Exofacial Protein Thiols as a Route for the Internalization of Gd(III)-Based Complexes for Magnetic Resonance Imaging Cell Labeling

Abstract

Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a large number of reactive exofacial protein thiols (EPTs) that can be exploited as anchorage points for suitably activated MRI probes. The probes are composed of a Gd(III) chelate (based on either DO3A or DTPA) connected through a flexible linker to the 2-pyridyldithio chemical function for binding to EPTs. GdDO3A-based chelates could efficiently label cells (up to a level of 1.2 x 10(10) Gd(III) atoms/cell), whereas GdDTPA-based chelates showed poor or no cell labeling ability at all. Among the GdDO3A based compounds, that having the longest spacer (compound GdL1A) showed the best labeling efficacy. The mechanism of EPT mediated cell labeling by GdL1A involves probe internalization without sequestration of the Gd(III) chelate within subcellular structures such as endosomes.

The yin of exofacial protein sulfhydryls and the yang of intracellular glutathione in in vitro transfection with SS14 bioreducible lipoplexes

“…Amid the plethora of potential candidates that have recently deserved special attention, exofacial membrane sulfhydryls have proven very useful for anchoring both bioreducible polyplexes and redox-sensitive polymeric capsules to the cell membrane [9,39]. Furthermore, different cell types have shown that display variable, physiological levels of reactive EPTs in the range of 4-62 nmol sulfhydryls/10 6 cells [42,43], although many factors can influence their content [44]. Under our experimental conditions HeLa, U87-MG and MG63 human cell lines exhibited similar amount of EPTs, attaining values of 25.5 ± 2.4, 22.3 ± 2.4, and 28.1 ± 2.2 nmol sulfhydryls/10 6 cells, respectively.…”

Synthesis and Characterization of Ligands and their Gadolinium(III) Complexes

“…The ligand was conjugated to, for example, aminooxy-terminated linear chain for tumor cells targeting [113]. Similar reaction strategy starting from l-cysteine, its transformation to l-S-(2-pyridylthio)cysteine, and further alkylation and deesterification resulted in bifunctional H 5 DTPA derivative, suitable for coupling with thiol moieties [114]. Direct C-alkylation of α-carbon of acetate pendant moiety was also used; for example, treatment of the H 5 DTPA pentamethyl ester with lithium diisopropylamide (LDA) in tetrahydrofuran (THF) followed by addition of benzylbromide in hexamethylphosphortriamide, (Me 2 N) 3 P = O (HMPA), afforded the H 5 DTPA derivative substituted by benzyl group on α-carbon atom of the acetate arm attached to the terminal amino group [115] (Scheme 3.21).…”

“…Coordination of amines to the lanthanide(III) ion is rather weak and, thus, amino group is mostly used as a spacer for attachment of other functionalities. The most common substituents, 2-aminoethyl and 3-aminopropyl groups, are usually attached to a macrocycle by alkylation with corresponding N-protected 2-aminoethyl-or 3-aminopropylhalogenide [114,[325][326][327]. The protecting groups such as carboxamide, phthalimide, or sulfonamide are introduced and removed by common methods of organic synthesis.…”

“…The free active group in the "primary" bifunctional ligand can be transformed into a "secondary" bifunctionality by appropriate substitution, as can be demonstrated by, for example, acylation of distant amino group of H 3 DOTA-CH 2 CH 2 NH 2 with 3-(2-pyridyldithio)propanoic acid [114], bringing protected disulfide moiety suitable for S-S transcoupling. Side maleinimido moiety was introduced analogously by acylation of side amino group of H 4 DOTA monoamide by β-maleinimidopropanoic acid N-hydroxysuccinimide ester [193].…”

“One-stop shop” spectral imaging for rapid on-site diagnosis of lung cancer: a future concept in nano-oncology

“…Exofacial protein thiols have been used as a route for internalization of gadolinium (III)-based complexes for labeling of subcellular structures, such as endosomes seen on magnetic resonance imaging. 49 Tc99 radiolabeling of adenoviral particles containing capsid …”