A Technique to Detect Extrapulmonary Tuberculosis via Superparamagnetic Nanoprobes

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. SPIO-MtbsAb synthesis

1. Synthesize SPIO-conjugated 2,2'-(ethylenedioxy)bisethylamine (EDBE) using previously reported methods.
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 Mycobacterium tuberculosis surface antibody (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 a 2.5 cm × 33 cm column and elute using a phosphate-buffered saline (PBS) buffer. Confirm Ab–nanoparticle complex (i.e., nanoprobe) using a bicinchoninic acid protein assay kit.

2. Particle morphology observation and relaxation tier measurement

1. Examine average particle size, morphology, and size distribution using a 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 relaxivities.

3. 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.

4. 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 described.
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 previously. Inject saline into mice as a 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 it in paraffin for serial sections at 5-10 µm. Stain tissue sections with the hematoxylin/eosin and Ziehl-Neelsen stains for acid-fast bacteria and with Berlin blue for ferric iron.

5. 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 the 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.