Role of the Spacer of Cationic Gemini Amphiphiles in the

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Role of the Spacer of Cationic Gemini Amphiphiles in the Condensation of DNA Cecilia Bombelli,†,‡ Stefano Borocci,§ Marco Diociaiuti,⊥ Francesca Faggioli,# Luciano Galantini,3 Paola Luciani,†,‡ Giovanna Mancini,*,†,‡ and Maria Grazia Sacco# Istituto di Metodologie Chimiche del CNR and Dipartimento di Chimica, Universita` degli Studi di Roma “La Sapienza”, P.le Aldo Moro 5, 00185 Roma, Italy, Centro di Eccellenza Materiali Innovativi Nanostrutturati per Applicazioni Chimiche, Fisiche e Biomediche, Dipartimento di Scienze Ambientali, Universita` della Tuscia, L.go dell’Universita` , 01100 Viterbo, Italy, Istituto Superiore di Sanita` , V.le Regina Elena 299, 00161 Roma, Italy, and Istituto di Tecnologie Biomediche del CNR, via Fratelli Cervi, 93, 20090 Segrate-Milano, Italy Received May 18, 2005. In Final Form: August 8, 2005 The condensation of calf thymus DNA into the cholesteric-like ψ-phase was observed by circular dichroism in liposome suspensions formulated with specific cationic gemini surfactants. The stereochemistry of the gemini spacer, the presence of specific functional groups, and the covalent link between the headgroups are fundamental issues in the condensation of DNA. Transmission electron microscopy images showed a multilamellar morphology, which corresponds with condensation.

Introduction The transfection of DNA in gene therapy depends on the possibility of obtaining its condensation; nucleic acids are, in fact, too large to efficiently cross the cellular membrane. Furthermore, DNA has to be protected from degradation by endogenous nucleases to enter undamaged in the cytosol of the target cell.1 Finally, it is necessary to neutralize the negative charge of DNA because an overall positive charge significantly improves the docking of the particle on the primarily negatively charged residues of the plasma membranes of the cell.2 In a variety of complex-forming and condensing agents, liposomes represent the most appealing nonviral carriers for genetic medicines because of their safety and versatility.3 The electrostatic interaction between cationic lipids and the phosphate backbone of DNA leads to the compaction of the macromolecule with modifications to its superficial structure and hydrophobic properties and, most importantly, reduces the size of the DNA and therefore promotes its penetration into the cell.1 Over the past years, many efforts have been devoted to the development of formulations for cationic liposomes, namely through the synthesis of different cationic amphiphiles (CAs) featuring low toxicity and different capabilities to mediate gene transfer.4,5 In the panorama of new amphiphiles, gemini surfactants,6 which feature * Corresponding author. E-mail: giovanna.mancini@ uniroma1.it. Tel: 00390649913078. Fax: 003906490421. † Istituto di Metodologie Chimiche del CNR and Dipartimento di Chimica, Universita` degli Studi di Roma “La Sapienza”. ‡ Centro di Eccellenza Materiali Innovativi Nanostrutturati (CEMIN). § Universita ` della Tuscia, L.go dell’Universita`. ⊥ Istituto Superiore di Sanita `. # Istituto di Tecnologie Biomediche del CNR. 3 Dipartimento di Chimica, Universita ` degli Studi di Roma “La Sapienza”. (1) Zhdanov, R. I.; Podobed, O. V.; Vlassov, V. V. Bioelectrochemistry 2002, 58, 53-64. (2) Bally, M. B.; Harvie, P.; Wong, F. M. P.; Kong, S.; Wasan, E. K.; Reimer, D. Adv. Drug Delivery Rev. 1999, 38, 291-315. (3) Pedroso de Lima, M. C.; Simo˜es, S.; Pires, P.; Faneca, H.; Du¨zgu¨nes¸ , N. Adv. Drug. Delivery Rev. 2001, 47, 277-294. (4) Paukku, T.; Lauraeus, S.; Huhtaniemi, I.; Kinnunen, P. Chem. Phys. Lipids 1997, 87, 23-29.

two hydrophobic chains and two polar headgroups linked by a spacer, seem quite promising. Their physicochemical features are different from those of conventional surfactants, and recent studies pointed out that opportunely designed cationic geminis exhibited high transfection efficiency.7-9 A correlation between gemini architecture and DNA compaction was recently reported;10 the design of new efficient condensing agents can, in fact, be accomplished by only a rationalization of the parameters responsible for an efficient condensation. However, despite several investigations in this direction,11-15 many issues are still under debate. To explore the role of the stereochemistry of the spacer of a cationic gemini surfactant in the condensation of DNA, we carried out an investigation on the condensation of calf thymus (CT) DNA by cationic liposomes formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholin (DMPC)16 and (5) Gaucheron, J.; Wong, T.; Wong, K. F.; Maurer, N.; Cullis, P. R. Bioconjugate Chem. 2002,13, 671-675. (6) Menger, F. M.; Littau, C. A. J. Am. Chem. Soc. 1991, 113, 14511452. (7) Camilleri, P.; Kremer, A.; Edwards, A.; Jennings, K. H.; Jenkins, O.; Marshall, I.; McGregor, C.; Nevelle, W.; Rice, S. Q.; Smith, R. J.; Wilkison, M. J.; Kirby A. J. Chem. Commun. 2000, 1252-1254. (8) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. J. M.; So¨derman, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; Garcı´a Rodrı´guez, C. L.; Gue´dat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; van Eijk, M. C. P. Angew. Chem., Int. Ed. 2003, 42, 1448-1457. (9) Bell, P. C.; Bergsma, M.; Dolbnya, I. P.; Bras, W.; Stuart, M. C.; Rowan, A. E.; Feiters, M. C.; Engberts, J. B. J. Am. Chem. Soc. 2003, 125, 1551-1558. (10) Karlsson, L.; van Eijk, M. C. P.; So¨derman, O. J. Colloid Interface Sci. 2002, 252, 290-296. (11) Zuidam, N. J.; Hirsch-Lerner, D.; Margulies, S.; Barenholz, Y. Biochim. Biophys. Acta 1999, 1419, 207-220. (12) Cherng, J.-Y.; Schuurmans-Nieuwenbroek, N. M. E.; Jiskoot, W.; Talsma, H.; Zuidam, N. J.; Hennink, W. E.; Crommelin, D. J. A. J. Controlled Release 1999, 60, 343-353. (13) Kennedy, M. T.; Pozharski, E. V.; Rakhmanova, V. A.; MacDonald, R. C. Biophys. J. 2000, 78, 1620-1633. (14) Uhrı´kova´, D.; Hanulova´, M.; Funari, S. S.; Lacko, I.; Devı´nsky, F.; Balgav×c6, P. Biophys. Chem. 2004, 111, 197-204. (15) Kamiya, H.; Yamazaki, J.; Harashima, H. Gene Ther. 2002, 9, 1500-1507. (16) DMPC was chosen because it features a relatively low Tc, although it is characterized, along with the investigated surfactants, by saturated alkyl chains. The choice of a saturated lipid allowed us to avoid the introduction of an additional parameter.

10.1021/la051324+ CCC: $30.25 © 2005 American Chemical Society Published on Web 09/28/2005

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Langmuir, Vol. 21, No. 23, 2005 Chart 1. Structure of the CAs Used

Letters Liposomes at different DMPC/CA ratios (9/1, 8/2, 7/3, 6/4, 5/5, and 4/6) were prepared at a constant total lipid concentration (85 µM for a +/- ) 1 charge ratio and 170 µM for a +/- ) 2 charge ratio). For liposomes containing surfactant 3, the total lipid concentration was varied by doubling the concentration of CAs to keep the total number of headgroups and tails constant.

Results and Discussion

any of the three diastereoisomers of the cationic gemini surfactant 2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane bromide (1; the enantiomers 1-SS and 1-RR and the meso form 1-M, according to the absolute configuration of C2 and C3). Moreover, we investigated the role of the methoxy groups by a comparison with 1,4-bis(N-hexadecyl-N,N-dimethylammonum)butane bromide (2) and the influence of the covalent link between the headgroups by a comparison with N,Ndimethyl-N-hexadecyl-N-(2-methoxy)ethylammonium bromide (3; Chart 1). The investigation was carried out by circular dichroism (CD) in solution and at a molecular level and by transmission electron microscopy (TEM) on the dehydrated samples for a morphological point of view. The information obtained was supported by quasi-elastic light scattering (QELS) measurements and gel electrophoresis. Materials And Methods Materials. DNA CT was purchased from Sigma Aldrich. DMPC was purchased from Avanti Polar. Gemini surfactant 1 was prepared by the quaternization, in refluxing acetone, of the corresponding isomer 2,3-dimethoxy-1,4-bis(N,N-dimethylamino)butane.17Gemini surfactant 2 was prepared by the quaternization, in refluxing acetone, of N,N,N′,N′-tetramethyl-1,4-butanediamine (Aldrich) with 1-bromohexadecane. Surfactant 3 was obtained by the quaternization of N-hexadecyl-N,N-dimethylamine with 2-bromoethylmethyl ether in refluxing acetone. DNA concentrations were expressed as mM and µM single-base concentrations and were determined by absorbance at 260 nm ( ) 6600 L/mol cm). Sample Preparation. DNA/DMPC/CA complexes (lipoplexes) were prepared by the addition, at room temperature, of known volumes of an aqueous 2 mM solution of CT DNA in HEPES buffer (5 mM HEPES and 0.1 mM EDTA at pH 7.4) to monodispersed suspensions of liposomes composed by different amounts of DMPC and CA. The concentrations of DNA were dictated by the concentration of CA to obtain neutral ([cationic headgroup]/[DNA single base]; +/- ) 1) or cationic (+/- ) 2) complexes. Monodispersed 100-nm liposomes were prepared by mixing appropriate amounts of the lipid stock solutions in dry chloroform to obtain the desired compositions. The lipid mixtures were dried to a film by rotary evaporation under reduced pressure. To remove the residual solvent, the samples were further maintained under high vacuum for at least 8 h. The resulting dry lipid films were then hydrated with an aqueous buffered solution (5 mM HEPES and 0.1 mM in EDTA at pH 7.4), vortexed vigorously, freezethawed, and then filtered under N2 pressure through 100-nm pore size polycarbonate membranes (Nucleopore, Pleasanton, CA) at 318 K (well above DMPC transition temperature), using a Lipex Biomembranes extruder according to the extrusion protocol.18 (17) Seebach, D.; Kalinowski, H. O.; Bastani, B.; Crass, G.; Daum, H.; Doerr, H.; DuPreez, N. P.; Ehrig, V.; Langer, W.; Nu¨ssler, C.; Oei, H.-A.; Schmidt, M. Helv. Chim. Acta 1977, 60, 301-325. (18) Hope, M. J.; Nayar, R.; Mayer, L. D.; Cullis, P. R. In Liposome Technology, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Bosca Raton, FL, 1992; Vol. 1, pp 123-139.

The CD spectra of condensed DNA are characterized by an enhanced negative ellipticity, an overall shift of the bands toward higher wavelengths, a flattening of the positive band, and the appearance of long “tails” above 300 nm.19,20 These chirooptical features, known as ψanomalies, have been ascribed to the supramolecular chiral order of a cholesteric-like phase, called the ψ-phase, in which DNA molecules are supposed to be tightly packed together to form highly condensed structures that exhibit negative CD signals because of a left-handed tertiary conformation.21 We carried out CD experiments on DNA/liposome complexes prepared at different DMPC/CA ratios (9/1, 8/2, 7/3, 6/4, 5/5, and 4/6), and we observed the features of condensed DNA only in the CD spectra of some of the lipoplexes at the 5/5 and 4/6 DMPC/CA ratios. Because the results of the condensation experiments carried out on all of the considered CAs at the 4/6 ratio were completely analogous to those at the 5/5 ratio, here we only report (Figure 1) the results relative to the formulations containing the minimum possible amount of synthetic amphiphiles (5/5 ratio). The CD spectra of DNA relative to lipoplexes formulated with gemini 1 at a +/- ) 2 charge ratio (reported in Figure 1) show the features of a ψ-phase; they show, in fact, an overall shift of the bands toward higher values of wavelengths, a more intense negative band, and a flattening of the positive one, with respect to the conservative spectrum of uncondensed DNA (solid line), relative to a buffered solution of CT DNA in the presence of DMPC vesicles (DMPC does not induce any conformational change on DNA, as shown in the Supporting Information (SI), in which the CD spectrum of naked DNA is reported). The three diastereomeric geminis induce analogous variations on the DNA spectrum at t ) 0 (Figure 1a), whereas 1-SS and 1-RR induce larger variations at t ) 24 h,22 namely, a more than 50% increase in the negative band and a more than 60% decrease in the positive one (Figure 1b). Gemini 2 and the single headgroup CA 3 do not induce any conformational change in the DNA in the complexes at either t ) 0 or t ) 24 h (Figure 1). On the other hand, in the neutral complexes (at a +/- ) 1 charge ratio) the presence of any of the geminis (1 and 2) has a very modest effect on the conformation of DNA at t ) 0, whereas it induces precipitation at t ) 24h; the presence of 3 in the formulation does not induce any appreciable effect on the conformation of DNA and on the solubility of the complexes at either t ) 0 or t ) 24 h (the CD spectra of neutral lipoplexes are available as SI). In summary, the chirooptical features of a DNA ψ-phase were induced only by gemini 1 in cationic lipoplexes, that (19) Keller, D.; C. Bustamante. J. Chem. Phys. 1986, 84, 2972-2980. (20) Simberg, D.; Danino, D.; Talmon, Y.; Minsky, A.; Ferrari, M. E.; Wheeler, C. J.; Barenholz, Y. J. Biol. Chem. 2001, 276, 47453-47459. (21) Zuidam, N. J.; Barenholz, Y.; Minsky, A. FEBS Lett. 1999, 457, 419-422. (22) No significant change was observed in the CD spectra that were recorded 10, 20, and 30 min after the addition of DNA to the liposome suspensions with respect to those performed at time t ) 0. Because there were no differences between the spectra acquired after 12 h and those acquired after 24 h, measurements were taken after 24 h for opportunity.

Letters

Figure 1. CD spectra of DMPC/CT/CA lipoplexes (1-SS: blue, long dash; 1-RR: red, dotted; 1-M: black, dash-dot-dot; 2: dark green, dash-dot; and 3: cyan, short dash) at a charge ratio of +/- ) 2 when t ) 0 (a) and at a charge ratio of +/) 2, when t ) 24 h (b). The solid line is the CD spectrum of uncondensed DNA (DNA/DMPC).

is, those featuring a +/- ) 2 ratio between the positive charges of the CAs and the negative charges of the DNA nucleotides. These results are not affected by any contribution of differential scattering; in fact, ψ-anomalies were also observed when the distance between the samples and the photomultiplier tube (PMT) was varied.23 The DNA complexation by cationic liposomes containing gemini surfactants was almost quantitative, especially at a +/- ) 2 charge ratio and after 24 h of incubation, as demonstrated by gel electrophoresis (picture available as SI). On the other hand, complexation by CA 3 was less efficient. As stated above, the condensation of DNA should protect the macromolecule from degradation by nucleases. We carried out a DNase I protection assay to verify the extent of protection of the DNA, which is mediated by cationic liposomes, from attack by nucleases. The intensity of the bands corresponding to the complexes exposed to DNase I demonstrated that DNA protection against DNase I was high in the complexes prepared with geminis at a +/- )2 charge ratio and incubated for 24 h and that the protection (picture available as SI) in complexes formulated with CA 3 was lower than that in the others. To correlate the effect of gemini structure on DNA conformation to the morphology of the complexes, we (23) Braun, C. S.; Jas, G. S.; Choosakoonkriang, S.; Koe, G. S.; Smith, J. G.; Middaugh, C. R. Biophys. J. 2003, 84, 1114-1123.

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carried out a number of TEM experiments on the charged lipoplexes. The TEM images of the formulations that induced strong variation in the CD spectrum of DNA, namely those containing either 1-SS or 1-RR at a +/- ) 2 charge ratio, show very organized and regular structures that resemble a net formed by long threads, with a number of multilamellar complexes within it (Figure 2a-c; enlarged images of Figure 1 are available in the SI). The TEM images of the same samples in the absence of DNA do not show the same organized structures; instead, they only show the presence of unilamellar liposomes (data not shown), which is expected on the basis of QELS measurements (Table S1 in SI). In the images of the formulation containing 1-M, one can still observe a residual organizationsalthough the net is not regular and the threads are larger (a full image is available in the SI)sas well as multilamellar systems (Figure 2d). On the other hand, the TEM images of the formulations that show no variation in the dichroic bands, namely, those containing 2 or 3, lack any organization (Figure 2e-f) and, most of all, lack the presence of the multilamellar systems that are yielded by gemini 1 and reported in Figure 2c. No relevant difference was revealed by the TEM images taken at t ) 0 and t ) 24 h for all samples. QELS measurements performed on the lipoplexes provide evidence of bigger aggregates with respect to the starting liposomes, but do not indicate the relevant differences between the types of lipoplexes (see Table S2 in SI). The described results demonstrate a fundamental role of the stereochemistry of the gemini in the condensation of CT DNA. Both chiral geminis, 1-SS and 1-RR, in fact, are able to condense CT DNA (under specific experimental conditions), whereas the meso form, 1-M, is definitely less efficient. Apparently, the configuration of the second and third carbon of the spacer governs the interactions of the ammonium groups with the phosphate groups of DNA. In principle, because the interactions of 1-SS and 1-RR with both DMPC and DNA are diastereomeric, these two surfactants could have also induced a different extent of DNA condensation, but the observed differences are within the experimental error. The collected evidence points to a cooperative effect of the ammonium-phosphate electrostatic interactions and the methoxy group interactions. These could be either steric or polar, but their fundamental role in CT DNA condensation is confirmed by the absence of condensation in the experiments carried out with the formulations containing gemini 2, in which we did not observed the formation of a condensed phase. Another important point is the role of the covalent link between the two ammonia headgroups of the geminis. In fact, cationic surfactant 3, which has a molecular structure that corresponds to half of gemini 1, is absolutely inefficient in condensing DNA. Another interesting result is the different extent of condensation observed by CD at t ) 0 and t ) 24 h, which demonstrates a slow condensation process. The slow process was confirmed by gel electrophoresis experiments and by the DNase I protection assay. The images obtained in TEM experiments on the dehydrated samples suggest that the condensation observed in solution by CD can be, to some extent, correlated to the presence of organized structures and multilamellar systems (Figure 2a-d). The two techniques give us two different perspectives of lipoplexes: by CD we have a molecular perspective on the conformation of DNA in the lipoplexes, whereas by TEM we have the view of the morphology of lipoplexes after dehydration. These results demonstrate the importance of a systematic screening of DNA condensing agents. The two

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Letters

Figure 2. TEM images of a liposome suspension containing CA 1-SS (a); 1-RR (b); 1-SS at higher magnification (c); 1-M (d); 2 (e); and 3 (f) at a +/- ) 2 charge ratio.

enantiomeric gemini surfactants of 1 were capable of condensing CT DNA under definite experimental conditions, and the condensation of DNA was correlated to the presence of organized lamellar systems. The same formulations have been investigated for transfection in vitro with a reporter gene, and the efficiency of transfection has been correlated to a high extent of condensation.24 The plasmid condensation experiments have shown that the extent of condensation depends on the structure of the spacer as well as on the topology of the DNA; in fact, it was found that the formulations that better condense the plasmid are different from those that better condense CT DNA. Other experiments are in due (24) Bombelli, C.; Faggioli, F.; Luciani, P.; Mancini, G.; Sacco, M. G. J. Med. Chem. 2005, 48, 5378-5382.

course to further investigate how and to what extent the topology, the size, and the sequence of DNA influence condensation by liposomes formulated with different gemini surfactants. Supporting Information Available: Experimental details (S2, S3); CD spectra of naked DNA and DNA complexed with DMPC vesicles and CD spectra of DNA complexed with liposomes DMPC/1, DMPC/2, DMPC/3 at a charge ratio of +/) 1 (S4); Lipid/DNA complexation assessed by gel electrophoresis (S5); Lipid/DNA resistance to enzymatic degradation assessed by gel electrophoresis (S6); full TEM images at lower magnification (S7-S9); QELS measurement tables (S10, S11). This material is available free of charge via the Internet at http://pubs.acs.org. LA051324+