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 
			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...

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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 ...

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Research

JoVE Journal

Bioengineering

9.3K Views.  Brooklyn College, The City University of New York. 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.

Video: A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Sustained Administration of &amp;#946;-cell Mitogens to Intact Mouse Islets &lt;em&gt;Ex Vivo&lt;/em&gt; Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

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 ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Optimized CRISPR-Based Molecular Diagnostics Protocol for Point-of-Care Use

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes ...

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...