Approach	Description
1. Enter circulation or target tissue
Parenteral administration	IV administration used for LNPs and other nanoparticles
Subcutaneous injection	GalNAc conjugates are administered subcutaneously and presumably reach the target tissue via lymphatics
Topical application	Used for targeting the skin, eye and mucosa
2. Avoid excretion
PEGylation	Increases the molecular weight of siRNA or delivery vehicle to avoid renal excretion
Cholesterol conjugation	Cholesterol-conjugated siRNAs bind to circulating lipoprotein particles
Nanoparticle formulation	Nanoparticles are above the renal filtration cut-off
3. Avoid nuclease degradation
Nucleic acid backbone modifications	Deoxynucleotides, phosphorothioate linkages and several other modifications of the ribonucleotides confer nuclease resistance
Nanoparticle formulation	Packaging within a delivery vehicle makes the RNA inaccessible to nucleases
4. Avoid immune recognition
Nucleic acid backbone modifications	2´-O-methyl and 2´-fluoro modifications block innate immune stimulation
PEGylation	Surface charge minimizes binding to phagocytic cells and other cells
5. Extravasation
Target tissues with leaky vessels	The liver and spleen have a fenestrated endothelium. Tumours can have leaky vessels
Endothelial transcytosis	Theoretically attractive approach to gain access to any tissue
Target endothelial or blood cells	No need to exit the vasculature
6. Cellular uptake
Targeting ligand	Conjugate siRNA or delivery vehicle to receptor-targeting moiety (ligand, aptamer or antibody fragment) for cell-specific uptake
Association with endogenous ligand	Cholesterol-conjugated siRNAs and LNPs bind to serum apolipoproteins conferring uptake in hepatocytes
7. Endosomal release
Membrane-destabilizing lipids	Lipid nanoparticles contain lipid bilayer-disrupting lipids that are activated by low endosomal pH
Membrane-destabilizing peptides and polymers	Masked endosomolytic peptides or polymers become unmasked (positively charged) in acidic endosomes and enhance endosomal escape of siRNA
Increase endosomal accumulation	Even if endosomal release is inefficient, efficient uptake can compensate for poor release, as only a few hundred cytosolic siRNAs are needed for maximal knockdown

IV, intravenous; GalNAc, N-acetylgalactosamine; LNP, lipid nanoparticle; PEG, polyethylene glycol.