Given the wide range of short peptide sequences, this method can be extended to many other nanoparticle designs. Peptide modification can make the system suitable for diagnostic purposes. Encapsulating anti-cancer platinum to drugs with biodegradable, low toxicity stabilizing agent is highly relevant for pharmaceutical applications.
Preliminary in-vivo studies with ovarian cancer are promising and will be further investigated. To begin, suspend 50 milligrams of cisplatin in four milliliters of ultra-pure water at 60 degrees Celsius. Add the silver nitrate solution drop-wise to the cisplatin solution and stir the mixture at 300 to 400 RPM for two hours at 60 degrees Celsius in the dark.
After that, use 10%by weight hydrochloric acid to confirm that there are no free silver ions in solution. If no additional silver chloride forms upon adding hydrochloric acid, centrifuge the mixture at 3, 220 times gravity for 10 minutes. Decant the supernatant and filter it through a 0.2 micrometer syringe filter.
Then, test the filtrate for platinum by applying two to three drops to 10 two chloride crystals. Platinum is present if the suspension turns dark brown or orange. Next, add 13.3 milligrams of sodium hydroxide in one milliliter of water into 94.2 milligram suspension of oleic acid in three milliliters of water at 60 degrees Celsius.
Stir the mixture for two hours at 60 degrees Celsius. Then, turn off the heat and continue stirring at room temperature for 16 to 24 hours to obtain the crude product as an oily, yellow-brown precipitant. Centrifuge the reaction mixture at 3, 220 times gravity for 10 minutes, and remove the supernatant.
Dry the products on a rotary evaporator at 25 degrees Celsius. Suspend the crude product in five milliliters of acetonitrile by vortexing and recovering the product by centrifugation. Repeat this washing process at least two three more times to obtain the pure, oleic acid platinum II conjugate as a pale yellow solid.
First, soak 5.7 grams of Fmoc protected L-phenylalanine functionalized Wang resin in 25 milliliters of dimethylformamide for one hour. Then, soak the resin in 15 milliliters of 20%piperidine in DMF for five minutes while shaking at 80 RPM to de-protect the amine. Drain the resin and repeat the de-protection for 20 minutes.
Wash the resin with DMF and isopropyl alcohol afterwards. Once de-protection has been confirmed, add Fmoc protected L-tyrosine TBTU and DIPEA and agitate the mixture for 16 to 24 hours to perform coupling. Wash resin with DMF and isopropyl alcohol.
Perform Kaiser test and continue with de-protection and wash as previously described. Couple and de-protect Fmoc protected lysine in the same way. Wash the resin with DMF, IPA, methanol, dichloromethane, and diethyl ether afterwards.
Set aside one gram of KYF-bearing resin for FITC modification. Soak the remaining resin in 95%trifluoroacetic acid. 2.5%triisopropylsilane and 2.5%water for three hours to cleave the tripeptide from the resin.
Precipitate the crude peptide with diethyl ether cooled to 20 degrees Celsius. Wash the precipitate three times by centrifugation in 30 to 40 milliliter portions of cold diethyl ether and dry it under vacuum. Next, mix the remaining gram of KYF resin with 120.1 milligrams of 6-acetohexanoic acid in 30 milliliters of DMF with TBTU and DIPEA.
Agitate the mixture for 16 to 24 hours at room temperature to obtain the azide modified tripeptide. Then, combine 253 milligrams of this resin with 3.78 milligrams of solid copper(I)iodide 71.9 milligrams of propargyl florisean and 2.24 milligrams of DIPEA. Let the mixture react while shaking for 24 hours.
Then, thoroughly wash the FITC modified KYF bearing resin. Cleave the FITC modified KYF from the resin and purify it in the same way as the unmodified tripeptide. To begin preparing the nanoemoulsion, dissolve 10 milligrams of the oleic acid platinum II conjugate in 1.5 milliliters of IPA.
Draw this solution into a 5 milliliter syringe, attach a 20 gauge needle, and mount the syringe on a syringe pump over the reaction area. Set the syringe pump to 0.1 milliliters per minute. For a nanoemulsion with FITC modified KYF, dissolve one milligram of FITC modified KYF and one milligram of unmodified KYF in 20 milliliters of water.
For a nanoemulsion with unmodified KYF, instead dissolve two milligrams of KYF in 20 milliliters of water. Heat the FITC-modified or unmodified KYF solution to 37 degrees Celsius. Ensure that the KYF solution is stirring at about 400 RPM, and that the syringe is aligned with the solution.
Then, start the syringe pump to begin adding the oleic acid platinum two conjugate drop wise. Once the conjugate has been added, reduce the stirring speed to 150 RPM and let the solution cool to room temperature. Continue stirring it for 16 to 24 hours.
Then, concentrate the nanoemultion in a 10, 000 Daltons centrifugal filter unit. Wash the nanoemulsion in a centrifugal filter unit three time with four milliliter portions of ultrapure water. And then store it as an aqueous solution at four degrees Celsius.
The final product is opalescent for KYF nanoparticles and fluorescent for FITC-modified nanoparticles. The droplets in a nanoemulsion prepared with unmodified KYF were spherical, well dispersed, and relatively uniform in size. Based on transmission electron microscopy images, the cores were about 107 nanometers in diameter.
The FITC-modified nanoemulsion, which appears here in green, could enter cells from various ovarian cancer cell lines. While the hydrodynamic diameter of both formulations increased during four months of storage in water, the overall dimensions were preserved. In addition, no opsonization of the emulsion was observed after a day of incubation in 20%fetal bovine serum.
The release of platinum from the emulsion was slowest at pH 7.4 with only 20.8%of the platinum released after four hours. The platinum release accelerated as the pH decreased. The platinum-containing KYF coated nanoemulsion reduced the viability of these ovarian cancer cell lines to a much greater degree than was achieved by carboplatin, the clinically relevant analog.
The nanoemulsion also showed a comparable or greater reduction in viability than cisplatin in five of the six cell lines. The KYF-coated nanoemulsion consistently induced a comparable or greater reduction in viability than the oleic acid platinum II conjugate alone. This is a simple, efficient method for encapsulating hydrophobic therapeutics within tripeptide base nanoparticle scaffold.
Short peptides are biodegradable, and have low toxicity, which is significant for medicine and biotechnology. Perform this procedure under the fume hood to avoid exposure to organic solvents. Platinum oleic acid and KYF platinum nanoemulsions have anti-cancer properties and therefore should be handled with special care.
This method is simple in terms of low filabilator activity and equipment, however it relies on proper application of the synthesis strategy, and underlying chemical concepts. Our method can be extended to deliver other small drug molecules. The nanoparticle core consists of oleic acid and therefore is suitable for encapsulation of hydrophobic agents.
