Freezing-Assisted Gene Delivery Combined with Polyampholyte Nanocarriers

Abstract

Physical methodologies such as electroporation and the gene-gun technology have been widely used for transfection; however, their applicability is limited because they lead to cell damage and low cell viability. Therefore, to address these limitations we developed a new freeze concentration-based gene transfection system that provides enhanced in vitro gene delivery compared to that provided by the commercially available systems. The system employs a facile freeze concentration step, whereby cells are simply frozen to very low temperatures in the presence of polymer-pDNA complexes. As part of system development, we also synthesized a low toxicity polyethylenimine (PEI)-based polyampholyte prepared through succinylation with butylsuccinic anhydride. In aqueous solution, this modified polyampholyte self-assembles to form small (20 nm diameter), positively charged (net surface charge of 35 mV), nanoparticles through a combination of hydrophobic and electrostatic interactions. Agarose gel electrophoresis analysis indicated that the polyampholyte nanoparticle was able to form a complex with pDNA that provided stability against nuclease degradation. Using transfection of HEK-293T cells, we demonstrated that a combination of polyampholyte: pDNA, at an appropriate ratio, and the freeze concentration method resulted in significant enhancement of GFP and luciferase expression compared to commercially available carriers. Endosomal escape of pDNA was also found to be increased when using the modified polyampholyte compared to branched PEI. This study suggests that the efficient combination of freeze concentration and the modified polyampholyte described here has great potential for in vitro gene therapy.

Enhanced Adsorption of a Protein–Nanocarrier Complex onto Cell Membranes through a High Freeze Concentration by a Polyampholyte Cryoprotectant

“…In our investigation, we found that 10% PLL-SA had remarkably higher adsorption (Figure G–L), and the same phenomenon was also observed for internalization. This is because the adsorption and internalization of proteins into the cells generally have a linear relationship, as the number of particles that adhere to the cell membrane will reflect that of particles being transferred inside the cells, as has been observed in our previous studies. ,,, Overall, this detailed study demonstrated the efficient use of the ice-crystal-based freezing approach to the internalization of proteins with the help of a unique polyampholyte cryoprotectant.…”

Bridging polymer chemistry and cryobiology

“…7b). Thus, we have reported a simple technique for freeze-thawing that allows macrophages to efficiently ingest antigens [55], introduce genes [56], and perform cell imaging by introducing quantum dots [57]. This method is inexpensive, novel, and does not require special equipment.…”

Hydrophobic Polyampholytes and Nonfreezing Cold Temperature Stimulate Internalization of Au Nanoparticles to Zwitterionic Liposomes

“…Polyampholytes including zwitterionic polymers also show promise in several other biomedical applications such as for cell preservation, antibiofouling, and tissue engineering. , These polyelectrolytes contain both cationic and anionic charges in the polymeric backbone. We previously developed new hydrophobic polyampholytes that can form self-assembled nanoparticles and efficiently transfer protein-based materials to the cytosol of cells. − These hydrophobic polyampholytes were easily designed by modifying the anionic functional group dodecylsuccinic anhydride (DDSA) and a large amount of succinic anhydride (SA) in ε-poly- l -lysine (PLL), which confers the hydrophobic polyampholytes with an anionic charge. Although hydrophobicity can enhance cell membrane affinity, it is nevertheless difficult to internalize proteins inside cells because of the negatively charged surface potential.…”

“…Although hydrophobicity can enhance cell membrane affinity, it is nevertheless difficult to internalize proteins inside cells because of the negatively charged surface potential. Thus, to achieve efficient delivery to cells, we also developed a novel low temperature-based freeze-concentration approach to act as a driving force to internalize materials inside cells efficiently. − Freeze concentration is a physicochemical phenomenon wherein water molecules are crystallized into ice so that the solutes present in the system are ejected, which thereby enhances the concentration. The increased concentration occurs across the periphery of the cell membrane resulting in adsorption of solutes.…”