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.
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.
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. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1 (IL-1) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1 cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan-and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.
Abstract: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 multiparameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDAapproved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.
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.
ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/β-catenin signalling, given its emerging importance in arthritis.MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain of function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types.ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/β-catenin signalling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages and chondrocytes.ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4hi lining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury.
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. Microscopically, some self-assembled structures exhibited a pronounced, diattenuation anisotropy signal, evident by the differential absorption of linearly polarized light, at the peak absorbance wavelength of the phenazine core. The measured anisotropy was well above the background anisotropy of endogenous cellular components, reflecting the self-assembly of condensed, insoluble complexes of ordered phenazine molecules. Chemical variations introduced at the R-imino position of the phenazine core led to idiosyncratic effects on the compounds‘ bioaccumulation behavior, as well as on the morphology and organization of the resulting intracellular structures. Beyond clofazimine, these results demonstrate how the self-assembly of membrane-permeant, orally-bioavailable small molecule building blocks can endow cells with unnatural structural elements possessing chemical, physical and functional characteristics unlike those of other natural cellular components.
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