Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging.

Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.

Weakly Supervised Identification and Localization of Drug Fingerprints Based on Label-Free Hyperspectral CARS Microscopy.

Understanding drug fingerprints in complex biological samples is essential for the development of a drug. Hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy, a label-free nondestructive chemical imaging technique, can profile biological samples based on their endogenous vibrational contrast. Here, we propose a deep learning-assisted HS-CARS imaging approach for the investigation of drug fingerprints and their localization at single-cell resolution. To identify and localize drug fingerprints in complex biological systems, an attention-based deep neural network, hyperspectral attention net (HAN), was developed. By formulating the task to a multiple instance learning problem, HAN highlights informative regions through the attention mechanism when being trained on whole-image labels. Using the proposed technique, we investigated the drug fingerprints of a hepatitis B virus therapy in murine liver tissues. With the increase in drug dosage, higher classification accuracy was observed, with an average area under the curve (AUC) of 0.942 for the high-dose group. Besides, highly informative tissue structures predicted by HAN demonstrated a high degree of similarity with the drug localization shown by the in situ hybridization staining results. These results demonstrate the potential of the proposed deep learning-assisted optical imaging technique for the label-free profiling, identification, and localization of drug fingerprints in biological samples, which can be extended to nonperturbative investigations of complex biological systems under various biological conditions.

Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging.

The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Differential Uptake of Antisense Oligonucleotides in Mouse Hepatocytes and Macrophages Revealed by Simultaneous Two-Photon Excited Fluorescence and Coherent Raman Imaging.

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time- and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex in vitro models and liver tissues to evaluate ASO distribution and activity.

Fluorescent nanodiamonds for characterization of nonlinear microscopy systems.

Characterizing the performance of fluorescence microscopy and nonlinear imaging systems is an essential step required for imaging system optimization and quality control during longitudinal experiments. Emerging multimodal nonlinear imaging techniques require a new generation of microscopy calibration targets that are not susceptible to bleaching and can provide a contrast across the multiple modalities. Here, we present a nanodiamond-based calibration target for microscopy, designed for facilitating reproducible measurements at the object plane. The target is designed to support day-to-day instrumentation development efforts in microscopy laboratories. The images of a phantom contain information about the imaging performance of a microscopy system across multiple spectral windows and modalities. Since fluorescent nanodiamonds are not prone to bleaching, the proposed imaging target can serve as a standard, shelf-stable sample to provide rapid reference measurements for ensuring consistent performance of microscopy systems in microscopy laboratories and imaging facilities.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

Highly purified extracellular vesicles from human cardiomyocytes demonstrate preferential uptake by human endothelial cells.

Extracellular vesicles (EVs) represent a promising cell-free alternative for treatment of cardiovascular diseases. Nevertheless, the lack of standardised and reproducible isolation methods capable of recovering pure, intact EVs presents a significant obstacle. Additionally, there is significant interest in investigating the interactions of EVs with different cardiac cell types. Here we established a robust technique for the production and isolation of EVs harvested from an enriched (>97% purity) population of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) with size exclusion chromatography. Utilizing an advanced fluorescence labelling strategy, we then investigated the interplay of the CM-EVs with the three major cellular components of the myocardium (fibroblasts, cardiomyocytes and endothelial cells) and identified that cardiac endothelial cells show preferential uptake of these EVs. Overall, our findings provide a great opportunity to overcome the translational hurdles associated with the isolation of intact, non-aggregated human iPSC-CM EVs at high purity. Furthermore, understanding in detail the interaction of the secreted EVs with their surrounding cells in the heart may open promising new avenues in the field of EV engineering for targeted delivery in cardiac regeneration.

Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling.

Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles. Here, we present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated d-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, our metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers.

Simultaneous label-free autofluorescence and multi-harmonic imaging reveals in vivo structural and metabolic changes in murine skin.

Simultaneous quantification of multifarious cellular metabolites and the extracellular matrix in vivo has been long sought. Simultaneous label-free autofluorescence and multi-harmonic (SLAM) microscopy has achieved simultaneous four-channel nonlinear imaging to study tissue structure and metabolism. In this study, we implemented two laser systems and directly compared SLAM microscopy with conventional two-photon microscopy for in vivo imaging. We found that three-photon imaging of adenine dinucleotide (phosphate) (NAD(P)H) in SLAM microscopy using our tailored laser source provided better resolution, contrast, and background suppression than conventional two-photon imaging of NAD(P)H. We also integrated fluorescence lifetime imaging with SLAM microscopy, and enabled differentiation of free and bound NAD(P)H. We imaged murine skin in vivo and showed that changes in tissue structure, cell dynamics, and metabolism can be monitored simultaneously in real-time. We also discovered an increase in metabolism and protein-bound NAD(P)H in skin cells during the early stages of wound healing.

The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice.

Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD) to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides) will be required. To investigate the dynamics and persistence of antisense 2'-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours-24 weeks). Oligonucleotide half-life was longer in heart (~65 days) compared with that in skeletal muscle, liver, and kidney (~35 days). Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days). Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3-8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2'-O-methyl phosphorothioate oligos used for the treatment of DMD.Molecular Therapy-Nucleic Acids (2014) 3, e148; doi:10.1038/mtna.2014.1; published online 18 February 2014.

Optimization of stress response through the nuclear receptor-mediated cortisol signalling network.

It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body's response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress.

Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays.

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.

Impact of transcription factor profile and chromatin conformation on human hepatocyte CYP3A gene expression.

Recent data have made it increasingly clear that the gene expression profile of a cell system, and its alteration in response to external stimuli, is highly dependent on both the higher order chromatin structure of the genome and the interaction of gene products in interpreting stimuli. To further explore this phenomenon, we have examined the role of both of these factors in controlling xenobiotic-mediated gene expression changes in primary and transformed human hepatocytes (HuH7). Using quantitative polymerase chain reaction, expression levels of several transcription factors implicated in the liver-specific regulation of the CYP3A gene family were examined in human adult and fetal liver RNA samples. These expression profiles were then compared with those obtained from both primary and transformed human hepatocytes, showing that, in general, cultured cells exhibit a distinct profile compared with either the fetal or adult samples. Transcriptome profiles before and after exposure to the CYP3A transcriptional activators rifampicin, dexamethasone, pregnane-16alpha-carbonitrile, and phenobarbital were subsequently examined. Whereas exposure to these compounds elicited a dose-dependent increase in CYP3A transcription in primary hepatocytes, no alteration in expression levels was observed for the hepatoma cell line HuH7. Alteration in the expression levels of pregnane X receptor and chicken ovalbumin upstream promoter transcription factor I, and the disruption of higher order chromatin within HuH7 cells altered CYP3A expression and/or activation by xenobiotics toward that observed in primary hepatocytes. These data provide potential roles for these two processes in regulating CYP3A expression in vivo.