ETD

• bio-conjugation
• contrast agents
• imaging
• lanthanides
• luminescence

In the last decades, lanthanide complexes have aĴracted increasing aĴention for bioimaging and
sensing applications, owing to their unique photophysical properties. Their emission arises
from Laporte forbidden 4f–4f transitions, which result in sharp and highly reproducible spectral
bands, large Stokes shifts and long luminescence lifetimes. Combined with their remarkable
stability in biological media, these features make lanthanide complexes particularly suitable for
advanced fluorescence microscopy techniques, including time-resolved imaging.
Efficient use of lanthanide complexes in biological systems, however, critically depends on
ligand design. In order to sensitize the lanthanide ion, the coordination environment must
include a chromophoric antenna capable of absorbing light and transferring the excitation
energy to the metal center. Aryl-alkynyl and aryl-picolinate groups have been widely employed
for this purpose, but their intrinsic lipophilicity often weakens biocompatibility and solubility,
requiring the introduction of additional hydrophilic substituents.
Beyond solubility, cellular delivery represents a further challenge. To overcome this limitation,
different strategies have been investigated, including conjugation with biomolecular vectors
such as antibodies, peptides, nucleic acids or carbohydrates. Glycoconjugation, is particularly
appealing for the central role of sugars and glycans in biology: sugars not only increase water
solubility, without adding charges, but may also promote selective accumulation in cancer cells,
whose altered metabolism is characterized by increased glucose uptake, a phenomenon known
as the Warburg effect. In this context, sugar moieties can be introduced through copper(I)-
catalyzed azide–alkyne cycloaddition (CuAAC), a prototypical “click chemistry” reaction.
Compared to classical glycosylation methods, CuAAC provides synthetic simplicity, high
chemoselectivity, mild conditions, and the incorporation of triazole linkages, which act as stable
amide bioisosteres, ensuring improved robustness of the complexes under physiological
conditions.
In parallel, bio-conjugation of lanthanide complexes with CPPs such as TAT, combined with coincubation with non-fluorescent oligopeptide dimers, has demonstrated to facilitate efficient
cellular uptake. However, this strategy is affected by an important problem: once internalized,
CPPs are often metabolized by amidases, which cleave the peptide fragment and leave the
complex unmodified. In such cases, solubility only depends on the antenna design, which must
be adequately functionalized to preserve stability and dispersion in the cytosol, that is necessary
to collect appropriate images.
Taken into account these observations, the present work aims to overcome the discussed
limitations through the synthesis of three europium complexes (Figure 1) based on two different
azamacrocycles, triazacyclononane, TACN, and tris-carboxymethyl-tetraazacyclododecane,
DO3A. All derivatives incorporate a mannose moiety, intended both as a hydrophilic
solubilizing group and as a potential targeting motif for selective internalization by cancerous
cells. One of the complexes additionally bears the TAT peptide as a CPP fragment, enabling a
direct comparison between sugar-mediated, peptide-mediated uptake mechanisms and the
dual contribution on the intracellular behavior.
The main goal is to evaluate how mannose conjugation and TAT functionalization can influence
the solubility and intracellular behavior of europium complexes, with particular aĴention to
their distribution in the cytosol and the resulting quality of two-photon microscopy (2PM)
images. The study integrates photophysical characterization of the europium complexes in
aqueous solutions, including assessment of brightness and luminescence lifetimes, with in-cell
imaging experiments and uptake analyses.
Through this combined approach, the project seeks to clarify the impact of mannose
functionalization on both solubility and intracellular behavior, contributing to the rational
design of new lanthanide-based probes for advanced bioimaging applications.