Gold nanoparticles (AuNPs) are currently intensively
exploited
in the biomedical field as they possess interesting chemical and optical
properties. Although their synthesis is well-known, their controlled
surface modification with defined densities of ligands such as peptides,
DNA, or antibodies remains challenging and has generally to be optimized
case by case. This is particularly true for applications like in vivo
drug delivery that require AuNPs with multiple ligands, for example
a targeting ligand and a drug in well-defined proportions. In this
context, we aimed to develop a calixarene-modification strategy that
would allow the controlled orthogonal conjugation of AuNPs, respectively,
via amide bond formation and copper(I)-catalyzed azide–alkyne
cycloaddition (CuAAC). To do this, we synthesized a calix[4]arene-tetradiazonium
salt bearing four PEG chains ended by an alkyne group (C1) and, after optimization of its grafting on 20 nm AuNPs, we demonstrated
that CuAAC can be used to conjugate an azide containing dye (N3-cya7.5). It was observed that AuNPs coated with C1 (AuNPs-C1) can be conjugated to approximately 600 N3-cya7.5 that is much higher than the value obtained for AuNPs
decorated with traditional thiolated PEG ligands terminated by an
alkyne group. The control over the number of molecules conjugated
via CuAAC was even possible by incorporating a non-functional calixarene
(C2) into the coating layer. We then combined C1 with a calix[4]arene-tetradiazonium salt bearing four carboxyl groups
(C3) that allows conjugation of an amine (NH2-cya7.5) containing dye. The conjugation potential of these bifunctional
AuNPs-C1/C3 was quantified by UV–vis
spectroscopy: AuNPs decorated with equal amount of C1 and C3 could be conjugated to approximately 350 NH2-dyes and 300 N3-dyes using successively amide
bond formation and CuAAC, demonstrating the control over the orthogonal
conjugation. Such nanoconstructs could benefit to anyone in the need
of a controlled modification of AuNPs with two different molecules
via two different chemistries.