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

Abstract

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) following oral administration of the drug in animals and humans. 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 nm to Ex:560-600 nm) and emission (Em: 560-580 nm to Em: 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(−) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and DAPI, cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently-tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.

Elasticity in Macrophage‐Synthesized Biocrystals

“…As such, formation of biocrystals mediated by Mφs leads to the development of elastic and curved crystalline elements with structural packing features that showcase the classical design rules designated for organic crystal flexibility [26–29] . The presence of CFZ biocrystals in the Mφ endows them with potent anti-inflammatory characteristics [37,38] and specific fluorescent [39] and photoacoustic [40,41] signatures for cellular optical tracking. Such dual therapeutic and diagnostic (theranostic) applications make biocrystals a potent cellular device that could be harnessed for biomedical applications.…”

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

“…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).…”

“…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).…”

“…Because CFZ is both highly lipophilic (LogP=7.66) (26, 27) and contains an ionizable amine group (apparent p K a =6.08)(28), it is poised to accumulates in adipose tissue, intracellular membranes, and acidic organelles such as lysosomes. In both human and animal models it has been shown that, following prolonged oral dosing, CFZ forms insoluble Crystal-Like Drug Inclusions (CLDIs) within macrophage lysosomes(15). …”

“…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.…”