Protonation Kinetics Compromise Liposomal Fluorescence Assay of Membrane Permeation

Area of Science:

• Pharmacokinetics and Drug Delivery
• Biophysical Chemistry
• Membrane Transport Phenomena

Background:

• Membrane permeation of weak acids and bases is pH-dependent.
• Fluorescence measurements using pH-sensitive dyes are common for studying drug permeation.
• Previous studies with aliphatic amines showed biexponential permeation kinetics into liposomes.

Purpose of the Study:

• To investigate the origin of biexponential kinetics observed in drug permeation studies.
• To analyze the influence of protonation rates on fluorescence-based drug permeability measurements.
• To emphasize the necessity of comprehensive kinetic modeling in drug permeation research.

Main Methods:

• Development of a kinetic rate model incorporating drug and dye protonation alongside permeation.
• Analysis of fluorescence data using the developed kinetic model.
• Comparison of model predictions with experimental observations.

Main Results:

• Biexponential kinetics in fluorescence measurements are strongly dependent on protonation rates.
• The faster kinetic component, often used for permeability coefficients, is influenced by these rates.
• The interplay between permeation and protonation kinetics affects the observed experimental readout.

Conclusions:

• Fluorescence studies using pH-sensitive proxies for drug permeation require careful consideration of protonation kinetics.
• Accurate determination of drug permeability coefficients necessitates comprehensive kinetic modeling.
• The study highlights potential limitations in interpreting fluorescence data without accounting for all relevant chemical processes.