概要

Preparation of Giant Vesicles Exhibiting Visible-light-induced Morphological Changes

Published: July 21, 2016

概要

The synthesis of ruthenium complex surfactants exhibiting photoisomerization in giant vesicles is described. The preparation and light irradiation of the giant vesicles are also described.

Abstract

We describe the preparation of giant vesicles that incorporate a photoresponsive ruthenium complex having two alkyl chains. The vesicles exhibited morphological changes when exposed to visible light. The ruthenium complex proximal-[Ru(L1)(L2)OH2](NO3)2, proximal2 (L1 is 4′-decyloxy-2,2′;6′,2″-terpyridine, L2 is 2-(2′-(6′-decyloxy)-pyridyl)quinoline) was prepared by a thermal reaction of Ru(L1)Cl3 and L2, followed by removal of a chloride ligand. In an aqueous solution and vesicle dispersions, proximal2 was reversibly photoisomerized to the distal isomer. Giant vesicles containing proximal2 were prepared by hydration of phospholipid films containing proximal2 in the dark at 80 °C. Giant vesicles were frequently found in the dispersions prepared from DOPC/proximal2 rather than from DPPC/proximal2 (DOPC is 1,2-dioleoyl-sn-glycero-3-phosphocholine, DPPC is 1,2-dipalmitoyl –sn-glycero-3-phosphocholine). The ratio of proximal2 and DOPC in the vesicle preparation was varied from 5:100 to 20:100. The light-induced morphological changes were observed for proximal2/DOPC in the presence of Na2SO4. However, they were highly suppressed in the presence of NaOH. Incubation of light-exposed vesicles at 45 °C in the dark induced reverse morphological changes. Morphological changes were observed under fluorescence microscopy using 635 nm (red) light. Rhodamine-DOPC [rhodamine-DOPC: 1,2-dioleoyl-sn-glycero-3-phos-phoethanolamine-N-(lissamine rhodamine B sulfonyl)] was used to fluorescently label the vesicles.

Introduction

Controlling the morphologies and shapes of macro- and meso-scale molecular assemblies by external stimuli has attracted considerable attention.1,2 In particular, the control of vesicle morphologies by remote stimuli such as light has potential applications for drug delivery.3 In this context, organic photochromic molecules with hydrophobic and hydrophilic moieties have been widely incorporated into liposomes and polymer vesicles.4,5,6,7,8 However, most of the assemblies require ultraviolet (UV) light to drive the morphological changes, and their applications are limited because UV light is strongly scattered in living tissues and induces DNA damage and cell death.

Alternatively, utilization of visible or near-infrared light in the phototherapeutic window (600-1000 nm) is more favorable because of abundant sunlight and its high transmission in tissues of living organisms. In this regard, ruthenium complexes with polypyridyl ligands are suitable visible-light-responsive surfactants. They exhibit a strong visible light absorption band (ε~104 M-1 cm-1) that induces ligand substitution9,10 and photoisomerization.11,12,13,14,15,16 Incorporation of the ruthenium complexes into vesicles will expand their applications because these complexes are also known as water oxidation catalysts17,18,19 and bioactive molecules.20,21 Recently, ruthenium complexes have been incorporated into vesicles.22,23,24 However, controlling morphologies of vesicles via visible-light absorption has remained challenging.

We have previously reported irreversible and reversible photoisomerization of mononuclear ruthenium aqua complexes having asymmetric bidentate ligands.25,26,27,28 Recently, we synthesized novel surfactants (proximal2, see Figure 1) that exhibit visible-light photoisomerization equilibria with distal2 by introducing an alkyl chain on each tridentate and bidentate ligand of the ruthenium aqua complex. Giant vesicles incorporating proximal2 undergo morphological changes under the irradiation of visible light in the phototherapeutic window.29 Herein, we describe the detailed syntheses of ruthenium complexes and the preparation of giant vesicles. The protocols will enable researchers to prepare, characterize, and utilize light-responsive giant vesicles.

Figure 1
Figure 1: Ruthenium complex surfactants. Reversible photoisomerization equilibrium between proximal- and distal- type complex of 1 (top) and 2 (bottom). Please click here to view a larger version of this figure.

Protocol

NOTE: Ru(tpy)Cl330, L129, 2-(2'-(6'-chloro)-pyridyl)quinoline29, proximal- 129 were synthesized as previously described. 1. Synthesis of 2-(2'-(6'-decyloxy)-pyridyl)quinoline (L2) Add 2-(2'-(6'-chloro)-pyridyl)quinoline (16.3 mg, 63 µmol), 1-decanol (0.1 mL), dimethyl sulfoxide (1 mL), KOH (0.12 g) to a 50…

Representative Results

We obtained high-purity proximal-2 to form spherical and giant multilamellar vesicles (proximal-2/DOPC, proximal-2: DOPC=20:100) 15-µm average diameters (see Table 1).29 Several layers were found inside the vesicles (Figures 2A and 2C). The inner spheres of the vesicles in Figures 2B, and 2D were darker than the outer spheres because of the concentric lipid layers. T…

Discussion

The ruthenium chloro complex proximal-[Ru(L1)(L2)Cl]+ was prepared by thermal synthesis of Ru(L1)Cl3 and a bidentate ligand L2 in the presence of triethylamine. The proximal isomer was the major product and a distal isomer and Ru(L1)22+ was a minor impurity. The crude product was purified with size-exclusion chromatography using methanol as an eluent. Coordinating solvents, such as wat…

開示

The authors have nothing to disclose.

Acknowledgements

The authors have no acknowledgements.

Materials

Triethylamine Wako Pure Chemical Industries, Ltd. 202-02646
Lithium Chloride Wako Pure Chemical Industries, Ltd. 125-01161
Chloroform Kanto Chemical Co. Ltd.  07278-03 Used for vesicle preparation
Chloroform Junsei Chemical Co. Ltd.  28560-0382 Used for ligand synthesis
Acetone Junsei Chemical Co. Ltd.  11265-0382
Ethanol Junsei Chemical Co. Ltd.  17065-0382
Ethyl Acetate Junsei Chemical Co. Ltd.  67150-0382
Hexane Junsei Chemical Co. Ltd.  31055-0382
Silica gel Kanto Chemical Co. Ltd.  37558-79 100-210 μm
1-decanol Tokyo Chemical Industry Co., Ltd. D0031 25 mL
Potassium hydroxide Kanto Chemical Co. Ltd.  32344-00
Sodium hydrixude Wako Pure Chemical Industries, Ltd. 197-02125
Dimethyl sulfoxide (DMSO) Kanto Chemical Co. Ltd.  10378-00
d-DMSO Sigma-Aldrich 166290100
CD3OD Kanto Chemical Co. Ltd.  25221-43
d-Acetone Kanto Chemical Co. Ltd.  01054-43
D2O Cambridge Isotope Laboratories, Inc. DLM-4-100
Ruthenium chloride n-Hydrate Wako Pure Chemical Industries, Ltd. 183-00823
2,2':6',2"-Terpyridine Sigma-Aldrich 234672-5G
0.1 mol/L Silver nitrate solution Wako Pure Chemical Industries, Ltd. 192-00855
Sodium sulfate Kanto Chemical Co. Ltd.  37280-00
1,2 Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) Wako Pure Chemical Industries, Ltd. 160-12781 100 mg, stored at -20°C
1,2 Dioleoyl-sn-glycero-3-phosphocholine (DOPC) Sigma-Aldrich P6354-100mg 100 mg, stored at -20°C
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) Avanti Polar Lipids, Inc. Avanti 810332p 5 mg, stored at -20°C
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) Avanti Polar Lipids, Inc. Avanti 810150c 1 mg, stored at -20°C
Dextran gel GE healthcare Japan 17009010 Sephadex LH-20
Amber glass vial Maruemu 0407-06
Septum Sigma-Aldrich Z564648-100EA
Heater Advantech DRM 320 DB
Silicon film AS ONE 6-9085-03 Thickness: 0.2 mm
Slide glass Matsunami S003130 76×26 mm, thickness: 0.8-1.0 mm
Cover glass Matsunami C218181 18×18 mm, thickness: 0.12-0.17 mm
Transfer pipette  Brand GMBH 704774
Round-bottom flask Vidtech 1500-05
Sonicator AS ONE 1-4591-32
Optical power meter OPHIR ORION/PD P/N 1Z01803
Oil bath Riko  MH-3D
Magnetic stirrer Riko  MSR-10
Diatomite Wako Pure Chemical Industries, Ltd. 537-02305 Celite 545
Evaporator Yamato RE-52
Glass funnel Kiriyama SB-21 10 mL, 21 mmφ
Bell jar Kiriyama VKB-200
Filter paper Kiriyama No.4 21 mmφ
Optical microscope KEYENCE VHX-5000
Confocal fluorescence microscope Olympus FV-1000

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記事を引用
Hirahara, M., Tsukamoto, A., Goto, H., Tada, S., Yagi, M., Umemura, Y. Preparation of Giant Vesicles Exhibiting Visible-light-induced Morphological Changes. J. Vis. Exp. (Pending Publication), e54817, doi: (2016).

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