Cytoplasmic Trafficking of Nanoparticles Delivers Plasmid DNA for Macrophage Gene-editing.

BACKGROUND: Successful delivery of gene-editing tools using nano-carriers is dependent on the ability of nanoparticles to pass through the cellular membrane, move through the cytoplasm, and cross the nuclear envelope to enter the nucleus. It is critical that intracellular nanoparticles interact with the cytoskeletal network to move toward the nucleus, and must escape degradation pathways including lysosomal digestion. Without efficient intracellular transportation and nuclear entry, nanoparticles-based gene-editing cannot be effectively used for targeted genomic modification. OBJECTIVE: We have developed nanoparticles with a low molecular weight branched polyethylenimine lipid shell and a PLGA core that can effectively deliver plasmid DNA to macrophages for gene editing while limiting toxicity. METHODS: Core-shell nanoparticles were synthesized by a modified solvent evaporation method and were loaded with plasmid DNA. Confocal microscopy was used to visualize the internalization, intracellular distribution and cytoplasmic transportation of plasmid DNA loaded nanoparticles (pDNA-NPs) in bone marrow-derived macrophages. RESULTS: Core-shell nanoparticles had a high surface charge of +56 mV and narrow size distribution. When loaded with plasmid DNA for transfection, the nanoparticles increased in size from 150 nm to 200 nm, and the zeta potential decreased to +36 mV, indicating successful encapsulation. Further, fluorescence microscopy revealed that pDNA-NPs crossed the cell membrane and interacted with actin filaments. Intracellular tracking of pDNA-NPs showed successful separation of pDNA- NPs from lysosomes, allowing entry into the nucleus at 2 hours, with further nuclear ingress up to 5 hours. Bone marrow-derived macrophages treated with pDNA/GFP-NPs exhibited high GFP expression with low cytotoxicity. CONCLUSION: Together, this data suggests pDNA-NPs are an effective delivery system for macrophage gene-editing.

The influence of differential burial preservation on the recovery of parasite eggs in soil samples from Korean medieval tombs.

The present study showed that ancient parasite eggs, not commonly present in soil samples from medieval Korean tombs, have been found in a very limited number of cases that satisfy certain archaeological requirements. In our paleo-parasitological examination of soil samples from medieval tombs encapsulated by a lime soil mixture barrier (LSMB), parasite eggs were more commonly detected in tombs that contained remains with clothes, hair, or brain tissue, though samples from not all such tombs contained eggs. Nonetheless, there was a close correlation between the preservation of certain types of cultural or human remains and the presence of ancient parasite eggs within medieval Korean LSMB tombs. Such remains, therefore, could be regarded as a strong predictor of well-preserved ancient parasite eggs in soil samples from LSMB tombs.