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Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes.

Edagwa BJ, Guo D, Puligujja P, Chen H, McMillan J, Liu X, Gendelman HE, Narayanasamy P - FASEB J. (2014)

Bottom Line: 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.Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, and.

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Related in: MedlinePlus

A) Schematic diagram of immunoisolation of NPs and M. smegmatis-containing endosomes. MDMs were treated with NPs or M. smegmatis side by side. MDMs were then washed in PBS and ruptured in homogenization buffer. Nuclei and unbroken cells were removed by centrifugation. Protein A/G paramagnetic beads conjugated to antibodies were incubated with the supernatants, and the beads containing endosomal compartments were washed and collected on a magnetic separator. Drug content of each compartment was determined by HPLC after sonication. For mycobacterium quantification, the compartments were diluted with sterile PBS (containing 10% human serum), treated with 0.25% SDS, and loaded onto agar plates for CFU counting. B–D) Following immunoisolation, RIF (B), INHP (C), and M. smegmatis (D) were quantitated in subcellular endosomal compartments by HPLC or CFU counting. E) Schematic diagram of intracellular pathways of NPs and M. smegmatis. RIF and INHP NPs (shown in green) and M. smegmatis (shown in red) are phagocytosed by MDMs and transported to early endosomes. Nanoparticles and mycobacteria are then either sorted into late endosomes (Rab 7) for release as secretory lysosome or into fast recycling (Rab14) or slow recycling (Rab 11) endosomes for eventual extracellular release.
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Figure 6: A) Schematic diagram of immunoisolation of NPs and M. smegmatis-containing endosomes. MDMs were treated with NPs or M. smegmatis side by side. MDMs were then washed in PBS and ruptured in homogenization buffer. Nuclei and unbroken cells were removed by centrifugation. Protein A/G paramagnetic beads conjugated to antibodies were incubated with the supernatants, and the beads containing endosomal compartments were washed and collected on a magnetic separator. Drug content of each compartment was determined by HPLC after sonication. For mycobacterium quantification, the compartments were diluted with sterile PBS (containing 10% human serum), treated with 0.25% SDS, and loaded onto agar plates for CFU counting. B–D) Following immunoisolation, RIF (B), INHP (C), and M. smegmatis (D) were quantitated in subcellular endosomal compartments by HPLC or CFU counting. E) Schematic diagram of intracellular pathways of NPs and M. smegmatis. RIF and INHP NPs (shown in green) and M. smegmatis (shown in red) are phagocytosed by MDMs and transported to early endosomes. Nanoparticles and mycobacteria are then either sorted into late endosomes (Rab 7) for release as secretory lysosome or into fast recycling (Rab14) or slow recycling (Rab 11) endosomes for eventual extracellular release.

Mentions: The trafficking of the nanoformulations through endosomal compartments was compared with subcellular sites of mycobacterial replication. Endosomal compartments from drug-loaded and infected MDMs were immunoisolated using magnetic beads coated with antibodies to Rab 5, 7, 11, and 14 compartments (46). Drug and mycobacterium levels were determined in each compartment. As shown in Fig. 6, the RIF and INHP NPs were distributed mainly to late endosomal (Rab 7) and recycling endosomal (Rab 11 and Rab 14) compartments (Fig. 6B, C, respectively). Similarly, mycobacteria were found in all the endosomal compartments (Fig. 6D), with the majority in Rab 7 (late) and Rab 14 (fast recycling) endosomes. These data demonstrate that the drug NPs traffic to the same subcellular compartments where the mycobacterium replicates (Fig. 6B–D).


Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes.

Edagwa BJ, Guo D, Puligujja P, Chen H, McMillan J, Liu X, Gendelman HE, Narayanasamy P - FASEB J. (2014)

A) Schematic diagram of immunoisolation of NPs and M. smegmatis-containing endosomes. MDMs were treated with NPs or M. smegmatis side by side. MDMs were then washed in PBS and ruptured in homogenization buffer. Nuclei and unbroken cells were removed by centrifugation. Protein A/G paramagnetic beads conjugated to antibodies were incubated with the supernatants, and the beads containing endosomal compartments were washed and collected on a magnetic separator. Drug content of each compartment was determined by HPLC after sonication. For mycobacterium quantification, the compartments were diluted with sterile PBS (containing 10% human serum), treated with 0.25% SDS, and loaded onto agar plates for CFU counting. B–D) Following immunoisolation, RIF (B), INHP (C), and M. smegmatis (D) were quantitated in subcellular endosomal compartments by HPLC or CFU counting. E) Schematic diagram of intracellular pathways of NPs and M. smegmatis. RIF and INHP NPs (shown in green) and M. smegmatis (shown in red) are phagocytosed by MDMs and transported to early endosomes. Nanoparticles and mycobacteria are then either sorted into late endosomes (Rab 7) for release as secretory lysosome or into fast recycling (Rab14) or slow recycling (Rab 11) endosomes for eventual extracellular release.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 6: A) Schematic diagram of immunoisolation of NPs and M. smegmatis-containing endosomes. MDMs were treated with NPs or M. smegmatis side by side. MDMs were then washed in PBS and ruptured in homogenization buffer. Nuclei and unbroken cells were removed by centrifugation. Protein A/G paramagnetic beads conjugated to antibodies were incubated with the supernatants, and the beads containing endosomal compartments were washed and collected on a magnetic separator. Drug content of each compartment was determined by HPLC after sonication. For mycobacterium quantification, the compartments were diluted with sterile PBS (containing 10% human serum), treated with 0.25% SDS, and loaded onto agar plates for CFU counting. B–D) Following immunoisolation, RIF (B), INHP (C), and M. smegmatis (D) were quantitated in subcellular endosomal compartments by HPLC or CFU counting. E) Schematic diagram of intracellular pathways of NPs and M. smegmatis. RIF and INHP NPs (shown in green) and M. smegmatis (shown in red) are phagocytosed by MDMs and transported to early endosomes. Nanoparticles and mycobacteria are then either sorted into late endosomes (Rab 7) for release as secretory lysosome or into fast recycling (Rab14) or slow recycling (Rab 11) endosomes for eventual extracellular release.
Mentions: The trafficking of the nanoformulations through endosomal compartments was compared with subcellular sites of mycobacterial replication. Endosomal compartments from drug-loaded and infected MDMs were immunoisolated using magnetic beads coated with antibodies to Rab 5, 7, 11, and 14 compartments (46). Drug and mycobacterium levels were determined in each compartment. As shown in Fig. 6, the RIF and INHP NPs were distributed mainly to late endosomal (Rab 7) and recycling endosomal (Rab 11 and Rab 14) compartments (Fig. 6B, C, respectively). Similarly, mycobacteria were found in all the endosomal compartments (Fig. 6D), with the majority in Rab 7 (late) and Rab 14 (fast recycling) endosomes. These data demonstrate that the drug NPs traffic to the same subcellular compartments where the mycobacterium replicates (Fig. 6B–D).

Bottom Line: 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.Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, and.

Show MeSH
Related in: MedlinePlus