Acoustic tweezing cytometry for mechanical phenotyping of macrophages and mechanopharmaceutical cytotripsy.

Macrophages are immune cells responsible for tissue debridement and fighting infection. Clofazimine, an FDA-approved antibiotic, accumulates and precipitates as rod-shaped, crystal-like drug inclusions within macrophage lysosomes. Drug treatment as well as pathophysiological states could induce changes in macrophage mechanical property which in turn impact their phenotype and function.

An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

Purpose: Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage.

Methods: Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function.

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 pH 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.

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.

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy.

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound.

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.

Macrophage-Mediated Clofazimine Sequestration Is Accompanied by a Shift in Host Energy Metabolism.

Prolonged (8 weeks) oral administration of clofazimine results in a profound pharmacodynamic response-bioaccumulation in macrophages (including Kupffer cells) as intracellular crystal-like drug inclusions (CLDIs) with an associated increase in interleukin-1 receptor antagonist production. Notably, CLDI formation in Kupffer cells concomitantly occurs with the formation of macrophage-centric granulomas. Accordingly, we sought to understand the impact of these events on host metabolism using H-nuclear magnetic resonance metabolomics.

Endocytic Transport of Polyplex and Lipoplex siRNA Vectors in HeLa Cells.

Purpose: siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency.

Methods: Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI).

Yoga practice improves executive function by attenuating stress levels.

Background: Prolonged activation of the hypothalamus-pituitary-adrenal system is thought to have deleterious effects on brain function. Neuroendocrine studies suggest that brain exposure to higher cortisol concentrations contribute to cognitive deficits as we age. Mind-body techniques such as yoga have shown to improve stress levels by restoring the body's sympathetic-parasympathetic balance.

Clofazimine Biocrystal Accumulation in Macrophages Upregulates Interleukin 1 Receptor Antagonist Production To Induce a Systemic Anti-Inflammatory State.

Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively.

Repositioning Clofazimine as a Macrophage-Targeting Photoacoustic Contrast Agent.

Photoacoustic Tomography (PAT) is a deep-tissue imaging modality, with potential clinical applications in the diagnosis of arthritis, cancer and other disease conditions. Here, we identified Clofazimine (CFZ), a red-pigmented dye and anti-inflammatory FDA-approved drug, as a macrophage-targeting photoacoustic (PA) imaging agent. Spectroscopic experiments revealed that CFZ and its various protonated forms yielded optimal PAT signals at wavelengths -450 to 540 nm.

Massive Bioaccumulation and Self-Assembly of Phenazine Compounds in Live Cells.

Clofazimine is an orally administered, FDA-approved drug that massively bioaccumulates in macrophages, forming membrane-bound intracellular structures possessing nanoscale supramolecular features. Here, a library of phenazine compounds derived from clofazimine was synthesized and tested for their ability to accumulate and form ordered molecular aggregates inside cells. Regardless of chemical structure or physicochemical properties, bioaccumulation was consistently greater in macrophages than in epithelial cells.

A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages.

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ.

Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition.

In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition, and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen.

Phagocytosed Clofazimine Biocrystals Can Modulate Innate Immune Signaling by Inhibiting TNFα and Boosting IL-1RA Secretion.

Clofazimine (CFZ) is an FDA-approved leprostatic and anti-inflammatory drug that massively accumulates in macrophages, forming insoluble, intracellular crystal-like drug inclusions (CLDIs) during long-term oral dosing. Interestingly, when added to cells in vitro, soluble CFZ is cytotoxic because it depolarizes mitochondria and induces apoptosis. Accordingly, we hypothesized that, in vivo, macrophages detoxify CFZ by sequestering it in CLDIs.

Intracellular trafficking of hybrid gene delivery vectors.

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking.

Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways.

Purpose: Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing.

Design of hybrid lipid/retroviral-like particle gene delivery vectors.

Recombinant retroviruses provide highly efficient gene delivery and the potential for stable gene expression. The retroviral envelope protein, however, is the source of significant disadvantages such as immunogenicity, poor stability (half-life of transduction activity of 5-7 h at 37 °C for amphotropic murine leukemia virus), and difficult production and purification. To address these problems, we report the construction of efficient hybrid vectors through the association of murine leukemia virus (MLV)-like particles (M-VLP) with synthetic liposomes comprising DOTAP, DOPE, and cholesterol (φ/M-VLP).

Effect of serum on transfection by polyethylenimine/virus-like particle hybrid gene delivery vectors.

Purpose: Murine leukemia virus-like particles (M-VLP) complexed with polymers to promote cellular uptake and endosomal escape represent a new class of effective gene delivery vectors. Building upon recent studies of viral-synthetic hybrid vectors, we report the effects of serum on the formation, activity and stability of PEI/M-VLP complexes.

Methods: M-VLP were produced by cells grown in serum-supplemented media (M-VLP-S), serum-free media (M-VLP-SF) or serum-free Opti-MEM® I (M-VLP-OM).