Evaluation of crotamine based probes as intracellular targeted contrast agents for magnetic resonance imaging

Rajendra Joshi; Kamal Sweidan; Deepti Jha; Irina Kerkis; Klaus Scheffler; Joern Engelmann

doi:10.1016/j.bmc.2022.116863

Evaluation of crotamine based probes as intracellular targeted contrast agents for magnetic resonance imaging

Bioorg Med Chem. 2022 Sep 1:69:116863. doi: 10.1016/j.bmc.2022.116863. Epub 2022 Jun 15.

Authors

Rajendra Joshi¹, Kamal Sweidan², Deepti Jha³, Irina Kerkis⁴, Klaus Scheffler⁵, Joern Engelmann³

Affiliations

¹ Department of Chemical Science and Engineering, School of Engineering, Kathmandu University, Dhulikhel, Nepal. Electronic address: rajendra.joshi@ku.edu.np.
² Department of Chemistry, The University of Jordan, Amman 11942, Jordan.
³ High-Field MR Center, Max Planck Institute for Biological Cybernetics, Tübingen, Germany.
⁴ Laboratory of Genetics Butantan Institute São Paulo, Brazil.
⁵ High-Field MR Center, Max Planck Institute for Biological Cybernetics, Tübingen, Germany; Department of Biomedical Magnetic Resonance, University of Tübingen, Germany.

PMID: 35752142
DOI: 10.1016/j.bmc.2022.116863

Abstract

Crotamine is a lysine and cysteine rich 42 amino acids long bio-active polypeptide, isolated from the venom of a South American rattlesnake, that can also be used as cell penetrating peptide. A facile synthetic scheme for coupling cargo molecules like fluorophores (carboxyfluorescein) or MRI probes (Gd-DO3A-based macrocycle) is presented. The toxicity, cellular internalization and steady-state accumulation after long-term incubation for 18 h, as well as magnetic resonance relaxivities and cellular relaxation rates of crotamine based probes were evaluated and compared to its shorter synthetic fragment CyLoP-1. The longitudinal relaxivity (r₁) of the conjugates of CyLoP-1 and crotamine is significantly lower in medium than in water indicating to the lower contrast enhancement efficacy of DO3A-based probes in biological samples. Carboxyfluorescein labeled crotamine did not exhibit toxicity up to a concentration of 2.5 µM. CyLoP-1 accumulated about four times better within the cells compared to crotamine. Fluorescence microscopy suggests different predominant uptake mechanisms for crotamine and CyLoP-1 in 3T3 cells. While crotamine is predominantly localized in vesicular structures (most likely endosomes and lysosomes) within the cell, CyLoP-1 is mainly homogeneously distributed in the cytosol. The cellular relaxation rate (R_{1, cell}) of the crotamine based probe was not significantly increased whereas the corresponding CyLoP-1-derivative showed a slightly elevated R_{1, cell}. This study indicates the potential of crotamine and in particular the shorter fragment CyLoP-1 to be useful for an efficient transmembrane delivery of agents directed to intracellular (cytosolic) targets. However, the applicability of the conjugates synthesized here as contrast agents in MR imaging is limited. Further improvement is needed to prepare more efficient probes for MRI applications, i.e., by replacing the DO3A- with a DOTA-based chelate.

Keywords: Cell penetrating peptide; Cellular internalization; Crotamine; CyLoP-1; Gadolinium; MRI.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Contrast Media* / metabolism
Crotalid Venoms* / metabolism
Crotalid Venoms* / toxicity
Crotalus / metabolism
Magnetic Resonance Imaging
Mice

Substances

Contrast Media
Crotalid Venoms
crotamine