Antioxidative Theranostic Iron Oxide Nanoparticles toward Brain

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Anti-oxidative theranostic iron oxide nanoparticles towards brain tumors imaging and ROS production Sophie Richard, Ana Saric, Marianne Boucher, Christian Slomianny, francoise geffroy, Sebastien Mériaux, Yoann Lalatonne, Patrice X. Petit, and Laurence Motte ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00558 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 14, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Anti-oxidative theranostic iron oxide nanoparticles towards brain tumors imaging and ROS production Sophie Richard,a Ana Saric, b,c Marianne Boucher,d Christian Slomianny,e Françoise Geffroy,d Sébastien Mériaux,d Yoann Lalatonne,f,g Patrice X. Petitb and Laurence Motte*,f. a. Laboratoire CSPBAT, CNRS UMR 7244 UFR SMBH, Université Paris 13 Sorbonne Paris Cité, F-93017 Bobigny, France b. Laboratoire de Toxicologie, Pharmacologie et Signalisation Cellulaire, INSERM U1124, Université Paris-Descartes, Centre Universitaire des Saints-Pères, F-75270 Paris Cedex 06, France c. Division of Molecular Medicine, Rudger Boskivic Institute, Zagreb, Croatia. d. Unité d'Imagerie par Résonance Magnétique et de Spectroscopie, CEA / DRF / I2BM / NeuroSpin, F-91191 Gif-sur-Yvette, France e. Inserm, U100,Laboratoire de physiologie cellulaire, Université Lille 1, F-59655 Villeneuve d’Ascq, France f. Inserm, U1148, Laboratory for Vascular Translational Science, UFR SMBH, Université Paris 13, Sorbonne Paris Cité, F-93017 Bobigny, France

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

g. Service de Médecine Nucléaire, Hôpital Avicenne Assistance Publique-Hôpitaux de Paris F93009 Bobigny, France. Keywords. Brain, Cancer, Magnetic Resonance Imaging, Nanoparticles, Toxicity, Targeting

Abstract. Gliomas are the most common primary brain tumor in humans. To date, the only treatment of care consists of surgical removal of the tumor bulk, irradiation and chemotherapy, finally resulting in a very poor prognosis due to the lack of efficiency in diagnostic. In this context, nanomedicine combining both diagnostic with Magnetic Resonance Imaging (MRI) and therapeutic applications is a relevant strategy referred to theranostic. Magnetic nanoparticles (NPs) are excellent MRI contrast agents because of their large magnetic moment, which induces high transverse relaxivity (r2) characteristic and increased susceptibility effect (T2*). NPs can be also used for drug delivery by coating their surface with therapeutic molecules. Preliminary in vitro studies show the high potential of caffeic acid (CA), a natural polyphenol, as a promising anticancer drug due to its anti-oxidant, anti-inflammatory and anti-metastatic properties. In this study, the anti-oxidative properties of iron oxide NPs functionalized with caffeic acid (γFe2O3@CA NPs) are investigated in vitro on U87-MG brain cancer cell lines. After intravenous injection of these NPs in mice bearing U87 glioblastoma, a negative contrast enhancement was specifically observed on 11.7 T MRI images in cancerous tissue, demonstrating a passive targeting of tumor with these nanoplatforms.

Glioma is the most frequent primary brain cancer (30%) in the central nervous system, with a median survival of 12-15 months.1,2 This poor prognosis is mainly related to the delayed detection of the pathology, since neurological signs and neuroimaging abnormalities appear at relatively late stages in the disease progression. In addition, the lack of efficiency in conventional


2

Page 3 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diagnostic and therapeutic approaches can be explained by the major challenge of delivering diagnostic agents and drugs to pathological brain tissues through the blood-brain barrier.3 In this context, nanomedicine devices based on iron oxide magnetic nanoparticles (NPs) represent a novel theranostic approach for neuro-oncology applications. Due to their superparamagnetic behavior, iron oxide NPs can be used as T2 contrast agents for magnetic resonance imaging (MRI), which is currently one conventional imaging tool for effective diagnosis and treatment monitoring of brain tumors. Furthermore, NPs of suitable hydrodynamic size can passively accumulate in the tumor interstitial space, this phenomenon being named the Enhanced Permeability and Retention (EPR) effect.4 However, this EPR effect is significantly weaker in the cranial microenvironment than in peripheral tumors: to further increase the NPs tumortargeting capacities, their surface can be modified by adding functionalized moieties, such as specific peptides for example.5 Despite the numerous advantages that iron oxide NPs could offer, several studies have already shown that the use of NPs causes adverse effects on biological cells, and in particular the generation of reactive oxygen species (ROS) has been described to be one of the main causes of NPs cytotoxicity.6-9 A recent paradigm shift has shown that certain NPs selectively induce autophagy in cancer cells but not in normal cells.10,11 This suggests that NPs can exhibit an intrinsic toxicity specifically towards cancer cells, which would lead to a high therapeutic effect.12 Another conventional therapeutic strategy is based on drugs that increase ROS generation and induce apoptotic damages in cancer cells. ROS are known to play an important role in oncogenesis: small changes in ROS production promote tumor formation by producing DNA mutations and pro-oncogenic signaling pathways, whereas large changes in ROS cause oxidative


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 29

stress resulting in cellular death. These contradictory effects have important implications for potential anticancer strategies, since both the suppression and the exacerbation of ROS represent promising therapeutic approaches.13 However, these approaches also induce various toxic side effects in normal tissues: the challenge for developing new anticancer strategies is therefore to improve the treatment efficacy while decreasing the potential toxicity for patients. To achieve this goal, many natural polyphenols have emerged as very promising anticancer bioactive compounds. Among them, caffeic acid (3,4-dihydroxy-cinnamic acid, CA) is an important phenolic acid present in medicinal plants, vegetables, bee propolis and beverages.14-21 Several studies have already shown that CA has positive biological effects such as anti-oxidant, antiinflammatory, anti-HIV, anti-tumor and anti-metastatic effects.22-30 In addition, CA can induce apoptosis of cervical carcinoma cells through the mitochondrial pathway.31 This class of compounds easily forms a bidentate binding with iron cations via the cathecolic function.32-38 Thus the conjugation of iron oxide nanoparticle with caffeic acid allows considering this nanoplatform as potential theranostic agent. Moreover, the terminal end function of CA, the carboxylic acid function, could be used for further insertion of various molecules of interest, such as peptides, antibodies and dyes, leading to the synthesis of multifunctional nanoplatforms. In previous works, we demonstrated that CA have a very strong affinity for iron oxide NPs: the resulting γFe2O3@CA NPs were efficient T2-shortening contrast agents for revealing vascularization in 7 T MRI images of mouse brain.39,40 In this study the anti-oxidative properties of γFe2O3@CA NPs are investigated in vitro on U87-MG cell lines of brain cancer. After intravenous injection of these NPs in mice bearing U87 glioblastoma, a negative contrast enhancement is specifically observed on 11.7 T MRI images in cancerous tissue, demonstrating a


4

Page 5 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


passive targeting of cerebral tumor with an anti-oxydative theranostic nanoplatform for ROS production. RESULTS AND DISCUSSION NP characterization The physicochemical properties of γFe2O3@CA NPs are deeply investigated through various methods and are resumed Figure 1 and in SI†. The morphology of NPs was found to be spherical as demonstrated by transmission electronic microscopy (TEM), and the crystalline size was about 9.6 nm. The standard deviation of 0.2 was deduced by simulating the diameter d distribution with a log-normal function (Figure 1A).41 The difference between infrared spectra of free CA molecules (red curve on Figure 1B), bare NPs (black curve) and after adsorption of CA onto γFe2O3 NPs (blue curve) indicated surface complexation with catechol bound to the oxide surface via two adjacent phenolic groups.42 The average number of CA per NP was evaluated to 1050 with thermogravimetric analysis (Supplementary Figure 1†), corresponding to a surface area of about 27.5 A2 for each CA. The high CA density on γFe2O3 surface is leading to highly stable dispersions over a broad range of pH (4–11) (Figure 1C), compared with the bare nanoparticles that were only stable in acidic media and agglomerated at physiological pH owing to an isoelectric point around pH 7 (Supplementary Figure 2†). At physiological pH, the hydrodynamic size was about 17 nm and was narrowly distributed (Supplementary Figure 3†) suggesting very low aggregation. The zeta potential was found -45 mV which confirms the CA grafting through the cathecol functions: the negative charges of the coated NPs are due to the presence of carboxylate end functions (pKa = 8.6, 11.5 for cathecol functions and 4.4 for COOH/COO-).43 Magnetic properties of γFe2O3@CA NPs were


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 29

characterized using a vibrating sample magnetometer (VSM). Field-dependent magnetism curve at room temperature was characteristic of superparamagnetism behavior (Figure 1D). The saturation magnetization was found equal to 48 emu.g-1. The temperature-dependent field cooled (FC) and zero field cooled (ZFC) magnetization curves (Figure 1E) indicated a blocking temperature (TB) around 230 K. The high TB value suggests dipolar magnetic interactions between NPs. Such result could be attributed to the small length of CA ligands favoring attractive magnetic dipolar interactions compared to repulsive steric and electrostatic interactions.44 The efficacy of γFe2O3@CA NPs as MRI contrast agents was assessed by measuring their longitudinal r1 and transverse r2 relaxivities. Proton transverse relaxation rates (R2) measured at 11.7 T are displayed in Figure 1F as a function of iron concentration. The transverse r2 relaxivities was deduced from the slope of this curve, and was equal 375 mM-1.s-1. This high relaxivity suggests a high T2 contrasting effect at this magnetic field intensity, demonstrating that γFe2O3@CA NPs can be efficient T2 contrast agents. In vitro evaluation With the rising use of nanosized materials due to the development of nanotechnology, harmful effects of NPs are of increasing concern.45-48 This is due to their small size and unique physicochemical properties that are not present in conventional bulk materials, surface area, charge, shape, solubility, surface chemistry, and so far. The uptake of NPs in cells is therefore an important factor to assess nanotoxicity. TEM investigations Figure 2 shows TEM images for control cells and after incubation with 10 nM γFe2O3@CA NPs. It is clearly observed that the NPs have been taken by the U87-MG cells and internalized into


6

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


endocytic vesicles within the cytoplasm. Despite this huge charge in term of NPs, there is no visible alteration of the other cellular compartments, i.e. the nuclei, mitochondria (M), endoplasmic reticulum (ER) and golgi neither apparent sign of perturbation affecting the cytoplasm. Determination of the mitochondrial membrane potential and ROS production associated with γFe2O3@CA NPs incubation Flow cytometry analysis has been recently used for determination of NPs cellular uptake.49-52 Light that is scattered in the Forward low angle SCatter (FSC) and in Side SCatter (90°, SSC) directions from the laser beam axis reflects the cell size and cell granulosity respectively. When NPs are taken up by cells, it is expected that the SSC intensity increases without any change of FSC intensity. The γFe2O3@CA NPs were incubated with U87-MG cell lines for 24h at various extracellular concentrations (ranging from 3.25 nM to 100 nM). The flow cytometric investigations are resumed in Figure 3. The γFe2O3@CA NPs inside the U87-MG cells (after incubation of 50 nM γFe2O3@CA NPs) were easily detectable by following the side scatter (90° angle light squatter, SSC) (Figure 3A). Cells containing γFe2O3@CA NPs exhibited a higher SSC than the control cells. This was confirmed by a titration of the SCC and FSC with different amounts of externally added γFe2O3@CA NPs (Figure 3B). If the FSC did not change, there was a significant linear increase in SSC following γFe2O3@CA NPs internalization, as soon as the external NP concentration was above 3.25 nM (Figure 3B). Indeed, the intensity of the SSC revealed that the particles were taken up by the cells in dose-, time-dependently manner.


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 29

Variations of the mitochondrial transmembrane potential (∆Ψm) were studied using 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)). This cyanine dye accumulates in the mitochondrial matrix under the influence of the ∆Ψm.20 The cellular viability was assessed by staining apoptotic nuclei with propidium iodide (PI). Figure 3C shows the biparametric analysis for the control cells and for cells treated with 50 nM of γFe2O3@CA NPs. Usually, the mitochondrial membrane potential (∆Ψm) is subject to variations as soon as the cells are subjected to stress, but in the tested experimental conditions (3.25 to 100 nM for 24h), no major change in ∆Ψm was observed (datas not shown). In order to detect any potential effect of γFe2O3@CA NPs, the possible perturbations of cellular ROS homeostasis (i.e., superoxide anions) were assessed. The control cells were stained with MitoSOX to define the basic level of ROS produced in cells devoid of NPs (Figure 4A). It appeared that in the initial conditions (γFe2O3@CA NPs, 50 nM), there was a significant decrease in ROS when the NPs were internalized for 24h (Figure 4A and 4B). Apparently, the γFe2O3@CA NPs were able to buffer the ROS within the cells: there was a highly significant shift from control cells at 28.1 to lower values 12.9 (Figure 4C). The bi-parametric representation, i.e. MitoSOX /TO-PRO-3, allowed deducing that the diminution of superoxide anions amount was associated with a stability of cellular viability. The histograms of MitoSOX (superoxide anions) mean values and the histograms of Annexin Vneg/ PIneg cellular population computed for both control and treated cells were another proofs confirming the anti-oxidant properties of γFe2O3@CA NPs at high doses (12.5 to 100 nM) in the context of fully viable cells


8

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(no changes in Annexin Vneg/ PIneg cellular population which stayed at around 97% viability), whereas low doses of γFe2O3@CA NPs (3.25 and 6.5 nM) had no effects whatever the test used (Figure 4D). Within the different measurements, the γFe2O3@CA NPs did not alter the ∆Ψm values neither the percentage of cells with high ∆Ψm (Figure 3C). However, the presence of γFe2O3@CA NPs (concentration ranging from 12.5 nM to 100 nM) clearly affected the superoxide anions homeostasis, in the sense that the particles have an anti-oxidant effect certainly due to the presence of caffeic acid at their surface. This was not the case at lower concentration, i.e. at 3.25 and 6.5 nM, where neither the side scatter, nor the ∆Ψm or the superoxide anions level changed. So, we were able to set the concentrations for which the γFe2O3@CA NPs were supposed to be devoid of any side effect on the cells where they were introduced (Figure 3). Impedance-based label-free test of toxicity (xCELLigence on cellular proliferation) The xCELLigence platform has been widely described as a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies.53 Indeed, such label-free technology, based on electrical impedance, allows the continuous monitoring of various cellular processes as proliferation and cytotoxicity. The device measures the relative cellular impedance changes over time (24h) at the cell surface to determine physiological changes. The cytotoxicity or the lack of toxicity, of the different NPs preparations was then evaluated using the xCELLigence real-time cell analyzer in 16-well plates. High doses of γFe2O3@CA NPs (50 nM) were first tested, before moving to lower concentration thought to be non-toxic (5 nM) as determined by flow cytometry analysis of loaded cells. The experiments needed the prior calibration of the cellular curve at different cell concentrations. For the U87-MG cells, 20.000


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 29

cells suspended in a volume of 200 µL were used for calibration. The control cells (Figure 5A, red line) adhered to the substrate and the cell index (CI) grew until reaching a plateau at around 20 hours where the cells started to proliferate. That was the time point chosen to inject the NPs, corresponding to an early proliferation state (Figure 5A). One can see that high concentration of γFe2O3@CA NPs (50 nM, black curve) induced a decrease in the slope of proliferating cells, accompanied by a lower CI maximal value and a faster reaching of this value (62h instead of 78h for the control cells). This result was confirmed when representing the histogram of cells proliferation slopes corresponding to each γFe2O3@CA NPs concentration (Figure 5B). One can observe only slight slope variations for concentrations between 1 and 10 nM, whereas a significant decrease of cells proliferation induced by γFe2O3@CA NPs can be evidenced for 10 and 50 nM concentrations. Nevertheless, from 1 to 25 nM, the attained CI maximal value was higher than for the control cells (Figure 5B). A second type of experiment has been conducted with the addition of NPs in the cells suspension before introducing these cells into the wells. So, the influence of NPs on the adhesion and early establishment of cells on the electrodes could be measured (Figure 5C). Compared to the additions made at the early proliferation phase, this second experiment established a clear cut-off between different concentrations of NPs. Apparently, the concentrations of 10, 25 and 50 nM γFe2O3@CA NPs had a huge effect on cells culture, since the cells were even not able to bind to the substrate and proliferate (Figure 5C). Moreover, they finally died in the culture media. For the lowest γFe2O3@CA NPs concentration values (1 and 5 nM), the proliferation curves were more or less similar to the one obtained with control cells.


10

Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In vivo MRI acquisitions The ability of the γFe2O3@CA NPs to target glioblastoma was tested in vivo on orthotopic U87MG tumor implanted in nude mouse brain. After intravenous injection of γFe2O3@CA NPs (200 µmol[Fe].kg-1 corresponding to 12 µmol[CA].kg-1) to U87-MG mouse, numerous dark spots, corresponding to the susceptibility effect induced by the iron content of NPs, appeared in mouse brain T2*-weighted image, and then disappeared slowly with time until 24h, where no dark spot remained within the contralateral healthy region (Figure 6A). This confirms that NPs circulated in cerebral blood vessels short time after injection, before being eliminated from blood stream by liver, spleen and kidneys. Contrast (C) between tumoral region and contralateral healthy region (Tum/Contra) was computed after manual segmentation of cerebral tumor (Figure 6B). The value of this Tum/Contra contrast before NPs injection was already greater than one, witnessing a higher vessels density (leading to a higher concentration of iron contained in blood hemoglobin) in tumor probably due to neoangiogenesis. Furthermore, the Tum/Contra contrast increased just after NPs injection until 20-30 min (Ct=30min / Ct=0 is equal to 2.3) showing a difference of NPs biodistribution between tumor and healthy tissue: this difference can be explained by a passive accumulation of NPs inside the tumor interstitial space due to the bloodbrain barrier enhanced permeability in neoangiogenic vessels (EPR effect). Indeed the hyperpermeable nature of the tumour vessels leads to a blood brain barrier leakage and this very aggressive pathology induces a loss of junction between the endothelial cells composing the BBB.54-56 Finally, after 30 min the Tum/Contra contrast started to decrease to reach a plateau at 45 min stable until 24 h. The contrast ratio Ct=45min / Ct=0 is equal to 1.2 and highlights the NPs retention within the tumoral tissue. This in vivo experiment demonstrates that γFe2O3@CA NPs can access to brain tumor, and then potentially act as anti-oxidant drugs for at least 45 min.


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Indeed within the literature

57,58

Page 12 of 29

caffeic acid derivatives are injected at a concentration between

50 nmol.kg-1 and 35 µmol.kg-1 (for one injection per day during 6 weeks) to observe a tumor regression. This γFe2O3@CA NPs brain tumor targeting observed at 12 µmol [CA].kg-1 allows envisaging an in vivo therapeutic effect.

Conclusion In summary, we synthesized γFe2O3@CA theranostic MRI/anti-oxidative nanoparticles. The high affinity of cathecol functions to iron oxide leads to high CA density on nanoparticle surface resulting in highly stable nanoplatforms within biological conditions. This article shows that flow cytometry analysis is a pertinent method to detect NPs within the cells due to the modification of side scatter (SSC). Indeed, the internalization of γFe2O3@CA NPs was revealed through a linear relationship between NPs and side cell scatter at 90° angle. Moreover, γFe2O3@CA NPs do not impact on the ∆Ψm values but an anti-oxidant effect is observed due to the presence of CA on NP surface for concentration up to 10 nM concentration as confirmed with the cell proliferation assays. The intravenous injection of NPs to mice intracranially xenografted with U87-MG tumors demonstrates that NPs circulate in the blood stream up to brain vessels, and passively accumulate in tumor tissue through an EPR effect. This proof-of-concept study will be completed by a pharmacokinetic investigation for quantitatively confirming the NPs accumulation in gliomas and determining their biodistribution. This new generation of nanoplatforms opens the way to


12

Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


promising theranostic approaches, combining the high MRI contrasting efficiency for an early and specific diagnosis and the anti-oxidative drugs delivery to induce a high therapeutic effect. ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge via the Internet at http://pubs.acs.org. Detailed experimental materials and methods; Determination of CA number per nanoparticle and iron concentration, Evaluation of hydrodynamic properties.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. REFERENCES (1) Jhanwar-Uniyal, M., Labagnara, M., Friedman, M., Kwasnicki, A., Murali, R. (2015) Glioblastoma: Molecular Pathways, Stem Cells and Therapeutic Targets, Cancers, 7, 538-555.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 29

(2) Persano, L., Rampazzo, E., Basso, G., Viola, G. (2013) Glioblastoma cancer stem cells : Role of the microenvironment and therapeutic targeting, Biochem. Pharmacol., 85, 612– 622. (3) Garbayo, E., Estella-Hermoso de Mendoza, A., Blanco-Prieto, M.J. (2014) Diagnostic and therapeutic uses of nanomaterials in the brain. Current Medicinal Chemistry, 21, 4100-4131. (4) Matsumura, Y., Maeda, H. (1986) A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Research,, 46, 6387-6392. (5) Kiessling, F., Huppert, J., Zhang, C., Jayapaul, J., Zwick, S., Woenne, E.C., Mueller. M.M., Zentgraf, H., Eisenhut, M., Addadi, Y., Neeman, M., Semmler, W. (2009) RGD-labeled USPIO inhibits adhesion and endocytotic activity of alpha v beta3-integrin-expressing glioma cells and only accumulates in the vascular tumor compartment, Radiology, 253, 462-469. (6) Soenen, S. J., Rivera-Gil, P., Montenegro, J.M., Parak, W. J., De Smedt, S. C., Braeckmans, K. (2011) Cellular toxicity of inorganic nanoparticles: common aspects and guidelines for improved nanotoxicity evaluation, Nano Today, 6, 446-465. (7) Mahmoudi, M., Hofmann, H., Rothen-Rutishauser, B., Petri-Fink, A. (2012) Assessing the in vitro and in vivo toxicity of superparamagnetic iron oxide nanoparticles, Chem Rev., 112, 23232338. (8) Liu, G., Gao, J., Ai, H., Chen, X. (2013) Applications and potential toxicity of magnetic iron oxide nanoparticles, Small, 9, 1533-1545.


14

Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) Manke, A., Wang, L., Rojanasakul, Y. (2013), Mechanisms of Nanoparticle-Induced Oxidative Stress and Toxicity, BioMed Research International, 2013, 12220-12235. (10) Wu, Y.N., Yang, L.X., Shi, X.Y., Li, I.C., Biazik, J.M., Ratinac, K.R., Chen, D.H., Thordarson, P., Shieh, D.B., Braet, F. (2011) The selective growth inhibition of oral cancer by iron coregold shell nanoparticles through mitochondria-mediated autophagy, Biomaterials, 32, 45654573. (11) Khan, M.I., Mohammad, A., Patil, G., Naqvi, S.A., Chauhan, L.K., Ahmad, I. (2012) Induction of ROS, mitochondrial damage and autophagy in lung epithelial cancer cells by iron oxide nanoparticles, Biomaterials, 33, 1477-1488. (12) Peynshaert, K., Manshian, B.B., Joris, F., Braeckmans, K., De Smedt, S.C., Demeester, J., Soenen, S.J. (2014) Exploiting intrinsic nanoparticle toxicity: the pros and cons of nanoparticle-induced autophagy in biomedical research, Chem Rev., 114, 7581-7609. (13) Sullivan L.B., Chandel N.S. (2014) Mitochondrial reactive oxygen species and cancer, Cancer Metab., 2, 1-12. (14) Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E.M., Efros, L., Caldwell, M., Estevez, V., Nakanishi, K. (1988) Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis, Experientia, 44, 230-232. (15) Shahidi, F., Naczk, M., (1995) Food phenolics: Sources, chemistry, effects, application, Technomic Publishing Co. Inc, Lancaster, PA, USA,


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 29

(16) Hsu, F.L., Chen, Y.C., Cheng, J.T. (2000) Caffeic acid as active principle from the fruit of Xanthium strumarium to lower plasma glucose in diabetic rats, Planta Med., 66, 228-230. (17) Simonetti, P., Gardana, C., Pietta, P. (2001) Caffeic acid as biomarker of red wine intake, Methods Enzymol. 335, 122-130. (18) Giovannini, L., Migliori, M., Filippi, C., Origlia, N., Panichi, V., Falchi, M., Bertelli, A.A., Bertelli A. (2002) Inhibitory activity of the white wine compounds, tyrosol and caffeic acid, on lipopolysaccharide-induced tumor necrosis factor-alpha release in human peripheral blood mononuclear cells., Int J Tissue React, 24, 53-56. (19) Naczk, M., Shahidi, F. (2004) Extraction and analysis of phenolics in food, J Chromatogr A., 1054, 95-111. (20) Almeida, A.A., Farah, A., Silva, D.A., Nunan, E.A., Gloria, M.B. (2006) Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria, J. Agric. Food Chem., 54, 8738-8743. (21) Lin, C.L., Chen, R.F., Chen, J.Y., Chu, Y.C., Wang, H.M., Chou, H.L., Chang, W.C., Fong, Y., Chang, W.T., Wu, C.Y., Chiu, C.C. (2012) Protective effect of caffeic acid on paclitaxel induced anti-proliferation and apoptosis of lung cancer cells involves NF-κB pathway, Int. J. Mol. Sci., 13, 6236-6245. (22) Nardini, M., D'Aquino, M., Tomassi, G., Gentili, V., Di Felice, M., Scaccini, C. (1995) Inhibition of human low-density lipoprotein oxidation by caffeic acid and other hydroxycinnamic acid derivatives, Free Radic Biol Med, 19, 541-552.


16

Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Chung, T.W., Moon, S.K., Chang, Y.C., Ko, J.H., Lee, Y.C., Cho, G., Kim, S.H., Kim, J.G., Kim, C.H. (2004) Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism, FASEB J., 18, 1670-1681. (24) Okutan, H., Ozcelik, N., Yilmaz, H.R, Zu, E. (2005) Effects of caffeic acid phenethyl ester on lipid peroxidation and antioxidant enzymes in diabetic rat heart, Clinical Biochemistry, 38, 191-196. (25) Chung, M.J., Walker, P.A., Hogstrand, C. (2006) Dietary phenolic antioxidants, caffeic acid and Trolox, protect rainbow trout gill cells from nitric oxide-induced apoptosis, Aquat. Toxicol., 80, 321-328. (26) Gülçin, I. (2006) Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid, Toxicology, 217, 213-230. (27) Yamada, Y., Yasui, H., Sakurai, H. (2006) Suppressive effect of caffeic acid and its derivatives on the generation of UVA-induced reactive oxygen species in the skin of hairless mice and pharmacokinetic analysis on organ distribution of caffeic acid in ddY mice, Photochem. Photobiol.,82, 1668-1676. (28) Sato, Y., Itagaki, S., Kurokawa, T., Ogura, J., Kobayashi, M., Hirano, T., Sugawara, M., Iseki, K. (2011) In vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid International Journal of Pharmaceutics, 403, 136-138.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 29

(29) Lombardo, G. M., Portalone, G., Chiacchio, U., Rescifina, A., Punzo, F. (2012) Potassium caffeate/caffeic acid co-crystal: the rat race between the catecholic and carboxylic moieties in an atypical co-crystal, Dalton Trans., 41, 14337-14344. (30) Ozturk, G., Ginis, Z., Akyol, S., Erden, G., Gurel, A., Akyol, O. (2012) The anticancer mechanism of caffeic acid phenethyl ester (CAPE): review of melanomas, lung and prostate cancers, Eur Rev Med Pharmacol Sci., 16, 2064-2068. (31) Chang, W.C., Hsieh, C.H., Hsiao, M.W., Lin, W.C., Hung, Y.C., Ye, J.C. (2010) Caffeic acid induces apoptosis in human cervical cancer cells through the mitochondrial pathway, Taiwan J. Obstet.Gynecol. 49, 419-424. (32) Stolnik, S., Illum, L., Davis S.S. (1995) Long circulating microparticulate drug carriers, Advanced Drug Delivery Reviews, 16, 195-214. (33) Zhu, B., Eurell, T., Gunawan, R. Leckband, D. (2001) Chain-length dependence of the protein and cell resistance of oligo(ethylene glycol)-terminated self-assembled monolayers on gold, J Biomed Mater Res. 56,, 406-416. (34) Veronese, F.M., Pasut, G. (2005) PEGylation, successful approach to drug delivery, Drug Discov Today, 10, 1451-1458. (35) Montet X., Weissleder R., Josephson L. (2006) Imaging pancreatic cancer with a peptidenanoparticle conjugate targeted to normal pancreas, Bioconjug Chem., 17, 905-911.


18

Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36) Gulley-Stahl,H., Hogan II, P. A., Schmidt, W. L., Wall, S. J., Buhrlage, A., Bullen, H. A. (2010) Surface complexation of catechol to metal oxides: an ATR-FTIR, adsorption, and dissolution study, Environ. Sci. Technol., 44, 4116-4121. (37) Mahmoudi, M., Lynch, I., Ejtehadi, M. R., Monopoli, M. P., Bombelli, F. B., Laurent, S. (2011) Protein-nanoparticle interactions: opportunities and challenges, Chemical Reviews., 111, 5610-5637. (38) Annapragada, A. (2015) Advances in nanoparticle imaging technology for vascular pathologies, Annual Review of Medicine, 66, 177-193. (39) Guénin, E., Lalatonne, Y., Bolley, J., Milosevic, I., Platas-Iglesias, C., Motte L. (2014) Catechol versus bisphosphonate ligand exchange at the surface of iron oxide nanoparticles: towards multi-functionalization, J. Nanopart. Res., 16, 1-13. (40) Richard, S., Boucher, M., Herbet, A., Lalatonne, Y., Mériaux, S., Boquet, D., Motte, L., (2015) Endothelin B receptors targeted by iron oxide nanoparticles functionalized with a specific antibody: toward immunoimaging of brain tumors, J. Mater. Chem. B, 3, 2939-2942. (41) de Montferrand, C., Lalatonne, Y., Bonnin, D., Lièvre, N., Lecouvey M., Monod, P., Russier, V., Motte, L. (2012) Size-dependent nonlinear weak-field magnetic behavior of maghemite nanoparticles, Small, 8, 1945-1956. (42) Bolley, J., Lalatonne, Y., Haddad, O., Letourneur, D., Soussan, M., Pérard-Viret, J., Motte, L. (2013) Optimized multimodal nanoplatforms for targeting α(v)β3 integrins, Nanoscale, 5,11478-11489.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 29

(43) Borges, F., Lima, J.L.F.C., Pinto, I., Reis, S., Siquet, C. (2003) Application of a Potentiometric System with Data-Analysis Computer Programs to the Quantification of Metal-Chelating Activity of Two NaturalAntioxidants: Caffeic Acid and Ferulic Acid, Helv. Chim.Acta, 86, 3081-3087. (44) Lalatonne, Y., Motte, L., Russier, V., Ngo, A.T., Bonville, P., Pileni, M.P. (2004) Mesoscopic Structures of Nanocrystals: Collective Magnetic Properties Due to the Alignment of Nanocrystals, J. Phys. Chem. B, 108, 1848-1854. (45) Oberdörster, G., Oberdörster, E., Oberdörster, J. (2005) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Health Perspect, 113, 823-839. (46) Oberdörster, G., Maynard, A., Donaldson, K., Castranova, V., Fitzpatrick, J., Ausman, K., Carter, J., Karn, B., Kreyling, W., Lai, D., Olin, S., Monteiro-Riviere, N., Warheit, D., Yang, H., (2005) Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy, Part. Fibre Toxicol., 2. (47) Schrand, A. M., Rahman, M. F., Hussain, S. M., Schlager, J. J., Smith, D. A., Syed, A. F. (2010) Metal-based nanoparticles and their toxicity assessment, Wiley Interdiscip. Rev.: Nanomed. Nanobiotechno, 2, 544-568. (48) Nel, A., Xia, T., Mädler, L., Li, N. (2006) Toxic potential of materials at the nanolevel, Science, 311, 622-627. (49) Suzuki, H., Toyooka, T., Ibuki, Y. (2007) Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis, Environ Sci Technol., 41, 3018-3024.


20

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(50) Ibuki, Y., Toyooka, T. (2012) Nanoparticle uptake measured by flow cytometry, Methods Mol Biol, 926, 157-66. (51) Toduka, Y., Toyooka, T., Ibuki, Y. (2012) Flow cytometric evaluation of nanoparticles using side-scattered light and reactive oxygen species-mediated fluorescence-correlation with genotoxicity, Environ Sci Technol, 46, 7629-7636. (52) Jiang, X., Foldbjerg, R., Miclaus, T., Wang, L., Singh, R., Hayashi, Y., Sutherland, D., Chen, C., Autrup, H., Beer, C. (2013) Multi-platform genotoxicity analysis of silver nanoparticles in the model cell line CHO-K1, Toxicol Lett, 222, 55-63. (53) Atienzar, F.A., Gerets, H., Tilmant, K., Toussaint, G., Dhalluin, S. (2013) Evaluation of impedance-based label-free technology as a tool for pharmacology and toxicology investigations, Biosensors (Basel), 3, 132-156. (54) Cuddapah, V.A., Robel, S., Watkins, S., Sontheimer, H., (2014) A neurocentric perspective on glioma invasion, Nat Rev Neurosci, 15, 455-465. (55) Gambarota, G., Leenders, W., (2011) Characterization of tumor vasculature in mouse brain by USPIO contrast-enhanced MRI, Methods Mol Biol.,771, 477-487 (56) Fang, J., Nakamura, H., Maeda, H., (2011) The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect, Adv Drug Deliv Rev., 63, 136-151.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 29

(57) Kudugunti, S.K., Vad, N.M., Ekogbo, E., Moridani, M.Y., (2011) Efficacy of caffeic acid phenethyl ester (CAPE) in skin B16-F0 melanoma tumor bearing C57BL/6 mice, Invest New Drugs, 29, 52-62. (58) Chiang, E.P., Tsai, S.Y., Kuo, Y.H., Pai, M.H., Chiu H.L., Rodriguez R.L., Tang F.Y., (2014) Caffeic acid derivatives inhibit the growth of colon cancer: involvement of the PI3-K/Akt and AMPK signaling pathways, PLoS One, 24 (59) Petit, P.X., Lecoeur, H., Zorn, E., Dauguet ,C., Mignotte, B., Gougeon, M.L. (1995) Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis, J. Cell. Biol., 130, 157-167. (60) Gendron, M.C., Schrantz, N., Metivier, D., Kroemer, G., Maciorowska, Z., Sureau, F., Koester, S., Petit, P.X. (2001) Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation, Biochem J., 353, 357-367.


22

Page 23 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of contents: The surface functionalization of γFe2O3 nanoparticles with caffeic acid molecules allows to obtain a theranostic agent combining MRI contrast agent and anti-oxidative properties 80x59mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Physicochemical characterizations of γFe2O3@CA NPs. (A) TEM (insert) and size distribution. (B) Infrared spectroscopy of free CA (red curve), bare of γFe2O3 NPs (black curve), and of γFe2O3@CA (blue curve). (C) Hydrodynamic diameter as a function of pH. (D) Magnetization curve at room temperature. (E) ZFC-FC magnetization curves. (F) Proton transverse relaxation rates (R2) measured at 7 T for various γFe2O3@CA NPs concentrations 119x114mm (300 x 300 DPI)


Page 24 of 29

Page 25 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. TEM images of control U 87-MG cells (A-B) and after 10 nM γFe2O3@CA NPs incubation (C-D) A and C: cell structure at low magnification and (B) Enlargement of an endocytic vesicle. ER : endoplasmic reticulum, M : mitochondria 119x90mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Flow cytometry analysis of the cells behavior in presence of various concentrations of γFe2O3@CA NPs. (A) Biparametric representation of 90° angle light scatter (SSC) vs Forward low angle light scatter (FSC) of non treated (left) and γFe2O3@CA NPs treated cells (right). (B) Analysis of changes in FSC and SCC of the cells treated with zero or 50 nM concentrations of γFe2O3@CA NPs. (C) Biparametric analysis of the propidium iodide staining versus the mitochondrial ∆Ψm for the control cells and cells treated with 50 nM of γFe2O3@CA NPs. 77x119mm (300 x 300 DPI)


Page 26 of 29

Page 27 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Biparametric analysis of the light scattering properties of the control cells (A) and cells treated with γFe2O3@CA NPs (50 nM, B), -(C) The monoparametric analysis focuses on changes in MitoSOX fluorescence between control and 50 nM of γFe2O3@CA NPs. (D) Combined histograms of superoxide anions detection (MitoSoX) and detection of viable cells percentage (as detected for corresponding annexin V-FITC/PI negativity) after treatment with various concentrations of γFe2O3@CA NPs. 77x89mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. RTCA profiles (xCELLigence measurements) generated by U87-MG cells with or without γFe2O3@CA NPs. (A) Effect of NPs on cell index curves in U87-MG cells. Cells growing in their normal culture medium (DMEM glutamax completed) were exposed for at least 120 h with none or different concentrations of NPs (ranging from 1 to 50 nM) in a volume of 200 µL just at the beginning of their proliferation curves. Cells indexes were normalized with the last point before compound addition. The normalized point is indicated by a vertical line. Each point of the curve was calculated from quadruplet values. Data represent the average of three experiments. For more details, please refer to supporting information data. (B) Histogram of the slope (1/h) values of proliferation of control or treated cells with γFe2O3@CA NPs. (C) RTCA profiles of cells growth when the γFe2O3@CA NPs are added before seeding the cells in the wells. The control curve is the reference and all the slopes of proliferation curves have been estimated towards this reference curve. (D) Enlargement of the curve (C) for the first 20 hours that allows estimating the alterations of the initial binding of cells to the electrode substrate. 106x113mm (300 x 300 DPI)


Page 28 of 29

Page 29 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Injection of γFe2O3@CA NPs to a U87-MG mouse model of glioblastoma. (A) T2*-weighted MR images of mouse brain pre and at different times post injection of γFe2O3@CA NPs. Tumor is pointed out with a red arrow. (B) Contrast follow-up between tumor and contralateral healthy zone versus time. (Upper right corner of B) Example of manual segmentation of tumor and contralateral healthy zone on one brain slice. 99x69mm (300 x 300 DPI)


Antioxidative Theranostic Iron Oxide Nanoparticles toward Brain

Recommend Documents