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 capture circulating progenitor cells⁶. While promising, these cell targeting approaches present drawbacks including lack of cell specificity, inconsistent cell retention, and off-target cell delivery.

The overall goal of the current method is to enable magnetically directed targeting of cells for a variety of cell delivery and sorting applications. Magnetic targeting allows for controlled delivery of specific cells to a specific target site with minimal off-target effects⁷. The magnetic fields can be generated by implanted or external devices to safely direct the movement of magnetically-labeled cells within the body⁸. Numerous research efforts have focused on magnetically directed targeting of stem cells to injured tissues such as the heart^9-14, retina¹⁵, lung¹⁶, skin¹⁷, spinal cord^18,19, bone²⁰, liver²¹, and muscle^22,23 in order to improve regeneration outcomes.

Magnetic targeting of cells has also been studied extensively as a means to endothelialize implantable cardiovascular devices. A uniform and complete endothelium provides a barrier between the device and circulating blood elements to mitigate thrombosis and inflammation. Endothelial cells can be delivered to the device either prior to implantation or via the vascular system following implantation. In both cases, magnetic fields are used to capture cells to the surface of the device and retain the cells when subjected to the shear stress generated by circulating blood. Magnetic vascular stents^24-27 and vascular grafts²⁸ have both been fabricated and tested for this purpose.

Magnetic cell targeting requires a strategy for labeling cells with magnetic carrier particles. These particles can be bound to the surface of cells via antibodies or ligand/receptor pairs or they can be endocytosed into the cells. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and readily endocytosed by a variety of cell types²⁹. These particles effectively render a cell responsive to magnetic fields and are naturally degraded over time. SPIONs provide a straightforward and safe means of magnetically labeling cells in culture for a variety of magnetic targeting and sorting applications. A method for synthesizing SPIONs with a magnetite (Fe₃O₄) core and poly(lactic-co-glycolic acid) (PLGA) shell is provided. In addition, a method for labeling cells in culture with SPIONs is provided.

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 and secure the center neck using a clamp and stand.
   2. Install a rubber septum into one of the round-bottom flask’s side necks and a reflux condenser with a rubber septum into the remaining side neck. Continuously run cold water through the reflux condenser.
   3. Puncture the round-bottom flask’s rubber septum with a needle connected to an N₂ gas line and puncture the reflux condenser’s rubber septum with a needle connected to a gas line running to a bubbler (i.e., flask with water) to visualize gas outflow.
   4. Install a blade paddle into the round-bottom flask’s center neck via a paddle adapter. Attach the blade paddle’s shaft to an overhead stirrer mounted onto a stand.
4. Purge the round-bottom flask with N₂ gas and leave N₂ gas flowing at a low but detectable rate.
5. Remove the reflux condenser from the round-bottom flask and add 1.000 g of iron(III) chloride, 0.6125 g of iron(II) chloride tetrahydrate, and 50 ml of de-gassed H₂O.
   CAUTION! iron(III) chloride and iron(II) chloride tetrahydrate are harmful – wear personal protective equipment.
6. Replace the reflux condenser and stir at 1,000 rpm while heating to 50 °C. Stirring under these conditions produces 10 nm diameter magnetite nanoparticles.
7. Once at 50 °C, add 10 ml of 28% ammonium hydroxide solution by injecting through the rubber septum in the round-bottom flask while still stirring.
   CAUTION! ammonium hydroxide is harmful – wear personal protective equipment.
   NOTE: The ammonium hydroxide solution is used to precipitate the magnetite and the solution should turn black.
8. Remove the rubber septum and N₂ gas line from the round-bottom flask and heat to 90 °C to boil off the ammonia gas while still stirring.
   NOTE: It is optional to maintain the flow of N₂ into the round-bottom flask by puncturing the reflux condenser’s rubber septum, however, oxidation of magnetite to maghemite is negligible during this step.
9. Once at 90 °C, add 1 ml of oleic acid to the round-bottom flask while still stirring. The oleic acid is used to coat the magnetite nanoparticles to form magnetite gel.
   CAUTION! oleic acid is harmful – wear personal protective equipment.
10. Replace the rubber septum and N₂ gas line onto the round-bottom flask and remove the reflux condenser.
11. Turn off heat and stir at 500 rpm for 2 hr.
12. Remove the round-bottom flask from the isomantle heater and decant any remaining liquid while using a strong magnet held against the bottom of the flask to retain the magnetite gel.
    CAUTION! handle the strong magnet with extreme care to avoid damage or injury.
13. Allow magnetite gel to air-dry O/N (optional).

2. Purification of Magnetite Gel

1. Add 40 ml of hexane into the round-bottom flask to dissolve the magnetite gel
   CAUTION! hexane is harmful – wear personal protective equipment and work in a fume hood.
2. Use a separatory funnel with 40 ml of de-gassed H₂O to remove residual H₂O from the magnetite solution.
   1. Slowly pour the magnetite solution onto the H₂O within the separatory funnel and gently swirl the two-phase liquid for 5 mins.
   2. Drain out and discard the lower aqueous fraction.
   3. Slowly add 40 ml of de-gassed H₂O to the separatory funnel such that it settles beneath the magnetite solution and gently swirl and drain as before.
   4. Repeat to wash for a third time.
3. Transfer magnetite solution to an Erlenmeyer flask, add a few spatulas worth of anhydrous sodium sulfate, and swirl to remove any remaining residual H₂O from the magnetite solution.
4. Filter the magnetite solution through 1 µm filter paper in a filter funnel to remove the sodium sulfate and residual H₂O.
   NOTE: Vacuum assistance is recommended.
5. Transfer the magnetite solution to a 50 ml evaporating flask and use a rotary evaporator to evaporate the hexane for 2 h with the following conditions: moderate rotation speed, vacuum applied, evaporating flask in a 50 °C water bath, and 24 °C water circulating through the condenser.
   NOTE: Optionally, store the magnetite gel prior to coating with PLGA.

3. Coating of Magnetite Nanoparticles with PLGA Shell

1. Dissolve 3.60 g of PLGA (75/25 blend) in 240 ml of ethyl acetate to create a 1.5% (m/v) solution. CAUTION: ethyl acetate is harmful – wear personal protective equipment and work in a fume hood.
2. Dissolve 25.00 g of Pluronic F-127 in 500 ml of de-gassed H₂O using a magnetic stirrer to create a 5.0% (m/v) solution.
   NOTE: Pluronic F-127 is a non-ionic amphiphilic block copolymer that acts as a biocompatible surfactant. It helps to stabilize the oil-in-water emulsion in step 3.3.2.
3. Using a microspatula, collect the magnetite gel into six 0.040 g aliquots within weighted glass vials. Perform the following coating and washing process for each aliquot.
   NOTE: The aliquots are necessary to ensure efficient handling and magnetic decantation, which will maximize purity and yield while minimizing degradation prior to freeze-drying in step 4.
   1. Add a 0.040 g aliquot of magnetite gel and 40 ml of the PLGA solution to a plastic beaker and sonicate in an ultrasonic cleaner for 10 mins.
   2. Add 80 ml of the Pluronic solution to the plastic beaker and immediately emulsify with a laboratory mixer at the highest setting for 7 mins to form the PLGA coating on the magnetite nanoparticles as an oil-in-water emulsion.
   3. Immediately dilute the SPION solution in 1 L of deionized H₂O and sonicate for 1 h in a chemical fume hood to evaporate the ethyl acetate.
   4. Place a strong magnet next to the SPION solution and gently stir to collect brownish SPIONs at the magnet.
      NOTE: It may be necessary to intermittently stir for several hours before the solution turns whitish indicating that most of the SPIONs have been collected.
   5. Decant the aqueous solution while retaining the SPIONs in the beaker with the magnet.
   6. Wash the SPIONs three times as follows.
      1. Suspend the SPIONs in 1 L of deionized H₂O.
      2. Sonicate the SPION solution for 20 mins.
      3. Place a strong magnet next to the SPION solution and gently stir to collect brownish SPIONs at the magnet. It may be necessary to intermittently stir for several hours before the solution turns clear indicating that most of the SPIONs have been collected.
      4. Decant the aqueous solution while retaining the SPIONs in the beaker with the magnet.
4. Collect the SPIONs synthesized from each of the six magnetite gel aliquots into a single weighted glass vial as an aqueous suspension. Optionally decant excess water magnetically as needed.

4. Freeze-drying of SPIONs

1. Freeze the SPION solution.
2. Freeze-dry the SPION solution O/N in a lyophilizer.
3. Weigh the freeze-dried SPIONs. Freeze-dried SPIONs can be stored at -20 °C until used for cell labeling.
   NOTE: Storage at -20 °C dramatically reduces degradation kinetics and increases shelf life.

5. Labeling of Cells with SPIONs

1. Suspend an aliquot of SPIONs in phosphate-buffered saline (PBS) at a concentration of 40 mg/ml and sonicate for 30 mins.
2. Add the SPION solution to a nearly confluent flask of cells at a concentration of 5 µl/ml of cell culture medium. Ensure even distribution by gently rocking the flask.
3. Incubate the cells for 16 hr at 37 °C.
4. Gently aspirate culture medium and wash cells twice with PBS.
5. Collect magnetically-labeled cells and use for experiments.
6. Unused SPION solution can be stored at 4 °C and should be used within a few months. Sonicate for 30 mins before each use.

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