Interaction of poly(L-lysine)-g-poly(ethylene glycol) with supported phospholipid bilayers

Abstract

Interactions between the graft copolymer poly(L-lysine)-g-poly(ethylene glycol), PLL-g-PEG, and two kinds of surface-supported lipidic systems (supported phospholipid bilayers and supported vesicular layers) were investigated by a combination of microscopic and spectroscopic techniques. It was found that the application of the copolymer to zwitterionic or negatively charged supported bilayers in a buffer of low ionic strength led to their decomposition, with the resulting formation of free copolymer-lipid complexes. The same copolymer had no destructive effect on a supported vesicular layer made up of vesicles of identical composition. A comparison between poly(L-lysine), which did not induce decomposition of supported bilayers, and PLL-g-PEG copolymers with various amounts of PEG side chains per backbone lysine unit, suggested that steric repulsion between the PEG chains that developed upon adsorption of the polymer to the nearly planar surface of a supported phospholipid bilayer (SPB) was one of the factors responsible for the destruction of the SPBs by the copolymer. Other factors included the ionic strength of the buffer used and the quality of the bilayers, pointing toward the important role defects present in the SPBs play in the decomposition process.

FIGURE 1

Schematic representation of the experimental procedure. (A) A supported phospholipid bilayer is formed on a SiO₂ surface by vesicle fusion in Ca²⁺ buffer. A bilayer composed of a DOPC (black headgroups): DOPS (white headgroups, negatively charged) mixture is used in this example; some experiments were performed with SPBs composed of DOPC alone. (B) After rinsing with the appropriate buffer(s), PLL-g-PEG is added and the interaction with the bilayer is monitored by QCM-D and fluorescence microscopy. The chemical structure of PLL-g-PEG and the types of lipids used are shown in the inset.

FIGURE 2

Addition of PLL-g-PEG in H1 buffer to zwitterionic (A) and negatively charged (B) bilayers. Arrows along the time axes indicate injections. The various stages of the experiment are indicated with schematic drawings where possible. (A) Zwitterionic bilayer (DOPC). Frequency decreases and dissipation factor increases upon addition of PLL-g-PEG, indicating copolymer adsorption. However, material is removed from the surface upon both further addition of the polymer and rinsing with H1 buffer (thick arrows). (B) Negatively charged bilayer (DOPC:DOPS 95:5). Initial adsorption of PLL-g-PEG is followed by a spontaneous desorption of material (encircled). For clarity and space reasons, some of the buffer exchange steps have been omitted. *1 and *2 indicate the start and endpoints used to calculate the frequency and dissipation shifts shown in Table 1.

FIGURE 3

Fluorescence signals arising from a DOPC:DOPS 9:1 bilayer doped with TRITC-PE (green) and from PLL-g-PEG-fluorescein (red) for an SPB alone (A, B) and for an SPB coated with PLL-g-PEG in EDTA buffer after rinsing away the excess polymer (C, D). The images were taken under identical conditions using the laser lines appropriate for each probe, and demonstrate that after the addition of PLL-g-PEG-fluorescein, the previously bare SPB (A, B) remains intact (C) and is coated with the polymer (D).

FIGURE 4

Time-lapse sequences of false-colored fluorescence images depicting the disruption of supported bilayers on SiO₂ (A–H) and the interaction of PLL-g-PEG in H1 with supported vesicular layers on TiO₂ (I–L). The green color corresponds to the lipids. (A–C) A DOPC bilayer, doped with 1% NBD-PC, before (A), a few seconds after (B), and 30 min after (C) the addition of PLL-g-PEG in H1 buffer. (D) The worms formed during the bilayer disruption were observed to collapse into globular structures (encircled). Two sequential images taken 45 min after PLL-g-PEG addition, 60 s apart, are shown. The collapse is instantaneous. (E–G) A DOPC:DOPS 9:1 bilayer, doped with 1% NBD-PC, before (E), 1 min after (F), and 5 min after (G) PLL-g-PEG addition in H1 buffer. (H) A fluorescence image demonstrating the colocalization of phospholipids (green) and PLL-g-PEG-fluorescein (red) in the worm-like structures and aggregates that form after the disruption of a of DOPC:DOPS 9:1 SPB. The polymer was added in H1 buffer. Some worms appear as double lines (one in green and one in red) in the images (white arrowheads) due to their movement during the time passed between the acquisition of the images in the two channels. Colocalization was also observed in the case of DOPC bilayers (not shown). (I–J) Zwitterionic (DOPC) vesicles adsorbed on the surface of TiO₂ form a supported vesicular layer (I). They are replaced on the surface by the adsorbing PLL-g-PEG (J). (K–L) The copolymer has no effect on the negatively charged (DOPC:DOPS 9:1) vesicles. The average intensity of the images is the same before (K) and after (L) the addition of the polymer. In both cases, the vesicles remain intact. Formation of wormlike structures is not observed. Vesicles were labeled with NBD-PC.

FIGURE 5

Interpretation of the bilayer disruption mechanism. Upon adsorption of the copolymer to a flat surface of an SPB, the PEG chains are compressed. This process is entropically costly, and the free energy of the system is lowered by bending the bilayer and lifting it off of the underlying substrate. The process involves bilayer rupture and is therefore thought to nucleate at pre-existing defects or be facilitated by osmotic and electrical gradients that destabilize the bilayer. When the disruption process is completed, most of the lipids are floating in solution in the form of worm-like lipid-PLL-g-PEG complexes. The copolymer also coats the SiO₂ substrate with the remains of bilayer.