Dual-Mode Imaging-Guided Synergistic Chemo- and

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Dual-mode Imaging-Guided Synergistic Chemo- and Magnetohyperthermia Therapy in a Versatile Nanoplatform to Eliminate Cancer Stem Cells Jinglong Tang, Huige Zhou, Jiaming Liu, Jing Liu, Wanqi Li, Yuqing Wang, Fan Hu, Qing Huo, Jiayang Li, Ying Liu, and Chunying Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06393 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on July 3, 2017

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ACS Applied Materials & Interfaces

Dual-mode Imaging-Guided Synergistic Chemo- and Magnetohyperthermia Therapy in a Versatile Nanoplatform to Eliminate Cancer Stem Cells

Jinglong Tanga,b,†, Huige Zhoua,b,†, Jiaming Liua,b, Jing Liua,b, Wanqi Lia, Yuqing Wanga, Fan Huc, Qing Huoc, Jiayang Lia, Ying Liua, Chunying Chena,b,* a

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety & CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190, China. b University of Chinese Academy of Sciences, Beijing 100049, China. c Department of Biomedical, Biochemical Engineering College of Beijing Union University, Beijing 100023, China. †

These authors contributed equally *Corresponding Author: Email address: [email protected]. Tel: +86-010-82545560; fax: +86-010-62656765

KEYWORDS: Fe3O4@PPr@HA hybrid nanoparticles, DAPT, photoacoustic imaging, magnetic resonance imaging, cancer stem cells.

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ABSTRACT: Cancer stem cells (CSCs) have been identified as a new target for therapy in diverse cancers. Traditional therapies usually kill the bulk of cancer cells, but are often unable to effectively eliminate CSCs, which may lead to drug resistance and cancer relapse. Herein, we propose a novel strategy: fabricating a multifunctional magnetic Fe3O4@PPr@HA hybrid nanoparticles and loading it with the Notch signaling pathway inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]S-phenylglycinet-butylester (DAPT) to eliminate CSCs. Hyaluronic acid (HA) ligands greatly enhance the accumulation of the hybrid nanoparticles in the tumor site and in the CSCs. Both hyaluronase in the tumor microenvironment and the magnetic hyperthermia effect of the inner magnetic core can accelerate the release of DAPT. This controlled release of DAPT in the tumor site further enhances the ability of the combination of chemo- and magnetohyperthermia therapy to eliminate cancer stem cells. With the help of polypyrrole-mediated photoacoustic and Fe3O4-mediated magnetic resonance imaging, the drug release can be precisely monitored in vivo. This versatile nanoplatform enables effective elimination of the cancer stem cells and monitoring of the drugs.


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INTRODUCTION Although much attention has been focused on genetic abnormalities that initiate cancer, there is now tremendous evidence that cancer cells are also highly affected by their microenvironment.1 Cancer stem cells (CSCs) are now considered to be one of the most important components in the tumor microenvironment.2 CSCs are endowed with the ability to self-renew and differentiate and are also involved in metastasis, angiogenesis, and drug resistance. Recent evidence indicates that heterogeneity in tumors may also be related to cancer stem cells.3-5 Because of the critical role of cancer stem cells, eradicating them in the tumor should be given priority. Current radio- and chemo-therapies kill the bulk of cancer cells, but are often unable to effectively eliminate CSCs, which may lead to drug resistance and relapse. CSCs use many of the same signaling pathways that are found in normal stem cells, such as the Notch, Wnt and Hedgehog signaling pathways. Therapeutic targeting of these pathways may provide a strategy to eradicate CSCs and suppress tumor regrowth. N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycinet-butylester (DAPT), as an inhibitor of γ-secretase, can effectively suppress the activity of γ-secretase and reduce the number of CSCs by inhibiting the Notch signal pathway.6-7 However, the two benzene rings and the tertiary butyl group in the chemical structure of DAPT make it hydrophobic, which in the physiological environment causes the molecules to aggregate. In preclinical studies, this aggregation resulted in indiscriminate biodistribution and low efficiency.8-11 The use of DAPT is also restricted by severe side effects, thus there is a need for a more precisely cancer-targeted therapy.12-13 Nanomedicine significantly extends the range of both anticancer drugs and treatment strategies14-20, and may be useful in eliminating CSCs.21-23 Major nanomedicine approaches to eliminating CSCs can be divided into the following three aspects: targeted drug delivery to CSCs24-25; destruction of CSCs 26-28, and specific inhibition of CSCs29. Various nanoparticles (NPs), including liposomes, dendrimers, polymeric nanoparticles, and magnetic inorganic nanoparticles17, 35-37, have been used as carriers to deliver drugs to the tumor site and to CSCs by passive and/or active targeting.35, 38 Recently, hyperthermia for cancer treatment has been widely explored and appears to be identified as an effective approach to eliminating CSCs both in vitro and in vivo.21, 27-28. In addition, nanocarriers may also provide an opportunity for imaging-guided therapy (i.e. X-ray computed tomography (CT), magnetic resonance imaging (MRI) or photoacoustic (PA) imaging).34-36 Polypyrroles (PPr) are widely used in bioelectronics and biomedical applications because of their good biocompatibility, high conductivity, outstanding stability, and simple synthesis procedures.37-38 Their hydrophobic properties and π-π stacking of polypyrrole nanomaterials make them a good candidate for delivery of hydrophobic drugs, especially aromatic ones.36, 39 The strong NIR absorption spectrum of polypyrrole (700-900 nm) allows its distribution to be monitored by photoacoustic imaging and also supports photothermal therapy for cancer.40 Although the photothermal approach can work in some cancer treatments, it still suffers from disadvantages: high doses of laser irradiation can damage bystander tissues, and the depth of laser penetration in tissues is limited.41 On the contrary, Fe3O4



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nanoparticle-mediated magnetic hyperthermia, which allows deep penetration in vivo, has been demonstrated to be an alternative and promising method for cancer treatment. When subjected to an alternating magnetic field, superparamagnetic Fe3O4 nanoparticles can convert electromagnetic energy into heat through oscillation of their magnetic moment. Fe3O4 nanoparticles also have negligible adverse effects on tissues 42-44 and permit T2-weighted magnetic resonance (MR) imaging, which is widely used in the clinic. Moreover, surface modification of superparamagnetic nanoparticles can reduce aggregation and nonspecific binding. Hyaluronic acid (HA), as a naturally occurring polysaccharide, has been extensively investigated for biomedical applications, because of its biocompatibility and biodegradability. In particular, HA can specifically bind to various cancer cells that over-express the CD44 receptor.45-46 Thus, HA has been conjugated onto various drug-loaded nanoparticles for use as a targeting moiety.9, 25

Here, we describe the design of a multifunctional magnetic nano-delivery system for DAPT, based on Fe3O4@PPr@HA layered hybrid nanoparticles (h-NPs) to eliminate cancer stem cells via both hyperthermia and chemotherapy (Figure 1A). The magnetic Fe3O4 core aids in inducing hyperthermia under an alternating current (AC) magnetic field, the PPr shell allows high loading of DAPT, and the HA enhances solubility and allows for targeting of the CSCs, Meanwhile, MR and PA imaging will help to confirm the tumor accumulation of h-NPs in vivo. The excellent water solubility of h-NPs with surface coverage by hyaluronic acid enhances the solubility of the hydrophobic DAPT in the physiological medium, which increases its accumulation in the tumor. HA ligands also act as targeting moieties improving the uptake of DAPT in CD44-positive cells.47 Under the dual actions of magnetic hyperthermia and chemotherapy, this novel DAPT-loaded nanoplatform can be applied to eradicate CSCs. EXPERIMENTAL SECTION Materials Ferric chloride hexahydrate (FeCl3•6H2O), ferrous chloride tetrahydrate (FeCl2•4H2O) and PVA were purchased from Sinopharm Chemical Reagent Co., Ltd. Chitosan quaternary ammonium salt (Mw = 100 kDa) (Chitosan-N-2-hydroxy-N,N,Ntrimethylpropan -1-ammonium chloride) abbreviated as CSQ, was purchased from the Dongying Tianhua Biological Additives Co. Ltd. (Shandong, China). Hyaluronic acid (HA, Mw = 6.8 kDa) was purchased from the Shandong Freda Biopharmaceutical Co., Ltd. (Shandong, China). DAPT was purchased from ApexBio Technology. Fluorescein isothiocyanate (FITC) were purchased from Beijing Lab lead Biotechnology Co., Ltd. (Beijing, China). Pyrrole monomer was purchased from TCI (Shanghai) Development Co., Ltd. All solvents and reagents were of analytical grade and used without further purification if not stated otherwise. Synthesis of Fe3O4@PPr@HA nanoparticles Fe3O4 nanoparticles were synthesized as previously described.48 Fe3O4@PPr


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nanoparticles were synthesized by an in situ chemical oxidation polymerization method.36-37 Briefly, 2.5 mg Fe3O4 nanoparticles were dispersed in 4 mL of 2 % (w/v) PVA aqueous solution and stirred at room temperature for 30 min. After adding 5 µL of pyrrole monomer, the solution was ultrasonicated for 10 min. Then, the reaction was triggered by the dropwise addition of FeCl3 aqueous solution (44.4 mg/mL, 1 mL). After stirring for 4 h in an ice bath, the polymerization was complete and the formation of Fe3O4@PPr nanoparticles was indicated by a dark green color. The nanoparticles were washed with hot water three times and then separated with a magnet to remove unreacted regents. HA was bound to the surface of Fe3O4@PPr by electrostatic interaction. Briefly, Fe3O4@PPr solution (1 mg/mL) was added to an HA aqueous solution (3 mg/mL, 5 mL) while sonicating by a probe at 90 W in an ice bath. The resulting Fe3O4@PPr@HA nanoparticles were separated by centrifugation and were washed three times with deionized water. FITC-labeled Fe3O4@PPr@HA was used in the intracellular localization study. The FITC-labeled HA was synthesized according to a previous report with a few modifications.49 Briefly, 50 mg HA powder was dissolved in 2 mL DMSO, and then 1 mg FITC was added. The mixture was reacted at 90 °C for 2 h in the dark. After cooling to room temperature, the solution was then dialyzed against distilled water for 3 d in darkness and lyophilized. Drug loading and release profiles Varying amounts of the DAPT methanol solution (10 mg/mL) were mixed with a Fe3O4@PPr solution (1 mg/mL) and incubated at 37 oC in an oscillation shaker overnight. Excess methanol and DAPT were removed by centrifugation and the pellet washed with deionized water three times. The supernatant liquor was collected and ultra-filtered with a 0.22 µm filter membrane. Then the unloaded DAPT and a series of DAPT acetonitrile solution were measured by HPLC; the drug load was obtained as the total mass minus the unloaded material. The encapsulation efficiency and loading content is defined as follows: Encapsulation efficiency (%)=(W0-Wt)/W0×100%; Loading content (%)=(W0-Wt)/Ws×100%, Where W0 and Wt are the weight of the initial DAPT and that of the total amount of DAPT detected in the supernatant after centrifugation, respectively. Ws is the weight of DAPT loaded particles after lyophilization. Each sample was assayed in triplicate. The release of DAPT from Fe3O4@PPy@DAPT@HA was studied by dialyzing samples at 37 oC in PBS (2 mM) at pH 7.4 and pH 5.0 with or without the presence of HAase (0.5 mg/mL) for different periods of time. The DAPT released from nanoparticles was collected and determined by HPLC. The release of DAPT under the AC magnetic field (635 kHz, 30 A) at different time points was measured in 3 mL PBS (2 mM) at pH 7.4 and pH 5.0. For each measurement, 0.2 mL sample was taken out every 5 min and centrifuged at 13000 rpm for 20 min. The amount of DAPT released in the supernatant was recorded by HPLC. Cell lines and animals Cell lines representing murine breast cancer cells (4T1) and human breast cancer



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cells (MDA-MB-231 and MCF-7) were maintained in our lab. Six week-old BALB/c mice and BALB/c nude mice were purchased from the Beijing Vital River Company. All animal experiments were conducted using protocols approved by the Institutional Animal Care and Use Committee at the Chinese Academy of Medical Sciences. Cell apoptosis and intracellular ROS level test In order to test apoptosis, the 4T1 cells were treated with 50 μg/mL h-NPs, 100 μg/mL h-NPs, 50 μg/mL h-NPs + AC, 50 μg/mL DAPT@h-NPs + AC, DAPT@h-NPs + laser (1 W/cm2). Cells without any treatment were regarded as control. Then, the cells were collected, washed three times with PBS, and dyed with an Annexin V-FITC/PI kit. Finally, the apoptosis and necrosis of cells were detected by flow cytometry (BD Biosciences, USA).

To test the intracellular reactive oxygen species (ROS) level, the 4T1 cells were treated with 50 μg/mL h-NPs and 50 μg/mL h-NPs + AC. Then, the cells were stained with a 10 μM ROS probe DCFH-DA (Molecular Probes, USA) in a serum-free DMEM medium. Finally, the treated cells were detached from the dishes, collected and measured by flow cytometry.

ALDH assay Cells were suspended in RPMI-1640 and seeded in 6-well culture plates at a density of 1 × 105 for 24 h. The medium was then changed, and cells were incubated for another 24 h with RPMI-1640 containing 49.25 μg/mL Fe3O4@PPr@DAPT@HA (equivalent to 19.62 μg/ml Fe3O4 and 10 μg/ml DAPT). Next, the cells were collected and treated by a moderate radio frequency heating machine (Shuangping SPG-10AB-II, 635 kHz, 30 A, China) for 15min. Then, the cells were re-suspended in RPMI-1640 medium and cultured for a further 12h. Single-cell suspensions were prepared for the aldefluor assay according to the manufacturer’s instructions (Stem CellTM Technologies). Briefly, 106 cells were suspended in 1 mL of assay buffer. 5 μL activated aldefluor substrate was added to the suspension, and an aliquot of 0.5 mL was immediately quenched with the specific ALDH inhibitor o diethylaminobenzaldehyde (DEAB). After incubation at 37 C for 40 min, the cells were centrifuged and re-suspended in 0.5 mL aldefluor assay buffer. ALDH-positive cells were counted with flow cytometry (Calibur, BD Bioscience, USA). Mammosphere formation assay After being treated by the AC magnetic field, the cells were cultured for another 12h. Then, the cells were washed with PBS and suspended in DMEM/F12 serum-free medium supplemented with basic fibroblast growth factor (bFGF, 20 ng/mL) and human recombinant epidermal growth factor (EGF, 20 ng/mL), 0.4% bovine serum albumin and 5 mg/mL insulin. Cells were seeded in ultra-low attachment 6-well plates (Costar, Corning Inc.) with 2000 cells/ well. After 7 days of culture, mammospheres were observed using a microscope (Olympus Co.). The number of mammosphere cells in each well was counted after they were trypsinized to single cells. Magnetic resonance imaging The effect of MR imaging for Fe3O4@PPr@HA NPs was evaluated using a 7.0 Tesla MR imaging machine (BioSpec 7.0 70/20, Bruker, German). Samples were diluted with water to give an iron concentration in the range of 0.07-0.56 mM before


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measurement. MR imaging of cells with varying concentrations of Fe was accomplished with the same 7.0 T MR imaging machine. For in vivo MR imaging, Balb/c-bearing 4T1 murine breast cancer tumors were intravenously injected with Fe3O4@PPr@HA (1.5 mg/mL, 200 μL). MR imaging was conducted on a 7.0 T MRI scanner equipped with a mouse body coil. The T2-weighted images were acquired at 4 h, 24 h and 48 h. Images obtained before injection were used as controls. MR images were acquired using a T2-Turbo RARE sequence with parameters as follows: TR=3000ms, TE=50ms, field of view=35×35mm, matrix size= 256×256, number of slices= 20, slice thickness= 1mm, flip angle=90˚, NEX=5. Photoacoustic imaging Samples with concentrations ranging from 1-10 μg/mL Fe3O4@PPr@HA were tested with a multispectral optoacoustic tomography system (MSOT incision 128, iTheraMedical, Germany). For in vivo photoacoustic imaging, 200 μL of Fe3O4@PPr@HA (1.5 mg/mL) solution was injected into the tail vain of 4T1 tumor-bearing Balb/c mice. The mice were then scanned at 1 h, 4 h, 8 h, 24 h and 48 h with the MSOT system. The main experimental parameters were five wavelengths for each slice from 680-900 nm, and the region of interest (ROI) is 20 mm. PA images obtained before injection were used as controls. Hyperthermia experiments in vivo Hyperthermia experiments in vivo were carried out using a moderate radio frequency heating machine. The mice (n = 5) were placed into the induction coil using a specially designed Teflon supporter so that tumors were located exactly in the region of the AC magnetic field. The hyperthermia therapy was conducted every three days by i.v. injection with 54.81 mg/kg Fe3O4@PPr@DAPT@HA (equal to 18.64 mg/kg Fe and 10 mg/kg DAPT according to the loading capacity). The tumor volume and mouse body weight were monitored daily. Immunofluorescence After being fixed overnight in 10% formalin buffer, the tumors were dehydrated and embedded in paraffin. For immunofluorescence, sections were rehydrated followed by heat-induced epitope retrieval and incubated for 10 min with 0.3% H2O2 in water to block endogenous peroxidase. Sections were then incubated with blocking buffer (10% bovine serum albumin, BSA) for 60 min at room temperature and then with a primary antibody (monoclonal CD44 antibody) diluted in blocking buffer overnight. The following day, the secondary antibodies conjugated with 488 Alexa dye were applied. The images were acquired with a fluorescence microscope and analyzed by Image J software. RESULTS AND DISCUSSION Synthesis and Characterization of Fe3O4@PPr@HA Positively charged Fe3O4 nanoparticles with positive charge were synthesized by high temperature co-precipitation according to a protocol previously described in the literature.48 TEM images and DLS data indicated that the Fe3O4 nanoparticles were



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uniform and their surface charge was positive (Figure S1A&S2). Fe3O4@PPr nanoparticles were prepared by an in situ chemical oxidation polymerization method in the presence of polyvinyl alcohol (PVA) as stabilizer. As shown in Figure 1B, PPr was deposited onto the Fe3O4 nanoparticles; the total size of the Fe3O4@PPr was 82 nm, which is consistent with their hydrodynamic diameter (Figure S1B). For better imaging performance, the ratio of Fe3O4 to PPr was optimized. T2-weighted MRI signal of Fe3O4@PPr nanoparticles was very weak when the input ratio of Fe3O4 to pyrrole monomer was less than 1:1.5 (Table S1). However, as the ratio of PPr decreased in the nanocomposites, the absorbance of the hybrid nanoparticles in near-infrared region under 808 nm laser irradiation also decreased (Figure S3A & B), which would reduce their performance as a PA contrast and photothermal agent. Thus, the ratio of Fe3O4: pyrrole=1:1.5 was used in the experiments. The ratio of Fe3O4 to PPr in the final Fe3O4@PPr product was 1:1 according to the Fe concentration from ICP-AES; the total mass was obtained by lyophilization. HA has been highlighted as a tumor-targeting moiety due to its high affinity to the CD44 receptor. It is a linear glycosaminoglycan consisting of alternating D-glucuronic acid and N-acetyl-D-glucosamine units.45 It is easy to attach the negative HA to the positive charged surface of Fe3O4@PPr because of the electrostatic attraction (Figure 1C). Although Fe3O4@PPr nanoparticles were stable in water in the presence of PVA, they tended to aggregate in media such as PBS or DMEM that mimic physiological conditions (Figure S4). The surface modification of nanoparticles with HA remedies the instability in a physiological medium. Moreover, the Fe3O4@PPr@HA nanoparticles could be stable in PBS for 2 weeks, while in the DMEM solution, the nanoparticles aggregated after 3 days. Interestingly, these nanoparticles is quite stable in DMEM containing 10% FBS, and this may attribute to the protein corona formation. This stabilization is essential for systemic administration in vivo. After the Fe3O4@PPr@HA h-NPs were synthesized, the optical and magnetic properties were tested with UV-vis-NIR absorbance spectrum and magnetic susceptibility. A high absorbance from 700 nm to 900 nm was observed from the UV-vis-NIR absorbance spectrum of Fe3O4@PPr@HA (Figure 1D). The field-dependent magnetization measurement curve of Fe3O4@PPr (Figure 1E) exhibited superparamagnetism, thus it could be used to be used as a T2-weighted MR imaging agent and for magnetic hyperthermia treatment. Previous studies have certified that PPr can act as a photoacoustic contrast and photothermal therapeutic agent40, and the Fe3O4 superparamagnetic nanoparticles can be used as MRI contrast and magnetic-mediated hyperthermia agents. Here, we utilize both functions simultaneously, combined into one nanoplatform for dual-modal imaging. Drug loading and release profiles DAPT, a novel antitumor chemotherapy drug, is insoluble in water and forms an unstable emulsion in 0.1% (w/w) Tween-80 solution (Figure S5). A stable suspension is obtained when it is loaded into h-NPs through hydrophobic interactions and π-π stacking.36, 40 The DAPT content and release from the h-NPs were analyzed by HPLC


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(Figure S6). As the relative amount of DAPT rose in the h-NPs, the loading capacity increased, but if the ratios of DAPT and Fe3O4@PPr was higher than 0.5 : 1, it could cause the aggregation of the h-NPs. Thus, the DAPT loading capacity has a maximum value of 31.3% (Figure 2A). Since the secondary amino groups in the chemical structure of DAPT are easily protonated, DAPT is more sensitive to acidic environments than neutral environment. Thus, the DAPT release is faster in acidic environments (Figure 2B, D). In the tumor microenvironment and lysosomes, there is usually plenty of HAase. When the surface HA is degraded by HAase under acidic conditions, DAPT is directly exposed, which also speeds up its release (Figure 2B). The release of DAPT from h-NPs is controlled not only by pH and enzyme conditions but also by the hyperthermia. The temperature of h-NP suspension ([Fe] 1 mg/mL) reached about 65 °C (Fig. 2C) in an AC magnetic field (635 kHz, 30A) (Figure S7), and this excellent heating performance can be attributed to the high specific absorption rate (SAR) value (421 W g-1) (Figure 2C). With the application of an AC magnetic field, the cumulative release of DAPT within one hour (17.2% and 27.0% at pH 7.0 and pH 5.0, respectively), is much higher than the release at 37 °C within a shaker (2.4% and 4.1%, respectively) (Figure 2B and D). There are two factors that may determine the magnetic-responsive drug release behavior: First,the produced heat may weaken the interaction between DAPT and PPr.50 Second, the HA ligand and drug molecules showed increased mobility at high temperatures, when the h-NPs underwent a thermal transition.43 These properties will facilitate precise control of the DAPT release in the tumor site. Cell viability and intracellular localization Before testing the efficiency of hyperthermia therapy with the h-NP nanoplatform, we evaluated its cytotoxicity. Cell viability assays were carried out for 4T1, MDA-MB-231 and MCF-7 tumor cells. The h-NPs showed no obvious toxicity to these cells at relative high concentrations (Figure 3B and S8). It is to be noted that DAPT also showed negligible toxicity to the 4T1 cells at our used concentration in vitro (Figure S9), which is in accordance with previous reports.51 Thus DAPT may not affect the proliferation of cancer cells but may regulate some compartments (like CSCs) in the tumor microenvironment. In order to explore the possible mechanism about the h-NPs induced cell death, we tested the cell apoptosis and necrosis after different treatments. Compared to the control group, the cell apoptosis and necrosis increased with the h-NP concentration increased. Moreover, when an AC magnetic field or laser irradiation was added, the cell apoptosis and necrosis ratio significantly enhanced. (Figure 3C and Figure S10) Thus, the hyperthermia caused the cell death possibly via apoptosis and necrosis. We further tested the intracellular ROS level after h-NPs incubation with or without AC magnetic field. The h-NPs induced the intracellular ROS level increasing, and when the AC magnetic field applied, the intracellular ROS reached to a higher level. (Figure 3D) The high ROS level may induce the cell apoptosis. Recent studies found that silica nanoparticles could increase the intracellular iron level and induce ferrosis, which can be used as potential anti-tumor agents.53-55 The iron oxide nanocarriers can also enhance the chemotherapy by generation of highly toxic ROS.56 The



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accumulation of ROS in cells may be helpful for the cancer therapy in vivo. To test the targeting efficacy of HA binding to cancer cell lines, we compared MDA-MB-231, MCF-7 and 4T1 cells in this study. Previous studies have proven that the MDA-MB-231 and 4T1 cell lines highly express the CD44 marker.25, 56-57 In this study, the h-NPs were labeled with the fluorescent probe FITC to test cell uptake and intracellular localization. The results showed the 4T1 and MDA-MB-231 cells took up more h-NPs via HA binding to the CD44 membrane protein than MCF-7 cells in the same condition (Figure 3E and S11). And the h-NPs were mainly located in the lysosome (Figure 3F) which is similar to most HA-modified nanomaterials.25, 47 Notably, the MDA-MB-231 and 4T1 cell lines possess an increased cancer stem cell subpopulation. In previous ALDH staining assays, the 4T1 cells showed the CSCs subpopulation is over 20% and that in the MDA-MB-231 cells showed approximately 10%, while that in the MCF-7 cells is only about 2%.56 The amount of CSCs subpopulation, which is always considered as CD44 positive subpopulation, is related to the malignance of tumor cells. For the high affinity of HA to CD44 receptor, the HA modified nanoparticles may be better targeting for CSCs. Because of the limitation of the laser penetration depth in biology tissues, which is usually decay exponentially (Figure S12), the laser can be easily blocked by the biological tissue. Thus, we used the Fe3O4 core to mediate magnetic hyperthermia therapy rather than using the PPr shell to mediate the photothermal therapy. To investigate the magnetic hyperthermia therapy with Fe3O4@PPr@DAPT@HA (DAPT@h-NPs), we used Live/Dead staining to evaluate the cell death ratio in vitro. The results indicated that the DAPT@h-NPs shows negligible toxicity, while under magnetic hyperthermia conditions they exhibits slight cytotoxicity; the 4T1 showed a nearly 30% death ratio after incubation with DAPT@h-NPs in the AC magnetic field (Figure 3A). CSCs elimination in vitro To investigate the influence of DAPT@h-NP-mediated magnetic hyperthermia on CSCs in 4T1 cells, the percentage of ALDH+ cells and mammosphere formation was evaluated. It has been proven in many types of cancer cells that ALDH is a specific intracellular marker of CSCs.56-57 After treatment with only DAPT@h-NPs or hyperthermia therapy, the percentage of ALDH+ cells decreased (Figures 4A and C). It is to be noted that the percentage of the ALDH+ cell subpopulation is lowest in the DAPT@h-NP-mediated hyperthermia therapy group, which indicates that synergistic hyperthermia-chemotherapy could more efficiently eliminate the CSCs. To further confirm the decrease the proportion of CSCs, we assayed the mammosphere formation ability of 4T1 cells after different treatments. In the breast cancer cells, the ability to form mammospheres under non-adherent culture conditions has been identified as another characteristic of CSCs. After DAPT@h-NP-mediated magnetic hyperthermia therapy, the cells were cultured for another 24 h and the dead cells were washed away. After that, the mammosphere formation assay was carried out and the mammosphere cells were counted after being trypsinized. As shown in Figures 4B and D, the h-NPs do not affect the mammosphere formation ability of 4T1 cells. However, when cells were treated by DAPT@h-NPs, hyperthermia therapy, or


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both, the mammosphere formation ability decreased significantly. The size of mammospheres in the DAPT@h-NP-mediated magnetic hyperthermia therapy group is the smallest of all the groups, which indicates the synergistic effects of chemo- and thermo- therapy. Statistically, the number of mammosphere cells in different groups is in accordance with the ALDH staining assay. A number of preclinical studies have demonstrated the potential use of magnetic hyperthermia as an effective anticancer treatment modality.50, 58 Recent studies also certify the hyperthermia therapy (i.e., carbon nanotube- and gold nanorod-mediated photothermal therapy) could effectively eliminate CSCs.28 With DAPT-loaded magnetic hybrid nanoparticles, the CSCs could be more efficiently eliminated by the combination of chemo- and magnetohyperthermia therapy. PA and MR dual-mode Imaging Before the DAPT@h-NPs were used in vivo for cancer therapy, we tested for accumulation of h-NPs in the tumor site. Utilizing the Fe3O4 magnetic core and PPr shell, MR and PA imaging were carried out to precisely monitor the biodistribution. The PPr shell, with its strong NIR absorption (Figure 1D) and good ability of photothermal conversion (Figure S13), could serve as a PA imaging agent. To examine the PA imaging capability and monitor the trace of h-NPs in vivo, multispectral optoacoustic tomography was performed in vitro and in vivo. The PA intensities with h-NPs were concentration-dependent with a good linear relationship, and the signal increased with increasing concentration (Figure 5B). The calculated slope suggests that h-NPs are good contrast agents for PA imaging.59 To monitor PA signal changes in vivo, tumor-bearing mice were intravenously injected with 200 μL of h-NPs (1.5 mg/mL), and the PA signal of tumors was recorded at different time intervals (Figure 5A). Before injection, the image showed no obvious signal in the tumor site. However, the signal intensity was increased and reached a peak value 4 to 8 hours after injection, and gradually disappeared after 24h. This result clearly shows that the h-NPs have satisfactory residence times in the tumor and are efficiently targeted to the tumor site in both a passive and an active manner endowed due to the surface HA ligand modification and appropriate particle size. In addition to the application for PA imaging, the h-NPs could also be used as T2-weighted MR imaging contrast agents because of the superparamagnetic Fe3O4 in the core. We measured the MRI contrast performance of h-NPs in vitro, calculating a transverse relaxivity (r2) of 127.17 mM-1 s-1 (Figure 5C). This r2 value is much higher than those of some similar Fe3O4 nanocomposites because of the optimization of the Fe3O4 core to PPr shell ratio. 36, 60 To some extent, a higher transverse relaxivity could also represent a better magnetic hyperthermia performance.55 In order to test the T2-weighted MRI images in cells, we incubated 4T1 cells with h-NPs with different Fe concentrations (0, 1, 5 μM). As shown in Figure 5C, with increasing Fe concentrations, the cell images became darker, indicating that the MR imaging signal was concentrations dependent. After tumor-bearing mice were intravenously injected with h-NPs (1.5 mg/mL, 200 μL), MR images of them acquired by using a mouse body coil on a 7.0 T scanner. The images showed a dramatic darkening effect in the



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tumor area, and the inner structure of the tumor became visible 4 h after the injection (Figure 5D). The results of the MR imaging is agreed with the PA imaging. After injection, the h-NPs gradually accumulated in the tumor site and the inner structure of the tumor could be observed, which means the h-NPs can penetrate the tumor. Tumor treatment in vivo Because the CSCs could initiate tumor formation and affect tumor growth, we evaluated the tumor growth in vivo after chemo- and magnetohyperthermia therapy. To better manage the magnetic-mediated hyperthermia therapy in vivo, we monitored the temperature change in the tumor site after h-NPs i.v. injection. The images recorded with an IR camera indicated that the temperature was raised about 6˚С in an AC magnetic field (Figure 6A). By using the Finite Element Modeling simulation, we concluded that the inner temperature of the tumor may reach as high as 45˚С, which is high enough for hyperthermia therapy (Figure 6B). During the antitumor treatment, the tumor volumes in different groups were recorded, and we conducted the treatment four times for each group. As shown in Figure 6C, tumor growth was remarkably suppressed in the DAPT@h-NPs and h-NPs+AC groups, and especially so in the DAPT@h-NPs+AC group. Conversely, these in the h-NPs, DAPT, and AC groups showed no significant difference from the control group. After the mice were sacrificed, the tumor weights also showed that the combination of chemo- and thermo- therapy could significantly inhibit tumor growth (Figure 6D and S14). The suppression of tumor growth may be partly attributed to the direct tumor cells killing and CSCs elimination by hyperthermia. On the other hand, the controlled release of DAPT in the tumor site during hyperthermia condition also led to a decrease in CSCs. We further evaluated the CSCs ratio in the tumors of different groups with anti-CD44 antibody immunofluorescence. As shown in Figures 6F and G, both the hyperthermia and DAPT@h-NPs treatments decreased the ratio of CSCs in tumor tissues, and the best effect was shown by the combined strategy. These results confirmed that the CSCs could be effectively eliminated by our nanoplatform. CSCs not only are able to initiate tumor formation, but seem to be a cross-link between angiogenesis and metastasis progression.61 We further tested the angiogenesis in the tumors after different treatments. We found that the blood vessels density in the tumor after DAPT@h-NP, h-NP+AC or DAPT@h-NP+AC therapy was dramatically reduced (Fig. S15). The reduced CSCs and angiogenesis are benefit for tumor inhibition. Meanwhile, the different treatments did not affect the growth of mice (Figure 6E) and further histopathological examination also indicated that the major organs functioned properly (Figure S16).This good biocompability of the h-NP makes it a good candidate for further applications in CSCs treatment. CONCLUSIONS CSCs are considered to play important roles in tumor initiation, metastasis, angiogenesis and drug resistance. We have synthesized a novel type of nanoplatform based on Fe3O4@PPr@HA hybrid nanoparticles that has high stability, good biocompability and excellent MR and PA contrast properties. With a high loading


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efficiency of DAPT, the DAPT@h-NPs exhibit effective elimination of CSCs and inhibit tumor growth, especially together with AC magnetic field-mediated hyperthermia. With h-NP-mediated MR and PA dual-mode imaging, the DAPT is visible in vivo and can be seen to target tumors. Overall, these results indicate that the novel magnetic hybrid nanoparticles present a bimodal imaging-guided hyperthermia therapy for elimination of the CSCs and encourage further explorations of this drug delivery system for cancer stem cell elimination.

ASSOCIATED CONTENT Supporting Information Additional figures and tables as described in the text including part of the characterization of the nanoparticles, HPLC analysis of DAPT, cytotoxicity and cellular uptake of h-NPs to MDA-MB-231 and MCF-7 cells and histological examinations of major organs. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION *Corresponding Authors: E-mail address: [email protected]. Tel: +86 10 82545560; fax: +86 10 62656765. Notes † These authors contributed equally. The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology of China (National Basic Research Program 2016YFA0201600), the National Natural Science Foundation of China (21320102003, 21403043), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), the CAS Key Research Program for Frontier Sciences (QYZDJ-SS-SLH022), the CAS Interdisciplinary Innovation Team and the National Science Fund for Distinguished Young Scholars (11425520).

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Figure 1. Synthesis and characterization of Fe3O4@PPr@HA nanoparticle (A) Schematic illustration to show the fabrication process of Fe3O4 @PPr@DAPT@HA hybrid nanoparticle (DAPT@h-NP) (B) TEM image of the synthesized Fe3O4@PPr@HA hybrid nanoparticles (Scale bar = 0.2 μm). Insert contains a higher resolution image (Scale bar = 20 nm); (C) Hydrodynamic size distribution of Fe3O4@PPr@HA hybrid nanoparticles; (D) UV-vis-NIR absorbance spectrum of Fe3O4@PPr@HA hybrid nanoparticles in water (100 μg/mL); (E) Magnetization loops of Fe3O4@PPr@HA hybrid nanoparticles at 300 K (emu per gram of Fe3O4). Insert: photos of Fe3O4@PPr@HA aqueous solutions without (left) and in the

presence of a magnet (right).


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Figure 2. Drug loading and release profiles under different conditions. (A) Influence of the input mass of DAPT on the encapsulation efficiency and loading capacity (based on 1 mg h-NPs); (B) In vitro drug release profiles of DAPT from DAPT@h-NPs in PBS (37˚С) at pH 7.4, pH 5.0 and pH 5.0 in the presence of hyaluronase (0.5 mg/mL); (C) The magnetic thermal profiles of h-NPs under an AC magnetic field. (D) Cumulative release of DAPT from DAPT@h-NPs in PBS at pH 7.4 and pH 5.0 under AC in 1 hour.



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Figure 3. Cell viability and cellular uptake experiments. (A) Live/Dead (green/red) staining of 4T1 cells after treatment by AC field, h-NPs, DAPT, DAPT@h-NPs and DAPT@h-NPs in AC field. (Scale bar = 100 μm). (B) Cell viability of 4T1 cells after treatment by h-NPs for 24 h. (C) Cell apoptosis and necrosis of different treatments including 50 μg/mL h-NPs, 100 μg/mL h-NPs, 50 μg/mL h-NPs + AC, 50 μg/mL DAPT@h-NPs + AC, DAPT@h-NPs + laser. (D) Intracellular ROS level after 50 μg/mL h-NPs and 50 μg/mL h-NPs + AC treatments. (E) Cellular uptake of FITC-labeled h-NPs in MCF-7, MDA-MB-231 and 4T1 cell lines after incubation 3 h were tested by confocal microscope. Fluorescence intensities of each cell were quantified by Volocity software. (F) Intracellular localization of h-NPs (green) and lysosomes (red) in murine 4T1 breast cancer cells. The nuclei were stained with hoechst33342 (blue). (Scale bar = 18 μm).


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Figure 4. Effect of DAPT@h-NP-mediated magnetic hyperthermia on ALDH+ cell subpopulation and mammosphere formation in 4T1 cells. (A) Flow cytometry analysis of ALDH+ cells in 4T1 cells after different treatments. (B) Mammosphere images acquired by a light microscope (Scale bar = 100 μm). (C) Statistical results of the percentage of ALDH+ in 4T1. **p< 0.01 compared to control group. (D) The number of cells was counted after the mammospheres were trypsinized to single cells. ** p< 0.01 compared to control group.



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Figure 5. The h-NPs-mediated photoacoustic and T2-weighted magnetic resonance multimodal imaging in vitro and in vivo. (A) In vivo PA imaging in 4T1 tumor-bearing mice before and after h-NPs injection at different time points. (Scale bar = 3mm). (B) The PA signal observed as a function of the h-NP concentrations (R2 = 0.97). Inset is the PA images of h-NPs with varied concentrations. (0, 1, 3, 5, 8 and 10 μg/mL) (C) Plot of R2 (1/T2) versus Fe concentration of corresponding relaxivity. (R2 = 0.99). Inserts are T2 weighted MR images with various Fe concentrations (0, 0.07, 0.14, 0.28, 0.56 mM) of h-NPs and MR images of 4T1 cells after co-incubation with h-NPs with different Fe concentrations (from left to right: 0, 1, and 5 mM). (D) T2-weighted MRI images of 4T1 tumor-bearing mice after i.v. injection at different time points.


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Figure 6. In vivo chemo- and magnetohyperthermia therapy. (A) Thermal images of 4T1-bearing mice with i.v. injection DAPT@h-NPs with the AC treatment for 15min. (B) Simulation of heating effect of tumor site under AC. (C) Tumor volume growth curves in different groups of mice after various treatments (n = 5). There were four treatments as the black arrows indicate. Groups: (i) PBS injection as control; (ii) h-NPs i.v. injection; (iii) DAPT i.v. injection only; (iv) DAPT@h-NPs i.v. injection; (v) AC only; (vi) h-NPs+ AC; and (vii) DAPT@h-NPs + AC. (D) Average weights of tumors collected from the mice after the mice were sacrificed. ** p

Dual-Mode Imaging-Guided Synergistic Chemo- and

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