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Distribution of DNA in Cationic Liposome Complexes Probed by Raman Microscopy Hiroshi Matsui* and Su Pan Center for Discovery of Drugs & Diagnostics and Department of Chemistry, University of Central Florida, Orlando, Florida 32816 Received March 22, 2000. In Final Form: June 19, 2000 The potential application of Raman microscopy to image the distribution of DNA inside lipid-DNA complexes was examined. To test this, we mixed DNA with DOTMA/DOPE liposome vesicles and imaged the distribution of DNA in the liposome complex by using the characteristic DNA guanine peak as a marker. The Raman intensity map indicates that the concentration of DNA molecules is higher near the core of the DOTMA/DOPE liposome vesicle. This result demonstrates that this technique is a viable technique to monitor DNA distribution inside liposomes.
The effectiveness of gene therapy depends on the successful delivery and expression of transgene DNA to intracellular targets.1,2 Liposome constructs, especially cationic versions for gene delivery, have proven to be efficient DNA delivery vehicles.3-9 An important factor in affecting this efficiency is the stability of the DNA-cationic liposome complex.10 The charge distribution of DNA within the liposome complex has a direct influence on the stability and activity of the transfection complex. Therefore, correlations between DNA conformations, their distributions in DNA-liposome complexes, and their transfection efficiency may provide insight to improve the design of delivery vehicles. To better understand the interactions between DNA and cationic liposomes, condensed forms of absorbed DNA have been extensively investigated on various lipid surfaces as model systems.11-13 These studies have shown * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Mulligan, R. C. Science 1993, 260, 926. (2) Crystal, R. G. Science 1995, 270, 404. (3) Behr, J. P.; Demeneix, B.; Loeffler, J. P.; Mutul, J. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6982. (4) Caplen, N. J.; Alton, E. W. F. W.; Middleton, P. G.; Dorin, J. R.; Stevenson, B. J.; Gao, X.; Durham, S. R.; Jeffery, P. K.; Hodson, M. E.; Coutelle, C.; Huang, L.; Porteous, D. J.; Williamson, R.; Geddes, D. M. Nat. Med. 1995, 1, 39. (5) Felgner, P. L.; Ringold, G. M. Nature 1989, 337, 387. (6) Nabel, G. J.; Nabel, E. G.; Yang, Z. Y.; Fox, B. A.; Plautz, G. E.; Gao, X.; Huang, L.; Shu, S.; Gordon, D.; Chang, A. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11307. (7) Smith, J. G.; Walzen, R. L.; German, J. B. Biochim. Biophys. Acta 1993, 1154, 327. (8) Zhu, N.; Liggitt, D.; Liu, Y.; Debs, R. Science 1993, 261, 209. (9) Rui, Y.; Wang, S.; Low, P. S.; Thompson, D. H. J. Am. Chem. Soc. 1998, 120, 11213. (10) Deshmukh, H. M.; Huang, L. New. J. Chem. 1997, 21, 113. (11) Fang, Y.; Yang, J. J. Phys. Chem. B 1997, 101, 441. (12) Zantle, R.; Baicu, L.; Artzner, F.; Sprenger, I.; Rapp, G.; Radler, J. O. J. Phys. Chem. B 1999, 103, 10300. (13) Matsui, H.; Pan, S. J. Phys. Chem. B 2000, 104, 8871. (14) Ma, C.; Bloomfield, V. A. Biophys. J. 1994, 67, 1678. (15) Fang, Y.; Hoh, J. H. J. Am. Chem. Soc. 1998, 120, 8903. (16) Hansma, H. G.; Golan, R.; Hsieh, W.; Lollo, C. P.; Mullen-Ley, P.; Kwoh, D. Nucleic Acids Res. 1998, 26, 2481. (17) Allen, M. J.; Bradbury, E. M.; Balhorn, R. Nucleic Acid Res. 1997, 25, 2221. (18) Ono, M. Y.; Spain, E. M. J. Am. Chem. Soc. 1999, 121, 7330. (19) Radler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810. (20) Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. R. Science 1998, 281, 78. (21) Dan, N. Biophys. J. 1996, 71, 1267. (22) Bandyopadhyay, S.; Tarek, M.; Klein, M. L. J. Phys. Chem. B 1999, 103, 10075.
Figure 1. Light micrograph of poly(dG-dC)‚poly(dG-dC)cationic liposome (DOTMA/DOPE) complexes. Table 1. Frequencies and Vibrational Assignments of Raman Spectra of poly(dG-dC)‚Poly(dG-dC) in Neat Condition and in Cationic Liposomes band position of poly(dGdC)‚poly(dGdC) (cm-1)
a
neat
in DOTMA/DOPE
assignmenta
594
594
681 781 1097 1175 1240 1260 1319 1362 1483 1577
681 781 1097 1175 1240 1260 1319 1362 1483 1577
C G (C3′-endo/syn) G (C2′-endo/anti) C/PO2- backbone PO2- backbone G C C G G G G
G, guanine; C, cytosine.
that cationic lipids condense DNA due to their electrostatic interactions.11,12 The same phenomenon was also observed on various charged surfaces.14-18 Recently, the structure of DNA complexed with the cationic lipid, dioleoyl trimethylammonium propane (DOTAP), and the helper lipid, dioleoyl phosphotidylethanolamine (DOPE), was probed by synchrotron X-ray diffraction in solution.19,20 In this (23) Twardowski, J.; Anzenbacher, P. Raman and IR Spectroscopy in Biology and Biochemistry; Ellis Horwood: New York, 1994. (24) Segers-Nolten, G. M. J.; Sijtsema, N. M.; Otto, C. Biochemistry 1997, 36, 13241. (25) Benevides, J. M.; Thomas Jr., G. J. Nucleic Acids Res. 1983, 11, 5747.
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Figure 2. Raman spectra of (a) poly(dG-dC)‚poly(dG-dC)cationic liposome complex, (b) neat poly(dG-dC)‚poly(dG-dC), and (c) neat cationic liposome.
study, DNA was found to be intercalated between cationic lipid bilayers, where the electrostatic interactions between cationic lipid headgroups and anionic DNA phosphate groups promoted lateral phase separation of DOTAP- and DOPE-rich phases within the complex. The result of this study is consistent with theoretical modeling.21,22 Although Raman spectroscopy offers a sensitive probe for DNA conformation changes by analyzing the characteristic vibrational frequencies,23-25 this technique has not been explored extensively to monitor DNA conformations in transfection complexes. Recently, we reported that various DNA conformations in multicationic liposomes were observed using Raman microscopy.13 In this report, we examined the potential application of Raman microscopy to image the distribution of DNA inside lipid-DNA complexes by imaging the Raman intensity of the characteristic DNA guanine peak. A confocal Raman microscope (LabRam, Jobin Yvon/ Horiba) was used to obtain two-dimensional Raman images. The 632.8-nm line of an air-cooled He-Ne laser was injected into an integrated Olympus BX 40 microscope and focused to a spot size of approximately 0.7 µm by a 80× long working distance objective. A holographic notch filter was used to reject the excitation laser line. The
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combination of an 1800 g/mm holographic grating and a 250-µm slit size provided the spectral resolution at 1.8 cm-1. Raman scattering was collected by an air-cooled 1024 × 256 pixel CCD detector. DNA-cationic liposome complexes comprised of poly(dG-dC)‚poly(dG-dC), obtained from Sigma, and N-[1-(2,3dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoyl phosphotidylethanolamine (DOPE) (1 mg/mL, 1:1 (w/w), Life Technologies) were used as a model transfection system. The liposomes were prepared in membrane-filtered water by bath sonication (Fisher Scientific FS20) for 30 min at room temperature and constant power output in closed centrifuge tubes. The poly(dG-dC)‚poly(dG-dC) was dissolved in 200 µL of water, pH 7.0, to obtain a concentration of 1.47 mg/mL. Transfection complexes were prepared by sonicating the poly(dG-dC)‚poly(dG-dC) solution with the DOTMA/DOPE liposome solution (1:1 charge ratio) for 30 min, followed by incubation at 4 °C overnight. Figure 1 is a light micrograph of the poly(dG-dC)‚poly(dG-dC)-cationic liposome complexes whose sizes vary between 500 nm and 5 µm in diameter. Parts b and c of Figure 2 show Raman spectra of poly(dG-dC)‚poly(dG-dC)-liposome complexes and neat poly(dG-dC)‚poly(dG-dC), respectively. The majority of the vibrational modes of poly(dG-dC)‚poly(dG-dC) appears in the poly(dG-dC)‚poly(dG-dC)-liposome complex spectrum. No major frequency shifts between these spectra were observed. The vibrational assignments are summarized in Table 1. This comparison indicates that poly(dG-dC)‚poly(dG-dC) does not undergo major conformation changes inside the DOTMA/DOPE liposome complex. Figure 2c shows the Raman spectrum of neat DOTMA/ DOPE liposomes. Two major peaks in the neat cationic liposome spectrum at 1442 and 1654 cm-1 are also observed in the poly(dG-dC)‚poly(dG-dC)-liposome complex spectrum. Recent experimental and theoretical studies in DNAcationic lipid complexes show that electrostatic interactions between cationic headgroups of the liposome complexes and anionic DNA phosphate groups promote DNAliposome complexation.22 Because strong intermolecular interactions should cause large vibrational frequency shifts that are detectable in Raman spectra, the lack of vibrational frequency shifts indicates that electrostatic interactions between the poly(dG-dC)‚poly(dG-dC) phosphates and the cationic liposome ammonium headgroups are much weaker than other intermolecular interactions.
Figure 3. Raman imagings of the poly(dG-dC)‚poly(dG-dC)-cationic liposome (DOTMA/DOPE) complex in a dotted square area in Figure 1: (a) Raman intensity mapping of a reference peak at 680 cm-1 for the poly(dG-dC)‚poly(dG-dC); (b) Raman intensity mapping of a reference peak at 1654 cm-1 for the DOTMA lipid molecules.
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Distribution of poly(dG-dC)‚poly(dG-dC) within the DOTMA/DOPE liposome complex (area inside the dotted square in Figure 1) was imaged by mapping the Raman intensity of the 680 cm-1 peak in the poly(dG-dC)‚poly(dG-dC) spectrum (Figure 3a), with the Z-axis representing the intensity of the 680-cm-1 peak. The poly(dG-dC)‚poly(dG-dC) molecules are distributed within a 2-µm radius inside the cationic liposome complex, with the concentration of poly(dG-dC)‚poly(dG-dC) molecules increasing significantly toward the core of the cationic liposome. The distribution of DOTMA was also imaged by Raman intensity mapping of the cationic lipid reference peak at 1654 cm-1 (Figure 3b). The cationic lipid molecules are distributed evenly, while the characteristic peak seen in the poly(dG-dC)‚poly(dG-dC) distribution (Figure 3a), is not observed in Figure 3b. The results reported here suggest that poly(dG-dC)‚ poly(dG-dC)-DOTMA/DOPE complexes contain a higher (26) Singhal, A.; Huang, L. Gene transfer in mammalian cells using liposomes as carriers; Birkhauser: Boston, 1994. (27) Thomas, B. N.; Safinya, C. R.; Plano, R. J.; Clark, N. A. Science 1995, 267, 1635.
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amount of poly(dG-dC)‚poly(dG-dC) molecules near the core of the complex. Since DOTMA/DOPE liposomes have been observed to form multilamellar vesicles26 and X-ray studies have shown that the lamellar structure of DNAliposome complexes consists of multiple lipid layers with DNA located between the lipid layers,19,27 our observations may suggest that the core of the complex has a densepacked multilamellar structure. In this report, DNA distribution in the DNA-liposome complex was imaged by Raman microscopy. Recently, we reported that liposomes with high cation density transform DNA from B-form to Z-form.13 Raman microscopy will be used to test whether distributions of various DNA forms can be imaged in highly cationic liposome vehicles. Acknowledgment. This work was supported by the University of Central Florida, Office of the Vice President for Research and Graduate Studies, I-4 Matching Fund. H.M. acknowledges Professor Harry Price (University of Central Florida) for useful discussions about lipid-DNA complex structure. LA000437K
