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Multifunctional Theranostic Nanoparticles Derived from Fruit Extracted Anthocyanins with Dynamic Disassembly and Elimination Abilities Caina Xu, Yanbing Wang, Haiyang Yu, Huayu Tian, and Xuesi Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b03525 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018
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Multifunctional Theranostic Nanoparticles Derived from Fruit Extracted Anthocyanins with Dynamic Disassembly and Elimination Abilities Caina Xu,‡,||,† Yanbing Wang,‡,§,||,† Haiyang Yu,‡,|| Huayu Tian,‡,§,||,* and Xuesi Chen‡,§,||,*
‡
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China
§
University of Science and Technology of China, Hefei, 230026, P. R. China
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Jilin Biomedical Polymers Engineering Laboratory, Changchun, 130022, P. R. China
ABSTRACT: Low toxic theranostic nanoparticles that can simultaneously achieve effective tumor accumulation and rapid renal clearance are highly desired for imaging contrast agents and photothermal therapy (PTT) in tumor diagnosis and therapy. Herein, we report a one-pot method for preparing multifunctional nanoparticles (FeAP-NPs) based on the coordination interaction of natural polyphenols (anthocyanins) extracted from fruits, FeIII ions, and poly(L-glutamic acid)-gmethoxy poly(ethylene glycol) copolymers. The FeAP-NPs possess the following favorable advantages: (1) The components of FeAP-NPs originate from the natural products, endogenous element, and poly(amino acid) derivatives, guaranteeing their safety for in vivo application. (2) FeAP-NPs exhibit excellent dual photoacoustic (PA)/magnetic resonance (MR) imaging capacity and high photothermal efficiency. (3) FeAP-NPs can overcome the intractable dilemma of enhanced permeability and retention (EPR) effect and renal clearance for nanomedicine through the dynamic disassembling ability, which induces the switch of the elimination pathway.
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Complete tumor ablation is realized by PTT in MCF-7 bearing nude mice under the precise guide of PA and MR imaging. The detailed evaluation of the safety, biodistribution and elimination behaviors of FeAP-NPs are conducted in vitro or in vivo. This work provides a promising comprehensive solution for nanomedicine clinical application. KEYWORDS:
anthocyanins,
photoacoustic
imaging,
magnetic
resonance
imaging,
photothermal therapy, EPR effect, dynamic disassembling Theranostic nanoparticles have potential use in cancer treatment owing to the growing use of contrast agents and drug carriers.1,2 Such nanoparticles are expected to change the landscape of biotechnology and pharmaceutical industries in the future.3 However, the translation of theranostic nanoparticles from bench to clinical practice is hindered by several challenges. Safety is one of the main issues in the clinical application of nanoparticles.4 Most nanoparticles could accumulate in the organs, leading to acute or chronic toxicity. Therefore, developing safe, reliable, biocompatible, and nontoxic nanoparticles remains a major challenge. In addition, nanoparticles that have multifunctional properties, and simultaneously accomplish diagnosis, monitoring, and treatment, are useful in tumor therapy.5-8 Moreover, nanoparticles with suitable size generally induce the EPR effect-mediated tumor accumulation.7 However, large-size nanoparticles with efficient EPR effect accumulate in reticuloendothelial systems (RES) for hours and even for months, inducing potential toxicity effects on the body.9,10 Although ultrasmall-size of nanoparticles (< 6 nm) are rapidly eliminated from the body via the renal system, they exert a weakened EPR effect on tumor tissues.9,11 The above-mentioned dilemma concerning EPR effect and renal clearance severely hampers the nanoparticles’ further clinical application. At present, there are only a few reports addressing this intractable dilemma. Above all, it is both appealing and practical to develop theranostic nanoparticles for simultaneously
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solving the following three concerns including safety, multifunction and the ability to overcome the dilemma of EPR effect and renal clearance.
Figure 1. Schematic illustration of construction and in vivo application of multifunctional theranostic nanoparticles derived from fruit extract. Herein, multifunctional nanoparticles (FeAP-NPs) were prepared through a simple approach using blue honeysuckle extracted anthocyanins (ACN), FeIII ions, and poly(L-glutamic acid)-gmethoxy poly(ethylene glycol) (PLG-g-mPEG) copolymers (Figure 1). Natural anthocyanins extracted from blue honeysuckle (Lonicera caerulea L.) present positive therapeutic and nontoxic properties against ophthalmic, inflammatory, and coronary diseases.12,13 ACN-based natural colorants are approved by the US Food and Drug Administration.14,15 Iron is an essential
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nutrient element in the metabolic reactions in the human body. PLG-g-mPEG copolymers, which improve the biocompatibility and physiological stability of FeAP-NPs, are derivatives of PLG that are currently undergoing in clinical trials in East Asia.16 Therefore, as-prepared nanoparticles possess the possibility to exhibit good biocompatibility. Moreover, the FeAP-NPs demonstrate both good T1 relaxivity and strong absorption in the near-infrared (NIR) region and can thus serve as a contrast agent for dual MR/PA imaging and a photothermal agent for PTT. Combined MR and PA imaging can realize the precise PTT accurately locating the tumor tissue. Furthermore, the FeAP-NPs possess in vivo dynamic disassembling ability initiated by deferoxamine mesylate (DFO), which is an iron chelator with iron chelation capability that clinically reduces excess iron in the body.17 The FeAP-NPs can be disassembled by DFO, and mechanism of DFO for FeAP-NPs disassembling may due to that the coordination between iron and ACN was destroyed by the chelation of iron and DFO. Thus the application of DFO for FeAP-NPs disassembling could regulate the hepatic clearance in vivo. This feature can be used for addressing the dilemma concerning EPR effect and renal clearance, and decreasing the accumulation time of FeAP-NPs in the liver. In this work, we performed detailed characterizations and experiments in vitro and in vivo, confirmed the properties of FeAP-NPs and their potential use in tumor diagnosis and treatment. RESULTS AND DISCUSSION ACN was obtained from blue honeysuckle, and the extraction procedure was carried out according to a previously reported method.18 Anthocyanin profiles in blue honeysuckle anthocyanin fractions were confirmed by its high-performance liquid chromatography (HPLC), high-performance liquid chromatography-tandem mass spectroscopy (HPLC-DAD-MSI-MS), the most abundant anthocyanin monomer in blue honeysuckle was cyanidin-3-O-glucoside
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(Figure S1-3), which accounted for more than 95 % of the total anthocyanins. PLG-g-mPEG copolymers were synthesized and purified as previously described.19 The structure of PLG-gmPEG was shown in Figure S4. The molecular weight of PLG-g-mPEG was 32,900 Da. Approximately 4 % of glutamic acid units was grafted by mPEG 5000 through 1H NMR spectrum calculation (Figure S5). The FeAP-NPs were prepared by sequentially mixing the solution containing PLG-g-mPEG, FeCl3 and ACN. A simple purification process by three centrifugation/redispersion cycles was then performed to remove excess PLG-g-mPEG, ACN and FeIII (Figure 2A). This one-pot procedure is facile, green and could be scaled up to dozens of grams, and the physicochemical characterizations of the FeAP-NPs kept unchanged in comparison with the small-scale FeAP-NPs (Figure S6). The FeAP-NPs were fairly stable in various solutions, under high temperature (60 oC, 30 min), and could remain stable for 6 months of preservation at 4 oC (Figure S7-9). As a consequence, the preparation process for FeAP-NPs could be a potential technology for further application in industrialization. In addition, several kinds of metal ions were selected to coordinate with ACN, the results showed that the color of metal-ACN complexes was different, in special, and color of metal-ACN complexes constructed by Fe and ACN was black (Figure S10). The Fe-ACN complexes exhibited the highest UV-vis absorption in 808 nm, which could be beneficial for photothermal therapy (Figure S11).20 First, the morphology of the FeAP-NPs was observed by transmission electron microscopy (TEM), which revealed that the spherical nanoparticles had an average diameter of 65 nm (Figure 2B). According to the results of the dynamic light scattering (DLS) measurement, the average hydrodynamic diameter of the FeAP-NPs was 73 nm (Figure 2C), which will be beneficial for tumor accumulation via EPR effect. The FeAP-NPs also will have longer retention time in tumor regions than free molecules or ultra-small nanoparticles, and will provide a much
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longer periods of time for tumor diagnosis and therapy due to the EPR effect.21,22 The zeta potential of the FeAP-NPs was -10.4 mV. The uptake by normal cells and opsonization by the immune system would be decreased by the negative potential.23 The XRD patterns of the FeAPNPs displayed amorphous properties with a broad humps caused by diffuse scattering (Figure S12), a rapid and kinetically dominated nonequilibrium process might contribute to the formation of these amorphous nanoparticles.24 Fourier transform infrared (FTIR) spectrum was performed for the characterization of the interaction between PLG-g-mPEG and FeIII (Figure 2D). The new bands at 1541 and 1595 cm-1 in the spectrum of PLG-g-mPEG + FeIII, which also existed in the spectrum of FeAP-NPs, could be attributed to the carboxyl (-COO)/FeIII interactions.25,26 The characteristic band at 1107 cm-1 (C-O-C stretching) in the spectra of PLG-gmPEG, PLG-g-mPEG + FeIII and FeAP-NPs illustrated the existence of PLG-g-mPEG in FeAPNPs.25,27 The UV-vis absorption spectrum of the FeAP-NPs showed a new absorbance band at 596 nm compared with the spectrum of ACN and PLG-g-mPEG, which indicated the presence of coordination between ACN and FeIII (Figure 2E).22,24 Furthermore, the XPS results clearly showed the existence of C, O, N, and Fe elements in the FeAP-NPs, and the Fe 2p signals were 712.2 and 725.7 eV, suggesting the presence of ferric species in the FeAP-NPs (Figure S13). The SEM-EDS analysis verified that the FeAP-NPs were composed of C, O, Fe, and N elements, and the weight percentage of Fe element in the FeAP-NPs was 2.3 % (Figure S14). The Fe and N elements were quantitatively analyzed by ICP-MS, and the elemental analysis, and the results were listed in Table S1, and the weight percentage of Fe was consistent with EDS analysis. The molar ratio of ACN to FeIII was 2.4:1, which might be attributed to the coexistence of the biscomplex and tris-complex states of the FeIII and ACN complexes.28 The above results suggested that FeAP-NPs were successfully prepared through the coordination interaction among PLG-g-
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mPEG, FeIII and ACN.
Figure 2. Preparation and characterization of FeAP-NPs. A) Schematic illustration for the preparation and structure of FeAP-NPs. B) TEM image of the FeAP-NPs (scale bar, 100 nm). C) DLS measurement of the FeAP-NPs. D) FTIR spectra of PLG-g-mPEG, PLGg-mPEG + FeIII and FeAP-NPs. E) UV-vis absorption spectra of FeAP-NPs, ACN and
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PLG-g-mPEG. Given that the FeAP-NPs exhibited a broad and strong absorption between 680 and 900 nm (Figure 2E), the FeAP-NPs could induce optoacoustic signals as a multispectral optoacoustic tomography (MSOT) contrast agent. As shown in Figure 3A and B, the PA signals were enhanced by the increased concentrations of FeAP-NPs. For in vivo PAI, MCF-7 tumor-bearing Balb/c nude mice were intravenously injected with FeAP-NPs (100 µL, 250 µg mL-1), and PA signals were acquired at different time intervals (Figure 3C and D). An obvious PA signal was observed in at the tumor region at 6 h post injection, and the tumor region of the mice exhibited the highest PA signal level at 24 h post injection.
Figure 3. PAI and MRI evaluation of FeAP-NPs. A) PA images of FeAP-NPs at different concentrations in vitro. B) Linear relationship between PA signals and concentrations of
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FeAP-NPs. C) PA images of mice bearing tumors at various time points after intravenous injection of FeAP-NPs in vivo. The circles indicate the tumors. D) Quantification of PA signals in tumor at various time points after intravenous injection of FeAP-NPs in vivo. E) MR images of FeAP-NPs at different concentrations in vitro. F) R1 relaxivity of FeAP-NPs as a function of the FeIII molar concentration in the solution. G) MR images of mice bearing tumors at various time points after intravenous injection of FeAP-NPs in vivo. H) Quantification of MR signals in tumors at various time points after intravenous injection of FeAP-NPs in vivo. The Fe content was used to define the concentration of the FeAP-NPs in all experiments. On the other hand, it was reported that nanoparticles based on the FeIII and polyphenol interactions had MR imaging capability.29 The T1-weighted MRI images of the FeAP-NPs exhibited higher image contrast (r1=4.61 mM-1s-1) than that of Fe3+/GA/PVP complex nanoparticles (r1=2.16 mM-1s-1), Fe-GA-PEG (r1=3.5 mM-1s-1) and a commercial MRI T1 contrast agent (Gd-DTPA, r1=3.69 mM-1s-1), suggesting that the FeAP-NPs could be used as a highly efficient T1 image contrast agent (Figure 3E and F).30-32 To evaluate the MR imaging properties of FeAP-NPs in vivo, the FeAP-NPs (100 µL, 250 µg mL-1) were intravenously injected into mice. The strongest MR imaging signals in the tumor region were observed at 24 h post injection. Furthermore, the accumulation of FeAP-NPs in the tumor was also confirmed with Cy5-labeled FeAP-NPs with the highest fluorescence intensity at 24 h post injection (Figure S15-17), and the fluorescent signals of free ACN will not interfere with fluorescent signals of Cy5-labeled FeAP-NPs (Figure S18). The PA, MR, and fluorescence images indicated that the FeAP-NPs could be gradually accumulated in the tumor, and that the high accumulation of FeAP-NPs in the tumors could be mainly attributed to the EPR effect of nanoparticles (Figure
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3D, 3H, Figure S17). Moreover, the mPEG grafted PLG copolymers that coordinated in the FeAP-NPs were beneficial to the long-term blood circulation of the nanoparticles (Figure S19), and contributed to effective tumor accumulation. These findings strongly demonstrated that the as-prepared FeAP-NPs possessed excellent PA/MR imaging ability and generate high-resolution, sensitivity, and specific images for tumor diagnosis, implying the potential use of as-prepared FeAP-NPs as guides for precise tumor treatment.
Figure 4. Photothermal properties of FeAP-NPs. A) Temperature increasing curves after laser irradiation (808 nm, 0.8 W cm-2, 10 min) at different FeAP-NPs concentrations (Curve a: 0 µg mL-1, b: 5 µg mL-1, c: 10 µg mL-1, d: 20 µg mL-1, e: 25 µg mL-1, f: 50 µg mL1
). B) Temperature variations of FeAP-NPs over five cycles of heating under laser
irradiation (808 nm, 0.8 W cm-2) and natural cooling. C) The heating and cooling curve of
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FeAP-NPs at 50 µg mL-1 under laser irradiation (808 nm, 0.8 W cm-2) for 10 min. D) Cell viability of MCF-7 cells treated with different concentrations of FeAP-NPs and laser irradiation (808 nm, 0.8 W cm-2, 6 min). E) Fluorescence images of Calcein AM (green, live cells) and PI (red, dead cells) co-stained MCF-7 cells after laser irradiation (808 nm, 0.8 W cm-2, 6 min). Scale bar, 100 µm. F) Time-dependent tumor growth curves of the mice after different treatments. G) Representative photos of tumors 26 days after treatments. The FeAP-NPs had a wide and strong absorption capacity in the near-infrared (NIR) region. Following the Lambert-Beer law, the extinction coefficient was measured to be 67 L g-1 cm-1 at 808 nm (Figure S20), which was significantly higher than that of CuFeSe2 nanocrystals at 808 nm (5.8 L g-1 cm-1) and Au nanorods (13.9 L g-1 cm-1), implying a strong NIR absorption capability.33,34 This phenomenon motivated us to evaluate potential PTT for tumors. The 808 nm laser irradiation with deep penetration for tumor tissues was used for the determination of the photothermal effects of FeAP-NPs. After laser irradiation at 0.8 W cm-2 for 10 min (Figure 4A), the temperature of the FeAP-NPs aqueous solution (25 µg mL-1) was increased to 55.7 oC and efficiently killed tumor cells for 4-6 min.35 The FeAP-NPs exhibited high photostability in water with 808 nm NIR laser irradiation for over 50 min (Figure 4B). The calculated photothermal conversion efficiency (η) of the FeAP-NPs was 36.7 % (Figure 4C and Figure S21), which was lower than that of previously reported Fe3+/GA/PVP complex nanoparticles and Fe(III)-gallicacid nanoparticles,21,32 but higher than that of Au nanorods.36 The photothermal effects of the FeAP-NPs were then assessed for their phototoxicity on cancer cells. Herein, we tested the cell viability of MCF-7, HeLa, and HepG2 cells with or without laser irradiation at various concentrations of FeAP-NPs. At first, the toxicity of free ACN with different concentrations was measured using MTT method (Figure S22). The results showed that the cell viability of MCF-7
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cells was correlative with different concentrations of ACN, and free ACN possessed certain toxicity only at high concentration. In contrast, after the cells were incubated with FeAP-NPs for 48 h without laser irradiation, no obvious cytotoxicity was observed, and all the three types of tumor cells showed more than 95 % cell viability with an Fe concentration range of 5-50 µg mL-1 (Figure S23, S25 and S27). This finding indicated that the FeAP-NPs were nontoxic to tumor cells at a broad concentration range. By contrast, all three types of tumor cells incubated with FeAP-NPs exhibited a dose-dependent phototoxicity after 808 nm laser irradiation at 0.8 W cm-2 for 6 min (Figure 4D, Figure S24 and S26). The PTT effect of free ACN at various concentrations were also investigated (Figure S28), and the results showed that the temperature was not enough difficult to satisfy the requirement for of PTT, which indicated that the coordination of Fe and ACN made a great contribution to the excellent PTT efficiency of FeAPNPs. The PTT efficacy of the FeAP-NPs on the tumor cells was further confirmed by Calcein AM and propidium iodide (PI) staining (Figure 4E, Figure S29-30). The staining results were consistent with the MTT assay, demonstrating the significant photothermal ablation effect of FeAP-NPs on the tumor cells. In view of the excellent PTT efficiency in vitro and high tumor accumulation behavior of the FeAP-NPs encouraged us to investigate the in vivo tumor ablation effects in mice. The MCF-7 tumor-bearing Balb/c nude mice were intravenously injected with FeAP-NPs (100 µL, 250 µg mL-1), and the tumors were then irradiated at a power density of 0.8 W cm-2 for 6 min with 808 nm laser at 24 h post injection. The changes of the tumor surface temperature were recorded with an infrared thermal camera (Figure S31). The surface temperatures of the tumors rapidly increased to 51.3 oC under NIR laser irradiation within 6 min. After being subjected to photothermal treatment for 2 days, the tumors of the mice treated with FeAP-NPs under laser
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irradiation left only black scars in the initial tumor regions (Figure S32). Finally, the black scars were completely eliminated at 14 days post photothermal treatment. Notably, for the mice injected with FeAP-NPs and treated with laser irradiation, all the tumors completely disappeared after photothermal treatment (Figure 4F, 4G and Figure S32), without any recurrence of tumors even after 60 days of observation. Moreover, no significant change in body weight was recorded during the treatment period (Figure S33). Hematoxylin and eosin (H&E) stained section of the major organs revealed no obvious damage or inflammatory lesion after the photothermal treatment (Figure S34). The values of the liver function markers (ALT, AST and ALP) and kidney function markers (UA, CRE and BUN) of the group treated with FeAP-NPs under laser irradiation exhibited no significant difference from the values in healthy mice (Figure S35-36). The above results clearly demonstrated the excellent tumor ablation effects of FeAP-NPs without obvious acute toxicity on the major organs of the experimented mice. These promising advantages could be attributed to the following features. First, the components of the FeAP-NPs originated from naturally extracted ACN, endogenous Fe element and poly(glutamic acid) derivatives, all of which possess good biocompatibility. As such, in vivo damages were considerably minimized. Second, the FeAP-NPs could efficiently accumulate in the tumors because of the appropriate size inducing the EPR “dividends”. Third, the dual PAI/MRI ability of the FeAP-NPs could detect the accurate spatiotemporal information regarding the tumor accumulation of the nanoparticles. Such information would guide the PTT at the most appropriate time and the most precise position. Lastly, the high photothermal conversion efficiency of the FeAP-NPs provided a powerful effect against the tumors. Taken together, the FeAP-NPs exhibited significant tumor treatment effects while mitigating safety concerns.
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Despite the high accumulation in tumor sites, a considerable amount of FeAP-NPs accumulates in the liver because of high RES retention (Figure S37). Such accumulation is an inevitable defect that affects relatively large nanoparticles that possess the EPR effect.37,38 Large nanoparticles can be cleared through hepatobiliary and mononuclear phagocyte systems (MPS). However, hepatobiliary clearance is a relative slow elimination pathway lasted for hours and even for weeks, whereas the clearance of nondegradable nanoparticles through MPS can last for months and even for years.39,40 Therefore, uncertain safety concerns and chronic damages still exist in the liver parenchyma. Currently, two primary strategies have been used to regulate or accelerate the hepatic clearance pathway. One strategy is to design ultra-small nanoparticles, which has rapid renal clearance ability, but exhibits a poor EPR effect into tumor tissues.41,42 The other strategy is to produce the degradable nanoparticles, which could control the hepatic clearance pathway.41-43 However, a robust elimination way is still needed to be developed.
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Figure 5. Dynamic disassembling properties of FeAP-NPs with DFO. A) The disassembling phenomenon for FeAP-NPs by adding DFO. B) TEM image of the disassembled FeAP-NPs (scale bar, 100 nm). Insert showed that DLS measurement of the size of the disassembled FeAP-NPs was 3.2 nm. C) The UV-vis absorption spectra of FeAP-NPs, FeAP-NPs + DFO and FeCl3 + DFO. D) The process for the dynamic disassembling ability of Fe-ACN complexes in the presence of DFO with time increasing (The UV-vis absorption values were detected at 596 nm). E) The coordination complexes structures of Fe-ACN and Fe-DFO. Controllable splitting of these relatively large nanoparticles into smaller sizes or deforming these nanoparticles into their original components might be a meaningful strategy to solve this problem. It is excited to find that the dynamic disassembling ability of the FeAP-NPs is rapidly initiated by DFO. As demonstrated in Figure 5A, the black FeAP-NPs quickly changed into orange-brown after DFO was added. The TEM images revealed no obvious nanoparticles in the disassembled solution (Figure 5B). The UV-vis absorption of the FeAP-NPs solution markedly changed after the disassembling occurred (Figure 5C). The specific UV-vis absorption at 596 nm that reflected the coordination between iron and ACN disappeared, and the UV-vis spectrum of the DFO-treated FeAP-NPs solution exhibited a similar profile to the spectrum of the mixture solution of FeCl3 and DFO. Then the process for the dynamic disassembling ability of iron and ACN in the presence of DFO was traced (Figure 5D). The UV-vis absorption at 596 nm was gradually reduced with the time extending, which indicated that the coordination between iron and ACN was destroyed by DFO. Moreover, the disassembling could be observed within a few minutes (Video S1). Therefore, the mechanism for the dynamic disassembling ability of the FeAP-NPs was presumed due to the destruction of the coordination between iron and ACN by the chelation of iron and DFO (Figure 5E). Consequently, the FeAP-NPs could be easily
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disassembled by DFO. These results indicated that the DFO-initiated disassembling of FeAPNPs could be exploited for the regulation of hepatic clearance in vivo.
Figure 6. The imageable in vivo disassembling of FeAP-NPs detected by PAI and MRI after DFO injection. A) Schematic illustration for imaging timelines of FeAP-NPs in vivo. B) PA images of the liver with or without the injection of DFO (30 h after the injection of FeAPNPs, 6 h after the injection of DFO). C) Quantitative PA signals analysis of the liver at different time in vivo. D) MR images of the liver with or without the injection of DFO (30 h after the injection of FeAP-NPs, 6 h after the injection of DFO). E) Quantitative MR signals analysis of the liver (30 h after the injection of FeAP-NPs, 6 h after the injection of DFO). In the view of the changes of the UV-vis absorption and the destruction of the coordination interaction between FeIII and ACN after FeAP-NPs disassembling, the PA and MR imaging signals of the FeAP-NPs could be weaken or disappear. These phenomena are confirmed in Figure S38-39. The PA and MR imaging signals were undetectable after disassembling of the FeAP-NPs at different concentrations. These behaviors provided a robust real-time detection strategy for the disassembling of the FeAP-NPs in vivo. The changes of the PA and MR images
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as well as the quantitative analyses in the mice liver were shown in Figure 6, and the schematic depiction of the treatment timelines was shown in Figure 6A. According to the distribution studies of the mice tumors (Figure 3 and Figure S17), the highest accumulation was achieved at 24 h post injection of FeAP-NPs. PTT was also completed at the same time (Figure 4). So it was the best time to inject DFO to disassemble FeAP-NPs in vivo at 24 h, and thus switch the nanoparticles’ elimination pathway. At 30 h post injection of the FeAP-NPs, i.e., 6 h post DFO injeciton, the PA signals in the liver were significantly decreased comparing with the live of the mice without DFO injection (Figure 6B). The PA signals in the liver were quantified at different time point during 0-168 h both in mice with DFO injection and without DFO injection. As shown in Figure 6C, the PA signals in the liver were markedly reduced at 6 h post DFO injection, and nearly restored to the levels before FeAP-NPs injection (healthy mice level). By contrast, the PA signals in the liver of mice without DFO injection were gradually decreased and showed significantly higher level than that in the mice with DFO injection up to 168 h. Moreover, the changes of the MR signals in the liver of mice exhibited the same tendency as the PA signals at 30 h post injection of FeAP-NPs (6 h post DFO injection) (Figure 6D). Quantitative analysis revealed that the MR signals in the liver of the mice without DFO injection were almost twice higher than that of the mice treated with DFO at 6 h post DFO injection (Figure 6E). The above results suggested that the FeAP-NPs could be effectively disassembled in the liver of mice by intravenous injection of DFO. The imageable disassembling of FeAP-NPs would be very helpful to decrease the patients’ worries about the nanoparticles’ in vivo fates in further clinical application. The FeAP-NPs could be disassembled in vivo. Thus, the excretion of disassembled FeAP-NPs from the body should be tracked. The elimination behaviors of FeAP-NPs were studied by
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analyzing the Fe content in the liver, kidney, feces, and urine of the mice. The experiments were conducted in accordance with the timeline listed in Figure 7A. Interestingly, high Fe contents were detected in the liver at 30 and 48 h without DFO injection, whereas Fe content was significantly decreased in the livers of the mice with DFO injection, suggesting that the FeAPNPs were quickly cleared in the liver by DFO injection (Figure 7B). This finding indicated that the accumulation time of Fe in the liver could be markedly shortened by in vivo disassembling strategy, thus the potential damage for the liver was greatly reduced. Furthermore, the areaunder-the-curve values in the liver (AUCLiver) was used to quantify the interaction between Fe and liver (Figure S40). AUCLiver was the area under the curve in a plot of Fe content in liver versus time, and it could be calculated by linear trapezoidal method from Figure 7B. The AUCLiver for the mice without DFO injection was approximately thrice higher than that for the mice treated with DFO. This result indicated that the time of Fe exposure to liver could be greatly reduced even after the FeAP-NPs accumulated in the liver. This feature was beneficial for protecting the liver from long-term potential damages of the FeAP-NPs used in vivo. The Fe content of kidneys in the mice treated with DFO was significantly increased at 30 h (Figure 7C), which supposed that Fe elimination pathway was more likely to switch from hepatobiliary clearance to renal clearance. Then, the Fe contents in the collected feces and urine were measured and quantified (Figure 7D, E). The Fe eliminated via the feces of the mice without DFO injection was gradually increased to 34.5 % ID from 24 h to 168 h post FeAP-NPs injection, whereas that of the mice with DFO injection increased to only 15.0 % ID over the same period. This finding indicated that DFO injection could cause 19.5 % ID Fe reduction in the feces via hepatobiliary clearance within 6 days. In comparison, an increase to 16.3 % ID was observed in the urine via the renal excretion pathway from 24 h to 168 h post FeAP-NPs
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injection. This phenomenon was likely due to the reason that the FeAP-NPs were disassembled in the liver by DFO. The Fe was returned to the general circulation, and eventually excreted via renal clearance (Figure 7F).
Figure 7. The switchable elimination pathways of FeAP-NPs in vivo. A) Schematic illustration for analyzing the Fe content in the liver, kidney, feces and urine from the mice. The biodistribution of Fe element in B) liver and C) kidney of FeAP-NPs injected mice with or without injection of DFO. The accumulative Fe levels in D) feces and E) urine of FeAPNPs injected mice with or without injection of DFO. The feces and urine were collected from 24 to 168 h post FeAP-NPs injection. F) The elimination pathways of the liver and kidney in the mice with or without the injection of DFO. In general, the nanoparticles in the liver could be endocytosed by macrophages and endothelial cells, and some of them have the chance to enter into the bile via the biliary system, and finally
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be excreted into duodenum and out of the body through the feces.44 The excised liver, gall bladder, and intestinal system were directly observed to study the elimination behaviors of FeAP-NPs in the mice (Figure S41). We found that the liver and the gall bladder with FeAP-NPs were blackened at 6 h post FeAP-NPs injection, and the intestinal system started to blacken at 48 h post injection. These results indicated that the FeAP-NPs could be cleared out by hepatobiliary clearance to the intestinal system and then the feces. Moreover, the Cy5 labeled FeAP-NPs showed the same elimination behaviors (Figure S42). In addition, we further confirmed that the FeAP-NPs could be disassembled by DFO even after they were encapsulated by the cells (Figure S43). The FeAP-NPs that were accumulated in the liver, either in the blood stream or encapculated by cells, could be disassembled after DFO injection. The blackened liver and gall bladder returned to their original color after the DFO triggered disassembling in vivo (Figure S41). These findings strongly confirmed the dynamic disassembling ability of the FeAPNPs triggered by DFO in vivo. The elimination pathway of the Fe accumulated in the liver could be effectively switched from hepatobiliary clearance to renal clearance. This strategy provided an ideal solution for overcoming the dilemma concerning EPR effect and renal clearance for nanomedicine. Finally, the effects of DFO injection on the health conditions of the mice were monitored. No significant differences were detected for serum transferrin, Fe content in the liver, kidney, feces, and urine of the mice with DFO injection relative to those of healthy mice (Figure S44-46), suggesting that the DFO administrated in the dosage we selected in our experiments could not cause anemia and would not disturb the endogenous Fe level in the mice. The liver and kidney function markers and the serum biochemistry assay showed no distinct changes in the mice after DFO injection, further confirming the absence of liver and renal dysfunction or other toxicity
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caused by DFO injection (Figure S47 and Table S2). These results indicated that the switch of the elimination pathways could not affect the health conditions of the mice, and DFO could be used as an efficient disassembling agent for nanoparticle clearance in vivo. CONCLUSION In summary, we successfully prepared multifunctional theranostic nanoparticles by coordination interaction among fruit extracted natural polyphenols (ACN), FeIII ions, and PLG-gmPEG copolymers. The as-prepared FeAP-NPs possessed excellent dual PA/MR imaging properties, and could effectively accumulate in the tumor via the EPR effect because of their suitable size. The FeAP-NPs had a high photothermal conversion efficiency, and they completely ablated the tumors in MCF-7 bearing nude mice. Furthermore, we demonstrated that the FeAPNPs exhibited the DFO triggered dynamic disassembling ability in vivo, which could be exploited to switch the elimination pathway of the Fe accumulated in liver from hepatobiliary clearance to renal clearance. The intractable dilemma concerning EPR effect and renal clearance for nanomedicine was reasonably addressed. This attractive and practical strategy presented in this work has potential in clinical translation in the field of tumor diagnosis and therapy. MATERIALS AND METHODS Materials. Anthocyanins were extracted from blue honeysuckle (Lonicera caerulea L.) which were harvested in northern China between August and September of 2016. FeCl3·6H2O was purchased from J&K scientific (Shanghai, China). Methoxy poly(ethylene glycol) (mPEG 5000) with number-average molecular weight (Mn) of 5000 g mol-1 was obtained from Sigma-Aldrich (Saint Louis, MO, USA). The γ-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) was purchased from Shanghai Yeexin Biochem&Tech Co., Ltd., China. Deferoxamine mesylate
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(DFO) was obtained from Sigma-Aldrich (Saint Louis, MO, USA). Cyanine 5 NHS ester (Cy5 NHS) was purchased from Lumiprobe Corporation (Broward, FL, USA). Calcein acetoxymethyl ester (Calcein AM) and propidium iodide (PI) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma-Aldrich (Saint Louis, MO, USA). Characterizations. Transmission electron microscopy (TEM) images were achieved by using a JEOL JEM-1011 TEM (Tokyo, Japan) with an accelerating voltage of 100 kV. The hydrodynamic diameters of nanoparticles were measured by dynamic laser scattering (DLS) method using a WyattQELS instrument (DAWN EOS, Wyatt Technology Co., Santa Barbara, CA, USA). The fourier transform infrared (FTIR) spectra were captured with a Bio-Rad Win-IR instrument (Bio-Rad Laboratories Inc., Cambridge, MA, USA). The absorbance spectra were measured with ultraviolet-visible (UV-Vis) spectrophotometer (2401PC, Varian). The powder Xray diffraction (XRD) analyses were measured on a D8 ADVANCE diffractometer (Bruker Co., Germany), and the diffraction data was recorded in the 2θ range of 10-60 degree. The X-ray photoelectron spectroscopy (XPS) measurement were performed on a VG ESCALAB MKII spectrometer (VG Scientific Ltd, UK). The SEM-EDS mapping of FeAP-NPs was characterized with an XL-30 ESEM FEG Scanning Electron Microscope (Philips, Netherlands). The Fe and N elemental analyses were performed on an ELAN 9000/DRC ICP-MS system (PerkinElmer, USA) and vario EL cube (Elementar, Germany), respectively. Extraction of anthocyanins. Anthocyanins in blue honeysuckle fruits were extracted with 70 % (v/v) ethanol containing 0.3 % TFA (v/v) for 48 h at 4 °C. Re-extraction was replicated twice, and then the final extractions were filtered, evaporated by the rotary evaporator (0.1 MPa, 50 °C).
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The anthocyanin extracts were subjected to a liquid-liquid extraction using ethyl acetate in order to remove the less polar non-anthocyanin flavonoids. The water phase was applied to an Amberlite XAD-7HP column, followed by washing with water containing 0.1 % (v/v) TFA. And then the column was eluted with 30 % ethanol containing 0.1 % (v/v) TFA, the eluate was monitored at 530 nm. The anthocyanin-rich eluate was collected, evaporated by the rotary evaporator (0.1 MPa, 50 °C), freeze-dried in a vacuum freeze drier, and resulting in the anthocyanin-rich extracts. HPLC-MS/MS analysis. For the characterization of the anthocyanins in blue honeysuckle anthocyanin extracts, a Waters 2695 liquid chromatography system connected with a PDA detector and a Waters Quattro premier XE triple quadrupole mass spectrometer (Micromass, Waters Corp., Milford, MA, USA). Data acquisition and processing were performed using Masslynx software (Version 4.1). The separation was performed on a Waters symmetryShield C18 column (4.6 × 150 mm i.d., 5 µm). The anthocyanins were detected at 530 nm. The mobile phase solvents consisted of anhydrous methanol (solvent A) and water/formic acid (98:2, v/v, solvent B). The gradient program was as follows: from 85 % to 80 % B in 10 min, from 80 % to 75 % B in 20 min, from 75 % to 30 % B in 15 min, from 30 % to 85% B in 10 min, and then isocratic elution for 5 min. The total run time was 60 min. The flow rate was 0.5 mL min-1. After the anthocyanin solution passed through HPLC DAD detector, it was directed to the MS system. The MS measurements were conducted in positive mode with the following parameters: capillary 3 kV; cone voltage 10 V, source block temperature 120 °C, desolvation temperature 350 °C. The mass spectra were recorded over the range m/z 200 to 1000 using a scan time of 1 s with an interscan time of 0.1 s. Cyanidin-3-O-glucoside was used as an external standard.
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Preparation of FeAP-NPs. The FeAP-NPs were prepared by a one-pot method. Briefly, the FeCl3·6H2O aqueous solution (1 mL, 100 mg mL-1) was added to 44 mL of PLG-g-mPEG aqueous solution (2.6 mg mL-1) and stirred for 1 h at room temperature, and then the 5 mL of ACN aqueous solution (10 mg mL-1) was added to the above solution and stirred for further 12 h at room temperature. The reaction solution was centrifuged at 10,000 rpm for 10 min by laboratory centrifuge (Sigma, St Louis, MO, USA), and the precipitate was redispersed using ultrasonic
instrument
(120 W,
JY
92-IIN,
Scientz,
Ningbo,
China).
After
three
centrifugation/redispersion cycles were performed to remove excess PLG-g-mPEG, ACN and FeIII, the FeAP-NPs were obtained for further use. The percentage of Fe ion and ACN exploited to form the FeAP-NPs was 3.5 %, and 27.6 %, respectively. The calculation results are shown in Table S3. PA imaging of FeAP-NPs. To test the linearity of PA signals, various concentrations of FeAPNPs were used for PA signal detection in vitro. A multispectral optoacoustic tomography (MSOT) scanner equipment was employed to accomplish PA imaging (MSOT inVision 128, iThera Medical GmbH, Munich, Germany). For PA imaging in vivo, MCF-7 tumor-bearing Balb/c nude mice were injected with FeAP-NPs (100 µL, 250 µg mL-1) via tail vein. The PA signals of tumor were recorded with multispectral process scanning at different time points (0, 6, 12, 24, 36 and 48 h). For the determination of disassembling for FeAP-NPs in vivo, the mice were injected with FeAP-NPs (100 µL, 250 µg mL-1) via tail vein, followed by injection of DFO (10 mg kg-1) at 24 h post injection via tail vein. The PA signals of the liver were captured at 0, 24, 30, 48, 72, 96 and 168 h, respectively. All animal experiments in this study were complied with the guidelines for laboratory animals established by Jilin University.
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MR imaging of FeAP-NPs. To measure the T1 relaxation properties, the FeAP-NPs with different Fe concentrations were measured using a Siemens Prisma 3.0 T MR scanner (Erlangen, Germany) with gradient strength up to 80 mT/m. For in vivo MRI, MCF-7 tumor-bearing Balb/c nude mice were injected with FeAP-NPs (100 µL, 250 µg mL-1) via tail vein. The MR signals of the tumor were captured with a Siemens Prisma 3.0 T MR scanner at different time points (0, 6, 12, 24, 36 and 48 h). For the determination of disassembling for FeAP-NPs in vivo, the mice were injected with FeAP-NPs (100 µL, 250 µg mL-1) via tail vein, followed by injection of DFO (10 mg kg-1) at 24 h post injection via tail vein. The MR signals of the liver were acquired at 30 h. Modification of PLG-g-mPEG for labeling Cy5. First, PLG-g-mPEG was modified by adipic dihydrazide (ADH) according to the procedure in Figure S15. Briefly, 1 g PLG-g-mPEG was dissolved in 50 mL MilliQ water, 46.8 mg of ADH and 60 mg of 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC·HCl) were added into above solution. The reaction mixture was adjusted by 0.1 M HCl to pH at 4.75 and maintained for 1 h, and then adjusted by 0.1 M NaOH to pH at 7.0 for 3 days dialysis against MilliQ water. Finally, the PLG-g-mPEG was successfully modified according to the 1H NMR spectrum (Figure S16). For labeling Cy5, the FeAP-NPs were prepared as above method using the ADH modified PLG-g-mPEG. Finally, 25 µL of Cy5 NHS (1 mg mL-1) was added to the 1 mL FeAP-NPs (250 µg mL-1) with stirring for overnight, and labeled FeAP-NPs were performed three centrifugation/redispersion cycles for removing the excess free Cy5. In vivo fluorescence imaging. The MCF-7 tumor-bearing Balb/c nude mice were injected with Cy5 labeled FeAP-NPs (100 µL, 250 µg mL-1) via tail vein. The tumors of mice were excised at 6, 12, 24 and 48 h post administration. The fluorescence distribution in tumors were visualized by a Maestro in vivo Imaging System (Cambridge Research & Instrumentation, Inc., USA). The
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fluorescence distribution in liver, gallbladder and intestines were obtained at 6, 24 and 48 h post injection, respectively. The color changes of liver, gallbladder and intestines after the injection of FeAP-NPs with or without DFO at different time were also captured by camera. Pharmacokinetic studies. SD rats were injected with FeAP-NPs (500 µL, 250 µg mL-1) via tail vein. The blood samples were collected from the orbital venous plexus at 0.5, 1, 2, 3, 6, 12, 24, 48 h post injection. These samples were digested with concentrated aqueous HNO3, and the amount of Fe was measured using ICP-MS (PerkinElmer, USA), and the untreated rats were used as a control. FeAP-NPs assisted PTT. The photothermal performance of FeAP-NPs was investigated by the temperature changes under the laser irradiation. 200 µL different concentrations of FeAPNPs solution were irradiated by an 808 nm laser at 0.8 W cm-2 for 10 min. The temperature changes were recorded every 10 s by the infrared imaging camera (FLIR E5; FLIR System AB, Täby, Sweden). To investigate the photostability, FeAP-NPs (50 µg mL-1) were irradiated by 808 nm laser at 0.8 W cm-2 for 10 min, followed by cooling to room temperature, and this procedure was repeated for five cycles. The photothermal conversion efficiency (η) was calculated according to the previous methods.30 For in vitro PTT, MCF-7, HeLa, HepG2 cells were incubated with FeAP-NPs at different concentrations and then irradiated by 808 nm laser at 0.8 W cm-2 for 6 min. The cells were incubated for further 24 h and washed with PBS twice. Subsequently, the viability of cells was measured at 492 nm using a Bio-Rad 680 Microplate Reader using MTT method. For further confirming in vitro PTT, the cells were incubated with FeAP-NPs at different concentrations and irradiated by an 808 nm laser at 0.8 W cm-2 for 6 min, and stained with Calcein AM and PI.
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For the PTT in vivo, when the tumor volume reached 70 mm3, the mice were randomly divided into four groups. The MCF-7 tumor-bearing Balb/c nude mice were injected with FeAP-NPs (100 µL, 250 µg mL-1) via tail vein. The tumors were irradiated with or without 808 nm laser irradiation at 0.8 W cm-2 for 6 min after 24 h injection. After PTT, the mice treated with FeAPNPs were injected DFO (10mg kg-1). Infrared thermal images were acquired for studying the photothermal effect of FeAP-NPs in vivo. The tumor sizes were measured with a calliper every other day for 26 days or 60 days depending on the different groups. The tumor volume was calculated through the following formula: tumor volume (V) = (tumor length) × (tumor width)2/2. The relative tumor volumes were calculated as V/V0, where V0 is the initiated tumor volume. The major organs (heart, liver, spleen, lung and kidney) were collected and stained with hematoxylin and eosin (H&E) for histological examination. During the treatment, the body weights of mice were recorded every other day. The blood of the mice was extracted for the hematology analysis, and the healthy mice were used as a control. The alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), uric acid (UA), creatinine (CRE) and blood urea nitrogen (BUN) levels of mice in the serum of mice were measured by enzyme linked immunosorbent assay (ELISA, Lengton Bio, Shanghai, China). Dynamic disassembling ability of FeAP-NPs. 200 µL DFO (100 mg mL-1) was dropwise added into FeAP-NPs (1.8 mL, 50 µg mL-1) solution at the room temperature, the color change of the FeAP-NPs solution was monitored by a camera. The change of UV-vis absorption spectra was measured with ultraviolet-visible (UV-Vis) spectrophotometer (2401PC, Varian) during the FeAP-NPs disassembling. Biodistribution and clearance studies in vivo. For the biodistribution of FeAP-NPs, the MCF-7 tumor-bearing Balb/c nude mice were intravenously injected with FeAP-NPs (100 µL,
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250 µg mL-1). The heart, liver, spleen, lung, kidney and tumor of mice were collected at 0, 6, 12, and 24 h, respectively. Tissue samples were digested with concentrated aqueous HNO3, and the amount of Fe was measured using ICP-MS (PerkinElmer, USA). For the elimination pathways studies, the mice were cared in the metabolism cages. The mice were intravenously injected with FeAP-NPs (100 µL, 250 µg mL-1), followed by injection with DFO (10mg kg-1) via tail vein at 24 h post FeAP-NPs injection. The liver, kidney, urine and feces of mice were collected at 0, 24, 30, 48, 72, 96 and 168 h, respectively. All samples were digested with concentrated aqueous HNO3, and the amount of Fe was measured using ICP-MS (PerkinElmer, USA). The disassembling of FeAP-NPs in macrophage cells. RAW 264.7 cells were cultured in the DMEM supplemented with 10 % fetal bovine serum (FBS). FeAP-NPs (250 µg mL-1) were added into the cells with the final concentration at 25 µg mL-1. Cells were incubated with the FeAP-NPs for 6 h. The cells were collected in the cube, and then DFO (25 mg mL-1) were added into cells to the final concentration at 2.5 mg mL-1, the color changes of the cells in the cube were recorded by camera, and the cube of cells without DFO addition was as a control. The toxicity studies of DFO in vivo. To determine the toxicity of DFO in vivo, the healthy mice (Balb/c mice) were injected with DFO (10 mg kg-1) and raised in the metabolism cages, and then the blood, liver, kidney, urine and feces of mice were collected at 0, 6, 24, 48, 72 and 168 h, respectively. A part of blood samples was used for blood biochemistry and complete blood panel analysis. The rest of blood samples was centrifuged at 6000 rpm for 10 min to collect the serum. The transferrin, ALT, ALP, AST, UA, CRE and BUN levels in the serum of mice were measured by enzyme linked immunosorbent assay (ELISA, Lengton Bio, Shanghai, China). The tissues samples were digested with concentrated aqueous HNO3, and the amount of Fe was measured using ICP-MS (PerkinElmer, USA).
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Statistical Analyses. All the experiments were carried out at least three times, and the data were expressed as mean ± standard deviation. All the statistical analysis was performed with the unpaired t-tests and repeated-measures (GraphPad Software).
***p
< 0.001 was considered to
indicate highly significant differences. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed characterizations of cyanidin-3-O-glucoside, PLG-g-mPEG and FeAP-NPs, biological evaluations, the physiological and pathological characterizations in vivo, the disassembling of FeAP-NPs in macrophage cells, the toxicity studies of DFO in vivo, Figures S1-S47, Tables S1S4, and Video S1. AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] (H.T.). *E-mail:
[email protected] (X.C.). ORCID Huayu Tian: 0000-0002-2482-3744 Xuesi Chen: 0000-0003-3542-9256 Author Contributions †
C.X. and Y.W. contributed equally to this work. C.X., Y.W., H.T. and X.C. conceived and
designed the experiments. H.Y. contributed materials; C.X., Y.W. and H.T. analyzed the data and wrote the manuscript. C.X. and Y.W. conducted all the experiments with help from H.Y., and all authors commented on the manuscript. ACKNOWLEDGMENTS The authors are thankful to the National Natural Science Foundation of China (51503200,
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21474104, 51520105004, and 51390484), National program for support of Top-notch young professionals, Jilin province science and technology development program (20160204032GX, 20170520142JH and 20180414027GH) for financial support of this work. REFERENCES (1) Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161−171. (2) Zhang, C.; Ni, D.; Liu, Y.; Yao, H.; Bu, W.; Shi, J. Magnesium Silicide Nanoparticles as a Deoxygenation Agent for Cancer Starvation Therapy. Nat. Nanotechnol. 2017, 12, 378−386. (3) Farokhzad, O. C.; Langer, R. Impact of Nanotechnology on Drug Delivery. ACS Nano 2009, 3, 16−20. (4) Zhang, Y.-N.; Poon, W.; Tavares, A. J.; McGilvray, I. D.; Chan, W. C. Nanoparticle-Liver Interactions: Cellular Uptake and Hepatobiliary Elimination. J. Controlled Release 2016, 240, 332−348. (5) Cheng, X.; Sun, R.; Yin, L.; Chai, Z.; Shi, H.; Gao, M. Light-Triggered Assembly of Gold Nanoparticles for Photothermal Therapy and Photoacoustic Imaging of Tumors in Vivo. Adv. Mater. 2017, 29, 1604894. (6) Ye, M.; Han, Y.; Tang, J.; Piao, Y.; Liu, X.; Zhou, Z.; Gao, J.; Rao, J.; Shen, Y. A Tumor-Specific Cascade Amplification Drug Release Nanoparticle for Overcoming Multidrug Resistance in Cancers. Adv. Mater. 2017, 29, 1702342. (7) Zhang, L.; Gao, S.; Zhang, F.; Yang, K.; Ma, Q.; Zhu, L. Activatable Hyaluronic Acid Nanoparticle as a Theranostic Agent for Optical/Photoacoustic Image-Guided Photothermal Therapy. ACS Nano 2014, 8, 12250−12258. (8) Gao, S.; Zhang, L.; Wang, G.; Yang, K.; Chen, M.; Tian, R.; Ma, Q.; Zhu, L. Hybrid Graphene/Au Activatable Theranostic Agent for Multimodalities Imaging Guided Enhanced Photothermal Therapy. Biomaterials 2016, 79, 36−45. (9) Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Ipe, B. I.; Bawendi, M. G.; Frangioni, J. V. Renal Clearance of Nanoparticles. Nat. Biotechnol. 2007, 25, 1165−1170. (10) Park, J.-H.; Gu, L.; Von Maltzahn, G.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Biodegradable Luminescent Porous Silicon Nanoparticles for in Vivo Applications. Nat. Mater. 2009, 8, 331−336. (11) Liu, J.; Yu, M.; Zhou, C.; Yang, S.; Ning, X.; Zheng, J. Passive Tumor Targeting of Renal-Clearable Luminescent Gold Nanoparticles: Long Tumor Retention and Fast Normal Tissue Clearance. J. Am. Chem. Soc. 2013, 135, 4978−4981. (12) Bridle, P.; Timberlake, C. Anthocyanins as Natural Food Colours-Selected Aspects. Food Chem. 1997, 58, 103−109. (13) Waterhouse, A. L. Wine and Heart Disease. Chem. Ind. 1995, 5, 338 −341. (14) Francis, F. J.; Markakis, P. C. Food Colorants: Anthocyanins. Crit. Rev. Food Sci. Nutr. 1989, 28, 273−314. (15) Delgado-Vargas, F.; Jiménez, A.; Paredes-López, O. Natural Pigments: Carotenoids, Anthocyanins, and Betalains-Characteristics, Biosynthesis, Processing, and Stability. Crit. Rev. Food Sci. Nutr. 2000, 40, 173−289.
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