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

Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2007; 104(25):10340-5. DOI: 10.1073/pnas.0703919104
Source: PubMed


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.

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    • "In this paper we have used luciferin-conjugated CPPs (luciferin– CPPs) to evaluate CPP uptake mechanisms. Previously we have used a semi-biological real-time uptake kinetics assay [18] [19] to characterize CPPs based on their uptake kinetics profiles [17]. In the latter study we showed that CPPs can be divided into two distinct groups —the fast internalization group (TP10, MAP and Tat) whose uptake kinetics profile resembled the behavior of membrane permeable free luciferin, and the slow uptake group (TP10(Cys), pVec, M918, penetratin and EB1) whose uptake profile was more consistent with the uptake rates conventionally observed in case of endocytosis. "
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    ABSTRACT: Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.
    Biochimica et Biophysica Acta 12/2011; 1818(3):502-11. DOI:10.1016/j.bbamem.2011.11.020 · 4.66 Impact Factor
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    • "Therefore, for therapeutic purposes the challenge remains in identifying the route yielding a biological response, which may not be the predominant one and to correlate the uptake pathway with a biological response associated with a specific cargo (Wadia et al., 2004; Gros et al., 2006). For that purpose, several approaches have been described, by using biological reporters (Wadia et al., 2004; Lebleu et al., 2008) or phenotypic (Morris et al., 2007a) assays enabling to follow shuttling and release of the cargo in real time in cultured cells (Lee et al., 2008) or in animal models (Wender et al., 2007) Although it remains difficult to establish a general scheme for CPP uptake mechanism, there is a consensus that the first contacts between the CPPs and the cell surface take place through electrostatic interactions with proteoglycans, and that the cellular uptake pathway is driven by several parameters including: (i) the nature and secondary structure of the CPP; (ii) its ability to interact with cell surface and membrane lipid components; (iii) the nature, type and active concentration of the cargo; and (iv) the cell type and membrane composition (Figure 1). "
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    ABSTRACT: The recent discovery of new potent therapeutic molecules that do not reach the clinic due to poor delivery and low bioavailability have made of delivery a key stone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). CPPs were first discovered based on the potency of several proteins to enter cells. Numerous CPPs have been described so far, which can be grouped into two major classes, the first requiring chemical linkage with the drug for cellular internalization and the second involving formation of stable, non-covalent complexes with drugs. Nowadays, CPPs constitute very promising tools for non-invasive cellular import of cargo and have been successfully applied for in vitro and in vivo delivery of therapeutic molecules varying from small chemical molecule, nucleic acids, proteins, peptides, liposomes and particles. This review will focus on the structure/function and cellular uptake mechanism of CPPs in the general context of drug delivery. We will also highlight the application of peptide carriers for the delivery of therapeutic molecules and provide an update of their clinical evaluation.
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