Efficiency — what it means and how to control it

Binding: the carrier must attach to the plasma membrane without being washed away by serum proteins. Cationic complexes bind electrostatically to the anionic glycocalyx; the number of particles per cell surface — not the total dose — drives the first step.

Uptake: most non-viral reagents enter through endocytosis, which is clathrin-, caveolin- or macropinocytosis-dependent according to particle size, charge and cell type. Particles smaller than 200 nm favour clathrin routes; larger aggregates drift toward macropinocytosis and often correlate with toxicity.

Endosomal escape: this is the true bottleneck. Under 2% of internalised plasmid typically reaches the cytosol, because cargo is either recycled back to the surface or degraded after endosome–lysosome fusion. Escape strategies are chemistry-specific: the proton sponge effect for PEI, cone-shaped helper lipids such as DOPE for lipoplexes, and ionisable lipid fusion for LNPs.

Nuclear access: plasmid DNA must cross the nuclear envelope, so it is efficient mainly in dividing cells where the envelope transiently disassembles. mRNA, siRNA and RNP act in the cytoplasm and are therefore far less dependent on cell-cycle status — a key reason RNP formats win in non-dividing cells.

Different readouts answer different questions. A fluorescent reporter gives the fraction of cells that received cargo; a luminescent reporter (luciferase) gives total protein produced and is more sensitive to strong single-cell expression; qPCR gives transcript levels independent of translation.

Always pair a positive control carried through the same workflow with your test condition. Normalising to a control reagent lets you compare runs and operators instead of chasing absolute percentages that shift with passage number and confluency.

Efficiency has a maximum in the reagent-to-cargo ratio, and pushing past it usually costs viability rather than gaining delivery. Build a two-dimensional matrix in advance: three reagent volumes × three nucleic-acid amounts in the same plate, with a no-reagent and a no-cargo control in every corner.

Because expression kinetics differ by format, harvest at several timepoints. Plasmid peak expression normally lands at 48–72 h, siRNA at 24–48 h, and CRISPR editing phenotypes often only stabilise at 72–96 h when the residual protein has been diluted by division.