In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy

Strategies for Labeling Proteins with PARACEST Agents

Reactive surface lysine groups on the chimeric monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 – 10.1 chelates were added per 3G4 molecule and between 5.6 – 5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53 μs for the non-conjugated chelate to 65–77 μs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73–75 μs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein

Europium(III) Macrocyclic Complexes with Alcohol Pendant Groups as Chemical Exchange Saturation Transfer Agents

Paramagnetic lanthanide(III) complexes that contain hyperfine-shifted exchangeable protons offer considerable advantages over diamagnetic molecules as chemical exchange saturation transfer (CEST) agents for MRI. As part of a program to investigate avenues to improve the sensitivity of such agents, the CEST characteristics of europium(III) macrocyclic complexes having appended hydroxyethyl groups were investigated. The CEST spectrum of the asymmetrical complex, EuCNPHC3+, shows five distinct peaks for each magnetically nonequivalent exchangeable proton in the molecule. The CEST spectra of this complex were fitted to NMR Bloch theory to yield exchange rates between each of six exchanging proton pools (five on the agent plus bulk water). Exchange between the Eu3+-bound hydroxyl protons and bulk water protons was slow in dry acetonitrile but accelerated incrementally upon stepwise addition of water. In pure water, exchange was too fast to observe a CEST effect. The utility of this class of europium(III) complex for CEST imaging applications is ultimately limited by the small chemical shifts induced by the hydroxyl-appended ligands of this type and the resulting small Δω values for the exchangeable hydroxyl protons

Near-Infrared Fluorescence Lifetime pH-Sensitive Probes

We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricarbocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive probe. The pKa of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole ring resulted in a compound with pKa ∼ 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-position shifted the pKa to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity. This sensitivity stemmed from distinct lifetimes for protonated (∼1.16 ns in acidic DMSO) and deprotonated (∼1.4 ns in basic DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescence-lifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living organisms further augments the potential of these lifetime-based pH probes

<i>In vivo</i> fluorescence imaging of xenograft mouse with high HER2 expressing human tumor model (NCI-N87) after injection of the HER2-nonspecific Affibody® (His6-Z_Taq:GS-Cys) conjugated to Dylight750.

<p>(<b>A</b>) Fluorescence intensity map at the tumor region. (<b>B</b>)Fluorescence intensity map at the contralateral site (<b>C</b>) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (<b>D</b>) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The data in Figs. (<b>D</b>) and (<b>H</b>) are the average data of three mice. Markers show the average and bars show the standard deviation. (<b>E</b>)Fluorescence lifetime map at the tumor region. (<b>F</b>) Fluorescence lifetime map at the contralateral site. (<b>G</b>) The difference of fluorescent lifetime at the tumor and the contralateral site mapped on the tumor region. (<b>E</b>) Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time.</p

<i>In-vitro</i> image of HER2 positive cancer cells (SKBR3) exposed to HER2-Affibody-Dylight750 in (A–C) PBS and (D–F) cell culture with 10% FBS.

<p>Figures (<b>A</b>) and (<b>D</b>) show the intensity and (<b>B</b>) and (<b>E</b>) show the lifetime image. The histogram of the lifetime distribution has been shown in figures (<b>C</b>) and (<b>F</b>) for PBS and 10% FBS in cell culture media, respectively.</p

Effect of (A) pH and (B) BSA concentration on the lifetime of DyLight750 conjugated to HER2 specific Affibody probe.

<p>Effect of (A) pH and (B) BSA concentration on the lifetime of DyLight750 conjugated to HER2 specific Affibody probe.</p

An example of fitting results obtained by SPCImage software, (ver. 3.2, Becker & Hickl GmbH) for measurements at the tumor (A) and contralateral (B) sites, 1 hour after injection of HER2-specific Affibody conjugated to Dylight750 in a mouse with high HER2 expressing human tumor model (BT-474).

<p>X axis is the amplitude and y axis is the measurement time (ns). Blue and green graphs show the measurement data and impulse response function of the system, respectively. The fitting was based on single exponential decay model. Parameter a1 shows the relative of amplitude in single exponential decay model and t1 is the lifetime (in picoseconds). The small graph at the bottom is the fitting error and the red line shows the fitted curve.</p

<i>In vivo</i> fluorescence imaging of xenograft mouse with high HER2 expressing human tumor model (BT-474) after injection of HER2-specific Affibody® (His6-Z_HER2:GS-Cys) conjugated to Dylight750.

<p>(<b>A</b>) Fluorescence intensity map at the tumor region. (<b>B</b>)Fluorescence intensity map at the contralateral site (<b>C</b>) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (<b>D</b>) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The fluorescent intensity was averaged over 16 pixels at the contralateral site. The data in Figs. (<b>D</b>) and (<b>H</b>) are the average data of four mice. Markers show the average and bars show the standard deviation. (<b>E</b>) Fluorescence lifetime map at the tumor region. (<b>F</b>) Fluorescence lifetime map at the contralateral site. (<b>G</b>) The difference of fluorescence lifetime at the tumor and the contralateral site mapped on the tumor region. (<b>E</b>)Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time. All lifetime and fluorescence intensity maps in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031881#pone-0031881-g002" target="_blank">figures 2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031881#pone-0031881-g004" target="_blank">4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031881#pone-0031881-g005" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031881#pone-0031881-g006" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031881#pone-0031881-g007" target="_blank">7</a> are from measurements at 1 hour after the injection of Affibody probe. In all measurements, the photons were counted over two seconds integration time, <i>t₀</i>. To exclude saturation effect of the 16 bit camera, in the brightest pixels, where the corresponding limit of 65536 counts was reached before time <i>t₀</i>, we have renormalized the data by multiplying photon counts, measured for lower integration time <i>t</i>, by factor <i>t₀/t.</i></p

Comparison of autofluorescence intensity at the tumor and contralateral sites.

<p>The autofluorescence intensity map at the (<b>A</b>) tumor and (<b>B</b>) contralateral sites before injection. Comparison of the autofluorescence intensity before injection and the fluorescence signal after injection of (<b>C</b>) HER2 specific Affibody-Dylight750 and (<b>D</b>) HER2 non-specific Affibody-Dylight750 in 3 mice with HER2 positive tumor (NCI-N87 tumor carcinoma) at the tumor and contralateral sites .</p