Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient

Area of Science:

• Nanomedicine
• Drug Delivery Systems
• Biophysics

Background:

• Doxorubicin (DOX) is a widely used chemotherapy drug.
• Liposomes are common drug delivery vehicles for DOX.
• Controlling the physical state of DOX within liposomes is crucial for drug efficacy and release.

Purpose of the Study:

• To investigate the physical state of doxorubicin (DOX) inside liposomes.
• To understand the role of buffer composition (citrate vs. lactobionic acid) on DOX aggregation.
• To correlate DOX physical state with liposomal drug release.

Main Methods:

• Cryogenic electron microscopy (cryo-EM) for visualizing DOX structures.
• Small-angle X-ray diffraction to analyze fiber aggregation patterns.
• Circular dichroism (CD) and 13C-NMR for molecular interactions and dynamics.
• In vitro drug release studies under various conditions.

Main Results:

• DOX formed linear, curved, and circular fiber bundles within citrate liposomes at high concentrations (200-300 mM) and even at lower concentrations (~20 mM).
• Cryo-EM and CD indicated >99% of DOX was in fiber bundles in citrate liposomes.
• DOX remained disaggregated in lactobionic acid (LBA) liposomes at ~20 mM but formed uncondensed fibers at ~200 mM.
• Citrate likely electrostatically bridged DOX fibers, forming bundles.
• DOX release was slow (<4% at 1h) from liposomes containing DOX fibers, but rapid leakage occurred from LBA liposomes with disaggregated DOX due to osmotic stress.

Conclusions:

• DOX self-assembles into ordered fiber structures within liposomes, particularly when buffered with citrate.
• Citrate plays a key role in stabilizing DOX fiber bundles, influencing drug release kinetics.
• The physical state of DOX within liposomes significantly affects its release profile and potential for osmotic stress-induced leakage.