Magnetofection-Based Transfection In Vitro: A Magnetic Field-Assisted Technique to Deliver Plasmids Into Primary Mouse Neuronal Cells Using Magnetic Nanoparticles
Transcrição
Begin with fluorescent protein-encoding plasmid DNA or pDNA suspension in media. Add biocompatible, cationic polymer-coated magnetic nanoparticles to the pDNA suspension and incubate.
The positively-charged cationic polymers electrostatically interact and conjugate with negatively-charged pDNA, resulting in pDNA condensation and formation of 'magnetic polyplexes' with an overall positive charge.
Now, transfer the polyplex mixture into a multi-well plate containing adherent primary mouse neuronal cell culture. Mount the multi-well plate over a magnetic plate and incubate.
The magnetic field generated by the magnetic plate causes the polyplexes to sediment and accumulate over the neuronal cell surface. This close contact with the cell surface facilitates the non-specific binding of positively-charged polyplexes to negatively-charged cell membrane-associated proteoglycans.
Subsequently, the polyplexes get internalized via endocytosis – a process wherein the local regions of the cell membrane invaginate and pinch off to form membrane-bound vesicles called endosomes.
Inside the cytoplasm, the polyplex's cationic polymers induce proton accumulation in the endosome. The simultaneous influx of chloride ions to maintain charge neutrality increases endosomal ionic strength, leading to water influx.
Eventually, the endosomes swell and rupture due to osmotic pressure, releasing polyplexes into the cytoplasm. The condensed pDNA within the polyplexes facilitate increased cytosolic motility, while the cationic polymers protect pDNA from cytoplasmic nuclease degradation.
Following dissociation of the polyplexes, the liberated pDNA – during the appropriate cell-cycle phase when the nuclear membrane temporarily disassembles – get translocated to the nucleus. Cells successfully transfected with pDNA express fluorescent proteins.
