Cationic Type I Amphiphiles As Modulators of Membrane Curvature

Langmuir 2008, 24, 11743-11751

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Cationic Type I Amphiphiles As Modulators of Membrane Curvature Elastic Stress in Vivo Marcus K. Dymond and George S. Attard* School of Chemistry, UniVersity of Southampton, Highfield, SO17 1BJ. U.K. ReceiVed June 6, 2008. ReVised Manuscript ReceiVed August 14, 2008 Recently we proposed that the antineoplastic properties observed in vivo for alkyl-lysophospholipid and alkylphosphocholine analogues are a direct consequence of the reduction of membrane stored elastic stress induced by these amphiphiles. Here we report similar behavior for a wide range of cationic surfactant analogues. Our systematic structure-activity studies show that the cytotoxic properties of cationic surfactants follow the same pattern of activity we observed previously for alkyl-lysophospholipid analogues, indicating a common mechanism of action that is consistent with the theory that these amphiphiles reduce membrane stored elastic stress. We note that several of the cationic surfactant compounds we have evaluated are also potent antibacterial and antifungal agents. The similarity of structure-activity relationships for cationic surfactants against microorganisms and those we have observed in eukaryotic cell lines leads us to suggest the possibility that the antibacterial and antifungal properties of cationic surfactants may also be due to modulation of membrane stored elastic stress.

Introduction Recently,1 we presented our hypothesis for the mechanism of action of the antineoplastic alkyl-lysophospholipid (ALP) and alkylphosphocholine (APC) analogues. These compounds are typified by the chemotherapeutic products Mitelfosine and Edelfosine (hexadecylphosphocholine (HDPC) and ET-18-OMe, respectively). Currently the consensus is that these compounds achieve their cytotoxicity through inhibition of the translocation enzyme CTP:phosphocholine cytidylylphosphotransferase (CCT),2-4 a key enzyme in the biosynthetic pathway of phosphatidylcholine (PC) phospholipids. We postulated that the biological activity of ALP and APC analogues stems from their ability to reduce the stored membrane curvature elastic stress, which is a lipid bilayer property that plays a role in maintaining the functionality of several membrane associated proteins. In model bilayer systems the activity of CCT correlates with increasing and decreasing stored elastic stress.5 Type I amphiphiles, which induce decreases in membrane stored elastic stress, HDPC and octaethyleneglycol monohexadecyl ether (C16EO8)5 and 1-hexadecyltrimethylammonium bromide (CTAB)6 all reduce the activity of CCT in vitro. That CCT is the key target for these compounds appears to be due to the reliance of most mammalian cells on producing PC lipids exclusively from dietary choline via phosphocholine, which is converted into CDP-choline by CCT.7 Cells with a shortage of PC undergo apoptosis, probably due to their inability to meet the requirement of doubling their lipid mass prior to mitosis.8 Cytotoxicity data in the literature, together with cytotoxicity * To whom correspondence should be addressed. Phone: +4423 8059 3019. Fax: +4423 8059 3781. E-mail: [email protected]. (1) Dymond, M.; Attard, G.; Postle, A. D. J. R. Soc. Interface. 10.1098/rsif. 2008.0041. (2) Geilen, C. C.; Wieder, T.; Orfanos, C. E. AdV. Exp. Med. Biol. 1996, 416, 333–336. (3) Jackowski, S.; Boggs, K. In Choline, Phospholipids, Health, and Disease, [International Congress on Phospholipids], 7th, Brussels, Sept., 1996, Zeisel, S. H.; Szuhaj, B. F.,. EdsACOS Press: Champaign, 1998; pp 30-44. (4) Jimenez-Lopez, J. M.; Carrasco, M. P.; Segovia, J. L.; Marco, C. Eur. J. Biochem. 2002, 269, 4649–4655. (5) Attard, G. S.; Templer, R. H.; Smith, W. S.; Hunt, A. N.; Jackowski, S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9032–9036. (6) Sohal, P. S.; Cornell, R. B. J. Biol. Chem. 1990, 265, 11746–50. (7) Kent, C. Biochim. Biophys. Acta 1997, 1348, 79–90. (8) Jackowski, S. J. Biol. Chem. 1994, 269, 3858–67.

data for compounds we synthesized, are in excellent agreement with our hypothesis.1 In devising tests for our hypothesis we introduced a set of rules-of-thumb, based on the structural characteristics of the amphiphilic molecules, which could be used to predict relative changes in the cytotoxicity of amphiphilic compounds. These rules-of-thumb were elaborated from the likely effects that the molecular structure of an amphiphile would have on the lateral stress profile of a membrane as shown schematically in Figure 1. Integral to this method is the concept of amphiphile typology; by definition type I amphiphiles form ‘normal’ topology phases, type 0 amphiphiles form bilayer phases and type II amphiphiles form ‘inverse’ topology phases. Using this approach we showed that our theory that amphiphile cytotoxicity is mediated through a reduction in membrane curvature elastic stress is consistent with structure-activity characteristics of classic type I amphiphiles, like CTAB and C16EO8, as well as ALP and APC analogues. In this paper we report detailed and systematic structure-cytotoxicity relationships of cationic type I amphiphiles in HL-60 cells. Previously our studies of these cationic amphiphiles were limited to structural changes in the hydrocarbon chain region; here we describe how changes in both headgroup and hydrocarbon chains affect cytotoxicity. All the cationic amphiphilic compounds were synthesized in our laboratories and their aggregation properties, as exemplified by the critical micelle concentration (CMC) and lyotropic liquid crystal behavior, were characterized. As we noted previously, measurement of both the CMC and lyotropic liquid crystal phase behavior is necessary to infer the extent to which lowering of stored curvature elastic energy contributes to cytotoxicity. This is because at concentrations that are near or above the CMC, the cytotoxicity of many amphiphiles is largely due to cell lysis and reflects the detergent action of the compounds. Studies of the lyotropic liquid crystal phase behavior of amphiphiles affords an empirical way of determining amphiphile typology, and this is key to determining an amphiphilic compound’s ED50 through its effect on stored membrane elastic stress.

10.1021/la8017612 CCC: $40.75  2008 American Chemical Society Published on Web 09/17/2008

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The local lipid environment is modeled as the fraction of molecules that have a positive spontaneous curvature, c0(λ). Increasing c0(λ) decreases E(λ), since c1 is negative; kB is the Boltzman constant and T is the temperature. The quantity E(λ) is analogous to the ED50 that is measured in experiments on cytotoxicity. Furthermore, in eq 2, the composition of the amphiphiles that surround the protein binding domain is dependent on the partition constants of antineoplastic amphiphiles between the membrane and extracellular solution. Under conditions of cell culture, antineoplastic amphiphiles that remain in the extracellular medium are likely to interact with the complex mix of proteins and fatty acids in the growth medium to produce a pool of complexed lipids. However, even under these conditions, the partitioning of amphiphiles into cell membranes will still be driven by the hydrophobic effect and stored elastic energy, although the composition of growth medium will influence the actual value of the partition coefficient. To a first approximation, we would expect n(λ) to be a linear function of λ for amphiphiles that contain a single hydrophobic chain. The functional dependence of c0(λ) on λ is not known, but phenomenologically it is reasonable to assume a power law dependence, λγ, in which the exponent γ < 1. Thus, eq 2 assumes the general form:

E(λ) ) E0exp{ζ(A + Bλ)(C + Dλγ)}

(3)

where the constants ζ g 0, A g 0, B < 0, C > 0, and D > 0 depend on the chemical structure of the amphiphile. The term Dλγ is proportional to the torque tension: Figure 1. Lateral stress profile of an amphiphile in a monolayer. The sum of the areas R, β and γ must be zero for a monolayer to form. If area R < γ then the amphiphile will be type I, γ < R then the amphiphile will be type II and if γ ) R then the amphiphile will be type 0. The rules-of-thumb we proposed for predicting amphiphile toxicity stem from combining γ and R. Once in the membrane, compounds characterized by large γ and small R will be most active; structural changes leading to smaller γ or larger R will decrease activity. The concentration of a compound in the membrane is dictated by the molecular hydrophobicity. Structural changes that change the relative magnitude of γ or R may also change the compounds solubility and its ED50.

Our hypothesis is that the extent of the type I character of an amphiphile is directly related to the inhibitory potency of the amphiphile. The type I character is quantified by the torque tension, τ:

τ ) κc0(λ)

(1)

where κ is the membrane bending rigidity and c0(λ) is the spontaneous curvature of the amphiphile. For a type I amphiphile c0 is positive. Equation 1 enables us to model the extent of cell survival, E(λ), in terms of changes in curvature elastic stress, such that type I amphiphiles lead to cell death because they inactivate some key protein (e.g., CCT) whose activity increases as a function of stored elastic stress.9

{

1 n(λ)κA 2c1c0(λ) - c21 2 E(λ) ) E0exp kBT

(

)

}

(2)

where A is the cross-sectional area per molecule, c1 is the principal curvature at the interface (with the convention that an interface with negative curvature curves toward water), n(λ) is the number of moleculesscomprising both lipids and type I amphiphiless surrounding the binding domain of the target protein (e.g., CCT), λ denotes the length of the hydrophobic chain and E0 is a constant. (9) Helfrich, W. Z. Naturforsch., C 1973, 28, 693–703.

τ ∝ Dλγ

(4)

The first part of eq 3 accounts for the observation that amphiphiles with longer hydrocarbon chains partition more readily into membranes than amphiphiles with shorter chains. While this should result in enhanced cytotoxicity, the second part of eq 3 highlights the fact that as λ increases, so the spontaneous curvature of the membrane will become less positive, thereby activating the target protein and counteracting cytotoxicity. Conversely, for amphiphiles with short chains, eq 3 predicts that their lower partitioning will be counteracted by their higher type I characteristics (larger τ values) which drive the spontaneous curvature to become more positive. The possibility that stored curvature elastic energy is a target for a class of compounds, typified by type I amphiphiles, is evidenced by the increasing number of enzymes that appear to be modulated by curvature elastic stress in vivo, many of which are involved in lipid biosynthesis. Examples include diglucosyl diacylglycerol synthase that synthesizes the major bilayer lipid diglucosyldiacylglycerol in Acholeplasma laidlawaii,10,11 phospholipase A212 and phospholipase C,13 CCT, glycerol-3-phosphate O-acyltransferase14 and diacylglycerol kinase.15 Simulations of lipid biosynthetic networks have shown that the lipid dependence of certain mammalian enzymes leads to a novel integrative feedback mechanism that allows homeostatic control of membrane stored curvature elastic energy.16 Enzymes that are involved in lipid biosynthesis and are regulated by membrane lipid composi(10) Dahlqvist, A.; Nordstroem, S.; Karlsson, O. P.; Mannock, D. A.; McElhaney, R. N.; Wieslander, A. Biochemistry 1995, 34, 13381–9. (11) Karlsson, O. P.; Dahlqvist, A.; Wieslander, A. J. Biol. Chem. 1994, 269, 23484–90. (12) Zidovetzki, R.; Laptalo, L.; Crawford, J. Biochemistry 1992, 31, 7683– 91. (13) Rao, N. M.; Sundaram, C. S. Biochemistry 1993, 32, 8547–52. (14) Green, P. R.; Bell, R. M. J. Biol. Chem. 1984, 259, 14688–94. (15) Thomas, W. E.; Glomset, J. A. Biochemistry 1999, 38, 3310–3319. (16) Beard, J.; Attard, G. S.; Cheetham, M. J. J. R. Soc. Interface 2008, 5, 533–543.

Cationic Type I Amphiphiles

tion are also found in prokaryotic cells and fungi, and there is evidence that analogous integrative feedback lipid biosynthesis control mechanisms also exist in these organisms. It is also increasingly apparent that a large number of enzymes that are not directly involved in lipid synthesis may be regulated (or folded) by membrane stored elastic stress; these include both extrinsic and intrinsic membrane proteins as reviewed.17,18 Representative examples include: the transmembrane protein bacteriorhodopsin,19-21 the γ-Aminobutyric acid (GABA) receptor GABAA22 and OmpA.18 Hence it is plausible that other organisms will be susceptible to compounds that decrease stored elastic energy. The particular biological targets most sensitive to curvature elastic stress reduction in nonmammalian systems are a matter of speculation; however the most probable targets are enzymes involved in lipid homeostasis. It is clear that CCT cannot be a universal target because, unlike many mammalian cell lines, most microorganisms do not rely on exclusively on CCT for PC biosynthesis.

Materials and Methods Materials. CTAB and HDPC were purchased from Fluka and Alexa Biochemicals respectively. Hanks Balanced Salt Solution (HBSS), HBSS (without phenol red), RPMI Medium (with Glutamax-1 and HEPES (25 mM)), fetal calf serum and antibioticantimiotic solution (10.000 units/mL penicillin G sodium, 10.000 µg/mL streptomycin sulfate, 25 µg/mL amphotericin B as Fungizone in 0.85% saline) were purchased from Invitrogen. CellTiter 96 AQueous One Solution Cell Proliferation Assay reagent was purchased from Promega. TLC of synthesized compounds was performed using reverse phase DC-Alufolien RP-18F254s (Merck) or normal phase Alugram SIL G/UV 254 (Machery-Nagel) soaked in a 6% solution of sodium bromide (Fisher Scientific) in methanol (Fisher Scientific) using the method of Bluhm et al.23 Synthesis of Type I Amphiphiles. The quaternary ammonium amphiphiles synthesized in this study were prepared using several methods reported in the literature and a method using acetonitrile as solvent developed from these. Piperidinium amphiphiles 13 and 14 were prepared by the methods of Hauser24 and Landquist.25 Quinuclidinium amphiphiles 2-5 and compound 6 were synthesized by the method of Imamura.26 Bola-amphiphiles 20 and 21 were synthesized by adapting the method of Bhattacharya.27 The remaining compounds 7-12 and 15-19 were prepared using the following method: equimolar quantities of n-alkyl halide and tertiary amine were reacted by dropwise addition of the alkyl halide to a refluxing solution of the tertiary amine in acetonitrile. Reflux was continued under dry nitrogen for approximately 16 h. Excess solvent was removed by rotary evaporation and the crude product was crystallized four times from ethanol/ethyl acetate. The product was dried in vacuo and lyophilized through four freeze-thaw cycles. Compound purity was assessed by TLC using reverse phase TLC and normal phase plates soaked in 6% w/v sodium bromide,23 melting point analysis, NMR spectroscopy, mass spectrometry and mi(17) Ces, O.; Mulet, X. Signal Transduction 2006, 6, 112–132. (18) Marsh, D. Biophys. J. 2007, 93, 3884–3899. (19) Allen, S. J.; Curran, A. R.; Templer, R. H.; Meijberg, W.; Booth, P. J. J. Mol. Biol. 2004, 342, 1293–1304. (20) Curran, A. R.; Templer, R. H.; Booth, P. J. Biochemistry 1999, 38, 9328– 9336. (21) Booth, P. J.; Riley, M. L.; Flitsch, S. L.; Templer, R. H.; Farooq, A.; Curran, A. R.; Chadborn, N.; Wright, P. Biochemistry 1997, 36, 197–203. (22) Sogaard, R.; Werge, T. M.; Bertelsen, C.; Lundbye, C.; Madsen, K. L.; Nielsen, C. H.; Lundbk, J. A. Biochemistry 2006, 45, 13118–13129. (23) Bluhm, L. H.; Li, T. Tetrahedron Lett. 1998, 39, 3623–3626. (24) Hauser, E. A.; Niles, G. E. J. Phys. Chem. 1941, 45, 954–9. (25) Landquist, J. K. J. Chem. Soc., Perkin Trans. 1 1976, 454–6. (26) Imamura, K.; Nogami, T.; Shirota, Y. Bull. Chem. Soc. Jpn. 1987, 60, 3499–504. (27) Bhattacharya, S.; De, S. J. Chem. Soc., Chem. Commun. 1995, 651–2. (28) Dymond, M. K. PhD, UniVersity of Southampton, Southampton, UK, 2001.

Langmuir, Vol. 24, No. 20, 2008 11745 croanalysis, in the case of novel compounds. All measurements were indicative of product purity greater than 98%; full synthetic details are available.28 Determination of CMC and Lyotropic Liquid Crystal Mesophases. The CMC of amphiphiles was determined from surface tension measurements using a CSC-Du Nouy Precision tensiometer 70535 (CSC Scientific Company, Inc.) fitted with a custom-made glass heating-jacket. The surface tension of different solutions of the amphiphiles in pure water were measured and the CMC determined as previously reported.1 Lyotropic liquid crystal phase diagrams were prepared from known compositions of amphiphile and pure water according to the method already described.1 Cell Culture. HL-60 cells were cultured at 37 °C in 5% CO2 in RPMI medium with Glutamax-1 and HEPES (25 mM) supplemented with 10% fetal calf serum and 5% antibiotic-antimiotic solution (10.000 units/mL penicillin G sodium, 10.000 µg/mL streptomycin sulfate, 25 µg/mL amphotericin B as Fungizone in 0.85% saline). Counts of viable cells were performed using a hemocytometer; aliquots of cells were mixed with trypan blue solution, and cells that stained blue were assumed to be nonviable and omitted from the cell count. Cells were seeded at 2 × 104 viable cells in 50 µL of medium per well in a 96 (8 × 12) well assay plate. Compounds were dissolved in pure water and sterilized by passing through a 2 µm filter, 50 µL of compound solutions at 10 concentrations was added to separate wells in the assay plate, replicating each 4 times. Cells were incubated for 72 h before CellTiter 96 AQueous One Solution Cell Proliferation Assay reagent (20 µL) was added. Cells were incubated for a further 3 h before the absorbance of each well was measured using a Titertek Multiscan R plus 96 well plate reader at 492nm. The absorbance readings for each of the wells were converted to percentage survival with respect to the absorbance of control cells, which were grown under identical culture conditions but without the active compounds. Mean survival values for each concentration of a compound were plotted on a graph and the concentration at which 50% of the cells had been killed (the ED50) was determined from these graphs (available as Supporting Information). Each group of compounds assayed included HDPC to enable normalization of respective ED50 values to HDPC, using eq 5. cpd

EDnorm 50 )

1 ED50 × n EDref

(5)

50

where EDcpd 50 is the ED50 concentration of the compound under study, n is the number of seed cells and EDref 50 is the ED50 of HDPC, which is measured in the same set of experiments as the compound under study.

Results The cytotoxicity, lyotropic liquid crystal properties and CMCs were determined for a representative set of the amphiphiles synthesized. Cytotoxicity Studies of Type I Amphiphiles. Table 1 shows the ED50 values determined for each of the compounds investigated; in each case HDPC was assayed as a reference compound. The compounds studied may be grouped into several headgroup types: the dimethylammonium derivatives (compounds 1 and 15-21, structures in Figure 2), the quinuclidinium derivatives (compounds 2-5), the piperidinium derivatives (compounds 6-12) and the pyridinium derivatives (compounds 13 and 14). Figure 2 shows the structure-activity plots for homologues in each of these groups. Characterization of Lyotropic Liquid Crystal Properties. Figure 3 shows phase diagrams for a representative selection of the cationic amphiphiles prepared in pure water; these provide a reliable guide to the typology of a given amphiphile. This is because the complex mix of proteins, fatty acids and salts that

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Table 1. ED50 Values for Cationic Type I Amphiphiles

compound

EDcpd 50 (µM)

EDref 50 HDPC (µM)

EDnorm 50 (per seed cell) × 10-6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1.58 ( 0.10 0.83 ( 0.05 1.12 ( 0.15 1.99 ( 0.15 7.10 ( 0.08 1.99 ( 0.05 2.43 ( 0.16 7.05 ( 0.30 11.2 ( 0.10 1.58 ( 0.11 1.78 ( 0.12 2.14 ( 0.10 1.99 ( 0.10 1.20 ( 0.25 1.68 ( 0.10 0.60 ( 0.10 1.86 ( 0.28 3.15 ( 0.10 7.85 ( 0.25 9.80 ( 0.30 7.05 ( 0.20

22.4 ( 1.50 19.9 ( 1.50 19.9 ( 1.50 19.9 ( 1.50 19.9 ( 1.50 26.9 ( 1.20 26.9 ( 1.20 26.9 ( 1.20 26.9 ( 1.20 26.9 ( 1.20 26.9 ( 1.20 26.9 ( 1.20 19.9 ( 1.00 19.9 ( 1.00 19.0 ( 0.60 19.0 ( 0.60 19.0 ( 0.60 24.0 ( 1.65 24.0 ( 1.65 19.9 ( 0.60 19.9 ( 0.60

3.59 ( 0.47 2.37 ( 0.32 3.23 ( 0.66 5.72 ( 0.86 2.05 ( 0.38 3.12 ( 0.22 3.81 ( 0.42 11.0 ( 0.96 17.5 ( 0.94 2.48 ( 0.28 2.78 ( 0.31 3.34 ( 0.30 5.68 ( 0.57 3.45 ( 0.88 5.28 ( 0.48 1.88 ( 0.37 5.86 ( 1.06 5.55 ( 0.55 13.8 ( 1.37 29.3 ( 1.78 21.1 ( 1.23

is present in cell culture media generally only affects the composition and temperature ranges over which particular liquid crystalline phases are present, rather than the nature of the phase. Possible exceptions are systems that contain negatively charged lipids. We note that when making contact with the rules-ofthumb we use here, it is the relative behavior of different compounds that is considered. In order to confirm that for our systems the phase topology is not affected by the culture medium we carried out representative contact preparations of the amphiphiles using culture medium as the contacting phase (results not shown). N-Alkylquinuclidinium Series. 1-Hexadecylquinuclidinium bromide (compound 2, Figure 2) is clearly a type I amphiphile, as evidenced by the normal topology hexagonal phase that dominates its phase behavior. Homologues having shorter chain length (λ) (14 and 12 carbon units, compounds 3 and 4) exhibit a phase behavior that confirms the type I properties of this series. Since the area R in Figure 1 decreases as λ decreases while the area γ remains effectively unchanged, it can be concluded that the homologues with the shorter alkyl chains are more strongly type I than the longer chain homologues (i.e., they are characterized by a larger spontaneous curvature, c0). Also as the chain length decreases we would expect a decrease in hydrophobicity, and this is observed, in optical microscopy contact studies, as a decrease in Krafft temperature with λ, (results not shown). Comparison of compound 5 to compound 2 shows the effect of substituting a hydrogen on the quinuclidinium headgroup by a hydroxyl group. The overall phase behavior of 5 is broadly the same as that of 2, indicating that 5 is also a type I amphiphile. However the lower Krafft point of 5 reflects its lower hydrophobicity. The broader hexagonal phase in 5 indicates that this compound is more strongly type I than compound 2, presumably because its bulkier, more polar headgroup increases the area γ in the curvature elastic energy profile. Interheadgroup hydrogen bonding involving the hydroxyl moiety, which would act to reduce γ, appears to have a negligible effect on the aggregation behavior of the amphiphile. N-Methylpiperidinium Series. The phase diagram of compound 6, 1-hexadecylmethylpiperdinium bromide, is dominated by a normal topology hexagonal phase, indicating that this is a type I amphiphile. Its shorter chain homologues 7-9 (compounds with chain lengths of 14, 12 and 10 carbon units) are also type I compounds, with the shortest chain homologue (compound 9) having the strongest type I characteristics.

Bola-Amphiphile Series. The importance of phase studies in assigning the typology of amphiphiles is illustrated by the bolaamphiphiles 20 and 21. Although these compounds may be thought of as analogues of compound 1 (CTAB), which is a type I amphiphile,29 the presence of the alkyl chain linking the two molecules makes it impossible to predict the typology of 20 and 21. However, the phase diagram of 21 shows it to be unambiguously a type I amphiphile. This means that compound 20 is also type I although the shorter linking chain of will make it slightly less hydrophobic than 20, and possibly slightly less type I (due to a reduction in the steric component of γ). N-Alkylpyridinium Series. The phase behavior of the pyridinium compound 13 shows it to be a type I amphiphile. Similarly, contact preparation studies of compound 14 confirm that this too is a type I amphiphile (data not shown). The presence of the nitrile group is predicted to increase headgroup-headgroup repulsion either due to increased hydration or to unfavorable dipole-dipole interactions, thereby increasing the magnitude of γ. Consequently it is expected that compound 14 will be slightly more type I than compound 13. N,N-Dialkylammonium Series. For compounds that have two alkyl chains, it is commonly the case that as the second alkyl chain is lengthened (keeping the first identical) the amphiphile initially becomes more strongly type I, since very short alkyl chains tend to increase headgroup repulsion due to steric effects. However, once the chain becomes sufficiently long to have conformations that extend into the hydrophobic region, then the chain will increase the magnitude of R and hence decrease the type I characteristics of the amphiphile. Thus for compounds 6 and 10-12 there will be a point where the headgroup size is at a maximum, corresponding to the amphiphile with the strongest type I behavior. Likewise for compounds 15-19, it is predicted that the second alkyl chain will have a maximal effect on headgroup cross-section and enhance the type I characteristics of the amphiphile when it is short enough to contribute to steric interactions in the headgroup region. This prediction is supported by the phase behavior of amphiphiles 15 and 19. Compound 15 is clearly a type I amphiphile as evidenced by the temperature/ composition extent of its normal topology hexagonal phase. However, the presence of a significant bicontinuous cubic phase is noted, which is an indicator that the homologues in this series of double chain compounds become less type I, and progressively type 0 as the length of the second chain is increased. Thus the phase behavior of compound 19, which has the longest secondary alkyl chain in this series, is dominated by a lamellar phase.30 Consequently this compound is a type 0 amphiphile. CMC and Ratios of ED50/CMC. Table 2 shows the CMC concentrations and the ED50/CMC ratios for the amphiphiles we have studied. The CMC of single chain amphiphiles can be estimated using eq 6.

log10 CMC ) A + Bλ

(6)

where λ is the alkyl chain length (number of carbon atoms) and A > 0 and B < 0 are constants that depend on the chemical structure of the headgroup. The reliability of this method for CMC prediction allows us to use the CMC values for the homologues in a series that have the longest and shortest alkyl chains to estimate the CMC of homologues with intermediate chain lengths. For example, in the quinuclidinium headgroup series the CMC of compound 3, in which λ ) 14, is estimated by using the experimentally determined values for compounds 2 and 4, which have λ ) 12 and 16, respectively. Likewise for the methylpiperidinium (29) Hertgel, G.; Hoffmann, H. Prog. Colloid Polym. Sci. 1988, 76, 123–31.

Cationic Type I Amphiphiles

Langmuir, Vol. 24, No. 20, 2008 11747

Figure 2. Structure-activity relationships in cationic type I amphiphiles, normalized ED50 are quoted. Arrows and annotations summarize the structure-activity comparisons made in the discussion.

headgroup series, the magnitude of the CMC of compounds 7 (λ ) 14), and 8 (λ ) 12) were estimated from the experimental CMC of compound 6 (λ ) 16) and compound 9 (λ ) 10). The relationship between CMC and alkyl chain length in the case of amphiphiles with two alkyl chains is of the form shown in eq 6, but with λ representing the total number of carbon atoms in the two chains. However, since apposed segments of the alkyl chains are effectively inaccessible to water, the slope, B, of the CMC vs λ curve is typically 0.6 times31 that for amphiphiles with single chains. On the basis of this relationship we estimated the CMC of intermediate chain lengths from curves fitted to experimentally derived CMC values for double chain surfactants with the same headgroup. CMC values for the other compounds were taken from the literature as referenced in Table 2 except for compounds 5, 14 and 21, which were estimated on the basis of the CMC values and Krafft temperatures for structurally similar amphiphiles. For example, compound 5 has a lower Krafft

temperature than 4, so it would be expected that the CMC of 5 would be slightly higher than for 4, reflecting the higher solubility of the quinuclidinol headgroup. A similar argument holds when comparing compounds 14 and 13. In the case of 21, its CMC can be estimated from that of 20 by taking into account the maximum possible decrease in the CMC as a consequence of adding two methylene groups. Hence the CMC of 21 is estimated to be 2.5 times lower than that of 20. In our work we use the ratio of ED50/CMC as a metric for the extent to which cell lysis/detergency is responsible for cell death. Specifically when the ED50 value approaches the CMC, detergent effects are likely to dominate the cytotoxicity of the amphiphile. Conversely, when the ratio of ED50/CMC is much less than one, we expect that cell lysis will not contribute significantly to the cytotoxic effects of the amphiphile. Table 2 shows the ratio of ED50/CMC for the amphiphiles we studied. From these data we can conclude that

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Figure 3. Lyotropic liquid crystal phase diagrams of type I cationic amphiphiles, where LI, HI, VI, LR, LII, S + W are micellar solution, hexagonal phase, cubic phase, lamellar phase, inverse micellar solution, and solid plus water, respectively.

out of the twenty one compounds investigated only three, compounds 18, 19 and 21, could be acting through a lytic mechanism since their ED50/CMC ratios are 0.47 and 6.6 and 0.12 respectively. However since both compound 18 and 19 are type 0 amphiphiles30 it is unlikely that they will induce significant cell lysis. In the case of compound 21 there is a possibility that cell death is due to lysis although it should be noted that the estimate of CMC as 2.5 times less than compound 20 is probably an over estimate because the extra carbon atoms do not form part of the hydrocarbon chain but rather the headgroup.

Discussion Structure-Activity Relationships in Type I Amphiphiles. Having established the typology of the amphiphiles we synthesized, together with the fact that most, if not all, compounds (30) Haas, S.; Hoffmann, H.; Thunig, C.; Hoinkis, E. Colloid Polym. Sci. 1999, 277, 856–867. (31) Kopecky, F. F., T; Kopecka, B; Kaclik, P. Acta Fac. Pharm. UniV. Comenianae 2007, LIV, 84–94.

are all acting nonlytically, i.e., at concentrations well below the CMC, we can proceed to test the predictions of our hypothesis against the experimental data. Equation 3 highlights that the fundamental relationship between ED50 and chemical structure of an amphiphile is dependent on two properties: the molecular hydrophobicity (which determines the quantity of active drug in the membrane) and the type I character, as embodied by the torque tension τ (which determines the effect of the molecule, once in the membrane, on stored elastic stress). The predictions of eq 3 as rules-of-thumb are summarized as follows; a structural change that increases hydrophobicity, evidenced by changes in CMC and the lyotropic liquid crystal phases, will increase the amount of drug within the cells. Increases in R, the hydrocarbon chain region will decrease the type I character of an amphiphile, thereby increasing the torque tension and increasing the ED50. Conversely, increases in γ, the headgroup region will increase the type I character of an amphiphile and decrease the torque tension together with the ED50 resulting in greater drug potency. Therefore by considering the structural

Cationic Type I Amphiphiles

Langmuir, Vol. 24, No. 20, 2008 11749

Table 2. CMC Values and ED50/CMC Ratios for Cationic Type I Amphiphiles

a

compound

CMC (µM)

ED50/CMC ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1000a 1033 ( 31 3582a 19246 ( 577 >1033a 1271 ( 38 3574a 10107a 283421 ( 850 27100a 2590a 247 ( 7 795 ( 24 >795a 799 ( 24 159a 33.4 ( 6 5.6a 130 161 ( 5 64a

1.6 × 10-3 9.1 × 10-4 3.6 × 10-4 1.2 × 10-4