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Fenton-Reaction-Accelerable Magnetic Nanoparticles for Ferroptosis Therapy of Orthotopic Brain Tumors Zheyu Shen, Ting Liu, Yan Li, Joseph Lau, Zhen Yang, Wenpei Fan, Zijian Zhou, Changrong Shi, Chaomin Ke, Vladimir Iosifovich Bregadze, Swadhin Mandal, Yijing Liu, Zihou Li, Ting Xue, Guizhi Zhu, Jeeva Munasinghe, Gang Niu, Aiguo Wu, and Xiaoyuan Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06201 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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Fenton-Reaction-Accelerable Magnetic Nanoparticles for Ferroptosis Therapy of Orthotopic Brain Tumors

Zheyu Shen,†,‡ Ting Liu,§ Yan Li,Δ Joseph Lau,‡ Zhen Yang,‡ Wenpei Fan,‡ Zijian Zhou,‡ Changrong Shi,§ Chaomin Ke,§ Vladimir I. Bregadze,‖ Swadhin K. Mandal,○ Yijing Liu*,‡, Zihou Li,† Ting Xue,† Guizhi Zhu,‡ Jeeva Munasinghe,# Gang Niu,‡ Aiguo Wu,*,† Xiaoyuan Chen*,‡

†

CAS Key Laboratory of Magnetic Materials and Devices, & Key Laboratory of Additive

Manufacturing Materials of Zhejiang Province, & Division of Functional Materials and Nanodevices, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, 1219 Zhong-guan West Road, Ning-bo, Zhe-jiang 315201, China. ‡

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and

Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States. §

State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular

Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, 361102, China. Δ

Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University,

Ningbo 315211, China. ‖

A.N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilov

Str. 28, Moscow 119991, Russia. ○

Department of Chemical Sciences, Indian Institute of Science Education and Research-Kolkata,

Mohanpur-741246, India. # Mouse

Imaging Facility, National Institute of Neurological Disorder and Stroke, National Institutes of

Health, Bethesda, Maryland 20892, United States.

Corresponding Authors *E-mail: [email protected] 1

ACS Paragon Plus Environment

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*E-mail: [email protected] *E-mail: [email protected]

2


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ABSTRACT Cancer is one of the leading causes of morbidity and mortality in the world. But more cancer therapies are needed to complement existing regimens due to problems of existing cancer therapies. Herein, we term ferroptosis therapy (FT) as a form of cancer therapy, and hypothesize that the FT efficacy can be significantly improved via accelerating the Fenton reaction by simultaneously increasing the local concentrations of all reactants (Fe2+, Fe3+, and H2O2) in cancer cells. Thus, Fenton-reaction-accelerable magnetic nanoparticles, i.e., cisplatin (CDDP) loaded Fe3O4/Gd2O3 hybrid nanoparticles with conjugation of lactoferrin (LF) and RGD dimer (RGD2) (FeGd-HN@Pt@LF/RGD2), were exploited in this study for FT of orthotopic brain tumors. FeGd-HN@Pt@LF/RGD2 nanoparticles were able to cross the blood-brain barrier (BBB) because of its small size (6.6 nm) and LF-receptor-mediated transcytosis. FeGd-HN@Pt@LF/RGD2 can be internalized into cancer cells by integrin αvβ3-mediated endocytosis, and then release Fe2+, Fe3, and CDDP upon endosomal uptake and degradation. Fe2+ and Fe3+ can directly participate in the Fenton reaction, while the CDDP can indirectly produce H2O2 to further accelerate the Fenton reaction. The acceleration of Fenton reaction generates reactive oxygen species to induce cancer cell death. FeGd-HN@Pt@LF/RGD2 successfully delivered reactants involved in the Fenton reaction to the tumor site, and led to significant inhibition of tumor growth. Finally, the intrinsic magnetic resonance imaging (MRI) capability of the nanoparticles was used to assess and monitor tumor response to FT (self-MRI-monitoring).

KEYWORDS ferroptosis

therapy,

orthotopic

glioblastoma,

Fenton

reaction,

magnetic

nanoparticles,

self-MRI-monitoring.

3


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Cancer is one of the leading causes of morbidity and mortality worldwide. Conventional cancer treatment often entails a combination of debulking surgery,1 radiotherapy,2,3 and chemotherapy.4-7 Advancements in the understanding of cancer biology and biomedical engineering have led to the development of alternative treatment strategies, including high intensity focused ultrasound (HIFU),8,9 immunotherapy,10-12 gene therapy (GT),13-15 photodynamic therapy (PDT),16-18 photothermal therapy (PTT),19-22 and magnetic hyperthermia (MHT).23-25 Several of these strategies are approved for cancer indications, while others are being investigated in clinical or preclinical studies. Although these cancer therapies are efficacious, they also have limitations. For example, surgery is ineffective for late stage cancers. Radiotherapy and chemotherapy can be toxic to normal cells and tissues resulting in serious side effects. HIFU therapy is not efficient for the ablation of deep-seated tumors because ultrasound waves are attenuated rapidly with increased penetration depth.26 Immunotherapy is costly, requires the identification of suitable biomarkers amidst tumor heterogeneity, and relies on the host immune system which is likely weakened by first-line treatment. GT has biosafety concerns resulting from poor target specificity and immune response against viral vectors. The application of PDT or PTT is limited by the penetration depth of light. Finally, low accumulation of magnetic nanomaterials at the tumor site and heat resistance of cancer cells are the primary concerns of MHT.27 Therefore, to improve patient outcomes, more cancer therapies are needed to complement existing regimens. Ferroptosis is a form of non-apoptotic cell death that is dependent on iron. It was, for the first time, termed in 2012 by Dixon et al.28,29 Many studies interrogating the cellular signaling pathways regulating ferroptosis soon followed.30-32 The mechanism of cell death is mediated by the production and accumulation of reactive oxygen species (ROS) via iron-based Fenton reaction.30-32 Herein, we term ferroptosis therapy (FT) as a form of cancer therapy. Although FT was not termed until now, a few studies tried to utilize iron-based nanomaterials for cancer therapy based on ferroptosis,33-36 which is essentially FT. The reported FT mechanism is ROS generation induced by 4


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iron-based nanomaterials via Fenton reaction since iron is one of the reactants of the reaction. Amorphous iron nanoparticles (AFeNPs) were reported to be most effective because iron is more readily released from AFeNPs in the acidic tumor microenvironment than other crystalline iron-based nanoparticles.36 Even so, the dosage of iron required for FT induction in tumor-bearing mice was very high (i.e., 75.0 mg iron/kg mice), suggesting that FT efficacy was low.36 We hypothesize that the FT efficacy in cancer cells can be significantly improved by simultaneously increasing the local concentrations of all reactants in the Fenton reaction (Fe2+, Fe3+, and H2O2) (Figure 1). In this study, we synthesized cisplatin (CDDP) loaded Fe3O4/Gd2O3 hybrid nanoparticles with conjugation of lactoferrin (LF) and RGD peptide dimer (RGD2), i.e., FeGd-HN@Pt@LF/RGD2, and monitored FT response in an orthotopic glioblastoma model using magnetic resonance imaging (MRI). Currently, with standard of care, the 2-year survival rate for glioblastoma patients is only around 27 % and the median survival is only ~10-11 months.37,38 To the best of our knowledge, no research has been reported for FT of brain tumors. Presumably, this is because: 1) the rate of Fenton reaction in vivo is low resulting in suboptimal FT efficacy, and 2) therapeutic drugs, including iron-based nanomaterials, are difficult to transport across the blood-brain barrier (BBB). Our FeGd-HN@Pt@LF/RGD2 nanoparticles are designed to cross the BBB by LF receptor-mediated transcytosis, and be internalized by cancer cells upon integrin αvβ3 (RGD2 receptor) binding. Fe2+, Fe3+, and CDDP can be released from the FeGd-HN@Pt2@LF/RGD2 nanoparticles after endocytosis in late endosome, yielding two of the three reactants that can directly accelerate the Fenton reaction. In addition, the released CDDP can activate NADPH oxidases (NOXs), convert NADPH to NADP+, release electrons, generate O2.-, and form H2O2.39-41 H2O2 serves as another substrate for the Fenton reaction. We show that these nanoparticles have a high FT efficacy for an orthotopic brain tumor model by delivering all reactants involved in the Fenton reaction (Fe2+, Fe3+ and H2O2) to the tumor site to accelerate the reaction.

5


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RESULTS AND DISCUSSION Synthesis and Characterization of Nanoparticles Due to the advantages of water phase synthesis method for other core-shell nanoparticles,42-44 our Fe3O4/Gd2O3 hybrid nanoparticles (FeGd-HN) were synthesized by a two-step co-precipitation technique under stabilization of poly(acrylic acid) (PAA). The Gd/Fe molar ratio of FeGd-HN was measured to be 0.48 by inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 5100). Next, cisplatin (CDDP) was reacted with FeGd-HN generating cisplatin-loaded FeGd-HN (i.e., FeGd-HN@Pt) via the reaction between CDDP and –COOH of PAA (Figure 1a). The Pt loading contents (i.e., weight of the loaded Pt/weight of the FeGd-HN@Pt * 100%) and Pt loading efficiencies (i.e., weight of the loaded Pt/weight of the feeding Pt * 100%) were determined utilizing ICP-OES and are summarized in Table S1. Based on the data, higher feeding amount of CDDP resulted in higher Pt loading content but lower Pt loading efficiency. As FeGd-HN@Pt4 and FeGd-HN@Pt5 dispersions were not stable during storage at room temperature, only FeGd-HN@Pt1-3 were subjected to MRI measurements. The longitudinal relaxivity (r1) or transverse relaxivity (r2) were acquired from slopes of the linear relationships shown in Figure S1. It was found that a higher feeding amount of CDDP resulted in lower r1 and higher r2/r1. Because high r1 and low r2/r1 benefit T1-weighted MRI, and high Pt loading content is preferred for FT application, FeGd-HN@Pt2 was considered the optimal sample among FeGd-HN@Pt1-3. FeGd-HN@Pt2 was conjugated with LF and RGD2 to form FeGd-HN@Pt2@LF/RGD2. The T1-weighted MR images of FeGd-HN@Pt2@LF/RGD2 with various CGd showed good dependence on concentration gradient (Figure S2) indicating efficient contrast enhancement by FeGd-HN@Pt2@LF/RGD2. The r1 and r2 values of FeGd-HN@Pt2@LF/RGD2 were measured at 7.0 T (Figure S1) and 1.5 T (Figure S3), and compared with FeGd-HN@Pt2 (Table S2). It was concluded

6


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that the FeGd-HN@Pt@LF/RGD2 is an excellent T1 contrast agent due to its high r1 (56.57 mM-1 s-1) and low r2/r1 (1.25) (1.5 T). The high r1 can be ascribed to the dotted-core-shell morphology of our magnetic nanoparticles (Figure 1). That’s because the special morphology results in a very large specific surface area, which causes more naked metal on the nanoparticle surface. The naked metal can interact with the proton in H2O of the inner sphere and then leads to a high r1 value (inner-sphere mechanism). TEM images showed that both FeGd-HN@Pt2 and FeGd-HN@Pt2@LF/RGD2 were well-dispersed in water (Figure 2a-c). Based on TEM images, the average particle diameter was determined to be 6.3 and 6.6 nm for FeGd-HN@Pt2 and FeGd-HN@Pt2@LF/RGD2, respectively. It is well established that particles that are small are more readily transported across the BBB.45,46 Cu, Gd, Fe, O and Pt peaks were found in the energy dispersive X-ray spectrum (EDS) of FeGd-HN@Pt2 (Figure 2d). The Cu peaks result from the copper grid. Gd, Fe, O and Pt are all components of the nanoparticles. Therefore, our magnetic nanoparticles are Fe3O4/Gd2O3 hybrid nanoparticles. Dynamic light scattering (DLS) results (Figure 2e) provided evidence for the mono-dispersion of our FeGd-HN@Pt2 and FeGd-HN@Pt2@LF/RGD2 nanoparticles due to their very narrow size distributions. The hydrodynamic diameter (dh) was determined to be 9.8 and 14.7 nm for FeGd-HN@Pt2 and FeGd-HN@Pt2@LF/RGD2, respectively. The larger dh of FeGd-HN@Pt2@LF/RGD2 compared with that of FeGd-HN@Pt2 can be attributed to the successful conjugation of LF and RGD2. Thermogravimetry (TGA) and differential thermogravimetry (DTG) curves (Figure S4) indicated continuous weight loss, which usually results from the organic molecules. This result confirmed the adhesion of LF and RGD2 on the magnetic nanoparticles and their abrasion at high temperatures. Field-dependent magnetization curve of FeGd-HN@Pt2@LF/RGD2 (Figure S5) indicated a very low magnetization. The saturation magnetization (Ms) value was determined to be only 8.6 emu/g. The low Ms value contributes to a low r2 value, which is good for T1-weighted MRI.47,48 Release behaviors of 7


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Fe or Pt from FeGd-HN@Pt2@LF/RGD2 were assessed at 37 oC at pH 7.4 and pH 5.5 to predict behavior at physiological blood pH and at the pH of late endosomes (Figure S6). At pH 7.4, that release of Fe and CDDP were negligible, while both Fe and CDDP were readily released at pH 5.5. This suggests that the FeGd-HN@Pt2@LF/RGD2 will not prematurely release its payload until the intended subcellular location is reached.

Cellular Uptake of Nanoparticles and MRI of Cells To confer cancer selectivity for FeGd-HN@Pt2@LF/RGD2, we conjugated RDG2 onto the particle surface. RDG2 is a nanomolar affinity ligand for integrin avβ3, an angiogenesis marker that has been explored for targeting brain tumors.49 In this study, laser scanning confocal microscope (LSCM), flow cytometry, and ICP were used to investigate uptake of the FeGd-HN@Pt2@LF/RGD2 nanoparticles by U-87 MG cells (a human primary glioblastoma cell line, avβ3 integrin positive) and MCF-7 cells (a human breast cancer cell line, avβ3 integrin negative).48,50 The blue, green, and red signals of the LSCM images (Figure S7-9) indicate the nucleus (Hoechst 33258), cytoskeleton (phalloidin-FITC), and rhodamine 6G conjugated-nanoparticles (R6G), respectively. It was observed that R6G@FeGd-HN@Pt2@LF/RGD2 was uptaken by U-87 MG cells, but not by MCF-7 cells. For R6G@FeGd-HN@Pt2, nanoparticles without LF and RDG2, uptake was low in both tested cell lines. Similar results were obtained from the flow cytometry assay (Figure 3a, b). R6G@FeGd-HN@Pt2@LF/RGD2 incubated U-87 MG cells had significantly higher relative fluorescence intensity (4.1±0.8) than R6G@FeGd-HN@Pt2 incubated U-87 MG cells or R6G@FeGd-HN@Pt2@LF/RGD2 incubated MCF-7 cells (1.1±0.4, 1.7±0.4) (* P < 0.01) (Figure 3c). ICP was also used to measure the amount of FeGd-HN@Pt2@LF/RGD2 and FeGd-HN@Pt2 that could be internalized by U-87 MG cells (Figure 3d). The internalized Gd levels in U-87 cells were 711±76 and 143±14 fg/cell for FeGd-HN@Pt2@LF/RGD2 and FeGd-HN@Pt2, respectively (** P < 8


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0.001). Collectively, the qualitative and quantitative results demonstrate that the RGD2 can bind with avβ3 integrin expressed by U-87 MG cells, and that binding can induce endocytosis. T1-weighted MR images (Figure S10 a, b) showed that the FeGd-HN@Pt2@LF/RGD2 treated U-87 MG cells were brighter than the Magnevist (i.e., Gd-DTPA chelate) treated U-87 MG cells and control cells without contrast agent.

The difference (2.88±0.24, 1.95±0.18) was statistically significant (* P

< 0.01) (Figure S10 c). The higher MRI efficiency of our FeGd-HN@Pt2@LF/RGD2 nanoparticles on cells than commercial Magnevist can be attributed to its high r1 value (56.57 mM-1 s-1) and low r2/r1 ratio (1.25).

Mechanism and Efficiency of Ferroptosis Therapy on Cancer Cells In this study, because FeGd-HN@Pt@LF/RGD2 nanoparticles were used to generate ROS to induce FT of cancer, ROS is the key factor that indicates the FT efficacy. So, the intracellular ROS was verified by a DCF-DA (2,7-dichlorofluorescein diacetate) assay in conjunction with LSCM (Figure S11, S12) and flow cytometry (Figure S13). In live cells, DCF-DA is first converted into a non-fluorescent intermediate that can be oxidized by intracellular ROS to produce fluorescent DCF. The DCF fluorescence intensity signal from FeGd-HN@Pt2@LF/RGD2-treated U-87 MG cells was similar to the FeCl3/FeSO4 + CDDP treated cells, and higher than cells treated with FeCl3/FeSO4 or CDDP alone (Figure S11). In comparison, untreated cells (control) (Figure S11) almost did not generate DCF fluorescence. The cells pretreated with iron chelator deferoxamine mesylate (DFO) or ROS scavenger N-acetyl-L-cysteine (NAC) showed decreased DCF fluorescence intensity (Figure S12). The decrease in DCF fluorescence was also quantitatively measured by flow cytometry (Figure S13)

showing

relative

intensities

of

4.9±0.5,

3.1±0.3,

and

2.2±0.4

for

FeGd-HN@Pt2@LF/RGD2-treated U-87 MG cells, treatment plus DFO, and treatment plus NAC (* P < 0.01). These results provide support that FeGd-HN@Pt2@LF/RGD2 nanoparticles can generate 9


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intracellular ROS through the Fenton reaction, and that this effect can be inhibited by DFO (chelating Fe2+ and Fe3+) or by NAC (scavenging intercellular ROS). The FT efficacy was further directly evaluated on MCF-7 and U-87 MG cells with MTT assays (Figure S14). FeCl3/FeSO4 (molar ratio 2:1) showed low cytotoxicity to both MCF-7 and U-87 MG cells with CFe ranging from 31.3 to 1000 μM (Figure S14a, b). Cell viability was > 90% at the tested concentrations for both cell lines, indicating the low FT efficiency of free Fe ions. For MCF-7 cells, the cytotoxicity of FeGd-HN@Pt2@LF/RGD2 nanoparticles was higher than that of free Fe ions, but lower than that of free CDDP (Figure S14b). This is likely due to the low uptake of nanoparticles by MCF-7 cells (Figure S8, S9, Figure 3). However, exposure to FeGd-HN@Pt2@LF/RGD2 nanoparticles was highly toxic for U-87 MG cells. The toxicity was higher than that of the free CDDP at 15.2 and 7.59 μM of CCDDP, with the differences being statistically significant (* P < 0.01). These results indicate high FT efficacy of our FeGd-HN@Pt2@LF/RGD2 nanoparticles because of the synergistic action of Fe2+, Fe3+, and CDDP in the Fenton reaction. After co-incubation with iron chelator DFO or ROS scavenger NAC, the FeGd-HN@Pt2@LF/RGD2 nanoparticles showed lower cytotoxicity (** P < 0.01, Figure S14c), which indicates that the cytotoxic effect can be blocked by DFO or NAC.

Transport Across the BBB In Vitro and In Vivo The transportability across the BBB of our FeGd-HN@Pt2@LF/RGD2 was investigated in vitro (Figure 4) and in vivo (Figure S15, S16, Figure 5). Figure 4a is a schematic illustration of the in vitro BBB model. HBEC-5i cells (human brain microvascular endothelial cell line) were cultured on a porous membrane with a mean pore size of 0.4 µm. During the cell culture process, the transendothelial electrical resistance (TEER) values were monitored. When the TEER values are larger than 200 Ω.cm2, the tight junctions of the in vitro models are considered to be similar with that of the in vivo BBB.51 10


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Our FeGd-HN@Pt2@LF/RGD2 nanoparticles were then added into the apical chamber. After a 12 h incubation period, the nanoparticle concentrations in both chambers were measured by ICP. It was found that 41.0±0.9% of our FeGd-HN@Pt2@LF/RGD2 nanoparticles crossed the in vitro BBB model, which was significantly higher than the FeGd-HN@Pt2@RGD2 nanoparticles without LF conjugation (13.6±1.9%) (* P < 0.001) (Figure 4b). Furthermore, a blocking study was also performed by incubating FeGd-HN@Pt2@LF/RGD2 in the apical chamber in the presence of excess LF. LF blockade decreased the transportability of FeGd-HN@Pt2@LF/RGD2 nanoparticles from 41.0±0.9% to 14.6±1.2% (Figure 4b). These results demonstrate that LF-mediated transcytosis is crucial for the transportability of our FeGd-HN@Pt2@LF/RGD2 nanoparticles across the BBB. T1-weighted MR images of normal mouse brains pre- or post-treatment with the Magnevist or FeGd-HN@Pt2@LF/RGD2 nanoparticles are shown in Figure S15 and Figure S16. The coronal slices with the highest MRI signal at each time point are shown in Figure 5a, b. The intensity of MRI signals remained relatively constant for Magnevist from 0 to 60 min, indicating its poor transportability across the BBB. The MRI signal intensity of FeGd-HN@Pt2@LF/RGD2 increased overtime, peaking at 12 h post injection. Quantitative analysis of the MR images using SNR is shown in Figure 5c, d, which is calculated from the following equations: SNR = SImean / SDnoise, ΔSNR = (SNRpost - SNRpre) / SNRpre × 100 %. The highest ΔSNR was 81±21% and 33±14% for FeGd-HN@Pt2@LF/RGD2 (at 12 h post injection)

and

Magnevist

(at

10

min

post

injection),

which

demonstrates

that

our

FeGd-HN@Pt2@LF/RGD2 nanoparticles is better suited for imaging the brain than Magnevist.

T1-Weighted MRI of Subcutaneous Tumors The T1-weighted MRI performance of FeGd-HN@Pt2@LF/RGD2 nanoparticles was evaluated on subcutaneous tumors in comparison with FeGd-HN@Pt2 nanoparticles. The MRI signal in tumors (axial orientation) peaked at 8 h post injection of the FeGd-HN@Pt2 nanoparticles, and at 12 h post 11


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injection of the FeGd-HN@Pt2@LF/RGD nanoparticles (Figure 6a, b). The time delay in reaching peak signal can be explained by the longer blood circulation time of FeGd-HN@Pt2@LF/RGD. As mentioned, FeGd-HN@Pt2@LF/RGD has a hydrodynamic particle size of 14.7 nm, which is larger than that of for FeGd-HN@Pt2 (9.8 nm) (Figure 2). The maximum ΔSNR was 308±43% or 329±37% for FeGd-HN@Pt2 or FeGd-HN@Pt2@LF/RGD2, respectively (Figure 6c, d). The slightly higher ΔSNR of FeGd-HN@Pt2@LF/RGD2 (i.e., higher tumor contrast of MRI images) is likely due to the active targeting of RGD2. The coronal T1-weighted MR images with strongest signal in tumor or liver at different time points post injection of our FeGd-HN@Pt2@LF/RGD nanoparticles (Figure 7a, b) show that the MRI signal reached maximum for both tumor and liver at 12 h post injection, which is in complete agreement with the axial MRI results (Figure 6b, d). The highest ΔSNR was 291±41% and 223±65% for tumor and liver, respectively (Figure 7c, d). The higher ΔSNR in tumors than livers indicated that our FeGd-HN@Pt2@LF/RGD2 nanoparticles had higher accumulation in tumor than in liver. The accumulation in tumor is a result of the high enhanced permeability and retention (EPR) effect52,53 with a small particle size (dh = 14.7 nm), as well as the active targeting effect by RGD2.

MRI-Guided Ferroptosis Therapy (FT) of Orthotopic Brain Tumors To evaluate the FT efficacy of our FeGd-HN@Pt2@LF/RGD2 nanoparticles on an orthotopic brain tumor model, luciferase imaging and diffusion-weighted MRI (DW-MRI) were used. Based on bioluminescence images, FeGd-HN@Pt2@LF/RGD2 was able to inhibit tumor growth compared to FeGd-HN@Pt2@RGD2, FeGd-HN@Pt2, FeGd-HN, and Saline (Figure 8a-e). The survival of the mice treated with FeGd-HN@Pt2@LF/RGD2 was 100% at 18 days post injection, compared to 20%, 0%, 0%, and 0% for FeGd-HN@Pt2@RGD2, FeGd-HN@Pt2, FeGd-HN, and Saline, respectively (Figure 8f). Quantitative analysis of the tumor growth (Figure 8g) shows that the orthotopic brain tumors 12


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treated with FeGd-HN@Pt2@RGD2, FeGd-HN@Pt2, FeGd-HN, or Saline grew quickly, but the tumor burden in mice treated with our FeGd-HN@Pt2@LF/RGD2 nanoparticles remained relatively constant. These

results

demonstrate

that

our

Fenton-reaction-accelerable

and

BBB-transportable

FeGd-HN@Pt2@LF/RGD2 nanoparticles have a high FT efficacy on orthotopic brain tumors. Diffusion weighted MRI (DW-MRI), which can quantify the water diffusion enhancement induced by cell death before tumor size or shape change is visible,54 was also used to monitor and guide FT. The apparent diffusion coefficient (ADC) value of the orthotopic brain tumors at 48 h post injection of FeGd-HN@Pt2@LF/RGD2 was much higher compared to pre-injection (Figure 9a). However, the DW-MRI images after 24 h or 72 h (Figure S17) do not show much differences. The ADC values decreased for FeGd-HN@Pt2@RGD2, FeGd-HN@Pt2, FeGd-HN, or Saline groups (Figure 9a). ∆ADC, calculated from ∆ADC = (ADCprost-ADCpre)/ADCpre × 100%, were measured to be 32.1±5.1%, -13.1±10.4%,

-14.9±6.6%,

-14.8±7.3%,

or

-17.9±10.3%

for

FeGd-HN@Pt2@LF/RGD2,

FeGd-HN@Pt2@RGD2, FeGd-HN@Pt2, FeGd-HN, and Saline (Figure 9b). For our study, a positive differential in ADC value correlated to treatment response, while a negative differential was indicative of null treatment response. The results show that the intrinsic MRI capability of the nanoparticles can be used to predict and monitor tumor response to FT (i.e., self-MRI-monitoring). Immunohistochemistry analysis of the orthotopic brain tumors stained with Ki-67 antibody (Figure S18) showed significant decrease in Ki-67 expression after treatment with FeGd-HN@Pt2@LF/RGD2 compared to the other groups (FeGd-HN@Pt2@RGD2, FeGd-HN@Pt2, FeGd-HN, or Saline). This result shows FT treatment by FeGd-HN@Pt2@LF/RGD2 can reduce the tumor cell proliferation of in our orthotopic model. In addition, our FeGd-HN@Pt2, and FeGd-HN@Pt2@LF/RGD2 nanoparticles were not toxic to mice organs (Figure S19), indicating good biocompatibility of our nanoparticles.

CONCLUSIONS 13


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In summary, we term ferroptosis therapy (FT) as a form of cancer therapy and hypothesize that the FT efficacy could be significantly improved via accelerating the Fenton reaction by simultaneously increasing the local concentrations of all reactants (Fe2+, Fe3+, and H2O2) in cancer cells. We synthesized Fe3O4/Gd2O3 hybrid nanoparticles to load CDDP, and conjugated them to LF and RGD dimer to confer BBB transportability and cancer selectivity. FeGd-HN@Pt@LF/RGD2 showed excellent T1-weighted MRI properties, and exerted tumoricidal effect in vitro on U87-MG cells. When evaluated in an orthotopic brain tumor model, FeGd-HN@Pt@LF/RGD2 successfully inhibited tumor growth and extended survival time in mice compared to treatment controls. Higher uptake of the nanoparticles was observed in tumor compared to liver. Moreover, the intrinsic MRI capability of the nanoparticles was used to monitor tumor response to FT. Collectively, our data shows that FT is a viable treatment strategy for cancer, and that our FeGd-HN@Pt2@LF/RGD2 nanoparticles can deliver high FT efficacy on orthotopic brain tumors.

EXPERIMENTAL METHODS Synthesis of Fe3O4/Gd2O3 Hybrid Nanoparticles (FeGd-HN). The Fe3O4/Gd2O3 hybrid nanoparticles (FeGd-HN) were synthesized using a water phase method, which is widely used for other core-shell nanoparticles.55,56 The O2 dissolved in PAA (Mw = 1.8 k) solution (160 mL, 0.4 %) was removed via N2 bubbling (~ 1 h). After that, the PAA solution in round-bottom flask with a reflux condenser was put in oil bath at 102 oC. 3.2 mL of mixture of iron precursors (500 mM FeCl3 plus 250 mM FeSO4) and 48 mL of ammonia solution (NH3.H2O, 28 %) were then respectively charged to PAA solution. After 30 min of reaction, 3.2 mL of Gd(NO3)3 (500 mM) and 24 mL of NH3.H2O were respectively charged. The FeGd-HN was obtained after further 60 min of reaction. The cooled FeGd-HN solution was finally subjected to dialysis (Mw cut-off 6-8 kDa) for purification, and ultrafiltration (Mw cut-off 10 kDa) for concentration. 14


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Synthesis of Cisplatin-Loaded FeGd-HN (FeGd-HN@Pt). 0.2~3.0 mL of CDDP (4.0 mg/mL in DMF) was mixed with 5.0 mL of FeGd-HN aqueous solutions (CFe = 5.50 mM, CGd = 2.69 mM, pH = 8.5) under magnetic stirring. After 72 h, the FeGd-HN@Pt was rinsed thrice by ultrapure water and concentrated via ultrafiltration (Mw cut-off 10 kDa). Finally, Fe, Gd and Pt concentrations in FeGd-HN@Pt were determined using ICP-OES. Synthesis of FeGd-HN@Pt2@LF/RGD2. The LF and RGD2 were grafted to FeGd-HN@Pt2 utilizing EDC/NHS, which activate carboxyl groups to react with primary amine group. EDC (10 μL) and NHS (50 μL, 13 mg/mL) were mixed with FeGd-HN@Pt2 (5.0 mL, CFe=2.80 mM, CGd=1.37 mM, CPt=0.34 mM). Then, LF (200 μL, 0.125 mM) and RGD2 (100 μL, 3.8 mM) were charged to the reaction solution. The FeGd-HN@Pt2@LF/RGD2 was obtained after 16 h. After 3 times of purification utilizing ultrafiltration (Mw cut-off 10 kDa), the final FeGd-HN@Pt2@LF/RGD2 sample was dissolved in Milli-Q water (5.0 mL, pH 7.4). In the obtained FeGd-HN@Pt2@LF/RGD2 solutions, the Fe, Gd and Pt concentrations were determined using ICP-OES.

ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials and methods: protocols for in vitro and in vivo experiments. Table S1, S2: synthesis conditions and characterization results. Figure S1: r1 or r2 measurements (7.0 T). Figure S2: T1-weighted MR images of nanoparticle solutions (7.0 T). Figure S3: r1 or r2 measurements (1.5 T). Figure S4: TGA and DTG curves. Figure S5: H – M curve. Figure S6: in vitro release behaviors of Fe or Pt. Figure S7-S9: LSCM images of cells. Figure S10: T1-weighted MR images of cells. Figure S11, S12: evaluation of intracellular ROS generation via DCF-DA assay observed by LSCM. Figure S13: Evaluation of 15


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intracellular ROS generation via DCF-DA assay measured by flow cytometry. Figure S14: evaluation of the ferroptosis therapy efficiency. Figure S15, S16: in vivo BBB studies. Figure S17: ADC parametric maps of the orthotopic brain tumor-bearing mice. Figure S18: immunohistochemistry analysis of the orthotopic brain tumors. Figure S19: histological analyses of main organs from the normal mice (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] ORCID Zheyu Shen: 0000-0002-0350-375X Aiguo Wu: 0000-0001-7200-8923 Xiaoyuan Chen: 0000-0002-9622-0870

ACKNOWLEDGMENTS This research is financially supported in part by the Zhejiang Provincial Natural Science Foundation of China, Youth Innovation Promotion Association of the Chinese Academy of Sciences (2016269) (Z. S.), Zhejiang Province Public Welfare Technology Application Research Project (2017C33129), National Natural Science Foundation of China (Grant Nos. 51761145021 (BRICS Research Project), U1501501, and U1432114), the National Key Research & Development Program (Grant Nos. 2016YFC1400600, 2018YFD0800302), Intramural Research Program (IRP), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH) (Grant No. ZIA 16


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EB000073). V.I.B. thanks Russian Foundation of Basic Research (Grant RFBR – BRICS country No. 17-53-80099). S. K. M. thanks BRICS Research Project-BGNCDT for financial support. A. W. thanks Hundred Talents Program of Chinese Academy of Sciences (2010-735). We also thank Dr. M.A. Aronova (NIBIB/NIH) for helpful discussions and help with TEM/EDS.

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Figure 1. (a): Design and synthesis of our Fenton-reaction-accelerable magnetic nanoparticles, i.e., cisplatin-loaded Fe3O4/Gd2O3 hybrid nanoparticles with conjugation of lactoferrin (LF) and RGD2 23


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(FeGd-HN@Pt@LF/RGD2). (b): Mechanism illustration for the ferroptosis therapy (FT) of orthotopic brain tumors with self-MRI-monitoring.

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Figure 2. (a-c): TEM images of FeGd-HN@Pt2 (a), and FeGd-HN@Pt2@LF/RGD2 (b, c). The average particle size was 6.3 and 6.6 nm for FeGd-HN@Pt2 and FeGd-HN@Pt2@LF/RGD2 as measured from the TEM images. (d): EDS of FeGd-HN@Pt2. (e): Size distribution of FeGd-HN@Pt2 (dh = 9.8 nm), and FeGd-HN@Pt2@LF/RGD2 (dh = 14.7 nm).

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Figure 3. Measurements of the nanoparticle uptake in cells. (a, b): MCF-7 or U-87 MG cells incubated with nanoparticles analyzed by flow cytometry. The cells without nanoparticle treatment served as controls. (c): The relative intensity (i.e., mean fluorescence intensity ratio) of the nanoparticle-treated cells compared to untreated cells. Mean ± SD, n = 3. * P < 0.01. (d): Internalized amounts of the nanoparticles by U-87 MG cells measured by ICP. Mean ± SD, n = 3. ** P < 0.001. 1 femtogram (fg) = 10-15 g.

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Figure 4. Studies of the in vitro BBB model. (a): Schematic illustration of the in vitro BBB model for evaluation

of

the

BBB

permeability

of

nanoparticles.

(b):

Distribution

of

FeGd-HN,

FeGd-HN@Pt2@LF/RGD2, or FeGd-HN@Pt2@LF/RGD2 plus LF block in apical chamber or basolateral chamber. Mean ± SD, n = 4. * P < 0.001.

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Figure 5. T1-weighted MRI images of mouse normal brains (without tumors) pre or post intravenous injection of (a) Magnevist, or (b) FeGd-HN@Pt2@LF/RGD2 (CGd = 5.0 mg/kg mice). (c, d): Quantitative analysis of the T1-weighted MR images in (a, or b) using SNR.

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ACS Nano

Figure 6. (a, b): T1-weighted MRI images of the nude mice bearing U-87 MG tumors pre or post IV administration of (a)FeGd-HN@Pt2, or (b) FeGd-HN@Pt2@LF/RGD2 (CGd = 5.0 mg/kg mice) Strongest signal in tumor was observed for FeGd-HN@Pt2@LF/RGD2 at 12 h. The slice orientation is axial. (c, d): Corresponding quantitative analysis of the MRI images in (a, b) showing the changes in tumor signals (ΔSNR).

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Figure 7. (a, b): T1-weighted MRI images of the nude mice bearing U-87 MG tumors pre or post IV administration of FeGd-HN@Pt2@LF/RGD2 nanoparticles (CGd = 5.0 mg/kg mice). Coronal slices showing the highest signal in (a) tumor or (b) liver are displayed. (c, d): Corresponding quantitative analysis of the MRI images in (a, b) showing the signal changes in (c) tumor or (d) liver (ΔSNR).

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Figure 8. (a-e): Bioluminescence images of orthotopic brain tumor-bearing mice (i.e., Luc-expressing U-87

MG

tumors)

after

(b)FeGd-HN@Pt2@RGD2

treatment nanoparticles,

with (c)

(a)

FeGd-HN@Pt2@LF/RGD2

FeGd-HN@Pt2

nanoparticles,

nanoparticles, (d)FeGd-HN

nanoparticles, or (e) Saline. (f): Percent survival of the mice bearing orthotopic brain tumors after treatment. Treatment with FeGd-HN@Pt2@LF/RGD2 significantly extended the survival of

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tumor-bearing mice. (g): Growth curves of the orthotopic brain tumors in different treatment groups. (mean ± SE, n = 5).

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Figure 9. Therapeutic efficacy of FT in orthotopic brain tumor-bearing mice measured by DW-MRI. (a): ADC parametric maps of the orthotopic brain tumor-bearing mice before or after treatment with FeGd-HN@Pt2@LF/RGD2, FeGd-HN@Pt2@RGD2, FeGd-HN@Pt2, FeGd-HN, or Saline. (b): Quantitative analysis of the ADC parametric maps showing ∆ADC values of different groups. ∆ADC = (ADCprost-ADCpre)/ADCpre × 100%.

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Table of Contents

Fenton-reaction-accelerable magnetic nanoparticles can be used for ferroptosis therapy of orthotopic brain tumors by delivering all reactants involved in the Fenton reaction (Fe2+, Fe3+ and H2O2) to the tumor site. The intrinsic MRI capability of the nanoparticles can be used to monitor tumor response to FT (self-MRI-monitoring).

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Fenton-Reaction-Acceleratable Magnetic ... - ACS Publications

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