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.
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, proximal–2 (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, proximal–2 was reversibly photoisomerized to the distal isomer. Giant vesicles containing proximal–2 were prepared by hydration of phospholipid films containing proximal–2 in the dark at 80 °C. Giant vesicles were frequently found in the dispersions prepared from DOPC/proximal–2 rather than from DPPC/proximal–2 (DOPC is 1,2-dioleoyl-sn-glycero-3-phosphocholine, DPPC is 1,2-dipalmitoyl –sn-glycero-3-phosphocholine). The ratio of proximal–2 and DOPC in the vesicle preparation was varied from 5:100 to 20:100. The light-induced morphological changes were observed for proximal–2/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.
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 (proximal–2, see Figure 1) that exhibit visible-light photoisomerization equilibria with distal–2 by introducing an alkyl chain on each tridentate and bidentate ligand of the ruthenium aqua complex. Giant vesicles incorporating proximal–2 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: 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.
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)
2. Synthesis of proximal -2
3. Standard conditions for preparation of vesicles
4. Preparation of Plates
5. Morphological changes of giant vesicles under visible light irradiation
6. Morphological changes of giant vesicles under red light irradiation
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. The vesicles containing proximal–2 displayed various morphological changes under the irradiation of visible light (λ > 380 nm). The vesicle diameter in Figure 2A both increased and then decreased, while that in Figures 2B was distorted and had budding. The morphological changes were not usually observed for vesicles prepared from DOPC alone. Most of the morphological changes depended on the amount of proximal–2 (Table 1). Changes were also observed for proximal–2/DPPC vesicles (DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine). In contrast, proximal–1/DOPC vesicles did not show visible-light-induced morphological changes.
Figure 4 shows vesicles prepared in the presence of Na2SO4 (Figure 4A) and NaOH (Figure 4C). The photoinduced morphological changes were frequently observed for the vesicles containing Na2SO4 (Figure 4B) while those were not observed for the vesicles containing NaOH (Figure 4D). Figure 5 shows photo- and thermal-induced morphological changes of vesicles of proximal–2/DOPC. The budded vesicles under light irradiation recovered the original spherical vesicle after incubation in the dark.
The morphological changes in proximal–2/DOPC/rhodamine-DOPC vesicles under red light (635 nm) irradiation are depicted in Figure 6. We observed budding of granule vesicles from the vesicle edges, which is similar to that observed when exposed to visible light (λ > 380 nm).
Entry | Change from standard conditionsa | Average size/ μm | Morphological change / % |
1 | none | 15 | 79 |
2 | proximal-1, 10 nmol | 20 | 11 |
3 | no proximal-2 | 24 | 8 |
4 | proximal-2, 10 nmol | 18 | 80 |
5 | proximal-2, 5 nmol | 22 | 33 |
6 | DPPC | 16 | 50 |
7 | 500 nmol Na2SO4 | 15 | 80 |
8 | 1000 nmol NaOH | 21 | 10 |
9 | 100 nmol NaOH | 27 | 27 |
a In standard conditions, vesicles are prepared from DOPC (100 nmol), proximal-2 (20 nmol, 20 mol%), and water (100 μL). |
Table 1: Dependence of morphological changes on vesicle preparation parameters. The percentages of vesicles showing morphological changes were calculated from vesicles (>10 mm) under visible light irradiation (λ > 380 nm, 120 mWcm−2) for 30 min.
Figure 2: Microscope images of vesicles under irradiation with a 100 W halogen lamp (λ>380 nm, 120 mWcm-2). A) and B): proximal–2/DOPC (DOPC: 100 nmol, proximal–2: 20 nmol (20 mol%), water 0.1 mL). C) Vesicles prepared from DOPC alone (DOPC: 100 nmol, water 0.1 mL). D): proximal–2/DPPC (DPPC: 100 nmol, proximal–2: 20 nmol (20 mol%), water 0.1 mL). Parts reproduced from ref29 with permission of John Wiley and Sons, Inc. Please click here to view a larger version of this figure.
Figure 3: Microscope images of vesicles before light exposure. A), proximal–2/DPPC (DPPC: 100 nmol, proximal–2: 20 nmol (20 mol%), water 0.1 mL). B), proximal–2/DOPC (DOPC: 100 nmol, proximal–2: 20 nmol (20 mol%), water 0.1 mL). Please click here to view a larger version of this figure.
Figure 4: Microscope images of vesicles under the irradiation with a 100 W halogen lamp (λ>380 nm, 120 mWcm-2) in the presence of ionic compounds. A) and B), proximal–2/DOPC (DOPC: 100 nmol, proximal–2: 20 nmol (20 mol%), NaOH: 1000 nmol, water 0.1 mL). C) and D), proximal–2/DOPC (DOPC: 100 nmol, proximal–2: 20 nmol (20 mol%), Na2SO4: 500 nmol, water 0.1 mL). Left panels: before light irradiation and right panels: after light irradiation for 27 min with a 100 W halogen lamp. Please click here to view a larger version of this figure.
Figure 5: Photo- and thermal-induced morphological changes. proximal–2/DOPC (DOPC: 100 nmol, proximal–2: 20 nmol (20 mol%), water 0.1 mL). The vesicle dispersions were irradiated under visible light at 25 °C (top) and then incubated in the dark at 45 °C (bottom). Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 6: Morphological changes of giant vesicles exposed to 635-nm light. Confocal fluorescence microscope (A and C), and digital microscope (B and D) images of giant vesicles containing DOPC (100 nmol), proximal–2 (20 nmol), and rhodamine-DOPC (10 nmol) under irradiation with a diode laser (635 nm, 20 mW). The fluorescence microscopy was acquired with excitation at 559 nm excitation. Scale bar: 30 µm. Reproduced from ref29 with permission of John Wiley and Sons, Inc.
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 water and acetonitrile, should not be used. Slow dropping of the eluent (3-4 drops per minute) is required to separate the product from impurities. The product purification can be performed under room light because proximal-[Ru(L1)(L2)Cl]+ does not photoisomerize in methanol. The aquation of proximal-[Ru(L1)(L2)Cl]+ 세스 proximal-[Ru(L1)(L2)OH2]2+ (proximal–2) should be performed in the dark to prevent photoisomerization of the product.
Giant vesicles were prepared by simple hydration of lipid films containing the phospholipids and proximal–2. The DOPC and proximal–2 vesicles were spherical and multilamellar, while those obtained from DPPC and proximal–2 were slightly distorted as depicted in Figure 2. More giant vesicles were formed from proximal–2/DOPC than from proximal–2/DPPC, as depicted in Figure 3. The hydration temperature of the films should be more than 50 °C; giant vesicles were not formed after room-temperature hydration. The hydration time was varied over 5-24 hours with no significant differences in vesicle morphologies. After film hydration, the vesicle-containing samples were stored in the dark at 4 °C and used within a week. The vesicles can be prepared in the presence of ionic compounds such as Na2SO4 and NaOH, as depicted in Table 1. As shown in Figures 4C and 4D, the morphological changes were highly suppressed in the presence of NaOH (Figure 4C and 4D). The results arise from the formation of ruthenium-hydroxo complex (Ru-OH), which has been reported as inactive for photoisomerization.28
We previously reported that the mixture of proximal- and distal-2 in the photostationary state displayed thermal back isomerization to the proximal isomer in an aqueous solution at 45 °C. In the vesicle dispersions, vesicles were irradiated under the visible light at 25 °C, and then incubated in the dark at 45 °C as depicted in Figure 5. The vesicles displayed budding from the edge under the light irradiation due to the photoisomerization to distal–2. The budded vesicles were recovered to the original spherical vesicles after incubating the vesicle in the dark at 45 °C for 30 min. The back morphological change may arise from the thermal back isomerization of distal–2 세스 proximal–2.
Fluorescence microscopy was used to examine the fluorescent surfactants rhodamine-DOPC and fluorescein-DOPC [fluorescein-DOPC is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) ammonium salt]. No fluorescence was observed for proximal–2/DOPC/fluorescein-DOPC vesicles because of the overlapping emission band of the fluorescein dye with a metal-to-ligand charge-transfer absorption band of proximal–2. In contrast, rhodamine-DOPC can be used in fluorescence experiments because of its red emission (575 nm). The percentage of the rhodamine-DOPC (20 mol %DOPC) was higher than previous studies6 because the absorption of rhodamine-DOPC overlapped with that of proximal–2.
Common troubleshooting tips for the protocols are: (a) clean the amber vials before the preparation; (b) gently evaporate the chloroform from the lipids and the ruthenium complex under nitrogen gas flow; and (c) protect the samples from light before the measurements.
Generally, giant vesicles have been prepared from simple hydration, electroformation, or centrifugation methods.31 Electroformation methods have been widely used for the preparation of giant unilamellar vesicles. However, we did not adopt the method in order to avoid redox reactions of the ruthenium complex under the electric field. In this protocol, we prepared giant multilamellar vesicles by hydration of lipid films with distilled water or aqueous solutions containing 10-4 M ionic compounds. It should be noted that it is difficult to prepare giant vesicles of proximal–2/DOPC in a highly concentrated aqueous solution of ionic compounds (> 10-2 M). The red light responsive vesicles of proximal-2/DOPC are contrastive to the UV-light responsive vesicles reported so far.6,7,8 We are now trying to prepare the giant vesicles containing the ruthenium complex under physiological conditions.
The authors have nothing to disclose.
The authors have no acknowledgements.
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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