Immunoisolation of nanoparticles containing endocytic vesicles for drug quantitation.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2013; 991:41-6. DOI: 10.1007/978-1-62703-336-7_5
Source: PubMed

ABSTRACT Cell-mediated nanoparticle delivery has recently emerged as an efficacious method of delivering therapeutic agents across physiological barriers. Use of cells as nanodelivery vehicles requires accurate assessment of their loading capacity and identification of intracellular compartments where nanoparticles are sequestered. This is of great interest since specific endocytic trafficking routes can ultimately influence the mode of nanoparticle release and their efficacy and function. Here, we describe a technique that allows for the isolation of individual populations of nanoparticle-containing endosomes for subsequent quantitative analysis and more accurate description of where nanoparticles are stored on a subcellular level.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Eradication of Mycobacterium tuberculosis (MTB) infection requires daily administration of combinations of rifampin (RIF), isoniazid [isonicotinylhydrazine (INH)], pyrazinamide, and ethambutol, among other drug therapies. To facilitate and optimize MTB therapeutic selections, a mononuclear phagocyte (MP; monocyte, macrophage, and dendritic cell)-targeted drug delivery strategy was developed. Long-acting nanoformulations of RIF and an INH derivative, pentenyl-INH (INHP), were prepared, and their physicochemical properties were evaluated. This included the evaluation of MP particle uptake and retention, cell viability, and antimicrobial efficacy. Drug levels reached 6 μg/10(6) cells in human monocyte-derived macrophages (MDMs) for nanoparticle treatments compared with 0.1 μg/10(6) cells for native drugs. High RIF and INHP levels were retained in MDM for >15 d following nanoparticle loading. Rapid loss of native drugs was observed in cells and culture fluids within 24 h. Antimicrobial activities were determined against Mycobacterium smegmatis (M. smegmatis). Coadministration of nanoformulated RIF and INHP provided a 6-fold increase in therapeutic efficacy compared with equivalent concentrations of native drugs. Notably, nanoformulated RIF and INHP were found to be localized in recycling and late MDM endosomal compartments. These were the same compartments that contained the pathogen. Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.-Edagwa, B. J., Guo, D., Puligujja, P., Chen, H., McMillan, J., Liu, X., Gendelman, H. E., Narayanasamy, P. Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes.
    The FASEB Journal 08/2014; 28(12). DOI:10.1096/fj.14-255786 · 5.48 Impact Factor