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

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.