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Langmuir 2007, 23, 8988-8994

Thermotropic and Hydration Studies of Membranes Formed from Gemini Pseudoglyceryl Lipids Possessing Polymethylene Spacers Santanu Bhattacharya*,†,‡ and Avinash Bajaj† Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India, and Chemical Biology Unit of JNCASR, Bangalore 560 064, India ReceiVed March 6, 2007. In Final Form: May 10, 2007 Membrane formation from gemini pseudoglyceryl lipids bearing n-C14H29 and n-C16H33 chains has been reported. These lipid aggregates have been characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), high sensitivity differential scanning calorimetry (DSC), and Paldan fluorescence studies. The length of the spacer between the cationic ammonium headgroups has been varied from -(CH2)3- (propandiyl) to -(CH2)12- (dodecandiyl) in these lipids. All gemini lipids were found to generate stable suspensions in aqueous media. Electron microscopic studies revealed the smaller size of the gemini lipid aggregates as compared to their monomeric lipid counterparts. DLS measurements showed that the gemini lipid suspensions with a -(CH2)8- spacer length were bigger in size than that of other analogues. DSC studies suggest the unusual behavior of the gemini lipids bearing -(CH2)3- propanediyl spacer based lipids. These observations were consistent irrespective of the hydrocarbon chain lengths of the lipids. Paldan fluorescence based hydration studies showed that the hexadecyl chain based gemini lipid aggregates bearing a -(CH2)12- spacer were the most hydrated in their gel states among all the gemini lipid series investigated herein.

Introduction Cationic liposome mediated gene delivery is at the forefront of scientists’ attention because of its various advantages like low toxicity, minimal immunogenicity, easy production, high DNA carrying capacity, etc. as compared to its viral vector counterparts.1 Since Felgner and co-workers2 exploited the cationic lipid N-[2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) as a vehicle for the transfer of DNA into eukaryotic cells, several new cationic lipids have been synthesized with the ultimate goal of obtaining efficient non-viral carrier systems.3-11 Highly charged naked DNA is not able to permeate across the negatively charged plasma membrane of cells because of the hydrophilic nature of the DNA. This is due to the presence of a negative charge imposed by the phosphate backbone of DNA.12 Modulation of molecular structures of cationic lipids13 at the headgroup region, chain backbone, linkages, and hydrocarbon * Corresponding author. E-mail: [email protected]; phone: (91)80-2293-2664; fax: (91)-80-2360-0529. † Indian Institute of Science. ‡ Chemical Biology Unit of JNCASR. (1) Miller, A. Angew. Chem., Int. Ed. 1998, 37, 1768. (2) 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. (3) Guenin, E.; Herve, A.-C.; Floch, V.; Loisel, S.; Yaouanc, J.-J.; Clement, J.-C.; Ferec, C.; Des Abbayes, H. Angew. Chem., Int. Ed. 2000, 39, 629. (4) Zhu, J.; Munn, R. J.; Nantz, M. H. J. Am. Chem. Soc. 2000, 122, 2645. (5) Choi, J. S.; Lee, E. J.; Jang, H. S.; Park, J. S. Bioconjugate Chem. 2001, 12, 108. (6) Heyes, J. A.; Niculescu-Duvaz, D.; Cooper, R. G.; Springer, C. J. J. Med. Chem. 2002, 45, 99. (7) Hulst, R.; Muizebelt, I.; Oosting, P.; Van der Pol, C.; Wagenaar, A.; Smisterova, J.; Bulten, E.; Driessen, C.; Hoekstra, D.; Engberts, J. B. F. N. Eur. J. Org. Chem. 2004, 835. (8) Majeti, B. K.; Singh, R. S.; Yadav, S. K.; Bathula, S. R.; Ramakrishna, S.; Diwan, P. V.; Madhavendra, S. S.; Chaudhuri, A. Chem. Biol. 2004, 11, 427. (9) Chittimalla, C.; Zammut-Italiano, L.; Zuber, G.; Behr, J.-P. J. Am. Chem. Soc. 2005, 127, 11436. (10) Jewell, C. M.; Hays, M. E.; Kondo, Y.; Abbott, N. L.; Lynn, D. M. J. Controlled Release 2006, 112, 129. (11) Matsui, K.; Sando, S.; Sera, T.; Aoyama, Y.; Sasaki, Y.; Komatsu, T.; Terashima, T.; Kikuchi, J.-I. J. Am. Chem. Soc. 2006, 128, 3114. (12) Zabner, J.; Fasbender, A. J.; Moninger, T.; Poellinger, K. A.; Welsh, M. J. J. Biol. Chem. 1995, 270, 18997. (13) Bhattacharya, S.; Bajaj, A. Curr. Opin. Chem. Biol. 2005, 9, 647.

chains influences the aggregation and transfection properties of lipid suspensions.14-22 These parameters in turn dictate the properties of cationic lipids toward their ability to induce gene transfer.23,24 Gemini surfactants possessing two surfactant units joined by a spacer are known to show unusual aggregation properties, and many studies have shown the impressive transfection properties of gemini surfactants.25-28 Gemini lipids are the new emerging class of lipids, where two lipid molecules are covalently attached through a spacer. Several years ago, we reported the synthesis and aggregation properties of gemini lipids bearing n-C16H33 chains.29,30 We observed that the length of a flexible spacer dramatically affected the aggregation behavior of the gemini lipids in water.29,30 Recently, we have reported the aggregation behavior of gemini lipids possessing oxyethylene-type spacers.31 Spacer segments between the headgroups induce exceptional effects in the aggregation and gene transfection properties of (14) Duzgunes, N.; Ilarduya, C. T.; Simoes, S.; Zhdanoc, R. I.; Konopka, K.; Lima, M. C. P. D. Curr. Med. Chem. 2003, 10, 1213. (15) Hirko, A.; Tang, F.; Hughes, J. A. Curr. Med. Chem. 2003, 10, 1185. (16) Kumar, V. V.; Singh, R. S.; Chaudhuri, A. Curr. Med. Chem. 2003, 10, 1297. (17) Lima, M. C. P. D.; Neves, S.; Filipe, A.; Duzgunes, N.; Simoes, S. Curr. Med. Chem. 2003, 10, 1221. (18) Liu, D.; Ren, T.; Gao, X. Curr. Med. Chem. 2003, 10, 1307. (19) Miller, A. D. Curr. Med. Chem. 2003, 10, 1195. (20) Nakanishi, M. Curr. Med. Chem. 2003, 10, 1289. (21) Nicolazzi, C.; Garinot, M.; Mignet, N.; Scherman, D.; Bessodes, M. Curr. Med. Chem. 2003, 10, 1263. (22) Niculescu-Duvaz, D.; Heyes, J.; Springer, C. J. Curr. Med. Chem. 2003, 10, 1233. (23) Bhattacharya, S.; Dileep, P. V. J. Phys. Chem. B 2003, 107, 3719. (24) Bhattacharya, S.; Dileep, P. V. Bioconjugate Chem. 2004, 15, 508. (25) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. J. M.; Soderman, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; Garcia Rodriguez, C. L.; Guedat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; Van Eijk, M. C. P. Angew. Chem., Int. Ed. 2003, 42, 1448. (26) Bombelli, C.; Borocci, S.; Diociaiuti, M.; Faggioli, G.; Galantini, L.; Luciani, P.; Mancini, G.; Sacco, M. G. Langmuir 2005, 21, 10271. (27) Bombelli, C.; Faggioli, F.; Luciani, P.; Mancini, G.; Sacco, M. G. J. Med. Chem. 2005, 48, 5378. (28) Bello, C.; Bombelli, C.; Borocci, S.; Di Profio, P.; Mancini, G. Langmuir 2006, 22, 9333. (29) Bhattacharya, S.; De, S.; George, S. K. Chem. Commun. 1997, 2287. (30) Bhattacharya, S.; De, S. Chem.sEur. J. 1999, 5, 2335. (31) Bhattacharya, S.; Bajaj, A. J. Phys. Chem. B 2007, 111, 2463.

10.1021/la700654w CCC: $37.00 © 2007 American Chemical Society Published on Web 07/13/2007

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Figure 1. Molecular structures of pseudoglyceryl monomeric and gemini lipids studied in this paper.

membranes. All gemini lipid aggregates formed smaller aggregate morphologies as compared to their monomeric counterparts. Thermotropic studies show the high Tm of lipid aggregates possessing a -(CH2)3- spacer segment, and gemini lipid suspensions possessing longer spacer lengths showed greater hysteresis as compared to lipids possessing shorter spacers. All gemini lipid suspensions showed very diverse hydration behavior depending upon the length of the spacer and hydrocarbon chain lengths. In gel states, as sensed by Paldan, the n-C16H33 chain based gemini lipids were found to be more hydrated than n-C14H29 based gemini lipids, and among n-C16H33 chain based gemini lipids, lipids bearing a longer -(CH2)12- spacer were found to be most hydrated.

gemini lipids.32 Lipid aggregate morphologies, phase transition temperature (Tm), and hydration properties often determine the biological applications of lipid aggregates.24 There is no report in the literature describing the influence of the length of the spacer segment on aggregate, thermotropic, and interfacial hydration properties of the gemini lipid aggregates. Transfection properties of lipid aggregates involve lipid-DNA complexation (lipoplex formation), which is governed by electrostatic interactions (ion-pairing). These interactions are very much dependent upon the hydration of the lipids during cationic-lipid complex formation, as the spine of hydration at the DNA backbone appears to influence the ion-pairing. Similarly, thermal properties of membranes are also important as the lipid aggregates with higher melting temperatures are less susceptible to DNA complexation at ambient conditions. Here, we present the results of thermotropic and hydration studies of the membranes formed from two sets of pseudoglyceryl gemini lipids bearing n-C14H29 or n-C16H33 hydrocarbon chains. All the lipids form stable suspensions in water, and the lipid aggregates have been characterized by transmission electron microscopy. Using high sensitivity differential scanning calorimetry (DSC), thermal properties of the resulting aggregates have been investigated. The membrane surface hydration of the lipid aggregates was examined using Paldan as a membrane soluble probe for sensing hydration. Interestingly, the examination of temperature-dependent surface hydration of the lipid aggregates revealed the differences in the solid and fluid phases of the

Two series of gemini lipids possessing n-C14H29 and n-C16H33 hydrocarbon chains have been synthesized following procedures as described previously.30 Lipids with polymethylene spacers varying in length from -(CH2)3- (propandiyl) to -(CH2)12(dodecandiyl) had been used (Figure 1). Lipid suspensions from all gemini lipids were prepared by repeated freeze-thawing the hydrated thin films of individual lipids. All the gemini lipids were found to be dispersed easily in water. All lipid suspensions formed clear solutions and possessed a long shelf life when stored at 4 °C. Aggregate Morphology and Sizes. Transmission electron micrographs of all lipid aggregates prepared in aqueous media have been shown in Figures 2 and 3. Negatively stained electron micrographs of gemini lipid aggregates showed the existence of vesicle-like aggregates that were considerably smaller in size as compared to that of their monomeric lipid counterparts. Gemini lipids (16)2-5-(16)2 and (16)2-12-(16)2 produced some irregular morphologies as well. The lipid suspensions in water as prepared as stated previously were further characterized using dynamic light scattering (DLS). The average hydrodynamic diameters of each lipid aggregate as obtained from DLS are given in Table 1. DLS measurements showed that lipid aggregates with a -(CH2)8spacer length were bigger in size than that of other analogues (Figure 4). Gemini lipids (14)2-3-(14)2 and (14)2-5-(14)2 were found to have larger hydrodynamic diameters than their corresponding lipid aggregates bearing n-C16H33 chains. Hydrodynamic

Results and Discussion

Figure 2. Negative stain transmission electron micrographs of aqueous suspensions of lipids (a) DTTMA (14)2, (b) (14)2-3-(14)2, (c) (14)2-5-(14)2, (d) (14)2-8-(14)2, and (e) (14)2-12-(14)2.

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Figure 3. Negative stain transmission electron micrographs of aqueous suspensions of lipids (a) DHTMA (16)2, (b) (16)2-3-(16)2, (c) (16)2-5-(16)2, (d) (16)2-8-(16)2, and (e) (16)2-12-(16)2.

Figure 4. Effect of the spacer length on the hydrodynamic diameter of the gemini lipid aggregates. Table 1. Hydrodynamic Diameters of Lipid Aggregates Obtained from DLS Measurements lipid

size (nm)

lipid

size (nm)

DTTMA (14)2 (14)2-3-(14)2 (14)2-5-(14)2 (14)2-8-(14)2 (14)2-12-(14)2

146 84 92 140 52

DHTMA (16)2 (16)2-3-(16)2 (16)2-5-(16)2 (16)2-8-(16)2 (16)2-12-(16)2

92 48 40 180 56

diameters obtained using DLS were found to be smaller as compared to the aggregates observed from the electron microscopic studies, presumably because of the aggregate fusion in the later stages in the presence of a staining agent. Thermal Properties of the Lipid Aggregates. To determine the phase transition temperature (Tm) of the solid-gel to fluid liquid-crystalline phase, individual lipid suspensions were examined by DSC. The Tm was found to depend strongly upon the hydrocarbon chain lengths and the spacer length between the headgroups. All the gemini lipid suspensions showed wellbehaved sharp and reversible gel to liquid-crystalline phase transitions. For most of the lipid suspensions, only one phase transition was observed. (32) Bajaj, A.; Kondiah, P.; Bhattacharya, S. J. Med. Chem. 2007, 50, 2432.

The thermotropic parameters as obtained from DSC for all the gemini lipids and their monomeric lipid aggregates are summarized in Table 2. Monomeric lipid DTTMA (14)2 bearing tetradecyl chains shows Tm at ∼29.5 °C. With the incorporation of a propandiyl -(CH2)3- spacer between the headgroups (14)23-(14)2, the Tm increased dramatically to ∼43 °C, whereas with a further increase in the spacer length to pentandiyl -(CH2)5(14)2-5-(14)2 led to a decrease in the Tm to ∼28.6 °C. Lipids (14)2-8-(14)2 and (14)2-12-(14)2 showed Tm values of 30.6 and 29.6 °C, respectively. Similarly, the incorporation of a -(CH2)3spacer between cationic ammonium headgroups dramatically increased the Tm from ∼46 to ∼59 °C in the case of the n-C16H33 chain based gemini lipid (16)2-3-(16)2. An increase in the number of polymethylene units brought about decreases in the Tm. This observation was quite consistent with gemini lipids bearing n-C14H29 chains with a -(CH2)3- spacer, as gemini lipid (14)23-(14)2 showed a Tm value of ∼43 °C as compared to the monomeric lipid DTTMA (14)2, which has a Tm near 29.5 °C. Similarly, with an increase in the spacer length, the Tm decreased. Gemini lipids bearing a -(CH2)5- spacer showed a Tm value comparable to that of the monomeric lipid, irrespective of the hydrocarbon chain length (Figure 5). In aqueous dispersions of the gemini lipids bearing a polymethylene spacer, the two covalently charged -N+Me2 headgroups should attempt to maintain a critical distance between them to minimize the coulombic repulsions. But since this situation would create a rather unfavorable contact of the hydrophobic -(CH2)m- spacer chain with bulk water, a separation based on a compromise of the two opposing tendencies resulted.33 In the case of gemini lipids bearing a -(CH2)3- spacer, the short spacer brings each pseudoglyceryl lipid unit close to each other, leading to efficient packing, which increases the Tm of gemini lipids (14)2-3-(14)2 and (16)2-3-(16)2. Gemini lipid (14)2-3-(14)2 aggregates show pre-transitions as well both during the heating and cooling scans. It may be because of the presence of either different aggregate morphologies (polymorphs) present in the lipid membranes or because of the pre-transition of the lipid membranes. Gemini lipid aggregates bearing a -(CH2)5- spacer on the other hand showed a Tm value comparable to their monomeric lipid counterparts because the distance between the

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Table 2. Thermotropic Parameters as Obtained from DSC Studies with Various Lipid Suspensions (1 mM) Tm (°C)a lipid DTTMA (14)2 (14)2-3-(14)2 (14)2-5-(14)2 (14)2-8-(14)2 (14)2-12-(14)2 DHTMA (16)2 (16)2-3-(16)2 (16)2-5-(16)2 (16)2-8-(16)2 (16)2-12-(16)2 a

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

∆Hc (kcal/mol)

∆S (cal/K mol)

up scan

down scan

up scan

down scan

up scan

down scan

CUb

29.6 43.2 28.6 30.6 29.6 45.8 59.3 46.2 48.6 47.3

26.3 40.2 26.3 23.7 22.5 43.3 55.7 43.5 40.4 39.8

8.1 14.7 15.3 17.8 18.2 12.1 18.7 23.2 31.6 22.5

26.6 26.3 26.6 20.8 31.8 19.4 18.9 38.4 53.3 34.6

0.027 0.047 0.051 0.059 0.060 0.038 0.056 0.073 0.098 0.069

0.089 0.084 0.089 0.070 0.107 0.061 0.057 0.121 0.170 0.107

210 41 84 59 76 81 80 31 56 96

Maximum deviation was (0.1 °C. b Size of the cooperativity unit (CU).

Figure 5. (a and c) Thermotropic phase transitions as evidenced by the DSC on heating scans. Thermograms 2-5 have been successively raised from the baseline by 2 (kcal/K mol) steps for clarity. (b and d) Thermotropic phase transitions as evidenced by the DSC on cooling scans. Thermograms 2-5 have been successively lowered from the baseline by 2 (kcal/K mol) steps for clarity.

cationic ammonium headgroups in this lipid closely resembles the distance (thermodynamic separation) between cationic ammonium headgroups in the case of monomeric lipid aggregates. With a further progressive increase in the spacer length, there are no dramatic effects on the Tm values. Figure 6 shows the effect of the spacer length on the Tm values in the case of both n-C14H29 and n-C16H33 chain based gemini lipids. A temperature lag was observed in the thermograms of the cooling scans (liquid-crystalline to gel phase transition) for all the suspensions of the gemini lipids. Such a hysteresis was, however, found to depend very much upon the length of the spacer. Gemini lipids possessing longer spacers such as octanediyl [-(CH2)8-] and dodecanediyl [-(CH2)12-] units showed maximum hysteresis (∼7-8 °C), whereas other gemini lipids with a shorter spacer (m ) 3, 5) manifested lower hysteresis. Such a temperature lag is indicative of a first-order lipid phase transition.34 This temperature lag increases with an increase in the length of the (33) De, S.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. 1996, 100, 11664. (34) Marsh, D. Chem. Phys. Lipids 1991, 57, 109.

Figure 6. Dependence of thermotropic phase transition (Tm) of lipid suspensions on the spacer length as designated by m-values.

polymethylene spacer. A temperature lag of as much as ∼8 °C was observed during the cooling scan of the lipids bearing -(CH2)8and -(CH2)12- spacers (Figure 5).

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Table 3. Summary of Fluorescence Characteristics of Paldan in Lipid Aggregates (1 mM) λex (nm)a lipid DTTMA (14)2 (14)2-3-(14)2 (14)2-5-(14)2 (14)2-8-(14)2 (14)2-12-(14)2 DHTMA (16)2 (16)2-3-(16)2 (16)2-5-(16)2 (16)2-8-(16)2 (16)2-12-(16)2

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

λem (nm)b

GP

24 °C

70 °C

24 °C

70 °C

24 °C

70 °C

fwhmc (nm)

Tm

353 356 357 356 352 355 363 363 364 363

351 355 356 355 354 352 355 355 360 355

445 454 453 455 458 445 466 461 471 483

483 484 485 466 469 469 486 483 483 487

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

106 117 107 104 102 95 117 103 112 97

d d 28 d d 40 61 45 d 45

a Probe was excited at 350 nm. b Emission monitored at 440 nm. c Full width at half-maxima (nm) at 24 °C. d Could not be measured accurately due to the absence of any detectable inflexion.

This suggests that a highly stabilized liquid-crystalline-like fluid phase might exist in these lipid aggregates. It is possible that, upon melting, these lipid aggregates become significantly hydrated due to spacer segment induced disruption of lipid packing in membranes. Cooling of the melted lipid aggregates at its full hydration does not appear to allow the release of the water molecules associated with these aggregates prior to its solidification to the gel state. It is therefore reasonable to infer that the presence of longer polymethylene units in the gemini lipids might help the retention of water molecules particularly in their melted state. Such hydrated melts resist a loss of water molecules prior to their solidification to the gel state during cooling. These observations strongly depend on the length of the spacer irrespective of the hydrocarbon chains of the lipids examined. In general, the enthalpy of transition of the heating scan was higher for all the gemini lipid suspensions in comparison with their corresponding monomer. Interestingly, all the lipids showed a higher enthalpy contribution during their cooling scan (fluid to solid phase transition) than the heating scan. Similarly, all the gemini lipid suspensions showed higher entropies of transition as compared to that of their monomeric counterparts, and cooling scans showed higher entropic contributions than heating scans. All the thermotropic transitions for the gemini lipids were found to be less cooperative than that of the monomeric lipids from the gel to liquid-crystalline transition from the liquid-crystalline to gel state as well. This could be due to the presence of a spacer segment in the lipid twin units in gemini lipids, leading to certain disruptions in the inter-lipid organizations in the membranes. Polarity of the Membrane Interfaces. To determine the interfacial hydration, we performed a steady-state fluorescence emission of a polarity sensitive fluorophore, Paldan,35,36 which is the palmitoyl derivative of the much studied polarity sensitive fluorophores Prodan and Laurdan. Since the chain length of Paldan matches closely with that of the lipid chain lengths examined herein, the fluorophore is expected to correctly report the hydration or polarity at the linkage region of the present set of both monomeric and gemini lipid aggregates. The fluorescence characteristics of Paldan (not shown here) were similar to that of its lauroyl analogue, Laurdan.36 Paldan, like Prodan or Laurdan, was found to be highly sensitive to the polarity of the medium. The important parameters obtained from the experiments using Paldan fluorescence with lipid aggregates are given in Table 3. Gemini lipid aggregates bearing n-C14H29 chains showed λem in the range of 450-458 nm in their rigid, gel states, whereas gemini lipid aggregates bearing n-C16H33 chains showed enhanced red-shifted λem in the range of 465-485 nm in their rigid, gel (35) Weber, G.; Davis, F. J. Biochemistry 1979, 18, 3075. . (36) Lakowicz, J. R.; Bevan, D. R.; Maliwal, B. P.; Cherek, H.; Balter, A. Biochemistry 1983, 22, 5714.

state. This bathochromic shift in the emission λmax of a lipid aggregate is probably a consequence of increased water penetration into the lipid interior.38 This indicates that gemini lipid aggregates bearing hexadecyl chains sense a greater hydration at the membrane interfaces, and among them, lipid (16)2-12(16)2 aggregates were found be most hydrated in the gel state. Among gemini lipids (16)2-m-(16)2, lipids with longer spacers are more hydrated as we have observed maximum red-shift in the emission spectra. Upon melting, there were further bathochromic shifts in all gemini lipid aggregates. Similarly, a greater red-shift in the case of monomeric lipid (14)2 indicates the more hydrated nature of the monomeric lipid bearing n-C14H29 chains. In the solid gel phases, gemini lipid aggregates bearing n-C14H29 chains are found to be equally hydrated independent of the spacer length. The excitation spectra of Paldan in these lipid aggregates were similar to that of the monomeric lipid analogue both in the gel and in the liquid-crystalline phase (Table 3). This further confirms the fact that the emission characteristics of Paldan in these novel gemini lipid aggregates are purely due to the excess solvent (water) mediated stabilization of the excited state and not due to any inherent differences in the ground states of these lipid aggregates.39 Generalized polarization (GP) is taken as a measure of the extent of hydration in the case of dimethylaminonaphthalene based fluorescent probes such as Prodan and Laurdan.39,40 Lipids in their gel states exhibit a high value of GP, which decreases upon melting to the liquid-crystalline state due to the increased water penetration to the bilayer interior during melting. The GP values obtained with the various lipid aggregates here are given in Table 3. It was observed that the water penetration as sensed by Paldan was higher for the gemini lipids bearing hexadecyl chains as compared to that of their monomeric counterparts. Of greater importance is the dramatically lower GP value exhibited by aggregates of lipid 162-12-162 as compared to the aggregates of its other gemini lipid and monomeric counterparts. In the case of gemini lipids bearing tetradecyl chains, lipid aggregates gave GP values slightly lower than that of their monomeric lipid aggregates in the gel state. All gemini lipid aggregates bearing hexadecyl chains possessed a lower GP value than that of the monomeric DHTMA (16)2 aggregates both in their gel and in their melted states. In general, with an increase in the length of the spacer, the GP values decreased, and the (37) Parasassi, T.; Conti, F.; Gratton, E. Cell. Mol. Biol. 1986, 32, 103. (38) Parasassi, T.; Di Stefano, M.; Loiero, M.; Ravagnan, G.; Gratton, E. Biophys. J. 1994, 66, 763. (39) Parasassi, T.; De Stasio, G.; Ravagnan, G.; Rusch, R. M.; Gratton, E. Biophys. J. 1991, 60, 179. (40) Parasassi, T.; De Stasio, G.; d’Ubaldo, A.; Gratton, E. Biophys. J. 1990, 57, 1179.

Thermotropic and Hydration Studies of Membranes

Figure 7. Effect of the spacer chain length of polymethylene units of gemini lipid aggregates on the GP in solid gel states.

Figure 8. Effect of the length of polymethylene spacer units on GP in the fluid-melted state of the gemini lipid aggregates.

hydration increased in the case of gemini lipids bearing n-C16H33 chains (Figures 7 and 8). In the case of aggregates of gemini lipids bearing n-C14H29 chains, the GP value increased from the propandiyl [-(CH2)3-] spacer to the octanediyl [-(CH2)8-] spacer and then decreased. Earlier, it has been shown that gemini lipids bearing dodecandiyl [-(CH2)12-] spacers formed interdigitated and tilted bilayers, where the spacer formed loops inward in their membranous aggregates.30 This in turn allowed for the penetration of more water molecules at the lipid-water interface, which led to greater hydration in the case of the gemini lipids possessing a -(CH2)12- spacer, (16)2-12-(16)2. On the other hand, the gemini lipids bearing short spacers did not allow more hydration of the interfaces in their aggregates. Moreover, the emission spectrum of Paldan in these gemini lipids was comparatively broader. This is indicative of the existence of fluorescence emissions from multiple excited states, arising probably from differential solvent relaxation. In the melted state, all gemini lipids (16)2-m-(16)2 bearing n-C16H33 chains were found to be more hydrated as compared to their monomeric lipid aggregates of (16)2. In the case of gemini lipids possessing tetradecyl chains, (14)2-3-(14)2 and (14)2-5-(14)2 were found to be more hydrated than (14)2 aggregates, and (14)2-8-(14)2 and (14)2-12-(14)2 were found to be less hydrated in their melted states (Figures 7 and 8). The GP versus T profiles in Figures 9 and 10 show systemic breaks related to the main chain thermotropic phase transition processes for individual lipid assemblies. Comparison with the monomeric lipid (16)2 revealed that the incorporation of a single -(CH2)3- unit (16)2-3-(16)2 between the cationic ammonium headgroups increased the gel to the liquid-crystalline phase transition temperature of the assemblies reported by Paldan. In the case of other gemini lipids, the gel to liquid-crystalline phase transition temperature was lower than that for the

Langmuir, Vol. 23, No. 17, 2007 8993

Figure 9. Changes in the GP of Paldan doped lipid aggregates as a function of temperature.

Figure 10. Changes in the GP of Paldan doped lipid aggregates as a function of temperature.

monomeric lipid (16)2. The breaks in the GP versus T plots (i.e., phase transition temperatures) were comparable to the Tm values observed under DSC studies as explained earlier.

Conclusion Although gemini lipids are pervasive in nature, very few groups have dealt with them extensively; thus, gemini lipids provide ample opportunity to unleash and unravel different properties of the lipids that are often unknown to researchers. It is known that cationic dimeric surfactants possess unusual physical, chemical, and biological properties. Therefore, it is quite expected that these surfactant characteristics can be extrapolated to lesser known gemini lipids. The long-term objective of the development of these synthetic gemini lipids is to find their use in gene therapy and drug delivery. To understand the exact nature, morphology, aggregation behavior, and hydration of the gemini lipids, we investigated the aggregation, thermotropic, and hydration behavior of pseudoglyceryl lipids possessing polymethylene spacers and different hydrocarbon chain lengths. All gemini lipids were found to generate stable suspensions in aqueous media. Electron microscopic studies revealed the smaller size of the gemini lipid aggregates as compared to their monomeric lipid counterparts. DLS measurements showed that the gemini lipid suspensions with a -(CH2)8- spacer length were larger in size than that of other homologues. Thermotropic studies showed that the incorporation of a -(CH2)3- spacer between cationic ammonium headgroups dramatically increased the Tm for gemini lipid aggregates irrespective of the hydrocarbon chain lengths and that a further increase in the number of polymethylene units brought about decreases in Tm. In the case of gemini lipids

8994 Langmuir, Vol. 23, No. 17, 2007

possessing -(CH2)8- and -(CH2)12- spacers, a highest temperature lag of ∼8 °C was observed during the cooling scan of the lipids, which showed the highly hydrated nature of these lipid aggregates in the liquid-crystalline phase. Hydration studies indicated that gemini lipid aggregates bearing hexadecyl chains sense greater hydration at membrane interfaces and that among them, lipid (16)2-12-(16)2 aggregates were found be most hydrated in the gel state. Overall, it has been shown that the aggregation, thermotropic, and hydration properties of the pseudoglyceryl gemini lipids depend both upon the length of the spacer between the headgroups and the hydrocarbon chain lengths. Elucidation of these properties could be helpful in the design of new synthetic lipid systems to understand their membrane level properties and their further use in gene delivery. Experimental Section Materials and Methods. All reagents, solvents, and chemicals used in this study were of the highest purity available. The solvents were dried prior to use. Column chromatography was performed using a 60-120 mesh silica gel. NMR spectra were recorded using a Jeol JNM λ-300 (300 MHz for 1H and 75 MHz for 13C) spectrometer. The chemical shifts (δ) are reported in ppm downfield from the internal standard, TMS, for 1H NMR and 13C NMR. Mass spectra were recorded on a Kratos PCKompact SEQ V1.2.2 MALDI-TOF spectrometer or on a MicroMass ESI-TOF spectrometer or Shimadzu table-top GC-MS or ESI-MS (HP1100LC-MSD). Infrared (IR) spectra were recorded on a Jasco FT-IR 410 spectrometer using KBr pellets or neat. Two series of gemini lipids possessing n-C14H29 and n-C16H33 hydrocarbon chains have been synthesized following procedures as described previously.30 Liposome Preparation. The individual lipid was dissolved in chloroform in autoclaved Wheaton glass vials. Thin films were made by evaporation of the organic solvent under a steady stream of dry nitrogen. The last traces of organic solvent were removed by keeping these films under vacuum overnight. Freshly autoclaved water (MilliQ) was added to the individual film such that the final concentration of the cationic lipid was ∼1 mM. The mixtures were kept for hydration at 4 °C for 10-12 h and were repeatedly freeze-thawed (ice-cold water to 70 °C) with intermittent vortexing to ensure optimal hydration. Sonication of these suspensions for 15 min in a bath sonicator at 70 °C afforded cationic liposomes as evidenced from transmission electron microscopy. Liposomes were prepared and kept under sterile conditions. Formulations were found to be stable and if stored frozen, possessed a long shelf life. Transmission Electron Microscopy. Lipid suspensions of cationic lipids (1 mM) were examined under transmission electron microscopy by negative staining using 1% uranyl acetate. A 15 µL sample of the suspension was loaded onto Formvar-coated, 400 mesh copper grids and allowed to remain for 1 min. Excess fluid was removed from the grids by touching their edges with filter paper, and 15 µL of 1% uranyl acetate was applied on the same grid after which the excess stain was similarly wicked off. The grid was air-dried for 30 min, and the specimens were observed under TEM (JEOL 200-CX) operating at an acceleration voltage of 120 keV. Micrographs were recorded at a magnification of 4000-20 000×. Dynamic Light Scattering. Unilamellar vesicles (1 mM) were prepared in pure water (Millipore) as mentioned under liposome preparation. They were diluted to 0.33 mM and were used for dynamic light scattering measurements. Experiments were performed using a DynaPro molecular sizing instrument, which employed an incident laser beam of ∼830 nm wavelength. An interfaced autocorrelator was used to generate the full auto-correlation of the scattered intensity. The time-correlated function was analyzed by the method of cumulants, and calculations yielded specific distribution of particle size populations. The values reported are the averages of two independent experiments, each of them having 10 sub-runs. Differential Scanning Calorimetry. Multilamellar vesicles of 1 mM concentration were prepared in degassed water as mentioned

Bhattacharya and Bajaj previously, and their thermotropic behavior was investigated by high sensitivity DSC using a CSC-4100 model, multicell differential scanning calorimeter (Calorimetric Science Corporation). A baseline thermogram was obtained using degassed water (0.5 mL) in all the ampoules, including the reference cell to normalize cell-to-cell differences. Samples were taken in the cells such that the differences in weight with the baseline experiment to the sample run were less than 0.001 g in the respective ampoules. The measurements were carried out in the temperature range of 20-90 °C. The scan rate was 20 °C per hour for all the lipids. The thermograms for vesicular suspensions were obtained by subtracting the respective baseline thermogram from the sample thermogram using the software CpCalc provided by the manufacturer. The peak position in the plot of the excess heat capacity versus temperature on the heating scan and cooling scan was taken as the solid-like gel-to-fluid phase transition temperature and fluid-to-gel phase transition temperature, respectively, for each amphiphilic suspension. The molar heat capacities, calorimetric enthalpies (∆H), and entropies (∆S) were also computed using the same software as reported. The size of the cooperativity unit (CU) for the phase transition of each lipid was determined using the formula CU )

∆HvH ∆Hc

where ∆HvH is the van’t Hoff enthalpy and ∆Hc is the calorimetric enthalpy.40 The van’t Hoff enthalpy was calculated from the equation ∆HvH )

6.9Tm2 ∆T1/2

where ∆T1/2 is the full width at half maxima of the thermogram and Tm is the phase transition temperature.42 Paldan Fluorescence. Paldan, a palmitoyl analogue of Prodan, was synthesized by appropriate modification of literature procedures.35,36 The synthesis of Paldan was accomplished by the following procedure. First, 2-methoxy naphthalene was palmitoylated using C16H33COCl in the presence of AlCl3 to afford 6-palmitoyl-2-methoxy naphthalene. The conversion of the methoxy compound to the corresponding diemthylamino derivative (Paldan) was performed following the procedure of Weber and Davis and Balter et al.35,36 The spectral properties of Paldan (not shown here) were very similar to that of its other analogues (Prodan and Laurdan). All the fluorescence experiments were carried out on sonicated lipid suspensions using a lipid-to-probe ratio of 1000:3. The width of the excitation and emission slit was 5 nm. Generalized polarization of emission (GP) was calculated using the equation GPem )

I440 - I490 I440 + I490

where I440 and I490 represent the fluorescence intensities at 440 and 490 nm.38,39 For the GP values reported herein, an excitation wavelength of 350 nm was used. For excitation, the spectra wavelength was kept at 440 nm unless specified otherwise. The full width at half maxima (fwhm) was calculated from the emission spectra during Paldan fluorescence studies, by Lorentzian fitting of the emission spectra using ORIGIN software.

Acknowledgment. This work was supported by the Department of Biotechnology, Government of India, New Delhi, India. A.B. thanks CSIR for a senior research fellowship. Note Added After ASAP Publication. This article was published ASAP on July 13, 2007 with an incorrect version of Figure 3. The correct version was published on July 17, 2007. LA700654W (41) Hinz, H. J.; Sturtevant, J. M. J. Biol. Chem. 1972, 247, 6071. (42) Sturtevant, J. M. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3963.