Rational Approach To Select Small Peptide Molecular Probes Labeled with Fluorescent Cyanine Dyes for in Vivo Optical Imaging

Abstract

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging, and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application Keywords near-infrared; contrast agent; extravasation; kurtosis; protein bindingThe development of a molecular probe in optical imaging is a complex process that requires substantial and concerted efforts from different disciplines, including chemistry, biology, physics, and engineering. From a chemistry point of view, a typical probe for molecular imaging consists of a targeting moiety, such as peptides, oligosaccharides, small organic molecules and a covalently linked fluorophore. Probes designed for deep tissue and noninvasive optical imaging are different from those for in vitro or cellular studies where desirable features include good fluorescence intensity in the visible range and decent cell

Lasing in blood

“…When BSA/ICG = 3, nearly all ICG molecules are bound to BSA and fluorescence starts to saturate. Our fluorescence results not only correlate well with our laser experiments, but also are consistent with a recent finding that a BSA molecule can bind at most one ICG molecule at a time [35,36]. Note that the slight reduction in fluorescence (and hence increase in the ICG lasing threshold) when an excessive amount of BSA was added may be attributed to the additional absorption and scattering loss from BSA.…”

“…S4). The slightly lower lasing threshold for HSA than for the BSA counterpart (2.3 μJ/mm 2 ) is due to the relatively high binding affinity of ICG to HSA [35] (hence the ICG quantum yield) as well as the red-shifted fluorescence (and the gain) spectrum (see Fig. S2).…”

Targeted non-covalent self-assembled nanoparticles based on human serum albumin

“…[8] In line with this, different cyanine dyes have been selected to control the rate of extravasation of targeting peptides from blood vessels into tissue by their affinity for HSA. [9]…”

Identification of a Novel Indoline Derivative for in Vivo Fluorescent Imaging of Blood-Brain Barrier Disruption in Animal Models

“…It has been shown that LMW fluorescent tracers that have a high affinity to serum proteins and can emit strong fluorescence in the presence of serum proteins are advantageous for CBV imaging because these tracers are unlikely to extravasate from intact BBB and are able to provide the high-contrast fluorescent imaging of CBV. 23 We first measured the fluorescence properties of EB and ICG (Supporting Information Figure 1A and D) in the absence or presence of BSA (Supporting Information Figure 1B and E). The maximum fluorescence intensity (FI max) of EB in the absence and presence of BSA was 6 and 1112, respectively ( Table 1).…”