STABILITY AND MOBILITY OF MAGNETIC NANOPARTICLES IN

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STABILITY AND MOBILITY OF MAGNETIC NANOPARTICLES IN BIOLOGICAL ENVIRONMENTS DETERMINED FROM DYNAMIC MAGNETIC SUSCEPTIBILITY MEASUREMENTS Ana C. Bohórquez, Mythreyi Unni, Sayali Belsare, Andreina Chiu Lam, Lori Rice, Christine Pampo, Dietmar W. Siemann, and Carlos Rinaldi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00419 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Figure 1. Experimental approach to evaluate magnetic nanoparticle stability and mobility in whole blood and tumor tissue explants ex-vivo using DMS measurements. 1) Cancer cell inoculation in mice. 2) Tumor harvest and blood collection 30 days after cancer cell inoculation. 3) Blood sample preparation. 4) Tumor tissue sample preparation. 5) Dynamic magnetic susceptibility (DMS) measurements performed at room temperature. 133x99mm (300 x 300 DPI)

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Figure 2. Dynamic magnetic susceptibility (DMS) spectra and lognormal diameter distributions for thermally blocked cobalt ferrite nanoparticles coated with polyethyleneimine (PEI) in phosphate buffered saline (PBS), whole blood, and tumor tissue. a) DMS spectra- open markers represent measurements immediately after contact (t = 0 h) while closed markers represent measurements 24 hours after contact (t = 24 h). b) Averaged lognormal hydrodynamic diameter distributions obtained from 3 experimental replicates by fitting DMS spectra to the Debye model of susceptibility. Branch PEI polymer molecular weight of ~25 kDa and PEI-coated MNPs surface charge in 1 mM KNO3 at pH 7.4 measures 23 ± 1 mV. 194x76mm (300 x 300 DPI)

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Figure 3. Dynamic magnetic susceptibility (DMS) spectra and lognormal diameter distributions for thermally blocked cobalt ferrite nanoparticles coated with carboxymethyl dextran (CMDx) in phosphate buffered saline (PBS), whole blood, and tumor tissue. a) DMS spectra- open markers represent measurements immediately after contact (t = 0 h) while closed markers represent measurements 24 hours after contact (t = 24 h). b) Averaged lognormal hydrodynamic diameter distributions obtained from 3 experimental replicates by fitting DMS spectra to the Debye model of susceptibility. CMDx polymer molecular weight of ~10 kDa and CMDxcoated MNPs surface charge in 1 mM KNO3 at pH 7.4 measures -30 ± 2 mV. 202x76mm (300 x 300 DPI)

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Figure 4. Dynamic magnetic susceptibility (DMS) spectra and lognormal diameter distributions for thermally blocked cobalt ferrite nanoparticles coated with poly (ethylene glycol) (PEG) in phosphate buffered saline (PBS), whole blood, and tumor tissue. a) DMS spectra- open markers represent measurements immediately after contact (t = 0 h) while closed markers represent measurements 24 hours after contact (t = 24 h). b) Averaged lognormal hydrodynamic diameter distributions obtained from 3 experimental replicates by fitting DMS spectra to the Debye model of susceptibility. Modified PEG polymer molecular weight of ~2 kDa and PEG-coated MNPs surface charge in 1 mM KNO3 at pH 7.4 measures 0 ± 1 mV. 77x29mm (300 x 300 DPI)

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Figure 5. Comparison of nanoparticle stability and mobility obtained from features of their DMS spectra. a) Changes in average hydrodynamic diameter for cobalt ferrite nanoparticles coated with PEI, CMDx, and PEG over 24 hours in water, PBS, blood, and tumor tissue explants determined from fitting the out-of-phase susceptibility spectra to the Debye model. b) Changes in the initial susceptibility, normalized with respect to that obtained in PBS, for cobalt ferrite nanoparticles coated with PEI, CMDx, and PEG over 24 hours in PBS, blood, and tumor tissue explants. 76x27mm (300 x 300 DPI)

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Figure 6. Comparing stability and mobility for commercial Bionized NanoFerrite (BNF particles) in blood and tissue from features of their dynamic magnetic susceptibility spectra. a) Changes in hydrodynamic diameters for BNF particles of two different sizes over 24 hours in water, PBS, blood, and tumor tissue explants determined from fitting the out-of-phase susceptibility spectra to the Debye model. b) Changes in the initial susceptibility, normalized with respect to that obtained in PBS for BNF 80 and BNF 100 particles over 24 hours in PBS, blood, and tumor tissue explants to monitor loss of particle mobility. BNF 80 and 100 particles coated with hydroxyethyl starch (HES) of unknown molecular weight and surface charge in 1 mM KNO3 at pH 7.4 measures -4 ± 1 mV and -6 ± 1 mV, respectively. 76x28mm (300 x 300 DPI)

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Figure 7. Representative transmission electron microscopy images of polyethyleneimine (PEI), carboxymethyl dextran (CMDx), and BNF 100 magnetic nanoparticles injected into tumor tissue explants and incubated for 30 min at 25°C. Highly electron dense features correspond to MNPs. Annotations: C cells; D – disintegrating cell membrane; L – lysosomes; MVBs – multivesicular bodies; N - cell nucleus. a) Clustered PEI-coated magnetic nanoparticles embedded with disintegrating cell membrane adjacent to a cell; b) Zoom in showing PEI-coated magnetic nanoparticles associated to an orphan MVB along with disintegrating cell membrane; c) Tumor xenograft stromal tissue with associated CMDx-coated magnetic nanoparticles; d) Zoom in of CMDx-coated magnetic nanoparticle clusters; e) Cell showing endocytosis of HES-coated magnetic nanoparticles (BNF 100), MVB formation and lysosome formation with co-localized magnetic nanoparticles; f) Two adjacent cells with MVB and lysosome formation and co-localized HES-coated magnetic nanoparticles (BNF 100). Scale bar 100 nm. 175x250mm (300 x 300 DPI)

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Figure 8. Using dynamic magnetic susceptibility (DMS) spectra to assess stability and mobility of magnetic nanoparticles (MNPs) in biological environments. a) Experimental DMS spectra (markers) and fit to model (dashed line). b) Illustration of DMS spectra for magnetic nanoparticles that are dispersed (solid lines) and that are aggregated (dashed lines). c) Normalized out-of-phase susceptibility spectra of MNPs suspended in biological fluids with increased concentrations of macromolecules. Increasing crowding of the nanoscale environment leads to greater hydrodynamic drag on the particles and a decrease in the peak frequency of the out-of-phase susceptibility. 294x76mm (300 x 300 DPI)

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Page 1 of 30 STABILITY AND MOBILITY OF MAGNETIC NANOPARTICLES IN BIOLOGICAL ENVIRONMENTS DETERMINED FROM DYNAMIC MAGNETIC SUSCEPTIBILITY MEASUREMENTS Ana C. Bohórquez 1, Mythreyi Unni2, Sayali Belsare1, Andreina Chiu-Lam2, Lori Rice3, Christine Pampo3, Dietmar Siemann3, and Carlos Rinaldi1,2 1

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, 1275 Center

Drive, Biomedical Sciences Building, Gainesville, FL 32611; 2Department of Chemical Engineering, University of Florida, 1030 Center Drive, Gainesville, FL 32611; 3Department of Radiation Oncology, University of Florida, Gainesville, FL 32610 ABSTRACT Low tumor accumulation following systemic delivery remains a key challenge for advancing many cancer nanomedicines. One obstacle in engineering nanoparticles for high tumor accumulation is a lack of techniques to monitor their stability and mobility in situ. One way to monitor the stability and mobility of magnetic nanoparticles biological fluids in situ is through dynamic magnetic susceptibility measurements (DMS), which under certain conditions provide a measure of the particle’s rotational diffusivity. For magnetic nanoparticles modified to have commonly used biomedical surface coatings, we describe a systematic comparison of DMS measurements in whole blood and tumor tissue explants. DMS measurements clearly demonstrated that stability and mobility changed over time and from one medium to another for each different coating. It was found that nanoparticles coated with covalently grafted, dense layers of PEG were the only ones to show good stability and mobility in all settings tested. These studies illustrate the utility of DMS measurements to estimate the stability and mobility of nanoparticles in situ, which can provide insights that lead to engineering better nanoparticles for in vivo use.

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Page 2 of 30 KEYWORDS Magnetic nanoparticles, nanoparticle stability, nanoparticle mobility, dynamic magnetic susceptibility, AC susceptibility, biological environments, blood, tumor tissue, Bionized NanoFerrite

INTRODUCTION Nanoparticles have tremendous potential for diagnostic and therapeutic use. However, such use often requires engineering them to be stable and mobile in biological environments. In contact with biological environments, nanoparticles can lose their stability and mobility due to interactions with ions and macromolecules, which can lead to their aggregation or sedimentation. Unfortunately, there are few methods available to directly assess nanoparticle stability and mobility in biological environments. Commonly, optical scattering techniques are used to assess nanoparticle stability in simple fluids. However, due to light scattering by biological components, these optical scattering techniques cannot be applied to biological environments such as whole blood and tissues. One could track particle motion using fluorescent tags, but this also requires optical access and only allows analysis of a comparatively small number of particles at a time.1-2 Finally, x-ray scattering techniques, such as x-ray photocorrelation spectroscopy, could potentially be applied to directly measure nanoparticle mobility in biological fluids and tissues, but unfortunately can only be carried out at highly specialized national laboratory facilities.3 We recognize the need for novel and accessible methods that provide insight into nanoparticle stability and mobility in biological environments in situ. Therefore, these accessible methods could provide unique insight and guide development of nanoparticles for biomedical applications. Colloidal stability studies of many nanoparticles being developed for in vivo use are often made in aqueous solutions meant to mimic biological environments, such as buffered and isotonic solutions,4-9 cell culture media,4-5,

10-12

diluted solutions of blood,13 serum,14 and plasma,6,

9, 11

artificial lysosomal

fluid,15 artificial cerebrospinal fluid,16 diluted solutions of cationic and anionic proteins,17 and concentrated protein solutions.18 These studies have suggested protein-corona formation, nanoparticle aggregation, nanoparticle dissolution, irreversible desorption of anchoring ligands, and reduction of

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Page 3 of 30 particle mobility due to protein aggregation and gelation.12,

18-20

Importantly, even though the first

biological barrier that nanoparticles encounter after systemic delivery is whole blood, there are limited studies in whole blood to assess the stability and mobility of nanoparticles. Blood plasma proteins can adsorb onto the surface of nanoparticles as a function of particle physicochemical properties, thus causing nanoparticle aggregation and/or activating an immune response. The activation of the immune system can result in low tumor accumulation following systemic delivery.21-22 Hence, nanoparticle-blood interactions may result in altered pharmacokinetics and biodistribution, compromising cancer-targeting and nanoparticle therapeutic effect.20, 22-23 In the context of magnetic nanoparticles (MNPs) in biological environments, one can envision that characterizing the response of nanoparticles to applied magnetic fields could provide a means to evaluate their stability and mobility. Indeed, various magnetism based approaches have been proposed to detect, quantify, and evaluate MNPs in biological systems. These magnetism based approaches include electron paramagnetic resonance spectroscopy,24 magneto-optical birefringence,11, dependent out-of-phase susceptibility,15,

28-30

magnetism in situ,15,

31

25-27

temperature-

magnetic particle spectrometry

(MPS),32-33 and dynamic magnetic susceptibility measurements (also known as AC susceptibility measurements) to assess the rotational diffusivity of the nanoparticles. Because of our interest in using dynamic magnetic susceptibility (DMS) measurements for assessing mechanical properties and nanoparticle interactions at nanoscale, we want to characterize the stability and mobility of magnetic nanoparticles in biological environments by means of their dynamic response to externally applied magnetic fields. Magnetic nanoparticles relaxing only by the Brownian mechanism of physical particle rotation in the presence of a externally applied magnetic fields produce a dynamic magnetic response that can deliver precise information about the hydrodynamic particle size distribution and the local viscosity of the surrounding media.34-36 Inorganic nanoparticle cores with high values of the magnetocrystalline anisotropy constant, such as cobalt ferrite (180 – 300 kJ m-3), suspended in fluids with viscosities below 1 Pa·s have exhibited a Brownian relaxation response for diameters as small as 8 nm.34 Similarly, there are

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Page 4 of 30 reports of thermally blocked iron oxide nanoparticles37-38 that also respond to small amplitude oscillating magnetic fields by physical rotation. In the past, we have studied nanoparticle-protein interactions for suspensions of cobalt ferrite MNPs in diluted17, and concentrated protein solutions18 in situ via DMS measurements. Recently, Soukup et al.39 used DMS measurements to probe the magnetic response of iron oxide magnetic nanoparticles in live cells, and these studies were extended to other inorganic cores such as bacterially synthesized zinc and cobalt-doped magnetite nanoparticles.40 In this contribution, we prepared MNPs modified to have a variety of commonly reported biomedical surface coatings, including, polyethyleneimine (PEI), carboxymethyl dextran (CMDx), and polyethylene glycol (PEG). Then, we applied DMS measurements to study the stability and mobility of these surface coated MNPs in simple fluids, whole blood, and tumor tissue explants. We also evaluated the stability and mobility of commercially obtained hydroxyethyl starch coated MNPs (Bionized NanoFerrite) used in biomedical applications. Therefore, the goal of this work was to conduct a systematic study comparing DMS measurements made in water, phosphate buffered saline (PBS), whole blood, and tumor tissue explants to assess how surface coating impacted particle stability and mobility in these environments. Our results confirmed that the nature of the nanoparticle’s coating affects stability and mobility in whole blood and tumor tissue over time. Furthermore, colloidal stability in simple fluids such as water and PBS do not extend to stability in whole blood, and good stability/mobility in whole blood does not guarantee a higher stability/mobility in tumor tissue for future biomedical applications. A schematic representation for our experimental approach to evaluate magnetic nanoparticle stability and mobility in whole blood and tumor tissue explants using DMS measurements can be seen in Figure 1.

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Figure 1. Experimental approach to evaluate magnetic nanoparticle stability and mobility in whole blood and tumor tissue explants ex-vivo using DMS measurements. 1) Cancer cell inoculation in mice. 2) Tumor harvest and blood collection 30 days after cancer cell inoculation. 3) Blood sample preparation. 4) Tumor tissue sample preparation. 5) Dynamic magnetic susceptibility (DMS) measurements performed at room temperature.

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Page 6 of 30 RESULTS AND DISCUSSION The particle coatings studied herein were selected due to their relevance in several biomedical applications.41 First, a non-viral gene transfection agent polyethyleneimine (PEI) was studied as a representative of a polycationic coating with a large buffering capacity at physiological conditions.42 PEIcoated MNPs have been used in magnetofection to improve DNA transfection efficiency43 and for targeted gene delivery.44-45 Second, nanoparticles were coated with carboxymethyl dextran (CMDx), as these have been used extensively for magnetic cell labeling,46 magnetic resonance imaging47 and studies of cancer treatment by magnetic fluid hyperthermia.48-52 CMDx-coated MNPs have shown significant non-specific interactions in biological environments due to their overall negative surface charge, phagocytic cell uptake, and biodegradability. Third, we evaluated nanoparticles coated with covalently grafted, dense layers of polyethylene glycol (PEG).53 PEG is a synthetic polymer commonly used to obtain particles that have good colloidal stability in aqueous environments, low surface charge, and low affinity for protein adsorption.54 Finally, we studied hydroxyethyl starch (HES) coated nanoparticles (BNF, Bionized NanoFerrite nanoparticles from MicroMod Partikeltechnologie GmbH, Rostock, Germany) that have been used for in vitro and in vivo magnetic fluid hyperthermia with results suggesting poor tumor accumulation and/or mobility.55-61 The detailed description of materials, particle synthesis, and surface modification methods are included the Supporting Information. A summary of the physicochemical characteristics of the particles used in this study, such as zeta potential, estimated amine number per particle, and hydrodynamic diameter can be found in Table S1. Size distribution estimations via dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and dynamic magnetic susceptibility (DMS) measurements were in good agreement as can be seen in Table S1. Transmission electron microscopy (TEM) micrographs, DMS spectra, and equilibrium magnetization curves at 300 K for the magnetic nanoparticles used in this study can be found in Figures S1 and S2 in the Supporting Information. Of note, the DMS spectra in Figure S1

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Page 7 of 30 and S2 confirm that the nanoparticles follow the Debye model with a single relaxation time and are therefore suitable for the experiments discussed hereafter. DMS measurements were applied to monitor stability and mobility of magnetic nanoparticle in water, PBS, whole blood, and tumor tissue (n = 3). By stability, here we refer to particles that do not aggregate, as evident by a hydrodynamic diameter determined from DMS measurements that does not change appreciably. By mobility, here we refer to the ability of the particles to rotate in response to the oscillating magnetic field applied during a DMS measurement, as evident by an initial magnetic susceptibility value ( x0 ) that does not change appreciably. The assessment of stability and mobility of MNPs was conducted per each type of polymer coating (PEI, CDMx, PEG and HES) and all samples were loaded with the same magnetic mass, which enables direct comparison of DMS spectra intensity in PBS, whole blood, and tumor tissue explants. Figure 2, 3 and 4 show representative DMS spectra and corresponding hydrodynamic diameter distributions for the PEI-, CMDx-, and PEG-coated MNPs. In the figures, open symbols correspond to measurements immediately after adding the particles to a given medium, whereas closed symbols correspond to measurements taken 24 hours after addition of the particles.

Figure 2. Dynamic magnetic susceptibility (DMS) spectra and lognormal diameter distributions for thermally blocked cobalt ferrite nanoparticles coated with polyethyleneimine (PEI) in phosphate buffered

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Page 8 of 30 saline (PBS), whole blood, and tumor tissue. a) DMS spectra- open markers represent measurements immediately after contact (t = 0 h) while closed markers represent measurements 24 hours after contact (t = 24 h). b) Averaged lognormal hydrodynamic diameter distributions obtained from 3 experimental replicates by fitting DMS spectra to the Debye model of susceptibility. Branch PEI polymer molecular weight of ~25 kDa and PEI-coated MNPs surface charge in 1 mM KNO3 at pH 7.4 measures 23 ± 1 mV.

The DMS spectra for PEI-coated MNPs did not show appreciable changes over 24 hours when suspended in PBS. This suggests that PEI-coated MNPs were stable in PBS for 24 hours. In contrast, the DMS spectra for PEI-coated MNPs in whole blood and in tumor explants showed an immediate decrease in signal intensity (relative to PBS, recall that all samples had the same magnetic mass), with a further decrease after 24 hours. Of note, negative susceptibility values observed in Figure 2 are a measurement artifact of the instrument at high frequencies when signal from the samples is low. These values were not included when fitting the DMS spectra to the Debye model of susceptibility. Additionally, the peak location in tumor tissue was seen to shift with time. These observations suggest that the PEI-coated MNPs lose mobility in both biological environments and that they significantly aggregate in tumor tissue. The latter is further illustrated in Figure 2b, where it is seen that the hydrodynamic diameter distribution for the PEI-coated MNPs in tumor explant increased, whereas the hydrodynamic diameter distributions did not appreciably change in water, PBS, and whole blood. Due to the positive surface charge of these particles, we attribute these observations of loss of mobility and aggregation to electrostatic interactions with proteins present in blood and the tumor tissue (and missing from PBS and water). Reductions in particle mobility due to attractive electrostatic interactions has been observed with cationic particles suspended in hyaluronic acid solutions and bovine vitreous humor ex-vivo using single particle tracking (SPT) microscopy analysis.62 Positively charged particles have shown a heterogeneous diffusion pattern in the vitreous humor, where a small fraction remains mobile and most immobilized particles were attached to collagen fibrils in the vitreous humor. Also, we have shown in earlier work that when particle rotation is restricted due to particle aggregation in protein solutions and polymer melts an increase in

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Page 9 of 30 hydrodynamic diameter Dh leads to a dramatic change in relaxation time, shifting the Brownian relaxation peak to very low frequencies.17, 63

Figure 3. Dynamic magnetic susceptibility (DMS) spectra and lognormal diameter distributions for thermally blocked cobalt ferrite nanoparticles coated with carboxymethyl dextran (CMDx) in phosphate buffered saline (PBS), whole blood, and tumor tissue. a) DMS spectra- open markers represent measurements immediately after contact (t = 0 h) while closed markers represent measurements 24 hours after contact (t = 24 h). b) Averaged lognormal hydrodynamic diameter distributions obtained from 3 experimental replicates by fitting DMS spectra to the Debye model of susceptibility. CMDx polymer molecular weight of ~10 kDa and CMDx-coated MNPs surface charge in 1 mM KNO3 at pH 7.4 measures -30 ± 2 mV.

DMS spectra and corresponding hydrodynamic diameter distributions for CMDx-coated MNPs are shown in Figure 3. Here we see that there was no appreciable change in DMS spectra for the CMDxcoated particles in PBS and whole blood. Signal intensities and peak locations are similar at 0 h and 24 h in both media. Furthermore, the corresponding hydrodynamic diameter distributions are similar to that in water and do not change over 24 hours. This suggests very good stability and mobility for these particles in PBS and whole blood. However, the DMS spectra for these particles in tumor tissue explants shows a slight decrease in signal intensity at t = 0 h and then a more significant decrease over 24 hours. We

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Page 10 of 30 suspect the initial slight decrease is an experimental artifact due to small sample losses when nanoparticles are infused into the tissue sample. However, the subsequent loss in signal intensity over 24 hours is indicative of some loss of particle mobility in the tissue environment. The corresponding hydrodynamic diameter distributions in Figure 3b suggest that the particles do undergo some aggregation or change in size over the 24 hour period. This could be due to nanoparticle association and/or internalization with cells and other extracellular matrix components, which are absent in the whole blood sample. Prior experiments with similar particles have shown non-specific uptake of CMDx-coated particles in MDA-MB-231 cells in vitro.52 Overall, these measurements suggest that while CMDx-coated particles may be suitable for intravenous delivery, they will face significant challenges penetrating into tumor tissue, as they lose their mobility and suffer an apparent increase in size in these environments, possibly due to aggregation or association with cells and extracellular matrix components.

Figure 4. Dynamic magnetic susceptibility (DMS) spectra and lognormal diameter distributions for thermally blocked cobalt ferrite nanoparticles coated with poly (ethylene glycol) (PEG) in phosphate buffered saline (PBS), whole blood, and tumor tissue. a) DMS spectra- open markers represent measurements immediately after contact (t = 0 h) while closed markers represent measurements 24 hours after contact (t = 24 h). b) Averaged lognormal hydrodynamic diameter distributions obtained from 3 experimental replicates by fitting DMS spectra to the Debye model of susceptibility. Modified PEG

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Page 11 of 30 polymer molecular weight of ~2 kDa and PEG-coated MNPs surface charge in 1 mM KNO3 at pH 7.4 measures 0 ± 1 mV.

DMS spectra and corresponding hydrodynamic size distributions for PEG-coated MNPs are shown in Figure 4. Here we see that the signal intensity and peak locations do not change appreciably over the 24 hour observation period in PBS, whole blood, and tumor tissue explant. Indeed, DMS intensities are similar for PBS and whole blood, and slightly lower for the tumor tissue explant sample. Again, we believe this slight decrease in signal intensity is an experimental artifact due to small losses during particle infusion into the tumor sample. The corresponding hydrodynamic size distributions show excellent agreement for measurements in water, PBS, whole blood, and tumor tissue explant. Further, there was not much change in the size distributions over the 24 hours of the experiment. We note that in vitro uptake studies for iron oxide nanoparticles coated with PEG by similar methods suggest negligible nonspecific uptake (< 0.1 pg/cell) in epithelial human breast cancer cells. Collectively, these measurements suggest excellent and prolonged stability and mobility of the PEG-coated MNPs in the biological environments tested.

Figure 5. Comparison of nanoparticle stability and mobility obtained from features of their DMS spectra. a) Changes in average hydrodynamic diameter for cobalt ferrite nanoparticles coated with PEI, CMDx, and PEG over 24 hours in water, PBS, blood, and tumor tissue explants determined from fitting the outof-phase susceptibility spectra to the Debye model. b) Changes in the initial susceptibility, normalized

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Page 12 of 30 with respect to that obtained in PBS, for cobalt ferrite nanoparticles coated with PEI, CMDx, and PEG over 24 hours in PBS, blood, and tumor tissue explants.

Figure 5 shows a comparison of nanoparticle stability and mobility obtained from features of the corresponding DMS spectra for PEI, CMDx and PEG-coated MNPs in water, PBS, whole blood, and tumor tissue explants. Considering the average hydrodynamic diameters and corresponding standard deviations in Figure 5a, only the PEG-coated particles showed consistent size and stability in all environments tested. Furthermore, the largest change in hydrodynamic diameter occurred for PEI- and CMDx-coated MNPs in tumor tissue explants. Figure 5b shows initial susceptibility values for all nanoparticles and environments, normalized with respect to the corresponding values in PBS. The initial susceptibility is proportional to the volume fraction of particles in the sample that can respond to the applied oscillating magnetic field and is therefore indicative of the fraction of particles that remain mobile in the sample. Normalization against the measurements in PBS (t= 0 h) allows direct comparison of changes in particle mobility for the various coatings. Again, the PEG-coated MNPs had the best mobility characteristics in all environments tested. Significant losses in mobility are evident for the PEI-coated MNPs in whole blood and tumor tissue explants and for the CMDx-coated MNPs in tumor tissue explants. Figure 6 shows a comparison of stability and mobility features for commercially obtained hydroxyethyl starch (HES)- coated nanoparticles with two representative diameters (BNF 80 nm and BNF 100 nm, according to the manufacturer), obtained from analysis of their DMS spectra in water, PBS, whole blood, and tumor tissue explants. The corresponding physicochemical properties of the particles are summarized in Table S1, and the DMS spectra and hydrodynamic diameter distributions are shown in Figures S3 and S4 in the Supporting Information. First, we note that transmission electron microscopy (TEM) micrographs show that the BNF particles consist of relatively large and non-spherical crystals that are significantly aggregated. This is probably the reason why the particles show magnetically blocked behavior and Brownian relaxation (see Figure S2) even though they consist of iron oxide.64 Judging from

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Page 13 of 30 Figure 6, BNF 80 and BNF 100 particles are stable in PBS but both particles aggregated and lost mobility in whole blood and tumor tissue explants, as seen from the increase in hydrodynamic size and reduction in normalized initial susceptibility, both immediately and after 24 hours for particles suspended in these environments. These observations would suggest that BNF particles would significantly aggregate and lose mobility when delivered intravenously, which would result in short circulation times and poor tumor accumulation. The observations in Figure 6 further suggest that even in the case of direct intra-tumoral injection, it can be expected that BNF particles would lose their mobility. These observations are in line with reports in the literature of poor tumor distribution of BNF particles in vivo.56

Figure 6. Comparing stability and mobility for commercial Bionized NanoFerrite (BNF) particles in blood and tissue from features of their dynamic magnetic susceptibility spectra. a) Changes in hydrodynamic diameters for BNF particles of two different sizes over 24 hours in water, PBS, blood, and tumor tissue explants determined from fitting the out-of-phase susceptibility spectra to the Debye model. b) Changes in the initial susceptibility, normalized with respect to that obtained in PBS for BNF 80 and BNF 100 particles over 24 hours in PBS, blood, and tumor tissue explants to monitor loss of particle mobility. BNF 80 and BNF 100 particles coated with hydroxyethyl starch (HES) of unknown molecular weight and surface charge in 1 mM KNO3 at pH 7.4 measures -4 ± 1 mV and -6 ± 1 mV, respectively.

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Page 14 of 30 To complement the DMS measurements reported above, we performed electron microscopy (EM) observations of the distribution of the MNPs in tumor tissue explants. Four representative particles with different coatings were studied: PEI-coated MNPs (positively charged), CMDx-coated MNPs (negatively charged), and neutrally charged PEG-coated MNPs and BNF 100 nanoparticles. Samples were fixed 30 minutes after infusion of the nanoparticles into the tissue samples and subsequently processed for EM. Representative micrographs are shown in Figure 7 and in Figures S5-S8 in the Supporting Information. Particles with poor stability/mobility in tumor explants, as determined from the DMS measurements, were readily observed through EM as small to large aggregates. Figures 7ab and S5 show that PEI-coated MNPs are found mainly as large aggregates of many particles. Similarly, Figures 7ef and S7 show that BNF 100 particles are found mainly as large aggregates of many particles. In contrast, Figures 7cd and S6 suggest that CMDx-coated nanoparticles were found as single particles or small aggregates of 2-5 particles. We had extreme difficulty imaging PEG-coated particles in these samples, and when found they were seen to be single particles (see Figure S8). Overall the observations from EM analysis are in line with the results of the DMS measurements. PEI-coated MNPs had the most significant change in hydrodynamic diameter and loss of mobility in tissue explants, followed by the BNF 100 particles, and then the CMDx-coated MNPs. In contrast, the DMS spectra for the PEG-coated particles suggested that these particles retained their initial hydrodynamic size and mobility in tumor tissue explants, consistent with their appearance as single entities in the EM micrographs.

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Page 15 of 30

Figure 7. Representative transmission electron microscopy (TEM) images of polyethyleneimine (PEI), carboxymethyl dextran (CMDx), and Bionized NanoFerrite (BNF) particles injected into tumor tissue explants and incubated for 30 min at 25°C. Highly electron dense features correspond to MNPs. Annotations: C - cells; D – disintegrating cell membrane; L – lysosomes; MVBs – multivesicular bodies; N - cell nucleus. a) Clustered PEI-coated magnetic nanoparticles embedded with disintegrating cell

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Page 16 of 30 membrane adjacent to a cell; b) Zoom in showing PEI-coated magnetic nanoparticles associated to an orphan MVB along with disintegrating cell membrane; c) Tumor xenograft stromal tissue with associated CMDx-coated magnetic nanoparticles; d) Zoom in of CMDx-coated magnetic nanoparticle clusters; e) Cell showing endocytosis of hydroxyethyl starch (HES)-coated particles (BNF 100), MVB formation and lysosome formation with co-localized magnetic nanoparticles; f) Two adjacent cells with MVB and lysosome formation and co-localized HES-coated particles (BNF 100). Scale bar 100 nm.

Of the various coatings assessed, it was found that particles coated with the positively charged polymer PEI had the worst stability/mobility characteristics – their DMS signal dropped significantly immediately upon dispersion in blood or injection in tumor tissue explants. This was to be expected given the nanoparticle’s positive zeta potential and the prevalence of negatively-charged proteins and macromolecules in biological environments, which would lead to adsorption onto and aggregation of the nanoparticles. Nanoparticle- tumor tissue interactions were confirmed by EM of fixed and microtomed tumor tissue fragments containing PEI-coated nanoparticles. These MNPs showed strong aggregation in tissue after particle contact ex-vivo and were found typically associated with necrotic disintegrating cell membranes, as can be seen in Figure S5. Also, we observed evidence of association of PEI-coated MNPs with orphan multivesicular bodies (MVBs), but not colocalized inside those MVBs. Electron microscopy (EM) images of CMDx-coated MNPs (Figures 7cd) show agreement with inferences made from DMS measurements. CMDx-coated MNPs appear as small aggregates but are not restricted inside cell structures, which could be the reason we observe particle mobility via DMS measurements even after 24 hours in tumor tissue explants. Additionally, CMDx-coated MNPs were identified in several cell structures and in dark necrotic disintegrating cell membranes, but not in tumorassociated macrophages or MVBs (this is in contrast with BNF 100 nanoparticles discussed below). Interestingly, prolonged stability/mobility in blood did not guarantee stability/mobility in tumor tissue for the CMDx-coated MNPs. This was evident in the DMS measurements of nanoparticles coated with

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Page 17 of 30 CMDx, where the particles appeared to retain stability/mobility in whole blood but the experienced loss of mobility and increasing hydrodynamic diameter when injected into tumor tissue after 24 hours. Similar observations were made for the commercially obtained Bionized NanoFerrite (BNF) particles, which quickly lost stability/mobility when injected into tumor tissue, according to DMS measurements. Figures 7ef and S7 show BNF 100 particles taken up by a tumor-associated macrophage (M), internalized in MVBs and lysosomal compartments. Interestingly, we observed that particle association with tumor tissue differs between CMDx-coated MNPs and BNF 100 particles, even though both coatings are carbohydrates and, perhaps, facilitate particle uptake in cells. It was observed that BNF 100 particles had significant cell recognition in tumor tissue fragments in contrast to CMDx coatings. This could be because hydroxyethyl starch (HES), consisting of two carbohydrate polymers (amylose and amylopectin), has a similar structural composition as glycogen and this leads to cell recognition.65 PEG-coated nanoparticles retain their mobility in tumor tissue explants as was shown by DMS measurements, which correlates with the presence of single particles dispersed in tissue confirmed under electron microscopy. We did not observe the presence of particle aggregates, which we attribute to the dense covalently bonded PEG layer grafted to our magnetic nanoparticles. A PEG shell minimizes protein adsorption responsible for particle aggregation, particle interactions with cell membranes that could lead to cell membrane disruption or nonspecific endocytosis uptake of particles into tumor cells as compare with PEI-, CMDx- and HES-coated nanoparticles used in this study.

CONCLUSIONS The studies reported here demonstrate that the stability and mobility of magnetic nanoparticles in complex biological environments, such as whole blood and tissue, can be studied in situ using dynamic magnetic susceptibility (DMS) measurements, and that the inferences made from DMS measurements correlate with electron microscopy observation of nanoparticles in tumor tissue explants. Furthermore, the reported studies illustrate the important influence of nanoparticle surface coating on nanoparticle stability and mobility in biological environments. All particles tested were stable against aggregation in water and

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Page 18 of 30 in PBS. However, they showed very different stability and mobility characteristics in whole blood and in tumor tissue explants. Interestingly and importantly, stability and mobility over 24 hours in whole blood did not ensure stability and mobility in tumor tissue explants, as was observed for CMDx-coated nanoparticles. As expected, positively charged nanoparticles, coated with PEI, showed poor stability and mobility in blood and tissue, as would be expected based on electrostatic interactions with negatively charged proteins and macromolecules prevalent in these environments. Our DMS measurements suggest that commercially obtained HES-coated particles (BNF particles) significantly aggregate and lose mobility in whole blood and in tumor tissue explants. This is an important observation as these particles have been used in several studies of nanoparticle distribution in tumors and of magnetic fluid hyperthermia,55-56,

59-60

and the observed aggregation and loss of mobility potentially impacts the

interpretation of these studies. Finally, DMS measurements suggest that nanoparticles coated with covalently grafted, dense layers of PEG retained stability and mobility in whole blood and tumor tissue explants for a period of 24 hours. Collectively, these observations suggest the potential of DMS measurements to provide valuable insights into the role of nanoparticle surface coatings on their ability to navigate biological environments and can serve to screen nanoparticles intended for biomedical applications.

EXPERIMENTAL PROCEDURES Nanoparticle Characterization A Hitachi 7600 transmission electron microscope (Hitachi High Technologies America, Pleasanton, CA) equipped with a MacroFire slow-scan CCD camera (Optronics, Goleta, CA) and AMT Image capture software (Advanced Microscopy Techniques, Danvers, MA) operating at 120 kV acceleration voltage was used to image the nanoparticles. Stock samples were applied onto an ultrathin Formvar-carbon-coated nickel grid and air dried. The histogram of particle size distribution was fitted to a lognormal size distribution to estimate the number-weighted mean diameter and the geometric deviation of the MNPs.

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Page 19 of 30 The hydrodynamic size distribution and surface charge of nanoparticles in solution were estimated by dynamic light scattering (DLS) using a particle analyzer (Zeta PALS, Brookhaven Instruments) with ~633 nm laser excitation. The nanoparticle stock solutions were prepared in water, filtered through 0.22 µm nylon syringe filter twice prior to measurements and then diluted to ~1% v/v with adjusted ionic strength using 1 mM KNO3 at pH 7.4. Volume-weighted size distributions were fitted to a lognormal distribution of hydrodynamic diameters as shown in Equation (1).

nv ( Dh ) =

 − ln 2 y  1 exp   2 2π Dh ln σ g  2 ln σ g 

(1)

where nv ( Dh ) is the probability density of particles having a hydrodynamic diameter of Dh , Dhpv is the volume-weighted median diameter and ln σ g is the geometric deviation of the lognormal distribution. Then, the arithmetic mean diameter, D P , and standard deviation, σ , of the resulting diameter distributions were calculated using Equations (2) and (3) as follow:

 ln 2 σ g DP = exp  ln Dhgv +  2 

  , 

σ = exp(ln 2 σ g − 1)

(2)

(3)

Nanoparticle tracking analysis (NTA) was performed using a Nanosight LM-10 system (Malvern Instruments) with 405 nm laser excitation. Particle stock solutions in water were diluted in KCl 0.1 M by a 106 dilution factor and filtered through 0.22 µm nylon syringe filter. Samples for analysis were injected into the sample chamber using a sterile syringe, and video capture was initiated immediately. All measurements were collected at room temperature with the camera level to the maximum value. For each measurement, three videos were taken with a video acquisition time auto defined and analyzed (50-100 particles per frame, 108-109 particles per mL) using NTA 3.1 software. Number-weighted size distributions were fitted to a lognormal distribution and converted to volume-weighted size, nv ( Dh ) .

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Page 20 of 30 DMS measurements using a commercial AC susceptometer (Dynomag system, Acreo Swedish ICT) were performed at room temperature. During measurements, the amplitude of the excitation field varied between 0-0.5 mT, and the frequency interval was evaluated in a range of 10 Hz to 160 kHz. All DMS measurements were fitted to the Debye’s model of susceptibility66 with the assumption that the particles follow a lognormal size distribution and the viscosity of the fluid carrier assumed as water. The magnetic core size and saturation magnetization of MNPs stocks in water were determined by measuring the response of the equilibrium magnetization obtained under the application of a DC field at 300 K in a magnetic range from 7T to -7T using a Quantum Design MPMS-3 Superconducting Quantum Interference Device (SQUID) magnetometer. The Langevin equation weighted using a lognormal size distribution was used to calculate the magnetic diameter of the nanoparticles.

Analysis of Dynamic Susceptibility Spectra to Assess Particle Stability and Mobility The rotational motion of thermally blocked magnetic nanoparticles during DMS measurements provides a straightforward way to obtain information about their stability and mobility by estimating the apparent hydrodynamic diameter, and the percentage of mobile particles. Assuming the particles follow a lognormal volume-weighted distribution of hydrodynamic diameters, nv ( Dh ) , the out-of-phase component in the Debye model, χ " , can be written as 18, 67

χ 0,hpv Ωτ hpv y 3 D n ( Dh )dy ; y = h χ"= ∫ 2 2 6 v 1 + Ω τ hpv y Dhpv 0 ∞

τ hpv =

3 πη Dhpv

(4)

2 k BT

Here, χ 0,hpv and τ hpv are the volume-weighted average initial susceptibility and relaxation time, respectively. Ω is the frequency; k B is the Boltzmann’s constant and T is the temperature. The dynamic susceptibility spectra according to the Debye model and Debye fit for MNPs with a single relaxation time can be seen in Figure 8a. Analysis of DMS spectra for magnetic nanoparticles in water,

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Page 21 of 30 isotonic solutions, and other buffers can be used as reference medium for comparison between MNP stability in complex biological environments and aqueous solutions under the same particle concentration. The particle mobility obtained via DMS measurements in complex biological fluids might vary depending on the state of dispersion of the MNPs as illustrated in Figure 8b. Complex biological environments can lead to nanoparticle-protein corona formation, facilitating polymer desorption and perhaps leading to protein-bridging-mediated nanoparticle aggregation.12,

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The initial susceptibility χ 0,hpv may be

reduced significantly upon particle aggregation and subsequent precipitation, as illustrated in Figure 8b, with a decrease in the magnitude of the low-frequency in-phase susceptibility and a high-frequency tail at the low end of the frequency range of the out-of-phase susceptibility (black dashed line in Figure 8b). The relation between particle mobility and changes in initial susceptibility can be explained more clearly using Equation (5)

χ0,hpv =

π 18

φµ0

M d 2d 3 , kT T

(5)

where the initial susceptibility is proportional to the magnetic volumetric fraction φ and the cube of the magnetic core diameter d of particles remaining in suspension. In Equation (5) M d is the domain magnetization, k B is the Boltzmann constant and T is the temperature. In another scenario, if particles are colloidally stable and mobile in suspension but their local environment changes, say due to gelation or increased concentration of macromolecules, these changes could also be monitored using DMS measurements. An increase in the relaxation time and a corresponding decrease in rotational diffusivity will shift the peak of the out-of-phase susceptibility towards a lower frequency without a reduction in the magnitude of the out-of-phase susceptibility peak frequency as can be seen in Figure 8c.

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Figure 8. Using dynamic magnetic susceptibility (DMS) spectra to assess stability and mobility of magnetic nanoparticles (MNPs) in biological environments. a) Experimental DMS spectra (markers) and fit to model (dashed line). b) Illustration of DMS spectra for magnetic nanoparticles that are dispersed (solid lines) and that are aggregated (dashed lines). c) Normalized out-of-phase susceptibility spectra of MNPs suspended in biological fluids with increased concentrations of macromolecules. Increasing crowding of the nanoscale environment leads to greater hydrodynamic drag on the particles and a decrease in the peak frequency of the out-of-phase susceptibility.

Animal and Tumor Model A subline of MDA-MB-231 cells71 was cultured in vitro in DMEM-high glucose media supplemented with 10% fetal bovine serum, 0.0025 wt. % L-asparagine, 1% v/v L-Glutamine and 1% v/v penicillin-streptomycin. Cells were cultured and incubated at 37°C in a 5% CO2 atmosphere. A cell suspension of ~6 x 106 MDA-MB-231 cells in 100 µL phosphate buffered saline (PBS) was injected subcutaneously into the fourth mammary fat pad of 6-week old female NOD-SCID mice (Envigo). After six weeks of post cell inoculation, a tumor of ~1000 mm3 in volume was formed. Tumor growth was monitored every three days using caliper measurements. Animal handling and procedures were approved and performed according to the guidelines of the Institutional Animal Care and Use Committee (AICUC) of The University of Florida.

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Page 23 of 30 Blood Collection and Tumor Harvesting Mice were randomly selected for particle mobility assessments when tumors reached a size of 800-1000 mm3 in volume. Blood was collected by cardiac puncture using sodium citrate-wetted syringes (0.1 v/v %) and 25G needles and transferred into heparin blood collection tubes. Then blood collection tubes were placed in a tube rotator with slow rolling motion to avoid clot formation and hemolysis. Whole blood was used immediately to perform DMS measurements. Tumors were harvested under sterile conditions, kept on ice and used the same day. Processing consisted of transferring the tumor into a petri dish containing PBS and cutting fragments using a disposable biopsy punch (10-12 fragments per tumor ~100mm3) to assess particle mobility in tumor tissue using DMS measurements or nanoparticletumor tissue visualization using electron microscopy (EM) from selected particle coatings.

Assessment of Particle Mobility Using Dynamic Magnetic Susceptibility Measurements Particle stability and particle mobility were assessed using a commercial AC susceptometer (Dynomag system, Acreo Swedish ICT). The amplitude of the excitation field varied between 0-0.5 mT, and the oscillating magnetic field frequency was swept from of 10 Hz to 160,000 Hz during measurement. Blood and harvested tumor tissue hydrated with PBS were used immediately. Briefly, 10 µl of polymer coated MNPs suspended in PBS were mixed gently with 90 µL of whole blood and immediately tested in the AC susceptometer (t = 0 h). For tumor tissue sample evaluation, a tumor fragment was placed in a polycarbonate capsule size 4 (L = 13.9 mm x W = 5.05 mm, Quantum Design) and a hole was opened in the closed capsule using a 26G needle. Then 10 µL of polymer coated MNPs suspended in PBS were loaded in a 25 µL Hamilton syringe coupled with a Hamilton needle 26G 0.75” 12° bevel and slowly injected into the tumor fragment placed inside the polycarbonate capsule. After infusion, the needle was left in the tissue for 5 min to avoid disruption of nanoparticle distribution and then removed carefully. The tissue fragments were immediately tested in the AC susceptometer (t = 0 h). Next, whole blood and tumor tissue samples were measured at t = 24 h as a second evaluation time point. One mouse was used per

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Page 24 of 30 replicate. Three replicates were evaluated per particle/time condition. All DMS measurements were made at 25°C. Dynamic magnetic susceptibility data was fitted to the Debye model of susceptibility assuming the particles follow a lognormal size distribution and the viscosity of water, obtaining as the fit parameters the lognormal diameter distribution and geometric deviation, which represents a measure of the particle stability in PBS, mice blood, and tumor tissue. Along with the obtained hydrodynamic diameter distributions, initial susceptibility values of MNPs in mice blood and tumor tissue were normalized with initial susceptibility values in PBS (isotonic buffer solution) at t = 0 h to assess changes in particle mobility. The apparent arithmetic mean diameter from Equation (2) and standard deviation from Equation (3), were estimated to evaluate particle polydispersity in the biological milieu as a function of time.

Tissue Preparation for Particle Visualization using Transmission Electron Microscopy (EM) Tumor tissue explants (~100 mm3) after MNPs infusion were incubated for 30 min at 25°C for particle visualization using electron microscopy (EM). Then, particle transport was stopped by fixation overnight at 4°C. The fixation cocktail was a mixture of 4% formaldehyde – 2% glutaraldehyde in PBS (primary fixation). For embedding into Epon resin, fixed tumor tissue fragments injected with MNPs were washed with PBS to remove fixation cocktail and cut into small pieces (~1 mm3). Afterward, a postfixation treatment with 1% osmium tetraoxide/ 2-mercaptoethanol (2-ME) in 0.1M Na cacodylate was performed. Then, samples were washed, dehydrated through a graded series of ethanol, and embedded with Quetol 651 resin following procedures that have been previously published.72 Ultrathin sections of samples (70 nm) containing well-preserved ducts were mounted on uncoated copper grids. Sections with and without uranyl acetate (alcoholic 8%)/ Venable’s lead citrate stains were examined using a Hitachi 7600 transmission electron microscope (Hitachi High Technologies America, Pleasanton, CA) equipped with a MacroFire slow-scan CCD camera (Optronics, Goleta, CA) and AMT Image capture software (Advanced Microscopy Techniques, Danvers, MA) operating at 120 kV acceleration voltage.

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Page 25 of 30 ACKNOWLEDGMENTS The authors would like to thank Dr. Melissa Cruz-Acuña, and Dr. Lorena Maldonado-Camargo for providing the PEI-coated magnetic nanoparticles. The authors thank Irayme Labrada for training in mice blood collection. We are also grateful to Dr. Sharon Matthews and the University of Florida College of Medicine Electron Microscopy Core Laboratory for assistance with tissue sample preparation and training for electron microscopy (EM).

ASSOCIATED CONTENT Supporting Information Description of materials, synthesis of magnetic nanoparticles and quantification of amine groups onto the surface of magnetic nanoparticles (MNPs). Representative transmission electron microscopy images (TEM), dynamic susceptibility spectra (DMS) in water and magnetization saturation measurements of polyethyleneimine (PEI), carboxymethyl dextran (CMDx) and polyethylene glycol (PEG) coated MNPs and BNF 80 and BNF 100 MNPs. DMS spectra and lognormal diameter distributions for BNF 80 and BNF 100 particles in phosphate buffered saline (PBS), blood and tumor tissue explants. Representative TEM images of PEI, CMDx, PEG and HES-coated MNPs in tumor tissue ex-vivo after 30 min of incubation at 25°C.

ABBREVIATIONS MNPs,

Magnetic

Nanoparticles;

DMS,

Dynamic

Magnetic

Susceptibility;

PEI,

polyethyleneimine, CMDx, carboxymethyl dextran; PEG, polyethylene glycol; HES, hydroxyethyl starch; BNF, Bionized NanoFerrite; TEM, Transmission Electron Microscopy.

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Page 26 of 30 AUTHOR INFORMATION Author Contributions A.C.B and C.R conceived and designed the experiments. A.C.B performed the particle stability and mobility experiments in water, PBS, whole blood and tumor tissue explants using DMS measurements and particle visualization in tumor tissue explants by transmission electron microscopy (TEM) and data analysis. M.U carried out magnetic measurements using a Quantum Design MPMS-3 Superconducting Quantum Interference Device (SQUID) magnetometer and data analysis, and TEM characterization of single core MNPs.

A.C.B and S.B performed hydrodynamic diameter

characterizations using dynamic light scattering and nanoparticle tracking analysis, zeta potential measurements and amine groups quantification and data analysis. A.C.B, A.C.L, L.R, C.P, and C.R prepared Animal Care and Use Committee (AICUC) protocols and monitored tumor inoculated animals during the experiments. A.C.B and C.R wrote the manuscript. A.C.B, M.U, S.B, A.C.L, L.R, C.P, D.S and C.R discussed the results and commented on the manuscript.

Notes The authors declare no competing financial interest.

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Page 27 of 30 REFERENCES 1. Braeckmans, K.; Buyens, K.; Bouquet, W.; Vervaet, C.; Joye, P.; Vos, F. D.; Plawinski, L.; Doeuvre, L.; Angles-Cano, E.; Sanders, N. N.; Demeester, J.; Smedt, S. C. D., Sizing Nanomatter in Biological Fluids by Fluorescence Single Particle Tracking. Nano Letters 2010, 10 (11), 4435-4442. 2. Rocker, C.; Potzl, M.; Zhang, F.; Parak, W. J.; Nienhaus, G. U., A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. Nat Nanotechnol 2009, 4 (9), 577-80. 3. Li, T.; Senesi, A. J.; Lee, B., Small Angle X-ray Scattering for Nanoparticle Research. Chemical Reviews 2016, 116 (18), 11128-11180. 4. Creixell, M.; Herrera, A. P.; Latorre-Esteves, M.; Ayala, V.; Torres-Lugo, M.; Rinaldi, C., The effect of grafting method on the colloidal stability and in vitro cytotoxicity of carboxymethyl dextran coated magnetic nanoparticles. Journal of Materials Chemistry 2010, 20 (39), 8539-8547. 5. Santiago-Rodriguez, L.; Lafontaine, M. M.; Castro, C.; Mendez-Vega, J.; Latorre-Esteves, M.; Juan, E. J.; Mora, E.; Torres-Lugo, M.; Rinaldi, C., Synthesis, Stability, Cellular Uptake, and Blood Circulation Time of Carboxymethyl-Inulin Coated Magnetic Nanoparticles. J Mater Chem B Mater Biol Med 2013, 1 (22), 2807-2817. 6. Jedlovszky-Hajdu, A.; Bombelli, F. B.; Monopoli, M. P.; Tombacz, E.; Dawson, K. A., Surface Coatings Shape the Protein Corona of SPIONs with Relevance to Their Application in Vivo. Langmuir 2012, 28 (42), 14983-14991. 7. Xu, Y.; Qin, Y.; Palchoudhury, S.; Bao, Y., Water-soluble iron oxide nanoparticles with high stability and selective surface functionality. Langmuir 2011, 27 (14), 8990-7. 8. Wang, W. T.; Ji, X.; Na, H. B.; Safi, M.; Smith, A.; Palui, G.; Perez, J. M.; Mattoussi, H., Design of a MultiDopamine-Modified Polymer Ligand Optimally Suited for Interfacing Magnetic Nanoparticles with Biological Systems. Langmuir 2014, 30 (21), 6197-6208. 9. Aires, A.; Cabrera, D.; Alonso-Pardo, L. C.; Cortajarena, A. L.; Teran, F. J., Elucidation of the Physicochemical Properties Ruling the Colloidal Stability of Iron Oxide Nanoparticles under Physiological Conditions. Chemnanomat 2017, 3 (3), 183-189. 10. Mondini, S.; Leonzino, M.; Drago, C.; Ferretti, A. M.; Usseglio, S.; Maggioni, D.; Tornese, P.; Chini, B.; Ponti, A., Zwitterion-Coated Iron Oxide Nanoparticles: Surface Chemistry and Intracellular Uptake by Hepatocarcinoma (HepG2) Cells. Langmuir 2015, 31 (26), 7381-90. 11. Lartigue, L.; Wilhelm, C.; Servais, J.; Factor, C.; Dencausse, A.; Bacri, J.-C.; Luciani, N.; Gazeau, F., Nanomagnetic Sensing of Blood Plasma Protein Interactions with Iron Oxide Nanoparticles: Impact on Macrophage Uptake. Acs Nano 2012, 6 (3), 2665-2678. 12. Moore, T. L.; Rodriguez-Lorenzo, L.; Hirsch, V.; Balog, S.; Urban, D.; Jud, C.; Rothen-Rutishauser, B.; Lattuada, M.; Petri-Fink, A., Nanoparticle colloidal stability in cell culture media and impact on cellular interactions. Chemical Society Reviews 2015, 44 (17), 6287-6305. 13. Janko, C.; Zaloga, J.; Pottler, M.; Durr, S.; Eberbeck, D.; Tietze, R.; Lyer, S.; Alexiou, C., Strategies to optimize the biocompatibility of iron oxide nanoparticles - "SPIONs safe by design". Journal of Magnetism and Magnetic Materials 2017, 431, 281-284. 14. Yallapu, M. M.; Chauhan, N.; Othman, S. F.; Khalilzad-Sharghi, V.; Ebeling, M. C.; Khan, S.; Jaggi, M.; Chauhan, S. C., Implications of protein corona on physico-chemical and biological properties of magnetic nanoparticles. Biomaterials 2015, 46, 1-12. 15. Gutierrez, L.; Romero, S.; da Silva, G. B.; Costo, R.; Vargas, M. D.; Ronconi, C. M.; Serna, C. J.; VeintemillasVerdaguer, S.; Del Puerto Morales, M., Degradation of magnetic nanoparticles mimicking lysosomal conditions followed by AC susceptibility. Biomed Tech (Berl) 2015, 60 (5), 417-25. 16. Timbie, K. F.; Afzal, U.; Date, A.; Zhang, C.; Song, J.; Wilson Miller, G.; Suk, J. S.; Hanes, J.; Price, R. J., MR imageguided delivery of cisplatin-loaded brain-penetrating nanoparticles to invasive glioma with focused ultrasound. Journal of Controlled Release. 17. Bohorquez, A. C.; Rinaldi, C., In Situ Evaluation of Nanoparticle-Protein Interactions by Dynamic Magnetic Susceptibility Measurements. Particle & Particle Systems Characterization 2014, 31 (5), 561-570. 18. Bohórquez, A. C.; Yang, C.; Bejleri, D.; Rinaldi, C., Rotational diffusion of magnetic nanoparticles in protein solutions. Journal of Colloid and Interface Science 2017, 506, 393-402. 19. Amstad, E.; Textor, M.; Reimhult, E., Stabilization and functionalization of iron oxide nanoparticles for biomedical applications. Nanoscale 2011, 3 (7), 2819-43. ACS Paragon Plus Environment

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Page 28 of 30 20. Lazarovits, J.; Chen, Y. Y.; Sykes, E. A.; Chan, W. C., Nanoparticle-blood interactions: the implications on solid tumour targeting. Chem Commun (Camb) 2015, 51 (14), 2756-67. 21. Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; et al., Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 2013, 8 (10), 772-81. 22. Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W., Analysis of nanoparticle delivery to tumours. Nature Reviews Materials 2016, 1 (5). 23. Dobrovolskaia, M. A.; Aggarwal, P.; Hall, J. B.; McNeil, S. E., Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Molecular Pharmaceutics 2008, 5 (4), 487-495. 24. Chertok, B.; Cole, A. J.; David, A. E.; Yang, V. C., Comparison of electron spin resonance spectroscopy and inductively-coupled plasma optical emission spectroscopy for biodistribution analysis of iron-oxide nanoparticles. Mol Pharm 2010, 7 (2), 375-85. 25. Hasmonay, E.; Bee, A.; Bacri, J. C.; Perzynski, R., pH effect on an ionic ferrofluid: Evidence of a thixotropic magnetic phase. Journal of Physical Chemistry B 1999, 103 (31), 6421-6428. 26. Wilhelm, C.; Gazeau, F.; Roger, J.; Pons, J. N.; Salis, M. F.; Perzynski, R.; Bacri, J. C., Binding of biological effectors on magnetic nanoparticles measured by a magnetically induced transient birefringence experiment. Physical Review E 2002, 65 (3). 27. Kober, M.; Moros, M.; Grazu, V.; de la Fuente, J. M.; Luna, M.; Briones, F., Transient magnetic birefringence for determining magnetic nanoparticle diameters in dense, highly light scattering media. Nanotechnology 2012, 23 (15). 28. Lazaro, F. J.; Abadia, A. R.; Romero, M. S.; Gutierrez, L.; Lazaro, J.; Morales, M. P., Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection. Biochim Biophys Acta 2005, 1740 (3), 434-45. 29. Gutiérrez, L.; Mejías, R.; Barber, D. F.; Veintemillas-Verdaguer, S.; Serna, C. J.; Lázaro, F. J.; Morales, M. P., Ac magnetic susceptibility study ofin vivonanoparticle biodistribution. Journal of Physics D: Applied Physics 2011, 44 (25), 255002. 30. Ruiz, A.; Gutierrez, L.; Caceres-Velez, P. R.; Santos, D.; Chaves, S. B.; Fascineli, M. L.; Garcia, M. P.; Azevedo, R. B.; Morales, M. P., Biotransformation of magnetic nanoparticles as a function of coating in a rat model. Nanoscale 2015, 7 (39), 16321-16329. 31. Mazuel, F.; Espinosa, A.; Luciani, N.; Reffay, M.; Le Borgne, R.; Motte, L.; Desboeufs, K.; Michel, A.; Pellegrino, T.; Lalatonne, Y.; Wilhelm, C., Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels. Acs Nano 2016, 10 (8), 7627-7638. 32. Arami, H.; Ferguson, R. M.; Khandhar, A. P.; Krishnan, K. M., Size-dependent ferrohydrodynamic relaxometry of magnetic particle imaging tracers in different environments. Med Phys 2013, 40 (7), 071904. 33. Gandhi, S.; Arami, H.; Krishnan, K. M., Detection of Cancer-Specific Proteases Using Magnetic Relaxation of Peptide-Conjugated Nanoparticles in Biological Environment. Nano Lett 2016, 16 (6), 3668-74. 34. Calero-DdelC, V. L.; Santiago-Quinonez, D. I.; Rinaldi, C., Quantitative nanoscale viscosity measurements using magnetic nanoparticles and SQUID AC susceptibility measurements. Soft Matter 2011, 7 (9), 4497-4503. 35. Roeben, E.; Roeder, L.; Teusch, S.; Effertz, M.; Deiters, U. K.; Schmidt, A. M., Magnetic particle nanorheology. Colloid and Polymer Science 2014, 292 (8), 2013-2023. 36. Remmer, H.; Dieckhoff, J.; Tschöpe, A.; Roeben, E.; Schmidt, A. M.; Ludwig, F., Dynamics of CoFe2O4 Single-Core Nanoparticles in Viscoelastic Media. Physics Procedia 2015, 75, 1150-1157. 37. Astalan, A. P.; Jonasson, C.; Petersson, K.; Blomgren, J.; Ilver, D.; Krozer, A.; Johansson, C., Magnetic response of thermally blocked magnetic nanoparticles in a pulsed magnetic field. Journal of Magnetism and Magnetic Materials 2007, 311 (1), 166-170. 38. Landers, J.; Roeder, L.; Salamon, S.; Schmidt, A. M.; Wende, H., Particle-Matrix Interaction in Cross-Linked PAAmHydrogels Analyzed by Mossbauer Spectroscopy. Journal of Physical Chemistry C 2015, 119 (35), 20642-20648. 39. Soukup, D.; Moise, S.; Cespedes, E.; Dobson, J.; Telling, N. D., In Situ Measurement of Magnetization Relaxation of Internalized Nanoparticles in Live Cells. Acs Nano 2015, 9 (1), 231-240. 40. Moise, S.; Cespedes, E.; Soukup, D.; Byrne, J. M.; El Haj, A. J.; Telling, N. D., The cellular magnetic response and biocompatibility of biogenic zinc- and cobalt-doped magnetite nanoparticles. Scientific Reports 2017, 7. ACS Paragon Plus Environment

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Bioconjugate Chemistry

Page 29 of 30 41. Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Elst, L. V.; Muller, R. N., Magnetic iron oxide nanoparticles: Synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chemical Reviews 2008, 108 (6), 2064-2110. 42. Wiseman, J. W.; Goddard, C. A.; McLelland, D.; Colledge, W. H., A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo. Gene Therapy 2003, 10 (19), 1654-1662. 43. Park, J. W.; Bae, K. H.; Kim, C.; Park, T. G., Clustered Magnetite Nanocrystals Cross-Linked with PEI for Efficient siRNA Delivery. Biomacromolecules 2011, 12 (2), 457-465. 44. Lee, Y.; Lee, S. H.; Kim, J. S.; Maruyama, A.; Chen, X.; Park, T. G., Controlled synthesis of PEI-coated gold nanoparticles using reductive catechol chemistry for siRNA delivery. J Control Release 2011, 155 (1), 3-10. 45. Lee, H. J.; Nguyen, Y. T. C.; Muthiah, M.; Vu-Quang, H.; Namgung, R.; Kim, W. J.; Yu, M. K.; Jon, S.; Lee, I. K.; Jeong, Y. Y.; Park, I. K., MR Traceable Delivery of p53 Tumor Suppressor Gene by PEI-Functionalized Superparamagnetic Iron Oxide Nanoparticles. Journal of Biomedical Nanotechnology 2012, 8 (3), 361-371. 46. Schwalbe, M.; Pachmann, K.; Hoffken, K.; Clement, J. H., Improvement of the separation of tumour cells from peripheral blood cells using magnetic nanoparticles. Journal of Physics-Condensed Matter 2006, 18 (38), S2865-S2876. 47. Li, W.; Tutton, S.; Vu, A. T.; Pierchala, L.; Li, B. S. Y.; Lewis, J. M.; Prasad, P. V.; Edelman, R. R., First-pass contrastenhanced magnetic resonance angiography in humans using ferumoxytol, a novel ultrasmall superparamagnetic iron oxide (USPIO)-based blood pool agent. Journal of Magnetic Resonance Imaging 2005, 21 (1), 46-52. 48. Rodriguez-Luccioni, H. L.; Latorre-Esteves, M.; Mendez-Vega, J.; Soto, O.; Rodriguez, A. R.; Rinaldi, C.; TorresLugo, M., Enhanced reduction in cell viability by hyperthermia induced by magnetic nanoparticles. International Journal of Nanomedicine 2011, 6, 373-380. 49. Alvarez-Berríos, M. P.; Castillo, A.; Mendéz, J.; Soto, O.; Rinaldi, C.; Torres-Lugo, M., Hyperthermic potentiation of cisplatin by magnetic nanoparticle heaters is correlated with an increase in cell membrane fluidity. IInternational Journal of Nanomedicine 2013, 11, 1003-1013. 50. Creixell, M.; Bohorquez, A. C.; Torres-Lugo, M.; Rinaldi, C., EGFR-Targeted Magnetic Nanoparticle Heaters Kill Cancer Cells without a Perceptible Temperature Rise. Acs Nano 2011, 5 (9), 7124-7129. 51. Alvarez-Berrios, M. P.; Castillo, A.; Rinaldi, C.; Torres-Lugo, M., Magnetic fluid hyperthermia enhances cytotoxicity of bortezomib in sensitive and resistant cancer cell lines. International Journal of Nanomedicine 2014, 9, 145-153. 52. Domenech, M.; Marrero-Berrios, I.; Torres-Lugo, M.; Rinaldi, C., Lysosomal Membrane Permeabilization by Targeted Magnetic Nanoparticles in Alternating Magnetic Fields. Acs Nano 2013, 7 (6), 5091-5101. 53. Barrera, C.; Herrera, A. P.; Bezares, N.; Fachini, E.; Olayo-Valles, R.; Hinestroza, J. P.; Rinaldi, C., Effect of poly(ethylene oxide)-silane graft molecular weight on the colloidal properties of iron oxide nanoparticles for biomedical applications. J Colloid Interface Sci 2012, 377 (1), 40-50. 54. Otsuka, H.; Nagasaki, Y.; Kataoka, K., PEGylated nanoparticles for biological and pharmaceutical applications. Advanced Drug Delivery Reviews 2003, 55 (3), 403-419. 55. Bordelon, D. E.; Cornejo, C.; Gruettner, C.; Westphal, F.; DeWeese, T. L.; Ivkov, R., Magnetic nanoparticle heating efficiency reveals magneto-structural differences when characterized with wide ranging and high amplitude alternating magnetic fields. Journal of Applied Physics 2011, 109 (12). 56. Giustini, A. J.; Ivkov, R.; Hoopes, P. J., Magnetic nanoparticle biodistribution following intratumoral administration. Nanotechnology 2011, 22 (34), 345101. 57. Giustini, A. J.; Perreard, I.; Rauwerdink, A. M.; Hoopes, P. J.; Weaver, J. B., Noninvasive assessment of magnetic nanoparticle-cancer cell interactions. Integrative Biology 2012, 4 (10), 1283-1288. 58. Giustini, A. J.; Petryk, A. A.; Hoopes, P. J., Ionizing radiation increases systemic nanoparticle tumor accumulation. Nanomedicine: Nanotechnology, Biology and Medicine 2012, 8 (6), 818-821. 59. Petryk, A. A.; Giustini, A. J.; Gottesman, R. E.; Trembly, B. S.; Hoopes, P. J., Comparison of magnetic nanoparticle and microwave hyperthermia cancer treatment methodology and treatment effect in a rodent breast cancer model. International Journal of Hyperthermia 2013, 29 (8), 819-827. 60. Toraya-Brown, S.; Sheen, M. R.; Baird, J. R.; Barry, S.; Demidenko, E.; Turk, M. J.; Hoopes, P. J.; Conejo-Garcia, J. R.; Fiering, S., Phagocytes mediate targeting of iron oxide nanoparticles to tumors for cancer therapy. Integrative Biology 2013, 5 (1), 159-171. ACS Paragon Plus Environment

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Page 40 of 39

Page 30 of 30 61. Toraya-Brown, S.; Sheen, M. R.; Zhang, P.; Chen, L.; Baird, J. R.; Demidenko, E.; Turk, M. J.; Hoopes, P. J.; ConejoGarcia, J. R.; Fiering, S., Local hyperthermia treatment of tumors induces CD8+ T cell-mediated resistance against distal and secondary tumors. Nanomedicine: Nanotechnology, Biology and Medicine 2014, 10 (6), 1273-1285. 62. Martens, T. F.; Vercauteren, D.; Forier, K.; Deschout, H.; Remaut, K.; Paesen, R.; Ameloot, M.; Engbersen, J. F. J.; Demeester, J.; De Smedt, S. C.; Braeckmans, K., Measuring the intravitreal mobility of nanomedicines with single-particle tracking microscopy. Nanomedicine 2013, 8 (12), 1955-1968. 63. Sierra-Bermudez, S.; Maldonado-Camargo, L. P.; Orange, F.; Guinel, M. J. F.; Rinaldi, C., Assessing magnetic nanoparticle aggregation in polymer melts by dynamic magnetic susceptibility measurements. Journal of Magnetism and Magnetic Materials 2015, 378, 64-72. 64. Ahrentorp, F.; Astalan, A.; Blomgren, J.; Jonasson, C.; Wetterskog, E.; Svedlindh, P.; Lak, A.; Ludwig, F.; van Ijzendoorn, L. J.; Westphal, F.; et al., Effective particle magnetic moment of multi-core particles. Journal of Magnetism and Magnetic Materials 2015, 380, 221-226. 65. Brandley Brian, K.; Schnaar Ronald, L., Cell-Surface Carbohydrates in Cell Recognition and Response. Journal of Leukocyte Biology 1986, 40 (1), 97-111. 66. W.T. Coffey, P. J. C. a. Y. P. K., On the Theory of Debye and Néel Relaxation of Single Domain Ferromagnetic Particles. Advances in Chemical Physics 1993, 83. 67. Connolly, J.; Pierre, T. G. S., Proposed biosensors based on time-dependent properties of magnetic fluids. Journal of Magnetism and Magnetic Materials 2001, 225, 156-160. 68. Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S., Protein-Nanoparticle Interactions: Opportunities and Challenges. Chemical Reviews 2011, 111 (9), 5610-5637. 69. Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A., Biomolecular coronas provide the biological identity of nanosized materials. Nature Nanotechnology 2012, 7 (12), 779-786. 70. Treuel, L.; Eslahian, K. A.; Docter, D.; Lang, T.; Zellner, R.; Nienhaus, K.; Nienhaus, G. U.; Stauber, R. H.; Maskos, M., Physicochemical characterization of nanoparticles and their behavior in the biological environment. Physical chemistry chemical physics : PCCP 2014, 16 (29), 15053-67. 71. Minn, A. J.; Kang, Y. B.; Serganova, I.; Gupta, G. P.; Giri, D. D.; Doubrovin, M.; Ponomarev, V.; Gerald, W. L.; Blasberg, R.; Massague, J., Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. Journal of Clinical Investigation 2005, 115 (1), 44-55. 72. Ellis, E. A., Quetol 651: Not Just a Low Viscosity Resin. Microscopy Research and Technique 2016, 79, 50-57.

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