Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

Summary

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

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^....

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

1. 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.
2. A....

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.......

Discussion

Similar to relevant studies, our findings regarding SPIO-MtbsAb nanoprobes demonstrated a significant specificity for Mtb^27,28. 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........

Materials

List of materials used in this article

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