Artemisinin-loaded Mesoporous Nanoplatform for pH-responsive

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Artemisinin-loaded Mesoporous Nanoplatform for pHresponsive Radical Generation Synergistic Tumor Theranostics Lidong Liu, Yuxin Liu, Liyi Ma, Fang Mao, Anqi Jiang, Dongdong Liu, Lu Wang, Qi Jia, and Jing Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18320 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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ACS Applied Materials & Interfaces

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Artemisinin-loaded Mesoporous Nanoplatform for pH-responsive Radical Generation Synergistic Tumor Theranostics By Lidong Liu, † Yuxin Liu, † Liyi Ma, Fang Mao, Anqi Jiang, Dongdong Liu, Lu Wang, Qi Jia and Jing Zhou*

Prof. J. Zhou, L. D. Liu, Y. X. Liu, L. Y. Ma, F. Mao, A. Q. Jiang, D. D. Liu, L. Wang and Q. Jia. Department of Chemistry, Capital Normal University, Beijing 100048, PR China E-mail: [email protected]

KEYWORDS:

mesoporous,

artemisinin,

photothermal

therapy,

synergistic

therapeutic, hypoxic

ABSTRACT: The development of novel and effective cancer treatments will greatly contribute to prolonging and improving patient lives. In this study, a multifunctional nanoplatform was designed and developed based on mesoporous NiO nanoparticles (mNiO) and terbium (Tb) complexes as an artemisinin (ART) vehicle, T2-weighted contrast agent and luminescence imaging probe. mNiO is a novel pH-responsive material that can degrade and release nickel ions (Ni2+) under the acidic tumor microenvironment. Endoperoxide bridge bond in the structure of ART tends to react with Ni2+ to produce radicals that can kill tumor cells. Based on its excellent

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page 2 of 36 near-infrared (NIR) absorbance, mNiO can also be considered as a novel photothermal conversion agent for cancer photothermal therapy (PTT). Compared with free ART or PTT only, this novel agent showed remarkably enhanced antitumor activity in cultured cells and in tumor mice models, owing to the hypoxic tumor microenvironment impelling synergistic therapeutic action. These results provide a novel way of using promising natural drugs-based nanoplatform for synergistic therapy of tumor.

INTRODUCTION Cancer is a life-threatening disease, and great effort has been made to treat the various forms of this serious malady over the last several decades.1-3 However, among the various therapies,4-8 chemotherapy is still most used clinically. Although many varieties of antitumor drugs have been developed and applied as chemotherapy, most causes side effects in patients, including physical and mental pain, and many have low therapeutic efficacy.9-11 Unlike artificial drugs, natural drugs extracted from herbs exhibit less side effects. As a plant-derived sesquiterpene lactone, artemisinin (ART) has been considered as a traditional Chinese medicine for centuries.12-14 ART has been widely used to treat a variety of malignancies and while it is famous as a malaria treatment, there are reports where ART was used to inhibit malignant tumors as well.15,16 Unlike traditional antitumor drugs, the main mechanism of the antitumor activity of ART is that the endoperoxide bridge of this compound can be cleaved in a process mediated by the


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page 3 of 36 Fenton-like effects of ions (such as Fe2+, Mn2+ and Ni2+), generating free radicals that kill cancer cells.17 Thus, the antitumor activities of ART-based drugs have a close relationship with Fenton ions. As a radical-induced therapeutic drug, ART has certain shortcomings. It is difficult to achieve local treatment, and it relies on the speed cancer cell proliferation far more than traditional drugs as well. Photothermal therapy (PTT) is an emerging therapeutic modality that has attracted recent attention due to its effective and selective treatment properties.18-29 PTT has been reported to show excellent anti-tumor effects in vivo,30-35 which are accompanied by fewer side effects compared with conventional treatments.36-38 Therefore, the combination of these two treatment modalities could complement each other to realize a relatively better therapeutic effect on tumor with fewer side effects to normal cells.39-42 The pH value of the tumor microenvironment is generally lower than that of blood or normal tissues.43 Thus, there is an opportunity to develop effective pH-sensitive drug carriers.44-46 Hypoxia is another common feature of all solid tumors; as tumors grow, the interior of the tumor becomes removed from the blood supply, leading to hypoxic conditions.47-52 The hypoxia microenvironment of tumor will inevitably lead to some problems mainly increase hypoxic resistance to traditional tumor treatment including chemotherapy.53-56 Therefore, it is necessary to develop therapeutic agents that are less dependent upon oxygen concentration. This idea came into being is that utilizing the pH-response behavior of mNiO, assisted by the free radical process of ART to overcome the hypoxic or make it indepedent on the hypoxia state of tumor, could ultimately solve series of problems above and achieve better therapeautic effect.



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page 4 of 36 In this work, a multifunctional nanoplatform was designed and developed based on ART-loaded mesoporous NiO nanoparticles (mNiO). The mNiO is a new pH-responsive material that is stable at physiological pH 7.4, but can degrade and release Ni2+ under acid tumor microenvironments. The release of Ni2+ can cause the endoperoxide bridge of ART to break down and produce free radicals. Even under hypoxic conditions, this radical-induced therapy is independent of oxygen, which will benefit the treatment of hypoxic tumors. Meanwhile, mNiO is also a photothermal conversion agent based on its near infrared (NIR) absorbance properties. Moreover, mNiO is a T2-weighted contrast agent and luminescence imaging probe when loaded with Tb complexes, allowing multifunctional imaging. In this study, we explored the synergistic effects of these radical and photothermal therapeutic properties on tumor cells (Scheme 1).

RESULTS AND DISCUSSION Synthesis and Characterization. Mesoporous NiO nanoparticles (mNiO) were acquired by calcinning the precursor Ni(OH)2 nanoparticles. The related characterizations of the precursor were shown in Supporting Information (Figure S1, 2). The morphology and size of mNiO were characterized by transmission electron microscope (TEM) and scanning electron microscope (SEM) analysis (Figure 1A, B), which showed that they are almost circular plates, with a size of approximately 150 nm. The crystalline structure of mNiO was deduced using high-resolution transmission electron microscope (HRTEM), selective area electronic diffraction


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page 5 of 36 (SAED) (Figure 1C, D) and powder X-ray diffraction (XRD). The XRD pattern showed that mNiO has cubic NiO phase corresponding to the Standards files 47-1049 (Figure 1E). X-ray photoelectron spectroscopy (XPS) of mNiO indicated characteristic peaks for nickel 2p, 853.7 eV (NiO) (Figure 1F). Energy-dispersive X-ray analysis (EDXA) revealed compositions of Ni and O, respectively (Figure S3). The Brunauer–Emmett–Teller (BET) surface area of mNiO was measured to be 98.4141 m2 g−1 and the pore volume was approximately 0.3035 cm3 g−1 (Figure 1G). The average pore-size was calculated to be around 3.33 nm which showed a rough surface with pores inside. These data confirmed that mNiO have been prepared. The ultraviolet-visible-near infrared (UV–vis–NIR) spectra of mNiO solution exhibited broad absorbance in the 400–900 nm region (Figure 1H). In Vitro and In Vivo Luminescence Imaging and MRI. Tb-based complexes have been used as luminescence probes for many years.57,58 To equip mNiO with luminescence imaging properties, typical Tb-diethylene triamine pentacetate acid complex (Tb-DTPA) was synthesized and adsorbed into mNiO (mNiO-Tb). Fourier transform infrared spectroscopy (FTIR) indicated the vibrational band around 1634 cm−1 and 1398 cm−1, attributing to carbonyl and alkyl groups of DTPA. The peaks in the mNiO-Tb spectrum showed a partial shift to the low wavenumber region, which could be attributed to the coordination between DTPA and Tb (Figure S4). The FTIR results confirmed that Tb-DTPA was successfully absorbed into the nanoparticles. Given that Tb-DTPA was absorbed into mNiO, the hydrodynamic diameters and zeta potential of the acquired nanoparticles were also measured (Figure S5). The zeta



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page 6 of 36 potential decreased from 3.61 mV of mNiO to −1.34 mV of mNiO-Tb, which also confirmed that mNiO-Tb was obtained. The hydrodynamic diameters of mNiO and mNiO-Tb were 277 nm and 310 nm, respectively. These results indicated that this method of generating mNiO-Tb was effective. The luminescence properties of mNiO-Tb was then tested. Upon 350 nm laser irradiation, both free Tb-DTPA and mNiO-Tb emitted luminescence centered at 485 nm, 542 nm, 584 nm, and 621 nm, corresponding to the transition between 5D4→7F6, 5

D4→7F6, 5D4→7F4, and 5D4→7F3, respectively (Figure S6). Compared with mNiO,

mNiO-Tb exhibited characteristic absorption peaks of the Tb complex (Figure 1I). These results indicated that the Tb complex was successfully absorbed into mNiO and would provide favorable luminescence imaging potential. Bluish-green luminescence was observed by the naked eye under a UV lamp, which confirmed strong luminescence intensity in the 480–570 nm range. Owing to the strong green luminescence emission, mNiO-Tb appeared green, which was further converted to the CIE chromaticity coordinates (Figure S7). As an important parameter, the relaxivity of T2-weighted magnetic resonance imaging (MRI) contrast agent was calculated for evaluating the capacity of MRI.59 With mNiO-Tb concentration increasing, the transversal relaxation time (T2) of water protons was significantly and accordingly shortened from 1240.4 ms to 159.5 ms. The transverse relaxivity (r2) and T2-weighted MR images were also measured as a function of mass concentration under 0.5 T magnetic fields. The r2 value for mNiO-Tb was 6.30 (mg mL−1)-1 s−1, which was calculated from Figure 1J. As the mass


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page 7 of 36 concentration of mNiO-Tb solution increasing, the T2-weighted MRI signal intensity increased, resulting in darker images (Figure 1J inset). These results, especially the high r2 value, suggested that mNiO-Tb was a promising T2-weighted MRI contrast agent. Photothermal Properties. Furthermore, the photothermal properties of the mNiO-Tb solution (2 mg mL−1) were detected by exposing to NIR laser irradiation (785 nm, 2.0 W cm−2), and the temperature changes were detected by an infrared thermal imaging camera. The photothermal images indicated that the temperature of the solution increased from 21.2°C to 58.9°C within 15 min (Figure 2A), which could be applied to cause tumor cells with irreversible damage. Besides, the color of the mNiO-Tb solution became darker as the concentration increased (Figure S8). The photothermal conversion effect of different concentrations of the mNiO-Tb solution (0-1.0 mg mL−1) were detected under the same laser irradiation. It is notable that the temperature increased by 10.8-28.5°C with the increase of the concentration, but no obvious increase for water (Figure 2B). Herein, these results indicated that the mNiO-Tb nanomaterials can convert the 785 nm laser energy into the thermal energy, followed by the absorbance, high heating rate and the photothermal conversion efficiency (η) value (48.88%) (Figure 2C, D and Equations 1). Given this ability to convert NIR to heat energy, mNiO-Tb has the potential to be a photothermal agent for clinical applications. The repeatability of the photothermal conversion of mNiO-Tb was then explored. The photothermal cycles of the mNiO-Tb (2 mg mL−1) was measured by laser on/off



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page 8 of 36 switch within 30 min (Figure 2E). Moreover, the absorbance curves of the mNiO-Tb remained the same before/after laser irradiation (Figure 2F) and stranded for 60 days (Figure 2G), which demonstrated excellent reproducibility and photostability of mNiO-Tb. Thus, mNiO-Tb was investigated to be a promising photothermal agent according to the data above. ART Delivery and Radical Release. ART, a traditional Chinese natural drug, showed potent antitumor activities after metal ion-mediated cleavage of its endoperoxide bridge.17 ART can be absorbed into mNiO-Tb due to the mesoporous structure of the nanoparticle. mNiO-Tb-ART was synthesized by simply stirring, and FTIR of mNiO-Tb-ART revealed peaks at 1737 cm−1 and 1116 cm−1. The corresponding peak (1737 cm−1) could attributed to the stretching vibrations of the ɣC=O bond and inner ester (Figure S4). And the stretching vibrations of the ɣC−O bond of ART was indicated by the peak at 1116 cm−1. These results confirmed that ART was loaded into the mNiO-Tb nanoparticles. In order to obtain the ART loading capacity, we measured the absorbance of ART in supernatant, and the loading capacity (ART/NiO-Tb [wt/wt] = 1 : 1) was quantified as 43.3% (Figure 3A). Moreover, by changing the ART quantity (0–11 mg), the loading rate changed from 0% to 59.7% (Equations (2) and Figure S9). Compared with some other silica, carbon, MOFs-based or nanocarriers 57-61 with pores structure, the loading capacity of mNiO is proper (59.7%) which probably partly due to the mesoporous properties with high specific surface areas and the approximate pore volume. As shown in Figure 3B, ART release from mNiO-Tb-ART was time and pH-dependent. It


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page 9 of 36 was clear that the release rate at pH 6.4 was higher than that at pH 7.4, probably because of the partial degradation of the oxide in acidic conditions based on a neutralization reaction. To test our hypothesis, a degradation experiment was performed. To this end, nickel oxide degradation was detected in buffer solutions (pH 4.4, 5.4, 6.4 and 7.4) by UV-vis-NIR absorption (Figure 3C). The maximum degradation was at pH 4.4, while neutral conditions showed little degradation. In contrast, there was a certain degree of decomposition in pH 6.4 condition. There was no obvious degradation in neutral condition (pH 7.4), and the pH value of the tumor is generally 6.456, so it can be speculated that our materials can degrade well in the tumor area. In addition, the release process of Ni2+ was measured by ICP-MS directly in buffer solutions with different pH values (Figure S10), which is also investigated from the UV-Vis-NIR measurements. In conclusion, the degree of nickel oxide degradation increased as the buffer solution acidity increased, the degradation rate was highest in acidic conditions compared with the neutral condition (pH 7.4) and the photothermal effect indeed could promote the process of drug release to a certain extent (Figure S11). To test the luminescence properties of the nanomaterial during the degradation process, the luminescence changes of mNiO-Tb-ART under acidic conditions (pH 6.4) were monitored. Intriguingly, we found that the luminescence intensity increased over time, and inferred that this was due to the degeneration of Förster resonance energy transfer (FRET) (Figure 3D). The specific process that allows this is as follows: as the Tb-complex is wrapped into the mNiO surface, the luminescence energy can be



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page 10 of 36 transferred from Tb-complex to mNiO when excited by UV irradiation, which resulted in high efficient luminescence quenching. However, when NiO were decomposed under acidic conditions, the Tb complex was released from the mNiO, which decreased the FRET efficiency and hence enhanced luminescence. Additionally, changes in the MRI signal were also monitored in same acidic conditions. As shown in Figure 3E, the signal decreased slightly over time, which can be illustrated by the enhancement of spin-canting effect. When mNiO-Tb-ART were degraded to Ni2+, the T2-weighted imaging effect will be partially weakened, gradually enhancing the brightness of the inset image in the corresponding graph (Figure S12). In conclusion, the above phenomena suggest that mNiO-Tb-ART is degraded under acidic conditions, which result in the change of imaging effects. It has been reported that Fenton metal ions can interact with ART and increase its antitumor efficacy by producing free radicals.17,65-67 We hypothesized that Ni2+ could possess the similar influence as these Fenton ions, and enhance the antitumor efficacy of ART in vitro by the pH-responsive Ni2+ release of the drug delivery system. To test our hypothesis, following radical detection experiments was performed. The free radicals were evaluated by detecting the luminescence signal from DCFH-DA (2,7-Dichlorodi-hydrofluorescein diacetate), which reacts with free radicals to exhibit stronger luminescence (Figure 3F, G). Among the blank group (Figure 3G-a) and control groups (Figure 3G-b to f), the mNiO-Tb-ART (pH 6.4) group (Figure 3G-g) exhibited the highest luminescence signals, which was similar to positive control group (Rosup group, Figure 3G-h). This phenomenon can be attributed to the free


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page 11 of 36 radicals generated by released Ni2+ and ART. It is notable that mNiO (pH 6.4) group (Figure 3G-c) showed stronger fluorescence than blank group (Figure 3G-a) probably

due to the partial generation of hydroxyl radicals by endogenous H2O2 reacted with Ni2+ according to some previous work68,69. At the meanwhile, the ART group (Figure 3G-f) also showed strong luminescence, which may contribute to the endoperoxide bridge, further illustrating the ART was the essential part for radical generating when compared with mNiO (pH = 6.4) group (Figure 3G-c). Brighter luminescence of mNiO-Tb-ART (pH 6.4) group (Figure 3G-g) than that of mNiO group (Figure 3G-b) and mNiO (pH 6.4) group (Figure 3G-c) were clearly displayed, directly showed that the Ni2+ induced ART radical in the nanosystem, which also indicated the radicals that play a role in the treatment come from artemisinin in the nanosystem. Cell Luminescence Imaging and in vivo MRI. The potential application of mNiO-Tb-ART for luminescence imaging was firstly investigated in cells, evaluated by a luminescence microscope using mercury lamp as excitation source (excitation at 370 nm). Intense cell luminescence was observed after incubation with mNiO-Tb-ART for 24 h at 37°C (Figure 4A). The cell luminescence images demonstrated that the mNiO-Tb-ART can be uptake by cells in acidic condition and the result indicated that mNiO-Tb-ART could play a role in the field of cell imaging. Luminescence imaging possesses unique properties, such as real-time and accurate tracking when applied at the cellular level.35 However, its penetration ability is not high enough, as optical signals are easily absorbed by surrounding tissues, while MRI is welll-known for its properties of high resolution and three dimensional details,70-75



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page 12 of 36 and the combination of these two technologies can make the imaging information more comprehensive and accurate.76 To determine its utility for in vivo tumor MRI, the mNiO-Tb solution (10 mg kg−1) was intravenously injected into the HeLa tumor mice models. The relative MRI signals in the organs (Figure 4B) was then measured, in which the mNiO-Tb uptake and retention were obviously demonstrated in tumor and liver sites within 2 h (n = 3). As shown in Figure 4D, T2-weighted MR signals were increased by 17.24% and 27.34% in liver and tumor sites, respectively. This suggested mNiO-Tb may passively target tumors due to the nanomaterial’s enhanced permeability and retention effect, confirming mNiO-Tb acts as a T2-weighted MR contrast agent for in vivo MRI. In conclusion, this material can be used as a diagnostic probe in dual-mode imaging during treatment. Synergistic Therapy. Prior to in vivo studies, we conducted preliminary in vitro experiments to test the application of the nanomaterial as a treatment modality. HeLa cancer cells were treated in six groups (blank, laser, ART, mNiO-ART, mNiO+laser, mNiO-Tb-ART+laser). As shown in Figure 4F, mNiO-Tb-ART+laser group exerted the highest therapeutic effect on HeLa cells when evaluated with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The lower cell viability (33.85%) of all HeLa cells survived in this group compared with the relatively high survival rate in other groups. Additionally, the mNiO-Tb+laser group also showed low cell viability, demonstrating the photothermal property of mNiO. The therapeutic effect of the mNiO-Tb-ART group indicated that mNiO degradation promoted the production of free radicals by ART in cancer cells, which increased


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page 13 of 36 intracellular lipid hydroperoxides and induced apoptosis. The relative cell viability experiments (Figure S13) of the last three groups (mNiO-Tb-ART, mNiO+laser and mNiO-Tb-ART+laser) with a set of different concentrations of ART and nanocomposite, and with disparate pH values (7.4 and 6.4) for 24 h also indicated the better therapy effect in acidic condition. As shown in Figure S14, Calcein-AM/PI cell staining in four groups (blank, mNiO, mNiO-ART, mNiO-ART+NIR) also demonstrated the significant synergistic effect in vitro. These results demonstrated synergistic effects in the mNiO-Tb-ART+laser group from combined photothermal and radical-induced therapy. These synergistic antitumor effects were further investigated in

HeLa

tumor-bearing nude mice. Mice were separated into six experimental groups (once the tumor volume reached 700 mm3): the laser (785 nm, 2.0 W cm−2 , 4 min), ART only (80 µg mL-1), mNiO-Tb-ART, mNiO-Tb+laser and mNiO-Tb-ART+laser and PBS-blank. The nanomaterials were mainly distributed within tumors after 24 h with the 10 mg kg−1 dosage for each injection, and the temperature of tumor reached up to 47.2 °C upon NIR irradiation (Figure 4C and 4E). These results suggest that this technique has photothermal therapeutic potential. While tumors in the laser and blank groups showed similar growth rates, as relative tumor volume (V/V0) reached 2.1 within 11 d (Figure 4G inset). However, most tumors in the mNiO-Tb-ART+laser group nearly disappeared by day 14, with a V/V0 of approximately 0.3, confirming the strong antitumor effects (Figure 4G). Although tumors in the ART group, mNiO-Tb-ART group and mNiO-Tb+laser group display a slight volume decreased,



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page 14 of 36 the variation of tumor volume in the mNiO-Tb-ART+laser group showed apparently tumor growth suppression compared with the others. Thus, this obvious effect was ascribed to the combined radical-induced and photothermal effects, which together improved therapeutic efficacy. The photos of mice/tumor after different treatments were obtained by digital camera (Figure S15) and it is also worth noting that the body weight of the mice barely changed within treatment process (Figure S16) suggesting the stable condition of mice during the treatment and verifying the PTT therapeutic efficacy. These results suggested synergetic therapy was achieved. Cytotoxicity. To further ensure the safety before application, the cytotoxicity of nanomaterials was studied in vitro. MTT assay, as an vital data, indicated that mNiO-Tb in HeLa cells with low toxicity. Most of all HeLa cells survived (85%), even at a high mNiO-Tb concentration (10 mg mL−1) after 48 h incubation (Figure 5A). Collectively, our results showed that mNiO-Tb itself was not noticeably toxic to cells. As Ni2+ showed no apparent release in neutral conditions, such as in normal tissues, ion release could only be observed after degradation in acidic microenvironments, such as tumors. The toxicity of mNiO-Tb (20 mg kg−1) in vivo experiments were also evaluated its potential utility. No death or obvious abnormal behaviors (retarded movement or lethargy) were observed, even receiving high-dose mNiO-Tb. The serum biochemical assays was investigated in the mNiO-Tb-injected mice on days 1, 7, and 30, as well as the complete blood tests. Liver and kidney function indices were all normal (Figure 5B-F),

suggesting

that

no

biodysfunction

was


occurred.

Furthermore,

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page 15 of 36 hematoxylin-eosin (H&E)-stained images demonstrated that the tissues were normal as same as the PBS-injection (blank) group (Figure 5G). These results corresponded to the in vitro results and demonstrated low toxicity of mNiO-Tb. These findings further indicated that mNiO-Tb could be a tool for antitumor therapy with reduced toxicity and good biocompatibility. CONCLUSION In conclusion, ART and Tb-complex loaded novel mNiO were successfully developed as a multifunctional nanoplateform for cancer theranostics. With outstanding performance in both T2-weighted MRI (r2 = 6.30 (mg mL−1)-1 s−1) and luminescence imaging, this nanoplateform can serve as an efficient dual modality probe for monitoring ART delivery and PTT. It is noteworthy that the obtained mNiO can degrade in acidic tumor microenvironment and release free Ni2+, which benefit the controlled release of ART. More importantly, the release Ni2+ played an essential role in the generation of free radicals by clear the endoperoxide bridge in ART molecule, killing tumor cells more efficiently with the assistance of PTT. Both the in vitro and in vivo experiment strongly confirmed this valuable finding. This work further highlighted the potential application of ART in cancer therapy and encouraged the development of multifunctional pH-responsive drug delivery nanovehicles.

EXPERIMENTAL SECTION Materials. Artemisinin (ART), nickel nitrate hexahydrate (Ni(NO3)2⋅6H2O) and oleylamine (OM) were purchased from Shanghai Energy Chemical Co., Ltd. Sodium



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page 16 of 36 hydroxide (NaOH), ethanol and cyclohexane were purchased from Sinopharm Chemical Reagent Co., Ltd. Diethylenetriaminepentaacetic acid (DTPA) and terbium nitrate hexahydrate (Tb(NO3)3⋅6H2O) were purchased from Shanghai Macklin Biochemical Co., Ltd. Synthesis of Mesoporous NiO (mNiO). The mesoporous NiO nanoparticles (mNiO) were obtained by calcinning Ni(OH)2 nanoplates, which were synthesized by a hydrothermal reaction according to reported hydrothermal method.77 In a typical experiment, 1 mL aqueous solution containing 0.5 g Ni(NO3)2.6H2O was added into 9 mL OM with gently magnetic stirring for 10 min at room temperature (RT). The reaction was then treated at 160 ℃ for 18 h after transferring into a Teflon-lined stainless steel autoclave, and allowed to cool to RT naturally. The resulting precipitate was collected by centrifugation at high speed (12,000 rpm) for 2 minutes and washed with ethanol and cyclohexane for several times. The Ni(OH)2 nanoplates precursor were finally dried in vacuum at 70 oC, and then carefully calcined at 400 °C in a quartz tube furnace for 5 h. Finally, mNiO were obtained. Preparation of mNiO-Tb. To get mNiO loaded with Tb complex (mNiO-Tb), 100 mg Tb(NO3)3·6H2O and 100 mg DTPA were added into 5 mL ethanol (50%) in flask, afterward, the pH value of the reaction system was adjusted to 7.4 with carefully adding 1 M NaOH aqueous solution and stirred at RT for 12 h. The mixture was then stored under 4 oC for next step use. The acquired solution was dropwise added into a mortar which containing 50 mg mNiO. In this process, continuous grinding is necessary until the obtained materials becoming dry powder.


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page 17 of 36 Characterization. TEM and HRTEM images were taken on a Tecnai G2F30 transmission electron microscope with a field emission gun operating at 300 kV. SEM images and EDXA spectra were obtained on a Hitachi SU8010 Scanning electron microscope, with a field emission gun operating at 20 kV. Samples were dispersed in cyclohexane and water dropped on the surface of a sillicon pellet. XRD pattern was performed on a Rigaku DMAX 2500 power diffractometer with ultra 18 KW Cu radiation. XPS was performed on Thermo escalab 250Xi. The specific surface areas were measured by nitrogen adsorption/desorption instrument (3H-2000PS1, BeiShiDe company) by using Brunauer-Emmett-Teller (BET) method, and the pore volume and pore size were calculated from the adsorption branch of the isotherm based on Barrett-Joyner-Halenda

BJH

model.

Ultraviolet-visible-infrared

spectroscopy

(UV-vis-NIR) light absorption analysis was performed with a UV-3600 UV-vis-NIR spectrophotometer from SHIMADZU Company. Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a Bruker TENSOR 27 FT-IR Spectrometer. Dynamic light scattering (DLS) and zeta potential experiments were carried out on an ALV-5000 spectrometer. The concentration of nickel ion was measured by inductively coupled-mass spectroscopy (ICP-MS) (Agilent 7500ce ICP-MS). ART Delivery Experiment and Releasing Experiment. To obtain the mNiO-Tb-ART, 5 mg NiO-Tb nanoparticles were stirred in ART ethanol solution (1 mg mL-1, 5 mL) for 24 hours at RT. The resulting nanoparticles were then collected by centrifugation and washing with distilled water several times to remove free ART. For



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page 18 of 36 acquiring ART loading rate, the supernatants in whole process were collected together, and drug loading content was measured by the UV-vis-NIR spectrophotometer. The mNiO-Tb-ART nanosuspension was added into about tenfold ethanol and sonicated for minutes to make sure that the loading ART was completely dissolved, then the nanosuspension was centrifuged at 12,000 rpm for 5 min to separate ART and mNiO-Tb to test the amount of ART attached on mNiO-Tb. Subsequently, the obtained solution was hydrolyzed by a five-fold sodium hydroxide solution (0.2%) for half an hour using water bath (50 oC). The efficiency of ART-loading and the quantity of mNiO were further measured at 291 nm and 785 nm by UV test, respectively. All washing solutions were collected, and the loading rate was approximately 45.46% (wt/wt) when the mass ratio is 1:1 (ART: mNiO) based on UV-vis-NIR absorption technique. This result confirmed incorporation of ART into the mNiO was successful. Then, we transferred the supernatant contain ART into sodium hydroxide aqueous solution at water bath (50°C, 30 min). After the hydrolytic process of ART, UV-absorbing compound appeared and the absorption peak was detected to be 291 nm. Loading content = (weight of ART in mNiO-Tb-ART) / (weight of mNiO-Tb+ART) The releasing assay was performed at 37.0 oC. The as-prepared material was then dispersed in 5 mL aqueous buffer solutions (pH 7.4 and pH 6.4 citric Na2HPO4-C6H8O7 buffer) at RT. 500 µL of suspension was imbibed at predetermined time. Then diluting the solution to 3 mL and the delivery of ART from the nanocomposites to the buffer solution was monitored via the absorbance band of ART


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page 19 of 36 at 291 nm. In the same way, we further tested the NIR sensitive drug release behavior of mNiO-Tb-ART in four groups (pH 7.4, pH 7.4+NIR, pH 6.4 and pH 6.4+NIR) at RT. Relaxation Rate in vitro and in vivo. The T2-weighted magnetic resonance (MR) images in vitro and in vivo were performed using a 3 T Siemens Magnetom Trio running on Siemens’Syngo software version B15. For in vitro experiment, we used several 2.0 mL centrifuge tubes containing different concentrations of mNiO-Tb solution (5, 10, 20, 40, and 80 µg mL−1) for testing. Mice models bearing HeLa tumor were intravenously injected with mNiO-Tb-ART (10 mg kg-1). Luminescence Properties. The photoluminescence spectrum was recorded in spectrometer (F-7000, HITACHI) with the Xe lamp as excitation source. In typical experiment, the quartz cuvette containing 3 mL of mNiO-Tb suspension liquid was set at sample holder, with the excitation wavelength 370 nm, and the slice 5 nm, in which the spectrum was collected in range from 400 nm to 700 nm. Cell Culture Experiments. HeLa cells were provided by the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. During experiment process, 1 × 105 cells well-1 were seeded in glass coverslips for 24 h attaching. For use in the experiments, a certain amount of HeLa cells were seeded in 10 mm plastic culture dish. HeLa cells were cultured in DMEM high glucose medium containing 10% Fetal Bovine Serum at 37 °C under 5% CO2 atmosphere. In vitro Luminescence Imaging. HeLa cells were incubated with mNiO-Tb solution (0.5 mg mL -1) for 4 hours and the luminescence imaging was obtained in



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page 20 of 36 optical microscopic, in which the bright photo was took with halogen lamp and the luminescence imaging was recorded with the mercury lamp as excited source. The images were captured using a luminescence microscope with an excitation laser (370 nm) showed the green luminescence in the region at 520-550 nm. Photothermal Imaging in vitro. To evaluate the photothermal property, the acquired mNiO solution (2 mg mL-1, 2 mL) in bottles was exposed to a 785 nm laser (2.0 W cm-2) irradiation for 900 s. Meanwhile, the IR thermographic camera (FLIR E40) was used to record the photothermal data during the experiment. Degradation Studies and Radical Detection. mNiO-Tb-ART (0.125 mg mL-1) was dispersed in 10 mL buffer solution with different pH value (4.4, 5.4, 6.4, 7.4), and the UV-vis-NIR spectra were then taken at different time points. The degradation process of mNiO-Tb-ART was reflected by the UV-vis-NIR spectra. Besides, the pH sensitive of released Ni2+ was measured directly by ICP-MS within 40 min in the same way. The DCFH-DA method was carried out to detect the intracellular radical level. To investigate pH-sensitive behavior of radical, cells were incubated with mNiO, Ni2+, mNiO (pH 6.4), mNiO-Tb-ART, ART and mNiO-Tb-ART (pH 6.4) as experiment group, the blank group and Rosup group were set as control groups. We used the luminescence microscope with excitation laser (488 nm) during this process. Photothermal Therapy. The similar set of HeLa cell samples were exposed to the 785 nm laser (2.0 W cm-2) for 4 min. For the cell uptake studies, the cells were grown on 96-well cell culture plate, treated with different nanoparticles in six groups (blank,


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page 21 of 36 laser, ART, mNiO-Tb-ART, mNiO+laser, mNiO-Tb-ART+laser). The ART (80 µg mL-1) solution was prepared and used for ART only group. And the cell viability of samples was measured after 4 h and 24 h by following the method described in MTT assay. Furthermore, the cell viability was also measured in mNiO-Tb-ART, mNiO+laser and mNiO-Tb-ART+laser with a set of concentration (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg mL-1) of nanocomposite under the pH value 7.4 and 6.4 for 24 h. Four groups of HeLa cells (PBS (blank), mNiO, mNiO-ART, and mNiO-ART+NIR) were cultured with medium to evaluate the synergistic therapy effect. The HeLa cells were then stained with Calcein-AM (excited at 490 nm) and propidium podide (PI, excited at 535 nm). We finally used a microscopy to observe the cell-staining images. In vivo experiment with animals obeyed the protocol as approved by IACUC. HeLa tumor mice models were divided into six groups for treatments when the tumor volume reached to 700 mm3 (PBS-blank, ART only, laser only, mNiO-Tb-ART, mNiO-Tb+laser and mNiO-Tb-ART+laser groups). The mice were gas anesthetized and injected by caudal vein with mNiO-Tb-ART (10 mg kg-1). After 1 h, the tumor section was exposed to the irradiation of 785 nm laser (2.0 W cm-2) for 4 min. The treatment were given every 2 or 3 days and the tumor volume (length/2×width2) was recorded during the experiment. MTT Assay. HeLa cells were seeded into 96-well cell culture plate (90 µL well-1, 105 mL-1), incubated for 12 h. After that, phosphate buffer solution (PBS) solution containing different concentrations of mNiO-Tb (0, 2, 4, 6, 8, 10 mg mL-1, 10 µL



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page 22 of 36 well-1) were added into left and right half part of 96 well plate for 24 h and 48 h, the 10 µL MTT solution (5 mg mL-1) was then added to each well for incubating another 4 h. The 96-well plate was then standing for 30 min after the addition of dimethylsulfoxide (DMSO, 100 µL well-1). Then the absorption value of each well was measured at 690 nm and we calculated the viability of cells growth by the formula.

 mean Abs.valueof treatmentgroup   × 100 % Cell viability(%) =   mean Abs.valueof control group 

Statistical Analysis. Data are presented as mean ± SD and the statistical significance was performed using ANOVA (p < 0.05). ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge at the ACS Publications website or from the author.

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

Contributions †L.L. and Y.L. contributed equally to this work.


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ACKNOWLEDGMENTS We thank Zhigao Lu from Institute of Chemistry, Chinese Academy of Sciences (ICCAS), for his help on the in vivo experiments; Luoyuan Li from Renmin University of China, for her help on FT-IR measurements; Jiahuan Ren and Peng Gao from Department of Chemistry, Capital Normal University, for their help on laser light path adjustment and the N2 adsorption–desorption measurements, respectively. The authors thank the funding of National Natural Science Foundation of China (21301121), Youth Innovative Research Team of Capital Normal University, and Beijing

Talent

Foundation

Outstanding

Young

Individual

Project

(2015000026833ZK02), Project of Construction of Scientific Research Base by the Beijing Municipal Education Commission, Yanjing Young Scholar Development Program of Capital Normal University, Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five–year Plan (IDHT20180517), Project of Construction of Scientific Research Base by the Beijing Municipal Education Commission and Opening Project of Shanghai Key Laboratory of Magnetic Resonance.

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Scheme 1. Schematic illustration of the synthesis of mNiO-Tb-ART nanoplatform for the tumor microenvironment-responsive enhanced synergistic tumor theranostics.


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Figure 1. Characterization of the mNiO. (A) SEM image of mNiO (scale bar: 100 nm). (B) TEM image of a single nanoparticle (inset, scale bar: 50 nm). (C) HRTEM image of mNiO: scale bar: 5 nm. (D) SAED images of mNiO: scale bar: 5 1/nm. (E) XRD pattern of pure mNiO for cubic phase, and the standard data of NiO (JCPDS card No. 47-1049) (F) X-ray photoelectron spectroscopy (XPS) of mNiO indicated characteristic peaks for nickel 2p, 853.7eV (NiO). (G) The N2 adsorption–desorption isotherms and the pore size distribution of mNiO (inset graph). (H) Normalized UV-vis-NIR absorption spectra of mNiO with different concentration. Inset: Standard curve of absorbance at 785 nm. (I) Luminescence spectra of Tb (Tb-DTPA), mNiO and mNiO-Tb, excited by 370 nm at room temperature. (J) Relaxation rate (1/T2) versus different concentrations of mNiO-Tb; Color-mapped phantom MR images of the mNiO-Tb (inset).



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Figure 2. Assessment of photothermal effect and photostability of mNiO-Tb. (A) The digital image of materials and the photothermal images for mNiO-Tb solution (2 mg mL−1) irradiated by 785 nm NIR laser (2.0 W cm-2, 0-15 min). (B) Temperature changes of the mNiO-Tb (0–1.0 mg mL−1) upon 785 nm laser irradiation (2.0 W cm−2). (C) The temperature change of the mNiO-Tb solution. (D) Cooling time of mNiO-Tb vs −ln θ obtained from the cooling period of (C). (E) The temperature change of the mNiO-Tb solution (2 mg mL−1) for laser on and off switches. (F) The photostability of mNiO-Tb solution (2 mg mL−1) and digital images before and after 785 nm irradiation. (G) The photostability of mNiO-Tb solution standing after 1 d, 7 d, 15 d, 30 d and 60 d.


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Figure 3. The ART delivery and radical release experiments. (A) The loading rate of ART in mNiO-Tb-ART. (B) ART released from mNiO-Tb-ART in pH 7.4 and 6.4. (C) Investigation on the degradation process of mNiO in different pH condition and time. The relative luminescence (D) and MR (E) signal change of mNiO-Tb-ART within time under pH 6.4. The relative intensity (F) and images (G) of intracellular fluorescence from the radical production by DCFH-DA were showed in these groups, (a) blank, (b) mNiO, (c) mNiO (pH 6.4), (d) Ni2+, (e) mNiO-Tb-ART, (f) ART, (g) mNiO-Tb-ART (pH 6.4) and (h) Rosup.



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Figure 4. Imaging and synergistic effect of mNiO-Tb. (A) The luminescence images of HeLa cells after 4 h incubation using a luminescence microscope. (B) The in vivo T2-weighted MRI of pre-injection and 2 h post-injection with mNiO-Tb (10 mg kg−1 injection). (D) The relative MR signal in tumor, liver, kidney and spleen with mNiO-Tb (10 mg kg−1) post-injection (0, 30, 60, 90 and 120 min). The photothermal images (C) and temperature difference diagram (E) in vivo after intravenous injection of mNiO-Tb-ART (10 mg kg−1), exposed to the irradiation of 785 nm laser (2.0 W cm-2, 4 min). (F) Synergistic tumor therapy in vitro (The ART solution (80 µg mL-1) was used in ART only group and the laser power was 2.0 W cm−2). (G) In vivo tumor volume changes on different groups (blank, ART, laser, mNiO-Tb-ART, mNiO-Tb+laser and mNiO-Tb-ART+laser) after different treatment. Results are expressed as mean ± SD (n = 3). Error bars are standard error of the mean. *p < 0.05, **p < 0.01.


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Figure 5. Evaluation of biosafety in vitro and in vivo. (A) In vitro MTT assays of HeLa cells, cell viability vs incubated mNiO-Tb concentration for 24 and 48 h, normalized by the cell samples without mNiO-Tb incubation (blank). Serum study results obtained from mice injected with mNiO-Tb (10 mg kg-1) different time post-injection (1 d, 7 d and 30 d) and mice receiving no injection (blank). (B) ALT (alanine aminotransferase) and AST (aspartate aminotransferase); (C) TBIL (total bilirubin); (D) TP (total protein); (E) ALB (albumin); (F) CREA (creatinine). (G) H&E-stained heart, kidney, liver, lung, and spleen sections from mice PBS-injection (blank) and 1 d, 7 d, and 30 d post-injection with mNiO-Tb (20 mg kg-1).



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page 36 of 36 Table of Content


Artemisinin-loaded Mesoporous Nanoplatform for pH-responsive

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