A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

Summary

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.

Abstract

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 ± 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.

Introduction

In recent years, there has been a growing interest in the engineering of nanoparticles for such biomedical applications as drug delivery and bioimaging^1,2,3,4. 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

Protocol

1. Synthesis of the Oleic Acids–Platinum(II) Conjugate

1. Activation of cisplatin
   1. Suspend 50 mg (0.167 mmol) of cisplatin in 4 mL of water (e.g., nanopure) at 60 °C.
   2. 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 °C. The white precipitate of AgCl will form indicating the progress of the reaction.
   3. 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).
   ....

Results

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.......

Discussion

Critical steps in the nanoemulsion synthesis include adjusting the molar ratio of the substrates, maintaining temperature and flow rate control during oleic acids–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.......

Materials

List of materials used in this article

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