J. Phys. Chem. B 2008, 112, 15673–15677
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In-Situ Observation of the Inside-to-Outside Molecular Transport of a Liposome Joon Heon Kim*,† and Mahn Won Kim*,‡ AdVanced Photonics Research Institute, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea, and Department of Physics, Korea AdVanced Institute of Science and Technology, Daejeon 305-701, Korea ReceiVed: August 25, 2008; ReVised Manuscript ReceiVed: October 14, 2008
The first in-situ and real-time observation of the molecular transport from inside to outside of a liposome was shown by using the second harmonic generation (SHG) technique. The transport of an organic cationic molecule across the liposome bilayer could be switched on and off using the structural change of the lipid bilayer caused by temperature change. This approach can be helpful for the understanding and control of the molecular transport in the liposome vehicle. 1. Introduction A liposome is a spherical vesicle composed of a lipid bilayer which is the simple model system for the biomembrane.1 Because of its unique structure, it has been studied for various applications including the nonviral drug delivery systems.1,2 In these applications, the main issue is to design the vehicle which can protect the contained materials during delivery and then release them at the target in the controlled way. Therefore, the understanding and proper control of the molecular transport from inside to outside of a liposome should be one of the most important factors. Among many ways to observe the molecular transport in the liposome, the optical second harmonic generation (SHG) is an in-situ surface-specific technique which enables the real-time observation of the molecular adsorption and transport without disturbing the system.3-7 Because SHG is a second-order nonlinear optical technique, it is forbidden in the centrosymmetric media, and the molecules dispersed in the bulk liquid cannot contribute to it.8 In the case of the liposome, the orientation of molecules adsorbed on the outer and inner surfaces should be opposite to each other due to the bilayer symmetry. Therefore, the SH electric field from the liposomes of several hundred nanometers size is proportional to the difference of the number of adsorbates on the outer and inner surfaces:3 E2ω(t) ∝ [nout(t) - nin(t)]. This makes it possible to observe the transport of adsorbates across the lipid bilayer by measuring the time dependence of the SH signal. The transport kinetics of the triphenyl cationic dye, malachite green (MG), through the liposome bilayer in various conditions of the surface charge density,4 cholesterols,5 counterions,6 or temperature7 have been extensively studied by the SHG technique because of its large second-order nonlinear hyperpolarizability generating the strong SHG signal and its partial hydrophobicity enabling its direct transport across the lipid bilayer. All these studies have been done for the molecular transport from outside to inside of the liposome because of the relative easy initial condition for the outside-to-inside transport which can be attained by just injecting liposome solution to dye solution. However, it is possible for the transport across * To whom correspondence should be addressed. E-mail: joonhkim@ gist.ac.kr,
[email protected]. † Advanced Photonics Research Institute. ‡ Korea Advanced Institute of Science and Technology.
the liposome bilayer to be dependent on the transport direction because the environments of both sides of liposome bilayer can be different due to the confinement of the inner volume. Therefore, it is necessary to investigate the inside-to-outside transport in the liposomes for the applications such as the drug delivery system. 2. Basic Idea To study the inside-to-outside molecular transport, we need to make the inside concentration of dye much higher than its outside concentration. The simplest way to achieve this condition might be mixing MGs with lipids before the extrusion9,10 and then diluting the extruded solution. However, in this way, there is possibility that many MG molecules can be lost by adsorbing on the polycarbonate membrane filter during extrusion. Furthermore, the interaction between MGs and lipids may affect the extrusion process to make a structure different with that made purely by lipids. For this reason, we tried to add MG molecules after the extrusion of the liposomes. Different from the microscopy experiment using the individual giant unilamellar vesicle (GUV) with the size of several tens of micrometers,11 it is impossible to directly inject dye solution inside many liposomes of size less than micrometers at the same time. To solve this problem, we first equilibrated the dye concentrations at both sides and then removed dyes at the outside of the liposome. The liposome was made of the saturated lipids, distearoylphosphatidylglycerol (DSPG), which has a gel-to-fluid phase transition above the room temperature.12 A dye can transport across only the fluid phase of the lipid bilayer, not the gel phase.7 Therefore, we can switch on and off the transport by changing temperature. First, we mixed the liposome solution and the dye solution at the gel phase of the lipid bilayer (step A in Scheme 1). At this step, the equilibrium was obtained between the dyes in the bulk and the ones adsorbed on the outer surface of the liposome. Then, we increased temperature to the fluid phase of the lipid bilayer (step B in Scheme 1). This makes dyes transport through the lipid bilayer to get the equilibrium between the numbers of dyes on both surfaces of the liposome bilayer. After equilibrium, we decreased temperature to the gel phase of the lipid bilayer to make no more dyes transport across the lipid bilayer. Now, we have to eliminate dyes outside the liposome to make the inside dye concentration higher than the outside one. A
10.1021/jp8075657 CCC: $40.75 2008 American Chemical Society Published on Web 11/13/2008
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SCHEME 1: Basic Sequencea
a Temperature was changed to switch on and off the transport of MG across the liposome bilayer. MG can pass through the fluid phase of the DSPG lipid bilayer at 50 °C, while it cannot pass through the gel phase at 20 °C.
simple addition of buffer to reduce the outside dye concentration is ineffective because it also reduces the liposome concentration, resulting in the decrease of the total SH intensity. Dialysis, often used for the dilution of small molecules without changing the concentration of large particles, is also ineffective because it takes too long time (order of several hours) to get enough difference of the dye concentrations on both sides of the lipid bilayer. To overcome these problems, we used clay particles to eliminate dyes outside the liposome for the SHG. Montmorillonite clay particles have a disk shape with ∼450 nm diameter and ∼3 nm thickness, which is composed of alumina and silica sheets.13 Its basal surfaces are negatively charged due to the isomorphous substitution of aluminum or silicon in the crystal lattice by other lower valence metal ions, while the edge surfaces are positively charged in neutral or acidic pH due to the broken and hydrolyzed Al-O and Si-O bonds. When these clay particles are mixed with the organic cationic dyes, most dyes are adsorbed on the basal surfaces due to the electrostatic attraction.14,15 The dyes adsorbed on the basal surfaces of the clay particles cannot contribute to the SHG signal because the thickness of the clay particle is very small compared to the wavelength so that SHG from the dyes on each side cancel each other. Therefore, the adsorption of dyes on the clay particles has the same effect as the elimination of dyes with regard to the SHG intensity. This application of the clay particles has previously been used for the study on the transfer kinetics of organic cationic dyes from polystyrene microspheres.15 By adding clay particles to the liposome and dye solutions in the gel phase of the lipid bilayer, we can decrease the number of dyes in the bulk outside the liposome (step C in Scheme 1). This induces desorption of the dyes from the outer surface of the liposome, but the dyes adsorbed on the inner surface cannot transport to the outer surface because the lipid bilayer is in the gel phase. Consequently, we can have the liposomes with the inner surface dye concentration much higher than the outer one. After that, we increased temperature to above the gel-fluid phase transition temperature, which induced the transport of dyes from inside to outside of the liposomes (step D in Scheme 1). 3. Experimental Methods Sample Preparation. The liposome was made of anionic DSPG (distearoylphosphatidylglycerol) lipids using the extrusion technique.9,10 The dry lipid film was hydrated and extruded using 1 mM NaCl solution at 60 °C, and the average size of the liposomes was measured to be ∼132 nm by the dynamic light scattering. The clay particle, Na-montmorillonite, was stored in 0.5 M NaCl solution until it was used. This solution was centrifuged at 14 100 rpm for 10 min, washed, and resuspended with Milli-Q water until no more chloride ion was detected with
Figure 1. (a) Change of the SHG intensity and (b) temperature in three different samples are plotted with respect to time. In the sample P1, 0.2 mL of the clay solution (1 mg/mL) was injected into 2 mL of the MG-only solution (MG 6 µM) at 20 °C. In sample P2, 0.2 mL of the clay solution (1 mg/mL) was injected into 2 mL of the liposome and MG mixtures (DSPG 20 µM and MG 6 µM) at 20 °C. In sample P3, the temperature of 2 mL of the liposome and MG mixtures (DSPG 20 µM and MG 6 µM) was increased from 20 to 50 °C. After the outside-to-inside MG transport was equilibrated, the solution temperature was returned to 20 °C, and then 0.2 mL of the clay solution (1 mg/mL) was injected. After MGs were desorbed from the outer surface of the liposome, the solution temperature was increased again from 20 to 50 °C to induce the inside-to-outside MG transport.
silver nitrate test.15 Finally, the clay particles were resuspended in the 1 mM NaCl solution to make a clay concentration of 1 mg/mL stock solution. All the solutions used in the SHG experiment including liposomes, MGs, and clays were made of 1 mM NaCl solution to have the same ionic strength. SHG Setup. The fundamental light pulses with 100 fs at 840 nm generated from the Ti:sapphire laser were focused into the sample contained in the 1 cm optical path length rectangular quartz cuvette. The generated SHG light was collected by the photon counting method at the scattering angle of 90° relative to the incident light direction. The 0.2 mL of the clay solution (1 mg/mL) in the glass syringe was injected within 1 s to the 2 mL of the liposome and dye mixtures (DSPG 20 µM and MG 6 µM) in the cuvette. Each solution was temperature controlled, and a magnetic stirring bar was used for a rapid mixing. 4. Results and Discussion In the first sample (P1 of Figure 1), we injected the clay solution to the MG-only solution (MG 6 µM) as a reference. The intensity at SH wavelength was slightly decreased but did not show the time dependence. From this, we can make sure that MG molecules adsorbed on the clay particles cannot contribute to SHG. The detected SH intensity from this sample
Inside-to-Outside Molecular Transport of Liposome
Figure 2. (a) Change of the SH field induced by the adsorption of MGs on the outer surface of the liposome at 20 °C. 0.4 mL of the liposome solution was injected into 1.6 mL of the MG solution to get 2 mL of mixture with the final concentration of DSPG 20 µM and MG 6 µM. The apparent time constant by the single-exponential fitting was ∼67 s. (b) Decay of the SH field in the sample P2 due to the desorption of MGs from the outer surface of the liposome at 20 °C was fitted by the single-exponential decay function. The apparent time constant was ∼130 s.
mostly comes from the two-photon fluorescence tail of MG and linearly proportional to the total MG concentration.4 Therefore, the slight decrease of the SH intensity when mixed with clays should be from the dilution of the total MG concentration. For samples P2 and P3 of the experiments using clays, we first mixed MG solution with liposome solution and then equilibrated it for more than 2 h at room temperature before using. During mixing, the SHG field increased with an exponential time constant of about 67 s due to the adsorption of MGs on the outer layer of liposomes (Figure 2a). The SH field was obtained byE2ω ) (I2ω - Ibg)1/2/Iinput, where I2ω is the intensity at the SH frequency from the sample, Ibg is the background SH intensity from the 6 µM of MG-only solution, and Iinput is the relative input intensity. In the second sample (P2) to observe the desorption of MG from the outer surface of the liposome, we injected 0.2 mL of the clay solution into 2 mL of the MG and liposome mixture (DSPG 20 µM and MG 6 µM) at 20 °C in which MG had
J. Phys. Chem. B, Vol. 112, No. 49, 2008 15675 adsorbed only on the outer surface of liposomes. In this case, the SH intensity was a little bit dropped at the moment of injection and then continuously decreased to the equilibrium value. We can estimate the desorption rate of MG from the outer surface of the liposome by fitting the time dependence of the SH field to the exponential decay function. The SH field could be obtained by E2ω ) (I2ω - Ibg)1/2/Iinput, where Ibg is the background SH intensity estimated from the data of the 6 µM MG-only solution considering the concentration change due to 10% dilution after the injection of the clay solution. The SH field was well fitted by the single-exponential decay function with the decay time constant of 130 s (Figure 2b). This value is a bit larger than that for the adsorption process of 67 s. In the third sample (P3 of Figure 1), we increased temperature of the MG and liposome mixture (DSPG 20 µM and MG 6 µM) to 50 °C, which is above the gel-fluid phase transition temperature. The gel-fluid phase transition temperature of the pure DSPG bilayer is 54.4 °C (ref 12), but the adsorption of dyes can shift it to lower temperature. It has been measured to be about 48 °C for the MG-to-lipid ratio of 20:6 (ref 7). At this temperature, we could observe the SH intensity decay corresponding to the outside-to-inside transport of MGs. The decay of the SH field in this region could be fitted by the singleexponential function with the time constant of ∼122 ( 38 s (Figure 3a). After the transport reached the equilibrium, we decreased temperature to 20 °C (gel phase) to block any more transport of MGs across the liposome bilayer. Now, with injecting clay solution at this temperature, we could observe the increase of the SH intensity which might come from the decrease of the number of MG molecules at the liposome outer surface without a change of the number of MG molecules at the inner surface. On the way of desorption, we increased temperature up to 50 °C to induce the inside-to-outside transport of MGs. As the phase of the lipid bilayer changed to the fluid phase, the SH intensity started to decrease. This should be due to the decrease of the number of MG molecules at the inner surface by the inside-to-outside transport of MGs. As far as we know, this is the first in-situ and real-time observation by SHG of the molecular transport across the lipid bilayer from inside to outside. The decay of the SH field could be well fitted by the single-exponential function (Figure 3b). However, when we waited enough time in the desorption process to reach the equilibrium before inducing the inside-tooutside transport, we could find that the SH field increased up to the value higher than the initial value at time zero (Figure 4a). Because the number of MGs on the inner layer of liposome cannot exceed the initial number of MGs on the outer layer, this means that some process other than desorption must be involved in the increase of the SH field. The possible aggregation or fusion of the liposomes induced by the interaction between clays and liposomes could be responsible for this large increase of the SH field in a long time scale. Because the SH field is linearly proportional to the number of MGs per liposome while proportional to the square root of the number of liposomes,8 the increase of the number of MGs per liposome by fusion can increase the SH field. Actually, if we stop the magnetic stirring bar in the MG and liposome solution mixed with clays, we can observe very large aggregates formed on the bottom of the cuvette within several hours. However, this additional process must be much slower than the desorption process. In sample P2 of Figure 1, we saw that a singleexponential decay function could well describe the decay of the SH field to the background level for at least 600 s. This
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Figure 3. (a) Decay of the SH field in the sample P3 due to the outsideto-inside transport was fitted by the single-exponential decay function. The apparent time constant for this sample was 108 s, but the averaged value for six samples gives 122 ( 38 s. (b) Decay of the SH field in the sample P3 due to the inside-to-outside transport was fitted by the single-exponential decay function. The apparent time constant for this sample was 134 s.
means that this additional process is not dominant at the initial stage of mixing. Since the outside-to-inside transport was made in the condition that MGs initially existed only in the liposome outer surface, it is necessary for the proper comparison of two transport time constants to induce the inside-to-outside transport in the condition that MGs existed only in the inner surface. To check when the desorption of MGs is completed, we induced the inside-to-outside transport at several different times after the injection of the clays and measured the SH decay time constant at each case (Figure 4c). As we induced the inside-to-outside transport at the longer elapsed time from the injection of the clays, the decay time constant became smaller. However, at the elapsed time longer than ∼15 min, the decay time constant did not decrease any more, even if the additional process causing the increase of the SH signal was still going on at this time. Because the surface density of MGs will not change much by the aggregation or fusion of the liposomes and the transport rate mainly depends on the surface density of MGs,7 we can expect that the desorption of MGs is almost completed within 15 min and the transport time constant of about 80 s obtained
Figure 4. (a) Changes of the SH field and (b) the corresponding temperature profiles for the inside-to-outside transport at several different elapsed times from the clay injection were plotted. Here, the elapsed time represents the time interval between the clay injection and the temperature increase to induce the inside-to-outside transport. (c) The inside-to-outside transport time constant for the decay of the SH field was plotted against the elapsed time from the clay injection. The averaged outside-to-inside transport time constant for six samples (122 ( 38 s) was also plotted for comparison.
after 15 min represents the value for the complete desorption of MGs from the liposome outer surface. This value is a bit small but in the same order of magnitude compared to the outside-to-inside transport time constant (∼122 s). This means that the transport rate of MGs across the liposome bilayer was not much affected by the confinement of the inner space of the liposome. 5. Conclusions In summary, the transport of a partially hydrophobic MG molecule from inside to outside of the liposome has been
Inside-to-Outside Molecular Transport of Liposome observed in situ and in real time using the SHG technique and the clay particles. By controlling the phase of the liposome bilayer using temperature change, we could switch on and off the transport of MG molecules. In addition, the clay particles effectively reduce the number of MG molecules outside the liposome, which induces desorption of MG molecules from the outer surface of the liposomes. This makes MG concentration inversion between the inner and outer surfaces of the liposome, which is required for the study of the inside-to-outside transport. This method enables us to measure all of the adsorption, desorption, outside-to-inside transport, and inside-to-outside transport in the same liposome sample. The measured insideto-outside transport time constant was in the same order of magnitude as the outside-to-inside one. The extensive measurements of the transport rates in both directions for various conditions will be helpful for the better understanding of the transport dynamics of this system and for the development of the practical drug delivery systems. Acknowledgment. This work was supported by the Ministry for Health, Welfare and Family affairs through the Korea Health 21 R & D Project and by the Ministry of Knowledge and Economy of Korea through the Ultrashort Quantum Beam Facility Program.
J. Phys. Chem. B, Vol. 112, No. 49, 2008 15677 References and Notes (1) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (2) Lian, T.; Ho, R. J. Y. J. Pharm. Sci. 2001, 90, 667. (3) Srivastava, A.; Eisenthal, K. B. Chem. Phys. Lett. 1998, 292, 345. (4) Liu, Y.; Yan, E. C. Y.; Eisenthal, K. B. Biophys. J. 2001, 80, 1004. (5) Yan, E. C. Y.; Eisenthal, K. B. Biophys. J. 2000, 79, 898. (6) Shang, X.; Liu, Y.; Yan, E.; Eisenthal, K. B. J. Phys. Chem. B 2001, 105, 12816. (7) Kim, J. H.; Kim, M. W. Eur. Phys. J. E 2007, 23, 313. (8) Wang, H.; Yan, E. C. Y.; Borguet, E.; Eisenthal, K. B. Chem. Phys. Lett. 1996, 259, 15. (9) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55. (10) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161. (11) Bucher, P.; Fischer, A.; Luisi, P. L.; Oberholzer, T.; Walde, P. Langmuir 1998, 14, 2712. (12) Zhang, Y.-P.; Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 1997, 72, 779. (13) VanOlphen, H. An Introduction to Clay Colloid Chemistry; Wiley: New York, 1963. (14) Yan, E. C. Y.; Eisenthal, K. B. J. Phys. Chem. B 1999, 103, 6056. (15) Yan, E. C. Y.; Liu, Y.; Eisenthal, K. B. J. Phys. Chem. B 2001, 105, 8531.
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