We are very happy to announce that Konrad Thorsteinsson, a PhD student in our lab, just got a paper accepted in Analytical Chemistry. The publication is based on work he began during his master thesis in our group. Check it out here: FRET-Based Assay for the Quantification of Extracellular Vesicles and Other Vesicles of Complex Composition.
The paper presents a novel method for the quantification of vesicles, small liquid enclosed lipid bubbles. Vesicle-type structures are widely used in nature as nanometer-sized containers. In the human body extracellular vesicles (EVs), carrying nucleic acids and proteins, are involved in a multitude of processes, such as immune response regulation and inflammation. EVs have also been linked to the spread of cancer. Bacteria also produce EVs to both communicate and propagate disease. Some viruses have a similar lipid envelope structure which helps them to evade host immune response. Inspired by nature’s vesicles, artificial vesicles have been investigated as vehicles for delivering drugs, nucleic acids, and proteins to their site-of-function in the body.
A major hurdle in research involving vesicles of both biological and artificial origins is the lack of adequate methods to characterize them, in particular to quantify them. For biological particles, a commonly reported measure is the total protein content. This method can be significantly affected by free protein in the sample, or even different protein/particle ratios between samples. Another method is nanoparticle tracking analysis, NTA, where the particles are illuminated with a laser and individual particles which are detected via the light they scatter are counted. The problem with this method is that smaller particles (<70 nm) are very difficult to detect, especially if there are larger particles in the sample as well; which is often the case. Given that EVs have been observed down to 30 nm, there is a need for size-independent methods to accurately quantify EVs.
Konrad’s a new method to characterize vesicle samples, is based on quantifying the total lipid membrane surface area of the sample using artificial liposomes labelled with a FRET-fluorophore pair. FRET is an abbreviation for Förster Resonance Energy Transfer, a phenomenon where one type of fluorophore (donor) transfers its fluorescence energy to another fluorophore of a different species (acceptor). This energy transfer is extremely sensitive to the distance between the donor and acceptor fluorophores, and even small changes in distance can drastically decrease the transfer efficiency.
In this method, sonication is used to drive the fusion between vesicles containing a FRET-fluorophore pair and the sample vesicles. As the vesicles fuse together and the membranes are mixed, the distance between the FRET fluorophores increases, decreasing the efficiency of the FRET. A direct relationship was observed, with higher amounts of sample membrane causing a greater shift. By comparing the degree of spectral shift measured in a sample to a calibration curve, made with vesicles of known concentration, the total lipid surface area of the sample can be quantified.
The method is robust, it can be easily integrated into most labs with minimal investment and is applicable to quantify wide variety of vesicles of both artificial and biological origins. This method has therefore the potential of becoming a powerful tool in a wide variety of research areas ranging from virology, immunology, oncology, as well as the development of lipid-based drug delivery vehicles.