To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.
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Method Article
Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection
In This Article
Summary
Abstract
A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.
Introduction
Globally, extrapulmonary tuberculosis (ETB) represents a significant proportion of tuberculosis (TB) cases. Nevertheless, ETB diagnosis is often missed or delayed because of its insidious clinical presentation and poor performance on diagnostic tests; false results include sputum smears negative for acid-fast bacilli, lack of granulomatous tissue on histopathology, or failure to culture Mycobacterium tuberculosis (Mtb). Relative to typical cases, ETB occurs less frequently and involves little liberation of the Mtb bacilli. In addition, it is usually localized at difficult-to-access sites, such as lymph nodes, pleura, and osteoarticular areas....
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Protocol
All protocol regarding animal experiment follows the standard operating procedures for laboratory animal breeding in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (8th Edition, 2011) and is approved by the institutional animal care and use committee.
1. SPIO nanoparticle synthesis
- Prepare dextran-coated iron oxide magnetic nanoparticles by vigorously stirring a mixture of dextran T-40 (5 mL; 50% w/w) and aqueous FeCl3×6H2O (0.45 g; 2.77 mmol) and FeCl2×4H2O (0.32 g; 2.52 mmol) solutions at room temperature.
- A....
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Results
SPIO-MtbsAb nanoprobe synthesis and characterization
SPIO nanoparticles were designed to conjugate with MtbsAb. The dextran stabilized on the surface of SPIO nanoparticles was crosslinked by epichlorohydrin. SPIO nanoparticles were subsequently incorporated with EDBE to activate primary amine functional groups at the dextran ends. SA was then conjugated to form SPIO-EDBE-SA. SPIO-MtbsAb nanoprobes formed in the final step through the conjugation of MtbsAb with SPIO-EDBE-SA in the presence of the coup.......
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Discussion
Similar to relevant studies, our findings regarding SPIO-MtbsAb nanoprobes demonstrated a significant specificity for Mtb27,28. The subcutaneous Mtb granuloma was found 1 month after TB injection in the mouse models. The typical TB granulomatous histology findings included lymphocyte infiltration, presence of epithelioid macrophages, and neovascularization. Acid-fast bacilli were scattered in the TB lesions, corroborating the MtbsAb immunohistochemistry findings........
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Disclosures
None of the authors has any proprietary interest in the materials examined in this study.
Acknowledgements
The authors are thankful for the financial support from the Ministry of Economy Taiwan (grants NSC-101-2120-M-038-001, MOST 104-2622-B-038 -007, MOST 105-2622-B-038-004) to perform this research work. This manuscript was edited by Wallace Academic Editing.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate | Sigma-Aldrich | ||
| 1-hydroxybenzotriazole | Sigma-Aldrich | ||
| dextran(T-40) | GE Healthcare Bio-sciences AB | ||
| epichlorohydrin, 2,2'-(ethylenedioxy)bis(ethylamine) | Sigma-Aldrich | ||
| ferric chloride hexahydrate | Fluka | ||
| ferrous chloride tetrahydrate | Fluka | ||
| Human monocytic THP-1 | |||
| M. bovis BCG | Pasteur Mérieux | Connaught strain; ImmuCyst Aventis | |
| MRI | GE medical Systems | 3.0-T, Signa | |
| NH4OH | Fluka | ||
| NMR relaxometer | Bruker | NMS-120 Minispec | |
| Sephacryl S-300 | GE Healthcare Bio-sciences AB | ||
| Sephadex G-25 | GE Healthcare Bio-sciences AB | ||
| SPECTRUM molecular porous membrane tubing, 12,000 -14,000 MW cut off | Spectrum Laboratories Inc | ||
| TB surface antibody- Polyclonal Antibody to Mtb | Acris Antibodies GmbH | BP2027 | |
| transmission electron microscope | JEOL | JEM-2000 EX II |
References
- Small, P. M., et al. Treatment of tuberculosis in patients with advanced human immunodeficiency virus infection. New England Journal of Medicine. 324, 289-294 (1991).
- Alvarez, S., McCabe, W. R. Extrapulmon....
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