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Lipid Bilayer Disruption by Polycationic Polymers: The Roles of Size and Chemical Functional Group Almut Mecke,† Istva´n J. Majoros,§ Anil K. Patri,§ James R. Baker, Jr.,§ Mark M. Banaszak Holl,*,‡,§ and Bradford G. Orr*,†,§ Departments of Physics and Chemistry and the Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor, Michigan 48109
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Received March 8, 2005. In Final Form: July 8, 2005 Polycationic polymers are used extensively in biology to disrupt cell membranes and thus enhance the transport of materials into the cell. The highly polydisperse nature of many of these materials makes obtaining a mechanistic understanding of the disruption processes difficult. To design an effective mechanistic study, a monodisperse class of polycationic polymers, poly(amidoamine) (PAMAM) dendrimers, has been studied in the context of supported dimyristoylphosphatidylcholine (DMPC) lipid bilayers using atomic force microscopy (AFM). Aqueous solutions of amine-terminated generation 7 (G7) PAMAM dendrimers caused the formation of 15-40-nm-diameter holes in lipid bilayers. This effect was significantly reduced for smaller G5 dendrimers. For G3, no hole formation was observed. In addition to dendrimer size, surface chemistry had a strong influence on dendrimer-lipid bilayer interactions. In particular, acetamideterminated G5 did not cause hole formation in bilayers. In all instances, the edges of bilayer defects proved to be points of highest dendrimer activity. A proposed mechanism for the removal of lipids by dendrimers involves the formation of dendrimer-filled lipid vesicles. By considering the thermodynamics, interaction free energy, and geometry of these self-assembled vesicles, a model that explains the influence of polymer particle size and surface chemistry on the interactions with lipid membranes was developed. These results are of general significance for understanding the physical and chemical properties of polycationic polymer interactions with membranes that lead to the transport of materials across cell membranes.
Introduction Polycationic polymers are used extensively to disrupt cell membranes and allow the transport of material into cells.1-5 Artificial polymers commonly employed include PEI, PLL, DEAE-dextran, and PAMAM dendrimers.6,7 Interestingly, several classes of natural polycationic polymers appear to play a similar role including the cellpenetrating peptides or CPPs.8-14 The details of action of * Corresponding authors. (B.G.O.) E-mail:
[email protected]; Phone: (734)973-2971; Fax: 734 764 2193. (M.M.B.H.) E-mail:
[email protected]; Phone: (734)763-2283; Fax: (734)763-2307. † Department of Physics. ‡ Department of Chemistry. § Michigan Nanotechnology Institute for Medicine and Biological Sciences. (1) Bielinska, A.; Kukowska-Latallo, J. F.; Johnson, J.; Tomalia, D. A.; Baker, J. R., Jr. Nucleic Acids Res. 1996, 24, 2176-2182. (2) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 72977301. (3) Haensler, J.; Szoka, F. C., Jr. Bioconjugate Chem. 1993, 4, 372379. (4) Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.; Tomalia, D. A.; Baker, J. R., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897-4902. (5) Tang, M. X.; Redemann, C. T.; Szoka, F. C., Jr. Bioconjugate Chem. 1996, 7,, 703-714. (6) Behr, J. P. Chimia 1997, 51, 34-36. (7) Tomlinson, E.; Rolland, A. P. J. Controlled Release 1996, 39, 357372. (8) Fischer, P. M.; Krausz, E.; Lane, D. P. Bioconjugate Chem. 2001, 12, 825-841. (9) Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U. Trends Pharmacol. Sci. 2000, 21, 99-103. (10) Snyder, E. L.; Dowdy, S. F. Pharm. Res. 2004, 21, 389-393. (11) Nishihara, M.; Perret, F.; Takeuchi, T.; Futaki, S.; Lazar, A. N.; Coleman, A. W.; Sakai, N.; Matile, S. Organic Biomol. Chem. 2005, 3, 1659-1669. (12) Perret, F.; Nishihara, M.; Takeuchi, T.; Futaki, S.; Lazar, A. N.; Coleman, A. W.; Sakai, N.; Matile, S. J. Am. Chem. Soc. 2005, 127, 1114-1115.
both the proteins and polymers are only partially understood and indeed have been the subject of considerable debate in the literature.15-28 This study focuses on common properties of these polymersssize, chemical functionality, and chargesand examines the role that they play in the disruption of a model membrane system, a supported lipid bilayer. The goal of this study is the development of a general model that can explain the role that these three properties of polymers play in membrane disruption. (13) Sakai, N.; Takeuchi, T.; Futaki, S.; Matile, S. ChemBioChem 2005, 6, 114-122. (14) Sakai, N.; Matile, S. J. Am. Chem. Soc. 2003, 125, 14348-14356. (15) Christiaens, B.; Grooten, J.; Reusens, M.; Joliot, A.; Goethals, M.; Vandekerckhove, J.; Prochiantz, A.; Rosseneu, M. Eur. J. Biochem. 2004, 271, 1187-1197. (16) Derossi, D.; Calvet, S.; Trembleau, A.; Brunissen, A.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1996, 271, 18188-18193. (17) Drin, G.; Cottin, S.; Blanc, E.; Rees, A. R.; Temsamani, J. J. Biol. Chem. 2003, 278, 31192-31201. (18) Fischer, D.; Li, Y.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. Biomaterials 2003, 24, 1121-1131. (19) Hong, S.; Bielinska, A. U.; Mecke, A.; Keszler, B.; Beals, J. L.; Shi, X.; Balogh, L.; Orr, B. G.; Baker Jr., J. R.; Banaszak Holl, M. M. Bioconjugate Chem. 2004, 15, 774-782. (20) Moreau, E.; Domurado, M.; Chapon, P.; Vert, M.; Domurado, D. J. Drug Targeting 2002, 10, 161-173. (21) Murthy, N.; Robichaud, J. R.; Tirrell, D. A.; Stayton, P. S.; Hoffman, A. S. J. Controlled Release 1999, 61, 137-143. (22) Ottaviani, M. F.; Daddi, R.; Brustolon, M.; Turro, N. J.; Tomalia, D. A. Langmuir 1999, 15, 1973-1980. (23) Ottaviani, M. F.; Matteini, P.; Brustolon, M.; Turro, N. J.; Jockusch, S.; Tomalia, D. A. J. Phys. Chem. B 1998, 102, 6029-6039. (24) Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. J. Biol. Chem. 2003, 278, 585-590. (25) Scheller, A.; Wiesner, B.; Melzig, M.; Bienert, M.; Oehlke, J. Eur. J. Biochem. 2000, 267, 6043-6050. (26) Thomas, J. L.; Tirrell, D. A. Acc. Chem. Res. 1992, 25, 336-342. (27) Vive`s, E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 1601016017. (28) Zhang, Z. Y.; Smith, B. D. Bioconjugate Chem. 2000, 11, 805814.
10.1021/la050629l CCC: $30.25 © 2005 American Chemical Society Published on Web 09/30/2005
Lipid Bilayer Disruption by Polycationic Polymers
Poly(amidoamine) (PAMAM) dendrimers29,30 provide a highly controllable model system for exploring the interactions between polycationic polymers and membranes because they have excellent monodispersity, well-defined shape and size, and surfaces that are readily chemically functionalized.31 Previous studies have addressed the biocompatibility of dendrimers by investigating their interaction with lipid vesicles22,23,28,32,33 as well as cultured cells.18,19,34-37 It was found that dendrimers are able to cause a disruption of these membranes and that the interaction is strongest for dendrimers of higher generations carrying positive charges. Atomic force microscopy (AFM) studies revealed that generation 7 (G7) PAMAMs have the ability to create holes in lipid bilayers and that this effect can be influenced by a change in macromolecular architecture.38 Despite this substantial body of literature indicating that dendrimers disrupt supported lipid bilayers and vesicles, one should be careful in directly interpreting these data as applicable to far more complex mammalian cell membranes. For example, typical mammalian cell membranes contain approximately 50% protein, a variety of lipids, intercalated cholesterol, and are coated by sugars. Nevertheless, experiments on KB and Rat2 cells in vitro indicate that the membrane disruption and resulting permeability are consistent with the dendrimer-mediated formation of holes in the cell membrane.19 In search of an underlying mechanism for this behavior, AFM experiments of dendrimers interacting with supported lipid bilayers have been performed. The size, chemical functionality, and charge of the dendrimer have been varied to examine the effect upon hole formation. A model that explicitly includes the interaction between dendrimer and lipid was used to estimate the stability and expected size of the resulting dendrimer/lipid assemblies. Recent studies published by Futaki, Sakai, and Matile have pointed to the importance of counteranion effects on the ability of polycationic polymers, specifically polyargine, to disrupt vesicle and cell membranes.11-14 Unfortunately, supported lipid bilayers are themselves sensitive to the ions present in solution, so we cannot vary the anions present in our system and ascribe any observed changes to the dendrimer/bilayer interaction alone. Therefore, we have not varied the counteranion for these experiments. For the amine-terminated dendrimers protonated by water in solution, the counteranion will be hydroxide. (29) Tomalia, D. A.; Baker, H.; Dewald, J. R.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17, 117-132. (30) Tomalia, D. A.; Baker, H.; Dewald, J. R.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Macromolecules 1986, 19, 2466-2468. (31) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665-1688. (32) Karoonuthaisiri, N.; Titiyevskiy, K.; Thomas, J. L. Colloids Surf., B 2003, 27, 365-375. (33) Ottaviani, M. F.; Favuzza, P.; Sacchi, B.; Turro, N. J.; Jockusch, S.; Tomalia, D. A. Langmuir 2002, 18, 2347-2357. (34) Jevprasesphant, R.; Penny, J.; Jalal, R.; Attwood, D.; McKeown, N. B.; D’Emanuele, A. Int. J. Pharm. 2003, 252, 263-266. (35) Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorenz, K.; Frey, H.; Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R. J. Controlled Release 2000, 65, 133-148. (36) Quintana, A.; Raczka, E.; Piehler, L.; Lee, I.; Myc, A.; Majoros, I.; Patri, A. K.; Thomas, T.; Mule´, J.; Baker Jr., J. R. Pharm. Res. 2002, 19, 1310-1316. (37) Tajarobi, F.; El-Sayed, M.; Rege, B. D.; Polli, J. E.; Ghandehari, H. Int. J. Pharm. 2001, 215, 263-267. (38) Mecke, A.; Uppuluri, S.; Sassanella, T. M.; Lee, D. K.; Ramamoorthy, A.; Baker, J. R., Jr.; Orr, B. G.; Banaszak Holl, M. M. Chem. Phys. Lipids 2004, 132, 3-14.
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Figure 1. Structure of an amine-terminated poly(amidoamine) (PAMAM) dendrimer, generation 1. Table 1. Properties of PAMAM Dendrimers dendrimer type G7-amine G7-acetamide G5-amine G5-acetamide G3-amine
maximum number of molecular diameter diameter in nmc end-groupsa weight in Daa in nmb 512 512 128 128 32
116 491 138 014 28 825 34 206 6909
8.0-8.2
13
4.3-6.6
10
3.5-4.1
7
a
Theoretical values. b Based on experimental values from Prosa et al.,47 Jackson et al.,46 and Uppuluri et al.57 c Based on molecular dynamics simulations from Lee et al.48 and Mecke et al.49
Experimental Section This study investigated PAMAM dendrimers with amineterminated (R-NH2, generations 3, 5, and 7) and acetamideterminated39 (R-NHC(O)CH3, generations 5 and 7) branches, respectively (notation: G#-amine, G#-acetamide). All polymer samples were provided by the Center for Biologic Nanotechnology, University of Michigan, Ann Arbor, MI. Some properties of these dendrimers are summarized in Figure 1 and Table 1. The preparation of supported lipid bilayers as well as the AFM imaging procedures are described in detail elsewhere.38 Briefly, supported DMPC bilayers were formed by vesicle fusion on freshly cleaved mica. All images were taken in tapping mode on a Nanoscope IIIa multimode AFM (Digital Instruments, Santa Barbara, CA) equipped with a liquid cell and standard silicon nitride cantilever (spring constant 0.32 N/m, length 100 µm). All experiments were performed in DI water (resistivity 18 MΩ cm, type 1). The temperature inside the liquid cell during imaging was determined to be ∼28 °C and therefore above the geltransition temperature of supported DMPC bilayers.38,40 As initially formed, bilayers frequently contained holes resulting from incomplete vesicle fusion (Figures 2a, 4a, 5a, and 6a). The presence of these holes is quite useful for verifying bilayer formation and thickness (∼5 nm for DMPC40). Once it was confirmed that a bilayer had formed, 20-30 µL of a dilute dendrimer solution was injected into the sample volume of approximately 100 µL for a final concentration in the range of 1.4-42 pM polymer at pH 6. The corresponding total number of functional end-groups (i.e., amine or acetamide) in the sample volume ranged from 0.5 to 5 nM. Note that the concentration of lower-generation dendrimers was increased to reach at least the same molar concentration of functional groups as for the case of high-generation dendrimers. Imaging continued for up to 2 h after injection in order to observe the interaction of the dendrimers with the bilayer. (39) Majoros, I. J.; Keszler, B.; Woehler, S.; Bull, T.; Baker, J. R., Jr. Macromolecules 2003, 36, 5526-5529. (40) Tokumasu, F.; Jin, A. J.; Dvorak, J. A. J. Electron Microsc. 2002, 51, 1-9.
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Figure 2. AFM height images of the DMPC bilayer (a) before adding dendrimers, (b) 20 min after exposure to G7-amine, and (c) 60 min after exposure to G7-amine. Gray areas: top surface of the lipid bilayer. Black areas: substrate supporting the lipid bilayer. The corresponding step height between the substrate and the top of bilayer is about 5 nm. Color height scale: 0-20 nm.
Figure 3. AFM height images of the same area of the DMPC bilayer (a) 12 to (c) 19 min after exposure to G5-amine. Color height scale: 0-20 nm.
Figure 4. AFM height images of the DMPC bilayer (a) before adding dendrimers as well as (b) 3 and (c) 5 min after adding G3-amine. In these images, the lipid molecules form two bilayers (seen in gray and white), each with a thickness of about 5 nm. Note that dendrimer adsorption occurs at lipid boundaries of both layers (see arrows in panel c). The color contrast in the lower left corner of panel c has been adjusted to better show dendrimers adsorbed to the bilayer edge.
Results and Discussion 1. AFM Imaging of DMPC Bilayers Exposed to PAMAM Dendrimers. The effect of decreasing dendrimer generation (size) on the ability to form holes in lipid bilayers is illustrated in Figures 2-4. Adding G7amine PAMAMs to the lipid bilayer caused the formation of small, isolated holes (typical diameters range from 15 to 40 nm) in previously intact parts of the bilayer. Hole formation occurred within 2 min (i.e., faster than the time between two consecutive AFM scans38). Figure 2 shows AFM images of a DMPC lipid bilayer at three different time points before and after the exposure to G7-amine. Once the holes had formed, their position and size changed very little for time periods of up to 1 h (Figure 2b and c). Some erosion of the bilayer was observed at the edges of existing bilayer defects. G5-amine dendrimers had a greatly reduced ability to remove lipid molecules from the surface (Figure 3), even when the concentration of charged end-groups was increased five times as compared to the case of G7. Although G5-amine dendrimers removed lipids, they did
so more slowly and mostly from the edges of existing bilayer defects, as can be seen in Figure 3a-c (12 min after adding dendrimers to 19 min after adding dendrimers, respectively). This resulted primarily in the growth of existing defects rather than the formation of isolated small holes as in the case of G7. When the size of the dendrimers was reduced still further, they were no longer able to remove lipids from the surface (Figure 4). G3-amine PAMAMs adsorbed preferentially to bilayer edges forming a layer approximately 1.5 nm in height along the boundary of the lipid bilayer as indicated by arrows in panel 4c. These first three experiments all involved dendrimers carrying a positive charge because their surface primary amines were determined to be protonated at pH
