Preassembled Fluorescent Multivalent Probes for the Imaging of Anionic Membranes

Abstract

A new self-assembly process known as Synthavidin (synthetic avidin) technology was used to prepare targeted probes for near-infrared fluorescence imaging of anionic membranes and cell surfaces, a hallmark of many different types of disease. The probes were preassembled by threading a tetralactam macrocycle with six appended zinc-dipicolylamine (ZnDPA) targeting units onto a linear scaffold with one or two squaraine docking stations to produce hexavalent or dodecavalent fluorescent probes. A series of liposome titration experiments showed that multivalency promoted stronger membrane binding by the dodecavalent probe. In addition, the dodecavalent probe exhibited turn-on fluorescence due to probe unfolding during fluorescence microscopy at the membrane surface. However, the dodecavalent probe also had a higher tendency to self-aggregate after membrane binding, leading to probe self-quenching under certain conditions. This self-quenching effect was apparent during fluorescence microscopy experiments that recorded low fluorescence intensity from anionic dead and dying mammalian cells that were saturated with the dodecavalent probe. Conversely, probe self-quenching was not a factor with anionic microbial surfaces, where there was intense fluorescence staining by the dodecavalent probe. A successful set of rat tumor imaging experiments confirmed that the preassembled probes have sufficient mechanical stability for effective in vivo imaging. The results demonstrate the feasibility of this general class of preassembled fluorescent probes for multivalent targeting, but fluorescence imaging performance depends on the specific physical attributes of the biomarker target, such as the spatial distance between different copies of the biomarker and the propensity of the probe-biomarker complex to self-aggregate.

Figure 1

Pre-assembly of fluorescent probes is achieved by threading a macrocycle (red) with appended targeting units (green) onto a linear scaffold (blue) with a single fluorescent squaraine station S or double squaraine station S3S.

Figure 2

Structures of the four pre-assembled fluorescent probes used in this study; non-targeted 6C ⊃ S and 2(6C) ⊃ S3S, and targeted 6Z ⊃ S and 2(6Z) ⊃ S3S.

Figure 3

Representation of 2(6Z) ⊃ S3S adopting a folded and self-quenched conformation in aqueous solution. Upon association with anionic liposomes the probe unfolds with turn-on fluorescence.

Figure 4

Fluorescence spectra for 6Z ⊃ S (left) and 2(6Z) ⊃ S3S (right) (250 nM) in the presence of either zwitterionic (100% POPC) or anionic (20% POPS, 80% POPC) liposomes, total phospholipid 1 mM, Ex: 645 nm.

Figure 5

Illustration of FRET induced by membrane association of fluorescent probe 6Z ⊃ S (shown) or 2(6Z) ⊃ S3S (not shown) to anionic liposomes containing 1% DiIC₁₈ (Ex/Em 480/568 nm) as the fluorescence energy acceptor. There is no probe association with zwitterionic (100% POPC) liposomes.

Figure 6

FRET titration spectra for 6Z ⊃ S (left) and 2(6Z) ⊃ S3S (right) in the presence of anionic liposomes (20% POPS, 79% POPC, 1% DiIC₁₈; total lipid concentration 2 μM). Probe concentrations during the titration ranged from 0–800 nM, Ex: 480 nm

Figure 7

Titration isotherms for relative quenching of DiIC₁₈ fluorescence at 568 nm (F/F₀) due to FRET caused by association of 6Z ⊃ S (left) or 2(6Z) ⊃ S3S (right) with the anionic liposomes (Ex: 645 nm). The unit for the x-axis is total concentration of ZnDPA targeting unit in the sample.

Figure 8

Change in fluorescence at 720 nm for probes 6Z ⊃ S (left) and 2(6Z) ⊃ S3S (right) during addition to anionic liposomes (20% POPS, 79% POPC, 1% DiIC₁₈). TP1 and TP2 are transition points 1 and 2, respectively.

Figure 9

Generalized picture showing that a pre-assembled probe with two linked squaraines is folded and self-quenched in aqueous solution. Upon association with a target membrane surface, the probe unfolds with turn-on fluorescence but additional probe association with the surface leads to probe self-aggregation and quenching.

Figure 10

Fluorescence microscopy images of dead and dying PAIII cells that were caused by cell pre-treatment with staurosporine (500 nM). All cells were stained with deep-red6Z ⊃ S (left), or 2(6Z) ⊃ S3S (right) (1 μM) and costained with blue nuclear indicator Hoechst33342 (3 μM) and green live-cell indicator CalceinAM (5 μM). Scale Bar = 10 μM.

Figure 11

Fluorescence micrographs of S. chromofuscus, E. coli, L. major, and T. cruzi, stained with 6C ⊃ S, 6Z ⊃ S, or 2(6Z) ⊃ S3S (5.0 μM). Brightfield (upper panel) and deep-red probe fluorescence (lower panel). The fluorescence intensity of each image in a row is scaled to the image with 2(6Z) ⊃ S3S.

Figure 12

Biodistribution of 6C ⊃ S or 6Z ⊃ S in tumor-bearing rats at 24 hours after probe dosing (20 nmol). (top) Fluorescence images of excised tumors (IVIS Cy5 filter set, Exposure 3s, f-Number 2, Binning 2). (middle) Quantification of tumor fluorescence as mean pixel intensity (MPI) (n=3, p<0.05). (top) Probe biodistribution measured as MPI for each excised organ.