Multistimuli-Regulated Photochemothermal Cancer Therapy Remotely

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Multi-Stimuli-Regulated Photo-Chemothermal Cancer Therapy Remotely Controlled via Fe5C2 Nanoparticles Jing Yu, Yanmin Ju, Lingyun Zhao, Xin Chu, Wenlong Yang, Yonglu Tian, Fugeng Sheng, Lei Zhang, Jian Lin, Fei Liu, Yunhe Dong, and Yanglong Hou ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b04706 • Publication Date (Web): 25 Nov 2015 Downloaded from http://pubs.acs.org on November 26, 2015

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Multi-Stimuli-Regulated Photo-Chemothermal Cancer Therapy Remotely Controlled via Fe5C2 Nanoparticles †

Jing Yu, Yanmin Ju,

╟, †

⊥

‡

†

†

†

Sheng,|| Jian Lin, Fei Liu, Yunhe Dong, and Yanglong Hou*, †

†

§

Lingyun Zhao,* , Xin Chu, Wenlong Yang, Yonglu Tian, Fugeng †

Department of Materials Science and Engineering, College of Engineering, Peking University,

Beijing 100871, China ╟

College of Life Science, Peking University, Beijing 100871, China

‡ Key Laboratory of Advanced Materials, Ministry of Education, School of Material Science & Engineering, Tsinghua University, Beijing, 100084, China §

||

Laboratory Animal Centre, Peking University, Beijing 100871, China

Department of Radiology, Affiliated Hospital of The Academy of Military Medical Sciences,

Beijing 100071, China ⊥

Synthetic and Functional Biomolecules Center, Department of Chemical Biology, College of

Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

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KEYWORDS stimuli-responsive, iron carbides nanoparticles, photo-chemothermal therapy, magnetic targeting ABSTRACT Stimuli-controlled drug delivery and release is of great significance in cancer therapy, endowing the stimuli-responsive drug carrier highly demanded. Herein, a multi-stimulicontrolled drug carrier was developed by coating bovine serum albumin on Fe5C2 nanoparticles (NPs). With a high loading of anticancer drug doxorubicin, the nanoplatform provides a burst drug release when exposed to near-infrared (NIR) light or acidic condition. In vitro experiment demonstrated a NIR-regulated cell inhibition which is ascribed from cellular uptake of the carrier and the combination of photothermal therapy and enhanced drug release. The carrier is also magnetic field responsive, which enable the target drug delivery under the guidance of magnetic field, and monitor the theranostic effect by magnetic resonance imaging. In vivo synergistic effect demonstrates that the magnetic-driven accumulation of NPs can induce a complete tumor inhibition without appreciable side effect to the treated mice by NIR irradiation, due to the combined photo-chemotherapy. Our results highlight the great potential of Fe5C2 NPs as a remote-controlled platform for photo-chemothermal cancer therapy.

Nanoparticle-based stimuli-responsive cancer therapy has received tremendous attention in recent years, as it can offer the merits of precision for spatial-, temporal- and dose-controlled drug release through a remote stimulation, and can conveniently switch the ‘on and off’ drug release on necessary conditions.1-4 Compared with other stimuli, such as magnetic field,5, 6 heat,7 and ultrasound,8, 9 light is the most flexible one to alter the treated site and the intensity applied, and thereby to achieve the on-demand drug release.10, 11, 12


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The most commonly used light-sensitive molecules are coumarin and o-nitrobenzyl. However, they need UV light to excite, which is harmful to living tissues and also with poor tissue penetration.13-15 The near-infrared (NIR) light with wavelength at 650-950 nm, on the contrary, is at the minimal absorbance region by skin and tissue.16 In the meanwhile, NIR light is at the tissue-transparent window, endowing a relatively deeper tissue penetration and lower phototoxicity on skin, enabling it an attractive stimulus for the controlled therapy, especially for the superficial tumors.13, 17, 18 With the application of NIR to the light absorbing agent, heat would generate to realize the photothermal therapy (PTT) for the direct cancer cell killing on the one hand.19-24 On the other hand, it would be a wise strategy to design a heat-responsive drug delivery system under the control of NIR by co-embedding PTT agents and drugs within temperature-sensitive carriers, or conjugating drugs to PTT agent with a heat sensitive linker.25-28 Such a multifunctional nano-platform can offer the opportunity of synergistic effect by the combination of PTT with drug release for the enhanced cancer therapy. Making full use of the differences between tumorous environment and normal tissue, pH value is applied to be another widely used stimulus for cancer theranostics.29-31 It is well-documented that the pH value in tumors is more acidic than that in blood and normal tissue, commonly about 6.5-6.8; while an even greater pH decrease can be found in some intracellular compartments, in which the pH values are about 5.0.32 By either loading anti-cancer drugs into some pH-sensitive polymers or hollow/porous nanoparticles (NPs), or associating drugs to carriers through a pHsensitive bond, tumor-selective drug release have been achieved.33-35 Another barrier hinders the development of cancer therapy is the reduced dosage to the desired part, which is highly dependent on the targeting technique. Commonly used targeting approaches nowadays mainly includes passive targeting and active targeting.36-38 However, passive targeting


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may fail in some hypovascular tumors, such as prostate cancer or pancreatic cancer, due to the absence of enhanced permeability and retention effect,39 while active targeting lacks of universality owing to the one-to-one correspondence of receptor and ligand. Magnetic targeting, with the protocol of attractive response of magnetic nanoparticles (MNPs) to an external magnetic field, is different from these two targeting approach. When exposed to an external magnetic field, MNPs can be magnetized, moved under magnetic driving, and concentrated at a specific site.40-42 This physical interaction is not confined by the specific receptor expression, showing promise as a more general tumor targeting technique, particularly work well for the surperficial tumor.43 Herein, we developed functionalized MNPs as a novel multifunctional drug carrier that can remotely control the drug delivery by two kinds of stimuli for combined cancer therapy. Carboncoated iron carbide (Fe5C2) NPs, which have been proved to be good candidates for magnetic resonance imaging (MRI) and light absorbing agents, are employed as the nano-vehicle.44, 45 They were coated with bovine serum albumin (BSA) for better water solubility and biocompatibility. Doxorubicin (DOX), a clinically used anticancer drug was effectively loaded through electrostatic interaction onto the surface of MNPs. This nano-platform showed a double controlling of the drug delivery by both pH value and NIR light, and exhibited a tumor targeting through magnetic field. Upon cellular uptake of MNPs into endosomes or lysosomes, a burst DOX release is activated by the low pH, in the meanwhile, a triggered DOX escape from these organelles to nuclei could be modulated by NIR irradiation. Except as one of the stimuli for drug delivery, the possible synergist effect between PTT and chemotherapy can be realized for an enhanced anti-tumor effect through NIR irradiation. The tumor-site specific targeting can be noticed by locating a magnet at the tumor site, which showed a significant accumulaton of both


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NPs and drugs to the tumor in vivo, and could be monitored by T2-weighted MRI. Therefore, the multifunctional MNPs can achieve a combined therapy to eliminate tumor under NIR laser irradiation, and no systemic side effects were observed during the treatment. Our result first demonstrated that Fe5C2 NPs is a promising drug carrier that not only could be modulated by two stimuli, but also could achieve a combined cancer therapy by a single particle.

Results and discussion Fe5C2 NPs were synthesized by a bromide-induced method following our previously reported protocol.46 Representative TEM image shows the as-synthesized Fe5C2 NPs have a core/shell structure, with an overall size of about 20 nm (Figure 1a). X-ray diffraction (XRD) data reveal that the chemical structure is Fe5C2 (JCPDS no. 36-1248) (Figure S1). To enhance the biocompatibility and water-solubility, bovine serum albumin (BSA) was selected to modify the assynthesized NPs. Due to the negative charge of both Fe5C2 NPs and BSA, the modification process was realized by a two-step strategy.47, 48 Fe5C2 NPs were first coated with a positive charged dopamine (Fe5C2-DOPA), followed by coating BSA through electrostatic interaction (Fe5C2-BSA). Observations from zeta-potential analysis well support the successful coatings, which reveals a positive-charge of Fe5C2-DOPA (12.0mV) and a negative-charge of Fe5C2-BSA (-13.5 mV) (Fig. 1b). No obvious morphological changes were found during the modification (Figure S2), while the overall size increased from 28.81 nm to 78.64 nm as characterized by dynamic light scattering (DLS) (Figure S3). Fe5C2 NPs possessed a strong magnetic property with saturation magnetization (Ms) of 125 emu/g(Fe), and even after surface coating, they still showed a high Ms of 112.74 emu/g(Fe) (Figure 1c). Therefore, it is highly feasible that Fe5C2-BSA NPs could act as a T2-weighted MRI contrast


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agent and a carrier for magnetic targeting. Additionally, according to our previously study, Fe5C2 NPs have a carbon coating, which endows a high absorption at the NIR region. By transferring the absorbed energy into heat, Fe5C2 NPs are in great potential for PTT (Figure S4).44 Under an 808 nm NIR laser irradiation, Fe5C2-BSA NPs aqueous solution at different concentrations showed an obvious temperature elevation, which is much higher than water (Figure 1d). Considering the ions in blood such as chlorides and phosphates may etch the NPs, TEM image of NPs was taken after being exposed in culture medium with 10% FBS for 1 month, which shows the morphology still maintained with a core-shell structure (Figure S5), indicating the lifetime of NPs for at least 1 month for remote controlling. As mentioned above, Fe5C2-BSA NPs are negative in charge, enabling the absorption of positively charged drug through electrostatic interaction.49, 50 DOX, a clinically used anticancer drug was used here as a drug model, and loaded onto surface of Fe5C2-BSA NPs by simple mixing (Fe5C2-BSA-DOX NPs). After removal of free DOX by centrifugation, the amount of loaded DOX was quantified by using UV/vis absorbance, which showed a distinguished DOX peak appearing at around 480 nm over the background of the carrier (Figure 2a). Compared with Fe5C2-DOPA NPs, a much improving in the loading capacity was achieved by Fe5C2-BSA-DOX NPs, with a saturated loading capacity of 12.3% (Figure 2a & b), illustrating Fe5C2-BSA NPs are good candidates as drug carriers. The drug releasing behaviors of Fe5C2-BSA-DOX NPs under different pH values were investigated by dialyzing Fe5C2-BSA-DOX NPs in pH 5.4 or 7.4 buffers at body temperature (37 ℃). The released DOX was collected and measured by UV-vis spectrum. Within 48 h, about 88% of DOX was released at pH 5.4 compared with only 22% released at pH 7.4 (Figure 2c), as the interaction between DOX and Fe5C2-BSA are weakened due to the changing of their


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electrification when pH value decreased. Also, DOX is protonized at acidic environment, which is more hydrophilic and tends to be released. To investigate the drug release in blood, the release studies was then done in culture medium with 10% fetal bovine serum (FBS). Interestingly, the released drug decreased from 22% to 15% (Figure 2c). This is because the DOX was loaded by interacting with BSA. When Fe5C2-BSA-DOX NPs were exposed to culture medium, FBS adsorbed onto DOX, protecting it from detaching from NPs. Due to the consideration that the drug desorption is an endothermic process, we hypothesize that local heating may induce an increased drug release. Fe5C2-BSA-DOX NPs at pH 7.4 were then incubated at 50 ℃ for 48 h to measure their drug release. Compared with drug releasing at 37 ℃, the improved temperature can significantly promote the drug release up to about 49% (Figure 2c). Considering the photothermal effect of Fe5C2 NPs, an on-off pattern of DOX releasing was further carried out. A burst release was observed when NPs was irradiated under 808 nm NIR, indicating the release dosage could be tuned by remote controlled NIR light (Figure 2d). As described above, the alteration of pH value from 7.4 to 5.4 also accelerates drug release, implying an extra- and intracellular stimuli-responsive release of DOX by Fe5C2-BSA-DOX NPs (Figure 2d). It highlights the great potential of Fe5C2-BSA-DOX NPs as carrier for multi-stimuli-triggered drug release. To investigate the stimuli-controlled enhanced drug delivery in vitro, confocal fluorescence test was used to study the cellular uptake and drug release of Fe5C2-BSA-DOX NPs on SK-OV-3 cells, a kind of ovarian cancer cells, which is the 5th leading cause of death for women.51 With the increasing in the incubation time, the red fluorescence from DOX inside cells appears gradually after 1 h (Figure S6a). By staining nucleus with DAPI, it suggested that most DOX were observed in the cytoplasm, which is different from free DOX that can diffuse to nucleus


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(Figure S6b & c). To confirm the localization of Fe5C2-BSA-DOX NPs in cells, lysosomes were stained with LysoTracker Green. After 4 h incubation, Fe5C2-BSA-DOX NPs exhibited well colocalization with lysosomes (Figure 3a), substantiating the cellular uptake of NPs by endocytosis and entrapment within endo/lysomal comparments.52,

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The cellular TEM also

confirmed the presence of NPs in lysosomes (Figure 3b). Due to the acidic environment of lysosomes and the burst release of DOX under low pH condition, we suggested that most DOX are released as free drug in lysosomes. However, most of the released DOX are confined within lysosomes by the membrane, and cannot reach to nucleus to interact with DNA. Fortunately, some previous studies showed that a mild photothermal heating could destroy the cell membrane and increase their permeability to enhance drug delivery.54, 55 We suggested the heat could also improve the permeability of organelle membrane. Moreover, as seen from cellular TEM, MNPs within lysosomes remain their unique core/shell structures (Figure S7), makes it possible to raise temperature by NIR irradiation. Taking these advantages, SK-OV-3 cells were incubated with Fe5C2-BSA-DOX NPs at 37 ℃ for 2 h, and washed with DPBS for 3 times to remove excess MNPs, followed by irradiating with an 808 nm NIR (0.8 W/cm2, 5 min), and re-cultured at 37 ℃ for 30 min. Compared with that incubated in dark, the red fluorescence after irradiation was more in cytosol, which is distinguishable from the green fluorescence emitting from LysoTracker, suggesting the escape of DOX from lysosomes (Figure 3c). Further prolonging the re-incubation time to 2 h, DOX fluorescence inside the nuclei was significantly enhanced after NIR irradiation, indicating the diffusion of the escaped DOX from cytosol to the nuclei (Figure 3d, Figure S8 & S9). From these phenomena, the mechanism for controlled DOX delivery can be described in Scheme 1. Fe5C2-BSA-DOX NPs were first engulfed into lysosomes through an endocytosis mechanism, and release the drug as free form at the acidic condition. Regulated by


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NIR irradiation, Fe5C2 NPs could raise the local temperature and enhance the permeability of lysomal membrane to release the free DOX to cytosol. These released DOX molecules were then diffused to nuclei. For the reason that SK-OV-3 cells are sensitive to DOX and NIR irradiation can enhance delivery of DOX to nuclei, it is suggested that the cancer cell apoptosis can be induced under NIR irradiating.56 As examined by quantifying the cell viability using CCK8 assay (Figure 4a), almost no obvious cytotoxicity for Fe5C2-BSA NPs (0-50 µg Fe/mL) or NIR (808 nm, 0.8 W/cm2, 5 min) alone was observed, demonstrating Fe5C2-BSA NPs can be considered as a nontoxic carrier and NIR under such condition was safe. A significant improvement in cell-killing effect could be realized by Fe5C2-BSA-DOX NPs under NIR irradiation, compared with Fe5C2BSA-DOX NPs alone or Fe5C2-BSA NPs with NIR. The low cytotoxicity for Fe5C2-BSA-DOX NPs could be attributed to the confined release of DOX to cytosol, and the low cell death rate caused by Fe5C2-BSA with NIR may be due to the low temperature increase to kill cells directly by PTT alone. As it is expected, PTT can combine with the previously discussed enhanced DOX delivery, and induce a synergistic effect in cell-killing. Although, under a low concentration, Fe5C2-BSA-DOX with NIR showed a lower toxicity than free DOX, it can remotely control the drug release to reduce fewer side effects on unexpected parts, and when the MNPs are at a high concentration, the synergistic effect endows it higher cell inhibition even better than free DOX. The cell viability assay was also investigated on NIH 3T3 cells, macrophage (RAW 264.7 cells) and SK-OV-3 cells (Figure 4b), which showed that Fe5C2-BSA-DOX NPs are with low cytotocity to all these cell lines, demostrating that it is an ideal candidate for drug delivery. From the in vitro assays, it is suggested that the higher concentration of NPs accumulated in the tumor site, the better of the synergistic effect will be. Benefiting from the high magnetic


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property of Fe5C2-BSA NPs, magnetic targeting was applied to direct MNPs to the tumor site. In addition, Fe5C2 NPs have been proved to be an excellent contrast agent in T2-weighted MRI,44, 45 and thus, Fe5C2-BSA-DOX NPs could be served for imaging-guided therapy. Here, Fe5C2-BSADOX NPs were intravenously injected through tail vein into SK-OV-3 ovarian tumor bearing mice (dose of DOX=0.4 mg/kg, Fe=10 mg/kg), which have a magnet of 0.3 T located at the tumor site. MR images of the tumor were obtained before and 1 days, 3 days and 7 days postinjection, which shows an obvious signal dropping at the tumor sites after administration (Figure S10). Particularly in the 3rd day, the tumor shows the greatest hypo-intensities, indicating the maximum NPs were accumulated in tumors. Compared to that without magnetic field, an enhanced darkening was observed in the targeting group, suggesting a much higher level of MNPs accumulated in the tumor site by magneti targeting (Figure 5a). The mice were then sacrificed and tumor tissue was dissected for Prussian blue staining. More positively stained areas are observed in the targeted group (Figure S11), confirming the enhanced delivery of MNPs to the target site. To investigate whether DOX has been delivered to the tumor site together with MNPs, fluorescence of DOX in tumor tissue was examined by fluorescence microscopy. A remarkable red fluorescence was observed at the tumor site of mice in the magnetic targeting group, while indiscernible fluorescence signal was observed in the Fe5C2BSA-DOX group, and also, almost no fluorescence from the free DOX-injected mice was detected (Figure 5b). All these results demonstrated that Fe5C2-BSA-DOX NPs was an effective carrier to deliver drugs to the tumor site under the guidance of magnetic field. To investigate the bio-distribution of Fe5C2-BSA-DOX NPs in animal model, the concentrations of Fe levels in tissue samples were measured after intravenous injection. Major organs including tumors were harvested 3 days after intravenous injection. ICP-AES was used to


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quantify the amount of iron in various organs. As shown in Figure 5c, the concentrations of MNPs are very low in heart and kidney. Despite that most MNPs accumulated in liver, magnetic targeting endows much higher tumor uptake and lower liver uptake of Fe5C2-BSA-DOX NPs, indicating the reduced influence of MNPs on normal tissues. Our earlier data have convinced that Fe5C2-BSA-DOX NPs could enhance the therapeutic effect under a mild NIR irradiation, we proposed the accumulation of Fe5C2-BSA-DOX NPs at the tumor site would improve the therapeutic effect by the combination of PTT and chemotherapy. The tumor-bearing mice were intravenously injected with Fe5C2-BSA-DOX NPs (dose of Fe=10 mg/kg), and a magnet was located at the tumor site in the magnetic targeting group. 3 days after injection, the tumors were irradiated by an 808 nm laser at a power density of 0.8 W/cm2 for 10 min. an IR thermal camera was used to monitor the temperature changes during irradiation. Mice treated with Fe5C2-BSA-DOX NPs by magnetic targeting showed an quick local temperature rising to above 60 ℃ at the tumor region, while the tumor temperatures of mice without magnetic field exhibited much lower heat rising (to about 40 ℃) (Figure 6a). Encouraged by the fascinating magnetic targeting and photothermal effect by Fe5C2-BSADOX NPs, we then investigate the therapeutic effect on SK-OV-3-tumor-bearing mice. When the tumor sizes reached to 500 mm3, mice were divided into seven groups with five mice in each group. Fe5C2-BSA-DOX, Fe5C2-BSA, DOX or saline were administrated by an intravenous injection. A magnetic was placed at the tumor site for magnetic targeting, and in the NIR treated group, the tumors were exposed to an 808 nm laser (0.8 W/cm2) for 5 min. The tumor region in the mice injected with Fe5C2-BSA-DOX or Fe5C2-BSA together with magnetic target and NIR irradiation became whitish, suggesting disruption of blood perfusion.57 One mouse from each group was sacrificed on the 4th day post-treatment, and hematoxylin and eosin (H&E) staining


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was applied on the tumor (Figure 6b). Apparent degenerative changes of coagulative necrosis appeared on mouse treated with PTT mediated by Fe5C2-BSA NPs and Fe5C2-BSA-DOX NPs under the guidance of magnetic field, while less necrotic areas were observed in the mouse subjected to PTT by Fe5C2-BSA-DOX NPs without magnetic field. No obvious malignant necrosis was shown in the other four groups. Tumor growth rate was monitored every three days (Figure 6c). As expected, compared with the control group, an effective inhibition of tumor was observed in the Fe5C2-BSA with NIR treated group under the guidance of magnetic field due to the PTT effect, but their tumors began to increase again after 21 days (red line). In comparison, in the group injected with Fe5C2-BSA-DOX NPs under laser irradiation and magnetic guidance (black line), a complete eradication of tumor was achieved without relapse within 27 days, as it is a result of the combination of PTT and triggered DOX release under NIR. On the contrary, Fe5C2-BSA-DOX NPs treated with magnetic targeting but in the absence of NIR irradiation exhibits a much lower therapeutic effect, which can be attributed to either the lower DOX release or the lack of the PTT effect (fuchsia line). Without magnetic field, Fe5C2-BSA-DOX NPs showed lower tumor inhibition even with NIR irradiation, since much fewer MNPs were transported to the tumor (light blue line). No significant differences were induced in the free DOX or NIR treated group, which suggested neither the free DOX at applied dose nor NIR radiation alone was effective in inhibiting the tumor growth (green or dark blue group). From the above results, we can conclude that the enhanced antitumor activity by Fe5C2-BSA-DOX NPs with NIR and magnetic field was a synergism between magnetic targeting, PTT effect and the increased drug release. Considering that body weight is an important parameter to evaluate the systemic toxicity of material, body weight of mice was measured during the treatments. As given in Figure 5d, body


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weight in the Fe5C2-BSA-DOX or Fe5C2-BSA with magnetic and NIR group decreased for the first three days, which might be the weight of the ablated tumor. However, it increased dramatically afterword, and the overall body weight in all groups increased except for the free DOX-treated one, indicating that the Fe5C2-BSA-DOX NPs could reduce the adverse side effects of traditional anticancer drugs (Figure 6d). Mice were then sacrificed at the 27th day to get the digital photos of tumor and examination on any effects on major organs by investigating H&E staining. The result then reveals that only the tumor in the targeted chemo-hyperthermia group disappeared completely and no significant abnormality in major organs by these treatments was observed (Figure S12 & S13). Although, further investigations are still needed to warrant Fe5C2BSA-DOX a safe biocompatible material, our preliminary results suggested that it is with low toxicity to mice within 1 month, and maybe an ideal candidate for multi-stimuli-regulated photochemothermal therapy.

Conclusion In summary, we developed a novel type of multi-stimuli-triggered drug delivery system based on BSA-functionalized Fe5C2 NPs. It shows enhanced DOX loading efficiency, high photothermal effect, and a facile controlling of the drug release by both pH value and NIR light. After engulfing into cells, the photothermal heating, on the one hand, resulted in the improvement in the drug release due to the lysosome/endosome disruption, on the other hand, caused the direct cell killing by PTT. The combined therapy was confirmed both in vitro and in vivo, which exhibited excellent synergistic therapeutic efficacy. Tumor targeting can also be achieved through magnetic field, which dramatically increased the specific drug delivery to the tumor site and reduced the premature release in normal organs, and this selective delivery can be


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monitored by T2-weighted MRI. This drug delivery platform thus turns out to be a promising drug carrier, that can be controlled by multi stimuli and with great potential for photochemothermal cancer therapy.

Materials and methods Synthesis of Fe5C2 NPs. Fe5C2 NPs were synthesized following our previously reported method.46 Briefly, 14.5 g octadecylamine and 0.113 g cetyltrimethylammonium bromide were mixed, and degassed under a flow of N2. The mixture was heated to 120 °C and injected with Fe(CO)5 (0.5 mL, 3.6 mmol) under a N2 blanket, then heated to 180 °C at 10 °C/min and kept for 10 min. After that, the mixture was further heated to 350 °C at 10 °C/min and kept for 10 min before it was cooled to room temperature. The product was washed with ethanol and hexane and collected for further characterization. Modification of Fe5C2 NPs. The modification was carried out according to a reported protocol with modification.47, 48 Briefly, 20 mg of Fe5C2 NPs were dispersed in 30 mL of chloroform. Then, 120 mg of dopamine in 12 mL of dimethyl sulfoxide (DMSO) was added to the solution. The mixture was stirred to form a homogeneous solution and heated to 70 ℃ for 1 h. After cooling down, the dopamine coated Fe5C2 NPs (Fe5C2-DOPA NPs) were collected by centrifugation, washed twice with chloroform/DMSO 1:1 mixture, and dispersed in DMSO (20 mg/mL) with the aid of sonication. Afterwards, the product was added dropwise to bovine serum albumin (BSA) solution in water (12 mg/mL, 1:7 v/v ratio) with sonication to obtain a homogeneous solution. The final product (Fe5C2-BSA NPs) was collected by centrifugation, and washed with water twice.


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Loading Fe5C2 NPs with DOX. DOX was dissolved in DMSO at a concentration of 16 mg/mL, and Fe5C2-DOPA NPs were dissolved in DMSO with concentration of 20 mg/mL. Different volumes of the former solution (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL) were added into 1 mL latter solution respectively, and the mixtures were sonicated for 10 min. Half of each mixture was further added dropwise to 15 mL BSA aqueous solution (12 mg/mL) with sonication for 15 min (forming Fe5C2-BSA-DOX NPs), while the left half mixure continue sonicating for 15 min (forming Fe5C2-DOPA-DOX NPs). The final product was collected by centrifugation, and washed with PBS twice, and dispersed in 10 mL PBS. Loading of DOX on Fe5C2-BSA-DOX were quantified using a UV–vis spectrophotometer (UV2550, Shimadzu, Japan) using the absorbance peak at λ = 480 nm by subtracting the absorbance of Fe5C2-BSA at that wavelength. The loading capacity was calculated as following:

DOX Releasing Efficiency of Fe5C2-BSA-DOX NPs. The release of DOX from Fe5C2-BSADOX NPs was studied by dialyzing the sample at 37 °C or 50 °C in the dark in NaHCO3/Na2CO3 buffer (2 mM) at pH 5.4, and 7.4, or in culture medium containing 10% FBS for different periods of time. DOX released was collected and determined by UV–vis absorbance spectroscopy at λ = 480 nm. For the multiple stimuli-triggered DOX release, Fe5C2-BSA-DOX NPs were dialyzed in the buffer solution with pH value of 5.4 or 7.4 at room temperature. DOX released was collected every 1 h. At the time period of 2 h, 5 h and 8 h, an optical-fiber-coupled power-tunable diode laser (continuous wave) with wavelengths of 808 nm and power intensity of 0.8 W/cm2 was employed for 5 min. After irradiating, the released DOX was collected immediately, and measure the dosage by using UV-vis spectrometry at λ = 480 nm.


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Characterization. Transmission electron microscopy (TEM) was carried out on an FEI Tecnai T20 microscope. X-ray diffraction (XRD) patterns were recorded on a Rigaku DMAX-2400 Xray diffractometer equipped with Cu Kα (λ = 1.5405 Å) radiation. Magnetization was measured by a superconducting quantum interference device (SQUID). Dynamic light scattering (DLS) was measured using a particle size analyzer (ZetaPALS, Brookhaven Instruments, Holtsville, NY). The concentrations of Fe were quantified using an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Profile, Leeman, USA). Cell culture. Human ovarian carcinoma cell line (SK-OV-3) was obtained from Cancer Institute and Hospital Chinese Academy of Medical Science. All cell culture related reagents were purchased from Invitrogen. Cells were cultured in RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under 5% CO2 with 100% humidity. Confocal Laser Scanning Microscope Study. SK-OV-3 cells were seeded at a density of 1 × 105 cells/well in a 24-well plate over glass coverslips, and incubated at 37 °C under 5% CO2 overnight. After incubated with Fe5C2-BSA-DOX or free DOX ([DOX]=5 µg/mL) for 4 h at 37 °C, the culture medium was carefully aspirated, and washed with Dulbecco’s phosphate buffered saline (DPBS) for three times, followed by fixed with 1 mL of 4% paraformaldehyde for 15 min. Cells on coverslips were mounted in Vectashield antifade mounting medium with DAPI. To investigate the colocalization of DOX and lysosomes, cells were stained with 1 mM Hoechst 33342 (Invitrogen) for 20 min and 1 mM LysoTracker Green DND-26 (Invitrogen) for 5 min after incubation with Fe5C2-BSA-DOX for 4 h. To investigate the photothermally controlled drug delivery by NIR irradiation, cells were treated with Fe5C2-BSA-DOX ([DOX]=5 µg/mL) for 2 h. After washing with DPBS for 3 times, cells were irradiated by an 808 nm laser at a


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power density of 0.8 W/cm2 for 5 min, and re-cultured at 37 °C for 30 min. Cells without NIR irradiation were used as control. Stained the nuclei with 1 mM Hoechst 33342 for 20 min and lysosomes for 1 mM LysoTracker Green DND-26 for 5 min. Prolonging the re-cultured time to 2 h, cells were fixed with 4% paraformaldehyde, and stained with DAPI. Cells were imaged by using confocal laser scanning microscopy (Zeiss Axio Observer A1, Germany) under laser excitation at 488 nm (for DOX), 364 nm (for DAPI or Hoechst 33342), and 515 nm (for LysoTracker). Cellular TEM Imaging. SK-OV-3 cells were incubated with Fe5C2-BSA-DOX ([Fe]=10 µg/mL) for 4h, and washed with DPBS for three times, then collected by centrifugation. The cell pellets were fixed in DPBS solution containing 2% gluteraldehyde and 2.5% paraformaldehyde for 2 h, and postfixed in 1% osmium tetrosxide, then washed by sodium cacodylate buffer and dehydrated with gradient alcohol, and replaced by propylene oxide and embedded in Epon 812. Semithin sections (1um) were cut, stained by methylene blue, and localized by a microscope. Ultrathin sections were stained with uranylo acetate and leaed citrate, and examined under JEM1400 electron microscope. Cytotoxicity of Fe5C2-BSA-DOX NPs. The in vitro cytotoxicity of Fe5C2 NPs was evaluated by standard CCK8 assay. SK-OV-3 cells were seeded into 96-well cell culture plates at 5 × 103 cells per well and incubated overnight at 37 ℃ under 5% CO2. After removing the culture medium fresh culture medium containing various concentrations of Fe5C2-BSA-DOX, Fe5C2BSA and free DOX were added, followed by further incubation for 24 h. These MNPs are equal in concentration, with [DOX]= 20, 10, 2, 0.2, 0.02 µg/mL, and [Fe]=50, 25, 5, 0.5, 0.05µg/mL, respectively. For the NIR irradiated groups, the cells were irradiated by an 808 nm laser (0.8 W/cm2) for 10 min before the following 24-incubation. In the NIR only group, 0.1 mL culture


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medium was added before irradiation. The standard CCK8 assay was carried out to determine the cell viabilities relative to the control untreated cells.

Animals and tumor model. All experiments involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Peking University, Beijing China. 4-5 weeks old Balb/c nude mice, with the average weight of 20 g, were provided by the Beijing Center for Disease Control and Prevention, Beijing, China. Mice were maintained in SPF animal house under a 12 h light and 12 h darkness cycle and were fed a standard laboratory diet and tap water ad libitum. Mice were injected with 107 SK-OV-3 cells (0.2 mL cells in 1640 culture medium without FBS) subcutaneously at the right axillary region. In vivo MRI. When the tumor volumes reached about 500 mm3, two mice were administered an intravenous injection via tail vein of Fe5C2-BSA-DOX (10 mg Fe/kg). A permanent magnet was located at the tumor site in the magnetic targeting one. MR images were acquired before and 3 days after injection. The T2-map images were obtained in a clinic 3 T MRI scanner (Philips), and the sequence is TR = 1200 ms, TE =30.2 ms, slice thickness = 2.5 mm. Biodistribution. 9 mice with tumor volumes of 500 about mm3 were randomly divided into 3 groups: (1) Fe5C2-BSA-DOX NPs (200 µL, 10 mg Fe/kg) injected group with a permanent magnet located at the tumor site; (2) Fe5C2-BSA-DOX NPs (200 µL, 10 mg Fe/kg) injected group; (3) control group injected with saline. 3 days after injection, mice were sacrificed, and major organs including tumors were collected, weighted and lysed with aqua regia. ICP-AES was applied to calculate the concentration of Fe. The levels of Fe5C2-BSA-DOX in various organs are presented as the percentage of injected dose per gram of tissue (% ID/g).


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In vivo temperature measurement during NIR irradiation and photothermal therapy. Two SK-OV-3 tumor bearing mice were intravenously injected with Fe5C2-BSA-DOX (10 mg Fe/kg), respectively, and a permanent magnet was located at the tumor site in the magnetic targeting one. 3 days after injection, the mice were received an NIR irritation (808nm, 0.8W/cm2) for 10 min. The thermographic maps of the tumor tissues were obtained by thermal imaging camera during illumination (0, 1, 3, 5, 10 min). Photothermal ablation was performed when the tumors reached to about 500 mm3. 7 groups, with 5 mice in each group were involved in the current study: (1) Fe5C2-BSA-DOX (10 mg Fe kg-1, 0.4 mg DOX kg-1) with NIR irradiation and magnet on tumor site; (2) Fe5C2-BSA (10 mg Fe kg-1) with NIR irradiation and magnet on tumor site; (3) Fe5C2-BSA-DOX (10 mg Fe kg-1, 0.4 mg DOX kg-1) with NIR irradiation; (4) Fe5C2-BSA-DOX (10 mg Fe kg-1, 0.4 mg DOX kg-1) with magnet on tumor site; (5) free DOX injected only; (6) NIR irradiated only; (7) control group injected with saline. 808 nm NIR laser irradiation was carried out 3 days after injection at an output power 0.8 W/cm2 for 5 min. Tumor sizes and body weights were measured every 3 days during the treatment. Tumor volume was calculated according to the formula of (a×b2)/2, where a and b are the long and short diameters of the tumor, respectively. Histological evaluation. 3 days after treatment, one mouse from each group were euthanized, and tumor was harvested from the necropsy; 27 days after treatment, mice from each group were euthanized and major organs were recovered, followed by fixing with 10% neutral buffered formalin. After the organs were embedded in paraffin and sectioned at 5 mm, hematoxylin and eosin (H&E) or Prussian blue staining were performed for histological examination. The slides were observed under optical microscope (OLYMPUS BX51, CCD: DP70). Frozen sections were also performed on the tumor tissues with the purpose for microscopic analysis on the local


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deposition of DOX by magnetic targeting. The tumor tissues were firstly embedded and frozen by an OCT (Jung, Tissue freezing medium, Leica). Cross sections of 10 µm thickness were cut using a cryomicrtome (Leica) and mounted on the glass slides. The slides were then observed under a confocal microsope. Statistical analysis. Results are expressed as means ± standard deviation (SD).

Figure 1. (a) TEM image of Fe5C2 NPs. (b) Zeta potential of dopamine-coated Fe5C2 NPs (Fe5C2-DOPA, red column), Fe5C2-BSA NPs (blue column) and DOX loaded Fe5C2-BSA NPs (Fe5C2-BSA-DOX, green column). (c) Room-temperature magnetic hysteresis loops of Fe5C2


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NPs before (black line) and after (red line) modification. (d) Temperature profiles of water and aqueous dispersions of Fe5C2 NPs at different concentrations determined by Fe content (0.01, 0.1, 1.0 mM) at the laser density of 0.8 W/cm2.

Figure 2. (a) UV-vis absorbance spectra of Fe5C2-DOPA NPs, Fe5C2-DOPA-DOX NPs, Fe5C2BSA NPs and Fe5C2-BSA-DOX NPs. (b) Comparison of drug loading capacity of Fe5C2-DOPADOX NPs and Fe5C2-BSA-DOX NPs. Error bars are based on standard deviations of triplicated samples. (c) Drug release profile of Fe5C2-BSA-DOX NPs under pH 5.4 at 37 ℃, pH 7.4 at 50


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℃, pH 7.4 at 37 ℃, and pH 7.4 at 37 ℃ in culture medium with 10% BSA. Error bars are based on standard deviations of triplicated samples. (d) Sequential stimuli-responsive DOX release profile of Fe5C2-BSA-DOX NPs with NIR irradiation under pH 5.4 or pH 7.4. Arrowed parts are the time point when NIR irradiating was carried out for 5 min. The NIR wavelength was 808 nm and power was 0.8 W/cm2. Error bars are based on standard deviations of triplicated samples.

Figure 3. (a) Fluorescence image of SK-OV-3 cells treated with Fe5C2-BSA-DOX NPs for 4 h. Nuclei were stained with Hoechst 33342 and lysosomes were stained with LysoTracker Green. Scale bars are 20 µm. (b) TEM image of SK-OV-3 cells treated with Fe5C2-BSA-DOX NPs for 4 h. (b2) is the high magnification for the rectangle area in (b1). Scale bar in (b1) is 5 µm, in (b2) is 200 nm. (c,d) Fluorescence images of SK-OV-3 cells treated with Fe5C2-BSA-DOX NPs for 2 h followed by irradiation/non-irradiation (808 nm, 0.8 W/cm2, 5 min). Cells were re-incubated for (c) 30 min and (d) 2 h after treating. Nuclei in (c) were stained with Hoechst 33342, and in (d)


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were stained with DAPI. Scale bars are in (c) are 20 µm and in (d) are 50 µm. Lysosomes in (c) were stained with Lysotracker Green, and the arrowed part is cytosol.

Scheme 1. Schematic illustration for the light-triggered drug delivery.

Figure 4. (a) Cell viability tested on SK-OV-3 cells. NIR wavelength was 808 nm, with intensity of 0.8 W/cm2. 20 µg DOX/mL in Fe5C2-BSA-DOX NPs contains 100 µg Fe/mL, and Fe5C2BSA-DOX NPs and Fe5C2-BSA NPs are at the same Fe concentration. Error bars are based on standard deviations of six parallel samples. (b) Cell viability of Fe5C2-BSA-DOX NPs tested on SK-OV-3 cells, Macrophage and NIH 3T3 cells. Error bars are based on standard deviations of six parallel samples.


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Figure 5. (a) T2-weighted MR images of SK-OV-3 tumor bearing mice before and after intravenous injection of Fe5C2-BSA-DOX NPs under the guidance of magnetic field (top) and without magnetic field (bottom). Tumors are marked by the white arrow. (b) Fluorescence images of the tumors by a froze section. The left column is the fluorescence from DOX, the middle column is the bright field, and the right column is their merged figure. The scale bars are 200 µm. (c) Biodistribution of Fe5C2-BSA-DOX NPs in mice 3 days after intravenous injection. The Fe amounts in tissue samples were measured by ICP-AES. Error bars are based on standard deviations of three parallel samples.


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Figure 6. (a) Infrared thermal image of tumor bearing mice exposed to the NIR laser (808 nm, 0.8 W/cm2, 10 min) after intravenously inject with Fe5C2-BSA-DOX with (top) or without (bottom) magnetic targeting. (b) H&E stained tumor sections collected from different groups of mice 3 days post treatment: (b1) Fe5C2-BSA-DOX NPs with magnetic field and NIR; (b2) Fe5C2BSA NPs with magnetic field and NIR; (b3) Fe5C2-BSA-DOX NPs with NIR; (b4) Fe5C2-BSADOX NPs with magnetic field; (b5) free DOX only; (b6) NIR only; and (b7) saline. Scale bars are 50 µm. (c) Tumor growth curves of different groups of tumor-bearing mice after treatment. The tumor volumes were normalized to their initial sizes. Error bars represent the standard deviations


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of 5 mice per group. (d) Body weight of mice in different groups. The error bars represent the standard deviations of 5 mice per group.

AUTHOR INFORMATION Corresponding Author * Corresponding to [email protected]; and [email protected]; ACKNOWLEDGMENT This work was supported in part by National Natural Science Foundation of China (NSFC) (51125001, 51172005, 81421004, 61078073, 81172182, and 21105120), the Research Fellowship for International Young Scientists of the National Natural Science Foundation of China (51450110437), the Doctoral Program (20090001120010), the Natural Science Foundation of Beijing (2122022), NSFC/RGC Joint Research Scheme (51361165201), Beijing Project of Science and Technolofy (Z141100003814012) and Opening Project of Beijing National Laboratory for Molecular Science (20140119). Supporting Information mainly include the following information: XRD pattern of Fe5C2 NPs, TEM image of Fe5C2 NPs after modification, hydrodynamic diameter of Fe5C2 NPs and Fe5C2BSA NPs, ultraviolet-visible-near-infrared optical absorption spectra of Fe5C2 NPs, TEM image of Fe5C2-BSA NPs exposed in culture medium with 10% FBS for 1 month, fluorescence images of SK-OV-3 cells incubated with Fe5C2-BSA-DOX NPs and free DOX for different time schedule, relativel fluorescence in nuclei SK-OV-3 cells incubated with Fe5C2-BSA-DOX NPs and free DOX for different time schedule, cellular TEM image of SK-OV-3 cells treated with Fe5C2-BSA-DOX NPs for 4 h, DOX fluorescence images of SK-OV-3 cells treated with Fe5C2-


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BSA-DOX NPs with or without irradiation, relative fluorescence in the nuclei of SK-OV-3 cells treated with Fe5C2-BSA-DOX NPs for 2 h, T2-weighted MR images of SK-OV-3 tumor bearing mice before and after intravenous injection of Fe5C2-BSA-DOX NPs for different time points, representative Prussian blue stained tumor sections images 3 days after injecting with Fe5C2PSA-DOX with or without magnetic targeting, photographs at day 27 of representative mice from different groups, H&E stained images of major organs collected from different groups of mice. Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website at DOI: Supplementary Figures 1-13 (PDF)

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SYNOPSIS


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Multistimuli-Regulated Photochemothermal Cancer Therapy Remotely

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