Electron microscopy of therapeutic and diagnostic nanoparticles

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We have analyzed bionanoparticles that are designed for labeling cells and then imaging those cells in animal models by in vivo magnetic resonance imaging. These nanocomplexes, comprising three FDA-approved drugs (heparin, protamine and ferumoxytol), can be taken up into human cell lines, and detected when implanted into rodents. The major component of the ferumoxytol component is superparamagetic iron oxide nanoparticle (SPIONP), which provides MRI contrast for diagnostic imaging. We have performed electron tomography and energy-filtered transmission electron microscopy (EFTEM) to determine the distribution of the three constituents within the individual nanocomplexes using element-specific signals. The protamine component was imaged with the nitrogen signal, the heparin component with the sulfur signal, and the surrounding shell of ferumoxytol with the iron signal. Electron tomography was also employed to visualize the three-dimensional organization of the ferumoxytol nanoparticles within the approximately 200-nm diameter nanocomplexes. Our analysis showed that the nanocomplexes contained a homogeneous soft core consisting of approximately a 1:1 mass ratio of protamine and heparin, consistent with a balancing of the positive charge on protamine with the negative charge on heparin. Scanning electron microscopy combined with energy-dispersive x-ray (EDXS) spectroscopy has enabled us to characterize the morphology and composition of another nanocomplex: DNA-inorganic hybrid nanovaccines (hNVs) developed for efficient uptake into antigen-presenting cells, enabling prolonged tumor retention, and potent immuno-stimulation and cancer immunotherapy. hNVs were self-assembled from concatemer CpG analogs and magnesium pyrophosphate, which renders hNVs resistant to nuclease degradation and thermal denaturation, both of which are demanding characteristics for effective vaccination and the storage and transportation of vaccines. EDXS analysis confirmed that the hybrid nanovaccines contained pyrophosphate.