pubs.acs.org/Langmuir © 2010 American Chemical Society
Interactions between DNA and Poly(amido amine) Dendrimers on Silica Surfaces Marie-Louise Ainalem,*,† Richard A. Campbell,†,‡ and Tommy Nylander† †
‡
Physical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, and Institut Laue-Langevin, 6 rue Jules Horowitz, BP 156, 38042 Grenoble Cedex 9, France Received December 15, 2009. Revised Manuscript Received March 27, 2010
This study increases the understanding at a molecular level of the interactions between DNA and poly(amido amine) (PAMAM) dendrimers on solid surfaces, which is a subject of potential interest in applications such as gene therapy. We have used in situ null ellipsometry and neutron reflectometry to study the structure of multilayer arrangements formed by PAMAM dendrimers of generation 2 (G2), 4 (G4), and 6 (G6) and DNA on silica surfaces. Specifically, we adsorbed cationic dendrimer layers, then we condensed DNA to form dendrimer-DNA bilayers, and last we exposed further dendrimer molecules to the interface to encapsulate DNA in dendrimer-DNA-dendrimer trilayers. The dendrimer monolayers formed initially result in the deformation of the cationic adsorbates as a result of their strong electrostatic attraction to the hydrophilic silica surface. The highest surface excess and most pronounced deformation occurs for the G6 molecules due to their relatively large size and high surface charge density. G6-functionalized surfaces give rise to the highest surface excess of DNA during the bilayer formation process. This result is explained in terms of the high number of charged binding sites in the G6 monolayer and the low electrostatic repulsion between DNA and exposed patches of silica surface due to the relatively thick G6 monolayer. The binding strengths of the silica-dendrimer and dendrimer-DNA interactions are demonstrated by the high stability of the interfacial bilayers during rinsing. For the formation of trilayers of dendrimers, DNA, and dendrimers, G2 adsorbs as a smooth layer while G4 and G6 induce the formation of less well-defined structures due to more complex DNA layer morphologies.
Introduction The potential applications of gene therapy have increased the efforts to find synthetic vectors to carry DNA as efficiently as a viral vector but with lower cytotoxicity.1-3 One promising nonviral vector is the poly(amido amine) (PAMAM) dendrimer which induces DNA condensation through electrostatic interactions.4-7 The size of PAMAM dendrimers increases with generation, Gn (the number of functional primary amine groups is given by 2nþ2), and while higher generation dendrimers adopt a spherical shape in aqueous solutions, lower generation dendrimers are more disk-like.8-12 The efficiency and cytotoxicity are linked to the structure and properties formed upon condensing DNA with the vector in use, which in turn is controlled by the intermolecular interactions. We have previously shown that DNA condensation using PAMAM dendrimers is a cooperative process where the condensing agent has a preference to bind to preformed dendrimer-DNA *Corresponding author. E-mail:
[email protected]. (1) Brown, M. D.; Schatzlein, A. G.; Uchegbu, I. F. Int. J. Pharm. 2001, 229, 1–21. (2) Glover, D. J.; Lipps, H. J.; Jans, D. A. Nat. Rev. Genet. 2005, 6, 299–310. (3) Pouton, C. W.; Seymour, L. W. Adv. Drug Delivery Rev. 2001, 46, 187–203. (4) Bielinska, A. U.; Chen, C. L.; Johnson, J.; Baker, J. R. Bioconjugate Chem. 1999, 10, 843–850. (5) Boas, U.; Heegaard, P. M. H. Chem. Soc. Rev. 2004, 33, 43–63. (6) Braun, C. S.; Vetro, J. A.; Tomalia, D. A.; Koe, G. S.; Koe, J. G.; Middaugh, C. R. J. Pharm. Sci. 2005, 94, 423–436. (7) Dufes, C.; Uchegbu, I. F.; Schatzlein, A. G. Adv. Drug Delivery Rev. 2005, 57, 2177–2202. (8) Lee, I.; Athey, B. D.; Wetzel, A. W.; Meixner, W.; Baker, J. R. Macromolecules 2002, 35, 4510–4520. (9) Naylor, A. M.; Goddard, W. A.; Kiefer, G. E.; Tomalia, D. A. J. Am. Chem. Soc. 1989, 111, 2339–2341. (10) Paulo, P. M. R.; Lopes, J. N. C.; Costa, S. M. B. J. Phys. Chem. B 2007, 111, 10651–10664. (11) Svenson, S.; Tomalia, D. A. Adv. Drug Delivery Rev. 2005, 57, 2106–2129. (12) Tomalia, D. A. Prog. Polym. Sci. 2005, 30, 294–324.
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aggregates rather than free DNA molecules.13,14 The morphology of the formed aggregates is controlled by the interaction between DNA and the surface of the dendrimer. Quantitative data on this interaction is not easily accessible in a bulk solution experiment. Here we have adsorbed the dendrimers on surfaces, which has allowed us to study quantitatively the interaction between the DNA and dendrimers by using in situ null ellipsometry and neutron reflectometry. Ellipsometry allows kinetic tracking of the surface excess and layer thickness with a time resolution of just a few seconds, and neutron reflectometry enables the extraction of the compositional distribution of a surface layer perpendicular to the plane of incidence with A˚ngstr€ om resolution. PAMAM dendrimers affect the integrity of phospholipid bilayers,15-17 and experimental18-20 and theoretical21,22 studies have displayed their deformation into flat disks when adsorbed to solid surfaces, for example, silica. Adsorption of dendrimers is (13) Ainalem, M. L.; Carnerup, A. M.; Janiak, J.; Alfredsson, V.; Nylander, T.; Schillen, K. Soft Matter 2009, 5, 2310–2320. € (14) Orberg, M. L.; Schillen, K.; Nylander, T. Biomacromolecules 2007, 8, 1557– 1563. (15) Ainalem, M. L.; Campbell, R. A.; Khalid, S.; Gillams, R. J.; Rennie, A. R.; Nylander, T. J. Phys. Chem. B 2010, accepted. (16) Erickson, B.; DiMaggio, S. C.; Mullen, D. G.; Kelly, C. V.; Leroueil, P. R.; Berry, S. A.; Baker, J. R.; Orr, B. G.; Holl, M. M. B. Langmuir 2008, 24, 11003– 11008. (17) Mecke, A.; Lee, D. K.; Ramamoorthy, A.; Orr, B. G.; Holl, M. M. B. Langmuir 2005, 21, 8588–8590. (18) Betley, T. A.; Holl, M. M. B.; Orr, B. G.; Swanson, D. R.; Tomalia, D. A.; Baker, J. R. Langmuir 2001, 17, 2768–2773. (19) Li, J.; Piehler, L. T.; Qin, D.; Baker, J. R.; Tomalia, D. A.; Meier, D. J. Langmuir 2000, 16, 5613–5616. (20) Muller, T.; Yablon, D. G.; Karchner, R.; Knapp, D.; Kleinman, M. H.; Fang, H. B.; Durning, C. J.; Tomalia, D. A.; Turro, N. J.; Flynn, G. W. Langmuir 2002, 18, 7452–7455. (21) Konieczny, M.; Likos, C. N. Soft Matter 2007, 3, 1130–1134. (22) Mecke, A.; Lee, I.; Baker, J. R.; Holl, M. M. B.; Orr, B. G. Eur. Phys. J. E 2004, 14, 7–16.
Published on Web 04/29/2010
DOI: 10.1021/la9047177
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governed by electrostatic interactions and the surface excess increases with increasing ionic strength, pH and surface charge which can be explained by a reduction in the interdendrimer electrostatic repulsion.23,24 At higher pH, the dendrimers become less protonated while the charge of the silica surface increases. The same trend is observed for other cationic polyelectrolytes.25,26 Quantitative agreement between theory and experiment has been obtained with the extended random sequential adsorption (RSA) model which involves the electrostatic interactions between two dendrimers and the substrate.27 Dendrimers have, in addition, been used together with oppositely charged polyelectrolytes for the layer-by-layer28 preparation of multilayer films by successive adsorption of the two oppositely charged components.29,30 Multilayers and multilayered microcapsules containing DNA,31-34 as well as DNA and phosphourous dendrimers,35 have also recently been studied. The microcapsules act as promising gene carriers and understanding DNA-surface interactions is critical in, for example, DNA computing.36,37 In the present work, we have studied dendrimer-DNA interactions through the sequential build-up in solution of adsorption layers on planar silica surfaces. Experiments were carried out using the following sequence: (i) exposure to a fresh silica surface of PAMAM dendrimers of a specific generation, (ii) a rinse with pure salt solution, (iii) exposure of linearized plasmid DNA of 4331 basepairs (bp), (iv) another rinse with pure salt solution, (v) a second exposure of dendrimers of the same generation as before, and (vi) a last rinse with pure salt solution. Parallel experiments were carried out involving PAMAM dendrimers of generation 2 (G2), 4 (G4), and 6 (G6). The rinsing steps were important for two reasons: to ensure that dendrimer-DNA aggregates did not preform in solution and then adsorb to the interface (i.e., to constrain the study to interactions of free molecular species in solution with the functionalized surface), and to allow us to gather information on the reversibility of the various interfacial interactions. This work demonstrates the buildup of surface-supported PAMAM dendrimer-DNA trilayers, which is of relevance to potential applications that aim to protect DNA against degradation (as has been observed for aggregates in solution7). To the knowledge of the authors, DNA interactions with surface-supported PAMAM dendrimer layers on either planar or spherical support have not previously been studied. (23) Pericet-Camara, R.; Cahill, B. P.; Papastavrou, G.; Borkovec, M. Chem. Commun. 2007, 266–268. (24) Pericet-Camara, R.; Papastavrou, G.; Borkovec, M. Langmuir 2004, 20, 3264–3270. (25) Meszaros, R.; Thompson, L.; Bos, M.; de Groot, P. Langmuir 2002, 18, 6164–6169. (26) Popa, I.; Cahill, B. P.; Maroni, P.; Papastavrou, G.; Borkovec, M. J. Colloid Interface Sci. 2007, 309, 28–35. (27) Cahill, B. P.; Papastavrou, G.; Koper, G. J. M.; Borkovec, M. Langmuir 2008, 24, 465–473. (28) Decher, G.; Hong, J. D. Makromol. Chem. Macromol. Symp. 1991, 46, 321– 327. (29) Khopade, A. J.; Caruso, F. Langmuir 2003, 19, 6219–6225. (30) Kim, B. S.; Lebedeva, O. V.; Kim, D. H.; Caminade, A. M.; Majoral, J. P.; Knoll, W.; Vinogradova, O. I. Langmuir 2005, 21, 7200–7206. (31) Lu, Z. Z.; Wu, J.; Sun, T. M.; Ji, J.; Yan, L. F.; Wang, J. Biomaterials 2008, 29, 733–741. (32) Pei, R. J.; Cui, X. Q.; Yang, X. R.; Wang, E. K. Biomacromolecules 2001, 2, 463–468. (33) Ren, K. F.; Ji, J.; Shen, J. C. Biomaterials 2006, 27, 1152–1159. (34) Vinogradova, O. I.; Lebedeva, O. V.; Vasilev, K.; Gong, H. F.; GarciaTuriel, J.; Kim, B. S. Biomacromolecules 2005, 6, 1495–1502. (35) Kim, B. S.; Lebedeva, O. V.; Koynov, K.; Gong, H. F.; Caminade, A. M.; Majoral, J. P.; Vinogradova, O. I. Macromolecules 2006, 39, 5479–5483. (36) Liu, Q. H.; Wang, L. M.; Frutos, A. G.; Condon, A. E.; Corn, R. M.; Smith, L. M. Nature 2000, 403, 175–179. (37) Ogihara, M.; Ray, A. Nature 2000, 403, 143–144.
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Ainalem et al. Table 1. Physical Data of PAMAM Dendrimers of Varying Generation, Ga G
M (g mol-1)
nNH3þ
r (A˚)
RH (A˚)
F (g cm-3)
c (μg mL-1)
2 4 6
3256 16 14.5 13.6 1.2 61.7 14 215 64 22.5 24.5 1.2 67.3 58 048 256 33.5 38.0 1.2 68.7 a G denotes the dendrimer generation and nNH3þ specifies the number of primary amines on the dendrimer surface. The geometrical radius as reported by the manufacturer is r. The hydrodynamic radius, RH, is adapted from Ainalem et al. and was obtained using dynamic light scattering.13 The dendrimer density F used was extracted from Kouskoumvekaki et al.41 The final dendrimer concentration used for the buildup of trilayers is given by c and corresponds to 1.83 1017 functional surface groups (charges) per milliliter.
Experimental Section Materials. G2, G4, and G6 PAMAM dendrimers with ethylenediamine cores, dissolved in methanol, were purchased from Sigma. Before use, the methanol was removed under reduced pressure and the dendrimers were resolubilized in aqueous solutions containing the same concentration of ionizable groups, that is 1.83 1017 primary amine functional surface groups per milliliter (which under the conditions used, in agreement with previous studies, are expected to be protonated).38-40 Table 1 lists the dendrimer size, hydrodynamic radius and solution concentration as a function of dendrimer generation. The dendrimer concentrations used result in the maximum surface excess, i.e., a value corresponding to a plateau in the adsorption isotherm. Luciferase plasmid DNA of 4331 bp (Promega) was amplified, linearized and purified as described in detail by Ainalem et al.13 A final DNA concentration of 100 μg mL-1 was used which corresponds to a concentration of 1.83 1017 DNA phosphate groups per milliliter. All experiments were performed in 10 mM sodium bromide (NaBr, Aldrich) using aqueous solutions of Milli-Q purified water and/or D2O from Euriso-top, C. E. Saclay, France for the G2 and G6 experiments at ILL or from ARMAR Chemicals, Switzerland for the G4 experiment at NIST. Ellipsometry. In situ null ellipsometry was performed using an automated Rudolph Research thin-film ellipsometer, type 43603200E equipped with high precision stepper motors and operating at a wavelength of 4015 A˚ with an angle of incidence of 67.96°.42,43 Experimental Procedure. The silica surfaces used as substrates for the adsorption studies were silicon wafers with a thermally grown oxide layer (∼300 A˚) and were provided by Bo Thuner (Department of Chemistry, IFM, Link€ oping University, Sweden). The wafers were cleaned according to the protocols described by Vandoolaeghe et al44 and were subsequently stored in ethanol. The substrates were dried under reduced pressure (0.02 mbar) and treated in an air-plasma cleaner (Harrick Scientific Corp., model PDC-3XG) for 5 min prior to the start of an experiment. The sample cell was a ∼5 mL trapezoidal cuvette of optical glass in which the silica surface was vertically arranged. The experimental setup was equipped with magnetic stirring (300 rpm) and the temperature was 25.0 ( 0.1 °C. A peristaltic pump (Ole Dich Instrument makers ApS, Hvidovre, Denmark) allowed for a ∼5 mL min-1 flow of the electrolyte solution. At the start of (38) Cakara, D.; Kleimann, J.; Borkovec, M. Macromolecules 2003, 36, 4201– 4207. (39) El-Sayed, M.; Kiani, M. F.; Naimark, M. D.; Hikal, A. H.; Ghandehari, H. Pharm. Res. 2001, 18, 23–28. (40) Milhem, O. M.; Myles, C.; McKeown, N. B.; Attwood, D.; D’Emanuele, A. Int. J. Pharm. 2000, 197, 239–241. (41) Kouskoumvekaki, I. A.; Giesen, R.; Michelsen, M. L.; Kontogeorgis, G. M. Ind. Eng. Chem. Res. 2002, 41, 4848–4853. (42) Landgren, M.; Jonsson, B. J. Phys. Chem. 1993, 97, 1656–1660. (43) Tiberg, F.; Harwigsson, I.; Malmsten, M. Eur. Biophys. J. 2000, 29, 196–203. (44) Vandoolaeghe, P.; Rennie, A. R.; Campbell, R. A.; Thomas, R. K.; Hook, F.; Fragneto, G.; Tiberg, F.; Nylander, T. Soft Matter 2008, 4, 2267–2277.
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an experiment, the optical properties of the substrate were determined with high accuracy to obtain reliable measures of the refractive index and thickness of the oxide layer as well as the complex refractive index of Si. This process was accomplished by measuring the ellipsometric angles, Δ and Ψ, of the bare surface in two ambient media with different refractive indices, air and water.43 During the substrate characterization, the instrument was further calibrated using four-zone averaging which was used to correct the recorded data of Δ and Ψ during the measurements.45 Such correction is made to account for defects in the optical configuration of the instrument. Addition of dendrimers or DNA was accomplished by injecting low-volume aliquots of stock solution into the cuvette. Data Evaluation. Our setup and measurement procedure, described in detail elsewhere,42,48 allows measurements of changes to the parameters Δ and Ψ for monomolecular layers adsorbed on oxidized silicon wafers. The data are used to calculate numerically the adsorbed layer thickness and its refractive index. An optical model comprising four layers, Si-SiO2-X-solvent, was used under the assumption that the layer is isotropic and that its boundaries are planar where X corresponds to the adsorbed layer of dendrimer, dendrimer-DNA or dendrimer-DNAdendrimer.42 The surface excess (Γ) was determined using de Feijters formula,45 eq 1, where the mean refractive index (nf) and the mean layer thickness (df) were calculated numerically from the recorded data based on the optical model of homogeneous layers and planar interfaces Γ ¼
ðnf - n0 Þdf dn=dc
ð1Þ
where n0 = 1.3423 which is the refractive index of the bulk solution and dn/dc is the refractive index increment as a function of the bulk concentration. The value of dn/dc at λ = 4015 A˚ used was 0.20 g cm-3 and relates to the experimentally determined value of the dendrimer molecules (independent of generation) at λ = 5893 A˚.46 It is noted that the dn/dc value for DNA is reported to be 0.185-0.197 g cm-3.47 Thus, the assumption that the dn/dc of DNA is the same as for dendrimers introduces only a minor ( G2, which matches the trend in the dendrimer surface excess. The fact that the number of adsorbed particles decreases with increasing dendrimer generation is consistent with the increasing interdendrimer electrostatic repulsion, i.e., the higher charge per unit area of G4 and G6 compared to G2. Here it is noteworthy that also the adsorbed amount of dendrimers on silica from ellipsometry is similar to and agrees well with that obtained by neutron reflectometry. We conclude that the adsorption is likely to be controlled by electrostatic forces which is in agreement with Cahill et al. proposing that the adsorption of dendrimers is best described using the extended three-body RSA model which involves two dendrimers and the charged surface.27 Our interpretation that the affinity to the surface of G2 is the lowest of the systems studied is supported by the large amount of material that leaves the surface during rinsing. The dendrimer surface excess decreased during rinsing for both G2 (pronounced) and G4 (slight) but no change was measured for G6. These observations can be explained as the larger dendrimers have a higher surface charge density and therefore have stronger electrostatic attraction to the silica surface, which is in excellent agreement with Longtin et al., showing that completely reversible adsorption occurred for low generations, low pH values and high ionic strengths, while irreversible adsorption occurred for high generations, high pH values, and low ionic strengths.67 The adsorbed dendrimer layer thickness reduced upon rinsing for G2 but not for G4 or G6. We propose that this further deformation is due to an increase in surface contact after a reduction in the interdendrimer repulsion from the lower surface excess. Bilayer Formation: Adsorption of DNA to a Dendrimer Monolayer on a Silica Surface. The surface excess of DNA bound to dendrimer monolayers on silica surfaces increases with the generation of dendrimer, as shown by the good agreement of results from the two experimental techniques considering the differences in experimental setup and flow conditions. The total adsorbed amount after rinse using ellipsometry (Table 3) is 1.0, 1.9, and 3.6 mg m-2 for G2, G4, and G6, respectively while the corresponding neutron reflectometry data (sum of the corresponding values in Table 6) is 0.85, 2.2, and 3.0 mg m-2. It follows that the larger dendrimers give rise to the more cationically charged surface due to the higher surface density of charged primary amine groups which in turn results in a stronger electrostatic attraction with negatively charged DNA molecules. Here it is important to note that DNA adsorbs to the dendrimer monolayer but not to a hydrophilic silica surface as a result of the charge reversal caused by the adsorption of cationic dendrimer molecules, which is a process observed for other cationic polymers.68 A further reason for the increased surface excess of DNA bound to higher generation dendrimer layers may be the thickness of the dendrimer layer which follows the trend G6>G4>G2; i.e., there is greater separation of the DNA molecules from exposed patches of the similarly charged silica surface for the higher generation dendrimer layers. The total thickness values are in reasonably good agreement for the two techniques used: 56, 56, and 75 A˚ for DNA in the presence of G2, G4 and G6, respectively, from ellipsometry and 26, 60, and 55 A˚ from neutron reflectometry. DNA layers are thicker and more extended for G4 and G6 than for G2 which is a result that might seem unexpected. Here, we should bear in mind that DNA is a semiflexible polyelectrolyte and that surface charge compensation during polyelectrolyte adsorption occurs in a thin layer adjacent to the surface. Thus, (67) Longtin, R.; Maroni, P.; Borkovec, M. Langmuir 2009, 25, 2928–2934. (68) Samoshina, Y.; Nylander, T.; Shubin, V.; Bauer, R.; Eskilsson, K. Langmuir 2005, 21, 5872–5881.
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a high surface charge density (like for the higher generation dendrimers) might kinetically trap the DNA chain in, for example, an extended conformation as has been observed in bulk solutions where the condensation of DNA results in toroidal structures for aggregates when using G2, yet disordered structures for higher generation dendrimers.13,69 The high stability during rinsing of DNA bound to the dendrimer monolayers and indeed of the dendrimer-DNA bilayer bound to the silica surfaces reveals that the electrostatic interactions in play are strong and that that the adsorption can be considered practically irreversible due to slow dynamics.70 Trilayer Formation: Adsorption of Dendrimers to a Dendrimer-DNA Bilayer on a Silica Surface. The driving force for the adsorption of a second dendrimer layer is proposed to be electrostatic and due to charge reversal obtained upon the adsorption of the DNA. The thickness of the second dendrimer layers, formed on dendrimer-DNA bilayers bound to silica surfaces, increases for higher dendrimer generations. On the one hand, the thickness of the G2 layer is comparable to that of the dendrimer monolayers of the equivalent generation on silica surfaces; on the other hand the thickness of the G6 layer is about 4 times higher than after its monolayer adsorption. The adsorbed conformations of G4 and G6 cannot be clearly elucidated due to the high interfacial roughness. This suggests that perhaps due to lateral inhomogeneity in the dendrimer-DNA bilayers, the second addition of dendrimers penetrated into the DNA layer and resulted in the formation of more complex structures. We note that when comparing the total adsorbed amounts from ellipsometry (Table 3) of 2.3, 3.7, and 7.2 mg m-2 for G2, G4 and G6, respectively, with that of 1.8, 4.5, and 6.2 mg m-2 from neutron reflectometry (sum of the corresponding values in Tables 6 and 7), a reasonably good agreement is obtained. The total layer thickness is furthermore 68, 97, and 210 A˚ using ellipsometry and 46, 168, and 163 A˚ using neutron reflectometry. In this case, discrepancies between the measures of total interfacial thickness using the two techniques may be attributed to the fact that it was appropriate to use a one layer model to evaluate the ellipsometry data, while a more elaborate multilayer model could be applied to the neutron reflectometry data. During adsorption processes, the chemical potential near the surface builds up and hinders further adsorption as a result of electrostatic and steric repulsion between incoming components and those already adsorbed. The higher surface excess of dendrimers adsorbed to the dendrimers-DNA bilayer compared with that to the silica surface is consistent with our previous interpretation that the dendrimer surface excess depends on both the dendrimer generation and the surface characteristics; i.e., in this case the number of charged sites on the relatively rough dendrimer-DNA bilayer is higher than that on a planar silica surface which results in an increased adsorption.63,71 The change in layer structure upon rinsing the dendrimer-DNA-dendrimer trilayers was analyzed only using ellipsometry where the layer thickness and surface excess both decreased for all generations. The most pronounced effect was observed with the G6 system for which no change was observed when rinsing the dendrimer monolayer on silica. It follows therefore, given the strong electrostatic interaction of the second dendrimer layer with the dendrimer-DNA bilayer, that rinsing the trilayer structure (69) Carnerup, A. M.; Ainalem, M. L.; Alfredsson, V.; Nylander, T. Langmuir 2009, 25, 12466–12470. (70) Holmberg, K.; J€onsson, B.; Kronberg, B.; Lindman, B. Surfactants and Polymers in Aqueous Solutions, 2nd ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2003. (71) van Duijvenbode, R. C.; Koper, G. J. M.; Bohmer, M. R. Langmuir 2000, 16, 7713–7719.
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Scheme 1. Proposed Model for the Build-up of Trilayers Using G2, G4, and G6 Dendrimers, Respectively, and DNA, on Planar Silica Surfaces a
a Adsorption of dendrimers to silica surfaces results in their deformation. Rinsing induces partial removal of adsorbed G2 and G4 dendrimers. Further deformation of the remaining dendrimers is most apparent for G2. DNA adsorption to the dendrimer monolayers results in interfacial layers that are parallel with the surface but not necessarily completely flat for G2, and have the highest surface excess for G6. The most pronounced increase in surface excess upon a further addition of dendrimers to the dendrimers-DNA bilayers is observed for G6.
possibly induces the removal of dendrimer-DNA aggregates in some form. Additional studies are, however, required to identify the composition of the aggregates removed.
Conclusions This study shows that DNA condensation by PAMAM dendrimers of generations 2, 4, and 6 can be supported on macroscopic flat silica surfaces. The intramolecular interactions between the dendrimers, DNA molecules and silica surfaces have been studied using two surface-specific techniques, in situ null ellipsometry and neutron reflectometry, and changes to the structure of the interface have been discussed in terms of the driving forces that govern the various interactions with respect to the generation of dendrimer. A central point in our interpretations is that the size and surface charge density of the dendrimers increase with generation (G6>G4>G2). We have demonstrated that the dendrimers adsorb as monolayers on hydrophilic silica surfaces and that the extent of the accompanying deformation depends strongly on the generation. While the surface excess increases with dendrimer generation, the larger molecules (G4 and G6) flatten more extensively than the smaller molecules (G2). We have explained these results, as well as the reversibility of the binding of the lower generation dendrimers upon rinsing, in terms of a balance between the electrostatic attraction of the cationic dendrimers to the negatively charged surfaces and electrostatic repulsion between lateral adsorbates of like charge.
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DNA adsorbs to hydrophilic silica surfaces functionalized with a monolayer of cationic dendrimer molecules of all generations studied to form solid-supported dendrimer-DNA bilayers. The highest surface excess of DNA is obtained for G6, which we propose to be the result of the highest surface charge density of the adsorbed cationic dendrimers and the largest medial separation between the negatively charged DNA molecules and exposed patches of the hydrophilic silica surface. DNA adopts the most extended conformations on top of G4 and G6 layers which we speculate may be caused by kinetic trapping of the DNA conformation by the higher charge density of the larger dendrimers. The strength of the DNA binding to the dendrimer monolayer has been shown by the high stability of the bilayers during rinsing. Dendrimer-DNA-dendrimer trilayers result from an additional exposure of dendrimer molecules to the dendrimer-DNA bilayers. This step results in a greater amount of dendrimer adsorption than took place originally on the bare silica surfaces. The surface excess of added dendrimers is highest for the G6 system, which is an observation we rationalize in terms of a higher number of negatively charged binding sites exposed on the rough bilayers compared with on flat silica surfaces. The interfacial behavior of DNA is important in a vast number of applications, which includes gene therapy, diagnostic tools, and their use in sensor devices such as DNA chips or microarrays. The unique structure of the dendrimers has furthermore made them promising surface-functionalizing agents for various applications. We conclude by presenting Scheme 1 which depicts the proposed interfacial multilayer structures created on silica surfaces by their stepwise interactions with PAMAM dendrimers (generation 2, 4 or 6, respectively), then DNA, and last the same generation of dendrimers again. Acknowledgment. The sixth EU framework program is acknowledged for work as being a part of a EU-STREP project with NEST program (NEONUCLEI, Contract 12967). The Linneaus center of excellence on Organizing Molecular Matter through the Swedish Research Council is also thanked for financial support. Travel grants, enabling the neutron experiments to be performed, were received from the Swedish Research Council (VR). The neutron research facilities NCNR, Gaithersburg, MD, and ILL, Grenoble, France, are thanked for allocation of reflectivity beam time. Adrian R. Rennie (Uppsala University), Sushil Satija (NIST) and Bulent Akgun (NIST) are acknowledged for help and assistance during experiments and analysis. Supporting Information Available: A table giving parameters obtained for fits to the neutron reflectivity profiles of the bare substrates and a figure showing Ψ and Δ as a function of time. This material is available free of charge via the Internet at http://pubs.acs.org.
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