. Vials containing this solution were removed from the oil bath after (A) 15, (B) 30, (C) 60, and (D) 120 min and cooled in air overnight. All scale bars are 500 nm.

change in the morphology with extended heating. After 15 min (Figure 6A) one sees elongated lenticular structures with multiple fibers protruding from the ends. The edges of these structures are disordered. (A related morphology was observed previously in studies of the self-assembly of PFS74-b-P2VP74 in 2-propanol.35) Upon further annealing of the PFS40-b-PBLG30 sample in DMF, this disorder slowly disappeared. The objects became shorter and wider. After 120 min (Figure 6D), only highly uniform truncated elliptical micelles were observed. Additional images are presented in Figure S10. Analysis of Samples Annealed at 75 °C by XRD and by 1 H NMR. We anticipated that the evolution of micelle morphology seen in Figure 6 was related to an increase in 2610

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Figure 7. X-ray diffraction patterns obtained for PFS40-b-PBLG30 micelles. Vials containing the samples (5 mg/2 mL) were annealed in DMF at 75 °C for different times [15 min (red), 30 min (blue), 60 min (green), and 120 min (purple)] and then cooled to room temperature. The “t = 0” sample (black) was dissolved in THF, precipitated in methanol, and dried under vacuum. It was not annealed.

Table 3. Sizes of the Lamellar Micelles Formed by PFS-b-PBLG Block Copolymers in DMF BCP

no. of micelles

Ln [μm]

Lw/Ln

Wn (W′n)a [μm]

Ww/Wn (W′w/W′n)

havb [nm]

PFS40-b-PBLG30 PFS90-b-PBLG30 PFS90-b-PBLG80

136 101 137

4.7 ± 0.4 1.3 ± 0.2 3.7 ± 0.3

1.006 1.019 1.006

0.46 ± 0.05 (0.34 ± 0.04) 0.25 ± 0.06 1.00 ± 0.06 (0.74 ± 0.08)

1.013 (1.014) 1.053 1.004 (1.008)

11.2 ± 0.9 12.8 ± 1.2 18.1 ± 1.2

a

Wn, number-average width at the center (widest point) of the lamellar micelles; W′, width at the micelle terminus. bhav values were calculated for single micelles shown in Figure 8, averaging more than 100 data points for each micelle using Microsoft Excel.

Analysis of the Micelle Geometry. Length and Width. In order to obtain a deeper understanding of the self-assembly process, we analyzed the shapes of the micelles formed by the different BCPs at 90 °C. From multiple TEM images, we measured lengths (Li) and widths (Wi) of individual micelles and analyzed these data using the image analysis software, ImageJ, published by the National Institute of Health, as described in our previous publications.39 The parameters of 136 micelles of PFS40-b-PBLG30, 137 micelles of PFS90-b-PBLG80, and 101 micelles of PFS90-b-PBLG30 were measured. The number-average micelle length (Ln) and width (Wn) and the weight-average micelle length (Lw) and width (Ww) were calculated using eqs 1 and 2, where N is the number of micelles examined in each sample. Because the structures are wider at the center than at the ends, we consider separately their widths at the widest point of each structure and the widths at the ends of the lamellae. For example, the average width at the centers of the micelles of PFS40-b-PBLG30 (Wn = 0.47 μm) is almost 30% larger than the average width at their termini (W′n = 0.34 μm) (see Figure 4B). In Table 3, we report these values of Ln, Lw/Ln, Wn, and Ww/Wn. N

Ln =

∑i = 1 NL i i N

∑i = 1 Ni

N

Lw =

2 ∑i = 1 NL i i

N

Wn =

∑i = 1 NW i i N

∑i = 1 Ni

As the images in Figure 4 suggest, the micelles of PFS40-bPBLG30 and of PFS90-b-PBLG80 are uniform in size, with values of Lw/Ln and Ww/Wn on the order of 1.01. The average length of the micelles is Ln = 4.8 μm for PFS40-b-PBLG30, and Ln = 3.7 μm for PFS90-b-PBLG80. The average width of micelles of PFS90-b-PBLG80 (Wn = 1.0 μm) is twice larger than the average width of PFS40-b-PBLG30 (Wn = 0.47 μm). The micelles of PFS90-b-PBLG30 are the smallest (Ln = 1.3 μm, Wn = 0.25 μm) and are somewhat more polydisperse (Lw/Ln = 1.02 and Ww/ Wn = 1.05) than those formed by the other two block copolymers. Height. To complement our analysis of the micelle lengths and widths, we measured their height profiles using intermittent contact mode AFM. Individual micelles of PFS40-b-PBLG30 (Figures 8A and 8B), PFS90-b-PBLG30 (Figures 8D and 8E), and of PFS90-b-PBLG80 (Figures 8G and 8H) were examined, and their 3D and height images are presented. The height profiles of a diagonal section of the micelles, marked with a dashed line on the corresponding height images B, E, and H, are shown in Figures 8C, 8F, and 8I, respectively. The 3D images and the height profiles show that all of the micelles are relatively flat through their length and width. The average heights (hav) were calculated from the height profiles presented in Figures 8C, 8F, and 8I by averaging more than 100 points for each micelle using Microsoft Excel, and they are given in Table 3. We calculated hav for one micelle of each type, but we assume that the micelles are uniform in their thickness as they are uniform in other two dimensions. The measured hav values show that the micelle thickness increased with the overall

N

∑i = 1 NL i i

(1)

N

Ww =

2 ∑i = 1 NW i i N

∑i = 1 NW i i

(2) 2611

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Figure 8. AFM images of the micelles obtained in intermittent contact mode. (A) 3D image, (B) height image, and (C) height profile of the micelle of PFS40-b-PBLG30; (D) 3D image, (E) height image, and (F) height profile of the micelle of PFS90-b-PBLG30; (G) 3D image, (H) height image, and (I) height profile of the micelle of PFS90-b-PBLG80. Height profiles correspond to the sections marked with dashed line on the height images. Scale bars of (A, B, D, E, G, and H) are 2.0 μm; vertical scale bars on (C, F, and I) are 10 nm. Contact mode images are given in Figure S12.

5, 6, and 8 were prepared from purified diblock copolymers. However, when we used an unpurified BCP sample containing traces of PFS homopolymer, we observed the formation of isolated micelles mixed with stacks of lamellar objects (see the example of PFS90-b-PBLG80 in Figure S13C,D). To test whether the presence of PFS homopolymer affects the selfassembly, we carried out an experiment in which we premixed a sample of PFS40-b-PBLG80 (0.5 mg) with 20 wt % PFS40-Cp (0.1 mg). The polymers were dissolved and mixed in a few drops of THF. The THF was evaporated, and DMF (3 mL) was added. The mixture then was heated at 90 °C for 30 min and allowed to cool to room temperature outside the oil bath. TEM analysis after 3 days showed the formation of large (ca. 10 μm in diameter) flower-like structures (Figure 9). Some isolated lamellar micelles were also observed. The inset of Figure 9 shows one flower-like structure at higher magnification. It appears to consist of a stack of platelets, which points to cocrystallization of the homopolymer and the block copolymer. It may be that PFS homopolymer incorporated into the platelet crystal promotes multiple nucleation sites on the platelet surface in much the same way we observed in the micelles of the purified PFS90-b-PBLG80 (cf. Figures 5A and 8G). Another possible explanation involves the growth of lamellar micelles of PFS40-b-PBLG80 from aggregated tiny crystals of PFS homopolymer in a way we recently reported for elongated micelles of PFS53-b-PI637.42

length of the BCPs. This observation is in agreement with the previously reported height analysis of cylindrical micelles.41 One important conclusion that can be drawn from the AFM images in Figure 8 and the micelle heights presented in Table 3 is that the PFS chains within the micelle core are folded. The contour length of PFS40 is ca. 25 nm, and that of PFS90 is ca. 55 nm.14h These values are much larger than the heights reported in Table 3, which include the contribution of the PBLG corona. While it is difficult to separate the contributions of the core and corona to these thickness values, it is unlikely that the PFS core itself contributes more than 10 nm to the thickness. We speculate that in PFS40 the PFS chains undergo 2−3 folds per chain and in PFS90 5−6 folds per chain. Chain segments in the fold most likely contribute to the amorphous component of the semicrystalline PFS core. On the micelle of PFS90-b-PBLG80 shown in Figure 8G, there is a small flat region, which is higher than the rest of the micelle (its height profile is presented Figure S11). It is likely one of the higher contrast regions observed in TEM images of these micelles as shown in Figure 5A. The average height of this region is 31.7 ± 1.3 nm, 14 nm higher than the average height of the micelle calculated from the height profile in Figure 6I (18.1 ± 1.2 nm). This higher region is somewhat less than twice the height of an individual lamellar micelle. For that reason, we suspect that it is not formed by stacking of two micelles formed independently in the solution. More likely it results from growth of a second layer nucleated by a defect on the surface of the micelle. Self-Assembly in the Presence of PFS Homopolymer. The isolated well-defined platelet micelles seen in Figures 4D,

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SUMMARY AND CONCLUSIONS We prepared a series of PFS-Cp and PBLG-Mal endfunctionalized homopolymers and used them for the synthesis 2612

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To rationalize these results, we invoke the idea that polymer molecules in less crystalline domains dissolve at lower temperatures than polymers in more crystalline domains, a phenomenon related to “self-seeding” of semicrystalline polymers.14f,g,45 We assume that when a solution of the less ordered structures formed initially was heated to 75 °C, about half the polymer dissolved. These polymers would no longer be soluble when the solution was cooled and would grow epitaxially off the edges of the crystals that survived in the annealed solution. If the polymer in the fiber-like appendages at the ends of the micelle seen in Figure 6A,B dissolve preferentially at 75 °C, they may crystallize more uniformly as the solutions were cooled. The XRD peak may then reflect the contribution of the polymer that crystallizes out upon cooling. This fraction would be independent of the annealing time. DMF is a helicogenic solvent for PBLG.40 This factor may play an important role in the formation of uniform truncated elliptical micelles. From our previous work we know that PFS polymers with random-coil coronae (e.g., PFS−PI,14d PFS− PDMS,34 and PFS-P2VP35) with similar block ratios do not lead to this morphology. It is possible that the helical corona leads to less steric hindrance at the micelle surface and therefore allows the PFS core to pack better in the lamellar micelle core. We also examined the self-assembly of one block copolymer sample premixed with PFS homopolymer (PFS40-b-PBLG80 with 20 wt % PFS40-Cp). Here we found mesoscale flower-like structures consisting of stacked plates pointing radially. These structures were different in size and shape from those formed by the block copolymer itself under identical conditions and suggest that cocrystallization of the homopolymer and PFS block copolymer core played an important role in structure determination.

Figure 9. TEM images of micelles formed from a mixture of (PFS40-bPBLG80 + 20 wt % PFS40-Cp). Inset: one of the flower-like structures at higher magnification. Smaller isolated lamellar structures are circled with a dashed line.

of PFS-b-PBLG copolymers by Diels−Alder coupling. Unreacted homopolymer was removed by a series of selective precipitations. We believe that Diels−Alder coupling will allow synthesis of many novel PFS-based block copolymers in the future. We examined the self-assembly of these BCPs in DMF. Three polymers, PFS40-b-PBLG30, PFS90-b-PBLG30, and PFS90b-PBLG80, with a PBLG block shorter than PFS block, formed remarkably uniform truncated elliptical micelles. By AFM, the micelles were seen to be uniformly flat with an occasional second layer most likely induced by secondary nucleation or due to the presence of a screw dislocation. The thickness of the micelle platelets as determined by AFM increased in the following order: PFS90-b-PBLG80 > PFS90-b-PBLG30 > PFS40-bPBLG30; i.e., the micelles formed by the longer polymers were thicker. Self-assembly experiments with PFS40-b-PBLG30 at 75 °C, monitored over time, showed that the initially formed less ordered structures evolved into uniform truncated elliptical structures over about an hour. In a subsequent set of experiments carried out under similar conditions, we monitored the evolution of the X-ray diffraction of these micelles. Samples annealed for 15 min or longer in DMF and then dried showed a broad peak corresponding to a reflection at 6.4 Å, characteristic of crystalline PFS. Curiously, the peak shape and peak intensity showed no further change for samples annealed longer than 15 min, suggesting no change in the degree of crystallinity of the PFS core of the micelles upon annealing. 1H NMR measurements at 25 °C showed only weak and broad signals associated with the Cp rings of PFS, consistent with low mobility of the PFS polymer in the micelle core. When this solution was heated to 75 °C, these protons became sharp and much stronger in intensity. When compared to the integration of the benzyl peaks of the PBLG corona, we found that about half of the ferrocene groups were mobile.

■

EXPERIMENTAL SECTION

Instrumentation and Sample Preparation. 1H NMR (400 MHz) spectra were recorded on a Varian Hg 400 or a Varian VnmrS 400 spectrometer with a 45° pulse width and 10 s delay time at 25 °C. TEM images were obtained using a Hitachi H-7000 TEM instrument operated at 75 kV. For TEM analysis, 1 drop of solution was deposited on a Formvar-carbon-coated TEM grid. The excess solution was removed by touching the edge of the drop with a KimWipe; the grid was left to dry for 1 h in a fume hood. Polymer molecular weights and molecular weight distributions (Đ = Mw/Mn) were determined by GPC. For analysis of PFS-Cp immediately after the synthesis (in Bristol), we used a Viscotek VE 2001 triple-detector gel permeation chromatograph equipped with automatic sampler, pump, injector, inline degasser, column oven (30 °C), styrene/divinylbenzene columns with pore sizes of 500 and 100 000 Å, and VE 3580 refractometer. THF (Fisher) containing 0.025% butylated hydroxytoluene as a stabilizer was used as the eluent, at a flow rate of 1.0 mL/min. The calibration employed polystyrene standards (Aldrich). Later studies (in Toronto) of PFS-Cp and of PFSPBLG BCPs employed a Viscotek GPC MAX liquid chromatography equipped with three columns (PolyAnalytik), a triple detector array, and a UV detector. The universal calibration employed polystyrene standards (Agilent). Stabilizer-containing THF was used as the eluent (column temperature 35 °C, flow rate 1.0 mL/min). For PBLG homopolymers, GPC measurements were run on a system comprising a Waters 515 HPLC pump, Agilent PLgel 3 μm columns, and a Viscotek VE 3580 refractive index (RI) detector. N-Methyl-2pyrrolidone (NMP) was used as the eluent at a flow rate of 0.6 mL/min at 80 °C. The columns were calibrated with poly(methyl methacrylate) standards. 2613

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Intermittent contact mode AFM images were recorded using the MFP-3DTM stand-alone atomic force microscope. Height and amplitude images of PFS40-b-PBLG30, PFS90-b-PBLG80, and PFS90-bPBLG30 micelle samples, prepared on silicon wafers, were obtained by utilizing ac-mode. Scans were performed using a Pointprobe-Silicon SPM-sensor (Nanoworld) tip with a resonance frequency of 320 kHz and force constant of 42 N/m. The tip radius of curvature was 20 nm. All images resulted from scan rates ranging between 0.5 and 1 Hz. Contact mode AFM images were recorded using the DimensionTM 3100 scanning probe microscope (SPM). Scans were performed using a nonconductive silicon nitride tip−cantilever system (Veeco) with a resonance frequency of 4−10 kHz and spring constant of 0.01 N/m. The loading force during contact mode imaging was 1.014 nN. All images resulted from scan rates ranging between 0.5 and 1 Hz. The samples for AFM were prepared by deposition of a drop of a DMF solution of micelles on a silicon wafer, which then was left to dry in a fume hood. The X-ray powder diffraction patterns of all samples were collected on Bruker AXS D8 diffraction system. A high-power point focus Cu Kα radiation with a graphite monochromator was used. For all measurements the dry bulk sample (ca. 3 mg) was placed on a square aluminum sample holder (side length = 1.3 cm) with a cone hole (diameter on one side = 4 mm; diameter on the other side = 2 mm). The thin side of the hole was blocked with Scotch tape. Details are shown in Figure S15. For the first X-ray diffraction measurement, a sample of 15 mg of purified PFS40-b-PBLG30 was annealed in 2 mL of DMF at 90 °C for 2 h, aged 3 days at room temperature, then precipitated in methanol, and dried for 2 h under vacuum. The diffraction image was captured on a Hi-Star GADDS area 2D detector. The diffraction pattern was collected in the transmission mode in a single frame, which covered a range of 24° 2θ. For the time-evolution XRD analyses, a sample of purified PFS40-bPBLG30 (ca. 10 mg) was dissolved in THF (0.3 mL), added to methanol (1.5 mL) to precipitate the polymer, and dried under vacuum for 1 h. In Figure 7, we denote this sample as “t = 0”. Samples of this material (ca. 5 mg) were placed in vials, suspended in DMF (2.0 mL), and then heated at 75 °C in an oil bath for various times. After removal from the bath, the samples were cooled to room temperature and allowed to age overnight. Then most of the DMF was removed by evaporation in a flow of air to a volume of ca. 0.2 mL. Then each sample was precipitated with methanol and dried under vacuum. XRD traces were run on 3.0 ± 0.2 mg of each sample for annealing times of t = 15, 30, 60, and 120 min. A single frame with constant exposure time of 400 s was used for measuring each sample. The obtained 2D diffraction images were then integrated within 2θ range of 8°−30° with step size of 0.005°. The XRD-patterns were further processed with Bruker AXS Eva 8.0 data processing software. 1 H NMR analysis of the micelles at 25 and 75 °C was performed on a Bruker 600 instrument, with 16 scans acquired with a delay time of 10 s. A solution in DMF of the PFS40-PBLG30 micelles shown in Figure 6A (0.25 mg/mL, annealed at 75 °C for 15 min) was placed in an NMR tube containing a sealed capillary with DMSO-d6 as the lock material. After recording the spectrum at 25 °C the NMR probe was heated to 75 °C. Spectra were recorded at 15 min intervals over 120 min. The DMF solvent signals were partially suppressed using a wet 1D pulse sequence.44 Diels−Alder Conjugation Reactions. Experimental details for the synthesis of PFS-Cp samples and PBLG-Mal samples are presented in the Supporting Information. PFS40-b-PBLG30. Solid 9.5 mg (1 μmol) of PFS40-Cp and 5.75 mg (0.833 μmol) of PBLG30-Mal were placed in a screw-capped vial, and 1 mL of DCE was added. The cap on the NMR tube was sealed with Teflon tape, and the vial was placed in the oil bath preheated at 75 °C. After 18 h, the crude DCE solution was concentrated with a stream of air from an initial volume of 1 mL to a final volume of ca. 0.1 mL by solvent evaporation. Then the concentrated solution was added via pipet to an Eppendorf tube containing DMF (2 mL) at room temperature. An orange solid precipitated. The DMF solution (also orange) was separated from the orange solid by centrifugation, and the

orange solution was dried by DMF evaporation. The remaining solid was dissolved in 0.1 mL of THF and then added to a methanol/DMF 8:2 mixture (2 mL) at room temperature in another Eppendorf tube. An orange solid precipitated, leaving a transparent colorless methanol/ DMF solution. The residue was separated by centrifugation and dried under vacuum (ca. 10 Torr, 40 °C, 2 h). The overall yield of this reaction after purification was 9.4 mg (68%). PFS25-b-PBLG80 was prepared using the same procedure. The other BCPs (PFS90-b-PBLG30, PFS40-b-PBLG80, and PFS90-bPBLG80) were prepared using very similar reaction with one difference: CD2Cl2 was used as the solvent, and the reaction temperature was set to 50 °C. Purification protocol was similar. The yields were the following: 32% for PFS90-b-PBLG30, 30% for PFS40-bPBLG80, and 35% for PFS90-b-PBLG80. Self-Assembly. For the self-assembly experiments PFS25-b-PBLG80 and PFS40-b-PBLG30 were used as obtained. For the self-assembly of PFS90-b-PBLG30, PFS40-b-PBLG80, and PFS90-b-PBLG80, an additional purification procedure was applied. Small portions (0.2 mg) of these BCPs were obtained by collecting the THF eluent with the polymers after passing through GPC columns and evaporation of the solvent.

■

ASSOCIATED CONTENT

* Supporting Information S

Additional experimental details, additional NMR spectra, GPC traces, TEM and AFM images, XRD spectrum of the sample annealed at 90 °C. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (I.M.). *E-mail [email protected] (M.A.W.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The Toronto authors thank NSERC Canada for their financial support. G.M. is grateful to Dr. Jiawen Zhao for the help with PFS-Cp synthesis and to Dmitry Pichugin for the help with the high-temperature NMR analysis and solvent peaks suppression. We thank Prof. Julius Vancso (U. Twente, Netherlands) for his helpful comments.

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REFERENCES

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dx.doi.org/10.1021/ma402441y | Macromolecules 2014, 47, 2604−2615