Title

Interaction of Cell-Penetrating Macromolecules With Model Membranes

Date of Award

2007

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Peter Butko

Advisor Department

Chemistry and Biochemistry

Abstract

The general class of cationic cell-penetrating macromolecules (CPMs) is defined by their ability to translocate into the living cells in a rapid manner by as yet unknown mechanism. While our long-term goal is the elucidation of the mechanism, the present work focused on the first phase of the translocation, which is binding of CPMs to the model cell membrane. We used fluorescence spectroscopy to study the interaction between CPMs and the lipid bilayers and monolayers labeled with various fluorescence probes. The two CPMs we studied are protein transduction domain (PTD) of HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat) and lower generations polyamidoamine (PAMAM) dendrimers G1 and G4 (generation 1, 4) labeled with various fluorophores. Kinetic analysis of the interaction between TAT-PTD and lipid bilayer, showed two apparent dissociation constants. While the value of one dissociation constant (Kd1), was found to be independent of the negative charge density, the value of the second dissociation constant (Kd2), decreased linearly with increasing negative charge density from zero to 25 mol%, suggesting a non-electrostatic and electrostatic nature of this interaction, respectively. However, salt studies with compact dendrimers showed the interactions to be completely electrostatic. Fluorescence resonance energy transfer (FRET), quenching, temperature-dependence and pyrene-labeled SUV experiments suggested that TAT-PTD is always on the outer surface of the lipid bilayer. Similar studies with dendrimers suggested a dendrimer-induced aggregation of lipid vesicles. Both TAT-PTD and PAMAM dendrimers preferentially bound to the membrane in the liquid state; only in that state could they gather multiple negatively charged lipid molecules, causing CPM-induced phase separation of lipids into distinct microdomains. Experiments with dendrimers showed a significant increase in surface pressure when initial pressure was below 20 mN/m, and no significant increase at physiologic surface pressure (> 29 mN/m). Hence, dendrimers are able to insert into monolayers at low pressure, but not at physiologic one. We conclude that the two studied CPM interact predominantly with the polar surface of the model lipid membrane, without significant disruption of membrane integrity. The electrostatics and membrane fluidity are the most important physicochemical parameters that govern the membrane binding of these CPM.