R. Ragheb, D. Griffiths
Keywords: nanoparticle tracking analysis, size, concentration, fluorescence
Summary:Purpose: When considering a nanoparticle-based drug it is critical to characterize the size of these vectors, as it has a considerable effect on their pharmacokinetics or their efficiency and ability to reach their target within the body. 1 Equally, it is also imperative to have an idea of the concentration of nano-objects and more importantly the dosage of nano-objects loaded with the drug of interest. Here we describe the use of Nanoparticle Tracking Analysis (NTA) from the NanoSight product range within Malvern Instruments for size and concentration measurements of drug delivery nanoparticles. In addition, by using a fluorescently tagged drug molecule, it was possible to determine how many drug delivery nanoparticles had successfully been loaded with drug molecules. Methods: Polylactic Acid (PLA) particles (Adjuvatis) were designed, synthesized and loaded with a drug of interest conjugated to a fluorescent label (Coumarin6) (PLA-Coum). PLA-coum nanoparticles (diluted 100x with pure water) was loaded into the laser sample chamber with 1 mL syringe. The chamber was loaded onto the instrument and the image focused. A 60 second video (captured using camera level 7) was collected in light-scatter (standard) mode (total particle population measured). With the sample chamber remaining on the instrument, the camera level was increased to 16, a 430nm long pass filter was inserted into the optical path, the image refocused and a second 60 second video was taken to obtain fluorescence-mode data (only particles emitting a fluorescence signal measured). The videos were then processed using Detection Threshold 4 for both scatter and for fluorescent mode to generate size-distribution profiles. Results: A mixture of labelled and non labelled PLA nanoparticles were expected as a result of the method of sample production. NTA data for these particles both under scatter mode (Fig.1, blue line) and fluorescence mode (Fig.1, red line) were obtained. The distance moved by each particle from frame to frame of the video was used to generate a diffusion coefficient and consequently the size of the particle while the average number of particles in each frame of the video was used to give a measure of sample concentration. From these parameters a size distribution profile graph was generated for each measurement mode. Conclusions: NanoSight NTA measurements enabled a fast evaluation of the size and concentration of polylactic acid nanoparticles. When loaded with fluorescently labelled drug molecules, the subpopulation of particles containing the fluorophore could be distinguished from the total population giving valuable information on the efficiency of the loading process. These data can be used to identify improved loading processes in a quick and easy manner saving time and effort for drug delivery researchers. Figure 1. Overlay of the size distribution profiles for PLA-coum nanoparticles in scatter mode (blue line) and fluorescent mode (red line) (x axis = diameter (nm), y axis = concentration (particles per mL)). References: (1) McNeil S., Methods in Molecular Biology 2011, 697 (1): 3-8.