4.10. Multiple particle tracking

MPT was used to measure MSD of fluorescently labeled DNA-loaded nanocomplexes ex vivo in normal rat brain slices or orthotopically established F98 tumor slices as previously reported [22]. Briefly, rat brains were harvested from healthy (8–10 weeks) or F98 GBM-bearing rats (10 days after tumor inoculation), incubated in aCSF for 10 min and sliced using a Zivic brain matrix slicer (Zivic Instruments, Pittsburgh, PA). The resulting 1.5 mm coronal slices were placed on custom-made slides. Subsequently, we injected 0.5 μl of fluorescently labeled nanocomplex solution into the cerebral cortex at a depth of 1 mm using a 50 μl Hamilton Neuro Syringe (Hamilton, Reno, NV) mounted on a stereotactic frame. Nanocomplex trajectories were recorded over 20 s at an exposure time of 66.7 ms by an Evolve 512 EMCCD camera (Photometrics, Tucson, AZ) mounted on an inverted epifluorescence microscope (Axio Observer D1; Carl Zeiss, Hertfordshire, UK) equipped with a 100×/1.46 NA oil-immersion objective. Movies were analyzed with a custom-made automated particle tracking MATLAB script to extract x, y-coordinates of nanocomplex centroids over time and calculate the MSD of individual nanocomplexes as a function of timescale [56]. Median MSD was determined based on the measured MSD of individual nanocomplexes; individual MSD values were not ensemble-averaged given their inherently heterogeneous, non-Gaussian distribution [56,79].