Tuning the Mechanical Properties of Recombinant Protein-Stabilized

Feb 24, 2016 - Gas bubbles enhance contrast in ultrasound sonography and can also carry and deliver therapeutic agents. The mechanical properties of t...
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Tuning the Mechanical Properties of Recombinant Protein-Stabilized Gas Bubbles Using Triblock Copolymers Yeongseon Jang,†,‡ Woo-Sik Jang,† Chen Gao,† Tae Soup Shim,†,§ John C. Crocker,† Daniel A Hammer,*,†,∥ and Daeyeon Lee*,† †

Department of Chemical and Biomolecular Engineering and ∥Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States S Supporting Information *

ABSTRACT: Gas bubbles enhance contrast in ultrasound sonography and can also carry and deliver therapeutic agents. The mechanical properties of the bubble shell play a critical role in determining the physical response of gas bubbles under ultrasound insonation. Currently, few methods allow for tailoring of the mechanical properties of the stabilizing layers of gas bubbles. Here, we demonstrate that blending of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) amphiphilic triblock copolymer with a recombinant protein, oleosin, enables the tuning of the mechanical properties of the bubble stabilizing layer. The areal expansion modulus of gas bubbles, as determined by micropipette aspiration, depends on the structure as well as the concentration of PEO-PPO-PEO triblock copolymers. We believe our method of using a mixture of PEO-PPO-PEO and oleosin can potentially lead to the formation of microbubbles with stabilizing shells that can be functionalized and tailored for specific applications in ultrasound imaging and therapy. The physical response of microbubbles in an ultrasound field also depends strongly on the mechanical properties of the shell material.2,9,10 Thus, it is critical to be able to characterize and tailor the mechanical properties of the microbubble shell. Such a capability would ultimately lead to better understanding of the interactions between ultrasound and microbubbles and the tailoring of microbubble functionality and properties for specific applications. Although approaches to improve the stability and functionalization of microbubbles using different types of stabilizing agents have been actively pursued, few methods have been developed to tailor the mechanical properties of the bubble shell.11−14 In this report, we use micropipette aspiration to characterize the elastic properties of the oleosin-based bubble shell and demonstrate that the areal expansion modulus of the oleosinstabilized gas bubbles can be controlled by blending different PEO-PPO-PEO triblock copolymers with oleosin. Micropipette aspiration is a sensitive method that has been widely used to study the mechanical properties of various membranes and complements other mechanical characterization techniques such as atomic force microscopy.15−19 Good agreement between results obtained from micropipette aspiration and those from other techniques has been described.20 To date, however, only a few groups have used this method to characterize the properties of gas bubbles covered with stabilizing agents.21 We show that the elastic properties of

G

as-filled microbubbles drastically enhance the contrast in ultrasonography and can also deliver therapeutic agents in cancer treatment.1−4 Significant effort has been focused on developing materials to stabilize and encapsulate the gaseous core for a variety of biomedical applications. The stabilizing layers surrounding microbubbles are typically composed of soft materials such as phospholipids, proteins, or polymers.3 Stabilizers that can impart functionality and properties to microbubbles are highly desirable for tailoring their properties for diagnostic and therapeutic applications. Toward this goal, our group has recently developed highly monodisperse microbubbles whose surfaces can be stabilized and functionalized with various motifs using a combination of microfluidics and surfactant engineering.5 We used a mixture of oleosin, which is a natural surfactant protein that has a structure analogous to amphiphilic triblock copolymers, and a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer to stabilize monodisperse microbubbles prepared using microfluidics. Because oleosin is made recombinantly, it not only acts to stabilize microbubbles, but can also be widely engineered to incorporate additional functionalities to microbubbles. The direct incorporation of recognition motifs, for example, could enable the targeted delivery of microbubbles to specific sites, enhancing their therapeutic functionality.6,7 Microfluidics allows for precise tuning of microbubble size, in the range of 1−10 μm, which is known to be an important factor that controls the echogenicity and therapeutic performance of microbubbles under ultrasound insonation.5,8 © XXXX American Chemical Society

Received: January 21, 2016 Accepted: February 19, 2016

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in multiple holes can merge to form one big gas bubble. An indepth study on the effect of the microhole array geometry on the bubble size and size distribution is currently underway and will be reported in the future. Using an array with 50 μm holes that are spaced by 50 μm, we prepare bubbles stabilized with a mixture of a (PEO)78(PPO)30-(PEO)78 triblock copolymer (subscripts denote the number of repeat unit of each monomer; this triblock copolymer is also known as Pluronic F68) and oleosin-30G.

the oleosin-based stabilizing layer surrounding gas bubbles depends on the concentration as well as the composition of PEO-PPO-PEO triblock copolymers. We believe this simple approach provides a unique opportunity to enhance our quantitative understanding on the effect of the mechanical properties of bubble shell on the echogenicity and therapeutic efficacy of microbubbles under ultrasound insonation.

Figure 1. Templated bubble formation on an array of holes made from PDMS. (a) Schematic illustration of the method using an array of holes to generate gas bubbles that are tens of micrometers in diameter. (b) Optical microscope images of arrays with different diameters (left, 30 μm; middle, 50 μm; right, 100 μm). The depth of holes is 40 μm. Gas bubbles generated using the three different PDMS hole arrays. Oleosin-30G solution (1 mg/mL) is used. The average radius of bubbles from each hole array is denoted in each microscope.

To enable reliable micropipette aspiration, stable bubbles in the size range of several tens of micrometers (e.g., 50−100 μm) are required, owing to limitations in pipet fabrication and the resolution of optical microscopy. To produce such gas bubbles without using a large amount of material, we develop a new method of preparing gas bubbles based on templated bubble formation using an array of holes that are lithographically defined in a slab of polydimethylsiloxane (PDMS; Figure 1). A small aliquot of a solution containing oleosin and a PEO-PPOPEO triblock copolymer is placed atop an array of holes. During this step, air becomes trapped in the holes due to the hydrophobicity of PDMS. Subsequently, spherical bubbles covered with the amphiphilic agents are formed by applying weak vacuum to the solution. Spherical gas bubbles rise and get collected at the apex of the pendant drop as shown in Figure 1a. An important advantage of this method is that relatively uniform bubbles in the size range of several tens of microns can be formed and that these bubbles can be prepared with a very small amount of stabilizing agents. Moreover, as compared to other methods of bubble preparation such as agitation and sonication,8 this approach allows for control over the bubble size, which depends on the geometric factors such as the volume of individual holes (Figure 1b), the aspect ratio of the hole and interhole distance. A single bubble does not necessarily originate from a single hole but rather gas trapped

Figure 2. Stability of gas bubbles. Optical microscopy of gas bubbles generated from four different surfactant solutions composed of (a) 1 mg/mL (PEO)78-(PPO)30-(PEO)78, (b) 1 mg/mL oleosin-30G, (c) 1 mg/mL oleosin-30G and 10 mg/mL (PEO)78-(PPO)30-(PEO)78, and (d) 1 mg/mL oleosin-30G and 20 mg/mL (PEO)78-(PPO)30-(PEO)78 upon collection (upper panels) and 60 min after collection (lower panels). (e) Changes in the average radius (Ravg) of gas bubbles stabilized with solutions of different compositions as a function of time after collection. 372

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ACS Macro Letters Both oleosin and (PEO)78-(PPO)30-(PEO)78 are essential for bubble manufacture; oleosin is essential for bubble stability, and (PEO)78-(PPO)30-(PEO)78 is essential for monodispersity.5 Oleosin-30G is a recombinantly engineered variant of oleosin in which the hydrophobic core has been truncated to 30 amino acids, and five bulky hydrophobic residues replaced with glycines to enhance the flexibility of chains; additionally, the Nterminal and C-terminal hydrophilic domains, have been trimmed to 42 and 63 residues, respectively.5−7 The stability of gas bubbles prepared using the templates is observed under different concentrations of (PEO)78-(PPO)30-(PEO)78 in the solution, keeping the oleosin concentration fixed. Gas bubbles formed with a solution containing only (PEO)78-(PPO)30(PEO)78 are highly unstable due to dissolution, whereas those formed with just oleosin-30G show high stability against dissolution (Figure 2a,b). The addition of (PEO)78-(PPO)30(PEO)78 to oleosin does not significantly alter the stability, as shown in Figure 2c−e. These observations are consistent with our prior results that showed the long-term stability of microbubbles formed with a mixture of (PEO)78-(PPO)30(PEO)78 and oleosin-30G using microfluidics.5 The stability of gas bubbles is also investigated by varying the ratio of oleosin-30G and (PEO)78-(PPO)30-(PEO)78 while maintaining the total concentration of the two molecules constant at 1 mg/mL (Figure S1 in Supporting Information). As the oleosin-30G content is decreased to below 0.5 mg/mL in the solution, bubbles become less stable, as indicated by increasing bubble size over time due to coalescence and coarsening. Moreover, we find that highly stable bubbles can be formed using pure oleosin-30G solutions as long as the concentration of oleosin-30G is above 0.75 mg/mL (≈50 μM; Figure S2 in Supporting Information). These results indicate that oleosin-30G is a critical component for bubble stabilization and that the addition of (PEO)78-(PPO)30-(PEO)78 up to the concentration of 20 mg/mL has little impact on the bubble stability. To understand the effect of (PEO)78-(PPO)30-(PEO)78 on the mechanical properties of oleosin-30G-stabilized bubbles, we perform micropipette aspiration as shown in Figure 3. Micropipette aspiration consists of applying suction on a single bubble (radius ≈ 50−60 μm) through a glass capillary with a constant cross-section (radius ≈ 8−10 μm) and observing its shape changes as a function of suction pressure (Figure 3a).19,22 Assuming the interface is fluid, the membrane tension (T) can be related to the suction pressure (Pp) and radii of bubble (Rb) and pipet (Rp) following eq 1, T=

R b·R p·Pp 2(R b − R p)

(1)

The areal stain (α), which is the ratio of area change to initial area, depends on the projected length of the gas bubble into the pipet (L) as expressed in eq 2,19,22,23 α=

Figure 3. Micropipette aspiration of oleosin-30G-stabilized bubble. (a) Images of oleosin 30G-stabilized bubbles during micropipette aspiration (inner radius of the pipet (Rp) = 8.0 μm). (b) Typical T−α curves of a gas bubble prepared in a mixture of 1 mg/mL oleosin30G and 10 mg/mL (PEO)78-(PPO)30-(PEO)78 (●, Rp = 10.5 μm, Rb = 53.2 μm; ⧫, Rp = 9.5 μm, Rb = 46.6 μm; ▲, Rp = 9.6 μm, Rb = 45.2 μm). (c) Elastic region of a T−α curve for a gas bubble made with a mixture of 1 mg/mL oleosin-30G and 10 mg/mL (PEO)78-(PPO)30(PEO)78. The slope of T vs α defines the area expansion modulus (Ka). Since each aspiration results in a different y-intercept (i.e., different grip tensions), the nonzero intercept is shifted to the origin to facilitate comparison between results from different experiments.

2πR pL(1 − R p/R b) 4πR b2

(2)

An experimental determination of the relationship between T and α based on T = Ka × α permits the estimation of the areal expanding modulus (Ka) of the bubble shell. Figure 3b shows a representative response of an oleosin-30Gstabilized bubble to areal expansion. To initiate micropipette aspiration, it is necessary to “grab” and “hold” a gas bubble firmly to the tip of the micropipette, which requires some 373

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ACS Macro Letters negative pressure.23 By relating the tension upon the protrusion of the gas bubble into the micropipette to the areal strain (at 0 < α ≤ 0.01), it is possible to characterize the properties of the stabilizing layer. The linear portion of the curve corresponds to the elastic region, and the slope gives the modulus of elasticity for areal expansion. We confirm that there is no significant effect of bubble size on the tension-strain curve when the radius of bubbles is kept between 50 and 70 μm (Figure S3 in Supporting Information). We observe that oleosin-30G-stabilized bubbles exhibit a yield behavior, characterized by a maximum in the stress−strain curve followed by gas budding in the micropipette (Figure S4). Such a budding behavior has previously been observed with micropipette-aspirated GUVs of phospholipids and diblock copolymers and is known to be driven by the minimization of the bending energy.24,25 Although these behaviors are of significant interest, we focus on characterizing and comparing the linear elasticity of bubbles in this current study. The areal expansion modulus is obtained by determining the slope of the linear region in the T versus α curve after accounting for the nonzero intercept (due to the initial grip tension), as shown in Figure 3c.23 To our surprise, as the concentration of (PEO)78-(PPO)30(PEO)78 is increased, the slope of the linear region of the T versus α curve and, in turn, the areal expansion modulus of the stabilizing layer, increase as shown in Figure 4; that is, (PEO)78(PPO)30-(PEO)78 stiffens the stabilizing layer of oleosin-30G protein-stabilized bubbles. This result is somewhat counterintuitive because gas bubbles stabilized with pure (PEO)78(PPO)30-(PEO)78 are very unstable (Figure 1a); thus, it is highly likely that (PEO)78-(PPO)30-(PEO)78 itself forms a very soft stabilizing layer on the bubble surface. We do not clearly understand the mechanism by which (PEO)78-(PPO)30-(PEO)78 is able to stiffen the oleosin-layer on the bubble surface. However, prior studies on the effect of (PEO)78-(PPO)30-(PEO)78 on cell membranes provide some interesting observations. (PEO)78-(PPO)30-(PEO)78 has been used for several decades as a cell membrane sealing agent to protect cells against external shocks. It has been proposed that (PEO)78-(PPO)30-(PEO)78 reseals the injured cell membrane and enhances the functional recovery of cells against electrical, thermal, or other membrane-damaging purtubations.26−28 Also, (PEO)78-(PPO)30-(PEO)78 has been shown to increase the mean bursting membrane tension as well as the mean elastic area compressibility modulus of hybridoma cells.26 Despite these interesting observations, the exact molecular mechanisms behind membrane stiffening effect of (PEO)78-(PPO)30(PEO)78 remain unresolved. Intrigued by the effect of (PEO)78-(PPO)30-(PEO)78 on the elasticity of oleosin-30G-stabilized gas bubbles, we blend different types of PEO-PPO-PEO triblock copolymers with oleosin-30G and study their impact on the bubble mechanics. We use (PEO)51-(PPO)31-(PEO)51 (Pluronic F77, Mw = 6600 g/mol), (PEO)37-(PPO)56-(PEO)37 (Pluronic P105, Mw = 6500 g/mol), and (PEO)13-(PPO)30-(PEO)13 (Pluronic L64, Mw = 2900 g/mol), while keeping the molar ratio of oleosin30G and PEO-PPO-PEO in the bubble preparation solution constant at 1:18. These triblock copolymers are chosen as they are commercially available and also known to stabilize aqueous foams effectively. Interestingly, all of the oleosin-30G stabilized bubbles blended with PEO-PPO-PEOs, except the ones blended with (PEO)78-(PPO)30-(PEO)78, exhibit softening behaviors, as

Figure 4. Areal expansion modulus of gas bubbles made with different amounts of (PEO)78-(PPO)30-(PEO)78. (a) T vs α of gas bubbles with (▲) 1 mg/mL oleosin-30G, (●) 1 mg/mL oleosin-30G and 10 mg/ mL (PEO)78-(PPO)30-(PEO)78, and (■) 1 mg/mL oleosin-30G and 20 mg/mL (PEO)78-(PPO)30-(PEO)78. (b) Changes in areal expansion modulus (Ka) of oleosin-30G-stabilized gas bubbles as a function of the concentration of (PEO)78-(PPO)30-(PEO)78 in the bubble preparation solution.

shown in Figure 5. In particular, (PEO)13-(PPO)30-(PEO)13 reduces the areal expansion modulus of the oleosin-30Gstabilizing layer by more than 50%. Softer bubbles are seen to yield at higher areal strains; however, the yield tension of all bubbles is around 30 dyn/cm regardless of the structure of the blended PEO-PPO-PEO triblock copolymer. We infer correlations between the molecular structure of PEO-PPO-PEO triblock copolymer and the changes in the expansion modulus of the stabilizing layer. (PEO)78-(PPO)30(PEO) 78 , (PEO) 51 -(PPO) 31 -(PEO) 51 , (PEO) 13 -(PPO) 30 (PEO)13, and oleosin-30G all have approximately the same number of monomeric units in the hydrophobic domains. As the length of the hydrophilic domain of the PEO-PPO-PEO triblock copolymer becomes shorter, the areal expansion modulus of the bubble stabilizing layer is further reduced. These observations point to the conclusion that triblock copolymers with short hydrophilic domains lead to softening of the oleosin-30G-based stabilizing layer on the bubble surface. An analogous inference can be made by comparing the softening behavior of bubbles made with PEO37-PPO56-PEO37 and PEO51-PPO31-PEO51. These two polymers have about the same molecular weight but have very different hydrophilic− hydrophobic monomer ratios. Again, the triblock copolymer with shorter hydrophilic domains leads to more significant 374

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as the concentration of PEO-PPO-PEO. In general, PEO-PPOPEO with short hydrophilic blocks softens the stabilizing layer on the bubble surface. Somewhat unexpectedly, stiffening of oleosin-30G stabilized gas bubbles can be achieved by using PEO-PPO-PEO with long hydrophilic domains. We believe this method of blending PEO-PPO-PEO with oleosin provides a simple yet versatile method to tailor the mechanical properties of gas bubbles, which will have important implications in controlling their response to ultrasound and thus their echogenicity and therapeutic functionality. Moreover, such an approach could potentially be used to tailor the mechanical properties of other types of membranes structures (e.g., vesicles, worm-like micelles, bilayer sheets, etc.) that are formed by the self-assembly of amphiphilic peptides and proteins such as elastin and oleosin.6,29,30

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00057. Experimental details, the effect of blend ratio of oleosin30G and PEO-PPO-PEO on the bubble stability (Figure S1), the effect of the concentration of oleosin-30G on the bubble stability (Figure S2), the effect of bubble size on the tension−strain curve (Figure S3), budding behavior of a oleosin-30G-stabilized gas bubble during micropipette aspiration with a narrow micropipette (Figure S4). Molecular weight and structure of PEO-PPO-PEO triblock copolymers used in the present study, and the corresponding mechanical properties of oleosin-30Gstabilized gas bubbles blended with PEO-PPO-PEO (Table S1; PDF).

Figure 5. Tailoring the mechanical properties of oleosin-30Gstabilized bubbles by blending different PEO-PPO-PEO triblock copolymers. (a) T−α curves of oleosin-stabilized bubbles blended with different PEO-PPO-PEO triblock coplymers. Filled circle symbol indicates pure oleosin-30G-stabilized bubbles, and empty symbols indicate the blended bubbles with different PEO-PPO-PEO triblock copolymers (x, y denote the number of repeat unit of each monomer in (PEO)x-(PPO)y-(PEO)x). The blend ratio of oleosin-30G to PEOPPO-PEO triblock copolymer is 1:18 in molar concentrations. (b) The areal expansion modulus of bubble stabilizing layers with different PEO-PPO-PEO triblock copolymers.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Addresses ‡

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, U.S.A. § Department of Chemical Engineering, Ajou University, Suwon 16499, South Korea.

softening of the bubble stabilizing layer. The longer hydrophobic domain of PEO37-PPO56-PEO37 compared to that of PEO51-PPO31-PEO51 may also contribute to the softening behavior. Although we do not clearly understand the physical mechanism behind the inferred correlation between the molecular structure of PEO-PPO-PEO triblock copolymers and the changes in the mechanical properties of oleosin-30Gstabilized gas bubbles, our results point to the fact that the hydrophilic domain of the added triblock copolymers plays a significant role in determining the overall mechanical properties of oleosin-30G-stabilized gas bubble and that the protein layer on the bubble surface can even be stiffened by the addition of an appropriately structured triblock copolymer surfactant. The physical mechanism behind the observed trends warrants future investigations. In this work, we have demonstrated that the mechanical properties of gas bubble-stabilizing layer based on a recombinant protein, oleosin-30G, can be tailored by using PEO-PPO-PEO triblock copolymers. The areal expansion modulus of oleosin-30G-stabilized gas bubbles, as determined using micropipette aspiration, depends on the structure as well

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Berkman Opportunity Fund, the Africk Fund, and Penn MRSEC (NSF DMR-1120901). Synthesis of oleosin was supported by NSF DMR-1309556. Training for micropipette aspiration and interpretation of results, as well as the activities of W.-S.J., was supported by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES), under Award No. DE-SC0007063.

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