Self-Assembly of Biological Membranes into 200–400 nm Aqueous Compartments

Copyright © 2010 American Scientific Publishers All rights reserved Printed in the United States of America

Journal of Nanoscience and Nanotechnology Vol. 10, 1–6, 2010 Vol. 10, 3085-3090, 2010

Self-Assembly of Biological Membranes into 200–400 nm Aqueous Compartments Aditya Mittal1 ∗ and Rahul Grover2 1

School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India 2 Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

Keywords: Liposomes, CMC, Bilayer, Organelle, Phospholipids

1. INTRODUCTION Self assembly of amphipathic molecules into membrane compartments encapsulating specific aqueous environments is central to biology. A mechanistic understanding of this self-assembly is a major goal in research on basic cell biology of organelle formation,1–3 synthetic biology for protocell generation,4 and development of liposome based drug delivery systems.5–8 While the advent of liposomes in the late 1960s 9 10 opened up several exciting promises, many are not yet fulfilled. This is because even today assembly of liposomes as either model systems for research in cell biology or as gene/drug delivery vehicle is more of an art than science. The seminal contributions of Tanford11 12 in characterizing the hydrophobic effect for amphipathic assembly in aqueous systems have resulted in a strong understanding of simple structures like micelles. Definition and utilization of a straightforward experimental ∗

Author to whom correspondence should be addressed.

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parameter called Critical Micellar Concentration (CMC) has enabled insights into self-assembled structures formed by single-chain amphiphiles. However, no such parameteric characterization exists to date that provides a direct understanding of membrane bilayer formation, and even more important, formation of compartments encapsulating aqueous contents. This is because of the difficulties and limitations in experimentally studying stoichiometric constraints playing a role in amphipathic self-assembly leading to bilayer formation. For example, even the trivial task of pipetting of a mixture of amphipathic molecules (leading to bilayer formation) into an aqueous environment suffers from the limitation of spontaneous self-assembly at the pipette tip, if somehow the interaction of the amphipathic molecules with the pipette itself is minimized along with minimizing the use of organic solvent for solvating the amphipathic mixture. Thus, with the aim of directly probing into mechanistics of bilayer formation leading to aqueous compartments, we designed an experimental system that overcomes limitations of stoichiometric studies on such

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Membrane formation by amphipathic mixtures in aqueous environments is central to eukaryotic biology. Formation of aqueous compartments enclosed by membranes is of immense importance in designing liposomal systems for pharmaceutical applications. It is also continuously controlled within the dynamic environment of a living cell and, during cell division. In spite of over four decades of research on protein-free lipid bilayers, membrane compartment formation is still an art rather than science. This is because the experimental efforts to date have been aimed at making aqueous compartments from different lipid mixtures in different buffers and solutions of different ionic strengths. Thus, even similar methodologies produce varying results in different laboratories. In this work, we provide for the first time, experimental parameters of minimum hydration volume and maximum possible volumes for aqueous entrapments formed by DOPE:DOPC:Chol stoichiometries similar to intracellular environments and those used in pharmaceutical research for liposomal systems. We define a new experimental parameter of “Critical Compartmentalization Concentration” for formation of membrane-bound 200–400 nm aqueous compartments by these amphipathic mixtures in the simplest possible controlled environment of pure water. We report the first experimental insights into “equations” governing self-assembly leading to formation of membrane compartments encapsulating aqueous volumes. Our work opens a completely new avenue for engineering of aqueous compartment and liposomal preparations using known biological lipids in different aqueous environments.

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Self-Assembly of Biological Membranes into 200–400 nm Aqueous Compartments

amphipathic systems. The most remarkable feature of our work is the simplicity of the experimental design, the clarity of the read-out from experiments and the robustness of the data obtained. We report, for the first time, parameters like the minimum hydration volume, maximum possible volume for aqueous entrapment, and the stoichiometric concentrations of amphipathic mixtures for maximizing their molar partitioning for the formation of membranebound aqueous compartments between 200–400 nm diameter. Utilizing stoichiometric amounts of DOPE, DOPC and cholesterol that are similar to intracellular environments and those used in drug delivery research for liposomal systems, we experimentally define a new parameter of “Critical Compartmentalization Concentration” for formation of membrane-bound aqueous compartments by these amphipathic molecules in the simplest controlled aqueous environment, i.e., pure water.

membrane bilayers? To answer this question, we devised a straight forward experimental strategy shown in Figure 1(a). Using the standard reverse phase evaporation method, we deposited a film (on the wall of a round bottom (a)

3. RESULTS 3.1. Experimental Design and Readout How can one “titrate” mixtures of amphipathic molecules that are known to form aqueous compartments from

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Liposomes/Membrane compartments were formed by taking stoichiometric mixtures of di-oleoylphosphatidylethanolamine (DOPE), di-oleoylphosphatidylcholine (DOPC) and cholesterol, all procured from Sigma Aldrich Chemicals Pvt Limited, India. The mixtures were dissolved in chloroform and the organic phase removed by reverse phase evaporation.13 200–400 nm liposomes were formed by optimizing the number of freeze and thaw cycles14 15 to eight. The liposomal suspension was then passed through a 0.22 m PES syringe membrane filter by mild manual pressure pressure. Single filter was used to pass the sample for lower hydration volume (VH  = 5 and 10 ml samples. Two to three filters were used to pass VH ≥ 20 ml since samples clogged the filters after passing a certain suspension volume of the solution. Liposomes, encapsulating methylene blue (MB) solution, stuck in filter assembly were recovered by flushing the filter with 0.5% SDS solution, and the recovered material assumed to be a part of retentate while accounting for material balance. Absorbance at 664 nm indicated the MB concentration in solution. All experiments were conducted at 28–30  C, at which temperature the lipid mixtures utilized are established to be in liposomal bilayer phases. Phospholipid material balance was done by using the inorganic phosphate assay.16 The amount of the phospholipids present were estimated in samples (before passing through membrane), retentate and permeate for every experiment.

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2. MATERIALS AND METHODS

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VH (ml) Fig. 1. Experimental design and readout: (a) shows the experimental design. A film of the phospholipids and cholesterol mixture was deposited on the bottom of a round bottom flask by evaporating the organic solvent. This lipid film was then hydrated by vigorous shaking in different volumes of distilled water (dH2 O) containing methylene blue (MB) as a marker. Liposomes/Compartments trapping MB containing dH2 O were prepared from the suspension using freeze and thaw method. The liposome/compartment suspension was passed through a 0.22 mm cut-off membrane attached to a syringe, while applying only manual pressure (i.e., not high pressure). This resulted in separation of fluid with liposomes/compartments larger than ∼200 nm into retentate, and the remaining water with “free” lipids into permeate. The liposomes/compartments formed were not visible under a microscope using 10X × 100X magnification, implying they were smaller than ∼400 nm. (b) Volume of permeate (VP ) and retentate (VR , inset) as a function of different hydration volumes of dH2 O (VT  used for making liposomes/compartments from three different DOPE:DOPC:Chol compositions, 30:50:20 (white bars), 40:40:20 (grey bars) and 50:30:20 (black bars). All data is mean ± standard deviation (n = 3).

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Self-Assembly of Biological Membranes into 200–400 nm Aqueous Compartments

3.2. Membrane Compartment Formation Depends on Hydration Volume To investigate the increase in VR , we plotted it as a function of VH , as shown by Figures 2(a, c and e) for the different amphipathic mixtures. To our pleasant surprise, all the three curves are hyperbolic, with a logarithmic dependence of VR on VH . With excellent regression coefficients for the logarithmic correlation, we were able to achieve a straightforward value for the minimum hydration volume (VH-Min ) for the given molar amounts and stoichiometries J. 1–6, 2010 2010 J. Nanosci. Nanotechnol. 10, 3085-3090,

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VH (ml) Fig. 2. Membrane compartment formation depends on hydration volume: Retentate volume (VR  depends strongly on the hydration volume for three different lipid compositions (DOPE:DOPC:Chol). (a) shows VR = 19044Ln(VH  − 1403 (r 2 = 09581) for DOPE:DOPC:Chol = 30:50:20 (, (b) shows the double reciprocal plot of the data in (a) with linear dependance given by (1/VR  = 28027 1/VH  + 0093 r 2 = 09881), (c) shows VR = 28297Ln(VH  − 33882 r 2 = 09749) for DOPE:DOPC:Chol = 40:40:20 (), (d) shows the double reciprocal plot of the data in (c) with linear dependance given by (1/VR  = 343311/VH  + 00406 (r 2 = 09949), (e) shows VR = 14229Ln(VH  − 06471 (r 2 = 09548) for DOPE:DOPC:Chol = 50:30:20 (, (f) shows the double reciprocal plot of the data in (e) with strong linear dependance given by (1/VR  = 301471/VH  + 01202 (r 2 = 09601). (g) shows that MB trapped in membrane compartments, i.e., in retentate (plain bars) has a much higher concentration than untrapped/free MB, i.e., in permeate (bars with lines) for the three different lipid compositions (color scheme same as in Fig. 1). Total MB mole balance yielded less than 10% loss for all the experiments. All data is mean ± standard deviation (n = 3).

of each of the three amphipathic mixtures by extrapolating the logarithmic fit towards VR = 0, as shown in Table I. This minimum hydration volume represents the minimum volume of an aqueous solution, for a fixed amount of

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flask) of amphipathic mixtures known to form liposomes, or organelles and/or small aqueous compartments in living cells. Then we introduced a measured volume of distilled water, with methylene blue (MB) dissolved in it as an indicator, into the round bottom flask. We call this the volume of hydration (VH ) for the amphipathic mixture. By simply changing VH , we were able to “titrate” the amphipathic mixture. Using the standard freeze and thaw method, we formed liposomes or aqueous compartments (membrane structures) entrapping water with MB. Now, instead of “extruding” liposomes with high pressure extrusion, we simply separated the liposomes/compartments formed spontaneously, by using a membrane filter as shown. This gave us a population of liposomes/aqueous compartments larger than 200 nm in the retentate, and monomeric or sub200 nm structures in permeate. The liposomes/aqueous compartments were smaller than 400 nm since we could not detect any of them using the highest achievable magnification in an optical microscope. Thus, the retentate, having blue appearance (because of encapsulated aqueous MB) had liposomes/compartments between 200– 400 nm diameters. The permeate had non-liposomal amphipathic mixture, probably monomeric or in form of some other phase structures, that does not encapsulate aqueous volumes. This is because of the well-characterized difficulty in creating stable sub-200 nm protein-free structures with the amphipathic mixtures used by us due to membrane curvature constraints in absence of stabilizing proteins.17 The first experimental read-out was remarkably simple, and yet extremely robust. Figure 1(b) shows that the permeate volume (VP ), as expected, increases with VH . However, surprisingly, for all the three stoichiometries of the amphipathic mixtures, even the retentate volume (VR ) increased with increasing VH (inset of Fig. 1(b)). VR comprises predominantly of the volume encapsulated by liposomes/compartments and the hydration layers associated with the outer peripheries of the structures. Apparently, more liposomes/compartments or larger liposomes/compartments (but still smaller than 400 nm) were forming with increasing the “dilution” of the amphipathic mixtures.

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Self-Assembly of Biological Membranes into 200–400 nm Aqueous Compartments Table I. Minimum hydration volume (VH-Min ) required to form membrane compartments and maximum total volume of membrane compartments (VComp-Max ) that can be formed, with the specific lipid compositions. Lipid composition DOPE:DOPC:Chol 30:50:20 40:40:20 50:30:20

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3.3. Membrane Compartment Formation by Phospholipids Having reliably calculated VH-Min and VComp-Max , while confirming MB encapsulation into membrane compartments, we also carried out mass balance of the phospholipids for two of our amphipathic compositions. Figure 3(a) shows that no phospholipids were lost in the experimental system, i.e., all phospholipids were distributed either into membrane compartments between 200–400 nm (retentate) or into the permeate phase. Interestingly, the phospholipids (a) 5

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an amphipathic mixture to form membrane-bound compartments encapsulating aqueous contents. To exploit the hyperbolic appearance of the experimental data, we prepared double reciprocal plots for the three mixtures shown in Figures 2(b, d and f). Again, all the three data sets showed excellent straight line fits for these plots. This allowed us to predict the maximum possible volume that can be entrapped in 200–400 nm membrane compartments (VComp-Max ) using the Y -intercept of the fits, as shown in Table I. Of course, this volume also includes the hydration layer on the surface of the membrane compartments, however, that is expected to be much smaller as compared to the volume entrapped inside. Thus, we have achieved the very first experimental measures of how much minimum hydration is required to form liposomes and/or membrane compartments between 200–400 nm, and the maximum volume that would be allowed to be entrapped in these compartments. Of course, the volume entrapped differs for the three mixtures due to (a) different numbers of 200– 400 nm membrane compartments and, (b) variations in sizes between 200 nm and 400 nm that can be formed by the three different mixtures. An important control for assessing the reliability of the above measurements was MB mass balance. Figure 2(g) shows the MB concentration, in terms of solution absorbance, measured for both retentate and permeate. While we were able to account for the all important MB mass balance, it was surprising to see that MB was more concentrated in the retentate (plain bars), and diluted in the permeate (bars with lines). In absence of any known physico-chemical interaction of MB with the components of our amphipathic mixtures, the only explanation we have is that while water was able to diffuse out of the liposomes/membrane compartments even on mild manual pressures applied to the syringe apparatus, MB (molecular weight ∼320 Da) was still trapped inside due to the protein-free lipid bilayers not having any channels/holes for it to escape.

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Fig. 3. Membrane compartment formation by phospholipids: (a) shows phospholipid (i.e., DOPE + DOPC) mole balance for two compositions, 30:50:20 (white) and 50:30:20 (black) with moles in retentate (plain bars) and permeate (bars with lines). Dashed line shows the total initial phospholipid (see Table I legend also). (b, c) show that total concentration of phospholipids forming 200–400 nm membrane compartments (in retentate) has a power-dependence on hydration volume ([PR  = 45975VH−10439 , r 2 = 09938, for 30:50:20 composition, ; [PR  = 52364VH−11016 , r 2 = 09985, for 50:30:20 composition, ). (d, e) show fraction of phospholipids in membrane compartments (retentate) as a function of total phospholipid concentration (in the second step of Fig. 1(a)). The observed linear dependence (fR = 00623PT  + 0728, r 2 = 01428, ; fR = 01741PT  + 0655, r 2 = 08581, ) predicts all phospholipids to be in membrane compartment form (fR = 1) at 4.37 mM for 30:50:20 composition and at 1.98 mM for 50:30:20 composition. All data is mean ± standard deviation (n = 3).

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Self-Assembly of Biological Membranes into 200–400 nm Aqueous Compartments

3.4. Critical Compartmentalization Concentration (CCC) Our final goal was to identify a parameter analogous to the well established parameter of CMC for amphipathic systems resulting in formation of membrane compartments. The standard plot used for deriving CMC is that of the amphiphile concentration in free and micellar forms as a function of total amphiphile (aqueous) concentration. Therefore, we plotted the phospholipid concentration in membrane compartments and in permeate (free) as a function of total phospholipid concentration as shown in Figure 4. Remarkably, the free and self-assembled data for the membrane compartment system was found to be the reverse of that established for micellar systems, in which the free amphiphile concentration initially remains higher than the amphiphile concentration in micellar form. Therefore, as expected, the aggregation equation used for selfassembly of amphipathic systems into micellar aggregates did not fit the experimental data for membrane compartment formation (dotted curves, Fig. 4). Interestingly, the J. 1–6, 2010 2010 J. Nanosci. Nanotechnol. 10, 3085-3090,

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[PT] (mM) Fig. 4. Critical Compartmentalization Concentration (CCC): (a) and (b) show phospholipid concentration in membrane compartments [PComp ], closed symbols, and free phospholipid [PFree ], open symbols, as a function of total initial (aqueous) phospholipid concentration, for the amphipathic mixtures of (DOPE:DOPC:Chol) 30:50:20 and 50:30:20 respectively. Dotted curves show the best possible fits to the data using the generally accepted scheme of amphipathic self-assembly, i.e., nX↔Xn . Smooth lines show straight line fits to the same data, which are clearly better for both (a) and (b) compared to the conventionally assumed scheme. (a) shows that membrane compartments for DOPE:DOPC:Chol as 30:50:20 has [PComp  = 07992PT  − 00114 (r 2 = 0996), and [PFree  = 01015PT  + 0019 (r 2 = 06248). Intersection of the two straight lines provides the “Critical Compartmentalization Concentration” for this phospholipid mixture as 0.044 mM, along with 20% cholestrol. (b) shows that membrane compartments for DOPE:DOPC:Chol as 50:30:20 has [PComp  = 08656PT  − 00369 (r 2 = 09958), and [PFree  = 01473PT  + 00355 (r 2 = 08791).

experimental data was fit well with simple straight lines. Therefore, the intersection of straight lines fits for the free amphiphile and amphiphile in membrane compartments provides a straightforward parameter that we define as the “Critical Compartmentalization Concentration” (CCC). Thus, CCC is that concentration at which DOPE and DOPC, along with the fixed molar ratio of cholesterol, with stoichiometries of 30:50:20 or 50:30:20, will form

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concentration in the retentate [PR ] showed a direct relationship with the hydration volume (Figs. 3(b, c)). It is clear from our results that the final (aqueous) concentration of phospholipids in membrane compartments is related to the hydration volume of the original phospholipid mixture by the relation [PR  ∼ 5/VH , regardless of the actual amphipathic stoichiometry. This is an important result towards understanding of membrane compartment assembly, since it shows a direct (strong and robust) relationship for predicting the number of 200–400 nm membrane compartments that would form in a given aqueous environment, provided the amphipathic constituents are capable of assembling into compartmental phases. Consequences of this relationship are relevant not only to de novo organelle formation inside the micro-aqueous intracellular environments, but also for achieving controlled formation of liposomes for different applications. An even more interesting relationship emerged when we calculated the fraction of phospholipids in membrane compartments and plotted them as a function of the total phospholipid concentration in our system (this intrinsically accounts for the “titration” using different hydration volumes in step 2 of Fig. 1(a)). We found that the fraction of phospholipids forming membrane compartments is linearly dependent on the total phospholipid concentration (Figs. 3(d, e)). This allowed us to extrapolate the straight lines to predict that most of the phospholipids would be found to be in the membrane compartment phase at a concentration of ∼4.4 mM for one amphipathic mixture and ∼2 mM for the other amphipathic mixture. Of course, these predictions are true only in presence of well defined stoichiometric amounts of cholesterol along with the phospholipids, which is a general feature of organelle formation in eukaryotic cell biology in any case.

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200–400 nm membrane compartments. Importantly, even a sophisticated “aggregation” equation system leading to membrane compartment formation, development of which is beyond the scope of this work, would require intersection of tangential straight lines as the CCC. The CCC for phospoholipids in the mixture of DOPE:DOPC:Chol of 30:50:20 was found to be 0.044 mM and that for 50:30:20 was found to be 0.1008 mM.

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4. CONCLUSIONS Intracellular membrane environment is very dynamic. Membrane vesicles comprising of DOPE, DOPC and cholesterol are formed and utilized continuously. From continuous membrane trafficking, to de novo golgi formation, to vesiculation and self-assembly during cell division, to sensing of different membrane curvatures by specialist proteins, to segregation of substrates and products of all enzymatic reactions, formation of aqueous membrane bound compartments is central to all activities inside a living cell. Furthermore, formation of liposomes as drug delivery vehicles is more of an art even after over four decades of their first descriptions. In this work, we report a remarkably novel and simple experimental design that has allowed us to provide the first parametric and direct insights into formation of membrane compartments entrapping aqueous volumes. Without the need of sophisticated instrumentation, we have successfully utilized our experimental system to define some key parameters like the minimum hydration volume, the maximum entrapment volume, concentration of amphiphiles at which maximum number of amphiphiles are in the compartment phase, and the “Critical Compartmentalization Concentration” for amphipathic mixtures that routinely form intracellular aqueous compartments and are also utilized as liposomes. Of course, while we have conducted our experiments in the simplest controlled aqueous environment of pure water, the same can be done with ease in other aqueous environments (e.g. varying ionic strengths, buffer systems etc.) to derive the desired parameters. Our results, while providing scientific insights towards engineering of liposomal

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preparations, also show how small stoichiometric variations of the abundant intracellular amphipathic species of DOPE, DOPC and cholesterol can control the type and number of membrane-bound aqueous compartments in the dynamic environment of a eukaryotic cell. Acknowledgments: This work was supported by funding from the Council of Scientific and Industrial Research, Government of India through the scheme 37/127206EMRII, and the Department of Science and Technology, Government of India through the scheme SR/FTP/ETA-29 awarded to Aditya Mittal. Rahul Grover acknowledges stipend support from IIT Delhi. We thank Vibhuti Gupta for her experimental efforts with bacterial lipids during the evolution of this work.

References and Notes 1. R. Behnia and S. Munro, Nature 438, 597 (2005). 2. H. T. McMahon and J. L. Gallop, Nature 438, 590 (2005). 3. R. W. Klemm, C. S. Ejsing, M. A. Surma, H.-J. Kaiser, M. J. Gerl, J. L. Sampaio, Q. de Robillard, C. Ferguson, T. J. Proszynski, A. Shevchenko, and K. Simons, J. Cell Biol. 185, 601 (2009). 4. S. Rasmussen, M. A. Bedau, L. Chen, D. Deamer, D. Krakauer, N. H. Packard, and P. F. Stadler, Protocells: Bridging Nonliving and Living Matter, MIT Press, Cambridge, MA (2009), p. 684. 5. S. M. Johnson, A. D. Bangham, M. W. Hill, and E. D. Korn, Biochim. Biophys. Acta 233, 820 (1971). 6. D. J. Crommelin and G. Storm, J. Liposome Res. 13, 33 (2003). 7. K. Mukherjee, J. Sen, and A. Chaudhuri, FEBS Letters 579, 1291 (2005). 8. E. Pupo, A. Padron, E. Santana, D. Sotolongo, and E. Hardy, J. Control Rel. 104, 379 (2005). 9. A. D. Bangham, Chem. Phys. Lipids 8, 386 (1972). 10. F. Szoka and D. Papahadjopoulos, Biochemistry 75, 4194 (1978). 11. C. Tanford, The Hydrophobic Effect: Formation of Micelles and Biological Membranes, John Wiley & Sons, Inc., New York, USA (1973), p. 200. 12. C. Tanford, Science 200, 1012 (1978). 13. S. Vemuri and C. T. Rhodes, Pharm. Acta Helv. 70, 95 (1995). 14. J. D. Castile and K. M. Taylor, Int. J. Pharm. 188, 87 (1999). 15. V. Gupta, R. Gupta, R. Grover, R. Khanna, V. Jangra, and A. Mittal, J. Nanosci. Nanotechnol. 8, 2328 (2008). 16. N. B. Ames, Methods in Enzymology 8, 115 (1960). 17. L. V. Chernomordik and M. M. Kozlov, Annu. Rev. Biochem. 72, 175 (2003).

Received: 1 October 2009. Accepted: 4 November 2009.

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