Interaction between Plasmid DNA and Cationic Polymers Studied by

Anal. Chem. 1999, 71, 801-805

Interaction between Plasmid DNA and Cationic Polymers Studied by Surface Plasmon Resonance Spectrometry Thijs Wink,*,† Joris de Beer,† Wim E. Hennink,‡ Auke Bult,† and Wouter P. van Bennekom†

Department of Pharmaceutical Analysis and Human Toxicology and Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Pharmacy, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands

A universal and novel strategy for the immobilization of polymers has been developed for studying the interaction between plasmid DNA and synthetic polymers with surface plasmon resonance spectrometry. The introduction of thiol moieties in polymers has been applied for a reliable determination of apparent kinetic rate constants between plasmid DNA and the nonviral carrier polymers. Thiolated poly(L-lysine) and poly[(2-dimethylamino)ethyl methacrylate] yielded reproducible sensor surfaces, contrary to the nonthiolated polymers. The knowledge of the kinetic parameters may play a crucial role in the development of nonviral carrier systems for gene therapy, because the dissociation rate constant is strongly correlated to the effectiveness of cell transfection. In surface plasmon resonance (SPR) spectrometry,1-4 the interaction process between unlabeled (bio)molecules is studied, while one of the binding partners is immobilized upon a sensor surface. SPR monitors the refractive index (n) in the so-called evanescent field layer, which penetrates only a few hundred nanometers into the solution from the sensor surface. An experimental setup is shown in Figure 1. The reflected light intensity (IR) is measured as a function of the angle of incidence θi, and a sharp minimum in IR is observed at the resonance angle θres (see inset, Figure 1). A change in n at the gold surface results in a shift of the resonance angle (∆θres), which is almost linearly proportional to the amount of (bio)molecules bound. In gene therapy, genetic material (e.g., plasmids that encode for a therapeutic protein) is delivered into a cell for the treatment of a variety of diseases.5,6 Its net negative charge prevents DNA from crossing a biological membrane. Interactions of DNA with * Corresponding author: (e-mail) [email protected]. † Department of Pharmaceutical Analysis and Human Toxicology. ‡ Department of Pharmaceutics. (1) Kretschmann, E.; Raether, H. Z. Naturforsch. 1968, 23A, 2135-2136. (2) Raether, H. Surface Plasmons on Smooth and Rough Surfaces and on Gratings; Springer-Verlag: Berlin, 1988. (3) Salamon, Z.; Macleod, H. A.; Tollin, G. Biochim. Biophys. Acta 1997, 1331, 117-129. (4) Salamon, Z.; Macleod, H. A.; Tollin, G. Biochim. Biophys. Acta 1997, 1331, 131-152. (5) Tomlinson, E.; Rolland, A. P. J. Controlled Release 1996, 39, 357-372. (6) Wagner, E.; Curiel, D.; Cotten, M. Adv. Drug Delivery Rev. 1994, 14, 113135. 10.1021/ac980679d CCC: $18.00 Published on Web 01/20/1999

© 1999 American Chemical Society

small counterions,7 (model)proteins,8 drugs,9 cationic liposomes,10,11 cationic lipids,12,13 and cationic polymers14-16 have been tested extensively for possible application in gene therapy. Prerequisites are that DNA must (1) have affinity for the carrier to effectively cross the cell membrane and, moreover, (2) dissociate sufficiently in the cell. For the rational design of nonviral carriers, detailed knowledge of the DNA-carrier interaction is of paramount importance. Polymers readily immobilize upon a (bare) gold surface by means of physical adsorption. A disadvantage is the impossibility of creating a reproducible, well-defined sensor surface, because the polymer will randomly be attached. An improvement for the immobilization process is expected by exploiting the high affinity of thiols for gold.17-20 Recent articles describe the attachment of polymers21,22 and DNA23,24 upon self-assembled alkanethiol monolayers. Also, thiolated DNA25,26 and biotinylated DNA, bound to (7) Stigter, D. Biophys. J. 1995, 69, 380-388. (8) Zama, M.; Ichimura, S. Biochem. Biophys. Res. Commun. 1971, 44, 936942. (9) Fritzsche, H. Nucleic Acids Res. 1994, 22, 787-791. (10) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810814. (11) Dan, N. Biophys. J. 1997, 73, 1842-1846. (12) Zuidam, N. J.; Barenholz, Y. Biochim. Biophys. Acta 1998, 1368, 115-128. (13) Mahato, R. I.; Rolland, A.; Tomlinson, E. Pharm. Res. 1997, 14, 853-859. (14) Cherng, J.-Y.; Van de Wetering, P.; Talsma, H.; Crommelin, D. J. A.; Hennink, W. E. Pharm. Res. 1996, 13, 1038-1042. (15) Van de Wetering, P.; Cherng, J.-Y.; Talsma, H.; Hennink, W. E. J. Controlled Release 1997, 49, 59-69. (16) Ganachaud, F.; Elaı¨ssari, A.; Pichot, C.; Laayoun, A.; Cros, P. Langmuir 1997, 13, 701-707. (17) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481-4483. (18) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (19) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (20) Wink, Th.; Van Zuilen, S. J.; Bult, A.; Van Bennekom, W. P. Analyst 1997, 122, 43R-50R. (21) Advincula, R.; Aust, E.; Meyer, W.; Knoll, W. Langmuir 1996, 12, 35363540. (22) Frey, B. L.; Corn, R. M. Anal. Chem. 1996, 68, 3187-3193. (23) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (24) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-4956. (25) Piscevic, D.; Lawall, R.; Veith, M.; Liley, M.; Okahata, Y.; Knoll, W. Appl. Surf. Sci. 1995, 90, 425-436. (26) Caruso, F.; Rodda, E.; Furlong, D. N.; Haring, V. Sens. Actuator B 1997, 41, 189-197.

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Figure 1. Experimental setup for SPR measurements. The inset shows the shift in resonance angle when plasmid DNA forms a complex with the immobilized polymer.

an avidin-functionalized self-assembled monolayer,27,28 have been used in hybridization studies. In the present paper, we utilize SPR spectrometry for studying cationic polymer-DNA interactions. Emphasis will lie on the interaction of the pCMV-LacZ plasmid DNA and a synthetic polymer (poly[(2-dimethylamino)ethyl methacrylate], pDMAEMA), which has proven to be an effective vector in gene transfection systems.14 Poly(L-lysine) is used as a reference. It is shown that by partial thiolation of a polymer a reproducible sensor surface is created. Furthermore, the interaction of plasmid DNA at different pH values with these immobilized polymers has been studied. The apparent association and dissociation reaction rate constants ka and kd are determined. EXPERIMENTAL SECTION Chemicals and Materials. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) was purchased from Fluka (Bornem, Belgium) and purified by distillation under reduced pressure. Aminoethyl methacrylate (AEMA) was purchased from Polysciences Inc. (Warrington, Pa). Poly(L-lysine) hydrobromide (MW 36.600) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Sigma (Zwijndrecht, The Netherlands). N-Succinimidyl 3-(2-pyridylthio)propionate was obtained from Pharmacia Biotech (Woerden, The Netherlands). Dithiothreitol (DTT) was obtained from Acros (Beerse, Belgium). (27) Caruso, F.; Rodda, E.; Furlong, D. N. Anal. Chem. 1997, 69, 2043-2049. (28) Okahata, Y.; Kawase, M.; Niikura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288-1296.

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Figure 2. Structure of 5%-thiolated pDMAEMA.

HEPES-buffered saline (HBS) contained 20 mM HEPES and 150 mM NaCl. The pH was adjusted by either HCl or NaOH to 5.4, 7.4, or 8.8. Index-matching fluid (Cargille Labs, Inc., Cedar Grove, NJ) and sensor disks (gold) were purchased from Intersens Instruments (Amersfoort, The Netherlands). The sensor disks were stored under nitrogen at room temperature. Thiolated Polymer Synthesis. A copolymer of DMAEMA and aminoethyl methacrylate hydrochloride salt (ratio of DMAEMA to AEMA is 95:5 (mol/mol) (see Figure 2)) was prepared essentially as described by Van de Wetering et al.15 The primary

Figure 3. Binding curves of (a) nonthiolated poly(L-lysine) and (b) nonthiolated pDMAEMA and (c, d) 5%-thiolated poly(L-lysine) and pDMAEMA, respectively, onto a bare gold sensor surface. m°, millidegrees.

amino groups of AEMA were derivatized with N-succinimidyl 3-(2pyridylthio)propionate and reduced with DTT, yielding 5%-thiolated pDMAEMA. The copolymer solution was purged with nitrogen and stored at 4 °C under nitrogen. Poly(L-lysine) was derivatized, yielding 5%, in a similar procedure. All SPR experiments were performed with freshly derivatized polymers. Preparation of Plasmid. The plasmid pCMV-LacZ, containing a bacterial LacZ gene preceded by a nuclear localization signal under control of a CMV promoter, was amplified and purified as described by Cherng et al.14 SPR Measurements. The experiments were performed with an IBIS (Intersens Instruments, Amersfoort, The Netherlands). The configuration is presented in Figure 1. Via a hemicylindrical lens, p-polarized laser light (λ ) 670 nm) was directed to the bottom side of the sensor surface and the intensity of the reflected light was detected by a photodiode. The angle of incidence was varied using a vibrating mirror with a frequency of 44 Hz. In each cycle, SPR curves were scanned on the forward and backward movement of the mirror. In an adjustable interval time, the minimums in reflectance were determined and averaged. Binding curves were obtained by processing this minimum in reflectance in real time. Between the sensor disk and the lens, index-matching fluid was used to obtain an optical entity. The small inner diameter of the cuvette (∼5 mm) in relation to the diameter of the gold surface (∼17 mm) made it possible to perform measurements upon five nonoverlapping spots.29 During measurements, the solution was constantly mixed. The temperature was kept constant at 25.0 ( (29) Wink, Th.; Van Zuilen, S. J.; Bult, A.; Van Bennekom, W. P. Anal. Chem. 1998, 70, 827-832.

0.1 °C. No attempts were performed to regenerate the sensor disks. Sample solutions were introduced to the sensor surface using an autosampler and injected by means of a controllable automatic aspirate-dispense-mixing pipet (Figure 1). Nonthiolated and thiolated poly(L-lysine) and pDMAEMA were immobilized upon the sensor surface through physical adsorption. In the initial adsorption experiments, the cuvette was filled with 100 µL of polymer solution (50 µg/mL in 20 mM HBS). Each polymer was allowed to adsorb until equilibrium (30-60 min). Then, unbound polymer was washed away with HBS buffer. The reversibility of the polymer/DNA binding was investigated, using a homemade flow-through cuvette (to be published) in a flow injection experiment. The 5%-thiolated pDMAEMA in HBS (pH 7.4) was immobilized upon the sensor surface, and 50 µL of a 20 µg/mL plasmid DNA solution was injected. The running buffer was replaced by HBS (pH 8.8). Again, after changing the running buffer to HBS (pH 7.4), DNA was injected. Plasmid Binding Study. The polymers were adsorbed overnight onto the gold surface. After washing in buffer, the disks were mounted upon the lens. Plasmid DNA (pCMV-LacZ), dissolved in concentrations between 2 and 10 µg/mL in HBS buffer, pH 7.4, was allowed to interact with the immobilized polymer. Also, experiments were carried out at different pH. From the binding curves, the kinetic parameters were determined using the (β release of the) Kinetic Evaluation software, version 2.0, from Intersens Instruments. Thickness Calculations. The thickness of the polymer and plasmid DNA layers was calculated according to the theoretical approximations of the dispersion relation of surface plasmon Analytical Chemistry, Vol. 71, No. 4, February 15, 1999

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Figure 4. Interaction plots of plasmid DNA with (a) nonthiolated poly(L-lysine) and (b) 5%-thiolated pDMAEMA. m°, millidegrees. Table 1. Measured Average Shift in Resonance Angle (in mdeg) and Calculated Average Thickness (in Å) of the Polymer Layer upon the Sensor Surface

Figure 5. Reversibility of plasmid DNA/pDMAEMA complex formation. After the pH of HBS buffer is changed from 7.4 to 8.8, the complex dissociates. m°, millidegrees.

oscillations as described by Pockrand,30 while the index of refraction (n) should be estimated. RESULTS AND DISCUSSION Immobilization Experiments of the Polymers. The adsorption curves of various polymers in HBS (pH 7.4) are presented in Figure 3. Figure 3a shows the typical, nonreproducible immobilization of nonthiolated poly(L-lysine). The amount of bound polymer, after a washing procedure, shows large differences. Also, an increase in signal was observed, probably due to rearrangement of the polymer upon the surface. Comparable irreproducibility was found for nonthiolated pDMAEMA (Figure 3b). In contrast, thiolated polymers were more orderly immobilized (Figure 3c and d). The variation in the amount of bound 5%-thiolated pDMAEMA was relatively small and for 5%-thiolated poly(L-lysine) the difference was even smaller. From these figures, it can be concluded that 5% thiolation of the polymers greatly improved the immobilization reproducibility. Plasmid DNA Binding Experiments. Next, we investigated the interaction between plasmid DNA and an overnight immobilized polymer layer. Figure 4a shows the interaction with nonthiolated poly(L-lysine), which follows an irregular binding pattern. At higher concentrations of plasmid DNA, stacked layers were formed. The interaction of plasmid DNA with 5%-thiolated pDMAEMA (Figure 4b) shows a straightforward pattern. Binding (30) Pockrand, I. Surf. Sci. 1978, 72, 577-588.

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polymer

av shift ( SD

n

av thickness ( SD

nonthiolated poly(L-lysine) 5%-thiolated poly(L-lysine) nonthiolated pDMAEMA 5%-thiolated pDMAEMA

219 ( 47 215 ( 1 206 ( 38 237 ( 21

4 3 4 3

10.6 ( 2.3 10.4 ( 0.05 10.0 ( 1.8 11.5 ( 1.0

of plasmid DNA to pDMAEMA or poly(L-lysine), when dissolved in HBS, pH 7.4 or pH 5.4, showed no substantial differences. However, in HBS at pH 8.8, almost no binding of plasmid DNA with pDMAEMA was observed, even up to a concentration of 10 µg/mL. The explanation is that at pH 8.8 this polymer is almost uncharged (pKa of pDMAEMA ∼7.5),31 so the electrostatic interaction with plasmid DNA is insignificant, while lowering the pH value increases interaction forces. A less pronounced influence on the binding with poly(L-lysine) (pKa ∼ 10.0)32 was observed, even when the pH was raised to 12. The interaction between plasmid DNA and 5%-thiolated pDMAEMA was investigated using an experimental flow injection setup33 coupled with the SPR apparatus. The results are shown in Figure 5. Raising the pH caused a dramatic effect upon the interaction. Also, plasmid DNA could repeatedly interact with the polymer. A drift in baseline was observed after changing the pH of the running buffer from 8.8 to 7.4, probably due to slow swelling of the polymer. Calculated Thickness of the Polymer and Plasmid Layers. The calculated average thickness of the polymer layers is summarized in Table 1, assuming n ) 1.49.34 The maximum shift of plasmid DNA is ∼300 mdeg, corresponding to an increase in thickness of 19 Å (here, n ) 1.45 for plasmid DNA19). This is in agreement with the thickness found by Caruso et al.26 and Piscevic et al.25 Apparent Association and Dissociation Rate Constants. From the experimental binding curves of plasmid DNA and the (31) Van de Wetering, P.; Zuidam, N. J.; Van Steenbergen, M. J.; Van der Houwen, O. A. G. J.; Underberg, W. J. M.; Hennink, W. E., submitted for publication. (32) Angeles Garcı´a del Vado, M.; Echevarrı´a Gorostidi, G. R.; Santos Blanco, J. G.; Garcı´a Blanco, F. J. Mol. Catal. 1997, 123, 9-13. (33) Hoogvliet, J. C.; Reijn, J. M.; Van Bennekom, W. P. Anal. Chem. 1991, 63, 2418-2423. (34) Brandrup, J.; Immergut, E. H. Polymer Handbook, 3rd ed.; Wiley: New York, 1989; p VI/455.

Table 2. Calculated Apparent Rate Constants and Affinity of Various Polymers with Plasmid DNA in HBS, pH 7.4 at 25.0 ˚C polymer

ka ( SD (M-1 s-1)

kd ( SD (s-1)

KA ( SD (M-1)

nonthiolated poly(L-lysine) 5%-thiolated poly(L-lysine) nonthiolated pDMAEMA 5%-thiolated pDMAEMA

1.0((0.2) × 107 5.8((0.01) × 106 9.2((0.3) × 106 4.6((0.4) × 106

1.2((0.1) × 10-3 4.4((0.2) × 10-4 1.4((0.1) × 10-2 1.2((0.1) × 10-2

8.3((0.2) × 109 1.3((0.2) × 1010 6.5((0.3) × 108 3.8((0.4) × 108

apparent affinity (KA) is hardly affected upon thiolation for both polymers. The differences in pKA values of the polymers explain the higher KA value for poly(L-lysine) at pH 7.4. These experiments demonstrate that pDMAEMA has a higher kd compared to poly(L-lysine), which may explain the substantial higher transfection efficiency in gene therapy.14 This is in agreement with an improvement of transfection efficiency by reducing the number of positive charges in poly(L-lysine), as described by Erbacher et al.37

Figure 6. Plot of ks () ka[plasmid DNA] + kd) versus concentration of plasmid DNA for the polymers studied.

various polymers, we were able to determine the apparent association and dissociation rate constants. The results are presented in Figure 6. It was our aim to quantitatively compare the interaction kinetics of plasmid DNA with either poly(L-lysine) or pDMAEMA, being aware of the restriction of the method.35 Data were fitted using a biphasic association model.36 The apparent ka, kd, and affinity constant KA () ka/kd) are summarized in Table 2. Although standard deviations in Table 2 seem comparable, it is not quite realistic. Using the kinetic evaluation software, entire interaction curves of plasmid DNA on both thiolated polymers could be fitted for all data points over a period up to 3600 s. Data of nonthiolated polymers could be fitted only in a very critical interval, the first ∼100 s, making results considerably less reliable. For nonthiolated and both thiolated polymers, it is seen in Table 2 that the apparent association rates (ka) have comparable magnitudes. Upon thiolation, a reduction of a factor ∼2 is observed. The dissociation rate (kd) of poly(L-lysine) is somewhat more influenced upon thiolation than pDMAEMA. The dissociation rates of poly(L-lysine) and pDMAEMA are lowered upon thiolation, indicating the formation of a more stable complex between plasmid DNA and immobilized polymer. Surprisingly, the (35) Schuck, P.; Minton, A. P. Anal. Biochem. 1996, 240, 262-272. (36) O’Shannessy, D. J.; Brigham-Burke, M.; Soneson, K. K.; Hensley, P.; Brooks, I. Methods Enzymol. 1994, 240, 323-349. (37) Erbacher, P.; Roche, A. C.; Monsigny, M.; Midoux, P. Biochim. Biophys. Acta 1997, 1324, 27-36.

CONCLUSIONS It is demonstrated that a thiol-derivatized polymer, in contrast to a nonthiolated polymer, is immobilized in a reproducible manner upon a gold surface. This is of paramount importance for reliable determination of apparent association and dissociation rate constants between immobilized polymer and plasmid DNA. The binding processes of plasmid DNA onto an immobilized polymer layer can be monitored with SPR. The calculated average thickness of the polymer layers was ∼10 Å (see Table 1), and for plasmid DNA 19 Å, which is in good agreement with the expected thickness for a monolayer of both polymer and DNA, respectively. Plasmid DNA was bound mainly by means of Coulombic or electrostatic interaction. When the polymer was uncharged at higher pH, association with plasmid DNA was not observed. With the presented immobilization method, the binding kinetics of plasmid DNA and cationic polymers can be studied. Therefore, the development of nonviral carriers, based upon modulation of the dissociation rate constant, can beneficially use this method. ACKNOWLEDGMENT Dr. W. N. E. van Dijk-Wolthuis and Dr. N. J. Zuidam from the Department of Pharmaceutics, Faculty of Pharmacy, Utrecht University, are acknowledged for preparation of the polymers and plasmid DNA, respectively. We gratefully thank Ing. P. J. H. J. van Os and S. J. A. van Boetzelaer from the Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, for the flow injection experiments. Received for review June 23, 1998. Accepted December 2, 1998. AC980679D

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