Protein‐Sized Bright Fluorogenic Nanoparticles Based on Cross‐Linked Calixarene Micelles with Cyanine Corona

Abstract

The key challenge in the field of fluorescent nanoparticles (NPs) for biological applications is to achieve superior brightness for sizes equivalent to single proteins (3-7 nm). We propose a concept of shell-cross-linked fluorescent micelles, where PEGylated cyanine 3 and 5 bis-azides form a covalently attached "corona" on micelles of amphiphilic calixarene bearing four alkyne groups. The obtained monodisperse NPs of 7 nm size increase their fluorescence quantum yield as a function of viscosity reaching 15% in glycerol. In the on-state they are ~2-fold brighter than quantum dots (QD-585), being the smallest PEGylated organic NPs of this high brightness. FRET between cyanine 3 and 5 cross-linkers at the surface of NPs suggests their integrity in physiological media, organic solvents and living cells, where they rapidly internalize showing excellent imaging contrast. Calixarene micelles with cyanine corona constitute a new platform for development of protein-sized ultrabright fluorescent NPs.* andrey.klymchenko@unistra.fr. Presently, we propose a concept of protein-sized fluorescent NPs based on calixarene micelles that are shell-cross-linked by fluorescent bi-functional dyes via Cu-catalysed "click" chemistry. In one recent report, a surfactant bearing three acetylene groups was polymerized using non-fluorescent cross-linker, while the non-reacted alkynes were further modified with fluorescent mono-functional dyes. Europe PMC Funders Group[15] In our strategy, we designed fluorescent cross-linkers to enable both micelle cross-linking and introduction of the fluorescent units. To this end, we selected cyanine 3 and 5 dyes, because of their excellent optical properties [16] and symmetric structure ( Figure 1). They were modified with two azide groups for crosslinking of the micelle and two hydrophilic PEG(8) chains to create its biocompatible shell. We call this design "cyanine corona", similarly to well-known protein corona that modifies the surfaces of a variety of NPs. (Figure 1). They are the first protein-sized NPs combining fluorogenic response to viscosity with superior brightness compared to quantum dots (QD-585).The calixarene amphiphile CX8TP at 150 μM concentration in aqueous solution (75 mM sodium sulfate) forms micelles of 5 nm according to dynamic light scattering (DLS) ( Figure S1). These micelles were further reacted with 2 eq of the cross-linkers Cy3L or Cy5L in the presence of copper catalyst for 24h at 30 °C. Then these reaction mixtures were dialyzed and their pick absorbance was compared to that before the dialysis. We found that after the reaction only a fraction (≤50%) of the cross-linkers was removed, whereas the control samples without added Cu-catalyst lost nearly all cross-linkers after the dialysis ( Figure 1A). These results provide an evidence for the successful cross-linking of micellar NPs with Cy3L and Cy5L. The absorption data suggested the cross-linking yields of 59 and 49% for Cy3L and Cy5L dyes, respectively. Moreover, the IR spectrum of the cross-linked micelles (CX/C...

Revisiting Fluorescent Calixarenes: From Molecular Sensors to Smart Materials

“…Furthermore, 104 was found preferentially in the cell cytoplasm and exhibited low In another investigation, Klymchenko and co-workers explored the possibility of developing calix[4]arene-based protein-sized fluorescent nanoparticles (NPs) by tagging cyanines (Cy3L or Cy5L) onto the calixarene core 106 through a Cu(I)-mediated click reaction (Figure 22). 219 The resulting micelles 106−Cy3L/Cy5L exhibited a homogeneous particle size of approximately 7 nm and a relatively broad bathochromically shifted emission band. The fluorescence quantum yield of the micelles was found to be dependent on the viscosity of the medium and was lower than those of free Cy3L and Cy5L.…”

Self-Assembly of Calix[4]arene-Based Amphiphiles Bearing Polyethylene Glycols: Another Example of “Platonic Micelles”

“…Furthermore, employing the calix [4]arene backbone in amphiphiles led to the formation of uniform micelles which was used in gene delivery 19 and bioimaging. 20 To explain this Platonic nature (i.e., monodispersity and a preference for adopting certain discontinuous N agg values), we considered the best packing of the unit spherical surface by equally sized cap objects such that the distance between the objects can be maximized 21,22 because the thermodynamic stability of micelles relies on how efficiently the micelle hydrophobic core is covered with the hydrophilic part of the amphiphile. Considering the repulsive interactions between the spherical caps, the arrangement where the total energy is minimized is the optimal one.…”

A Multifunctional Hybrid[4]arene-Based Macrocyclic Amphiphile: Self-Assembly, Tunable LCST Behavior, and Construction of Fluorescent Nanoparticles for Cell Imaging

“…On the other hand, the recognition sites of the macrocyclic host can form a complex with functional molecules as guests via host−guest interactions, 36 endowing the MCAs with a variety of material properties. To date, macrocyclic amphiphiles based on traditional macrocyclic hosts, including cyclodextrins, 37 calixarenes, 38 cucurbiturils, 39 and pillararenes, 40 have been widely investigated and applied in numerous fields such as a light-harvesting system, 32 cell agglutination, 41 and adsorption of explosives. 42 However, at present, the development of MCAs has been hindered.…”