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Characterization of Magnetic Nanoparticles in Biological Matrices Katie R Hurley, Hattie Ring, Hyunho Kang, Nathan D Klein, and Christy L. Haynes Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02229 • Publication Date (Web): 11 Sep 2015 Downloaded from http://pubs.acs.org on September 15, 2015

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Analytical Chemistry

Characterization of Magnetic Nanoparticles in Biological Matrices Katie R. Hurley1, Hattie L. Ring1,2, Hyunho Kang1, Nathan D. Klein1, and Christy L. Haynes1* 1

Department of Chemistry, University of Minnesota, 207 Pleasant St SE, Minneapolis, MN

55455, United States of America 2

Center for Magnetic Resonance Research, University of Minnesota, 2021 Sixth Street SE,

Minneapolis, MN 55455, United States of America AUTHOR BIOGRAPHIES: Katie R. Hurley is a PhD candidate working with Professor Christy Haynes. Her graduate work has focused on the development of core/shell iron oxide/mesoporous silica nanoparticles as theranostic agents. Hattie L. Ring is a postdoctoral associate working with Professor Christy Haynes and Professor Michael Garwood. Her postdoctoral research has focused on in vivo imaging of core/shell iron oxide/mesoporous silica nanoparticles with magnetic resonance imaging. Hyunho Kang is a graduate student working with Professor Christy Haynes. His research interests include the biomedical application of porous and magnetic materials.

ACS Paragon Plus Environment

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Nathan D. Klein is an undergraduate researcher working with Professor Christy Haynes. His research interests include the use of dark field TEM to probe nanoparticle characteristics. Christy L Haynes is the Elmore H. Northey Professor of Chemistry. Her research interests span analytical, materials, and biological chemistry, and especially the interfaces between those.

KEY WORDS: Magnetic nanoparticles, nanoparticle characterization, transmission electron microscopy, SQUID magnetometry, Mössbauer spectroscopy

FEATURE SUMMARY: This Feature describes several methods for the characterization of magnetic nanoparticles in biological matrices such as cells and tissues. The article focuses on sample preparation and includes several case studies where multiple techniques were used in conjunction.

Introduction The interactions between magnetic nanoparticles (NPs) and biological matrices such as cells, tissues, or whole organisms provide an intriguing area of study. Magnetic NPs can be used in vitro or in vivo intentionally as MRI contrast agents1,2, cell sorting materials3, or as a component of therapy.4–6 They can also naturally occur within biological matrices as in magnetotactic bacteria7 or various vertebrates such as pigeons, salmon, and even humans, where magnetic NPs have been found in teeth or brain matter.8–10 While iron oxide NPs are the most common naturally occurring magnetic nanomaterial in vivo, engineered NPs such as cobalt ferrite11 and manganese oxide12 are also likely to be found in biological matrices either through intentional administration or environmental exposure. Given a pristine sample (i.e. purified, capable of


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being suspended in media, etc.) of magnetic NPs, a large number of characterization techniques would be available.13,14 Several common examples are listed in Table 1. Table 1. Common characterization techniques for pristine magnetic NPs. Technique

Information Acquired

Electron microscopy (transmission, TEM; scanning, SEM)

morphology, crystallinity, size distribution, composition15

X-ray diffraction (XRD)

crystal structure, size16

Dynamic light scattering (DLS)

hydrodynamic diameter17

Zeta potential measurement

surface charge18

Thermal analysis (differential scanning calorimetry, thermogravimetric analysis, etc.)

surface coverage, thermal stability, nature of surface functionalization19

Infrared spectroscopy (IR)

nature of surface functionalization20

Nuclear magnetic resonance spectroscopy (NMR)

longitudinal and transverse relaxivity21

Inductively coupled plasma-mass spectrometry (ICP-MS)

concentration in suspension22

Superconducting quantum interference device magnetometry (SQUID)

magnetic properties23

Characterizing the location, morphology, and magnetic properties of these NPs while they are embedded in a biological system is important for a fundamental understanding of biology, to glean synthetic insights for novel nanomaterial synthesis, and for therapeutic efficacy and toxicity monitoring. However, due to the complexity of the biological matrix, the study of these systems is not always clear cut. Experimental design must take into account sample preparation considerations, especially because the body contains large amounts of endogenous iron in the form of hemoglobin or ferritin. This feature article covers versatile techniques for characterizing magnetic NPs in biological specimens with a focus on special consideration for sample preparation and the need for


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complementary techniques. The feature concludes with a discussion of two case studies demonstrating the use of many techniques to form a complete picture of magnetic NP/biological interactions.

Transmission Electron Microscopy Overview One of the most common characterization techniques for NPs in general is transmission electron microscopy (TEM). By passing a focused electron beam through an extremely thin sample, researchers can gain insight into NP localization, size, morphology, composition, crystallinity, and other characteristics.24,25 A summary of various electron/sample interactions and their resultant information is given in Figure 1. Transmitted electrons (those that undergo very little scattering or energy loss as they encounter the sample) pass through a system of electromagnetic lenses and encounter the CCD camera, thus appearing as bright signal in a digital image. Contrast comes from the lack of signal from highly scattered electrons that never reach the CCD. Scattering largely occurs when an electron impinges on a high mass element (Zcontrast) or a strongly crystalline area (diffraction contrast). Thus, magnetic NPs, which are often both high-mass and highly crystalline compared to the carbonaceous cell or tissue material, will appear darker than the biological matrix. Once the NPs are identified, the high resolution of TEM enables researchers to draw conclusions about cell internationalization26–28, membrane association29, NP aggregation state30, etc. High-resolution TEM (HRTEM) can even reveal atomic-scale information regarding nanoparticle degradation.31 Diffraction patterns collected from scattered electrons show composition-dependent rings that enable crystal structure identification.26,27,32,33 Sometimes, when electrons collide inelastically with the sample, they eject


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inner-sphere electrons from the sample. Electrons in outer shells drop down to fill the void, and the accompanying energy is emitted in the form of an x-ray. In a process called energy dispersive x-ray spectroscopy (EDS), the energy of those x-rays can be quantified and related back to an elemental signature.26 The electron that underwent inelastic collision but was still transmitted can also provide information. In electron energy loss spectroscopy (EELS), electrons are sorted by energy to generate a plot of intensity vs. energy. Peaks at certain energies can provide information about the electronic environment of an atom, including oxidation state.32,34

Figure 1. Electron-sample interactions in TEM. High-mass or highly crystalline samples like magnetic NPs scatter electrons more effectively than most biological material, resulting in images with dark contrast for NPs (left panel). Electrons that collide inelastically with the sample cause both an element-specific x-ray (measured with EDS) and a transmitted electron with a lower energy (measured with EELS, right panel). Sample preparation


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To image NPs contained in cells or tissues, the sample typically is embedded in a polymer resin which is cured to a hard block and sliced into thin sections with a microtome. Samples can undergo one of at least two preparation pathways ahead of polymer embedding. The first involves cryogenic high pressure freezing and freeze substitution before polymer curing. Depending on experimental conditions, this process can take anywhere from hours to several days.35 The second involves cell fixation, dehydration, and infiltration with resin at room temperature and can take up to a week.36 High pressure freezing can result in better preservation of cell ultrastructure35, but requires more complex equipment (a high pressure freezer or plunge freezing apparatus) than the more traditional room temperature process. Sample preparation and staining is a delicate process which requires practice and dexterity.37 Exceptionally hard or brittle samples (i.e. bones or teeth) have a higher probability of shattering during microtome slicing.10,32 Alternative techniques such as focused ion beam cutting of an ultra-thin sample have been developed to avoid this concern.32 Samples that are thin to begin with, such as some bacterial species, can be prepared as a whole mount without resin embedding, although NP internalization becomes a more complex question when imaging a projection of the whole bacteria as opposed to a discrete slice.26,27 Limitations The most important limitation of TEM is its sampling volume. The extreme magnifications make it difficult if not impossible to obtain a truly representative sample, which is why it is important to pair TEM with a correlative technique whenever possible. Beyond sampling concerns, it is sometimes difficult to even locate the NP of interest when examining features on the scale of cells, organs, or organisms. Green and colleagues summarize the issue nicely: “Light microscopy and transmission electron microscopy work at such different scales that some


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components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy.”38 Several techniques can be used to solve this ‘needle in a haystack’ problem. When working with crystalline NPs, dark field mode can be used to readily identify NPs at low magnification through their strong diffraction signal.39 For large tissues or organs that contain magnetic NPs, researchers can use magnetism to their advantage by physically sectioning progressively smaller tissue slices and checking each for response to an external rare earth magnet.26 Finally, large tissues can be entirely embedded in resin and cut progressively into 1 µm thick slices. Each slice can be visualized with light microscopy, and when NPs are identified (either through a stain such as Prussian blue or as a dark feature), the next slice from the block can be cut to TEM specifications (60-70 nm thick).40 Recent Advances As researchers aim to record images from nano/bio interfaces in their natural context, techniques such as cryogenic TEM and liquid-cell TEM have been applied. In cryo-TEM, an aqueous specimen is vitrified such that amorphous water captures the cell and/or nanoparticles in their native state.41 This technique has recently been used to visualize the formation of iron-rich nanoparticles on planktonic bacteria42 and to probe the interactions between silver nanoparticles and lipid bilayer vesicles.43 Liquid-TEM is a technique in which a liquid sample is sealed between electron-transparent windows and visualized in the electron microscope.44 This technique has recently become more popular as microfabrication techniques and aberrationcorrected microscopes have improved. Cell-nanoparticle interactions can be probed in fixed45,46 or live47 cells. Once a liquid cell technique has been developed for a given microscope or lab, liquid-TEM can reduce the time scale of routine measurements such as multiple exposures for


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varying nanoparticle surface charge or concentration because the sample preparation time is reduced from days to hours.47 Case Study Recently, TEM was used very effectively in a study by Fdez-Gubieda et al. on the formation of magnetite biominerals in the magnetotactic bacteria Magnetospirillum gryphiswaldense.27 In this study, researchers performed time-resolved structural and magnetic experiments aimed at determining the phases of iron oxide NPs (here called magnetosomes) during mineralization. Bacteria samples at various time points after exposure to 100 µM Fe(III)- citrate were fixed with formaldehyde and placed on a TEM grid as a whole mount. Imaging studies revealed a wealth of information, including magnetosome size distribution, number density, and localization relative to one another at each time point. The authors determined that the number and size of the magnetosomes increased over time, and that they gradually oriented together into small subunits and then larger chains. By using a correlative technique called x-ray absorption near edge structure (XANES) spectroscopy, which is theoretically similar to EELS48, researchers were able to determine that the magnetosomes formed through a two-step process. In the first step, magnetosomes largely consisted of phosphorous-rich ferrihydrite structures during a period of slow iron accumulation and then, in a second step, were converted to a more reduced and crystalline form, Fe3O4 (magnetite). Importantly, the authors were able to use knowledge of magnetosome size and number density from TEM images to estimate the mass of iron present in each bacterium, which was then used to convert qualitative XANES observations into quantitative conclusions.

Mössbauer Spectroscopy


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Overview Mössbauer spectroscopy is a versatile analytical technique intended to study the chemical, structural, and magnetic characteristics of materials by measuring nuclear energy level transitions with gamma rays.49 Depending on the environmental condition of the nuclei in a sample, the energy of a given nuclear transition will change compared to a probe nuclei of the same element (hyperfine interactions). The energy levels of the sample nuclei absorbing the gamma ray can be modified by isomer shift (related to s-electron environment), quadrupole splitting (related to charge distribution and electronic arrangement) and magnetic splitting (related to magnetic ordering).49 By adjusting the energy of probing gamma rays via the Doppler effect, researchers measure the wavelength of these energy splittings, which in turn gives information about the oxidation state, magnetic properties, and crystallinity of the sample.49 Among several isotopes which behave according to the Mössbauer effect,

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Fe is the most

common and popular isotope due to its very low energy gamma ray and long-lived excited states.50 Regarding crystallinity, a correlation between Mössbauer parameters (isomer shift and quadrupole splitting) and crystal structure parameter have been investigated via iron ions in silicate minerals.51 Among several crystal structure parameters of average metal-oxygen distance, volume per oxygen in a unit cell and polyhedral volume, the correlation between isomer shift and polyhedral volume delimits the shift ranges for tetrahedral, octahedral and 5coordinatied Fe3+ ions. Regarding magnetism, iron-containing magnetic NPs experience hyperfine interaction in the presence of a magnetic field (‘Zeeman splitting’).49 This behavior can be shown in the spectra as magnetic sextets. As shown in Figure 2, non-magnetic materials or superparamagnetic particles at room temperature have a characteristic doublet spectrum, indicating a lack of magnetic splitting.52,53 When the temperature is low enough, magnetic


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splitting for superparamagnetic particles (the characteristic sextet) becomes apparent as there is not enough thermal energy to cancel each NP’s magnetic moment.54 In a biological matrix, Mössbauer spectroscopy is not sensitive to most organic materials and is able to separate the spectra of exogenous iron in NPs from the endogenous iron in vivo (i.e. ferritin or hemoglobin) based on oxidation state and magnetic behavior.54 Thus, researchers can distinguish the injected or applied NPs from naturally presented iron in the spectra. In addition to the study of magnetic NP oxidation state and crystal structure, time-dependent Mössbauer spectroscopy measurements can show biodegradation processes of magnetic NPs as sextets gradually become doublets, representing a loss of magnetic character.55,56

Figure 2. Nuclear energy levels of

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Fe in different local environments and related Mössbauer

spectra. From the left, unperturbed state, isomer shift, quadrupole splitting, and magnetic splitting respectively. The arrow in (b) indicates the change in peak position from the unperturbed state. I is the nuclear angular momentum spin quantum number and mi is the magnetic quantum number. Sample preparation


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Mössbauer spectroscopy measurements require a solid sample so that recoil-free nuclear transitions can occur as gamma rays from a probe isotope interact with the sample.57 To study the samples in a biological matrix, magnetic NPs are introduced into biological systems, the media containing the sample is extracted, and the samples are lyophilized.53,58 If solid samples can be obtained from biological suspensions, samples can be gathered on filter paper as a dry paste and placed directly into the instrument.59 Mössbauer isotope-rich samples can be introduced to achieve better signal to noise ratios and resolutions.60 Limitations Lifetime of the excited states and low-lying excited states are key factors for a Mössbauer isotope, and thus the number of isotopes that can be used in Mössbauer spectroscopy is limited. 57

Fe is the most common isotope, and 119Sn and 121Sb are also frequently studied. Unfortunately,

isotopes of other elements likely to be present in magnetic NPs such as Co and Mn do not display Mössbauer activity, although small amounts of Fe can be doped in to enable the study of these materials.61 Because of the low percentage of 57Fe in natural iron minerals, there is a limit in the sensitivity for dilute samples.62 In addition, several minerals containing iron can have similar Mössbauer characteristics, and thus, spectra. For example, silicate minerals of pyroxene, amphibole, and mica with Fe2+ in the octahedral state have nearly identical Mössbauer parameters (isomer shift and quadrupole splitting), making their spectra indistinguishable without the use of additional techniques.63 The instrument itself requires a new annual radioactive source and daily liquid helium, making maintenance costly. The data collection process for Mössbauer spectroscopy is slow. Taking more than a hundred hours to collect data for a single spectrum is not unusual.64 Finally, no identification of NP localization within a structure can be acquired via Mössbauer spectroscopy.


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Case Study Recently, the biodegradation of magnetic NPs in different organs of mice has been studied, taking advantage of Mössbauer spectroscopy.53,55,56 In one study, Panchenko and co-workers injected magnetic NPs into mice and investigated their time-dependent biodegradation.55 Mice livers were extracted at various times following i.v. injection (two hours, two days, two weeks, and two months), and the Mössbauer spectra of

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Fe nuclei in the samples were measured with

and without an external magnetic field at room temperature and at 78K without the external magnetic field. The authors noticed that, along with the sextets from the injected NPs, intense doublet lines appeared in the spectra with increasing time. After two hours in vivo, samples displayed this doublet only in the spectrum collected at room temperature without the external magnetic field, suggesting that the spectra were from the exogenous NPs. In contrast, doublets appeared in all three spectra after two months, leading to the conclusion that the doublet then indicated non-magnetic iron. To further investigate the nature of the non-magnetic iron species, the authors calculated the concentrations of superparamagnetic and paramagnetic components from the spectra using the Debye model and measured the isomer shift parameters for each. The researchers concluded that the paramagnetic and superparamagnetic components retained their isomer shift and quadrupole splitting, and their concentrations increased, whereas the concentrations of NP components decreased with increasing time. From the isomer shift and quadrupole splitting, it was inferred that the doublet patterns were associated with ironcontaining proteins present during the biodegradation process, making it possible to distinguish NPs from the iron-based background signal rather than overestimating the retention of injected NPs.


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SQUID Magnetometry Overview Superconducting quantum interference device (SQUID) magnetometry is a standard measurement method for magnetic materials characterization. A number of books describe the details of SQUID detection.23 In brief, a SQUID consists of a coil made with superconducting wire and Josephson Junctions. Typically, a change in magnetic field (flux) can be detected with a coil by measuring the change in current caused by inductance of the coil. For a coil made with superconducting wire, the current through the wire is unable to change. Josephson Junctions are weak links within the superconducting wire, typically a thin layer of non-superconducting material that superconducting electrons can tunnel through. The critical current that passes through the Josephson Junctions is heavily dependent on magnetic flux and alters the current flowing through the superconducting coil. This results in the high sensitivity flux-to-voltage conversion detectable with a SQUID. Two characteristic magnetization measurements are acquired with a SQUID. A fielddependent magnetization curve is measured with the temperature held constant while the external magnetic field is adjusted (0 – 7T for commercial systems).65 In the usual data representation, the magnetic induction of the material (B) is plotted against the applied field strength (H). The resulting curve can be used to determine magnetic material properties such as the saturation magnetization, remanant induction, coercivity, and magnetic susceptibility. Typical data acquired from these measurements are shown in Figure 3. Temperature-dependent magnetization is measured at a low magnetic field over a variable temperature range (2 - 400K)65, indicating the magnetic phase of a material. An important property for this measurement is the Curie temperature, which is the temperature at which the magnetic character of a material transitions


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from ferromagnetic to paramagnetic. Overall, the information from these measurements can be combined to obtain information about the inherent strength of a magnet, which gives insight on materials properties such as oxidation state, anisotropy, size, and shape.

Figure 3. A hysteresis loop of a (top) ferromagnetic and (bottom) paramagnetic material as it would appear in a SQUID measurement. Typical magnetic properties measured, such as the saturation magnetization, coercivity, remanent induction, and magnetic susceptibility are marked.


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Sample Preparation SQUID magnetometry is a versatile method capable of measuring many sample types including powders, crystals, thin films, liquids, and gases. Samples are typically packed into a gelatin capsule40,66, although modified NMR and EPR tubes have been utilized due to their lowsusceptibility glass.67,68 Liquid and cell samples are often diluted with glycerol to avoid strain caused on the sample if crystallization of the liquid carrier occurs.66 Alternatively, liquid samples can be loaded onto filter paper before measurement.69,70 Tissue samples typically undergo freeze drying where either the sample is kept intact or cut into smaller pieces before placement in a gelatin capsule.40,71 Sample sizes can vary based on the magnetic susceptibility of sample; however, in the case of tissue samples, smaller organs such as the spleen may require combining several samples to obtain a sufficient signal.66 Limitations SQUID measurements assess the bulk magnetic properties of the sample and, therefore, do not give localization information. Furthermore, when interacting with biological samples, the interparticle interactions can become convoluted with the sample matrix. However, information regarding inter-particle interactions can be obtained by comparing the known characteristics of a magnetic NP in an aqueous suspension to a sample in a biological matrix.72 Conventional SQUID detection requires the use of cryogenic cooling of the superconducting coil, increasing the cost of operation. Other SQUID applications: Scanning SQUID Biosusceptometry (SSB) The high magnetic sensitivity of SQUIDs makes it feasible to detect very low concentrations of magnetic NPs in vivo, which is typically performed by scanning SQUID biosusceptometry (SSB). SSB utilizes a scanning coil connected to a SQUID. Recently, this was actualized by


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Chieh et. al. when they demonstrated scanning SQUID measurements, obtaining in vivo and ex vivo imaging of NPs in rat liver and heart.73 This method has since been used to establish a comprehensive model of magnetic NP metabolism.74 Alternatives to SQUID for biosusceptability measurements Other magnetic measurement techniques with sensitivities comparable to SQUID are capable of measuring the small magnetic fields produced in vivo. These methods are mentioned briefly below. NV-diamond Magnetometry: The photoluminescence of a nitrogen-vacancy (NV) defect in a diamond is highly sensitive to magnetic field changes. Therefore, through optical detection, the magnetic field is observed.75 Images similar to those acquired through SSB can be acquired using two methods with NV-diamond magnetometery. Most commonly, a nanodiamond is attached to the tip of an atomic force microscope probe and scans over a sample to obtain a magnetic image.76 Alternatively, fluorescence microscopy can be used to scan an ensemble of diamonds with NV center-defects near the surface which is placed on the sample of interest.77 In biological matrices, this method has been applied toward the detection magnetic NPs produced by bacteria.78 Magnetic Force Microscopy: This technique is based on atomic force microscopy (AFM), where the measured force is caused by a magnetized tip and a magnetic sample. The standard silicon tip on the cantilever of the AFM is coated with a magnetic layer, such as iron.79 The convoluted measurement method makes it difficult to obtain a quantitative value for the magnetic field present; however, quantitative analysis has been performed.80,81 The high resolution of this technique has made it versatile toward the analysis of magnetic NPs in biological matrices (both ex vivo8 and in vitro81,82).


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Table 2. Techniques for biosusceptability measurements in vitro or in vivo SSB

NV-diamond NV-diamond Magnetometry (probe) Magnetometry (ensemble)

Magnetic Force Microscopy

Sensitivity

1.3 pT/√Hz 83

10 nT/√Hz 75

50 pT/√Hz 75

N/A

Resolution

1 cm73

~ 10 nm75

~ 500 nm 75

20 nm 79

Benchtop 75

in vitro 75

in vitro and ex vivo

Application in vivo73

Comprehensive Case Study 1: Naturally Occurring Magnetite in Pigeons One largely qualitative study by Walcott, Gould, and Kirschvink demonstrates several complementary techniques to provide a comprehensive understanding of magnetic particles in biological samples, specifically examining pigeon heads and necks.84 Several species of pigeon demonstrate homing tendencies and are able to return home from distant and unfamiliar locations, a trait thought to be possible in part because of some ability to sense magnetic fields. To determine whether the mechanism behind this sense involves the presence of permanent super-paramagnetic materials, Walcott and colleagues measured induced magnetic remanence in samples of pigeon heads and necks using SQUID magnetometry. An external permanent magnet was used to induce remanence at both room and liquid nitrogen temperatures before analysis with the SQUID magnetometer. When remanence was found, the sample was then subdivided to further pinpoint the location of magnetic activity. The authors successfully located a region containing magnetic material between the brain and skull of the pigeons. They then set about characterizing the material more thoroughly. By analyzing the decay of the remanence as the tissues warmed from liquid nitrogen temperature, the authors were able to conclude that the material was not likely


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superparamagnetic as there was no sudden drop in remanence during the warming process. Using electron microscopy, they determined the dimensions of the crystalline material in the tissue. Because most naturally occurring magnetic materials aside from maghemite and magnetite would display superparamagnetism at similar dimensions, the authors also gleaned insight as to the material’s identity. Light microscopy was used to investigate aggregates of the crystals, which were black in color, suggesting they were composed of magnetite. Another technique used was electron probe analysis, wherein the sample is probed with an electron beam and the energy of emitted x-rays is measured to determine elemental composition. This technique was used to confirm the strong presence of iron in the material. Finally, the material was heated to the Curie temperature, and the measured temperature closely matched with the Curie point of magnetite, further supporting Walcott and colleagues’ identification of the primary magnetic component of the material as magnetite. Other studies have investigated the presence of magnetic or iron-containing materials in pigeons using different techniques as well. Hanzlik et al. used SQUID magnetometry, selected area electron diffraction, TEM (both bright and dark field), as well as zero-field cooled and field cooled curves.25 In two separate studies, Fleissner et al. utilized light microscopy with Prussian blue staining and immunohistology, TEM, as well as x-ray fluorescence and XANES.85,86 While the role that these structures play as magnetoreceptors, if any, is uncertain87,88, these investigations show a wide range of techniques used to analyze magnetic NPs in biological samples.

Comprehensive Case Study 2: Monitoring Iron Oxide NP Degradation in vivo


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Some techniques presented in this feature were used by Levy et al. to better understand the biodegradation of iron oxide40 NPs in vivo. While many NPs demonstrate pH- or enzymedependent degradation in vitro, it is not clear that a given NP system will behave similarly in the complex in vivo environment.89 Studying NP degradation in vivo is essential for understanding how the body processes NPs, especially those that are expected to have long residence times (e.g. significant amounts of gold NPs have been found in the livers of exposed mice up to 120 days following injection).90 However, studying degradation in vivo is a complex task due to endogenous species, varied biodistribution, etc. Studies can require extensive tissue workup to recover NP building blocks such as polymers91, or they might necessitate the development of entirely new instruments.92 Fortunately, magnetic NPs are able to be studied through less invasive methods as demonstrated by Levy40 and others.93 In this study, Levy et al. endeavored to study the biodegradation mechanism of maghemite iron oxide NPs following i.v. injection at two different doses in mice. A detailed understanding of the degradation mechanism requires knowledge of NP magnetism, localization, morphology, and concentration over time. To accomplish this task, the authors excised liver and spleen tissues from treated mice at 1, 7, 30, 60, and 90 days following injection and prepared the samples for analysis by ferromagnetic resonance (FMR), inductively coupled plasma optical emission spectroscopy (ICP-OES), SQUID, and TEM. As iron oxide NPs degrade, they dissolve into iron ions94 which may be complexed by a number of species within the body (hemoglobin, ferritin, transferrin, etc.).95 Compared to the original NP, these degraded species should have a loss of magnetism. In this study, the authors exploited that fact by quantifying iron both by FMR and ICP-OES. FMR, a spectroscopic technique which measures the response of a material’s magnetic dipole to an external magnetic


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field, can quantify the amount of superparamagnetic iron through a calibration curve.96 ICPOES, on the other hand, is indiscriminate of magnetic properties and simply quantifies total iron content in a sample. The combination of these two techniques gives insight into the difference between endogenous iron species and iron oxide NP degradation products, both of which are non-superparamagnetic iron species. At the designated time points, organ samples were excised, rinsed, finely sliced, dried, and weighed before analysis. In Figure 4, panels (a), (b), and (c) show trends in FMR and ICP-OES iron quantitation in mice livers and spleens over time. From the FMR data, it is clear that superparamagnetic iron content decreases over time and that the decrease is steeper for the liver than the spleen. The authors suggest that iron oxide NPs are broken down in the liver and that their byproducts (free or sequestered iron ions) are transported to the spleen. It should be noted that the changes in superparamagnetic iron content in the liver and spleen are on the same order of magnitude as the error bars of the ICP-OES data (panel b), indicating that ICP-OES alone would not have been sensitive to the change in iron type in either organ at this NP dose.


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Figure 4. FMR, ICP-OES, and TEM data from ref. 40. (a-c) Iron concentration in the spleen and liver of mice after injection with iron oxide (maghemite) NPs. The combination of FMR and ICP-OES can distinguish between superparamagnetic and non-superparamagnetic iron (both endogenous and exogenous). (d-g) TEM with EDS and diffraction mode show that the electron dense spheres in the spleen are actually iron-containing crystalline NPs on day 1. (h-j) After 7 days, some iron has been transferred to poorly-crystalline, fingerprint-like ‘whorls’ (yellow arrows in h) which are indicative of ferritin iron storage proteins. Adapted from Biomaterials Volume 32, Levy, M. et al. “Long Term in vivo Biotransformation of Iron Oxide Nanoparticles” pp. 3988-3999, Copyright 2011, with permission from Elsevier.40


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SQUID data under various conditions gives insight into the magnetic transformations of the NPs (see Levy et al. Figure 3).40 Consistent with the FMR data, the magnetization of the spleen and liver samples decreases over time. Interestingly, field-dependent magnetization curves at 300K indicate that the size distribution of superparamagnetic NPs is not affected over time (i.e. those particles that are still superparamagnetic are not gradually getting smaller in a uniform way). In addition, at the final time point, a change in magnetic character was seen, indicating the presence of iron-containing sequestration compounds such as ferritin. The magnetic signal of these species was too weak to be observed until all traces of superparamagnetc iron had disappeared. To further investigate this result, the authors used TEM. Portions of the organs to be analyzed were cut into 1 mm3 regions and processed via fixation, staining, dehydration, and embedding in epoxy resin. Areas of interest were broadly identified via fluorescence microscopy with a methyl II – azure blue stain, and smaller slices for TEM analysis were acquired from these larger areas. Wide-field images identified areas of prominent iron oxide NP uptake – usually in macrophages. On day 1, higher magnification imaging along with EDS and diffraction-mode imaging confirmed the morphology, elemental content, and crystallinity of the IONPs (see Figure 4 d-g). By day 7, the morphology of some IONPs had changed substantially, resulting in “fingerprint like whorls” which the authors identified as ferritin particles. Additional ferritin particles, identified by their low crystallinity and octahedral shape (Figure 4 h-j) are packed together in lysosomes. These trends are born out at longer time points. At 90 days, no discrete NPs can be visualized and all the EDS-identified iron is present in ferritin-like structures. These TEM data, along with the dramatic loss in superparamagnetic iron and magnetization, indicate that the exogenous iron oxide NPs degraded to iron ions and were sequestered in iron storage proteins


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such as ferritin. This work is an excellent example of the combination of multiple characterization techniques to piece together a thorough picture of the nano/bio interface in the case of magnetic NPs.

Conclusion The question of how magnetic NPs interact with biological environments will only become more important in the future as the creation and application of engineered nanoparticles accelerates further. To thoroughly understand the nano/bio interface, researchers need to utilize a variety of correlative techniques, paying careful attention to sample preparation considerations. It is of particular importance that NPs are recognized as dynamic structures, capable of oxidation, dissolution, and even re-deposition as they interact with the complex in vivo environment.

AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT K.R.H. acknowledges funding from a University of Minnesota Doctoral Dissertation Fellowship. H.L.R acknowledges funding from a University of Minnesota “MN Futures” seed grant. H.K. and N.D.K. acknowledge the University of Minnesota for funding.


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credit to Ella Marushchenko 352x458mm (72 x 72 DPI)


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