Nattawut Sinsuebphon

Comparison of NIR FRET pairs for quantitative transferrin-based assay

Transferrin (Tfn) is commonly used as a drug delivery carrier for cancer treatment. Tfn cellular internalization can be observed by Förster resonance energy transfer (FRET), which occurs when two fluorophores - donor and acceptor - are a few nanometers apart. Donor fluorescence lifetime can be used to sense and quantify FRET occurrence. In FRET state, the donor is quenched leading to a significant reduction in its lifetime. In this study, donor and acceptor near-infrared (NIR) fluorophore-labeled Tfn were used to quantify cellular internalization in breast cancer cell line (T47D). Based on donor lifetime, quantum yield and spectral data, seven NIR FRET pairs were chosen for this comparison. Performance of the different NIR FRET pairs was evaluated in vitro in multiwell plate settings and by analyzing the relationship between quenched donor fraction and acceptor:donor ratio. Additionally, we performed brightness comparison between each pairs. Several parameters, such as brightness, lifetime, R0 and FRET donor population values are used to identify the most suitable NIR FRET pair for in vivo studies in preclinical settings.

Comparison of illumination geometry for lifetime-based measurements in whole-body preclinical imaging

Macroscopic fluorescence lifetime imaging (MFLI) has been proved to be an accurate tool to quantify Förster resonance energy transfer (FRET) lifetime-based assessment of receptor-ligand engagement in vitro and in vivo. Herein, we report on the quantitative comparison of MFLI for whole-body preclinical studies in transmittance and reflectance geometries. The comparative study was conducted for both in vitro and in vivo conditions. FRET quantification performance in both geometries was similar in high fluorescence concentration samples. However, the reflectance geometry performed better at low fluorescence concentration. In addition, reflectance geometry could acquire subsurface imaging of the main whole-body organs of small animals without being compromised by tissue attenuation.

Selection of Temporal Gates for Bi-Exponential Fluorescence Lifetime Imaging

Fluorescence lifetime imaging is a commonly-used molecular imaging technique for preclinical and clinical studies of cells or tissues. Herein, the accuracy of lifetime imaging in the case of a bi-exponential model is investigated based on limited time data points. An in silico investigation shows that estimation accuracy of the model parameters involved (lifetimes and fractional amplitudes) can be maintained while reducing the number of time-gates used to eight. Recommendations of which time gates to use are also made based upon the presented analysis.

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short lifetime near-infrared dyes and time-gated imaging

We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores’ short (sub-nanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time-gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from non-specific binding. Our method, implemented in a freely-available software, has the advantage of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging, and could be extended to applications as diverse as image guided-surgery or optical tomography.

Deep tissue imaging of target engagement in live animals (Conference Presentation)

Targeted drug delivery in cancer treatment has been a major area of development in the past decades. However, there is a great need in preclinical studies to not only assess the drug distribution but also monitor and quantify target engagement in vivo to ensure maximal drug delivery efficacy. Macroscopic Fluorescence Lifetime Imaging of Forster Resonance Energy Transfer (MFLI-FRET) is a unique non-invasive imaging methodology to monitor in vivo receptor-target interactions and directly discriminate between unbound and internalized ligands in pre-clinical studies. In this study, we capitalized on the homodimeric nature of the transferrin receptor (TfR) to quantify transferrin (Tf) internalization into cancer cells by measuring FRET between receptor-bound Tf-labeled donor and acceptor near-infrared (NIR) fluorophore pairs. We found a strong correlation between FRET levels and Tf internalization into tumor cells despite the significant heterogeneity of tumors regarding their size and cellular density. In contrast, no correlation between MFLI-FRET and TfR levels was observed, underscoring the insufficient link between receptor density and intracellular drug delivery. Additionally, we compared results of in vivo MFLI-FRET imaging with intensity-based FRET (using the IVIS pre-clinical optical imaging system). Intensity-based imaging failed to provide reliable and consistent results, showing significantly higher FRET quantification in negative control samples. Overall, we demonstrated that MFLI-FRET enables real time in vivo information on receptor ligand engagement in deep tissues, conversely to current commercial systems. Hence, MFLI-FRET is well positioned to play a critical role in accelerating the optimization of targeted drug delivery efficacy in pre-clinical studies.

Near infrared fluorescence lifetime FRET imaging of target engagement at multiscale (Conference Presentation)

Monitoring the binding of protein ligands and therapeutic antibodies to their respective receptors (target engagement) is crucial to compound prioritization in anti-cancer targeted drug screening. However, current in vivo optical imaging techniques cannot distinguish between co-localization and actual receptor-ligand binding at the tumor region. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The homodimeric nature of TfR allows for measuring fluorescence lifetime FRET (FLI-FRET) to quantitate the TfR-Tf binding and internalization into cancer cells, based on the reduction of donor fluorophore lifetime. Near infrared (NIR) FLI-FRET has been used to directly visualize and quantitate TfR-Tf binding and internalization by providing the fraction of donor-labeled entity that is interacting with its respective receptor. NIR FLI-FRET has been validated at multiscale, using both in vitro microscopy as well as in vivo macroscopy whole-body deep imaging assays using different NIR FRET pairs. Accuracy of NIR FLI-FRET quantitation has been compared between fluorescence intensity and lifetime measurements using both microscopy and macroscopy fluorescence imaging. NIR FLI-FRET employs well-characterized quantitative lifetime-based metrics, standard in FRET microscopy, but with the additional benefit of a seamless multiscale technological platform. In summary, we have successfully demonstrated quantitative imaging of receptor-mediated binding and uptake of Tf using NIR FLI-FRET microscopy and macroscopy imaging in vitro and in vivo, respectively. This novel approach can be extended to other receptors, currently targeted in oncology. Hence, NIR FLI-FRET can find numerous applications in pre-clinical drug delivery and targeted therapy assessment and optimization.

Quantitative imaging of receptor-ligand engagement in intact live animals

ABSTRACT Maintaining an intact tumor environment is critical for quantitation of receptor‐ligand engagement in a targeted drug development pipeline. However, measuring receptor‐ligand engagement in vivo and non‐invasively in preclinical settings is extremely challenging. We found that quantitation of intracellular receptor‐ligand binding can be achieved using whole‐body macroscopic lifetime‐based Förster Resonance Energy Transfer (FRET) imaging in intact, live animals bearing tumor xenografts. We determined that FRET levels report on ligand binding to transferrin receptors conversely to raw fluorescence intensity. FRET levels in heterogeneous tumors correlate with intracellular ligand binding but strikingly, not with ubiquitously used ex vivo receptor expression assessment. Hence, MFLI‐FRET provides a direct measurement of systemic delivery, target availability and intracellular drug delivery in preclinical studies. Here, we have used MFLI to measure FRET longitudinally in intact and live animals. MFLI‐FRET is well–suited for guiding the development of targeted drug therapy in heterogeneous tumors in intact, live small animals. Graphical abstract Figure. No caption available.

Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

Fluorescence lifetime FRET non-invasive imaging of breast cancer xenografts provides a measure of target engagement in vivo (Conference Presentation)

Fluorescence Lifetime Förster Resonance Energy Transfer (FLIM-FRET) is a unique non-invasive imaging platform to monitor and quantify in vivo target engagement in pre-clinical studies. FLIM FRET is a valuable tool in targeted drug delivery due to its nanoscale-range molecular resolution that detects near-infrared labeled ligand binding to dimerized receptors followed by their uptake into cancer cells in vivo. Various imaging platforms, including PET, lack the ability to directly discriminate between unbound and internalized ligands. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The dimeric nature of TfR allows for the quantification of Tf internalization into cancer cells by measuring FLIM FRET between receptor-bound Tf donor and acceptor NIR fluorophore pairs, based on the reduction of donor fluorophore lifetime in live mice. We analyzed tumor morphology, the level of expression of TfR, estrogen receptor (ER) and Tf accumulation in human breast cancer tumor xenografts. We found a remarkable heterogeneity of breast cancer tumors regarding their size, cell density, TfR and ER expression and Tf uptake. The results of this study confirm a strong correlation between in vivo NIR FLIM FRET and ex vivo evaluation of Tf uptake into tumor tissues, thus validating FD% as a robust measure of the target engagement of TfR-Tf in tumor cells in vivo.

Wide-field lifetime-based FRET imaging for the assessment of early functional distribution of transferrin-based delivery in breast tumor-bearing small animals

Targeted drug delivery is a critical aspect of successful cancer therapy. Assessment of dynamic distribution of the drug provides relative concentration and bioavailability at the target tissue. The most common approach of the assessment is intensity-based imaging, which only provides information about anatomical distribution. Observation of biomolecular interactions can be performed using Förster resonance energy transfer (FRET). Thus, FRET-based imaging can assess functional distribution and provide potential therapeutic outcomes. In this study, we used wide-field lifetime-based FRET imaging for the study of early functional distribution of transferrin delivery in breast cancer tumor models in small animals. Transferrin is a carrier for cancer drug delivery. Its interaction with its receptor is within a few nanometers, which is suitable for FRET. Alexa Fluor® 700 and Alexa Fluor® 750 were conjugated to holo-transferrin which were then administered via tail vein injection to the mice implanted with T47D breast cancer xenografts. Images were continuously acquired for 60 minutes post-injection. The results showed that transferrin was primarily distributed to the liver, the urinary bladder, and the tumor. The cellular uptake of transferrin, which was indicated by the level of FRET, was high in the liver but very low in the urinary bladder. The results also suggested that the fluorescence intensity and FRET signals were independent. The liver showed increasing intensity and increasing FRET during the observation period, while the urinary bladder showed increasing intensity but minimal FRET. Tumors gave varied results corresponding to their FRET progression. These results were relevant to the biomolecular events that occurred in the animals.