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 test with 10% HCl for the presence of free Ag+ ion in solution. The test should be negative (no additional AgCl precipitate should form).</li>
		<li>Centrifuge the reaction mixture at 3,220 x g for 10 min and remove the white precipitate of AgCl.</li>
		<li>Collect the supernatant and filter it via a 0.2 &#956;m syringe filter. Test the supernatant for the presence of platinum by applying 2-3 drops of the solution via Pasteur pipette to SnCl2 crystals. The test is positive if the color of coordinate complex of tin with platinum is dark yellow/orange.</li>
		<li>Use the supernatant with activated Pt(II) for the second step.</li>
	</ol>
	</li>
	<li>Reaction of oleic acid with activated Pt(II)
	<ol>
		<li>Suspend 94.2 mg of oleic acid (0.333 mmol) in 3 mL of water at 60 &#176;C.</li>
		<li>Add 13.3 mg (0.333 mmol) of NaOH in 1 mL of water, and mix with the solution of activated Pt(II) from step 1.1</li>
		<li>Stir the reaction for 2 h at 60 &#176;C and next at room temperature overnight. The crude product is an oily brown/yellow precipitate.</li>
		<li>Centrifuge the reaction mixture at 3,220 x g for 10 min and remove the supernatant. Dry the crude product at 25 &#176;C using a rotary evaporator. Purify the product by multiple washes with acetonitrile. The final color of pure oleic acids&#8211;Pt(II) conjugate is pale yellow.</li>
	</ol>
	</li>
</ol>
2. Synthesis of KYF-Pt-NE, and the FITC-labeled Nanoemulsion FITC-KYF-Pt-NE
<ol>
	<li>Synthesis of the KYF tripeptide and the fluorescently labeled tripeptide FITC-KYF
	<ol>
		<li>Synthesize the KYF using standard solid-state peptide chemistry. Use the following standard coupling conditions to attach each amino acid: Wang resin (2.19 mmol), Fmoc protected amino acid (4.38 mmol), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (4.38 mmol) and diisopropylethylamine (DIPEA) (8.76 mmol). Dissolve the amino acids with TBTU in dimethylformamide (DMF) and DIPEA.</li>
		<li>Soak 5.7 g of Fmoc-L-Phe 4-alkoxybenzyl alcohol resin (0.382 meq/g) in 25 mL of DMF for 1 h prior to use.</li>
		<li>Deprotect the amine group of L-phenylalanine amino acid with 15 mL of 20% piperidine/DMF solution for 5 min, discard the solvent and repeat the wash with 20 min cycle.</li>
		<li>Wash the resin for 1 min with the following solvents: DMF, isopropyl alcohol (IPA), DMF, IPA, DMF, IPA, DMF, DMF. Discard the solvent after each wash.</li>
		<li>Perform the Kaiser test to determine the presence of free NH2 group on the resin (see substeps) and if positive (the resin seed is purple), add the Fmoc-L-tyrosine amino acid and perform the coupling overnight.
		<ol>
			<li>Prepare Kaiser test solutions in separate bottles.</li>
			<li>Dissolve 5 g of ninhydrin in 100 mL of ethanol.</li>
			<li>Dissolve 80 g of phenol in 20 mL of ethanol.</li>
			<li>Mix 2 mL of 0.001 M aqueous solution of potassium cyanide with 
			98 mL of pyridine.</li>
			<li>Add 2-3 drops of each Kaiser test solutions to sample and heat in boiling water for 5 min.</li>
		</ol>
		</li>
		<li>Upon successful coupling of second amino acid, perform the Kaiser test and if negative proceed with deprotection protocol (steps 2.1.3 to 2.1.5). Repeat the process with the Fmoc-phenylalanine amino acid.
		<ol>
			<li>Upon coupling of all amino acids, wash the resin for 1 min with 5 mL of DMF, IPA, DMF, methanol, dichloromethane and diethyl ether, after each wash discard the solvent. Save the resin for further processing.</li>
			<li>Use half of the resin for the next steps (2.1.7-2.1.9) to modify the peptide with FITC. To obtain unmodified KYF tripeptide, follow the procedure starting at 2.1.10.</li>
		</ol>
		</li>
		<li>Modify the N-terminal amino acid of the KYF to KYF-N3 with 6-azidohexanoic acid. To this end, mix 1 g (0.382 mmol) of KYF Wang resin and 120.1 mg (0.764 mmol) of 6-azidohexanoic acid with 245.2 mg (0.764 mmol) of TBTU and 197.1 mg (1.528 mmol) of DIPEA in 30 mL of DMF. Stir the reaction overnight at room temperature.</li>
		<li>Obtain the KYF-FITC from the synthesis of the KYF-N3 with propargyl fluorescein via a click reaction. To this end, mix 253 mg (0.097 mmol) of the KYF-N3 Wang resin with 3.78 mg (0.019 mmol) of CuI solid, 71.9 mg (0.193 mmol) of propargyl fluorescein, and 2.24 mg (0.017 mmol) of DIPEA. The reaction should change color from green to brown.</li>
		<li>After 24 h, wash the resin for 1 min alternately with 5 mL of DMF and IPA five times, methanol and water thrice, DMF and water thrice, and with dichloromethane and diethyl ether thrice. Discard the solvent after each wash.</li>
		<li>Cleave the KYF-FITC or KYF peptide from the resin with a solution of trifluoroacetic acid (TFA)/triisopropylsilane (TIPS)/H2O at the ratio of 95/2.5/2.5 over 3 hours.</li>
		<li>Precipitate the crude peptide in a cold diethyl ether, wash thrice with cold ether, and then dry under the vacuum.</li>
	</ol>
	</li>
	<li>Synthesis of FITC-KYF-stabilized nanoemulsion with Pt(II) (FITC-KYF-Pt-NE) and KYF-Pt-NE
	<ol>
		<li>Dissolve 10 mg (0.0126 mmol) of oleic acids&#8211;Pt(II) conjugate in 1.5 mL of isopropanol and place in a 5 mL syringe.</li>
		<li>Place the syringe with oleic acids&#8211;Pt(II) conjugate in a syringe pump and set the flow to 0.1 mL/min.</li>
		<li>In order to synthesize the FITC-KYF-Pt-NE, dissolve 1 mg (0.00105 mmol) of FITC-KYF and 1 mg (0.00219 mmol) of KYF (1:2 molar ratio of FITC-KYF:KYF) in 20 mL of water and adjust the temperature of the solution to 37 &#176;C. Cover the walls of the container with aluminum foil to avoid photobleaching of the FITC fluorophore. To synthesize the KYF-Pt-NE, dissolve 2 mg (0.0044 mmol) of KYF tripeptide in 20 mL of water and adjust the temperature of the solution to 37 &#176;C.</li>
		<li>Add the oleic acids&#8211;Pt(II) conjugate dropwise to the solution of FITC-KYF/KYF or KYF tripeptide. Perform this step under the hood.</li>
		<li>Stir the solution overnight at room temperature to evaporate organic solvents and to allow the self-assembly of FITC-KYF-Pt-NE or KYF-Pt-NE.</li>
		<li>Concentrate the FITC-KYF-Pt-NE or KYF-Pt-NE in a centrifugal concentrator (10k MWCO), and wash thrice with 4 mL of the nanopure water.</li>
		<li>Store the aqueous solutions of KYF-Pt-NE and FITC-KYF-Pt-NE at 4 &#176;C.</li>
		<li>Analyze for platinum content using atomic absorption spectroscopy (AAS), following the manufacturer&#8217;s guide48.
		<ol>
			<li>Prepare platinum standards in 10% HCl solution for the calibration curve (effective range for AAS is between 100 to 1,200 ppb (parts per billion)).</li>
			<li>Dissolve 50 &#181;L of KYF-Pt-NE solution from step 2.2 in 100 &#181;L of aqua regia (a mixture 3:1 of concentrated hydrochloric acid and nitric acid) and leave at room temperature overnight. Add 850 &#181;L of water to reach a final sample volume of 1 mL. Analyze the sample using AAS. The final acid concentration should be 10% in all analyzed samples.</li>
			<li>Record the reading of Pt concentration in ppb, and compute the final platinum content in the sample (account for sample dilution and initial volume of nanoemulsion).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Confocal Imaging of the Cellular Uptake of FITC-KYF-Pt-NE
<ol>
	<li>Seed 6 ovarian cancer cell lines (A2780, CP70, SKOV3, OV90, TOV21G, and ES2), into 4 well-chamber confocal dishes at the density of 4.7 x 104 cells per chamber, and pre-culture overnight at 37 &#176;C.</li>
	<li>After 24 h, wash the cells thrice with phosphate buffer saline (PBS) and incubate with FITC-KYF-Pt-NE in cell culture medium (see step 6.1 for cell line specific details) for 15 min at 37 &#176;C.</li>
	<li>After incubation, remove the media and wash the cells three times with PBS.</li>
	<li>Fix the cells with cold methanol for 5 min at -20 &#176;C and wash three times with 1 mL of PBS.</li>
	<li>Permeabilize the cells with 1 mL of 0.1% Triton-X for 10 min at room temperature and wash three times with 1 mL of PBS.</li>
	<li>Incubate the cells for 90 min with LAMP1 antibody conjugated with Alexa Fluor 647 (1 mL) at 1:50 dilution in PBS at room temperature. Next, wash the cells 3 times with PBS.</li>
	<li>Dilute DAPI stock solution (1 mg/mL) to 1 &#956;g/mL in PBS. Then, add 1 mL of the diluted solution to each chamber and incubate for 15 min at room temperature. Wash the cells 3 times with PBS.</li>
	<li>Mount coverslips on a slide using mounting medium.</li>
	<li>Image the cells using live cell confocal microscope at excitation wavelength of 405 nm, 488 nm and 633 nm. Set the detection parameters as follow: laser power from 0.2% and no more than 1%, Pinole 1 Airy unit, Gain master 650-750, Digital offset 0.</li>
	<li>Open image in imaging software. Under displayed image, select Graphics and select Insert Scale Bar, to insert the scale bar to the image.</li>
</ol>
4. Drug Release Studies
<ol>
	<li>Carry out the drug release studies in PBS. Adjust the pH values of three PBS buffers to 7.4, 6.8 and 5.0 respectively.</li>
	<li>Dilute 5 &#956;L of KYF-Pt-NE in 180 &#956;L of appropriate pH PBS buffer, transfer to 3.5 kDa MWCO mini dialysis cup and incubate at 37 &#176;C in PBS.</li>
	<li>Remove buffer from each three mini dialysis tubes at 2, 4, 6, 24, 48, and 140 h, and measure the platinum concentrations by AAS. Prepare all samples according to step 2.2.8 but adjust the final volume of the sample to 500 &#181;L.</li>
</ol>
5. Cell Culture Methods
<ol>
	<li>Culture cell lines A2780, CP70, SKOV-3, OV-90, TOV-21G, ES-2 in cell culture medium (DMEM) supplemented with 10% (A2780, CP70, SKOV-3, ES-2) or with 15% (TOV-21G, OV-90) fetal bovine serum (FBS), with L-Glutamine and penicillin/streptomycin. Grow all cells in a 5% CO2, water saturated atmosphere at 37 &#176;C.</li>
	<li>Seed 3 x 105 cells in each 96-well plate and pre-culture overnight for in vitro incubation experiments. Prepare the KYF-Pt-NE, cisplatin, and carboplatin solutions in water. Adjust the concentration of Pt(II) in KYF-Pt-NE to match the concentration of carboplatin and cisplatin for each cell line. Incubate for 72 hours at 37 &#176;C.</li>
	<li>After incubation, evaluate the cellular viability using a colorimetric assay (the MTT Cell Proliferation assay). Briefly, remove medium and add 110 &#956;L of 10% MTT in medium to each well and incubate for 2 h at 37 &#176;C. Then add 100 &#956;L detergent to each well and incubate for 5 h at 37 &#176;C.</li>
	<li>Check the absorbance at 570 nm by using plate reader. Analyze results using statistical analysis with Z-test and P-test.</li>
</ol>

Authors do not have a conflict of interest to disclose.

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&lt;br/&gt; 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&amp;rsquo;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&amp;rsquo;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&amp;rsquo;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&amp;rsquo;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&amp;rsquo;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 ...

a-tripeptide-stabilized-nanoemulsion-of-oleic-acid

Research

JoVE Journal

Bioengineering

9.3K Views.  Brooklyn College, The City University of New York. Gezien het brede scala van korte peptide sequenties, kan deze methode worden uitgebreid tot vele andere nanodeeltjes ontwerpen. Peptide modificatie kan het systeem geschikt maken voor diagnostische doeleinden. Het inkapselen van anti-kanker platina aan geneesmiddelen met biologisch afbreekbare, lage toxiciteit stabiliserende agent is zeer relevant voor farmaceutische toepassingen.Voorlopige in-vivo studies met eierstokkanker zijn veelbelovend en zullen verder worden onderzocht. Om te b...

Video: A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

Engineering

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Chemistry

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Triplet Fusion Upconversion Nanocapsule Synthesis

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has significant implications for producing high energy light beyond a material's surface. However, the deployment of UC materials has been stymied due to poor material solubility, high concentration requirements, and oxygen sensitivity, ultimately resulting in reduced light output. Toward this end, nanoencapsulation has been a popular motif to circumvent these challenges, but durability has remained elusive in organic solvents. Recently, a nanoencapsulation technique was engineered to tackle each of these challenges, whereupon an oleic acid nanodroplet containing upconversion materials was encapsulated with a silica shell. Ultimately, these nanocapsules (NCs) were durable enough to enable triplet fusion upconversion-facilitated volumetric three-dimensional (3D) printing. By encapsulating upconversion materials with silica and dispersing them in a 3D printing resin, photopatterning beyond the surface of the printing vat was made possible. Here, video protocols for the synthesis of upconversion NCs are presented for both small-scale and large-scale batches. The outlined protocols serve as a starting point for adapting this encapsulation scheme to multiple upconversion schemes for use in volumetric 3D printing applications.

This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Evaluating Immunogenicity of Nanoemulsion&#45;Encapsulated Retinoic Acid in Mice

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid System for Mucosal Vaccination

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

Samenvatting

Meer video's verkennen