An Intelligent and Tumor-Responsive Fe2+ Donor and Fe2+-

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An Intelligent and Tumor-responsive Fe2+ Donor and Fe2+-dependent Drugs Cotransport System Huijuan Zhang, Qianqian Chen, Xiaoge Zhang, Xing Zhu, Jianjiao Chen, Hongling Zhang, Lin Hou, and Zhenzhong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11839 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 26, 2016

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ACS Applied Materials & Interfaces

An Intelligent and Tumor-responsive Fe2+ Donor and Fe2+-dependent Drugs Cotransport System Huijuan Zhang1,a,b,c, Qianqian Chen1,a, Xiaoge Zhanga, Xing Zhua, Jianjiao Chena, Hongling Zhang a, Lin Hou*,a,b,c, Zhenzhong Zhang*,a,b,c a

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China

b

Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province

c

Collaborative Innovation Center of New Drug Research and Safety Evaluation , Henan Province,

Zhengzhou, China 1

Huijuan Zhang and Qianqian Chen contributied equally to this work.

* Corresponding Author: Email address: [email protected] (Zhenzhong Zhang), [email protected] (Lin Hou); Tel.: +86 371 6778 1910. Fax: +86 371 6778 1908. Mailing address: No.100, Kexue Road, Zhengzhou 450001, China

Abstract Fe2+ plays an essential role for artemisinin (ART)-based drugs in anti-cancer therapy. As a result, it’s important to realize these two agents’ cotransport for improving anti-tumor efficacy. We utilized a kind of alternating magnetic field (AMF) and tumor responsive materialmesoporous Fe3O4 (mFe3O4) to encapsulate ART. After that, the outer surface of mFe3O4 was capped with multifunctional hyaluronic acid (HA) which was used not only as smart gatekeeper but also as tumor targeting moiety. In vitro and in vivo studies proved that ART can be encapsulated in HA-mFe3O4 and protected by HA coating which could effectively avoid premature release during in vivo circulation. HA-mFe3O4/ART could be taken up by MCF-7 tumor cells via CD44 receptor-mediated endocytosis and locate at acidic lysosome. Subsequently, “HA gate” could be degraded by acidity and hyaluronidase. Then this system synchronously released Fe2+ and ART at the same site. Fe2+ can non-enzymatically convert ART to ROS for killing cancer cells. Under AMF irradiation, HA-mFe3O4 could not only 1 ACS Paragon Plus Environment


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effectively convert electromagnetic wave into heat for tumor thermal therapy, but also generate high levels of reactive oxygen species (ROS) for tumor dynamic therapy. These results demonstrated that the anti-tumor efficacy of HA-mFe3O4/ART in vivo significantly enhanced 3.7 times compared with free ART. Combining with AMF, it further improved 3.9 times (V/V0 of 0.11), suggesting the successful combined application of HA-mFe3O4/ART and AMF for tumor treatment. It is believed that HA-mFe3O4/ART is a promising system for Fe2+-dependent drugs to improve their therapeutic effect. Keywords: Fe2+ donor, Fe2+-dependent drugs, gate-controlled cotransport system, tumor responsive drug release, alternating magnetic field

1. Introduction Artemisinin (ART) is a sesquiterpene lactone compound extracted from sweet wormwood Artemisia annuaL, which contains the specific endoperoxide bridge. It has been used widely to treat against malaria. In recent years, studies have demonstrated that ART also have a strong killing effect on tumor cells1. Fe2+ within tumor cells can react with the peroxide bridge structure (-O-O-) of ART to produce free radicals or electrophilic compounds2. Such products are strong alkylating agents, which can induce cell death by attacking cell membrane structures, direct oxidation of protein molecules or destruction of DNA structure3. As a class of Fe2+-dependent antitumor agent4, Fe2+ plays an essential role in tumor cell killing for artemisinin-based drugs. So how to make these drugs achieve the greatest degree of anti-tumor effect? Designing Fe2+ and Fe2+-dependent drugs cotransport system will effectively solve this issue. It has been reported that transferrin-mediated (the only endogenous protein capable of transport iron ions) ART delivery system could achieve these two substances cotransport5. Yang et al. prepared transferrin-modified ART analogs and found they had good anti-tumor effect on human hepatoma (HepG2) and lung cancer (A549) cell lines6. We also developed a 2 ACS Paragon Plus Environment

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similar transferrin-mediated ART analogs delivery system (HA-C60-Tf/AS) for tumor treatment7. In these previous studies, we found that although such cotransport systems could improve the anti-tumor effect of ART analogs to some extent, there were also shortcomings such as limited transport capacity of Fe2+, low drug loading efficiency and single action mechanism. Therefore, the general idea of this study is to design a simple and effective drug delivery system to solve the above problems. Compared with the solid Fe3O4 nanoparticles, mesoporous Fe3O4 nanoparticles (mFe3O4) have a larger specific surface area, smaller density, greater interior space and better surface properties8. mFe3O4 can be used as carrier material for bio-catalysis, drug delivery or adsorptive separation. In addition, mFe3O4 also has the alternating magnetic field (AMF)-heat transfer characteristics9. Furthermore, in this study, our results showed that mFe3O4 combining with AMF could induce heat and ROS generation in tumor cells synchronously. This feature can be extensively explored as magnetic fluid thermal treatment and ROS therapy for cancer. However, the magnetic dipole interaction, van der Waals forces and quantum size effect tend to cause mFe3O4 particles in the solution quick coagulation10. Polymer modification is an effective way to improve their stability. Due to its hydrophilic, biocompatible, biodegradable, non-immunogenic and tumor targeting (receptors: CD44) properties, hyaluronic acid (HA) has been used widely in the novel drug and gene delivery systems11,12. HA-modified nanoparticles can not only increase their hydrophilicity and stability, but also extend the blood circulation time and improve tumor targeting ability13. Moreover, organic polymer, such as HA, can coat onto the surface of mesoporous materials by a pH sensitive bond to construct pH-responsive intelligent controlled-release system. This "nanogate" can protect drug from leakage during systemic circulation. When drug delivery systems reach the tumor sites, pHresponsive bonds break and mesoporous turn on to release the entrapped drug molecules14.

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Thus the purpose of this study is to build an intelligent Fe2+ and Fe2+-dependent drugs cotransport system, as shown in Fig.1. In this system, mFe3O4 was employed as the matrix and HA was chosen as a polymer modified material. ART was the model drug. This system had the following characteristics. (1) It can realize Fe2+ donor and Fe2+-dependent drug synchronous cotransport; (2) It can achieve an effective drug loading and door remote control release at the designated tumor site; (3) It can reach positioning multi-mechanism treatment for tumor, with unique advantages like deeper penetration depth, remote-control and no damage. 2. Experimental Section 2.1 Materials Sodium dodecylbenzene sulfonate (SDBS, ≥ 98%), glycol (99.9%), ferric chloride (FeCl3•6H2O, >99.0%), sodium acetate (NaAC, >99%), sodium hydroxide(NaOH, >99%) absolute ethanol (C2H5OH, >99.7%), p-hydrazinobenzoic acid (HBA, 99.8% ), cysteamine (CYS, 99.8%), dimethyl sulfoxide (DMSO, 99%) N-(3-dimethylamino propyl -N′ethylcarbodiimide) hydrochloride (EDC•HCl, 98.5% ), N-Hydroxysuccinimide (NHS, 99%), glutathione (GSH, 99% ), hyaluronidase (HAase, 99%) and sulforhodamine B (SRB, 99% ) were used as purchased from Sigma-Aldrich (St Louis, MO, USA). Artemisinin (ART, >99.0%) was purchased from Create-Life Biotech Limited Company (Zhengzhou, Henan, China). Sodium hyaluronate (HA, MW≈12000, >98%) was bought from Bloomage Freda Biopharm Co. Ltd. (Jinan, Shandong, China). Penicillin, streptomycin and fetal bovine serum were bought from Life Technologies (Carlsbad, CA, USA). All reagents were of analytical purity and used without further purification. 2.2 Preparation of Mesoporous Fe3O4 Nanoparticles (mFe3O4) The mFe3O4 nanoparticles were prepared by the hydrothermal method. The 0.16 g of SDBS and 16 mL of glycol were mixed in a beaker and then thoroughly stirred at room temperature for 60 min to dissolve. After that, 0.54 g of FeCl3•6H2O and 0.93 g of NaAC 4 ACS Paragon Plus Environment

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were added into the above solution and then transferred to a polytetrafluoroethylene reaction kettle, following a hydrothermal reaction for 12h at 180 °C. The black mFe3O4 nanoparticles were collected by the magnet separation and then washed using distilled water and absolute ethanol for several times. Those products were placed in vacuum drying oven and then dried at 60 °C for 12h to further characterization. 2.3 Preparation of HA Modified mFe3O4 (HA-mFe3O4) ① Synthesis of mFe3O4-HBA. 0.20g mFe3O4 nanoparticles were dispersed into 50mL PBS solution (0.01M，pH 7.4) to obtain a homogeneous aqueous dispersion (4mg/ml). Add appropriate amount of CYS and stir for 24h, followed by dialysis (MW=3,500) to remove unreacted CYS. Then mFe3O4-CYS were collected according to freeze-drying. After that, mFe3O4-CYS (50mg), HBA (50mg), EDC•HCl (80mg) and NHS (35mg) were added into PBS solution (50mL) and completely dissolved by ultrasound. The condensation reaction was carried out according to our previous method15. The intermediate product (mFe3O4-HBA) was obtained and stored in the dark.

Synthesis of HA-mFe3O4. HA was chemically grafted on

mFe3O4 by hydrazone bond. mFe3O4-HBA (50mg) and HA (100mg, MW=12,000) were both added into DMSO (50mL) and then thoroughly dissolved by ultrasound. After reaction for 24h at room temperature in the dark, the reaction solution was dialyzed to remove unreacted HA and DMSO. HA-mFe3O4 were freeze-dried and stored in the dark at 4 °C. 2.4 Characterization Morphological feature of nanoparticles were monitored by transmission electron microscopy (TEM). Surface analysis of HA-mFe3O4 was observed by scanning electron microscope (SEM). Specific surface areas were got according to Brunauer-Emmet-Teller (BET) and Barrett-Joyner-Halenda (BJH). X-ray photoelectron spectrum (XPS) was performed to identify the elements and chemical status. The spectral features were monitored with UV−vis and FT-IR spectrometers. Thermogravimetric analysis (TGA) was used to measure the weight loss of nanoparticles. Magnetic properties were measured by 5 ACS Paragon Plus Environment


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superconducting quantum interference device vibrating sample magnetometer (SQUID). The particle size and zeta potential were determined by a laser diffraction-based particle size analyzer (DLS). 2.5 ART Loading and Drug Content Determination 2mg/mL HA-mFe3O4 dispersion were prepared by ultrasonic techniques. Add different amount of ART which dissolved in ethanol and stir for 24h at room temperature. After that, the suspension was dialyzed for 48h (MW=3,500) in dialysate (distilled water: ethanol = 9:1) to remove those unloaded drugs. To measure loading efficacy, dilute HA-mFe3O4/ART nanosuspension with 10 times the volume of ethanol and sonicate to ensure loading drug dissociation completely. Centrifuge to separate HA-mFe3O4 and ART. Thereafter, hydrolyze ART with 4 fold volumes of NaOH (0.2%) for 30 min at 50±1°C and measure the absorbance at 292nm by the standard method. The ART loading efficiency (LE) and entrapment efficiency (EE) were calculated using formula (1) and (2), respectively: Loading efficiency (%) =

WLoaded ART × 100% WLoaded ART + WHA-mFe O

(1)

WLoaded ART × 100% WTotal ART

(2)

3

Entrapment efficiency (%) =

4

2.6 ART release study In vitro ①pH-sensitive release. The pH-sensitive drug release behaviors of HA-mFe3O4/ART were investigated in PBS containing 10% ethanol (pH=7.4, 5.5 and 4.0) for 12h. 4mL HAmFe3O4/ART dispersion (1.0mg/mL) was put into dialysis bags (MW=3,500) and subsequently immersed into 80mL release medium. Drug release investigation was carried out in an incubator shaker (37°C, 100 rpm). At pre-determined time intervals, 1mL dialysate was withdrawn and replaced by fresh buffers. Then dialysate was hydrolyzed according to 2.5 Section for ART quantitative analysis. ②hyaluronidase (HAase)-responsive drug release. The HAase-responsive ART release behaviors of HA-mFe3O4/ART were investigated in PBS


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containing 10% ethanol (pH=7.4, HAase: 0, 0.1, 10 and 100µg/ml) for 72h. The operation and calculation methods were as above (①) mentioned. 2.7 GSH and pH Sensitive Fe2+ Releasing from HA-mFe3O4 ①pH-sensitive Fe2+ release. HA-mFe3O4 dispersions (100µg/ml) were adjusted to pH 7.4, 5.5 and 4.0, respectively.. Shake these three samples on a horizontal shaker (37°C, 100 rpm). At 4, 6 and 12h, 50µl liquid was drawn and transferred to a 96-well plate. Then Iron Assay Kit (Sigma-Aldrich, MO, USA) was used to determine Fe2+ concentration. ②GSH-responsive Fe2+ release. Disperse HA-mFe3O4 in water (pH=5.5) with different GSH concentrations (,0, 2 and 5mM) to 100µg/ml. Shake samples on a horizontal shaker (37°C, 100 rpm). At predetermined time intervals, 50µl liquid was drawn and Iron Assay Kit was used to determine Fe2+ concentration. 2.8 Behavior of the HA-mFe3O4 upon AMF Trigger Magnetocaloric effect. Magnetocaloric effect of HA-mFe3O4 was performed in a plastic micro dish (d=3.0cm) using a

heating coil (5cm, 4 turns). HA-mFe3O4 water

dispersions (0, 0.1 and 0.25 mg/mL) were placed at the centre of the coil. Samples were heated for 20 min (488kHz, 40A). A Thermal imaging camera was used to record temperature changes. ②ROS generation. Intracellular ROS was measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, MCF-7 cells were seeded in 6 well plates with a density of 5×104 cells/well. After incubation for 24h, cells were treated with HA-mFe3O4 (50µg/mL) for a further 6h. Then medium was discarded and 10µM DCFH-DA was loaded. For AMF groups, the plates were then under AMF irradiation for 20min (488kHz, 40A). The images were recored by the Fluorescence Microscope (Zeiss LSM 510). 2.9 Cell uptake and Lysosomal accumulation Iron-specific Prussian blue staining was performed to evaluate cellular uptake of HAmFe3O4. MCF-7 cells were seeded in 6-well plates. After incubation for 24 h, cells were 7 ACS Paragon Plus Environment


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treated with mFe3O4 and HA-mFe3O4 at the concentration of 50µg/ml. At pre-determined time, Cells were washed and fixed with paraformaldehyde (4%, 20min), Thereafter cells were stained with prussian blue solution for 30min. After washed with water, cells were counterstained with neutral red solution for 5min. Furthermore, in order to explore the intracellular localization of nanoparticles, LysoTracker Red DND-99 was used to mark lysosome simultaneously. Fluorescence microscope was used to record results. 2.10 ART and Fe2+ Synchronous Release from HA-mFe3O4/ART in MCF-7 Cells Cells were collected after treated with ART and HA-mFe3O4/ART (ART: 50µg/ml) for 0.5, 1, 3, 6, 12 and 24h, respectively. Then cells were broken using a probe ultrasonic (400W, 20times). Add 3.5ml ethyl ether to extract ART. After centrifugation (8,000rpm, 5min), organic layer was transferred to a new tube and dried under nitrogen. Thereafter, samples were hydrolyzed with NaOH (0.2%) to measure ART content. What’s more, Fe2+ release amount in MCF-7 cells was determined at 6, 12 and 24 h. After incubated with HAmFe3O4/ART for predetermined time, cells were collected and broken using an ultrasonic cell disruption system (400 W, 20 times). After centrifugation at 8,000rpm for 5 min, the supernatant was taken for Fe2+ measurement. 2.11 Intracellular ROS levels MCF-7 cells were seeded in confocal dishes at a density of 5×104 cells/dish. Following incubation with ART and HA-mFe3O4/ART at the same ART concentrations (30µg/ml) for 0.5, 3 and 6h, DCFH-DA method was carried out to determine intracellular ROS level. 2.12 Cytotoxicity assay MCF-7 cells were plated in 96-well plates and then incubated for 24h. After that, cells were treated with various formulations of free ART, HA-mFe3O4 and HA-mFe3O4/ART at the same HA-mFe3O4 and ART concentrations (HA-mFe3O4: 18.6µg/ml and ART: 30µg/ml). For AMF groups, the plates were under AMF irradiation for 20min (488 kHz, 40A), following by a further incubation for 24h. The groups without AMF irradiation acted as the control groups. 8 ACS Paragon Plus Environment

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At last SRB method was carried out to analyze cell viability. Furthermore, after MCF-7 cells were treated under the aforementioned conditions, cells were stained with calcein acetoxymethyl ester (calcein AM, 1 µM, Molecular Probes, USA) and propidium iodide (PI) to distinguish the live cells from dead cells through a fluorescence microscope (Zeiss LSM 510). 2.13 DNA fragmentation detection MCF-7 cells were plated in 6-well plates and incubated with free ART, HA-mFe3O4 and HA-mFe3O4/ART at the same HA-mFe3O4 and ART concentrations (HA-mFe3O4: 18.6µg/ml and ART: 30µg/ml) for 24h. For AMF groups, the plates were exposed to AMF irradiation for 20min (488 kHz, 40A). After that, single cell gel electrophoresis (comet assay) was carried out according to our previous method to observe DNA damage16. 2.14 In Vivo Antitumor Studies All animal experiments were performed under a protocol approved by Henan laboratory animal center. The antitumor efficacy was evaluated using female mice (BALB/c), which was implanted 2×106 S180 cancer cells into the right shoulder (4weeks old; 18−22g). After tumor volumes were approximately 50-100mm3, tumor-bearing mice were randomly divided into nine groups (n=6): ①Control group (0.15 M NaCl, N.S.); ②Control group combined with AMF

(N.S.+AMF);

③HA-mFe3O4;

④HA-mFe3O4+AMF;

⑤ART;

⑥mFe3O4/ART;

⑦mFe3O4/ART+AMF; ⑧HA-mFe3O4/ART; ⑨HA-mFe3O4/ART+AMF. All formulations were administrated by intravenous injection via tail vein every two days. For all above groups, animals were treated with same doses of ART (25mg/kg) and mFe3O4 (15.6mg/kg). For AMF combining groups, AMF was placed on the top of tumor for 20min (488 kHz, 40A). On the second day, these groups experienced additional AMF irradiation for another 10min. Tumors were measured with calipers every two days. Tumor volume (V) was calculated according to the formula: V= [length× (width) 2]/2. At the end of experiment, tumor tissues of each group 9 ACS Paragon Plus Environment


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were taken out and soaked in 10% formalin solution, embedded with paraffin for hematoxylin and eosin (H&E) staining. Morphological changes were observed under microscope (Eclipse 80i, Nikon, Japan). 2.15 Toxicity Evaluation in vivo The viability, status and any visible toxicity changes at the injection sites were observed during pharmacodynamic experiments. The body weight of each mouse was monitored every other day and recorded as a function of time. At the end of experiment, heart, liver, spleen, lung, kidney, brain tissues of each group were taken out for H&E staining. 2.16 Biodistribution Studies ① NIR imaging. Noninvasive NIR imaging was used to visually monitor biodistribution of HA-mFe3O4. HA-mFe3O4 was labeled with IR783. The model mice were intravenously injected with IR783 solution and IR783-loaded HA-mFe3O4, with the same dosage of IR783 (2mg/kg). After 2, 8 and 24h, an in vivo imaging system FX PRO (Kodak, USA) was used to record the results. Futhermore, after injection for 8h, heart, liver, spleen, lung, kidney, brain and tumor tissues of each group were taken out and recorded fluorescence intensity. Biodistribution studies with HPLC. Tumor-bearing mice were treated by tail vein injection with ART, mFe3O4/ART and HA-mFe3O4/ART at a matched dose of 50 mg/kg. At times points, animals were killed and tissues (heart, liver, spleen, lung, kidney, brain and tumor) were homogenized in saline with W/V 1: 3. Samples were extracted by diethyl ether. After dried, the residue was redissolved with methyl alcohol. Finally, ART was determined by HPLC at 210nm, with mobile phase of acetonitrile/water: 52/48. 3. Results and Discussion 3.1 Synthesis and Characterization of mFe3O4 In this study, mFe3O4 nanoparticles were synthesized by hydrothermal method. TEM images indicated that synthesized mFe3O4 (about 250nm) exhibited spherical and porous structure (Fig.2A1-A3). Nitrogen adsorption–desorption isotherm of mFe3O4 was shown in 10 ACS Paragon Plus Environment

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Fig.2D. It was typical type IV isotherm with a H2 hysteresis loop according to the IUPAC classification. This was in line with the characteristics of mesoporous materials17,18. The surface area was calculated to be 64.2m2/g. There were two types of pores diameter. They were measured to be 4.3 and 49.7nm, allowing small drug molecules (such as ART) to diffuse into the hollow interior of mFe3O4. This was consistent with Fig.2A3. SEM images of mFe3O4 were given in Fig.2B. As seen in Fig.2B1, mFe3O4 was made up of many small Fe3O4 particles. Fig.2B2 demonstrated mFe3O4 nanoparticles had a uniform particle size distribution. The EDS spectra confirmed that obtained mesoporous material was composed of Fe and O elements (Fig.2C). There were no other element peaks corresponding to impurities observed, which implied the purity of mFe3O4. Besides, surface analysis of mFe3O4 flower was carried out by XPS. Fig.2E1 displayed the binding energies for O1s (564.1eV) and Fe2p of the samples19. Fig.2E2 showed two intense bands (724.4eV and 711.8eV). Those were assigned to Fe2p1/2 and Fe2p3/2, respectively. These peaks were all typical characteristics of Fe3O4 20. In this study, we intended to construct a kind of ART and Fe2+ ions cotransport system. All above characterizations indicated the successful synthesis of mFe3O4. Its excellent mesoporous structure and high surface to volume ratio can achieve high drug loading capacity. 3.2 Preparation and Characterization of HA-mFe3O4 Due to high surface energy and strong magnetic dipole–dipole attraction between particles, magnetic nanoparticles are prone to aggregates21. This limited their medical and biological applications. To overcome this obstacle, it is necessary to modify the surface of these magnetic nanoparticles. In this study, HA-mFe3O4, which had high water dispersibility and good biocompatibility, was synthesized. Firstly, CYS modified the surface of mFe3O4 by metal coordination-chelation interaction. Then HBA molecules with carboxyl groups were connected onto mFe3O4-CYS via amine bonds. Finally, HA with carbonyl group was grafted onto mFe3O4-HBA via pH-sensitive hydrazone bonds.



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Fig.3A showed the FT-IR spectra of HA-mFe3O4, mFe3O4 and HA. HA grafting onto mFe3O4 was confirmed by amide III (~1383.9 cm-1) vibrations, N–N (~991.2 cm-1) vibrations and C=N (1627.7 cm-1) vibrations. These peaks were consistent with hydrazone bond between mFe3O4-HBA and HA15. Fig.3B showed that mFe3O4 (c) had a wide absorption bands at 200-800nm, while synthetic mFe3O4-HBA (b) showed an enhanced absorption at 500-700nm. HA (d) only demonstrated a peak in the ultraviolet terminal. However, after HA grafting onto mFe3O4, there was a red-shift phenomenon (a) appearing. The relative amount of HA grafting onto mFe3O4 was tested by TGA. As Fig.3C shown, TGA curve of HAmFe3O4 showed two-stage weight loss. The first-stage weight loss occurred below 200°C, which was due to evaporation of the residual water in the sample. When temperature was higher than 200°C, main weight loss was due to the decomposition of HA. The grafting amount of HA was estimated about 7.6%. Fig.3D presented the magnetic properties of different nanoparticles. As indicated in Fig.3D, mFe3O4 and HA-mFe3O4 both showed typical superparamagnetic behaviors. The saturation magnetization of mFe3O4 and HA-mFe3O4 were 73.3 and 47.5emug-1, respectively. This difference was mostly attributed to the existence of HA. The obtained nanocarriers were well-dispersed in water (Fig.3D (b)) with an average size (246nm, Fig.3E) and negative charge (-22.4mV, Fig.3F). The zeta potential of HA-mFe3O4 was more negative than mFe3O4, which further testified HA layer coating (Fig.3F). 3.3 The characteristics of HA-mFe3O4/ART ART was physically encapsulated into HA-mFe3O4 by dialysis to evaluate drug loading capacity. Table.1 demonstrated drug loading efficiency increased from 32.91% to 55.87% along with increasing ART feeding amount, while entrapment efficiency demonstrated first increased and then decreased trend. Finally, a weight ratio of 2:1 (ART: HA-mFe3O4) was chosen for further studies with excellent drug-loading capacities (52.77%) and high entrapment efficiency (56.00%). A comparison between TGA curves of HA-mFe3O4 and HAmFe3O4/ART had been performed to validate this loading efficiency (~52%) (Fig.S1). 12 ACS Paragon Plus Environment

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mFe3O4 had a large internal space, wherein the hollow and pore structure can both load ART. So this carrier owned a high drug loading capacity. Acidity and high GSH concentration are distinct and varied characteristics of solid tumors compared to normal tissue22,23. What’s more, hyaluronidase (HAase) is rich in tumor microenvironment and endocytic vesicles including endosomes and lysosomes

24,25

. These

parameters are the most significant factors underlying the metabolism and physiology of solid tumors26. At present, a new targeting strategy is to develop “activable” or “activated” nanocarriers27. In this system, HA was adopted to coat the surface of mFe3O4 by hydrazone bond to prevent drug leakage. When HA-mFe3O4/ART reached tumor site, HA shell degraded under acidic and HAase-rich environment to release ART. In addition, we believe that after removal of HA, the naked and porous mFe3O4 can be as a tumor (low pH and high GSH content) responsive Fe2+ reservoir. To investigate pH-sensitive release behavior of HA-mFe3O4/ART, three acidity solutions (pH=7.4, 5.5 and 4.0) were chosen as external stimuli. The released ART was determined by UV-vis absorption spectroscopy at 292nm. Real-time release profile was seen in Fig.4A. The leakage amount of ART from HA-mFe3O4/ART still remained at a low level (11.0%) when incubated for 12h; whereas ART leakage increased to over 27.0% and 34.4% when the system was exposed to pH 5.5 and 4.0 solutions for 12h, respectively. This phenomenon could be interpreted that acid cleaved hydrazone bonds, leading to HA removal from the surfaces of HA-mFe3O4. Thus, loaded ART released quickly from the system. Next, HAase was employed as another trigger. As seen in Fig.4B, without HAase, HA-mFe3O4/ART revealed just about 46% drug release within 72h. In contrast, HAase accelerated ART release. When HAase concentration was 10µg/ml, obvious increased ART release (81%) was observed. All these results proved that ART can be encapsulated in HA-mFe3O4 and protected by HA coating effectively to avoid premature release during in vivo circulation. After HA-



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mFe3O4/ART reached tumor site (acidity and HAase-rich), “HA gate” was degraded and drug release accelerated. In our study, we found mFe3O4 can act as a tumor sensitive Fe2+ reservoir, owing to pH and GSH responsiveness. As Fig.4C shown, external acid stimuli could induce Fe2+ release from HA-mFe3O4. Fe2+ release amount was acidity-dependent and time-dependent. For pH4.0 group, Fe2+ release amount reached 13.65µg/ml within 12 hours. However, only very little Fe2+ (0.81µg/ml) released in neutral solution (pH 7.4). Furthermore, GSH was employed as another trigger. As Fig.4D shown, GSH addition could accelerate Fe2+ release， even reach 24.9µg/ml within the same time interval. This characteristic may be related with the special mesoporous structure and electron valence state of mFe3O4. On one hand, because of mesoporous, H+ and GSH could enter into the interior of nanocarrier, which could increase contact area. The loose structure would be easily decomposed and release Fe2+ ions slowly. On the other hand, as Fig.2 (E2) shown, there were two electron valence states in mFe3O4, Fe2+ and Fe3+. H+ could react with mFe3O4 to generate Fe2+ ions according to metathesis reaction. GSH could also react with mFe3O4 by means of redox reactions, in which Fe3+ was reduced to Fe2+ ions 28. Based on above results, HA-mFe3O4/ART system owned tumor responsive ART and Fe2+ release behavior, implying that HA-mFe3O4/ART could be utilized to locally release Fe2+ and Fe2+-dependment drug synchronously. In brief, this tumor-responsive system could be stable in blood circulation (pH 7.4, without GSH and HAase), while release ART and Fe2+ in the specific tumoral environment (acidic, with GSH and HAase). This characteristic made it maximize anti-tumor effect and minimize side effect for cancer therapy. 3.4 Heat and ROS Generation of HA-mFe3O4 upon AMF Trigger Magnetic nanoparticles can realize hyperthermia and tissues ablation under a highfrequency magnetic field, reported as magnetic fluid hyperthermia (MFH)

29

. Fe3O4

nanoparticles coated with organic biopolymers can improve their stability and heat induction 14 ACS Paragon Plus Environment

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ability with lowered cytotoxicity30. In this study, we explored the behavior of HA-mFe3O4 under AMF irradiation. Fig.5B showed concentration and time dependent, gradual increase in temperature under AMF irradiation. From Fig.5A, we could observe that the temperature of HA-mFe3O4 dispersion (0.25mg/ml) with AMF (488 kHz, 40A) could increase 12.6°C in 20min, while the control group just showed a very slight temperature increase. This property made HA-mFe3O4 high potential to be used for cancer hyperthermia therapy. The surfaces of iron oxide nanoparticles are capable of catalytically generating ROS through Fenton and Haber-Weiss reactions. In both cases, iron oxide nanoparticles act as a homogeneous and heterogeneous catalyst for H2O2 degradation into free radicals31. As we know, there is much H2O2 in tumor. Decomposition of H2O2, which can be catalyzed by Fe3O4, generates ROS such as hydroxyl radical (OH•)

32

. As Fig.5C and D shown, HA-

mFe3O4 nanoparticles induced intracellular ROS production (10.11%) at 50µg/ml. Under AMF exposure, ROS amount increased to 48.72%, while control group did not show significant changes (1.32%). This result demonstrated that HA-mFe3O4 combining with AMF could induce tumor intracellular ROS production. These ROS could oxidize various organic molecules including membrane lipids, DNA and proteins, leading to tumor cell damage30. The possible explanation is that ROS generation corresponds with Fenton reaction. Fenton reaction has been proved to be temperature dependent with an increased activity31,33. When exposed to AMF, HA-mFe3O4 absorbed the energy from magnetic field and converted into heat, which had been testified in Fig.5A. This could accelerate Fenton reaction to increase ROS amount. As mentioned above, HA-mFe3O4 combining with AMF could induce heat and ROS synchronously. This feature can be extensively explored as magnetic fluid thermal treatment and ROS therapy for cancer. 3.5 Locate in lysosomes and release ART and Fe2+ synchronously Intracellular uptake of HA-mFe3O4 was evaluated using MCF-7 cells by iron-specific Prussian blue staining. Fig.6A showed HA-modified mFe3O4 nanoparticles accumulated more 15 ACS Paragon Plus Environment


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extensively in the cells. In comparison, there were only a few internalized blue particles in unmodified mFe3O4 group. These results demonstrated the high specificity and affinity of HA-mFe3O4 to MCF-7 cells. Because ART and Fe2+ release were both related with pH, the intracellular localization of HA-mFe3O4 was important. We stained lysosome using LysoTracker Red DND-99 to elucidate the intracellular localization of this nanocarrier. The result was shown in Fig.6B. After incubation with HA-mFe3O4 for 6h, we obviously observed its specific accumulation in lysosomes. Furthermore, these nanoparticles showed an evident agglomeration in lysosomes. Functionalization of mFe3O4 with HA had been confirmed by zeta potential measurement (Fig.3F). Table.2 showed the zeta potential change of HA- mFe3O4 suspension (50µg/ml) at different pH values. HA-mFe3O4 showed a high negative potential (-20.6mV) at pH 7.4,(8h), due to the presence of negatively charged HA grafting onto mFe3O4. This modification gave the nanocarriers good stability in aqueous suspension. The particle size was 252.8 nm with a PDI of 0.18 (pH 7.4, 8h). However, under acidic condition, zeta potential of HA-mFe3O4 decreased to -10.64 (pH 5.5, 8h) and -7.35mV (pH 4.0, 8h), respectively. The particle size also changed to 337.9nm with a PDI of 0.32 (pH 5.5, 8h) and 387.2nm with a PDI of 0.43 (pH 4.0, 8h), respectively. It demonstrated that zeta potential of HA-mFe3O4 tended to reduce as pH decreased. This resulted in HA-mFe3O4 aggregation to form larger particles. HA was grafted onto mFe3O4 by pH-sensitive hydrazone bond. After HA-mFe3O4 entered into lysosome, hydrazone bond ruptured and then HA was removed. Zeta potential of nanoparticles decreased. mFe3O4 without HA protection would attract each other and result in clusters. This may be one possible explanation for HA-mFe3O4 agglomeration in intracellular acidic compartments. After “ HA gate” removal, ART and Fe2+ would release synchronously. Next, we cultured MCF-7 cells with ART and HA-mFe3O4/ART (ART: 50µg/ml, HA-mFe3O4: 31µg/ml) for 0.5, 1, 3, 6, 12 and 24h, respectively. Then we determined ART and Fe2+ amount 16 ACS Paragon Plus Environment

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in MCF-7 cells. As Fig.6C shown, after entering into cancer cells, the drug concentration in ART group decreased sharply while HA-mFe3O4/ART group demonstrated a slow and gentle downward trend. ART concentration in HA-mFe3O4/ART group was higher than that in ART control group after 0.5h. This was due to that the metabolism of ART in MCF-7 cells was quick so that most ART will soon be metabolized once it entered cells. However, because of slow and sustained drug-release characteristics of ART from HA-mFe3O4, HA-mFe3O4/ART system would increase intracellular accumulation and retention of ART in cancer cells. Furthermore, Fe2+ amount was determined after incubation with HA-mFe3O4/ART for 6, 12 and 24h. As Fig.6D shown, there was a little Fe2+ (12.5nmol/105 cells) in blank cancer cells. After co-cultured with HA-mFe3O4/ART, Fe2+ increased to 81.4nmol/105 cells at 24h. Fe2+ concentration was positively correlated with the incubating time. Thus, HA-mFe3O4/ART system could release ART and Fe2+ synchronously in response to the acidic stimuli of lysosomes. As we know, Fe2+ ions play an essential role for ART in killing tumors. ART contains specific endoperoxide bridge which tends to react with Fe2+ to generate ROS1. Those ROS species can alkylate vital cellular components such as haem, glutathione, DNA, proteins or membranes to kill cancer cells7. So we determined ROS in MCF-7 cells induced by ART and HA-mFe3O4/ART at 1, 3 and 6h. As Fig.7 shown, in ART group, the fluorescence intensity was strongest at 0.5h. Then green fluorescence was getting weak. This was due to rapid drug metabolism. While in HA-mFe3O4/ART group, a significant increased ROS intensity was observed. As demonstrated in Fig.6C and D, ART and Fe2+ could slowly and sustained release from HA-mFe3O4/ART. Then these two substances reacted to produce ROS. So there was a lasting and higher ROS level in acid-loving cancer cells. 3.6 Anti-tumor effect in vitro Firstly, the cytotoxicity of nanocarriers was determined by SRB method. As shown in Fig.8A, this drug vehicle had low cytotoxicity against cancer cells. Next, we investigated the 17 ACS Paragon Plus Environment


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cytotoxicity of ART and HA-mFe3O4/ART on MCF-7 cells. A time-dependent cytotoxicity was observed. As Fig.8A shown, cell viability in ART group displayed a slight decrease over time. While in HA-mFe3O4/ART group, it showed a sustained and dramatically decreases from 24 to 96h. This enhanced cytotoxicity was mainly due to ART and Fe2+ synchronous release from HA-mFe3O4/ART, leading to more generation of local ROS within MCF-7 cells. It also further proved that released Fe2+ was vital for ART to exert its anticancer ability. Wang et al. developed novel Fe3O4@C@MIL-100(Fe) (FCM) nanoparticles for synchronously delivery of DHA and Fe (Ⅲ) for cancer therapy. Results suggested that the intracellular Fe (Ⅲ) will be reduced further to ferrous ion and interact with DHA to enhance its cytotoxicity34. As testified in Fig.5A, HA-mFe3O4 combining with AMF could induce heat and ROS generation synchronously. This feature can be extensively explored as magnetic fluid thermal treatment and ROS therapy for cancer. Wang et al. have proved combing chemotherapy with thermal therapy of ART-loaded nanoparticles (d-MOFs@ART) can enhance cancer treatment efficacy of ART significantly35. In order to verify this property, an AMF (488kHz, 40A) was adopted. Result was shown in Fig.8B. For ART and ART+AMF groups, there was no significant difference in cell survival. However, for HA-mFe3O4 and HA-mFe3O4/ART, in comparison to those groups without AMF, groups combining with AMF presented a greatly enhanced cytotoxicity. The cell viability of HA-mFe3O4/ART decreased from 62.5% to 42.7% after AMF irradiation, indicating that AMF could be used as a remote-control approach to improve anti-tumor effect of HA-mFe3O4/ART. Calcein AM/PI staining was conducted to classify the live and dead cells. As shown in Fig.8C, the untreated blank cells showed green nuclei and uniform chromatin, which imply that they were live and healthy cells. In ART group, a few cells showed orange or red color, suggested they were suffering from apoptosis 36. In comparison to ART, there were increased cells stained by red color. When exposed to AMF, most cells (red color) showed chromatin 18 ACS Paragon Plus Environment

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condensation with shrunken and irregular shape. Finally, cancer cells were lysed and stained with ethidium bromide (EB, 2%) to identify DNA damage. Images were analyzed by CASP software. Tail DNA content represented DNA damage degree. As shown in Fig.8D and E, there was a little tail DNA (15.7±2.7%) in ART treated cells. While HA-mFe3O4/ART caused 34.2±4.9% DNA damage, about double of ART group. Moreover, with AMF irradiation, HAmFe3O4/ART induced cell damage up to 57.8±5.7%. These results were well in agreement with cell viability assay as mentioned above. 3.7 In Vivo Anti-tumor Studies To investigate anti-tumor efficacy of HA-mFe3O4/ART in vivo, comparative efficacy study was conducted. Fig.9C and D showed changes of relative tumor volume (V/V0) as a function of time. As Fig.9C shown, tumor size of mice steadily increased over time in N.S. or HA-mFe3O4 groups. However, with AMF irradiation, tumor volume significantly reduced with V/V0 of 2.16±0.56 (HA-mFe3O4+AMF). This result indicated that HA-mFe3O4 combining with AMF had an effective antitumor effect. This effect could also be proved by H&E-staining. As seen in Fig.9E, tumor cells in N.S. and N.S.+AMF groups were in good condition of rapid proliferation and cell-to-cell closely spaced with a big cell size. In HAmFe3O4 group, there was slight nuclei pyknotic phenomenon. But it clearly observed in HAmFe3O4+AMF group that more tumor cells began to shrink and became smaller with clear gap emerging among cells, indicating HA-mFe3O4 was toxic to tumor cells under AMF irradiation. The state of cells had begun to change badly. Next, we investigated anti-tumor efficacy of various ART formulations. As Fig.9D shown, ART, mFe3O4/ART and HA-mFe3O4/ART resulted in V/V0 of 1.61%, 0.77% and 0.43%, respectively. The data of ART and mFe3O4/ART groups suggested that co-delivery of Fe2+ and ART could enhance anti-tumor effect significantly in vivo. The data of mFe3O4/ART and HA- mFe3O4/ART groups suggested that as tumor targeting molecule HA could make more drug delivery system reach tumor site. When HA-mFe3O4/ART reached targeted site, the tumor-sensitive carrier can generate and 19 ACS Paragon Plus Environment


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release Fe2+. Then ART and Fe2+ could react in the same loci for ROS generation. So mice treated with HA-mFe3O4/ART showed better tumor suppression effect than ART group. Moreover, HA-mFe3O4/ART group showed V/V0 of 0.43%, while HA-mFe3O4/ART combining AMF showed V/V0 of 0.11. It demonstrated successfully combined application of HA-mFe3O4/ART and AMF for tumor treatment in vivo. This therapeutic efficacy was also evaluated by H&E staining (Fig.9E). In these groups, nucleus atrophy, necrosis and fragmentation were seen varying degrees. Symptoms including obvious necrosis, karyotheca dissolving, and nucleolus disappearing was the most typical in HA-mFe3O4/ART group. For HA-mFe3O4/ART+AMF group, there was almost no tumor cell existing. These results indicated that the co-delivery system of Fe2+ and ART combining with AMF could get the best anti-tumor effect in vivo. In tumor site, HA-mFe3O4 could generate ROS under AMF irradiation. After HA-mFe3O4/ART reached tumor, AMF was used to locally and directionally irradiate the targeted site. Then ROS induced by HA-mFe3O4 and ART could play a role at the same site. Comparing with other physical stimulation, such as laser, AMF owned unique advantages like deeper penetration depth, remote-control non-invasive and no damage. 3.8 Safety Studies in vivo As reported, a good drug delivery system should have better therapeutic effect and less toxic or side effects. So next, safety studies of HA-mFe3O4/ART were carried out. Allowing for high toxicity usually leads to weight loss, the safety profiles of different ART formulations were evaluated by measuring the changes in body weight over time, as shown in Fig.9A and B. There was no obvious change in the body weight for all formulations, implying that HAmFe3O4/ART would not cause significant systemic toxicity. Then organ toxicity was demonstrated by H&E staining. As shown in Fig.10, there were no obvious pathological changes in any impartment organs. 3.9 Biodistribution Study


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Visualized distribution of HA-mFe3O4 was realized by NIR optical imaging technique. Fig.11A showed real-time images of IR783 and IR783-loaded HA-mFe3O4 in tumor-bearing mice at 2, 8 and 24h post-injection. The fluorescence signal of IR783 in control group was mainly in liver tissue and little in tumor, revealing a bad tumor targeting ability. What’s more, IR783 was eliminated from the body so quickly that the signals were hard to see at 24h postinjection. Excitingly, HA-mFe3O4/IR783 exhibited much stronger fluorescence intensity in tumor regions for quite a long time. As time increased, a preferential accumulation in tumor was obvious rather than liver or other tissues within 24h post-injection. At 8h post-injection, mice were killed and tissues were taken out. The fluorescence intensity of various organs was seen in Fig.11B and C. In IR783 group, the NIR dye mainly accumulated in liver, while in HA-mFe3O4/IR783 group, the dye most retained in tumor. In order to quantitatively evaluate the distribution of this system in vivo, HPLC was used to determine ART concentration remaining in different organs. The results were summarized in Fig.11D. After administration, ART and its formulations could soon distribute in various tissues. 2h later, these three groups all exhibited a high ART concentration in tumor site. HA-mFe3O4/ART formulations kept a high and effective drug level in tumor site up to 8h. 4. Conclusion In summary, our work highlighted an intelligent and tumor-responsive Fe2+ and Fe2+dependent drugs cotransport system (HA-mFe3O4/ART) for cancer treatment. HAmFe3O4/ART could preferentially accumulate in tumor tissues with enhanced targeting ability. After HA-mFe3O4/ART located in tumor, HA took off to open the "gate". Then the tumorspecific acid and reducible environment further enhanced Fe2+ ions and ART synchronous release locally. Both in vitro and in vivo studies confirmed the combined chemomagnetotherapy effect simultaneously driven by an AMF irradiation. Based on above results, it is anticipated that the comprehensive medical concept of this versatile nanoplatform will facilitate further anti-tumor application of ART analogs. 21 ACS Paragon Plus Environment


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Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 81573364 and 81273451). Supporting Information TGA curves of HA-mFe3O4 and HA-mFe3O4/ART References (1) Ho, W. E.; Peh, H. Y.; Chan, T. K.; Wong, W. S. Artemisinins: Pharmacological Actions beyond AntiMalarial. Pharmacol. Ther. 2014, 142(1), 126-139. (2) Ganguli, A.; Choudhury, D.; Datta, S.; Bhattacharya, S.; Chakrabarti, G. Inhibition of Autophagy by Chloroquine Potentiates Synergistically Anti-Cancer Property of Artemisinin by Promoting ROS Dependent Apoptosis. Biochimie 2014, 107 Pt B, 338-349. (3) Li-Weber, M. Targeting Apoptosis Pathways in Cancer by Chinese Medicine. Cancer Lett. 2013, 332(2), 304-312. (4) Chen, J.; Guo, Z.; Wang, H. B.; Zhou, J. J.; Zhang, W. J.; Chen, Q. W. Multifunctional Mesoporous Nanoparticles as pH-Responsive Fe2+ Reservoirs and Artemisinin Vehicles for Synergistic Inhibition of Tumor Growth. Biomaterials 2014, 35(24), 6498-6507. (5) Righeschi, C.; Coronnello, M. M.; Leto, I.; Bergonzi, M. C.; Bilia, A. R. Transferrin-Targeted Stealth Liposomes Loaded with Artemisinin: The Trojan Horse to Enhance Its Selectivity and Anticancer Activity. Planta Med. 2014, 80(16), 1363-1363. (6) Yang, Y.; Zhang, X. M.; Wang, X. F.; Zhao, X. M.; Ren, T. R.; Wang, F.; Yu, B. Enhanced Delivery of Artemisinin and Its Analogues to Cancer Cells by Their Adducts with Human Serum Transferrin. Int. J. Pharm. 2014, 467(1-2), 113-122. (7) Zhang, H. J.; Hou, L.; Jiao, X. J.; Ji, Y. D.; Zhu, X. L.; Zhang, Z. Z. Transferrin-Mediated Fullerenes Nanoparticles as Fe2+-Dependent Drug Vehicles for Synergistic Anti-Tumor Efficacy. Biomaterials 2015, 37, 353-366. (8) Kumari, M.; Pittman, C. U.; Mohan, D. Heavy Metals [Chromium (VI) and Lead (II)] Removal from Water Using Mesoporous Magnetite (Fe3O4) Nanospheres. J. Colloid Interface Sci. 2015, 442, 120-132.


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(29) Patra, S.; Roy, E.; Karfa, P.; Kumar, S.; Madhuri, R.; Sharma, P. K. Dual-Responsive Polymer Coated Superparamagnetic Nanoparticle for Targeted Drug Delivery and Hyperthermia Treatment. ACS Appl. Mater. Interfaces 2015, 7(17), 9235-9246. (30) Patil, R. M.; Thorat, N. D.; Shete, P. B.; Otari, S. V.; Tiwale, B. M.; Pawar, S. H. In Vitro Hyperthermia with Improved Colloidal Stability and Enhanced SAR of Magnetic Core/Shell Nanostructures. Mater. Sci. Eng., Proc. Conf. 2016, 59, 702-709. (31) Wydra, R. J.; Oliver, C. E.; Anderson, K. W.; Dziubla, T. D.; Hilt, J. Z. Accelerated Generation of Free Radicals by Iron Oxide Nanoparticles in the Presence of An Alternating Magnetic Field. RSC Adv. 2015, 5(24), 18888-18893. (32) Sahu, N. K.; Gupta, J.; Bahadur, D. PEGylated FePt-Fe3O4 Composite Nanoassemblies (CNAs): in Vitro Hyperthermia, Drug Delivery and Generation of Reactive Oxygen Species (ROS). Dalton Trans. 2015, 44(19), 9103-9113. (33) Zhang, D.; Wang, Y. X.; Niu, H. Y.; Meng, Z. F. Degradation of Norfloxacin by Nano-Fe3O4/H2O2. Huan Jing Ke Xue 2011, 32(10), 2943-2948. (34) Wang, D. D.; Zhou, J. J.; Chen, R. H.; Shi, R. H.; Xia, G. L.; Zhou, S.; Liu, Z. B.; Zhang, N. B.; Wang, H. B.; Guo, Z.; Chen, Q. W. Magnetically Guided Delivery of DHA and Fe Ions for Enhanced Cancer Therapy Based on pH-Responsive Degradation of DHA-Loaded Fe3O4@C@MIL-100(Fe) Nanoparticles. Biomaterials 2016, 107, 88-101. (35) Wang, D. D.; Zhou, J. J.; Chen, R. H.; Shi, R. H.; Zhao, G. Z.; Xia, G. L.; Li, R.; Liu, Z. B.; Tian, J.; Wang, H. J.; Guo, Z.; Wang, H. B.; Chen, Q. W. Controllable Synthesis of Dual-MOFs Nanostructures for pH-Responsive Artemisinin Delivery, Magnetic Resonance and Optical Dual-Model Imaging-Guided Chemo/Photothermal Combinational Cancer Therapy. Biomaterials 2016, 100, 27-40. (36) Qin, J. B.; Peng, Z. Y.; Li, B.; Ye, K. C.; Zhang, Y. X.; Yuan, F. K.; Yang, X. R.; Huang, L. J.; Hu, J. Q.; Lu, X. W. Gold Nanorods as A Theranostic Platform for in Vitro and in Vivo Imaging and Photothermal Therapy of Inflammatory Macrophages. Nanoscale 2015, 7(33), 13991-14001.



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Table 1. Characteristics of Drug-Loaded HA-mFe3O4 (n =3). Loading efficiency

Entrapment efficiency

(wt %)

(wt %)

1:1

32.91±1.28

49.10±2.85

2:1

52.77±2.01

56.00±4.50

3:1

55.87.±0.78

42.22±1.35

weight ratio of ART to HA-mFe3O4

Table 2. The size distribution and zeta potential of HA-mFe3O4 aqueous dispersion with different pH at 2, 4, 8 and 12h. Time (hours)

Size/nm

2 4 8 12

241.6 248.6 252.8 261.5

pH=7.4 PDI Zeta/mV 0.12 0.15 0.18 0.21

-22.5 -21.3 -20.6 -19.4

pH=5.5 Size/nm PDI Zeta/mV 262.4 307.5 337.9 373.9

0.19 0.24 0.32 0.47

-16.3 -13.1 -10.64 -8.95


pH=4.0 Size/nm PDI Zeta/mV 288.6 333.3 387.2 432.8

0.21 0.29 0.43 0.62

-14.6 -10.06 -7.35 -6.07

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Figures:

Figure 1. The intelligent and tumor-responsive Fe2+ and Fe2+-dependent drug cotransport system.



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Figure 2. Characterization of mFe3O4. A: TEM images with different scales. B: SEM with different scales. C: EDS analysis of mFe3O4. D: N2 adsorption-desorption curves and pore diameter distribution. E: XPS spectra of mFe3O4.


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Figure 3. Characterization of HA-mFe3O4. A: FT-IR spectra of a) HA-mFe3O4, b) mFe3O4, c) HA; B: UVvis spectra of a) HA-mFe3O4, b) mFe3O4-HBA, c) mFe3O4, d) HA; C: TGA curves of HA-mFe3O4, mFe3O4 and HA; D: Hysteresis curves of different nanoparticles and water dispersibility of a) mFe3O4, b) HAmFe3O4; E: Particle size distribution of HA-mFe3O4; F: Apparent zeta potential of HA-mFe3O4, mFe3O4 and HA.



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Figure 4. A: The release curve of HA-mFe3O4/ART in different acidic conditions; B: The release curve of HA-mFe3O4/ART in PBS containing different concentrations of hyaluronidase; C: pH-sensitive Fe2+ release characteristic of HA-mFe3O4 (pH=7.4, 5.5 and 4.0); D: GSH-responsive Fe2+ release characteristic of HA-mFe3O4 with different GSH concentrations (pH= 5.5, GSH: 0, 2 and 5mM).


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Figure 5. A: IR thermal images of HA-mFe3O4 exposed to AMF at different concentrations for 20min. B: The temperature change curves of HA-mFe3O4 exposed to AMF at different concentrations for 0, 5, 10, 15 and 20min. C: Fluorescence microscopic images of intracellular ROS production by DCFH-DA staining in MCF-7 cells: a) Blank cells; b) Blank cells +AMF; c) HA-mFe3O4 and d) HA-mFe3O4+AMF. D: Detection of intracellular ROS production by flow cytometer.



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Figure 6. A: Prussian blue staining results; B: Lysosomal localization characteristic of HA-mFe3O4; C: The remaining amount of drug in the cells at different times for ART and HA-mFe3O4/ART groups; D: The Fe2+ release amount of HA-mFe3O4/ART in MCF-7 cells at different times.


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Figure 7. Fluorescence microscopic images of intracellular ROS production by DCFH-DA staining in MCF-7 cells at different times.



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Figure 8. A: Cell inhibition of ART, HA-mFe3O4 and HA-mFe3O4/ART on MCF-7 cells for 24, 48, 72 and 96h; B: Cell inhibition of ART, HA-mFe3O4 and HA-mFe3O4/ART combining with AMF on MCF-7 cells for 24h; C: Live cells and dead cells were stained with calcein AM (green) and PI (red); D: Images of single cell gel electrophoresis for DNA damage; E: Tail DNA percentage of various groups.


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Figure 9. A and B: Changes of body weight of mice in different groups during treatment; C and D: Tumor growth trend chart of mice in different treatment groups within 15 days; E: H&E stained tumor tissues harvested from the mice with different treatments.



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Figure 10. H&E stained tissues harvested from the mice with different treatments; A: N.S.; B: N.S.+AMF; C: HA-mFe3O4; D: HA-mFe3O4+AMF; E: ART; F: mFe3O4/ART; G: mFe3O4/ART+AMF; H: HAmFe3O4/ART and I: HA- mFe3O4/ART+AMF.


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Figure 11. A: In vivo NIR imaging of tumor-bearing mice intravenous injected with free IR783 solution and IR783-loaded HA-mFe3O4 carriers at 2, 8 and 24h post injection (a: IR783; b: HA-mFe3O4/IR783); B: NIR imaging of various tissues at 8h post injection(a: IR783; b: HA-mFe3O4/IR783); C: Fluorescence intensity statistics of various tissues; D: Tissue distribution of ART, mFe3O4/ART and HA-mFe3O4/ART at 2 and 8h post injection determined by HPLC.



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TOC Graphic 299x110mm (72 x 72 DPI)

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An Intelligent and Tumor-Responsive Fe2+ Donor and Fe2+-

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