This PDF file contains the front matter associated with SPIE Proceedings Volume 10475, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Nanomedicine can kill tumor cells or block the growth and spread of cancers by maximizing drug accumulation in tumor tissues. How much drug will selectively accumulate in tumor tissues depends on physical parameters such as vessel permeability in tumor microenvironments. On the other hand, by properly choosing the size of nanomedicine, the accumulation of drugs in normal tissues will be reduced. In this study, we measured the permeability rate of vessels in different strain of mice in vivo with two-photon fluorescent angiography. We used FITC / TRITC / TEXAS RED labeled dextran to investigate size-dependent permeation from vessels in normal tissues. We choose nanoparticles of 40kDa, 70 kDa, 150 kDa and 2000 kDa, and selected three strains of wild-type Balb/C, ICR, C2J mice. We found , even at the same size, the vascular permeability rate of dextran still vary with the dye-conjugates and the strain of mice. One of our results shows that ICR mice have the biggest vessel hole in normal tissues. Choosing wrong nanoparticle size will cause their leaking in normal tissues. This study will provide a new threshold and guideline to reduce the accumulation of nanomedicine in normal tissues.

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 Förster 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.

One of the primary functions of human skin is to provide a mechanical barrier and interface with the outside world, owing to its unique structure and composition. Indeed, the most superficial layer of the epidermis, the stratum corneum, is a selectively porous structure composed of thin layers of protein-rich corneocytes joined together by lipids such as ceramides. The overall impermeability of the stratum corneum is crucial for homeostasis, but also hampers the penetration of beneficial topical agents into the skin. Specifically, hydrophilic compounds typically do not permeate through the epidermis, while hydrophobic compounds can readily be delivered through lipid-containing routes such as sebaceous gland ducts or the spacing between corneocytes. A proper understanding of stratum corneum structure and composition is therefore of great benefit for the design of topical formulations in order to properly optimize the delivery of active compounds to the skin. To this aim, coherent Raman scattering imaging techniques including both coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopies can be used to study the chemical composition and structure of the stratum corneum, as these modalities are sensitive to unique vibrational modes of specific chemical groups such as lipids, proteins, and water. These metrics can further be used to measure uptake and efficacy of topical compounds in order to optimize formulation design.

Approximately 29 million Americans have diabetes, and 86 million are living with prediabetes, increasing the risk of developing type 2 diabetes. Complications of wound healing in diabetic patients represent a significant health problem. Impaired diabetic wound healing is characterized by reduced collagen production and diminished angiogenesis. During the proliferative phase of wound healing, the injured tissue undergoes angiogenesis, re-epithelialization, and fibroplasia. Monitoring the development of new blood vessels, metabolic changes, and collagen deposition, is critical to elucidate the process of diabetic wound healing and to improve the development of therapeutic drugs. This study employs a custom-built multimodal microscope where Optical Coherence Tomography Angiography (OCTA) is used for studying neovascularization, Fluorescence Lifetime Imaging Microscopy (FLIM) for NADH/FAD assessment, Second Harmonic Generation (SHG) microscopy for analyzing collagen deposition, and Coherent anti-Stoke’s Raman Scattering (CARS) microscopy for visualizing water/lipid distribution, all together to non-invasively follow closure of a skin wound in healthy diabetic (db/db) mice treated with placebo and angiogenesis-promoting topical formulation (GlaxoSmithKline). The (db/db) mouse model presents hyperglycemia, obesity, and delayed wound healing that is pathologically similar to human type 2 diabetes mellitus. In this ongoing study, the animals are treated once daily for 14 days after wounding. Images of the wound and surrounding areas are taken at different time points for 28 days. In this experiment, the wound healing process is investigated to gain deeper understanding of the drug mechanism. The capability to non-invasively monitor wound healing mechanisms can become a valuable tool in development of new drug compounds for diabetic wound care.

Topical drug application often relies on the well-established models of drug absorption and distribution in tissues. While optical methods are good in probing first several hundred micrometers of tissues, deeper penetration is challenging. Diffusional optical imaging and photoacoustic imaging can extend the imaging depth, but both of those methods rely on incoherent light scattering and absorption, thus limiting application of coherent optical techniques. One of the modern trends is to use beam-shaping optics to pre-compensate for wavefront’s distortions; however, this approach is the most efficient for low scattering medium and for systems where “guiding star” is available or can be easily introduced into the system. We have recently discovered a minimally invasive approach, which allows substantial penetration depth enhancement and, as we discovered recently, coherent light propagation for distances exceeding tens and hundreds of scattering lengths of the tissue [1]. 1. . J. V. Thompson, B. H. Hokr, W. Kim, C. W. Ballmann, B. Applegate, J. Jo, A. Yamilov, H. Cao, M. O. Scully, and V. V. Yakovlev, “Enhanced coupling of light into a turbid medium through microscopic interface engineering,” Proceedings National Academy of Sciences USA (2017) doi: 10.1073/pnas.1705612114.

Accurate measurement of the response of cancer cells to therapeutic agents is confounded by many variables. Lysate-based assays such as CellTiter-Glo® (CTG) are commonly used but rely on a surrogate of cell number (ATP in the case of CTG) and provide limited information on the phenotypic response to treatment. To characterize drug response at a single-cell level, we developed the DeepDyeDrop assay, a high throughput minimally disruptive microscopy-based assay. In brief, the assay involves plating cells in 384-well plates, delivering treatments of interest, and staining and fixing the treated plates at an appropriate time point. Nuclei are stained with Hoechst, dead cells with a membrane impermeable LIVE/DEAD (LDR) dye, cells progressing through S phase of the cell cycle with EdU, and cells in mitosis with a conjugated phospho-histone H3 (pH3) antibody. Images are acquired with a Perkin Elmer Operetta microscope, nuclei are segmented based on the Hoechst signal, and the intensity of all signals within the nuclear area are measured. After segmentation, we first identify dead cells based on the Hoechst and LDR signals. The remaining live cells are then assigned to the phases of the cell cycle based on their DNA content, and EdU and pH3 intensities. To quantify the differences among treatments, we relied on normalized growth rate inhibition (GR) metrics, which allow for the comparison of cell lines with variable division rates. We added new theoretical framework to the standard GR metrics to account for differential responses within a treated population of cells to independently score the contributions of the cytostatic and cytotoxic components to the overall response. By conducting the assay on a panel of breast cancer cell lines treated with a library of kinase inhibitors, we observed a wide range of responses that reflect drug mechanisms of action.

The recent introduction of small molecule inhibitors of cyclin-dependent kinases (CDK) 4/6 to the clinic has improved the treatment of hormone receptor positive breast cancer, and shown promise in other malignancies. The three clinically-used CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, are reported to be broadly similar although recent data suggest that abemaciclib has distinct single-agent activity in patients and a unique toxicity profile. Key questions are: How do these drugs differ at the molecular level? Should such differences inform their use in the clinic? Can these three agents be used interchangeably or should patient stratification differ between them? We started to address these questions by characterizing the spectrum of kinases inhibited by palbociclib, ribociclib, and abemaciclib, and the functional consequences of their inhibition in cell lines using multiple methods. First, in vitro biochemical profiling of the CDK4/6 inhibitors confirmed abemaciclib to be more potent on target, but less selective than either palbociclib or ribociclib. We used profiling by mRNA sequencing to capture differences in transcriptional response; mass spectrometry-based proteomics to infer changes in kinase activity following treatment, and imaging-based dose response, and immunofluorescence assays to characterize the phenotypic responses of breast cancer cell lines to the three CDK4/6 inhibitors. We propose that multi-omic approaches are required to fully elucidate the spectrum of targets relevant to drug mechanisms of action in cells, and we expect such understanding to inform clinical use of these drugs.

The safety and efficacy of an investigational topical minocycline gel (BPX-01) has recently been studied in a Phase 2b trial for the treatment of acne vulgaris. As part of the drug development process, there was a need to determine if minocycline was delivered to the target tissue compartments, including the epidermis, hair follicle, and the sebaceous gland. While it was easier to demonstrate delivery on an ex vivo human skin model with an infinite dose, it was initially challenging to verify low-dose delivery with conventional fluorescence microscopy due to the high autofluorescence inherent to human skin. An integrating sphere screening approach was implemented along with conventional fluorescence microscopy to quantitatively and qualitatively assess endogenous fluorescence concurrently from numerous human facial skin specimens. Donor tissues were cut into 50-µm frozen sections, mounted onto microscope slides, and positioned on an inverted fluorescence microscope, sandwiched between the microscope’s 40x high NA objective lens and an external integrating sphere. The tissue sections were illuminated with UV excitation centered at 386 nm. For the first time, it was found that random samples from >40 human facial skin donors produced at least 5× differential in measurable autofluorescence. This observation has significant implications for the use of 2PEF microscopy and FLIM to visualize/quantify drug distribution; the endogenous autofluorescence may limit the detectability of the minocycline signature. Our studies indicated that a single daily dose of BPX-01 was detected in low autofluorescence skin specimens with FLIM, thus validating a novel imaging modality for future pharmacokinetic studies.

Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a phasor approach to fluorescence lifetime microscopy (FLIM). In phasor analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the phasor plot. Since every fluorophore has a specific decay trace, we can identify a specific molecule by its position in the phasor plot. To demonstrate the feasibility of this visualization and quantification method, the human facial skin samples treated with various concentrations of BPX-01 were investigated using the phasor approach to FLIM. The unique signature of minocycline in FLIM phasor analysis was successfully differentiated from the endogenous fluorescence of human tissue. Furthermore, by sorting the individual pixels of minocycline signature in FLIM image, the distribution of minocycline within human facial skin can be visualized and quantified. Based on these results, we believe that the visualization and quantification method using a phasor approach to FLIM can play an important role in future pharmacokinetics and pharmacodynamics analyses.

Tools for rapid therapeutic selection for pancreatic ductal adenocarcinoma (PDAC) patients are vital to improved survival as PDAC is the third leading cause of cancer death, with an average life expectancy of 5-7 months post diagnosis. Treatment options are limited to surgery and chemotherapy, where Gemcitabine has been the cornerstone of therapy since 1997. However, Gemcitabine resistance prevents PDAC cure and mechanisms of resistance are not fully understood. Yet, with average survival times still <1 year, improved treatment selection and early assessment of response are urgently needed. Our group has recently developed a promising imaging based technology that will improve patient specific treatment selection and provide a platform for assessment of newly developed therapeutics with improved efficacy prediction compared to drug distribution alone. We have established a spatially-resolved, quantitative imaging methodology termed paired agent imaging (PAI), that enables quantification of drug target sites at the single cell level, allowing direct visualization of drug target availability (DTA) in situ, a parameter directly related to therapeutic efficacy. Using PAI, we examined the mechanisms of cancer therapy resistance on a cell-by-cell basis with the goal of utilizing explants for rapid, patient-specific therapy selection as they can predict treatment response in <5 days. As proof of concept we have studied Erlotinib, synthesizing paired targeted and untargeted fluorescently labeled derivatives. Overall, we anticipate that our spatially resolved maps of DTA will be predictive of therapeutic efficacy in patient PDAC explants, providing a platform technology for patient specific therapy selection <1 week after resection or biopsy.

Pancreatic ductal adenocarcinoma (PDAC) has the poorest five-year survival rate of any cancer. Surgical resection is the only curative treatment but, is only possible in 15-20% of cases. Systemic therapies that demonstrate some efficacy are limited to chemotherapeutics, such as Gemcitabine, Abraxane (nab-Paclitaxel) and FOLFIRINOX, a multidrug chemotherapy regimen. Numerous studies have tried to apply multi-agent therapies to advance standard of care for metastatic PDAC. However, consistent improvement in survival remains poor. Of broader interest, there is an urgent need for improved treatment selection and early assessment of therapeutic responses as mean survival time following diagnosis is less than one year. Paired agent imaging (PAI) is a spatially-resolved, quantitative imaging methodology, which enables quantification of bound and unbound drugs on a cell by cell basis, using a targeted and control (untargeted) imaging agent. PAI has the potential to provide a spatially resolved map of drug engagement and treatment efficacy for PDAC. Herein, we have developed fluorescently labeled, paired targeted and control Gemcitabine, which have been characterized for their similarity to the parent Gemcitabine using competitive binding, liquid chromatography mass spectroscopy based uptake and pharmacokinetic studies, and cytotoxicity assays. Using our validated targeted and control agents, we have generated maps of targeted and untargeted Gemcitabine in murine models of PDAC, demonstrating that therapeutic efficacy is correlated to the bound drug fractions. We anticipate that PAI using fluorescently labeled targeted and control drug derivatives will be useful for future studies to prediction therapy combinations for personalized PDAC therapy.

Photodynamic inactivation (PDI) is a promising alternative for the treatment of infectious diseases, and the combination of indocyanine green (ICG) and extracorporeal infrared light has shown optimistic results against pneumonia in vitro and in vivo. However, the pharmacokinetics and the possible side effects of the pulmonary delivery via nebulization have not been fully investigated. This study assessed the distribution of the photosensitizer within the lungs and to other organs of mice, and monitored the fluorescence of ICG in the thorax in the presence and absence of the activating light. The excitation wavelength was 780 nm and detection focused on the emission between 795 and 890 nm. Experiments demonstrated that the amount of fluorescence detected from outside the body was significantly higher after the nebulization of ICG, and reduced after the illumination, allowing for the monitoring of the PDI in real time. The fluorescence remained detectable in the mice for at least 24 hours, and was present in the lungs, stomach, liver, small and large intestines, bladder, spleen and heart after this time.