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 pre-assembled 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. But 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 pre-assembled probes have sufficient mechanical stability for effective in vivo imaging. The results demonstrate the feasibility of this general class of pre-assembled 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.

Non‐Covalent Assembly Method that Simultaneously Endows a Liposome Surface with Targeting Ligands, Protective PEG Chains, and Deep‐Red Fluorescence Reporter Groups

“…The second major difference between POPC @CSP⊃6C and POPC @CSP⊃6Z liposomes was the fluorescence brightness. As shown in Figure B the fluorescence intensity of POPC @CSP⊃6Z liposomes was about four times lower, which matches previous observations that fluorescent ZnDPA complexes have a tendency to become self‐aggregated and self‐quenched on a membrane surface . As shown below, relief of this self‐quenching effect can be exploited to achieve target‐activated turn‐on fluorescence.…”

“…As shown in Figure A the threading of 6C was complete within a few seconds, whereas the threading of 6Z required ≈20 minutes (Figure A). Previous studies have shown that the threading of 6Z by a dispersed, water‐soluble squaraine is fast so the slow formation of POPC @CSP⊃6Z must be a liposome surface effect. The most likely explanation is surface promoted association of the ZnDPA units in the newly docked 6Z with neighbouring CSP conjugates, driven by Lewis acid/base interactions between the Zn II cations and the electron‐rich squaraine oxygen atoms, as reported for related metal cation/dye association systems .…”

“…In short, a deep‐red fluorescent squaraine dye is encapsulated by a water‐soluble tetralactam macrocycle to give an extremely stable, threaded complex that is well suited for fluorescence imaging. To date, we have pre‐assembled fluorescent molecular probes that selectively target bone, dead/dying mammalian tissue, and various microbial species . One of the major advantages of Synthavidin self‐assembly is the capability of forming threaded complexes with long PEG chains appended to the ends of the squaraine dye .…”

Non‐Covalently Pre‐Assembled High‐Performance Near‐Infrared Fluorescent Molecular Probes for Cancer Imaging

“…Indeed, preliminary threading studies showedt hat near-quantitative threading occurred when H wasm ixed with one molar equivalent of R or C,o rH3H was mixed with two equivalents of R or C.I nF igure 1a,b are representative absorption spectra of the squaraine dyes before and after probe pre-assembly.T he absorption profile for free dimeric H3H is extremelyb road due to foldingo f the structure ands tacking of its two squaraine chromophores. [20,22] But after addition of macrocycle, near-quantitative threading and probe pre-assembly was indicated by the appearance of diagnostic narrow andr ed-shifted squaraine absorbance maxima. In Figure1c,d are two representative sets of absorption spectra showing the spectralc hanges that occur during the probe pre-assemblyp rocess.…”

“…Thisf inding is consistent with our previous work showingt hat Synthavidin pre-assembledp robesh ave sufficiently high kinetica nd thermodynamic stability to be effective biological imaging agents. [20][21][22] The cancert argeting capabilities of R'H and 2(R)'H3H were assessed using the integrin expressing cell lines OVCAR-4 and A549. OVCAR-4 is an excellent cell model of high-grade serous ovarian carcinoma because it has avery similar genomic sequence and response to chemotherapeutic drugs.…”