Induced dye leakage by PAMAM G6 does not imply dendrimer entry into vesicle lumen

Abstract

Dendrimers are polymers with unique properties that make them promising in a variety of applications such as potential drug and gene delivery systems. Polyamidoamine (PAMAM) dendrimers, in particular, have been widely investigated since they enter rapidly into cells. The entry mechanism, however, is still not yet fully clarified as both passive and active uptake have been proposed. In this work we focus on understanding passive uptake, for which simple cell model systems are used in order to ensure that only dendrimer-lipid interactions are probed. We developed protocols for investigating independently the effect of the dendrimer on lipid bilayer integrity, in terms of permeability of small dyes and effective dendrimer translocation. This was achieved by the use of membrane labeled giant unilamellar vesicles (GUVs) either containing Alexa 488 hydrazide in the vesicle lumen or FITC-labeled PAMAM G6 dendrimers. Vesicle integrity and dendrimer-membrane binding were then assessed by fluorescence microscopy. The importance of membrane fluidity and charge was investigated using GUVs composed of various lipid compositions. A quartz crystal microbalance with dissipation was used to probe the effect of dendrimers on the rigidity of vesicle layers. The results indicate that PAMAM dendrimers can locally alter the membrane properties. An increased bilayer permeability towards soluble small dyes but no effective translocation, where PAMAM dendrimers could dissociate from the lipid membrane into the vesicle lumen, was observed. To our knowledge this is the first time it is shown that PAMAM G6 dendrimer does not effectively translocate the lipid bilayer although it readily interacts with the model membrane, regardless of lipid membrane properties. However, bilayer charge and fluidity modulate the dendrimer interaction in agreement with previous reports. The results clearly highlight the importance of the choice of the model system when investigating nanoparticles interaction with lipid membranes.

A mechanistic view of lipid membrane disrupting effect of PAMAM dendrimers

“…The domains containing more dendrimers should have more significant change in the molecular packing of surrounding lipids. Åkesson et al got similar results when investigating giant liposomes by fluorescent microscopy [27].…”

“…They showed that dendrimers attach to the surface of the vesicles and can bridge neighboring vesicles until liposomes collapse in lamellar phase in which dendrimers are located between the layers. Later they investigated the effect of dendrimers on giant unilamellar vesicles (GUVs) by fluorescent microscopy and on supported lipid bilayers by quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectivity [27][28][29]. Interestingly they have found that the dendrimers enhance the permeability of the membranes for small molecules without hole formation or passive translocation across the membrane.…”

Molecular analysis of interactions between dendrimers and asymmetric membranes at different transport stages

“…6 Although passive and endocytosis pathways have been proposed, the exact mechanism of the transport process is only partially understood. 7 In cells, dendrimers can interact with biomembranes, such as the membrane of an endosome (membrane-bounded compartment inside cells) 8 and a cell membrane. 9,10 Therefore, interactions between PAMAM dendrimers and biomembranes at different transport stages should be understood.…”

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

“…The vesicles were immobilized on the coverslip surface via an adapted biotin-streptavidin binding procedure [37,38] (Fig. 1d), which is described in more detail in the SI [33].…”