Name: A Tripeptide-Stabilized Nanoemulsion of Oleic Acid (Video) | JoVE Journal
Uploaded: 2019-02-27T13:00:00.000+00:00
Duration: 10 min 42 s
Description: Learn to synthesize a KYF tripeptide-stabilized nanoemulsion of oleic acid-platinum(II) conjugate for anti-cancer drug delivery. This method enables encapsulation of hydrophobic therapeutics using biodegradable, low-toxicity stabilizing agents.

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5 minutes

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

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 ± 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 Zeta...

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 ...

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The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

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

Learn to synthesize a KYF tripeptide-stabilized nanoemulsion of oleic acid-platinum(II) conjugate for anti-cancer drug delivery. This method enables encapsulation of hydrophobic therapeutics using biodegradable, low-toxicity stabilizing agents.

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

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