Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Our unique drug delivery system, which uses 20 nanometer gold particles in two specific short peptides, can stabilize gold nano particles in physiological solution and enhance its cellular uptake, and thus, effectively deliver peptide-based or other drugs into cells. Our GNP peptide hybrid is easy to synthesize and stable. By using gold nanoparticle peptide hybrid as drug carriers, we can transfer peptide drugs into the cells effectively and in abundance.

We treated ischemia-reperfusion injury in the lungs with our GNP/PKCi hybrid. GNP brings PKC inhibitor peptides into the cytoplasm. This delivery method was able to reduce more severe lung injury using a lower dosage of PKCi than other delivery methods.

To begin this procedure, retrieve the peptides from a freezer at minus 20 degrees Celsius, and thaw them at room temperature. Use a micro scale to weigh 0.01 gram of each thawed peptide, and transfer each peptide into a separate 50 milliliter conical tube. Add 18.74 milliliters of deionized water to the P2 tube, and 16.93 milliliters of deionized water to the P4 tube.

Add 8.21 milliliters of 50%acetonitrile diluted in deionized water to the PKC delta inhibitor tube. Vortex the peptide solutions briefly. Then place the tubes into a sonicator at 40 megahertz for five minutes.

After this, transfer the solutions to a biosafety cabinet. It is critical to put the 50 mL conical tube in a sonicator for five minutes. If this step is skipped, the GNP/PKCi will form aggregations.

Transfer one milliliter of each peptide solution to its own new 50 milliliter conical tube. Add 19 milliliters of deionized water to the tubes containing P2 and P4.And add 19 milliliters of 50%acetonitrile to the tube containing PKC delta inhibitor. Add 475 milliliters of the P2 solution, 25 microliters of the P4 solution, 500 microliters of the PKC delta inhibitor solution to a 15 milliliter tube.

Then add nine milliliters of a 20 nanometer GNP solution to the same tube. Remove the combined solution from the biosafety cabinet and wrap the tube in aluminum foil. Place the tube on a shaker overnight.

The next day, return the samples to the biosafety cabinet. Transfer one milliliter aliquots of the GNP/PKCi solution into 1.5 milliliter micro tubes. Centrifuge the micro tubes at a micro centrifuge at 15294 times g and at four degrees Celsius for 30 minutes.

Transfer the micro tubes back into the biosafety cabinet, and remove the supernatent from each tube, while ensuring the GNP pellet is not disturbed. Resuspend each pellet in the desired solvent according to the concentration required. To assess the GNP/PKCi hybrid solubility, pour 0.5 milliliters of the GNP/PKCi solution into an accrual cuvette.

Place the cuvette on a UV-Vis spectrophotometer and test the peak absorption. In this study, protein kinase C-delta inhibitor is conjugated with gold nanoparticles to form a GNP/PKCi hybrid. Care must be taken with evaluating the biophysical properties of the hybrid, as gold nanoparticles tend to aggregate in a solvent.

When the nanoparticles aggregate, the color of the solution will change from pink to purple. The samples are analyzed with a UV-Vis spectrophotometer which is able to detect with a high degree of sensitivity. If the hybrid is not aggregated, the peak of an absorption should be at 525 nanometers.

If the GNP is aggregated, the peak of absorption will be shifted to the right. As can be seen, when aggregates have formed, the delta optical density decreases. When formulating the GNP peptide hybrids, it is important to remember the ratio of peptides to GNP solution should be one to nine.

This GNP/PKCi formula can be used for ischemia-reperfusion injury in organ systems other than the lung. Any peptides can attach to GNP if those peptides have a cysteine residue at the N-terminus. This means GNP peptide hybrid can be used to transport other peptide drugs into the cell.

Rationally designed peptides are new technology that can brought specific protein-protein interaction inside those cells. This technique, developed here, can be used for delivery of new peptide drugs or for modified versions of existing drugs.