This talk will introduce the conference and discuss the program for the day. There will be an emphasis on areas of focus in the program, as well as potential new areas of interest for the conference. The talk will additionally introduce the panel discussion.

The failure rate of drug development is high, especially in Phase III clinical trials after significant time and monetary investment. This can be observed in oncology trials, where drug failure rates are much higher than non-oncological trials due to inadequate improvement in overall survival. To address this problem, we propose the use of fluorescence paired-agent imaging (PAI) to monitor drug-receptor interactions in vivo in individual patients. Therapeutic agents can be conjugated to fluorophores used for measuring target-specific interactions. Our preclinical progress will be discussed for both oncology and non-oncology applications with promising translation to clinical applications in the future.

The effectiveness of antibody therapeutics relies on in vivo drug pharmacology, intrinsic parameters of tumor cells, and tumor microenvironment factors. An understanding of the antibody-target-microenvironment interactions will improve patient selection and development of new targeted therapeutics. Using optically labeled therapeutic antibodies systemically delivered to patients prior to surgical resection, we were able to develop a novel analytical method to measure therapeutic behavior of these agents and their cellular targets at single cell resolution within intact human tumors. We identified two major subtypes of CAFs as well a unique enrichment of extracellular matrix components with the tumor. The spatial arrangement of ECM proteins were also associated with reduced therapeutic antibody penetration. Our findings were further supported by spatial transcriptomics of adjacent tissue slices and public scRNA seq data. This study provides a new framework for interrogating drug pharmacology in conjunction with tumor biology, opening new avenues for dosing optimization, biomarker identification, and the development of new stromal-targeting therapies to improve treatment outcomes.

Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of active pharmaceutical ingredients within the skin, with superior chemical specificity and spatial and temporal resolution. Here, we present a method to study topical formulations on ex vivo human skin using two modalities, SRS and near-infrared light (NIR) transmission. NIR transmission is used to compensate for the SRS signal variance caused by differences in skin thickness and formulation properties. Optical co-registration of the two modalities enables recording the variance in each pixel. The developed method helps to evaluate the cutaneous pharmacokinetics of tretinoin from tretinoin-containing solution and cream formulations.

We applied Raman microscopy to observe the response of intracellular molecules after drug administration. We found that the dynamics change in heme protein redox states and distributions can be imaged without labelling. We also applied alkyne-tag Raman imaging to detect the uptake of drugs and quantitatively compare the efficiency under different drug administration conditions.

Understanding drug fingerprints in complex biological samples is essential for drug development. We demonstrate a deep learning-assisted hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging approach for identifying drug fingerprints at single-cell resolution. The attention-based deep neural network, Hyperspectral Attention Net (HAN), highlights informative spatial and spectral regions in a weakly supervised manner. Using this approach, drug fingerprints of a hepatitis B virus therapy in murine liver tissues was investigated. Higher classification accuracy was observed with increasing drug dosage, reaching an average AUC of 0.942. Results demonstrate the potential for label-free profiling and localization of drug fingerprints in complex biological samples.

Nicotinamide Adenine dinucleotide (NAD+) deficiency have shown to cause pathogenesis of age-related functional decline and diseases. Investigational studies have demonstrated improvements in age-associated pathophysiology and disease conditions. However, invasive methods such as immunohistochemistry, metabolic assays, and PCR currently used to measure cell metabolism render cells unviable and unrecoverable for longitudinal studies and are incompatible with in-vivo dynamic observations. We report a non-invasive optical technique to interrogate the upregulation of NAD+ in keratinocytes (both in-vitro and ex-vivo) upon administration of NMN. Our technique exploits intrinsic autofluorescence of cells and tissues using multiphoton microscopy. Keratinocytes (HaCat cells) was treated with four concentrations of NMN drug (50, 250, 500 and 1000 µg). Using multi-photon microscopy, we demonstrate that fluorescence of the endogenous NADH in the HaCat cells show a decreasing trend in both the average fluorescence lifetime (Tm) and the Free unbound NADH (T1), while we see an increasing trend in the NADH:NAD+ cellular redox ratio, with increasing dosage of NMN administration. A similar trend in the fluorescence of endogenous NADH was also seen in human ex-vivo skin upon delivery of NMN using NMN loaded dissolvable microneedle patches. NMN was successfully incorporated into the dissolvable microneedle patches using the solvent casting method. After successful delivery of 400 – 1600 µg of NMN from the dissolvable microneedle patches through human skin in-vitro (Franz cells), we show a promising, minimally invasive, alternative delivery system for the NAD+ precursor molecule that can enhance patient compliance and therapeutic outcomes.

The use of metallic nanoparticles in applications ranging from drug delivery to consumer electronics has exploded in the last two decades. Although this broad range of use cases has brought about technological revolutions in multiple fields, the effects of widespread production and subsequent human exposure to these nanoparticles have yet to be fully understood. New imaging techniques are a critical part of developing a more complete understanding of chronic exposure and biodistribution. Here we present a novel label free luminescence imaging technique to analyze the biodistribution, content, and biological context of metallic nanoparticles using multiphoton luminescence.

Development of a simple, label-free screening technique capable of precisely and directly sensing interaction-in-solution over a size range from small molecules to large proteins such as antibodies could offer an important tool for researchers and pharmaceutical companies in the field of drug development. In this work, we present a thermostable Raman interaction profiling (TRIP) technique that facilitates low-concentration and low-dose screening of binding between protein and ligand in physiologically relevant conditions. TRIP was applied to eight protein–ligand systems, and produced reproducible high-resolution Raman measurements, which were analyzed by principal component analysis. TRIP was able to resolve time-depending binding between 2,4-dinitrophenol and transthyretin, and analyze biologically relevant SARS-CoV-2 spike-antibody interactions. Mixtures of the spike receptor–binding domain with neutralizing, nonbinding, or binding but nonneutralizing antibodies revealed distinct and reproducible Raman signals. TRIP holds promise for the future developments of high-throughput drug screening and real-time binding measurements between protein and drug.

Failure to fully understand the molecular expression and tumor heterogeneity across a patient’s tumor can lead to administration of ineffective therapies that increase patient morbidity and healthcare costs. The -omics era has made it possible to identify several new molecular markers involved in cancer development, survival, invasion and even predicting treatment response. We are developing an entirely new nano-based molecular imaging strategy that has the potential to offer both high content molecular expression and spatial profiling in a single histology image. We have created an expansive library of 26 SERS nanoparticle (NP) batches, each bearing a unique spectral fingerprint with exceptional multiplexing capabilities. Spectral deconvolution was successfully demonstrated with a mixture of all 26 SERS NPs in a single imaging pixel both in vitro and in vivo. This opens up new opportunities to efficiently interrogate the heterogeneous molecular expression found within and across patient tissues offering clinicians a new multiplexed molecular imaging tool with the potential to predict how well a patient is likely to respond to given therapies based on their unique profile.

Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy is a nonlinear multimodal optical imaging technique with sub-micron spatial resolution, enabling 3-D visualization and analysis of live cells, complex in vitro models, and tissues. SLAM microscopy detects NAD(P)H and FAD autofluorescence as well as second and third harmonic generation signals simultaneously from biological samples. It can be used for a wide range of applications in cell-to-clinic pharmaceutical research. To run proof-of-concept, longitudinal, and clinical studies of interest to GSK project teams, the GSK Center for Optical Molecular Imaging (COMI) was established in 2015. Based on promising results from these studies, GSK contracted with spin-out start-up, LiveBx, to design and develop the first portable SLAM microscope, and is currently being used for studies on-site at GSK. In this presentation, major milestones and challenges in translating the SLAM technology from academia to industry and key learnings from this process will be shared from multiple perspectives.

Pharmaceutical development of solid-state formulations requires testing for uniformity and stability of active pharmaceutical ingredients and excipients. Solid-state properties such as component distribution and grain size are crucial factors of dissolution profile, which greatly affect drug efficacy and toxicity, and can only be analyzed spatially by chemical imaging (CI) techniques. Current CI techniques such as near infrared and confocal Raman spectroscopy do not offer high chemical and spatial resolution at speeds feasible for integration into the pharmaceutical quality control and quality assurance processes. We demonstrate fast chemical imaging by epi-detected sparse spectral sampling stimulated Raman scattering to quantify API and excipient degradation and distribution.

Quantifying intracellular drug target validation and target engagement has presented significant challenges, as drugs tend to accumulate non-specifically due to variations in drug affinity, biodistribution, pharmacokinetics, and metabolism. We have developed a ratiometric imaging approach, termed intracellular Paired Agent Imaging (iPAI), for estimating the concentration of protein receptors. This innovative technique holds great potential for revitalizing quantitative intracellular protein receptor imaging, offering a potential solution to quantify the availability of intracellular drug targets. Our approach involves the utilization of fluorophore-labeled small molecule therapeutics as imaging agents. We show it is now possible to use small molecule fluorophore labeled therapeutic inhibitors to visualize their targeted proteins intracellularly, providing a chemical tool kit to generate maps of bound and unbound inhibitors at the single-cell level both in vitro and ex vivo. iPAI platform provides a powerful new opportunity to study numerous other drugs in cells and tissues, resulting in a comprehensive spatial view of drug distribution and binding for improved personalized therapy.

The limit of detection (LoD) is the lowest concentration of an analyte that can be reliably observed with a sufficient degree of confidence. This presentation will discuss a method to determine the LoD for detecting an exogenous analyte in the Stratum Corneum, using classical Raman spectroscopy and a least-squares fit analysis method. We will show how measurement dependent parameters like the signal-to-noise of the spectra, available spectral information, and the quality of the fit influence the LoD.

Macroscopic fluorescence lifetime Forster resonance energy transfer (MFLI FRET) imaging presents an analytical tool to non-invasively quantify drug-receptor engagement in tumors and other organs in preclinical studies. Near infrared (NIR) fluorescence lifetime FRET acts as a direct reporter of drug-target engagement via measuring the fraction of labeled-donor antibody drugs undergoing binding to their respective target receptor using in vitro microscopy or in vivo macroscopy. We show that NIR MFLI FRET measurements correlates with drug-receptor binding in tumor cells, but strikingly, not with ubiquitously used ex vivo receptor expression assessment. Our study demonstrates that MFLI FRET is a powerful non-invasive imaging approach to study the impact of tumor microenvironment on drug delivery and drug-target engagement in intact live tumor xenografts.

Delivery of gold nanoparticles (AuNPs) to retinal ganglion cells is gaining attention as a therapeutic and diagnostic approach for retinal diseases. However, intravitreal injection of AuNPs is invasive and thus is not optimal. Focused ultrasound with microbubbles (FUS) is a non-invasive method for systemic delivery of viral vectors to retinal Müller glia; however, whether metallic nanoparticles of various sizes and shapes can be delivered via FUS remains unknown. Here, we report FUS-assisted delivery of AuNPs of varying shapes and sizes to retinal ganglion cells. FUS can also deliver dextran (70kDa) to the retinal layer, especially the retinal ganglion cell layer and inner nuclear layer cells. Two-photon microscopic imaging of AuNPs injected into the retinal ganglion cell layer confirms that spherical- and rod-shaped AuNPs with maximum dimensions <80nm are effectively delivered without damage. The amount of detected AuNPs varies with size. Spherical nanoparticles of small diameter (10nm) are ~20-fold more abundant than larger nanoparticles (55nm). Our findings provide a novel approach for delivering nanometer-sized metallic and organic nanomaterials without damaging retinal tissue