Name: Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection (Video) | JoVE Journal
Uploaded: 2020-02-16T12:00:00.000+00:00
Duration: 9 min 54 s
Description: Learn to synthesize superparamagnetic iron oxide nanoprobes for detecting Mycobacterium tuberculosis using MRI techniques. This method enables precise imaging of extrapulmonary tuberculosis at low concentrations without inducing autoimmune responses.

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5 minutes

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.

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
<ol>
	<li>Prepare dextran-coated iron oxide magnetic nanoparticles by vigorously stirring a mixture of dextran T-40 (5 mL; 50% w/w) and aqueous FeCl3&#215;6H2O (0.45 g; 2.77 mmol) and FeCl2&#215;4H2O (0.32 g; 2.52 mmol) solutions at room temperature.</li>
	<li>Add NH4OH (10 mL; 7.5% v/v) rapidly.</li>
	<li>Further stir the black suspension for 1 h; subsequently, centrifuge at 17,300 x g for 10 min and then remove the aggregates.</li>
	<li>Separate the final SPIO products from unbound dextran T-40 by gel filtration chromatography20.</li>
	<li>Load the reaction mixture (total volume = 5 mL) into a 2.5 cm &#215; 33 cm column and elute with a buffer solution containing 0.1 M Na acetate and 0.15 M NaCl at pH 7.0.</li>
	<li>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.</li>
</ol>
2. SPIO-MtbsAb synthesis
<ol>
	<li>Synthesize SPIO-conjugated EDBE using previously reported methods21,22.</li>
	<li>Synthesize SPIO-EDBE-succinic anhydride (SA).
	<ol>
		<li>Stir an alkaline solution (5 M NaOH; 10 mL)) of SPIO-EDBE and SA (1 g; 10 &#181;mol) at room temperature for 24 h.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Finally, add 100 &#956;L of SPIO-EDBE-SA (4 mg/mL of Fe) to 400 &#956;L 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.</li>
	<li>Finally, separate the solutions from the unbound antibody through gel filtration chromatography.</li>
	<li>Load the reaction mixture (5 mL) on 2.5 cm &#215; 33 cm column and elute using a PBS buffer. Confirm Ab&#8211;nanoparticle complex (i.e., nanoprobe) using a bicinchoninic acid protein assay kit23.</li>
</ol>
3. Particle morphology observation and relaxation tier measurement
<ol>
	<li>Examine average particle size, morphology, and size distribution using transmission electron microscope at a voltage of 100 kV.
	<ol>
		<li>Drop-cast the composite dispersion onto a 200-mesh copper grid and air dry at room temperature before loading it onto the microscope.</li>
	</ol>
	</li>
	<li>Measure the relaxation time values (T1 and T2) of the nanoprobes using the NMR relaxometer at 20 MHz and 37.0 &#176;C &#177; 0.1 &#176;C.
	<ol>
		<li>Calibrate the relaxometer before each measurement.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Cell imaging
<ol>
	<li>Cultivate human monocytes THP-1 in RPMI 1640 with 10% fetal bovine serum, 50 &#181;g/mL gentamycin sulfate, 100 units/mL penicillin G sodium, 100 &#181;g of streptomycin sulfate, and 0.25 &#181;g/mL fungizone in a 5% CO2 incubator at 37 &#176;C.</li>
	<li>Incubate SPIO-MtbsAb nanoprobes (2 mM) with 106 colony forming units (CFU) of Mycobacterium bovis BCG preincubated with 1 &#215; 107 activated monocytes in microcentrifuge tubes (1 mL) in a 5% CO2 incubator at 37 &#176;C for 1 h.</li>
	<li>Centrifuge tubes at 200 x g and discard the supernatant. Redissolve pellets in the medium (200 &#181;L).</li>
	<li>Scan the samples using a fast gradient echo pulse sequence (Repetition time (TR) = 500; Echo time(TE) = 20; Flip angle = 10&#176;) through 3.0-T MRI to determine the nanoprobe&#39;s specificity and sensitivity21,22.</li>
</ol>
5. BCG (Bacillus Calmette&#8211;Gu&#233;rin) inoculation
<ol>
	<li>Reconstitute the lyophilized vaccine or bacterial stock in Sauton&#39;s medium and then dilute the stock with saline until properly dispersed as previously described24.</li>
	<li>Inoculate a live attenuated strain of M. bovis BCG, obtained from ADIMMUNE (Taipei, Taiwan) (Connaught strain; ImmuCyst Aventis, Pasteur M&#233;rieux) 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.</li>
	<li>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 &#181;m. Stain tissue sections with the hematoxylin/eosin and Ziehl-Neelsen stains for acid-fast bacteria24 and with Berlin blue for ferric iron25.</li>
</ol>
6. In vivo MRI
<ol>
	<li>Inject ketamine (80 mg/kg of body weight) and xylazine (12 mg/kg body weight) subcutaneously into mice for animal anesthesia.</li>
	<li>Inject SPIO-TbsAb probes (2 nmol/200 &#181;L) 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).</li>
	<li>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.</li>
	<li>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] &#215; 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 scan21,22.</li>
</ol>

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<li>Lee, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-paramagnetic+iron+oxide+nanoparticles+for+use+in+extrapulmonary+tuberculosis+diagnosis%5BTitle%5D)+AND+%22Clinical+Microbiology+and+Infection%22%5BJournal%5D)">Super-paramagnetic iron oxide nanoparticles for use in extrapulmonary tuberculosis diagnosis.</a> Clinical Microbiology and Infection. 18, 149-157 (2012).</li>
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</ol>

None of the authors has any proprietary interest in the materials examined in this study.

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....

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 essential2,3,4.
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-&#947;, 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 ETB6,7,8. 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 tool6,10,11. Other diagnostic methods typically used for TB meningitis, such as polymerase chain reaction, are still too insensitive to confidently exclude clinical diagnosis12,13. 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 vivo14,15. 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 examination18,19. 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.

<table><tbody><tr><td>(benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>1-hydroxybenzotriazole</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>dextran(T-40)</td><td>GE Healthcare Bio-sciences AB</td><td></td><td></td></tr><tr><td>epichlorohydrin, 2,2&#039;-(ethylenedioxy)bis(ethylamine)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>ferric chloride hexahydrate</td><td>Fluka</td><td></td><td></td></tr><tr><td>ferrous chloride tetrahydrate</td><td>Fluka</td><td></td><td></td></tr><tr><td>Human monocytic THP-1</td><td></td><td></td><td></td></tr><tr><td>M. bovis BCG</td><td>Pasteur M&amp;eacute;rieux</td><td>Connaught strain; ImmuCyst Aventis</td><td></td></tr><tr><td>MRI</td><td>GE medical Systems</td><td>3.0-T, Signa</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>Fluka</td><td></td><td></td></tr><tr><td>NMR relaxometer</td><td>Bruker</td><td>NMS-120 Minispec</td><td></td></tr><tr><td>Sephacryl S-300</td><td>GE Healthcare Bio-sciences AB</td><td></td><td></td></tr><tr><td>Sephadex G-25</td><td>GE Healthcare Bio-sciences AB</td><td></td><td></td></tr><tr><td>SPECTRUM molecular porous membrane tubing, 12,000 -14,000 MW cut off</td><td>Spectrum Laboratories Inc</td><td></td><td></td></tr><tr><td>TB surface antibody- Polyclonal Antibody to Mtb</td><td>Acris Antibodies GmbH</td><td>BP2027</td><td></td></tr><tr><td>transmission electron microscope</td><td>JEOL</td><td>JEM-2000 EX II</td><td></td></tr></tbody></table>

synthesis, characterization, and application of superparamagnetic iron oxide nanoprobes for extrapulmonary tuberculosis detection

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

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.

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...

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

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

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Research

JoVE Journal

Medicine

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This video demonstrates a technique for the detection of extrapulmonary tuberculosis. Superparamagnetic iron oxide (SPIO) nanoprobes, conjugated to Mycobacterium-specific antibodies, are injected into a mouse previously infected with Mycobacterium bovis BCG, labeling the granuloma and providing a better imaging contrast for its detection.

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Learn to synthesize superparamagnetic iron oxide nanoprobes for detecting Mycobacterium tuberculosis using MRI techniques. This method enables precise imaging of extrapulmonary tuberculosis at low concentrations without inducing autoimmune responses.

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection (Video) | JoVE Journal

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

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