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

Rzeczycki, Phillip; Yoon, Gi Sang; Keswani, Rahul K.; Sud, Sudha; Stringer, Kathleen A.; Rosania, Gus R.

doi:10.1364/boe.8.000860

Cited by 17 publications

(28 citation statements)

References 41 publications

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“…Under all loading conditions, the less mature bone marrow monocytes were free of cargo. At a population level, these trends were confirmed by multi-parameter image-based cytometric analyses(16) (Figure 1c, d). …”

Section: Resultssupporting

confidence: 53%

“…Multi-parameter polarization, brightfield, and fluorescence imaging and analyses of different macrophage subpopulations were performed using a custom-built imaging system, as previously described(29). Brightfield, fluorescence, and polarization images were acquired on adherent cells.…”

Section: Methodsmentioning

confidence: 99%

“…Due to their ubiquitous presence and high rates of endocytosis, pinocytosis, and phagocytosis, they are recognized to play a major role in the clearance of drug nano- or microparticles from the circulation(9, 10). Potentially, this action could be exploited for the delivery of small molecule drugs(11–13) , (14), particularly those that are poorly soluble because they are prone to form insoluble aggregates that may ultimately accumulate in macrophages(15, 16). Furthermore, cationic, amphiphilic molecules, particularly weakly basic molecules, can become trapped within lysosomes following protonation in the acidic lysosomal microenvironment(17–19).…”

Section: Introductionmentioning

confidence: 99%

“…To study the in vivo, cargo storage capacity of macrophages in an intact, living organism, we have co-opted an FDA-approved and biocompatible antibiotic, clofazimine (CFZ)(15, 16). CFZ is a weakly basic anti-mycobacterial agent that is clinically used to treat leprosy and multi-drug resistant tuberculosis(20–22).…”

Section: Introductionmentioning

confidence: 99%

“…Experimentally, CLDIs were microscopically monitored to determine the numbers of drug-sequestering macrophages in different organs. From these same organs, CLDIs were then biochemically isolated and the drug content chemically analyzed to determine the average amount of drug per CLDI-containing cell(15, 29). Additionally, we modeled the subcellular transport and precipitation properties of CFZ using a physiologically based pharmacokinetics approach to assess whether thermodynamics alone could explain the selective accumulation and stabilization of the drug in macrophage lysosomes.…”

Section: Introductionmentioning

confidence: 99%

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An Expandable Mechanopharmaceutical Device (1): Measuring the Cargo Capacity of Macrophages in a Living Organism

et al. 2018

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Purpose: Clofazimine (CFZ) is an FDA-approved, poorly soluble small molecule drug that precipitates as crystal-like drug inclusions (CLDIs) which accumulate in acidic cytoplasmic organelles of macrophages. In this study, we considered CLDIs as an expandable mechanopharmaceutical device, to study how macrophages respond to an increasingly massive load of endophagolysosomal cargo. Methods: First, we experimentally tested how the accumulation of CFZ in CLDIs impacted different immune cell subpopulations of different organs. Second, to further investigate the mechanism of CLDI formation, we asked whether specific accumulation of CFZ hydrochloride crystals in lysosomes could be explained as a passive, thermodynamic equilibrium phenomenon. A cellular pharmacokinetic model was constructed, simulating CFZ accumulation driven by pH-dependent ion trapping of the protonated drug in the acidic lysosomes, followed by the precipitation of CFZ hydrochloride salt via a common ion effect caused by high chloride concentrations. Results: While lower loads of CFZ were mostly accommodated in lung macrophages, increased CFZ loading was accompanied by organ-specific changes in macrophage numbers, size and intracellular membrane architecture, maximizing the cargo storage capabilities. With increasing loads, the total cargo mass and concentrations of CFZ in different organs diverged, while that of individual macrophages converged. The simulation results support the notion that the proton and chloride ion concentrations of macrophage lysosomes are sufficient to drive the massive, cell type-selective accumulation and growth of CFZ hydrochloride biocrystals. Conclusion: CLDIs effectively function as an expandable mechanopharmaceutical device, revealing the coordinated response of the macrophage population to an increasingly massive, whole-organism endophagolysosomal cargo load.

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Section: Resultssupporting

confidence: 53%

Section: Methodsmentioning

confidence: 99%