A Single Thermoresponsive Diblock Copolymer Can Form Spheres, Worms or Vesicles in Aqueous Solution

Abstract It is well‐known that the self‐assembly of AB diblock copolymers in solution can produce various morphologies depending on the relative volume fraction of each block. Recently, polymerization‐induced self‐assembly (PISA) has become widely recognized as a powerful platform technology for the rational design and efficient synthesis of a wide range of block copolymer nano‐objects. In this study, PISA is used to prepare a new thermoresponsive poly(N‐(2‐hydroxypropyl) methacrylamide)‐poly(2‐hydroxypropyl methacrylate) [PHPMAC‐PHPMA] diblock copolymer. Remarkably, TEM, rheology and SAXS studies indicate that a single copolymer composition can form well‐defined spheres (4 °C), worms (22 °C) or vesicles (50 °C) in aqueous solution. Given that the two monomer repeat units have almost identical chemical structures, this system is particularly well‐suited to theoretical analysis. Self‐consistent mean field theory suggests this rich self‐assembly behavior is the result of the greater degree of hydration of the PHPMA block at lower temperature, which is in agreement with variable temperature 1H NMR studies.


Introduction
Block copolymer self-assembly in solution has been known for more than fifty years. [1] Many copolymer mor-phologies have been reported, including spheres,worms,rods, vesicles,l amellar platelets,d isks,t oroids,s tomatocytes and framboidal vesicles. [2] Potential applications include drug delivery,n anoencapsulation, membranes,b iocompatible hydrogels,chemotaxis and diesel soot dispersion in engine oils. [3] Fort he self-assembly of amphiphilic AB diblock copolymer chains in aqueous solution, spheres, [2c] worms [2f] or vesicles [4] are by far the most common copolymer morphologies.S uch nano-objects are now readily accessible via polymerizationinduced self-assembly (PISA), which can be conducted in concentrated aqueous media. [5] This is largely owing to the development of controlled radical polymerisation techniques such as reversible addition-fragmentation chain transfer (RAFT) polymerization, [6] which has enabled the convenient synthesis of aw ide range of well-defined functional block copolymers. [7] In particular, many examples of thermoresponsive watersoluble block copolymers have been reported in the literature. [4,8] Recently,PISA formulations have provided various examples of thermally-induced worm-to-sphere,v esicle-tosphere or vesicle-to-worm transformations. [9] In the case of certain aqueous dispersions of thermoresponsive diblock copolymer nano-objects,aworm-to-sphere or vesicle-tosphere transition occurs on cooling.I nc ontrast, for diblock copolymer nano-objects dispersed in non-aqueous media, aw orm-to-sphere or vesicle-to-worm transition occurs on heating. Both phenomena can be explained in terms of surface plasticization of the insoluble structure-directing block;t his leads to as ubtle change in the packing parameter [10] that drives each morphological transition. [11] Herein we report an ew thermoresponsive AB diblock copolymer which exhibits remarkable self-assembly behavior: asingle copolymer composition that can form either spheres, worms or vesicles in aqueous solution depending solely on the temperature (see Figure 1). These two thermal transitions are again attributed to surface plasticization of the hydrophobic block, which becomes significantly more hydrated on lowering the aqueous solution temperature.T he hydrophilic stabilizer block is awell-known highly biocompatible polymer that has been extensively studied by others in the context of drug delivery applications:p oly(N-(2-hydroxypropyl) methacrylamide) [PHPMAC]. [12] This water-soluble polymer has been prepared by RAFT polymerization (see Figure S1a) with good control over its molecular weight distribution (MWD) being achieved. [13] Thehydrophobic structure-directing block is poly(2-hydroxypropyl methacrylate), which has been used for many PISA formulations based on RAFT aqueous dispersion polymerization. [9b,14] Ther emarkably subtle difference in chemical structure (see Figure 1) between these two types of monomer repeat units aids theoretical analysis of this system using self-consistent mean field theory, [15] because it ensures very similar segment volumes for these two components when using alattice model.
Systematic variation of the target degree of polymerization (DP or y)for the hydrophobic PHPMA block from 140 to 220 led to the formation of either spheres,w orms or vesicles at ambient temperature (22 8 8C) as judged by transmission electron microscopy (TEM) studies (see Figure S3). On cooling from 70 8 8Cto228 8C, the physical appearance of some of the aqueous copolymer dispersions changed from amilky-white free-flowing fluid to either agel or aless turbid fluid. Based on our prior experience, [5b, 9a, 16] this suggested the likelihood of at least one thermally-induced morphological transition. In particular, the PHPMAC 41 -PHPMA 180 copolymer dispersion exhibited three distinct physical states over ac onvenient temperature range:aw eakly turbid fluid was obtained at 4 8 8C, asoft free-standing gel was formed at 22 8 8C and am ilky-white free-flowing dispersion was observed at 50 8 8C. Accordingly,the as-synthesized 10 %w/w PHPMAC 41 -PHPMA 180 aqueous dispersion was diluted to 0.20 %w /w using deionized water, with each dilution being conducted at either 4 8 8C( with the aid of ar efrigerator), 22 8 8C( ambient temperature) or 50 8 8C(with the aid of an oven), after allowing 24 hf or equilibration. These dilute copolymer dispersions were analyzed by TEM (see Figure 1). Remarkably,this single PHPMAC 41 -PHPMA 180 diblock copolymer can form either spheres,w orms or vesicles simply by varying the aqueous solution temperature:t his involves crossing both the vesicle/ worm and worm/sphere phase boundaries within ar elatively narrow temperature range.S imilar behavior has been recently reported by Delaittre and co-workers [17] for poly(2ethyl-2-oxazoline)-PHPMA diblock copolymer nanoparticles.H owever,i nt his prior study morphological transitions were only shown to be reversible at ar elatively high copolymer concentration (20 %w /w): the spheres formed at low temperature became kinetically-trapped for thermal cycles performed in dilute solution (e.g.0 .1 %w /w copolymer). Moreover,the sole characterization data provided were TEM images obtained for dried diluted dispersions.I nt he current study,p reliminary experiments indicated significant kinetic differences for the interconversion between spheres, worms and vesicles,e ven for 10 %w /w aqueous copolymer dispersions.M ore specifically,t he worm-to-sphere transition occurred relatively quickly (within 45 min, according to rheological studies), whereas the complementary sphere-toworm transition typically required rather longer time scales (hours). This is not particularly surprising given the former transition involves ad issociative mechanism (most likely via worm budding), whereas the latter requires ac ooperative associative mechanism (i.e.m ultiple sphere-sphere fusion events). [9c] Similar temporal differences were also observed for the vesicle-to-worm and worm-to-vesicle transitions (data not shown).
Small-angle X-ray scattering (SAXS) is widely recognized as apowerful characterization technique for block copolymer nano-objects. [9a,g, 18] Unlike DLS,S AXS experiments can be performed on 10 %w /w copolymer dispersions and established models exist for the detailed analysis of spheres, [19] worms [19] and vesicles. [20] Moreover,X -ray scattering is averaged over millions of nano-objects so the resulting structural information is far more statistically robust than that obtained from TEM image analysis.I nv iew of these advantages,w ep erformed as eries of SAXS experiments. Initially,t he following sample preparation protocol was adopted:1 0% w/w copolymer dispersions were equilibrated at the desired temperature for 24 hand then diluted ten-fold with deionized water equilibrated at the same temperature. Ther esulting dilute dispersions were then immediately transferred to the pre-equilibrated temperature-controlled Linkam capillary cell and data were collected over 30 min. This series of experiments yielded three characteristic SAXS patterns,s ee Figure 2. There is no structure factor at ac opolymer concentration of 1.0 %w /w,w hich simplifies the data analysis.T he low q gradients for the patterns collected at 4 8 8C, 22 8 8Ca nd 50 8 8Ca re consistent with the presence of spheres,w orms and vesicles,r espectively. [21] Closer inspection suggests the presence of as ignificant proportion of dissolved copolymer chains ( % 30 vol%) in addition to spheres at 4 8 8C. Similar findings have been reported for PHPMA-based diblock copolymers at 3-5 8 8C by Kocik and co-workers. [22] Data fits to these SAXS patterns provided the mean sphere radius,worm cross-sectional radius, overall vesicle radius and vesicle membrane thickness (see Table S1). Importantly,t hese SAXS data fits indicated that the volume fraction of solvent within the weakly hydrophobic PHPMA block increases on cooling from 50 8 8Ct o48 8C.
Although fitting the SAXS data recorded at 22 8 8C provides am ean worm length, it is emphasized that our laboratory SAXS instrument does not provide access to sufficiently low q to enable accurate determination of this parameter.I na ddition, SAXS studies were conducted on 10 %w /w aqueous copolymer dispersions.P rominent structure factors were observed at this higher concentration for each morphology (data not shown), which complicates detailed analysis.N evertheless,t hermal cycling experiments (e. g. 22 8 8Ct o4 8 8Ct o2 2 8 8Ca nd 22 8 8Ct o5 0 8 8Ct o2 2 8 8C) confirmed the thermoreversible nature of these morphological transitions because the initial and final SAXS patterns were remarkably similar at 10 %w/w (see Figure S4).
During PISA syntheses,s pheres can be efficiently converted into worms within tens of minutes at elevated temperature. [16,24] However,t his is because monomer swelling confers relatively high copolymer chain mobility under such conditions.I nc ontrast, the morphological transitions observed in the present study occur in the absence of any unreacted monomer and are instead facilitated by the variable degree of hydration of the weakly hydrophobic PHPMA block. This interpretation is consistent with the relatively high volume fraction of water associated with this block indicated by the x sol values obtained from SAXS data fits.I np rinciple,v ariable temperature 1 HNMR studies can provide further evidence for the hydrated nature of the structure-directing PHPMA chains.
Accordingly,a2.0 %w /w aqueous dispersion of PHPMAC 41 -PHPMA 180 diblock copolymer was freeze-dried overnight and then redispersed in cold D 2 O( initially at 4 8 8C, following the protocol reported by Kocik et al. [22] )b efore warming up to ambient temperature.This dispersion was then used for 1 HNMR spectroscopy studies at 50 8 8C, 22 8 8Cand 4 8 8C (with 24 hb eing allowed at each temperature to achieve the preferred equilibrium morphology). Unfortunately,t he very similar chemical structures of the PHPMACa nd PHPMA blocks means that there is just one unique signal for the latter block. This corresponds to the two oxymethylene protons attached to the methacrylic ester at % 4.1 ppm, which can be distinguished (but not fully resolved) from the two azamethylene protons of the PHPMACs tabilizer block at % 3.9 ppm (see Figure S5). Ther elatively weak nature of the former signal indicates that the hydrophobic PHPMA block is only partially solvated at either 22 8 8Cor508 8C. However, this feature becomes more discernible at 4 8 8C, which is consistent with the variable temperature 1 HNMR studies reported by Blanazs et al. for ac losely-related PHPMA-based diblock copolymer. [9a] This greater degree of hydration at lower temperature is consistent with the observed change in morphology for the thermoresponsive PHPMAC 41 -PHPMA 180 diblock copolymer indicated in Figures 1a nd 2 and has been rationalized in terms of as urface plasticization effect. [5b] An as-synthesized 10 %w /w aqueous dispersion of the PHPMAC 41 -PHPMA 180 diblock copolymer vesicles was prepared at 50 8 8Cp rior to being subjected to temperaturedependent rheological studies.T he rheometer was preheated to 50 8 8Ca nd loaded with this low-viscosity dispersion, which was then slowly cooled at arate of 0.5 8 8Ch À1 (see Figure 3). A local maximum in viscosity was observed at around 14 8 8C, which indicated the formation of weakly interacting worms.  [19] and amodel for dissolved copolymer chains [23] for the SAXS pattern recorded at 4 8 8C, (ii)aworm-likem icelle model [19] for that recorded at 22 8 8Cand (iii)avesicle model [20] for that recorded at 50 8 8C. [N.B. Forc larity,the black and red curves are offset by arbitrary factors of 10 2 and 10 4 , respectively].
Cooling further to 2 8 8Cl ed to the formation of low-viscosity, non-interacting spheres.
However,heating this cold dispersion of spheres revealed significant hysteresis (data not shown). This is because the dissociation of vesicles to form first worms and then spheres occurs on ar elatively short time scale,b ut the reverse pathway is highly cooperative (e.g.worms are formed via the stochastic 1D fusion of multiple spheres [25] )and hence subject to relatively slow kinetics.E quilibrium copolymer morphologies can be eventually achieved on heating, but this requires relatively long time scales (typically many hours to days, depending on the copolymer concentration). Based on recent work by Warren and co-workers, [26] we anticipate that the viscosity maximum observed in Figure 3should be tunable by systematically varying the mean DP of the PHPMA block. Indeed, this hypothesis is supported by tube inversion studies of as eries of five PHPMAC 41 -PHPMA x diblock copolymers where x varies from 140 to 220 (see Figure S6). Theoretical calculations for the effect of varying this parameter from 50 to 300 are also shown in the Supporting Information (see Figure S7). However, further rheological experiments to verify these predictions are beyond the scope of the current study.
In order to understand the underlying mechanism that drives these morphological transitions,w ep erformed calculations of the equilibrium properties of these self-assembled structures using numerical lattice computations based on the self-consistent field (SCF) theory developed by Scheutjens and Fleer. [15] This approach is based on Flory-Huggins lattice theory [27] and small-system thermodynamics. [28] It utilizes at heoretical mean field model, where the block copolymers and the solvent molecules are distributed over at hreedimensional lattice,w hile accounting for concentration gra-dients in one direction. Given as pecific copolymer composition, the change in the configurational entropy upon mixing polymer and solvent is calculated via astep-weighted random walk while the enthalpy of mixing is modeled via aset of pair interaction parameters,also known as c parameters. [15b, 27] This approach enables various thermodynamic properties to be calculated, including the preferred copolymer morphology, the mean aggregation number for the self-assembled copolymer chains (g), and the surface area occupied by each copolymer chain at the core/corona interface (s), as well as concentration profiles for all components inside and outside the self-assembled structure.
Fort he present work, three c parameters must be considered: c HPMA-W , c HPMAC-HPMA and c HPMAC-W .B oth c HPMAC-HPMA and c HPMAC-W are held constant:the former parameter is taken to be unity to ensure inter-block segregation and, according to the literature,the latter parameter has anumerical value of 0.48. [29] This condition is necessary because,ifthe blocks were not segregated, SCF modeling predicts macroscopic phase separation rather than colloidally-stable diblock copolymer nano-objects. c HPMA-W has been estimated to be 0.83 at room temperature utilizing the method proposed by Lindvig et al. [30] This approach uses the Hansen solubility parameter and is consistent with experimental values obtained for similar molecules. [31] To simulate temperature variation, the c parameter of the thermoresponsive block (c HPMA-W )i sv aried between 0.50 and 1.50 with as tep size of 0.02. This interval is considered most relevant for morphology transitions because for c HPMA-W < 0.50 the copolymer is expected to be fully soluble,while the copolymer morphology is expected to be kinetically frozen for such ar elatively long hydrophobic PHPMA block if c HPMA-W > 1.50. Fors uch theoretical calculations,t he diblock copolymer chains are assumed to be perfectly uniform in chain length. Recently, Ianiro et al. reported that ar easonably narrow chain length distribution has an egligible effect on their self-assembly in solution. [32] Thee quilibrium morphology is determined at each step by performing the calculations for lattices with different geometries (spherical, cylindrical and flat). The preferred copolymer morphology corresponds to the geometry with the lowest critical micellization concentration (CMC), since the Gibbs free energy of micellization [33] can be approximately expressed as: According to our SCF calculations (see Figure 4), the PHPMAC 41 -PHPMA 180 copolymer preferentially assumes as pherical morphology if 0.70 < c HPMA-W < 0.78, while cylindrical (or worm-like) micelles are the thermodynamically preferred state for the 0.78 < c HPMA-W < 0.88 interval. For c HPMA-W > 0.88, SCF theory predicts vesicle formation.
Although more precise knowledge of the interaction parameters and their temperature dependence is required for a quantitative comparison between our model and the experimental data, these results qualitatively describe the morphological transitions that are observed experimentally.
Theinterfacial tension (g)atthe core-corona interface of the diblock copolymer micelles may be estimated from the interaction parameter between the solvent and the solvophobic block c HPMA-W .I tf ollows from theory [34] that g % (c HPMA-W ) 1/2 .I nt urn, the solvophobic block-water interaction c HPMA-W increases with temperature,which is consistent with the 1 HNMR spectra shown in Figure S5. Hence,a tl ow temperatures (4 8 8C), g is small and the copolymer morphologies are characterized by ah igh equilibrium value for the interfacial surface area (s)[see Eq. (S2)-(S4) in the Supporting Information].T his results in much lower steric repulsion between the chains in the core-forming block, which reduces the degree of chain stretching and hence favors as pherical morphology.A th igher temperature,t he increase in g necessitates ar eduction in s (see Figure 4a). This results in expulsion of water molecules from the core (see Figure 4b) and in agradual increase in the end-to-end distance (s PHPMA ) of the core blocks (see Figure 4c). On further increasing s PHPMA ,c hain stretching becomes energetically too unfavorable to maintain the spherical morphology,r esulting in at ransition to form first worms at 22 8 8Ca nd then vesicles at 50 8 8C. These morphology transitions enable ar eduction of s PHPMA (see Figure 4c)a nd hence ar eduction in the overall free energy (DG)o ft he system. Thec opolymer packing parameter (P)c alculated according to Equation S8 (see Supporting Information) is plotted in Figure 4d. [35] The fractional values obtained for spheres,w orms and vesicles are consistent with the literature. [35] Conclusion In summary,w er eport an ew thermoresponsive amphiphilic diblock copolymer that can form spheres,w orms or vesicles in aqueous solution simply by varying the solution temperature.T his unprecedented self-assembly behavior is driven by the variable degree of hydration of the core-forming poly(2-hydroxypropyl methacrylate) block, which enables ,c )the average end-to-endd istance of the PHPMA block (s PHPMA )a nd d) the molecular packing parameter P as afunction of c HPMA-W ,ascalculated for PHPMAC 41 -PHPMA 180 .D ashed lines mark the sphere/worm and worm/vesicleb oundaries. two phase boundaries to be crossed within arelatively narrow temperature range.T heoretical analysis of this new diblock copolymer system using self-consistent mean field theory supports our experimental observations.F inally,w ee nvisage that the worm-to-vesicle thermal transition reported herein should provide new opportunities for the convenient loading of nanoparticles,p roteins or enzymes within vesicles.M oreover, the vesicle-to-worm transition that is observed on cooling could provide as uitable (and tunable) release mechanism for such payloads.