Understanding Membranes through the Molecular ... - ACS Publications

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Understanding Membranes through the Molecular Design of Lipids Santanu Bhattacharya*,†,‡,§ and Joydeep Biswas† †

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India, ‡Chemical Biology Unit, JNCASR, Bangalore 560 064, India, and §J. C. Bose Fellow, DST, New Delhi, India Received April 4, 2009. Revised Manuscript Received August 26, 2009

Lipids are amphiphilic molecules that are composed of hydrophilic and hydrophobic regions. A typical membranous aggregate (vesicles, water-filled lipid nanospheres) is formed upon the self-organization of lipids in water from a diverse collection of amphiphiles producing a dynamic supramolecular structure that shows phase behavior and ordering as required for specific biological functions. The determination of various physical properties of lipid aggregates is the key to determining structure-function relationships. Over the years, we have designed and synthesized a wide variety of lipid molecular systems for the investigation of their membrane-forming properties and have used them for purposes such as gene delivery and enzyme activation. In this feature article, we focus on our work on various types of lipids including ion-paired amphiphiles, cholesterol-based lipids, aromatic lipids, macrocyclic lipids containing disulfide tethers, cationic dimeric lipids, and so forth. The emphasis is on experimental design and bottom-line conclusions.

Introduction The lipid bilayer is the scaffolding that joins proteins and carbohydrates to form functional cell membranes. Cellular membranes, such as plasma membranes, also provide a semipermeable barrier that separates the cell from its surroundings. Animal cells are packed with membranous structures that constitute the structural frameworks of the cellular organelles, such as the endoplasmic reticulum, Golgi apparatus, mitochondria, nucleus, and so forth.1 Lipid membranes have been described in the fluidmosaic model as a 2D fluid that organizes the integral and peripheral membrane proteins to perform their physiological functions.2 In addition to their structural functions, a dynamic view of lipid membranes is emerging as their active role in cell physiology is being revealed.3 A key feature of biological membranes in living tissues is the spontaneous self-organization of lipids (and other protein and carbohydrate components) from diverse amphiphiles producing metastable structures required to sustain the biological functions of a living organism.4,5 Lipid membranes are important as starting materials for various applications that range from controlled release and drug delivery6 to nanotechnology.7 Because membranes are intricate molecular assemblies, an interdisciplinary approach involving biochemistry, organic and physical chemistry, cell biology, and soft matter physics is necessary to study them.8 In this feature article, we focus primarily on our work on the polar lipid design aimed at investigating structure-property relationships in membranes on the molecular level. Toward the *Corresponding author. Tel: (91)-80-22932664. Fax: (91)-80-22930529. E-mail: [email protected]. (1) Czub, J.; Baginski, M. Biophys. J. 2006, 90, 2368. (2) Singer, S. J.; Nicolson, G. L. Science 1972, 175, 720. (3) Harder, T. Curr. Opin. Immunol. 2004, 16, 353. (4) Derdak, S. V.; Kueng, H. J.; Leb, V.; Neunkirchner, A.; Schmetterer, K. G.; Bielek, E.; Majdic, O.; Knapp, W.; Seed, B.; Pickl, W. F. Proc. Natl. Acad. Sci. U.S.A 2006, 103, 13144. (5) Simons, K.; Ikonen, E. Nature 1997, 387, 569. (6) Allen, T. M.; Cullis, P. R. Science 2004, 303, 1818. (7) Evans, E.; Bowman, H.; Leung, A.; Needham, D.; Tirrell, D. Science 1996, 273, 933. (8) Fantini, J. Cell. Mol. Life Sci. 2003, 60, 1027.

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later part of this feature article, we discuss how we utilize some of them for applications involving DNA complexation and gene transfer across different cell lines and enzyme activation.

Features of Lipid Molecules and Properties of Their Bilayer Membranes All polar lipid molecules are amphiphilic in nature and can be classified on the basis of their molecular features (Figure 1). The most ubiquitous structures of monomeric amphiphiles have one or multiple polar headgroups connected to one hydrophobic tail (a). These include fatty acid salts, lysophospholipids, common surfactants such as cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), and even multiheaded surfactants, typically with one or more water-soluble counterions.9 The majority of the naturally occurring lipid molecules have one polar headgroup connected to two hydrophobic chains via a suitable linkage (e.g., ester, ether, or amide) (b).10 Such a molecular structure favors the formation of bilayer organization because having two hydrocarbon chains may, but need not, be better aligned; if so, this increases the order parameter and leads to the tighter packing of lipid chains. Single-chain amphiphiles bearing large hydratable headgroups also form bilayer membranes.10c Another form of lipid is possible when single-chain amphiphiles of complementary charges form an ion pair (c).11 For such molecules, the counterions are located at the interface of the amphiphilic aggregates. Another class of amphiphile (dimer) has been called gemini surfactants by Menger, where flexible or rigid spacers connect two headgroups (d).12 (9) (a) Handbook of Applied Surface and Colloid Chemistry; Holmberg, K., Ed.; Wiley: New York, 2002; Vol. 1. (b) Haldar, J.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. Angew. Chem. Int. Ed. 2001, 40, 1228. (c) Bhattacharya, S.; Haldar, J. Langmuir 2004, 20, 7940. (10) (a) Kunitake, T. Angew. Chem., Int. Ed. 1992, 31, 2137. (b) Bhattacharya, S.; Haldar, S. Langmuir 1995, 11, 4748. (c) Bhattacharya, S.; Acharya, S. N. G. Langmuir 2000, 16, 87. (11) (a) Fukuda, H.; Kawata, K.; Okuda, H.; Regen, S. L. J. Am. Chem. Soc. 1990, 112, 1635. (b) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371. (12) (a) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. 2000, 39, 1906. (b) Zana, R. Adv. Colloid Interface Sci. 2002, 97, 205. (c) De, S.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. 1996, 100, 11664.

Published on Web 10/20/2009

Langmuir 2010, 26(7), 4642–4654

Bhattacharya and Biswas

Invited Feature Article

Effect of Cholesterol in Lipid Membranes

Figure 1. Schematic of various amphiphilic molecules: (a) monomeric, (b) lipidic, (c) ion-paired, (d) gemini (dimeric), and (e) bolaamphiphilic.

When two ends of a hydrocarbon chain are functionalized with charged headgroups, the resulting molecules are called bolaamphiphiles (e).13 These terms will be used often in this feature article, and the short description along with a cartoon representation here will allow nonspecialist readers to understand them in the right context. From a physical standpoint, the lipid bilayer may be considered to be a liquid-crystal, which manifests phase transitions depending on both the temperature (thermotropic) and the lipid concentration (lyotropic).14 Phase transitions for some polar lipids occur from the lamellar, ordered gel Lβ phase through the intermediate rippled gel Pβ phase to the liquid-crystalline LR phase whereas other polar lipids have no rippled phase and still others melt directly from a crystalline to a liquid-crystalline phase.9 Such phase transitions have been studied in detail by means of techniques such as differential scanning calorimetry (DSC), transmission electron microscopy (TEM), X-ray diffraction (XRD), neutron diffraction, infrared and fluorescence spectroscopy, and nuclear magnetic resonance (NMR).4 The thermally induced main phase transition occurs at a temperature designated as Tm. It is accompanied by a change in enthalpy (ΔH) because rearrangement chain-melting requires energy and thus can be observed as a peak in excess specific heat in a DSC heating scan.15 At Tm, fatty acyl chains of the lipids melt, and also the other type of lipid chains (alkyl, alkenoyl, oxy- or branched chains etc.) undergoes an order-disorder transition (i.e., chain-melting phase transition). During this process, their conformation changes from the straight all-trans state of the solid gel phase to the more flexible, kinked s-gauche conformation of the liquiddisordered fluid phase. The transition from the solid to the fluid state is reflected in the properties and structure of the lipid bilayers at different levels of molecular organization (i.e., at the level of lipid hydrocarbon chains, individual lipid molecules, and the supramolecular assembly as a whole). Augmented freedom of movement of the acyl chains is observed from an expansion of the cross-sectional area and, to a lesser extent, an increment in the volume occupied by acyl chains as well as a reduction of the membrane thickness.16 In keeping with the above, both the Tm and the ΔH (area of the transition peak) in the DSC trace are enhanced with increasing length of the fatty acid chains for saturated lipids with more or less symmetric chains whereas the introduction of cis double bonds into the fatty acyl chains has the opposite effect on these parameters. (13) Fuhrhop, J.-H.; Koning, J. Membranes and Molecular Assemblies: The Synkinetic Approach; Royal Society of Chemistry: London, 1994. (14) (a) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta 2000, 1469, 159. (b) de Vries, A. H.; Yefimov, S.; Mark, A. E.; Marrink, S. J. Proc. Natl. Acad. Sci. U.S.A 2005, 102, 5392. (15) McElhaney, R. N. Chem. Phys. Lipids 1982, 30, 229. (16) Heimburg, T. Biochim. Biophys. Acta 1998, 1415, 147.

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Cholesterol is present in most animal cell membranes, accounting for nearly 20-40 mol % of the total lipids in a plasma membrane.17a Cholesterol interacts noncovalently with lipids within the cell.17a The structure of the cholesterol molecule is very rigid and flat, which favors solid, ordered conformations of the vicinal fatty acid chains of the phospholipids in cell membranes. However, because its molecular shape is quite different from that of other lipids, it disrupts the packing order of the neighboring lipids. Accordingly, to fit better into lipid bilayers, cholesterol induces a so-called liquid-ordered phase that is intermediate between the Lβ and LR phases. In the liquidordered phase, the lipids undergo rapid lateral diffusion similar to that of lipids in their fluid bilayers and the conformational order of their acyl chains resembles that of the gel-phase membranes.18 Thus, cholesterol molecules modulate the hydrocarbon chain fluidity of the other lipid components in membranes. This increase in fluidity alters the thermotropic phase-transition temperature in the lipid bilayer region of the cell membrane and also influences (increases or decreases) their permeability behavior. Depending on the lipid chain length, type, and packing, the incorporation of cholesterol into the phospholipid membrane increases the thickness of the bilayer and diminishes the permeability to various degrees.17b,c,18 Cholesterol has also been connected to the formation of ordered lipid rafts in mammalian cell membranes that are believed to include hydrogen bonds with sphingolipids.19a-c Cholesterol and sphingomyelin are the main components of lipid rafts. They are more ordered and tightly packed than the surrounding bilayer, float freely in the membrane bilayer, and increase the bilayer permeability.19

Interaction of Cholesterol with Lipids in Membranes To understand the interactions of bilayer-forming lipids (Figure 2A) with neutral membrane component cholesterol, we synthesized lipids differing in the chain-headgroup linkage region.20a,b Unlike naturally occurring 1,2-dipalmitoyl phosphatidyl choline (DPPC), three lipids (1-3) employed in this study did not have any functional linkages such as ester or amide groups between the hydrocarbon chains and the respective lipid backbones. However, in the other three lipids (4-6), amide- or ester-based connectors attach the long alkyl chains near the polar cationic headgroup. Small unilamellar vesicles (SUVs) formed from each of the PC and cationic lipids with or without varying amounts of cholesterol were examined by measuring the steady-state fluorescence anisotropy (r) of a membrane-soluble probe, DPH (1,6-diphenylhexatriene) as a function of temperature. The anisotropy due to membranedoped DPH indicates that molecular order in the lipid bilayer packing is strongly affected upon inclusion of cholesterol. This effect was similar regardless of lipid charge. The changes in the values of r induced by cholesterol incorporation into the membranes were the least in the gel state but more pronounced (17) (a) Roche, Y.; Gerbeau-Pissot, P.; Buhot, B.; Thomas, D.; Bonneau, L.; Gresti, J.; Mongrand, S.; Perrier-Cornet, J.-M.; Simon-Plas, F. FASEB J. 2008, 22, 3980. (b) Mouritsen, O. G.; Zuckermann, M. J. Lipids 2004, 39, 1101. (c) Block, M. C.; Van Deenen, L. L. M.; De Gier, J. Biochim. Biophys. Acta 1977, 464, 509. (18) (a) Yeagle, P. L. Biochim. Biophys. Acta 1985, 822, 267. (b) Darke, A.; Finer, E. G.; Flook, A.-G.; Phillips, M.-C. J. Mol. Biol. 1972, 63, 265. (19) (a) Crane, J. M.; Tamm, L. K. Biophys. J. 2004, 86, 2965. (b) Silvius, J. R. Biochim. Biophys. Acta 2003, 1610, 174. (c) Bacia, K.; Schwille, P.; Kurzchalia, T. Proc. Natl. Acad. Sci. U.S.A 2005, 102, 3272. (20) (a) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta 1996, 1283, 21. (b) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta 2000, 1467, 39. (c) McMullen, T. P. W.; McElhaney, R. N. Biochemistry 1997, 36, 4979. (d) Wang, T.-Y.; Silvius, J. R. Biophys. J. 2000, 79, 1478.

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Figure 2. (A) Structures of cationic lipids and phospholipids used for interaction with cholesterol in membranes. (B) Changes observed in 1H NMR line widths upon addition of various quantities (mol %) of cholesterol to phosphatidylcholine membranes. (C) Structures of cholesterol, cationic lipids, and phospholipids showing that hydrogen bonding of cholesterol is feasible only with both ester carbonyl and phosphate groups of DPPC.

after the onset of a thermal phase transition. The amidecontaining lipid (4) was more tightly packed in the solid gel state as a consequence of interlipidic intermolecular hydrogen bonding than its counterparts (5, 6) with ester linkages. For all of the lipids (1-6), the rigidity increased steadily with an increase in cholesterol content in the vesicular lipid/cholesterol mixture in the solid-to-fluid coexistence region. The interactions of cholesterol with different cationic lipids in excess water were also examined in multilamellar vesicles (MLVs) using proton magnetic resonance spectroscopy. In all cases, the methylene 4644 DOI: 10.1021/la9011718

proton line widths in the NMR spectra respond to the addition of cholesterol to the vesicles. Hydrophobic association of the lipid and cholesterol restricts chain -(CH2)n- motion but leaves the terminal CH3 groups relatively mobile. The line widths of -(CH2)n- protons of the hydrocarbon chains in the NMR spectra of all of the lipids broadened on the addition of cholesterol to the vesicles (Figure 2B). The effect of cholesterol on the efflux rates of entrapped carboxyfluorescein (CF) from the PC vesicles was studied to determine how linkage types and headgroup charges of lipids Langmuir 2010, 26(7), 4642–4654



influence the packing and organization of the lipid-cholesterol coaggregates. Upon incremental incorporation of cholesterol into vesicles of 2 and DPPC (7), the CF leakage rates were progressively reduced, an observation consistent with cholesterol-induced stiffening of the resulting membranes. The effects of cholesterol in membranes thus did not depend on the hydrogen bonding interaction with the chain-backbone linkage of lipids. Measurement of the trans-membrane OH- ion permeation rates from cholesterol-doped cationic lipid unilamellar vesicles using entrapped dye riboflavin also demonstrated that the addition of cholesterol to the cationic lipid vesicles with lipids 1 and 3-6 reduced the leakage rates irrespective of the lipid molecular structure. Cholesterol-induced changes of membrane properties such as lipid order, line-width broadening, efflux rates, bilayer widths, and so forth did not depend on the ability of the lipids (1-3) to participate in the hydrogen bonding interactions with the 3β-OH of cholesterol.20b Lipid-cholesterol interactions are subtle in general20c and may involve hydrogen bonds, at least when sphingolipids are involved.20d However, in the specific instances with our synthetic lipids (e.g., 1-3), they contain no functional groups (fatty acid ester carbonyl) that can participate in hydrogen bonding with cholesterol in the same fashion as DPPC (7). When a phosphatidylcholine headgroup is replaced by an -Nþ(CH3)3 cationic headgroup (e.g., in 1 and 3), the possibility of hydrogen bonding with 3β-OH of cholesterol totally disappears, especially when there is no formal linkage in the chain-backbone region of the lipids (Figure 2C). These findings thus emphasize the importance of hydrophobic interactions between the lipid and cholesterol and further demonstrate that it is not necessary to explain the observed cholesterolinduced effects in membranes only on the basis of hydrogen bonding interactions between the 3β-OH of cholesterol and the lipid chain-backbone linkage region or headgroup region.

In the fourth group, 11a-d, a cationic center was introduced into the steroid backbone via an increasing number of OEG spacer units between the steroid backbone and the cationic headgroup. In the fifth series, 12a-b, the OEG segments of one amphiphile each of series 10 and 11 were replaced by the same length of polymethylene chain. Vesicle formation from the aqueous suspensions of these compounds was confirmed by TEM and dye entrapment studies. Fluorescence anisotropy and XRD studies revealed remarkable control of membrane characteristics by both the length and location of the oxyethylene segment. Values of r from 20 to 70 °C obtained using doped DPH showed no phase transition for aqueous suspensions of these cholesteryl amphiphiles (0.1 mM) unlike DPPC (Figure 3B), probably because these amphiphiles are more rigid than hydrocarbon chain-based lipids that display chain-melting transitions in r versus T plots (Figure 3B). Cationic derivatives 10a-d where the OEG segments are attached to the cholesteryl backbone through an -NþMe2 center show a steady decrease in r value at 20 °C as the n value increases (Figure 3C). The attachment of a PEG segment to a cationic center in these systems seems to result in the formation of a large headgroup, which limits the lateral distance of closest approach of the neighboring monomers.22 A headgroup with greater bulk leads to a greater intermonomer separation, which in turn leads to looser membrane packing. In cationic derivatives 11a-d where the -NþMe3 center is attached to the steroid backbone through varying lengths of OEG segments, the anisotropy at 20 °C increases as n increases (Figure 3C). This reversal in trend may be due to the dual nature of the PEG segment as well as its specific location in the bilayer membranes of these systems. These efforts illustrate a step toward the generation of thermally stable organized assemblies of controllable order and thickness.

Cholesterol-Based Lipids

To investigate how these rigid lipids influence the vesicular properties of DPPC, two types of cationic cholesteryl lipids, one where the headgroup is attached to the steroid by an ester linkage (10a-d) and the second where the headgroup is attached to the steroid by an ether linkage (11a-d), were selected.22b A third type of neutral cholesteryl lipid bearing an OEG segment (9d) was also included. OEG segments when inserted in other type of lipids bring about significant changes in their membrane hydration properties.22c-e The interaction of these lipids with DPPC membranes was examined by fluorescence anisotropy, TEM, and DSC. In DPPC membranes, each cholesteryl lipid fills in between DPPC lipids in their vesicular structures in aqueous media (no evidence of domain formation), and because of the presence of a rigid framework, they restricted the chain motion of the DPPC and DPPC cholesteryl lipids mixture in membranes and there is no evidence of domain formation. The DSC and temperature-dependent fluorescence anisotropy data suggest that the changes induced by each cationic cholesterol derivative in the properties of DPPC membranes are dependent on the details of their molecular structure. In general, ester-linked amphiphiles such as 10a or 10d rigidify the fluid phase more than the others. Increasing the concentrations of these amphiphiles not only abolished the phase transition and depressed the transition temperature but also increased the curvature of the resulting mixed DPPC membranes as evident from TEM studies. However, it is not apparent why ether- and ester-based amphiphiles

Cholesterol itself is weakly amphiphilic because its hydroxyl group is polar and the steroid skeleton and its isopentanyl tail at the C-17 position are hydrophobic. Its aggregates in water have been reported to be very small micelles21a although a recent report suggests that cholesterol actually forms small crystals.21b To examine whether the attachment of a charged or polar nonionic group to cholesterol brings about a new type of lipid membranes, we synthesized a number of cholesterol-based neolipids. In the first and second groups, 8a-c and 9a-d, of neolipids, nonionic oligo(ethylene glycol) (OEG) [-(OCH2CH2OH)n-] appendages were covalently introduced either via a succinate spacer or by direct attachment to the 3β-OH group of cholesterol.22a A third set of cholesterol derivatives, 10a-c, was prepared in which the location of the cationic charge was conserved but the headgroup was modified with increasing numbers of OEG units. (21) (a) Castanho, M. A. R. B.; Brown, W.; Prieto, M. J. E. Biophys. J. 1992, 63, 1455. (b) Uskokovic, V. Steroids 2008, 78, 356. (22) (a) Bhattacharya, S.; Krishnan-Ghosh, Y. Langmuir 2001, 17, 2067. (b) Ghosh, Y. K.; Indi, S. S.; Bhattacharya, S. J. Phys. Chem. B 2001, 105, 10257. (c) Bhattacharya, S.; Dileep, P. V. Bioconjugate Chem. 2004, 15, 508. (d) Bhattacharya, S.; Dileep, P. V. J. Phys. Chem. B 2003, 107, 3719. (e) Dileep, P. V.; Antony, A.; Bhattacharya, S. FEBS Lett. 2001, 509, 327. (23) (a) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A 1987, 84, 7413. (b) Felgner, J. H.; Kumar, R.; Sridhar, C. N.; Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L. J. Biol. Chem. 1994, 269, 2550. (c) Felgner, P. L.; Ringold, G. M. Nature 1989, 337, 387. (d) Bhattacharya, S.; Bajaj, A. Chem. Commun. 2009, 4632. (24) Ghosh, Y. K.; Visweswariah, S. S.; Bhattacharya, S. FEBS Lett. 2000, 473, 341. (25) Ghosh, Y. K.; Visweswariah, S. S.; Bhattacharya, S. Bioconjugate Chem. 2002, 13, 378.

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Interaction of Cholesteryl Lipids with DPPC Membranes

(26) Bajaj, A.; Mishra, S. K.; Kondaiah, P.; Bhattacharya, S. Biochim. Biophys. Acta 2008, 1778, 1222. (27) Bhattacharya, S.; Bajaj, A. Biochim. Biophys. Acta 2008, 1778, 2225.

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Figure 3. (A) Molecular structures of various cholesterol-based cationic lipids with different linkers and headgroups. (B) Fluorescence anisotropy (r) versus temperature (°C) with DPH for different amphiphiles. [Amphiphile] = 0.1 mM, [DPH] = 1 μM, and pH 6.8. (C) Plots of fluorescence anisotropy (r) at 20 °C with n for -(CH2CH2O)n- units in 10a-d (0) and 11a-d (b).

influence curvature differently. Presumably, it could be due to the different way in which the ester-linked cholesteryl amphiphiles (10) associate themselves with the host DPPC molecules as compared to the ether-based amphiphiles (11a-c). This is probably due to the presence of dipole-induced dipole interactions that hold the ester and carbonyl oxygen atoms of the DPPC and cholesteryl amphiphile monomers intimately to each other. In the series 11a-d, the -(CH2-CH2-O)n- unit is located between the cholesteryl backbone and the -NþMe3 headgroup. Here it acts almost like a hydrophobic spacer where an increase in the n value in 11 results in an increase in the length of the bilayer as evidenced from the XRD data (Figure 4). Depending on its location in the cholesteryl lipid monomer, the oxyethylene group shows either hydrophilic or hydrophobic character. In the series 10a-d, the OEG segment is located outside the hydrophobic portion of the membrane. It takes on hydrophilic character and remains in a random orientation, thereby increasing the headgroup bulk. Increases in the bulkiness of the headgroup increases the intermonomer separation, as reflected in the r values, which induces interdigitation as supported by XRD data (Figure 4). In general, the ester-linked lipids that support structured water in the interfacial region tend to rigidify the fluid phase more than others. Also, at higher concentration, they induce a curvature in the 4646 DOI: 10.1021/la9011718

Figure 4. Plots of membrane thickness as obtained from XRD with n-value -(CH2CH2O)n- units in the 10a-d (0) and 11a-d (b) series.

DPPC membranes. Importantly, these cholesteryl amphiphiles behave less like cholesterol in that their incorporation into DPPC not only abolishes the phase transition but also depresses the phase-transition temperature. The differential behavior of esterversus ether-based cholesteryl lipids in phosphatidylcholine lipid Langmuir 2010, 26(7), 4642–4654



Figure 5. Stereochemical representation of the structure of the cationic cholesteryl lipids with different linker groups.

membranes is noteworthy. This probably originates from the dipole-induced dipole interactions that hold the cholesteryl ester and carbonyl groups in DPPC together in membranes.

Gene Transfer Mediated by Cholesterol-Derived Lipids The successful implementation of gene therapy in a clinical setting depends on the development of efficient and safe gene delivery vehicles known as gene transfection vectors. In gene therapy, the vectors that are generally used are of two types: viral and nonviral. The potency of viral transfection vectors is superior to that of their nonviral counterparts. However, the adverse immunogenic consequences associated with the use of virus-based vectors have made the nonviral gene delivery systems the vectors of choice. Among the existing classes of nonviral gene transfection reagents, cationic lipids23a-d possess distinct advantages: (a) a welldefined molecular structure and hence the possibility of improved design through structure-function studies; (b) easy preparation; (c) convenient handling; (d) the ability to inject large lipid/DNA complexes; and (e) insignificant immunogenic reactions. To examine the potential of our cationic cholesteryl lipids toward gene transfection, we synthesized cationic cholesterol derivatives with three linkage types, an ester (10a), an ether (11a) and a urethane (14), between the cationic headgroup and the steroid backbone (Figure 5).24 To study the effect of headgroup hydration on gene transfer, a hydroxyethyl group at the level of the lipid headgroup was incorporated (13). Each of these lipids was mixed with DOPE (1,2-dioleoyl phosphatidyl ethanolamine) to prepare formulations for the gene-transfer study. Comparisons of optimal transfection efficacies showed that lipids with ester and urethane linkages were less efficient than their ether counterpart (11a), which was ∼6 times more efficient than 14. Lipids 13 and 11a showed transfection efficiencies that were nearly 10 times greater than the commercially available genetransfer reagent, lipofectin. Lipid 11a was also found to be more efficient than another commercially available highly efficient formulation, lipofectamine, especially in the presence of serum. In addition, lipid 13 induced transfection efficiently without requiring a helper lipid (DOPE). This increased its usefulness as a gene-transfer agent significantly because a single stable lipid suspension is always preferred as a formulation over that based on a lipid mixture. Influence of Inserted OEG Segments in Lipids on Gene Transfection On the basis of the above findings, we undertook a detailed study as to how lipid hydration influences gene transfer by selecting different cationic cholesteryl lipids 10a-d (ester-linked) and 11a-d (ether-linked) (Figure 3A).25 Also, varying lengths of OEG units in lipids were tested to determine the relationship between headgroup hydration and transfection efficiency. Langmuir 2010, 26(7), 4642–4654

Depending on its location in the cholesteryl lipid monomer, the OEG units show either hydrophilic or hydrophobic character. In the series 10a-d, the OEG segment is located outside the hydrophobic portion of the membrane, adopting hydrophilic character, and remains in a random orientation, thereby increasing headgroup bulk. In contrast, in 11a-d, it acts almost like a hydrophobic spacer as an increase in the n value in 11 results in an increase in the length of the bilayer as evidenced from the XRD data. As stated earlier, these aggregates are thermally insensitive22b because no solid-to-fluid transition is observed by temperature-dependent fluorescence anisotropy measurements or by DSC. In ester-linked series 10a-d, the transfection efficiency decreased gradually with an increasing number of oxyethylene units attached to the cationic center. The introduction of oligooxyethylene units as spacers in the linker region of the lipid monomer in the series 11a-d also decreased the transfection efficiency (Figure 6). Thus optimal transfection with this class of lipids requires a positive charge located as close as possible to the steroid skeleton of the cytofectins, which is also not hydrated.

Influence of Headgroup on Gene Transfection To examine the possibility of further improving the gene transfection efficiency, we synthesized eight novel cholesterolbased cationic lipids possessing different headgroups and one ethylene oxide linkage between the headgroup and the cholesteryl backbone (Figure 7).26 Each lipid formed a stable suspension both as a neat lipid or as co-vesicles with 1:2 or 1:3 lipid/DOPE mixtures. The transfection efficiency of each cationic lipid was studied in HeLa cells. Chol-DMAP (18) possessing one pyridinium group and a tertiary amine showed efficient transfections in the presence as well as in the absence of DOPE. Cationic lipid Chol-PR (19) having two tertiary amine functionalities in the headgroup showed optimized transfection without a helper lipid, DOPE. In the presence of serum, formulation Chol-DMAP (18) was found to be very efficient. For formulations Chol-NMP (21) and Chol-PYR (16), more than 70% of cells were transfected as determined from FACS analysis at N/P ratios of 3 and 4. However, these formulations were found to be very cytotoxic because only 60% of cells were found to be viable at these N/P ratios. Formulations Chol-NMe (11a) and Chol-DMAP (18) had a high transfection efficiency (80%) with a low cytotoxicity at an N/P ratio of 3. Between the Chol-PR (19) and Chol-PRþ (20) formulations, the cell viability decreased in Chol-PRþ (20) because there is introduction of positive charge. Ethidium bromide exclusion assays and SDS-induced release of DNA from lipid-DNA lipoplexes were performed to get a clear idea of the transfection activities of the different cationic lipids. Modifications of the lipid headgroups influenced the binding of the liposomes with DNA. Thus, the liposomes of 11a were found to bind more strongly with DNA, compared to DOI: 10.1021/la9011718

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Figure 6. Optimized transfection efficiency [normalized to lipofectamine (LA)] of various cholesterol-based cationic lipids possessing -(CH2CH2O)n- units in (A) 10a-d and (B) 11a-d.

Figure 7. (A) Molecular structures of cholesterol-based cationic lipids with different headgroups. (B) Transfection efficiencies of various cholesterol-based cationic lipids with different headgroups compared with two very potent commercially available reagents, effectene and lipofectamine 2000.

other lipid based liposomes in this series. All of the other cationic lipids that contain cyclic headgroups (Figure 7B) did not allow efficient binding with DNA.

Cholesterol-Linked Lipopolymers We recently introduced lipopolymers with variable fractions of cholesteryl units attached to PEIs (polyethyleneimines) with three 4648 DOI: 10.1021/la9011718

different molecular weights [MW = 800 (P8), 1200 (P12), and 2000 (P20)] via an ether-based linkage with cholesterol.27 We investigated their interactions with phospholipid membranes (Figure 8).27 Although PEI itself is very polar and does not readily associate with phospholipid membranes, cholesterol tethering leads to their efficient partitioning into dipalmitoyl phosphatidylcholine membranes. The aqueous suspensions of Langmuir 2010, 26(7), 4642–4654



Figure 9. Molecular structure of natural dimeric lipid, cardiolipin.

Figure 8. General structure of the PEI-cholesterol-based lipopolymers.

these cholesteryl-PEI lipopolymers possessing the highest cholesterol grafting (∼57 mol %) among P8 derivatives showed helical aggregates of 20 nm width with twists, whereas a lower loading of cholesteryl units in P8 (∼29 mol %) showed vesicular aggregates of 50-130 nm in size. Lipopolymeric suspensions derived from P12 afforded vesicular aggregates of 80-120 nm in size. Among P20-derivatized PEI-chol lipopolymers, the ones with maximum cholesterol grafting showed nanofibers of 100 nm size such as P8 derivatives. Lower cholesterol loading into P20 derivatives afforded vesicular aggregates 100 nm in size. These cholesteryl-PEI lipopolymers interact with lipid molecules in DPPC membranes as does cholesterol, although the lateral location of the cholesterol units in the polymer and in membranes depends on the extent of cholesterol loading per PEI monomer and the molecular weight of the lipopolymer. In membranes, the cholesteryl-PEI lipopolymers quench the motion of DPPC acyl chains and broaden the thermal phase transition. However, this broadening was dependent upon the type of lipopolymer and the extent of cholesterol incorporation. Detailed analysis of the fluorescence anisotropy and DSC data indicates that the nature of perturbation induced by PEI-chol lipopolymers was dependent on the molecular weight of the PEI used and the % cholesterol grafted to PEI. In general, PEI-chol lipopolymers rigidifed the liquid-crystalline phase of the membranes without any noticeable effect on the gel phase, unlike natural cholesterol, which is known to fluidize the gel phase of membranes. The rigidification of the liquid-crystalline phase of the membranes depends on the percentage of cholesterol grafted to PEI. Electron microscopy studies on the lipopolymer-doped DPPC liposomes showed the vesicular nature of the aggregates of different sizes depending upon the lipopolymer. Thus, PEI-chol lipopolymers bearing a low percentage of cholesterol grafting hardly showed any effect on the liquid-crystalline phase of the DPPC membranes. Each of the lipopolymers (Figure 8) showed high transfection efficiency as co-liposomes with DOPE.28 Transfection studies in HeLa cells show that all of the lipopolymers are better transfecting agents than commercially available 25 kDa PEI. The lipopolymers also display high serum compatibility. Transfection efficacy and serum compatibility were dependent upon the molecular weight of PEI used for lipopolymer synthesis and the percentage of cholesterol grafting on lipopolymers. Higher cholesterol grafting in such PEIs showed optimized transfection at a lipopolymer/DOPE ratio of 1:1 whereas lipopolymers with the

Pseudoglyceryl Lipid Dimers To generate novel analogues of cardiolipin and to explore their membrane-forming properties, we prepared nine cationic dimeric lipids, 25a-i (Figure 10), in which the two Me2Nþ ion headgroups are separated by a variable number of polymethylene units [-(CH2)m-].30

(28) Bajaj, A.; Kondaiah, P.; Bhattacharya, S. Bioconjugate Chem. 2008, 19, 1640.

(29) Hubner, W.; Mantsch, H. H.; Kates, M. Biochim. Biophys. Acta 1991, 1066, 166. (30) Bhattacharya, S.; De, S.; George, S. K. Chem. Commun. 1997, 2287.

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Figure 10. Molecular structures of the monomeric and dimeric cationic pseudoglyceryl lipids with polymethylene spacers.

least amount of cholesterol incorporation showed maximum transfection activities in the absence of DOPE. The lipopolymer formulations are much less toxic than commercially available 25 kDa PEI. Optimized formulations of the lipoplexes synthesized in this study had the highest transfection activities among those known for nonviral delivery reagents.

Dimeric Lipids with Four Chains Over the past few years, the gemini surfactants, formed by two amphiphilic units linked with a spacer at the level of headgroups, have attracted significant interest because of the possibility of tailoring the molecular structure by changing headgroup structure, spacer, and tail lengths. Many gemini surfactants that possess unusual aggregation and biological properties have been prepared.12 Gemini surfactants also exhibit enhanced surface activity compared to those of the corresponding monomeric counterparts. Naturally occurring glycerol-bridged dimeric phosphatidic acid, cardiolipin (Figure 9), which show high metabolic activity in heart and skeletal muscles, is a good example of a gemini phospholipid.29

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Table 1. Unit Bilayer Thicknesses Obtained from XRD Studies of Self-Supporting Cast Films of Dimeric Cationic Pseudoglyceryl Lipids with n-C16H33 Chains (25a-i) and Their Proposed Packing31 unit bilayer thickness (A˚) lipid m value

observeda

calculatedb

proposed packing

0 3 4 5 6 12 16 20 22

45.9 49 bilayer 45 49 bilayer 45.9 49 bilayer 45.9 49 bilayer 44.2 49 bilayer 31.3 49, 16.8,c 31d tilted and interdigitated bilayer 29.6 49, 21.9,c 31d tilted and interdigitated bilayer monolayer-bilayer 29.8 49, 27c monolayer-bilayer 30.8 49, 29c a As obtained from reflection XRD of cast films. b Lengths of two molecular layers of lipids as obtained from energy-minimized CPK models (INSIGHT). c Length of unit monolayer-bilayer arrangement of lipids as obtained from energy-minimized CPK models. d Length of unit tilted, interdigitated bilayer arrangement of lipids as obtained from energy-minimized CPK models.

TEM and dynamic light scattering (DLS) measurements of their aqueous dispersions indicated the formation of vesiculartype aggregates from these dimeric lipids. The vesicle sizes and morphologies were found to depend strongly on the mvalue, the method, and the thermal history of vesicle preparation. Information on the thermotropic properties of the resulting vesicles was obtained from microcalorimetry. Microcalorimetry shows that the Tm values of these vesicles have a nonlinear dependence on the spacer chain length (m value). As the m value increased from 3 to 16, the Tm values progressively decreased from 57 to 41 °C at a constant chain length of 25a-i. At m values of 20 and 22, Tm values increased to >66 °C. Temperature-dependent fluorescence anisotropy due to membrane-doped DPH also showed inflections (not shown here) that were in agreement with the Tm values obtained by calorimetry. X-ray diffraction of the cast films of the lipid dispersions revealed that these lipids organize in three different ways (i.e., regular bilayer, tilted and interdigited bilayer, and monolayer-bilayer hybrid-type organizations) depending on the m value. Membrane thicknesses for the lipids of m = 3-6 remain in the range of 44-46 A˚, whereas for lipids with higher m values the membrane thickness decreased to 30-31 A˚ (Table 1). Each of these lipid vesicles entrapped riboflavin, demonstrating the presence of inner aqueous compartments.31 We employed neutral dye riboflavin as it has been used in other lipid vesicles, and information on such marker partitioning into/binding onto cationic and neutral lipid bilayers is available.32a The rates of permeation of OH- under an imposed transmembrane pH gradient also depend significantly on the m value. This again demonstrate how spacer chains in these lipids influence their packing organization, which in turn renders the resulting vesicles tight or leaky. The EPR spin-probe method with doxylstearic acids 5NS, 12NS, and 16NS spin-labeled at the 5, 12, and 16 positions of stearic acid, respectively, was used to establish the chain-flexibility gradient and homogeneity of these bilayer assemblies. Evidence of strong lipidic chain interdigitation was seen in the EPR studies. This type of EPR behavior is reported in the literature,32b indicating the formation of the interdigitated lipid phases in phosphatidylcholine (PC) and phosphatidylglycerol (PG). Hence, these results may be attributed to the formation (31) Bhattacharya, S.; De, S. Chem.-Eur. J 1999, 5, 2335. (32) (a) Kunitake, T.; Okahata, Y.; Yasunami, S. Chem. Lett. 1981, 10, 1397. (b) Boggs, J. M.; Rangaraj, G. Biochim. Biophys. Acta 1985, 816, 221.

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Figure 11. Dependence of the thermotropic phase transition (Tm) of dimeric cationic pseudoglyceryl lipid (with n-C16H33 and nC14H29 chains) suspensions on the spacer length as designated by m values.

of fully interdigitated lipid bilayers by 25h compared with the noninterdigitated gel-phase lipid bilayers by 23, 25a, and 25f. The apparent fusogenic propensities of these bipolar tetraether lipids were investigated in the presence of Na2SO4 with a fluorescenceresonance energy-transfer fusion assay. Na2SO4 helps in the fusion and intermixing of membrane lipids, and the rate of fluorescence decay was found to vary for membranes with changes in the m values of the lipids. Small unilamellar vesicles formed from monomeric 23 and three representative dimeric lipids were also studied using fluorescence anisotropy and 1 H NMR spectroscopic techniques in the absence and presence of varying amounts of cholesterol. These membranes still showed a reduced but perceptible thermal phase transition at high cholesterol content. Because the freedom of motion and packing of four independent hydrocarbon chains are critical to their membrane-level properties, this study shows the covalent attachment of two lipid monomers by a polymethylene chain upon exceptional changes in their thermal, permeability, fusogenic, and packing properties with respect to membrane formation.33 The properties of membranes formed from dimeric lipids possessing n-C14H29 and n-C16H33 chains were compared closely. The lipid aggregates were characterized using TEM, DLS, highsensitivity DSC, and PALDAN fluorescence studies. In this study, the variation in the length of the spacer between the cationic ammonium headgroups was m = 3-12. EM studies revealed that dimeric lipid aggregates of 24a-d and 25a, c, e, and f are smaller than their monomeric analogs 22 and 23, respectively. DLS studies showed that the dimeric lipid suspensions with a -(CH2)8- spacer were larger than that of other dimeric lipid analogues. Dimeric lipids with an n-C16H33 chain had higher phasetransition temperatures than those with n-C14H29 chains (Figure 11). DSC studies show that dimeric lipids with -(CH2)8and -(CH2)12- spacers have the high temperature lag of ∼8 °C during the cooling scan of the lipid aggregates in aqueous media. These observations are consistent with the significantly higher hydration level in their melted liquid-crystalline phases because they indicate the preferential existence of fluid lipid membranes in the hydrated state (Table 2). The determination of apparent hydration at aggregate interfaces using a PALDAN probe (33) Bhattacharya, S.; Bajaj, A. Langmuir 2007, 23, 8988.

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Table 2. Thermotropic Phase Transitions as Obtained from DSC Studies and Generalized Polarization (GP) of Dimeric Lipid Aggregates (1 mM) as Sensed by a Membrane-Soluble Probe, PALDAN Tm (°C)a lipids

spacer (-m-)

up scan

down scan

29.6 26.3 43.2 40.2 28.6 26.3 30.6 23.7 29.6 22.5 45.8 43.3 59.3 55.7 -(CH2)346.2 43.5 -(CH2)548.6 40.4 -(CH2)847.3 39.8 -(CH2)12a The maximum deviation was (0.1 °C.

22 24a 24b 24c 24d 23 25a 25c 25e 25f

-(CH2)3-(CH2)5-(CH2)8-(CH2)12-

GP 24 °C

70 °C

0.19 0.07 0.11 0.14 0.07 0.22 -0.08 -0.08 -0.14 -0.32

-0.24 -0.30 -0.34 -0.33 -0.10 -0.02 -0.32 -0.43 -0.28 -0.41

Figure 12. Molecular structures of the cationic pseudoglyceryl dimeric lipids with different oligo-oxyethylene spacers.

revealed that dimeric lipid aggregates with n-C16H33 chains have greater hydration than dimeric lipids possessing n-C14H29 chains (Table 2). Interestingly, lipid aggregates with n-C16H33 chains and -(CH2)12- spacers were found to be more hydrated than other dimeric lipids in the gel state. Hence, the aggregation, thermotropic, and hydration studies clearly show that the membranelevel properties of the pseudoglyceryl dimeric lipids with polymethylene spacers in aqueous media depend upon both the length of the spacer between the headgroups and the length of the main chain. To explore this kind of lipid systems further, the corresponding lipid dimers with oligo-oxyethylene spacers were also synthesized (Figure 12).34 These surfactants formed stable vesicular aggregates in water as confirmed by TEM studies. The presence of oxyethylene-based spacers between the cationic headgroups of the dimeric lipids change the membrane properties. Thus, aggregates of lipid 26a (i.e., a dimeric lipid with a -CH2-CH2-O-CH2-CH2spacer) had the highest Tm (54 °C), close to that of the dimeric lipid having n-C16H33 chains and a -(CH2)3- spacer (25a). In this series, lipid aggregates of 26a possess the highest Tm and the lowest zeta potential. In contrast, the lipid aggregates of 26b possess the lowest Tm (44.2 °C), comparable to that of monomer 23, and the highest zeta potential. Thus, the increase in the number of oxyethylene units lowers the Tm in these lipid membranes and increases the membrane thickness from 49 to 56 A˚. What are the molecular structure-membrane relationships among Tm, the zeta potential, and the membrane thickness? For example, an increase in the spacer length in the series 26a-c should increase the separation between headgroups. This in turn should reduce the headgroup packing density and lower the zeta potential more than for the dimeric lipids with polymethylene spacers of the same length. The oxygens in the oxyethylene spacer should have a stronger interaction with water (34) Bhattacharya, S.; Bajaj, A. J. Phys. Chem. B 2007, 111, 2463.

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Figure 13. Molecular structures of the gemini ion-paired amphiphiles.

than a methylene in the (CH2)m spacer, and this interaction would prevent the oxyethylene spacers from forming a U-like shape. U shapes would permit tighter packing of the headgroups, higher surface charge densities, and hence higher zeta potentials. This is also reflected in their comparative gene-transfer properties.35 Thus, the transfection efficiency of oxyethylene-based dimeric lipids was much higher for some of the formulations when doped with DOPE than for their polymethylene-based dimeric lipid counterparts.35

Ion-Paired Dimeric Lipids36 One of our early goals was to synthesize gemini surfactants with lipophilic single-chain, negatively charged counterions. Accordingly, eight dicationic gemini surfactants were synthesized by joining two CTAB-like moieties with different lengths of spacer polymethylene groups (Figure 13).37 The bromide counterions of these geminis were then replaced by palmitate counterions via an ion-exchange procedure.37 All of the ion-paired gemini amphiphiles (IPA) formed vesicular aggregates in aqueous media as evidenced by the TEM (transmission electron microscopy) experiments. Both DSC and fluorescence methods indicated that the vesicles of gemini IPA 27a with the shortest spacer [-(CH2)2-] in the series had the highest chain melting temperature (Tm ∼74 °C from the DSC method and ∼76 °C from the fluorescence method), where the fluorescence method measured the monomer vibrational intensity ratio (I3/I1) of bilayer-doped pyrene as a function of temperature. In contrast, gemini IPA 27h with the longest spacer [-(CH2)12-] had the lowest phasetransition temperature (∼39 °C from the DSC method and ∼38 °C from the fluorescence method).38 Molecular modeling studies suggested that as the number of methylene units in the spacer increases, the [-(CH2)n-] chain between the two headgroups of the gemini IPA starts to loop into the membrane interior to minimize unfavorable water contacts. In this process, the hydrocarbon chain packing within the assembly becomes impaired, leading to the disruption of the packing order within the individual dimeric IPA itself and also of the neighboring lipids in the membranous assembly. Thus, in this process the phasetransition temperature of the gemini IPAs decreases with the increase in spacer length. The fluorescence anisotropy values (r) due to membrane-doped DPH for vesicular 27b-g were quite high (r ≈ 0.3) compared to that of 27h (r ≈ 0.23) at 20-30 °C in their solid gel states. However, the r value for vesicular 27b beyond melting was higher (0.1) compared to any of those for 27c-h (similar to 0.04-0.06). The entrapment of a small water-soluble solute, riboflavin, by the individual unilamellar vesicular aggregates and their sustenance under an imposed transmembrane pH gradient have also been examined. These results showed that all such lipid vesicles (35) Bajaj, A.; Paul, B.; Kondaiah, P.; Bhattacharya, S. Bioconjugate Chem. 2008, 19, 1283. (36) Soussan, E.; Cassel, S.; Blanzat, M.; Rico-Lattes, I. Angew. Chem., Int. Ed. 2009, 48, 274. (37) Bhattacharya, S.; De, S. J. Chem. Soc., Chem. Commun. 1995, 651. (38) Bhattacharya, S.; De, S. Langmuir 1999, 15, 3400.

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Figure 14. Molecular structures of the aromatic-based bolaphile/amphiphile ion pairs.

could entrap riboflavin and that generally the resistance to OHpermeation decreased with the increase in the n value, supporting the notion that the membrane packing started loosening with an increase in the n value. Finally, on the basis of the calculated structures of these lipids and the above observations, we concluded that the membrane properties of these lipids can be modulated by changing the spacer chain lengths of these IPAs. To examine whether a change in the shape of the amphiphiles has a bearing on their properties with respect to membrane formation, we prepared four IPAs with aromatic-core-based bolaform counterions (Figure 14).39 In this series of bis(hexadecyl trimethyl ammonium) phenyl-1,2-, 1,3-, and 1,4-di(oxyundecanoate) amphiphiles 28-31, the alkyl chains of the bolaphilic counterions originate from the different isomeric positions of the central phenyl ring. TEM indicated that all four amphiphiles formed vesicle membranes upon suspension in water. Membrane properties were examined by microcalorimetry, temperature-dependent fluorescence anisotropy measurements, and UV-vis spectroscopy. The Tm values for vesicular 28, 29, 30, and 31 were 31, 38, 12, and 85 °C, respectively. Thus, vesicles of 30 had the lowest phase-transition temperature (∼12 °C) whereas those of 31 had the highest Tm value (∼85 °C). Interestingly, the Tm values for 29 and 31 were also found to depend on their concentration. The entrapment of small solutes and the release capability were also examined to demonstrate that these bilayers formed enclosed vesicles. Using these IPA vesicles, it was possible to encapsulate up to 2.28% riboflavin in water. X-ray diffraction of the cast IPA films indicated that the bilayer widths of these aggregates ranged from 33 to 47 A˚. XRD (39) Bhattacharya, S.; De, S. Chem. Commun. 1996, 1283.

4652 DOI: 10.1021/la9011718

experiments and molecular modeling studies revealed that 28 preferred a tilted bilayer arrangement whereas 29, 30, and 31 preferred supported interdigitated organizations. In 29, the two undecanoate chains generate from a core catechol unit at an angle of ∼60° in their extended form. After ion pairing with cetyl trimethyl ammonium (CTAþ), the long chains propagated in an angular fashion, which led to a loose packing of the bilayer arrangement. Hence, 29 adopted a tilted arrangement to minimize the contact of hydrocarbon chains with water. In 30, the two undecanoate chains from the central resorcinol unit span an angle of 120° in their extended form. Hence, the intramonomer van der Waals interactions between the long chains are significantly looser than their counterparts. In 31, the two undecanoate chains propagate from the core quinol unit at an angle of 180° in their extended form and span the bilayer. This conformational arrangement helps optimize van der Waals contacts upon ion pairing with CTAþ and also during interaromatic π-stacking association in the membranous assembly.40 The phase-transition temperatures and the permeabilities of these vesicular aggregates are strongly influenced by the geometry of the bolaamphiphilic counterion. This is a novel approach for fine-tuning vesicular properties and suggests new recipes for the preparation of stable vesicular structures that may be of practical value. In particular, mixed bilayers composed of bacterial lipid (bolaamphiphile) and fatty acids or mammalian lipids (monopolar amphiphile) such as lysophosphatidates could be tailored to have useful biological properties.36 (40) Bhattacharya, S.; De, S.; Subramanian, M. J. Org. Chem. 1998, 63, 7640.

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Figure 16. Histogram showing the activity of PLA2s on DAG 32Figure 15. Molecular structures of the macrocyclic lipids containing the disulfide tether.

Macrocyclic Lipids with a Disulfide Tether Diacylglycerols (DAGs) are phospholipid precursors that also play an important role in controlling the activity of membranebound enzymes such as protein kinase C (PKC) and phospholipase A2 (PLA2).41 Certain DAGs induce membrane fusion via conversion of the bilayer to hexagonal or other nonbilayer phase structures that affect membrane curvature. Such changes within membranes are thought to create defects within bilayers that make the substrate (phospholipids) more accessible to the membrane-bound enzymes and may be important in understanding the biological action of DAGs. We developed a general synthetic method for preparing certain disulfide-tethered macrocyclic diacylglycerols (32-37) in which a disulfide linkage was used to “stitch” together the two hydrocarbon chains (Figure 15).42 Interchain linking through a disulfide tether also provided the extra advantage of converting these macrocycles into the corresponding open-chain thiol analogues via a simple dithiothreitol-mediated reduction step. Access to the open-chain analogue allows further investigation to compare the effects of macrocyclization systematically. The macrocyclic DAGs (32-37) and their reduced open-chain analogues were used to investigate the effect of imposing chain restriction within the membrane bilayer. 31P NMR of DPPC vesicles in which 1 mol % DAG 32 was doped indicates the formation of isotropic phases.43a These phases disappear upon the opening of the macrocycle by the reduction of the disulfide tether. The thermal properties of DPPC membranes containing various amounts of individual macrocyclic DAGs (32-37) as well as their nonmacrocyclic analogues (32-35, 37) were investigated using DSC. These studies indicate that the co-operativity of the thermal melting process of the mixed bilayer system is enhanced upon relaxation of the macrocyclic DAG to the open-chain version at the Tm.43b The co-operativity unit (CU) [ΔHv/ΔHc, where ΔHv is the van’t Hoff enthalpy and ΔHc is the calorimetric enthalpy]43c gives information about the number of lipid molecules that undergo melting together. When the DAGs are compatible with host membrane phospholipids, they are more homogeneously distributed in the membrane and the CU of the resulting transition should be high. Cooperativity is greater when the disulfide macrocycle is reduced to the open-chain form, making it more adaptable to the DPPC bilayer. Biochemical studies establish that the macrocyclization of DAG 32 results in an enhancement of the activity of both types of PLA2s examined herein. Multilamellar vesicles consisting of substrates DPPC (5 mM) and sn-2-NBD-hexanoyl-PC (5 μM) (41) Cunningham, B. A.; Tsujita, T.; Brockman, H. L. Biochemistry 1989, 28, 32. (42) Ghosh, S.; Easwaran, K. R. K.; Bhattacharya, S. Tetrahedron Lett. 1996, 37, 5769.

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incorporated DPPC vesicles. Blue bars correspond to bee venom PLA2, and shaded red bars correspond to cobra venom PLA2.

and doped with different quantities of DAGs ranging from 5 to 30 mol % were prepared in a solution of 50 mM Tris 3 HCl and 10 mM Ca2þ at pH 7.4 (Figure 16). Phospholipase A2 activity using 0.5 μg of PLA2 (either cobra venom or bee venom) was used, and the reaction mixtures were kept at 45 °C (above Tm of DPPC) for 5 min. After incubation for the requisite time, the reaction was stopped by the addition of 1.88 mL of 2:1 (v/v) MeOH/CHCl3. The resulting mixture was separated into organic and aqueous phases. An aliquot of the aqueous phase was then removed, and its fluorescence emission at 530 nm (λexcit = 470 nm) was used to estimate the amount of NBD-hexanoic acid produced upon the hydrolysis of PC lipids by PLA2. It is likely that DAG 32 functions to activate PLA2 by causing an increasing fraction of enzyme to be at the membrane surface. There is a variation in the thermotropic phase properties of diacylglycerol analogue-DPPC complexes in which the extent of chain perturbation of the DAG has been modulated by using macrocyclic DAGs with different extents of chain restriction. When the macrocyclic DAGs were doped in DPPC, they destabilize the regular lamellar arrangements of the DPPC membrane in aqueous media and thus the thermal properties of the DPPC bilayer and fluidize the resulting membranes at ambient temperature. Both dicaproyl and its macrocyclic counterpart 34 impart comparable destabilization effects upon incorporation into DPPC vesicles in terms of their thermal transition behavior. This indicates that the effects of restricting chain motions with shorterchain DAG are different from the DAGs with longer chain lengths, which are comparable to that of DPPC.

Aromatic Segments as Part of Lipid Molecules Lipids containing aromatic rings are rare in eukaryotic organisms, although their presence has been demonstrated in marine sponges.44 Lipids formed in some plants contain catechol, resorcinol, or hydroquinone groups alkylated with long hydrocarbon chains. These lipid molecules have both aromatic rings and straight hydrocarbon chains of a length depending on the lipid source. We synthesized the first examples of analogues of diacyl phosphatidylcholine (38-40) that contained aromatic units at various locations along their hydrocarbon chains.45 They were synthesized from sn-glycerophosphocholine obtained from natural lecithin that was extracted from egg yolk and (43) (a) Bhattacharya, S.; Ghosh, S.; Easwaran, K. R. K. J. Org. Chem. 1998, 63, 9232. (b) Ghosh, S.; Swaminathan, C. P.; Surolia, A.; Easwaran, K. R. K; Bhattacharya, S. Langmuir 2000, 16, 9729. (c) Mabrey, S.; Sturtevant, J. M. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3862. (44) Kozubek, A.; Tyman, J. H. P. Chem. Rev. 1999, 99, 1. (45) Bhattacharya, S.; Subramanian, M.; Hiremath, U. Chem. Phys. Lipids 1995, 78, 177.

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Figure 17. Molecular structures of phosphatidylcholine lipids with fatty acid chains bearing aromatic units.

subjected to hydrolysis using 0.1 M Bu4NþOH- in MeOH to afford free sn-glycerophosphocholine [(R)-GPC] using fatty acid anhydrides possessing aromatic segments (Figure 17).46 TEM studies revealed the formation of multiwalled vesicular structures from their aqueous suspensions in HEPES buffer. DSC studies of these aromatic-based phospholipid suspensions did not show the presence of any peak because of the chainmelting transition from 5 to 90 °C. When individual aromaticbased phospholipids were doped into DPPC, the phase transition of DPPC was lowered depending upon the percentage of all three aromatic-based phospholipids incorporated, suggesting that phospholipids containing aromatic units retain a fluidlike melted state at lower temperatures in bilayers. In summary, a general and adaptable method has been developed for the synthesis of novel phospholipid analogues bearing acyl chains with aromatic rings that are unsaturated and oxidatively stable. Such synthetic phospholipids should become valuable tools in membrane receptor research, enzymology, and bioseparation techniques.

Concluding Remarks Herein we have discussed primarily our work on polar lipid design aimed at investigating structure-property relationships in membranes on the molecular level. We have shown how cholesterol interacts with other lipids (both phospholipids and cationic lipids) in membranes. Cholesterol-induced effects in membranes are due to its hydrophobic interactions with other lipids in membranes and are not necessarily caused by the hydrogen bonding interactions between the 3β-OH of cholesterol and the lipid chain-backbone ester linkage or headgroup phosphate region. Cholesterol-based lipids can be developed by the attachment of hydrophilic groups. These form thermally stable organized assemblies of controllable order and thickness upon suspension in water. Cholesteryl amphiphiles behave less like cholesterol in that their incorporation into DPPC not only abolishes the phase transition but also depresses the phasetransition temperature. Cholesteryl amphiphiles manifest their usefulness as efficient gene-transfer agents. The cholesterolbased lipopolymers formed different types of aggregates (e.g., helical tapes, vesicles, etc.) and are much less toxic as compared to commercially available 25 kDa PEI. These formulations also have very high gene-transfer efficiency. We also developed lipid (46) Bhattacharya, S.; Subramanian, M. Tetrahedron Lett. 2002, 43, 4203.

4654 DOI: 10.1021/la9011718

dimers possessing four hydrocarbon chains where each lipid unit is separated by spacer chains made of polymethylene or oligo-oxyethylene segments. These modifications are reflected in their properties upon membrane formation. Analogously ion-paired dimeric lipid vesicles were also developed that could entrap riboflavin, and generally the resistance to OH- permeation decreased with the increase in the spacer polymethylene chain length. This suggests that the membrane packing loosens with the increase in the spacer chain length. Other types of ionpaired lipids were prepared from aromatic-core-based bolaphile/amphiphile ion pairs. The phase-transition temperatures and the permeabilities of these vesicular aggregates could be strongly influenced by the geometry of the bolaamphiphilic counterion. We developed macrocyclic diacylglycerols (DAGs) with a disulfide tether. The effects of restricting chain motion with shorter-chain DAG are different from those for DAGs with longer chains that are comparable to that of DPPC. One such disulfide DAG was able to activate the phospholipase A2 activity in phospholipid vesicles. We also developed phospholipid analogues bearing fully acyl chains with aromatic rings. Although these phospholipids are unsaturated, they have been found to have excellent oxidative stability and long shelf life, making them useful for the reconstitution of membrane proteins and membrane-bound enzymes. To reap the benefits of the functional attributes of a biological cell membrane, we must continue to design multifarious lipid analogues endowed with increasingly sophisticated and specific information for a desired purpose. Events such as exoand endocytosis, cellular adhesion, cell sorting, raft formation, immune recognition, cyto-differentiation, cell disruption, and budding at the molecular level are among the challenges that one needs to understand. Progress in this direction will not improve our understanding of the complex subject, but additional studies will be carried out on hitherto unknown systems for novel applications in medicinal chemistry, immunology, and signal transduction processes. Note Added after ASAP Publication. This article was published ASAP on October 20, 2009. References 26 and 43(a) have been modified. The correct version was published on November 2, 2009. Acknowledgment. J.B. thanks the CSIR, New Delhi, for the award of a research fellowship. Langmuir 2010, 26(7), 4642–4654

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