By Bruce B. Weiner, Ph.D.
Vesicles as well as micelles, and microemulsions were samples of interest in the early days of what became dynamic light scattering1. This occurred in the 1970’s even before the invention of the word nanotechnology and the first biotech revolution. Not until the 1990’s, however, were DLS instruments small enough, powerful enough, and automated enough to be routinely used for these proposed targeted drug delivery systems.
Liposomes are synthetic versions of natural vesicles such as exosomes, microvesicles, ectosomes, prostasomes, and several others that are distinguished by their biogenesis2. Liposomes were a hot topic in the 1980’s and 1990’s as they were thought to be excellent candidates for targeted drug delivery. The problem was, not being natural to the body, they were attacked by white blood cells, cut open, and their relatively high dose of internal drugs spilled into the blood system usually far from the targeted (cancerous, for example) organ. While some improvements have been made by adding surface moieties that fool the white blood cells, it is not clear if this will yet become a serious candidate for targeted drug delivery.
Exosomes, microvesicles, and liposomes are core- shell particles: The core is water with drugs (liposomes) or cytoplasmic proteins, small RNAs, cell-specific receptors, and surface markers (exosomes and microvesicles). The shell is a lipid layer. Since the liposomes are manmade and do not have the natural markers to fool white blood cells, they were attacked.
Table 1: Properties and Origins
|Exosome||Homogeneous, 40-100 nm diameter, Exosomal surface markers CD9, CD63, Alix, flotillin-1, etc.||Release from cells when multi-vesicular bodies fuse with plasma membrane|
|Micro vesicles||Heterogeneous, 50-1,000 nm diameter. No unique markers||Fission directly from plasma membrane|
|Liposomes||Homogeneous, 50-250 nm diameter. Some with surface chemistry, many without||Synthetically made. No natural defense in bodily fluids.|
Particle Sizing: A sample prepared, initially, from FBS (fetal bovine serum) was prepared and diluted in PBS at pH 7.4. A 50 uL disposable BI-SM50 cell was used and inserted into the Brookhaven 90Plus. Temperature was set at 25 °C and allowed to equilibrate for several minutes. Three, three-minute runs were made at 90° scattering angle, all with the same results within a few percent.
The Effective Diameter was 131.5 nm and the PDI (polydispersity index) was 0.208. A PDI of 0.025 or less indicates a narrow size distribution. And the size above 100 nm also strongly suggests polydispersity.
The intensity-, volume-, and number-weighted size distributions were calculated. As shown in the graphs, the result suggests not a broad unimodal size distribution but a bimodal, with one mode around 80 nm and the other three times larger around 240 nm.
It is well-known that by intensity a relatively few, large particles will dominate the intensity-weighted size distribution and also shift the Effective Diameter higher. But when recast in terms of the volume- weighted distribution, the smaller particles will increase their percent contribution. Finally, when recast yet again to compare to number counting instruments such as electron microscopes, the smaller particles can easily dominate. In this case, when rounded to the nearest ½%, there is apparently no significant contribution of the larger particles. And the remaining 80 nm particles are in the expected size range for exosomes.
Given that exosomes and microvesicles are another means of transferring genetic material between cells, they are candidates for another attempt at targeted drug delivery and for study in the transfer of pathogenic proteins and RNAs. Being able to study their size and state of aggregation quickly and repeatably using DLS helps in their property characterization. This application note demonstrates that capability.
Zeta Potential: Charges at the surface of a particle in a liquid setup a distribution of charge density around the particle. This results in electrostatic potential differences, one of which is known as zeta potential. If the zeta potential is too close to zero, there is no electrostatic repulsion between particles and therefore the always-present, naturally occurring attractive forces will causes particles to aggregate leading to instability. To remain stable, electrostatically stabilized particles must have a finite zeta potential, usually greater than 20 to 30 millivolts (mV). However, since zeta potential also depends on the free salt ion concentration, the zeta potential can be lower in a high salt concentration environment and there is still enough repulsion to keep the particles apart.
Zeta potential is calculated from the determination of electrophoretic mobility: charged particles in the liquid between electrodes move when a field is applied. Measuring the movement is very difficult in high salt, not the least because the current may be so high as to cause heating and chemical changes. Measurements in physiological saline or PBS are particularly difficult for this reason.
The Brookhaven ZetaPALS is uniquely suited to such difficult measurements since it applies low current in high salt and uses phase analysis light scattering to measure with great sensitivity very small movements of charged particles.
Sample preparation was the same as described above under particle sizing. The electrodes are inserted into a square plastic cell with about 1,250 µL of suspension. The temperature was set at 25 C and the sample was allowed to equilibrate for five minutes.
The average measured conductance was 27,000 µS. For comparison, normal measurements are made from 1 to 10 millimolar of 1:1 electrolyte. These concentrations have conductance in the range of 320 to 3200 µS.
Because passing too much current for too long can irreparably change the sample and/or heat the liquid, either effect destroying the measurement of electrophoretic motion, measurements were made at 1, 5, 10, 20, 30, 30, 30 and another 30 cycles, where a cycle corresponds to 1 second with the field on in one direction and another second where it is reversed to avoid polarization of the electrodes. The cycle- weighted average of the first 126 cycles was – 17.7 mV; whereas, the average of the last 30 cycle measurement was –3.36 mV, indicating the sample had been irreversibly changed by the passage of too much current.
Zeta Potential Summary:
Measurements in high salt such as saline or PBS are very difficult. The Brookhaven ZetaPALS has been accomplishing this since the 1990’s using Phase Analysis Light Scattering. In this note, results on an Exosome in PBS indicates a zeta potential of – 17.7 mV, perhaps not quite enough for complete stability as shown by the aggregates in size that were measured as well as the monomer size.
1 Degiorgio, V., Corti, M., Giglio, M., editors, “Light Scattering in Liquids and Macromolecular Solutions”, 1979, Plenum Press, New York. See Section II, p. 111 on micelles, p. 125 on vesicles, and p. 139 on microemulsions.
2 Lee, Y., El Andaloussi, S., Wood, M.J.A., Human Molecular Genetics, 2012, Vol. 21, Rev. Issue 1, R125- R134.