Targeting the Inside of Cells with Biologicals: Chemicals as a Delivery Strategy

Abstract

Delivering macromolecules into the cytosol or nucleus is possible in vitro for DNA, RNA and proteins, but translation for clinical use has been limited. Therapeutic delivery of macromolecules into cells requires overcoming substantially higher barriers compared to the use of small molecule drugs or proteins in the extracellular space. Breakthroughs like DNA delivery for approved gene therapies and RNA delivery for silencing of genes (patisiran, ONPATTRO ® , Alnylam Pharmaceuticals, Cambridge, MA, USA) or for vaccination such as the RNA-based coronavirus disease 2019 (COVID-19) vaccines demonstrated the feasibility of using macromolecules inside cells for therapy. Chemical carriers are part of the reason why these novel RNA-based therapeutics possess sufficient efficacy for their clinical application. A clear advantage of synthetic chemicals as carriers for macromolecule delivery is their favourable properties with respect to production and storage compared to more bioinspired vehicles like viral vectors or more complex drugs like cellular therapies. If biologicals can be applied to intracellular targets, the druggable space is substantially broadened by circumventing the limited utility of small molecules for blocking protein–protein interactions and the limitation of protein-based drugs to the extracellular space. An in depth understanding of the macromolecular cargo types, carrier types and the cell biology of delivery is crucial for optimal application and further development of biologicals inside cells. Basic mechanistic principles of the molecular and cell biological aspects of cytosolic/nuclear delivery of macromolecules, with particular consideration of protein delivery, are reviewed here. The efficiency of macromolecule delivery and applications in research and therapy are highlighted.

Targeting the Inside of Cells with Biologicals: Toxin Routes in a Therapeutic Context

“…lipid- or polymer-based reagents). Chemicals may therefore allow a slightly more versatile choice of cargoes, having been applied for both nucleic acid delivery and the direct delivery of proteins, although mainly applied for nucleic acid delivery [ 6 , 145 , 276 ]. However, nucleic acids have also already been described as a potential cargo when delivering via toxin routes [ 277 , 278 ].…”

“…These results show how the efficiency of cytosolic delivery was rather limited for TAT. Cytosolic delivery methods based on chemicals are also known to have very limited delivery efficiency in the low single percentage range (1–2% reaching the cytosol [ 128 ], ~ 3.5% reaching the cytosol [ 129 ] and 1–5% of nucleic acids reaching the nucleus [ 130 ]), although delivery by chemicals is already used for approved drugs, such as coronavirus disease 2019 vaccines [ 6 , 131 – 135 ]. Therefore, provided the low efficiency of delivery to the cytosol is sufficient for the application, delivery via toxin routes might be just as suitable for therapeutic strategies as approved drugs using chemicals for delivery.…”

“…The cell culture medium had been renewed after transfection of the colon cancer cell line with guide RNA-encoding plasmids [ 125 ]. Removal of transfection reagents might be a relevant detail in this context because the delivery of proteins by certain chemicals has been demonstrated [ 6 , 144 , 145 ]. In addition, Cas9 together with nucleic acids have been delivered to cells by various chemicals [ 6 , 146 – 149 ].…”

“…Removal of transfection reagents might be a relevant detail in this context because the delivery of proteins by certain chemicals has been demonstrated [ 6 , 144 , 145 ]. In addition, Cas9 together with nucleic acids have been delivered to cells by various chemicals [ 6 , 146 – 149 ]. Furthermore, histidine residues have been suggested to potentially enhance cytosolic delivery [ 150 – 152 ].…”

“…Many intracellular delivery methods for macromolecules are associated with some degree of membrane damage, for example, many chemicals that are used as carriers for macromolecule delivery or electroporation as a delivery method [ 5 , 6 ]. Delivery of macromolecules in a way that does not result in damage to membranes might be less toxic for the cells.…”