Molecular additives: all gain, no pain in gene delivery

Non-viral gene delivery systems face a persistent challenge: achieving high transfection efficiency while maintaining acceptable cytotoxicity. Cationic vectors effectively condense nucleic acids into complexes and promote cellular uptake, yet their positive surface charge drives cytotoxic interactions with cellular membranes and serum components. This review examines how molecular additives, auxiliary charged molecules incorporated during complexation, address this fundamental trade-off through ternary complex formation. We systematically analyze three functional classes of additives based on their primary mechanisms. Additives enhancing transfection efficiency, including oligoamines (spermine, spermidine) and modified polypeptides (carboxymethylated poly-L-histidine), boost gene delivery by enhancing proton sponge activity, improving endosomal escape, and stabilizing nucleic acid interactions via dehydration effects and hydrogen bonding. Additives enhancing viability, comprising synthetic polyacids (polyglutamic acid, polyaspartic acid) and polysaccharides (alginate), reduce cytotoxicity by neutralizing surface charge, preventing protein corona formation, and minimizing nonspecific membrane interactions while maintaining colloidal stability. Dual function glycosaminoglycans (hyaluronic acid, heparin, chondroitin sulfate) simultaneously address both parameters by coupling electrostatic shielding with receptor-mediated cellular uptake via CD44, HARE, and RHAMM, enabling targeted delivery despite their reduced surface charge. Beyond cataloging these additives, we provide mechanistic insights into how assembly pathways, stoichiometric ratios, and molecular interactions govern complex performance. This analysis establishes rational design principles for optimizing the efficiency-cytotoxicity balance in non-viral gene delivery, demonstrating that strategic additive selection enables the development of safer and more effective vectors. • Molecular additives tune efficiency-cytotoxicity in non-viral gene delivery • Oligoamines boost transfection via buffering, endosomal escape and cargo stability • Polyacids/polysaccharides cut cytotoxicity by charge neutralization and corona block • Glycosaminoglycans provide dual benefits via charge shielding and receptor targeting • Assembly pathway and stoichiometry critically drive complex structure and performance