Abstract
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.
Highlights
Alterations in signal transduction pathways that affect the cell’s inflammatory response[10]
Because of its physicochemical properties, CFZ is highly prone to precipitation in acidic endolysosomal compartments in macrophages, where it self-assembles into a highly ordered CFZ-H+Cl− biocrystal, possessing many interesting optical and biomechanical features
We performed computational simulations and experiments to probe the relationship between CFZ’s pH-dependent solubility properties, the formation of salt and free base crystals, and the molecular, ion transport mechanisms responsible for this mechanopharmaceutical phenomenon, which could be exploited for developing other weakly basic small molecule building blocks as cell-targeted, self-assembling mechanopharmaceutical devices[41,42]
Summary
Alterations in signal transduction pathways that affect the cell’s inflammatory response[10]. Given that macrophages express high levels of proton pumping vacuolar ATPase (V-ATPase) and chloride channels on their lysosomal membranes[12,13,14], and that weak bases are prone to accumulate inside lysosomes[15], we probed whether the expression levels of these lysosomal membrane proteins in macrophages actively drive the accumulation of CFZ-H+Cl− and its self-assembly in these cells. Using a well-established and published lysosomal ion regulation model[16], we studied how proton pumping and chloride transport mechanisms influenced cell-type specific stabilization of CFZ-H+Cl− inside lysosomes. In addition to verifying model predictions and further refining our understanding of the computational simulation results, we performed experiments using pharmacological inhibitors of V-ATPase[17] and chloride channels[18] to probe the biological mechanism underpinning the massive accumulation and self-assembly of the building block
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