Interaction of Poly(amidoamine) Dendrimers with Supported Lipid Bilayers and Cells: Hole Formation and the Relation to Transport Seungpyo Hong, §,3 Anna U. Bielinska, |,3 Almut Mecke, ⊥,3 Balazs Keszler, |,3 James L. Beals, |,3 Xiangyang Shi, |,3 Lajos Balogh, §,|,3 Bradford G. Orr, †,⊥,3 James R. Baker, Jr., |,3 and Mark M. Banaszak Holl* ,†,‡,§,X,3 Programs in Applied Physics, Biophysics, and Macromolecular Science and Engineering, Departments of Internal Medicine, Physics, and Chemistry, and Center for Biologic Nanotechnology, University of Michigan, Ann Arbor, Michigan 48109. Received February 17, 2004; Revised Manuscript Received May 4, 2004 We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine- terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide- terminated PAMAM dendrimers. INTRODUCTION Poly(amidoamine) (PAMAM) dendrimers have demon- strated great promise for a variety of biomedical applica- tions. This class of polymers has a number of favorable properties including well-defined chemical structure, globular shape, low polydispersity index (close to 1.0), biocompatibility, and controlled terminal functional groups. Amine-terminated PAMAM dendrimers have proven to be effective as a nonviral cell transfection agent (1, 2). Modification of the PAMAM dendrimer surface functional groups with targeting compounds, fluorescent groups, and drugs have produced promising imaging and thera- peutic agents (3-7). The success of PAMAM dendrimers in biomedical applications such as those mentioned above raises the question regarding how PAMAM dendrimers interact with cell membranes at the molecular level. The complex structure of cell membranes has prompted several re- search groups to employ phosphatidylethanolamine- containing vesicles as model systems. Efficient cross- membrane transport and membrane disruption were observed with a strong dependence on dendrimer size (generation), chemical structure of the dendrimer, and composition of the model membranes (8-10). Based upon these studies, the internalization mechanism of den- drimers into cells has been explained as a dendrimer- mediated endocytosis. Endocytosis is a complex multistep process and can be generally described in terms of cell transfection efficiency. A cationic polymer (e.g. poly- (lysine) or PAMAM dendrimer) is proposed to bind with the outer cell membrane and be internalized into the cell through endocytosis. The polymer then exits from the endosome, which is a step that controls the transfection efficiency (11). The electrostatic interaction between the polymer and the lipid bilayer membrane and concomitant osmotic imbalance have been understood as important driving forces for polycation-mediated endocytosis (10, 12). Other oligomers that may follow similar pathways include the cell-penetrating peptides (CPPs) such as Tat and oligoarginine (13, 14). The model systems studied to date show good evidence for membrane disruption by PAMAM dendrimers (8, 10). * To whom correspondence should be addressed. Phone: (734) 763-2283. Fax: (734) 763-2307. E-mail: mbanasza@umich.edu. † Program in Applied Physics. ‡ Program in Biophysics. § Program in Macromolecular Science and Engineering. | Department of Internal Medicine. ⊥ Department of Physics. X Department of Chemistry. 3 Center for Biologic Nanotechnology. 774 Bioconjugate Chem. 2004, 15, 774-782 10.1021/bc049962b CCC: $27.50 © 2004 American Chemical Society Published on Web 06/22/2004