Lipid nanoparticles, comprising stable complexes between synthetic cationic lipids and anionic nucleic acids, are currently the most widely used non-viral delivery system for nucleic acid drugs and vaccines. The advantages of lipid systems include ease of production, biodegradability, protection of entrapped nucleic acids from nuclease degradation and renal clearance, promotion of cellular uptake, and endosomal escape. Since the first cationic lipids successfully delivered plasmids into cells in 1987, many more have been synthesized and tested as nucleic acid carriers. Cationic lipids differ from natural lipids by having an ionizable (cationic) head group in place of the zwitterionic or anionic head group of the natural lipids. They include two hydrophobic alkyl chains or a cholesterol moiety, a positively charged polar head group, and a linker joining them. Ionizable lipids are positively charged inside the cell but are neutral in the bloodstream due to pH differences, and they are less toxic than nonionizable cationic lipids. Examples of cationic lipid structures used as vectors for gene delivery are provided in Figure , and a comprehensive list, including the chemical structures of the ̴ 50 most frequently used cationic lipids in drug delivery , is available. The head groups of cationic lipids are typically amine derivatives such as primary, secondary, and tertiary amines (e.g., DOGS, DC-Chol), quaternary ammonium (e.g., DOTMA, DOTAP, DORIE, DMRIE), combinations of amines (e.g., DOSPA, GAP-DLRIE), and amidinium salts (e.g., diC14-amidine) (Figure ). Guanidine and imidazole groups 279 as well as pyridinium, piperazine, and amino acid headgroups such as lysine, arginine, ornithine, and tryptophan have also been used. Headgroups with multiple cationic charges such as DOSPA, DOGS 282 may be more efficient than single-charged lipids 283 since the multiple charges of the headgroups condense nucleic acids. However multivalent cationic lipids bind strongly to the nucleic acid preventing intracellular release, and they tend to form toxic micelles. Combinations of quaternary amines and polyamines enhance transfection efficiency. The first cationic lipid to combine these two functionalities, Lipofectamine, is the long-standing gold standard in nucleic acid delivery efficiency. It comprises DOSPA formulated with the helper lipid dioleoyl phosphatidylethanolamine (DOPE). Complexation with cationic lipids stabilizes nucleic acids, protects them from nuclease degradation, and facilitates delivery to the target cells. Lipid nanoparticles carrying nucleic acids adsorb to the cell membrane, are endocytosed, and release the nucleic acids into the cell. Cell membranes have negative charges that attract the cationic lipid nanoparticles and drive the adsorption and fusion of the lipid nanoparticle with the cell membrane. The anionic lipids of the cells are thought to neutralize the charge of the cationic lipid carriers, thereby eliminating the electrostatic interactions between the lipid carriers and the nucleic acids and facilitating the release of the nucleic acids. Neutralization of the cationic lipids also destroys the nanoparticles by promoting the formation of nonlamellar structures 286 , which are associated with the efficacy of cationic lipid carriers in delivering nucleic acids into cells. Despite their benefits, lipid nanoparticles show cell toxicity, stimulate the release of systemic inflammatory cytokines, and lipid aggregates may accumulate in the liver and spleen causing hepatotoxicity. Figure . Examples of cationic lipids used as nucleic acid carriers. (An extensive list with the structures of the most frequently used cationic lipids in lipid nanoparticle pharmaceutical formulations according to the CAS Content Collection is available) Nearly 20 years after scientists first prepared liposomes (the first-generation lipid nanoparticles), they found similar ̴ 100 nm extracellular lipid vesicles in most eukaryotic cells. They are exosomes that form naturally in the endosomes and are released from cells normally or as result of some pathologies. They are formed by invaginations of the endosomal membrane and are subsequently released in the extracellular space via fusion with the plasma membrane. Although their functions are largely unknown, they are believed to be associated with important physiological processes such as the regulation of the intercellular communications and signaling, and of transmission of macromolecules between cells. Exosomes, which are secreted by most cells, may be important in intercellular communication and signaling, and in the transport of proteins, lipids, and nucleic acids between cells. Since they are rich in mRNAs and small RNAs and can transport their contents to recipient cells, they are considered good candidates for RNA delivery. Exosome-mediated delivery of β-secretase-1 siRNA (targeting the enzyme β-secretase, which is important in Alzheimer's disease) to the brain resulted in highly efficient β-secretase-1 gene knockdown in the mouse brain cortex. Electroporation has been applied to load exosomes with exogenous siRNA.