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Highly Compacted DNA-Polymer Complexes Obtained via New Polynorbornene Polycationic Latexes with Lactobionate Counterion S. Franceschi,§ O. Bordeau,§ C. Millerioux,§ E. Perez,§ P. Vicendo,§ I. Rico-Lattes,§,* and A. Moisand† Laboratoire I.M.R.C.P., UMR CNRS No 5623, Universite´ Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France, and Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 route de Narbonne, 31077 Toulouse Cedex, France Received July 25, 2001. In Final Form: October 21, 2001 Receptor-mediated gene transfer is an attractive method for the targeting of DNA in specific tissues. Membrane lectins have been found at the surface of many cells and are good candidates for gene transfer by DNA-glycoconjugate complexes as specific carriers. However, to be useful for gene therapy, it is critical that the DNA be complexed and aggregated in very small particles. We describe here the properties of a new polycationic polynorbornene, with lactobionate counterions, that spontaneously forms small and stable latexes in water. Furthermore, because of lactobionate effect, the complexation with DNA lead to highly compacted aggregates with a diameter of around 10-20 nm. By the use of transmission electron microscopy, gel electrophoresis, and zeta potential measurements, we have studied both polymer organization and DNA-polymer complex formation.
Introduction The introduction of a foreign gene in a cell is of great interest for gene therapy. Many viral and nonviral techniques are available to deliver genes. Virus-transfection techniques use either retroviruses or adenoviruses.1-3 However the immunogenic effect, causes the main limits for these transfection methods. The risks of viral-based vectors have led to the development of various synthetic vectors including cationic lipids,4-7 linear cationic polymers,8-10 and branched cationic polymers such as polyethylenimine11 and polyamidoamines.12 Rational chemical design of improved vectors requires a better knowledge of the multistage process by which the nonviral vectors promote transgene expression in eucaryotic cells. Initially there is an interaction of the synthetic vector with DNA to form the complex which may penetrate into the cell via an endocytosis pathway.13 * To whom correspondence should be addressed. Tel: (33) 05 61 55 62 70. fax: (33) 05 61 55 81 55. email:
[email protected]_tlse.fr. § Laboratoire I.M.R.C.P., Universite Paul Sabatier. † Institut de Pharmacologie et de Biolgie Structurale, CNRS. (1) Crystal, R. G. Science 1995, 270, 404 and references therein. (2) Wilson, J. M. New Engl. J. Med. 1996, 334, 1185. (3) Michael, S. I.; Curiel, D. T. Gene therapy 1994, 1, 223 and references therein. (4) Behr, J.; Demeneix, B.; Loeffler, J.; Perez-Mutul, J. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 69982. (5) Schwartz, B.; Benoist, C.; Abdallah, B.; Scherman, D.; Behr, J. P. Hum. Gene Ther. 1995, 6, 1515. (6) Felgner, P. L.; Tsai, Y. J.; Sukhu, L.; Wheeler, C. J.; Maathorpe, M.; Marshall, J.; Cheng, S. H. Ann. N. Y. Acad. Sci. 1995, 772, 126. (7) Remy, J. S.; Kichler, A.; Mordvinov, V.; Schuber, F.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1744. (8) Hansma, H. G.; Golan, R.; Hsieh, W.; Lollo, C. P.; Mullenley, P.; Kwoh, D. Nucleic Acids Res. 1998, 26, 2481. (9) Midoux, P.; Mendex, C.; Legrand, A.; Raimond, J.; Mayer, R.; Monsigny, M.; Roche, A. C. Nucleic Acids Res. 1993, 21, 871. (10) Felgner, P. L. Adv. Drug Delivery Rev. 1990, 5, 163. (11) Boussif, O.; Lezoualc’h, F.; Zanta, M. A., Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297. (12) Tang, M. X.; Redmann, C. T.; Szoka, F. C. J. Bioconjugate Chem. 1996, 17, 703. and references therein. (13) Behr, J. P. Acc. Chem. Res. 1993, 26, 274.
All these processes have been extensively studied in detail and show that complexation and aggregation of DNA in very small particles is the basis for preparing efficient vehicles for gene delivery.13,14 Recently, we described the aggregation properties of a new linear polycationic polynorbornene with acetate counterions.15 This polymer was formed by the coupling of acetic acid and a neutralized ammonium chloride norbornene.16,17 It spontaneously forms stable latexes in water and compacts DNA into very small aggregates because of the counterion effect on the hydrophobic packing of the norbornane units. Receptor-mediated gene transfer is an attractive method to target DNA to specific cellular receptors and/or tissues. Membrane lectins (sugar-specific receptors) have been found at the surface of many cells such as hepatocytes, monocytes, macrophages, lymphocytes, etc. These lectins are good candidates for gene transfer by DNA/glycoconjugate complexes as specific carriers.18,19 Hepatocytes are interesting targets for gene therapy applications because these cells express galactose-specific membrane lectins that may be used as selective receptors to deliver genes into the liver.20,21 To try to improve the compaction of DNA, but also to elaborate a new vehicle for targeted gene transfer into hepatocytes, we investigated the influence of a sugar counterion like lactobionate, a disaccharide with a galactose subunit. (14) Wagner, E.; Cotten, M. Curr. Opin. Biotechnol. 1993, 4, 705. (15) Noe¨l, T.; Franceschi, S.; Perez E.; Vicendo P.; Rico-Lattes I. Langmuir 2000, 16, 8990. (16) Janiak, C.; Lassahn, P. G. J. Mol. Catal. A: Chemical 2001, 166, 193. (17) Serre, E.; Vicendo P.; Perez E.; Noel, T.; Rico-Lattes, I. Langmuir 1999, 15, 6956. (18) Sharon, N. Advances in Immunology; Dixon F. J., Kunkel H. G., Eds; Academic Press: New York, 1983; p 213. (19) Perales, J.; Ferkol, T.; Molas, M.; Hauson, R. W. Eur. J. Biochem.1994, 226, 255. (20) Zanta, M. A.; Bouissif, O.; Adib, A.; Behr, J. P. Biconjugate Chem. 1997, 8, 839. (21) Klink, D. T.; Chao, S.; Glick, M. C.; Scanlin, T. F. Mol. Ther. 2001, 3, 831.
10.1021/la011168g CCC: $22.00 © 2002 American Chemical Society Published on Web 02/01/2002
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Scheme 1. Synthesis of Polycationic Polynorbornene with Lactobionate Counterions
Polymeric9,22 or amphiphilic7,23 carriers bearing covalently bonded sugar residue have already been used for targeted gene transfer. We described here, for the first time, an example of sugar counterions electrostatically bonded to the polymeric backbone. Experimental Section Materials. All the solvents and chemicals were reagent grade and were used without further purification. Agarose was purchase from Sigma and was for routine use. Plasmid pCMV-βgal (7740 base pairs) encoding β galactosidase was a generous gift from Dr. M. Defais (IPBS, Toulouse, France). Plasmid was amplified in Escherichia coli, extracted by alkaline lysis and purified by column separation from a Quiafilter Plasmid Maxi Kit (Quiagen). Plasmid integrity was confirmed by gel electrophoresis in 0.8% agarose. DNA concentration was determined by absorbance at 260 nm (50 µg/(mL/absorbance unit)). Synthesis and Characterization of Polycationic Polynorbornene with Lactobinate Counterions. This polymer was obtained by neutralization, with NaOH, of the polynorbornene with chloride counterions. The amine precursor polymer precipitated as a white powder. 0.5 g of this amine polymer in a methanol solution was then treated with 1.92 g (1.1 eq) of lactobionic acid in solution (MeOH/H2O: 4/1 v/v). The mixture was heated to 40 °C for 6 days. Then, the polymer slowly precipitated and after successive washings with methanol, polynorbornene with lactobionate counterions was isolated in 50% yield as a white powder (Scheme 1). Titration with NaOH solution showed that in this case the degree of quaternisation was 45%. These polymers were characterized as previously reported polynorbornene with acetate counterions:15 1H NMR (D O): δ 0.90-2.90 (m, norbornane), 3.20-5.0 2 (lactobionate part) ppm. 13 C NMR (D2O): δ 61.54-103.93 (lactobionate part), 178.92 (COO-) ppm. Methods. Agarose Gel Electrophoresis. A reaction mixture of 20 µL containing 1 µg of DNA and various amounts of polymers was used to obtain NH3+/PO4- ratios varying from 0.5 to 4.0 DNA/polymer complexes were made (22) Bettinger, T.; Remy, J. S.; Erbacher, P. Bioconjugate Chem. 1999, 10, 558. (23) Fuhrhop, J. H.; Tank, H. Chem. Phys. Lipids 1987, 43, 193.
by adding different solutions of polymer to DNA solubilized in 10 mM phosphate buffer (pH 7.4). After 30 min incubation at room temperature, 5 µL of electrophoresis sample buffer (250mM Hepes pH 7.45, 75% glycerol, 0.005% bromophenol blue) was added. The samples were run through a 0.8% agarose horizontal electrophoresis slab gel, overnight at 20 mA using a 90mM Tris buffer system (pH 8.0). DNA was visualized using ethidium bromide staining. Light Scattering and Zeta Potentials. Observations were performed on a Malvern Instrument Zetasizer 3000. The zeta potentials of DNA-polymer complexes formed with varying amounts of polycationic polynorbornene were determined using an electrophoretic light scattering technique. Measurements were made on complexes in water. For each formulation, mean particle electrophoretic mobility was measured in a thermostatically controlled microelectrophoresis cell equilibrated at 25 °C at a frequency of 1000 Hz. The size and distribution of objects in solution were obtained by measuring the light scattered by particles illuminated with a laser beam (θ ) 90°). Electron Microscopic Measurements. The polymer (C ) 10 µM in norbornane units; phosphate buffer pH 7.4) and DNA-polymer complex solutions were studied by electron microscopy. For the DNA-polymer complexes, the final DNA solution was 10 µM in nucleotide units. Compactions were induced by the addition to the DNA solution, of the polynorbornene with lactobionate counterions. The solutions were studied at NH3+/PO4- ratios varing from 0.5 to 3.0. Before placing the complexes on the TEM grids, grids were glow-discharged (110 mV, 60 s). A drop of sample solution was left on the carbon-coated copper grids for 1 min. Samples were negatively stained with a phosphotungstate solution (1% w/w; pH 7). Observations were performed at 60 kV with transmission electron microscope (Philips EM 301). Results 1- Organization of Polycationic Polynorbornene with Lactobionate Counterions. This polymer was observed by transmission electron microscopy using a staining technique and uniformaly latex particles were obtained. The latex particles had diameters of ap-
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Figure 1. Transmission electron micrographs of lactobionate polynorbornene (10 µM in norbornane units; phosphate buffer pH 7.4).
proximately 20-40 nm for the major part of the population (Figure 1a). Some particles, however, had a larger diameter (approximately 150-300 nm) probably resulting from aggregation of the smaller particles (Figure 1b). Electron microscopy confirmed that these particles are stable upon dilution. They presented the same size distribution at the polymer concentration used for the DNA compaction assay. Light scattering analysis confirmed the largest size aggregates observed by transmisssion electron microscopy had diameters of approximately 35 and 350 nm. The latex particles consisted of a packed core formed by the norbornane units, surrounded by the hydrated lactobionate counterions. This is in agreement with 13C NMR results since by this method we did not observe any signal of free norbornane units.24,25
2- Complexation of DNA by Lactobinate Polynorbornene. Agarose Gel Electrophoresis. The electrophoresis was performed to evaluate the efficiency of the polymer to bind DNA (Figure 2). It was able to inhibit DNA migration on an agarose gel. At a polymer-DNA ratio varying from 0.5 to 1.8, a fraction of the plasmid DNA was still free to migrate in the gel in the same way as the noncomplexed initial DNA (it is clearly visible but reduced in intensity). At a ratio of two, all the DNA was complexed and no longer able to migrate into the gel. Also, up-to-this ratio, no staining of DNA was observed on the gel except in the starting well. By this method, we demonstrated (24) Puech, L.; Perez, E.; Rico-Lattes, I.; Bon, M.; Lattes, A.; Moisand, A. New J. Chem. 1997, 21, 1235. (25) Puech, L.; Perez, E.; Rico-Lattes, I.; Bon, M.; Lattes, A.; Moisand, A. New J. Chem. 1997, 21, 1229.
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Figure 2. Effect of lactobionate polynorbornene on the electrophoretic mobility of pCMV-βgal plasmid: lane 1, Plasmid pCMV-βgal; lane 2 to 11: complexation of DNA with various amounts of polymer at different charge ratios varying from 0.5 to 4. Lane 2, NH3+/PO4- of 0.5; lane 3, 1; lane 4, 1.2; lane 5, 1.4; lane 6, 1.6; lane 7, 1.8; lane 8, 2; lane 9, 2.5; lane 10, 3; lane 11, 4.
NH3+/PO4- ratio of three. The diameter of these highly compacted structures was measured to be approximately 10-20 nm. Discussion
Figure 3. Zeta potential of DNA-polymer complexes as a function of their charge ratio.
that this lactobionate polymer immobilized DNA at an NH3+/PO4- ratio of 2. These results were confirmed by zeta potential measurements. Zeta Potential. The zeta potentials were measured for the polymer and for the polymer-DNA complexes in water. For the polymer, the value was +54 mV. The zeta potential of polymer-DNA complexes at different NH3+/PO4- ratios is shown in Figure 3. This curve indicates a sigmoı¨d variation of the resulting complex from a negative charge (DNA alone) to a positive charge as the quantity of polymer is increased. The curve is consistent with expectation; being negative at low polymer ratios, passing from zero at an NH3+/PO4- ratio of 1.9 and rising to a maximum of +25 mV above a charge ratio of three. These data indicate that there was very little free polycation present; almost all of the polycation became complexed with DNA until the negative charge of the DNA had been neutralized. In fact, the positive charge excess of the DNA-polymer complex is critical to allow interaction with the negatively charged cell membranes for its entrance into cells. Therefore, to enable specific gene transfer mediated by lactobionate counterions into hepatoma cells, it is again necessary to maintain an excess positive charge (lactobionate counterions). So, to improve the efficiency of the transfection, the DNA-polymer ratio is generally maintained above neutralization. Electron Microscopy. Lactobionate polymer-DNA complexes were characterized using transmission electron microscopy at NH3+/PO4- ratios varying from 0.5 to 3.0. No changes in the size or the shape of the particles were observed. Figure 4 shows spherical dense particles uniformly dispersed corresponding to these complexes at an
Organization of Lactobionate Polycationic Polynorbornene. In agreement with recent results obtained with acetate counterions,15 lactobionate counterions also enabled the spontaneous formation of small particles of latexes in water. Similar results were already described in our laboratory with polynorbornene bearing covalently bonded sugar residues.24,25 In all these cases, the latexes are spherical particles with packed cores formed by the norbornane units, surrounded by hydrophilic moieties (sugar or acetate). However, in the case of the lactobionate counterions, the particles are much smaller ( mainly 2040 nm) than those obtained with the acetate counterion (90 and 330 nm). This strong decrease in the size of the latexes may result from the combination of both the size and the degree of hydration of the two counterions.26-28 By analogy with the influence of counterion on the size of the micellar aggregate; the smaller the counterion is, the stronger the attachment to the polymer and the greater the aggregating power.26-28 Thus, if hydration was not involved, the latexes should increase in size as the counterion becomes smaller. Furthermore, this phenomenon is increased by the large difference of hydration between the two counterions. In the case of micelles, it was observed that hydrated ions are less closely attached to the aggregates than unhydrated ones and therefore contribute less to their formation. Lactobionate is a large and very hydrated counterion, so the particles of lactobionate polynorbornene do not grow as large as the polymer with acetate counterion and we observed very small dense particles. This counterion effect was already observed in the difference of packing between an ammonium chloride polynorbornene and an ammonium acetate polynorbornene, with no latex formation with chloride counterions.17 2- DNA Complexation with Polymer. Changing the counterions also has an important effect on the size of DNA-polymer complexes. The complexes formed with lactobionate polynorbornene were the smallest to be obtained without having to use the salt effect.29-32 Such methods have been described by some authors to obtain (26) Anacker, E. W.; Ghose, H. M. J. Phys. Chem. 1963, 67, 1713. (27) Anacker, E. W.; Ghose, H. M. J. Am. Chem. Soc. 1968, 90, 3161. (28) Wagner, K.; Harries, D.; May, S.; Kahl, V.; Ra¨dler, J. O.; BenShaul, A. Langmuir 2000, 16, 303. (29) Pollard, H.; Remy, J. S.; Loussouarn, G.; Demolombe, S.; Behr, J. P.; Escande, D. J. Biol. Chem. 1998, 273, 7507. (30) Ferkol, T.; Perales, J. C.; Mularo, F.; Hanson, R. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 101.
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Figure 4. Transmission electron micrograph of DNA-polymer complexes at an NH3+/PO4- ratio of 3 (phosphate buffer pH 7.4).
poly(L-lysine)-DNA complexes with small diameters of approximately 10-30 nm.30 Compaction of DNA in very small particles is fundamental for in-vivo targeted gene transfer and efficient internalization into cells. It was observed that when the size of the DNA-complex particles exceeded the limit for efficient transport through the capillary system, the DNA was taken up by macrophages from the spleen and the liver.19 Also, the size of the DNA-complex is critical in the design of a DNA delivery vehicle for receptor-mediated endocytosis, because receptors are not able to internalize ligands above certain size limits.19 In the case of the polymer-DNA complex, it is more difficult to explain the role of the lactobionate ion on the size of the particles. Before complexation with DNA, the diameter of the particles is approximately 20-40 nm and decreases to 10-20 nm after complexation. This phenomenon may be partly explained by the fact that lactobionate is a large and hydrated counterion weakly attached to the polymer and can be easily exchanged, leading to a better interaction with DNA. Also, the complexation started from small polymer aggregate and consequently the interaction with DNA was more direct when facilitating the reorganization of the complex into smaller particles. (31) Wolfert, M. A.; Seymour, L. W. Gene Therapy 1996, 3, 269. (32) Dunlap, D. D.; Maggi, A.; Soria, M. R.; Monaco, L. Nucl. Acids Res. 1997, 25, 3095.
Conclusion We have synthesized a new polycationic polynorbornene with lactobionate counterions. It was obtained after neutralization of an ammonium-chloride polynorbornene and reaction with lactobionic acid. This lactobionate polymer spontaneously forms stable latexes in water with a diameter of approximately 20-40 nm. Lactobionate is a large and highly hydrated counterion and is thus less closely attached to the polymer backbone so the particles cannot grow as large as the acetate polymer. Also, because lactobionate is weakly attached to the polymer, the latexes are very small and the complexation with DNA leads to highly compacted aggregates with a diameter of approximately 10-20 nm. Furthermore, the addition of higher concentrations of polymer does not change the size or the shape of the particles. This observation is very important for specific gene transfer into hepatoma cells, it is necessary to maintain a sufficient concentration of lactobionate ions around the particles of complex. Acknowledgment. The authors express their thanks to Dr. M. Defais (IPBS, Toulouse) for the Plasmid pCMVβgal, to Mr. L. Datas for electronic microscopy studies (UPS, Toulouse) and to Mr L. Labadie for physicalchemical measurements. LA011168G
