Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Samuel Effman; Shawn Avidan; Zhen Xiao; Vicki Colvin

doi:10.3791/63140

JoVE Journal > Chemistry

Chimie

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Published: February 05, 2022

doi:

10.3791/63140

Samuel Effman*¹, Shawn Avidan*¹, Zhen Xiao*¹, Vicki Colvin^1,2

¹Department of Chemistry,Brown University, ²Center for Biomedical Engineering, School of Engineering,Brown University

Summary

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

Abstract

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

Introduction

The inclusion of manganese as a dopant in an iron oxide lattice can, under the appropriate conditions, increase the material's magnetization at high applied fields as compared to pure iron oxides. As a result, manganese ferrite (Mn_xFe₃_-xO₄) nanoparticles are highly desirable magnetic nanomaterials due to their high saturation magnetization, strong response to external fields, and low cytotoxicity¹^,²^,³^,⁴^,⁵. Both single domain nanocrystals as well as clusters of these nanocrystals, termed multidomain particles, have been investigated in diverse biomedical applications, including drug delivery, magnetic hyperthermia for cancer treatment, and magnetic resonance imaging (MRI)⁶^,⁷^,⁸. For example, the Hyeon group in 2017 used single domain manganese ferrite nanoparticles as a Fenton catalyst to induce cancer hypoxia and exploited the material's T₂contrast for MRI tracking⁹. It is surprising in light of these and other positive studies of ferrite materials that there are few in vivo demonstrations as compared to pure iron oxide (Fe₃O₄) nanomaterials, and no reported applications in humans⁹^,¹⁰.

One immense challenge faced in translating the features of ferrite nanomaterials into the clinic is the generation of uniform, non-aggregating, nanoscale clusters¹¹^,¹²^,¹³^,¹⁴. While conventional synthetic approaches to monodomain nanocrystals are well developed, multidomain clusters of the type of interest in this work are not easily produced in a uniform and controlled fashion¹⁵^,¹⁶. Additionally, ferrite composition is usually non-stoichiometric and not simply related to the starting concentration of the precursors and this can further obscure systematic structure-function characterization of these materials⁹^,¹²^,¹³^,¹⁷. Here, we address these issues by demonstrating a synthetic approach that yields independent control over both the cluster dimension and composition of manganese ferrite nanomaterials.

This work also provides a means to overcome the poor colloidal stability of ferrite nanomaterials¹⁸^,¹⁹^,²⁰. Magnetic nanoparticles are generally prone to aggregation due to strong particle-particle attraction; ferrites suffer more from this problem as their larger net magnetization amplifies particle aggregation. In relevant biological media, these materials yield large enough aggregates that the materials rapidly collect, thereby limiting their routes of exposure to animals or people²⁰^,²¹^,²². Hilt et al. found another consequence of particle-particle aggregation in their study of magnetothermal heating and dye degradation²³. At slightly higher particle concentrations, or increased time of exposure to the field, the effectiveness of the materials was reduced as materials aggregated over time and the active particle surface areas decreased. These and other applications would benefit from cluster surfaces designed to provide steric barriers that precluded particle-particle interactions²⁴^,²⁵.

Here we report a synthetic approach to synthesize manganese ferrite clusters (MFCs) with controllable dimensions and composition. These multidomain particles consist of an assembly of primary manganese ferrite nanocrystals that are hard aggregated; the close association of the primary nanocrystals enhances their magnetic properties and provides for an overall cluster size, 50-300 nm, well matched to the optimum dimensions for a nanomedicine. By changing the amount of water and manganese chloride precursor, we can independently control the overall diameter and composition. The method utilizes simple and efficient one-pot hydrothermal reactions that allow for frequent experimentation and material optimization. These MFCs can be easily purified into a concentrated product solution, which is further modified by sulfonated polymers that impart colloidal stability. Their tunability, uniformity, and solution phase stability are all features of great value in applications of nanomaterials in biomedical and environmental engineering.

Protocol

1. Synthesis of MFCs with control over MFCs' overall diameter and ferrite composition

Wash and thoroughly dry all glassware to be used in the synthesis. The amount of water in the synthesis impacts the dimensions of the MFCs, so it is crucial to ensure the glassware has no residual water in it¹⁶^,²⁶.
1. To wash the glassware, rinse with water and detergent and scrub with a flask brush to remove debris. Thoroughly rinse to remove all detergent and finish with a rinse of deionized water.
2. To dry the glassware, shake water droplets off the surface of the glassware and place into an oven at 60°C until completely dry.
3. Rinse the polyphenylene-lined (PPL) reactors with 37% hydrochloric acid to remove any debris from previous use. To do this, place the reactors and their caps in a large beaker and fill with hydrochloric acid until the reactors are completely submerged. Let this sit for 30 min before pouring out the hydrochloric acid. Continuously rinse the beaker containing the reactors with water for 1-2 min, and then place the reactors in the oven to dry.
Use an automatic pipette to transfer 20 mL of ethylene glycol into a 50 mL beaker with a magnetic stir bar.
Weigh out the required amount of iron(III) chloride (FeCl₃·6H₂O, solid) to achieve a final concentration of 1.3 mM and add it to the beaker. Put the beaker on a stir plate and turn it on at 480 rpm to begin continuous stirring of the beaker.
NOTE: As this is a hydrate, it must be measured and added quickly to avoid unwanted absorption of water from the ambient air.
Weigh 250 mg of polyacrylic acid (PAA, Mw ~6,000, powder) and add it to the beaker. After the addition of PAA, the solution becomes opaque and slightly lighter in color.
Weigh 1.2 g of urea (CO(NH₂)₂, powder) and add it to the beaker.
Using a pipette, add 0.7 mM manganese(II) chloride (MnCl₂·6H₂O aq, 3.5 M, 0.2 mL) to the beaker.
Finally, using a pipette add the required amount (0.5 mL) of ultra-pure water to the beaker.
Let the solution stir for 30 min and notice the color change. It will present as a translucent, dark orange color.
Transfer the reaction mixture into the polyphenylene lined (PPL) reactor. Note that after the solution has stirred some solids may have accumulated on the sides of the beaker.
1. Use a magnet (cubic permanent rare earth magnet, 40 x 40 x 20 mm, hereafter referred to as a "magnet" for all separation and magnetic collection procedures) to drag the stir bar around the walls of the beaker to ensure any solids that have accumulated on the sides are dispersed into the reaction solution.
2. Once the solution is mixed and ready, transfer it into the 50 mL PPL lined reactor.
3. Use a clamp and lever to seal the reactor in the stainless-steel autoclave as tightly as possible. Clamp the reactor vessel to a stable surface, and using a rod inserted into the cap as a lever, push the reactor to seal. Note that the sealed reactor should not be able to be opened by hand. This is crucial as the high-pressure environment of the oven requires a tight seal on the reactor.
Place the reactor into an oven for 20 h at 215 °C.
After the hydrothermal reaction is done, remove the reactor from the oven and allow it to cool down to room temperature. The pressure of the oven will enable the reactor to be opened by hand. Note that at this point, the reactor will contain the MFC product dispersed in ethylene glycol with other impurities, such as unreacted polymer, and will be an opaque black solution. The product will be isolated in the following steps.

2. Magnetic separation and purification of MFCs

Place 200 mg of steel wool into a glass vial. Fill the glass vial halfway with the reaction mixture from the reactor. Fill the rest of the vial with acetone and shake well. Note that the steel wool increases the magnetic field strength in the vial and will help magnetic separation of the nanoclusters from the solution.
Place the vial on a magnet for magnetic collection to occur. The result will be a translucent solution with precipitate at the bottom.
1. Pour off the supernatant solution while the MFCs are magnetically trapped by the steel wool by holding the magnet to the bottom of the vial while pouring. Ethylene glycol will be mostly removed in this step.
2. Start washing with the low ratio of acetone to water and increase the ratio in subsequent washes until pure. Do this 3-4 times.
Remove the vial from the magnet and fill it with water. Shake well to dissolve the MFCs. Now the product will be fully dispersed in water.
Repeat the previous two steps several times until the aqueous solution of the MFCs produces no bubbles when shaken. The result will be a dark, opaque ferrofluid that will respond strongly to magnets.
NOTE: In a typical synthesis with 20 mL of ethylene glycol, approximately 80 mg of MFC product will be obtained.

3. Surface functionalization of MFCs toward ultra-high colloidal stability

NOTE: The synthesis of nitro-dopamine and Poly(AA-co-AMPS-co-PEG) can be found in our previous work¹⁶. The copolymer is made through free radical polymerization. Add 0.20 g of 2,2′-Azobis(2-methylpropionitrile) (AIBN), 0.25 g of acrylic acid (AA), 0.75 g of 2-Acrylamido-2-methylpropane sulfonic acid (AMPS), and 1.00 g of Poly(ethylene glycol) methyl ether acrylate (PEG) in 10 mL of N,N-Dimethylformamide (DMF). Heat the mixture in a 70 °C water bath for 1 h and transfer it to a dialysis bag (Cellulose Membrane, 3 kDa) in water. The weight ratio of AA, AMPS, and PEG is 1:3:4. Polymerization for these monomers has a 100% conversion rate as confirmed by freeze drying and weighing.

Combine 10 mL of purified nanoparticles (around 100 mg) in a 20 mL vial with 10 mL of saturated N-[2-(3,4-dihydroxyphenyl)ethyl]nitramide (nitro-dopamine) solution (~1 mg/mL). Wait for 5 min.
Wash the nitro-dopamine coated MFCs using magnetic separation. Pour out the pale-yellow supernatant. Add water and shake vigorously. Then, pour out water using the magnet to retain the product. Repeat this washing several times leaving the dark brown collection in the vial.
NOTE: Prepare a aqueous solution with a concentration of 20 mg/mL, a buffer solution with a concentration of 100 mg/mL, and a poly(AA-co-AMPS-co-PEG) polymer solution with a concentration of 20 mg/mL.
Mix 1 mL of EDC solution, 1 mL of MES buffer, and 3 mL of the polymer solution. Lightly stir by swirling the mixture, and let it sit for approximately 5 min. It should be a clear and colorless solution when fully combined.
Add this mixture to the MFC collection and place the vial in an ice bath. Lower the probe sonicator into the solution, and then turn it on (250 watts of power at 20 kHz).
1. After a 5 min sonication treatment, add roughly 5 mL of ultra-pure water to the vial while the sonicator is still running. Continue monitoring the vessel to ensure that no product spills. Maintain the ice in the ice-water mixture as some of the initial ice will melt due to the intensity and heat of the sonication.
2. Allow the mixture to sonicate for an additional 25 min, for a total of 30 min.
Place the vial on top of a magnet to separate the MFCs and pour out the supernatant solution.
Wash the modified MFCs with deionized water several times.
Fill the vial containing the MFCs with ultra-pure water. Pipette this fluid into a vacuum filtration system with a 0.1 µm polyethersulfone membrane filter to remove any irreversibly aggregated MFCs. Make sure to flush the walls of the funnel to minimize any loss of product.
Vacuum filter the solution. Repeat this process 2-3 times. The result will be a purified aqueous solution of monodispersed MFCs.
NOTE: Roughly 10% of the product will be irreversibly aggregated and this material will remain on the filter and should be discarded.

Representative Results

After hydrothermal treatment, the reaction mixture turns into a viscous black dispersion as can be seen in Figure 1. What results after purification is a highly concentrated MFC solution that behaves like a ferrofluid. The fluid in the vial responds within seconds when placed near a handheld magnet (<0.5 T), forming a macroscopic black mass that can be moved around as the magnet is placed at different locations.

This synthesis yields products whose dimension and ferrite composition depend on the amount of water added and the ratio of manganese to iron precursor in the reaction mixture. Figure 2 illustrates how the cluster morphology depends on water and precursor concentration; it also details the reaction conditions used to obtain the samples listed in Table 1. We find that the MFC diameter is affected by the amount of water added, and the MFC composition depends on the ratio of iron and manganese in the precursors. Both parameters can thus be independently controlled to make a library of MFCs with distinct dimensions and manganese content.

While this is a very simple synthetic procedure, errors in the method execution may lead to failed products. Figure 3 depicts samples with irregular MFC morphologies. In Figure 3A, odd-shaped MFCs result if water is completely excluded from the reaction environment. The lack of water hinders the dynamic assembly of the primary nanocrystals and results in a very broad distribution of nanocluster dimension and non-spherical shapes¹⁶. The samples shown in Figure 3B had insufficient reaction time (6-12 h) and as a result did not have sufficient primary nanocrystal growth. These poor results demonstrate that an appropriate amount of the reactant, as well as reaction time, is necessary to achieve consistent and uniform clusters.

After completion of the hydrothermal synthesis, the ferrite MFCs were separated and purified using magnetic separation. A magnet was placed under the solution to force their collection at the bottom of the vessel. Impurities and non-magnetic by-products formed in the synthesis, along with the excess solvent, could then be decanted to yield pure and monodispersed MFCs²⁷. Figure 4 illustrates the time required for nearly complete magnetic collection of the MFCs with and without the addition of steel wool. The steel wool placed in the vial during magnetic separation increases the gradient of the magnetic field within the vial, allowing for a much faster separation²⁸.

The MFCs purified using magnetic separation show a high degree of uniformity compared to those purified using a more conventional ultracentrifugation process. Figure 5 shows the size distribution of MFCs obtained using magnetic separation (A and B) compared to those using ultracentrifugation (5,000 g for 30 min) (C and D). Magnetic separation results in a narrower cluster diameter distribution as compared to ultracentrifugation and is the preferred purification strategy for the MFCs.

The as-synthesized MFCs are coated with polyacrylate (PAA), which provides a negatively charged surface and some degree of interparticle repulsion that prevents interparticle aggregation (Figure 6A). However, by performing a ligand replacement reaction with nitrodopamine (Figure 6B), we can replace the PAA coating with a copolymer coating of P(AA-co-AMPS-co-PEG), which allows for greater stability in higher ionic strength solutions. Figure 7 shows the schematic of this surface functionalization process. The colloidal stability of the MFCs dispersed in a PBS buffer is apparent in Figure 8. As-synthesized MFCs coated with PAA rapidly aggregate and separate from the solution within 30 min and are of little use in biological applications. In contrast, MFCs functionalized with a polysulfonate coating remained well dispersed in this solution for over 2 days without any sign of aggregation. The post-synthesis surface modification described here provides a route for forming homogeneous solutions of MFCs appropriate for introduction into biological environments.

Figure 1: The schematic for the synthesis of manganese ferrite nanoclusters. The reagents, iron(III) chloride, manganese(II) chloride, polyacrylic acid (PAA), urea, water and ethylene glycol are combined under hydrothermal conditions to produce the manganese ferrite nanoclusters. This product forms a stable colloidal solution in pure water as shown in the middle. The amount of water added in the synthesis and the ratio of manganese to iron in the precursors is used to tune the cluster size and ferrite composition, respectively. After magnetic separation, the nanoclusters form a ferrofluid as shown in the right, indicating they are highly responsive to even small applied magnetic fields. Please click here to view a larger version of this figure.

Figure 2: Transmission electron microscope (TEM) images of the manganese ferrite nanoclusters and their diameter distributions. In images A-D, the cluster diameter (Dc) increases as a result of reducing the amount of water added in the synthesis. The average cluster diameter is 31, 56, 74, and 120 nm for A, B, C, and D, respectively, with a constant composition of Mn_0.15Fe_2.85O₄. In images E-H, the ferrite composition monotonically changes in proportion to the Mn/Fe ratio of the precursors. Despite their different compositions, a nearly equivalent cluster diameter is achieved. Our synthesis allows for independent control over both the cluster diameter and ferrite composition, both features that are important for the magnetic properties of nanoscale ferrites. Please click here to view a larger version of this figure.

Label in Figure 2	H₂O (mL)	FeCl₃ (mmol)	MnCl₂ (mmol)	Ferrite Composition	Dc (nm)
A	1.5	1.3	0.7	Mn_0.15Fe_2.85O₄	34
B & G	0.7	1.3	0.7	Mn_0.15Fe_2.85O₄	56
C	0.5	1.3	0.7	Mn_0.15Fe_2.85O₄	74
D	0.1	1.3	0.7	Mn_0.15Fe_2.85O₄	120
E	1.3	2	0	Fe₃O₄	56
F	0.6	1.5	0.5	Mn_0.06Fe_2.94O₄	56
H	2	1	1	Mn_0.6Fe_2.4O₄	55

Table 1: Reaction conditions for the synthesis of the nanocluster samples shown in Figure 2. Other synthesis parameters are: 20 mL ethylene glycol, 250 mg PAA, and 1.2 g urea. The reaction mixtures are hydrothermally heated at 200 °C for 20 h. For A, B, C, and D, decreasing the water content while keeping other parameters constant resulted in clusters of larger diameters. For E, F, G, and H, increasing the ratio of MnCl₂to FeCl₃in the initial reaction mixture resulted in clusters with higher proportions of manganese in the cluster structure. Varying the amount of water E, F, G, and H at the same time allows for clusters of different composition but near equivalent diameters.

Figure 3: TEM images of failed and incomplete reactions. The small, low contrast features observed in these images are primary nanocrystals that have not developed into nanoclusters. The sample in Figure 3A was prepared with no additional water, while the material shown in Figure 3B had an insufficient, four-hour, reaction time. Please click here to view a larger version of this figure.

Figure 4: Comparison of magnetic separation of nanoclusters. Comparison of magnetic separation of nanoclusters without (A) and with (B) the addition of steel wool in the container. Steel wool increases gradient of the magnetic field inside the vial to allow for faster magnetic separation of the nanoclusters. As a result, it is possible to scale-up the production of nanoclusters efficiently without sacrificing sample quality. Please click here to view a larger version of this figure.

Figure 5: Comparison of ultracentrifugation and magnetic separation. Comparison of ultracentrifugation (A,B) and magnetic separation (C,D) and their impact on the uniformity of the purified clusters. A and C are the TEM images of the purified clusters, and B and D are the size distributions of the clusters in A and C, respectively. The y axis represents the number of clusters counted, and for each sample, a total of 150 clusters were surveyed. Please click here to view a larger version of this figure.

Figure 6: The structure of poly(acrylic acid) (PAA) (A) and nitro-dopamine (B) used in the surface modification step. The initial PAA coating used in synthesis is not ideal in biological or acidic media due as the carboxylic acid is easily protonated. Nitro-dopamine is used to replace the PAA coating creating a functional group to anchor a sulfonated copolymer. Please click here to view a larger version of this figure.

Figure 7: Schematics of the cluster surface modification process. (A) original PAA coating, (B) intermediate nitro-dopamine coating, and (C) the final P(AA-co-AMPS-co-PEG) coating. In (C), the blue, red, and green curves represent the AA, AMPS, and PEG units, respectively. The composition of the cluster can be either Fe₃O₄ or Mn_xFe₃_-xO₄. Please click here to view a larger version of this figure.

Figure 8: Surface functionalization of the nanoclusters with polysulfonate leads to materials that are colloidally stable under many different aqueous conditions. Clusters with two different surface coatings, as-synthesized PAA coated (A) and P(AA-co-AMPS-co-PEG) surface functionalized (B) are dissolved in the PBS buffer solution that is relevant for biological settings and observed for their colloidal stability over time. Please click here to view a larger version of this figure.

Discussion

This work demonstrates a modified polyol synthesis of manganese ferrite nanocrystals clustered together into uniform nanoscale aggregates²⁹. In this synthesis, iron(III) chloride and manganese(II) chloride undergo a forced hydrolysis reaction and reduction, forming molecular Mn_xFe₃_-xO₄. These ferrite molecules form primary nanocrystals under the high temperature and high pressure in the reactors, ultimately assembling into spherical aggregates termed here magnetite ferrite clusters (MFCs). Without sufficient reaction time or sufficient water, the aggregation process cannot fully complete leading to non-uniform, poorly formed particles. Conversely, given enough time and enough water, the metal oxide crystallization and assembly process is complete and yields a uniform spherical cluster comprising tens to hundreds of primary nanocrystals.The primary nanocrystals in these materials are hard aggregated, sharing some crystalline interfaces, leading to a high initial susceptibility, and pronounced magnetic response even to the small fields available from handheld permanent magnets²⁷. As a result, these materials have great potential for applications in drug delivery, magnetic hyperthermia, magnetic resonance imaging, and magnetic particle imaging³⁰^,³¹^,³².

We find that the amount of water added to the initial reaction mixture controls the diameter of the assembled clusters. As the water content in the reactants is increased, the diameter of the clusters and the number of aggregated primary nanocrystals decreases. The optimal range is 0.8 M to 5.0 M water, conditions that yield, respectively, cluster diameters ranging from 150 nm to 30 nm. Water has an important role in this process because it is necessary to ensure rapid hydrolysis of the metal precursors, more rapid aggregation of primary crystallites, and consequently smaller clusters¹⁶. Because the synthesis is extraordinarily sensitive to water, reactants handled under ambient conditions of variable humidity could absorb different amounts of water from the air. This could affect the subsequent dimensions and morphology of the product. While the humidity control in most research laboratories (e.g., 30%-60% RH) is sufficient to minimize this issue, this is one source of systematic error in the reported procedure. Control of the manganese to iron ratio in the product is achieved by varying the ratio of manganese to iron precursors. This is surprising as in many hydrothermal reactions the doping level of products is often not simply related to the stoichiometry of the starting materials⁴^,⁶^,⁸^,¹²^,¹³^,¹⁷. For these conditions, however, the product composition is well predicted by the ratio of the metal precursors. Taken together, independent control of both the cluster diameter as well as its composition is possible through straightforward manipulation of the starting reactant mixtures.

Often the purification of nanoparticles from the reaction media is the most time-consuming and intricate step in generating high quality materials. Ultracentrifugation is often applied for this purpose and while this is effective at separating nanoparticles from molecular by-products, it is poorly suited for removing unwanted solid products. When applied here to the purification of nanomaterials, the ultracentrifugation produces relatively polydisperse particles with variable dimensions and shapes. It is far more efficient to take advantage of the magnetic response of these materials by applying magnetic separation to improve the uniformity and purity of the final product. We speed up magnetic separation by creating very high gradients of magnetic fields within macroscopic vial using steel wool immersed in the solution and a rare-earth permanent magnet applied outside of the sample containers. This arrangement permits uniform samples to be recovered in under thirty minutes with high yields (~90%). It is important to match the amount of steel wool introduced to the solution to the anticipated MFC cluster diameters. For example, a MFC with an average diameter of 40 nm requires between 100 to 200 mg of steel wool for a rapid separation, while larger materials may require much less or even no steel wool. It is well established that smaller of magnetic nanoparticles are less responsive to applied fields by virtue of their smaller magnetic volume¹⁵^,¹⁷^,²⁶. The magnetic separation process thus provides a means to sharpen the uniformity of these materials as smaller clusters are not retained as efficiently by the process¹⁶. Using this magnetic separation method not only saves time in the laboratory, but it also results in products with greater uniformity in diameter.

Although the as-synthesized MFCs are stable in pure water, they exhibit poor colloidal stability in solutions with lower pH or higher ionic strength. Manganese ferrites have large magnetization densities, and as a result for these diameters the clusters possess magnetic dipoles that lead to interparticle attraction. The native polyacrylate coating used during the formation of the materials imparts a negative charge to the particle surfaces and helps to prevent particle-particle aggregation. However, at lower pH the carboxylic groups are fully protonated in effect removing the electrostatic repulsion needed to maintain homogeneous MFC dispersions; alternatively, in higher ionic strength media, the charge repulsion is reduced leading to more particle aggregation. Aggregation of the MFCs creates macroscopic materials that are not homogeneously dispersed in the solution making it challenging to use the materials in vivo or in applications that require large and available nanoparticle surfaces. For these reasons, we introduce a second polymer into the reaction to replace the original PAA coating. The copolymer, P(AA-co-AMPS-co-PEG), includes neutral polyethylene glycol (PEG) to provide biocompatibility and some degree of steric hindrance. Additionally, the polysulfonate component (PAMPS) offers both a greater charge density than the polyacrylate as well as a functional group that has a much lower pKa and consequently a greater working pH range (pKa ~ 1.2)²⁴. Manganese ferrite clusters modified with these surface coatings show dramatically increased stability in acidic and biological media. The procedure to ensure the correct surface modification is detailed, however, and must be followed carefully to ensure that the samples are effectively coated. Specifically, the method requires constant monitoring of the reaction mixture while it is treated with a probe sonicator to ensure homogeneous, complete replacement of the initial polyacrylate coating. It is also important to use appropriately sized glassware to minimize any product loss during vigorous sonication and apply an ice bath to the sonication mixture to minimize thermal degradation of the polymers caused by probe sonication.

In conclusion, this method allows for the quick and efficient production of manganese ferrite clusters (MFCs) with tunable diameters and manganese to iron compositions. Reactant water content as well as the ratio of iron to manganese are important parameters in defining the material product characteristics. A simple magnetic separation technique utilizing a handheld magnet and steel wool provides an efficient means to purify the product after synthesis yielding more uniform clusters. Finally, a sulfonated PEG copolymer is applied to the materials to ensure they remain non-aggregating in a variety of different pH and ionic strength media. The increased magnetic responsiveness of these manganese-doped iron oxides compared to pure iron oxide (Fe₃O₄) nanomaterials makes it simpler, cheaper, and easier to develop devices for applying external fields to manipulate the materials in vivo. Their improved surface coatings are also important as applications for magnetic nanoparticles in drug delivery, water remediation, and advanced imaging systems all require materials that are non-aggregating and homogeneous in a variety of biological and environmental media.

Divulgations

The authors have nothing to disclose.

Acknowledgements

This work was generously supported by Brown University and the Advanced Energy Consortium. We gratefully thank Dr. Qingbo Zhang for his established synthetic method of iron oxide MFCs.

Materials

0.1 Micron Vaccum Filtration Filter	Thermo Fisher Scientific	NC9902431	for filtration of aggregated clusters after synthesis and surface coating to achieve a uniform solution
2-Acrylamido-2-methylpropane sulfonic acid (AMPS, 99%)	Sigma-Aldrich	282731-250G	reagent used in copolymer to surface coat nanoclusters and functionalize them for biological media
2,2′-Azobis(2-methylpropionitrile) (AIBN)	Sigma-Aldrich	441090-100G	reagent used in copolymer making as the free ridical generator
4-Morpholineethanesulfonic acid, 2-(N-Morpholino)ethanesulfonic acid (MES)	Sigma-Aldrich	M3671-250G	acidic buffer used to stabilize nanocluster surface coating process
Acrylic acid	Sigma-Aldrich	147230-100G	reagent used in copolymer to surface coat nanoclusters and functionalize them for biological media; anhydrous, contains 200 ppm MEHQ as inhibitor, 99%
Analytical Balance	Avantor	VWR-205AC	used to weigh out solid chemical reagents for use in synthesis and dilution
Digital Sonifier and Probe	Branson	B450	used to sonicate nanocluster solution during surface coating to break up aggregates
Dopamine hydrochloride	Sigma-Aldrich	H8502-25G	used in surface coating for ligand exchange reaction
Ethylene glycol (anhydrous, 99.8%)	Sigma-Aldrich	324558-2L	reagent used as solvent in hydrothermal synthesis of nanoclusters
Glass Vials (20mL)	Premium Vials	B1015	container for nanocluster solution during washing and surface coating as well as polymer solutions
Graduated Beaker (100mL)	Corning	1000-100	container for mixing of solid and liquid reagents during hydrothermal synthesis (to be transferred into autoclave reactor before oven)
Handheld Magnet	MSC Industrial Supply, Inc.	92673904	1/2" Long x 1/2" Wide x 1/8" High, 5 Poles, Rectangular Neodymium Magnet low strength magnet used to precipitate nanoclusters from solution (field strength is increased with steel wool when needed)
Hydrochloric acid (ACS grade, 37%)	Fisher Scientific	7647-01-0	for removing leftover nanocluster debris and cleaning autoclave reactors for next use
Hydrothermal Autoclave Reactor	Toption	TOPT-HP500	container for finished reagent mixture to withstand high temperature and pressure created by the oven in hydrothermal synthesis
Iron(III) Chloride Hexahydrate (FeCl3·6H2O, ACS reagent, 97%)	ACS	236489-500G	reagent used in synthesis of nanoclusters as source of iron (III) that becomes iron (II) in finished nanocluster product (keep dry and weigh out quickly to avoid water contamination)
Labware Washer Brushes	Fisher Scientific	13-641-708	used to wash and clean glassware before synthesis
Magnetic Stir Plate	Thermo Fisher Scientific	50093538	for mixing of solid and liquid reagents during hydrothermal synthesis
Manganese chloride tetrahydrate (MnCl2·4H2O, 99.0%, crystals, ACS)	Sigma-Aldrich	1375127-2G	reagent used in synthesis of nanoclusters as source of manganese
Micropipette (100-1000μL)	Thermo Fisher Scientific	FF-1000	for transferring liquid reagents such as water and manganese chloride
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)	Sigma-Aldrich	25952-53-8	used in surface coating to assist in ligand exchange of copolymer (keep bulk chemical in freezer and diluted solution in refrigerator)
N,N-Dimethylformamide (DMF)	Sigma-Aldrich	227056-2L	reagent used in copolymer making as the solvent
Polyacrylic acid sodium salt (PAA, Mw~6,000)	PolyScience Inc.	06567-250	reagent used in hydrothermal synthesis to initially coat the nanoclusters (eventually replaced in surface coating step)
Poly(ethylene glycol) methyl ether acrylate	Sigma-Aldrich	454990-250ML	reagent used in copolymer to surface coat nanoclusters and functionalize them for biological media; average Mn 480, contains 100 ppm BHT as inhibitor, 100 ppm MEHQ as inhibitor
Reagents Acetone, 4L, ACS Reagent	Cole-Parmer	UX-78920-66	used as solvent to precipitate nanoclusters during washing
Single Channel Pipette, Adjustable 1-10 mL	Eppendorf	3123000080	for transferring ethylene glycol and other liquids
Steel Wool	Lowe's	788470	used to increase the magnetic field strength in the vial to aid in precipitation of nanoclusters for washing and surface coating
Stirring Bar	Thomas Scientific	8608S92	for mixing of solid and liquid reagents during hydrothermal synthesis
Table Clamp	Grainger	29YW53	for tight sealing of autoclave reactor to withstand high pressure of oven during hyrothermal synthesis
Urea (ACS reagent, 99.0%)	Sigma-Aldrich	U5128-500G	reagent used in hydrothermal synthesis to create a basic solution
Vaccum Filtration Bottle Tops	Thermo Fisher Scientific	596-3320	for filtration of aggregated clusters after synthesis and surface coating to achieve a uniform solution
Vacuum Controller V-850	Buchi	BU-V850	for filtration of aggregated clusters after synthesis and surface coating to achieve a uniform solution
Vacuum Oven	Fisher Scientific	13-262-51	used to create high temperature and pressure needed for nanocluster formation in hydrothermal synthesis

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Effman, S., Avidan, S., Xiao, Z., Colvin, V. Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition. J. Vis. Exp. (180), e63140, doi:10.3791/63140 (2022).