The effect of stabilizer on the mechanical response of double-emulsion-templated polymersomes.

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The Effect of Stabilizer on the Mechanical Response of Double-Emulsion-Templated Polymersomes Woo-Sik Jang, Seung Chul Park, Miju Kim, Junsang Doh, Daeyeon Lee,* Daniel A. Hammer*

Recent studies have shown that polymersomes templated by microfluidic double-emulsion possess several advantages such as high monodispersity and encapsulation efficiency compared with those generated based on thin-film rehydration and electroformation. Stabilizers, including bovine serum albumin (BSA) and polyvinyl alcohol (PVA), have been used to enhance the formation and stability of double emulsions that are used as templates for the generation of polymersomes. In this work, the effect of stabilizers on the mechanical response of doubleemulsion-templated polymersomes using micropipette aspiration is investigated. It is demonstrated that the existence of stabilizers results in the inelastic response in polymersomes in the early stage of solvent removal. However, aged polymersomes that have little residual solvent show elastic behavior. Polymersomes prepared from PVA-stabilized double emulsions have noticeably lower area expansion moduli than polymersomes prepared from stabilizerfree and BSA-stabilized double emulsions, suggesting that PVA is incorporated in the bilayer membrane of polymersomes. Dr. W.-S. Jang, Dr. S. C. Park, Dr. M. Kim, Prof. D. Lee, Prof. D. A. Hammer Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 220 South 33rd Street 311A Towne Building, Philadelphia, PA 19104-6315, USA E-mail: [email protected]; [email protected] Dr. M. Kim Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-Gu, Gyeongbuk 790–784 , Republic of Korea Prof. J. Doh Department of Mechanical Engineering, School of Interdisciplinary Bioscience and Bioengineering (I-Bio), Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-Gu, Gyeongbuk 790–784 , Republic of Korea Prof. D. A. Hammer Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street Suite 240 Skirkanich Hall, Philadelphia, PA 19104–6321, USA

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1. Introduction Polymersomes are synthetic vesicles whose membrane is self-assembled from amphiphilic diblock copolymers.[1–13] The hydrophobic block of amphiphilic diblock copolymers constitutes the core of the bilayer membrane, while the hydrophilic block forms the brush that protects membrane from aqueous environments. Similar to lipid-based vesicles (liposomes), polymersomes can encapsulate hydrophilic compounds within the aqueous lumen and at the same time be loaded with hydrophobic species in the hydrophobic core of the membrane. The membrane of polymersome is generally several times thicker than that of a liposome, owing to larger molecular weight of polymer amphiphiles, which leads to their superior mechanical stability over liposomes.[3] These features make polymersomes ideal microcapsules that can be used for drug delivery and also as model templates to mimic cells and

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DOI: 10.1002/marc.201400472

The Effect of Stabilizer on the Mechanical Response of Double-Emulsion-Templated Polymersomes

Macromolecular Rapid Communications www.mrc-journal.de

organelles[1,7,11,14,15] and be used for protocells that can perform cell-like functions.[16–20] Polymersomes can be made by thin-film rehydration[3,7,13] and electroformation.[1,21–23] More recently, microfluidic double emulsions[9,12,24–29] have proven to be versatile for generating highly monodisperse polymersomes with high encapsulation efficiency. In this method, monodisperse water-in-oil-in-water (W/O/W) double emulsions are formed by a microfluidic device and subsequently used as templates to form polymersomes. To increase the stability of double emulsions and polymersomes, stabilizing agents such as bovine serum albumin[12] (BSA) or polyvinyl alcohol[24,26–29] (PVA) are often added to the outer aqueous phase of W/O/W double emulsions. Given that these stabilizers are highly surface active, it is plausible that these agents will incorporate themselves into the membrane of double-emulsion-templated polymersomes. Despite many recent reports describing the fabrication, characterization, and application of doubleemulsion-templated polymersomes,[9,11,24,27–31] the effect of stabilizers on the membrane property is poorly understood. Verifying the effects of stabilizers on the mechanical properties of polymersome membranes, in particular, would be important for understanding their stability and composition for numerous applications. In this present study, we investigated the effect of various stabilizers on the mechanical properties of double-emulsion-templated polymersomes made of poly(ethylene oxide)-b-poly(butadiene) diblock copolymers. We prepared three types of polymersomes, including those made from stabilizer-free, BSA-stabilized, and PVA-stabilized double emulsions and characterize their mechanical response using micropipette aspiration. We show that the stabilizers indeed change the mechanical response of polymersomes and their influence changes over time as solvent gradually dissolves from the emulsions. Our work emphasizes the importance of understanding the effect of stabilizers on the physical properties of polymersome membranes.

2. Experimental Section The amphiphilic diblock copolymer, poly(ethylene oxide)-bpoly(butadiene), was purchased from Polymer Source (Montreal, Canada). The weight-averaged molecular weights (MW ) of polyethylene glycol and butadiene blocks were 1.3 and 2.5 kg mol−1, respectively. The polydispersity index of polymer is 1.05. We refer to this polymer as OB29. Chloroform, hexane, phosphate-buffered sailine (PBS), sucrose, BSA, and PVA (87%–89% hydrolyzed, average MW = 13–23 kg mol−1) were purchase from Sigma– Aldrich (St. Louis, MO). W/O/W double emulsions were formed using a microfluidic device.[32] Three syringe pumps (PHD, Harvard Apparatus) were used to control the flow rates of two aqueous and one oil

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phases for the microfluidic device. The inner aqueous phase was 290 mOsm sucrose. The middle phase contained 2 mg mL−1 OB29 within the mixture of chloroform (28 vol%) and hexane (72 vol%). Three different outer aqueous phases were prepared: 290 mOsm PBS without stabilizer, 290 mOsm PBS with 1 wt% BSA, or 290 mOsm PBS with 2 wt% PVA. The amphiphilic diblock copolymer is a surfactant itself. Thus, it is possible to stabilize polymersomes without extra stabilizers in the continuous phase. For BSA-stabilized polymersomes, we tested a wide range of BSA concentrations. BSA concentrations exceeding 1 wt% were found to be detrimental for polymersome preparation. When BSA concentration was lower than ≈ 0.7 wt%, the yield of stable polymersomes was not as high as 1 wt% BSA. As for PVA, we use 2 wt% because this concentration is widely used by researchers in this area to stabilize various double emulsions generated using microfluidics.[33] Flow focusing of middle and inner phases by continuous phase in microfluidic devices results in doubleemulsion formation. Details regarding the device configuration have been presented elsewhere[32] and are provided by Supporting Information. Double emulsions were collected in a reservoir containing 290 mOsm PBS. Collected double emulsions were stored in a 20 mL vial at room temperature. We measured the diameter of the vesicles we formed to be 74.5 ± 5.5 μm. Micropipettes were made in a micro-puller (David Kopf Instruments, Tujunga CA) and finished in a micro-forge (Technical Products International INC., St. Louis MO). The typical inner diameter of micropipette is 12 μm but the exact inner diameter varied from experiment to experiment. Stored polymersomes were dispersed in experimental chamber, comprised two cover glasses, filled with osmolality matched PBS. Weak negative pressure is initially applied to grab the target polymersome. Subsequently, the captured polymersome is lifted up to detach from substrate. We performed multiple aspirations (3–5) with single polymersome and three different polymersomes were tested for each experiment.

3. Results and Discussion We use micropipette aspiration to characterize the mechanical response and properties of OB29 polymersomes made from W/O/W double emulsions. Micropipette aspiration[1,3,9,11,34–42] applies suction on a microcapsule through a narrow glass capillary and correlates the areal strain (α) to the membrane tension (TV), which is measT ured using a custom-made manometer: α = α 0 + KV where a α0 is a term representing thermal undulation of the membrane and Ka is the area expansion modulus.[37,39,43] The full expression for the tension–strain equation is provided in Supporting Information. Initial tension releases α0 and results in a finite deformation, which is demonstrated by the nonzero intercept on x-axis. When the effect of thermal undulation on the membrane behavior is released by initial tension, the relationship between TV and α can be expressed as TV = K a ⋅α


(1)

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where Ka represents the area expansion modulus. However, a small negative pressure is required to grab and hold polymersomes firmly with micropipette; this initial pressure causes initial nonzero intercepts on y-axis. An initial nonzero intercept does not necessarily indicate quantitative mechanical properties such as bending rigidity. Therefore, we shifted the TV versus α curves to origin for simplicity. The detailed procedure for making vesicles is provided in the Supporting Information. OB29 polymersomes were prepared by dissolving the polymer in the oil phase of W/O/W double emulsions and subsequently removing the solvent via evaporation from the oil phase. Studies have shown that the slow removal of the solvent results in dewetting of the middle phase and the formation of a patch that remains on the polymersome surface.[9] To avoid the influence of such a patch in determining the mechanical response of polymersomes, we performed micropipette aspiration on the patch-free regions of polymersomes. The tension–areal strain relationship of polymersomes made from stabilizer-free, BSA-stabilized, and PVA-stabilized W/O/W double emulsions was measured over a period of 2 weeks after the polymersomes were made. We measured the properties of vesicles over areal strains ranging from 0.1 to 0.2. Typically, 2- and 5-day-old polymersomes could be aspirated up to α ≅ 0.2. In contrast, 8- and 13-day-old samples generally ruptured when α exceeded ≈ 0.13. Figure 1a,b show the representative aspiration results of polymersomes made without stabilizer, aged from 2 to 8 days, respectively. Membrane tension produced by micropipette suction resulted in a linear increase in areal strain and showed a reversible recovery upon decreasing membrane tension, irrespective of aging, which is the characteristics of purely elastic membranes.[9] Furthermore, Figure 1a,b clearly illustrate that the slope of TV versus α curve, thus Ka, increases markedly with aging time. Ka continues to increase over a period of 8 days and eventually reaches its maximum at the previously reported value of Ka for solvent- and stabilizer-free polymersomes made from OB29, as seen in Figure 1c.[9] Our previous work showed that an increase in Ka with age reflects the gradual evaporation of residual solvent from the doubleemulsion template, and the current results are consistent with those findings.[9] We note that the response of the 2and 5-day-old polymersomes does not change upon multiple cycles of aspiration and release, which indicates that aspiration does not facilitate solvent removal. The mechanical response of OB29 polymersomes templated with BSA-stabilized double emulsions under micropipette aspiration is different from that of polymersomes made without any stabilizers. In the case of 2-day-old polymersomes, the response during aspiration and relaxation showed a linear tension–strain behavior

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Figure 1. Representative TV versus α behavior of a) 2- and b) 8-day-old polymersomes made without stabilizer. The aspiration and release curves follow same path for both young and mature polymersomes. Best fits are shown by black-dashed line. c) The evolution of Ka with age of the polymersomes. Red dashed line represents previously reported Ka for solvent- and stabilizerfree polymersomes from OB29 amphiphilic diblock copolymer.[9]

during aspiration; however, during the release of tension, the response displayed curvature, with less tension required for a given strain, indicating some type of





Figure 2. a) Representative TV versus α behavior of 2-day-old BSA-stabilized polymersomes. Significant hysteresis was observed. To determine the best fits for aspiration and release curves, we used nonlinear least square fitting of the first- and second-order polynomial equations, respectively. b) The energy dissipation associated with each aspiration cycle decreases with increasing aspiration cycles. c) Representative TV versus α behavior of an 8-day-old BSA-stabilized polymersome. Aspiration and release curves show little difference irrespective of the number of aspiration cycles. d) Ka from 2-, 5-, 8-, and 13-day-old BSA-stabilized polymersome converges to previously reported Ka for solvent- and stabilizer- free polymersome made from OB29.

inelastic/plastic deformation taking place during aspiration. The evidence for inelastic deformation can be more clearly seen by determining the energy dissipation during one cycle of aspiration and release, which can be calculated by taking the difference between the work associatedαwith aspiration and release: Waspiration − Wreleasing ( where W = ∫ TV dα). Figure 2a shows the representative α aspiration and release curves from BSA-stabilized polymersome. Multiple aspiration and release curves are provided in Supporting Information. Figure 2b shows that there is significant amount of energy dissipation during one cycle of aspiration/release. Overall, the dissipated energy during each cycle decreases with increasing numbers of aspiration cycles, indicating that the extent of inelasticity decreases upon multiple loading–unloading cycles. Tension vs. areal strain shows linear behavior with increasing slopes as the number of aspiration/ release cycles is increased. In contrast, the release curves exhibit nonlinear behavior with the curvature decreasing with increasing number of aspiration cycles. Since the 2

1


aspiration curve eventually coincides with the release curve, the slope of release curve from third-aspiration cycle likely represents the true elastic modulus of the membrane, Ka = 85.1 ± 5.6 dyne cm−1. The differences in intercept likely reflect changes in the bending modulus,[4] where the bending modulus appears to decrease with each aspiration. However, these intercepts do not represent the quantitative mechanical properties. Polymersomes made from BSA-stabilized double emulsions showed purely elastic response of repeated aspiration/ release cycles after 5 days of aging. At this age, aspiration and release curves show little difference irrespective of the number of aspiration cycles as shown in Figure 2c. The modulus of these polymersomes after 5 days also reached a plateau and showed a similar value as stabilizer-free Ka. Polymersomes made from PVA-stabilized double emulsions showed mechanical response that is markedly different from the stabilizer-free and BSA-stabilized cases. Figure 3a shows the representative TV versus α response for 2-day-old OB29 polymersomes made from


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Figure 3. a) Representative TV versus α behavior of 2-day-old PVA-stabilized OB29 polymersomes. To determine the best fits for aspiration and release curves, we used nonlinear least square fitting of the second-order polynomial equation, which provides a good description of the observed behavior: TV = A ⋅ α 2 + B ⋅ α + C where A, B, and C are fitting variables. The aspiration and release curves show the repeatable negative and positive curvature, respectively. b) The energy dissipation associated with 2 and 5-day-old PVA-stabilized OB29 polymersome. c) Representative TV versus α behavior of 8-day-old PVA-stabilized OB29 polymersome. Similar to stabilizer-free and BSA-stabilized polymersome, after 8-day-old aging, polymersomes made from PVA-stabilized double emulsions exhibit repeatable linear loading–unloading behaviors. d) Ka for 8- and 13-day-old PVA-stabilized polymersome. Ka’s for 8- and 13-day-old samples are significantly lower than the values we obtained for stabilizer- and solvent-free polymersomes made from OB29.

PVA-stabilized double emulsions exhibit repeatable linear PVA-stabilized double emulsions. The aspiration and loading–unloading behavior as seen in Figure 3c, sugrelease curves show hysteresis with repeatable negagesting a purely elastic response. However, corresponding tive and positive curvature, respectively. Unlike polyKa’s for 8- and 13-day-old samples are significantly lower mersomes made from BSA-stabilized double emulsions, dissipated energy does not change significantly upon than the values we obtained for stabilizer- and solventrepeated aspiration and release. However, dissipation free polymersomes made from OB29, Figure 3d. Table 1 energy of these polymersomes is significantly larger than summarizes the Ka with respect to the polymersome type that observed for 2-day-old polymersomes made from and aging time. BSA-stabilized double emulsions. The average dissipated Polymersomes made from stabilizer-free double emulenergy for 2-day-old BSA-stabilized and PVA-stabilized sions exhibited reversible aspiration-release behaviors polymersomes are 0.33 ± 0.18 and 0.52 ± 0.08, respectively. This result indiTable 1. Ka with respect to the polymersome type and aging time. cates that OB29 polymersomes made from PVA-stabilized double emulsions Ka [dyne cm−1] are more inelastic than those tem2-day-old 5-day-old 8-days-old 13-day-old plated by BSA-stabilized double emulStabilizer-Free 67.0 ± 7.6 83.6 ± 3.0 94.7 ± 3.1 93.6 ± 5.4 sions. Similar to stabilizer-free and BSA85.1 ± 5.6 94.4 ± 3.2 93.9 ± 3.2 97.2 ± 2.8 stabilized polymersomes, after 8 days BSA-Stabilized of aging, polymersomes made from

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PVA-Stabilized

n. a.

n. a.


82.6 ± 5.9

79.7 ± 7.4




regardless of aging time, whereas polymersomes made from BSA- or PVA-stabilized double emulsions showed inelastic response in the early stages of solvent removal. These results suggest that stabilizers influence the mechanical response by possibly being incorporated or interacting with the polymersome membranes when residual solvent is present. However, the manner by which inelasticity is manifested in these two types of polymersomes is quite different. Polymersomes made from BSAstabilized double emulsions respond to repeated aspiration/release, whereas those made from PVA-stabilized double emulsions do not; that is, apparent stiffness of polymersomes upon aspiration increases with increasing number of aspiration/release cycles. Also, tension does not return to its initial value when strain is reduced to 0, which suggests that BSA conveys a significant resistance to bending. The hysteretic closed-loop aspiration/ release behavior that is exhibited by 2 and 5-day-old polymersomes made from PVA-stabilized double emulsions is reminiscent of the stress–strain relationship of viscoelastic solids without plasticity.[44] The molecular mechanisms behind these different responses, however, are not clear and warrant future investigations. Interestingly, all three types of polymersomes showed purely elastic behavior once solvent was completely removed. Ka’s for polymersomes prepared from stabilizerfree and BSA-stabilized double emulsions, in fact, converge to previously reported stabilizer- and solvent-free Ka’s. These results indicate that BSA does not strongly affect the mechanical properties of mature polymersome membranes upon the complete removal of the solvent. We confirmed that when fluorescently labeled BSA was used to stabilize double emulsions, little fluorescence signal is detected in the membrane of resulting polymersomes, whereas strong signal is observed in the patches of polymersomes as seen Figure 4. These results suggest that BSA affect the properties of the polymersomes membrane in the early stages of solvent removal but eventually segregates to the patches upon complete removal of the solvent, rendering the properties of the membrane similar to those made without stabilizers. The segregation of material into the patches of microcapsules resulting from the dewetting of the oil phase in double emulsions has previously been observed.[45] In contrast to polymersomes made from BSA-stabilized double emulsions, the Ka of polymersomes made from PVA-stabilized double emulsions is smaller than Ka of stabilizer- and solventfree polymersomes. These results strongly suggest that PVA is incorporated into the polymersome bilayer membrane and significantly affects its mechanical properties even after the removal of solvent, possibly by inducing some hydration, which would soften the membrane. Furthermore, we also observed another piece of evidence that indicates the influence of PVA on the membrane


Figure 4. a) Optical and b) fluorescent images for polymersomes made with a fluorescently labeled BSA. After several days, BSA is incorporated with patches on membrane. No fluorescent signal was detected from polymersome membrane. White arrows point out the patches from broken polymersomes.

properties is very different from that of BSA. The patches on 13-day-old stabilizer-free and BSA-stabilized polymersomes show wrinkles on the surface, whereas that on the 13-day-old PVA-stabilized polymersomes appear smooth (figure is provided by Supporting Information). These observations are consistent with our results on the mechanical properties of polymersomes. The moduli of stabilizer-free and BSA-stabilized polymersomes are quite similar to each other, whereas that of PVA-stabilized polymersomes is very different.

4. Conclusion In this work, we characterized and compared the mechanical response of polymersomes prepared from double emulsions with and without commonly used stabilizers— PVA and BSA. We showed that the presence of a stabilizer can induce inelastic responses in polymersomes in the


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early stages of solvent removal. With increasing aging time, polymersomes show elastic behavior; however, polymersome prepared from PVA-stabilized double emulsions has significantly lower modulus than polymersomes prepared from stabilizer-free and BSA-stabilized double emulsions, indicating that PVA is likely incorporated into the membrane of polymersomes and affects their mechanical response. Our study clearly illustrates that composition of the aqueous phase that are used for polymersome preparation can have significant impact on the mechanical response of resulting polymersomes. Understanding the effect of stabilizers on the mechanical properties of polymersomes could potentially lead to novel applications such as chemomechanically-triggered release from polymersomes. Furthermore, our finding potentially provides a new method to control the physicochemical properties of polymersomes made from one type of amphiphilic diblock copolymers by prudently choosing the stabilizer use to generate double emulsions. Acknowledgements: This work was supported in its entirety by the Biomolecular Materials program at the U.S Department of Energy, Office of Basic Energy Science, Division of Materials Science (DE−FG02−11ER46810). The authors especially indebted to Dr. Eric Johnston for micropipette aspiration training. Received: August 22, 2014; Revised: October 13, 2014; Published online: December 16, 2014; DOI: 10.1002/marc.201400472 Keywords: bovine serum albumin; microfluidic double emulsion; micropipette aspiration; polymersome; polyvinyl alcohol

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