Name: a tripeptide-stabilized nanoemulsion of oleic acid
Uploaded: 2019-02-27T13:00:00
Description: Watch this Scientific Journal Video about A Tripeptide-Stabilized Nanoemulsion of Oleic Acid at JoVE.com

We gratefully acknowledge financial support from the National Cancer Institute, grant SC2CA206194. No competing financial interests are declared.

1. Synthesis of the Oleic Acids&#8211;Platinum(II) Conjugate
<ol>
	<li>Activation of cisplatin
	<ol>
		<li>Suspend 50 mg (0.167 mmol) of cisplatin in 4 mL of water (e.g., nanopure) at 60 &#176;C.</li>
		<li>Add dropwise 55.2 mg (0.325 mmol) of AgNO3 in 0.5 mL of water to the solution of cisplatin and stir the reaction for at least 2 h at 60 &#176;C. The white precipitate of AgCl will form indicating the progress of the reaction.</li>
		<li>To determine if the activation reaction is completed, perform the tes...

Critical steps in the nanoemulsion synthesis include adjusting the molar ratio of the substrates, maintaining temperature and flow rate control during oleic acids&#8211;Pt(II) addition, providing sufficient time for self-assembly, and purifying the product using a centrifugal concentrator column. These parameters influence the size and morphology of the KYF-Pt-NE; thus, it is particularly important to maintain the proper molar ratio and adjust the synthetic conditions correctly.
The ratio of t...

In recent years, there has been a growing interest in the engineering of nanoparticles for such biomedical applications as drug delivery and bioimaging1,2,3,4. The multifunctionality of nanoparticle-based systems often necessitates incorporating multiple components within one formulation. The building blocks that are based on lipids or polymers often differ in terms of their physicochemical properties as well as their biocompatibility and biodegradability, which ultimately might affect the function of the nanostructure1,5,6. Biologically derived materials, such as proteins and peptides, have long been recognized as promising components of multifunctional nanostructures due to their sequence flexibility7,8. Peptides self-assemble into highly ordered supramolecular architectures forming helical ribbons9,10, fibrous scaffolds11,12, and many more, thus paving the way to building biomolecule-based hybrid nanostructures using a bottom-up approach13.
Peptides have been explored for applications in medicine and biotechnology, especially for anticancer therapy14 and cardiovascular diseases15 as well as for antibiotic development16,17, metabolic disorders18, and infections19. There are over a hundred of small-peptide therapeutics undergoing clinical trials20. Peptides are easy to modify and fast to synthesize at low cost. In addition, they are biodegradable, which greatly facilitates their biological and pharmaceutical applications21,22. The use of peptides as structural components includes the engineering of responsive, peptide-based nanoparticles and hydrogel depots for controlled release23,24,25,26,27, peptide-based biosensors28,29,30,31, or bio-electronic devices32,33,34. Importantly, even short peptides with two or three amino-acid residues that include phenylalanine were found to guide the self-assembly processes35,36,37 and create stabilized emulsions38.
Platinum-based drugs, owing to their high efficacy, are used in many cancer treatment regimens, both alone and in combination with other agents39,40. Platinum compounds induce DNA damage by forming monoadducts and intrastrand or interstrand cross-links. The Pt-DNA lesions are recognized by the cellular machinery and, if not repaired, lead to cellular apoptosis. The most important mechanism, by which Pt(II) contributes to cancer cell death, is the inhibition of DNA transcription41,42. However, the benefits of platinum therapy are diminished by systemic toxicity of Pt(II) that triggers severe side effects. This leads to lower clinical dosing of Pt(II)43, which often results in sub-therapeutic concentrations of platinum reaching the DNA. As a consequence, the DNA repair that follows contributes to cancer cell survival and acquiring Pt(II) resistance. The platinum chemo-resistance is a major problem in anticancer therapy and the main cause of treatment failure44,45.
We have developed a stable nanosystem that encapsulates the Pt(II) agent in order to provide a shielding effect in systemic circulation and to diminish the Pt(II)-induced side effects. The system is based on an oleic acids-Pt(II) core stabilized with a KYF tripeptide to form a nanoemulsion (KYF-Pt-NE)46. The building blocks of KYF-Pt-NE, the amino acids of the tripeptide as well as the oleic acid, have the Generally Recognized As Safe (GRAS) status with Food and Drug Administration (FDA). The KYF-Pt-NE is prepared by using a nanoprecipitation method47. In short, the oleic acids-Pt(II) conjugate is dissolved in an organic solvent and then added dropwise to an aqueous KYF solution (Figure 1) at 37 &#176;C. The solution is stirred for several hours to allow self-assembly of the KYF-Pt-NE. The nanoemulsion is concentrated in 10 kDa centrifugal concentrators and washed three times with water. The chemical modification of the KYF with a fluorophore allows the synthesis of fluorescent FITC-KYF-Pt-NE suitable for biomedical imaging.

<table>
	<tbody>
		<tr>
			<td>2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium 
			tetrafluoroborate (TBTU)</td>
			<td>ANASPEC INC.:</td>
			<td>AS-20376</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>4-well chamber confocal dish</td>
			<td>Lab-Tek II, Thermo Fisher Scientific</td>
			<td>154526</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>6-bromohexanoic acid</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>24477</td>
			<td>Click modification for peptide</td>
		</tr>
		<tr>
			<td>A2780</td>
			<td>Generously doanted by professor John Martignetti from The Mount Sinai Hospital</td>
			<td></td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Barnstead Nanopure</td>
			<td>Thermo Fisher</td>
			<td>D11901</td>
			<td>water filtration system</td>
		</tr>
		<tr>
			<td>BUCHI rotavapor R-3</td>
			<td>Buchi</td>
			<td>Z568090</td>
			<td>For solvent removal and sample drying</td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>eppendorf</td>
			<td>5811F</td>
			<td>For platinum complex separation</td>
		</tr>
		<tr>
			<td>Cis-dichlorodiamineplatinum (II) 99%</td>
			<td>Acros Organics</td>
			<td>19376-0050</td>
			<td>in vitro tests</td>
		</tr>
		<tr>
			<td>CP70</td>
			<td>Generously doanted by professor John Martignetti from The Mount Sinai Hospital</td>
			<td></td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Digital water bath</td>
			<td>VWR</td>
			<td>97025-134</td>
			<td>For warming up media for cell culture</td>
		</tr>
		<tr>
			<td>Dynamic Light Scattering (DLS)</td>
			<td>Brookhaven Instrument Corporation</td>
			<td></td>
			<td>For nanoparticle size measurments</td>
		</tr>
		<tr>
			<td>ES-2</td>
			<td>ATCC</td>
			<td>CRL-1978</td>
			<td>ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Fmoc-L-Lys(Boc)-OH 99.79%</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>00493</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Fmoc-L-Phe 4-alkoxybenzyl alcohol resin (0.382 meq/g),</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>01914</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Fmoc-LTyr(tBu)-OH 98%</td>
			<td>Alfa Aesar</td>
			<td>H59730</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>HERACELL 150i CO2 incubator</td>
			<td>Thermo Scientific Fisher</td>
			<td></td>
			<td>incubator</td>
		</tr>
		<tr>
			<td>High pressure syringe pump</td>
			<td>New Era</td>
			<td>1010-US</td>
			<td>For platinum complex addition in nanoparticle synthesis</td>
		</tr>
		<tr>
			<td>Hotplate/stirrer</td>
			<td>VWR</td>
			<td>12365-382</td>
			<td>For sample stirring and heating</td>
		</tr>
		<tr>
			<td>LAMP-1 Antibody(cojugated with Alexa Fluor 647)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-18821 AF647</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>N,N-diisopropylethylamine (DIPEA)</td>
			<td>Oakwood Chemical</td>
			<td>005027</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Ninhydrin 99%</td>
			<td>Alfa Aesar</td>
			<td>A10409</td>
			<td>Kaiser test</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>01421</td>
			<td>For platinum complex synthesis</td>
		</tr>
		<tr>
			<td>OV90</td>
			<td>ATCC</td>
			<td>CRL-11732</td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>For cell wash</td>
		</tr>
		<tr>
			<td>Permount mounting medium</td>
			<td>Fisher Chemical</td>
			<td>SP15-100</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Fisher Chemical</td>
			<td>A92500</td>
			<td>Kaiser test</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Fisher Chemical</td>
			<td>A248-25</td>
			<td>negative stain for TEM</td>
		</tr>
		<tr>
			<td>Piperidine 99%</td>
			<td>BTC</td>
			<td>219260-2.5L</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Platinum AAS standard soultion</td>
			<td>Alfa Aesar</td>
			<td>88086</td>
			<td>1000ug/ml for calibration curve</td>
		</tr>
		<tr>
			<td>Propargyl bromide 97%</td>
			<td>Alfa Aesar</td>
			<td>L10595</td>
			<td>For alkyne modification of fluoresceine</td>
		</tr>
		<tr>
			<td>Scientific biological cabinet</td>
			<td>Thermo Scientific Fisher</td>
			<td>1385</td>
			<td>Bio-hood for cell culture</td>
		</tr>
		<tr>
			<td>Self-Cleaning Vacuum System</td>
			<td>Welch</td>
			<td>2028</td>
			<td>Vacuum pump for rotavapor</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Acros Organics</td>
			<td>19768-0250</td>
			<td>Cisplatin activation</td>
		</tr>
		<tr>
			<td>SKOV3</td>
			<td>ATCC</td>
			<td>HTB-77</td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S313-1</td>
			<td>For platinum complex synthesis</td>
		</tr>
		<tr>
			<td>Tin (II) chloride</td>
			<td>Sigma Aldrich</td>
			<td>208256</td>
			<td>Test for Platinum presence</td>
		</tr>
		<tr>
			<td>TOV21G</td>
			<td>ATCC</td>
			<td>CRL-11730</td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid 99% (TFA)</td>
			<td>Alfa Aesar</td>
			<td>L06374</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Triisopropylsilane (TIPS)</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>01966</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma Aldrich</td>
			<td>T8787-100ML</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>Uranine powder 40%</td>
			<td>Fisher Scientific</td>
			<td>S25328A</td>
			<td>For alkyne modification of fluoresceine</td>
		</tr>
		<tr>
			<td>Vivaspin 20 (10000 MWCO)</td>
			<td>Sartorious</td>
			<td>VS2001</td>
			<td>For Nanoparticle wash and condensation</td>
		</tr>
		<tr>
			<td>VWR Inverted Microscope</td>
			<td>VWR</td>
			<td>89404-462</td>
			<td>For cell culture monitoring</td>
		</tr>
	</tbody>
</table>

a tripeptide-stabilized nanoemulsion of oleic acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

Representative TEM image of KYF-Pt-NE prepared using this protocol is shown in Figure 2A. The KYF-Pt-NEs are spherical in morphology, well dispersed, and uniform in size. The core diameter of the KYF-Pt-NEs, measured directly from three TEM images with a minimum of 200 measurements done, is 107 &#177; 27 nm. The hydrodynamic diameter of KYF-Pt-NE, analyzed using dynamic light spectroscopy (DLS), was found to be 240 nm with a polydispersity index of 0.156. The...

Watch this Scientific Journal Video about A Tripeptide-Stabilized Nanoemulsion of Oleic Acid at JoVE.com

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

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.

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