doi.org/10.26434/chemrxiv.11845584.v2

Complex Relationship Between Signal Intensity Properties in MagneticParticle Imaging (MPI) and Iron Oxide Nanoparticle DegradationJulia Guzy, Shatadru Chakravarty, Foster Buchanan, Haoran Chen, Jeffrey M. Gaudet, Jeremy Hix,Christiane L. Mallett, Erik Shapiro

Submitted date: 18/02/2020 • Posted date: 19/02/2020Licence: CC BY-NC-ND 4.0Citation information: Guzy, Julia; Chakravarty, Shatadru; Buchanan, Foster; Chen, Haoran; M. Gaudet,Jeffrey; Hix, Jeremy; et al. (2020): Complex Relationship Between Signal Intensity Properties in MagneticParticle Imaging (MPI) and Iron Oxide Nanoparticle Degradation. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.11845584.v2

Magnetic particle imaging (MPI) is an exciting new biomedical imaging technology that usessuperparamagnetic nanoparticles as an imaging tracer. MPI is touted as a quantitative imaging modality butMPI signal properties have never been characterized for nanoparticles undergoing biodegradation. Here wecharacterize the nature of the MPI signal properties as a function of degradation of various magnetic particleformulations. We show that MPI signal properties can increase or decrease as a function of nanoparticleformulation and chemical environment and that long-term in vitro experiments only roughly approximatelong-term in vivo MPI signal properties. Data are supported by electron microscopy of nanoparticledegradation. Knowledge of MPI signal property changes during nanoparticle degradation will be critical indesign and interpretation of all MPI experiments. Further, we demonstrate for the first time, an environmentallysensitive MPI contrast mechanism opening the door to smart contrast paradigms in MPI.

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Complex relationship between signal intensity properties in magnetic particle imaging (MPI)

and iron oxide nanoparticle degradation

Julia Guzy,1* Shatadru Chakravarty,1* Foster J. Buchanan,1 Haoran Chen,1 Jeffrey M. Gaudet,2,3

Jeremy M.L. Hix,1 Christiane L. Mallett,1 and Erik M. Shapiro1

1 Department of Radiology and Institute for Quantitative Health Science and Engineering,

Michigan State University, East Lansing, MI 48824, USA

2 Institute for Quantitative Health Science and Engineering, Michigan State University, East

Lansing, MI 48824, USA

3 Magnetic Insight, Alameda, CA 94501, USA

*Equal first author contribution

Correspondence should be addressed to:

Erik M. Shapiro

MSU Radiology

846 Service Rd

East Lansing, MI 48824

shapir86@msu.edu

Phone: 517-884-3270

Fax: 517-432-2849

Abstract

Magnetic particle imaging (MPI) is an exciting new biomedical imaging technology that

uses superparamagnetic nanoparticles as an imaging tracer. MPI is touted as a quantitative

imaging modality but MPI signal properties have never been characterized for nanoparticles

undergoing biodegradation. Here we characterize the nature of the MPI signal properties as a

function of degradation of various magnetic particle formulations. We show that MPI signal

properties can increase or decrease as a function of nanoparticle formulation and chemical

environment and that long-term in vitro experiments only roughly approximate long-term in

vivo MPI signal properties. Data are supported by electron microscopy of nanoparticle

degradation. Knowledge of MPI signal property changes during nanoparticle degradation will be

critical in design and interpretation of all MPI experiments. Further, we demonstrate for the first

time, an environmentally sensitive MPI contrast mechanism opening the door to smart contrast

paradigms in MPI.

Keywords: Magnetic Particle Imaging; nanoparticles; iron oxide; biodegradation; contrast

agents.

Introduction

Scheme 1: Iron oxide embedded magnetic nanoparticles can undergo a variety ofphysicochemical changes as they degrade which can affect magnetic particle imaging (MPI)signal properties. Iron oxide nanocrystals can dissolve, which causes MPI signal to decrease.Nanoparticles can decompose, and depending on the nature of the iron oxide nanocrystals andsecondary biophysical interactions (nanocrystal aggregation or dispersion), MPI signal can eitherincrease or decrease in complex fashion.

Magnetic particle imaging (MPI) is a biomedical imaging modality that employs

superparamagnetic iron oxide nanoparticles as the imaging probes. Images are produced with a

spatial varying magnetic field containing a unique Field Free Region (FFR) to localize the MPI

signal 1. MPI generates signal by measuring the magnetization response of superparamagnetic

nanoparticles subjected to an oscillating magnetic field. This signal can be quantified as peak

signal intensity or total signal within a region of interest. The magnetization response of

superparamagnetic particles follows the classic Langevin magnetization curve. The derivative of

this magnetization curve yields a point spread function (PSF) whose full width at half height is

related to the effective spatial dimensions of the signal and is often referred to as the

“nanoparticle resolution”. The observed image resolution is influenced by both the hardware,

wherein higher gradient strength improves resolution; and the nanoparticle resolution.

Nanoparticles with high saturation magnetization and steep Langevin curves produce high signal

intensity with narrow PSF or “high resolution”, while nanoparticles with low saturation

magnetization and broad Langevin curves produce low signal intensity with wide PSF or “low

resolution” 2. This relationship seems to hold true for nanoparticles that are <15 nm, with Neel

relaxation being the dominant signal producing magnetization principle; nanoparticles >15 nm

begin to lose signal and resolution due to not reaching magnetization saturation under the

applied oscillating field caused by Brownian relaxation of the larger nanoparticles 3-6.

In principle, like other tracer-based imaging modalities such as nuclear medicine, MPI

signal can be equated to quantity of tracer, given that the signal is properly calibrated 7. In this

context, one can therefore use MPI to quantitatively measure the amount of iron oxide

nanoparticles in a given anatomical location, such as delivery of iron oxide nanoparticles

harboring drugs or cells containing iron oxide nanoparticles (recently reviewed in 2 and 8). This

gives MPI a significant potential advantage of MPI over MRI regarding the quantification of

targeted iron oxide nanoparticles for theranostics or magnetically labeled cells, as MRI has

difficulty in quantifying these due to complex changes in molar relaxivity as magnetic

nanoparticles cluster and/or degrade. Yet, for MPI, it is similarly unknown how the relationship

between MPI signal and nanoparticle quantity changes when particles degrade. Presumably, as

nanoparticles disintegrate and iron oxide nanocrystals dissolve, MPI signal will change, but in

which direction and by how much has not been thoroughly studied, though the details above

suggest that MPI signal could either increase or decrease depending on the size of the

nanoparticle and the physical arrangement of the nanocrystal cores. Coincident with this,

nanoparticle degradation could change the shape of the Langevin curve, resulting in changes to

nanoparticle resolution.

In this work we performed serial in vitro and in vivo MPI on various forms of iron oxide

nanoparticles and measured the change in MPI signal and nanoparticle resolution as a function

of nanoparticle degradation and iron oxide dissolution, as well as against the background of

nanoparticle in vivo biological clearance. Transmission electron microscopy (TEM) was

performed on samples to characterize the physical properties of the nanoparticles in an attempt

to relate these properties to MPI signal characteristics. The findings of this work are critical to

interpreting MPI data, particularly where long term biological processes are being studied.

Additionally, this work demonstrates the first enzymatic manipulation of MPI signal and

nanoparticle resolution, suggesting the potential for environmentally sensitive MPI paradigms.

Results and Discussion

Experiment 1: Effect of buffer composition and pH

In living subjects, iron oxide nanoparticles can disintegrate in multiple simultaneous

ways; the nanoparticle can disassemble, iron oxide nanocrystals can dissolve, or nanoparticles

and/or nanocrystals can be excreted. Each of these processes is likely to produce unique

changes in MPI signal and nanoparticle resolution as defined above. As a first experiment, we

measured the change in total signal intensity within a standardized region of interest, peak

signal intensity and nanoparticle resolution of four different iron oxide nanoparticles with

varying suspected degradation rates, in solutions that mimicked extracellular conditions

(phosphate buffered saline, pH 7.4), harsh endosomal/lysosomal conditions (50 mM citrate

buffer, pH 5.5) or mild endosomal/lysosomal conditions (10 mM citrate buffer, pH 6.5), during

incubation at 37℃ for 4200 hours. The four different nanoparticles used were: 1) Ferucarbotran

(Vivotrax, Magnetic Insight, Alameda, CA), which is a dextran coated iron oxide nanoparticle

with a diameter of 62 nm, 2) 45 kDa molecular weight PLGA-encapsulated iron oxide

nanoparticles (8 nm iron oxide nanocrystals) with ~ 45% Fe w/w, with a diameter of ~ 150-200

nm (CLIPs, Shape Scientific, East Lansing, MI), 3) Magnefy particles, an inert polymer coated

nanoparticle with ~ 45% Fe w/w and a diameter of 0.747 µm (Bangs Laboratories, Fishers, IN),

and 4) MPIOs, a polystyrene coated particle with ~ 45% Fe w/w, with a diameter of 1.63 µm

(Bangs Laboratories, Fishers, IN). Citrate is a strong iron leachant, so it was hypothesized that

disintegration of nanoparticles would release iron oxide nanocrystals which could be dissolved

by the citrate. For all samples, particles were withdrawn to achieve 1 mg of iron of each

respective nanoparticle, added to small plastic PCR tubes and then diluted with solution to

reach 500 µL. Samples were placed in a tube rotator, to prevent settling of the nanoparticles,

and incubated at 37℃ for the duration of the experiment.

Figure 1: A-C) Normalized MPI signal and D-F) Nanoparticle resolution for Bangs, Magnefy,Vivotrax and CLIPs during degradation over 4200 hours in PBS, pH 7.4 (A,D), citrate buffer, pH6.5 (B,E), and citrate buffer, pH 5.5 (C,F). Error bars represent standard deviations of singlemeasurements in 3 different samples. G) TEM of various nanoparticles under conditions aslabeled. Scale bars as indicated. H) MPI images of 3 samples of nanoparticles as a function ofincubation time as labeled. Note different scales for each nanoparticle on scale bars.

Serial 2D MPI in isotropic mode was performed on a Magnetic Insight Momentum MPI

system with a gradient strength of 5.7 T/m (Magnetic Insight, Alameda, CA). Within each image,

the regions of interest that included the samples were extracted and the sum of the image

signal was obtained. Using the RELAX module, we acquired relaxometry scans which are used to

measure nanoparticle resolution. For the most caustic condition of 50 mM citrate buffer, pH 5.5,

each particle exhibited varied reduction in total and peak MPI signal intensity over the 4200

hours, and changes in nanoparticle resolution. The MPI signal intensity decrease due to particle

degradation and iron oxide nanocrystal dissolution predictably followed our previous MRI-based

study 9, with the signal from the highly biodegradable Vivotrax completely disappearing at

~1200 hours, the signal intensity of the Magnefy completely disappearing at ~3500 hours, the

signal intensity of the slowly biodegradable CLIPs reducing to ~50% at ~3000 hours and 40% at

4200 hours, and the signal intensity of the inert MPIOs reducing to 80% at 4200 hours.

Nanoparticle resolution for Vivotrax remained essentially constant until near complete loss of

signal, nanoparticle resolution for Magnefy particles decreased steadily until near complete loss

of signal, nanoparticle resolution for CLIPs varied within a small range until near the end of the

experiment, and nanoparticle resolution for MPIO remained completely constant throughout

the entire experiment. Decrease in signal intensities and changes in nanoparticle resolution

were significantly less in the more mild 10 mM citrate buffer, pH 6.5. For PBS, MPI signal and

nanoparticle resolution did not change for the first 1500 hours for any particle. Unpredictably,

Vivotrax exhibited a 30% increase in total signal intensity at 1500 hours, coincident with a drop

in peak signal intensity of ~30%, and a doubling in nanoparticle resolution that was maintained

through 4200 hours.

TEM was performed on all four particle types incubated in the three different solutions,

at the beginning and end of the experiment. It was found that TEM produced better images

following dialysis of nanoparticles against water for 48 hours prior. In general, for all four

particle types, TEM confirms nanoparticle decomposition during the experiment, albeit to

different degrees depending on the nanoparticle type and the incubation solution. For Vivotrax,

TEM shows that iron oxide nanocrystals are completely dissolved at pH 5.5 while in pH 6.5 and

in PBS some nanocrystals remain. For Magnefy particles, at the beginning of the experiment,

TEM shows large spherical nanoparticles with a coating of much smaller nanoparticles. For

CLIPs, at the beginning of the experiment, TEM shows ~ 100 nm spherical polymer encapsulated

iron oxide nanoparticles. At the end of the experiment, TEM shows loss of particle structure at

pH 5.5. CLIPs appear to be structurally intact in PBS at the end of the experiment. For MPIOs,

TEM shows intact spherical polymer encapsulated iron oxide particles both at the beginning and

end of the experiment. As such, the disappearance in signal intensity for Vivotrax and small

decrease in signal intensity for CLIPs in 50 mM citrate buffer, pH 5.5, and the lack of signal

intensity changes for these particles in 10 mM citrate buffer, pH 6.5 and PBS, is well explained

by the decomposition of the nanoparticles and dissolution of the iron oxide nanocrystals in

citrate buffer at pH 5.5. Magnefy particles are a proprietary product of Bangs Laboratories, and

little information is publicly available. For MPIOs, the lack of signal intensity change in pH 6.5 or

PBS is explained by inertness of the particles. The small decrease in signal intensity at pH 5.5

could be explained by some dissolution of the iron oxide nanocrystals from the interior of the

MPIOs or iron oxide nanocrystals that may have resided at the surface of the MPIOs.

Experiment 2: Effect of particle coating

Figure 2: A) Normalized MPI signal for Vivotrax over 187 hours in acetate buffer, pH 6.5, withand without the addition of dextranase. Error bars represent standard deviations of singlemeasurements in 3 different samples. B) Nanoparticle resolution for Vivotrax over 187 hours inacetate buffer, pH 6.5, with the addition of dextranase. C) MPI images of 3 samples of Vivotraxat 187 hours with and without dextranase. Note the elongation of the MPI signal in theDextranase samples with respect to samples without enzyme, reflecting the worsening of thenanoparticle resolution.

To probe the unexpected robust increase in total signal intensity, peak signal intensity

decrease, and decrease in nanoparticle resolution of the Vivotrax in PBS following incubation,

we devised an experiment to rapidly decompose Vivotrax in an attempt to isolate the effects of

particle disintegration from other variables that could have presented themselves during long

term incubation. To rapidly decompose Vivotrax, we adapted an experiment previously

performed with dextran coated iron oxide nanoparticles for MRI, called REACTION or RElease

ACTivated Iron Oxide Nanoparticles 10, where dextranase is used to rapidly degrade the dextran

coating of nanoparticles to release the encapsulated iron oxide nanocrystals. Here, we

incubated particles with dextranase in 10 mM acetate buffer, pH 5.6 for ~ 1 week to degrade the

dextran and release the iron oxide nanoparticles. 2D MPI was performed in default mode and

indeed, the same 30% increase in total signal intensity and 30% decrease in peak signal intensity

was measured in solution containing dextranase, while Vivotrax incubated without dextranase

had no change in signal intensity. Dynamic light scattering measured an increase in nanoparticle

size from 75 nm for intact Vivotrax to 330 nm for degraded Vivotrax, suggesting that removal of

the dextran coating resulted in aggregation of the nanocrystal cores. A broadening in FWHH was

measured in the relaxometry curves, precisely matching that measured in the long-term

degradation experiment.

We hypothesize that the increase in total signal intensity, decrease in peak signal

intensity, and broadening of the nanoparticle resolution is the result of a decreased

magnetization response of the particles as they increase in size. As demonstrated by Tay et al.,

with standard MPI excitation techniques larger particles do not achieve magnetic saturation and

the Langevin model is not applicable 6. This results in a broadening of the particles resolution

and a decrease in the observed peak signal intensity as a larger magnetic field change is

required to saturate the magnetization. However, the particles are subjected to strong fields as

the FFR moves away from the sample ultimately saturating all of the particles as measured by a

higher net signal intensity in the images. This was tested by measuring the signal response to

decreasing RF excitation amplitude from 15 mT to 9 mT. Signal decreased on average by 8% for

dextranase treated Vivotrax compared to a 2% average increase for control Vivotrax at the lower

excitation amplitude. These results support the hypothesis that the observed signal change of

the treated Vivotrax is due to a decreased magnetization response of the resulting particles.

Experiment 3:

Figure 3: A-C) Normalized MPI signal and D-F) TEM for CLIPs comprised of (A,D) 45 kDa PLGAand 8 nm iron oxide nanocrystals over 1000-1250 hours, (B,E) 5 kDa PLGA and 25 nm iron oxidenanocrystals, and (C,F) 45 kDa PLGA and 25 nm iron oxide nanocrystals, in PBS, pH 7.4,phosphate buffer, pH 6.5, and citrate buffer, pH 5.5. Error bars represent standard deviations ofsingle measurements in 3 different samples.

Effect of polymer molecular weight and nanocrystal size

To further probe the change in MPI signal as a function of degradation, we next

performed a series of experiments to compare the change in MPI signal for CLIPs as a function

of PLGA molecular weight and size of iron oxide nanocrystal. In the long-term degradation

experiments performed in Experiment 1, CLIPs were fabricated with 45 kDa molecular weight

PLGA and 8 nm iron oxide nanocrystals and remained largely intact, especially in PBS and at pH

6.5, during the course of the experiment. We hypothesized that nanoparticle degradation would

be accelerated by using 5 kDa molecular weight PLGA, as PLGA nanoparticle degradation is

faster with lower molecular weight polymer 11, and as recently demonstrated by Zhu et al 12. To

test this hypothesis, PLGA encapsulated iron oxide nanoparticles were fabricated as previously

described 9, 13 using 5 kDa MW PLGA and 8 nm iron oxide nanoparticles and were incubated in

the solutions used by Zhu et al. 12, PBS and phosphate buffer, pH 6.5, and in the harsh 50 mM

citrate buffer, pH 5.5 we used in Experiment 1. MPI was performed over ~1250 hours. Firstly, as

compared to PLGA encapsulated iron oxide nanoparticles fabricated from high molecular weight

PLGA containing 8 nm iron oxide nanocrystals, particles made from low molecular weight PLGA

exhibited more rapid decrease in total signal intensity in PBS, phosphate buffer, pH 6.5, and 50

mM citrate buffer, pH 5.5. For the acidic solutions, this can be explained by more rapid

degradation of the PLGA particles and dissolution of the iron oxide 9, 13. This is supported by the

TEM images of these particles at the end of the experiment, where at pH 6.5, nanoparticles

have decomposed, but iron oxide nanocrystals remain, whereas at pH 5.5, iron oxide

nanocrystals appear to be nearly dissolved. The total signal intensity in PBS is statistically

indifferent (p>0.05; except at 336 hours, p=0.04) from that measured at pH 6.5, with TEM

similarly demonstrating complete decomposition of the PLGA particles with only iron oxide

nanocrystals remaining. This suggests that PLGA encapsulated iron oxide nanoparticles

composed of 5 kDa MW PLGA are generally unstable in physiologically meaningful solutions.

Next, we fabricated PLGA encapsulated iron oxide nanoparticles with 5 kDa molecular

weight PLGA containing 25 nm iron oxide nanocrystals and measured the total signal intensity in

the same three solutions. We hypothesized that similar to the previous particles, and as shown

by Zhu, et al. 12, that 5 kDa PLGA particles would rapidly degrade, and that due to the release of

the iron oxide nanocrystals, total signal intensity would increase for particles incubated in both

PBS and at pH 6.5, and would decrease quickly in pH 5.5. The reason for the increase in total

signal intensity has been attributed to an increase in net magnetization as a result of reduced

Brownian relaxation of the nanocrystals 6. Indeed, for particles incubated in PBS and phosphate

buffer, pH 6.5, we measured 2.7-fold and 3.3-fold MPI signal increase over 981 hours for both

PBS and phosphate buffer, pH 6.5, respectively, but at a much slower rate than reported by Zhu

et al. 12, with higher magnitude change in total signal intensity. Total signal intensity changes for

particles incubated in PBS or at pH 6.5 become statistically different from baseline (p<0.05) at

51 hours and total signal intensity changes at pH 6.5 become statistically different (p<0.05) from

PBS at 332 hours incubation time. The samples incubated in 50 mM citrate buffer, pH 5.5

exhibited near instant reduction in total signal intensity essentially to baseline presumably as

the iron oxide nanocrystals were dissolved by the citrate. TEM of samples at the end of the

experiment confirm that for PBS and pH 6.5, intact PLGA particles no longer remain and that

only iron oxide nanocrystals remain, while at pH 5.5, no iron oxide nanocrystals are seen. Again,

this suggests that 5 kDa PLGA particles are generally unstable when encapsulating iron oxide

nanocrystals, or that 25 nm iron oxide nanocrystals are destabilizing when encapsulated in low

molecular weight PLGA.

Lastly, to explore the effect of PLGA molecular weight on the stability of nanoparticles

when harboring 25 nm iron oxide nanoparticles, we fabricated PLGA encapsulated iron oxide

nanoparticles with 45 kDa MW PLGA and 25 nm iron oxide nanoparticles. Based on the results

of our experiments in Experiment 1, we hypothesized that PLGA encapsulated iron oxide

nanoparticles with 45 kDa MW PLGA would degrade more slowly, resulting in slower change in

MPI signal intensities in the three solutions (increases in PBS and at pH 6.5; decrease at pH 5.5).

Indeed, over the course of the experiment, the total signal intensity increased more slowly as

compared to PLGA encapsulated iron oxide nanoparticles with 5 kDa MW PLGA, with particles

incubated in PBS and at pH 6.5 both achieving 2.3-fold increase in total signal intensity at 1051

hours. Unexpectedly, the total signal intensity of nanoparticles incubated at pH 5.5 increased

nearly 2-fold by 240 hours and leveled off between 1.6-1.8-fold increase in total signal intensity

for the length of the experiment, with data points from 334 hours until 1051 hours being

statistically indistinguishable (p>0.05).

Experiment 4: MPI signal change in vivo

Figure 4: A) Normalized in vivo MPI signal in mouse liver following injection with either Vivotrax(n=2 mice) or Magnefy (n=2 mice) over 3082 hours. Data is individually plotted with in vitro datafor Vivotrax and Magnefy for reference. B) In vivo MPI of individual mice; one injected withVivotrax and one injected with Magnefy. Note the difference in the scales of the scale bars.Time points for MPI is as indicated.

Lastly, we performed a set of in vivo MPI on mice injected with the fast degrading

Vivotrax (n=2) and the slow degrading Magnefy (n=2) to determine how well the in vitro

experiments predict in vivo biodegradation. The hypothesis, supported by work from Keselman

et al. 14, was that following injection of nanoparticles, the Kupffer cells of the liver would

phagocytose nanoparticles and digest the nanoparticles, resulting in a decrease in total signal

intensity. 2D MPI in isotropic mode was performed on isoflurane anesthetized mice according to

an approved MSU IACUC protocol in accordance with AALAS guidelines for animal experiments.

Indeed, immediately after injection, mice had high total signal intensity at the location of the

liver. Over the course of 3082 hours, mice injected with each particle exhibited decrease in total

signal intensity, with Vivotrax signal decaying faster (31.7 d half-life) than Magnefy particles

(36.2 d half-life). In comparing in vitro experiments to in vivo experiments, Vivotrax exhibited

slower MPI signal loss in vivo as compared to Vivotrax incubated in vitro in 50 mM citrate buffer,

pH 5.5 (13.2 d half-life), presumably due to slower nanoparticle degradation and iron oxide

nanocrystal dissolution in vivo. Perhaps this suggests that 50 mM citrate buffer, pH 5.5 is too

harsh a buffer to properly simulate endosomal/lysosomal digestion. This has also been observed

by Barrow et al. 15. Surprisingly, for the Magnefy particles, the rate of MPI signal change in vitro

in 50 mM citrate buffer, pH 5.5 was slower (69.5 d half-life) than in vivo. In comparison to the in

vivo signal clearance measured in Keselman et al. 14, the in vivo degradation rate for Vivotrax

measured in this study was significantly longer (31.7 d vs 5.6 d).

Limitations of this study

In vitro experiments: For these experiments, samples were incubated at 37℃ in small plastic

tubes and underwent either rotating (Experiment 1) or constant rocking (Experiment 2 and 3). It

is possible that physical forces during incubation influence time scales for decomposition, and

that slower or faster degradation kinetics will be measured in samples subjected to variable

forces, such as samples incubated in rotators or shaken faster or slower. Another limitation is

the choice of buffers we used, especially for mimicking lysosomal conditions. We and others 9, 13,

16-18 have used acidic citrate buffer as a rudimentary lysosomal environmental mimic, but other

lysosomal mimic buffers have been described 19-21 and would likely influence degradation

kinetics. A third limitation is that these experiments were performed on the first commercial

Magnetic Insight MPI system with a gradient strength of 5.7 T/m, and that MPI technology is

rapidly evolving, and that different magnitude of results would be obtained by using other MPI

systems or power settings.

In vivo experiments: For in vivo experiments, in parallel to biodegradation, iron oxide

nanoparticles can be excreted by the animal in waste. An attempt to image fecal waste at

different time points in our study failed to detect MPI signal, though others have been

successful 22. But while the ability to simply use MPI to measure true in vivo biodegradation is

our studies was not possible, it points to a general limitation in the ability of biomedical imaging

to study in situ biodegradation of nanomaterials where excretion occurs in parallel. As such,

highly informative and well-planned in vitro degradation experiments take on increased

importance.

Impact of the complex relationship of iron oxide degradation and MPI signal intensity properties

on biomedical MPI studies: There is a vast number of proposed uses for MPI in biomedical

research and perhaps soon in human health, but to date, the two most commonly reported

experiments are: 1) tracking the delivery and persistence of magnetic nanoparticles in a

particular location, usually a tumor, following delivery to an animal model, for drug delivery

and/or imaging purposes, and 2) tracking the delivery and persistence of magnetically labeled

cells in a particular location, following administration 23. For tracking magnetic nanoparticles for

drug delivery, when using nanoparticles with slow degradation kinetics, the impact of

nanoparticle degradation-induced changes in signal properties are likely to be negligible for

acute studies, though changes in signal properties due to aggregation/clustering/binding could

present themselves. When using nanoparticles with fast degradation kinetics, such as dextran

coated iron oxide nanoparticles, changes in signal properties due to nanoparticle degradation

could manifest themselves within 4 days. This means that in the hours and days following the

injection and accumulation of relatively inert theranostic iron oxide nanoparticles in a tumor, for

example, the ability to use MPI to measure the quantity of iron oxide nanoparticles at that site

should be straightforward, providing again, that the signal has been well calibrated, whereas

reliable measurements for rapidly biodegradable nanoparticles can only be made within the

first 2-3 days. Regarding the composition of PLGA encapsulated iron oxide nanoparticles, we

identified that large iron oxide nanocrystals within low molecular weight PLGA were highly

destabilizing, causing rapid decomposition of the nanoparticle, as evidenced by very rapid

dissolution of the iron oxide nanocrystals in acidic citrate buffer and parallel signal behavior of

these particles in both PBS and acidic phosphate buffer.

For the second set of popular experiments, where cells are labeled either in vitro or in

vivo with magnetic particles, similar as above, reliable measurements of cell numbers, enabled

by the surrogate measurement of iron oxide nanoparticle MPI signal, can be made for inert

particles for several weeks, but for rapidly biodegrading particles, reliable measurements can

only be made within the first 2-3 days. Thus, like MRI-based cell tracking, the ability of MPI to

reliably measure the persistence of magnetically labeled cells long term, is likely dependent on

the type of particle employed for labeling, notwithstanding the complexity of free nanoparticles

in the vicinity from dead cells impacting the ability to actually measure cells - the so-called

‘bystander effect’.

In conclusion, we performed exhaustive and comprehensive experiments measuring

properties of MPI signal as a function of iron oxide nanoparticle and degradation environments.

We demonstrated complex relationships between MPI total signal, peak signal intensity and

nanoparticle resolution for different types of iron oxide nanoparticles in different conditions,

which will inform MPI users on interpretation of MPI data especially in longitudinal studies. A

comparison as made with in vivo degradation experiments in rodents where we demonstrated

the challenges in simply using MPI (and allude to a general problem in biomedical imaging) to

study in situ biodegradation where parallel elimination of the very materials under study are

happening. We also demonstrated, for the first time, an environmentally sensitive MPI imaging

strategy where enzymatic degradation of a nanoparticle coating can produce robust changes in

MPI signal properties. This initial exhibition should encourage many more environmentally

sensitive contrast paradigms, similar to the plethora of environmentally sensitive imaging

paradigms one finds for MRI.

Acknowledgements

Authors are grateful for helpful conversations with Dr. Daniel Hensley and Dr. Patrick Goodwill,

Magnetic Insight. Authors Chakravarty and Shapiro have pending patents on the PLGA-

encapsualted iron oxide nanoparticles described herein and Shapiro is Founder and President of

Shape Scientific, supplier of the iron oxide nanocrystals and CLIPs used in these studies.

References

1. Gleich, B.; Weizenecker, J., Tomographic imaging using the nonlinear response of magnetic particles. Nature 2005, 435 (7046), 1214-7.2. Knopp, T.; Gdaniec, N.; Moddel, M., Magnetic particle imaging: from proof of principle to preclinical applications. Phys Med Biol 2017, 62 (14), R124-R178.3. Ferguson, R. M.; Minard, K. R.; Krishnan, K. M., Optimization of nanoparticle core size for magnetic particle imaging. J Magn Magn Mater 2009, 321 (10), 1548-1551.4. Biederer, S.; Knopp, T.; Sattel, T. F.; Ludtke-Buzug, K.; Gleich, B.; Weizenecker, J.; Borgert, J.; Buzug, T. M., Magnetization response spectroscopy of superparamagnetic nanoparticles for magnetic particle imaging. J Phys D Appl Phys 2009, 42 (20).5. Rauwerdink, A. M.; Weaver, J. B., Viscous effects on nanoparticle magnetization harmonics. Journal of Magnetism and Magnetic Materials 2010, 322 (6), 609-613.6. Tay, Z. W.; Hensley, D.; Ma, J.; Chandrasekharan, P.; Zheng, B.; Goodwill, P.; Conolly, S., Pulsed Excitation in Magnetic Particle Imaging. Ieee T Med Imaging 2019, 38 (10), 2389-2399.7. Lu, K.; Goodwill, P. W.; Saritas, E. U.; Zheng, B.; Conolly, S. M., Linearity and Shift Invariance for Quantitative Magnetic Particle Imaging. Ieee T Med Imaging 2013, 32 (9), 1565-1575.8. Talebloo, N.; Gudi, M.; Robertson, N.; Wang, P., Magnetic Particle Imaging: Current Applications in Biomedical Research. J Magn Reson Imaging 2019.9. Granot, D.; Nkansah, M. K.; Bennewitz, M. F.; Tang, K. S.; Markakis, E. A.; Shapiro, E. M., Clinically viable magnetic poly(lactide-co-glycolide) particles for MRI-based cell tracking. Magn Reson. Med 2014, 71 (3), 1238-1250.10. Granot, D.; Shapiro, E. M., Release activation of iron oxide nanoparticles: (REACTION) a novel environmentally sensitive MRI paradigm. Magn Reson Med 2011, 65 (5), 1253-1259.11. Makadia, H. K.; Siegel, S. J., Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel) 2011, 3 (3), 1377-1397.12. Zhu, X. J.; Li, J. F.; Peng, P.; Nassab, N. H.; Smith, B. R., Quantitative Drug Release Monitoring in Tumors of Living Subjects by Magnetic Particle Imaging Nanocomposite. Nano Letters 2019, 19 (10), 6725-6733.13. Nkansah, M. K.; Thakral, D.; Shapiro, E. M., Magnetic poly(lactide-co-glycolide) and cellulose particles for MRI-based cell tracking. Magn Reson Med 2011, 65 (6), 1776-1785.14. Keselman, P.; Yu, E. Y.; Zhou, X. Y.; Goodwill, P. W.; Chandrasekharan, P.; Ferguson, R. M.; Khandhar, A. P.; Kemp, S. J.; Krishnan, K. M.; Zheng, B.; Conolly, S. M., Tracking short-term biodistribution and long-term clearance of SPIO tracers in magnetic particle imaging. Phys Med Biol 2017, 62 (9), 3440-3453.15. Barrow, M.; Taylor, A.; Fuentes-Caparros, A. M.; Sharkey, J.; Daniels, L. M.; Mandal, P.; Park, B. K.; Murray, P.; Rosseinsky, M. J.; Adams, D. J., SPIONs for cell labelling and tracking using MRI: magnetite or maghemite? Biomater Sci-Uk 2017, 6 (1), 101-106.16. Bennewitz, M. F.; Williams, S. S.; Nkansah, M. K.; Shapiro, E. M., Poly(lactic-co-glycolic acid) encapsulated gadolinium oxide nanoparticles for MRI-based cell tracking. J. Nanosci. Nanotechnol 2013, 13 (6), 3778-3783.17. Bennewitz, M. F.; Lobo, T. L.; Nkansah, M. K.; Ulas, G.; Brudvig, G. W.; Shapiro, E. M., Biocompatible and pH-Sensitive PLGA Encapsulated MnO Nanocrystals for Molecular and Cellular MRI. Acs Nano 2011, 5 (5), 3438-3446.

18. Arbab, A. S.; Wilson, L. B.; Ashari, P.; Jordan, E. K.; Lewis, B. K.; Frank, J. A., A model of lysosomal metabolism of dextran coated superparamagnetic iron oxide (SPIO) nanoparticles: implications for cellular magnetic resonance imaging. NMR Biomed 2005, 18 (6), 383-389.19. Russier, J.; Menard-Moyon, C.; Venturelli, E.; Gravel, E.; Marcolongo, G.; Meneghetti, M.; Doris, E.; Bianco, A., Oxidative biodegradation of single- and multi-walled carbon nanotubes. Nanoscale 2011, 3 (3), 893-6.20. Carreira, A. C.; de Almeida, R. F. M.; Silva, L. C., Development of lysosome-mimicking vesicles to study the effect of abnormal accumulation of sphingosine on membrane properties. Sci Rep 2017, 7 (1), 3949.21. Plan Sangnier, A.; Van de Walle, A. B.; Curcio, A.; Le Borgne, R.; Motte, L.; Lalatonne, Y.; Wilhelm, C., Impact of magnetic nanoparticle surface coating on their long-term intracellular biodegradation in stem cells. Nanoscale 2019, 11 (35), 16488-16498.22. Tay, Z. W.; Chandrasekharan, P.; Zhou, X. Y.; Yu, E.; Zheng, B.; Conolly, S., In vivo tracking and quantification of inhaled aerosol using magnetic particle imaging towards inhaled therapeutic monitoring. Theranostics 2018, 8 (13), 3676-3687.23. Bulte, J. W. M., Superparamagnetic iron oxides as MPI tracers: A primer and review of early applications. Adv Drug Deliv Rev 2019, 138, 293-301.

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Complex relationship between signal intensity properties in magnetic particle imaging (MPI)

and iron oxide nanoparticle degradation

Julia Guzy,1* Shatadru Chakravarty,1* Foster J. Buchanan,1 Haoran Chen,1 Jeffrey M. Gaudet,2,3

Jeremy M.L. Hix,1 Christiane L. Mallett,1 and Erik M. Shapiro1

1 Department of Radiology and Institute for Quantitative Health Science and Engineering,

Michigan State University, East Lansing, MI 48824, USA

2 Institute for Quantitative Health Science and Engineering, Michigan State University, East

Lansing, MI 48824, USA

3 Magnetic Insight, Alameda, CA 94501, USA

*Equal first author contribution

Correspondence should be addressed to:

Erik M. Shapiro

MSU Radiology

846 Service Rd

East Lansing, MI 48824

shapir86@msu.edu

Phone: 517-884-3270

Fax: 517-432-2849

Materials and methods

In Vitro Sample Preparation for Experiment 1

Four types of superparamagnetic iron oxide particles were suspended in three different

types of solutions. Experiments were performed in triplicate (N=3 for each sample in medium).

The solutions used were: 1) phosphate-buffered saline, pH of 7.4, 50 mM sodium citrate, pH 5.5,

20 mM sodium citrate, pH 6.5. Nanoparticles used were: 1) Ferucarbotran (Vivotrax, Magnetic

Insight, Alameda, CA), which is a dextran coated iron oxide nanoparticle with a diameter of 62

nm, 2) PLGA-encapsulated iron oxide nanoparticles with ~ 45% Fe w/w, with a diameter of ~ 150

nm (CLIPs, Shape Scientific, East Lansing, MI), 3) Magnefy particles, an inert polymer coated

nanoparticle with ~ 45% Fe w/w and a diameter of 0.747 µm (Bangs Laboratories, Fishers, IN),

and 4) MPIOs, a polystyrene coated particle with ~ 45% Fe w/w, with a diameter of 1.63 µm

(Bangs Laboratories, Fishers, IN).

For all samples, particles were withdrawn to achieve 1 mg of iron of each respective

nanoparticle, added to Eppendorf tubes and then diluted with solution to reach 500 µL.

Samples were placed in a tube rotator, to prevent settling of the nanoparticles, and incubated at

37℃ for the duration of the experiment.

In vitro MPI Imaging

All scans were performed on a Magnetic Insight Momentum MPI system. For Experiment

1, serial Isotropic 2D scans with a gradient strength of 5.7 T/m were performed on samples, and

the images were analyzed using VivoQuant software (Invicro, Boston, MA). Within each image,

the regions of interest that included the samples were extracted. All ROIs were circular with a

diameter of 45 pixels, and the sum of the image signal in each of the regions was obtained.

Relaxometry scans were also performed on samples using the RELAX module, from which

relaxometry graphs were created and analyzed in Excel. In Experiments 2 and 3, 2D MPI was

performed in Default mode, and ROIs were 50 pixels. Relaxometry was performed as in

Experiment 1.

Fabrication of other PLGA encapsulated iron oxide nanoparticles

PLGA encapsulated iron oxide nanoparticles were synthesized using established

published procedures using 5 or 45 kDa PLGA with either 8 nm diameter iron oxide

nanoparticles synthesized by thermal decomposition from Iron(III) acetylacetonate (Shape

Scientific, East Lansing, MI) or with 25 nm diameter iron oxide nanoparticles (Ocean Nanotech,

San Diego, CA).

In vivo MPI Imaging of Mice

Mice (n=2 for each nanoparticle type, 4 mice total), were injected via tail vein with

either Vivotrax or Magnefy particles at xx mg. Mice then underwent serial periodic MPI of the

liver until 3082 hours, using isotropic imaging protocol. For MPI, mice were anesthetized with

isoflurane via nosecone to effect, and revived after each but the final scan session. Within each

image, the total image signal was measured.

Scanning electron microscopy and transmission electron microscopy of nanoparticles

For SEM analysis, NP dispersions were placed on 10 mm x 10 mm silicon wafers attached

on top of aluminum stubs. The solution was then allowed to dry in air. Sample stubs were

sputter coated with iridium with an approximate thickness of 2.7 nm (Quorum Technologies,

Laughton, East Sussex, England BN8 6BN). Finally, these sample stubs were vacuum dried for 48

h prior to imaging on a JEOL 7500F Scanning Electron Microscope with a cold field emission

emitter. For TEM analysis of the nanoparticles, square mesh, carbon support film on copper

grids (CF300-Cu, 300 mesh, standard thickness, Electron Microscopy Sciences, USA) were used.

A homogenous aqueous suspension of the NPs was prepared and 10 µL was gently placed on

the grid. The particles were allowed to settle for 30 minutes and the residual aqueous drop was

absorbed using paper. The resulting grid was air dried prior to imaging at 200 kV on a JEOL,

2200FS Transmission Electron Microscope. SEM and TEM of samples at the end of the

experiments were performed on samples that had been dialyzed against DI water for 48 hours

prior to sample preparation to avoid crystallization of salts.

download fileview on ChemRxivMagnetic particle imaging to measure iron oxide nanoparti... (39.50 KiB)

Complex relationship between signal intensity properties in magnetic particle imaging (MPI)

and iron oxide nanoparticle degradation

Julia Guzy,1* Shatadru Chakravarty,1* Foster J. Buchanan,1 Haoran Chen,1 Jeffrey M. Gaudet,2,3

Jeremy M.L. Hix,1 Christiane L. Mallett,1 and Erik M. Shapiro1

1 Department of Radiology and Institute for Quantitative Health Science and Engineering,

Michigan State University, East Lansing, MI 48824, USA

2 Institute for Quantitative Health Science and Engineering, Michigan State University, East

Lansing, MI 48824, USA

3 Magnetic Insight, Alameda, CA 94501, USA

*Equal first author contribution

Correspondence should be addressed to:

Erik M. Shapiro

MSU Radiology

846 Service Rd

East Lansing, MI 48824

shapir86@msu.edu

Phone: 517-884-3270

Fax: 517-432-2849

New concepts

Magnetic particle imaging (MPI) is an exciting new biomedical imaging technology that

uses superparamagnetic nanoparticles as an imaging tracer. In this work, we describe two new

concepts of general interest to the nanotechnology and imaging fields. The first new concept is

a thorough characterization of the effect of magnetic nanoparticle degradation on the signal

properties in MPI. We show that MPI signal properties can increase or decrease as a function of

nanoparticle formulation and chemical environment and that long-term in vitro experiments

only roughly approximate long-term in vivo MPI signal properties. Knowledge of MPI signal

property changes during nanoparticle degradation will be critical in design of new magnetic

particles for MPI and interpretation of all MPI experiments. The second new concept we

introduce is that MPI contrast can be environmentally sensitive. We demonstrate for the first

time, that an environmental stimulus, in this case an enzyme, can modulate MPI signal

properties, by altering the properties of magnetic particles. This first demonstration of an

environmentally sensitive MPI contrast mechanism opens the door to smart contrast paradigms

in MPI.

download fileview on ChemRxivMagnetic particle imaging to measure iron oxide nanoparti... (36.50 KiB)