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</...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+folic+acid-PEG+nanoparticles+for+noninvasive+imaging+of+folate+receptor+by+MRI.">Targeted folic acid-PEG nanoparticles for noninvasive imaging of folate receptor by MRI.</a> Journal of Biomedical Materials Research Part A. 87, 165-175 (2008).</li><li>Chen, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Herceptin-dextran+iron+oxide+nanoparticles+for+noninvasive+imaging+of+HER2/neu+receptors+using+MRI.">Targeted Herceptin-dextran iron oxide nanoparticles for noninvasive imaging of HER2/neu receptors using MRI.</a> Journal of Biological Inorganic Chemistry. 14, 253-260 (2009).</li><li>Weissleder, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+superparamagnetic+iron+oxide:+an+intravenous+contrast+agent+for+assessing+lymph+nodes+with+MR+imaging.">Ultrasmall superparamagnetic iron oxide: an intravenous contrast agent for assessing lymph nodes with MR imaging.</a> Radiology. 175, 494-498 (1990).</li><li>Wang, J., Wakeham, J., Harkness, R., Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+are+a+significant+source+of+type+1+cytokines+during+mycobacterial+infection.">Macrophages are a significant source of type 1 cytokines during mycobacterial infection.</a> Journal of Clinical Investigation. 103, 1023-1029 (1999).</li><li>Angra, P., Ridderhof, J., Smithwick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+different+strengths+of+carbol+fuchsin+in+Ziehl-Neelsen+staining+for+detecting+acid-fast+bacilli.">Comparison of two different strengths of carbol fuchsin in Ziehl-Neelsen staining for detecting acid-fast bacilli.</a> Journal of Clinical Microbiology. 41, 3459 (2003).</li><li>Woods, A. E., Ellis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Histopathology- A Complete Reference. 1st edn. , 6-11 (1994).</li><li>Lee, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-paramagnetic+iron+oxide+nanoparticles+for+use+in+extrapulmonary+tuberculosis+diagnosis.">Super-paramagnetic iron oxide nanoparticles for use in extrapulmonary tuberculosis diagnosis.</a> Clinical Microbiology and Infection. 18, 149-157 (2012).</li><li>Lee, H., Yoon, T. J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+detection+of+bacteria+using+core-shell+nanoparticles+and+an+NMR-filter+system.">Ultrasensitive detection of bacteria using core-shell nanoparticles and an NMR-filter system.</a> Angewandte Chemie International Edition. 48, 5657-5660 (2009).</li><li>Fan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Popcorn-shaped+magnetic+core-plasmonic+shell+multifunctional+nanoparticles+for+the+targeted+magnetic+separation+and+enrichment,+label-free+SERS+imaging,+and+photothermal+destruction+of+multidrug-resistant+bacteria.">Popcorn-shaped magnetic core-plasmonic shell multifunctional nanoparticles for the targeted magnetic separation and enrichment, label-free SERS imaging, and photothermal destruction of multidrug-resistant bacteria.</a> Chemistry. 19, 2839-2847 (2013).</li><li>Nishie, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+imaging+of+human+monocytic+cellular+activity+using+superparamagnetic+iron+oxide.">In vitro imaging of human monocytic cellular activity using superparamagnetic iron oxide.</a> Computerized Medical Imaging and Graphics. 31, 638-642 (2007).</li><li>von Zur Muhlen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superparamagnetic+iron+oxide+binding+and+uptake+as+imaged+by+magnetic+resonance+is+mediated+by+the+integrin+receptor+Mac-1+(CD11b/CD18):+implications+on+imaging+of+atherosclerotic+plaques.">Superparamagnetic iron oxide binding and uptake as imaged by magnetic resonance is mediated by the integrin receptor Mac-1 (CD11b/CD18): implications on imaging of atherosclerotic plaques.</a> Atherosclerosis. 193, 102-111 (2007).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;-(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&#233;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>NH4OH</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

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 co...

Watch this Scientific Journal Video about Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection at JoVE.com

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

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 ...

Globally, extrapulmonary tuberculosis diagnosis is often delayed, as it requires a surgical intervention for confirmation due to non-specific clinical presentation and a poor performance on diagnostic tests. To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we have developed super-paramagnetic iron oxide nanoprobes for detecting extrapulmonary tuberculosis. TB-SPIO nanoprobes can provide precise magnetic resonance images at low concentrations due to their paramagnetic properties without inducing any autoimmune response.TB-SPIO nanoprobes enable the targeting and detection of Mycobacterium tuberculosis infection. They can also be applied as MRI contrast agent for the detection of other diseases. Be sure to study the protocol carefully before attempting the protocol, as each part of the experiment utilizes a specific core technology.Visual demonstrate of the method can help pupils to understand how to implement the process, especially for some of the more complicated experiments. To prepare dextran-coated iron oxide magnetic nanoparticles, vigorously combine five milliliters of dextran T40 with 45 milligrams aqueous ferric chloride hexahydrate and 32 milligrams of of ferrous chloride tetrahydrate solutions at room temperature. Rapidly add 10 milliliters of ammonium hydroxide and use a stir bar to stir the resulting black suspension for one hour at room temperature.At the end of the stirring incubation, centrifuge the solution and remove the aggregates. To separate the final SPIO products from the the unbound dextran T40, load the entire five-milliliter reaction mixture into a 2.5 by 33-centimeter gel filtration chromatography column and elute the dextran with 0.1-molar of sodium acetate and 0.15-molar of sodium chloride buffer solution. Then, 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 nanometers according to standard protocols.To create SPIO-conjugated Mycobacterium tuberculosis surface antibodies, first synthesize SPIO-EDBE-succinic anhydride by mixing 10 milliliters of an alkaline solution of SPIO-EBDE with one gram of succinic anhydride for 24 hours at room temperature. The next day, use 12, 000 to 4, 000 molecular weight cutoff molecular porous membrane tubing to dialyze the solution with 26-hour changes of two liters of distilled water per change. After the last change, add 100 microliters of the resulting solution to 400 microliters of 4.5 milligrams per milliliters TB surface antibody, followed by the addition of 1-hydroxybenzotriazole and benzotriazol-1-yloxy tripyrrolidinophosphonium hexafluorophosphate as catalysts at room temperature for 24 hours with stirring.Separate the solutions from the unbound antibody through gel filtration chromatography using PBS as the buffer. To determine the average particle size, morphology, and size distribution, drop-cast the composite dispersion onto a 200-mesh copper grid and air-dry the sample at room temperature before loading onto a transmission electron microscope for imaging at 100 kilovolts. To measure the relaxation time values of the nanoprobes, use a nuclear magnetic resonance relaxometer at 20 megahertz and 37 degrees Celsius plus or minus one degree.For nanoprobe pulsed cell imaging, first cultivate human THP-1 monocytes according to standard cell culture protocols. When the cells have reached the appropriate stage of activation, preincubate one times 10 to the seven monocytes with one times 10 to the sixth colony-forming units of Mycobacterium bovis BCG for one hour. At the end of the incubation, transfer the cells to one-milliliter microcentrifuge tubes and add two millimolar of SPIO Mycobacterium tuberculosis surface antibody-conjugated nanoprobes to each tube for a one-hour incubation at 37 degrees Celsius and 5%carbon dioxide.At the end of the incubation, collect the cells by centrifugation and resuspend the pellets in 200 microliters of fresh medium. Then, scan the samples using a fast gradient echo pulse sequence through 3.0-T magnetic resonance imaging to determine the specificity and sensitivity of the nanoprobes. To inoculate experimental animals with M.bovis BCG, first reconstitute the lyophilized vaccine or bacterial stock in Sauton's medium and dilute the reconstituted stock solution with saline.Next, load 100 microliters of the solution into one one-milliliter syringe per animal and inject the entire volume of bacteria intradermally into the left or right dorsal scapular skin of each mouse. For in vivo magnetic resonance imaging of live nanoprobe-injected animals, acquire baseline T2-weighted fast spin-echo images of each anesthetized experimental animal before injecting two nanomolar of SPIO-TB antibody probes suspended into 200 microliters of saline into the tail vein of each mouse. Then, image the animals again immediately after the injection and every five minutes for the next 30 minutes.At the end of the imaging session, quantitatively analyze all of the magnetic resonance images using the signal intensity as a measurement of the defined regions of interest in comparable locations of an M.tuberculosis granuloma center and the back muscle adjacent to a granulomatis area. Then, use the formula to calculate the relative signal enhancements using the signal intensity measurement before and zero to three hours after the injection of the contrast agents. One month after inoculation, collect the tissue from the intradermal inoculation site of each animal and stain the formalin-fixed paraffin-embedded five to 10-micrometer thick tissue samples with hematoxylin and eosin, Ziehl-Neelsen stains for acid-fast bacteria, and Berlin blue for ferric iron.Transmission electron microscopy imaging of SPIO-TB surface antibody nanoprobes reveals that the nanoprobes exhibit a well-dispersed appearance with an average size of 3.8 plus or minus 0.4 nanometers per core. M.bovis BCG and acid-fast bacterial strain can be detected through Ziehl-Neelsen staining. After isolation and culture with probes containing ferric iron, the bacteria can be identified through Berlin blue staining.The TB-targeting degree of SPIO-TB surface antibody nanoprobes can be determined through T2-weighted magnetic resonance imaging as indicated by a decrease in the signal intensity in the presence of the nanoprobes that occurs in a concentration dependent manner. In nanoprobe-injected animals, the T2-weighted enhancement of signal reduction in the M.tuberculosis granulomatis areas is approximately 14-fold higher than that of the control sites. One month after infection, an organized subcutaneous granuloma can be observed in nanoprobe-injected C57 black 6 mice with new blood vascularization and lymphocyte and epithelioid macrophage aggregates noted within these lesions.Positive M.tuberculosis expression is also observed in the granulomatous areas with acid-fast bacilli staining positive at the lesion site. Berlin blue, a ferric iron positive stain, can also be used to determine the sensitivity of the probes to TB.For in vivo magnetic resonance image of life, nanoprobe-injected animals acquire baseline T2-weighted fast spin-echo image of each anesthetized experimental animal before injecting the SPIO-TB antibody probes. Now that we have established the SOP technique to detect extrapulmonary tuberculosis, these antibodies can be used to specifically diagnose extrapulmonary tuberculosis.

synthesis-characterization-application-superparamagnetic-iron-oxide

Research

JoVE Journal

Medicine

5.6K Views.  Taipei Medical University-Shuang Ho Hospital. Globally, extrapulmonary tuberculosis diagnosis is often delayed, as it requires a surgical intervention for confirmation due to non-specific clinical presentation and a poor performance on diagnostic tests. To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we have developed super-paramagnetic iron oxide nanoprobes for detecting extrapulmonary tuberculosis. TB-SPIO nanoprobes can provide precise magnetic resonance images at low concentrations due to their parama...