Vector-free intracellular delivery by reversible permeabilization

Abstract

Despite advances in intracellular delivery technologies, efficient methods are still required that are vector-free, can address a wide range of cargo types and can be applied to cells that are difficult to transfect whilst maintaining cell viability. We have developed a novel vector-free method that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying composition, properties and size. A permeabilizing delivery solution was developed that contains a low level of ethanol as the permeabilizing agent. Reversal of cell permeabilization is achieved by temporally and volumetrically controlling the contact of the target cells with this solution. Cells are seeded in conventional multi-well plates. Following removal of the supernatant, the cargo is mixed with the delivery solution and applied directly to the cells using an atomizer. After a short incubation period, permeabilization is halted by incubating the cells in a phosphate buffer saline solution that dilutes the ethanol and is non-toxic to the permeabilized cells. Normal culture medium is then added. The procedure lasts less than 5 min. With this method, proteins, mRNA, plasmid DNA and other molecules have been delivered to a variety of cell types, including primary cells, with low toxicity and cargo functionality has been confirmed in proof-of-principle studies. Co-delivery of different cargo types has also been demonstrated. Importantly, delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner. Unlike some other vector-free methods, adherent cells are addressed in situ without the need for detachment from their substratum. The method has also been adapted to address suspension cells. This delivery method is gentle yet highly reproducible, compatible with high throughput and automated cell-based assays and has the potential to enable a broad range of research, drug discovery and clinical applications.

Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts

“…As an example, significant membrane defects have been reported in membranes exposed to only 1% butanol 1329 . One case where ethanol is used for intracellular delivery purposes was report by O’Dea et al Ethanol sprayed with an atomizer was used to reversibility permeabilize cells for intracellular delivery of proteins, mRNA, and plasmids 1330 .…”

A Comprehensive Review on Intracellular Delivery

“…used ethanol as a permeabilizing agent for intracellular delivery purposes and demonstrated that the 25% v/v concentration of this alcohol provides the optimal permeabilization condition. [ 20 ] Studies have shown that ethanol remains at the water–lipid interface to induce disordering effect on phospholipid acyl chains leading to partially destroy the plasma membrane bilayer structure. [ 20,89 ]…”

“…[ 19 ] Once chemical modifications, extensive mechanical forces, or high‐intensity energies are introduced to the cells, the plasma membrane will experience the perturbation state, which triggers an increase in membrane deformation and permeability of the exogenous cargo. [ 20 ] Upon intracellular cargo delivery, the cell would reseal the disruptions through the active membrane and cytoplasmic recovery processes, which largely depend on the cell type, pore size, temperature and the factors presented in the extracellular medium. [ 21 ] Studies have proposed up to six different plasma membrane resealing mechanisms (e.g., exocytosis) that are implicated in active membrane repair.…”

Biomanufacturing for clinically advanced cell therapies

“…4d), effectively controlling loading. Avectas has a delivery efficiency of ~53% and a cell survival of 78% (for comparison, the efficiency and cell survival are 93% and 73%, respectively for electroporation) 94 . For most methods, effective delivery must be balanced with maintenance of cell viability to provide a scalable solution (Avectas is also developing a closed continuous system for GMP manufacturing).…”