Multilamellar Thermoresponsive Emulsions Stabilized with

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Multilamellar Thermoresponsive Emulsions Stabilized with Biocompatible Semicrystalline Block Copolymers Anna Manova,† Jekaterina Viktorova,† Jens Köhler,†,‡ Stefan Theiler,‡ Helmut Keul,†,‡ Alexey A. Piryazev,¶ Dimitri A. Ivanov,¶,∥ Larisa Tsarkova,*,§ and Martin Möller†,‡ †

DWI − Leibniz-Institut für Interaktive Materialien, 52056 Aachen, Germany Institut für Technische und Makromolekulare Chemie and §Institut für Physikalische Chemie, RWTH Aachen University, 52056 Aachen, Germany ¶ Faculty of Fundamental Physical and Chemical Engineering, Moscow State University, Moscow, Russia ∥ Institut de Sciences des Matériaux de Mulhouse, (IS2M), CNRS UMR 7361, F-68057 Mulhouse, France ‡

S Supporting Information *

ABSTRACT: We demonstrate specific interface-templated crystallization behavior of biocompatible amphiphilic poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) block copolymers enabling triggered shaping of the curvature of the oil/water interface and controlled phase inversion, including the formation of stable multiple emulsions. Water-born anisotropic micelles of PEO-b-PCL block copolymers selfassemble at the oil−water interface in a multilayer form and undergo conformational rearrangements into unique semicrystalline multilamellar shells, for which curvature (type of emulsion) can be tuned by the molecular architecture (volume fractions of the blocks) and/or by the temperature. The latter trigger affects both the solubility of the PEO block in water and the semicrystalline state of the PCL block. Remarkably, multilamellar semicrystalline shells provide both long-term stability and enhanced barrier properties of toluene−water emulsions, as well as the fast change of the bending, leading to thermo-induced phase inversion. These findings signify the development of novel practical mechanisms for controlled triggered encapsulation and release systems.

E

cosmetic industries,15−17 a detailed interfacial stabilization mechanism has not been evaluated yet. Water-born micellar PEO-b-PCL emulsifiers can be viewed as solvent-soluble PEO corona grafted on both sides of the PCL lamella crystals (Figure 1a).18 This shape anisotropy of the micelles leads to their kinetic instability in a form of secondary aggregation and polymorphism in water solutions.19,20 At the same time, interfacial adsorption of amphiphilic polymeric micelles at the liquid−liquid interface proceeds via a Pickering mechanism, assuring effective stabilization of emulsions due to the huge contribution of the adsorption energy of the particles.21 However, PEO-b-PCL micelles at the oil−water interface cannot be considered invariable and are anticipated to undergo conformational transformations and interface-templated lateral cocrystallization of the PCL cores (Figure 1a). Here we demonstrate a unique self-assembly behavior of PEO-b-PCL micellar building blocks into semicrystalline multilamellar structure templated by the toluene/water inter-

ngineering liquid−liquid interfaces with polymer molecules toward multicompartment micro- and nanoparticulate systems is of significant research interest which is dictated by practical needs in diverse fields ranging from templates for spatially confined chemical conversions,1,2 encapsulation,3 and triggered release.4 An example of triggered emulsification behavior without high (mechanical) energy input, which finds practical use on industrial scales, is the preparation of concentrated, fine, and monodisperse emulsions stabilized with nonionic ethoxylated emulsifiers using a phase inversion temperature method.5,6 Another application example concerns functional multilamellar emulsions based on templated interfacial cocrystallization of amphiphilic emulsifiers and synthetic ceramides7 which have shown efficiency in the recovery of the skin barrier function.8 A particular challenge in developing complex functional emulsions focuses on the design of polymeric emulsifiers with a dual-responsive behavior, e.g., pH and temperature sensitivity,4,9−11 as well as with specific interactions of biohybrid macromolecular architectures.12−14 Although poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) micelles have been proposed for designing of biocompatible emulsions for applications in the food and © XXXX American Chemical Society

Received: October 18, 2015 Accepted: January 6, 2016

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Figure 1. (a) Schematic illustration of the interfacial assembly of PEO-b-PCL shape-anisotropic micelles. (b) (I−III) Photos of 1:1 toluene (T)/ water (W) emulsions emulsified at 50 °C with a polymer concentration in water of 0.1 mg/mL: I - PEO113-b-PCL18, II - PEO113-b-PCL40, and III - PEO113-b-PCL79. Sketches illustrate the conformation of the anisotropic micelles at the toluene/water interface. (c) Phase diagram of the type of emulsions stabilized with micelles of PEO-b-PCL as a function of the wt % fraction of the PCL block (Table 1) versus temperature of emulsification at standard agitation conditions: crosses − emulsification failed, filled circles − oil-in-water emulsions, open circles − oil-continuous-phase multiple emulsions; half-filled circles − mixed emulsion containing oil-continuous-phase emulsion with fractions of water-continuous-phase emulsion.

that the differences in the emulsion appearance can be attributed to the effect of the micelle shape, which is in turn defined by the volume composition of the block copolymer (Table 1). Emulsification with PEO113-b-PCL18 micelles failed, as seen from almost complete macrophase separation of toluene and water phases (Figure 1b,I). Slight turbidity of the lower water phase (Tyndall effect), caused by the presence of (solubilized) micelles, indicates relatively high solubility of these micelles in water. Figure 1b,II displays emulsion stabilized with the PEO113-bPCL40 block copolymer of a symmetric composition. It consists of excess water phase and emulsified phase, which is a water-continuous-phase emulsion coexisting with a fraction of oil-continuous-phase emulsion (evident as macroscopic waterin-toluene drops at the border between the water phase and emulsified phase), with a toluene fraction above ∼70% so that a high internal-phase volume fraction emulsion is formed.25 An excess water phase appears immediately after emulsion preparation by creaming, i.e., upward sedimentation of oil droplets with smaller than water density. Emulsion stabilized with the PEO113-b-PCL79 block copolymer (Figure 1b,III) consists of a small fraction of water phase at the bottom, excess toluene phase on top, and a middle emulsion phase with droplets of water comprising 80% of the dispersed fraction in a continuous toluene phase (a fluorescent microscopy image of the emulsion is shown in Figure S2). Results in Figure 1b clearly demonstrate the influence of the fraction of the hydrophobic PCL block on the emulsification efficiency with PEO-b-PCL micelles, which can be partially explained along the lines of the Pickering stabilization mechanism. The advantages of using PEO-b-PCL micelles as an alternative to inorganic particles for stabilization of emulsions are associated with their intrinsic amphiphilicity so that there is no need to modify the surface with surfactants as is typically done in the classical Pickering approach. Instead, the hydrophobicity of the micelle surface is controlled via the tethering density of the swollen PEO chains on the hydrophobic PCL crystallite, i.e., by the polymer architecture. Additionally, PEO-b-PCL micelles are intrinsically small (below the 100 nm range, Figure S3) which provides an effective stabilization of the emulsion interface.21 Since the hydrodynamic diameter of PEO-b-PCL micelles is mainly defined by the length of the swollen PEO chains, and shows only weak dependence on the length of the core PCL block

face even at high volume fractions of toluene, a good solvent for PCL. Emulsion droplets with such multilamella shells show unprecedented long-term stability and enhanced barrier properties. The interfacial curvature (type of emulsion) can be tuned by the micellar structure, i.e., by the block copolymer composition, and/or by the emulsification temperature. Importantly, a fast phase transition from water-continuousphase emulsion to oil-continuous-phase emulsion can be thermally triggered, indicating a controlled bending elasticity of the semicrystalline multilamellar interfacial layer,22 signifying the development of novel triggered encapsulation and release systems. To screen the molecular architecture effect, a series of PEOb-PCL block copolymer emulsifiers (Table 1) were synthesized Table 1. PEO-b-PCL Block Copolymers

a

polymer

ratio EO/CL

Mna [g/mol]

Mnb [g/mol]

Mw/Mna

PCL wt %

PEO113-b-PCL18 PEO113-b-PCL40 PEO113-b-PCL79

113/18 113/40 113/79

8900 15700 21200

7055 9680 14000

1.11 1.11 1.18

29 48 64

SECTHF.

b1

H-NMRChloroform.

by ring-opening polymerization of ε-caprolactone using monomethoxy poly(ethylene glycol) as the macroinitiator and a tin-based catalyst.23 The molecular weight of PEO block was fixed by a molecular weight of the macroinitiator of 5000 g/ mol, while the fraction of the hydrophobic PCL block was varied from the minority 29 wt % to the majority 64 wt % fraction. The selected composition and total molecular weights were chosen to compromise between the upper oligomer range dictated by the biocompatibility of PEO, on one side, and the chain length of the PCL block allowing for crystallization. A more detailed screening over the volume fractions and shorter PEO block is reported elsewhere.20 The initial micellar solutions have been prepared by stirring the block copolymer in water20,24 at a temperature above the melting point of the PCL block (Tm ∼ 47−58 °C, according to differential scanning calorimetry (DSC) data in Figure S1). Figure 1b displays photos of one-week-old toluene/water emulsions (toluene phase contains Nile red fluorescent dye) prepared at identical emulsification conditions (not optimized in terms of the input energy and emulsifier concentration), so 164

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Figure 2. (a−f) POM micrographs of oil-in-water (a,b,d) and multiple water-in-oil (c,e,f) emulsions stabilized with PEO113-b-PCL40 and PEO113b-PCL79 micelles (0.1 mg/mL concentration in water phase) at indicated temperatures. All images show a Maltese cross, characteristic of uniaxial crystal systems. The images illustrate more than one year-aged emulsion. The scale bar is 100 μm.

cooling, indicating possible trapping of metastable conformational states. We note that we used 50 times lower concentration of the polymeric emulsifier (0,1 mg/mL) as compared to what is typically reported for emulsions stabilized with amphiphilic polymers,25,26 and still the emulsions remained practically unchanged over the time periods of more than a year (Figure 2), confirming the existence of a robust physical barrier both against coalescence and against isothermal mass transfer (Ostwald ripening). The type of emulsions was identified by optical and fluorescence microscopy. At utilized emulsification conditions, stable toluene/water emulsions can not be formed if the PCL fraction is below 30 wt %, independent of the temperature of emulsification. For other molecular compositions, the water-continuous phase and mixed emulsions are stabilized at room temperature, while higher PCL fraction and higher emulsification temperatures above TmPCL promote the formation of toluene-continuous-phase emulsions (Figure S2), as well as of mixed and multiple emulsions, suggesting the ability of PEO113-b-PCL40 and PEO113-bPCL79 copolymers to stabilize interfacial curvatures of both types. Closer insights into such bifunctional interfacial behavior of PEO-b-PCL micelles were derived from the analysis of aged emulsions with polarized optical microscopy (POM). A striking observation from POM images in Figure 2a−f is the presence of a so-called “Maltese cross” interference figure which is a characteristic appearance of uniaxial crystal systems with two vibration directions.27 Accordingly, the radiant halo with a Maltese cross in POM images of emulsion droplets strongly suggests a liquid crystalline/multilamellar structure of the droplets’ shells. Furthermore, emulsions with a continuous toluene phase reveal a multiple character in that large water droplets contain down to micrometer-size toluene droplets showing a Maltese cross (Figure 2c,e,f). Another observation is the distortion of the original round droplet shapes in toluenecontinuous-phase emulsions (as in Figure S2) into a network of thickened planar adhesion junctions between macroscopically large water droplets (Figure 2c,e,f), leading to the observed

(Figure S3), poor emulsification efficiency of PEO113-b-PCL18 micelles is due to their high hydrophilicity caused by the dense tethering of the corona PEO chains. The low energy gain due to the adsorption energy of the PEO113-b-PCL18 micelles can be compensated by increasing particle concentration and reducing the diffusion-limited adsorption step (Figure S4). As can be seen in Figure S5, using higher concentrations of this polymer allows both increasing the fraction of the emulsified toluene and decreasing the size of the toluene droplets, indicating the importance of the stage of the micelle rearrangement within the interfacial layer in the stabilization mechanism. The ability of the micelles to stabilize negative (oil-in-water) or/and positive interfacial curvature can be explained in analogy with the packing parameter of surfactants or amphiphilic block copolymers (Figure 1b). The suggested model relates the shape anisotropy of adaptive micelles to the macroscopic droplet curvature, assuming that PEO-b-PCL micelles adopt interfacetemplated planar lamellar morphology already during the emulsification procedure. We note that toluene is a good solvent for the PCL block; still the high local concentration of the PCL in the interfacial layer supports its semicrystalline state. Additionally to the demonstrated above effect of the molecular architecture, temperature change turned out to be a powerful tool to alter the structure of the emulsions stabilized by PEO-b-PCL micelles. Temperature variation affects the solubility of the PEO segments in water, similar to the widely exploited thermal switchability of ethoxylated surfactants,5,6 and at the same time alters the semicrystalline/molten state of the PCL block. The phase diagram in Figure 1c summarizes the stability and type of emulsions stabilized with 0.1 mg/mL of PEO-b-PCL block copolymer micelles with the varied fraction of the PCL block observed for indicated temperature of emulsification (at standard emulsification conditions). Once prepared at the indicated temperature, the emulsions have been cooled, stored, and analyzed at room temperature. In all cases the initial visual appearance of the emulsions did not noticeably change upon 165

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ACS Macro Letters high viscosity of toluene-continuous-phase emulsions stabilized with PEO-b-PCL micelles. All these results strongly indicate that PEO-b-PCL micelles reorganize within interfacial layers into semicrystalline shells with multilamella structure. The mechanism of such assembly can be explained by considering multilayer adsorption of amphiphilic particles.28 As depicted in Figure 3b, due to the

Figure 4. Confocal fluorescent micrographs and photos of emulsion stabilized with PEO113-b-PCL79 at room temperature (a,c) and the same emulsion after heat shock (b,d). Scale bar is 100 μm. Sketches of the temperature triggered conformational change of the PEO113-bPCL79 interfacial layer.

Figure 3. (a) Snapshots of the droplets at indicated drop-age; (b) related schematics illustrate the growth of the multilamellar shell by a proposed mechanism of multilamellar structure formation. (c) Lorentz-corrected SAXS curve measured on water-continuous-phase emulsion stabilized with 0.5 wt % PEO113-b-PCL79 micelles dissolved in the toluene phase.

fast phase inversion into emulsion with oil-continuous phase, confirmed both by fluorescent microscopy and by the visual appearance of the inverted emulsion (Figure 4b,d). Interestingly, the inversion retains even after cooling the emulsion back to room temperature so that the transition is not reversible. Schematics in Figure 4 depict the responses of both blocks to the temperature trigger, leading to the spontaneous change of the bending sign of the shell: an increase of the partitioning of the lamella in the adjacent toluene phase due to the melting of the PCL chains and larger swelling of the PEO and shrinking of the water-adjacent PEO corona due to the decrease of its solubility in water. The irreversibility of this transition upon cooling can be attributed to the freezing of the trapped conformational states within the alternating sheets of semicrystalline PCL and amorphous PEO blocks, while the outer lamella sheets satisfy the asymmetric wetting conditions. Such multilamellar organization suggests unique properties for encapsulation and controlled release both of hydrophobic8 and of hydrophilic drugs. Important for potential applications is that the reported here interfacial crystallization and related thermoresponsive properties of biocompatible emulsifiers are valid for other types of oils, e.g., for polydimethylsiloxanes, suitable for pharmaceutical nanoemulsions.31 In particular, Park et al. reported an elegant approach to tune the melting temperature of the emulsion by incorporating a cosurfactant in the water phase.32 In summary, we have demonstrated the interface-templated crystallization of amphiphilic micelles of biocompatible PEO-bPCL block copolymers in the presence of high volume fractions of toluene, a good solvent for the PCL block. This crystallization behavior allows multiple triggers in shaping the curvature of the oil/water interface toward formation of stable multiple emulsions and controlled phase inversion. The interfacial curvature (type of emulsion) can be tuned by the volume fraction of the hydrophobic block and by the temperature trigger, so that a fast phase inversion between water-continues-phase emulsion and water-in-toluene emulsion is induced by thermal shock. We proposed a model which relates the macroscopic droplet curvature to the mean

fixed tethering density of the PEO chains, part of the hydrophobic PCL block is exposed to water, promoting adsorption of the next layer of micelles. Finally strong van der Waals interactions between lamella crystallites29 lead to their cocrystallization and formation of multilamellar structure with alternating sheets of crystalline PCL block and amorphous PEO block (sketches in Figure 4). To evaluate the time scale of the PEO-b-PCL rearrangement at the toluene−water interface we performed time-resolved pendant drop measurements of the surface tension (Figure 3 and Figure S6), focusing on the kinetics of the shell growth around the water droplet immersed in the toluene phase with an excess of PEO113-b-PCL79 micelles.30 Transparent skin of macroscopic size was detected within 15 min of the drop aging time (Figure 3a), accompanied by the poor fitting of the droplet shape (Figure S6). These observations suggest that submicrometer thickness of the PEO-b-PCL shell is achieved during the first minutes of the drop age, providing enhanced shell stability toward coalescence. A direct confirmation of the suggested multilamellar interfacial organization was evaluated from the small-angle Xray scattering (SAXS) measurements of water-continuousphase emulsions stabilized with 0.5 wt % of PEO113-b-PCL79. The scattering pattern is presented in Figure 3c and reveals a relatively broad and weak interference peak at a distance of approximately 25 nm, well in agreement with a thickness of a dried lamella layer of 7 nm measured by scanning force microscopy on self-assembled lamella structures on the mica surface (Figure S7). The interference also reveals an even weaker second-order reflection. The observed interference maximum is likely to correspond to the interlamellar spacing of swollen lamella. Another remarkable property of the studied emulsions is the temperature-triggered phase transition which is depicted in Figure 4. Fluorescence microscopy and photoimages in Figure 4a and c, respectively, show water-continuous phase emulsion stabilized with PEO113-b-PCL79, originally prepared at RT (Figure 1c). Mild stirring and heating to 70 °C results in the 166

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and Science of the Russian Federation (contract No. 14.604.21.0079 (RFMEFI60414X0079, 30.06.14)).

spontaneous curvature of semicrystalline building blocks within the in-plane lamella sheets and considered conformational changes of PEO and PCL blocks in response to the triggers. The long-term stability of the emulsion droplets is attributed to the multilamella structure of alternating semicrystalline PCL and amorphous PEO block copolymers in the interfacial layer.

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EXPERIMENTAL SECTION

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ASSOCIATED CONTENT

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Micellar solutions of block copolymers have been prepared by dissolving the polymer in Milli-Q water under vigorous stirring with a magnetic stirrer at 70 °C for 20 min. The quality of dispersion and aging of micelles have been controlled via dynamic light-scattering measurements.20 For emulsification, equal volumes of toluene, containing 1 μmol/L of Nile Red, and of Milli-Q water, containing 0.1 mg/mL (unless otherwise stated) of a PEO-b-PCL, were pre-equilibrated at the emulsification temperature (RT, 50 or 70 °C), mixed, and agitated with a digital Ultraturrax (T25 Ika) at 6000 rpm for 5 min. After preparation, emulsions were cooled slowly to RT and stored in tightly closed flasks. Steady-state optical examination of emulsions has been done at RT with the bright-field method in transmitted light and with the polarization contrast method in incident light (Carl Zeiss Axioplan-2 with AxioCam MRc digital camera). The sample chamber was assembled from a 1 × 1 cm2 silicon wafer substrate and cover glass slide sealed with commercial grease adhesive. Keyence Biozero BZ-8000E with TexasRed standard filter (excitation: 560/40 nm) and confocal laser scanning microscope Leica TCS SP8 were used for visualization of the “Nile Red” fluorescent dye. Tensiometric measurements at the toluene/water interface have been done with the pendant drop method using Krüss Drop Shape Analyzer − DSA100. Small-angle X-ray scattering (SAXS) measurements were performed on a Xenocs WAXS/SAXS machine equipped with a beam generator Genix3D (λ = 1.54 Å) with a beam size of 300 × 300 μm2. Further details can be found in the Supporting Information.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00743. Figures S1−S7: DSC heating curves of PEO-b-PCL block copolymers, fluorescence microscopy, and optical microscopy images of emulsions, hydrodynamic diameters of micelles, interfacial tension measurements, and scanning force microscopy of self-assembled PEO-b-PCL lamella (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was financially supported by BMBF (Grant number 03X2515C). We thank A. Kühne for assistance with the confocal fluorescent microscopy imaging and W. Sager for helpful discussions. DI and AP thank the Ministry of Education 167

DOI: 10.1021/acsmacrolett.5b00743 ACS Macro Lett. 2016, 5, 163−167