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 areas¹. Thus, invasive procedures for obtaining adequate clinical specimens, which makes bacteriological confirmation risky and difficult, are essential^2,3,4.

Commercially available antibody detection tests for ETB are unreliable for clinical detection because of their wide range of sensitivity (0.00-1.00) and specificity (0.59-1.00) for all extrapulmonary sites combined⁵. Enzyme-linked immunospot (ELISPOT) assays for interferon-γ, culture filtrate protein (CFP), and early secretory antigenic target (ESAT) have been used for diagnosing latent and active TB. However, the results vary between different disease sites for diagnosing ETB^6,7,8. In addition, skin PPD (purified protein derivative) and QuantiFERON-TB frequently provided false negative results⁹. QuantiFERON-TB-2G is a whole blood immune reactivity assay, which does not require a specimen from the affected organ and this may be an alternative diagnostic tool^6,10,11. Other diagnostic methods typically used for TB meningitis, such as polymerase chain reaction, are still too insensitive to confidently exclude clinical diagnosis^12,13. These conventional tests demonstrate insufficient diagnostic information to discover the extrapulmonary infection site. Thus, novel diagnostic modalities are clinically required.

Molecular imaging aims at designing novel tools that can directly screen specific molecular targets of disease processes in vivo^14,15. Superparamagnetic iron oxide (SPIO), a T2-weighted NMR contrast agent, can significantly enhance the specificity and sensitivity of magnetic resonance (MR) imaging (MRI)^16,17. This new functional imaging modality can precisely sketch tissue changes at the molecular level through ligand-receptor interactions. In this study, a new molecular imaging probe, comprising SPIO nanoparticles, was synthesized to conjugate with Mtb surface antibody (MtbsAb) for ETB diagnosis. SPIO nanoprobes are minimally invasive to tissues and bodies under examination^18,19. Furthermore, these nanoprobes can demonstrate precise MR images at low concentrations due to their paramagnetic properties. In addition, SPIO nanoprobes appear elicit least allergic reactions because the presence of iron ions is part of normal physiology. Here, the sensitivity and specificity of the SPIO-MtbsAb nanoprobes targeting ETB were evaluated in both cell and animal models. The outcomes demonstrated that the nanoprobes were applicable as ultrasensitive imaging agents for ETB diagnosis.

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. Add NH₄OH (10 mL; 7.5% v/v) rapidly.
3. Further stir the black suspension for 1 h; subsequently, centrifuge at 17,300 x g for 10 min and then remove the aggregates.
4. Separate the final SPIO products from unbound dextran T-40 by gel filtration chromatography²⁰.
5. Load the reaction mixture (total volume = 5 mL) into a 2.5 cm × 33 cm column and elute with a buffer solution containing 0.1 M Na acetate and 0.15 M NaCl at pH 7.0.
6. Collect the purified dextran-coated iron oxide magnetic nanoparticles in the void volume and assay the column eluates for iron and dextran at 330 and 490 nm by using hydrochloric acid and the phenol/sulfuric acid methods²⁰, respectively.

2. SPIO-MtbsAb synthesis

1. Synthesize SPIO-conjugated EDBE using previously reported methods^21,22.
2. Synthesize SPIO-EDBE-succinic anhydride (SA).
   1. Stir an alkaline solution (5 M NaOH; 10 mL)) of SPIO-EDBE and SA (1 g; 10 µmol) at room temperature for 24 h.
   2. Dialyze the solution with 20 changes of 2 L of distilled water using molecular porous membrane tubing (12,000-14,000 MW cutoff). 6 h for each change.
3. Finally, add 100 μL of SPIO-EDBE-SA (4 mg/mL of Fe) to 400 μL of 4.5 mg/mL MtbsAb to synthesize SPIO-MtbsAb by using 1-hydroxybenzotriazole and (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate as catalysts and stir the solution at room temperature for 24 h.
4. Finally, separate the solutions from the unbound antibody through gel filtration chromatography.
5. Load the reaction mixture (5 mL) on 2.5 cm × 33 cm column and elute using a PBS buffer. Confirm Ab–nanoparticle complex (i.e., nanoprobe) using a bicinchoninic acid protein assay kit²³.

3. Particle morphology observation and relaxation tier measurement

1. Examine average particle size, morphology, and size distribution using transmission electron microscope at a voltage of 100 kV.
   1. Drop-cast the composite dispersion onto a 200-mesh copper grid and air dry at room temperature before loading it onto the microscope.
2. Measure the relaxation time values (T₁ and T₂) of the nanoprobes using the NMR relaxometer at 20 MHz and 37.0 °C ± 0.1 °C.
   1. Calibrate the relaxometer before each measurement.
   2. Record the r₁ and r₂ values from the eight data points generated through inversion-recovery and the Carr-Purcell-Meiboom-Gill pulse sequence, respectively, to determine r₁ and r₂ relaxivities²⁰.

4. Cell imaging

1. Cultivate human monocytes THP-1 in RPMI 1640 with 10% fetal bovine serum, 50 µg/mL gentamycin sulfate, 100 units/mL penicillin G sodium, 100 µg of streptomycin sulfate, and 0.25 µg/mL fungizone in a 5% CO₂ incubator at 37 °C.
2. Incubate SPIO-MtbsAb nanoprobes (2 mM) with 10⁶ colony forming units (CFU) of Mycobacterium bovis BCG preincubated with 1 × 10⁷ activated monocytes in microcentrifuge tubes (1 mL) in a 5% CO₂ incubator at 37 °C for 1 h.
3. Centrifuge tubes at 200 x g and discard the supernatant. Redissolve pellets in the medium (200 µL).
4. Scan the samples using a fast gradient echo pulse sequence (Repetition time (TR) = 500; Echo time(TE) = 20; Flip angle = 10°) through 3.0-T MRI to determine the nanoprobe's specificity and sensitivity^21,22.

5. BCG (Bacillus Calmette–Guérin) inoculation

1. Reconstitute the lyophilized vaccine or bacterial stock in Sauton's medium and then dilute the stock with saline until properly dispersed as previously described²⁴.
2. Inoculate a live attenuated strain of M. bovis BCG, obtained from ADIMMUNE (Taipei, Taiwan) (Connaught strain; ImmuCyst Aventis, Pasteur Mérieux) at a volume of 0.1 mL/mouse (i.e., 10⁷ CFU) intradermally into the left or right dorsal scapular skin of mice, as described previously²³. Inject saline into mice as negative control. Monitor animals daily after BCG inoculation.
3. Sacrifice animals 1 month after bacteria inoculation using carbon dioxide euthanasia. Harvest the tissue from the intradermal inoculation site. Fix the tissue in 10% formalin and embed in paraffin for serial sections at 5-10 µm. Stain tissue sections with the hematoxylin/eosin and Ziehl-Neelsen stains for acid-fast bacteria²⁴ and with Berlin blue for ferric iron²⁵.

6. In vivo MRI

1. Inject ketamine (80 mg/kg of body weight) and xylazine (12 mg/kg body weight) subcutaneously into mice for animal anesthesia.
2. Inject SPIO-TbsAb probes (2 nmol/200 µL) into tail veins of mice. MR image mice before and immediately after probe injection and then every 5 min for 30 min to acquire T2-weighted fast spin-echo images (TR = 3000; TE = 90; field of view = 8).
3. Quantitatively analyze all MR images using signal intensity (SI), a measurement of defined regions of interest in comparable locations of an Mtb granuloma center and the back muscle adjacent to a granulomatous area.
4. Calculate relative signal enhancements using the SI measurement before (SIpre; control) and 0-3 h after (SIpost) injection of the contrast agents using the formula
   
   [(SIpost - SIpre)/SIpre] × 100
   
   where SIpre is the SI of the lesion on the pre-enhanced scan and SIpost is the SI of the lesion on the post-enhanced scan^21,22.

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 co...

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

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