Superior transfection efficiency of phagocytic astrocytes by large chitosan/DNA nanoparticles

Mechanism study of why astrocytes isolated from experimental autoimmune encephalomyelitis (EAE)-induced B6 mice or after being exposed to inflammatory factors had the highest transfection efficiency to larger-sized, but not compacted, pspCS/pDNA particles.

Phosphorylatable short peptide conjugated chitosan (pspCS) was compounded with plasmid DNA (pDNA) at different N:P ratios to form pspCS/pDNA particles of different size and zeta potentials. These pspCS/pDNA particles were used for the transfection of astrocytes isolated from either EAE induced or healthy B6 mice. Transfection efficiency and cell permeability of the particles were determined by the internalization of radio [H3]-labeled plasmid and the expression of a luciferase reporter gene respectively. Phagocytosis of EAE-astrocytes was determined by the internalization of FITC labeled dextran beads. By comparing the transfection efficiency of differently-sized pspCS/pDNA particles to normal and phagocytic astrocytes, with or without cytochalasin D, a phagocytosis inhibitor, in the presence, the contribution of phagocytosis to cell permeability and transfection efficiency was evaluated.

In vivo EAE-induction or in vitro inflammatory factors treatment transferred normal astrocytes to be phagocytic astrocytes which underwent phagocytosis, had the highest cell permeability and transfection efficiency to larger-sized pspCS/pDNA particles formed at lower N:P ratios. When phagocytosis was inhibited by cytochalasin D, both cell permeability and transfection efficiency of phagocytic astrocytes to larger were significantly decreased. Thereafter, particle size, not zeta potential, was verified as the key factor for determining whether the particles could be phagocytosed. In addition phagocytosis was successfully induced in ARPE-19 cells as well, which also improved the transfection efficiency of larger pspCS/pDNA particles.

A generally accepted concept is that the internalization of cationic polymer/pDNA particles, chitosan-DNA complex for instance, is mainly through the procedure of endocytosis of the transfected cells. More compacted particles with higher zeta potential were used to be considered had higher cell permeability and transfection efficiency. However, here we reported that phagocytosis is another important procedure for determining internalization and transfection efficiency of cationic polymer/pDNA nanoparticles, especially for advanced transfection efficiency of large pspCS/pDNA particles. Thus, for gene delivery applications, the environmental condition of the target cells should be seriously considered for selecting an appropriate gene transfer strategies.