Effect of Inflammation on lipid nanoparticle performance

Characterization of nanoparticles was conducted on various samples for the purpose of analyzing the concentration of the samples and their particle counts. The samples studied were liposomes composed of phosphatidylcholine and phosphatidylserine (PSPC), and platelet microparticles. Nanoparticle characterization was conducted on these samples in order to better understand the results of surface plasmon resonance (SPR) analyses that were previously conducted on the same samples to test their binding ability to the Lox-1 protein. The data collected by SPR analysis showed that the PSPC sample had the greatest binding to the SPR sensor chip. However, the particle count or concentration of a sample is known to influence the accumulation of the sample. To measure particle count, samples were diluted in filtered deionized water and analyzed using a Zeta View instrument. The Zeta Viewer is equipped with a camera that collects up to 11 images at high magnification to analyze particle size and density. Results showed that PSPC had a mean concentration of 7E+13 particles per mL with a mean diameter of 213.7 nm and polydispersity of 209.5 nm – 217.1 nm (range). Platelet microparticles were found to have a mean concentration of 9.93E+10 particles per mL with a mean diameter of 176.9 nm and polydispersity of 176 nm – 178.5 nm. These results indicate that PSPC liposomes had a 1000-fold particle count compared to the platelet microparticles. Future experiments will require Zeta View analysis of the particle count and subsequent SPR tests with several dilutions, to calculate the binding constant of each sample type.

Hello, my name is Peter Touma, and today I’ll be sharing my undergraduate research with you all. I hope you enjoy. My research from this semester consisted of a nanoparticle analysis using a ZetaView instrument in one of the labs at the Manassas SciTech campus. Micro particles are nano sized extra cellular vesicles that are released from cells that are known to carry procoagulant surfaces with an anionic charge due to the presence of phosphatidylserine within them. The long term objective of the project is to measure the binding affinity of these micro particles to scavenger receptors, such as lectin-like oxidized lipoprotein receptor one, or simply called lox-1. To this end, the goal of the experiment was to measure the diameter and concentration of synthetically made lipid nanoparticles that contain phosphatidylserine and phosphatidylcholine, as well as platelet-derived particles from a human donor. To prepare the samples, liposomes containing phosphatidylcholine and phosphatidylserine were prepared and platelet microparticles were collected from a healthy consenting donor. The lipids were then suspended in PBS, extruded using varying filter sizes to form liposomes, and stored at 4 degrees Celsius until use in the experiment. The platelet microparticles were previously generated from another experiment using, again, a human donor. A citrated blood sample was centrifuged at 200 xg for five minutes at room temperature to first clear the sample’s red and white blood cells. Then the supernatant was centrifuged again, this time at a higher degree of 1500 xg for 10 minutes to pellet the platelets. These platelets were then washed and citrated isotonic saline and then resuspended in half the original volume of in isotonic saline with two millimolar CaCl2. The platelet samples were then pipetted into half milliliter aliquots and incubated for 30 minutes at 37 degrees Celsius in the presence of thrombin. The resulting platelet clot was then centrifuged again, and 25 microliters of the supernatant-containing platelet microparticles was frozen for use in this experiment. To conduct the experiment, the samples were first diluted in filtered water before injecting into the ZetaView machine. This is to ensure proper analysis, and it goes with the standard operating procedure of the machine. Four cycles were conducted using the microparticles, one of which was at first omitted but then later added back into the study to see a comparison with and without it. And three cycles were carried out using the PSPC liposomes. The initial cycle was recorded and used in the data, but the data was interpreted both with and without it to see the difference that or any possible difference that may arise. The purpose of collecting the data that we collected was the concentration in the particle size of particles. And this was just the overall particle count of our samples. Here we have three scatter plots, one depicting the platelets with cycle A, or the initial cycle, one of the platelets without the initial cycle, and one of the PSPC liposomes alone. Seen in the scatter plots, the PSPC liposomes had an overall larger diameter or size than the platelets. And the average particle size of the platelets seemed to increase when we removed the initial cycle from the analysis. Here, we have a table. Here in this table, we see, again, the mean particle size of our samples, as well as the concentration of our samples as well. And it’s followed by the dilution factors for each cycle, as well as the particles for frames and numbers of channels included for analysis. The number of channels included is important because the ZetaView uses 11 total channels, and not all of them are typically seen to be readable for the machine. Well, here we see that the liposomes actually had 1,000-fold higher concentration than the platelet micro particles at 7 times 10 to the 13th particles per milliliter. And we also see that the concentration of platelet microparticles increased, as did the particle size when removing the initial cycle. So using the ZetaView analysis, we obviously found out that the particle size and concentration of the PSPC liposomes was greater than the micro particles. The platelet micro particles also showed an increase in size and concentration when omitting the first cycle from the data, showing that the collected data initial cycle may be quite different than from the other cycles. With the measurements taken in this study, it will be beneficial to conduct further analysis and further analyze results from a previous SPR experiment or surface plasmid resonance so that we can better understand the binding affinities of both of these particles onto the lectin 1 protein, the lox1 protein. Typically, it’s best to conduct SPR analysis with similar particle counts and concentrations. So nanoparticle tracking analysis, such as this experiment, should usually be done prior to SPR and will be done prior to SPR for future experiments. That is all I had. I hope that you enjoyed it. Thank you for your time.