J. Phys. Chem. B 2001, 105, 10257-10265
10257
Thermal Lipid Order-Disorder Transitions in Mixtures of Cationic Cholesteryl Lipid Analogues and Dipalmitoyl Phosphatidylcholine Membranes Yamuna Krishnan Ghosh, Shantinath S. Indi, and Santanu Bhattacharya* Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India ReceiVed: October 25, 2000; In Final Form: April 30, 2001
Two types of cationic cholesteryl amphiphiles, one where the headgroup is attached to the steroid by an ester linkage and the second by an ether linkage, were synthesized. A third type of cholesteryl lipid bearing an oligoethylene glycol segment was also prepared. Each of these synthetic lipids generated vesicle-like aggregates with closed inner aqueous compartments from their aqueous suspensions. We examined their interaction with L-R-dipalmitoyl phosphatidylcholine (DPPC) membranes using fluorescence anisotropy, transmission electron microscopy (TEM), and differential scanning calorimetry (DSC). When included in membranes, the synthetic cholesteryl lipids were found to quench the chain motion of the acyl chains of DPPC. This suggests that these cationic cholesteryl derivatives act as filler molecules despite modification at the headgroup level from the molecular structure of natural cholesterol. Careful analyses of DSC and fluorescence anisotropy data suggest that the nature of perturbation induced by each of these cationic cholesterol derivatives is dependent on the details of their molecular structure and provides significant information on the nature of interaction of these derivatives with phospholipid molecules. In general, amphiphiles that support structured water at the interfacial region tend to rigidify the fluid phase more than others. Importantly, these cholesteryl amphiphiles behave less like cholesterol in that their incorporation in DPPC not only abolishes the phase transition but also depresses the phase transition temperature.
1. Introduction Synthetic cationic cholesterol derivatives are currently the focus of attention of many workers. This is because several of such compounds are being employed for diverse purposes such as gene therapy,1-4 enzyme inhibition,5 or in medicinal applications.6 Cholesterol derivatives such as 3β[N-(N′,N′-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol), which has been used as a potent gene delivery agent is generally employed as a complex with dioleoyl phosphatidylethanolamine (DOPE).3 Several other steroid-based cationic lipid formulations are also being used as transfection agents.7-11 It has been shown that despite chemical modification of parent cholesterol structure, cholesterol derivatives continue to interact strongly with natural lipids in membranes and affect the permeability and in vivo tissue distribution properties.12,13 However, it is not known how such positively charged cholesterol derivatives alter phospholipid-based membrane organizations although it is obvious that they modify the membrane surface charge characteristics. Examination of the physical nature of the interactions of cationic cholesteryl lipid with natural phospholipid components in mixed membranes of defined composition may be relevant to understanding the mechanism by which these types of molecules manifest their biological activities. Although the interaction between natural cholesterol and phosphatidylcholine lipids in membranes has been the subject of extensive research,14-25 there has been no attempt to examine the effect of cationic cholesterol lipids on the physical chemical properties of dipalmitoyl phosphatidylcholine (DPPC) membranes. * Corresponding author and Swarnajayanti Fellow. Fax: +91-80-3600529, +91-80-360-0083. E-mail: sb@ orgchem.iisc.ernet.in. Also at the Chemical Biology Unit of JNCASR, Bangalore 560 012, India.
Earlier investigations from this laboratory attempted to elucidate mechanisms by which hydrocarbon-chain-based cationic lipids complex with nucleic acids and the factors that govern the release of the DNA from such complexes.26,27 In view of the emergence of cholesterol-based cationic lipids as efficient gene transfer agents, we have recently focused on developing cationic cholesteryl lipids. Indeed a few of these cationic cholesterol derivatives showed excellent transfection abilities across eukaryotic cells.28 In this paper we present the results of an investigation on the interaction of a number of different cationic cholesteryl amphiphiles (Figure 1) with membranes of a natural phospholipid, dipalmitoyl phosphatidylcholine (DPPC). In particular, we examined the physical changes in membranes comprising mixtures of these cationic amphiphiles and DPPC by using electron microscopy (TEM), differential scanning calorimetry (DSC), and fluorescence anisotropy using the probe 1,6-diphenylhexatriene (DPH). These results show that each of the cationic amphiphiles forms membranous aggregates in water. Although the nature of the interaction with DPPC of a few derivatives was generally similar to that of cholesterol, significant differences were observed with other derivatives. Possible reasons for such differential effects conferred by some of the cholesteryl amphiphiles are presented. 2. Experimental Section 2.1. Materials. All the reagents and chemicals used in this study were of the ACS grade. The amphiphiles (shown in Figure 1) used in the present study have been synthesized according to a procedure described elsewhere,29 and the IR, 1H NMR, mass spectral, and elemental analyses data for the newly synthesized amphiphiles were consistent with their given structures. Dipalmitoyl phosphatidylcholine (DPPC) was purchased from
10.1021/jp003940e CCC: $20.00 © 2001 American Chemical Society Published on Web 10/03/2001
10258 J. Phys. Chem. B, Vol. 105, No. 42, 2001
Ghosh et al.
Figure 1. The molecular structures of cationic cholesteryl lipids 1a-c and 2a-c and neutral cholesteryl derivative, 3.
Avanti Polar lipids (Birmingham, AL) and 1,6-diphenyl-hexa1,3,5-triene (DPH) was obtained from Sigma (St. Louis, MO). Water was distilled and deionized in a Milli-Q apparatus from Millipore. 2.2. Vesicle Preparation. Separate solutions of DPPC and DPPC containing the desired amount of a given cationic cholesteryl amphiphile in CHCl3 were evaporated to dryness in Wheaton vials under a stream of dry nitrogen gas and then under high vacuum to prepare thin films of lipid containing cholesteryl lipid on the walls of the vials. Each lipid film sample was dispersed in water (Millipore), and the resulting solution was left for hydration for ca. 4 h at 4 °C. The suspension was thawed to 65 °C for 15 min, vortexed for 5 min, and then frozen to 0 °C for 20 min. Each sample was subjected to 5 freezethaw cycles to ensure optimal hydration. 2.3. Transmission Electron Microscopy. Vesicles were made by adding a known cholesteryl amphiphile from stock solutions in CHCl3, evaporating the organic solvent to form a film and samples were prepared as mentioned above in water (Millipore) at pH ∼ 6.8. The concentration of the amphiphile or amphiphile/DPPC was maintained at 5 × 10-5 M for all samples. A 15 µL sample of the vesicular solution was loaded onto Formvar-coated, 400 mesh copper grids and allowed to remain for 1 min. Excess fluid was wicked off the grids by touching their edges to filter paper, and 15 µL of 0.5% uranyl acetate was applied on the same grid for a after which the excess stain was similarly wicked off. The grid was air-dried for 10 min, and the specimens were observed under a bright-field TEM (JEOL 100 CX II) operating at an acceleration voltage of 80 kV. 2.4. Fluorescence Anisotropy Measurements. The samples for fluorescence measurements were prepared by mixing 1.5 mg of DPPC, the appropriate amounts of cationic cholesteryl amphiphile, and DPH to give a 250:1 DPPC/DPH molar ratio, all of them in CHCl3 solution. After evaporation to form a thin film, 4 mL of water was added and multilamellar vesicles were formed by freeze-thaw treatment as described earlier. The fluorescence emission spectrum was recorded by excitation of membrane-doped DPH at 360 nm. The maximum peak-height of the emission was recorded at 430 nm. The slit widths of both the excitation and emission window were kept at 5 nm. The fluorescence intensity of the emitted light polarized parallel (I|) and perpendicular (I⊥) to the excited light was recorded with temperature. There was no polarization due to light scattering since the dilution of DPH-doped vesicles had no effect on the fluorescence anisotropy. The fluorescence anisotropy values (r) at various temperatures with different lipid coaggregates were measured by employing Perrin’s equation, r ) (I| - G ‚I⊥)/(I| + 2‚G ‚I⊥), where G represents the instrumental correction factor. The apparent gel-to-liquid crystalline phase transition temperatures were calculated from the midpoints of the breaks related to the temperature-dependent r-values. The temperature range for phase transition was calculated from two temperature points, which marked the beginning and the end of the phase transition process.
2.5. Differential Scanning Calorimetry. The lipid mixtures (1.5 mg of DPPC and appropriate amounts of cholesteryl amphiphiles) for DSC were prepared as solutions in CHCl3. Then the solvent from each solution was evaporated first under a stream of N2. The last traces of CHCl3 were eliminated by evaporation under high vacuum. After the addition of 2.0 mL of water (Millipore), multilamellar vesicles were formed upon vortexing in a benchtop vibrator for 5 min keeping the samples at ∼65 °C for 15 min. Vortexing was continued until a homogeneous and uniform suspension was obtained. This was frozen to 0 °C for 20 min and then again thawed. After ca. five freeze-thaw cycles, 0.5 mL of each of these suspensions was loaded inside cells of a multicell differential scanning calorimeter, Calorimetry Sciences Corp. (CSC, Provo, UT) with a reference cell containing water (Millipore). The heating rate was maintained at 0.5 K/min. 3. Results 3.1. Transmission Electron Microscopy (TEM). 3.1.1. Cholesteryl Lipid Aggregates. Direct comparison of the molecular structures between 1a and 2a show that they differ in terms of the mode of linkage (ester vs ether) which attaches the NMe3+ residue to the 3β-OH of cholesterol. Compound 1b is similar to 1a except that an oligoethylene glycol unit is incorporated to the cationic NMe2+ center making the headgroup in 1b more susceptible to hydration in water. To see whether the introduction of an unsaturated fatty acid residue such as cis-9-octadecenyl (oleyl) chain30 has any effect on its properties, 1c was synthesized. Compounds 2a-c are related in that they are all cationic and the NMe3+ unit is connected either via a short -(CH2)2- spacer (2a), or through a long, hydrophobic -(CH2)11- spacer (2c), or via a long oligooxyethylene spacer (2b) linked to the 3β-OH of the cholesterol moiety. In all of these an ether linkage is maintained. As a control, a neutral cholesteryl derivative, 3, with a nonionic, wettable headgroup -(OCH2CH2)4-OH was also synthesized. All the cholesteryl amphiphiles shown in Figure 1 formed stable suspensions in aqueous media. Transmission electron microscopy revealed the formation of closed aggregates. Typical diameters of the aggregates spanned from 135 to 375 nm in diameter. Representative micrographs for 1a, 1c, and 2c are shown in Figure 2C, F, and I. While most of the aggregates showed vesicle-like organizations, giant organizations or tubular structures were also seen (see for example, Figure 2I). Each of the aggregates from individual cholesteryl lipids also entrapped a water-soluble dye, methylene blue, with entrapment capacities that ranged from 0.5 to 4% (not shown). Taken together these results confirm the existence of closed, inner aqueous compartments in these aggregates. 3.1.2. Cationic Cholesteryl Amphiphile-DPPC Coaggregates. Blends of cholesterol amphiphiles 1a, 1b, 1c, and 2c doped at 10 and 30 mol-% each in DPPC (at 0.05 mM total lipid concentrations) were investigated by TEM. Aggregates of DPPC alone prepared as described showed the presence of long, open lamellar type of structures (not shown). The DPPC
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J. Phys. Chem. B, Vol. 105, No. 42, 2001 10259
Figure 2. Comparison of negatively stained TEM images of different aggregates. (A, B) 10, 30 mol-% of 1a in DPPC and (C) 1a alone; (D, E) 10, 30 mol-% of 1c in DPPC and (F) 1c alone; (G, H) 10, 30 mol-% of 2c in DPPC and (I) 2c alone. In all cases total [lipid] ) 5 × 10-5 M; pH ) 6.8.
formulations containing 10 mol-% of any cholesteryl amphiphile also generated mainly unclosed, long lamellae of variable dimensions (Figure 2A, D, and G) although a few closed aggregates were also seen. However, on increasing the amount of cholesteryl lipid such as 1a or 1b to an extent of 30 mol-% in DPPC membranes, curved and closed membranous aggregates were quite obvious (Figure 2B and E). Interestingly, even after inclusion of 30 mol-% of 2c in DPPC (Figure 2H) aggregates with closed microstructures were not seen. 3.2. Fluorescence Anisotropy. 3.2.1. Cholesteryl Amphiphiles. While the membranes based on fatty acyl phosphatidylcholine DPPC manifested a sharp inflection at ∼ 42 °C indicating the gel-to-liquid crystalline phase transition, for neat aqueous suspensions of cholesteryl lipids 1-3, irrespective of their headgroup structures, no evidence of a typical melting temperature (Tm) profile was seen. For neat 1-3 only gradual monotonic decreases of r-values with increase in temperature were observed after which a plateau was reached (Figure 3). This is not surprising as the major contribution to the solid-tofluid phase transition in membranes composed of saturated fatty acid-based lipid molecules originates due to thermally induced
s-trans f s-gauche isomerization of the hydrocarbon chains. This leads to sharp increases in the membrane disorder at Tm. In contrast the membranes generated from the cholesterol-based lipids (1-3), where the fused ring system of the steroid avoids hydrocarbon chain splay, are much more rigid than their counterparts with long hydrocarbon chains. Thus they do not show any phase transition. Even the membranes from cholesteryl lipid molecules that also contain oleyl chains (1c) did not show any detectable phase transition. This is notable since the lipids containing oleyl chains generally have Tm much lower than the lipids with saturated hydrocarbon chains. In lipids containing unsaturated hydrocarbon chains such as oleyl residue, the presence of a cis-olefinic type of geometry introduces kink in the hydrocarbon chain packing. As a consequence the lipid packing in the resulting bilayers even at room-temperature becomes “loose”. In the membranes of 1c, despite the presence of oleyl chains, the fluorescence anisotropy values did not fall below 0.15 (Figure 3), signifying that considerable rigidity is maintained over the examined temperature range of 20-60 °C. 3.2.2. Cationic Cholesteryl Amphiphile-DPPC Coaggregates. Inclusion of cationic cholesteryl amphiphiles affected the
10260 J. Phys. Chem. B, Vol. 105, No. 42, 2001
Ghosh et al. TABLE 1: Thermal Phase Transition Parameters of DPPC and Its Coaggregates with Various Cholesteryl Amphiphiles As Determined from Fluorescence Anisotropy Measurementsa lipid DPPC 1a 1b 1c 2a 2b 2c
Figure 3. Temperature dependance of fluorescence anisotropy value (r) of DPH doped in membranes composed of neat cholesteryl amphiphiles 1c, 2c, and 3 in comparison with DPPC.
phase transition of DPPC depending on the molecular structure of the added amphiphile. Thus the Tm due to DPPC as detected by DPH fluorescence was nearly abolished by the addition of >10 mol-% of 1a (Figure 4). However, the phase transition was not abolished even when ∼20 mol-% of either 1b, or 2a, or 2c was included in DPPC membranes. When naturally occurring cholesterol is incorporated in DPPC membranes, below its Tm, 10 mol-% of cholesterol induced a pronounced decrease in fluorescence anisotropy (r). Thereafter with increasing mol-% of cholesterol, only a modest decrease in r was observed (not shown). In contrast, 10 mol-% of any one of the cationic cholesterol amphiphiles 1a, 1c, 2a, and 2b (Table 1) did not markedly fluidize the membrane below Tm. However, upon addition of >20 mol-% of this cationic cholesteryl amphiphile, there was a progressive decrease in anisotropy. On the other hand, addition of either 1b or 2c affected fluidization of the membrane in a significant manner in their gel states. Compound 1b showed a marked decrease in r-value at 10 mol-% (see Table 1) followed by a modest decrease in r with increasing mol-%. Thus the addition of cholesteryl amphiphiles could be considered to induce fluidization of the gel phase similar to cholesterol.31 The pertinent data are given in Table 1. Above Tm, i.e., in the fluid state, natural cholesterol evidenced appreciable rigidification of the bilayer membrane showing regular increase in r with increasing mol-% of cholesterol in DPPC.31 Interestingly, none of the cholesteryl amphiphiles, except for 1b and 1c, showed a regular increase in bilayer rigidity with increasing mol-% which is a pattern characteristic of cholesterol. Addition of either 1a or 2a in DPPC showed marked increases in r-value as compared to cholesterol in DPPC above Tm. While 2a showed an abrupt increase in rigidity at 30 mol-%, 1a and 2b showed rather regular increases in r till 20 mol-% and remained invariant thereafter. Interestingly, inclusion of either 2c (Figure 4) and 3 (not shown) did not result in significant rigidification of the fluid state of the DPPC membrane even though the inflections in the r vs T plots on the mixtures containing more than 20 mol-% amphiphile in DPPC were blurred. 3.3. Differential Scanning Calorimetry (DSC). 3.3.1. Aggregates of Cholesteryl Amphiphiles. DSC studies of the
3
% in DPPC
rb (25 °C)
Tmc,d (°C)
∆Tme (K)
r (Tm +10 °C)
100 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30
0.29 0.29 0.28 0.27 0.27 0.26 0.26 0.29 0.28 0.28 0.29 0.28 0.27 0.29 0.28 0.28 0.29 0.27 0.26 0.29 0.28 0.28
41.5 43.0 n n 37.0 34.0 n 40.5 n n 42.0 43.0 n 41.5 43.0 43.5 40.0 35.0 32.0 40.5 40.0 40.0
7.0 14.0 n n 20.0 n n 14.0 n n 10.0 18.0 n 14.0 18.0 18.0 19.0 20.0 22.0 11.0 18.0 24.0
0.09 0.14 0.23 0.24 0.1 0.12 0.15 0.12 0.15 0.18 0.09 0.11 0.2 0.09 0.17 0.15 0.09 0.1 0.1 0.09 0.1 0.11
a See text for experimental details; [DPPC] ) 1 mM; [DPH] ) 1 µM; pH ) 6.5 water (Millipore). b The accuracy of the r-value is (0.005. c Error in Tm is (1.0 °C. d “n”signifies no detectable break in the r-T plot. In such cases Tm for calculation of r(Tm + 10 °C) was chosen as the last identified temperature where a break was observed for a lower mol-% of amphiphile in DPPC. e ∆Tm values represent the temperature range for phase transition; error in ∆Tm is (2.0 K.
suspensions of each of the neat cholesteryl amphiphiles showed flat traces without any peak (not shown). This is consistent with fluorescence data due to DPH where no melting temperature (Tm) due to solid-to-fluid phase transition was also observed with the above aggregates. Hence it is reasonable to affirm that the peaks obtained due to doping DPPC membranes with amphiphiles 1-3 were due solely to the disordering of the fatty acid chains of DPPC (see below). 3.3.2. Cationic Cholesteryl Amphiphile-DPPC Coaggregates. Dispersal of DPPC or its mixture with the cholesteryl amphiphiles into pure water, by use of vortex mixing procedures, followed by five freeze-thaw cycles afforded multi-lamellar vesicles having solid-to-fluid thermal phase transition properties that are summarized in Table 2. The temperature, at which each of these membranes is half-converted into the fluid phase, was shown as Tm. The calorimetric enthalpy (∆HC) that was associated with chain melting for the palmitoyl chains in DPPC/ cholesteryl lipid coaggregates was, however, significantly lower than that of pure DPPC membranes. This is understandable as addition of cholesteryl amphiphiles into DPPC led to progressive broadening of the gel-to-liquid crystalline phase transition of DPPC leading to its eventual abolition in most of the instances. Transition enthalpy and entropy values generally decreased with the increase of the amount of cholesteryl amphiphile included in the coaggregates of DPPC (Table 2). Due to the complex nature of transition (Figure 6B and C), it is however less certain why transition enthalpy and entropy values of DPPC coaggregates with cholesteryl amphiphiles 2b or 2c were higher. One possibility is that amphiphiles 2b and 2c have long spacer chains that separate their cationic headgroup from the steroid backbone. Under such circumstances, the steroid part may be less effective in quenching the chain motions of palmitic acid chains in DPPC.
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TABLE 2: Thermodynamic Characterization of the Phase Transition Exhibited by DPPC and Its Coaggregates with Various Cholesteryl Amphiphiles As Determined from Differential Scanning Calorimetrya lipid DPPC 1a 1b 1c 2a 2b 2c 3
% in DPPCa
Tmb (°C)
∆Hcc (kcal/mol)
∆Sc (cal/K‚mol)
∆HvH (kcal/mol)
CU
100 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30
41.6 39.1 n n 37 35.1 n 39.9 39.1 n 40.7 39.3 n 39.5 35.4 42.2 40.1 36.0 33.6 40.8 38.5 38.3
8.5 5.2
27.0 16.7
2279 489
268 94
4.7 4.3d
15.2 14.0
231 70
49 16
5.1 4.4d
16.3 14.1
461 71
90 16
4.4 3.2d
14.0 10.2
635 438
144 137
7.2d 6.7d 2.7 7.8 3.3d 2.1d 5.6 2.9d 3.0
23.0 21.7 8.6 24.9 10.7 6.8 17.8 9.3 9.6
238 334 78 319 196 100 421 167 83
33 50 29 41 59 48 75 58 28
a See text for experimental details on DSC; [DPPC] ) 1 mM; scan rate used was 0.5 K/min. b Accuracy of Tm was (0.2 °C between successive runs of the same sample; two different sample preparations gave a difference of (1.5 °C; “n” signifies that no detectable phase transition was observed. c Calorimetric data are averages of two independent experiments; the error in ∆Hc is (0.2 kcal/mol. d The error in ∆Hc is (0.6 kcal/mol.
On the basis of their line widths at half-maximum excess specific that (∆T1/2), van’t Hoff enthalpies were determined. Comparison of the experimentally determined calorimetric enthalpy with the van’t Hoff enthalpy allows one to estimate the size of the molecular aggregates over which the motion of the DPPC molecules undergoing the phase transition is transmitted, i.e., the cooperativity of the melting process (CU ) ∆HVH/ ∆HC). As seen from the data in Table 2, the inclusion of cholesteryl amphiphiles in DPPC membranes make the melting of the resulting coaggregates less cooperative than the melting of pure DPPC bilayers. It should be noted that specific values of Tm, ∆HC, and CU, determined for DPPC membranes, are in good agreement with those reported eariler.32 The DSC traces containing thermograms of the DPPC membranes doped with 10, 20, and 30 mol-% of cholesteryl lipids 1-3 are shown in Figure 5, 6, and 7. Addition of cholesteryl amphiphiles led to progressive broadening of the gel-to-liquid crystalline phase transition of DPPC leading to its eventual abolition. Gradual decrease in the enthalpy of transition was also seen in each case. However, the concentrations of these amphiphiles required for complete abolition of the phase transition varied significantly as compared to cholesterol. While as high as 45 mol-% of cholesterol is required23 to completely quench all chain motions in DPPC, even
