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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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, 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.

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 (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-introduction-3455
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)

  1. 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 mL round bottom flask equipped with a stir bar.
  2. Heat and stir the reaction mixture in an 80 °C oil bath for 4 h.
  3. Transfer the reaction mixture to a separating funnel, and add chloroform (ca. 20 mL) and water (ca. 20 mL). Shake the funnel for 2-3 minutes and wait several hours for complete separation into two layers. Collect the bottom organic layer, add anhydrous magnesium sulfate to absorb the water in the chloroform, filter with folded filtration paper, and remove the solvent in a rotary evaporator at 40 °C to obtain the oily crude product.
  4. Purify the product with silica gel chromatography (1.5 cm×20 cm) using a mixed solvent (AcOEt/hexane/CHCl3, 1:5:5, v/v/v) as an eluent.29 The product band emits blue light in the silica gel under UV light (254 nm).
  5. Collect the fractions of the blue band eluted from the column to the glass vials, and remove the solvent in a rotary evaporator at 40 °C. Check the product purity with 1H and 13C NMR in CDCl3 referenced with tetramethylsilane (TMS). The oily product contains a small amount of 1-decanol as impurity.29

2. Synthesis of proximal -2

  1. Synthesis of [Ru(L1)Cl 3 ]
    1. Add RuCl3·3H2O (60 mg, 0.23 mmol), L1 (80 mg, 0.21 mmol), and ethanol (EtOH, 40 mL) to a 50 mL round bottom flask equipped with a stir bar.
    2. Reflux and stir the reaction mixture in an oil bath for 4 h.
    3. Collect the yellow-brown precipitate with vacuum filtration, and wash with water (ca. 5 mL).
    4. Dry the product in vacuum for a yield of approximately 98 mg (80% yield).
  2. Synthesis of [Ru(L1)(L2)Cl]Cl
    1. Add [Ru(L1)Cl3] (47.2 mg, 0.078 mmol), triethylamine (0.2 mL), EtOH (12mL), water (4mL), LiCl (50 mg), and purified product of L2 synthesized from 0.10 mmol of 2-(2'-(6'-chloro)-pyridyl)quinoline to a 50 mL round bottom flask equipped with a stir bar.
    2. Reflux the reaction mixture in an oil bath for 4 h.
    3. Filter the purple solution with diatomite (ca. 2 g) on the filter paper in a glass funnel to remove unreacted [Ru(L1)Cl3].
    4. Reduce the solvent to ca. 3 mL in a rotary evaporator at 45 °C, collect the purple precipitate by vacuum filtration, and wash with diethyl ether.
    5. Purify the crude solid (44.2 mg) with size exclusion chromatography on a dextran gel, using methanol as eluent (column length: 20 cm).29 Collect fractions of the second purple band eluted from the column to glass vials. Spot the eluted fractions on the thin layer chromatography plate (ca. 1 µL for each spot). Check the purity using a mixed eluent (MeOH/saturated aqueous solution of NaCl, 30:1, v/v). Repeat the purification process two or three times.
    6. Remove the solvent on a rotary evaporator at 45 °C and dry in vacuum to obtain 30.1 mg of the product (39% yield). Check the purity with by 1H and 13C NMR in CDCl3 referenced with TMS.29
  3. Synthesis of proximal- [Ru(L1)(L2)OH 2 ](NO 3 ) 2 ( proximal- 2)
    1. Add proximal-[Ru(L1)(L2)Cl]Cl (16.3 mg, 0.017 mmol), water (3 mL), acetone (10 mL), and an aqueous solution of 0.1 M AgNO3 (0.60 mL, 0.060 mmol) to a 50 mL round bottom flask equipped with a stir bar. Cover the flask with aluminum foil
    2. Reflux the reaction mixture in an oil bath for 2 h in the dark.
    3. Filter the purple solution with diatomite (ca. 2 g) on the filter paper in a glass funnel.
    4. Reduce the solvent to ca. 3 mL in a rotary evaporator at 45 °C, collect the purple solid, and wash it with water.
    5. Dry in vacuum to obtain 12.6 mg of the product (75% yield). Check the purity with 1H and 13C NMR in the mix solvent of d-acetone and D2O (1:1, v/v) referenced with TMS.29

3. Standard conditions for preparation of vesicles

  1. To prepare 0.5 mM stock solution A, dissolve 4.0 mg of proximal- 2 in 8.0 mL of chloroform. Store the stock solution in the dark and refrigerate.
  2. To prepare 1.0 mM stock solution B, dissolve 15.7 mg of DOPC in 20.0 mL of chloroform.
  3. To prepare 0.1 mM stock solution C, dilute 1 mg/mL solution of rhodamine-DOPC with 6.6 mL of chloroform.
  4. Mix 40 µL of stock solution A and 100 µL of stock solution B in an amber glass vial. For the fluorescence microscopy experiments, add 100 µL of stock solution C.
  5. Seal the vial with a rubber septum equipped with a nitrogen inlet and outlet, and dry the solution under nitrogen flow overnight.
  6. Remove the septum and heat the vial in an 80 °C oven for 30 min.
  7. Add 0.1 mL of pure water to the vial. Seal and incubate the vial at 80 °C overnight. The lipid film is gradually hydrated to yield a reddish-purple vesicle dispersion that settles to the bottom of the vial.
  8. Store the vial in a refrigerator in the dark. The vesicle dispersion should be used within a week.

4. Preparation of Plates

  1. Rinse glass plates with ethanol and acetone, sonicate in ethanol for 5 min, and dry at 50 °C for 20-30 min.
  2. Cut a silicon film (thickness = 0.2 mm) into a 20 mm×20 mm square with a knife.
  3. Make a 5 mm hole with a hole punch at the center of the film, and remove plastic covers.
  4. Wet one side of the silicon film with diluted detergent (0.3 %) and then wipe it with cleaning tissue.
  5. Attach the edge of the film to a glass plate, and slowly lay the film in order to extrude the bubbles.
  6. Slowly shake the amber vial containing the vesicles, and with a micropipette transfer a small drop (diameter ~1 mm) to the center of the hole on the glass plate.
  7. Place a cover glass (18 mm × 18 mm) on the vesicle dispersion.

5. Morphological changes of giant vesicles under visible light irradiation

  1. Perform experiments in the dark at a constant temperature of 25 °C.
  2. Put the glass plate with sample droplets under a digital microscope (700X), and acquire images.
  3. Expose the sample plate with emission from a halogen lamp (distance from the plate: 2.5 cm) at a constant intensity of 120 mWcm-2.

6. Morphological changes of giant vesicles under red light irradiation

  1. Perform experiments in the dark at a constant temperature of 25 °C.
  2. Put the glass plate with sample droplets containing DOPC, proximal-2, and rhodamine-DOPC under a confocal microscope.
  3. Acquire images with a confocal microscope. Transmit excitation light (559 nm) and emission light (575 nm) through the same objective.
  4. Turn on the LED laser (635 nm), and adjust its intensity to 20 mW.

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors have no acknowledgements.

Materials

NameCompanyCatalog NumberComments
TriethylamineWako Pure Chemical Industries, Ltd.202-02646
Lithium ChlorideWako Pure Chemical Industries, Ltd.125-01161
ChloroformKanto Chemical Co. Ltd. 07278-03Used for vesicle preparation
ChloroformJunsei Chemical Co. Ltd. 28560-0382Used for ligand synthesis
AcetoneJunsei Chemical Co. Ltd. 11265-0382
EthanolJunsei Chemical Co. Ltd. 17065-0382
Ethyl AcetateJunsei Chemical Co. Ltd. 67150-0382
HexaneJunsei Chemical Co. Ltd. 31055-0382
Silica gelKanto Chemical Co. Ltd. 37558-79100-210 μm
1-decanolTokyo Chemical Industry Co., Ltd.D003125 mL
Potassium hydroxideKanto Chemical Co. Ltd. 32344-00
Sodium hydrixudeWako Pure Chemical Industries, Ltd.197-02125
Dimethyl sulfoxide (DMSO)Kanto Chemical Co. Ltd. 10378-00
d-DMSOSigma-Aldrich166290100
CD3ODKanto Chemical Co. Ltd. 25221-43
d-AcetoneKanto Chemical Co. Ltd. 01054-43
D2OCambridge Isotope Laboratories, Inc.DLM-4-100
Ruthenium chloride n-HydrateWako Pure Chemical Industries, Ltd.183-00823
2,2':6',2"-TerpyridineSigma-Aldrich234672-5G
0.1 mol/L Silver nitrate solutionWako Pure Chemical Industries, Ltd.192-00855
Sodium sulfateKanto Chemical Co. Ltd. 37280-00
1,2 Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)Wako Pure Chemical Industries, Ltd.160-12781100 mg, stored at -20°C
1,2 Dioleoyl-sn-glycero-3-phosphocholine (DOPC)Sigma-AldrichP6354-100mg100 mg, stored at -20°C
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt)Avanti Polar Lipids, Inc.Avanti 810332p5 mg, stored at -20°C
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)Avanti Polar Lipids, Inc.Avanti 810150c1 mg, stored at -20°C
Dextran gelGE healthcare Japan17009010Sephadex LH-20
Amber glass vialMaruemu0407-06
SeptumSigma-AldrichZ564648-100EA
HeaterAdvantechDRM 320 DB
Silicon filmAS ONE6-9085-03Thickness: 0.2 mm
Slide glassMatsunamiS00313076×26 mm, thickness: 0.8-1.0 mm
Cover glassMatsunamiC21818118×18 mm, thickness: 0.12-0.17 mm
Transfer pipette Brand GMBH704774
Round-bottom flaskVidtech1500-05
SonicatorAS ONE1-4591-32
Optical power meterOPHIRORION/PD P/N 1Z01803
Oil bathRiko MH-3D
Magnetic stirrerRiko MSR-10
DiatomiteWako Pure Chemical Industries, Ltd.537-02305Celite 545
EvaporatorYamatoRE-52
Glass funnelKiriyamaSB-2110 mL, 21 mmφ
Bell jarKiriyamaVKB-200
Filter paperKiriyamaNo.421 mmφ
Optical microscopeKEYENCEVHX-5000
Confocal fluorescence microscopeOlympusFV-1000

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