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.

Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan

“…This term describes the fraction of drug released from the liposome and available for therapeutic activity [51]. The presence of both MU and MU-P in the tumor 48 hours after administration indicates the persistence of intact liposomes, and the presence of MU supports the hypothesis of sustained MU-P release from liposomes to inhibit HA synthesis (Figure 3D).…”

“…MU-P is rapidly dephosphorylated over the course of minutes to form MU in serum (Figure 1B) and in a number of tissue homogenates [51]. This conversion is slower in heat-inactivated serum, indicating a loss in endemic serum phosphatases (data not shown).…”

“…MU-P was passively encapsulated in a range of liposome formulations and its rate of release from each formulation was quantified using a simple in vitro assay [51]. Two MU-P encapsulating formulations, HSPC and POPC , were selected for further study (Figure 2A).…”

Designer lipids for drug delivery: From heads to tails

“…Interestingly, we found that at acyl chain lengths of C 16 and C 18 , SML liposomes are more stable in circulation than liposomes containing free cholesterol. Further, we found that these SML liposomes had improved uptake into and slower clearance from the liver and spleen compared to traditional liposomes [136]. These studies highlight the stability of SML systems in vivo and their potential utility as drug carrier systems.…”

“…SMLs are easily synthesized, commercially available, and recapitulate the biophysical properties of liposomes incorporating cholesterol [134–136]. Compared to liposomes formulated with free cholesterol, SMLs eliminate the phase transition of diacylphospholipids [135], exhibit similar permeability to entrapped hydrophilic molecules [135,136] and demonstrate similar membrane fluidity [136]. SMLs maintain these properties while preventing cholesterol transfer from the bilayer [135].…”