Inkjet-printed micro-calibration standards for ultraquantitative Raman spectral cytometry.

Herein we report the development of a cytometric analysis platform for measuring the contents of individual cells in absolute (picogram) scales; this study represents the first report of Raman-based quantitation of the absolute mass - or the total amount - of multiple endogenous biomolecules within single-cells. To enable ultraquantitative calibration, we engineered single-cell-sized micro-calibration standards of known composition by inkjet-printer deposition of biomolecular components in microarrays across the surface of silicon chips. We demonstrate clinical feasibility by characterizing the compositional phenotype of human skin fibroblast and porcine alveolar macrophage cell populations in the respective contexts of Niemann-Pick disease and drug-induced phospholipidosis: two types of lipid storage disorders. We envision this microanalytical platform as the foundation for many future biomedical applications, ranging from diagnostic assays to pathological analysis to advanced pharmaco/toxicokinetic research studies.

An Expandable Mechanopharmaceutical Device (3): a Versatile Raman Spectral Cytometry Approach to Study the Drug Cargo Capacity of Individual Macrophages.

PURPOSE: To improve cytometric phenotyping abilities and better understand cell populations with high interindividual variability, a novel Raman-based microanalysis was developed to characterize macrophages on the basis of chemical composition, specifically to measure and characterize intracellular drug distribution and phase separation in relation to endogenous cellular biomolecules. METHODS: The microanalysis was developed for the commercially-available WiTec alpha300R confocal Raman microscope. Alveolar macrophages were isolated and incubated in the presence of pharmaceutical compounds nilotinib, chloroquine, or etravirine. A Raman data processing algorithm was specifically developed to acquire the Raman signals emitted from single-cells and calculate the signal contributions from each of the major molecular components present in cell samples. RESULTS: Our methodology enabled analysis of the most abundant biochemicals present in typical eukaryotic cells and clearly identified "foamy" lipid-laden macrophages throughout cell populations, indicating feasibility for cellular lipid content analysis in the context of different diseases. Single-cell imaging revealed differences in intracellular distribution behavior for each drug; nilotinib underwent phase separation and self-aggregation while chloroquine and etravirine accumulated primarily via lipid partitioning. CONCLUSIONS: This methodology establishes a versatile cytometric analysis of drug cargo loading in macrophages requiring small numbers of cells with foreseeable applications in toxicology, disease pathology, and drug discovery.

Elasticity in Macrophage-Synthesized Biocrystals.

Supramolecular crystalline assembly constitutes a rational approach to bioengineer intracellular structures. Here, biocrystals of clofazimine (CFZ) that form in vivo within macrophages were measured to have marked curvature. Isolated crystals, however, showed reduced curvature suggesting that intracellular forces bend these drug crystals. Consistent with the ability of biocrystals to elastically deform, the inherent crystal structure of the principal molecular component of the biocrystals-the hydrochloride salt of CFZ (CFZ-HCl)-has a corrugated packing along the (001) face and weak dispersive bonding in multiple directions. These characteristics were previously found to be linked to the elasticity of other organic crystals. Internal stress in bent CFZ-HCl led to photoelastic effects on the azimuthal orientation of polarized light transmittance. We propose that elastic, intracellular crystals can serve as templates to construct functional microdevices with different applications.

Lithium chloride induces TNFα in mouse macrophages via MEK-ERK-dependent pathway.

Lithium (Li) is one of the currently prescribed drugs for bipolar disorders (BPDs) and has many neuro-regulatory and immune-modulating properties. Because many neuro-pathological diseases including BPDs have been associated with some level of inflammation, Li's effect on inflammation may have some crucial consequences. Even though Li has been shown to have pro- and anti-inflammatory activities in different cell models, mechanisms involved in these effects are not well understood. Moreover, Li's effect on inflammation in the presence of activators of Toll-like receptors (TLRs), especially TLR-2 (that activates MyD88-dependent pathway) and TLR-3 (that activates TRIF-dependent pathway) is not known. Here we tested the role of Li in the presence and absence of TLR2, and TLR3 on MAPK and NFκB pathways and the consequent production of tumor necrosis factor-α (TNFα) in Raw264.7 macrophages. Our results indicate that Li enhances TNFα production both in the absence and presence of TLR stimulation. Interestingly, Li differentially modulates MAPK and NFκB pathways in the absence and presence of TLR2/3 ligands. Our results further indicate that the effect of Li on TNFα occurs at the post-transcriptional level. Together, these studies demonstrate that Li induces TNFα production in macrophages and that it modulates signaling at different levels depending on the presence or absence of TLR2/3 stimulation.

Quantitative Raman chemical imaging of intracellular drug-membrane aggregates and small molecule drug precipitates in cytoplasmic organelles.

Raman confocal microscopes have been used to visualize the distribution of small molecule drugs within different subcellular compartments. This visualization allows the discovery, characterization, and detailed analysis of the molecular transport phenomena underpinning the Volume of Distribution - a key parameter governing the systemic pharmacokinetics of small molecule drugs. In the specific case of lipophilic small molecules with large Volumes of Distribution, chemical imaging studies using Raman confocal microscopes have revealed how weakly basic, poorly soluble drug molecules can accumulate inside cells by forming stable, supramolecular complexes in association with cytoplasmic membranes or by precipitating out within organelles. To study the self-assembly and function of the resulting intracellular drug inclusions, Raman chemical imaging methods have been developed to measure and map the mass, concentration, and ionization state of drug molecules at a microscopic, subcellular level. Beyond the field of drug delivery, Raman chemical imaging techniques relevant to the study of microscopic drug precipitates and drug-lipid complexes which form inside cells are also being developed by researchers with seemingly unrelated scientific interests. Highlighting advances in data acquisition, calibration methods, and computational data management and analysis tools, this review will cover a decade of technological developments that enable the conversion of spectral signals obtained from Raman confocal microscopes into new discoveries and information about previously unknown, concentrative drug transport pathways driven by soluble-to-insoluble phase transitions occurring within the cytoplasmic organelles of eukaryotic cells.

Quantitative chemometric phenotyping of three-dimensional liver organoids by Raman spectral imaging.

Confocal Raman spectral imaging (RSI) enables high-content, label-free visualization of a wide range of molecules in biological specimens without sample preparation. However, reliable quantification of the deconvoluted spectra is needed. Here we develop an integrated bioanalytical methodology, qRamanomics, to qualify RSI as a tissue phantom calibrated tool for quantitative spatial chemotyping of major classes of biomolecules. Next, we apply qRamanomics to fixed 3D liver organoids generated from stem-cell-derived or primary hepatocytes to assess specimen variation and maturity. We then demonstrate the utility of qRamanomics for identifying biomolecular response signatures from a panel of liver-altering drugs, probing drug-induced compositional changes in 3D organoids followed by in situ monitoring of drug metabolism and accumulation. Quantitative chemometric phenotyping constitutes an important step in developing quantitative label-free interrogation of 3D biological specimens.

Synthesis and Characterization of a Biomimetic Formulation of Clofazimine Hydrochloride Microcrystals for Parenteral Administration.

Clofazimine (CFZ) is a broad spectrum antimycobacterial agent recommended by the World Health Organization as a first line treatment for leprosy and second line treatment for multidrug resistant tuberculosis. Oral administration of CFZ leads to a red skin pigmentation side effect. Since CFZ is a weakly basic, red phenazine dye, the skin pigmentation side effect results from lipophilic partitioning of the circulating, free base (neutral) form of CFZ into the skin. Here, we developed a stable and biocompatible formulation of CFZ-HCl microcrystals that mimics the predominant form of the drug that bioaccumulates in macrophages, following long term oral CFZ administration. In mice, intravenous injection of these biomimetic CFZ-HCl microcrystals led to visible drug accumulation in macrophages of the reticuloendothelial system with minimal skin accumulation or pigmentation. In fact, no skin pigmentation was observed when the total amount of CFZ-HCl administered was equivalent to the total oral dose leading to maximal skin pigmentation. Thus, parenteral (injected or inhaled) biomimetic formulations of CFZ-HCl could be instrumental to avoid the pigmentation side effect of oral CFZ therapy.

Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructing sophisticated molecular devices inside the cells of living organisms. Here, using experimental and computational approaches, we probed the relationship between the biological mechanisms mediating lysosomal ion homeostasis and the self-assembly of a weakly basic small molecule building block (clofazimine) into a functional, mechanopharmaceutical device (intracellular Crystal-Like Drug Inclusions - "CLDIs") in macrophage lysosomes. Physicochemical considerations indicate that the intralysosomal stabilization of the self-assembled mechanopharmaceutical device depends on the pHmax of the weakly basic building block and its affinity for chloride, both of which are consistent with the pH and chloride content of a physiological lysosomal microenvironment. Most importantly, in vitro and in silico studies revealed that high expression levels of the vacuolar ATPase (V-ATPase), irrespective of the expression levels of chloride channels, are necessary and sufficient to explain the cell-type dependent formation, stabilization, and biocompatibility of the self-assembled mechanopharmaceutical device within macrophages.

The Physicochemical Basis of Clofazimine-Induced Skin Pigmentation.

Clofazimine is a weakly basic, Food and Drug Administration-approved antibiotic recommended by the World Health Organization to treat leprosy and multi-drug-resistant tuberculosis. Upon prolonged treatment, clofazimine extensively bioaccumulates and precipitates throughout the organism, forming crystal-like drug inclusions (CLDIs). Due to the drug's red color, it is widely believed that clofazimine bioaccumulation results in skin pigmentation, its most common side effect. To test whether clofazimine-induced skin pigmentation is due to CLDI formation, we synthesized a closely related clofazimine analog that does not precipitate under physiological pH and chloride conditions that are required for CLDI formation. Despite the absence of detectable CLDIs in mice, administration of this analog still led to significant skin pigmentation. In clofazimine-treated mice, skin cryosections revealed no evidence of CLDIs when analyzed with a microscopic imaging system specifically designed for detecting clofazimine aggregates. Rather, the reflectance spectra of the skin revealed a signal corresponding to the soluble, free base form of the drug. Consistent with the low concentrations of clofazimine in the skin, these results suggest that clofazimine-induced skin pigmentation is not due to clofazimine precipitation and CLDI formation, but rather to the partitioning of the circulating, free base form of the drug into subcutaneous fat.