Massive Bioaccumulation and Self‐Assembly of Phenazine Compounds in Live Cells

Abstract

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.

Development of background-free tame fluorescent probes for intracellular live cell imaging

“…However, after the probes were incubated in cells for 30 min, the formation of intracellular insoluble or precipitate-like form which is hardly washed out of cells was observed from some of the probes in H-group. It suggests that intracellular precipitation or aggregation due to the low water solubility may be one of the mechanisms behind the slow cellular efflux and high retention of probes in H-group 32 33 34 .…”

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

“…Previously, our lab has used polarization microscopy to study the optical properties of drug inclusions formed in vitro. Cells were incubated with CFZ and other phenazine derivatives to study the self-assembly of drug aggregates in live cells, revealing that, even after 72 hours, the self-assembly of CFZ aggregates within cells yields a measurable diattenuation signal [33]. Cultured cells, however, are not the optimal model for studying intracellular drug crystallization for a variety of factors, namely the length of time necessary for the crystals to form.…”

Elasticity in Macrophage‐Synthesized Biocrystals

“…Zuschriften photoelastic phenomena that is commonly observed with application of stress and development of strain. [31] In this particular study,l inear dichroism could be measured using hardware configured on an epifluorescence-brightfield microscope [32,33] (see the SI for protocol). Crystals were imaged under polarized light following which the anisotropy in transmittance and importantly,the azimuth (the polarization orientation that results in maximum transmittance) were computed ( Figure 5).…”