Use of microsphere technology for targeted delivery of rifampin to Mycobacterium tuberculosis-infected macrophages

Abstract

Microsphere technology was used to develop formulations of rifampin for targeted delivery to host macrophages. These formulations were prepared by using biocompatible polymeric excipients of lactide and glycolide copolymers. Release characteristics were examined in vitro and also in two monocytic cell lines, the murine J774 and the human Mono Mac 6 cell lines. Bioassay assessment of cell culture supernatants from monocyte cell lines showed release of bioactive rifampin during a 7-day experimental period. Treatment of Mycobacterium tuberculosis H37Rv-infected monocyte cell lines with rifampin-loaded microspheres resulted in a significant decrease in numbers of CFU at 7 days following initial infection, even though only 8% of the microsphere-loaded rifampin was released. The levels of rifampin released from microsphere formulations within monocytes were more effective at reducing M. tuberculosis intracellular growth than equivalent doses of rifampin given as a free drug. These results demonstrate that rifampin-loaded microspheres can be formulated for effective sustained and targeted delivery to host macrophages.

FIG. 1

In vitro release characteristics of rifampin-loaded microsphere formulations over a 6-day period in receiving fluid. Microsphere formulations 4, 5, and 6 are shown. The method used is described in Materials and Methods.

FIG. 2

Size distributions of rifampin-loaded microsphere formulations 5 (top) and 6 (bottom) as determined with a Malvern particle size analyzer. Data are plotted as volume percentile versus particle diameter.

FIG. 3

Scanning electron micrographs of rifampin-loaded microsphere formulation 5 taken at scope magnifications of ×500 (A) and ×2,000 (B). Bars, 10 μm.

FIG. 4

Effectiveness of rifampin-loaded microspheres (formulation 5) at reducing intracellular growth of M. tuberculosis H37Rv in murine J774 (A) and human MM6 (B) monocytic cell lines. Cell lines were infected with M. tuberculosis H37Rv as described in Materials and Methods and treated with rifampin-loaded microspheres containing 1.4 wt% rifampin (formulation 5) for 7 days. Samples were plated at 0 and 7 days to determine numbers of CFU. Rifampin concentrations for free drug were 0 (×), MIC (■), 3× MIC (▴), and 8× MIC (•). Rifampin concentrations of drug-loaded microspheres were MIC (□), 3× MIC (▵), and 8× MIC (○).

FIG. 5

Comparison of levels of antimycobacterial effectiveness of free rifampin versus equivalent doses delivered with rifampin-loaded microspheres. Three experiments were performed, i.e., experiments 1 (A), 2 (B), and 3 (C). J774 macrophages were infected with M. tuberculosis H37Rv and treated with no rifampin (○), 0.09 ± 0.002 μg of rifampin/well (n = 9) (■) as free drug, or 0.097 ± 0.008 μg of rifampin/well (n = 9) delivered in microsphere formulation no. 5 (□). Numbers of CFU were determined in triplicate at 0 and 7 days, as described in Materials and Methods. In each experiment, the difference between the mean value for the nontreated control and that for drug-treated sets was significant at P ≤ 0.001. Also, in each experiment the difference between the mean values for the cells treated with free rifampin versus those treated with rifampin-loaded microspheres was significant at P ≤ 0.001. Significance was determined by one-way analysis of variance, and a correction for multiple comparisons (posttest) was performed by the Tukey-Kramer multiple-comparisons test.