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The overall goal of this procedure is to show the preparation and characterization of a dendrimeric paramagnetic chelating which can be used as a contrast agent for magnetic resonance imaging and to demonstrate its performance in an imaging experiment. This method can help answer key questions in the field of MRI contrast agents such as the properties and the efficacy of dendrimeric contrast agents. The main advantage of this technique is convenient preparation of the efficient MRI contrast agent with high protein, thus ensuring its easy characterization.

This method can extend towards further structural modifications and functional optimizations of dendrimeric MRI contrast agents which is of great relevance for potential in vivo applications. The implications extend towards biomedical research and diagnostic imaging as the high molecular rate of dendrimeric contrast agents causes longer tissue retention times and substantially enhance the MRI signal. First, dissolve one gram of the macrocyclic precursor DO3A tributyl ester in five milliliters of DMF.

Add 0.67 grams of potassium carbonate and stir at room temperature for 45 minutes. Add 0.87 grams of tert-butyl-2-bromo-4 4-nitrophenyl butatnoate in portions over an hour and continue stirring the reaction mixture for 18 hours. Then, remove the DMF with bulb-to-bulb vacuum distillation at 40 to 60 degrees Celsius.

Purify the residue with silica gel column chromatography to obtain a round solid. Dissolve one gram of the solid in a mixture of 10 milliliters ethanol and 150 microliters of a seven normal ammonia solution in methanol. Add 150 milligrams of palladium on activated carbon and set up the bottle reactor in the hydrogenator.

Shake the mixture for 16 hours under a 2.5 bar hydrogen atmosphere. Pour a suspension of diatomaceous earth in ethanol into a sintered glass funnel Filter the reaction mixture through the diatomaceous earth to remove the palladium carbon catalyst. Remove solvent from the filtrate on a rotary evaporator.

Dissolve the resulting solid in 15 milliliters of dichloromethane and four equivalents triethylamine. To this, add 1.3 equivalents of thiophosgene and stir the mixture at room temperature for 16 hours. Remove the solvent from the reaction mixture on a rotary evaporator and purify the residue with silica gel column chromatography to obtain the DOTA-type monomer as a light brown solid.

Prepare 66.7 milligrams of G4 PAMAM dendrimer from a 10%solution in methanol by evaporating the solvent on a rotary evaporator at 40 degrees Celsius. Dissolve the dendrimer in four milliliters of DMF. Add 0.105 milliliters of triethylamine to the dendrimer solution and stir for 45 minutes at 60 degrees Celsius.

Then add 354 milligrams of the monomer in portions over an hour. Continue stirring at 45 degrees Celsius for 48 hours. Remove the solvent with bulb-to-bulb vacuum distillation.

Pack a size exclusion chromatography column by swirling lipophilic gel filtration medium in methanol for three hours at room temperature. Collect one milliliter fractions using methanol as the eluent. Analyze the fractions by TLC.

Remove solvent from the fractions containing the protected dendrimeric chelator on a rotary evaporator. Acquire a proton NMR spectrum of the compound and integrate the aromatic and aliphatic regions. Using these values, determine the number of protected DOTA-type macrocycles coupled to the G4 PAMAM dendrimer.

To de-protect the product, dissolve the solid in five milliliters formic acid and stir the mixture at 60 degrees Celsius for 36 hours. Remove the formic acid with a rotary evaporator. Freeze dry the residue removed from the rotary evaporator to obtain the dendrimeric chelator.

Using proton NMR, determine the number of macrocyclic units coupled to the dendrimer. Dissolve the freeze dried chelator in water. Use 0.1 molar sodium hydroxide to adjust the solution pH to 7.0.

Add a solution of 113 milligrams Gadolinium III chloride in one milliliter water drop-wise to the chelator solution over a four-hour period. Periodically adjust the pH to 7.0 with 0.05 molar sodium hydroxide. Stir the mixture at room temperature for 24 hours.

Then, add 158 milligrams of EDTA in portions over a four-hour period correcting the pH back to 7.0 as needed. Stir for another 24 hours at room temperature. Pack a size exclusion chromatography column with hydrophilic gel filtration medium swollen in water.

Concentrate the reaction mixture, load the mixture onto the column, and elute with deionized water. Centrifuge the purified sample in a three kilodalton centrifuge filter unit for 30 minutes at 1, 800 times g. Repeat the filtration about five times to remove excess Gadolinium and EDTA.

Confirm the absence of free Gadolinium ions in the filtrate with a xylenol orange test. Remove volatiles from the filtered sample and freeze dry the residue to obtain the dendrimeric contrast agent. Prepare a stock solution of DCA in water with a concentration between five and 10 millimolar.

To determine the precise DCA concentration, first combine 360 microliters of the stock solution with 60 microliters of a D2O and tert-butanol standard. Place 400 microliters of the sample in an outer NMR tube. Place a coaxial NMR insert tube containing a tert-butanol and water reference solution into the sample tube.

Acquire a proton NMR spectrum and measure the frequency shift between the tert-butanol signals of the reference and sample solutions. Calculate the DCA concentration using the shown formula, correcting for the added tert-butanol in the sample. Determine the concentration of a commercially available GdDOTA solution in the same way.

Place DCA and GdDOTA solutions in HEPES buffer and water controls in two sets of plastic vials. Close vials with caps ensuring that no air bubbles are present. Into a 60 milliliter syringe, horizontally load the 0.01 and 0.02 millimolar DCA samples, one of the 0.5 and the 1.0 millimolar GdDOTA samples and two of the water controls.

Repeat this procedure by loading the second set of samples into a second 60 milliliter syringe. Fill each syringe with one millimolar GdDOTA solution and cap the syringe tips. Place one of the syringes horizontally in an MRI scanner so that the samples are vertical with respect to the scanner.

Slide the syringes into the center of the scanner. Select the anatomical scan to position the syringe at the isocenter of the magnet. Then press the traffic light button to perform adjustments of initial scanning parameters and start the scan.

Choose the fast/slow angle shot method to perform T1 weighted imaging or the rapid acquisition with relaxation enhancement method to perform T2 weighted imaging. Use the localizer scan to select coronal slice for the vertically aligned samples. Optimize the contrast to noise acquisition parameters and acquire the image.

Use a circular ROI to determine average signal amplitude and standard deviation for the background and for each sample. Calculate the signal-to-noise ratio. NMR, MALDI-TOF MS, and elemental analysis indicated that an average of 49 macrocyclic units were conjugated to the G4 PAMAM dendrimer.

The concentrations and longitudinal and transverse relaxation times of DCA solutions were also determined with NMR. The obtained relaxivity values were compared to a commercially available and clinically used GdDOTA monomer. The DCA performance was also compared to the GdDOTA monomer.

Two sets of solutions had similar Gadolinium concentrations as determined by the number of conjugated macrocyclic units and the other two sets had similar molecular concentrations. In a T1 weighted imaging experiment with similar Gadolinium concentrations, the signal-to-noise ratio was slightly higher for DCA than for GdDOTA. When the solutions had similar concentrations by molecule, the DCA signal-to-noise ratio was about three times greater than that of GdDOTA.

In a T2 weighted imaging experiment, the signal-to-noise ratio of the DCA was significantly lower than that of the GdDOTA in both concentration pairs, particularly at higher DCA concentrations. After watching this video, you should have a good understanding of how to prepare purified and characterized dendrimer-based MRI contrast agent. Following this procedure, different types of dendrimeric and nano-sized MRI contrast agents can be prepared by modifying the units of the molecule and conveniently analyzed.

After mastering this technique, the researchers in the field of MRI contrast agent development should be able to prepare a range of specific probes to execute various functional studies of critical importance for contemporary molecular imaging.