Effect of lanthanide complex structure on cell viability and association

Abstract

A systematic study of the effect of hydrophobicity and charge on the cell viability and cell association of lanthanide metal complexes is presented. The terbium luminescent probes feature a macrocyclic polyaminocarboxylate ligand (DOTA) in which the hydrophobicity of the antenna and that of the carboxyamide pendant arms are independently varied. Three sensitizing antennas were investigated in terms of their function in vitro: 2-methoxyisophthalamide (IAM(OMe)), 2-hydroxyisophthalamide (IAM), and 6-methylphenanthridine (Phen). Of these complexes, Tb-DOTA-IAM exhibited the highest quantum yield, although the higher cell viability and more facile synthesis of the structurally related Tb-DOTA-IAM(OMe) platform renders it more attractive. Further modification of this latter core structure with carboxyamide arms featuring hydrophobic benzyl, hexyl, and trifluoro groups as well as hydrophilic amino acid based moieties generated a family of complexes that exhibit high cell viability (ED50 > 300 μM) regardless of the lipophilicity or the overall complex charge. Only the hexyl-substituted complex reduced cell viability to 60% in the presence of 100 μM complex. Additionally, cellular association was investigated by ICP-MS and fluorescence microscopy. Surprisingly, the hydrophobic moieties did not increase cell association in comparison to the hydrophilic amino acid derivatives. It is thus postulated that the hydrophilic nature of the 2-methoxyisophthalamide antenna (IAM(OMe)) disfavors the cellular association of these complexes. As such, responsive luminescent probes based on this scaffold would be appropriate for the detection of extracellular species.

Chart 1. Chemical Structures of Tb-DOTA-IAM(OMe) ([Tb-1]), Tb-DOTA-IAM ([Tb-2]⁻), and Tb-DOTA-Phen ([Tb-3])

Chart 2. Chemical Structures of Derivatives of [Tb-1] with Varying Charged and Hydrophobicitiesa

Scheme 1. Synthesis of Tb-DOTA-IAM(OMe) ([Tb-1]), Tb-DOTA-IAM ([Tb-2]⁻), and Tb-DOTA-IAM(OMe) Complexes with Varying Charge and Hydrophobicity ([Tb-4]³⁺–[Tb-10]^3–)

Reagents and conditions: (a) MnO₂, CH₃Cl, 60 °C, 20 h; (b) KOH (s), 230 °C, 1 h; (c) CH₃I, K₂CO₃, acetone, 56 °C, 16 h; (d) NH₂CH₃, CH₃OH, 65 °C, 18 h; (e) SeO₂, naphthalene, 215 °C, 2.5 h; (f) hydroxylamine hydrochloride, pyridine, 22 °C, 24 h; (g) Pd/C (10%), HCl, ethanol/H₂O, 5 bar, 16 h; (h) HATU, DIPEA, DMF, 22 °C, 40 h; (i) HCl, CH₃OH, 22 °C, 18 h; (j) Cs₂CO₃, CH₃CN, 40 °C, 18 h; (k) HCl, CH₃OH, 22 °C, 18 h; (l) BBr₃, CH₂Cl₂, 25 °C, 18 h; (m) CH₃OH, 65 °C, 16 h; (n) TbCl₃, H₂O/CH₃OH, pH 7, 45 °C, 48 h; (o) 0.2 M KOH, H₂O, 22 °C, 20 h.

Figure 1

Viability (24 h) of L6 myoblasts treated with 0–300 μM [Tb-1] (solid squares), [Tb-2]⁻ (open circles), and [Tb-3] (solid triangles) as determined with an MTT assay. Results are expressed as mean ± SD (n = 3).

Figure 2

Viability (24 h) of L6 myoblasts treated with 0–300 μM Tb complexes as determined with an MTT assay: (a) [Tb-1] (solid squares), [Tb-4]³⁺ (solid triangles), [Tb-5]³⁺ (open circles), and [Tb-6]³⁺ (open inverted triangles); (b) [Tb-7]³⁺ (solid squares), [Tb-8]³⁺ (solid triangles), [Tb-9] (open circles), and [Tb-10]^3– (open inverted triangles). Results are expressed as mean ± SD (n = 3).

Figure 3

Fluorescence microscopy images of representative L6 myoblasts treated with 200 μM [Tb-3] and [Tb-6] for 4 h at 37 °C: (a) fluorescence images, 60× objective, 325–375 nm excitation filter, 470–750 nm emission filter, 0.4 s exposure time; (b) bright field images, 0.04 s exposure time. Cells were rinsed with PBS and fixed with formaldehyde prior to imaging. Scale bars represent 10 μm.