biostudies-literatureHogarth CEngineering and Physical Sciences Research Council1042-1053https://www.ebi.ac.uk/biostudies/studies/S-EPMC12781925biostudies-literatureUnknown8(3)Lipid nanoparticle (LNP) formulations have emerged as a versatile platform for the delivery of therapeutics. However, achieving long-term stability and effective delivery of water-soluble small molecule drugs remains a challenge. In this study, we demonstrate a cryopreservable LNP formulation incorporating a hydrophobically modified bis-prodrug of lamivudine. By systematically varying the surfactant composition by combining a PEGylated surfactant (Brij S20) with an unPEGylated, zwitterionic lipid (Lipoid S100), we identify formulations that maintain colloidal stability following freeze-drying and redispersion in the presence of 10% w/v sucrose. Particle size measurements before and after lyophilisation indicate that surfactant ratio significantly impacts redispersibility, with 50/50 Brij/lipoid compositions offering the best performance. A core composition comprising the prodrug and tricaprin at either 1 : 1 or 3 : 1 ratio was evaluated, with the 3 : 1 formulation achieving redispersed particle sizes below 150 nm and low polydispersity. Enzymatic studies using porcine liver esterase confirm slow, sustained conversion of the bis-prodrug to active lamivudine over up to 9 weeks. This work highlights the opportunity of a prodrug-based strategy to formulate water-soluble APIs into stable, freeze-dried LNPs, enabling controlled, enzyme-responsive release. These findings offer insight into how surfactant composition influences freeze-drying compatibility and provide a platform for the development of LNP systems for small molecule delivery.Nanoscale advancesBis-prodrug cryopreserved lipid nanoparticles with enzymatically triggered release.PMC12781925EP/S012265/1Arnold KRannard SMcDonald TOHogarth CElkateb HfalseBis-prodrug cryopreserved lipid nanoparticles with enzymatically triggered release.Lipid nanoparticle (LNP) formulations have emerged as a versatile platform for the delivery of therapeutics. However, achieving long-term stability and effective delivery of water-soluble small molecule drugs remains a challenge. In this study, we demonstrate a cryopreservable LNP formulation incorporating a hydrophobically modified bis-prodrug of lamivudine. By systematically varying the surfactant composition by combining a PEGylated surfactant (Brij S20) with an unPEGylated, zwitterionic lipid (Lipoid S100), we identify formulations that maintain colloidal stability following freeze-drying and redispersion in the presence of 10% w/v sucrose. Particle size measurements before and after lyophilisation indicate that surfactant ratio significantly impacts redispersibility, with 50/50 Brij/lipoid compositions offering the best performance. A core composition comprising the prodrug and tricaprin at either 1 : 1 or 3 : 1 ratio was evaluated, with the 3 : 1 formulation achieving redispersed particle sizes below 150 nm and low polydispersity. Enzymatic studies using porcine liver esterase confirm slow, sustained conversion of the bis-prodrug to active lamivudine over up to 9 weeks. This work highlights the opportunity of a prodrug-based strategy to formulate water-soluble APIs into stable, freeze-dried LNPs, enabling controlled, enzyme-responsive release. These findings offer insight into how surfactant composition influences freeze-drying compatibility and provide a platform for the development of LNP systems for small molecule delivery.2026-01-01T00:00:00Z2026 Feb2026-06-16T07:30:46.456Z2026-06-16T03:10:16.721ZS-EPMC127819254152218010.1039/d5na00675a