Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Summary

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Abstract

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe₃O₄) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Introduction

Targeted delivery and capture of cells to specific sites within the body is desirable for a variety of biomedical applications. Delivery of neural stem cells to the brain by MRI-guided focused ultrasound has been proposed as a possible treatment option for neurodegenerative disease, traumatic brain injury, and stroke¹. Mesenchymal stem cells are being studied for their ability to deliver anti-cancer drugs to tumors due to their natural tumor-tropic properties^2,3. Cardiac stem cells have been delivered to the heart as a possible treatment for myocardial infarction^4,5. Vascular stents have been developed with CD34 antibodies to captu....

Protocol

1. Synthesis of Magnetite Gel

1. Wash all glassware by using concentrated hydrochloric acid followed by deionized water followed by ethyl alcohol. Allow to dry O/N, preferably in a drying oven.
   CAUTION! hydrochloric acid is harmful – wear personal protective equipment and work in a fume hood; ethyl alcohol is harmful – wear personal protective equipment.
2. Use a Dreschel bottle to de-gas 500 ml of deionized H₂O by gently bubbling N₂ gas for 30 mins.
3. Set-up the magnetite synthesis apparatus within a chemical fume hood.
   1. Place a 500 ml three-neck round-bottom flask within an isomantle heater&....

Results

Magnetite nanoparticles are approximately 10 nm in diameter as a result of stirring an aqueous solution of iron(III) chloride and iron(II) chloride tetrahydrate at 50 °C and 1,000 rpm (Figure 1). These results demonstrate successful synthesis of magnetite nanoparticles. It is important to verify the size and shape of magnetite nanoparticles taken from a small sample of the batch when attempting the synthesis for the first time. Transmission electron microscopy (TEM) is the preferred method for visualizin.......

Discussion

As with any nanoparticle synthesis protocol, the purity of the reactant chemicals is critical for achieving high quality SPIONs that will have minimal cytotoxic effects. It is therefore important to purchase very pure reagents including oleic acid (≥99%), iron(II) chloride tetrahydrate (≥99.99%), iron(III) chloride (≥99.99%), ethyl acetate (HPLC grade, ≥99.9%), hexane (HPLC grade, ≥97.0%), ammonium hydroxide (≥99.99%), and sodium sulfate (≥99.0%). It is of particular importance t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium Hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metal basis	Sigma-Aldrich	338818-100ML	Harmful reagent - wear personal protective equipment
Dreschel bottle, 500 ml	Ace Glass	5516-16
Ethyl Acetate, CHROMASOLVR Plus, for HPLC, 99.9%	Sigma-Aldrich	650528-1L	Harmful reagent - wear personal protective equipment & work in fume hood
Ethyl alcohol	Sigma-Aldrich	E7023	Harmful reagent - wear personal protective equipment
Evaporating flask, 50 ml, 24/40 joint	Sigma-Aldrich	Z515558	For use with rotoevaporator
Filter paper, 3 cm dia, grade 1	Fisher	09-805P	For use with glass filter funnel
Glass beakers, 1 L	Fisher	FB-101-1000	For washing SPIONs
Glass filter funnel, vacuum hose adapter, fits 24/40, 30 mL	Fisher	K954100-0344
Glass vial caps	Fisher	03-391-46	For use with glass vials
Glass vials, 2 ml	Fisher	03-391-44	For collecting magnetite gel & SPIONs
Hexane, CHROMASOLVR, for HPLC, ≥97.0% (GC)	Sigma-Aldrich	34859-1L	Harmful reagent - wear personal protective equipment & work in fume hood
Hydrochloric acid	Sigma-Aldrich	H1758	Harmful reagent - wear personal protective equipment & work in fume hood
Iron(II) chloride tetrahydrate, ≥99.99% trace metals basis	Sigma-Aldrich	380024-5G	Harmful reagent - wear personal protective equipment
Iron(III) chloride anhydrous, powder, ≥99.99% trace metals basis	Sigma-Aldrich	451649-1G	Harmful reagent - wear personal protective equipment
Isomantle heater, 500 mL	Voight Global	EM0500/CEX1
Laboratory mixer	Silverson	L5M-A
Lyophilizer	Labconco	7670520
Microspatulas	Fisher	21-401-25A	For transfering magnetite gel
NdFeB magnet, 1 in x 1 in x 1 in	Amazing Magnets	C1000H-M	Very strong magnet, handle with care
Oleic acid, ≥99% (GC)	Sigma-Aldrich	O1008-5G	Store in freezer; Harmful reagent - wear personal protective equipment
Overhead stirrer	IKA	2572201
Overhead stirrer clamp	IKA	2664000	For use with overhead stirrer
Overhead stirrer H-stand	IKA	1412000	For use with overhead stirrer
Phosphate buffered saline	Life Technologies	10010-023
Plastic beakers, 250 ml	Fisher	02-591-28
PLGA PURASORB PDLG (75/25 blend)	Purac	PDLG 7502	PDLG 7502A may be used as well; Store in freezer
Pluronic F-127 powder, BioReagent, suitable for cell culture	Sigma-Aldrich	P2443-250G
PTFE expandable blade paddle, 8 mm dia	SciQuip	SP4018
PTFE vessel adapter, fits 24/40, 8 mm dia paddle	Monmouth Scientific	PTFE Vessel Adaptor A480	For use with PTFE expandable blade paddle
Recirculating chiller	Clarkson	696613	For use with rotoevaporator
Reflux condenser, fits 24/40, 250 mm	Ace Glass	5997-133
Rotoevaporator	Clarkson	216949
Rubber septa, fits 24/40	Ace Glass	9096-56
Separatory funnel with stopper, 250 ml	Fisher	10-438E
Sodium sulfate ACS reagent, ≥99.0%, anhydrous, granular	Sigma-Aldrich	239313-500G
Three neck round bottom flask, angled, 24/40 joints, 500 ml	Ace Glass	6948-16
Ultrasonic cleaner perforated pan	Fisher	15-335-20A	For use with ultrasonic cleaner
Ultrasonic cleaner, 2.8 L	Fisher	15-335-20
Vacuum controller	Clarkson	216639	For use with rotoevaporator (optional)
Vacuum pump	Clarkson	219959	For use with rotoevaporator