Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.


Introduction
Surface binding or internalization of superparamagnetic iron oxide nanoparticles (SPIONs) has enabled magnetization of a variety of cell types for applications such as imaging and remote manipulation. 1 However, achieving sufficient cellular magnetization can be a challenge, particularly when the interaction between the SPION and the cell surface is weak. 2 In the past, prolonged exposure or high SPION concentrations have been employed as strategies to overcome this challenge. 3,4 Nevertheless, these strategies are problematic because they increase toxicity 5,6 and have very limited success in cell types with low internalization rates, such as lymphocytes. 7 To enhance cellular uptake of SPIONs, several surface functionalization approaches have been explored. For instance, antibodies have been used to promote receptor-mediated endocytosis, 8 while non-specific uptake can be achieved using transfection agents 9,10 or cell-penetrating species, such as HIV tat-peptide. 11,12 However, antibody and peptide functionalization approaches are limited by expensive reagents and complex synthetic preparation, while transfection agents can induce nanoparticle precipitation and adversely affect cell function.
1. Prepare ferritin standards of 0.06, 0.125, 0.25, 0.5 and 1 mg/ml in 50 mM Tris buffer using commercially available horse spleen ferritin. NOTE: The protein concentration of commercially available ferritin is stated on the bottle. If not, contact the supplier for this information. 2. Dilute magnetoferritin samples of unknown concentration in 50 mM Tris buffer until the color of the solution approximately matches the color of the 0.5 mg/ml standard. Make a note of the dilution factor. 3. Add 10 μl of each standard and magnetoferritin sample in triplicate into wells of a 96 well plate. 4. Add 200 μl of ready-made Bradford reagent to each well (refer to materials list for Bradford assay reagent details). 5. Incubate at room temperature for 8 minutes. 6. Measure the absorbance at λ = 595 nm using a microplate reader. 7. Plot the mean absorbance of the ferritin standards as a function of protein concentration and use the slope and intercept of the linear fit to calculate the concentration of the magnetoferritin sample. NOTE: Take into account the dilution factor that was used to adjust the magnetoferritin sample to the standard curve.

Magnetoferritin Cationization
1. General remarks NOTE: For magnetoferritin cationization, N,N-dimethyl-1,3-propanediamine (DMPA) was coupled to aspartic and glutamic acid residues on the MF surface using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). 1. Carry out the reaction at room temperature under stirring. The protocol given below is for the cationization of 10 mg of magnetoferritin in a total volume of 5 ml (protein concentration 2 mg/ml). However, scale up or down by keeping the protein:DMPA:EDC ratios consistent, as well as the volumes of the buffer and magnetoferritin solution. 2. Prior to the cationization reaction, dialyze the magnetoferritin samples into 50 mM phosphate buffer (pH 7) containing 50 mM NaCl. This is important to remove Tris elution buffer and avoid unwanted reactions between the Tris amine and the EDC-activated carboxylic acid residues. Determine the protein concentration after dialysis and adjust it to 4 mg/ml prior to cationization. containing 50 mM NaCl for 2 days at 4 °C, replacing the dialysis buffer at least three times during that period. NOTE: At this point, cationized magnetoferritin can be stored at 4 °C until further use.

Human Mesenchymal Stem Cell Labeling with Cationized Magnetoferritin
1. General remarks 1. Perform all cell culture using class II laminar flow cabinets and humidified incubators at 37 °C and 5% carbon dioxide atmosphere. 5. When the reservoir is empty, add 0.5 ml of magnetic separation buffer. 6. When the reservoir is empty, add further 0.5 ml of magnetic separation buffer. 7. Repeat one more time (the total volume of magnetic separation buffer used for washing should be 1.5 ml). This wash step elutes all non-magnetized cells from the column (non-magnetized cell fraction). 8. Remove the column from the magnet and place it in a fresh 15 ml centrifuge tube. Remove filter from the column reservoir. 9. Add 1 ml of magnetic separation buffer to the reservoir and immediately push through the column using the plunger supplied by the manufacturer. This elutes the magnetized cells from the column into the centrifuge tube (magnetized cell fraction). 6. Analyze with ICP-OES. 28 7. Normalize the measured iron content to the number of cells to determine a value for cellular iron content.

Representative Results
TEM was used to confirm nanoparticle mineralization inside the apoferritin cavity and determine the average core size (Figures 1A and 1B). Image analysis of unstained magnetoferritin samples gave an average core diameter of 8.2 ± 0.7 nm, and aurothioglucose stain confirmed the presence of nanoparticles within the protein cage. Note that the images show a magnetoferritin sample that was further purified using magnetic separation to isolate uniform nanoparticle cores. Magnetoferritin samples that were not magnetically purified have a slightly broader core size distribution. 29 Analysis of the structure of the magnetoferritin core using selected-area electron diffraction indicated the possible presence of the inverse spinel structure based on magnetite (Fe 3 O 4 ) and/or maghemite (γ-Fe 2 O 3 ), as well as the spinel structure due to Co 3 O 4 . Furthermore, Raman spectra revealed peaks attributed to Fe 3 O 4 , small amounts of γ-Fe 2 O 3 , and a cobalt ferrite (Figure 1 C). ICP-OES analysis of magnetoferritin showed an average of 102 μg of iron and 0.9 μg of cobalt per milligram of magnetoferritin.
A schematic is included, illustrating the subsequent cationization step (Figure 2 A). The hydrodynamic diameter of magnetoferritin and cationized magnetoferritin was 11.8 ± 1.1 nm and 12.5 ± 1.4 nm, respectively, as determined by dynamic light scattering. The cationization efficiency of covalent DMPA-coupling to magnetoferritin was assessed using zeta potentiometry and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The zeta potential changed from -10.5 mV for MF to + 8.3 mV for cationized magnetoferritin, confirming a change in surface potential from negative to positive ( Table 1). Mass spectrometry experiments found a subunit molecular weight of 20.1 kDa for native apo-ferritin and 21.1 kDa for cationized apo-ferritin (Figure 2 B). This mass increase corresponds to approximately 12 coupled DMPA molecules per protein subunit, and the cationization of 288 residues on the entire 24-subunit protein.
Magnetic saturation and susceptibility were measured using SQUID magnetometry, and transverse and longitudinal relaxivity were measured using magnetic resonance imaging. Magnetic properties were similar for magnetoferritin and cationized magnetoferritin, indicating that cationization had negligible impact on the magnetic properties of the enclosed SPION (Table 1). Furthermore, these properties are similar to other iron oxide based nanoparticles, 19,30 demonstrating that cationized magnetoferritin would be as suitable as conventional SPION-based MRI contrast agents in enhancing imaging contrast.
After a 30-minute exposure, the cell surface was densely covered with cationized magnetoferritin (Figure 3 A). However, after one week, no nanoparticles were found on the cell surface (Figure 3 B). Cationized magnetoferritin was remarkably effective at magnetically labelling hMSCs.
Notably, exposing the cells to cationized magnetoferritin for one minute resulted in the magnetization of 92% of the cell population and the delivery of 3.6 pg of iron per cell. Increasing the incubation time to 15 minutes resulted in the magnetization of the entire cell population (Figure 3  C).

Discussion
TEM of magnetoferritin samples stained with aurothioglucose revealed the successful mineralization of nanoparticles inside the protein cage. Electron diffraction and Raman analysis of the nanoparticle core indicated the presence of a cobalt ferrite, indicating successful doping of the nanoparticle core with cobalt. This demonstrates that mixed-oxide nanoparticles can successfully be mineralized within the apo-ferritin cavity. Furthermore, we have shown previously that cobalt doping can be varied by altering the amount of cobalt precursor added to the reaction mixture, which enables tuning of the magnetic properties. 18 Magnetoferritin synthesis can be performed in a variety of vessels, as long as they are tightly sealable and have access ports through which reactants can be introduced (e.g., a three-neck round bottom flask). The reaction temperature should be maintained at 65 °C either by placing the vessel in a water/oil bath or using a double-jacketed vessel. Here, we used a double-jacketed electrochemical cell setup to perform the synthesis. To guarantee successful synthesis, maintaining the correct pH and avoiding oxygen contamination of the aqueous solutions is crucial. Metal salt solutions should always be prepared freshly prior to use rather than in advance. Furthermore, commercial apoferritin solutions can vary in quality and affect synthesis outcome (e.g., size of nanoparticle core mineralized). It can help to dialyze the apoferritin solution into 50 mM HEPES buffer (pH 8.6) prior to synthesis to remove any residual reducing agent used by the manufacturer. It is useful to make a note of the batch number of the apo-ferritin solution used for synthesis, so it can be specifically requested from the manufacturer should additional material need to be purchased. Furthermore, the protein concentration of commercially available apo-ferritin should be stated on the bottle, which can be used to calculate the volume of apo-ferritin solution needed for synthesis. If this is not the case, contact the supplier for this information.
The advantage of gradual addition of metal salts and hydrogen peroxide -as presented here and in previous reports -is that mineralization of the nanoparticle core can be controlled such that different loading factors (i.e., nanoparticle sizes) can be achieved. 33 Furthermore, it is possible to purify magnetoferritin further using a magnetic separation column, e.g., a column packed with stainless steel powder secured inside an electromagnet. 34 Thus, highly monodisperse nanoparticle cores can be isolated from the bulk magnetoferritin sample. However, for magnetic cell labelling as presented here this is not required. A limitation of magnetoferritin synthesis is the relatively low synthesis yield of about 10%, and the relatively high cost of commercial apo-ferritin solutions. However, apo-ferritin may also be prepared from cheaply available horse spleen ferritin by following established de-mineralization protocols. 16 Cationization of magnetoferritin was achieved by adding a molar ratio of 250 molecules of DMPA and 50 molecules of EDC per negatively charged residue (calculations based on the amino acid sequence of horse spleen ferritin). This excess of reagent over protein resulted in high cationization efficiencies, comparable also to previously reported results for the cationization of ferritin. 35  apoferritin and cationized apoferritin were used because of the excessive molecular mass of the magnetoferritin core. To yield high cationization efficiencies, optimal pH is also crucial. EDC-mediated crosslinking is most effective under mildly acidic conditions, and we found that pH 5 yielded optimal cationization results for magnetoferritin. However, for other proteins cationization pH may need to be optimized. Cationization at or close to the isoelectric point of the protein should be avoided, because this may lead to severe precipitation.
Stem cell magnetization with cationized magnetoferritin was highly efficient and could be achieved using incubation times well below 30 minutes. Even a one-minute incubation resulted in a cellular iron content of 3.6 pg, which is within the reported range required to influence T2 and T2* contrast for MRI. 36,37 It is also remarkable that this efficient labeling is achieved with low extracellular iron concentrations. For example, previous studies using anionic nanoparticles report iron levels of 10 pg per cell after a 30-minute incubation period with 5 mM iron. 38 In comparison, incubation with a cationized magnetoferritin solution containing 0.5 μM protein corresponds to incubation with approximately 0.2 mM iron and also yields approximately 10 pg of iron per cell after 30 minutes. We were not able to clearly identify any endocytotic vesicles using TEM. However, previous studies using cationized ferritin found that internalization occurred within the first ten minutes of exposure. 39,40 Cationized ferritin could be localized in coated vesicles, indicating clathrin-or caveolin-dependent endocytosis. The same studies also reported that after 30 minutes of incubation, cationized ferritin was still present on the cell surface, as well as in multivesicular bodies, resembling lysosomes.
Further applications could include cationization of apo-ferritin cages loaded with other nanoparticles and/or functional molecules, such as anticancer drugs 41 or quantum dots 42 . Cationization of these ferritin constructs could result in faster and more efficient delivery of their cargo to cells.

Disclosures
The authors have nothing to disclose.