Determination of intraliposomal pH and its effect on membrane partitioning and passive loading of a hydrophobic camptothecin, DB-67

Abstract

The purpose of this work was to study the effect of pH on the liposomal encapsulation of a model camptothecin anti-tumor agent, DB-67, by considering the state of ionization and bilayer membrane/ water partitioning of the drug as a function of pH. A novel fluorescence method was developed to monitor intravesicular pH in liposomal formulations containing entrapped DB-67 by using the drug itself as a pH indicator. Fluorescence spectra were recorded in aqueous buffers and liposomes and used to estimate the ionization constant of the A-ring phenol of DB-67 (pKa 2 ) and shifts in ionization constants (pKa 1 and pKa 2 ) due to membrane binding. Bilayer/water partitioning studies by equilibrium dialysis were employed to show that DB-67 is highly membrane bound over the entire pH range examined though binding decreases with an increase in pH. The observed ionization constants of membrane-bound DB-67 obtained from the equilibrium dialysis experiments were consistent with observations from fluorescence measurements and previous permeability results. The pH dependence of DB-67 loading using a passive loading technique was found to reflect the pH dependence of membrane binding of the drug. This results in poor encapsulation efficiency of DB-67 at high pH, necessitating further development of formulation strategies to improve loading efficiency.

Effect of Cyclodextrin Complexation on the Liposome Permeability of a Model Hydrophobic Weak Acid

“…2). The ionization constant of membrane bound DB-67 was previously (6,7) shown to be approximately 1.5 pK a units higher than that for the free drug. Cyclodextrin complexation was also expected to shift the ionization constant of DB-67.…”

“…1) in a membrane impermeable ionized form was recently explored in order to develop prolonged release liposomal suspensions (5,6). However, a high intravesicular pH could not be maintained under physiological conditions due to the rapid dissipation of the trans-membrane pH gradient by carbonate buffer (CO 2 / H 2 CO 3 ) (5,7). In the present study, the ability of a liposomally entrapped, chemically modified cyclodextrin complexing agent (hydroxypropyl-β-cyclodextrin, HPβCD) to decrease the fraction of membrane permeable species and thereby prolong the liposomal retention of DB-67 was explored.…”

Enhanced active liposomal loading of a poorly soluble ionizable drug using supersaturated drug solutions

“…The assumptions of the present model are: (a) the lipid bilayer permeabilities of ionized species are negligible while both neutral and charged drug species cross the dialysis membrane with equal permeability coefficients; (b) SBE-CD and its complexes with AR-67 undergo transport across the dialysis membrane with identical permeability coefficients but liposomal uptake of these species is negligible; and (c) an instantaneous equilibrium exists between the various species inside the vesicle during the loading process [20,21,25]. All of the ionization constants and equilibrium constants for membrane binding of AR-67 were taken from previously published results [20,21,[25][26][27]. The permeability value for AR-67 lactone across the liposome was determined from model fitting of experimental data under various loading conditions.…”

Structural characterization of novel phospholipid lipid nanoparticles for controlled drug delivery

“…The excitation wavelength was fixed at 480 nm, and the emission spectra were recorded from 400 to 700 nm with a scanning speed of 100 nm/min and a wavelength accuracy of 1 nm. For fluorometric measurements, 3 ng nile red per 1 mg sample (corresponding to 0.000003% (nile red/w lipids)) was added [26].…”