Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases

Abstract

High-resolution imaging of molecules intrinsically involved in malignancy and metastasis would be of great value for clinical detection and staging of tumors. We now report in vivo visualization of matrix metalloproteinase activities by MRI and fluorescence of dendrimeric nanoparticles coated with activatable cell penetrating peptides (ACPPs), labeled with Cy5, gadolinium, or both. Uptake of such nanoparticles in tumors is 4-to 15-fold higher than for unconjugated ACPPs. With fluorescent molecules, we are able to detect residual tumor and metastases as small as 200 μm, which can be resected under fluorescence guidance and analyzed histopathologically with fluorescence microscopy. We show that uptake via this mechanism is comparable to that of other near infrared protease sensors, with the added advantage that the approach is translatable to MRI. Once activated, the Gd-labeled nanoparticles deposit high levels (30-50 μM) of Gd in tumor parenchyma with even higher amounts deposited in regions of infiltrative tumor, resulting in useful T 1 contrast lasting several days after injection. These results should improve MRI-guided clinical staging, presurgical planning, and intraoperative fluorescence-guided surgery. The approach may be generalizable to deliver radiationsensitizing and chemotherapeutic agents.Molecular navigation | dendrimeric nanoparticles | molecular amplification | targeted imaging agent | transgenic tumor model C linical cancer staging currently depends mainly on anatomical imaging with x-ray computed tomography (CT) and MRI. Some tumors can be imaged by PET of glucose uptake, but modest spatial resolution, high cost, exposure to radiation, and imperfect correlation of glucose uptake with malignancy limit the usefulness of PET and its more recent combination with CT. MRI is a particularly attractive imaging modality due to its moderate cost, relatively widespread availability, high spatial resolution tomography, excellent anatomical detail, and lack of radioactivity. Most clinical MRI is either T 1 -or T 2 -weighted, for which the standard contrast agents are, respectively, gadolinium (Gd) chelates and superparamagnetic iron oxide particles. The difficulty in using MRI for molecular imaging of specific biomolecules rather than for anatomy is sensitivity, because the detection limit is on the order of 10 −5 M Gd chelate or Fe, respectively (1, 2). Therefore several orders of magnitude of molecular amplification are necessary to detect tumor markers at low nanomolar abundance. T 2 -weighted MRI has the additional disadvantages that contrast is usually negative and the iron oxide particles are largely confined to the intravascular and reticuloendothelial compartments. Recently, there has been interest in designing T 1 magnetic resonance (MR) contrast agents that give information beyond that of a standard blood pool agent and detect tumor neovascularization (3, 4), folate receptor (5), and various antigens (6-8). Recent attempts at in vivo MRI of matrix metalloproteinase (MMP) activity have been based on...

Diagnosis and therapy are walking together on radiopeptides’ avenue

“…This capability can be further increased utilizing the effect on the blood-brain barrier permeability of contrast media for MRI and is therefore particularly interesting as a strategy for cerebral neoplasm [7].…”

Administration de molécules actives dans les cellules

“…Tsien a obtenu le prix Nobel de chimie en 2008 pour l'ensemble de ses travaux sur la conception de molécules fluorescentes et photolabiles [57]. Cette même approche a été généralisée à l'utilisation de nanoparticules de gadolinium, ce qui permet, outre une détection de la fluorescence, une imagerie de la tumeur par IRM [41]. D'autres efforts sont en cours de réalisation pour la détection d'ARNm cellulaires par la délivrance de PNA aux séquences complémentaires à ces ARNm, grâce à des CPP couplés à des agents de contraste IRM, tel le DOTA [44].…”

Ratiometric Activatable Cell‐Penetrating Peptides Provide Rapid In Vivo Readout of Thrombin Activation

“…Analogous ACPPs attached to dendrimers labeled with Gd chelates have given MRI contrast for primary tumors [18] and metastatic lymph nodes, [31] so we are analogously attempting to image thrombin activity in atherosclerotic plaques with MRI. Unfortunately, MRI does not have ready equivalents for FRET or ratiometric fluorescence imaging.…”

“…[15] Activatable cell-penetrating peptides (ACPPs) target various cargos, including fluorescent imaging agents, to sites of protease activity in vivo. [16][17][18][19] ACPPs consist of a polycationic cell-penetrating peptide attached to a cargo and a polyanionic inhibitory domain with a protease-cleavable linker. Probe activation and cargo uptake depend on localized proteolysis of the linker sequence that connects the polyanionic and polycationic domains, which converts the probe to an adherent form.…”