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Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

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.

SUBMITTER: Woldemichael T

PROVIDER: S-EPMC5811454 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

Woldemichael Tehetina T Keswani Rahul K RK Rzeczycki Phillip M PM Murashov Mikhail D MD LaLone Vernon V Gregorka Brian B Swanson Joel A JA Stringer Kathleen A KA Rosania Gus R GR

Scientific reports 20180213 1

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 - ...[more]

PMID: 29440773

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Project description:Clofazimine (CFZ) is a poorly soluble, weakly basic, small molecule antibiotic clinically used to treat leprosy and is now in clinical trials as a treatment for multidrug resistant tuberculosis and COVID-19. CFZ exhibits complex, context-dependent pharmacokinetics that are characterized by an increasing half-life in long term treatment regimens. The systemic pharmacokinetics of CFZ have been previously represented by a nonlinear, 2-compartment model incorporating an expanding volume of distribution. This expansion reflects the soluble-to-insoluble phase transition that the drug undergoes as it precipitates out and accumulates within macrophages disseminated throughout the organism. Using mice as a model organism, we studied the mechanistic underpinnings of this increasing half-life and how the systemic pharmacokinetics of CFZ are altered with continued dosing. To this end, M. tuberculosis infection status and multiple dosing schemes were studied alongside a parameter sensitivity analysis (PSA) to further understanding of systemic drug distribution. Parameter values governing the sigmoidal expansion function that captures the phase transition were methodically varied, and in turn, the systemic concentrations of the drug were calculated and compared to the experimentally measured concentrations of drug in serum and spleen. The resulting amounts of drug sequestered were dependent on the total mass of CFZ administered and the duration of drug loading. This phenomenon can be captured by altering three different parameters of an expansion function corresponding to key biological determinants responsible for the precipitation and the accumulation of the insoluble drug mass in macrophages. Through this analysis of the context dependent pharmacokinetics of CFZ, a predictive framework for projecting the systemic distribution and self-assembly of precipitated drug complexes as intracellular mechanopharmaceutical devices of this and other drugs exhibiting similarly complex pharmacokinetics can be constructed.

Project description:One of the greatest obstacles to clinical translation of bone tissue engineering is the inability to effectively and efficiently vascularize scaffolds. The goal of this work was to explore systematically whether architecture, at a scale of hundreds of microns, can be used to direct the growth of microcapillary-like structures into the core of scaffolds. Biphasic bioceramic patterned architectures were produced using silicone molds of 3D printed parts. Grooves and ridges were designed to have widths of 330 μm and 660 μm, with periodicities respectively of 1240 μm and 630 μm. Groove depth was varied between 150 μm and 585 μm. Co-cultures of human dermal microvascular endothelial cells (HDMECs) and human osteoblasts (hOBs) were used to grow microcapillary-like structures on substrates. Bioceramic architecture was found to significantly affect microcapillary-like structure location and orientation. Microcapillary-like structures were found to form predominantly in grooves or between convexities. For all patterned samples, the CD31 (endothelial cell marker) signal was at least 2.5 times higher along grooves versus perpendicular to grooves. In addition, the average signal was at least two times higher within grooves than outside grooves for all samples. Grooves with a width of 330 μm and a depth of 300 μm resulted in the formation of individual, highly aligned microcapillary-like structures with lengths around 5 mm. Extensive literature has focused on the role of nano- and micro-topography (on the scale below tens of microns) on cellular response. However, the idea that architecture at a scale much larger than a cell could be used to modulate angiogenesis has not been systematically investigated. This work shows the crucial influence of architecture on microcapillary-like structure self-assembly at the scale of hundreds of microns. Elucidating the precise correspondence between architecture and microcapillary-like structure organization will ultimately allow the engineering of microvasculature by tuning local scaffold design to achieve desirable microvessel properties.

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Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

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Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device.

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