A robust and quantitative method for tracking liposome contents after intravenous administration

Abstract

We introduce a method for tracking the rate and extent of delivery of liposome contents in vivo based on encapsulation of 4-methylumbelliferyl phosphate (MU-P), a profluorophore of 4-methylumbelliferone (MU). MU-P is rapidly dephosphorylated by endogenous phosphatases in vivo to form MU after leakage from the liposome. The change in fluorescence spectra when MU-P is converted to MU allows for quantification of entrapped (MU-P) and released (MU) liposome contents by fluorescence or by a sensitive high performance liquid chromatography assay. We define the "cellular availability" of an agent encapsulated in a liposome as the ratio of the amount of released agent in the tissue to the total amount of agent in the tissue; this parameter quantifies the fraction of drug available for therapy. The advantage of this method over existing technologies is the ability to decouple the signals of entrapped and released liposome contents. We validate this method by tracking the circulation and tissue distribution of MU-P loaded liposomes after intravenous administration. We use this assay to compare the cellular availability of liposomes composed of engineered phosphocholine lipids with covalently attached cholesterol, sterol-modified lipids (SML), to liposomes composed of conventional phospholipids and cholesterol. The SML liposomes have similar pharmacokinetic and biodistribution patterns as conventional phospholipid-cholesterol liposomes but a slower rate of contents delivery into the tissue. Thus, MU-P enables the tracking of the rate and extent of liposome contents release in tissues and should facilitate a better understanding of the pharmacodynamics of liposome-encapsulated drugs in animals.

Keywords: 4-methylumbelliferone; Drug release; Liposomes; Sterol-modified lipids.

Figure 1. Relevant Structures

Structures of 4-methylumbelliferone (MU), MU metabolite 4-methylumbelliferyl glucuronide (MU-G), MU prodrugs 4-methylumbelliferyl phosphate (MU-P) and 4-methylumbelliferyl sulfate (MU-S), carboxyfluorescein (CF), and HPLC standard 7-hydroxycoumarin (Std).

Figure 2. MU-P Reporter System

(A) MU-P is encapsulated in liposomes. Upon leakage from liposomes, MU-P is rapidly converted to MU by endogenous phosphatases. (B,C) Michaelis-Menten kinetics of MU-P and MU-S conversion in mouse serum.

Figure 3. Effects of Acyl Chain Length, Saturation, and Liposome Composition on Membrane Stability

CF (A) or MU-P (B) was passively encapsulated in (C) a range of CPL (○) or SML (Δ). Molar ratios of liposome formulations are given. Liposomes were incubated in HBS containing 30% serum. The percent of MU-P leaked from liposomes after 14 days is shown.

Figure 4. Evaluation of SML and CPL Stability in Circulation by Fluorescence

(A-H) MU-P, MU and DiD signals were measured in serum samples by fluorescence. The MU-P and DiD data is fit to a two-compartment model. MU was undetectable in serum at all time points. (I) The ratio of (AUC_MUP/AUC_DiD) for each formulation is shown.

Figure 5. SML Alter Delivery to the Liver and Spleen

(A) HPLC trace from the liver, spleen, and kidney of a mouse injected with liposomal MU-P. Percent of injected dose in the (B) liver and (C) spleen at 3 hours (left) and 48 hours (right) after administration. MU, MU-P and MU-G levels are shown and sum to the total amount of MU equivalents in the tissue.