Cryopreservation technology for improving the stability of liposomes and its precise drug monitoring in clinical drug research.

The instability of liposomes in blood samples during clinical drug research and drug monitoring results in the inability to accurately determine the actual drug concentrations in the body at the time of collection, mainly due to lipid deterioration, particle fusion or aggregation, and phase separation degradation, resulting in payload leakage. To improve drug monitoring accuracy, we developed a cryopreservation strategy in this study by innovatively combining cryoprotective agents (CPAs), such as L-proline, sucrose, and polyvinyl alcohol (PVA), to prevent liposomal leakage and maintain stability for reliable drug monitoring and clinical drug research applications. Doxorubicin liposomes were prepared, and the CPAs were tested at various concentrations and under different freeze‒thaw protocols in biological matrices, with the stability and leakage of the liposomes assessed. Each CPA contributes distinct stabilization mechanisms, with L-proline's osmoprotective ability, sucrose's hydrogen bonding, and PVA's steric hindrance to form a protective barrier. The optimized CPA combination demonstrated superior performance at 85% (v/v) by preserving liposomal integrity, offering the best cryoprotective effect for liposomes in plasma stored at -20 °C, achieving about 90% entrapment efficiency, compared to about 60% in the control group without CPAs. Mechanistic investigations confirmed that CPAs protect liposomes against mechanical stress, prevent membrane disruption, and reduce ice damage by inhibiting recrystallization and adjusting bilayer hydration. These findings offer practical solutions for accurate pharmacokinetic assessments and reliable personalized dosing, safer alternative for liposomal drug research, biobanking, and real-world therapeutic monitoring.