I.Reviakine - Liposome-TiO2 Interactions

Liposome - TiO₂ Interactions

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Most of the work described below was performed in collaboration with the the BioInterface Group at the Swiss Federal Institute of Technology (Zürich) within the context of the Doctoral thesis of Fernanda F. Rossetti Interactions of Lipidic Assemblies with Metal Oxide Surfaces and Brush-like Polyelectrolytes.

This page was jointly designed by F. F. Rossetti and I. Reviakine.

Why study liposome-surface interactions in general and in the case of TiO₂ specifically?

Supported lipidic systems (liposomes and bilayers) are useful

as model systems for studying cell membranes, especially with regards to cell-cell communication and protein-lipid interactions;
for studying crystallisation of membrane-binding (and possibly transmembrane) proteins;
as platforms for the design of biosensors based on transmembrane proteins.

Adsorption of liposomes to surfaces is the first step in supported bilayer formation, and surface-liposome interactions determine the faith of the surface adsorbed vesicles. We would like

to control the process of bilayer formation;
to prepare supported bilayers on a wide variety of surfaces, under controlled conditions;
to understand the basis of selectivity liposomes display with respect to various surfaces (e.g., TiO₂ vs SiO₂ vs mica vs gold; on all of these surfaces vesicles were found to behave differently).

Titanium is an important biomaterial: it is used in dental and orthopoedic prosthetics, vascular stents, hausing of heart valves (and some older heart valve models), ventricular assist devices, etc.

The biocompatible properties of titanium oxide (TiO₂) are partly responsible for the success of Ti as implant material, because the metal is covered with a thin oxide film undeer normal conditions and it is the oxide that the body "sees". We do not know the mechanisms of this biocompatibility.

Therefore it is interesting to investigate interactions between TiO₂ and various components of the living systems, from molecules and their assemblies (such as proteins and liposomes) to cells.

Furthermore, due to its high refractive index, TiO₂ is used sensors with optical detection methods. Our inability to form bilayers on its surface has limited us both in terms of applying certain surface-sensitive techniques (e.g., OWLS) to studying cell membranes, and in terms of biosensor design.

These are some of the reasons why we are interested in liposome-surface interactions. In this study, we have investigated interactions of zwitterionic phospholipids (e.g., DOPC) and negatively charged phospholipids (DOPS) with TiO₂ by quartz crystal microbalance, atomic force, and fluorescence microscopy.

Interactions between zwitterionic (dioleoyl phosphatidyl choline) liposomes and TiO₂:

On the left, contact mode AFM images of 100 nm (top pair) and 200 nm (bottom pair) DOPC liposomes adsorbed on TiO₂ are shown. The images are 5 x 5 um² and were taken in liquid, at low (left column) and high (right column) liposome concentrations, respectively. Individual liposomes are shown in insets. Objects of torroidal morphology were observed in the case of 200 nm vesicles (bottom inset). On the right, changes in the resonance frequency of a quartz crystal oscillating in shear-thickness mode (QCM-D) upon exposure to DOPC vesicles of various sizes, and the layer thickness extracted from the QCM-D response (blue rhombs), are plotted. It can be seen that larger vesicles deform to a larger extent.
The oxide film was prepared by reactive magnetron sputtering in both cases.

Based on quartz crystal microbalance and atomic force microscopy results, we conclude that PC liposomes adsorb on TiO₂ intact (i.e., without rupturing). Bilayer formation is not observed. Erik Reimhult (formerly from Gotheburg University of Technology, Sweden) has reached the same conclusion, but found the vesicle deformation to be linear in size. Details can be found in Reimhult et al. 2002 J. Chem. Phys. 117, 7401; 2003, Langmuir 19, 1681; and Reviakine et al. 2005 J. Chem. Phys. 122, 20471.

Effect of negatively charged lipids (phosphatidyl serine) on TiO₂ - liposome interactions:

Chages in the resonance frequency of a TiO₂-coated quartz crystal upon exposure to 100 vesicles nm vesicles of various compositions (QCM-D, right pannel).
Frequency and dissipation shifts (QCM-D) and fluorescence intensities were normalized to the values observed in the absence of PS and plotted as a function of PS contents (left pannel).

Both the magnitude of the frequency and dissipation shifts, and the fluorescence intensities, decrease as the contents of PS in the vesicles increases. Thus, in the absence of Ca²⁺, the more PS the vesicles have, the fewer of them are adsorbed. Surface potentials were calculated according to Eisenberg et al. 1979 Biochemistry 18, 5213, and Ohki et al. 1982 Biochemistry 21, 2127.

Effect of Ca²⁺ on the interactions between TiO₂ and negatively charged liposomes; supported bilayer formation:

Various stages of bilayer formation are indicated at the bottom of the figure (after Keller and Kasemo, PRL 2000): 1. Bare crystal. 2. Vesicle adsorption. 3. Decomposition of adsorbed vesicles. 4. Bilayer. The extrema in the QCM-D curves occur before the vesicles begin decomposing (red arrowhead).

In the presence of Ca²⁺, PS-containing vesicles once again adsorb to the oxide surface (blue and red curves). Frequency and disipation shifts due to 10% PS vesicles are smaller than those for PC alone. This is due to increased deformation (smaller thickness, stronger interactions with the surface) of vesicles that contain PS.

Vesicles that contain 20% PS or more form bilayers on TiO₂. This conclusion is drawn from the appearance of characterristic extrema in QCM-D curves (red arrowheads).
Bilayer formation is confirmed by FRAP measurements shown on the right hand side: a spot is bleached in the initially homogeneous bilayer containing fluorescently labeled PC. Fluorescence intensity recovers completely. Diffusion coefficient of PC ~ 2*10^-8 cm²/s was measured. No recovery is observed when vesicles composed of PC only are adsorbed on TiO₂.

TiO₂ - liposome interactions: summary.

Thie diagram on the left summarizes the vesice-TiO₂ interactions.

1. In the absence of Ca²⁺, there is an energy barrier that prevents negatively charged (PS-containing) vesicles from adsorbing.
2, 3. PC vesicles, and PS-containing vesicles in the presence of Ca²⁺, adsorb to the surface. The adhesion energy is not stong enough for the bilayers to form. Ca²⁺ reduces or abolishes the energy barrier. We have zeta-potential data that support this conclusion (not shown).
4. When enough PS (> 20 %) is present in the vesicles, bilayers form in the presence of Ca²⁺.

The adhesion strength increases from 2 to 4.

Curves 1 also have adhesive minima. We know that since vesicles that do manage to overcome the energy barrier and adsorb do not desorb upon rinsing. We can speculate that the presence of this adhesive minumum is caused by the PC-TiO₂ interactions which are not strongly affeted by Ca²⁺.

Two roles of Ca²⁺: 1. Reducing/abolishing the energy barrier (c.f. role of divalent cations and electrostatic interactions in stability of colloidal suspensions and their coagulation). 2. Mediating strong PS-TiO₂ interactions, that are responsible for increased adhesion strength and bilayer formation.