Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Summary

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Abstract

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles’ integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Introduction

There is increased interest in the magnetic features of iron oxide nanoparticles for a wide range of biomedical applications. Their response to magnetic resonance makes them reliable contrast agents for magnetic resonance imaging (MRI), an advantage in regenerative medicine where cells labeled with magnetic nanoparticles can be tracked in vivo following implantation¹. Using magnetic fields, cells can also be guided at a distance; this way, cellular spheroids^2,3, rings⁴, or sheets⁵ can be engineered magnetically and also remotely stimulated....

Protocol

1. Synthesis of magnetic nanoparticles

1. Core synthesis – microwave-assisted
   1. Dissolve 400 mg of iron(III) acetylacetonate (>99.9%) in 10 mL of benzyl alcohol (BA, 99.8%) within a 30 mL monowave glass vial.
   2. Increase the temperature of the suspension from 25 to 250 °C in 20 min (at a rate of 11.25 °C/min) and maintain it at 250 °C for 30 min using a microwave reactor.
   3. Transfer the resulting nanoparticles suspended in benzyl alcohol to a glass vial and separate the nanoparticles using a neodymium magnet for 30 min.
   4. Wash the precipitate in the previous 100 mL glass vial with 10 mL of dichlorom....

Results

Using the microwave-assisted synthesis, magnetic nanoparticles with a monodisperse 8.8 ± 2.5 nm core size are produced and coated with either citrate or PAA (Figure 1A). Stem cells are then incubated with these nanoparticles dispersed in culture medium at a given concentration for 30 minutes, resulting in their endocytosis and confinement within the cellular endosomes (Figure 1B). The magnetic stem cells are then suspended in medium, centrifuged, and the ce.......

Discussion

Using a fast and efficient microwave-based synthesis, magnetic nanoparticles can easily be synthesized, with controlled size, and further coated with given molecules. A critical step is to stock the iron salt and the benzyl alcohol under vacuum to keep a small dispersion in size. The benzyl alcohol acts as both as solvent and ligand at the same time allowing to directly obtain calibrated bare iron oxide without the need of additional ligands. After nanoparticles transfer in water the bare magnetic nanoparticles can be ea.......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA (1x)	Life Technologies	25300-054
Benzyl alcohol for synthesis	Sigma Aldrich	8.22259
Dexamethasone	Sigma	D4902	Prepare a 1 mM stock solution diluted in Ethanol 100% and store at -20°C
Dichloromethane ≥99% stabilised, GPR RECTAPUR	VWR Chemicals	23367
DMEM with Glutamax I	Life Technologies	31966-021	No sodium pyruvate, no HEPES
Ethanol absolute	VWR	20821.310
Fetal Bovine Serum	Life Technologies	10270-106
Formalin solution 10% neutral buffered	Sigma	HT5012
Hydrochloric acid, 1.0N Standardized Solution	Alfa Aesar	35640
Iron(III) acetylacetonate (> 99.9%)	Sigma Aldrich	517003
ITS Premix Universal Culture Supplement (20x)	Corning	354352
L-Ascorbic Acid 2-phosphate	Sigma	A8960	Prepare a fresh concentrated solution (25 mM) diluted in distilled water
L-Proline	Sigma	P5607	Prepare a 175 mM stock solution diluted in distilled water and store at 4°C
Mesenchymal Stem Cell (MSC)	Lonza	PT-2501
Monowave glass vial	Anton Paar	82723_us
Microwave reactor	Anton Paar	Monowave 300
MSCGM BulletKit medium	Lonza	PT-3001	For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium
PBS w/o CaCl2 w/o MgCl2	Life Technologies	14190-094
Penicillin (10.000U/mL)/Streptomicin (10.000µg/mL)	Life Technologies	15140-122
Poly(acrylic acid, sodium salt)	Sigma Aldrich	416010	MW = 1200 g/mol
RPMI medium 1640, no Glutamine	Life Technologies	31870-025	No sodium pyruvate, no HEPES
Sodium hydroxide, 1.0N Standardized Solution	Alfa Aesar	35629
Sodium pyruvate solution 100mM	Sigma	S8636
Sterile conical centrifuge tube	Falcon	352097	15 mL tubes
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher Scientific	25300054
Tri-sodium citrate	VWR	33615.268	Prepare a 1 M stock solution diluted in distilled water and store at 4°C
Tri-Sodium Citrate Dihydrate, Certified AR for Analysis	Sigma Aldrich	10396430
Ultra centrifugal filter	Amicon	AC S510024