The overall goal of this method is to increase solubility of hydrophobic compounds in aqueous solution for potential delivery into clinical settings. This method addresses a key limitation in the medical field which is a lack of use of hydrophobic compounds in clinical settings due to their poor solubility in aqueous systems such as the blood. The main advantage of this technique is that hydrophobic compounds can be dissolved in aqueous solutions without any significant use of toxic co-solvents.
First, label two 50 liter conical centrifuge tubes for each amino acid. Prepare a large two liter flask containing purified water. After calculating the amount of each amino acid for the desired concentrations, weight the appropriate amounts of amino acids into the respective centrifuge tubes using a spatula.
Using a seriological pipette, ad 40 milliliters of purified water to each centrifuge tube. After capping the tubes, vortex the samples until dissolved. Using a high performance analytical balance, weigh approximately one plus or minus 0.2 milligrams of peptide into the bottom of a 20 milliliter scintillation vial.
Record the exact weight of the peptide on the cap and screw the cap on the vial. Next, pipette the appropriate volume of amino acid solution into each vial in order to reach the desired concentration of self-assembling peptide Sonicate the samples for 10 minutes in a water bath sonicator at room temperature ensuring the solutions in the vial are completely immersed in the water bath. Prepare two drug stock solutions by combining one milligram of the desired of the desired drug with the appropriate amount of DMSO and another one milligram of the drug with ethanol.
Then, vortex both solutions for 15 seconds to completely dissolve the drug. Following this, label 1.5 milliliter micro-centrifuge tubes for each formulation with the self assembling peptide, amino acid, and co-solvent. Add 10 microliters of the drug DMSO stock solution or 20 microliters of the drug ethanol stock solution to the appropriate micro-centrifuge tubes.
Next, add 990 microliters of the self assembling peptide amino acid solutions to the micro-centrifuge tubes containing drug DMSO stock solution. Add 980 microliters of the peptide amino acid solutions to the micro-centrifuge tubes containing the drug ethanol stock solution. Vortex all drug formulations vigorously for 30 seconds.
Then, cover with aluminum foil and allow formulations to sit for 30 minutes before solubility testing. After 30 minutes, vortex the formulations vigorously for 30 seconds. Then, centrifuge the formulations at 14, 220 times G for one minute.
Following centrifugation, visual inspect the bottom of the micro-centrifuge tubes for precipitation. Out of the 40 formulations tested in this study, seven successfully dissolved the hydrophobic drug, curcumin. Two major trends were identified by grouping the formulations by components.
Ethanol seems to be a better co-solvent for dissolving curcumin, and positively charged amino acids, lysine, and arginine seem to be optimal components for dissolving curcumin. It is interesting to note the color change for formulations containing arginine and lysine which reveal curcumin is dissolved in the alkaline condition. Once mastered, this technique can be done in two to three hours with the proper equipment and if the amino acid solutions are prepared before-hand.
Following this procedure, soluble drug formulations can be further tested in vitro and in vivo in order to directly assess and compare the biological efficacy or safety of the dissolved drug. After it's development, this formulation method paved the way for in vivo delivery of a hydrophobic stock inhibitor called PP@for the prevention of acute lung injury. After watching this video, you should have a good understanding of how to apply a self-assembling peptide and amino acid combination to dissolve any given hydrophobic drug.
