Size Control and Fractionation of Ionic Liquid Filled Polymersomes

May 9, 2016 - Size Control and Fractionation of Ionic Liquid Filled Polymersomes with Glassy and Rubbery Bilayer Membranes. Soonyong So† and Timothy...
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Size Control and Fractionation of Ionic Liquid Filled Polymersomes with Glassy and Rubbery Bilayer Membranes Soonyong So† and Timothy P. Lodge*,†,‡ †

Department of Chemical Engineering & Materials Science and ‡Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: We demonstrate control over the size of ionic liquid (IL) filled polymeric vesicles (polymersomes) by three distinct methods: mechanical extrusion, cosolvent-based processing in an IL, and fractionation of polymersomes in a biphasic system of IL and water. For the representative ionic liquid (1ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide ([EMIM][TFSI])), the size and dispersity of polymersomes formed from 1,2-polybutadiene-b-poly(ethylene oxide) (PB− PEO) and polystyrene-b-poly(ethylene oxide) (PS−PEO) diblock copolymers were shown to be sensitive to assembly conditions. During mechanical extrusion through a polycarbonate membrane, the relatively larger polymersomes were broken up and reorganized into vesicles with mean size comparable to the membrane pore (100 nm radius); the distribution width also decreased significantly after only a few passes. Other routes were studied using the solvent-switch or cosolvent (CS) method, whereby the initial content of the cosolvent and the PEO block length of PS−PEO were systemically changed. The nonvolatility of the ionic liquid directly led to the desired concentration of polymersomes in the ionic liquid using a single step, without the dialysis conventionally used in aqueous systems, and the mean vesicle size depended on the amount of cosolvent employed. Finally, selective phase transfer of PS−PEO polymersomes based on size was used to extract larger polymersomes from the IL to the aqueous phase via interfacial tension controlled phase transfer. The interfacial tension between the PS membrane and the aqueous phase was varied with the concentration of sodium chloride (NaCl) in the aqueous phase; then the larger polymersomes were selectively separated to the aqueous phase due to differences in shielding of the hydrophobic core (PS) coverage by the hydrophilic corona brush (PEO). This novel fractionation is a simple separation process without any special apparatus and can help to prepare monodisperse polymersomes and also separate unwanted morphologies (in this case, worm-like micelles).

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INTRODUCTION

Various strategies have been utilized to prepare narrowly dispersed polymersomes with controlled size in aqueous suspensions.22 In mechanical approaches, such as extrusion, large polymersomes are ruptured and transformed into smaller polymersomes when forced through a membrane with wellcontrolled pore size. The extrusion method is widely used because it is a relatively rapid way to produce nearly monodisperse polymersomes without the introduction of contaminants.23−25 Other methods, such as inkjet printing, microfluidics, and nanoprecipitation involve a cosolvent, which can dissolve both vesicle forming polymers and a selective solvent.26−28 The basic mechanism for polymersome formation is the same as the solvent-switch or cosolvent (CS) method.29 Homogeneously dispersed droplets are produced in the selective solvent by the listed methods. Then, as the cosolvent in the droplets is removed, amphiphilic block copolymers selfassemble into polymersomes under quasi-equilibrium conditions, and narrowly sized polymersomes can be produced.

Amphiphilic diblock copolymers in selective solvents have been documented to self-assemble into more than 20 morphologies, including spherical micelles, worm-like micelles, vesicles, and large compound micelles.1−6 Multimorphological self-assembly of polymers in water, organic solvents, and ionic liquids (ILs) offers considerable potential to adapt copolymers for a range of applications, e.g., in biotechnology, catalysis, and separations.7−14 In many applications, however, not only the type of morphology but also the average size and size distribution affects performance. 15−17 Especially, precise control of polymersome diameter is desirable. For instance, in biological applications, the size distribution of vesicles is important for the efficiency of delivery because the circulation time of vesicles and the release rate of cargo are both size-dependent.18,19 In nanoreactor applications, a narrowly distributed, small vesicle size is required, both to render the system simpler in terms of analysis and characterization and for achieving higher reaction rates and better catalytic performance through higher collision frequencies among the reactants, catalysts, and the wall of the vesicle interior.20,21 © XXXX American Chemical Society

Received: March 10, 2016 Revised: April 28, 2016

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DOI: 10.1021/acs.langmuir.6b00946 Langmuir XXXX, XXX, XXX−XXX

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Langmuir Moreover, using separation techniques such as chromatography, extrusion, centrifugation, or field-flow fractionation, the aggregates can be separated based on their size.30−33 Here, two types of diblock copolymers, one possessing a rubbery and one a glassy membrane-forming block, were employed for polymersome preparation, and we controlled the size and dispersity of polymersomes in the IL. Two methods mechanical extrusion and cosolvent evaporationwere used as environmentally friendly and facile approaches for fabricating nanosized polymersomes of 1,2-polybutadiene-b-poly(ethylene oxide) (PB−PEO) and polystyrene-b-poly(ethylene oxide) (PS−PEO), exploiting the nonvolatility and high thermal stability of the IL. Furthermore, the number of extrusion passes in the mechanical approach and the content of cosolvent and hydrophilic block length in the cosolvent protocol were varied, and the concomitant effects on the size and distribution of polymersomes were studied. Additionally, using the interfacial tension-hindered phase transfer behavior, we demonstrate a third way to select one size of PS−PEO polymersomes from a broadly dispersed polymersome suspension. In a previous study, we explored the phase transfer behavior of various PS−PEO polymersomes between water and the common IL 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide ([EMIM][TFSI]).34 The phase transfer was shown to depend on a competition between the packing density of the PEO corona and the free energy of the outer interface of the PS membrane. Here, the interfacial tension between PS and the aqueous phase was varied with added salt in the aqueous phase, by which larger polymersomes were selectively separated to the aqueous phase.

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Table 1. Molecular Characterization of PB−PEO and PS− PEO Diblock Copolymers sample code

Mna (kg/mol)

MPEOb (kg/mol)

Đc

f PEOd

BO(9−2.5)e SO(10−3) SO(14−2.5) SO(18−2.5) SO(18−3) SO(18−3.6)

9 9.8 14.1 18.4 18.4 18.4

2.5 2.9 2.6 2.5 2.9 3.6

1.07 1.06 1.04 1.01 1.03 1.01

0.17 0.22 0.14 0.11 0.13 0.15

Number-average molecular weight of the ω-hydroxylpolybutadiene (PB-OH) and polystyrene (PS-OH) homopolymers determined by 1H NMR spectroscopy. bNumber-average molecular weight of PEO block from 1H NMR spectroscopy and the Mn of PB-OH or PS-OH. c Dispersity (Mw/Mn) of PB−PEO and PS−PEO determined by SEC (Agilent Technologies 1260 Infinity) equipped with a multiangle light scattering detector (Wyatt Technology Dawn Heleos-II), where Mw is weight-average molecular weight. dVolume fraction of PEO block from Mn of each block and the bulk densities: ρPB = 0.87 g/cm3, ρPS = 1.05 g/cm3, ρPEO = 1.13 g/cm3. eThe fraction of 1,2-addition of PB-OH was 91%. a

stirred vigorously (∼1000 rpm) at 150 °C under an argon atmosphere overnight, and the suspension was slowly cooled down to room temperature. Dilute polymersome suspensions (0.1 wt % of BO(9− 2.5) and SO(14−2.5)) from the TF method were extruded and pressed 1−25 times (Figure S1a) by using an extrusion device (LiposoFast-Basic Extruder, Avestin) through polycarbonate membranes with defined pore sizes of 200 nm diameter. For SO(14−2.5) polymersomes, 10 vol % of DCM was added as plasticizer for the glassy polystyrene membrane. In the other approaches, the solvent-switch or cosolvent (CS) method was used. The weighed polymers were dissolved in DCM (0.57 wt % polymers in 1 mL of DCM) followed by adding 1 mL of [EMIM][TFSI] to the solution at once. The order of mixing was chosen to produce narrowly dispersed polymersomes based on a previous study.28 The volatile DCM was selectively removed by N2 purging with 700 rpm stirring and drying under vacuum for a day at room temperature. It is known that the homogeneous conditions of the CS method favors the formation of relatively monodisperse polymersomes compared to the TF method.28,38 However, even with the CS method, the size and dispersity depend on the CS content and the relative length of the hydrophilic block. To study the effect of CS content, the initial content of DCM was varied from 17 to 60 vol % for SO(14−2.5) and from 30 to 60 vol % for BO(9−2.5). Three PS−PEO block copolymers with a common PS block length (Mn = 18 kg/mol), but different lengths of the PEO block (Mn = 2.5, 3.0, and 3.6 kg/mol), were used to study the effect of PEO block length on the polymersome size and distribution. The polymersomes from SO(18−2.5), SO(18− 3), and SO(18−3.6) were prepared through the CS protocol with an initial DCM content of 60 vol %. Phase Transfer of PS−PEO Polymersomes at Different NaCl Concentration. A suspension of PS−PEO was prepared by a CS protocol. Weighed polymers were dissolved in DCM followed by addition of [EMIM][TFSI] and subjected to vacuum to yield 0.5 wt % in the IL after CS removal. The initial DCM content was 50 vol %. Phase transfer of PS−PEO polymersomes was conducted by adding an equal volume of water or NaCl solution to an [EMIM][TFSI] suspension of PS−PEO. NaCl has poor solubility in [EMIM][TFSI]39,40 but is highly soluble in water. The NaCl concentration in water was varied from 0 to 0.2 M to control the surface tension of the aqueous phase. It is well-known that the surface tension (γ) of an aqueous solution increases linearly with the concentration of NaCl,41 and the solvent quality for PEO becomes poorer; for example, the LCST of PEO in water decreases in salted water.42 Two immiscible phases were contacted for at least 24 h with gentle stirring (∼100 rpm). Light Scattering. The mean size and dispersity of the polymersomes were analyzed by dynamic light scattering (DLS) with a

EXPERIMENTAL SECTION

Materials. PB−PEO and PS−PEO block copolymers were prepared via sequential anionic polymerization.35,36 Hydroxyl-terminated 1,2-polybutadiene (PB-OH) and polystyrene (PS-OH) homopolymers were synthesized in anhydrous tetrahydrofuran (THF) and cyclohexane, respectively, with sec-butyllithium (1.4 M in cyclohexane, Aldrich) as initiator. The PEO block was grown from PB-OH and PSOH in the same manner. Homopolymers were dissolved in anhydrous THF and converted to macroinitiators using electron transfer agents (diphenylmethylpotassium or potassium naphthalenide). Then, purified ethylene oxide monomer was added and reacted for 24 h at 45 °C. The polymer chains were terminated with concentrated deoxidized methanol at room temperature. The number-average molecular weight (Mn) was obtained by 1H NMR spectroscopy, and the dispersity (Đ = Mw/Mn) was measured by size exclusion chromatography (SEC) using THF as solvent, where Mw is the weight-average molecular weight. Mn of each block was used for the sample code as BO(Mn,PB−Mn,PEO) for PB−PEO and SO(Mn,PS− Mn,PEO) for PS−PEO. Polymers used in this study are listed in Table 1. The ionic liquid, [EMIM][TFSI], was prepared by combining 1-ethyl3-methylimidazolium bromide ([EMIM][Br], Io-Li-Tec, 99%) and lithium bis(trifluoromethylsulfonyl)imide ([Li][TFSI], HQ115, 3M) in water.37 An equimolar mixture of [EMIM][Br] and [Li][TFSI] was allowed to react at 70 °C for a day. The separation of the waterimmiscible product was followed by washing with deionized water at least three times. The [EMIM][TFSI] was purified via neutral alumina (Al2O3) column and stored in a desiccator before use. The cosolvent, dichloromethane (Stabilized/Certified ACS) (DCM), was purchased from Fischer Scientific and used as received. Size-Controlled Polymersome Preparation. For the mechanical approach, thin films of polymers (BO(9−2.5) and SO(14−2.5)) were prepared by drying the polymer solution in DCM under N2 purge and further dried under vacuum at 50 °C for 24 h to remove the remaining DCM. In order to produce an 0.5 wt % polymersome suspension, weighed [EMIM][TFSI] was added to the thin film and B

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Langmuir Brookhaven BI-200SM goniometer, a Brookhaven BI-9000AT correlator, and a polarized He−Ne laser (λ = 637 nm). Measurements were performed at different angles between 60° and 120° at λ = 637 nm and 25 °C. The average hydrodynamic radius (⟨Rh⟩) and the dispersity (μ2/Γ2) were evaluated from the average decay rate (Γ = Dmq2, where Dm is a mutual diffusion coefficient and q is the scattering vector) and the second cumulant (μ2) values, which were obtained from cumulant fitting of the normalized autocorrelation function, g(2)(q,t).43 The size distribution of the polymersomes was also obtained using a Laplace inversion program, GENDIST for the REPES (Regularized Positive Exponential Sum) algorithm.44 All samples for DLS were diluted to 0.01 wt % and filtered through a syringe filter (Millex-SV 5.0 μm) prior to measurement. For the scattering intensity experiment after the phase transfer, the aqueous phase was separated and diluted over 150-fold and filtered through a syringe filter. The measurements were conducted using the same system for DLS. Cryogenic Transmission Electron Microscopy (Cryo-TEM). The morphology and the size of PB−PEO and PS−PEO polymersomes were directly investigated with cryo-TEM. The samples were prepared in a FEI Vitrobot Mark III vitrification robot with 0.3 μL of suspension (0.1 or 0.5 wt %). Samples placed on a carbon-coated and lacey film-supported copper TEM grid were blotted once for 1.5 s with −1 mm of blot offset and 3 s drainage time. For viscous samples, such as IL suspensions of polymersomes, manual blotting was applied before blotting with the vitrification robot. After blotting, the samples were vitrified in liquid ethane (∼−180 °C) and then quickly moved and stored in liquid N2. The images were taken using a FEI Tecnai G2 Spirit BioTWIN, operating at 120 kV under liquid N2 cryogenic conditions with an Eagle 4 megapixel CCD camera. The size of the assemblies was analyzed using ImageJ software.

observed for PS−PEO polymersomes. In the bright-field cryo-TEM images, the probability of scattering is a primary source for the intensity (brightness) difference.51 The intensity increases as the probability of scattering by the specimen, which is proportional to the electron density and the thickness of species, decreases. Since the PB and PS membranes scatter fewer electrons than the IL, the membranes appear brighter than the interior and exterior of the polymersomes. In the cryoTEM images of BO(9−2.5) (Figure 1a), the polymersomes are polydisperse, with the size ranging from about 50 to 400 nm in diameter. Most of the vesicles observed are unilamellar vesicles consisting of a single closed bilayer, but a few multilamellar vesicles having an indefinite number of concentric bilayers are also present. The broad size distribution is also reflected in the DLS results. Cumulant fitting shows that μ2/Γ2 ≈ 0.41 with ⟨Rh⟩ ≈ 172 nm. The relatively large value of μ2/Γ2 indicates a broad distribution of polymersome sizes (when the particles are monodisperse, μ2/Γ2 = 0) as shown in Figure S1b. Extrusion through the membrane was conducted with a BO(9−2.5) suspension from the TF method. An extrusion device was employed to extrude the suspension through polycarbonate membranes with defined pore sizes of 100 nm in radius. This process was repeated up to 25 times to determine the optimum number of passes. As polymersome suspensions were driven through the polycarbonate membranes, the relatively large polymersomes were rapidly broken up into smaller ones, and the width of the size distribution also decreases with subsequent passes. The nanostructure of BO(9− 2.5) is retained even after 25 passes, and the sizes of all polymersomes are comparable to the radius of the membrane pores (100 nm) (see Figure 1b). Figure 2 summarizes the variation in the variation in ⟨Rh⟩ and μ2/Γ2 with the number of passes through the membrane. At the first pass, ⟨Rh⟩ and μ2/Γ2 decrease significantly from 172 to 113 nm and from 0.41 to 0.11, respectively, and then level off to roughly constant values (⟨Rh⟩ ∼ 99 nm, μ2/Γ2 ∼ 0.1) after three or more passes through the extruder. The substantially higher viscosity of [EMIM][TFSI] compared to water does not make this process easy, but neither does it preclude manual extrusion. Whereas the BO(9−2.5) polymersomes with rubbery PB bilayer membranes did pass through the extruder under manually applied pressure at room temperature, it was not possible to extrude SO(14−2.5) polymersomes with glassy hydrophobic segments directly. The estimated glass transition temperature (Tg) of 14 kg/mol PS homopolymers is around 90 °C.52 The fluidity of the membrane is an important factor to consider during extrusion, since the large polymersomes need to be ruptured when they are extruded through the pores.53 The fluidity of the membrane is governed by the molecular weight and the glass transition temperature (Tg) of the membrane forming block.54,55 Since Tg of the PS membrane is higher than room temperature, the minimum required force to overcome the line tension of the polymersomes might be much higher than the BO(9−2.5) counterpart. When a cloudy SO(14−2.5) suspension was driven through the extruder membrane, the extrudate turned optically clear, indicating loss of polymer material. However, when 10 vol % of DCM was added to the SO(14−2.5) suspension as a plasticizer for the PS bilayer membrane, the suspension remained cloudy even after several passes, and the scattering intensity of the extrudate actually increased compared to the pristine solution (see Figure S2). The scattering result indicates that the plasticization decreases the Tg of the PS membrane, and consequently the

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RESULTS AND DISCUSSION Size Control via Mechanical Approach. It has been demonstrated that amphiphilic block copolymers, such as polystyrene-b-poly(methyl methacrylate), poly(N-isopropylacrylamide)-b-poly(ethylene oxide), PB−PEO, and PS−PEO can self-assemble into micelles or vesicles in ILs.45−47 Especially, diblock copolymers with low volume fractions of the solvophilic block preferentially form vesicles in order to minimize the interfacial area between the membrane-forming block and the selective solvent.48 The polymers in this study have low volume fractions of PEO (≤0.2, except for SO(10− 3)), and all self-assemble into vesicles in [EMIM][TFSI]. The self-assembled nanostructure of BO(9−2.5) in [EMIM][TFSI] from the TF method is shown in Figure 1a. The vesicular structure was observed with bright circular regions, which represent the PB membranes in the dark IL matrix (phase contrast of BO(9−2.5) polymersomes also can be seen in Figure 1b). The phase contrast can be attributed to the electron density difference between the PB membrane and [EMIM][TFSI].7,49,50 A similar phase contrast was also

Figure 1. Cryo-TEM image of BO(9−2.5) polymersomes in [EMIM][TFSI] (a) from the TF method and (b) after 25 passes (0.1 wt %) through the membrane with pore radius of 100 nm. C

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Figure 2. (a) Evolution of hydrodynamic radius (Rh) and (b) size distribution (μ2/Γ2) of BO(9−2.5) with the number of passes through the 100 nm radius pore, which were measured by DLS at 90°.

Figure 3. Hydrodynamic radius (Rh) distribution of (a) polystyrene-b-poly(ethylene oxide) (SO(14−2.5)) and (b) 1,2-polybutadiene-bpoly(ethylene oxide) (BO(9−2.5)) vesicles at different cosolvent content. Rh increases as the initial ionic liquid content increases.

size of the extruder membrane. Block copolymers having a high Tg block (e.g., PS) cannot form polymersomes through the TF method in conventional solvents, which can make relatively large polymersomes,58,59 because the diffusion process of solvent into the high-Tg polymer film is significantly reduced. In ILs, however, the process temperature can be set well above the Tg of the glassy segments to accelerate the solvent diffusion, since the IL is a stable liquid even at high temperature (the melting temperature of [EMIM][TFSI] is −12 °C, and degradation occurs above 400 °C60). Size Control through Cosolvent Method. Effect of Cosolvent Content. In the TF method, the thin film is swollen by a selective solvent, and polymersomes form and diffuse into the IL.61,62 In contrast, the polymersomes through the CS method are homogeneously and instantaneously formed, when the amount of a selective solvent drops to the point where the solution turns cloudy.63 Therefore, the quality and content of a cosolvent in the suspension can affect the dimension of the formed polymersomes. The samples prepared by the CS method are labeled such that the number after the polymer code is the initial volume fraction of DCM in the suspension. For example, SO(14−2.5)60 means that the polymersomes in [EMIM][TFSI] were prepared by SO(14−2.5) and the initial suspension contained 60 vol % of DCM (and 40 vol % of [EMIM][TFSI]). The final suspension, however, had no DCM, which was removed under vacuum. The critical concentration of [EMIM][TFSI] at which the polymers start to self-assemble was determined by light scattering; the solutions turned cloudy at the point where the DCM content decreased to less than about 70 vol %. [EMIM][TFSI] was added to the polymer solution in DCM to control the initial DCM content from 17 vol % up to 60 vol %. Figure 3 shows the size distribution of each sample and illustrates the effect of cosolvent content on the polymersome

polymersomes are ruptured through the extrusion process rather than filtered out. As with BO, the extrusion of SO(14− 2.5) polymersomes with DCM added also produced narrowly dispersed polymersomes with size comparable to the extruder membrane. The ⟨Rh⟩ decreased from around 300 to 110 nm, and μ2/Γ2 also significantly decreased from 0.53 to 0.13, after nine passes. Interestingly, some of the extruded SO(14−2.5) polymersomes exhibited prolate shapes. Figure S3a shows SO(14−2.5) polymersomes after nine extrusion passes. Many of polymersomes in the images have elliptical shapes, including dumbbells, and the measured aspect ratio ranged from 1.1 to 2.3. The length of the minor axis ranged from ca. 100 to 200 nm. These cryo-TEM results indicate that while the size of the polymersomes decreased, the deformed shape did not completely recover to a spherical form after passing through the membrane pore, which has an aspect ratio of about 30. This frozen shape of the polymersomes is presumably also attributable to the high Tg of the PS membranes. In other words, the cosolvent made the PS membranes sufficiently compliant to enter the pores, but by the time they exited at the downstream end the kinetics of shape recovery was too slow. However, the spherical shape could be restored by annealing the suspension at 100 °C for 8 h, as shown in Figure S3b. One disadvantage of extrusion is the loss of suspension during the process.56 Furthermore, extruder membrane failure can occur by pressure buildup due to the blockage of the polycarbonate membrane pores by the polymersomes.57 Accordingly, longer processing times and higher pressures are needed. Despite these disadvantages, extrusion is still a powerful tool to obtain a desired polymersome size. Especially, the extrusion with ILs can be applied to various polymers regardless of their Tg. A crucial prerequisite for the extrusion to be useful is a population of vesicles initially larger than the pore D

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solvent for the polymers. As water content increased, the vesicle size also increased. They also demonstrated that the larger vesicles had higher size dispersity due to a reduced size dependence to the total interfacial energy change, which is proportional to −1/R2. Reversibility of Polymersome Size. PS−PAA polymersomes showed reversible size changes with solvent composition on the time scale of 1 min as reported by the Eisenberg group.66 Here, in contrast, the size of the PS−PEO polymersomes was not reversible. The reversibility was tested as shown in Figure 5. In step i, [EMIM][TFSI] was added to the polymer solution, and the solution turned cloudy as described in Figure 5a. Thereafter, all DCM was removed as depicted in step ii. Depending on the DCM content in step i, different sizes of polymersomes were prepared (larger in Figure 5a, smaller in Figure 5b). In step iii, even more DCM was added to the suspension of larger polymersomes (Figure 5a), to correspond to the initial amount in Figure 5b. Conversely, a smaller amount of DCM was added to the smaller vesicles in Figure 5b to match the step ii of Figure 5a. Then all of the DCM in the suspensions was evaporated as depicted in step iv. The final samples after these four steps were named and differentiated with the initial samples by adding “-to-(vol % of DCM in step iii)”. If this size change was thermodynamically reversible, the larger vesicles should shrink, and vice versa. But as shown in the size distributions (Figure 6a), smaller vesicles did not grow even when the suspension was interrupted by adding some of the CS. The size of SO(14−2.5)-17 was significantly decreased from 457 to 86 nm in radius, and the dispersity was also reduced to μ2/Γ2 = 0.12 from μ2/Γ2 = 0.35. However, SO(14− 2.5)-60 and SO(14−2.5)-60-to-17 had the same dimensions, with ⟨Rh⟩ = 68 nm and μ2/Γ2 = 0.13. Figure 6b shows a cryoTEM image of SO(14−2.5)-17-to-60 polymersomes. Compared to the image of SO(14−2.5)-20 polymersomes (Figure 4a), the size and dispersity of SO(14−2.5)-17-to-60 polymersomes significantly diminished. The average radius in the image is 75 ± 16 nm, which is close to that of SO(14−2.5)-60. It is known that the chain exchange rate (k (s−1)) of amphiphilic chains in a suspension is proportional to exp(−γ), where γ is interfacial tension between hydrophobic part and a solvent.67 Therefore, the size irreversibility of PS−PEO polymersomes is due to a kinetically locked-in structure when the IL content is dominant. At low DCM concentration, γ is not changed much; consequently, the polymersomes are frozen without size or morphology changes, whereas the size can change at high DCM concentration due to the increased mobility of PS membrane. A similar asymmetry in annealing of nonequilibrium BO spherical micelles in an IL was also reported previously.38,64

size and dispersity. As the initial DCM content was increased, the size and dispersity decreased for both SO(14−2.5) and BO(9−2.5) polymersomes. For example, ⟨Rh⟩ of SO(14−2.5)17 was 457 nm with dispersity μ2/Γ2 = 0.35, but that of SO(14−2.5)-60 was 68 nm with μ2/Γ2 = 0.11. This trend was the followed for BO(9−2.5) with rubbery bilayer membranes. Cumulant fitting gave ⟨Rh⟩ ≈ 253 nm and μ2/Γ2 ≈ 0.37 for BO(9−2.5)-30 but ⟨Rh⟩ ≈ 67 nm and μ2/Γ2 ≈ 0.13 for BO(9− 2.5)-60. Cryo-TEM images of SO(14−2.5)-20 and SO(14− 2.5)-60 in Figure 4 clearly show the effect of the DCM content

Figure 4. Cryo-TEM images of SO(14−2.5) polymersomes in [EMIM][TFSI] (0.5 wt %): (a) SO(14−2.5)-20; (b) SO(14−2.5)-60.

on the size and dispersity. Whereas the size of the SO(14−2.5)20 polymersomes varies from 87 to 275 nm in radius in Figure 4a, Figure 4b from SO(14−2.5)-60 shows relatively smaller polymersomes (mean radius 74 nm) and more narrowly dispersed (standard deviation 14 nm); see Supporting Information for cryo-TEM images of BO(9−2.5). These changes are tentatively explained by the solvent quality toward the membrane forming blocks, PS and PB, which can be estimated via solubility parameter (δ) differences between the two components, which is directly related to the interfacial tension. The solubility parameters of PS (δPS) and PB (δPB) are 18.6 and 17.1 MPa1/2, respectively, close to 20.2 MPa1/2 of DCM, but the solubility parameter of the IL, δIL, is 27.6 MPa1/2.64,65 Therefore, increasing the [EMIM][TFSI] content should increase the interfacial tension between the bilayer membranes and the solvent mixture. Since the total interfacial energy is proportional to the surface area of the polymersomes, the polymersome size will be larger in order to reduce the surface area per chain when more [EMIM][TFSI] is added. Similar observations were also made by Luo and Eisenberg.66 They studied the size changes of polystyrene-b-poly(acrylic acid) (PS−PAA) assemblies by varying the THF/dioxane content from 43.3% to 80% in mixtures with water. THF/ dioxane was used as a cosolvent, and water was a selective

Figure 5. Phase diagrams of SO polymers, and the DCM content along with the each step for the reversibility test for (a) SO(14−2.5)-17 to SO(14−2.5)-17-to-60 and (b) SO(14−2.5)-60 to SO(14−2.5)-60-to-17. E

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Figure 6. Size change of polymersomes after following the steps described in Figure 5: (a) size distiribution change measured by DLS; (b) cryoTEM image of SO(14−2.5)-17-to-60 in [EMIM][TFSI].

Effect of PEO Length on Polymersome Size. The effect of the solvophilic block was studied by systemically varying the PEO block length with a constant 18 kg/mol of PS-OH. With three different SO(18−2.5), SO(18−3), and SO(18−3.6), the CS method was used for the polymersome preparation. The initial DCM content was fixed at 60 vol % for all polymers. As shown in Figure 7, as the PEO length is increased, the

removed under vacuum, and the time for preparing the organic solvent-free polymersomes was reduced because there was no additional steps, such as dialysis. Fractionation of Larger Polymersomes Using Phase Transfer. We have previously studied the phase transfer behavior of polymersomes and micelles having PEO moieties in the biphasic systems of water and water-immiscible ILs.47,68,69 The lower critical solution temperature (LCST) behavior of PEO in water makes the system thermally responsive and reversible.70 Above the phase transfer temperature (Tt), micelles transfer to the IL phase, but below Tt, they prefer the aqueous phase. During the transfer process between two immiscible phases, the morphology of the polymersomes and micelles is unperturbed; similarly, the polymersomes retain the IL interiors without any loss.49,71 These characteristics of the phase transfer could have potential application in separations, delivery of hydrophobic materials, and reversible nanoreactors. However, the phase transfer in the biphasic system is influenced by the interfacial tension between the aqueous solvent and the polymersome membrane block; if it is too high, polymersomes may not transfer.34 Nontransferrable PS−PEO polymersomes could be transferred to the aqueous phase, if the interfacial tension between PS and the aqueous phase (γPS−water) was reduced by introducing THF to the aqueous phase. Here, in contrast, γPS−water was increased by adding NaCl, and larger polymersomes were preferentially transferred. The broadly dispersed SO(18−3.6) polymersomes in the IL (see Supporting Information for cryo-TEM images before the transfer) were contacted with an aqueous phases with various NaCl concentrations from 0 to 0.2 M. After at least 24 h, the phase transfer of SO(18−3.6) polymersomes reached a steady state under equilibrium as shown in Figure 8. Figure 8 clearly shows cloudiness changes of the aqueous and IL phases as a function of the salt concentration. Without NaCl, all of the

Figure 7. Effect of PEO block length on the size of SO polymersomes. The initial content of DCM was 60% for all polymers.

polymersome size decreased; cryo-TEM images are shown in Figure S5. The longer PEO blocks are more crowded for a given brush density, and therefore the curvature of the polymersomes should be larger with a longer PEO block. The size and dispersity of the polymersomes could be controlled by varying the CS content and the length of PEO block of PS−PEO. Both approaches successfully produced small and nearly monodisperse polymersomes in the IL. Compared to other systems with water as a selective solvent, the CS protocol with the IL was more efficient due to the nonvolatile IL. The organic cosolvent, DCM, was effectively

Figure 8. Images of [EMIM][TFSI] (bottom phase) and aqueous NaCl solution (upper phase) biphasic system with SO(18−3.6) polymersomes. The concentration of NaCl in the upper phase was varied from 0 to 0.2 M. The images were taken after contacting the two phases for at least 24 h. Initially, all of the polymersomes were in the IL phase, as in the image at 0.2 M NaCl. F

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Figure 9. Variation of (a) mean hydrodynamic radius (Rh) of SO(18−3.6) polymersomes in the upper phase and (b) their size dispersity (μ2/Γ2) at different NaCl concentrations from the DLS measurement.

polymersomes were transferred to the top water phase from the IL phase, and the bottom IL phase turned clear. As the salt concentration increased, the cloudiness of the aqueous phase decreased and the aqueous phase became clear at 0.2 M NaCl, whereas the cloudiness of the IL phase increased. Figure S7 shows the scattered intensity of the diluted aqueous phase. The scattered intensity diminishes with salt concentration and becomes the same as that without polymers (∼2 kcps). The visible cloudiness trend (Figure 8) and the scattered intensity of the aqueous phase (Figure S7) clearly indicate that the increased surface tension influenced the phase transfer of SO(18−3.6) polymersomes. The self-assembled structures and their dimensions are not changed during phase transfer even with the aqueous solution of NaCl, isomaltotriose, and THF as shown previously,34,72 because they are “frozen” and not able to equilibrate during transfer. The following DLS results with SO(18−3.6) at different NaCl concentrations also support that the size was not changed by the salt concentration. An aliquot of a 0.5 wt % aqueous suspension of SO(18−3.6) polymersomes was diluted to 0.01 wt % with pure water, 0.05 M NaCl, and 0.1 M NaCl solution to see the effect of added NaCl on the size of the polymersomes. Rh and μ2/Γ2 were not changed with salt concentration as Rh = 114 ± 0.9 nm, μ2/Γ2 = 0.19 ± 0.01 in pure water, Rh = 116 ± 0.9 nm, μ2/Γ2 = 0.19 ± 0.01 at 0.05 M NaCl, and Rh = 114 ± 0.5 nm, μ2/Γ2 = 0.18 ± 0.02 at 0.1 M NaCl. Moreover, since the samples were diluted over 150 times in pure water to analyze the size of the upper phase with the DLS measurement, the effect of NaCl on the size of polymersomes is negligible. As shown in Figure 9a, the average Rh of the polymersomes in the aqueous phase increases from 97 to 130 nm, but μ2/Γ2 decreases from 0.2 to 0.1 (Figure 9b), when the concentration of NaCl changed from 0 to 0.1 M. By controlling the surface tension of the aqueous phase in the biphasic system, more narrowly dispersed and larger polymersomes were selectively separated. This fractionation was also quantified using cryoTEM images (representative images are shown in Figure S8). The distribution of polymersome size is shown in Figure 10, based on measurements of more than 550 polymersomes with the aqueous suspensions at 0 and 0.1 M NaCl. The mean of the distribution moves to larger polymersome radius (R), and the polymersomes below R = 50 nm disappear. The size distribution was fitted with a log-normal distribution function,73 and the fitting parameters, average radius (⟨R⟩), and standard deviation (w) were obtained. The absolute values of ⟨R⟩ are different than Rh because the number-averaged value ⟨R⟩ is always smaller than the scattered intensity weighted Rh for polydisperse sample, but it is clear that relatively larger polymersomes could be found at higher NaCl concentration,

Figure 10. Size distribution of SO(18−3.6) polymersomes from cryoTEM images. The cryo samples were prepared with the aqueous suspension of the biphasic system without NaCl and with 0.1 M NaCl. The solid lines are fitted curve with a log-normal distribution function.

as shown in the distributions. ⟨R⟩ increased from 65 nm (w = 32 nm) to 87 nm (w = 28 nm), and the dispersity value ((w/ ⟨R⟩)2), which can be directly compared to the dispersity, μ2/Γ2, in DLS decreased from 0.24 to 0.10. The fractionation based on the size can be explained by the total free energy change (ΔGtot) of the polymersomes. ΔGtot can be written as a summation of three contributions from the membrane interface (ΔGinterface), the membrane (ΔGcore), and the corona (ΔGcorona) as59 ΔGtot = ΔGcore + ΔGinterface + ΔGcorona

(1)

Depending on the sign of ΔGtot, the phase transfer behavior can be estimated. When it is negative, the micelles will transfer to the aqueous phase, but when it is positive, complete phase transfer does not happen. Here ΔGtot can be simplified to two competing terms, a positive ΔGinterface and a negative ΔGcorona, due to negligible dimensional size changes of the assemblies:74 ΔGtot = ΔGinterface + ΔGcorona = (γPS − water − γPS − IL)A + ΔGcorona

(2)

where A is the outer surface area of an assembly, and γPS−water and γPS−IL are the interfacial tension between PS core membrane and water or the IL, respectively. As the NaCl concentration increases, γPS−water increases, and therefore the interfacial tension penalty, ΔγInterface = γPS−water − γPS−IL, increases, and ΔGcorona also increases due to increased hydrophobicity of PEO in the aqueous phase.42 To have phase transfer at high salt concentration, the increased interfacial tension penalty should be mitigated by covering the hydrophobic core with more densely packed corona chains. The degree of the coverage can be quantified by the reduced tethering density (σPEO), which is proportional to ΔGtot as shown in a previous study.34 G

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Figure 11. Cryo-TEM images of SO(10−3) assemblies in (a) the aqueous phase at 0 M NaCl, (b) the aqueous phase, and (c) the IL phase at 0.08 M NaCl after contacting the IL suspension of SO(10−3) assemblies with the aqueous solution at least 24 h. Scale bars: 200 nm.

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We showed that σPEO increases as Rh of polymersomes increases and has a critical value to determine the phase transfer of PS−PEO polymersomes, σPEO,c.34 The polymersomes can transfer above σPEO,c, but not below. As the salt concentration increases, σPEO,c is expected to shift to a higher value due to the increased interfacial tension penalty (see the Supporting Information for more details). As a result, polymersomes having σPEO less than a new σPEO,c would not undergo phase transfer. Using the changes of σPEO,c depending on the NaCl concentration, not only the fractionation of the polymersomes based on their size but also the fractionation based on their morphology is possible. As an example, polymersomes and worm-like micelles of SO(10−3) were separated. SO(10−3) polymers in [EMIM][TFSI] self-assembled into two different morphologies, polymersomes and worm-like micelles, as shown in Figure S10. Cloudiness was also used to determine the phase transfer of two kinds of assemblies because a suspension is cloudy in the presence of large assemblies.75 As shown in the experimental images (Figure S11), at 0 M NaCl, all of the assemblies transferred to the top water phase from the IL phase, and the bottom IL phase turned clear. After the phase transfer, SO(10−3) assemblies also maintained their polymersome and worm-like micelle structures, as shown in Figure 11a. Considering the medium of the polymersome suspension changes from the IL to water, the phase contrast in the image is changed due to the electron density difference; the dark interiors represent the encapsulated IL phase, the gray domains represent the PS membrane and micelle core, and the aqueous medium is lighter than the IL and PS cores. To have selective phase transfer of the polymersomes, σPEO,c was shifted by setting the NaCl content to 0.08 M NaCl, which is between σPEO of the polymersomes and the worm-like micelles. As the salt content increased, the aqueous phase became less cloudy, whereas the IL phase turned cloudy (Figure S11). This visible cloudiness change in each phase clearly shows that the controlled σPEO,c was effective in the phase transfer of SO(10−3) assemblies. To investigate the self-assembled morphologies in the aqueous and IL phase after controlling σPEO,c, the vitrified samples were prepared and imaged by cryoTEM. When the concentration of NaCl was increased to 0.08 M, only polymersomes could be seen in the aqueous phase, but worm-like micelles were not observed, as shown in Figure 11b. Interestingly, as shown in Figure 11c, the nontransferred wormlike micelles were mainly found in the cryo-TEM image of the IL phase at 0.08 M NaCl. Thus, the worm-like micelles could not transfer to the aqueous phase and remained in the IL phase due to the shifted σPEO,c, while polymersomes having higher σPEO than the new σPEO,c could transfer to the aqueous phase.

SUMMARY The size and dispersity of PS−PEO and PB−PEO polymersomes in [EMIM][TFSI] were controlled by mechanical extrusion and by changing the methodology of the polymersome formation. Rather precisely controlled size (⟨Rh⟩ ≈ 100 nm) with fairly narrow size distribution (μ2/Γ2 ≤ 0.2) was obtained by several passes through polycarbonate membranes. Through the CS methods approaches, relatively monodisperse small polymersomes ((⟨Rh⟩ ≈ 70 nm, μ2/Γ2 ≈ 0.1) were obtained with an organic solvent at high CS content. Use of a nonvolatile IL directly led to the organic solvent-free polymersomes under vacuum in a single step. Upon increasing the CS concentration or the PEO block length, the diameter and size dispersity of the polymersomes decreased. Furthermore, as-assembled polymersomes using the cosolvent method showed irreversible size change with additional DCM. Furthermore, we applied selective phase transfer behavior of PS−PEO vesicles in the biphasic system as a simple fractionation process based on the size of the polymersomes. The increased surface tension of the aqueous phase could increase the interfacial penalty for the phase transfer and shift the necessary value of σPEO,c to a higher value. The careful design of a biphasic system with tunable solvent quality (by tuning IL or surface tension of the aqueous phase) could possibly be applied to fractionation of other self-assembly systems of polymersomes, liposomes, or so-called “aquanelles”.1,18,76 This fractionation method is a new approach to achieve a desired size and morphology of assembly by simply controlling the salt content in the aqueous phase and has potential to be applied to the fields of biotechnology and nanoreactors.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b00946. Cryo-TEM images of PS−PEO polymersomes in [EMIM][TFSI] and water at different conditions, light scattering results, experimental images of PS−PEO polymersomes in the biphasic system, and the details of fractionation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (T.P.L.). Notes

The authors declare no competing financial interest. H

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation through Award DMR-1206459. Cryo-TEM measurements were carried out in the Characterization Facility, University of Minnesota, which has received capital equipment funding from the NSF through the UMN MRSEC program, under Award DMR-1420013. We acknowledge Dr. Robert Hickey and Prof. Yeshayahu Talmon for helpful discussions.

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DOI: 10.1021/acs.langmuir.6b00946 Langmuir XXXX, XXX, XXX−XXX