Experimental techniques

Our experimental techniques:

• Cryo-transmission electron microscopy (cryo-TEM)
• QCM-D
• Other techniques

Cryo-transmission electron microscopy (cryo-TEM)

Amphiphilic molecules, such as lipids, surfactants and copolymers, may via self-assembly form a variety of complex structures in dilute aqueous solution. These delicate and dynamic structures are often very sensitive to changes in temperature, concentration, and ionic strength. Consequently fixation, drying, and staining protocols of conventional electron microscopy may induce serious structural artefacts. Cryo-transmission electron microscopy (cryo-TEM) has evolved as an excellent tool for the investigation of these labile structures.

Aggregate structure in monoolein / oleic acid dispersion.

Cryo-TEM is ideally suited for visualization of structures in the size range of 5-500 nm and during the last 15 years we have successfully employed the technique for structural investigations of systems containing micelles, liposomes, and various related lipid/surfactant aggregates. In our experience the cryo-TEM method offers unique possibilities to retrieve detailed information on the aggregate level and constitutes a valuable complement to more indirect methods, such as light scattering, NMR, SAXS, and photophysical techniques.

Fibril-like aggregates formed upon incubation of Amyloid ß-peptide
with the polyunsaturated fatty acid docosahexaenoic acid (DHA).

The equipment at our microscopy unit is: transmission electron microscope Zeiss LIBRA-120 and environmental chamber for sample preparation.

QCM-D

The suspension of lipids in water can lead to the formation of several different structures, such as liposomes and lipid bilayer disks. These can be prepared in a way that mimics biological cell membranes or can be designed as to present optimal properties for applications in drug delivery.

One way of characterizing these structures and their interaction with other molecules (e.g., peptides, proteins, surfactants, etc.) is to immobilize them onto a sensing support. In our research we employ the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) for this purpose. This instrument is based on the piezoelectric properties of quartz, i.e., the capability of producing a mechanical deformation upon applying an electric potential to the material. When an AC potential is applied across electrodes attached to a quartz sensor crystal, the latter will oscillate. The oscillation resonance frequency of the sensor crystal will depend, among other parameters, on the total mass of the sensor. Therefore, upon adsorption of material on the sensor surface, changes in the frequency can be observed and the mass deposited can be quantified with high sensitivity (in the range of nanograms per square centimeter).

The QCM-D can also provide with information about the structural properties of the deposited film. This is achieved by determining the “dissipation factor” of the system. After the crystal has been made to oscillate at is resonance frequency, the AC potential is turned off. The amplitude of the oscillation will then decay. If the deposited film is soft, some of the oscillation energy will be lost due to the deformation of the layer and the oscillation frequency will decay faster. A measurement of the rate of this decay provides the dissipation factor, which can then be related to the viscosity and elasticity of the immobilized material.

QCM-D signals obtained for the formation of (left) an immobilized soft liposome layer containing large amounts of water and (right) a rigid, supported lipid bilayer.

Once the structures are immobilized, we can study their interaction with, e.g., antimicrobial peptides (AMPs). The binding of these peptides to the immobilized lipid structures increases the loaded mass on the sensor surface, which translates into frequency changes. Peptide/lipid membrane association isotherms have been obtained using this approach. It is also possible to determine if the association with the peptides changes the structural properties of the immobilized lipid layer.

QCM-D E1 available at our laboratory.

Other experimental techniques

The following experimental techniques and equipment are also available at our laboratory:

Dynamic Light Scattering (DLS)

Useful for determining the size of particles and nanostructures in suspension. We have a custom built DLS setup that allows, besides particle sizing, the study of light scattering at different angles. A Nicomp 380 Zeta Potential/Particle Sizer is also available. This instrument can, besides DLS, also perform z-potential determination experiments via Laser Doppler Velocimetry (LDV) and Phase Analysis Light Scattering (PALS).

Differential Scanning Calorimetry (DSC)

A CSC 4100 Multi-cell Differential Scanning Calorimeter is used in our laboratory to study phase transitions in self-associated lipid systems.

UV/Vis and Fluorescence Spectroscopy

A SPEX fluorolog 1650 0.2 m double spectrometer is available in-house. Experiments on liposome permeability, fluorescence anisotropy, liposome-liposome fusion, etc. can be performed with this instrument.

A HP 8453 UV/Vis spectrometer is also available.

Electrochemical methods

We use an Ivium CompactStat potentiostat to carry out electrochemical studies with supported lipid bilayers. The techniques used in our laboratory include, among others, cyclic voltammetry, electrochemical impedance spectroscopy, and chronoamperometry. The electrochemical measurements can be coupled with QCM-D determinations by using the electrochemistry module from Q-Sense.

Liposome preparation

We have equipment to prepare liposomes and other self-assembled structures by different methods, including extrusion, sonication, and detergent depletion.