Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Summary

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Abstract

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture and detection of circulating tumor cells (CTCs), and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Introduction

Hybrid nanoparticles consisting of different materials with distinct physicochemical properties can open new opportunities in biomedical applications including multimodal molecular imaging, therapy delivery and monitoring, new screening and diagnostic assays^1-3. The combination of plasmonic and magnetic properties in a single nanoparticle is of particular interest because it provides a very strong light scattering and absorption cross-sections associated with plasmon resonances and responsiveness to a magnetic field. For example, magneto-plasmonic nanoparticles were used to increase contrast in dark-field imaging of labeled cells by applying a temporal....

Protocol

1. Instrumentations and Glassware Preparation

1. Wear appropriate protective equipment, i.e., a lab coat, disposable gloves, and eye protection.
2. Connect a round-bottom flask to a condenser and immerse it in a silicone oil bath with a temperature monitoring by a thermometer. Place a source of heat (e.g., hot plate) under the oil bath (Figure 1). Use a thermometer capable of measuring the temperature higher than 260 °C.

2. Synthesis of Primary Hybrid Magneto-plasmonic Nanoparticles

1. Making Magnetic Core Nanoparticles
   1. Add 353.2 mg (1 mmol) iron(III) acetyl....

Results

A scheme for synthesis of immunotargeted magneto-plasmonic nanoclusters is shown in Figure 2. First, magnetic Fe₃O₄ iron oxide nanoparticles are synthetized via thermal decomposition method. Then, a thin ca. 1 nm gold shell is deposited on the iron oxide core particles via thermal decomposition. The primary ca. 6 nm hybrid nanoparticles serve as seeds to create magneto-plasmonic nanoclusters by utilizing an oil-in-water microemulsion approach. The nanoclusters are .......

Discussion

Critical steps in successful synthesis of magneto-plasmonic nanoclusters include making highly monodispersed primary gold shell/iron oxide core nanoparticles and directing self-assembly of the primary particles into nanoclusters. A molar ratio between the primary particles and surfactants play an important role in determining size distribution of the nanoclusters. Non-uniform size distribution of primary nanoparticles may cause formation of big aggregates during assembly of magneto-plasmonic nanoclusters. In addition, th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PYREX 50 ml round bottom boiling flask with short neck & 24/40 [ST] joint	Corning	4320A-50	Thermal decomposition reaction
PYREX 41 x 300 mm 5-bulb Allihn condenser with 24/40 [ST] outer/inner joints	Corning	2480-300	Thermal decomposition reaction
Silicone oil	Fisher	S159-500	Oil bath
Hot plate stirrer	Corning	PC-351	Heat the reacton with stirring function
Thermometer	ThermoWorks	221-092	Measure temperature
Iron(III) acetylacetonate	Fisher	AC11913-0250	Material for primary hybrid nanoparticles synthesis
Oleic acid 99%	Fisher	A195-500	Material for primary hybrid nanoparticles synthesis
Gold(III) acetate	Fisher	AA3974206	Material for primary hybrid nanoparticles synthesis
Hexane	Fisher	H292-1	Material for primary hybrid nanoparticles synthesis
Phenyl ether 99%	Fisher	AC13060-0025	Material for primary hybrid nanoparticles synthesis
1,2-Hexadecanediol 90%	Sigma	213748-50G	Material for primary hybrid nanoparticles synthesis
Oleylamine 70%	Sigma	O7805-100G	Material for primary hybrid nanoparticles synthesis
Sodium dodecyl sulfate	Fisher	BP166-100	Cluster synthesis
Sodium citrate dihydrate	Sigma	W302600	Cluster synthesis
Monoclonal anti-EGF receptor antibody	Sigma	E2156	Cell labeling specificity test
Monoclonal anti-HER2 antibody	Sigma	AMAB90627	Cell labeling specificity test
Sodium periodate	Sigma	311448	Oxidate Fc region of antibodies
Dithiolaromatic PEG6-CONHNH2	SensoPath Technologies	SPT-0014B	Heterofunctional linker for antibody conjugation to nanoclusters
Methoxy-PEG-thiol, 5 k	Creative PEGworks	PLS-604	Passivate the remaining gold surface after antibody conjugation
Amicon Ultra-4 centrifugal filter unit with Ultracel-10 membrane	Millipore	UFC801008	Protein purification
HEPES	Sigma	H3375	Buffer
PBS, 1x solution	Fisher	BP2438-20	Buffer
UV-Vis spectroscopy	BioTek	Synergy HT	Obtain spectrum
Centrifuge	Eppendorf	5810R	Separation
Transmission Electron Microscope	FEI	TECNAI G2 F20 X-TWIN	Obtain morphology of nanostructures
Upright microscope	Leica	DM6000	Obtain dark-field images
Sonicator	Branson	1510	Sonication
Carbon film 300 mesh grid	EMS	CF300-Cu	TEM imaging
96-well plate	Corning	09-761-145	UV-Vis reading plate