Article pubs.acs.org/Langmuir
Micrometer-Size Vesicle Formation Triggered by UV Light Tatsuya Shima,† Takahiro Muraoka,†,‡ Tsutomu Hamada,§ Masamune Morita,§ Masahiro Takagi,§ Hajime Fukuoka,† Yuichi Inoue,† Takashi Sagawa,† Akihiko Ishijima,† Yuki Omata,∥ Takashi Yamashita,∥ and Kazushi Kinbara*,† †
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai 980-8577 Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan § School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan ∥ Department of Pure and Applied Chemistry, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan ‡
S Supporting Information *
ABSTRACT: Vesicle formation is a fundamental kinetic process related to the vesicle budding and endocytosis in a cell. In the vesicle formation by artificial means, transformation of lamellar lipid aggregates into spherical architectures is a key process and known to be prompted by e.g. heat, infrared irradiation, and alternating electric field induction. Here we report UV-light-driven formation of vesicles from particles consisting of crumpled phospholipid multilayer membranes involving a photoactive amphiphilic compound composed of 1,4-bis(4-phenylethynyl)benzene (BPEB) units. The particles can readily be prepared from a mixture of these components, which is casted on the glass surface followed by addition of water under ultrasonic radiation. Interestingly, upon irradiation with UV light, micrometer-size vesicles were generated from the particles. Neither infrared light irradiation nor heating prompted the vesicle formation. Taking advantage of the benefits of light, we successfully demonstrated micrometer-scale spatiotemporal control of single vesicle formation. It is also revealed that the BPEB units in the amphiphile are essential for this phenomenon.
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fission and budding. Inspired by the dynamic and regulated behaviors of the biomembranes, topological changes of membranes in synthetic vesicles have been actively explored. Examples26−41 include fusion,26−28 division,29 and budding,30−34 triggered by heating, addition of membraneinteracting molecules, osmotic pressure, or photochemical reactions. In this article, on the basis of our serendipitous finding, we report a micrometer-size vesicle budding and formation from a shapeless particle, including an amphiphile bearing multiple chromophore units, triggered by ultraviolet (UV) or visible light irradiation. Taking advantage of the benefit to use finely focused light, micrometer-scale spatiotemporal control of the vesicle formation was demonstrated.
INTRODUCTION A vesicle is a self-closed supramolecular assembly of amphiphiles consisting of a lipid bilayer with an internal aqueous volume and a universal supramolecular architecture for building up the cells and organelles.1−3 In extant cells, the vesicle fission or budding plays important roles in vital activities, such as endocytosis and vesicle trafficking. Attachment of a coatomer−adaptor complex onto a cargo receptor in a biomembrane triggers the budding.4 As artificial procedures for vesicle formation,1−3,5−25 electroformation,6−11 gentle hydration,12,13 water/oil emulsion,14−17 microfluidic device,18−22 and infrared irradiation methods23−25 have been developed. Such diversified and efficient methods facilitate the use of vesicles as models of a plasma membrane and vehicles of drug-delivery systems. A synthetic lipid bilayer generally exists in either a liquidordered (Lo), liquid-disordered (Ld), or solid-ordered (So) phase, depending on the structure of lipids, composition, and temperature. Namely, a ratio of saturated/unsaturated bonds and length of the acyl chains of the lipid molecules largely influence their packing, where addition of cholesterol encourages formation of Lo-phase membrane. Meanwhile, biomembranes are consisting of multiple lipid components, where coexistent Lo- and Ld-phase are laterally separated to form domains.1−3 Thanks to the flexibility of Lo- and Ld-phases, biomembranes possess intriguing dynamic properties such as © 2014 American Chemical Society
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EXPERIMENTAL SECTION
General. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on 400 MHz FT NMR Bruker BioSpin AVANCE III 400 or 500 MHz FT NMR Bruker BioSpin AVANCE III 500, where the chemical shifts were determined with respect to tetramethylsilane (TMS) or a residual nondeuterated solvent as an internal standard. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF MS) spectrometry was performed in the reflector
Received: March 1, 2014 Revised: May 2, 2014 Published: June 4, 2014 7289
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mode with α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix on Bruker Daltonics REFLEX III spectrometer. Infrared spectra were recorded on JASCO FT/IR-4100 spectrometer equipped with JASCO ATR PRO 670H-S. Analytical thin layer chromatography (TLC) was performed on precoated, glass-backed silica gel (Merck 60 F254). Visualization of the developed chromatogram was performed by UV absorbance, Hanessian’s stain, or iodine. Light irradiation was performed with Asahi Spectra LAX-102 (100 W) or LAX-1000 (1 kW) xenon light source. Sonication for the preparation of lipid particles was performed with COSMO BIO Bioruptor UCD-250 cell homogenizer at 250 W. Microflow imaging (MFI) was performed with Brightwell Technologies DPA 4200 flow microscope. For the MFI measurement, 100 μL of a particle suspension in 200 mM sucrose (aqueous) was diluted with 9.9 mL of 200 mM sucrose (aqueous) followed by degassing for 30 min under reduced pressure at 25 °C. 900 μL of the suspension was taken by a plastic syringe, which was attached to the device. After initial flowing (0.2 μL), 0.42 μL of the suspension was analyzed. Preparation and Photoirradiation of the DOPC·1 Particles. A typical procedure: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 2.0 mM) and 1 (400 μM) were dissolved in a mixture of MeOH and CHCl3 (1.0/1.0, v/v) followed by evaporation of the solvents under Ar flow at 20 °C to leave a uniform thin film at the bottom of a glass test tube. The obtained film was placed under vacuum at 20 °C for longer than 1.5 h. The resulting film was then hydrated with an aqueous solution of sucrose and glucose mixture ([sucrose] = [glucose] = 100 mM; [DOPC] = 200 μM, [1] = 40 μM), which was immediately subjected to sonication (COS-MO BIO Bioruptor UCD-250 cell homogenizer, 250 W) for 15 min at 0−5 °C. Photoirradiation was performed with Asahi Spectra LAX-1000UV xenon light source (1 kW), while the light source of the microscope was used for the irradiation during the microscopic observations. Optical Microscopic Observations. Fluorescent and phase contrast microscopy was performed with Olympus BX-51 or IX-71 microscopes, where U-MWU2 mirror unit (excitation filter: 330−385 nm; emission filter: 420 nm; dichroic mirror: 400 nm) was used for fluorescence observation, and the excitation light through the filter (330−385 nm) was used for the photoirradiation. A Linkam Scientific Instruments Type 10021 temperature controlled stage or TOKAI HIT Thermo Plate MATS-1002RO was attached on Olympus IX-71 microscope with the same mirror unit as above for the observation under heated conditions. On a slide glass, a coverslip was placed over the object through a 0.1 mm thick silicon-based spacer. Apodized phase contrast microscopy with local light irradiation was performed with Olympus IX-71 microscope equipped with an objective lens (Nikon CFI S Plan Fluor ELWD ADM 40×) using a 405 nm laser (405 ± 5 nm, 90 mW, KIMMON KOHA KBL-90C-A) being focused at the back focal plane of the objective lens. Laser power from the objective lens was 0.2 mW. Confocal laser scanning microscopy was performed with an Olympus IX-81 microscope. Preparation of Cross-Sectioned Specimens and TEM Observations. The particles before and after the photoirradiation, fixed by glutaraldehyde, were postfixed in 2% OsO4, dehydrated in a graded series of alcohol, and then embedded in Epon 812 (TAAB Laboratories, UK). The epoxy resin block was then ultramicrotomed using a Sorvall Porter-Blum MT-2 ultra microtome equipped with a diamond knife at 25 °C. The ultrathin section was transferred onto a Cu mesh grid. Prior to the transmission electron microscopy (TEM) and 3D observations by transmission electron microtomography (TEMT), the ultrathin section was stained with lead citrate and uranyl acetate for 10 min. TEM and TEMT images were acquired by a JEOL JEM 1400 and JEOL JEM 2100F, respectively, operating at 120 kV equipped with a CCD camera. The tilt angle ranges from −64° to +64° by 1° increment.
consisting of alternatively arranged hydrophilic and hydrophobic units,42,43 we newly synthesized a cyclic amphiphile 1 composed of two fluorescent 1,4-bis(4-phenylethynyl)benzene (BPEB) units and four tetraethylene glycol chains, which are connected via methyl benzoate and azobenzene units. Initially, we tried to prepare micrometer-size vesicles including 1 following the conventional gentle hydration method.12,13 In fact, DOPC (2.0 mM) and 1 (400 μM) were dissolved in a mixture of MeOH and CHCl3 (1.0/1.0, v/v), followed by evaporation of the solvents by Ar flow at 20 °C to form a uniform thin film at the bottom of a glass test tube. The obtained film was placed under vacuum (6.7 × 10−2 Pa) at 20 °C over 1.5 h, which was then hydrated with an aqueous solution of a mixture of glucose and sucrose ([glucose] = [sucrose] = 100 mM; [DOPC] = 200 μM, [1] = 40 μM) at 37 °C for 3 h. The thin film seemed almost intact after hydration. Instead, a small quantity of shapeless particles was observed in the aqueous dispersion (Figure 1a).
Figure 1. Time-course phase-contrast microscopic observation of the light-triggered micrometer-size vesicle formation from a shapeless particle composed of DOPC and 1 in an aqueous solution of glucose and sucrose ([DOPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM). Snapshots were taken after irradiation (330−385 nm) at 20 °C for (a) 0, (b) 10, (c) 25, (d) 95, (e) 115, and (f) 175 s, respectively. The yellow arrows indicate the generated micrometer-size vesicles. Scale bars: 10 μm. The original video is available in the Supporting Information, Video S1.
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RESULTS AND DISCUSSION Preparation of Photoresponsive Particles and LightTriggered Micrometer-Size Vesicle Formation. In the course of our research on membrane-penetrating amphiphiles
Interestingly, when the particles in the dispersion were irradiated with UV light (330−385 nm) at 20 °C, using the 7290
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light source of the fluorescence microscope, micrometer-size vesicles were readily formed from their surfaces (Figure 1; the original video is available in the Supporting Information, Video S1). The vesicles kept growing on the surface accompanied by gradual dwindling of the particles as long as irradiation was continued. Along with the growth of the vesicles, they were eventually launched from the surface of the particles (Figures 1d−f). The generated vesicles mostly grown over 3.0 μm in diameter, and the maximum size is around 10 μm (Figure 1f). When the irradiation was paused, the growth of the vesicles synchronously stopped without shrinkage or deformation, and restart of irradiation induced further vesicle growth. Interestingly, some particles bent and deformed immediately after the UV irradiation, and the vesicles are formed thereafter (Figure 2;
microflow imaging (MFI), which gives digital images of micrometer to submicrometer-size objects in a flow cell, allows for overall visualization of the particles in the suspension. Indeed, MFI displayed that the aqueous suspension of the particles containing DOPC and 1 includes 1.0−1.9 μm objects as the most dominant species, with smaller quantities of larger objects (1.0−1.9 μm: 60.6% 2.0−2.9 μm: 13.1%, 3.0−3.9 μm: 5.3% etc; Figure 3, blue bars) before irradiation. After exposure
Figure 3. Distribution of the particle size evaluated by microflow imaging (MFI) of aqueous suspensions of shapeless particles composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM; blue bars) before and (red bars) after light irradiation (320 ± 10 nm, 60 min, 1.1 mW, 20 °C).
to UV light (320 ± 10 nm, 60 min, 1.1 mW, 20 °C), the populations of 1.0−1.9 and 2.0−2.9 μm objects decreased (55.9 and 12.0%, respectively; Figure 3, red bars), while those of >3.0 μm objects wholly increased (3.0−3.9 μm: 6.6% etc; Figure 3, red bars). As described above, the diameters of the generated vesicles are mostly larger than 3.0 μm. Thus, it is likely that the increase in population of large objects (>3.0 μm) in MFI images results from the vesicle formation, while reduction of the population of relatively small objects simultaneously occurs due to dwindling of the particles as observed in Figure 1. Such overall change of the particle-size distribution indicates that the vesicle formation takes place from the most particles contained in the suspension. Light-Driven Single Vesicle Formation with Micrometer-Scale Spatiotemporal Control. Taking advantage of the benefit to use light that enables spatial control of irradiation at a local spot, we challenged single vesicle formation from the micrometer-scale area on the surface of the photoresponsive particle. A 405 nm laser, focused with 1 μm diameter, was used as an excitation light source, where the laser beam (0.25 mW) was set on the stage of an apodized phase-contrast microscope. The edge of a particle composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM) was located at the focused irradiation spot (Figure 4a, the original video is available in the Supporting Information, Video S3) whereupon a single vesicle was readily formed at the spot and grew up to 6 μm in diameter through continuous irradiation for 70 s at 20 °C (Figures 4b,c). The generated vesicle was eventually detached from the particle (Figure 4d). Importantly, no vesicle formation was observed in other areas that are not exposed to the laser beam. Thus, the spatiotemporally controlled single vesicle formation at the micrometer-size limited area was successfully demonstrated by photoirradiation of the particle.
Figure 2. Time-course phase-contrast microscopic observation of a bending shapeless particle composed of DOPC and 1 in an aqueous solution of glucose and sucrose ([DOPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM) at the initial stage of the lighttriggered micrometer-size vesicle formation. Snapshots were taken after irradiation (330−385 nm) at 20 °C for (a) 0, (b) 2, (c) 5, and (d) 7 s. Schematic models of the particle are shown in the insets (a− c). The white arrows indicate the bending motion of the particle. Scale bars: 10 μm. The original video is available in the Supporting Information, Video S2.
the original video is available in the Supporting Information, Video S2). Importantly, we found that the exposure of the film to sonication (250 W, 15 min, 0−5 °C), immediately after the addition of the aqueous solution of the sugars to the casted film of DOPC containing 1, significantly encourages the formation of the particles dispersed in the aqueous phase. While the lighttriggered micrometer-size vesicle formation was observed even in deionized water without sugars, it took place more efficiently in the aqueous solution of the sugars.44 The particles prepared by a mixture containing a higher ratio of 1 (800 μM) to DOPC (2.0 mM) hardly responded to the light irradiation, while a mixture containing lower ratio of 1 (100 μM) to DOPC (2.0 mM) afforded only few particles. Hence, we adopted the ratio of [1]/[DOPC] = 0.20 for the subsequent experiments. It should be noted here that neither heating the aqueous suspension of the particles up to 80 °C in the dark nor irradiation by IR laser (0.29 mW, 1064 nm)23−25 induced vesicle formation, suggesting that heat has little effect on the vesicle formation from the particle. The above-mentioned microscopic studies visualize the formation of vesicles from each particle. On the other hand, 7291
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ization of the phospholipid component, we prepared the particles consisting of DOPC, 1, and 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE; [DOPC] = 400 μM, [1] = 80 μM, [NBD-PE] = 7.8 μM) dispersed in an aqueous media ([glucose] = [sucrose] = 100 mM). The resulting particles also generate micrometer-size vesicles upon irradiation with UV light. Confocal laser scanning micrographs (CLSM) of nonirradiated particles observed with excitation lights for 1 and NBD (405 and 473 nm, respectively) mostly overlapped with each other at each depth in the particle (Figure 5), indicating that 1 and NBD-PE are distributed over
Figure 4. Spatially controlled single micrometer-size vesicle formation on a particle composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM) observed by an apodized phase contrast microscopy at 20 °C. Micrographs were taken after irradiation of focused 405 nm laser beam for (a) 0, (b) 50, (c) 72 and (d) 80 s. In each micrograph, the irradiation spot is marked with a red circle, and a black arrow indicates the generated micrometer-size vesicle. Scale bars: 10 μm. The original video is available in the Supporting Information, Video S3.
Dependence of Micrometer-Size Vesicle Photogeneration on Lipid Phase. The deformation of the biomembrane is known to occur in the fluid liquid phase.1−3 Likewise, DOPC provides a liquid-phase membrane at 20 °C (DOPCTm = −20 °C), which is likely favorable for photogeneration of micrometer-size vesicles from the particle. In connection with this, we found that a mixture of 1 and 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC), forming a solid-phase membrane at 20 °C (DSPCTm = 55 °C), also gives the shapeless particles by the procedure described above ([DSPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM). However, upon irradiation (330−385 nm) of these particles at 20 °C, no micrometer-size vesicle formation (see the Supporting Information, Figures S1a,b) took place. In sharp contrast, irradiation at 65 °C, where DSPC forms a liquid-phase membrane, triggered the micrometer-size vesicle formation (see the Supporting Information, Figure S1d). Thus, it is likely that the lipid phase is an important factor in photogeneration of the micrometer-size vesicles. It should be noted here that no morphological change of the particles occurred simply by heating without irradiation (see the Supporting Information, Figure S1c), again indicating that thermal stimuli do not trigger the formation of micrometer-size vesicles from the particle. The importance of the lipid phase is also suggested by the result that the particles consisting of DSPC, cholesterol, and 1 ([DSPC] = 100 μM, [cholesterol] = 100 μM, [1] = 40 μM) also allowed the light-driven vesicle formation at 20 °C (see the Supporting Information, Figure S2), since incorporation of cholesterol into a solid-phase membrane is known to increase its fluidity.3 Thus, it is suggested that a liquid phase is required for the light-driven micrometer-size vesicle formation. Structural Analyses of Particles and Photogenerated Vesicles. The particles prepared from the mixture of DOPC and 1 emitted bright fluorescence upon excitation by 330−385 nm irradiation, indicating that the particles contain 1 (see the Supporting Information, Figure S3). For fluorometric visual-
Figure 5. Confocal laser scanning micrographs of a photoresponsive particle composed of DOPC, 1, and NBD-PE ([DOPC] = 400 μM, [1] = 80 μM, [NBD-PE] = 7.8 μM) dispersed in an aqueous solution of glucose and sucrose ([glucose] = [sucrose] = 100 mM) at 20 °C. (a) The top surface and the cross sections at depths of (b) 2.0 ± 0.2 and (c) 4.0 ± 0.2 μm of the particle were observed with (1) 405 nm and (2) 473 nm lasers for excitation of the BPEB unit of 1 and NBD units of NBD-PE to show blue and green emissions, respectively. Merged micrographs of (1) and (2) are displayed in (3). Scale bars: 20 μm.
the particle. Nevertheless, although the fluorescence intensity of NBD-PE seems to be almost independent of the depth, that of 1 gradually increases with the depth of the confocal section. Importantly, CLSM also displayed a number of voids in the particle. Transmission electron microscopic (TEM) and microtomographic (TEMT) observations of the nonirradiated particles ([DOPC] = 200 μM, [1] = 40 μM) revealed that they are enveloped with membranes (Figure 6a; see the Supporting Information, Figures S4a,b). Membrane-like architectures, surrounding the voids, were also observed inside the particle. The thickness of the membranes at the surface and the inner area is 21−37 nm, suggesting that they are multilayer of DOPC membranes.45 These observations revealed that the particles are membrane-enveloped objects which include intricate multilayer membranes inside. TEM observation of the photoirradiated particle displayed a vesicle attached on the surface, with a 4.7 nm thick membrane, corresponding to a unilamellar membrane (Figure 6b).46 Further irradiation of the particle resulted in disappearance of the enveloping membranes, and only fragmentary membranes remained in the particle (Figure 6c; see the Supporting Information, Figures S4c,d). Hence, it is 7292
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composed of DOPC and 2 responded to UV irradiation (330− 385 nm) at 20 °C to form vesicles (see the Supporting Information, Figures S6a,b). In contrast, the particles composed of DOPC and 3 did not show any vesicle formation upon UV irradiation at 20 °C (see the Supporting Information, Figures S6c,d). Thus, the BPEB units are likely the essential components enabling the light-triggered vesicle formation.
Another point that should be taken into account is the contribution of the azobenzene unit. Comparison of phasecontrast micrographs between Figure 1f and Figure S6b (see the Supporting Information) suggests that the particles composed of DOPC and 1 tend to form the vesicles more effectively than the ones composed of DOPC and 2. UV−vis and 1H nuclear magnetic resonance (NMR) spectroscopic analyses indicated that the azobenzene unit of 1 undergoes trans−cis isomerization upon photoirradiation (see the Supporting Information, Figures S7 and S8). This was also supported by a fluorescence lifetime study; a shorter fluorescence lifetime of 1 (1.0 ns, 330 nm excitation light) than that of 2 (1.42 ns, 330 nm excitation light) in THF suggests energy transfer from the BPEB units to the azobenzene unit of 1. At the photostationary state, the ratio between [trans-1] and [cis-1] was 83/17. It is well-known that, even at the photostationary state, a photochromic molecule such as azobenzene isomerizes between the two isomers reversibly during the light irradiation. It is considered that the photoisomerization of the azobenzene unit of 1 enhances the efficiency of the vesicle formation. The detailed mechanism of the light-triggered vesicle formation from the particle, including the roles of BPEB and azobenzene units, is currently being investigated.
Figure 6. Transmission electron micrographs of a plastic-embedded section of the particle composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM) (a) before and (b, c) after UV light (320 nm, 1 kW, 3 h, 20 °C) irradiation. Scale bars: 2.0 μm.
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likely that the enveloping membrane of the particle turns into the micrometer-size unilamellar vesicles. Under the condition of fluorescence microscopy, the membrane of the photogenerated micrometer-size vesicles showed fluorescence upon irradiation with 330−385 nm light, exciting the BPEB units of 1 (see the Supporting Information, Figure S5). This observation indicates that 1 is embedded in the lipid membrane of the photogenerated vesicles. Importance of Chromophore Units in Light-Driven Vesicle Formation. As demonstrated above, the vesicle formation takes place upon irradiation of the particles with 330−385 nm light, which can excite the chromophore units included in 1. To unveil the essential unit in 1 for the lighttriggered micrometer-size vesicle formation, we prepared 2 and 3, which lack the azobenzene and BPEB units, respectively. Mixtures of DOPC (2.0 mM) and 2 or 3 (400 μM) in an aqueous media ([glucose] = [sucrose] = 100 mM) afforded shapeless particles after sonication (250 W, 15 min, 0−5 °C), analogous to the mixture of DOPC and 1. The particles
CONCLUSION This work demonstrates light-triggered micrometer-size vesicle formation with micrometer-scale spatial control. In general, hydration of lipids under sonication is well-known to produce nanometer- to submicrometer-size vesicles.1−3 However, the addition of amphiphile 1 with DOPC dominantly provides micrometer-size shapeless particles. The shapeless particles are coated with the multilayer membranes that likely turn into the vesicles upon exposure to light. Thanks to the advantage of light, which acts as a contactless trigger, vesicle formation with micrometer-scale spatial control on the shapeless particle was successfully achieved. This phenomenon was also observed for other lipids like DSPC and the mixture of DSPC and cholesterol. Interestingly, lipid phase is an important factor for the transformation of the particles into the vesicles. Namely, upon photoirradiation, the shapeless particles composed of a solid-phase lipid show no response, while the particles made of 7293
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a liquid-phase lipid generate the vesicles. It is also revealed that the BPEB units of 1 are essential for the light-triggered vesicle formation. This methodology could provide a new approach for the vesicle development and manipulation of membranes.
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ASSOCIATED CONTENT
S Supporting Information *
Phase-contrast and fluorescent micrography and transmission electron microtomography of the particles composed of DOPC and 1, UV−vis and NMR spectra of 1, and UV−vis spectrum of 2. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel +81-22-217-5612; e-mail [email protected] (K.K.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Prof. Kouhei Tsumoto (The University of Tokyo) and Dr. Satoru Nagatoishi (The University of Tokyo) for helpful discussions. We also thank Ms. Ryoko Sugimoto (JAIST) for her support in preparation and microscopic studies of micrometer-size vesicles, Prof. Masatsugu Shimomura (Tohoku University) and Dr. Takahito Kawano (Tohoku University) for their support in confocal laser scanning microscopic observation, Dr. Akihiro Kishimura (The University of Tokyo), Dr. Satoshi Fukuda (The University of Tokyo Hospital), and Prof. Hiroshi Yabu (Tohoku University) for their support in TEM and TEMT experiments, and Prof. Atsushi Muramatsu (Tohoku University) and Prof. Kiyoshi Kanie (Tohoku University) for their support in polarized optical microscopic observation. TEM observations were conducted in Research Hub for Advanced Nano Characterization, The University of Tokyo, supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was partially supported by MEXT, Grants-inAid for Young Scientists S (21675003), Scientific Research on Innovative Areas “Spying minority in biological phenomena” (No. 3306), (23115003), and the Management Expenses Grants for National Universities Corporations to K.K. and Noguchi Foundation to T.M.
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REFERENCES
(1) Giant Vesicles, Perspectives in Supramolecular Chemistry; Luisi, P. L., Walde, P., Eds.; Wiley-Interscience: Weinheim, 2000; Vol. 6. (2) Structure and Dynamics of Membranes, 1st ed.; Lipowsky, R., Sackmann, E., Eds.; Elsevier: Amsterdam, 1995. (3) Liposomes: From Physics to Applications; Lasic, D. D., Ed.; Elsevier: Amsterdam, 1993. (4) Molecular Biology of the Cell, 5th ed.; Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., Eds.; Garland Science: New York, 2007. (5) Walde, P.; Cosentino, K.; Engel, H.; Stano, P. Giant Vesicles: Preparations and Applications. ChemBioChem 2010, 11, 848−865. (6) Shimanouchi, T.; Umakoshi, H.; Kuboi, R. Kinetic Study on Giant Vesicle Formation with Electroformation Method. Langmuir 2009, 25, 4835−4840. (7) Pott, T.; Bouvrais, H.; Méléard, P. Giant Unilamellar Vesicle Formation under Physiologically Relevant Conditions. Chem. Phys. Lipids 2008, 154, 115−119. 7294
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Porphyrin−Protein Polymersomes. J. Am. Chem. Soc. 2009, 131, 3872−3874. (32) Ishii, K.; Hamada, T.; Hatakeyama, M.; Nagasaki, T.; Takagi, M. Reversible Control of Exo- and Endo-Budding Transitions in a Photosensitive Lipid Membrane. ChemBioChem 2009, 10, 251−256. (33) Long, M. S.; Cans, A.-S.; Keating, C. D. Budding and Asymmetric Protein Microcompartmentation in Giant Vesicles Containing Two Aqueous Phases. J. Am. Chem. Soc. 2008, 130, 756−762. (34) Charras, G. T. A Short History of Blebbing. J. Microsc. 2008, 231, 466−478. (35) Diguet, A.; Yanagisawa, M.; Liu, Y.-J.; Brun, E.; Abadie, S.; Rudiuk, S.; Baigl, D. UV-Induced Bursting of Cell-Sized Multicomponent Lipid Vesicles in a Photosensitive Surfactant Solution. J. Am. Chem. Soc. 2012, 134, 4898−4904. (36) Hamada, T.; Sugimoto, R.; Vestergaard, M. C.; Nagasaki, T.; Takagi, M. Membrane Disc and Sphere: Controllable Mesoscopic Structures for the Capture and Release of a Targeted Object. J. Am. Chem. Soc. 2010, 132, 10528−10532. (37) Yanagisawa, M.; Imai, M.; Taniguchi, T. Shape Deformation of Ternary Vesicles Coupled with Phase Separation. Phys. Rev. Lett. 2008, 100, 148102. (38) Brückner, E.; Sonntag, P.; Rehage, H. Light-Induced Shape Transitions of Unilamellar Vesicles. Langmuir 2001, 17, 2308−2311. (39) Petrov, P. G.; Lee, J. B.; Döbereiner, H.-G. Coupling Chemical Reactions to Membrane Curvature: A Photochemical Morphology Switch. Europhys. Lett. 1999, 48, 435−441. (40) Käs, J.; Sackmann, E. Shape Transitions and Shape Stability of Giant Phospholipid Vesicles in Pure Water Induced by Area-toVolume Changes. Biophys. J. 1991, 60, 825−844. (41) Kunitake, T.; Nakashima, N.; Shimomura, M.; Okahata, Y.; Kano, K.; Ogawa, T. Unique Properties of Chromophore-Containing Bilayer Aggregates: Enhanced Chirality and Photochemically Induced Morphological Change. J. Am. Chem. Soc. 1980, 102, 6642−6644. (42) Muraoka, T.; Shima, T.; Hamada, T.; Morita, M.; Takagi, M.; Tabata, K. V.; Noji, H.; Kinbara, K. Ion Permeation by a Folded Multiblock Amphiphilic Oligomer Achieved by Hierarchical Construction of Self-assembled Nanopores. J. Am. Chem. Soc. 2012, 134, 19788−19794. (43) Muraoka, T.; Shima, T.; Hamada, T.; Morita, M.; Takagi, M.; Kinbara, K. Mimicking Multipass Transmembrane Proteins: Synthesis, Assembly and Folding of Alternating Amphiphilic Multiblock Molecules in Liposomal Membranes. Chem. Commun. 2011, 47, 194−196. (44) Tsumoto, K.; Matsuo, H.; Tomita, M.; Yoshimura, T. Efficient Formation of Giant Liposomes through the Gentle Hydration of Phosphatidylcholine Films Doped with Sugar. Colloids Surf., B 2009, 68, 98−105. (45) Caffrey, M.; Feigenson, G. W. Fluorescence Quenching in Model Membranes. 3. Relationship between Calcium Adenosinetriphosphatase Enzyme Activity and the Affinity of the Protein for Phosphatidylcholines with Different Acyl Chain Characteristics. Biochemistry 1981, 20, 1949−1961. (46) Rawicz, W.; Olbrich, K. C.; McIntosh, T.; Needham, D.; Evans, E. Effect of Chain Length and Unsaturation on Elasticity of Lipid Bilayers. Biophys. J. 2000, 79, 328−339.
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Micrometer-Size Vesicle Formation Triggered by UV Light Tatsuya Shima,† Takahiro Muraoka,†,‡ Tsutomu Hamada,§ Masamune Morita,§ Masahiro Takagi,§ Hajime Fukuoka,† Yuichi Inoue,† Takashi Sagawa,† Akihiko Ishijima,† Yuki Omata,∥ Takashi Yamashita,∥ and Kazushi Kinbara*,† †
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai 980-8577 Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan § School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan ∥ Department of Pure and Applied Chemistry, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan ‡
S Supporting Information *
ABSTRACT: Vesicle formation is a fundamental kinetic process related to the vesicle budding and endocytosis in a cell. In the vesicle formation by artificial means, transformation of lamellar lipid aggregates into spherical architectures is a key process and known to be prompted by e.g. heat, infrared irradiation, and alternating electric field induction. Here we report UV-light-driven formation of vesicles from particles consisting of crumpled phospholipid multilayer membranes involving a photoactive amphiphilic compound composed of 1,4-bis(4-phenylethynyl)benzene (BPEB) units. The particles can readily be prepared from a mixture of these components, which is casted on the glass surface followed by addition of water under ultrasonic radiation. Interestingly, upon irradiation with UV light, micrometer-size vesicles were generated from the particles. Neither infrared light irradiation nor heating prompted the vesicle formation. Taking advantage of the benefits of light, we successfully demonstrated micrometer-scale spatiotemporal control of single vesicle formation. It is also revealed that the BPEB units in the amphiphile are essential for this phenomenon.
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fission and budding. Inspired by the dynamic and regulated behaviors of the biomembranes, topological changes of membranes in synthetic vesicles have been actively explored. Examples26−41 include fusion,26−28 division,29 and budding,30−34 triggered by heating, addition of membraneinteracting molecules, osmotic pressure, or photochemical reactions. In this article, on the basis of our serendipitous finding, we report a micrometer-size vesicle budding and formation from a shapeless particle, including an amphiphile bearing multiple chromophore units, triggered by ultraviolet (UV) or visible light irradiation. Taking advantage of the benefit to use finely focused light, micrometer-scale spatiotemporal control of the vesicle formation was demonstrated.
INTRODUCTION A vesicle is a self-closed supramolecular assembly of amphiphiles consisting of a lipid bilayer with an internal aqueous volume and a universal supramolecular architecture for building up the cells and organelles.1−3 In extant cells, the vesicle fission or budding plays important roles in vital activities, such as endocytosis and vesicle trafficking. Attachment of a coatomer−adaptor complex onto a cargo receptor in a biomembrane triggers the budding.4 As artificial procedures for vesicle formation,1−3,5−25 electroformation,6−11 gentle hydration,12,13 water/oil emulsion,14−17 microfluidic device,18−22 and infrared irradiation methods23−25 have been developed. Such diversified and efficient methods facilitate the use of vesicles as models of a plasma membrane and vehicles of drug-delivery systems. A synthetic lipid bilayer generally exists in either a liquidordered (Lo), liquid-disordered (Ld), or solid-ordered (So) phase, depending on the structure of lipids, composition, and temperature. Namely, a ratio of saturated/unsaturated bonds and length of the acyl chains of the lipid molecules largely influence their packing, where addition of cholesterol encourages formation of Lo-phase membrane. Meanwhile, biomembranes are consisting of multiple lipid components, where coexistent Lo- and Ld-phase are laterally separated to form domains.1−3 Thanks to the flexibility of Lo- and Ld-phases, biomembranes possess intriguing dynamic properties such as © 2014 American Chemical Society
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EXPERIMENTAL SECTION
General. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on 400 MHz FT NMR Bruker BioSpin AVANCE III 400 or 500 MHz FT NMR Bruker BioSpin AVANCE III 500, where the chemical shifts were determined with respect to tetramethylsilane (TMS) or a residual nondeuterated solvent as an internal standard. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF MS) spectrometry was performed in the reflector
Received: March 1, 2014 Revised: May 2, 2014 Published: June 4, 2014 7289
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mode with α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix on Bruker Daltonics REFLEX III spectrometer. Infrared spectra were recorded on JASCO FT/IR-4100 spectrometer equipped with JASCO ATR PRO 670H-S. Analytical thin layer chromatography (TLC) was performed on precoated, glass-backed silica gel (Merck 60 F254). Visualization of the developed chromatogram was performed by UV absorbance, Hanessian’s stain, or iodine. Light irradiation was performed with Asahi Spectra LAX-102 (100 W) or LAX-1000 (1 kW) xenon light source. Sonication for the preparation of lipid particles was performed with COSMO BIO Bioruptor UCD-250 cell homogenizer at 250 W. Microflow imaging (MFI) was performed with Brightwell Technologies DPA 4200 flow microscope. For the MFI measurement, 100 μL of a particle suspension in 200 mM sucrose (aqueous) was diluted with 9.9 mL of 200 mM sucrose (aqueous) followed by degassing for 30 min under reduced pressure at 25 °C. 900 μL of the suspension was taken by a plastic syringe, which was attached to the device. After initial flowing (0.2 μL), 0.42 μL of the suspension was analyzed. Preparation and Photoirradiation of the DOPC·1 Particles. A typical procedure: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 2.0 mM) and 1 (400 μM) were dissolved in a mixture of MeOH and CHCl3 (1.0/1.0, v/v) followed by evaporation of the solvents under Ar flow at 20 °C to leave a uniform thin film at the bottom of a glass test tube. The obtained film was placed under vacuum at 20 °C for longer than 1.5 h. The resulting film was then hydrated with an aqueous solution of sucrose and glucose mixture ([sucrose] = [glucose] = 100 mM; [DOPC] = 200 μM, [1] = 40 μM), which was immediately subjected to sonication (COS-MO BIO Bioruptor UCD-250 cell homogenizer, 250 W) for 15 min at 0−5 °C. Photoirradiation was performed with Asahi Spectra LAX-1000UV xenon light source (1 kW), while the light source of the microscope was used for the irradiation during the microscopic observations. Optical Microscopic Observations. Fluorescent and phase contrast microscopy was performed with Olympus BX-51 or IX-71 microscopes, where U-MWU2 mirror unit (excitation filter: 330−385 nm; emission filter: 420 nm; dichroic mirror: 400 nm) was used for fluorescence observation, and the excitation light through the filter (330−385 nm) was used for the photoirradiation. A Linkam Scientific Instruments Type 10021 temperature controlled stage or TOKAI HIT Thermo Plate MATS-1002RO was attached on Olympus IX-71 microscope with the same mirror unit as above for the observation under heated conditions. On a slide glass, a coverslip was placed over the object through a 0.1 mm thick silicon-based spacer. Apodized phase contrast microscopy with local light irradiation was performed with Olympus IX-71 microscope equipped with an objective lens (Nikon CFI S Plan Fluor ELWD ADM 40×) using a 405 nm laser (405 ± 5 nm, 90 mW, KIMMON KOHA KBL-90C-A) being focused at the back focal plane of the objective lens. Laser power from the objective lens was 0.2 mW. Confocal laser scanning microscopy was performed with an Olympus IX-81 microscope. Preparation of Cross-Sectioned Specimens and TEM Observations. The particles before and after the photoirradiation, fixed by glutaraldehyde, were postfixed in 2% OsO4, dehydrated in a graded series of alcohol, and then embedded in Epon 812 (TAAB Laboratories, UK). The epoxy resin block was then ultramicrotomed using a Sorvall Porter-Blum MT-2 ultra microtome equipped with a diamond knife at 25 °C. The ultrathin section was transferred onto a Cu mesh grid. Prior to the transmission electron microscopy (TEM) and 3D observations by transmission electron microtomography (TEMT), the ultrathin section was stained with lead citrate and uranyl acetate for 10 min. TEM and TEMT images were acquired by a JEOL JEM 1400 and JEOL JEM 2100F, respectively, operating at 120 kV equipped with a CCD camera. The tilt angle ranges from −64° to +64° by 1° increment.
consisting of alternatively arranged hydrophilic and hydrophobic units,42,43 we newly synthesized a cyclic amphiphile 1 composed of two fluorescent 1,4-bis(4-phenylethynyl)benzene (BPEB) units and four tetraethylene glycol chains, which are connected via methyl benzoate and azobenzene units. Initially, we tried to prepare micrometer-size vesicles including 1 following the conventional gentle hydration method.12,13 In fact, DOPC (2.0 mM) and 1 (400 μM) were dissolved in a mixture of MeOH and CHCl3 (1.0/1.0, v/v), followed by evaporation of the solvents by Ar flow at 20 °C to form a uniform thin film at the bottom of a glass test tube. The obtained film was placed under vacuum (6.7 × 10−2 Pa) at 20 °C over 1.5 h, which was then hydrated with an aqueous solution of a mixture of glucose and sucrose ([glucose] = [sucrose] = 100 mM; [DOPC] = 200 μM, [1] = 40 μM) at 37 °C for 3 h. The thin film seemed almost intact after hydration. Instead, a small quantity of shapeless particles was observed in the aqueous dispersion (Figure 1a).
Figure 1. Time-course phase-contrast microscopic observation of the light-triggered micrometer-size vesicle formation from a shapeless particle composed of DOPC and 1 in an aqueous solution of glucose and sucrose ([DOPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM). Snapshots were taken after irradiation (330−385 nm) at 20 °C for (a) 0, (b) 10, (c) 25, (d) 95, (e) 115, and (f) 175 s, respectively. The yellow arrows indicate the generated micrometer-size vesicles. Scale bars: 10 μm. The original video is available in the Supporting Information, Video S1.
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RESULTS AND DISCUSSION Preparation of Photoresponsive Particles and LightTriggered Micrometer-Size Vesicle Formation. In the course of our research on membrane-penetrating amphiphiles
Interestingly, when the particles in the dispersion were irradiated with UV light (330−385 nm) at 20 °C, using the 7290
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light source of the fluorescence microscope, micrometer-size vesicles were readily formed from their surfaces (Figure 1; the original video is available in the Supporting Information, Video S1). The vesicles kept growing on the surface accompanied by gradual dwindling of the particles as long as irradiation was continued. Along with the growth of the vesicles, they were eventually launched from the surface of the particles (Figures 1d−f). The generated vesicles mostly grown over 3.0 μm in diameter, and the maximum size is around 10 μm (Figure 1f). When the irradiation was paused, the growth of the vesicles synchronously stopped without shrinkage or deformation, and restart of irradiation induced further vesicle growth. Interestingly, some particles bent and deformed immediately after the UV irradiation, and the vesicles are formed thereafter (Figure 2;
microflow imaging (MFI), which gives digital images of micrometer to submicrometer-size objects in a flow cell, allows for overall visualization of the particles in the suspension. Indeed, MFI displayed that the aqueous suspension of the particles containing DOPC and 1 includes 1.0−1.9 μm objects as the most dominant species, with smaller quantities of larger objects (1.0−1.9 μm: 60.6% 2.0−2.9 μm: 13.1%, 3.0−3.9 μm: 5.3% etc; Figure 3, blue bars) before irradiation. After exposure
Figure 3. Distribution of the particle size evaluated by microflow imaging (MFI) of aqueous suspensions of shapeless particles composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM; blue bars) before and (red bars) after light irradiation (320 ± 10 nm, 60 min, 1.1 mW, 20 °C).
to UV light (320 ± 10 nm, 60 min, 1.1 mW, 20 °C), the populations of 1.0−1.9 and 2.0−2.9 μm objects decreased (55.9 and 12.0%, respectively; Figure 3, red bars), while those of >3.0 μm objects wholly increased (3.0−3.9 μm: 6.6% etc; Figure 3, red bars). As described above, the diameters of the generated vesicles are mostly larger than 3.0 μm. Thus, it is likely that the increase in population of large objects (>3.0 μm) in MFI images results from the vesicle formation, while reduction of the population of relatively small objects simultaneously occurs due to dwindling of the particles as observed in Figure 1. Such overall change of the particle-size distribution indicates that the vesicle formation takes place from the most particles contained in the suspension. Light-Driven Single Vesicle Formation with Micrometer-Scale Spatiotemporal Control. Taking advantage of the benefit to use light that enables spatial control of irradiation at a local spot, we challenged single vesicle formation from the micrometer-scale area on the surface of the photoresponsive particle. A 405 nm laser, focused with 1 μm diameter, was used as an excitation light source, where the laser beam (0.25 mW) was set on the stage of an apodized phase-contrast microscope. The edge of a particle composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM) was located at the focused irradiation spot (Figure 4a, the original video is available in the Supporting Information, Video S3) whereupon a single vesicle was readily formed at the spot and grew up to 6 μm in diameter through continuous irradiation for 70 s at 20 °C (Figures 4b,c). The generated vesicle was eventually detached from the particle (Figure 4d). Importantly, no vesicle formation was observed in other areas that are not exposed to the laser beam. Thus, the spatiotemporally controlled single vesicle formation at the micrometer-size limited area was successfully demonstrated by photoirradiation of the particle.
Figure 2. Time-course phase-contrast microscopic observation of a bending shapeless particle composed of DOPC and 1 in an aqueous solution of glucose and sucrose ([DOPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM) at the initial stage of the lighttriggered micrometer-size vesicle formation. Snapshots were taken after irradiation (330−385 nm) at 20 °C for (a) 0, (b) 2, (c) 5, and (d) 7 s. Schematic models of the particle are shown in the insets (a− c). The white arrows indicate the bending motion of the particle. Scale bars: 10 μm. The original video is available in the Supporting Information, Video S2.
the original video is available in the Supporting Information, Video S2). Importantly, we found that the exposure of the film to sonication (250 W, 15 min, 0−5 °C), immediately after the addition of the aqueous solution of the sugars to the casted film of DOPC containing 1, significantly encourages the formation of the particles dispersed in the aqueous phase. While the lighttriggered micrometer-size vesicle formation was observed even in deionized water without sugars, it took place more efficiently in the aqueous solution of the sugars.44 The particles prepared by a mixture containing a higher ratio of 1 (800 μM) to DOPC (2.0 mM) hardly responded to the light irradiation, while a mixture containing lower ratio of 1 (100 μM) to DOPC (2.0 mM) afforded only few particles. Hence, we adopted the ratio of [1]/[DOPC] = 0.20 for the subsequent experiments. It should be noted here that neither heating the aqueous suspension of the particles up to 80 °C in the dark nor irradiation by IR laser (0.29 mW, 1064 nm)23−25 induced vesicle formation, suggesting that heat has little effect on the vesicle formation from the particle. The above-mentioned microscopic studies visualize the formation of vesicles from each particle. On the other hand, 7291
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ization of the phospholipid component, we prepared the particles consisting of DOPC, 1, and 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE; [DOPC] = 400 μM, [1] = 80 μM, [NBD-PE] = 7.8 μM) dispersed in an aqueous media ([glucose] = [sucrose] = 100 mM). The resulting particles also generate micrometer-size vesicles upon irradiation with UV light. Confocal laser scanning micrographs (CLSM) of nonirradiated particles observed with excitation lights for 1 and NBD (405 and 473 nm, respectively) mostly overlapped with each other at each depth in the particle (Figure 5), indicating that 1 and NBD-PE are distributed over
Figure 4. Spatially controlled single micrometer-size vesicle formation on a particle composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM) observed by an apodized phase contrast microscopy at 20 °C. Micrographs were taken after irradiation of focused 405 nm laser beam for (a) 0, (b) 50, (c) 72 and (d) 80 s. In each micrograph, the irradiation spot is marked with a red circle, and a black arrow indicates the generated micrometer-size vesicle. Scale bars: 10 μm. The original video is available in the Supporting Information, Video S3.
Dependence of Micrometer-Size Vesicle Photogeneration on Lipid Phase. The deformation of the biomembrane is known to occur in the fluid liquid phase.1−3 Likewise, DOPC provides a liquid-phase membrane at 20 °C (DOPCTm = −20 °C), which is likely favorable for photogeneration of micrometer-size vesicles from the particle. In connection with this, we found that a mixture of 1 and 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC), forming a solid-phase membrane at 20 °C (DSPCTm = 55 °C), also gives the shapeless particles by the procedure described above ([DSPC] = 200 μM, [1] = 40 μM, [glucose] = [sucrose] = 100 mM). However, upon irradiation (330−385 nm) of these particles at 20 °C, no micrometer-size vesicle formation (see the Supporting Information, Figures S1a,b) took place. In sharp contrast, irradiation at 65 °C, where DSPC forms a liquid-phase membrane, triggered the micrometer-size vesicle formation (see the Supporting Information, Figure S1d). Thus, it is likely that the lipid phase is an important factor in photogeneration of the micrometer-size vesicles. It should be noted here that no morphological change of the particles occurred simply by heating without irradiation (see the Supporting Information, Figure S1c), again indicating that thermal stimuli do not trigger the formation of micrometer-size vesicles from the particle. The importance of the lipid phase is also suggested by the result that the particles consisting of DSPC, cholesterol, and 1 ([DSPC] = 100 μM, [cholesterol] = 100 μM, [1] = 40 μM) also allowed the light-driven vesicle formation at 20 °C (see the Supporting Information, Figure S2), since incorporation of cholesterol into a solid-phase membrane is known to increase its fluidity.3 Thus, it is suggested that a liquid phase is required for the light-driven micrometer-size vesicle formation. Structural Analyses of Particles and Photogenerated Vesicles. The particles prepared from the mixture of DOPC and 1 emitted bright fluorescence upon excitation by 330−385 nm irradiation, indicating that the particles contain 1 (see the Supporting Information, Figure S3). For fluorometric visual-
Figure 5. Confocal laser scanning micrographs of a photoresponsive particle composed of DOPC, 1, and NBD-PE ([DOPC] = 400 μM, [1] = 80 μM, [NBD-PE] = 7.8 μM) dispersed in an aqueous solution of glucose and sucrose ([glucose] = [sucrose] = 100 mM) at 20 °C. (a) The top surface and the cross sections at depths of (b) 2.0 ± 0.2 and (c) 4.0 ± 0.2 μm of the particle were observed with (1) 405 nm and (2) 473 nm lasers for excitation of the BPEB unit of 1 and NBD units of NBD-PE to show blue and green emissions, respectively. Merged micrographs of (1) and (2) are displayed in (3). Scale bars: 20 μm.
the particle. Nevertheless, although the fluorescence intensity of NBD-PE seems to be almost independent of the depth, that of 1 gradually increases with the depth of the confocal section. Importantly, CLSM also displayed a number of voids in the particle. Transmission electron microscopic (TEM) and microtomographic (TEMT) observations of the nonirradiated particles ([DOPC] = 200 μM, [1] = 40 μM) revealed that they are enveloped with membranes (Figure 6a; see the Supporting Information, Figures S4a,b). Membrane-like architectures, surrounding the voids, were also observed inside the particle. The thickness of the membranes at the surface and the inner area is 21−37 nm, suggesting that they are multilayer of DOPC membranes.45 These observations revealed that the particles are membrane-enveloped objects which include intricate multilayer membranes inside. TEM observation of the photoirradiated particle displayed a vesicle attached on the surface, with a 4.7 nm thick membrane, corresponding to a unilamellar membrane (Figure 6b).46 Further irradiation of the particle resulted in disappearance of the enveloping membranes, and only fragmentary membranes remained in the particle (Figure 6c; see the Supporting Information, Figures S4c,d). Hence, it is 7292
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composed of DOPC and 2 responded to UV irradiation (330− 385 nm) at 20 °C to form vesicles (see the Supporting Information, Figures S6a,b). In contrast, the particles composed of DOPC and 3 did not show any vesicle formation upon UV irradiation at 20 °C (see the Supporting Information, Figures S6c,d). Thus, the BPEB units are likely the essential components enabling the light-triggered vesicle formation.
Another point that should be taken into account is the contribution of the azobenzene unit. Comparison of phasecontrast micrographs between Figure 1f and Figure S6b (see the Supporting Information) suggests that the particles composed of DOPC and 1 tend to form the vesicles more effectively than the ones composed of DOPC and 2. UV−vis and 1H nuclear magnetic resonance (NMR) spectroscopic analyses indicated that the azobenzene unit of 1 undergoes trans−cis isomerization upon photoirradiation (see the Supporting Information, Figures S7 and S8). This was also supported by a fluorescence lifetime study; a shorter fluorescence lifetime of 1 (1.0 ns, 330 nm excitation light) than that of 2 (1.42 ns, 330 nm excitation light) in THF suggests energy transfer from the BPEB units to the azobenzene unit of 1. At the photostationary state, the ratio between [trans-1] and [cis-1] was 83/17. It is well-known that, even at the photostationary state, a photochromic molecule such as azobenzene isomerizes between the two isomers reversibly during the light irradiation. It is considered that the photoisomerization of the azobenzene unit of 1 enhances the efficiency of the vesicle formation. The detailed mechanism of the light-triggered vesicle formation from the particle, including the roles of BPEB and azobenzene units, is currently being investigated.
Figure 6. Transmission electron micrographs of a plastic-embedded section of the particle composed of DOPC and 1 ([DOPC] = 200 μM, [1] = 40 μM) (a) before and (b, c) after UV light (320 nm, 1 kW, 3 h, 20 °C) irradiation. Scale bars: 2.0 μm.
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likely that the enveloping membrane of the particle turns into the micrometer-size unilamellar vesicles. Under the condition of fluorescence microscopy, the membrane of the photogenerated micrometer-size vesicles showed fluorescence upon irradiation with 330−385 nm light, exciting the BPEB units of 1 (see the Supporting Information, Figure S5). This observation indicates that 1 is embedded in the lipid membrane of the photogenerated vesicles. Importance of Chromophore Units in Light-Driven Vesicle Formation. As demonstrated above, the vesicle formation takes place upon irradiation of the particles with 330−385 nm light, which can excite the chromophore units included in 1. To unveil the essential unit in 1 for the lighttriggered micrometer-size vesicle formation, we prepared 2 and 3, which lack the azobenzene and BPEB units, respectively. Mixtures of DOPC (2.0 mM) and 2 or 3 (400 μM) in an aqueous media ([glucose] = [sucrose] = 100 mM) afforded shapeless particles after sonication (250 W, 15 min, 0−5 °C), analogous to the mixture of DOPC and 1. The particles
CONCLUSION This work demonstrates light-triggered micrometer-size vesicle formation with micrometer-scale spatial control. In general, hydration of lipids under sonication is well-known to produce nanometer- to submicrometer-size vesicles.1−3 However, the addition of amphiphile 1 with DOPC dominantly provides micrometer-size shapeless particles. The shapeless particles are coated with the multilayer membranes that likely turn into the vesicles upon exposure to light. Thanks to the advantage of light, which acts as a contactless trigger, vesicle formation with micrometer-scale spatial control on the shapeless particle was successfully achieved. This phenomenon was also observed for other lipids like DSPC and the mixture of DSPC and cholesterol. Interestingly, lipid phase is an important factor for the transformation of the particles into the vesicles. Namely, upon photoirradiation, the shapeless particles composed of a solid-phase lipid show no response, while the particles made of 7293
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a liquid-phase lipid generate the vesicles. It is also revealed that the BPEB units of 1 are essential for the light-triggered vesicle formation. This methodology could provide a new approach for the vesicle development and manipulation of membranes.
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ASSOCIATED CONTENT
S Supporting Information *
Phase-contrast and fluorescent micrography and transmission electron microtomography of the particles composed of DOPC and 1, UV−vis and NMR spectra of 1, and UV−vis spectrum of 2. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel +81-22-217-5612; e-mail [email protected] (K.K.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Prof. Kouhei Tsumoto (The University of Tokyo) and Dr. Satoru Nagatoishi (The University of Tokyo) for helpful discussions. We also thank Ms. Ryoko Sugimoto (JAIST) for her support in preparation and microscopic studies of micrometer-size vesicles, Prof. Masatsugu Shimomura (Tohoku University) and Dr. Takahito Kawano (Tohoku University) for their support in confocal laser scanning microscopic observation, Dr. Akihiro Kishimura (The University of Tokyo), Dr. Satoshi Fukuda (The University of Tokyo Hospital), and Prof. Hiroshi Yabu (Tohoku University) for their support in TEM and TEMT experiments, and Prof. Atsushi Muramatsu (Tohoku University) and Prof. Kiyoshi Kanie (Tohoku University) for their support in polarized optical microscopic observation. TEM observations were conducted in Research Hub for Advanced Nano Characterization, The University of Tokyo, supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was partially supported by MEXT, Grants-inAid for Young Scientists S (21675003), Scientific Research on Innovative Areas “Spying minority in biological phenomena” (No. 3306), (23115003), and the Management Expenses Grants for National Universities Corporations to K.K. and Noguchi Foundation to T.M.
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