Article pubs.acs.org/JPCB
Deformation of Lipid Membranes Containing Photoresponsive Molecules in Response to Ultraviolet Light Kazunari Yoshida,* Yasuhiro Fujii, and Izumi Nishio* Department of Physics and Mathematics, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5258, Japan S Supporting Information *
ABSTRACT: Recently, membrane deformation using photoresponsive molecules has been extensively studied toward controlling their shapes because light can supply energy without contacting the vesicles. In this study, photoresponsive pseudogem-bis(diphenylimidazole) [2.2]paracyclophane (pseudogem-bisDPI[2.2]PC) molecules were doped into dioleoylphosphatidylcholine (DOPC) membranes, and the deformation of the DOPC/pseudogem-bisDPI[2.2]PC vesicles was observed under ultraviolet (UV)-light irradiation. It was also found that the volume-to-surface area ratio of spherical vesicles was changed by UV irradiation. Further, we performed high performance liquid chromatography (HPLC) analysis of membrane components in order to clarify the absence of irreversible chemical reactions and UV-irradiation experiments under an osmotic pressure in order to investigate the volume change of the vesicles. Then, we calculated the time-correlation function of membrane fluctuation. Change in the relaxation time of the time-correlation function indicated that the photoisomerization of pseudogembisDPI[2.2]PC might decrease the membrane fluidity. We consider that decreasing fluidity is induced by physical entanglement between photochromic compounds and lipids. This technique of membrane deformation may be expected to be applied to various situations such as drug delivery systems (DDS).
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INTRODUCTION It is well-known that the amphipathic nature of the phospholipids causes them to form bilayers spontaneously in aqueous environments. Since the lipid bilayer is a main component of biological membranes such as cell membrane,1 the lipid bilayer vesicles have been widely studied as a simple model system of biological membranes. For example, properties of the membrane proteins2,3 and the interactions between toxic peptides and biological membranes4 have been studied using liposomes. In addition, the physicochemical properties of biological membrane have been studied using mesoscopic liposomal systems, such as phase-separated liposomes.5,6 In particular, deformation of the lipid bilayer vesicles has been intensively investigated to understand the mechanism of endocytosis, exocytosis, and the formation of cellular organelles with various shapes. These investigations have revealed that lipid membranes were deformed in response to various stimuli such as temperature,7 osmotic stress,8−11 pH,12,13 addition of proteins,14 and application of surfactant molecules.8,9,15,16 Interactions between photoresponsive molecules and lipid membranes have also been studied. Light is one of the best tools for manipulating soft materials because it can apply energy to the target without contacting. Several studies using photoresponsive surfactants have been reported.17,18 In the mesoscopic systems, light-induced shape change of the lipid membranes was demonstrated in the presence of pyrene.19 Recently, fast reaction of switching membrane shape20−22 and vesicular burst23 have been observed using light with specific © 2014 American Chemical Society
wavelength. Most of these studies aim to control the membrane shape and/or to release the inner molecules of vesicles because such techniques are expected to be applied to the drug delivery systems (DDS).24−28 In this study, we doped a photoresponsive pseudogembis(diphenylimidazole) [2.2]paracyclophane (abbreviated as pseudogem-bisDPI[2.2]PC, hereafter)29 into lipid bilayer membrane composed of dioleoylphosphatidylcholine (DOPC). We performed ultraviolet (UV)-light irradiation experiments on lipid membranes containing the pseudogembisDPI[2.2]PC. It was found that shape changes of the photoresponsive-molecule-doped membranes can be triggered by UV irradiation, and the volume-to-surface area ratio of the vesicle was changed. Then, we performed high performance liquid chromatography (HPLC) analysis and UV irradiation experiments under osmotic pressure. We also calculated the time-correlation function of the membrane fluctuation before and during UV irradiation. The results of the calculation suggested that the membrane stiffness was changed by photoisomerization of pseudogem-bisDPI[2.2]PC.
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EXPERIMENTAL SECTION Materials. Pseudogem-bisDPI[2.2]PC was purchased from Kanto Chemical Co., Inc. (Japan). Figure 1 shows the chemical
Received: December 28, 2013 Revised: March 6, 2014 Published: March 13, 2014 4115
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rate was set to 1 mL/min using a PU-2080 (JASCO, Japan). The optical density detected at λ = 254 nm was recorded using a UV-2075 (JASCO, Japan). Application of Osmotic Pressure. The vesicle suspension and sucrose aqueous solution were mixed in order to apply the osmotic pressure to the vesicles. The difference of sucrose concentration between inside and outside of the vesicles (Δc) was 0.5 mM. The water molecules inside the vesicles were intended to move to the outside of the lipid vesicles under this situation.
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Figure 1. Photoisomerization of pseudogem-bisDPI[2.2]PC. Cleavage of the C−N bond between the imidazole rings is caused by UV-light (λ ≤ 400 nm) irradiation. After stopping UV irradiation, the C−N bond is thermally formed again. This chemical reaction is reversible.
RESULTS Membrane Deformation under UV Irradiation. Figure 2 shows the typical response of DOPC/pseudogem-bisDPI[2.2]PC vesicles (vesicles 1−3) to UV irradiation. These vesicles were hydrated at 37 °C during vesicle formation. It is considered that the photoresponsive molecules are homogeneously distributed in the bilayer, because phase separation (pseudogem-bisDPI[2.2]PC rich dark region) was not observed by a fluorescence microscope, as shown in Figure S1 of the Supporting Information. Before UV irradiation, the surfaces of these vesicles were rather smooth and then all the vesicles were rippled by UV exposure. Parts a and b of Figure 2 show the deformation of gourd-shaped vesicles (vesicles 1 and 2), while part c shows the deformation of the spherical vesicle (vesicle 3). These vesicles were deformed by UV irradiation within 100 s. The number of drastically deformed vesicles is 40 out of 90 vesicles. Only 5% of the drastically deformed vesicles (n = 40) recovered their original shapes after stopping UV irradiation. In addition, there is no drastically deformed vesicle composed of only DOPC (n = 26). Figure 3a shows the effect of UV irradiation on a simple spherical vesicle (vesicle 4) which was hydrated at 50 °C during membrane formation. We defined the UV irradiation time of 0 s and post-irradiation time of 0 s as timings at which we started and stopped irradiation, respectively. The pre-irradiation time was represented by negative numbers. The shape of the membrane was changed with UV irradiation (shown in the upper row of Figure 3a) and recovered its shape after stopping UV irradiation (shown in the lower row of Figure 3a). However, no further significant deformation was observed with additional UV irradiation. In other words, the shape was recovered only once. We traced the membrane surface of this vesicle using 2D images of the cross section and calculated the barycenter of these traced points. Membrane displacement was defined as
structure and a scheme of photoisomerization of the molecule. The C−N bond of the molecule is cleaved by UV irradiation, and the conformation is changed with the bond cleavage. After irradiation ceases, the complete thermal bleaching is achieved within 200 ms in benzene at 25 °C.29 Dioleoylphosphatidylcholine (DOPC), sucrose, and organic solvents except methanol were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Methanol was obtained from Showa Chemical Industry Co., Ltd. (Japan). Ultrapure water was obtained using a WT101UV AUTOPURE (Yamato Scientific Co., Ltd., Japan). Preparation of Lipid Membranes Containing Photoresponsive Molecules. We prepared DOPC/pseudogembisDPI[2.2]PC vesicles by the natural swelling method.30 First, DOPC and pseudogem-bisDPI[2.2]PC were dissolved in a mixture of chloroform and methanol with a volume ratio of 2:1. We purged the organic solvent with N2 gas flow and then incubated it in a vacuum for more than 8 h in order to make dry-DOPC/pseudogem-bisDPI[2.2]PC film. Finally, the film were hydrated with ultrapure water for more than 24 h at 37 or 50 °C. The molar ratio of DOPC and pseudogem-bisDPI[2.2]PC was 7:3, while the concentration of the solutes of the vesicle suspension was 0.35 mM. It is considered that the pseudogembisDPI[2.2]PC molecules exist in the hydrophobic region of the bilayer because the molecule also has a hydrophobic nature. Microscopic Observation with an UV Irradiation System. The vesicle suspension was placed between a slide glass and a coverslip and was sealed with vacuum grease. We observed the vesicles using a bright-field microscope (Olympus BX40, Japan) equipped with a 40× objective lens and a metal halide lamp, which did not include the UV region (λ ≤ 400 nm). The vesicles were observed at room temperature (22.3 ± 0.5 °C), and the images of the vesicles were recorded by a digital camera PEN E-PL1 (Olympus, Japan). We irradiated the samples with a power of 540 μW at a focused spot on the stage in order to excite the pseudogem-bisDPI[2.2]PC using a Hg lamp (λmax = 365 nm) through a WU filter set (Olympus, Japan) and the objective lens. High Performance Liquid Chromatography (HPLC) Analysis. First, we prepared a DOPC/pseudogem-bisDPI[2.2]PC-vesicle suspension, and a part of the suspension was exposed to UV light (λmax = 365 nm) with a power density of 844 μW/cm2 for 20 min. The UV-exposed and nonexposed suspensions were mixed with acetonitrile (acetonitrile:vesicle suspension = 25:1 in volume). We performed HPLC analysis on both solutions with an ODS-3V column (5 μm, 4.6 × 250 mm2, GL Sciences Inc., Japan). Flow solvent was made from acetonitrile and distilled water (25:1 in volume), and the flow
Δr(t , θ ) ≡ r(t , θ ) − ⟨r(t , θ )⟩
(1)
where the radius of a vesicle r(t, θ) is the time-dependent distance between the barycenter and the membrane surface around the contour and ⟨r(t, θ)⟩ is the mean value of the radius in an angle range of 360°. Figure 3b shows Δr(t, θ) as a function of θ at an UV irradiation time of 0 s, 37 s, and the post-irradiation time of 35 s. The amplitude of Δr(t, θ) at an irradiation time of 37 s was larger than those of the two other timings. We performed the Fourier analysis of membrane waviness Δr(t, θ). Obtained Fourier amplitudes are shown as a function of wavenumber k in Figure 3c, where the wavenumber of k = 2 corresponds to an elliptic shape.21 Appearance of the k = 2 peak means that the spherical shape of the vesicle was changed into an ellipsoid with UV irradiation. Disappearance of this peak without appearance of other peaks means that spherical shape was recovered after stopping UV irradiation. 4116
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Figure 3. (a) Microscopic images of a vesicle which was hydrated at 50 °C during membrane formation. The negative number and positive numbers in the upper row represent the pre-irradiation time and the irradiation time, respectively. The numbers in the lower row also represent the post-irradiation time. Deformation of spherical vesicle with UV irradiation. The scale bar represents 20 μm. We named the vesicle of these images vesicle 4. (b) Change in membrane waviness of vesicle 4. Time dependent differences between the radius r(t, θ) and mean value of the radius ⟨r(t, θ)⟩ around the contour. (c) Fourier amplitude versus wavenumber k of membrane waviness. Wavenumber represents the numbers of waves in the membrane contour.
Figure 2. Microscopic images of vesicles which were hydrated at 37 °C during membrane formation. Examples of membrane deformation with UV irradiation. The scale bars represent 20 μm. The numbers at the upper left of each image show the UV irradiation time. We named the vesicles (a) vesicle 1, (b) vesicle 2, and (c) vesicle 3, respectively. These vesicles are rippled with UV exposure.
Reduced Volume of Vesicle. We calculated the reduced volume of vesicle 4, which represents a volume-to-surface area ratio.31 The reduced volume vR of a vesicle with a surface area A and a volume V is given by V (4π /3)r0 3
(2)
r0 ≡ (A /4π )1/2
(3)
vR ≡
is the radius of a sphere with the same surface area. The spherical shape of vesicle 4 changed into ellipsoid with UV irradiation, as shown in the microscopic images (Figure 3a). Figure 4 shows the reduced volume of vesicle 4 versus time. The irradiation time range is shown from 12 to 60 s in Figure 4. We calculated the surface area A and volume V as a body of
where
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each peak. The derivatives of pseudogem-bisDPI[2.2]PC generated by irreversible chemical reaction may create a large peak due to their speculated high absorbance at λ = 254 nm. We found only one peak that originates from the molecule on each chromatogram. Since the absorbance of DOPC at λ = 254 nm is significantly less than that of pseudogem-bisDPI[2.2]PC, it is considered that the other peak that originates from DOPC is not seen. Other minor peaks on the chromatograms are considered to originate from impurities other than derivatives of pseudogem-bisDPI[2.2]PC. The results show that UV irradiation does not induce an irreversible chemical reaction that might cause membrane deformation. Reversibility of membrane deformation (vesicle 4) is consistent with this result. Membrane Deformation with UV Irradiation under an Osmotic Pressure. Next, we discuss the change in permeability of water molecules through the membranes due to photoisomerization of the photochromic compounds. There is a possibility that the decreasing inner aqueous volume is induced by water efflux across the membrane and induces the vesicular deformation. In order to estimate the water permeability, we performed the UV-irradiation experiment on the vesicle under osmotic pressure. Figure 6a shows microscopic images of the vesicle under osmotic pressure (vesicle 5). Fission of vesicle 5 was observed within 100 s. The vesicle has an ellipsoidal shape until an irradiation time of 20 s. We regarded the vesicle at 70 s as an aggregate of two ellipsoidal vesicles and the vesicle at 99 s as an aggregate of three ellipsoidal vesicles. Since the drastic deformation induced by osmotic pressure is usually achieved for several hundred seconds,21,35 it is speculated that the rapid deformation of vesicle 5 may be induced by UV exposure. Parts b and c of Figure 6 show the surface area and the volume of vesicle 5 under osmotic pressure versus time, respectively. We calculated the surface area and volume of body of rotation using 2D images of the vesicle, assuming that the vesicle is an aggregate of ellipsoids. Surface area increased with UV irradiation (shown in Figure 6b), while volume was not significantly changed in spite of the osmotic pressure (shown in Figure 6c). If the passing of water molecules through the membranes were induced by photoisomerization of the photochromic molecules, the volume of the vesicle should be changed with UV irradiation. The present result suggests that the permeability of the water molecules is not influenced by photoisomerization of the pseudogem-bisDPI[2.2]PC molecules. In other words, it is confirmed that the volume change is not the cause of membrane deformation. In addition, the result shown in Figure 6b suggested that changing surface area is a possible cause of membrane deformation. It is considered that the change in reduced volume as shown in Figure 4 was induced by surfacearea change. Time-Correlation Function of Membrane Fluctuation. Finally, we discuss the membrane fluctuation using a timecorrelation function.36 We defined the difference between two perpendicular diameters D(t) of a vesicle as
Figure 4. Reduced volume of vesicle 4 versus time. We started UV exposure at the time of 12 s and stopped at 60 s.
rotation of 2D images of vesicle 4, assuming ellipsoidal-vesicle shape. Reduced volume decreased with UV irradiation and increased after stopping UV irradiation. The vesicle shape at UV irradiation times of 37 s (vR = 0.93) and 47 s (vR = 0.94) was ellipsoid. It was theoretically reported that the vesicle with vR ≈ 0.95 tends to have ellipsoidal shape because ellipsoid is the minimal energy shape.32 The behavior of the vesicle in the present study is in good agreement with this report.
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DISCUSSION HPLC Analysis. One of the candidates of the cause of membrane deformation is irreversible chemical reaction between hydrophobic chains of DOPC and imidazolyl radicals of pseudogem-bisDPI[2.2]PC, since there is a possibility that the vesicles are deformed by oxidative stress.33,34 UV irradiation generates imidazolyl radicals from the pseudogem-bisDPI[2.2]PC, as shown in Figure 1. If the irreversible chemical reaction occurred as a result of the UV irradiation, materials other than DOPC and pseudogem-bisDPI[2.2]PC should be generated. We performed HPLC experiments on UV-exposed and nonexposed vesicle solutions in order to confirm whether extra materials are generated or not. Parts a and b of Figure 5 show the chromatograms of UV-exposed and nonexposed vesicles dissolved in water−acetonitrile solvent, respectively. The numbers in the chromatograms show the retention time of
D(t ) ≡ Δr(t , 0) + Δr(t , π ) − Δr(t , π /2) − Δr(t , 3π /2)
(4)
as shown in Figure 7a. The elliptic solid line represents the membrane surface, while the dotted one shows a circular contour with a mean value of the membrane radius. Then, the time-correlation function was defined as
Figure 5. The optical density detected at 254 nm is plotted as a function of the retention time. (a) Chromatogram of UV-exposed vesicle solution. (b) Chromatogram of nonexposed vesicle solution. 4118
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Figure 6. (a) Upper images show a vesicle under osmotic stress (Δc = 0.5 mM) before UV irradiation. Lower images show membrane deformation with UV irradiation. The scale bar represents 10 μm. We named the vesicle of these images vesicle 5. (b) Surface area of vesicle 5 versus time. (c) Volume of vesicle 5 versus time.
Figure 7. (a) Definition of Δr(t, 0), Δr(t, π/2), Δr(t, π), and Δr(t, 3π/2). The elliptic solid line represents the membrane surface. The dotted line is drawn using the mean value of the membrane radii of all angles. (b) Normalized time-correlation function c(τ)/c(0) of vesicle 3 versus delay time.
c(τ ) =
1 N
N−1
were entangled in hydrophobic chains of lipids by photoisomerization. The entanglement may reduce fluidity of the membrane and bend the vesicle surface, as shown in Figure 2. If simply changing excluded volume of the molecules occurred, the relaxation time of the correlation function would be become shorter. This is because expanding the excluded volume disturbs the packing of the lipids and increases the membrane fluidity. Further, the surface area of the bended membrane is larger than that of the nonbended one. The surface area increasing as shown in Figure 6 can support this discussion. In addition, the entanglement may also disturb the recovering of the membrane shapes and deformation reversibility. The idea of entanglement is supported by the low recovering percentage and nonrepeated deformation.
∑ D(t )·D(t + τ) t=0
(5)
where τ is the delay time and N is the number of video frames used for the calculation. We calculated the time-correlation function c(τ) of D(t) using microscopic images of vesicle 3. Figure 7b shows the normalized time-correlation function c(τ)/ c(0) of vesicle 3 before and during UV exposure. The normalized time-correlation function was found to decay faster before than during UV irradiation. Vesicle fluctuation before UV irradiation was interpreted as a thermal fluctuation because the pseudogem-bisDPI[2.2]PC molecules were not excited by visible-light irradiation.29 Relaxation times of the timecorrelation function were increased with UV irradiation. The result indicates that the membrane stiffness was changed by UV irradiation. The results of the HPLC analysis suggest that there are no irreversible chemical reactions in the bilayer. It is considered that the change in stiffness is caused by physical entanglement between photoresponsive molecules and lipids. The molecules simply existed in the bilayer without entanglement before UV irradiation, and then, the molecules
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CONCLUSION In this study, we found that lipid membranes containing photoresponsive pseudogem-bisDPI[2.2]PC molecules were deformed by UV exposure, and the change of the reduced volume of spherical vesicles was induced by UV irradiation. It was also found that the decay time of the time-correlation function of membrane fluctuation was changed by UV 4119
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irradiation. In other words, the photoisomerizaiton of pseudogem-bisDPI[2.2]PC induced change in membrane stiffness. We considered that the change of stiffness was caused by entanglement between photoresponsive molecules and hydrophobic chains of lipids. The entanglements bend the membranes and increase the surface area of the vesicles. We expect that the deformation technique of the vesicles containing pseudogem-bisDPI[2.2]PC plays an important role in the biomimetic technologies such as self-reproduction37,38 and DDS.
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ASSOCIATED CONTENT
* Supporting Information S
Movies of the membrane deformation with UV irradiation and results of fluorescence microscopy and analysis. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. Junji Kimura (Aoyama Gakuin Univ.) and the members of his laboratory for technical support in the HPLC experiments. Significant advice for this work from Prof. Tsutomu Hamada (JAIST), Prof. Jiro Abe (Aoyama Gakuin Univ.), and Mr. Katsuya Mutoh (Aoyama Gakuin Univ.) is greatly appreciated.
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