Polymer Chemistry

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Cite this: DOI: 10.1039/c5py00919g

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Polymerization-induced self-assembly driving chiral nanostructured materials† Kamal Bauri, Amal Narayanan, Ujjal Haldar and Priyadarsi De* To prepare chiral nanostructures coated with bioactive molecules, a side-chain amino acid containing macromolecular chain transfer agent (macroCTA), poly(Boc-L-alanine methacryloyloxyethyl ester) (PBLAEMA), has been used as the steric stabilizer for the reversible addition–fragmentation chain transfer (RAFT) mediated dispersion polymerization of benzyl methacrylate (BzMA) in methanol at 65 °C. Gel permeation chromatography (GPC) analysis confirmed an efficient and well-controlled block copolymerization. A full spectrum of morphologies spanning spherical micelles, worm like micelles, fibres and vesicles could be attained by tuning (i) the length of the solvophobic block and (ii) the total solid content at which the block copolymerization is performed. Interestingly, a purely fibre phase morphology formed a thermoresponsive gel at room temperature above a critical fibre entanglement concentration, which underwent degelation upon heating because of the morphological transformation from anisotropic fibre to isotropic sphere. In actual fact, twisted nano-fibres have been formed through the hierarchical selfassembling of polymerization induced self-assembly (PISA) generated macromolecules in the gel state. Circular dichroism (CD) spectroscopy was used to elucidate chiroptical properties. Additionally, a high demanding wrinkle surface has been constructed preliminarily from this copolymer dispersion solution. Successful Boc-group expulsion facilitates the disassembly of vesicles to either worms or spheres with an

Received 14th June 2015, Accepted 3rd July 2015

appreciable cationic character below pH 7.0 as revealed by aqueous electrophoresis studies. Though the creation of nano-objects through PISA is well-known, fabricating chiral nanostructures with reactive

DOI: 10.1039/c5py00919g

handles and their hierarchical self-organization to have functional architectures is a less explored area. In

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this present work we were able to hybridize PISA, chirality and hierarchical self-assembling.

Introduction Molecular and macromolecular self-assembly into nanostructures of diverse shapes is ubiquitous in nature, for example the formation of the cell-membrane by phospholipids and the specific hydrogen bonding interaction between individual DNA strands to construct the double helix. Inspired from nature, polymer chemists have been engaged in the bespoke construction of synthetic polymeric nanoscale soft materials with desired nanostructures. These promising materials have a range of potential applications in the fields of nanomedicine (e.g. drug delivery and imaging), microelectronics and catalysis.1–3 Self-organization of block copolymers in selective solvents has been shown to be valuable for the fabrication of a wide variety of nanostructures. Spherical micelles

Polymer Research Centre, Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur-741246, Nadia, West Bengal, India. E-mail: [email protected] † Electronic supplementary information (ESI) available: GPC chromatograms, CD spectra, 1H NMR spectrum and DLS size distributions of various block copolymers. See DOI: 10.1039/c5py00919g

This journal is © The Royal Society of Chemistry 2015

are perhaps the most exploited polymeric soft material, although other types of higher ordered and more complex morphologies can also be accessed by the self-directed assembly process.4–11 Traditionally such self-assembled species have been generated by the post-polymerization processing of mostly AB-type diblock copolymers using either a solvent or pH switch, dialysis, or thin film rehydration. Such protocols are typically performed at very low copolymer concentrations (≤1 wt% is common).12–16 This makes the scaled-up production of block copolymer nano-objects rather problematic for industrial purposes. During the last six years there has been a prolific interest in polymerization-induced self-assembly (PISA) as a convenient way of making the desired block copolymer morphology which does not need any post-polymerization processing steps.17–28 PISA facilitates the formation of a myriad of macromolecular nano-architectures depending on the solvophilic/solvophobic volume ratio at much higher concentrations (up to 50 wt%) by using living radical polymerization techniques, such as atom transfer radical polymerization (ATRP),29,30 nitroxide mediated polymerization (NMP)31–33 and reversible addition–fragmentation chain transfer (RAFT) polymerization.17,20 The contri-

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butions from Pan,17 Armes34 and their co-workers to RAFT dispersion polymerization (RAFTDP) are significant. Whereas emulsion RAFT polymerization was led by Charleux,35–37 Monteiro,38 Cunningham39,40 and Hawkett41 and their co-workers. Among the above mentioned heterogeneous polymerization techniques, the RAFTDP is the most versatile PISA formulation, which can be performed in various dispersion media such as water,42 organic polar (lower alcohols such as methanol and ethanol)34 or non-polar solvents (n-alkanes).43–46 In each case, a molecularly dissolved vinyl monomer is used for the chain extension of a soluble macromolecular chain transfer agent (macroCTA) in a suitable solvent which is a nonsolvent for the budding second block, and insolubility of the second block induces the self-assembly process resulting in the formation of various nano-architectures. As of now, various block copolymer nano-objects, including nanospheres, nanorods, nanofibers and vesicles, have been prepared through the so-called PISA via macro-RAFT agent mediated dispersion polymerization. Interestingly, in some cases a block copolymer morphology transition, such as vesicle-to-largecompound vesicle, sphere-to-worm, worm-to-vesicle and rodto-vesicle have even been observed during the RAFT dispersion polymerization.21,47 There are also several reports of thermoresponsive co-monomer building blocks such as N-isopropylacrylamide (NIPAM)48–50 and N,N-diethylacrylamide (DEA)51,52 in certain aqueous systems where the well-established inverse temperature dependent solubility behaviour of poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEA) is exploited to induce phase separation and self-assembly as the polymerizations proceed. The previously mentioned processes are commonly known as the temperature directed morphology transformation (TDMT) or the polymerizationinduced thermal self-assembly (PITSA). In RAFTDP there are several parameters which dictate the size and morphology of the block copolymer nano-objects: (i) the degree of polymerization (DPn) of the solvophobic block,34 (ii) the chain length of the solvophilic block or the macro-RAFT agent,44 (iii) the monomer concentration at which the RAFT polymerization was conducted27,53 and (iv) the solvent character.22 Supramolecularly self-assembled nanostructures covered with bioactive building blocks have been actively explored as promising materials in the field of biotechnology.54 Nowadays, co-monomer building blocks comprising of biologically relevant molecules, such as sugar,55 nucleobases56 and amino acids,57 are either being used as solvophobic blocks or solvophilic segments to make in situ nano-objects through the RAFTDP formulation. Although various morphologies ranging from spherical micelles, cylindrical micelles and vesicles have been achieved by this well-known RAFTDP formulation, there is still significant scope and opportunity to exploit the macromolecular engineering of polymeric nano-architecture with reactive handles either in solvophobic cores or solvophilic shells which can be used to make them functional.58,59 Since amino acids are biomolecules and the constituent component of peptides and proteins, researchers have been making polymers from amino acids to mimic the property and function

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exhibited by these natural biomacromolecules. Chirality transcription from the molecular level to the macromolecular level has been observed in amino acid incorporated polymers.60–63 So the coating of chiral materials on different nanostructures generates a single entity which will combine the intriguing properties of both chiral materials and nanostructures. Therefore, in this present work we employed a side-chain amino acid containing methacrylate polymer with a chiroptical property, poly(tert-butyloxycarbonyl (Boc)-L-alanine methacryloyloxyethyl ester) (PBLAEMA), for the chain extension block copolymerization of benzyl methacrylate (BzMA) by RAFT dispersion formulation in methanol to cover a full spectrum of morphologies by the systematic variation of (i) the length of the core-forming PBzMA block and (ii) the total solid concentration at which the formulation is being prepared. Furthermore, the thermoreversible gelation behaviour and morphological transition of the Boc-group deprotection strategy have also been studied for the current system. The hierarchical self-organization of dispersed nanostructures has been observed to form twisted nano-fibres in the gel state and in surface wrinkling.

Experimental section Materials Boc-L-alanine (Boc-L-Ala-OH, 99%, Sisco Research Laboratories Pvt. Ltd, India), trifluoroacetic acid (TFA, 99.5%, Sisco), dicyclohexylcarbodiimide (DCC, 99%, Sigma), 4-dimethylaminopyridine (DMAP, 99%, Sigma), 2-hydroxyethyl methacrylate (HEMA, 97%, Sigma), anhydrous N,N′-dimethylformamide (DMF, 99.9%, Sigma), and anhydrous methanol (99.8%, Sigma) were used without any further purification. The 2,2′azobisisobutyronitrile (AIBN, 98%, Sigma) was recrystallized from methanol and BzMA (96%, Sigma) was passed through a basic alumina column to remove inhibitors/anti-oxidants. NMR solvents such as CDCl3 (99.8% D), CD3OD (99.8% D) and DMSO-d6 (99.8% D) were purchased from Cambridge Isotope Laboratories, Inc., USA. 4-Cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (CDP)64 and Boc-protected amino acid based monomer Boc-L-alanine methacryloyloxyethyl ester (Boc-L-Ala-HEMA) were synthesized as described elsewhere.63 The solvents, such as hexanes (mixtures of isomers), methanol (MeOH), acetone, ethyl acetate, tetrahydrofuran (THF), dichloromethane (DCM), etc., were purified by standard procedures. Characterization Polymer molecular weight and molecular weight distributions (dispersity, Mw/Mn, Đ) were determined by gel permeation chromatography (GPC) in THF using poly(methyl methacrylate) standards at 30 °C with a flow rate of 1.0 mL min−1. The system consisted of a PolarGel-M guard column (50 × 7.5 mm) and two PolarGel-M analytical columns (300 × 7.5 mm), a Waters Model 515 HPLC pump and a Waters Model 2414 refractive index (RI) detector. The 1H NMR spectroscopy was conducted on a Bruker AvanceIII 500 MHz spectrometer.


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Hydrodynamic diameter measurements were carried out using a dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instrument Ltd., UK) instrument equipped with a He–Ne laser beam at 658 nm. At least three measurements at 25 °C were made for each sample with an equilibrium time of 2 min before starting the measurement. Samples for size analysis were prepared by taking 50 µL of the dispersed polymer solution diluted with 1.95 mL MeOH followed by filtering through a 0.45 µm polytetrafluorethylene (PTFE) filter. Aqueous electrophoresis measurements were performed on 0.01% w/v aqueous copolymer solutions using the same Zetasizer Nano ZS instrument. Field-emission scanning electron microscopy (FE-SEM) images were recorded using a Carl Zeiss Sigma SEM instrument. Transmission electron microscopy (TEM) imaging was performed at 200 kV on a JEOL JEM 2100 HR electron microscope. No staining of polymer samples was done. Aggregate solutions were diluted with MeOH at 25 °C to generate a 0.2 wt% solution and the resulting diluted dispersion solution was drop casted either on silicon wafers for SEM or carbon grids for TEM image recordings followed by room temperature air drying and vacuum drying. Circular dichroism (CD) spectroscopic measurements were carried out in a JASCO J-815 CD spectrometer with a 400 µL quartz cuvette cell (1.0 mm pathlength) in acetonitrile. The rheological measurements were conducted on a TA-ARG2 rheometer using a steel parallel plate with a 40 mm diameter. The rheometer was equipped with a Peltier circulator thermo cube for use with a Peltier plate that helps to accurately control the temperature. A gap spacing of around 0.8 mm was used for all the measurements. Dynamic shear modulus values, storage modulus (G′) and loss modulus (G″) were recorded with varying temperature at a 5 °C min−1 heating rate and at a 1.0% fixed strain. Frequency sweep experiments were done with the same dispersed solution at 25 °C at a 1.0% fixed strain.

RAFT homopolymerization of Boc-L-Ala-HEMA Procedure for RAFT homopolymerization of Boc-L-Ala-HEMA is followed as described elsewhere in the literature.61,65 In a typical polymerization method, a 20 mL septum capped vial was charged with 2.0 g (6.64 mmol) Boc-L-Ala-HEMA monomer, 149.0 mg (0.36 mmol) CDP, 6.1 mg (36.8 µmol) AIBN and 8.0 g anhydrous DMF. Then the vial was air-tightened, purged with dry nitrogen for 20 min and finally placed in a preheated reaction chamber at 70 °C for 3.5 h. Polymerization was stopped by exposure to air while cooling in an icewater bath. Acetone (∼1.0 mL) was added to the polymer solution and the mixture was precipitated into excess cold hexanes. The polymer was then reprecipitated (×5) from acetone/hexanes and dried under vacuum at 30 °C for 6 h to obtain a dark-yellow powder polymer, PBLAEMA. The mean degree of polymerization (DPn) for this macroCTA has been calculated as 14 via 1H NMR spectroscopy by comparing the signal integration ratio at 4.1–4.5 ppm corresponding to the oxyethylene and chiral proton (5H) with CDP protons at 2.5 ppm.63


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RAFT dispersion polymerization of BzMA using PBLAEMA14macroCTA in methanol A typical RAFT dispersion polymerization for the synthesis of the PBLAEMA14-b-PBzMA50 block copolymer at 15 wt% total solid content was carried out as follows: in a 20 mL glass vial equipped with a magnetic spin bar, 91.0 mg (22.7 µmol) macroCTA (Mn,NMR = 4200 g mol−1, Mn,GPC = 4000 g mol−1, Đ = 1.2), 0.2 g (1.13 mmol) BzMA and 1.12 mg (6.81 µmol) AIBN were dissolved in 1.33 g anhydrous MeOH. The reaction mixture was sealed with a rubber septum cap, placed in an icebath and purged with dry nitrogen for 15 min. Finally the reaction mixture was placed in a preheated reaction block at 65 °C for 10 h. Polymerization was quenched by cooling the vial in an ice-water bath and exposing the solution to air. The block copolymer was isolated by precipitation into large excess of cold hexanes followed by vacuum drying at 40 °C for 8 h.

Results and discussion Kinetics of RAFT dispersion polymerization of BzMA Keeping in mind the disadvantage of using styrene for chain extension as reported by Armes et al.,66 herein, we have explicitly chosen BzMA as the solvophobic block to obtain a library of chiral nano-objects from the side-chain L-alanine containing methacrylate polymer (PBLAEMA) as macroCTA (Scheme 1) via RAFT polymerization. In recent reports the Armes group preferentially chose either BzMA or hydroxyl propyl methacrylate as the core forming monomer over styrene, since a high monomer conversion could be achieved by using all-methacrylic formulations.34,67 Initially, we prepared PBLAEMA14 by solution RAFT polymerization at 75% monomer conversion to attain high end group fidelity, which is indeed needed for acting as a macroCTA. Since PBLAEMA14 is soluble in methanol, we examined its activity as an effective steric stabilizer for the RAFT alcoholic dispersion polymerization of BzMA to synthesize a series of PBLAEMA14-b-PBzMAm

Scheme 1 Synthesis of diblock copolymers by RAFT alcoholic dispersion polymerization of BzMA using PBLAEMA as macroCTA and their subsequent Boc-group deprotection.

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block copolymers by varying several parameters to obtain confined nano-architectures. First, a systematic kinetic investigation was performed for RAFT dispersion polymerization of BzMA targeting the final block copolymer composition PBLAEMA14-b-PBzMA150 under [macroCTA]/[AIBN] = 1/0.3 at 10 wt% in methanol (Fig. 1). The initial reaction mixture was transparent, but with the progress of the reaction the solution started to become faintly bluish (Fig. 1A). The changes in the macroscopic phase indicate the formation of block copolymer nano-objects that can act as nano-reactors for the incoming monomers.66 Finally, the reaction mixture became milky white with an essentially full conversion achieved after 16 h. Polymerization mixtures were periodically taken out and monomer conversions were determined using 1H NMR spectroscopy by comparing the integral ratio of poly(benzyl methacrylate) (PBzMA) –CH2– peak at 4.9 ppm to BzMA vinyl (–CH2–) signal at 6.1 and 5.7 ppm. Fig. 1B shows the conversion versus time plot which is almost linear but the corresponding semi-logarithmic curve is slightly non-linear. This behaviour is frequently encountered in RAFTDP because of the apparent rate enhancement caused by the micellar nucleation mechanism.44 The evolution of the number average molecular weight (Mn,GPC) and dispersity (Đ) with monomer conversion was monitored by recording GPC from the aliquot which was diluted with measured amounts of THF. The molecular weight increases linearly with conversion and Đ remained below 1.20 throughout the polymerization (Fig. 1C). Theoretical molecular weights (Mn,theo) calculated based on conversion match nicely with the Mn,GPC values (Fig. 1C). The symmetric unimodal GPC chromatograms shifted smoothly towards lower elution volumes with increasing monomer consumption having little or no trace at high molecular weights and the low molecular weight region indicates no dead polymer species present formed by macromol-

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ecular radical coupling side reactions and inactive macroCTA impurities (Fig. 1D). From the above results relating to Fig. 1, it can be concluded that high quality block copolymers can be obtained by a well controlled RAFTDP having a very high blocking efficiency by this formulation. RAFTDP of BzMA with PBLAEMA14 macroCTA for variable DPn of the PBzMA block In this section we studied how the size and shape of chiral nano-objects evolves with the length of the solvophobic block, namely PBzMA, keeping the macroCTA length and monomer (M) 15 wt% constant. Table 1 gives a summary of the results for PBLAEMA14-b-PBzMAm (m = 22–140) diblock copolymers nano-objects formed by RAFTDP in methanol. Unimodal GPC chromatograms smoothly shifted towards the higher molecular weight side with increasing DPn of the core-forming PBzMA segment with no trace from the unreacted macroCTA or bimolecular termination even when higher [M]/[macroCTA] was targeted (Fig. 2A). 1H NMR spectra analysis for these block copolymers before precipitation and followed by gravimetric analysis of the final polymers indicated more than 90% monomer conversion after 14 h. The NMR number average molecular weight (Mn,NMR) and molecular composition of the resulting PBLAEMA14-b-PBzMAm block copolymers were determined by using the following formula: Mn,NMR = (DPn,BzMA of PBzMA segment × molecular weight (MW) of BzMA + Mn,NMR of macroCTA); where DPn,BzMA is the degree of polymerization of the core-forming block, which in turn can be estimated based on the DPn of macroCTA and comparing the integration (I) ratio of the peaks at δ = 4.80–5.00 ppm (2H, benzylic protons of BzMA units) to those at 4.00–4.45 ppm (5H, oxyethylene protons and chiral proton of alanine moieties) (Fig. 2B). Here we have used macroCTA with DPn = 14 and the DPn,BzMA for BzMA (m) can be

Fig. 1 (A) Digital images with the advancement of time (t ) highlighting the change in appearance of the solution for the PBLAEMA14-mediated RAFTDP of BzMA at 10 wt% solid content. (B) Monomer conversion-time and Ln[M]0/[M]-time plots ([M]0 and [M] are the concentrations of BzMA at t = 0 and t = t, respectively). (C) Evolution of the number average molecular weight (Mn,GPC and Mn,theo) and dispersity (Đ) with monomer conversion. (D) GPC chromatograms with the progress of time. The targeted diblock copolymer composition was PBLAEMA14-b-PBzMA150 and the [PBLAEMA macroCTA]/[AIBN] molar ratio was 1/0.3.

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Table 1 Summary of the results obtained from the synthesis of PBLAEMA14-b-PBzMAm block copolymer nano-objects at 15 wt% total solid content and their physiochemical characterization

Compositiona

[M]/[CTA]

Conv.a (%)

Mn, NMRa (g mol−1)

Mn,GPCb (g mol−1)

(Đ)b

Dhc (nm) (PDI)c

Morphold

PBLAEMA14-b-PBzMA22 PBLAEMA14-b-PBzMA30 PBLAEMA14-b-PBzMA48 PBLAEMA14-b-PBzMA60 PBLAEMA14-b-PBzMA75 PBLAEMA14-b-PBzMA84 PBLAEMA14-b-PBzMA100 PBLAEMA14-b-PBzMA140

25 34 50 65 80 92 105 150

90 90 95 92 93 91 95 93

8100 9500 12 600 14 800 17 400 19 000 21 800 28 900

7200 8900 11 600 13 300 15 600 17 200 18 800 25 700

1.10 1.10 1.15 1.17 1.17 1.16 1.21 1.27

24.0 (0.083) 43.0 (0.011) 142 (0.176) 154 (0.110) 162 (0.212) 168 (0.273) 190 (0.201) 260 (0.244)

S S+W W F F+V V+F V V

a

Determined by 1H NMR spectroscopy. b Obtained from GPC. c Measured from DLS. TEM, where S = sphere, W = worm, F = fibre, V = vesicle.

Fig. 2 (A) GPC RI chromatograms of PBLAEMA14-b-PBzMAm diblock copolymers made at 15 wt%, where m = 22, 30, 48, 60, 75, 100 and 140. (B) 1H NMR spectrum of PBLAEMA14-b-PBzMA60 block copolymer in CDCl3.

estimated as m¼

5 Ið4:805:00 ppmÞ DPn of macroCTA 2 Ið4:004:45 ppmÞ

FE-SEM studies have been carried out for the morphological characterization of block copolymer nano-objects. From the SEM images, nearly monodispersed spheres with an

d

Morphologies (Morphol) were obtained from SEM and

average diameter of about 20 nm were observed for PBLAEMA14-b-PBzMA22 (Fig. 3A). From Fig. 3B and 3C it can be seen that for the PBLAEMA14-b-PBzMA30 and PBLAEMA14-bPBzMA48 block copolymers small worms are formed by coalescing spherical objects, although for the latter composition worms are the predominant species. The hydrodynamic size (Dh) by DLS measurement for PBLAEMA14-b-PBzMA22 and PBLAEMA14-b-PBzMA30 is very close to the microscopic technique with small associated polydispersities (Fig. 4). An increase in DPn,BzMA for the PBzMA block to 60 resulted in the formation of an exclusive fibrilar morphology (Fig. 3D) as a consequence of which we observed a room temperature macroscopic thermoreversible gelation. The gelation behaviour is not retained when the composition was PBLAEMA14-bPBzMA75 due to the formation of very few vesicular structures in addition to the fibrils (Fig. 3E). But when the DPn,BzMA slightly changed from 75 to 84, mixed morphologies consisting of fibres and vesicles were observed with an apparent larger proportion of vesicular shapes in this case (Fig. 3F). Although the lengths of those worms/fibres are several nanometers, their diameters measured by SEM are comparable to the Dh values of their precursors spheres as expected. Again DLS

Fig. 3 SEM images of PBLAEMA14-b-PBzMAm diblock copolymers made at 15 wt%, where m = 22 (A), 30 (B), 48 (C), 60 (D), 75 (E), 84 (F), 100 (G) and 140 (H). TEM images of the same nano-objects for (I) m = 22 and (J) m = 100.


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Fig. 4 DLS intensity-average size distributions for the PBLAEMA14-bPBzMAm diblock copolymers made at 15 wt%, where m = 22, 30, 60, 75 and 140.

reported diameters of non-spherical nano-objects should be treated with caution because DLS measurements are based on the Stokes-Einstein equation which is only valid for hard sphere objects. An exclusive vesicular morphology has been seen when the experimental DPn,BzMA for the core forming block was 100 (Fig. 3G) or more than that (Fig. 3H). The size of the vesicle was found to increase with the increments in length of the core. From the TEM images we can also see that PBLAEMA14-b-PBzMA22 forms spherical micelles (Fig. 3I) and PBLAEMA14-b-PBzMA100 forms vesicles (Fig. 3J). Microscopyestimated sizes for the vesicular nano-objects are somewhat smaller compared to the sizes determined by DLS. This is common and is a direct reflection of the sizes being measured in two different conditions. So at a 15 wt% fixed total solid concentration there is a gradual evolution of morphologies from spheres to worms to fibres to vesicles as the length of the PBzMA chains is increased, with mixed phases always being observed between these pure phases. The evolution of morphology from sphere to worm to fibre to vesicle is a consequence of changes in the block copolymer packing parameter and summarized in Scheme 2.68 Effect of total solid concentration for a fixed PBLAEMA14-bPBzMAm composition For a fixed targeted block copolymer composition the final self-assembled morphology also depends on the copolymer concentration, since it influences the aggregation number which in turn can affect the molecular curvature and the final morphology. In this section we employed the same macroCTA targeting a specified block copolymer composition PBLAEMA14-b-PBzMA62 at different total solid contents ranging from 5–25 wt%. In all cases high monomer conversions could be attained ranging from 90% for the 5 wt% system to 97% for 25 wt% system (Table 2). 1H NMR analysis indicates an almost similar chain length of the solvophobic section, and unimodal symmetric GPC chromatograms at the same elution volume (Fig. S1†) which reveal the comparable molecular weight for this series of block copolymers. Although

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Scheme 2 Schematic representation of the general strategy for making various types of nano-objects by RAFT mediated dispersion polymerization.

these block copolymers possess near identical molecular compositions, they differed considerably both macroscopically as well as microscopically. After attaining a ∼90% conversion, a free-flowing yellow coloured solution was observed for the 5 and 10 wt% systems, while the 15, 20 and 25 wt% formulation samples appear as physical gels at ambient temperature. In characterizing dilute dispersions, only 5 wt% formulations gave a spherical morphology (Fig. 5A), whereas the remaining others exhibited worm or fibrilar morphologies as shown by SEM (Fig. 5B–E). It is noteworthy that although the 10 wt% sample self-assembled to a fibrilar morphology, it appeared as a sol rather than a gel at room temperature because above the critical worm/fibre entanglement concentration a gel can only be formed, which is greater than 10 wt% in this case. Thermoreversible gelation–degelation Among the PBLAEMA14-b-PBzMAm block copolymers synthesized at 15 wt%, a purely fibrilar phase was observed in the case of m = 60 (Fig. 3D), and m = 75 gave a slightly contaminated fibrilar shape with a very few vesicles (Fig. 3E). Interestingly, a macroscopic thermoinduced gelation–degelation behaviour was found for the former case (Fig. 6A), whereas the latter one gives a highly viscous solution. Again when we varied the total solid content for a fixed molecular composition, gelation was observed for the 15, 20 and 25 wt% formulations. The thermoreversible gelation behaviour, plausibly because of interworm/interfibre entanglements, has been proven as a common feature for RAFT dispersion systems both in polar and non-polar media.42,45,69 Rheological studies have been performed with the concentrated dispersion solution. At first, temperature variable studies were conducted with the 15 wt% sample and it was found that while cooling the hot solution the storage modulus (G′) crosses over the loss modulus (G″) at 45 °C indicating visco-elastic gel formation (Fig. 6B). Though this thermal transition is reversible, a little hysteresis is observed when the gel was heated. The frequency independent storage modulus confirmed a true gel at 25 °C


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Table 2 Summary of the results obtained for the synthesis of PBLAEMA14-b-PBzMA(56–60) block copolymer nano-objects made at variable wt% total solid contents and their physiochemical characterization

Compositiona

Conc. (wt %)

Conv.a (%)

Mn,NMRa(g mol−1)

Mn,GPCb(g mol−1)

(Đ)b

Dh c(nm), (PDI)c

Morphold

PBLAEMA 14-b-PBzMA56 PBLAEMA14-b-PBzMA57 PBLAEMA14-b-PBzMA59 PBLAEMA14-b-PBzMA58 PBLAEMA14-b-PBzMA60

5 10 15 20 25

90 92 95 94 97

14 100 14 200 14 600 14 400 14 800

12 800 12 900 13 100 13 100 13 300

1.39 1.53 1.35 1.19 1.19

82.0 (0.105) 110 (0.152) 125 (0.161) 200 (0.314) 256 (0.236)

S F+S F F F

a

Determined by 1H NMR spectroscopy. b Obtained from GPC. c Measured from DLS. d Morphology were obtained from SEM, where S = sphere, F = fibre.

Fig. 5 SEM images for PBLAEMA14-b-PBzMA(56–60) diblock copolymers made at different (5–25 wt%) total solid contents: (A) 5 wt%, (B) 10 wt%, (C) 15 wt%, (D) 20 wt% and (E) 25 wt%.

Fig. 6 (A) Digital images of the thermoreversible gelation–degelation. (B) Variation of storage modulus (G’) and loss modulus (G’’) with temperature during cooling and heating cycles. (C) Variation of G’ and G’’ with angular frequency at 25 °C (1.0% strain).

(Fig. 6C). There could be three possible reasons behind this thermoinduced gelation–degelation: (i) the complete disentanglement of the worm phase, (ii) a fundamental morphology transformation, or (iii) the molecular dissolution of the block copolymer chains at higher temperatures. A sample was extracted from the solution at 65 °C, which was then diluted with hot MeOH and rapidly drop casted on a silicon wafer for morphological characterizations. SEM images disclose that a fundamental morphology transformation from worm to sphere is responsible for this temperature induced phenomenon (Fig. 7A). A similar degelation mechanism has already been explored by Armes et al.45 In this context, the relevant questions are (a) why the morphology switches from fibre to sphere and (b) why spherical nano-objects cannot form gel? To answer the first question we performed variable temperature 1 H NMR studies. From Fig. 7B we can see that at 25 °C the


phenyl protons of the core forming segment are almost invisible, but at 63 °C the normalised intensity of those protons increases which results in a subtle shift of the relative volume fraction of the stabilizer and core-forming segments causing morphological transitions. Whereas the answer of the second point is, isotropic spheres do not get room to interact with each other efficiently whereas highly anisotropic fibres can do that very efficiently. If the length of the fibres is sufficiently high, then they can form gel through topological interactions alone (entanglements), even in the absence of putative crosslinks.70 Hierarchical self-organization and chiral properties of PBLAEMA14-b-PBzMAm Over the last few decades there has been a growing interest in designing wrinkled surfaces with soft materials. Engineered

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Fig. 8 SEM image of the film made of the PBLAEMA14-b-PBzMA60 diblock copolymer. The polymer dispersion was casted on a silicon wafer and dried at room temperature.

Fig. 7 (A) SEM images of PBLAEMA14-b-PBzMA60 diblock copolymer nano-objects recorded at 25 and 65 °C demonstrating thermo-induced morphology changes. (B) Variable temperature 1H NMR spectra for the same block copolymer recorded at 0.5 wt% concentration in CD3OD.

wrinkle surface patterns have been revealed to be potentially applicable to a variety of scientific research such as adhesive materials71 and creating prospective platforms for tissue engineering, enhanced cell growth and cell-based biosensors.72–74 It is desirable to make such surfaces from bioresourced materials and since amino acids are a bio-resourced material we tried to make such patterned film surfaces with the synthesized nano-objects coated with side-chain L-alanine containing polymers. An anisotropic aggregation can take place in this RAFT dispersion condition. Meanwhile, solvent conditions can induce the hierarchical self-assembly of block copolymer dispersions. If there are enough sites for bond formation within the dispersed particles, a wrinkle surface can be obtained through the anisotropic assembly during film formation.75 From the image (Fig. 8) we can see that the surface is completely covered with a fibrous morphology. The fibres are twisted and weaved together to cover the whole surface. Presently we are engaged in fabricating wrinkled surfaces from these as-synthesized chiral nano-objects by controlling the solvent evaporation rate, block copolymer composition, copolymer concentration, etc., and the results will be published in due course. Circular dichroism spectroscopy was carried out for the chiroptical characterizations of these block copolymers in acetonitrile (a good solvent for both blocks). The CD spectrum showed a positive CD signal peak at 216 nm and a negative peak at 238 nm that are assigned to the π → π* and n → π* transitions of the carbonyl group of the ester chromophore,

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respectively (Fig. S2†). This characteristic CD signal corresponding to the main chain absorption indicates an ordered arrangement of the amino acid pendants along the methacrylate backbone.60,61 In other words molecular chirality has been transcripted from the molecular level to the macromolecular level, displaying the overall chiral nature of the macromolecule. SEM images in Fig. 3D, 5C–E and 7A show the exclusive presence of network structures of one-handed helical nanofibres a few micrometers in length. This is the first report on the gelation-assisted formation of one-handed helical ropes through the hierarchical self-assembly of block copolymer nano-fibres by the RAFTDP technique. The hydrogen bonding interactions between the side-chain chiral amino acids in the fibres possibly provide such twisted morphologies in the gel state. We also prepared a block copolymer soft gel from an achiral amino acid (2-amino isobutyric acid) keeping the block ratio the same as L-alanine (data not shown here), where we could not find any such twisted fibres. We are currently investigating the helical-sense of nano-fibre gels by RAFTDP in detail taking the D-alanine moiety in the side-chain. Disassembly of vesicles due to the expulsion of Boc-groups To study the effect of Boc-group expulsion on the morphology of block copolymers in this section we have chosen two block the compositions which showed vesicular morphologies with different diameters. These two vesicle shaped nano-objects carrying Boc-groups on their periphery were subjected to deprotection by in situ TFA addition. The reaction mixture then was allowed to stir for 2 h (Scheme 1). Later these solution mixtures were transferred from methanol to aqueous media via dialysis retaining colloidal stability. The pH of the final solution was then adjusted to 2.0 after adding dilute HCl. The 1 H NMR study indicates the complete removal of Boc-groups as there is no peak at 1.43 ppm which corresponds to the tertbutoxy carbonyl protons (Fig. S3†). Morphologies of these Bocgroup deprotected nano-objects both in methanol and water were examined by SEM from their respective dispersed solu-


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tions. Surface charges of those particles were obtained by aqueous electrophoresis. From the SEM images it is seen that for a fixed block copolymer composition morphology changes from vesicles to either worms or spherical micelles (Fig. 9). First, the PBLAEMA14-bPBzMA150 block copolymer exists as vesicles (Fig. 9A) in dispersed methanol, but it changes morphology to worms both in methanol and water after the removal of the Boc-groups (Fig. 9C and 9D). When the PBLAEMA14-b-PBzMA100 polymer was deprotected, vesicles (Fig. 9B) turn into spherical micellar charged particles both in alcoholic and aqueous media (Fig. 9E and 9F). This morphological switching has also been proved by DLS (Fig. S4†). The likely reason for this morphological transformation is that the Boc-group removal from the vesicle surface obviously makes a difference in the effective volume of the amino acid segment and also the solvophilic/ solvophobic balance. After deprotection it contains positively charged species on the surface which exert repulsive coulombic interactions between them. Geometrically speaking, the higher the curvature the greater the average distance between the charged species. Thus there will always be a morphology transformation with increased curvature so as to take care of the electrostatic potential when the nano-object carries ionic species. In this case we presented (Fig. 9G) δr as the distance between the two polymeric chains (charged/uncharged) where δ1 < δ2 < δ3 < δ4. So the nano-particle will adapt to the morphology with higher δ (δ4 in this case). But the size of the transformed nano-objects in aqueous media is always higher than the alcoholic one. This is probably because of the higher hydration of the ammonium (–+NH3) groups in water compared to methanol. Surface charges of these particles in aqueous solution at pH 4.0 after transferring from the metha-

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nol solution were found to be +43 mV for the worms and +36 mV for the spherical ones. This result indicates the protonation of the primary amine group at a lower pH and the high colloidal stabilities of those nano-objects.

Conclusions Alcoholic RAFT dispersion polymerization of BzMA using PBLAEMA14 macroCTA as a solvophilic stabilising agent afforded near-monodispersed diblock copolymers, which selfassembled in situ into a continuum of morphologies including spherical and worm-like micelles, fibres and vesicles depending on the targeted block composition and the copolymer concentration. The nano-particle morphology evolves as the length of the core forming PBzMA block increases at a constant total solid content (15 wt%). The evolution in self-assembly behaviour was found to be somewhat less sensitive to the total solid content for a fixed solvophobic block length. We obtained a pure fibre phase produced thermoreversible physical freestanding soft gel at room temperature above a critical entanglement concentration; an outcome of microscopic fibre entanglement. Degelation occurred upon heating to a critical temperature due to fundamental nano-architectural transformation. CD spectroscopy confirmed the chiral characteristics of the prepared polymers. Through the hierarchical self-organization of these chiral nano-objects, twisted nano-fibres formed in the gel state and also a demanding wrinkle patterned surface was generated at some specific conditions. Interestingly, the conversion of masked structopendant reactive functional groups of the parent diblock copolymers to the free primary ammonium moiety was accompanied with a morpho-

Fig. 9 SEM images of PBLAEMA14-b-PBzMA150 (A) and PBLAEMA14-b-PBzMA100 (B) diblock copolymer vesicles and their corresponding in situ TFA induced morphology transitions to worm like micelles in methanol (C), in water (D) and to spherical micelles in methanol (E) and water (F). Illustration of the transformation of the vesicular nano-objects into either worm like or to spherical micelles when the Boc-groups are subjected to remove upon addition of TFA (G).


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logical switch from vesicles to either worms or spheres so as to increase copolymer curvature. These as-made nanostructures coated with a chiral entity may find applications in chiral recognition, asymmetric catalysis, surface coating, etc.62

Acknowledgements This work was supported by the Department of Science and Technology (DST), New Delhi, India [Project No.: SR/S1/OC-51/ 2010]. K. Bauri and U. Haldar acknowledge Council of Scientific and Industrial Research (CSIR), Government of India for their fellowships.

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