Real-time analysis of uptake and bioactivatable cleavage of luciferin-transporter conjugates in transgenic reporter mice

Abstract

Many therapeutic leads fail to advance clinically because of bioavailability, selectivity, and formulation problems. Molecular transporters can be used to address these problems. Molecular transporter conjugates of otherwise poorly soluble or poorly bioavailable drugs or probes exhibit excellent solubility in water and biological fluids and at the same time an enhanced ability to enter tissues and cells and with modification to do so selectively. For many conjugates, however, it is necessary to release the drug/probe cargo from the transporter after uptake to achieve activity. Here, we describe an imaging method that provides quantification of transporter conjugate uptake and cargo release in real-time in animal models. This method uses transgenic (luciferase) reporter mice and whole-body imaging, allowing noninvasive quantification of transporter conjugate uptake and probe (luciferin) release in real time. This process effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications.drug delivery ͉ imaging ͉ transgenic animals M olecular transporters § are agents which, when covalently linked to or complexed with a cargo, enable or enhance its entry into cells or tissues. Many types of transporters have been reported in recent years including peptides (1-5), peptoids (6), polyamines (7,8), oligocarbamates (9), dendrimers (10, 11), polysaccharides (12), steroids (13), cationic lipids (14, 15), guanidinoglycosides (16), and even nanotubes (17). These transporters operate through a variety of mechanisms and some through multiple mechanisms depending on cell type, cargo, and other variables (5,(18)(19)(20)(21)(22). Among the classes of transporters, oligoguanidine based transporters are particularly promising, providing excellent water solubility and at the same time the remarkable ability to rapidly cross the nonpolar membrane of a cell. These transporters have been used for the delivery of small molecules, peptides, proteins, nucleic acids, liposomes, and imaging agents (23-29) and have been modified to provide selective delivery of drugs into target cells and tissue (30,31). Significantly, an octaarginine transporter has been shown to facilitate uptake into tissue (32), including human skin (23). A conjugate of octaarginine and Cyclosporin A enabling uptake of the latter selectively into the skin and thereby effectively eliminating its systemic toxicity has been advanced into Phase II clinical trials (23).There are two overarching and interrelated challenges confronting the further advancement of this field: the development of methods to quantify the uptake of new or existing transporters in real-time in animal models and the identification and evaluation of linkers that would allow for controllable release (if required) of a free drug/probe from the transporter conjugate only after cel...

In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter

“…Related strategies have been employed for assaying enzyme activity (33)(34)(35)(36)(37)(38) and cell-penetrating peptide transport efficiency (39), but these strategies have not been used for smallmolecule detection. Alkylation of firefly luciferin at the phenolic position prevents signal production even when luciferin's reactive carboxylic acid moiety remains unaltered (40).…”

The design of guanidinium-rich transporters and their internalization mechanisms

“…Recently, we have also reported an imaging method that provides, for the first time, quantification of transporter-conjugate uptake and cargo release in real-time in both cells and animal models [103]. This method uses luciferase-transfected cells and transgenic (luciferase) reporter mice and whole-body imaging, allowing non-invasive quantification of transporter-conjugate uptake and probe (luciferin) release in real-time.…”

Overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters

“…As a prelude to animal studies with Taxol conjugates, a third small-molecule system, luciferin, the substrate for firefly luciferase, was used as a drug surrogate to compare the biodistribution and pharmacokinetics of the r8 drug surrogate conjugate and the surrogate alone. Luciferin and luciferin conjugated to r8 (7) (38,39), at concentrations equimolar to those used in the studies with Taxol (5 mg/kg), were delivered via i.p. injection into transgenic mice ubiquitously expressing firefly luciferase (Fig.…”