Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

This method can help answer key questions in protea mix and diagnostics, such as how to design probes that can detect the progression of diseases by measuring proteates'activity in vivo. The main advantage of this method is that the diagnositics developed will be noninvasive, and can actually amplify the detection of biological signals. This method can be applied to numerous diseases, because many proteuses are actually major drivers of pathogenesis.

In the chemical fume hood, transfer one ml of 30%ammonium hydroxide to a 15 ml conical tube. Place this tube into an ice bucket and let it chill for 30 minutes. Wash a 250 ml Ehrlenmeyer flask with deionized water.

Then add 10 ml of double distilled water. Submerge the flask in an ice bath resting on a heat plate. Using a plastic pipette, begin the flow of nitrogen gas into the double distilled water in the flask.

Bubble for 15 minutes to deoxygenate. Next, weigh out 4.5 g of dextrine in a 15 ml conical tube. Add water such that the final volume of the mixture including the dextrine is 20 ml.

Vortex vigorously to dissolve the dextrine. Weigh out 78.5 mg of iron 3 chloride hexahydrate and add this to the dextrine solution. Vortex to dissolve.

Using a 0.2 um filter, filter the resulting solution. After this, transfer 9 ml of the deoxygenated water to the 15 ml conical tube containing the 1 ml of chilled ammonium hydroxide. Return the conical tube to the ice bucket to chill.

Weigh out 73 mg of iron 2 chloride tetrahydrate and dissolve it in the remaining 1 ml of deoxygenated double distilled water. Use a 0.2 um filter to filter the solution. Then, transfer the dextrine iron 3 solution into the 250 ml Ehrlenmeyer flask.

Deoxygenate with nitrogen gas for 15 minutes on ice, while mixing at 1, 600 rpm. Cap the flask with a rubber septum. Using an 18 gauge needle, puncture the septum to flow nitrogen gas into the flask.

Insert a separate 18 gauge needle, which will be the flow outlet. Next, use a 1 ml syringe to add 467 ul of the iron 2 solution to the dextrine iron 3 solution. Stir at 1, 600 rpm.

Add the chilled dilute ammonium hydroxide solution dropwise to initiate the nucleation process. Make sure each drop mixes well before adding another. Stop the flow of nitrogen gas and remove the rubber septum.

Remove the ice bath and replace it with a bath of warm water. Heat the solution to 75 degrees Celsius and then incubate for 75 minutes. Remove the flask from the hot plate.

Doubly filter the solution through a 0.2 um filter and then a 0.1 um filter to remove any coarse particles. Using 100 kDa molecular weight cutoff concentrators, buffer exchange the particles into double distilled water to remove excess dextrine. If the flow through remains dark after two to three spins, replace the filters as they may have broken.

Next, resuspend the iron oxide nanoparticles in double distilled water at a concentration of 10 mg per ml. Add 1.6 volumes of 5 molar sodium hydroxide, then add 0.65 volumes of epichlorohydrine. Using a plate shaker, rigorously mix at room temperature for 12 hours.

Use a 20 ml syringe with an 18 gauge needle to transfer the iron oxide nanoparticle solution into a dialysis membrane with a 50 kDa molecular weight cutoff. Place the dialysis membrane into 4 liters of double distilled water. Incubate at room temperature overnight.

After determining the concentration, bring the iron oxide nanoparticles to a concentration of 5 mg per ml. Add ammonium hydroxide to reach 20%and shake at room temperature for at least 12 hours to amenate the surface of the iron oxide nanoparticles. Then, buffer exchange using 30 kDa molecular weight cutoff filters.

Use dynamic light scattering to determine the hydrodynamic radius of the iron oxide nanoparticles as outlined in the text protocol. After synthesizing a peptide substrate, aliquot 0.5 mg of iron oxide nanoparticles into the 10 kDa molecular weight cutoff spin filter tube. Buffer exchange into coupling buffer using a 10 kDa molecular weight cutoff spin filter as outlined in the text protocol.

Dissolve SIA in DMF to reach a concentration of approximately 30 mg per ml. Then, add SIA to the iron oxide nanoparticles at a mole ratio of 500 to one. After incubation and buffer exchange, bring the final product to a concentration of 1 mg per ml.

Mix the peptide of interest with 20 kDa thiol terminated polyethylene glycol, and then mix this peptide glycol solution with iron oxide nanoparticles. Cover the tubes in foil to prevent photo bleaching the fluorescent molecules. And incubate overnight at room temperature on a plate shaker set the to the highest speed.

Next, add L cystine at a molar ratio of 500 to one to the iron oxide nanoparticles. Incubate for one hour at room temperature on a shaker set to the highest speed. After this, make an 18 ul nanoparticle solution using PVS, and a 200 nanomolar concentration of peptide.

Add 2 ul of the protease of interest. Incubate in a plate reader for one hour at 37 degrees Celsius taking flourescence measurements every one to two minutes to monitor cleavage. Immediately after injecting the prepared nanosensor solution via tail vein injection, place the mice into metabolic cages, and note the time of injection for each mouse.

Combine 2 ul of urine with 5 ul of prepared magnetic beads. Using PVS, bring the final total volume to 50 ul. Incubate at room temperature for 60 minutes.

Next, wash twice using 50 ul of PVS each time, while using a magnetic separator to collect the magnetic beads after each wash. Elute twice with 32.5 ul of 5%glacial acetic acid. Use 35 ul of 2 molar tris to neutralize the pool dilution and achieve a final ph around 7.

After this, use a plate reader at appropriate excitation and emission wavelengths to quantify the urine flourescence. Calculate the concentration of fluorescent reporter against a ladder of known concentrations of free flourophor. In this study, activity based nanosensors are developed by conjugating protease substrates to a nanoparticle core.

The average diameter of iron oxide nanoparticles is observed to be between 40 and 50 nm. After pecolation, the circulation half life for this size range is seen to be approximately 6 hours. After successful peptide conjugation, spectral absorbance analysis reveals a distinct absorbance peak at the maximum excitation wavelength of the fluorescent reporter.

The ability of the probe to sense protease activity is then tested by incubating an aliquot of nanosensors with recombinant proteases. It is typical to observe substrate cleavage velocities that follow Michaelis-Menten kinetics. Nanosensors coated with a near infrared fluorescent reporter are then used to visualize the pharmacokinetics in mouse models of cancer by whole animal fluorescent imaging.

A fluorescent signal is seen to localize in the urine of mice, bearing LS 174 t colorectal, 60 to 90 minutes after intravenous administration of the nanosensors. Significantly lower fluorescent signals are observed in the bladders of control mice. It should be noted that the signal observed in the liver arises because particles are eventually scavenged by monocites and and macrophages in the ridiculoendophilial system, which are commonly found in the liver, spleen, and lymph nodes.

Once mastered, this technique can be done in four days, if it's performed properly. While attempting this procedure, it's important to remember that flourescence is the primary readout, so it's important to keep the peptide substrates with fluorescent markers in the dark. Following this procedure, other methods like biodistribution studies and in viva imaging can be performed in order to answer additional questions, like localization of particles to various organs.

After its development, this technique paved the way for researchers in the field of diagnostics to explore cancer biomarkers in mice. After watching this video, you should have a good understanding of how to synthesize and characterize iron oxide nanoparticles, use these to build nanosensors, then use the nanosensors in vivo. Don't forget that working with the chemicals involved in a nanoparticle synthesis can be extremely hazardous, and precautions such as personal protective equipment and working within a fume hood should always be taken while performing this procedure.