LETTER pubs.acs.org/Langmuir
Giant Vesicles with Membranous Microcompartments Yukihisa Okumura,* Takayuki Nakaya, Hiroshi Namai, and Koji Urita Department of Chemistry and Material Engineering, Shinshu University, 4-17-1 Wakasato, Nagano, Nagano 380-8553, Japan
bS Supporting Information ABSTRACT: Incubation of a cell-sized lipid membrane vesicle (giant vesicle, GV) in a diluted aqueous solution of neutral phosphate buffer salts or glucose transformed the GV to an oligovesicular vesicle (OVV) that encapsulates one or more smaller GVs. During the incubation, the membrane of flaccid vesicle invaginated and closed to form the inner vesicle of an OVV engulfing a part of the bulk aqueous phase. Using the GVto-OVV transformation, an OVV that has different aqueous contents in each membranous microcompartment was constructed.
’ INTRODUCTION Recently, construction of microchemical systems that mimic biological cells (“artificial cells”) or reconstituted biochemical systems on cell-sized lipid membrane vesicles (giant vesicles, GVs) has been appearing.1 In a biological cell, various membranous substructures are present as cell organelles, and these subsystems allow a cell to perform highly sophisticated functions that would otherwise be difficult or impossible to achieve with plasma membrane alone. Future studies of artificial cells or complex cell functions may need an advanced cell membrane model that has substructures or inner compartments. A GV that contains one or more smaller GVs, or an oligovesicular vesicle (OVV), has the required topological feature.2,3 OVVs have been known to form as a minor byproduct in GV preparation.4 Also, during morphological change of GV membrane under high osmotic stress, temperature change, or presence of a detergent, transformation to OVVs has been observed.3,5 7 In the present study, GV-to-OVV transformation was induced under much milder conditions compared with the previous cases: by incubation of preformed GVs with a small amount of neutral phosphate buffer salts or other common substances such as glucose at ambient temperature. In the previous examples of OVV formation, encapsulation into OVVs has rarely been explored. As an advanced cell membrane model, the contents of each aqueous compartment of an OVV must be controlled separately. In the present work, by using the morphological transformation of a preformed, substance-encapsulating GV as a key step, an OVV that has different contents in its aqueous compartments was constructed. ’ RESULTS AND DISCUSSION GVs were prepared by the electroformation.8 The formation chamber has two thin platinum wire electrodes placed in parallel (diameter 0.50 mm) and was equipped with an inlet and an outlet for substitution of an aqueous solution in the chamber (see the r 2011 American Chemical Society
Supporting Information, Figure S1 for the scheme). Egg yolk phosphatidylcholine (EggPC; Avanti Polar Lipids, Alabaster, AL, USA) was deposited on the electrode as a methanol solution (5.0 mM, 2.0 μL). After drying, the chamber was filled with MilliQ grade water (0.50 mL), and application of sinusoidal ac voltage (5.0 V peak-to-peak, 2.0 Hz) for 120 min yielded GVs attached to the electrode wire (typical diameter 10 50 μm). GVs were observed with an inverted microscope (Olympus IX-50, Tokyo, Japan) with a digital image enhancement system. At this point, approximately 500 GVs were present, and OVVs were only sparsely seen (less than 3% of the total GVs). In fact, electroformation has been known to yield less OVVs or other “defective” vesicles than other GV preparation methods.4 Then, 0.50 mL of a diluted neutral phosphate buffer solution (0.80 mM, pH 7.0) was slowly added to the aqueous phase in the formation chamber (0.50 mL) to make the final concentration 0.40 mM. After 120 min, newly formed OVVs were present among ordinary GVs (Figure 1a). For the evaluation of the OVV formation, typically 80 100 vesicles were randomly examined. Approximately 15 20% of the preformed GVs were transformed to OVVs. Most of the OVVs contained a single inner GV (Figure 1b), but some with multiple GVs were also seen (Figure 1c). The OVV formation process was further studied in the presence of green fluorescent polystyrene particles (F-PS; Fluoresbrite-YG Carboxylate Microspheres from Polyscience (Warrington, PA, USA; average diameter 200 nm)). The particles were suspended in the phosphate buffer and introduced as a small aliquot (0.50 mL of 0.066% w/w) into the chamber of preformed GVs. After 180 min, the bulk aqueous phase was replaced with pure water. Fluorescence observation showed nests of F-PS (Figure 2). The position of the particle nests exactly corresponded to that of the inner GVs. As the inner GV moved within Received: February 2, 2011 Revised: March 3, 2011 Published: March 11, 2011 3279
dx.doi.org/10.1021/la2004485 | Langmuir 2011, 27, 3279–3282
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Figure 1. Oligovesicular vesicles formed after incubation in a neutral phosphate buffer solution (0.40 mM, pH 7.0). (a) An OVV among other GVs. The electrode appears as a black shadow at the bottom. (b) Close-up view of an OVV with a single inner GV and (c) view with multiple ones. The bar indicates 20 μm.
Figure 2. Green fluorescent polystyrene particles (F-PS) in the inner GV of an OVV. Observation with (a) bright field, (b) bright field and fluorescence, and (c) fluorescence only. The bar indicates 20 μm.
the outer GV, the nest changed its position accordingly. The random movement of the particles, presumably by Brownian motion, was confined only to the interior of the inner GV. No particles were seen in the intermediate aqueous phase between the outer and inner GV membranes. No adsorption of the particles onto the GV membrane was visible. Approximately 60 70% of the OVVs possessed the F-PS. The F-PS always appeared as particle nests, indicating that the encapsulation should have occurred while the GVs were exposed to large population of F-PS. One of the key phenomena that lead to the OVV formation is fluctuation of GV membrane. After the electroformation, the fluctuation was barely visible. The addition of the buffer caused wobbling of the membrane in approximately 40% of the GVs (out of 80 100 GVs randomly examined). Then, some GVs became flaccid and were transformed to OVVs (Figure 3). During the process, a small amount of the external aqueous phase was engulfed and became the interior of the newly formed inner GV (also see Figure S2 of Supporting Information for another example of the OVV formation). Sometimes, the transformation was very slow and stopped before the completion. Then, it reversed from a discoid or even from a shape similar to Figure 3d (Figure 4). However, transformation from a once-formed OVV to a GV was not observed. The morphological transformation similar to Figure 3 or 4 was seen many times and was reproducible. The membrane of the OVV thus formed was no longer fluctuating. With the buffer, 25 40% of the fluctuating GVs were transformed to OVVs. The OVVs thus formed were stable, and no detectable change was seen in 3 h after their formation. Table 1 summarizes the OVV formation under various conditions. The optimum concentration of the phosphate buffer for the OVV formation was around 0.40 mM. At lower concentration (0.04 mM), the number of fluctuating GVs was clearly
Figure 3. Fluctuation and transformation of a GV. A fluctuating GV underwent large deformation that resulted in OVV formation. Images were taken at (a) the beginning and after (b) 40, (c) 50, (d) 80, (e) 120, and (f) 440 s. The electrode wire is present at the bottom of the frames. The bar indicates 20 μm.
Figure 4. Slow transformation of a GV to an OVV. This time, the transformation reversed at a point just before the completion and halted for a while (d f), and then again proceeded to form an OVV. Images are taken at (a) the beginning and after (b) 6, (c) 6.5, (d) 7, (e) 27, (f) 38, (g) 73, and (h) 77 min. The bar indicates 10 μm.
smaller than that at the optimum concentration. Higher buffer concentration (2.0 mM) resulted in many collapsed GVs probably due to high osmotic stress. An experiment using pure water in place of the buffer showed neither the fluctuation nor OVV formation, indicating that the deformation was not simply caused by the prolonged incubation. 3280
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Table 1. GV-to-OVV Transformation by Incubation with Various Aqueous Solutions substance incubated with preformed GVs none (pure water) neutral phosphate buffer
OVVs (%)
0.04 mM
0 0
0.40 mM
20
0.40 mM
13
2.0 mM
0a
NaCl
0.60 mM
9
KI
0.60 mM
4
phthalate buffer
glucose
a
GVs transformed to concentration
0.10 mM
7
0.75 mM 1.0 mM
5 0a
sucrose
1.0 mM
4
1,2-ethanediol
1.0 mM
5
dextran (MW ∼70 000)
0.036 wt %
0
Figure 5. OVV formed from a lipid mixture. GVs were prepared using POPC/POPG/cholesterol (90/10/40) and incubated in a neutral phosphate buffer solution (0.40 mM, pH 7.0). The bar indicates 20 μm.
Collapsed.
Various water-soluble substances were found to be effective in the induction of OVV formation (Table 1). Among the ones tested, neutral phosphate buffer was the most efficient agent, although salts of monovalent ions (NaCl or KI) or low molecular nonionic species (glucose, sucrose, or 1,2-ethanediol) also induced the transformation. Similar fluctuation of GV membrane and following transformation to an OVV has been known to occur under high osmotic stress (for example, 10 mM glucose),2,3,9 temperature change (around 42 43 °C),5 phase transition,7 or the presence of a detergent such as Triton X-100.3,6 It is somewhat surprising that rather low concentration of relatively common and inert substances at ambient temperature could trigger such the large morphological change in GVs. For construction of an OVVbased microchemical systems, a less intrusive method should be preferable. High temperature could cause damage to sensitive biomolecules. A trace of a detergent in membrane could become irremovable and permanently modify the membrane properties. Considering the diversity in the types of effective inducing agents, the present fluctuation is likely to be an osmotic phenomenon.2,9 In fact, when dextran (approximate molecular weight 70 000 obtained from Tokyo Kasei (Tokyo, Japan)) was used at the concentration that worked well with glucose (0.018% w/w), less GV fluctuation and failed OVV formation was observed probably due to insufficient osmotic stress. The fluctuation causes occasional large deformation and could produce a part of the membrane with high curvature and loose lipid packing (such as the edge of the hole in Figure3d). Reorganization of the membrane lipid molecules at the unstable part could result in membrane fusion and the OVV formation. The total area of the membrane of the OVV (the outer and the inner vesicles combined; Figure 3f) seems to be larger than that of the original GV (Figure 3a; this can be more clearly seen in Figure S2 of Supporting Information). This may in part be explained by membrane expansion under the conditions observed in the previous study of similar GV deformation.5 Also, GVs obtained by electroformation are associated with the lipid layer on the electrodes,8 which could provide the lipid necessary
Figure 6. OVV with heterogeneous inner aqueous compartments. (a) Bright field observation. (b) Fluorescence from RITC-Dex. (c) Fluorescence from encapsulated F-PS. The bar indicates 20 μm.
for the OVV formation. The GVs may later be detached from the lipid layer and the electrode. GV-to-OVV transformation described in this study also occurred with mixed lipids that are often used for model membranes. The diluted phosphate buffer worked well with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/1-palmitoyl2-oleoylphosphatidylglycerol (POPG) (90/10), POPC/cholesterol (100/40), and POPC/POPG/cholesterol (90/10/40) (Figure 5; the phospholipids and cholesterol were obtained from Avanti Polar Lipids and Wako Pure Chemicals (Tokyo, Japan), respectively). In all cases, the transformation was essentially the same as that with EggPC, and 15 18% of preformed GVs were transformed to OVVs. The insensitivity of the phenomenon to the presence of an anionic lipid in GV membrane suggests that contribution of interaction between the membrane and the electrolyte ions to the transformation should be small. Using the transformation in combination with replacement of the external aqueous phase, one may construct an OVV with heterogeneous inner aqueous compartments. To demonstrate the idea, GVs were prepared in an aqueous solution (0.040% w/ w) of rhodamine B isothiocyanate-dextran (RITC-Dex; average molecular weight 70 000 obtained from Sigma-Aldrich (St. Louis, MO, USA)). After removing the RITC-Dex remaining in the external aqueous phase by gently replacing with pure water, an equal volume of F-PS suspended in a phosphate buffer solution (0.80 mM) was then added. The following incubation (120 min) and replacement of the external F-PS with pure water yielded OVVs that possessed different contents in each aqueous compartments in a single OVV (Figure 6). The RITC-Dex was present in the intermediate aqueous phase between the inner and the outer vesicles (the inner GV appeared darker but not totally dark due to the overlapping fluorescence from the intermediate aqueous phase), and the F-PS particles were confined in the inner vesicle. Introduction of another chemical component to the external bulk aqueous phase 3281
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Langmuir produces a membrane-based microchemical system with ternary heterogeneous aqueous phases. The importance of a vesicular membrane model system with heterogeneous inner environment has begun to be recognized. An interesting system proposed by Keating and co-workers used separation of an aqueous two-phase system of poly(ethylene oxide)-dextran-water for microcompartmentation in GVs without inner membranous structures.10 The present study showed the possibility of constructing a model system that has membrane-separated heterogeneous microcompartments. The concept demonstrated here may also be valid with GV-to-OVV transformation that is induced by other triggers such as temperature change. We are presently working on development of a microchemical system based on the approach along with the further elucidation of the mechanism of the transformation from a GV to an OVV.
LETTER
(9) Hotani, H.; Nomura, F.; Suzuki, Y. Curr. Opin. Colloid Interface Sci. 1999, 4, 358–368. (10) (a) Long, M. S.; Cans, A.-S.; Keating, C. D. J. Am. Chem. Soc. 2008, 130, 756–76. (b) Domminak, L. M.; Gundermann, E. L.; Keating, C. D. Langmuir 2009, 26, 5697–5705.
’ ASSOCIATED CONTENT
bS
Supporting Information. A scheme for the formation chamber and pictures of another GV transformed to an OVV. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Address: Department of Chemistry and Material Engineering, Shinshu University, 4-17-1 Wakasato, Nagano, Nagano 3808553, Japan. Tel: 81-26-269-5399. Fax: 81-26-269-5424. E-mail:
[email protected].
’ ACKNOWLEDGMENT The authors are grateful to Mr. Shun Horiuchi for his assistance in some of the experiments. A part of this study was supported by Grant-in-Aids for Scientific Research (B) (16310099) from Japan Society for the Promotion of Sciences (JSPS). ’ REFERENCES (1) (a) Takiguchi, K.; Yamada, A.; Negishi, M.; Tanaka-Takiguchi, Y.; Yoshikawa, K. Langmuir 2008, 24, 11323–11326. (b) Merkle, D.; Kahya, N.; Schwille, P. ChemBioChem 2008, 9, 2673–2681. (c) Streicher, P.; Nassoy, P.; B€armann, M.; Dif, A.; Marchi-Artzner, V.; Brochard-Wyart, F.; Spatz, J.; Bassereau, P. Biochim. Biophys. Acta 2009, 1788, 2291–2300. (d) Sunami, T.; Hosoda, K.; Suzuki, H.; Matsuura, T.; Yomo, T. Langmuir 2010, 26, 8544–8551. (2) (a) Menger, F. M.; Lee, S. J.; Kieper, J. S. Langmuir 1996, 12, 4479–4480. (b) Girard, P.; Pecreaux, J.; Lenoir, G.; Falson, P.; Rigaud, J.-L.; Bassereau, P. Biophys. J. 2004, 87, 419–429. (3) Hamada, T.; Miura, Y.; Ishii, K.; Araki, S.; Yoshikawa, K.; Vestergaard, M.; Takagi, M. J. Phys. Chem. B 2007, 111, 10853–10857. (4) (a) Rodriguez, N.; Pincet, F.; Cribier, S. Colloids Surf., B 2005, 42, 125–130. (b) Kuribayashi, K.; Tresset, G.; Coquet, Ph.; Fujita, H.; Takeuchi, S. Meas. Sci. Technol. 2006, 17, 3121–3126. (5) K€as, J.; Sackman, E. Biophys. J. 1991, 60, 825–844. (6) Babnik, B.; Miklavcic, D.; Kanduser, M.; H€agerstrand, H.; Kralj-Iglic, V.; Iglic, A. Chem. Phys. Lipids 2003, 125, 123–138. (7) Leirer, C.; Wunderlich, B.; Myles, V. M.; Schneider, M. F. Biophys. Chem. 2009, 143, 106–109. (8) Angelova, M. I. In Giant Vesicles; Luisi, P. L., Walde, P., Eds.; John Wiley & Sons: Chichester, U.K., 2000; pp 27 36. 3282
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