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Phase effect of mixed-phospholipid layer on phospholipase D reaction-induced-vesicle rupture
Colloids and Surfaces B: Biointerfaces 97 (2012) 207–210
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Phase effect of mixed-phospholipid layer on phospholipase D reaction-induced-vesicle rupture Jin-Won Park ∗ Department of Chemical Engineering, College of Energy and Biological Science, Seoul National University of Science and Technology, 172 Gongreung 2-dong, Nowon-gu, Seoul 139-743, South Korea
a r t i c l e
i n f o
Article history: Received 6 February 2012 Received in revised form 13 April 2012 Accepted 21 April 2012 Available online 28 April 2012 Keywords: Biophysics Vesicles Phospholipase D Phosphatidylcholine Phosphatidylethanolamine Phase
a b s t r a c t Spherical phospholipid-bilayers, vesicles, were prepared through layer by layer using double emulsion technique, which allows the outer layer of the vesicles to be formed with two phospholipids that have different headgroups – phosphatidylcholine (PC) and phosphoethanolamine (PE). At the outer layer of the vesicles, the phospholipase D (PLD) reacted on the layer to convert phosphatidylcholine (PC) to phosphatidic acid (PA). The reaction induced the curvature change of the vesicles, which eventually led to the rupture of the vesicles. Response time from the PLD injection to the rupture was measured at each phase of the layer made with the different-composition lipids, using the fluorescence intensity change of pH-sensitive dye encapsulated in the vesicles. It was found that the retardation of the response time started at different composition up on the phase. At the liquid phase, the composition was 30% PC, while the composition at the solid phase was 20% PC. The difference was interpreted with the surface density of PC at the layer, which was varied with the phase. Furthermore, the difference was also caused by the size of PLD active-site, not PLD. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Phospholipase D (PLD), as one of membrane-active enzymes, is involved in various functions of cells, including membrane/vesicle trafficking, actin cytoskeleton rearrangements, glucose transport, superoxide production, secretion, cellular proliferation, and apoptosis [1,2]. Therefore, PLD is implicated in a range of diseases including cancer, inflammation, and myocardial disease [3–6]. By PLD action upon phosphatidylcholine (PC), PC is cleaved into alcohol and phosphatidic acid (PA, a potent mitogen) that may be essential for the formation of certain types of transport vesicles or may be constitutive vesicular transport to signal transduction pathways. The cleavage leads to the changes in the composition of membranes that could also play a role. PLD may have a physiological function through the further metabolism of PA to diacylglycerol (DAG) and lysophosphatidic acid {1,6}. Lipid layers are known as widely used models for cell surface and for investigating molecular events in membranes, because the preparation methodology for the layers has been well-established and very sensitive analytical techniques can be applied to investigate the events [7–10]. The lipid layers have been also useful to the biomedical research such as cell recognition, membrane-mediated catalysis, effects of anesthetics, and antimicrobial peptides [11–15].
∗ Tel.: +82 2 970 6605; fax: +82 2 977 8317. E-mail address: [email protected] 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.04.034
Furthermore, the actions of the phospholipases have been investigated at the layers, i.e. the effect of the enzymes on the wetting properties of the lipid layers and the configuration of the layers, and the activity of the enzymes [16–18]. The hydrolysis triggered by PLD is a critical step for the fusion that is essential for the cellular processes. The hydrolysis is found to induce the change in the composition of the membranes, which eventually induces the rupture of the vesicles. The change in the composition correlates to that in the geometry of the vesicle, because the hydrolysis results in the occurrence of the smaller headgroups at the outer layer of the vesicles. Therefore, the hydrolysis leads to the decrease in the curvature and the rupture is finally reached. Recently, it has been found how the PLD-induced-vesicle rupture occurred with respect to the phase of the vesicle layers [19]. However, little is known how the phase of the outer layer made with different lipids makes an effect on the PLD-induced vesicle rupture. The investigation of the effect may have a contribution to understanding the physical behavior of these enzymes in terms of quantitative analysis. In this work, we aim to investigate the effect of the outer mixed-lipid layer phase on the vesicle rupture. 2. Experiments Dioeloylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), oleoylhydroxyphosphatidylethanolamine (OHPE), palmitoylhydroxyphosphatidylethanolamine (PHPE), dioeloylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and dipalmitoylphosphatidic acid (DPPA)
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J.-W. Park / Colloids and Surfaces B: Biointerfaces 97 (2012) 207–210
were purchased from Avanti Polar Lipids (Alabaster, AL), and used without further purification. The DPPA was dissolved in 10 ml of tert-butyl methyl ether at 10 mg/ml, followed by adding 100 l DI water of 5 mM pyranine, 50 mM NaCl, and 1 mM CaCl2 at pH 9.0. Therefore, the micelles with DPPA were prepared by extrusion through the 50 nm pores of 78 diameter PTFE membranes above the transition temperature of the DPPA. Several drops (less than 10 l) of the micelle solution were added through 22-gauge needle inserted into the 10 ml aqueous solutions of 50 mM NaCl and 1 mM CaCl2 at pH 5.0, followed by dropping the 10 mg/ml tert-butyl methyl ether solution of a desired ratio of DOPC, DOPE, OHPE (or DPPC, DPPE, PHPE) continuously. The final lipid concentration of the aqueous solution is 1 mg/ml. During the addition, the aqueous solution was magnetically stirred and nitrogen stream was injected into the aqueous solution. After the solution was through the centrifugation (3700 × g) to remove the phospholipids that did not form the vesicles, the liposome solution was obtained from the supernatant of the solution. These procedures are well-known as a methodology to prepare vesicles [7]. For the confirmation of the vesicle formation, the diameter of the micelles was measured using ELS-8000 (Otsuka Electronics Co. Ltd., Osaka, Japan) before they were transferred to the aqueous solution. The viscosity and the refractive index of the tert-butyl methyl ether, required for the measurements, are 0.23 cP and 1.3686, respectively [20]. After the transfer of the micelles into the aqueous solution, the diameter of the vesicles was measured, too. The diameters of the micelles and the vesicles were 75 ± 10 nm and 80 ± 10 nm, respectively. These results indicated that the one lipid layer was formed on the micelle surface was on the micelle surface. Besides the change in the diameter, no leakage of the pyranine molecules indicated that each layer was not disturbed. Otherwise, the fluorescence intensity at 510 nm would be changed tremendously with the addition of several drops of aqueous solutions of 50 mM NaCl and 1 mM CaCl2 at pH 3.0 into the vesicle solution. Phospholipiase D (PLD) was purchased from Sigma Aldrich (St. Louis, MO). One nM was selected as a PLD concentration since the recent results showed that more than 1 nM of PLD concentration made little effect on the response time for the rupture [19]. Since the pyranine (pH-sensitive fluorescence dye) was encapsulated inside of the vesicles only, the rupture was monitored in real time using a Wallac Victor3 multi-well fluorimeter (Perkin Elmer, Waltham, MA). The tremendous difference in the intensity change between the vesicle solution with the PLD injection and that with only the buffer solution injection means the rupture of the vesicles, which was caused by PLC reaction. Therefore, the intensity was observed to investigate the effect of the outer mixed-lipid layer phase on the vesicle rupture on the PLD reaction. For each phase of the layer, the intensity was measured when the rupture occurred from the injection of the PLD molecules. The measurement was performed more than 5 times at each condition. The HPLC analyzer consisted of a Gel Silica 60 column (particle size 5 l; ID 47 mm; Length 15 cm; Tosoh Co., Tokyo, Japan) and an HPLC system (Waters Associates, Milford, MA) containing a Type 600 solvent delivery system, a Type U6K injector, a Type 490 variable wavelength detector and a Type 740 data module. Elution was performed with a solution of acetonitrile–methanol–85% phosphoric acid (130:5:1.7, v/v/v) at a flow rate of 1 ml/min at room temperature. Phospholipids, dissolved in methylene chloride, were injected into the HPLC system.
Fig. 1. Fluorescence intensity change after the addition of pH 3 DI water drops.
emission wavelengths. The change in the fluorescence intensity of the vesicle solution was observed with detergent (Tween 20) treatment. Without the treatment, the change was not found after the addition of pH 3 DI water drops (Fig. 1). Therefore, the encapsulation was successfully achieved. The desired ratio of the phospholipids at the outer layer was confirmed using the HPLC system. The peak area of each component was compared for the lipid solution before the centrifugation and for the supernatant from the solution after the centrifugation. The results are shown in Fig. 2. The intensity of the peak for every component was proportionally decreased after the centrifugation, compared to the intensity prior to the centrifugation. However, the intensity for PA remained after the centrifugation. The proportional decrease means that the ratio of the phospholipids at the outer layer still almost identical with that of the PC and PE solution added for liposome preparation. As expected, the more negative component, except lyso-PE, was corresponding to the shorter retention due to the surface interaction with the silica spheres whose surfaces had
3. Results and discussion Towards the investigation of the PLD reaction on the lipid layer, the pyranine molecules were dissolved only inside of the vesicles. The encapsulation of the molecules was confirmed using fluorometer (Perkin Elmer, Boston, MA) with 460 nm excitation and 520 nm
Fig. 2. HPLC peaks for each component, lipid solution before the centrifugation (above) and supernatant of the solution after the centrifugation (below).
J.-W. Park / Colloids and Surfaces B: Biointerfaces 97 (2012) 207–210
209
Fig. 3. Schematic diagram for the vesicles made with different components. (A) Vesicles made with pure DPPC and (B) vesicles made with DPPE and PHPE.
inherently negative dipoles. It was also expected that the retention time for lyso-PE was longest due to the molecular weight. The order of the peak for each component was consistent with that previously published [21,22]. PA peak after the centrifugation was identical with that before the centrifugation. From the invariance, it is found that the inner layer made with PAs remained during the centrifugation. This result is consistent with the confirmation of the encapsulation. Besides the composition, the PLD reaction are influenced by other factors such as the vesicle number, the PLD concentration, the ionic concentration of the vesicle solution, the phase of the inner layer, the vesicle radius, and the vesicle stability. Therefore, the factors were determined followed by the conduction of the experiments for the composition. The concentration of the phospholipids and the radius of the vesicles were constant in this research, and it is known that 10 mg/ml of the phospholipids are corresponding to the 107 –108 number of the vesicles [23]. The results from the previous research provided the information for the other factors [20]. At the 1 mg/ml solution of phospholipids, 1–10 nM PLD concentration led to little change in the reactivity. 50 mM NaCl and 1 mM CaCl2 , one of biomimetic conditions, was selected for the ionic concentration, because the reactivity was dependent on the concentration at lower concentration [6,24–26]. At the conditions determined above, the PLD-induced hydrolysis was independent of the inner-layer phase. Since solid phase is known more stable, DPPA was used instead of DOPA at room temperature [27]. The phase of the lipid layers was made with the phospholipids whose transition temperature was considered [28]. Dioleoyl lipids were used for the liquid phase at room temperature due to their transition temperature lower than room temperature, while dipalmitoyl lipids were for solid at room temperature. At a specific phase, the stability was adjusted identically for the vesicle of each composition. The stability was described in next paragraph, because it depended on the composition of the vesicles. Since all of factors were decided, the phase effect of each composition was investigated. As components for the outer layer, PC and PE were selected because only PC was reacted by PLD. However, if PC was replaced with PE only, the radius of the vesicles would be increased due to the difference in the geometries between PC and PE. Therefore, lyso-PE, OHPE or PHPE, was used for the vesicle preparation with PC and PE so that the radius of the vesicles made at a specific phase was little varied (Fig. 3). OHPE was for the liquid phase of the outer layer, while PHPE was for the solid phase. Since the PLD reaction was based on the curvature change of the vesicles, it was essential that the radius remained constant. The introduction of lyso-PE allowed only the composition effect on the PLD reaction found. For the investigation on the PLD reaction, 11 conditions (non-PC to pure PC by 10%) were considered. However, at a specific PC, it was also necessary to determine the ratio of DOPE to OHPE (or DPPE to PHPE) that might influence the stability of the vesicles. Therefore, the ratio
was adjusted because the vesicles of each composition were necessary found as stable as the vesicles made with pure PC. In order to find out the ratio of the identical stability for each phase, the permeability was observed at 5:1, 10:1, 15:1 ratios of DOPE to OHPE (or DPPE to PHPE) for each PC condition at each phase with measuring the fluorescence intensity change with respect to time (Fig. 4). The variance in the PC composition at a specific ratio of DOPE to OHPE (or DPPE to PHPE) made little effect on the stability. It was found that the stability of the vesicles at 15:1 ratio was almost identical with that of the vesicles made with pure PC. This result was consistent with the expectation from the geometries of the lipids in that the ratios of lipid volume to headgroup area and lipid length are 0.5–1, 1, and less than 0.33333 for PC, PE with two fatty-acid tail, and lyso-PE, respectively [29]. Reactivity was monitored at the various composition conditions for each phase of the outer mixed-lipid layer using the fluorescence response time. The time at pure DOPC vesicles was consistent with the previous result [19]. 30% DOPC to non-PC condition led to the gradual increase in the response time (Table 1), which means that the reactivity was decreased gradually from 30% DOPC to non-PC condition. However, if the outer layer was solid phase, the composition, which started to increase the response time, was 20% DPPC (Table 2). Since even the vesicles made with PE only have less than
Fig. 4. Fluorescence intensity change with respect to time, for the vesicles made with pure DPPC (above) and for the vesicles made at the different ratios of DPPE to PHPE (below).
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J.-W. Park / Colloids and Surfaces B: Biointerfaces 97 (2012) 207–210
Table 1 Response time from the PLD injection to vesicle rupture (s). PE consisted of DOPE and OHPE at the ratio 15:1 of DOPE:OHPE.
Response time (s)
Pure DOPC
90% DOPC 10% PE
80% DOPC 20% PE
70% DOPC 30% PE
60% DOPC 40% PE
50% DOPC 50% PE
40% DOPC 60% PE
30% DOPC 70% PE
20% DOPC 80% PE
10% DOPC 90% PE
Pure PE
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.9
1.3
2.7
50
Table 2 Response time from the PLD injection to vesicle rupture (s). PE consisted of DPPE and PHPE at the ratio 15:1 of DPPE:PHPE.
Responsec time (s)
Pure DPPC
90% DPPC 10% PE
80% DPPC 20% PE
70% DPPC 30% PE
60% DPPC 40% PE
50% DPPC 50% PE
40% DPPC 60% PE
30% DPPC 70% PE
20% DPPC 80% PE
10% DPPC 90% PE
Pure PE
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.8
1.6
More than two days
7% lyso-PE, the mean-molecular-area (MMA) of PE and PC only are considered for the lipid density calculation. MMA of DOPE and DOPC is presumably ∼0.8 nm2 [28,30]. At the 30% DOPC vesicles, the density of DOPC on the outer layer was one DOPC molecule per 2.7 nm2 . However, for DPPC, 20% DPPC was the composition corresponding to the density, because MMA of DPPE and DPPC is ∼0.5 nm2 . Considering the PLD gyration radius that is known around 4 nm, it was expected that the response time would be little changed at even 10% DOPC vesicles and 5–6% DPPC vesicles [31]. These discrepancies may be due to the size of the PLD active site. Previously, it was published that PLD had the PC hydrolysis site whose area was 1.5–2 nm2 [32]. Therefore, considering PC density for each phase and PLD active-site area, the compositions for each phase were consistent with the expectation. The composition difference between the phases was eventually caused by the lipid density difference between the phases. 4. Conclusion In this study, the phase effect of the outer mixed-lipid layer on the PLD-induced hydrolysis was investigated using the fluorescence intensity change. Prior to the investigation, the ratio of two fatty-acid tail PE to lyso-PE was found as a condition that the vesicles made with the mixed-lipids were as stable as those made with pure PC. For each phase, the PLD-induced rupture was interpreted in terms of the composition of each layer, especially the PC density on the outer layer of the vesicles. For the liquid phase of the outer layer, the vesicles made with more than 30% DOPC at the layer showed the PLD reactivity identical with the vesicles made with pure DOPC. From 30% DOPC to non-DOPC, the reactivity changed gradually. However, for the solid phase, the composition necessary for the reactivity different from the vesicles made with pure DPPC was 20% DPPC. This difference seems to be caused by the lipid density difference between the phases. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0010097). We thank all of members of Department of
Chemical Engineering at the Seoul National University of Science and Technology for help and valuable discussions. We thank Prof. D.J. Ahn, Dr. G.S. Lee, Mr. H. Choi, and Mr. C.S. Choi at the Korea University for valuable help. References [1] [2] [3] [4] [5] [6]
[7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
M. McDemott, M.J. Wakelam, A.J. Morris, Biochem. Cell Biol. 82 (2004) 225. J.H. Exton, Rev. Physiol. Biochem. Pharmacol. 144 (2002) 1. P. Huang, M.A. Frohman, Exp. Opin. Ther. Targets 11 (2007) 707. P.S. Tappia, M.R. Dent, N.S. Dhalla, Free Radical Biol. Med. 41 (2006) 349. S.A. Scott, P.E. Selvy, J.R. Buck, H.P. Cho, T.L. Criswell, A.L. Thomas, M.D. Armstrong, C.L. Arteaga, C.W. Lindsley, H.A. Brown, Nat. Chem. Biol. 5 (2009) 108. H.A. Brown, L.G. Henage, A.M. Preininger, Y. Xiang, J.H. Exton, in: H.A. Brown (Ed.), Lipidomics and Bioactive Lipids: Lipids and Cell Signaling, Academic Press, Amsterdam, Netherlands, 2007. R.R.C. New, Liposomes: A Practical Approach. Academic Press, New York, 1990. H.M. McConnell, T.H. Wats, R.M. Weis, A.A. Brian, Biochim. Biophys. Acta 864 (1986) 95. E. Sackmann, Science 271 (1996) 43. A.A. Brian, H.M. McConnell, Natl. Acad. Sci. U. S. A. 81 (1984) 6159. P.L. Giesen, G.M. Willems, H.C. Hemker, W.T. Hermens, J. Biol. Chem. 266 (1991) 18720. J.X. Mou, J. Yang, C. Huang, Z. Shao, Biochemistry 33 (1994) 9981. A. Miszta, R. Machan, A. Benda, A.J. Ouellette, W.T. Hermens, M. Hof, J. Pept. Sci. 14 (2008) 503. H.H.P. Fang, K.-Y. Chan, L.-C. Xu, J. Microbiol. Methods 40 (2000) 89; D. Mamelak, C. Lingwood, J. Biol. Chem. 276 (2001) 449. W.S. Cheow, K. Hadinoto, Colloid Surf. B: Biointerfaces 8 (2011), 214. M. Jurak, E. Chibowski, Appl. Surf. Sci. 256 (2010) 6304. C.H. Chen, S. Malkova, S.V. Pingali, F. Long, S. Garde, W. Cho, M.L. Schlossman, Biophys. J. 97 (2009) 2794. S. Chemburu, E. Ji, Y. Casana, Y. Wu, T. Buranda, K.S. Schanze, G.P. Lopez, D.G. Whitten, J. Phys. Chem. B 112 (2008) 14492. J.-W. Park, J. Membr. Biol. 244 (2011) 55. D.R. Lide, CRC Handbook of Chemistry and Physics: A Ready-Reference Book of Chemical and Physical Data, 85th ed., CRC Press, Boca Raton, 2005. Y. Kurumi, Y. Adachi, T. Ito, H. Kobayashi, T. Nanno, T. Yamamoto, J. Gastroenterol. 26 (1991) 628. J.A. Singleton, L.F. Stikeleather, JAOCS 72 (1995) 485. J.-W. Park, Bull. Kor. Chem. Soc. 28 (2007) 1709. L.M. Webb, A.T. Arnholt, M.E. Venable, Mol. Cell Biochem. 337 (2009) 153. W.S. Cheow, M.W. Chang, K. Hadinoto, J. Biomed. Nanotechnol. 6 (2010) 391. F. Furt, P. Moreau, Int. J. Biochem. Cell Biol. 41 (2009) 1828. J.-W. Park, J. Membr. Biol. 237 (2010) 107. J.-W. Park, D.J. Ahn, Colloid Surf. B: Biointerfaces 62 (2008) 157. G. Cevc, D. Marsh, Phospholipid Bilayers: Physical Principles and Methods, John Wiley and Sons, New York, 1987. J.-W. Park, Colloid Surf. B: Biointerfaces 75 (2010) 290. H.A. Brown, S. Gutowski, R.A. Kahn, P.C. Sternweis, J. Biol. Chem. 270 (1995) 14935. I. Leiros, F. Secundo, C. Zambonelli, S. Servi, E. Hough, Structure 8 (2000) 655.
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Phase effect of mixed-phospholipid layer on phospholipase D reaction-induced-vesicle rupture Jin-Won Park ∗ Department of Chemical Engineering, College of Energy and Biological Science, Seoul National University of Science and Technology, 172 Gongreung 2-dong, Nowon-gu, Seoul 139-743, South Korea
a r t i c l e
i n f o
Article history: Received 6 February 2012 Received in revised form 13 April 2012 Accepted 21 April 2012 Available online 28 April 2012 Keywords: Biophysics Vesicles Phospholipase D Phosphatidylcholine Phosphatidylethanolamine Phase
a b s t r a c t Spherical phospholipid-bilayers, vesicles, were prepared through layer by layer using double emulsion technique, which allows the outer layer of the vesicles to be formed with two phospholipids that have different headgroups – phosphatidylcholine (PC) and phosphoethanolamine (PE). At the outer layer of the vesicles, the phospholipase D (PLD) reacted on the layer to convert phosphatidylcholine (PC) to phosphatidic acid (PA). The reaction induced the curvature change of the vesicles, which eventually led to the rupture of the vesicles. Response time from the PLD injection to the rupture was measured at each phase of the layer made with the different-composition lipids, using the fluorescence intensity change of pH-sensitive dye encapsulated in the vesicles. It was found that the retardation of the response time started at different composition up on the phase. At the liquid phase, the composition was 30% PC, while the composition at the solid phase was 20% PC. The difference was interpreted with the surface density of PC at the layer, which was varied with the phase. Furthermore, the difference was also caused by the size of PLD active-site, not PLD. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Phospholipase D (PLD), as one of membrane-active enzymes, is involved in various functions of cells, including membrane/vesicle trafficking, actin cytoskeleton rearrangements, glucose transport, superoxide production, secretion, cellular proliferation, and apoptosis [1,2]. Therefore, PLD is implicated in a range of diseases including cancer, inflammation, and myocardial disease [3–6]. By PLD action upon phosphatidylcholine (PC), PC is cleaved into alcohol and phosphatidic acid (PA, a potent mitogen) that may be essential for the formation of certain types of transport vesicles or may be constitutive vesicular transport to signal transduction pathways. The cleavage leads to the changes in the composition of membranes that could also play a role. PLD may have a physiological function through the further metabolism of PA to diacylglycerol (DAG) and lysophosphatidic acid {1,6}. Lipid layers are known as widely used models for cell surface and for investigating molecular events in membranes, because the preparation methodology for the layers has been well-established and very sensitive analytical techniques can be applied to investigate the events [7–10]. The lipid layers have been also useful to the biomedical research such as cell recognition, membrane-mediated catalysis, effects of anesthetics, and antimicrobial peptides [11–15].
∗ Tel.: +82 2 970 6605; fax: +82 2 977 8317. E-mail address: [email protected] 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.04.034
Furthermore, the actions of the phospholipases have been investigated at the layers, i.e. the effect of the enzymes on the wetting properties of the lipid layers and the configuration of the layers, and the activity of the enzymes [16–18]. The hydrolysis triggered by PLD is a critical step for the fusion that is essential for the cellular processes. The hydrolysis is found to induce the change in the composition of the membranes, which eventually induces the rupture of the vesicles. The change in the composition correlates to that in the geometry of the vesicle, because the hydrolysis results in the occurrence of the smaller headgroups at the outer layer of the vesicles. Therefore, the hydrolysis leads to the decrease in the curvature and the rupture is finally reached. Recently, it has been found how the PLD-induced-vesicle rupture occurred with respect to the phase of the vesicle layers [19]. However, little is known how the phase of the outer layer made with different lipids makes an effect on the PLD-induced vesicle rupture. The investigation of the effect may have a contribution to understanding the physical behavior of these enzymes in terms of quantitative analysis. In this work, we aim to investigate the effect of the outer mixed-lipid layer phase on the vesicle rupture. 2. Experiments Dioeloylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), oleoylhydroxyphosphatidylethanolamine (OHPE), palmitoylhydroxyphosphatidylethanolamine (PHPE), dioeloylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and dipalmitoylphosphatidic acid (DPPA)
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J.-W. Park / Colloids and Surfaces B: Biointerfaces 97 (2012) 207–210
were purchased from Avanti Polar Lipids (Alabaster, AL), and used without further purification. The DPPA was dissolved in 10 ml of tert-butyl methyl ether at 10 mg/ml, followed by adding 100 l DI water of 5 mM pyranine, 50 mM NaCl, and 1 mM CaCl2 at pH 9.0. Therefore, the micelles with DPPA were prepared by extrusion through the 50 nm pores of 78 diameter PTFE membranes above the transition temperature of the DPPA. Several drops (less than 10 l) of the micelle solution were added through 22-gauge needle inserted into the 10 ml aqueous solutions of 50 mM NaCl and 1 mM CaCl2 at pH 5.0, followed by dropping the 10 mg/ml tert-butyl methyl ether solution of a desired ratio of DOPC, DOPE, OHPE (or DPPC, DPPE, PHPE) continuously. The final lipid concentration of the aqueous solution is 1 mg/ml. During the addition, the aqueous solution was magnetically stirred and nitrogen stream was injected into the aqueous solution. After the solution was through the centrifugation (3700 × g) to remove the phospholipids that did not form the vesicles, the liposome solution was obtained from the supernatant of the solution. These procedures are well-known as a methodology to prepare vesicles [7]. For the confirmation of the vesicle formation, the diameter of the micelles was measured using ELS-8000 (Otsuka Electronics Co. Ltd., Osaka, Japan) before they were transferred to the aqueous solution. The viscosity and the refractive index of the tert-butyl methyl ether, required for the measurements, are 0.23 cP and 1.3686, respectively [20]. After the transfer of the micelles into the aqueous solution, the diameter of the vesicles was measured, too. The diameters of the micelles and the vesicles were 75 ± 10 nm and 80 ± 10 nm, respectively. These results indicated that the one lipid layer was formed on the micelle surface was on the micelle surface. Besides the change in the diameter, no leakage of the pyranine molecules indicated that each layer was not disturbed. Otherwise, the fluorescence intensity at 510 nm would be changed tremendously with the addition of several drops of aqueous solutions of 50 mM NaCl and 1 mM CaCl2 at pH 3.0 into the vesicle solution. Phospholipiase D (PLD) was purchased from Sigma Aldrich (St. Louis, MO). One nM was selected as a PLD concentration since the recent results showed that more than 1 nM of PLD concentration made little effect on the response time for the rupture [19]. Since the pyranine (pH-sensitive fluorescence dye) was encapsulated inside of the vesicles only, the rupture was monitored in real time using a Wallac Victor3 multi-well fluorimeter (Perkin Elmer, Waltham, MA). The tremendous difference in the intensity change between the vesicle solution with the PLD injection and that with only the buffer solution injection means the rupture of the vesicles, which was caused by PLC reaction. Therefore, the intensity was observed to investigate the effect of the outer mixed-lipid layer phase on the vesicle rupture on the PLD reaction. For each phase of the layer, the intensity was measured when the rupture occurred from the injection of the PLD molecules. The measurement was performed more than 5 times at each condition. The HPLC analyzer consisted of a Gel Silica 60 column (particle size 5 l; ID 47 mm; Length 15 cm; Tosoh Co., Tokyo, Japan) and an HPLC system (Waters Associates, Milford, MA) containing a Type 600 solvent delivery system, a Type U6K injector, a Type 490 variable wavelength detector and a Type 740 data module. Elution was performed with a solution of acetonitrile–methanol–85% phosphoric acid (130:5:1.7, v/v/v) at a flow rate of 1 ml/min at room temperature. Phospholipids, dissolved in methylene chloride, were injected into the HPLC system.
Fig. 1. Fluorescence intensity change after the addition of pH 3 DI water drops.
emission wavelengths. The change in the fluorescence intensity of the vesicle solution was observed with detergent (Tween 20) treatment. Without the treatment, the change was not found after the addition of pH 3 DI water drops (Fig. 1). Therefore, the encapsulation was successfully achieved. The desired ratio of the phospholipids at the outer layer was confirmed using the HPLC system. The peak area of each component was compared for the lipid solution before the centrifugation and for the supernatant from the solution after the centrifugation. The results are shown in Fig. 2. The intensity of the peak for every component was proportionally decreased after the centrifugation, compared to the intensity prior to the centrifugation. However, the intensity for PA remained after the centrifugation. The proportional decrease means that the ratio of the phospholipids at the outer layer still almost identical with that of the PC and PE solution added for liposome preparation. As expected, the more negative component, except lyso-PE, was corresponding to the shorter retention due to the surface interaction with the silica spheres whose surfaces had
3. Results and discussion Towards the investigation of the PLD reaction on the lipid layer, the pyranine molecules were dissolved only inside of the vesicles. The encapsulation of the molecules was confirmed using fluorometer (Perkin Elmer, Boston, MA) with 460 nm excitation and 520 nm
Fig. 2. HPLC peaks for each component, lipid solution before the centrifugation (above) and supernatant of the solution after the centrifugation (below).
J.-W. Park / Colloids and Surfaces B: Biointerfaces 97 (2012) 207–210
209
Fig. 3. Schematic diagram for the vesicles made with different components. (A) Vesicles made with pure DPPC and (B) vesicles made with DPPE and PHPE.
inherently negative dipoles. It was also expected that the retention time for lyso-PE was longest due to the molecular weight. The order of the peak for each component was consistent with that previously published [21,22]. PA peak after the centrifugation was identical with that before the centrifugation. From the invariance, it is found that the inner layer made with PAs remained during the centrifugation. This result is consistent with the confirmation of the encapsulation. Besides the composition, the PLD reaction are influenced by other factors such as the vesicle number, the PLD concentration, the ionic concentration of the vesicle solution, the phase of the inner layer, the vesicle radius, and the vesicle stability. Therefore, the factors were determined followed by the conduction of the experiments for the composition. The concentration of the phospholipids and the radius of the vesicles were constant in this research, and it is known that 10 mg/ml of the phospholipids are corresponding to the 107 –108 number of the vesicles [23]. The results from the previous research provided the information for the other factors [20]. At the 1 mg/ml solution of phospholipids, 1–10 nM PLD concentration led to little change in the reactivity. 50 mM NaCl and 1 mM CaCl2 , one of biomimetic conditions, was selected for the ionic concentration, because the reactivity was dependent on the concentration at lower concentration [6,24–26]. At the conditions determined above, the PLD-induced hydrolysis was independent of the inner-layer phase. Since solid phase is known more stable, DPPA was used instead of DOPA at room temperature [27]. The phase of the lipid layers was made with the phospholipids whose transition temperature was considered [28]. Dioleoyl lipids were used for the liquid phase at room temperature due to their transition temperature lower than room temperature, while dipalmitoyl lipids were for solid at room temperature. At a specific phase, the stability was adjusted identically for the vesicle of each composition. The stability was described in next paragraph, because it depended on the composition of the vesicles. Since all of factors were decided, the phase effect of each composition was investigated. As components for the outer layer, PC and PE were selected because only PC was reacted by PLD. However, if PC was replaced with PE only, the radius of the vesicles would be increased due to the difference in the geometries between PC and PE. Therefore, lyso-PE, OHPE or PHPE, was used for the vesicle preparation with PC and PE so that the radius of the vesicles made at a specific phase was little varied (Fig. 3). OHPE was for the liquid phase of the outer layer, while PHPE was for the solid phase. Since the PLD reaction was based on the curvature change of the vesicles, it was essential that the radius remained constant. The introduction of lyso-PE allowed only the composition effect on the PLD reaction found. For the investigation on the PLD reaction, 11 conditions (non-PC to pure PC by 10%) were considered. However, at a specific PC, it was also necessary to determine the ratio of DOPE to OHPE (or DPPE to PHPE) that might influence the stability of the vesicles. Therefore, the ratio
was adjusted because the vesicles of each composition were necessary found as stable as the vesicles made with pure PC. In order to find out the ratio of the identical stability for each phase, the permeability was observed at 5:1, 10:1, 15:1 ratios of DOPE to OHPE (or DPPE to PHPE) for each PC condition at each phase with measuring the fluorescence intensity change with respect to time (Fig. 4). The variance in the PC composition at a specific ratio of DOPE to OHPE (or DPPE to PHPE) made little effect on the stability. It was found that the stability of the vesicles at 15:1 ratio was almost identical with that of the vesicles made with pure PC. This result was consistent with the expectation from the geometries of the lipids in that the ratios of lipid volume to headgroup area and lipid length are 0.5–1, 1, and less than 0.33333 for PC, PE with two fatty-acid tail, and lyso-PE, respectively [29]. Reactivity was monitored at the various composition conditions for each phase of the outer mixed-lipid layer using the fluorescence response time. The time at pure DOPC vesicles was consistent with the previous result [19]. 30% DOPC to non-PC condition led to the gradual increase in the response time (Table 1), which means that the reactivity was decreased gradually from 30% DOPC to non-PC condition. However, if the outer layer was solid phase, the composition, which started to increase the response time, was 20% DPPC (Table 2). Since even the vesicles made with PE only have less than
Fig. 4. Fluorescence intensity change with respect to time, for the vesicles made with pure DPPC (above) and for the vesicles made at the different ratios of DPPE to PHPE (below).
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J.-W. Park / Colloids and Surfaces B: Biointerfaces 97 (2012) 207–210
Table 1 Response time from the PLD injection to vesicle rupture (s). PE consisted of DOPE and OHPE at the ratio 15:1 of DOPE:OHPE.
Response time (s)
Pure DOPC
90% DOPC 10% PE
80% DOPC 20% PE
70% DOPC 30% PE
60% DOPC 40% PE
50% DOPC 50% PE
40% DOPC 60% PE
30% DOPC 70% PE
20% DOPC 80% PE
10% DOPC 90% PE
Pure PE
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.9
1.3
2.7
50
Table 2 Response time from the PLD injection to vesicle rupture (s). PE consisted of DPPE and PHPE at the ratio 15:1 of DPPE:PHPE.
Responsec time (s)
Pure DPPC
90% DPPC 10% PE
80% DPPC 20% PE
70% DPPC 30% PE
60% DPPC 40% PE
50% DPPC 50% PE
40% DPPC 60% PE
30% DPPC 70% PE
20% DPPC 80% PE
10% DPPC 90% PE
Pure PE
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.8
1.6
More than two days
7% lyso-PE, the mean-molecular-area (MMA) of PE and PC only are considered for the lipid density calculation. MMA of DOPE and DOPC is presumably ∼0.8 nm2 [28,30]. At the 30% DOPC vesicles, the density of DOPC on the outer layer was one DOPC molecule per 2.7 nm2 . However, for DPPC, 20% DPPC was the composition corresponding to the density, because MMA of DPPE and DPPC is ∼0.5 nm2 . Considering the PLD gyration radius that is known around 4 nm, it was expected that the response time would be little changed at even 10% DOPC vesicles and 5–6% DPPC vesicles [31]. These discrepancies may be due to the size of the PLD active site. Previously, it was published that PLD had the PC hydrolysis site whose area was 1.5–2 nm2 [32]. Therefore, considering PC density for each phase and PLD active-site area, the compositions for each phase were consistent with the expectation. The composition difference between the phases was eventually caused by the lipid density difference between the phases. 4. Conclusion In this study, the phase effect of the outer mixed-lipid layer on the PLD-induced hydrolysis was investigated using the fluorescence intensity change. Prior to the investigation, the ratio of two fatty-acid tail PE to lyso-PE was found as a condition that the vesicles made with the mixed-lipids were as stable as those made with pure PC. For each phase, the PLD-induced rupture was interpreted in terms of the composition of each layer, especially the PC density on the outer layer of the vesicles. For the liquid phase of the outer layer, the vesicles made with more than 30% DOPC at the layer showed the PLD reactivity identical with the vesicles made with pure DOPC. From 30% DOPC to non-DOPC, the reactivity changed gradually. However, for the solid phase, the composition necessary for the reactivity different from the vesicles made with pure DPPC was 20% DPPC. This difference seems to be caused by the lipid density difference between the phases. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0010097). We thank all of members of Department of
Chemical Engineering at the Seoul National University of Science and Technology for help and valuable discussions. We thank Prof. D.J. Ahn, Dr. G.S. Lee, Mr. H. Choi, and Mr. C.S. Choi at the Korea University for valuable help. References [1] [2] [3] [4] [5] [6]
[7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
M. McDemott, M.J. Wakelam, A.J. Morris, Biochem. Cell Biol. 82 (2004) 225. J.H. Exton, Rev. Physiol. Biochem. Pharmacol. 144 (2002) 1. P. Huang, M.A. Frohman, Exp. Opin. Ther. Targets 11 (2007) 707. P.S. Tappia, M.R. Dent, N.S. Dhalla, Free Radical Biol. Med. 41 (2006) 349. S.A. Scott, P.E. Selvy, J.R. Buck, H.P. Cho, T.L. Criswell, A.L. Thomas, M.D. Armstrong, C.L. Arteaga, C.W. Lindsley, H.A. Brown, Nat. Chem. Biol. 5 (2009) 108. H.A. Brown, L.G. Henage, A.M. Preininger, Y. Xiang, J.H. Exton, in: H.A. Brown (Ed.), Lipidomics and Bioactive Lipids: Lipids and Cell Signaling, Academic Press, Amsterdam, Netherlands, 2007. R.R.C. New, Liposomes: A Practical Approach. Academic Press, New York, 1990. H.M. McConnell, T.H. Wats, R.M. Weis, A.A. Brian, Biochim. Biophys. Acta 864 (1986) 95. E. Sackmann, Science 271 (1996) 43. A.A. Brian, H.M. McConnell, Natl. Acad. Sci. U. S. A. 81 (1984) 6159. P.L. Giesen, G.M. Willems, H.C. Hemker, W.T. Hermens, J. Biol. Chem. 266 (1991) 18720. J.X. Mou, J. Yang, C. Huang, Z. Shao, Biochemistry 33 (1994) 9981. A. Miszta, R. Machan, A. Benda, A.J. Ouellette, W.T. Hermens, M. Hof, J. Pept. Sci. 14 (2008) 503. H.H.P. Fang, K.-Y. Chan, L.-C. Xu, J. Microbiol. Methods 40 (2000) 89; D. Mamelak, C. Lingwood, J. Biol. Chem. 276 (2001) 449. W.S. Cheow, K. Hadinoto, Colloid Surf. B: Biointerfaces 8 (2011), 214. M. Jurak, E. Chibowski, Appl. Surf. Sci. 256 (2010) 6304. C.H. Chen, S. Malkova, S.V. Pingali, F. Long, S. Garde, W. Cho, M.L. Schlossman, Biophys. J. 97 (2009) 2794. S. Chemburu, E. Ji, Y. Casana, Y. Wu, T. Buranda, K.S. Schanze, G.P. Lopez, D.G. Whitten, J. Phys. Chem. B 112 (2008) 14492. J.-W. Park, J. Membr. Biol. 244 (2011) 55. D.R. Lide, CRC Handbook of Chemistry and Physics: A Ready-Reference Book of Chemical and Physical Data, 85th ed., CRC Press, Boca Raton, 2005. Y. Kurumi, Y. Adachi, T. Ito, H. Kobayashi, T. Nanno, T. Yamamoto, J. Gastroenterol. 26 (1991) 628. J.A. Singleton, L.F. Stikeleather, JAOCS 72 (1995) 485. J.-W. Park, Bull. Kor. Chem. Soc. 28 (2007) 1709. L.M. Webb, A.T. Arnholt, M.E. Venable, Mol. Cell Biochem. 337 (2009) 153. W.S. Cheow, M.W. Chang, K. Hadinoto, J. Biomed. Nanotechnol. 6 (2010) 391. F. Furt, P. Moreau, Int. J. Biochem. Cell Biol. 41 (2009) 1828. J.-W. Park, J. Membr. Biol. 237 (2010) 107. J.-W. Park, D.J. Ahn, Colloid Surf. B: Biointerfaces 62 (2008) 157. G. Cevc, D. Marsh, Phospholipid Bilayers: Physical Principles and Methods, John Wiley and Sons, New York, 1987. J.-W. Park, Colloid Surf. B: Biointerfaces 75 (2010) 290. H.A. Brown, S. Gutowski, R.A. Kahn, P.C. Sternweis, J. Biol. Chem. 270 (1995) 14935. I. Leiros, F. Secundo, C. Zambonelli, S. Servi, E. Hough, Structure 8 (2000) 655.