Gel phase formation in dilute triblock copolyelectrolyte complexes

Samanvaya Srivastava; Marat Andreev; Adam E. Levi; David J. Goldfeld; Jun Mao; William T. Heller; Vivek M. Prabhu; Juan J. de Pablo; Matthew V. Tirrell

doi:10.1038/ncomms14131

Gel phase formation in dilute triblock copolyelectrolyte complexes

Srivastava, Samanvaya; Andreev, Marat; Levi, Adam E.; Goldfeld, David J.; Mao, Jun; Heller, William T.; Prabhu, Vivek M.; de Pablo, Juan J.; Tirrell, Matthew V. 2017-02-23 00:00:00 ARTICLE Received 4 Oct 2016 | Accepted 1 Dec 2016 | Published 23 Feb 2017 DOI: 10.1038/ncomms14131 OPEN Gel phase formation in dilute triblock copolyelectrolyte complexes 1,2 1 1 1 1,2 3 Samanvaya Srivastava , Marat Andreev , Adam E. Levi , David J. Goldfeld , Jun Mao , William T. Heller , 4 1,2 1,2 Vivek M. Prabhu , Juan J. de Pablo & Matthew V. Tirrell Assembly of oppositely charged triblock copolyelectrolytes into phase-separated gels at low polymer concentrations (o1% by mass) has been observed in scattering experiments and molecular dynamics simulations. Here we show that in contrast to uncharged, amphiphilic block copolymers that form discrete micelles at low concentrations and enter a phase of strongly interacting micelles in a gradual manner with increasing concentration, the formation of a dilute phase of individual micelles is prevented in polyelectrolyte complexation-driven assembly of triblock copolyelectrolytes. Gel phases form and phase separate almost instantaneously on solvation of the copolymers. Furthermore, molecular models of self-assembly demonstrate the presence of oligo-chain aggregates in early stages of copo- lyelectrolyte assembly, at experimentally unobservable polymer concentrations. Our discoveries contribute to the fundamental understanding of the structure and pathways of complexation-driven assemblies, and raise intriguing prospects for gel formation at extra- ordinarily low concentrations, with applications in tissue engineering, agriculture, water puriﬁcation and theranostics. 1 2 Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, USA. Institute for Molecular Engineering, Argonne National 3 4 Laboratory, Lemont, Illinois 60439, USA. Biology & Soft Matter Division, Oak Ridge National laboratory, Oak Ridge, Tennessee 37831, USA. Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. Correspondence and requests for materials should be addressed to J.J.de.P (email: [email protected]) or to M.V.T. (email: [email protected]). NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 olyelectrolyte complexation—associative phase separation Results of oppositely charged polyelectrolytes in aqueous milieu— Structure and the disorder-order transition in PEC gels. Pcan be harnessed via intelligent macromolecular design Figure 1 highlights the similarities and differences among to drive self-assembly of nanoscale micelles with highly the structures formed via self-assembly of pairs of oppositely 1–4 hydrated cores . Electrostatic interactions between oppo- charged di- and triblock copolyelectrolytes comprising sitely charged chains lead to entropy gains from the release of poly(ethylene oxide) (PEO) and either guanidinium chloride the counterions associated with the chains that drive (cationic) or sodium sulfonate (anionic) functionalized 5,6 complexation . Conjugation of either or both polyelectro- poly(allyl glycidyl ether) (PAGE) blocks. The scattering intensity lytes with a neutral polymer block allows for molecular tuning (I(q), q is the wave vector) proﬁles from both di- and of the microphase separation and results in micelles of diverse triblock copolyelectrolyte assemblies, shown in Fig. 1a, indicate 7–9 10–12 13–15 16–18 shapes , including spherical , Janus , rod-like incipient ordering in the structures at high-polymer concentra- 19,20 21–23 and vesicular micelles . Substantial water content and tion f (10% by mass), with strong correlations among the 24–26 extremely low surface tension against water of liquid poly- PEC domains leading to prominent primary peaks in the electrolyte complexes facilitate encapsulation of hydrophilic small I(q) proﬁles. Each of the oppositely charged pairs of polyelec- 27 11,28,29 30–32 molecule drugs , nucleic materials and proteins trolytes studied were functionalized from the same PEO–PAGE in the micelle cores, enabling polyelectrolyte complex (PEC) block copolymer and were mixed in charge equivalent micelle carriers for targeted, encapsulated delivery of hydro- amounts. Therefore, any effects of charge or length mismatch on 29,33–40 philic cargos . the self-assembly were eliminated, thus producing strongly Increasing micelle concentrations above the overlap phase segregated model assemblies. The diblock polymers concentration leads to jamming of micelles resulting in highly were precisely synthesized to be nearly half the size of the 41–48 hydrated, viscoelastic solid materials . An evolution of triblock copolymers; therefore, the morphologies and arrange- ordered micellar arrangements and morphology with increa- ments of the PEC domains at high concentrations, where the singly denser packings, akin to those resulting from jamming PEO coronas began to impinge, regardless of whether these 49–51 of uncharged, amphiphilic block copolymer micelles have tethered corona chains were brushes, loops or bridges, were 42,45 been reported . Speciﬁcally, a disorder-order transition, expected to be near identical, leading to almost indistinguishable exempliﬁed by ordering of spherical PEC domains into a I(q) proﬁles. The faintly sharper peaks in diblock copolyelec- cubic lattice, followed by morphological transitions of trolyte gels could be attributed to fewer topological constraints PEC domains from spheres to hexagonally close-packed to self-assembly . Furthermore, salt is known to affect the 42,45,48 cylinders to parallel-stacked lamella are observed nature of such electrostatics-driven assemblies. However, the with increasing micelle concentrations, allowing for facile results reported here describe studies carried out with no added 44,45,47,52,53 tuning of the gel properties . In addition, growth salt; detailed investigations of salt effects on the structure 45–47 factors, nutrients and drugs can easily be encapsulated in the of similar materials systems have been reported earlier . 32,53 well-hydrated complex cores , and the electrostatic cross- Ordering of the PEC domains on a body-centred cubic links render excellent self-healing properties, as well as lattice was observed on increasing f from 10 to 40% by mass a swift responsiveness to variations in salt concentration gels in both the material systems, driven by compression 42,45 and pH (refs 42,43,45), making these PEC hydrogels extre- of the neutral midblock , which form swollen interstices mely attractive materials as tissue growth supports and between the complex domains. The scattering patterns for bioadhesives. the 40% by mass samples consist of scattering from strongly Complexation-driven self-assemblies are understood to correlated PEC cores in conjunction with the sharp primary 1 1 proceed in an analogous manner to uncharged, amphi- (q*B0.033 Å ), secondary (O2q*B0.046 Å ) and tertiary philic block copolymers, despite the stark differences among (O3q*B0.055 Å ) Bragg reﬂection peaks, corresponding the driving forces for self-assembly in each case. In this article, to an arrangement of the PEC cores in the body centred cubic we report the discovery of spontaneous interconnected (BCC) lattice. Such ordering transitions have been reported 45,46,48 gel-phase formation on solvation of oppositely charged earlier for similar material systems . Last, I(q) grew at ABA triblock copolyelectrolytes, and phase separation of the low q values (qo0.02 Å ) in both the ordered and the gel phases from the solution at very low polymer concentrations. disordered materials, attributable to the presence of structures An observable phase of individual ﬂower-like micelles is larger than individual micelles, either interconnected networks absent. The gel formation is driven by the enhanced propensities or jammed star-like micelle packings with strong correlations. of the neutral midblocks to form bridges between the These large-scale structures would scatter at inverse length PEC domains. The reported observations illustrate the long- scales corresponding to q values smaller than the range range structure-directing contributions of electrostatic forces, investigated in the experiments reported here, with only the in addition to their role in actuating complexation. AB diblock asymptotic power law scattering manifesting as an upturn at low copolyelectrolytes, expectedly, form discrete star-like micelles q range investigated in the current study. at low polymer concentrations. Furthermore, we demonstrate that at high-polymer concentration, both AB diblock and ABA triblock copolyelectrolytes assemblies have near identical Gel phases in low triblock copolyelectrolyte assemblies. Intri- scattering signatures; however, the evolution of scattering guing differences in the scattering proﬁles from di- and triblock patterns with increasing polymer concentrations follow distinct copolyelectrolyte self-assemblies appeared at polymer con- pathways. Our ﬁndings mark a signiﬁcant departure from centrations well below the micelle overlap concentrations (con- the established uncharged, amphiphilic block copolymer-like centrations corresponding to jamming of the unperturbed assembly mechanisms for complexation driven assemblages, micelles). At polymer concentrations below 2% by mass, diblock and will have implications in guiding the design principles copolyelectrolyte assemblies exhibited weak correlation among for PEC gels for many applications such as cell scaffolds for the PEC domains, exempliﬁed by a plateauing of the I(q) at low q tissue engineering, charge based ﬂocculating agents for water values (qo0.01 Å ) and indistinct correlation peaks. Contrarily, puriﬁcation, thin nutrient ﬁlms for agriculture and extremely triblock copolyelectrolyte assemblies exhibited a strong upturn sensitive theranostic probes. in I(q) at low q values, indicating the presence of large 2 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE structures in solution. The scattering intensities also exhibited It was noteworthy that the scattering proﬁles still coincided a shoulder, corresponding to strong correlations between the over moderate to high q values (q40.05 Å ), indicating that PEC domains. These correlations decayed with decreasing f, but the morphologies of the individual PEC domains were similar their presence at such low f was nevertheless surprising. for the di- and triblock copolyelectrolyte assemblies. Halving the size of the neutral block in both the di- and triblock copolyelectrolytes made the disparities in the scattering patterns from the two macromolecular architectures extremely apparent, as evident in Fig. 1b. Diblock copolyelectro- q * lyte assemblies exhibited scattering patterns characteristic of non-interacting isolated micelles: plateau at low q values 6 2q * 1 10 (qo0.01 Å ) transitioning to I(q) decaying with a 4 scaling 1 1 exponent at intermediate q values (0.03 Å oqo0.07 Å ), 3q * indicative of scattering from spherical PEC cores, and further I(q) decaying with a 2 scaling exponent at high q values (q40.1 Å ), suggestive of the scattering from the individual polymer chains. Scattering from triblock copolyelectrolyte assemblies, however, exhibited an upturn in the low q region, along with prominent primary and secondary peaks around 10 40 wt% 1 1 q values of 0.03 Å and 0.05 Å , respectively. These observa- tions, coupled with the f-invariant peak positions, strongly suggested that triblock copolyelectrolytes self-assembled into gel phases characterized by interconnected networks of PEC domains bridged by the neutral midblocks. The progression of structures of self-assemblies with 10 wt% f, as revealed by the scattering results, are depicted by a schematic in Fig. 2. Diblock copolyelectrolytes assemble into dilute solution of star-like micelles at low f; the micelles jam and eventually assemble into ordered structures with increasing f. Triblock copolyelectrolytes, contrarily, assemble 2 wt% into gel phases with interconnected networks of PEC domains, even at the lowest f investigated in the scattering experiments, with no discernible evidence of ﬂower-like micelles. The ﬁnite, non-percolating gels phase separate from the solution at low f, as was evident from negligible scattering from regions 1 wt% of the solution without any polymer networks. Increasing f leads to an expansion of the gel phases until they percolate –1 through the solution, followed by a disorder–order transition of the PEC domains. This is in sharp contrast to the typical assembly of uncharged, amphiphilic ABA triblock copolymers 0.5 wt% in a B-selective solvent, wherein ﬂower-like micelles form at –2 low polymer concentrations , and bridging among the micelles PEO -PAGE occurs only at signiﬁcantly high-polymer concentrations. 227 26 PAGE -PEO -PAGE It should be noted that network formation has been reported 27 455 27 –3 in isolated cases of telechelic polymer assemblies, wherein triblock copolymers with short solvophobic sticker ends form phase separated interconnected networks, existing in 55–59 equilibrium with ﬂower-like micelles . Structure characterization of the interconnected gel phases. Further identiﬁcation of the structural features of the interconnected networks in the low concentration gel phase was facilitated by extracting the structure factors and PEC domain characteristics via a description of the I(q) data with models 2 wt% Figure 1 | Scattering from di- and triblock copolyelectrolyte –1 self-assemblies. Neutron scattering proﬁles (I(q) versus wave vector q) –4 from self-assemblies comprising oppositely charged diblock (circles) and triblock (squares) copolyelectrolytes at various polymer concentrations. 0.5 wt% –2 The polyelectrolytes were oppositely charged functionalized forms of PEO -PAGE (a)PAGE -PEO -PAGE and PEO -PAGE and (b)PAGE -PEO - 114 27 27 455 27 227 26 30 227 PAGE and PEO -PAGE .In (a) diffraction peaks corresponding to PAGE -PEO -PAGE –2 30 114 27 30 227 30 –3 arrangements of complex domains in a BCC lattice are illustrated for the 40% by mass gel. In (b) various power law slopes are indicated by 5 6 7 8 2 3 4 5 6 7 8 2 3 4 0.01 0.1 corresponding lines. Error in the data are typically smaller than the symbols –1 q (A ) and therefore not shown in the ﬁgure. NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 3 I(q) (A.U.) I(q) (A.U.) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 and Diblock copolyelectrolytes Dilute micelles Jammed micelles Ordered micelles – – and Triblock copolyelectrolytes Phase separted gel phase Percolated gel phase Ordered gel phase Increasing polymer concentration Figure 2 | Structure evolution of complexation-driven assemblies. Diblock copolyelectrolytes assemble into star-like micelles. At low polymer concentration, discrete micelles remain suspended in the solution. With increasing polymer concentration, micelles jam, thus forming viscoelastic solids, and eventually order in cubic lattice structures. Triblock copolyelectrolytes are expected to form ﬂower-like micelles at extremely low concentrations (the expected ﬂower-like micelle is shown in the inset), but instead form interconnected gels that phase separate from the solution. Increasing polymer concentrations leads to growth of the networks until they percolate through the solution resulting in viscoelastic solids, followed by a disorder-order transition of the PEC domains. In the corresponding jammed/percolated and ordered phases, PEC domains have very similar size and arrangements, leading to nearly identical scattering patterns. 46,50 for block copolymer micelle scattering . The details of the including two charged end-groups comprising four beads model and the ﬁtting procedure can be found in the Supple- each and representing a PAGE -PEO -PAGE copolymer. 40 200 40 mentary Methods, and the ﬁts are shown in Supplementary AB diblock copolymers were correspondingly simulated as Fig. 1. Structure factors (S(q)), as obtained from the data ﬁts, 14 bead chains (with four bead charged end-groups) are shown in Fig. 3a for both di- and triblock copolyelectrolyte and represented the PAGE -PEO copolymers. The electro- 40 100 assemblies. While the primary S(q) peaks shifted to right static and repulsive forces were modelled by the Coulomb with increasing f for the diblock copolyelectrolyte micelles, interactions making use of the Ewald summation technique their q-invariance with varying f for triblock interconnected and a Lennard–Jones potential, respectively. The strengths gels was evident. The inverse of the primary peaks position of potentials were adjusted to produce aggregation statistics (q*) was used to estimate an average inter-domain spacing corresponding to experiments. The simulations were carried d ( ¼ 2p/q*). As shown in Fig. 3b,d, did not vary signiﬁcantly with out at constant volume conditions with an implicit solvent. varying f for the interconnected gels. A Langevin thermostat was employed to regulate temperature An assessment of the neutral block bridge conformations in the simulations. Detailed simulation methodology can be in the interconnected networks was obtained by approximating found in the Methods section. it to the surface-to-surface distance between the PEC domains. Simulations were initiated from random conﬁgurations A stretching ratio for the midblock polymers was deﬁned as and evolved toward more organized states of PEC self-assembly. SR ¼ (d 2R )/R , with R and R being the radius of the Structural analysis of the ﬁnal equilibrium conﬁgurations allowed c e-e,0 c e-e,0 PEC domains and the equilibrium end-to-end distance of the us to identify the individual PEC cores as well as larger neutral midblock, respectively. The SR values were found to be self-assembled structures. Snapshots from the simulation box, approximately around 1.3 irrespective of the polymer size or shown in Fig. 4a, corroborated with the experimental ﬁndings— concentration, as depicted in Fig. 3c, denoting a moderate diblock copolyelectrolytes self-assembled into star-like micelles but consistent (B30%) stretching of the bridging chains. It could that were dispersed throughout the simulations box, while be surmised from these observations that the f-independent triblock copolyelectrolytes assembled into interconnected structure of the interconnected network gels was strongly networks that did not span the simulation box and micro-phase inﬂuenced by the neutral midblock conformations. separated into gel aggregates. We did not observe macro-phase separation of the networks, though we believe it was due to relatively small size of the simulation boxes. Larger Molecular dynamics simulations of copolyelectrolyte assemblies. (though prohibitively computationally expensive) simulation Insights into the network formation and subsequent phase boxes would allow networks to diffuse and merge, thus forming separation were gained via coarse-grained molecular dynamics macro-phase separated regions. simulations. Polymers were simulated as bead-spring chains, Primarily ﬂower-like micelle (oligo-chain aggregate) popula- with individual beads connected to their neighbours via tions with negligible bridging among them were eventually harmonic springs. One bead represented 10 monomers, for revealed via simulations at extremely low concentration, inacces- both neutral and charged blocks. The conversion of length sible in laboratory experiments f (0.0075% by mass), as shown scales from simulation to real units was achieved following in Fig. 4b. However, bridging between micelles increased previously reported atomistic modelling of PEO chains . rapidly with increasing polymer concentration, and eventually ABA triblock copolymers were simulated as 28 bead chains, led to continuous connected gel phase structures at f, as low 4 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications + + + + NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE Diblock copolyelectrolytes Triblock copolyelectrolytes a a 0.36 wt% 0.36 wt% PAGE -PEO -PAGE 30 227 30 2 wt% 1 wt% 0.5 wt% PAGE -PEO -PAGE 27 455 27 2 wt% 1 wt% 0.5 wt% PEO -PAGE 227 26 1.0 0.0075 wt% 2 wt% 2 0.8 0.075 wt% 1 wt% 0.5 wt% 0.6 0 0.75 wt% 4.5 wt% 0.00 0.02 0.04 0.06 0.4 –1 q (A ) 0.2 0.0 5 67 8 23 45 67 8 2 3 45 67 8 23 45 0.01 0.1 1 Polymer conc. (wt%) Figure 4 | MD simulations reveal triblock copolyelectrolyte assembly into interconnected gels. (a) Snapshots of the simulation box showing 1.75 self-assembled structures comprising oppositely charged di- and triblock copolyelectrolytes. The polycation, polyanion and neutral blocks are depicted by red, blue and yellow coloured beads, respectively. 1.25 (b) Fraction of PEC cores forming isolated ﬂower-like micelles as a function of polymer concentration for triblock copolyelectrolyte assemblies. The 0.75 fraction approaches a near zero value at f ¼ 0.36% by mass. Insets: Snapshots of the simulation box depicting triblock copolyelectrolyte 0.5 1.0 1.5 2.0 assemblies at various polymer concentrations. The polycation, polyanion Polymer conc. (wt%) and neutral blocks are depicted by red, blue and grey coloured beads, respectively. PAGE -PEO -PAGE PAGE -PEO -PAGE 30 227 30 27 455 27 PAGE -PEO -PAGE PAGE -PEO -PAGE 40 227 40 51 455 51 Figure 3 | Structure of the interconnected gels. (a) Structure factor Neutral block conformation and stretching. Representative S(q) for di- and triblock copolyelectrolyte self-assemblies at distributions of the end-to-end distance (R ) of the neutral e-e various polymer concentrations. (b) Inter-domain distance d, and midblocks for the triblock copolyelectrolyte networks, shown (c) SR ( ¼ (d–2R )/R ) for the neutral midblock as a function of polymer c e-e,0 in Fig. 5a, exhibited a clear bimodal distribution with peaks concentration for various triblock copolyelectrolyte gels. In (a), successive on either side of the R corresponding to the looping e-e,0 S(q) plots for different concentrations are shifted vertically by 1 unit each, and bridging midblock chain populations, respectively. At the and collectively by 2 units for different polymer architectures. In (b), closed same time, R distribution for the neutral blocks in diblock e-e and open symbols denote data obtained from neutron and X-ray scattering, copolyelectrolyte micelles showed a unimodal distribution respectively. with a maximum at the R values (Supplementary Fig. 3). The e-e,0 fraction of midblock chains forming either loops or bridges is depicted in Fig. 5b. At the lowest f ( ¼ 0.0075% by mass), a population of exclusively ﬂower-like micelles corresponded as 0.075% by mass. All the ﬂower-like micelles were found to B100% midblocks forming loops. However, with increasing assimilated in a single network at f ¼ 0.36% by mass. Interest- f the loop fraction decreased (and the bridge fraction increased) ingly, the PEC domains in the ﬂower-like micelles observed at rapidly, and a substantial fraction (B30%) of chains formed the lowest concentrations also were composed of fewer chains bridges at f as low as 0.2% by mass, leading to formation than those in interconnected networks, as illustrated by of interconnected non-percolating gels. the growth of the aggregations numbers with increasing f, The structure-directing role of the midblock chains in shown in Supplementary Fig. 2, denoting an evolving micellar the networks, brieﬂy discussed in Fig. 3c, was exempliﬁed by structure with f. Bridging and gel phase formation coincided f-independent conformations of the midblock chains. The with the stabilization of the aggregation number, denoting stretching ratio, SR, for the bridge-forming midblock chains, the absence of the dilute ﬂower-like micelle solution phase in deﬁned in the simulations as R /R and plotted as a function e-e e-e,0 fully developed triblock copolyelectrolyte assemblies. of f in Fig. 5c, indicated nearly f-independent, moderate NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 5 SR d (A) S(q) Fraction of cores forming flower-like micelles ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 the complexation driven assemblies with the corresponding 0.04 uncharged, amphiphilic ABA triblock copolymers assemblies (shown in Fig. 5b,c as open symbols). Bridging and network 40-200-40 Triblock copol. formation comparable to triblock PEC networks was observed at B10-fold higher polymer concentrations in solvophobicity- 0.36 wt% 0.03 driven assemblies, as shown in Fig. 5b. Therefore, similar 3 wt% interconnected gel phase networks were obtained in both the cases, although at very different polymer concentrations. Owing to the larger polymer concentrations and consequently 0.02 larger networks in the latter case, the bridged gels percolated throughout the whole solution and did not phase separate. Further, at comparable f, complexation driven assemblies 0.01 exhibited noticeably higher bridge fractions, which could possibly contribute towards the higher fracture strength and moduli of the hydrogels at higher f. Interestingly, the bridge-forming midblocks were similarly stretched in both forms of assemblies, 0.00 denoting the similarity of structure-directing roles of the 0.0 0.5 1.0 1.5 2.0 2.5 midblocks in both the cases. R /R e-e e-e,PEO200 An analysis of the R of the loop forming fraction of e-e the midblock chains provides an insight into the physical underpinnings of this enhanced network formation and 1.0 the ensuing phase separation. Micellization in uncharged, 0.8 amphiphilic block copolymers is driven by the mutual attraction between the solvophobic blocks, and the entropy penalty for 0.6 f midblock loop formation in the case of triblock copolymers loop 0.4 assembling into ﬂower-like micelles is overcome by the energy bridge gains from expulsion of the solvophobic blocks from the solvent 0.2 as well as their afﬁnity towards each other . Conversely, 0.0 micellization in the systems presented here was driven by complexation between oppositely charged, fully water-soluble, 1.5 blocks on distinct chains. Upon complexation of one of the charged endblocks of a chain, there may not be a necessarily 1.0 strong driving force to recruit the other endblock into the same complex core, aided by the strongly hydrophilic nature 0.5 of the uncomplexed polyelectrolyte endblocks. Moreover, strong repulsion between the like-charged endblocks upon Loops Bridges 0.0 being brought in the near vicinity of each other, as evidenced 6 8 2 4 6 8 2 4 6 8 2 4 that accompanies the looped midblock by the small R e-e 0.01 0.1 1 populations (Fig. 5c), would provide further hindrance to Polymer conc. (wt%) loop formation of the midblocks unless both the endblocks are completely complexed. These factors may combine to lead Figure 5 | Neutral block conformations from MD simulations. to an enhanced propensity of the neutral midblocks to form (a) Distribution of the end-to-end distances of the neutral block in the bridges rather than loops, and thus drive phase separation of interconnected triblock copolyelectrolyte gels at f ¼ 0.36 and 3% by mass. the networks. (b) Fractions of neutral midblocks forming loops or bridges and (c) normalized end-to-end distance of the neutral midblocks forming bridges or loops as a function of polymer concentration in the Discussion interconnected gels. In (b) and (c), open symbols correspond to the In summary, spontaneous gel phase formation and an absence corresponding data from assembly of uncharged, amphiphilic triblock of well-dispersed micelle solution phase was observed in copolymers. complexation driven assemblies of oppositely charged ABA triblock copolyelectrolytes. At low polymer concentra- stretching of the chains. The SR determined from simulations tions (o2% by mass), the interconnected gels were found to is expected to be slightly larger than that estimated phase separate from the solution, denoting a novel transition from experiments given the approximation of SR as (d R )/R to a gel phase, existing between micellization at the extremely c 0 from the experimental data, and use of harmonic bond potential low critical micelle concentrations and the disorder-order in simulations. However, excellent agreement between the SR, transition above the micelle overlap concentrations. Molecular as well as its near invariance with f from both experiments dynamics simulations, on samples sufﬁciently large to demon- and simulations served as strong evidence for the midblock strate assembly of multiple cores and formation of larger inter- stretching entropy directing the equilibrium network structure. core structures, showed excellent agreement with experimental The f-independent domain sizes and conformations of the observation. The short-range repulsion (between the like-charged midblock chains also reveal a unique evolution of the gel endblocks) is presumed to drive the long-range attraction by structure with increasing polymer concentration, wherein biasing the placement of the like-charged blocks against the networks grew while preserving their internal structure until complexing in the same PEC cores, leading to network formation. they percolated through the whole solution. This emphasizes the role of the polyelectrolytes in deﬁning It is instructive to compare the extent of bridge formation, the structure of the micelles and their networks in addition to as well as stretching of the bridge-forming midblocks for driving the micellization via complexation. At the same time, the 6 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications R /R f , f p(R ) loop bridge e-e e-e e-e,0 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE role of neutral blocks goes beyond forming the coronae. of 5% by mass guanidinium functionalized PEO–PAGE with appropriate amounts of water and then adding the solution of 5% by mass sulfonate The stretching of the bridge-forming neutral blocks strongly functionalized PEO-b-PAGE to achieve a speciﬁc polymer concentration f, inﬂuenced the inter-domain spacings, thus directing the structure deﬁned in w/v units, and equal charge molar ratio. For the high polymer of the networks. These ﬁndings denote a signiﬁcant advance- concentration assemblies, 50% by mass stock solutions were used. The ment in our understanding of polyelectrolyte complexa- concentration and the order of mixing of the polymer stock solutions were found to have minimal impact on the structure of resultant assemblies. The low tions based assemblies, which were understood until now concentration assemblies were found to be stable for multiple days, and their to proceed analogous to uncharged, amphiphilic block copolymer stability was further probed by subjecting the solutions to overnight sonication. assemblies. The kinetics of formation of high-concentration structures in these materials In combination with excellent encapsulating propensities systems has been previously shown to be very fast, followed by structural evolution persisting over days owing to evolving packing . The low concentrations of PEC assemblies, triblock copolyelectrolyte gel formation structures were therefore expected to form quickly owing to a lack of packing at very low concentrations leads to new avenues for development frustration. Visual observations of the phase separated gels in some cases agreed of efﬁcient ﬂocculants for water treatment applications with these expectations, with structures forming over the time-scales of mixing of and theranostic carrier-probes. At the same time, our discovery the polymer solutions. will also aid in establishing the design criteria and tuning the long-term performance of the PEC hydrogels. Greatly Scattering measurements. Small-angle neutron and X-ray scattering measure- enhanced bridging among the micelle cores can be credited ments were conducted at EQSANS beamline at Spallation Neutron Source, Oak Ridge National Laboratory and beamline 12-ID-B at Advanced Photon Source, for signiﬁcantly higher equilibrium moduli as well as their Argonne National Laboratory, respectively. For neutron scattering measurements, slow evolution in triblock copolyelectrolyte gels as compared samples were sandwiched between quartz plates, sealed and exposed to neutrons to diblock copolyelectrolyte gels . Swelling and erosion for 40 min. Two sample detector distances of 2 m and 4 m were employed, and the characteristics of PEC hydrogels will also be notably inﬂuenced data sets collected were merged. Samples were sandwiched between Kapton tapes by the polymer architecture, with gels comprising diblock for X-ray scattering measurements, and the exposure times were limited to 0.1 s. All measurements were conducted at room temperature. copolyelectrolytes swelling inﬁnitely and dissolving in Certain commercial equipment and materials are identiﬁed in this paper in the surrounding medium, while hydrogels comprising triblock order to specify adequately the experimental procedure. In no case does such copolyelectrolytes swelling to a ﬁnite extent only. Thus, mixtures identiﬁcation imply recommendations by the National Institute of Standards of di- and triblock copolyelectrolytes can be employed to prepare and Technology (NIST) nor does it imply that the material or equipment identiﬁed is necessarily the best available for this purpose. hydrogels with precisely controlled moduli, morphology and spacing of cargo and nutrient loaded PEC domains, swelling, and in-media retention times. MD simulations. Coarse-grained molecular dynamics simulations, with polymers being represented by bead-spring chains were carried out. The individual beads in each chain were connected to their neighbours via harmonic spring and Methods represented 10 monomers, for both neutral and charged blocks. The electrostatic Materials. Di- and triblock copolymers comprising poly(ethylene oxide) and repulsive forces were modelled by the Coulomb interactions making use of the (PEO) and poly(allyl glycidyl ether), denoted herein as P(EO-AGE) and Ewald summation technique, and by a Lennard-Jones potential, respectively. P(AGE-EO-AGE), respectively, with varying degrees of polymerizations were Parameters for Lennard–Jones were adapted from conventional explicit water synthesized by anionic polymerization, and were subsequently functionalized simulation approach (rs ¼ 0.8). However, water beads were removed and 42,45 via thiol-ene click reactions, following previously reported protocols .The Langevin thermostat with constant volume was applied. Simulation time-step PEO initiator, AGE monomers, solvents and all the other reagents were set to 0.025 Lennard–Jones time units, and the systems were equilibrated for obtained from Sigma Aldrich and were used as received. B10 time-steps. The conversion to real concentration was calculated from atomistic simulations and yielded in 180 polymer beads for every PEO R to 1% by mass. The parameters for Coulomb potential was tuned 200 e-e Anionic polymer synthesis. AGE was puriﬁed by overnight stirring with to give aggregation number of PEC core on the order of 20–25 end blocks per calcium hydride (1 g per 25 ml AGE), followed by three freeze-pump-thaw cycles core, comparable to experimental observation. Supplementary Fig. 4 shows the and distillation using a Schlenk line. Reactions were set-up by dissolving relative strength of employed potentials. either PEO or PEO methyl ether (B20 g) for triblock and diblock copolymer, respectively, in tetrahydrofuran (THF, B200 ml) under a dry argon atmosphere, and then titrated with a solution of potassium naphthalenide dissolved in Code availability. The code generated during and/or analysed during the current THF until the solution had a light green coloration. Distilled AGE was then study are available from the corresponding author on reasonable request. added in appropriate amounts to initiate the polymerization, and the reaction was stirred for 48 h. The polymerization was terminated with degassed methanol (B10 ml), and precipitated in hexanes, followed by drying in vacuo. Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Polymer functionalization via click chemistry. Sodium sulfonate functionalization. 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Ernst at ANL during scattering experiments. This work was performed (2011). under the following ﬁnancial assistance award 70NANB14H012 from U.S. Department 35. Pittella, F. & Kataoka, K. in RNA Interference from Biology to Therapeutics of Commerce, National Institute of Standards and Technology as part of the Center for (ed. Howard, K. A.), 161–184 (Springer US, 2012). Hierarchical Materials Design (CHiMaD). A portion of this research used resources at 36. Kataoka, K., Harada, A. & Nagasaki, Y. Block copolymer micelles for drug the Spallation Neutron Source, a DOE Ofﬁce of Science User Facility operated by the Oak delivery: design, characterization and biological signiﬁcance. Adv. Drug. Deliv. Ridge National Laboratory. This research also used resources of the Advanced Photon Rev. 64, 37–48 (2012). Source, a U.S. Department of Energy (DOE) Ofﬁce of Science User Facility operated for 8 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE the DOE Ofﬁce of Science by Argonne National Laboratory under contract no. DE- Reprints and permission information is available online at http://npg.nature.com/ AC02-06CH11357. reprintsandpermissions/ How to cite this article: Srivastava, S. et al. Gel phase formation in dilute triblock copolyelectrolyte complexes. Nat. Commun. 8, 14131 doi: 10.1038/ncomms14131 (2017). Author contributions S.S. designed and performed the scattering experiments, with help from W.T.H. Polymer Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in synthesis was carried out by S.S., A.L. and D.J.G.; J.M. assisted in the synthesis. published maps and institutional afﬁliations. M.A. designed and performed computer simulations. All authors contributed to the interpretation of the data. S.S., M.A., A.L., J.J.d.P. and M.T. wrote the manuscript, with inputs from V.M.P. J.J.d.P. supervised the computational work. M.T. supervised the This work is licensed under a Creative Commons Attribution 4.0 experimental work. International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. Additional information To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. r The Author(s) 2017 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 9 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals http://www.deepdyve.com/lp/springer-journals/gel-phase-formation-in-dilute-triblock-copolyelectrolyte-complexes-COtuQQdRh3

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Publisher: Springer Journals
Copyright: Copyright © 2017 by The Author(s)
Subject: Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
eISSN: 2041-1723
DOI: 10.1038/ncomms14131
Publisher site: See Article on Publisher Site

Abstract

ARTICLE Received 4 Oct 2016 | Accepted 1 Dec 2016 | Published 23 Feb 2017 DOI: 10.1038/ncomms14131 OPEN Gel phase formation in dilute triblock copolyelectrolyte complexes 1,2 1 1 1 1,2 3 Samanvaya Srivastava , Marat Andreev , Adam E. Levi , David J. Goldfeld , Jun Mao , William T. Heller , 4 1,2 1,2 Vivek M. Prabhu , Juan J. de Pablo & Matthew V. Tirrell Assembly of oppositely charged triblock copolyelectrolytes into phase-separated gels at low polymer concentrations (o1% by mass) has been observed in scattering experiments and molecular dynamics simulations. Here we show that in contrast to uncharged, amphiphilic block copolymers that form discrete micelles at low concentrations and enter a phase of strongly interacting micelles in a gradual manner with increasing concentration, the formation of a dilute phase of individual micelles is prevented in polyelectrolyte complexation-driven assembly of triblock copolyelectrolytes. Gel phases form and phase separate almost instantaneously on solvation of the copolymers. Furthermore, molecular models of self-assembly demonstrate the presence of oligo-chain aggregates in early stages of copo- lyelectrolyte assembly, at experimentally unobservable polymer concentrations. Our discoveries contribute to the fundamental understanding of the structure and pathways of complexation-driven assemblies, and raise intriguing prospects for gel formation at extra- ordinarily low concentrations, with applications in tissue engineering, agriculture, water puriﬁcation and theranostics. 1 2 Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, USA. Institute for Molecular Engineering, Argonne National 3 4 Laboratory, Lemont, Illinois 60439, USA. Biology & Soft Matter Division, Oak Ridge National laboratory, Oak Ridge, Tennessee 37831, USA. Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. Correspondence and requests for materials should be addressed to J.J.de.P (email: [email protected]) or to M.V.T. (email: [email protected]). NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 olyelectrolyte complexation—associative phase separation Results of oppositely charged polyelectrolytes in aqueous milieu— Structure and the disorder-order transition in PEC gels. Pcan be harnessed via intelligent macromolecular design Figure 1 highlights the similarities and differences among to drive self-assembly of nanoscale micelles with highly the structures formed via self-assembly of pairs of oppositely 1–4 hydrated cores . Electrostatic interactions between oppo- charged di- and triblock copolyelectrolytes comprising sitely charged chains lead to entropy gains from the release of poly(ethylene oxide) (PEO) and either guanidinium chloride the counterions associated with the chains that drive (cationic) or sodium sulfonate (anionic) functionalized 5,6 complexation . Conjugation of either or both polyelectro- poly(allyl glycidyl ether) (PAGE) blocks. The scattering intensity lytes with a neutral polymer block allows for molecular tuning (I(q), q is the wave vector) proﬁles from both di- and of the microphase separation and results in micelles of diverse triblock copolyelectrolyte assemblies, shown in Fig. 1a, indicate 7–9 10–12 13–15 16–18 shapes , including spherical , Janus , rod-like incipient ordering in the structures at high-polymer concentra- 19,20 21–23 and vesicular micelles . Substantial water content and tion f (10% by mass), with strong correlations among the 24–26 extremely low surface tension against water of liquid poly- PEC domains leading to prominent primary peaks in the electrolyte complexes facilitate encapsulation of hydrophilic small I(q) proﬁles. Each of the oppositely charged pairs of polyelec- 27 11,28,29 30–32 molecule drugs , nucleic materials and proteins trolytes studied were functionalized from the same PEO–PAGE in the micelle cores, enabling polyelectrolyte complex (PEC) block copolymer and were mixed in charge equivalent micelle carriers for targeted, encapsulated delivery of hydro- amounts. Therefore, any effects of charge or length mismatch on 29,33–40 philic cargos . the self-assembly were eliminated, thus producing strongly Increasing micelle concentrations above the overlap phase segregated model assemblies. The diblock polymers concentration leads to jamming of micelles resulting in highly were precisely synthesized to be nearly half the size of the 41–48 hydrated, viscoelastic solid materials . An evolution of triblock copolymers; therefore, the morphologies and arrange- ordered micellar arrangements and morphology with increa- ments of the PEC domains at high concentrations, where the singly denser packings, akin to those resulting from jamming PEO coronas began to impinge, regardless of whether these 49–51 of uncharged, amphiphilic block copolymer micelles have tethered corona chains were brushes, loops or bridges, were 42,45 been reported . Speciﬁcally, a disorder-order transition, expected to be near identical, leading to almost indistinguishable exempliﬁed by ordering of spherical PEC domains into a I(q) proﬁles. The faintly sharper peaks in diblock copolyelec- cubic lattice, followed by morphological transitions of trolyte gels could be attributed to fewer topological constraints PEC domains from spheres to hexagonally close-packed to self-assembly . Furthermore, salt is known to affect the 42,45,48 cylinders to parallel-stacked lamella are observed nature of such electrostatics-driven assemblies. However, the with increasing micelle concentrations, allowing for facile results reported here describe studies carried out with no added 44,45,47,52,53 tuning of the gel properties . In addition, growth salt; detailed investigations of salt effects on the structure 45–47 factors, nutrients and drugs can easily be encapsulated in the of similar materials systems have been reported earlier . 32,53 well-hydrated complex cores , and the electrostatic cross- Ordering of the PEC domains on a body-centred cubic links render excellent self-healing properties, as well as lattice was observed on increasing f from 10 to 40% by mass a swift responsiveness to variations in salt concentration gels in both the material systems, driven by compression 42,45 and pH (refs 42,43,45), making these PEC hydrogels extre- of the neutral midblock , which form swollen interstices mely attractive materials as tissue growth supports and between the complex domains. The scattering patterns for bioadhesives. the 40% by mass samples consist of scattering from strongly Complexation-driven self-assemblies are understood to correlated PEC cores in conjunction with the sharp primary 1 1 proceed in an analogous manner to uncharged, amphi- (q*B0.033 Å ), secondary (O2q*B0.046 Å ) and tertiary philic block copolymers, despite the stark differences among (O3q*B0.055 Å ) Bragg reﬂection peaks, corresponding the driving forces for self-assembly in each case. In this article, to an arrangement of the PEC cores in the body centred cubic we report the discovery of spontaneous interconnected (BCC) lattice. Such ordering transitions have been reported 45,46,48 gel-phase formation on solvation of oppositely charged earlier for similar material systems . Last, I(q) grew at ABA triblock copolyelectrolytes, and phase separation of the low q values (qo0.02 Å ) in both the ordered and the gel phases from the solution at very low polymer concentrations. disordered materials, attributable to the presence of structures An observable phase of individual ﬂower-like micelles is larger than individual micelles, either interconnected networks absent. The gel formation is driven by the enhanced propensities or jammed star-like micelle packings with strong correlations. of the neutral midblocks to form bridges between the These large-scale structures would scatter at inverse length PEC domains. The reported observations illustrate the long- scales corresponding to q values smaller than the range range structure-directing contributions of electrostatic forces, investigated in the experiments reported here, with only the in addition to their role in actuating complexation. AB diblock asymptotic power law scattering manifesting as an upturn at low copolyelectrolytes, expectedly, form discrete star-like micelles q range investigated in the current study. at low polymer concentrations. Furthermore, we demonstrate that at high-polymer concentration, both AB diblock and ABA triblock copolyelectrolytes assemblies have near identical Gel phases in low triblock copolyelectrolyte assemblies. Intri- scattering signatures; however, the evolution of scattering guing differences in the scattering proﬁles from di- and triblock patterns with increasing polymer concentrations follow distinct copolyelectrolyte self-assemblies appeared at polymer con- pathways. Our ﬁndings mark a signiﬁcant departure from centrations well below the micelle overlap concentrations (con- the established uncharged, amphiphilic block copolymer-like centrations corresponding to jamming of the unperturbed assembly mechanisms for complexation driven assemblages, micelles). At polymer concentrations below 2% by mass, diblock and will have implications in guiding the design principles copolyelectrolyte assemblies exhibited weak correlation among for PEC gels for many applications such as cell scaffolds for the PEC domains, exempliﬁed by a plateauing of the I(q) at low q tissue engineering, charge based ﬂocculating agents for water values (qo0.01 Å ) and indistinct correlation peaks. Contrarily, puriﬁcation, thin nutrient ﬁlms for agriculture and extremely triblock copolyelectrolyte assemblies exhibited a strong upturn sensitive theranostic probes. in I(q) at low q values, indicating the presence of large 2 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE structures in solution. The scattering intensities also exhibited It was noteworthy that the scattering proﬁles still coincided a shoulder, corresponding to strong correlations between the over moderate to high q values (q40.05 Å ), indicating that PEC domains. These correlations decayed with decreasing f, but the morphologies of the individual PEC domains were similar their presence at such low f was nevertheless surprising. for the di- and triblock copolyelectrolyte assemblies. Halving the size of the neutral block in both the di- and triblock copolyelectrolytes made the disparities in the scattering patterns from the two macromolecular architectures extremely apparent, as evident in Fig. 1b. Diblock copolyelectro- q * lyte assemblies exhibited scattering patterns characteristic of non-interacting isolated micelles: plateau at low q values 6 2q * 1 10 (qo0.01 Å ) transitioning to I(q) decaying with a 4 scaling 1 1 exponent at intermediate q values (0.03 Å oqo0.07 Å ), 3q * indicative of scattering from spherical PEC cores, and further I(q) decaying with a 2 scaling exponent at high q values (q40.1 Å ), suggestive of the scattering from the individual polymer chains. Scattering from triblock copolyelectrolyte assemblies, however, exhibited an upturn in the low q region, along with prominent primary and secondary peaks around 10 40 wt% 1 1 q values of 0.03 Å and 0.05 Å , respectively. These observa- tions, coupled with the f-invariant peak positions, strongly suggested that triblock copolyelectrolytes self-assembled into gel phases characterized by interconnected networks of PEC domains bridged by the neutral midblocks. The progression of structures of self-assemblies with 10 wt% f, as revealed by the scattering results, are depicted by a schematic in Fig. 2. Diblock copolyelectrolytes assemble into dilute solution of star-like micelles at low f; the micelles jam and eventually assemble into ordered structures with increasing f. Triblock copolyelectrolytes, contrarily, assemble 2 wt% into gel phases with interconnected networks of PEC domains, even at the lowest f investigated in the scattering experiments, with no discernible evidence of ﬂower-like micelles. The ﬁnite, non-percolating gels phase separate from the solution at low f, as was evident from negligible scattering from regions 1 wt% of the solution without any polymer networks. Increasing f leads to an expansion of the gel phases until they percolate –1 through the solution, followed by a disorder–order transition of the PEC domains. This is in sharp contrast to the typical assembly of uncharged, amphiphilic ABA triblock copolymers 0.5 wt% in a B-selective solvent, wherein ﬂower-like micelles form at –2 low polymer concentrations , and bridging among the micelles PEO -PAGE occurs only at signiﬁcantly high-polymer concentrations. 227 26 PAGE -PEO -PAGE It should be noted that network formation has been reported 27 455 27 –3 in isolated cases of telechelic polymer assemblies, wherein triblock copolymers with short solvophobic sticker ends form phase separated interconnected networks, existing in 55–59 equilibrium with ﬂower-like micelles . Structure characterization of the interconnected gel phases. Further identiﬁcation of the structural features of the interconnected networks in the low concentration gel phase was facilitated by extracting the structure factors and PEC domain characteristics via a description of the I(q) data with models 2 wt% Figure 1 | Scattering from di- and triblock copolyelectrolyte –1 self-assemblies. Neutron scattering proﬁles (I(q) versus wave vector q) –4 from self-assemblies comprising oppositely charged diblock (circles) and triblock (squares) copolyelectrolytes at various polymer concentrations. 0.5 wt% –2 The polyelectrolytes were oppositely charged functionalized forms of PEO -PAGE (a)PAGE -PEO -PAGE and PEO -PAGE and (b)PAGE -PEO - 114 27 27 455 27 227 26 30 227 PAGE and PEO -PAGE .In (a) diffraction peaks corresponding to PAGE -PEO -PAGE –2 30 114 27 30 227 30 –3 arrangements of complex domains in a BCC lattice are illustrated for the 40% by mass gel. In (b) various power law slopes are indicated by 5 6 7 8 2 3 4 5 6 7 8 2 3 4 0.01 0.1 corresponding lines. Error in the data are typically smaller than the symbols –1 q (A ) and therefore not shown in the ﬁgure. NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 3 I(q) (A.U.) I(q) (A.U.) ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 and Diblock copolyelectrolytes Dilute micelles Jammed micelles Ordered micelles – – and Triblock copolyelectrolytes Phase separted gel phase Percolated gel phase Ordered gel phase Increasing polymer concentration Figure 2 | Structure evolution of complexation-driven assemblies. Diblock copolyelectrolytes assemble into star-like micelles. At low polymer concentration, discrete micelles remain suspended in the solution. With increasing polymer concentration, micelles jam, thus forming viscoelastic solids, and eventually order in cubic lattice structures. Triblock copolyelectrolytes are expected to form ﬂower-like micelles at extremely low concentrations (the expected ﬂower-like micelle is shown in the inset), but instead form interconnected gels that phase separate from the solution. Increasing polymer concentrations leads to growth of the networks until they percolate through the solution resulting in viscoelastic solids, followed by a disorder-order transition of the PEC domains. In the corresponding jammed/percolated and ordered phases, PEC domains have very similar size and arrangements, leading to nearly identical scattering patterns. 46,50 for block copolymer micelle scattering . The details of the including two charged end-groups comprising four beads model and the ﬁtting procedure can be found in the Supple- each and representing a PAGE -PEO -PAGE copolymer. 40 200 40 mentary Methods, and the ﬁts are shown in Supplementary AB diblock copolymers were correspondingly simulated as Fig. 1. Structure factors (S(q)), as obtained from the data ﬁts, 14 bead chains (with four bead charged end-groups) are shown in Fig. 3a for both di- and triblock copolyelectrolyte and represented the PAGE -PEO copolymers. The electro- 40 100 assemblies. While the primary S(q) peaks shifted to right static and repulsive forces were modelled by the Coulomb with increasing f for the diblock copolyelectrolyte micelles, interactions making use of the Ewald summation technique their q-invariance with varying f for triblock interconnected and a Lennard–Jones potential, respectively. The strengths gels was evident. The inverse of the primary peaks position of potentials were adjusted to produce aggregation statistics (q*) was used to estimate an average inter-domain spacing corresponding to experiments. The simulations were carried d ( ¼ 2p/q*). As shown in Fig. 3b,d, did not vary signiﬁcantly with out at constant volume conditions with an implicit solvent. varying f for the interconnected gels. A Langevin thermostat was employed to regulate temperature An assessment of the neutral block bridge conformations in the simulations. Detailed simulation methodology can be in the interconnected networks was obtained by approximating found in the Methods section. it to the surface-to-surface distance between the PEC domains. Simulations were initiated from random conﬁgurations A stretching ratio for the midblock polymers was deﬁned as and evolved toward more organized states of PEC self-assembly. SR ¼ (d 2R )/R , with R and R being the radius of the Structural analysis of the ﬁnal equilibrium conﬁgurations allowed c e-e,0 c e-e,0 PEC domains and the equilibrium end-to-end distance of the us to identify the individual PEC cores as well as larger neutral midblock, respectively. The SR values were found to be self-assembled structures. Snapshots from the simulation box, approximately around 1.3 irrespective of the polymer size or shown in Fig. 4a, corroborated with the experimental ﬁndings— concentration, as depicted in Fig. 3c, denoting a moderate diblock copolyelectrolytes self-assembled into star-like micelles but consistent (B30%) stretching of the bridging chains. It could that were dispersed throughout the simulations box, while be surmised from these observations that the f-independent triblock copolyelectrolytes assembled into interconnected structure of the interconnected network gels was strongly networks that did not span the simulation box and micro-phase inﬂuenced by the neutral midblock conformations. separated into gel aggregates. We did not observe macro-phase separation of the networks, though we believe it was due to relatively small size of the simulation boxes. Larger Molecular dynamics simulations of copolyelectrolyte assemblies. (though prohibitively computationally expensive) simulation Insights into the network formation and subsequent phase boxes would allow networks to diffuse and merge, thus forming separation were gained via coarse-grained molecular dynamics macro-phase separated regions. simulations. Polymers were simulated as bead-spring chains, Primarily ﬂower-like micelle (oligo-chain aggregate) popula- with individual beads connected to their neighbours via tions with negligible bridging among them were eventually harmonic springs. One bead represented 10 monomers, for revealed via simulations at extremely low concentration, inacces- both neutral and charged blocks. The conversion of length sible in laboratory experiments f (0.0075% by mass), as shown scales from simulation to real units was achieved following in Fig. 4b. However, bridging between micelles increased previously reported atomistic modelling of PEO chains . rapidly with increasing polymer concentration, and eventually ABA triblock copolymers were simulated as 28 bead chains, led to continuous connected gel phase structures at f, as low 4 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications + + + + NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE Diblock copolyelectrolytes Triblock copolyelectrolytes a a 0.36 wt% 0.36 wt% PAGE -PEO -PAGE 30 227 30 2 wt% 1 wt% 0.5 wt% PAGE -PEO -PAGE 27 455 27 2 wt% 1 wt% 0.5 wt% PEO -PAGE 227 26 1.0 0.0075 wt% 2 wt% 2 0.8 0.075 wt% 1 wt% 0.5 wt% 0.6 0 0.75 wt% 4.5 wt% 0.00 0.02 0.04 0.06 0.4 –1 q (A ) 0.2 0.0 5 67 8 23 45 67 8 2 3 45 67 8 23 45 0.01 0.1 1 Polymer conc. (wt%) Figure 4 | MD simulations reveal triblock copolyelectrolyte assembly into interconnected gels. (a) Snapshots of the simulation box showing 1.75 self-assembled structures comprising oppositely charged di- and triblock copolyelectrolytes. The polycation, polyanion and neutral blocks are depicted by red, blue and yellow coloured beads, respectively. 1.25 (b) Fraction of PEC cores forming isolated ﬂower-like micelles as a function of polymer concentration for triblock copolyelectrolyte assemblies. The 0.75 fraction approaches a near zero value at f ¼ 0.36% by mass. Insets: Snapshots of the simulation box depicting triblock copolyelectrolyte 0.5 1.0 1.5 2.0 assemblies at various polymer concentrations. The polycation, polyanion Polymer conc. (wt%) and neutral blocks are depicted by red, blue and grey coloured beads, respectively. PAGE -PEO -PAGE PAGE -PEO -PAGE 30 227 30 27 455 27 PAGE -PEO -PAGE PAGE -PEO -PAGE 40 227 40 51 455 51 Figure 3 | Structure of the interconnected gels. (a) Structure factor Neutral block conformation and stretching. Representative S(q) for di- and triblock copolyelectrolyte self-assemblies at distributions of the end-to-end distance (R ) of the neutral e-e various polymer concentrations. (b) Inter-domain distance d, and midblocks for the triblock copolyelectrolyte networks, shown (c) SR ( ¼ (d–2R )/R ) for the neutral midblock as a function of polymer c e-e,0 in Fig. 5a, exhibited a clear bimodal distribution with peaks concentration for various triblock copolyelectrolyte gels. In (a), successive on either side of the R corresponding to the looping e-e,0 S(q) plots for different concentrations are shifted vertically by 1 unit each, and bridging midblock chain populations, respectively. At the and collectively by 2 units for different polymer architectures. In (b), closed same time, R distribution for the neutral blocks in diblock e-e and open symbols denote data obtained from neutron and X-ray scattering, copolyelectrolyte micelles showed a unimodal distribution respectively. with a maximum at the R values (Supplementary Fig. 3). The e-e,0 fraction of midblock chains forming either loops or bridges is depicted in Fig. 5b. At the lowest f ( ¼ 0.0075% by mass), a population of exclusively ﬂower-like micelles corresponded as 0.075% by mass. All the ﬂower-like micelles were found to B100% midblocks forming loops. However, with increasing assimilated in a single network at f ¼ 0.36% by mass. Interest- f the loop fraction decreased (and the bridge fraction increased) ingly, the PEC domains in the ﬂower-like micelles observed at rapidly, and a substantial fraction (B30%) of chains formed the lowest concentrations also were composed of fewer chains bridges at f as low as 0.2% by mass, leading to formation than those in interconnected networks, as illustrated by of interconnected non-percolating gels. the growth of the aggregations numbers with increasing f, The structure-directing role of the midblock chains in shown in Supplementary Fig. 2, denoting an evolving micellar the networks, brieﬂy discussed in Fig. 3c, was exempliﬁed by structure with f. Bridging and gel phase formation coincided f-independent conformations of the midblock chains. The with the stabilization of the aggregation number, denoting stretching ratio, SR, for the bridge-forming midblock chains, the absence of the dilute ﬂower-like micelle solution phase in deﬁned in the simulations as R /R and plotted as a function e-e e-e,0 fully developed triblock copolyelectrolyte assemblies. of f in Fig. 5c, indicated nearly f-independent, moderate NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 5 SR d (A) S(q) Fraction of cores forming flower-like micelles ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 the complexation driven assemblies with the corresponding 0.04 uncharged, amphiphilic ABA triblock copolymers assemblies (shown in Fig. 5b,c as open symbols). Bridging and network 40-200-40 Triblock copol. formation comparable to triblock PEC networks was observed at B10-fold higher polymer concentrations in solvophobicity- 0.36 wt% 0.03 driven assemblies, as shown in Fig. 5b. Therefore, similar 3 wt% interconnected gel phase networks were obtained in both the cases, although at very different polymer concentrations. Owing to the larger polymer concentrations and consequently 0.02 larger networks in the latter case, the bridged gels percolated throughout the whole solution and did not phase separate. Further, at comparable f, complexation driven assemblies 0.01 exhibited noticeably higher bridge fractions, which could possibly contribute towards the higher fracture strength and moduli of the hydrogels at higher f. Interestingly, the bridge-forming midblocks were similarly stretched in both forms of assemblies, 0.00 denoting the similarity of structure-directing roles of the 0.0 0.5 1.0 1.5 2.0 2.5 midblocks in both the cases. R /R e-e e-e,PEO200 An analysis of the R of the loop forming fraction of e-e the midblock chains provides an insight into the physical underpinnings of this enhanced network formation and 1.0 the ensuing phase separation. Micellization in uncharged, 0.8 amphiphilic block copolymers is driven by the mutual attraction between the solvophobic blocks, and the entropy penalty for 0.6 f midblock loop formation in the case of triblock copolymers loop 0.4 assembling into ﬂower-like micelles is overcome by the energy bridge gains from expulsion of the solvophobic blocks from the solvent 0.2 as well as their afﬁnity towards each other . Conversely, 0.0 micellization in the systems presented here was driven by complexation between oppositely charged, fully water-soluble, 1.5 blocks on distinct chains. Upon complexation of one of the charged endblocks of a chain, there may not be a necessarily 1.0 strong driving force to recruit the other endblock into the same complex core, aided by the strongly hydrophilic nature 0.5 of the uncomplexed polyelectrolyte endblocks. Moreover, strong repulsion between the like-charged endblocks upon Loops Bridges 0.0 being brought in the near vicinity of each other, as evidenced 6 8 2 4 6 8 2 4 6 8 2 4 that accompanies the looped midblock by the small R e-e 0.01 0.1 1 populations (Fig. 5c), would provide further hindrance to Polymer conc. (wt%) loop formation of the midblocks unless both the endblocks are completely complexed. These factors may combine to lead Figure 5 | Neutral block conformations from MD simulations. to an enhanced propensity of the neutral midblocks to form (a) Distribution of the end-to-end distances of the neutral block in the bridges rather than loops, and thus drive phase separation of interconnected triblock copolyelectrolyte gels at f ¼ 0.36 and 3% by mass. the networks. (b) Fractions of neutral midblocks forming loops or bridges and (c) normalized end-to-end distance of the neutral midblocks forming bridges or loops as a function of polymer concentration in the Discussion interconnected gels. In (b) and (c), open symbols correspond to the In summary, spontaneous gel phase formation and an absence corresponding data from assembly of uncharged, amphiphilic triblock of well-dispersed micelle solution phase was observed in copolymers. complexation driven assemblies of oppositely charged ABA triblock copolyelectrolytes. At low polymer concentra- stretching of the chains. The SR determined from simulations tions (o2% by mass), the interconnected gels were found to is expected to be slightly larger than that estimated phase separate from the solution, denoting a novel transition from experiments given the approximation of SR as (d R )/R to a gel phase, existing between micellization at the extremely c 0 from the experimental data, and use of harmonic bond potential low critical micelle concentrations and the disorder-order in simulations. However, excellent agreement between the SR, transition above the micelle overlap concentrations. Molecular as well as its near invariance with f from both experiments dynamics simulations, on samples sufﬁciently large to demon- and simulations served as strong evidence for the midblock strate assembly of multiple cores and formation of larger inter- stretching entropy directing the equilibrium network structure. core structures, showed excellent agreement with experimental The f-independent domain sizes and conformations of the observation. The short-range repulsion (between the like-charged midblock chains also reveal a unique evolution of the gel endblocks) is presumed to drive the long-range attraction by structure with increasing polymer concentration, wherein biasing the placement of the like-charged blocks against the networks grew while preserving their internal structure until complexing in the same PEC cores, leading to network formation. they percolated through the whole solution. This emphasizes the role of the polyelectrolytes in deﬁning It is instructive to compare the extent of bridge formation, the structure of the micelles and their networks in addition to as well as stretching of the bridge-forming midblocks for driving the micellization via complexation. At the same time, the 6 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications R /R f , f p(R ) loop bridge e-e e-e e-e,0 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE role of neutral blocks goes beyond forming the coronae. of 5% by mass guanidinium functionalized PEO–PAGE with appropriate amounts of water and then adding the solution of 5% by mass sulfonate The stretching of the bridge-forming neutral blocks strongly functionalized PEO-b-PAGE to achieve a speciﬁc polymer concentration f, inﬂuenced the inter-domain spacings, thus directing the structure deﬁned in w/v units, and equal charge molar ratio. For the high polymer of the networks. These ﬁndings denote a signiﬁcant advance- concentration assemblies, 50% by mass stock solutions were used. The ment in our understanding of polyelectrolyte complexa- concentration and the order of mixing of the polymer stock solutions were found to have minimal impact on the structure of resultant assemblies. The low tions based assemblies, which were understood until now concentration assemblies were found to be stable for multiple days, and their to proceed analogous to uncharged, amphiphilic block copolymer stability was further probed by subjecting the solutions to overnight sonication. assemblies. The kinetics of formation of high-concentration structures in these materials In combination with excellent encapsulating propensities systems has been previously shown to be very fast, followed by structural evolution persisting over days owing to evolving packing . The low concentrations of PEC assemblies, triblock copolyelectrolyte gel formation structures were therefore expected to form quickly owing to a lack of packing at very low concentrations leads to new avenues for development frustration. Visual observations of the phase separated gels in some cases agreed of efﬁcient ﬂocculants for water treatment applications with these expectations, with structures forming over the time-scales of mixing of and theranostic carrier-probes. At the same time, our discovery the polymer solutions. will also aid in establishing the design criteria and tuning the long-term performance of the PEC hydrogels. Greatly Scattering measurements. Small-angle neutron and X-ray scattering measure- enhanced bridging among the micelle cores can be credited ments were conducted at EQSANS beamline at Spallation Neutron Source, Oak Ridge National Laboratory and beamline 12-ID-B at Advanced Photon Source, for signiﬁcantly higher equilibrium moduli as well as their Argonne National Laboratory, respectively. For neutron scattering measurements, slow evolution in triblock copolyelectrolyte gels as compared samples were sandwiched between quartz plates, sealed and exposed to neutrons to diblock copolyelectrolyte gels . Swelling and erosion for 40 min. Two sample detector distances of 2 m and 4 m were employed, and the characteristics of PEC hydrogels will also be notably inﬂuenced data sets collected were merged. Samples were sandwiched between Kapton tapes by the polymer architecture, with gels comprising diblock for X-ray scattering measurements, and the exposure times were limited to 0.1 s. All measurements were conducted at room temperature. copolyelectrolytes swelling inﬁnitely and dissolving in Certain commercial equipment and materials are identiﬁed in this paper in the surrounding medium, while hydrogels comprising triblock order to specify adequately the experimental procedure. In no case does such copolyelectrolytes swelling to a ﬁnite extent only. Thus, mixtures identiﬁcation imply recommendations by the National Institute of Standards of di- and triblock copolyelectrolytes can be employed to prepare and Technology (NIST) nor does it imply that the material or equipment identiﬁed is necessarily the best available for this purpose. hydrogels with precisely controlled moduli, morphology and spacing of cargo and nutrient loaded PEC domains, swelling, and in-media retention times. MD simulations. Coarse-grained molecular dynamics simulations, with polymers being represented by bead-spring chains were carried out. The individual beads in each chain were connected to their neighbours via harmonic spring and Methods represented 10 monomers, for both neutral and charged blocks. The electrostatic Materials. Di- and triblock copolymers comprising poly(ethylene oxide) and repulsive forces were modelled by the Coulomb interactions making use of the (PEO) and poly(allyl glycidyl ether), denoted herein as P(EO-AGE) and Ewald summation technique, and by a Lennard-Jones potential, respectively. P(AGE-EO-AGE), respectively, with varying degrees of polymerizations were Parameters for Lennard–Jones were adapted from conventional explicit water synthesized by anionic polymerization, and were subsequently functionalized simulation approach (rs ¼ 0.8). However, water beads were removed and 42,45 via thiol-ene click reactions, following previously reported protocols .The Langevin thermostat with constant volume was applied. Simulation time-step PEO initiator, AGE monomers, solvents and all the other reagents were set to 0.025 Lennard–Jones time units, and the systems were equilibrated for obtained from Sigma Aldrich and were used as received. B10 time-steps. The conversion to real concentration was calculated from atomistic simulations and yielded in 180 polymer beads for every PEO R to 1% by mass. The parameters for Coulomb potential was tuned 200 e-e Anionic polymer synthesis. AGE was puriﬁed by overnight stirring with to give aggregation number of PEC core on the order of 20–25 end blocks per calcium hydride (1 g per 25 ml AGE), followed by three freeze-pump-thaw cycles core, comparable to experimental observation. Supplementary Fig. 4 shows the and distillation using a Schlenk line. Reactions were set-up by dissolving relative strength of employed potentials. either PEO or PEO methyl ether (B20 g) for triblock and diblock copolymer, respectively, in tetrahydrofuran (THF, B200 ml) under a dry argon atmosphere, and then titrated with a solution of potassium naphthalenide dissolved in Code availability. The code generated during and/or analysed during the current THF until the solution had a light green coloration. Distilled AGE was then study are available from the corresponding author on reasonable request. added in appropriate amounts to initiate the polymerization, and the reaction was stirred for 48 h. The polymerization was terminated with degassed methanol (B10 ml), and precipitated in hexanes, followed by drying in vacuo. Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Polymer functionalization via click chemistry. Sodium sulfonate functionalization. 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Ernst at ANL during scattering experiments. This work was performed (2011). under the following ﬁnancial assistance award 70NANB14H012 from U.S. Department 35. Pittella, F. & Kataoka, K. in RNA Interference from Biology to Therapeutics of Commerce, National Institute of Standards and Technology as part of the Center for (ed. Howard, K. A.), 161–184 (Springer US, 2012). Hierarchical Materials Design (CHiMaD). A portion of this research used resources at 36. Kataoka, K., Harada, A. & Nagasaki, Y. Block copolymer micelles for drug the Spallation Neutron Source, a DOE Ofﬁce of Science User Facility operated by the Oak delivery: design, characterization and biological signiﬁcance. Adv. Drug. Deliv. Ridge National Laboratory. This research also used resources of the Advanced Photon Rev. 64, 37–48 (2012). Source, a U.S. Department of Energy (DOE) Ofﬁce of Science User Facility operated for 8 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14131 ARTICLE the DOE Ofﬁce of Science by Argonne National Laboratory under contract no. DE- Reprints and permission information is available online at http://npg.nature.com/ AC02-06CH11357. reprintsandpermissions/ How to cite this article: Srivastava, S. et al. Gel phase formation in dilute triblock copolyelectrolyte complexes. Nat. Commun. 8, 14131 doi: 10.1038/ncomms14131 (2017). Author contributions S.S. designed and performed the scattering experiments, with help from W.T.H. Polymer Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in synthesis was carried out by S.S., A.L. and D.J.G.; J.M. assisted in the synthesis. published maps and institutional afﬁliations. M.A. designed and performed computer simulations. All authors contributed to the interpretation of the data. S.S., M.A., A.L., J.J.d.P. and M.T. wrote the manuscript, with inputs from V.M.P. J.J.d.P. supervised the computational work. M.T. supervised the This work is licensed under a Creative Commons Attribution 4.0 experimental work. International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. Additional information To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. r The Author(s) 2017 NATURE COMMUNICATIONS | 8:14131 | DOI: 10.1038/ncomms14131 | www.nature.com/naturecommunications 9

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Gel phase formation in dilute triblock copolyelectrolyte complexes

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Gel phase formation in dilute triblock copolyelectrolyte complexes

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