Quantitative Study of the Structure of Multilamellar Giant Liposomes As

Oct 30, 2008 - properties of a model cell system based on giant liposomes. Introduction. Liposomes are small vesicles composed of lipid bilayer membra...
0 downloads 0 Views 1MB Size
Download PDF
13540

Langmuir 2008, 24, 13540-13548

Quantitative Study of the Structure of Multilamellar Giant Liposomes As a Container of Protein Synthesis Reaction Kazufumi Hosoda,†,⊥ Takeshi Sunami,‡,⊥ Yasuaki Kazuta,† Tomoaki Matsuura,† Hiroaki Suzuki,† and Tetsuya Yomo*,†,‡,§ Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka UniVersity, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan, Exploratory Research for AdVanced Technology (ERATO), Japan Science and Technology Agency, Tokyo 135-0064, Japan, and Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka UniVersity, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan ReceiVed July 28, 2008. ReVised Manuscript ReceiVed September 12, 2008 Liposomes are widely used as cell-sized compartments for encapsulation of biochemical reaction systems to construct model cell systems. However, liposomes are usually diverse in both size and structure, resulting in highly heterogeneous properties as microreactors. Here, we report the development of a strategy to investigate the internal structure of giant multilamellar vesicles (GMLVs) formed by the freeze-dried empty liposomes (FDEL) method as containers of an in Vitro transcription/translation system. To evaluate the occurrence of the protein synthesis reaction in GMLVs, we designed a cascade reaction system in which a synthesized enzyme hydrolyzes the fluorescent substrate, and thus the space where the reaction takes place in liposomes becomes fluorescent. We found that only a part of the liposome was reactable and not the entire internal volume, i.e., the hydrolysis reaction took place in only a part of the fractured compartment volumes in GMLVs. Simultaneous measurement of the whole internal volume of the liposomes and the quantity of reaction product of more than 100 000 liposomes using a fluorescence-activated cell sorter (FACS) revealed that the distribution of reactable volume was proportional to the whole internal volume regardless of the liposome size, i.e., the relation between the quantity of whole and reactable volume in GMLV was found to be scale-free. This information would allow us to reduce the geometric parameters of GMLV for quantitative analysis of reaction kinetics in liposomes. The present measurement and analysis method will be an indispensable tool for exploring high-dimensional properties of a model cell system based on giant liposomes.

Introduction Liposomes are small vesicles composed of lipid bilayer membranes, and are widely used in both basic and applied research in life sciences.1,2 Especially, giant liposomes (diameter >1 µm) are of particular interest as a cell model and a microreactor containing biochemical reaction systems in a cellsized volume.3-8 Reconstruction of such a cell model from defined biochemical components, with which the characteristics of the extant cells are expected to be elicited,9,10 is a challenging task for biochemists and chemical engineers. In practice, researchers attempt to encapsulate biochemical reaction systems in lipo* To whom correspondence should be addressed. Tel: +81-6-6879-4171. Fax: +81-6-6879-4151. E-mail: [email protected]. † Graduate School of Information Science and Technology, Osaka University. ‡ Japan Science and Technology Agency. § Graduate School of Frontier Biosciences, Osaka University. ⊥ These authors contributed equally to this work. (1) Luisi, P. L.; Walde, P. Giant Vesicles; John Wiley & Sons, Inc.: New York, 2000. (2) Torchilin, V.; Wessig, V. Liposomes: A Practical Approach, 2nd ed.; Oxford University Press: Oxford, 2003. (3) Kaneko, T.; Itoh, T. J.; Hotani, H. J. Mol. Biol. 1998, 284, 1671–1681. (4) Tsumoto, K.; Nomura, S. M.; Nakatani, Y.; Yoshikawa, K. Langmuir 2001, 17, 7225–7228. (5) Hanczyc, M. M.; Fujikawa, S. M.; Szostak, J. W. Science 2003, 302, 618–622. (6) Hanczyc, M. M.; Szostak, J. W. Curr. Opin. Chem. Biol. 2004, 8, 660–664. (7) Nomura, S.; Tsumoto, K.; Hamada, T.; Akiyoshi, K.; Nakatani, Y.; Yoshikawa, K. ChemBioChem 2003, 4, 1172–1175. (8) Noireaux, V.; Bar-Ziv, R.; Godefroy, J.; Salman, H.; Libchaber, A. Phys. Biol. 2005, 2, P1-P8. (9) Szostak, J. W.; Bartel, D. P.; Luisi, P. L. Nature 2001, 409, 387–90. (10) Luisi, P. L.; Ferri, F.; Stano, P. Naturwissenschaften 2006, 93, 1–13.

somes.4,7,11-15 For encapsulation of highly complex reactions consisting of a large number of components, including an in Vitro transcription/translation system,16,17 the choice of vesicle formation method is critical, as concentration and encapsulation efficiency of numbers of individual components in such dense suspension may vary over a wide range.12,18-20 Among the various vesicle formation methods proposed to date, the water-in-oil (W/O) emulsion method21,22 and freezedried empty liposomes (FDEL) method2,15,23,24 are the most promising candidates. In the former method proposed by Weitz’s group, unilamellar vesicles are produced by passing W/O emulsion droplets through a second oil-water interface. Unlike the simple (11) Yu, W.; Sato, K.; Wakabayashi, M.; Nakaishi, T.; Ko-Mitamura, E. P.; Shima, Y.; Urabe, I.; Yomo, T. J. Biosci. Bioeng. 2001, 92, 590–593. (12) Walde, P.; Ichikawa, S. Biomol. Eng. 2001, 18, 143–77. (13) Fischer, A.; Franco, A.; Oberholzer, T. ChemBioChem 2002, 3, 409–417. (14) Noireaux, V.; Libchaber, A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17669–17674. (15) Murtas, G.; Kuruma, Y.; Bianchini, P.; Diaspro, A.; Luisi, P. L. Biochem. Biophys. Res. Commun. 2007, 363, 12–17. (16) Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T. Nat. Biotechnol. 2001, 19, 751–5. (17) Kazuta, Y.; Adachi, J.; Matsuura, T.; Ono, N.; Mori, H.; Yomo, T. Mol. Cell. Proteomics 2008, 7, 1530–1540. (18) Monnard, P. A.; Oberholzer, T.; Luisi, P. Biochim. Biophys. Acta 1997, 1329, 39–50. (19) Gregoriadis, G.; McCormack, B.; Obrenovic, M.; Saffie, R.; Zadi, B.; Perrie, Y. Methods 1999, 19, 156–162. (20) Pupo, E.; Padron, A.; Santana, E.; Sotolongo, J.; Quintana, D.; Duenas, S.; Duarte, C.; de la Rosa, M. C.; Hardy, E. J. Controlled Release 2005, 104, 379–96. (21) Pautot, S.; Frisken, B. J.; Weitz, D. A. Langmuir 2003, 19, 2870–2879. (22) Yamada, A.; Yamanaka, T.; Hamada, T.; Hase, M.; Yoshikawa, K.; Baigl, D. Langmuir 2006, 22, 9824–9828. (23) Kirby, C.; Gregoriadis, G. Biotechnology 1984, 2, 979–984. (24) Kikuchi, H.; Suzuki, N.; Ebihara, K.; Morita, H.; Ishii, Y.; Kikuchi, A.; Sugaya, S.; Serikawa, T.; Tanaka, K. J. Controlled Release 1999, 62, 269–277.

10.1021/la802432f CCC: $40.75  2008 American Chemical Society Published on Web 10/30/2008

Reactable Volume in Giant Liposomes

hydration (natural swelling) method, this technique shows almost 100% encapsulation efficiency21 due to the low level of interaction between the membrane and suspended components upon encapsulation. Pore-forming antibiotic peptide was expressed and assembled in vesicles encapsulating an Escherichia colibased in Vitro transcription/translation system.14 Similar vesicle encapsulation methods using a microjet25,26 or a microfluidic system27 have been proposed. The FDEL method has long been used as a means of vesicle formation with high entrapment efficiency even for biomolecules.2,23 In this method, liposomes in suspension are lyophilized (freeze-dried) to form empty lamellae of dry lipid film. Upon rehydration, a suspension containing biochemical molecules permeates into the lamellae, which swell to form material-containing vesicles. This method provides facile, stable, and reproducible formation of lipid vesicles containing complex and dense reaction mixture. It is also important to note that this method is applicable for almost any combination of buffer and components. Therefore, we have been using this technique to encapsulate the in Vitro transcription/ translation system into liposomes. Functional proteins, such as green fluorescent protein (GFP),11,28,29 T7 RNA polymerase,28,29 Qβ RNA replicase, and β-galactosidase,30 were produced in liposomes, indicating that all biochemical components necessary were enclosed in the liposomes. The next step is the quantitative evaluation of the internal reaction within liposomes. Reactions in a small compartment volume may behave differently from those in bulk experiments, and such differences characterize the cell system. For example, because of the small number of copies of genomic DNA per cell, a large stochastic fluctuation in the phenotype of individual cells should be observed as demonstrated in the in ViVo studies.31-33 Furthermore, interactions between the cytosolic components and the membrane become significant as a result of the large surfaceto-volume ratio, which may result in the enhancement of reaction rates of membrane-bound molecules.34,35 Thus, in cellular systems, the occurrence and progress of the reaction should become a function of the compartment volume, and these features should be clarified using liposomes as a model cell system. However, as there is no morphological control in the self-assembly process, liposomes usually have multiple layers (multilamellar vesicles (MLVs)) and complex internal structures (multivesicular vesicles (MVVs)).1,2,36 Thus, liposomes have properties heterogeneous to those of microreactors, and the volume in which the reaction can take place may differ from the total internal volume. Therefore, for detailed analysis, statistical understanding of the properties of these vesicles as microreactors is necessary. The most straightforward method of examining the relationship between liposome structure and internal reaction is direct (25) Funakoshi, K.; Suzuki, H.; Takeuchi, S. J. Am. Chem. Soc. 2007, 129, 12608–12609. (26) Stachowiak, J. C.; Richmond, D. L.; Li, T. H.; Liu, A. P.; Parekh, S. H.; Fletcher, D. A. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4697–4702. (27) Tan, Y. C.; Hettiarachchi, K.; Siu, M.; Pan, Y. R.; Lee, A. P. J. Am. Chem. Soc. 2006, 128, 5656–5658. (28) Ishikawa, K.; Sato, K.; Shima, Y.; Urabe, I.; Yomo, T. FEBS Lett. 2004, 576, 387–390. (29) Sunami, T.; Sato, K.; Matsuura, T.; Tsukada, K.; Urabe, I.; Yomo, T. Anal. Biochem. 2006, 357, 128–136. (30) Kita, H.; Hosoda, K.; Sunami, T.; Ichihashi, N.; Matsuura, T.; Tsukada, K.; Urabe, I.; Yomo, T. ChemBioChem. 2008, 9, 2403–2410. (31) Elowitz, M. B.; Levine, A. J.; Siggia, E. D.; Swain, P. S. Science 2002, 297, 1183–1186. (32) Cai, L.; Friedman, N.; Xie, X. S. Nature 2006, 440, 358–362. (33) Bar-Even, A.; Paulsson, J.; Maheshri, N.; Carmi, M.; O’Shea, E.; Pilpel, Y.; Barkai, N. Nat. Genet. 2006, 38, 636–643. (34) Wang, D.; Gou, S. Y.; Axelrod, D. Biophys. Chem. 1992, 43, 117–137. (35) Axelrod, D.; Wang, M. D. Biophys. J. 1994, 66, 588–600. (36) Sato, K.; Obinata, K.; Sugawara, T.; Urabe, I.; Yomo, T. J. Biosci. Bioeng. 2006, 102, 171–178.

Langmuir, Vol. 24, No. 23, 2008 13541

observation by fluorescence microscopy.13,14,37 However, statistical analysis of a wide range of vesicle volumes, which ranges over several orders of magnitude, is difficult by optical microscopy. The size (volume) distribution of the liposome population has been evaluated by dynamic light scattering (DLS).38,39 However, the internal properties of liposomes that may affect the internal reaction cannot be clarified quantitatively. Another strategy is to use a fluorescence-activated cell sorter (FACS). With this technology, it has become possible to measure multiple properties of liposomes simultaneously at extremely high frequency (up to tens of thousands of events per second),36,40-43 and to sort the population of interest for subsequent observation and assays.29,36 In FACS measurement, with irradiation of the individual liposomes flowing through the orifice with a laser, the resulting forward and side scattering signals are correlated with the liposome size and the quantity of membrane, respectively. Using fluorescent markers, it is also possible to directly measure multiple properties of liposomes from the fluorescence intensity.36,42 In our previous studies, we showed that the cell-free synthesis of enzymes and their hydrolysis in liposomes can be detected from the high-dimensional data obtained by FACS analysis.28,29 In this study, we investigated the properties of giant liposomes formed by the FDEL method as containers of the in Vitro transcription/translation system using FACS. This attribute is strongly related to the geometrical characteristics of liposomes. In practice, the whole internal volume of the liposomes was quantified from the red fluorescence signal from a fluorescent protein encapsulated at a high concentration, while the volume where the reaction can take place (reactable volume) was evaluated from the green fluorescent signal from the final product of the hydrolysis reaction of the enzyme (β-glucuronidase) produced by the transcription/translation reaction. We showed that the green fluorescent signal was localized in only a part of the liposomes, and thus only part of the internal volume was reactable. By detailed statistical analysis, we found that such reactable volume was proportional to the whole internal volume of the liposome at a constant factor, and its volume distribution was identical when normalized by the whole volume. We also found that the average number of dominant reactable compartment in a liposome was almost 1, the volume of which was 16% of the entire volume on average. Our approach is capable of exploring the property of giant MLVs (GMLVs) as cell-sized containers for biochemical reactions involving multiple components.

Experimental Section Preparation of Freeze-Dried Empty Liposome Membranes. The procedure used for preparation of FDEL membranes was described in our previous reports.29,36 Briefly, 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC; Avanti Polar Lipids, Alabaster, AL), cholesterol (Nacalai Tesque, Kyoto, Japan), and 1,2-distearoylsn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)5000] (DSPE-PEG5000; NOF Corporation, Tokyo, Japan) dissolved in dichloromethane/diethyl ether (1:1, v/v) were mixed at a molar (37) Tamba, Y.; Yamazaki, M. Biochemistry 2005, 44, 15823–15833. (38) Maulucci, G.; De Spirito, M.; Arcovito, G.; Boffi, F.; Castellano, A. C.; Briganti, G. Biophys. J. 2005, 88, 3545–3550. (39) Woodbury, D. J.; Richardson, E. S.; Grigg, A. W.; Welling, R. D.; Knudson, B. H. J. Liposome Res. 2006, 16, 57–80. (40) Vorauer-Uhl, K.; Wagner, A.; Borth, N.; Katinger, H. Cytometry 2000, 39, 166–171. (41) Zazueta, C.; Ramirez, J.; Garcia, N.; Baeza, I. J. Membr. Biol. 2003, 191, 113–122. (42) Kageyama, Y.; Toyota, T.; Murata, S.; Sugawara, T. Soft Matter 2007, 3, 699–702. (43) Toyota, T.; Takakura, K.; Kageyama, Y.; Kurihara, K.; Maru, N.; Ohnuma, K.; Kaneko, K.; Sugawara, T. Langmuir 2008, 24, 3037–3044.

13542 Langmuir, Vol. 24, No. 23, 2008 ratio of 58:39:3 and dried using a rotary evaporator, followed by complete removal of solvent in the vacuum chamber. The dried lipid film was hydrated with Milli-Q water (12 mM lipid), and this suspension was subjected to vortex mixing for 20 s and sonication for 5 s. After passing through a polycarbonate filter with a pore size of 0.4 µm (Nuclepore Track-Etch Membranes; Whatman, Maidstone, Kent, U.K.), the suspension was dispensed into small aliquots (40 µL each), and freeze-dried overnight (Labconco Corp., Kansas, MO). After filling with Argon gas, the freeze-dried membranes were stored in a freezer. A scanning electron microscope image of the freezedried membrane is shown in the Supporting Information (Figure S1). Plasmid Preparation. Plasmid encoding β-glucuronidase (pETuidA) was prepared by inserting the uidA gene obtained by digesting pCA24NuidA (ASKA library JW1609, kindly provided by the National Institute of Genetics, Japan)44 with the restriction enzyme SfiI and ligating the insert into the plasmid pET-21a-SfiI also digested with the same enzyme. The plasmid pET-21a-SfiI was prepared by ligating pET-21a (Novagen, Madison, WI) digested with EcoRI and HindIII, with hybridized oligoDNA (hybrid of 5′-AATTCGAGGCCCTGAGGGCCAGGAGGCCTCCTGGCCTATGCGGCCGCA and 5′-AGCTTGCGGCCGCATAGGCCAGGAGGCCTCCTGGCCCTCAGGGCCTCG). Plasmid DNA encoding β-galactosidase (pET-bGal, Supporting Information) was also used to confirm that the identified properties of the internal compartments were not specific to the β-glucuronidase. In Witro Transcription/Translation Reaction and Subsequent Enzyme Activity Assay. The mixture for enzyme synthesis and subsequent hydrolysis reaction (hereafter, cascade reaction) was prepared in a test tube by adding plasmid DNA (pET-uidA) and 50 µM 5-(pentafluorobenzoylamino) fluorescein di-β-D-glucuronide (PFB-FDGlcU; Invitrogen, Carlsbad, CA) to 20 µL of the in Vitro transcription/translation system. This process was performed on ice to prevent unnecessary reaction. For the in Vitro transcription/ translation system, we used PURESYSTEM classic II (Post Genome Institute, Tokyo, Japan) or a modified version of this system developed in our laboratory (laboratory-made system) because of the insufficient stock of the commercial product. The laboratory-made system is an optimized version with increased translation efficiency (the components of the laboratory-made system are shown in the Supporting InformationTable S1), but there were no essential differences between the two systems, as will be discussed in the results of liposome experiments. The time course of changes in the green fluorescence intensity with incubation at 37 °C was measured with a real-time PCR system (Mx3005P QPCR system; Stratagene, La Jolla, San Diego, CA) at excitation and emission wavelengths of 492 and 610 nm, respectively. PFB-FDGlcU is converted into a fluorescent molecule, PFB-fluorescein, upon hydrolysis and emits green fluorescence. The peak wavelengths of excitation and emission spectra of PFB-fluorescein are 495 and 520 nm, respectively. We used an emission filter that passes the wavelength off the peak of the PFBfluorescein emission spectrum to keep the fluorescence intensity within the detection range of the PCR system. In Liposome Reaction. Liposomes containing the cascade reaction system were prepared by adding 10 µL of reaction mixture to an aliquot of freeze-dried membranes (final lipid concentration: 48 mM). The red fluorescent protein allophycocyanin (APC, 500 nM; Molecular Probes, Eugene, OR) was encapsulated together with the reaction mixture as a marker of the whole internal volume of liposomes. Then, aliquots of 1.5 µL of the liposome suspension were diluted 20-fold with dilution buffer (reaction mixture without DNA, substrate, and APC). Protease from Streptomyces griseus (Sigma-Aldrich Corporation, St. Louis, MO) was included in the dilution buffer at a final concentration of 1 mg/mL to inhibit any reaction that may occur outside the vesicles. We confirmed in test tube experiments that this protease concentration was sufficient to completely suppress the cascade reaction initiated by the plasmid DNA in the concentration range used in this study (data not shown). (44) Kitagawa, M.; Ara, T.; Arifuzzaman, M.; Ioka-Nakamichi, T.; Inamoto, E.; Toyonaga, H.; Mori, H. DNA Res. 2005, 12, 291–299.

Hosoda et al. All preparation procedures mentioned above were performed on ice. The reaction was then initiated by incubation at 37 °C, and the suspension was time-sampled and subjected to FACS measurement and microscopy observation. Measurement of Whole and Reactable Volumes of Liposomes by FACS. Prior to FACS measurement, the sampled liposome suspension was further diluted 10-fold with a sheath solution of FACS to reduce the measurement frequency to smaller than 20 000 events/s. Then, red and green fluorescent signals, reflecting the whole internal volume and the amount of product, respectively, were measured with the FACS (FACSAria; Becton Dickinson, San Jose, CA). We obtained 100 000 data samples under each measurement condition. Briefly, APC was excited with a HeNe laser (633 nm), and the emission was detected through a 660 ( 10 nm bandpass filter. The number of APC molecules was converted from the red fluorescent signal detected (FIR) using the linear relation between the intensity from fluorescent beads carrying a known amount of another fluorescent protein R-phycoerythrin (R-PE) (QuantiBRITE PE Quantitation kit; BD Biosciences Clontech, Palo Alto, CA) and APC (the intensity per single molecule of PE (c1) and the intensity ratio of APC to PE (c2) were 4.4 × 10-17 and 1.40, respectively). The quantity of the whole volume (Vw) was calculated by dividing the number of APC molecules by the concentration of encapsulated APC (CAPC ) 500 nM). The final equation used for conversion was Vw ) FIR/(c1 · c2 · NA · CAPC) ) 0.054FIR, where NA is Avogadro’s number. The observed reactable volume (Vr,obs) was derived from the intensity of the green fluorescent signal (FIG). PFB-fluorescein, the reaction product of β-glucuronidase enzyme reaction, was excited with a 488 nm semiconductor laser, and the emission was detected through a 530 ( 15 nm bandpass filter. For conversion, we first measured the linear constant (c3) between the red fluorescent intensities from 500 nM APC and the green fluorescent intensities from 5 µM fluorescein-tagged bovine serum albumin (BSA; albumin from bovine serum fluorescein conjugate; Invitrogen) (FIG,BSAF) encapsulated together in liposomes (Vw ) 0.054FIR ) c3 · FIG,BSAF ) 0.097FIG,BSAF, Supporting InformationFigure S2). Then, we derived the intensity ratio of the PFB-fluorescein to the fluorescein-tagged BSA using a fluorometer (c4 ) 0.88). Using these constants, the volume of the compartment where the reaction took place was calculated from FIG using the concentration of encapsulated substrate (50 µM). The final equation used for conversion was Vr,obs ) (c3/ c4)(5/50)FIG ) 0.011FIG. Microscopic Observation. Microscopic observation of the liposomes was carried out using an inverted light microscope (IX70; Olympus, Tokyo, Japan). The liposomes used for the microscopic observations were prepared by sorting the liposomes of interest by FACS that exhibited definite red and green fluorescence. The whole and reacted volumes of liposomes were estimated from the apparent diameter by assuming these volumes have spherical shape.

Results Measurement Principle of the Whole and Reactable Volumes in Liposomes. Here, we describe the measurement principle of the entire internal volume (whole volume, Vw) and the volume where the cascade reaction occurs (reactable volume, Vr) in individual liposomes based on the physical model illustrated in Figure 1. The cascade reaction system to be encapsulated consists of the plasmid DNA, the in Vitro transcription/translation system, and fluorogenic substrate (Figure 1a). The gene encoded on the plasmid DNA was transcribed and translated to produce the enzyme β-glucuronidase, which hydrolyzes the substrate (fluorescein di-β-D-glucuronide) to liberate green fluorescein. To investigate the occurrence of the reaction in liposomes, we measured Vw and the quantity of reaction product as shown in Figure 1b. The whole volume of a liposome, which was defined as the volume where the red fluorescent protein (500 nM APC) was encapsulated, was evaluated from the red fluorescent intensity, whereas the quantity of the reaction product was evaluated from the green fluorescent signal. When carried out in GMLVs, the

Reactable Volume in Giant Liposomes

Langmuir, Vol. 24, No. 23, 2008 13543

Figure 1. Strategy for quantifying the internal structure of liposomes. (a) Schematic of the cascade reaction to be encapsulated in liposomes. The enzyme β-glucuronidase was synthesized from the plasmid DNA by the in Vitro transcription/translation system, and the enzyme produced hydrolyzed the substrate that emits green fluorescence after reaction. (b) Schematic of determining the reactable volume in GMLV containing the cascade reaction system. Encapsulation of the plasmid DNA into a liposome at low or high concentration allows elucidation of quantitative information of the distribution of the reactable volume. As the reaction proceeds only in the compartment where DNA is present, a single reactable compartment volume is detected at low plasmid concentration, while the sum of multiple reactable compartments should be detected at higher plasmid concentration. Red fluorescent protein (APC) is encapsulated as a marker of the whole liposome volume.

reaction did not occur in all of the liposome volume, and the amount of green fluorescence product differed from the quantity of substrate encapsulated in the whole volume of liposomes, as discussed below. We investigated this mismatch between the whole and reacted volumes based on the following hypothesis: each liposome carries multiple internal compartments that are isolated from each other. As the components of the in Vitro transcription/translation system vary in their concentrations (the present system contains nearly 100 components with concentrations ranging from nanomolar to millimolar), it is likely that these components are not distributed uniformly in all internal compartments. Especially, encapsulation of components with concentrations in the nanomolar range is likely to be a stochastic event in compartments with a volume in the 1 fL range or smaller. Consequently, the reaction takes place only in the space where all of the components necessary are present (reactable volume). On the basis of this hypothesis, we studied the distribution of the fraction of the resulting reactable volume by varying the concentration of plasmid DNA, the material that initiates the reaction, as illustrated in Figure 1b. By designing the conditions such that the concentration of plasmid DNA in suspension is low (less than 1 plasmid/liposome on average), no or only one plasmid DNA will be encapsulated in a liposome. If the space where the plasmid DNA is encapsulated is reactable, the cascade reaction takes place upon incubation, which results in the appearance of a green fluorescent signal. Thus, among multiple internal compartments in GMVL, only a reactable part with the plasmid DNA should fluoresce (upper region of Figure 1b). In this case, the volume where the reaction was observed can be different from the sum of the reactable volume if a liposome contains multiple reactable spaces. On the other hand, when the plasmid DNA is encapsulated at larger concentration, the sum of the

reactable volumes should be observed (lower region of Figure 1b), as the plasmid DNA is likely to be distributed in all reactable spaces. In both cases, the volume where the reaction took place was calculated by dividing the amount of product (derived from the green fluorescence intensity) by the concentration of the loaded substrate. Here, this volume directly converted from the green fluorescence intensity is denoted as the observed reacted volume, Vr,obs, as it may differ from the total quantity of the reactable volume (Vr,total) depending on the plasmid concentration. To convert the reactable volume from the fluorescence intensity, the concentration of the product in Vr,obs must be the same as that of the loaded substrate. This can be achieved at the end point of the reaction, where all of the substrate encapsulated at high concentration (50 µM) in compartments was completely hydrolyzed. As FACS can measure both red and green fluorescent signals of individual liposomes, the relation between the whole and reactable volumes can be determined. Cascade Reaction In Vitro As described above, it is important that the β-glucuronidase produced by the transcription/translation reaction hydrolyze all of the fluorescent substrate present to quantify the reactable volume. Thus, we first examined the reaction in a test tube. The time courses of changes in the green fluorescence intensity of the reaction using the in Vitro transcription/translation system (PURESYSTEM classic II) are shown in Figure 2. In the reaction mixture with no plasmid DNA, no increase in fluorescence intensity was observed (+). However, the fluorescence intensity increased rapidly and saturated within 150 min in the mixture containing 0.21 nM plasmid DNA (b). This plateau matched the intensity of the fluorescent product at the same concentration prepared separately (gray solid line). Thus, we confirmed that all of the substrate (PFB-fluorescein) was hydrolyzed in this cascade reaction system from a small amount of plasmid DNA, which is a prerequisite in the model

13544 Langmuir, Vol. 24, No. 23, 2008

Figure 2. Time courses of the cascade reaction using the in Vitro transcription/translation system (PURESYSTEM classic II) in a test tube. b and + represent the normalized green fluorescent intensities of the reaction mixture with plasmid concentrations of 0.21 and 0 nM, respectively. The gray solid line shows the fluorescence intensity of the 50 µM reaction product (PFB-fluorescein) prepared separately.

Figure 3. Dot plot representation of the FACS measurement data on a logarithmic scale. Each dot represents the data of a single liposome. The x- and y-axes are the volumes converted from the green and red fluorescent intensities, respectively (Vr,obs and Vw). (a) Result of the reaction in liposomes without plasmid DNA after 6-h incubation. (b) Result of the reaction in liposomes with 0.21 nM plasmid DNA after 6-h incubation. In both plots, 30 000 data points from the total of 100 000 obtained are shown for clarity. The gray solid line represents the theoretical line of Vw ) Vr,obs. Liposomes in regions (i)-(iv) were sorted out and observed by fluorescent microscopy (see Figure 4).

proposed in Figure 1. We also confirmed that the plasmid concentration is the rate limiting factor of transcription and translation reaction in the range used in this study (Supporting InformationFigure S3). Thus, the amount of β-glucuronidase synthesized should depend on the plasmid concentration, although the concentration of PFB-fluorescein at the end point of the cascade reaction will not be affected, as all substrates are hydrolyzed (Supporting InformationFigure S4). FACS Measurements of Reactions in Liposomes. Next, we encapsulated the reaction mixture into liposomes composed of a mixture of POPC, cholesterol, and DSPE-PEG5000 at a molar ratio of 58:39:3 by the FDEL method. After incubation, the red and green fluorescent signals from individual liposomes were measured by FACS. Whole and observed reactable volumes (Vw and Vr,obs, respectively) were converted from the observed red and green fluorescent intensities, respectively, according to the procedure described in the experimental section. Two-dimensional (2D) dot plots of FACS measurement data, in which Vr,obs (xaxis) and Vw (y-axis) of 30 000 individual liposomes are represented in each dot, are shown in Figure 3. Both axes are on logarithmic scales to represent the wide range of liposome volumes that span over 4 orders of magnitude. The result of the control experiment is plotted in Figure 3a, showing the distribution of liposomes in which the reaction mixture without plasmid DNA (i.e., mixture containing 50 µM PFB-FDGlcU, PURESYSTEM classic II, and APC) was encapsulated and

Hosoda et al.

incubated for 6 h. In this case, Vr,obs in all liposomes was less than approximately 0.1 fL, while Vw was distributed over a wide range (mainly in 1 to 100 fL). As no green fluorescent product should be produced without plasmid DNA, green fluorescence intensity in this range was considered to be background noise, and liposomes with Vr,obs < 0.1 fL were judged as those in which no reaction proceeded (nonreacted liposomes) in the subsequent analysis. On the other hand, in the measurement of liposomes containing the complete mixture (i.e., the same mixture supplemented with 0.21 nM plasmid DNA encoding β-glucuronidase), a large population distinct from the nonreacted liposomes with Vr,obs > 0.1 fL was observed (Figure 3b). Hence, the population that appeared in this region represents the liposomes in which the enzyme reaction had proceeded (reacted liposomes). In the sequential measurement of time-sampled liposomes (Supporting Information, Figure S4), the distribution of Vr,obs did not change after 90 min. These observations indicated that the reaction in liposomes had completed (i.e., all substrate present was hydrolyzed) faster than the reaction in a test tube. We assumed that this discrepancy was due to the condensation effect from quantization of plasmid DNA. For example, if a single copy of DNA is encapsulated in 1 fL volume, the resulting concentration becomes 2 nM, regardless of the initial concentration (CDNA). Hence, we considered that the reaction had completed when the distribution of time-sampled liposomes showed no further changes. We performed the same experiment with 0.21 nM plasmid DNA encoding β-galactosidase, and obtained similar results (Supporting Information Figure S5), indicating that this phenomenon is not specific to the β-glucuronidase gene. There are two important conclusions that can be drawn from this observation. (1) We can distinguish two groups of liposomes in which the cascade reaction did or did not proceed. The reacted liposomes should have at least one reactable volume carrying plasmid DNA, while this condition did not occur in nonreacted liposomes. (2) Among the reacted liposomes, both Vw and Vr,obs showed diversity in their distributions, and even in the liposomes with identical Vw (corresponding to the horizontal section in Figure 3b), Vr,obs fluctuated by approximately 1 order. This distribution in Vr,obs is much greater than that caused by the error of FACS measurement and the fluctuation in encapsulation of the substrate, i.e., when 500 nM APC and 5 µM fluoresceintagged BSA were encapsulated together, the distribution showed a very narrow width with a slope of 1 on a logarithmic scale (Supporting Information Figure S2). Thus, when both green and red fluorescent materials are encapsulated at high concentration, they are uniformly encapsulated and their distribution shows narrow width. Hence, the wide distribution in Vr,obs is attributable to the heterogeneity in reaction volume in liposomes. To further understand the characteristics of this heterogeneity, we drew the theoretical line Vw ) Vr,obs calculated from the calibration of fluorescence intensities (gray solid line in Figure 3). Almost all data points were distributed on the upper left side of this line. This is the direct consequence of Vr,obs being smaller than Vw in all liposomes, which is additional evidence that the distribution in Vr,obs is due to the heterogeneity in reaction volume. Microscopic Observation. In the schematic shown in Figure 1b and the previous discussion, we assumed that the reaction proceeded only in the reactable compartments, which appeared as the wide distribution of Vr,obs smaller than Vw. We examined the occurrence of the cascade reaction in the partial compartment by optical microscopy. Ideally, the population of liposomes appearing in the region close to the line Vw ) Vr,obs in Figure 3b has Vr,obs close to Vw. Thus, most of the volume in a liposome should show green fluorescence. On the other hand, the population

Reactable Volume in Giant Liposomes

Langmuir, Vol. 24, No. 23, 2008 13545

Figure 4. Microscopic images of liposomes sorted from regions (i)-(iv) shown in Figure 3b. Upper images: bright field observation; lower images: fluorescence observation.

appearing in the upper left region has small Vr,obs as compared to Vw, and only a part of a liposome should show fluorescence. To confirm this, we sorted the liposomes in the four regions (i)-(iv) depicted in Figure 3b using FACS and observed the collected liposomes by fluorescence microscopy (Figure 4). On bright field observation, complicated internal structures were observed in the liposomes from regions (i) and (ii), while relatively univesicular liposomes were observed in (iii) and (iv). On fluorescence observation, liposomes fluoresced only in the partial volume as shown in (i) and (ii), while almost the whole volume fluoresced in (iii) and (iv). Averaged whole volumes estimated from the bright field images were 22, 48, 1.8, and 4.2 fL for (i)-(iv), respectively, which was in accordance with the range of assigned Vw in Figure 3b. Volumes approximated from the green areas were 1.8 fL for (i) and (iii) and 4.2 fL for (ii) and (iv), respectively, which also agreed with assigned Vr,obs values in Figure 3b. Therefore, we could confirm directly from the optical microscopy observations that the cascade reaction took place only inside the reactable compartment, and Vw and Vr,obs derived from the FACS measurements properly reflect the whole and reactable volumes as illustrated in Figure 1b. Cascade Reaction in Liposomes with Various Plasmid DNA Concentrations. In the model shown in Figure 1b, a single copy of plasmid DNA is expected to be encapsulated in each liposome when encapsulating a low concentration of DNA, while the number should increase with increasing concentration of plasmid DNA. Thus, the properties of the internal compartments can be elucidated by altering the plasmid DNA concentration. We obtained 100 000 liposome data from the experiments conducted at different original plasmid concentration (CDNA). These experiments were conducted using the laboratory-made in Vitro transcription/translation system (the time courses of this system in a test tube are plotted in the Supporting InformationFigure S6). The plots of Vw and Vr,obs for CDNA) 2.1, 0.21, and 0.021 nM, each sampled at incubation time t ) 120 and 180 min, are shown in Figure 5. Overall, the reacted and nonreacted liposomes

were still readily distinguishable, similar to Figures 3b and S3. Moreover, the distributions of the dots did not change after t ) 120 min (i.e., data plotted at t ) 120 and 180 min are essentially the same in Figure 5), indicating the reaction had completed before t ) 120 min under all conditions. From the data plotted in Figure 5, we can further derive important characters in this reaction system. The number of reacted liposomes at Vr,obs > 0.1 fL increased at larger CDNA, while their distributions appeared similar. In the following section, we discuss the first aspect, while the second is discussed in the subsequent section. Probability of DNA Encapsulation. We analyzed the probability of DNA encapsulation to confirm whether the plasmids were encapsulated into liposomes at single-copy level at low CDNA, as illustrated in Figure 1b. Figure 6a shows the fraction of reacted liposomes in all liposomes, which was derived from the 2D dot plot representation shown in Figure 5, at different CDNA plotted as a function of Vw. It was apparent that the fraction of reacted liposomes was larger at greater Vw and at higher CDNA. At low CDNA, where the average number of plasmids in solution is 0.126 and 0.0126 molecule/fL for CDNA ) 0.21 and 0.021 nM, respectively, encapsulation of DNA molecules into a reactable volume becomes a stochastic process (i.e., a Poisson process).45,46 Thus, the fraction of reacted liposomes is described by the probability in which at least one copy of plasmid DNA is encapsulated P(k g 1), where P(k) is the probability of k copies of plasmid being encapsulated. As encapsulation is a Poisson process, P(k g 1) can be formulated as a function of CDNA and Vw, which is written as

P(k g 1) ) 1 - exp(-εcapareacCDNAVw)

(1)

This equation is based on two assumptions: (1) the total reactable volume in a liposome (Vr,total) is proportional to Vw (45) Rissin, D. M.; Walt, D. R. Nano Lett. 2006, 6, 520–523. (46) Rondelez, Y.; Tresset, G.; Tabata, K. V.; Arata, H.; Fujita, H.; Takeuchi, S.; Noji, H. Nat. Biotechnol. 2005, 23, 361–365.

13546 Langmuir, Vol. 24, No. 23, 2008

Hosoda et al.

Figure 5. Dot plot representation of liposomes containing the cascade reaction system with different concentrations of plasmid DNA. (a-c) Results of CDNA ) 2.1, 0.21, and 0.021 nM, respectively. Figures in the upper (i) and lower (ii) columns are the measurement at incubation times of 120 and 180 min, respectively, at each concentration. The gray line in each plot shows the theoretical line of Vw ) Vr,obs. In all plots, 30 000 data points from the total of 100 000 obtained are shown for clarity.

Figure 6. Probability of DNA encapsulation. (a) Fraction of reacted liposomes P(k g 1) at different CDNA (2.1 (b), 0.21 (O), 0.021 (2), and 0 nM (+)). Gray lines represent fitting curves of eq 1. Error bars represent the standard errors of two separate experiments. (b) Apparent concentration of plasmid in liposomes estimated from the fitting in (a). Black solid line: εcap · areac ) 1; gray line: line with slope 1 fitted to the points at CDNA ) 0.021 and 0.21 using least-squares fitting on a logarithmic scale.

with the constant ratio areac (Vr,total ) areacVw), and (2) the plasmid encapsulation rate εcap, the ratio of the encapsulated concentration in Vr,total to the original concentration, CDNA, is independent of Vw. Therefore, εcapareacCDNAVw is the average number of DNA molecules encapsulated and initiated the cascade reaction in a liposome with the reactable volume Vr,total. We then fitted the data with eq 1. As shown in Figure 6a, this equation gave good fits to the experimental data (gray lines), suggesting that our two assumptions were reasonable. In Figure 6b, the average concentrations of encapsulated plasmid estimated from the fitting (εcapareacCDNA) were plotted against CDNA. Then, we fitted the two points at CDNA ) 0.021 and 0.21 nM in Figure 6b with a linear line of slope of 1 using the least-squares fitting on a logarithmic scale (gray line). Note that the error bars in Figure 6b represent the standard errors of two independent experiments, which are very small and invisible. As these two points lie almost on this line, we considered εcapareac to be a constant in this range of CDNA, which was derived to be εcapareac ) 0.43. This derivation indicates that approximately 40% of the plasmid DNA theoretically present in the volume Vw at concentration CDNA is encapsulated into Vr,total and consequently reacted. Meanwhile, the point at CDNA ) 2.1 nM deviated from this proportionality. This deviation at higher CDNA may be due

to the mutual electrostatic repulsion of DNA molecules that may have lowered the encapsulation rate εcap.18 Using this derived constant, we can estimate the probability of liposomes containing a given number (k) of copies of plasmid DNA as a function of Vw from the theory of the Poisson distribution of k occurrence P(k). As a result, the ratio of liposomes containing more than two plasmids among the reacted liposomes (i.e., P(k g 2)/P(k g 1)) becomes larger than 0.5 at Vw ) 6.6, 25, and 216 fL for conditions CDNA) 2.1, 0.21, and 0.021, respectively. Thus, more than half of the reacted liposomes with Vw smaller than these thresholds contain only a single copy of plasmid DNA, while liposomes with Vw larger than these thresholds are likely to contain more than two plasmids. The anticipated number of plasmids estimated here is related to the analysis based on our physical model (Figure 1b), which is discussed in the next section. Fraction of Reactable Volume in the Whole Volume. In this section, we discuss analysis of the relation between the distributions of reactable and whole volumes. To study the dependence of Vr,obs on Vw, we plotted the frequency distributions of Vr,obs/Vw with different Vw (Vw ∼ 1.9, 4.7, 12, and 29 fL; these are the representative values of bins on a logarithmic scale, i.e., 1.2-3.0, 3.0-7.5, 7.5-18, 18-45 fL, respectively) for CDNA ) 2.1 and 0.21 nM in Figure 7a and b, respectively. Each frequency distribution was normalized by the frequency averaged over the Vr,obs/Vw range of 2.4-fold around the peak. Surprisingly, all the normalized distributions agreed well in the range of Vr,obs/Vw > 0.05 regardless of Vw in both Figure 7a and b, except that deviations at Vr,obs/Vw < 0.05 were attributed to the background noise from the overlap on the population of nonreacted liposomes. Next, we plotted the dependence of the peak value in Vr,obs distributions (Vr,mode) on Vw on a logarithmic scale to compare the differences among three CDNA concentrations (CDNA ) 2.1, 0.21, and 0.021) in Figure 7c. In the figure, Vr,mode was proportional to Vw over 2 orders of magnitude at all plasmid concentrations used. Therefore, from Figure 7a-c, we can derive three conclusions: (1) there is one dominant reactable compartment in a liposome; (2) the scale of the reactable volume is proportional to the whole volume; and (3) the shape of the volume distribution of the reactable compartment is independent of the whole volume

Reactable Volume in Giant Liposomes

Figure 7. Fraction of reactable volume in the whole volume. (a, b) Normalized frequency distributions of the fraction of reacted volume Vr,obs/Vw at various Vw. (a,b) Distributions at CDNA ) 2.1 and 0.21 nM, respectively. In both plots, markers show the distributions at Vw ) 1.9 (+, green), 4.8 (2, red), 12 (O, blue), and 29 fL (b,black) (these values represent the bins on the log scale, i.e., 1.2-3.0, 3.0-7.5, 7.5-18, 18-45 fL, respectively). All distributions were normalized by each peak value averaged over Vr,obs/Vw of 2.4-fold. (c) Dependency of the mode of Vr,obs/Vw (Vr,mode) on Vw. Vw is shown on the left axis analogous to Figure 5. The original concentrations used were CDNA ) 2.1 (b, black), 0.21 (O, blue), and 0.021 nM (2, red). The black solid line represents the line of Vw ) Vr,obs. The gray line represents the line of Vr,mode )areac · Vw fitted to the data of CDNA ) 2.1 and 0.21 nM. The data at CDNA ) 0.021 nM were omitted for fitting because the frequency was too low.

(scale independent). The first conclusion is derived by the following deduction. In the physical model in Figure 1b, the quantity of Vr,obs should change at different CDNA if there are multiple reactable compartments on comparable scales. However, in Figure 7a and b, all the normalized distributions coincided, and Vr,mode and Vw were proportional (Vr,mode/Vw was constant), although the anticipated number of encapsulated plasmids was different at various values of CDNA and Vw as, estimated, as described in the previous section. Thus, there should be a single reactable volume present in a liposome, and so we can consider Vr,obs to be equal to Vr,total. Consequently, we can derive the second and third conclusions. The second indicates that our assumption Vr,total ) areacVw, was adequate, while the third implies that, as not only the average of Vr,total but also their distributions were identical, the relation of Vw and Vr is independent of scale. Next, we fitted the data points at CDNA ) 2.1 and 0.21 nM to the equation Vr,mode ) areacVw by the least-squares method on a logarithmic scale (gray line). From this fitting, we derived areac ) 0.16, indicating that 16% of the whole volume was reactable on average for all liposome sizes. This volume ratio corresponds to 50% in the ratio of diameter when assuming the liposomes are spherical. As we derived εcapareac ) 0.43 in the previous section, the encapsulation rate εcap was calculated to be 2.7. Thus, the average concentration of plasmid DNA in liposomes is 2.7 times larger than that in the original reaction mixture (i.e., before encapsulation, CDNA). The cause of this condensation effect is discussed below.

Discussion Simultaneous measurement of the whole volume and the reaction product indicated that the reaction proceeded only in

Langmuir, Vol. 24, No. 23, 2008 13547

the partial fraction volume in GMLV formed by the FDEL method. Statistical analysis revealed the geometrical properties of GMLV as a container of the protein synthesis system; the number of dominant reactable compartments in a liposome was almost one, the volume of which was 16% of the entire volume on average, and the scale of the reactable volume was proportional to the whole volume. The present measurement system allowed estimation of the average ratio of the reactable and whole volume, and the encapsulation rate. These results provide information necessary to gain a quantitative understanding of the protein synthesis reaction encapsulated in liposomes, used for reconstruction of a model cell system. First, our results allowed us to determine the reactable volume of liposomes by linear conversion from the whole volume. Second, the encapsulation rate was also shown to be independent of the whole liposome volume. These aspects found when the biomolecules are encapsulated in a small volume at low concentration are especially important for artificial cell studies as the numbers of genes and transcribed mRNA are often small in the extant cells.31,32 Although it has not been demonstrated directly, we deduced the physical condition of the nonreactable volume (84% of the total volume) is as follows. The membrane structure of liposomes formed by the FDEL method is multilamellar, and there are scattered and isolated small volumes present between the lamellae. Fluorescent proteins such as APC can fluoresce by themselves even in such small volumes, and therefore function as markers of the liposome volume. However, as the reaction mixture for the in Vitro transcription/translation contains nearly 100 components with a diverse range of concentrations, most of the scattered volumes do not contain all of the components necessary to produce the fluorescent product. We derived an encapsulation rate, εcap, of 2.7 at CDNA ) 0.21 and 0.021 nM. Although this value may include fluctuations in the whole volume estimation, the result can be interpreted as indicating that the resulting concentration of the plasmid in liposomes after encapsulation became greater than the original concentration. This may be due to the percolation effect. When a solution containing plasmid DNA is introduced into the dried liposome membrane (Supporting Information Figure S1), it tends to become trapped in the large volume in the highly porous structure, whereas water and small molecules should penetrate into the small spaces. Thus, as the trapped DNA was enclosed in the vesicles, the resulting concentration of DNA increased. At higher plasmid concentrations (CDNA ) 2.1 nM), εcap was approximately 1, indicating that this condensation effect did not occur. In either case, a derived encapsulation rate larger than 1 suggests the efficacy of the FDEL method for efficient material encapsulation. In conclusion, the present method for measurement and analysis of biochemical reactions in liposomes allowed us to assess the properties of giant multilamellar liposomes as a cell-sized artificial bioreactor for encapsulation of a complex biochemical reaction. This approach can be applied for characterizing liposomes prepared by different methods, which will provide useful information for the application of liposomes in life sciences, as well as for further understanding of the fundamental physics of liposome self-assembly. Particularly, the scale-independency of the distribution of the relative reactable volume found in the present study suggests the presence of a scale-free mechanism of liposome formation. Although the physical process is still unclear, our results provided new insight into the self-assembling and material

13548 Langmuir, Vol. 24, No. 23, 2008

encapsulation process of liposome formation that involves dynamic phenomena. Acknowledgment. We thank Dr. Kanetomo Sato and Kei Obinata for their assistance in the initial stages of the project, and Drs. Hiroshi Kita, Norikazu Ichihashi, and Taro Toyota for helpful discussion. This study was partially conducted in the Open Laboratories for Advanced Bioscience and Biotechnology (OLABB), Osaka University. This research was supported in part by the “Special Coordination Funds for Promoting Science and Technology: Yuragi Project” and “Global COE (Centers of Excellence) Program” of the Ministry of Education, Culture,

Hosoda et al.

Sports, Science, and Technology, Japan, and Noda Institute for Scientific Research. Supporting Information Available: A scanning electron microscopy image of the freeze-dried membrane is shown in Figure S1, the FACS data of liposomes encapsulating two fluorescent molecules is shown in Figure S2, the expression of GFP in the in Vitro transcription/ translation system is shown in Figure S3, the time course of 2D plots of the in liposome reaction is shown in Figure S4, the experiment with a different enzyme (β-galactosidase) is shown in Figure S5, and the time courses of the cascading reaction with the laboratory-made in Vitro transcription/translation system in a test tube are shown in Figure S6. This material is available free of charge via the Internet at http://pubs.acs.org. LA802432F