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 stimulated⁶, an asset in the development of scaffold-free tissues. The range of possibilities for these nanoparticles also includes drug delivery systems^7,8 and magnetic and photoinduced hyperthermal treatment to kill cancerous cells^9,10,11. For all these applications, the nanoparticles are integrated in the biological environment either by intravenous injection or via direct internalization in cells and are then left within, which brings into question their intracellular fate.

In vivo analyses conveyed a general understanding of the nanoparticles’ fate in the organism: upon injection in the blood stream, they are first captured mostly by the macrophages of the liver (Kupffer cells), spleen and bone marrow, are progressively degraded, and join the iron pool of the organism^{12,13,14,15,16,17,18,19}. Qualitative observations are only possible due to the circulation of the nanoparticles throughout the organism. Typically, transmission electronic microscopy (TEM) can be used to directly observe the nanoparticles and the presence of iron in the organs can be determined via dosage. More recently, their fate has been assessed directly on a pool of cells, meaning in close circuit with no iron escape, allowing a quantitative measurement of their biotransformations at the cell-level^20,21,22. Such measurements are possible via the analysis of the magnetic properties of the nanoparticles that are tightly linked to their structural integrity. Vibrating sample magnetometry (VSM) is a technique where the sample is vibrated periodically so that the coil-measurement of the flux induced provides the magnetic moment of the sample at the applied magnetic field. Such synchronous detection allows for a rapid measurement, which is an asset for determining the magnetic moments of a large number of samples^20,21,22,23. The macroscopic magnetic signature retrieved by VSM then gives a quantitative overview of the entire biological sample directly correlated to the nanoparticles' size and structure. In particular, it provides the magnetic moment at saturation (expressed in emu) of the samples, which is a direct quantification of the number of magnetic nanoparticles present in the sample, respectively to their specific magnetic properties.

It has been shown that the intracellular processing of magnetic nanoparticles is tightly linked to their structural features²⁰. These features can be controlled via optimal synthesis protocols. Each protocol presents advantages and limitations. Iron oxide nanoparticles are commonly synthesized in aqueous solutions via coprecipitation of iron ions²⁴. To overcome the limitations of nanoparticles size polydispersity, other synthesis methods such as polyol-mediated sol-gel methods have been developed²⁵. Nonaqueous approaches by thermal decomposition leads to the production of very well-calibrated iron oxide nanoparticles²⁶. However, the use of massive amounts of surfactants like oleylamine or oleic acid complicates their functionalization and water transfer for biomedical applications. For this reason, we synthesize such magnetic nanoparticles through a nonaqueous sol gel route leading to high crystallinity, purity and reproducibility²⁷. This protocol produces well-controlled size nanoparticles that can be tuned through temperature variation²⁸. Nevertheless, the microwave-assisted non-aqueous sol-gel route has an upper size limit of the obtained nanoparticles of around 12 nm. This procedure would not be adapted for applications using ferromagnetic particles at room temperature. In addition to the core synthesis, another main feature to be considered is the coating. Lying at the surface of the nanoparticle, the coating acts as an anchoring molecule, helping the targeted internalization of the nanoparticles, or it can protect the nanoparticle from degradation. Since benzyl alcohol acts as an oxygen source and a ligand at the same time, bare nanoparticles are produced without the need for additional surfactants or ligands. The nanoparticles are then easily surface functionalized after synthesis without a surfactant exchange process.

Herein, two types of nanoparticles are assessed that possess the same core and differ in the coating. The core is synthesized using a fast and highly efficient microwave based technique. The two coatings compared consist of citric acid, one of the most used as coating agent in biomedical applications^29,30, and polyacrylic acid (PAA), a polymeric coating with a high number of chelating functions. VSM magnetometry measurements are then used first to quantify the nanoparticle uptake by the cells, and then as a direct assessment of the nanoparticle structural integrity upon internalization in stem cells. Results demonstrate that the incubation concentration impacts nanoparticle uptake and that the coating influences their degradation, with the large number of anchoring molecules of PAA protecting the core from degradation.

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 dichloromethane, 1 M sodium hydroxide, ethanol, pH 7 water (three times) using a neodymium magnet for 5 min each step to pelletize nanoparticles and remove the supernatant.
   5. Adjust the nanoparticle suspension in water to pH 2 using 1 M hydrochloric acid, and then centrifuge in 100 kDa ultracentrifugal filters at 2,200 x g for 10 min.
   6. Remove the filtrate, add 12 mL of 10^-2 M hydrochloric acid to the nanoparticles solution and centrifuge in 100 kDa ultracentrifugal filters at 2,200 x g for 10 min (three times). Then dilute the nanoparticles solution within 10 mL of 10^-2 M hydrochloric acid before coating.
2. Coating
   1. Dilute 175 mg of citric acid and polyacrylic acid in water at pH 2 in a 100 mL glass vial and adjusted the pH to 2 with a 1 M hydrochloric acid solution.
   2. Add the 10 mL nanoparticles aqueous dispersion to the coating molecule solution, corresponding to a mass ratio of 5 between the coating molecule and the nanoparticles. Agitate for 2 h under magnetic stirring at room temperature.
   3. After the reaction, adjust the pH to 7 with 1 M sodium hydroxide.
   4. Centrifuge the nanoparticle solution 3 times with deionized (DI) water in 100 kDa ultracentrifugal filters at 2,200 x g for 10 min; remove the filtrate and dilute the nanoparticles solution in 12 mL of DI water.

2. Culture and magnetic labeling of stem cells

1. Culture human mesenchymal stem cells (MSC) in complete Mesenchymal Stem Cell Growth Medium (MSCGM) at 37 °C and 5% CO₂. When the cells are at 90% confluence, add 10 mL of trypsin-EDTA pre-warmed at 37°C per 150 cm² flask and leave for 2-3 minutes to detach the cells.
   1. To know when the cells are detached, observe them with a bright-field microscope. Resuspend the detached cells in MSCGM and divide them in four 150 cm² flasks. Amplify the cells this way until passage 4 to 5.
2. Prepare the solution of magnetic nanoparticles for cell labeling: disperse the chosen concentration of iron oxide nanoparticles in serum free Roswell Park Memorial Institute medium (RMPI-1640) without glutamine. RPMI is used for nanoparticle incubation (and the related rinsing steps) as it has a lower ionic strength than DMEM and better prevents nanoparticle aggregation events.
3. When the cells are at passage 4 or 5 and 90% confluent, remove the MSCGM medium, rinse the cells with serum free RPMI (without glutamine) and add 10 mL of iron oxide nanoparticles solution per 150 cm² culture flask, which is the minimum volume required to cover all the cells.
4. Incubate at 37 °C and 5% CO₂ for 30 min and then discard the nanoparticle solution. Wash once with serum free RPMI-1640 (no glutamine) and incubate with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (25 mL per 150 cm² flask) overnight at 37 °C and 5% CO₂ to allow a complete internalization of the nanoparticles.

3. Formation of stem cell-spheroids

1. Freshly prepare cell-spheroid culture medium composed of DMEM high glucose with L-glutamine supplemented with 50 µM L-ascorbic acid 2-phosphate, 0.1 µM dexamethasone, 1 mM sodium pyruvate, 0.35 mM L-proline, and 1% universal culture supplement containing Insulin, human Transferrin and Selenous acid (ITS-Premix).
2. Detach the magnetic MSCs by adding 10 mL of 0.05% trypsin-EDTA pre-warmed at 37 °C per 150 cm² flask for 2-3 minutes. When the cells are detached, immediately inactivate the trypsin by adding 1/3 of the volume of DMEM supplemented with 10% FBS and 1% penicillin-streptomycin pre-warmed at 37 °C.
3. Centrifuge the dissociated cells at 260 x g for 5 min, aspirate the media and re-suspend the cells in a small volume (less than 1 mL per 150 cm² flask) of cell-spheroid culture medium such as having 200,000 cells in about 50 µL of medium. Count the cell number using a hemocytometer and adjust the volume if needed.
4. Add 1 mL of freshly prepared cell-spheroid culture medium in a 15 mL sterile conical centrifuge tube and add the volume of resuspended solution corresponding to 200,000 cells.
5. Centrifuge these magnetically labeled cells at 180 x g for 3 min such as forming a cell pellet at the bottom of the tube and keep the supernatant, which is the cell culture medium.
6. Slightly open the tubes to allow air exchange and incubate at 37 °C with 5% CO₂ for up to 21 days, culture time along which the cells form a cohesive 3D structure that results in a fully formed spheroid. Change the medium twice a week.
7. On given days, wash the spheroids twice with cacodylate buffer made of 0.2 M cacodylate diluted in demineralized water and fix them using 2% glutaraldehyde in 0.1 M cacodylate buffer diluted in distilled water for 30 min at room temperature. Store the fixed spheroids in PBS until used for VSM measurement or TEM imaging. This fixation step stops all biological processes and allows long-term conservation.

4. Quantification of magnetic nanoparticles in solution and in cellulo using a vibrating sample magnetometer (VSM)

1. Place either a given volume of magnetic nanoparticle solution (maximum 10 µL) or a single cell-spheroid into the sample holder specifically designed to fit in the VSM.
2. Insert the sample in the VSM and scan for sample offset. Place the sample at the position corresponding to the magnetization maximum.
3. Perform the first measurement at a low magnetic field (between -1500 Oe and +1500 Oe) with a 20 Oe/s rate.
4. Perform a second measurement at a high magnetic field (between -30 000 Oe and +30 000 Oe) with a 200 Oe/s rate.

5. Transmission Electron Microscopy (TEM) analysis

1. For the nanoparticle solutions, deposit 10 μL of the aqueous solution onto a carbon-coated copper grid and let it dry at room temperature.
2. For the nanoparticles internalized in cells, contrast the fixed cell-spheroids with Oolong Tea Extract (OTE) at 0.5% diluted in 0.1 M cacodylate buffer, post fix with 1% osmium tetroxide containing 1.5% potassium cyanoferrate and then dehydrate in graded ethanol baths, included in Epon. Slice ultrasections of 70 nm and deposit them onto cooper grids.
3. Take images using an electron microscope at 80 kV with a magnification from 1k for the observation of entire cells to 40k for the observation of endosomal compartments.

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