Integration of Polymerization and Biomineralization as a Strategy to

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Integration of Polymerization and Biomineralization as a Strategy to Facilely Synthesize Nanotheranostic Agents Bing Xiao, XIAOXUAN Zhou, Hongxia Xu, Bihan Wu, Ding Hu, Hongjie Hu, Kanyi Pu, Zhuxian Zhou, Xiangrui Liu, Jianbin Tang, and Youqing Shen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07584 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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Integration of Polymerization and Biomineralization as a Strategy to Facilely Synthesize Nanotheranostic Agents

Bing Xiao, a,‡ Xiaoxuan Zhou, b,‡ Hongxia Xu, a Bihan Wu, a Ding Hu, a Hongjie Hu, b

aKey

Kanyi Pu, c Zhuxian Zhou, a Xiangrui Liu, a Jianbin Tang , a* Youqing Shen a

Laboratory of Biomass Chemical Engineering of Ministry of Education, Center

for Bionanoengineering, and Colledge of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang, 310027, China. bDepartment

of Radiology, Sir Run Run Shaw Hospital (SRRSH) of School of

Medicine, Zhejiang University, Hangzhou, Zhejiang, 310027, China. cSchool

of Chemical and Biomedical Engineering Nanyang Technological University,

Singapore 637457, Singapore. ‡The

authors contributed equally to this work.

*Corresponding Author: [email protected]

ABSTRACT: Integration of biological macromolecules with inorganic materials via biomineralization has demonstrated great potential for development of nanotheranostic agents. To produce multifunctionality, integration of multiple components in the biomineralized theranostic agents is required; however, how to efficiently and reproducibly implement this is challenging. In this report, a universal biomineralization strategy is developed by incorporation of oxidization polymerization into albumintemplated biomineralization for facile synthesis of nanotheranostic agents. A series of biomineralized polymers and manganese dioxide hybrid nanoparticles (PMHNs) can be synthesized via the polymerization of various monomers, including dopamine (DA), epigallocatechin (EGC), pyrrole (PY), and diaminopyridine (DP), along with the reduction of KMnO4 and formation of manganese dioxide nanoparticles in albumin templates. These biomineralized PMHNs demonstrate ultra-high MRI (longitudinal 1

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relaxivity up to 38 mM−1s−1) and ultrasonic (US) imaging contrasting capabilities and have excellent photothermal therapy (PTT) efficacy with complete ablation of orthotopic tumors. Moreover, these biomineralized hybrid nanoparticles can be effectively excreted through the kidneys, avoiding potential systemic toxicity. Thus, integration of polymerization into biomineralization presents a strategy for the fabrication of hybrid nanomaterials, allowing the production of multifunctional and biocompatible nanotheranostic agents via a facile one-pot method.

KEYWORDS: biomineralization, manganese dioxide nanoparticle, magnetic resonance imaging, photothermal therapy, renal clearance

2


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Nanotheranostics integrate diagnostic and therapeutic functions into a single nanoplatform and have shown great potential in multimodal diagnosis, imaging-guide therapy, and real-time monitoring of treatment for various diseases.1-4 For example, magnetic resonance imaging (MRI)-guided photothermal therapy (PTT)/photodynamic therapy (PDT) demonstrate greatly enhanced therapeutic efficacy by their ability to precisely spot lesions and finely tune the treatment parameters via real-time monitoring of the response upon treatment.5-13 However, the preparation of complex multifunctional nanotheranostic agents requires complicated multi-step reactions and is thus difficult to reproduce, thus limiting their translation into clinical application. In addition, most reported nanotheranostic agents are non-biodegradable and can not be effectively excreted by way of the kidneys, leading to a high risk of systemic toxicity.1416

Unfortunately, it is currently a considerable challenge to develop ideal nanoplatforms

with highly efficient theranostic performance and optimal renal clearance via simple and reproducible fabrication methods. Biomineralization is a facile method used to prepare functional nanomaterials by integrating biological macromolecules and inorganic materials.17-20 Expressly, albumin-based biomineralization has been used to easily and reproducibly produce biocompatible and biodegradable theranostic nanomaterials.18 Various nanostructures, including MnO2, 8, 21 Gd2O3, 22, 23 FeS2, 24 CuS, 25, 26 and Ag2S27 nanoparticles, have been prepared using albumin as the soft template. For instance, Liu et al. recently exploited human serum albumin (HSA)-based biomimetic mineralization to fabricate HSA/MnO2/Ce6 or cis-platinum prodrug nanotheranostic agents with an O2-generating function, to ameliorate tumor hypoxia and thus improve the photodynamic therapy against bladder cancer therapy.8 Additionally, Zhang et al. fabricated Gd/CuS@BSA nanoparticles for photoacoustic (PA) imaging, MRI, and PTT with a dual-metal doped/integrated biomineralization strategy.25 Moreover, Chen et al. prepared ultrasmall Ag2S nanodots for fluorescence and photoacoustic imaging plus simultaneous PTT using HSA as a nanocage.27 These pioneering studies offered fresh insights into the practical and versatile design of biomineralization-inspired nanotheranostic agents. However, to achieve multifunctional theranostic agents with enhanced theranostic and 3


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pharmaceutic properties, other functional organic components must be integrated into such biomineralized systems.28, 29 Nevertheless, how to efficiently and reproducibly integrate such functional materials in a biomineralized system is still a challenge. In this report, we develop a biomineralization strategy by integrating oxidization polymerization and biomineralization in albumin templates to easily synthesize nanotheranostic agents. A series of biomineralized polymer and manganese dioxide hybrid nanoparticles (PMHNs) were synthesized via the polymerization of various monomers, including dopamine (DA), epigallocatechin (EGC), pyrrole (PY), and diaminopyridine (DP) along with the reduction of KMnO4 and the formation of manganese dioxide nanoparticles in albumin templates. These biomineralized PMHNs demonstrated distinguished multimodal imaging contrasting capability and excellent PTT efficacy. Additionally, they exhibited excellent biocompatibility and kidney clearance. These multifunctional nanotheranostic agents have great potential in imaging-guided PTT for different cancers. More importantly, the integration of polymerization and biomineralization provides a strategy for fabricating hybrid nanomaterials, by which researchers can produce multifunctional and biocompatible nanotheranostic agents using a facile one-pot method.

RESULTS AND DISCUSSION Synthesis and Characterization of PMHNs The multifunctional nanotheranostic agents PMHNs were synthesized via integration of KMnO4-initiated polymerization and biomineralization in the presence of BSA. Briefly, KMnO4 was added to a mixed aqueous solution of BSA and monomers (DA, EGC, PY, or DP) at predesigned ratios and stirred at ambient temperature for 2 h. The PMHNs were purified by dialysis and collected by lyophilization. The dynamic laser scattering (DLS) measurement showed the average hydrodynamic diameters of the PMHNs from the different monomers were in the range of 7.0 to 60.9 nm (Figure 1a, Table S1), and transmission electron microscopy (TEM) indicated the PMHNs were spherical nanoparticles with sizes consistent with the DLS results (Figure S1). Of note, 4


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the sizes of the PMHNs could be easily tailored by tuning the mass ratio of the monomers, BSA, and KMnO4. For instance, the average diameters of PMHN-DA (No1-5) made from dopamine increased from 38.8 nm to 71.3 nm with an increase in the amount of KMnO4 while keeping the mass ratio of dopamine to BSA fixed at 1:2 (Table S2, Figure S2a). Additionally, three batches of PMHN-DA samples made from the same condition showed almost identical size distributions, which proved the reproducibility of the preparation (Figure S3). PMHN-DA (No 8) made from BSA, DA, and MnO2 at a mass ratio of 20:10:3, whose average hydrodynamic diameter was 60.9 nm, was selected for further evaluation in this study. The TEM revealed that PMHN-DA had a uniform spherical structure with an average diameter of ca. 60 nm (Figure 1b, Figure S2b), which conformed to the DLS results. In the high resolution TEM (HRTEM) images, there were some black nanodots with a diameter of 2.5 ± 0.7 nm dispersed in the PMHN-DA, and the interplanar distance of crystal lattice in the nanodot shown in the inset of Figure 1c was 0.24 nm, corresponding to the (100) lattice planes of MnO2.30 Moreover, in the X-ray photoelectron spectroscopy (XPS) pattern of PMHN-DA, there were two typical binding-energy peaks at 654.2 and 642.5 eV, corresponding to Mn (IV)2p1/2 and Mn (IV)2p3/2, respectively, which further confirmed the presence of MnO2 (Figure 1e). An inductively coupled plasma-mass spectroscopy (ICP-MS) was used to measure the Mn content in the PMHN-DA, which was 3.0%. Additionally, the FT-IR spectrum of the PMHN-DA reflected the characteristic peaks from the BSA and polydopamine (PDA) (Figure S4), and the CD spectra showed that PMHN-DA still maintained the secondary structure of BSA even after oxidation with KMnO4 (Figure 1f). Like natural or artificially synthesized melanin, PMHN-DA had a single-line electron spin resonance (ESR) spectrum and a single peak with a g-factor of 2, reflecting the distinctive paramagnetic property of the stable π-electron free radicals from the melanin-like polydopamine component in PMHN-DA (Figure 1g).

31-33

The elemental mapping

images, line scanning data, and energy dispersive spectrometry (EDS) of PMHN-DA along with the XPS survey spectrum further revealed the existence of C, O, N, and Mn elements in the structure of the PMHN-DA (Figure 1d, and Figure S5, S6, and S7). All 5


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of these characterizations suggested that PMHN-DA was comprised of BSA and a polydopamine hybrid matrix with spherical MnO2 nanodots dispersed in the matrix. With a zeta potential of -14.6 mV, the PMHN-DA were negatively charged in aqueous solution, and they had good colloidal stability in different environments, including deionized water, serum, RMPI-1640 culture medium, normal saline and phosphate buffer solution (PBS) with no precipitation in these media (Figure S8), and no obvious leakage of Mn ions in aqueous solution over 15 days (Figure S9). This high stability of PMHNDA could be ascribed to the simultaneous confinement of the MnO2 nanodots by both BSA and melanin-like polydopamine.

Photothermal Properties All PMHNs solutions showed a significant temperature increase under laser irradiation (808nm, 2 W cm-2), whereas no observable temperature increase was seen for water under the same laser exposure, indicating all PMHNs could be used as photothermal agents (Figure 2a and Figure S10). PMHN-DA at one-third the concentration of the other PMHNs showed nearly the greatest temperature increase, indicating it possessed the highest photothermal effect. The photothermal conversion efficiency (η) of PMHN-DA calculated from Figure 2b and 2c according to the reported method was 47.1%, which was evidently greater than that previous result for melaninlike polydopamine (~ 40%).31 The high η of PMHN-DA could be attributed to its strong absorption at the near infra-red (NIR) wavelength of 808 nm (Figure S11) and the manganese-induced enhancement in electron-transfer efficiency.34,

35

Moreover, the

photothermal effect of PMHN-DA was not impacted by laser exposure with, demonstrating no decrease in temperature rise after four rounds of repeated laser exposure (Figure 2d). Furthermore, there were no obvious variations in the UV-vis absorption, hydrodynamic diameter, polydispersity index (PDI), zeta potential, and relaxation time of the PMHN-DA solutions before and after NIR laser irradiation over 7 days (Figure S12, S13, and S14), supporting the outstanding photothermal stability of PMHN-DA. The excellent photothermal stability and high photothermal conversion efficiency indicated that these PMHNs were highly promising PTT agents. 6


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Moreover, the confocal laser scanning microscopy (CLSM) observation and flow cytometry analysis showed that PMHN-DA can be efficiently internalized by cancer cells (Figure S15), facilitating concentration-dependent hyperthermia in these cells. The in vitro PTT efficacy of PMHN-DA towards 4T1 cells was also explored through MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. As expected, the cytotoxicity of PMHN-DA upon exposure to the 808-nm NIR irradiation was very high, with an IC50 of 183.14 µg mL−1 (Figure 2e). It was confirmed by the CLSM observation of calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI) co-stained 4T1 cells and with PMHN-DA photothermal treatment (Figure 2f), in which all the cells treated with PMHN-DA under exposure to the 808-nm NIR irradiation died (shown in red), while those without laser irradiation remained alive (shown in green). The flow cytometry assays employing annexinV-fluorescein isothiocyanate (annexinV-FITC) and propidium iodide (PI) fluorescence staining indicated that PMHN-DA-mediated PTT mainly caused apoptosis-type cell death (Figure S16), which is similar to other PTT agents.36, 37

In vitro and in vivo MRI Performance The MnO2 nanoparticles in the PMHNs made them potential contrast agents (CAs) for MRI. The in vitro T1-weighted MRI and the longitudinal proton relaxation (r1) of the PMHNs were studied using a 3T MR scanner and a 0.52T NMR analyzer, respectively. The r1s of the PMHNs were obtained from the slopes of the curves of 1/T1 vs. [Mn]. The PMHNs made from different monomers had very different r1s, ranging from 13.15 to 38.14 mM−1s−1 (Figure 2g), and the PMHNs made from the same monomer dopamine but with different BSA, DA, and KMnO4 charge ratios also had very different r1s, ranging from 9.36 to 37.17 mM−1s−1 (Figure S17), and thus demonstrated different brightness in their T1-weighted MR images (Figure 2h). This indicated that the r1s of the PMHNs were very sensitive to their structure variables, including size, monomer type, and composition (Figure S1, S10). The highest r1 of 38.14 mM−1s−1 was found in PMHN-DA prepared at a BSA, DA, and KMnO4 charge ratio of 20:10:3 with a size of 60 nm (No 8, Table S2). It was nine folds of that for the 7


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currently used gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)38, namely, Magnevist and much higher than other documented MnO2-based nanotheranostic materials.39-42 The high r1s of the PMHNs was likely due to the size-dependent superparamagnetic property of the ultra-small MnO2 nanodots within them.43, 44 The saturation magnetization of PMHN-DA was two-times higher than that of albumin/MnO2 nanoparticles (AMNs) (Figure 2i), and the super high r1s made PMHNs efficient MRI contrast agents for use in the detection of cancer and other diseases. To examine the efficiency of PMHNs in contrasting the MRI of tumors in vivo, female Balb/c mice with 4T1 orthotopic breast cancer cells were then intravenously administered with PMHN-DA at the dose of 0.06 mmol kg-1 Mn and observed under a 3T MR clinical scanner. Figure 3a showed the axial MR images taken before the administration and at different time points after the administration. A greatly highlighted boundary was observed around the tumor contrasted by PMHN-DA during the entire course duration, which provided highly detailed edge imaging and precise differentiation between normal tissues and tumor. By calculating the changes of signalto-noise ratio (SNRpost/SNRpre) at the tumor boundary quantitatively, a 1.57-fold positive enhancement was found at 1 h post injection (Figure 3c). The MRI of the mice with intrahepatic metastases after intravenous injection of PMHN-DA at a dose of 0.06 mmol kg-1 Mn was also assessed to evaluate the feasibility of PMHN-DA for use in liver metastases imaging. In the MRI without CA, the metastasized tumors could barely be observed. Inversely, in the MRI contrasted by PMHN-DA, the hepatic regions where the metastasized tumors were extensively localized (which were verified by anatomy and hematoxylin and eosin (H&E) staining, as shown in Figure S18) were distinctly distinguished from the surrounding normal liver tissues, with the highest CNR in metastasized tumors at 30 min after injection (Figure 3b, d). Significantly, PMHN-DA was also found to greatly enhance the MRI of kidneys. Figure 3e and Figure S19 presented the MR images of healthy mice which were injected with a single dose (0.02 mmol Kg-1 Mn) of PMHN-DA, intravenously. There was clear boundary between the surrounding tissues and the renal parenchyma at 5 min after injection, and the renal parenchyma was clearly discriminated from the renal pelvis at 8


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8 h post injection. Even the renal medulla could be clearly distinguished, as it had a relatively strong signal in comparison with the renal cortex and pelvis (Figure S20). The extremely high efficiency at enhancing MRI of kidneys was likely due to the high distribution of PMHN-DA in the kidneys (Figure S26). In addition, the signal in the kidneys decayed to the original status without CA at 24 h post injection, indicating efficient clearance of PMHN-DA from the kidneys.

O2 Generation and in vivo US Imaging Since MnO2 can react with H2O2 as an oxidant to produce O2 in an acidic cancer microenvironment

8, 45

and melanin-like polydopamine has the superoxide dismutase

(SOD)-mimic catalytic ability to assist in the efficient transformation of O2•− to H2O2,46 we hypothesized that PMHN-DA in combination with MnO2 and melanin-like polydopamine could generate O2 more efficiently within tumors. The oxygen generation ability of PMHN-DA with H2O2 was examined at pH 6.5 by way of ultrasound imaging. While there were no bubbles in the tube with H2O2 alone, numerous bubbles were observed in the tubes with H2O2 and PMHN-DA, indicating that considerable oxygen was generated via the reaction between H2O2 and the MnO2 in PMHN-DA (Figure 3f). As a result, the tumors of the mice intravenously injected with PMHN-DA exhibited higher acoustic signal in the ultrasound imaging of tumors than those treated with BSA/MnO2 nanoparticles (AMNs) without polydopamine (Figure 3g and Figure S21), confirming PMHN-DA can be potentially used as a contrast agent for tumor ultrasound imaging.

In vivo PTT of PMHN-DA In vivo PTT efficacy was tested with a mouse model bearing orthotopic 4T1 tumors. When the size of the tumors was about 70 mm3, the mice were intravenously injected with a single dose of 3.25 mg Kg-1 PMHN-DA. The tumor received a 5-min laser irradiation (808 nm, 2 W cm−2) at 30 minutes after the injection, and the tumor sizes were measured in the subsequent 20 days. During laser irradiation, the temperature variation in the tumors was monitored through an infrared camera. Figure 4a and 4b 9


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showed that the surface temperature of the tumor site in the PMHN-DA+laser group sharply increased, while that from the control group injected with PBS barely increased. A laser exposure of 5 min made the temperature in the tumor site elevate up to 53 oC. As a result, the group treated with PMHN-DA plus laser irradiation showed complete tumor ablation without recurrence in 20 days, whereas the groups treated with PMHNDA without laser irradiation, laser irradiation alone, or PBS showed rapid tumor growth, with a nearly 18 fold increase in the tumor volume (Figure 4c and Figure S22). Additionally, the group treated with PMHN-DA plus laser irradiation showed no noticeable body weight loss (Figure 4d), indicating a low side effect. Notably, the group treated with PMHN-DA plus laser irradiation had a tumor-free survival up to 60 days, whereas the average life span of the mice in the other three control groups was shorter than 36 days (Figure 4e). These results demonstrated that PMHN-DA was a highly efficient PTT agent for use in cancer therapy.

Biosafety, Biodistribution, and Renal Clearance Biosafety is critical for the translation of nanotheranostic agents into clinical application. The MTT assays showed that PMHN-DA did not show significant cytotoxicity against either normal cells (3T3) or cancer cells (Hela, MCF-7, and 4T1) after 48 h incubation with cells without NIR irradiation at a high concentration up to 1000 µg mL−1 (Figure 2e and Figure S23). The Sprague Dawley (SD) rats intravenously injected with PMHN-DA at a dose of 0.06 mmol kg-1 Mn remained healthy over a 30day period with the same steady body weight growth as the control group (Figure S24). The complete blood panel and serum biochemistry of the treated rats were carefully analyzed and compared with the control group. There was no obvious difference in red blood cells, hemoglobin, hematocrit and platelets, levels of liver function markers (alkaline

phosphatase,

ALP;

aspartate

aminotransferase,

AST;

alanine

aminotransferase, ALT; albumin, ALB; and total protein, TP), or kidney function markers (urea nitrogen, UREA; uric acid, UA; and creatinine, CREA) between the control and the PMHN-DA-treated group, implying that PMHN-DA had no obvious hepatic or renal toxicity (Figure S25). Moreover, the H&E staining analysis showed 10


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that there was no inflammatory lesion or noticeable tissue damage in the major organs, including kidney, intestine, lung, spleen, liver and heart, in Balb/c mice 60 days after the intravenous injection of PMHN-DA at a dose of 0.06 mmol kg-1 Mn (Figure 4f). Both the in vivo and in vitro analysis demonstrated the excellent biocompatibility of PMHN-DA. Most nanotheranostic agents can not be effectively excreted from the body, which severely limit their translation into clinical application. The blood circulation profile of PMHN-DA was quantitatively tested by tracking the Mn content in blood samples at different times after intravenous injection of PMHN-DA (0.02 mmol kg-1 Mn). As shown in Figure S26a, a two-compartment model was fitted in the blood circulation curve, and the first (t1/2(α)) and the second (t1/2(β)) phases of circulation half-lives were 0.47 and 6.08 h, respectively. This moderate blood circulation time made it possible for PMHN-DA to accumulate in the tumors and also to be excreted out of body. The timedependent biodistribution experiment showed that PMHN-DA had a high accumulation in tumors of 5.08 % ID g-1 at 24 h post injection, which would be favorable for PTT of tumors (Figure S26b). Notably, PMHN-DA also demonstrated a high accumulation in the major organs, including the kidneys (12.66 % ID g-1) and liver (5.08 % ID g-1), at 24 h post injection, but this accumulation decreased with the increase in time (Figure S26c). After one week, the retention of Mn in the kidneys and liver decreased to a low level, suggesting a nearly complete clearance of PMHN-DA from the mouse body. To disclose the clearance pathway of PMHN-DA, the ICP-MS was then used to analyze the Mn content in the urine and feces of the mice with an intravenous injection of PMHN-DA (0.02 mmol kg-1 Mn). It was found that up to 25.56 % ID of PMHN-DA was detected in the urine and only a small portion of PMHN-DA (about 5.05 ± 0.46 % ID) was excreted through the binary pathway into feces in 12 h after injection (Figure S26d). The above results strongly demonstrated that PMHN-DA was mainly excreted via renal filtration, which was consistent with the MRI study (Figure 3e and S19). The PMHN-DA of 60 nm in size could be excreted from kidneys because it was likely degraded into small species by glutathione, as shown by TEM (Figure S27). The efficient renal clearance of PMHN-DA could substantially decrease the risk of systemic 11


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toxicity, especially liver toxicity.

CONCLUSIONS In summary, nanotheranostic PMHNs were efficiently prepared employing a onepot method via integration of biomineralization and polymerization of different monomers. With their hybrid three-component nanostructure, the prepared PMHNs possessed controllable size and tunable r1s. Among them, PMHA-DA made from dopamine showed unexpected superiorities, including excellent colloidal and optical stability, ultrahigh r1 (38.14 mM-1s-1), oxygen generation capability, and favorable photothermal conversion efficiency (47.1%). PMHA-DA could both greatly enhance the MRI of orthotopic tumors, liver metastases, and renal structures and also improve US imaging of tumors. In vivo PTT showed that PMHA-DA could completely ablate orthotopic tumors. Importantly, the PMHA-DA could also be efficiently excreted from the body via the kidneys and showed extremely high biocompatibility and biosafety both in vitro and in vivo. These PMHNs with distinguished imaging and PTT properties plus excellent biocompatibility represent promising nanotheranostic agents for multimodal

imaging-mediated

PTT

of

tumors.

Moreover,

the

integration

of

biomineralization and polymerization proposed in this study provide a strategy for preparing organic and inorganic hybrid multifunctional nanomaterials.

MATERIALS AND METHODS Materials. All reagents and chemicals used were of analytical grade and can be used without additional purification. Potassium permanganate (KMnO4) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Epigallocatechin (EGC), 2, 6-diaminopyridine (DP), pyrrole (PY) and dopamine hydrochloride (DA) were purchased from Tansoole Co., Ltd. (Shanghai, China). Cy5.5 (Cy5.5-NHS) were acquired from GE Healthcare (Piscataway, NJ). RPMI-1640 12


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medium was obtained from GE Healthcare Co., Ltd. Fetal bovine serum (FBS) was purchased from Gibco Life Technologies (AG, Switzerland). 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich. The deionized water (18.2 MΩ.cm) was purchased from a Milli7Q water purification system.

Synthesis of PMHNs. Through a facile and one-step method, the polymer and manganese dioxide hybrid nanoparticles (PMHNs) were synthesized. In brief, take PMHN-DA (No 8) as an example, 30 mg of KMnO4 dissolved in 2 mL of deionized water was dropwise added into 100 mg DA and 200 mg of BSA solution in 100 mL of water at a 5 min interval, and the mixture was stirred magnetically for 2 h at ambient temperature. The colorless solution turned deep brown over time. The excess reactants were finally removed by dialysis using the dialysis bag (molecular weight cut-off: 8-14 kDa), and the purified nanoparticles were obtained by lyophilization. The other PMHNDAs of different compositions were synthesized in the similar procedures by altering mass ratios of three reactants (Table S2).The other PMHNs made from different monomers (PMHN-EGC, PMHN-PY, and PMHN-DP) were synthesized with the same method for PMHN-DA except that the monomer (DA) was replaced by epigallocatechin (EGC), pyrrole (PY), and diaminopyridine (DP), respectively (Table S1).

Cellular Experiments. 4T1 murine breast cancer cells, MCF-7 murine breast cancer cells, HeLa human epithelial carcinoma cancer cells, and NIH3T3 murine embryonic fibroblast cells were obtained from American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) under 5% CO2 at 37 oC. Cytotoxicity assays: MCF-7, 4T1, Hela, and 3T3 cells which were seeded into 96well plates were incubated with PMHN-DA at different concentrations (1, 10, 25, 50, 100, 200, 400, 800, 1000 μg mL-1) for 48 h. The relative cell activities were determined through the standard MTT assays. 13


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Photothermal therapy assays: the seeded 4T1 cancer cells were incubated in 96well plates at 1 × 105/well until adhesion, which were added with PMHN-DA with different concentrations at 37 oC. The cells were then exposed to an 808 nm NIR laser for 5 min at the power density of 2 W cm-2. The standard MTT assay was subsequently applied to evaluate the relative cell viabilities after photothermal treatment. For the confocal imaging of live/dead stained cells, 4T1 cancer cells were incubated in the glass bottom dishes with PMHN-DA (0.75 mg mL-1) for 30 min at 37 oC, which were exposed to an 808 nm laser for 5 min at the power density of 2 W cm-2. PI (propidium iodide) and calcein AM (calcein acetoxymethyl ester) were used to stain the cells for 15 minutes, which were imaged through a Nikon A1 laser scanning confocal microscope. Cellular uptake observation: 4T1 cells were seeded and incubated in six well glass bottom plates at 1 × 105/well until adhesion. Each well was added with Cy5.5-labelled PMHN-DA (20 μL) and incubated for 2h, and subsequently observed by CLSM. To observe cellular uptake by the flow-cytometry, 4T1 cells seeded in 12-well plates (1 × 105/well) were incubated for different time periods (2 h, 4 h, 6 h and 12 h) with Cy5.5labelled PMHN-DA (20 μL), which were subsequently washed with PBS. The flowcytometry and CLSM were subsequently used to observe the treated cells. Tumor Models. The Institutional Ethical Committee of Animal Experimentation of Zhejiang University in China approved all the animal studies in this paper, and they were performed according to the guidelines established by the Institute for Experimental Animals of Zhejiang University. The SD rats and Balb/c mice (6-8 weeks) were acquired from SLAC Laboratory Animal (shanghai, China). The orthotopic 4T1 tumor model was constructed through the injection of 5×105 cells in 100 μL of serumfree RMPI-1640 medium into the mammary gland of each Balb/c mouse. The liver metastasis mouse model was made through the injection of 4T1 breast cancer cells (10×105 cells in 40 μL) to the parenchyma of spleen of Balb/c mice followed 5 min later splenectomy. Metal clips was used to close the left subcostal incision.

In Vivo Biosafety Assessment. SD rats were intravenously injected with PMHN-DA 14


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(0.06 mmol Kg-1 Mn) or without any treatment were divided into two groups (n=3), and weighed every other day. Blood was collected from the rats on days 1and 30. The serum was obtained by centrifugation. Subsequently, the complete blood panel and the serum biochemistry were analyzed.

In Vivo MR Imaging. A microMR imaging & analyzing system was used to test the T1s of the PMHN solutions with different concentrations (0-0.2 mM) at 32 oC. The longitudinal relaxivities (r1s) of PMHNs were obtained from the curves of the 1/T1 relaxation rate (s−1) vs Mn concentration (mM) curves as their slopes. A 3T clinical MRI scanner were exploited for MRI of the PMHN solutions. For in vivo MRI of orthotopic tumor, PMHN-DA (0.06 mmol Kg-1 in terms of Mn for each mouse) was injected intravenously to Balb/c mice grafted with 4T1 murine breast cancer tumors when their sizes reached 5-7 mm. For in vivo MRI of liver metastases, the contrast agent with the same dose of PMHN-DA was intravenously administered on the 12nd day after the model was constructed. For in vivo MRI of kidneys, PMHN-DA (0.02 mmol Kg-1 in terms of Mn for each mouse) was intravenously administered to normal Balb/c mice. Before and after injections, the MRI of the mice was conducted on a 3T clinical MRI scanner with a special animal-imaging coil. The T1-weighted MR signal intensities were collected from MR images through the selected regions of interest, followed by the calculation of signal to noise ratio (SNR). The scan parameters for T1 FSPGR were as below: matrix size = 320 × 192, field of view (FOV) = 60 × 60 mm2, slice thickness = 1 mm, number of excitations (NEX) = 3, time to echo (TE) =21 ms, repetition time (TR) = 400 ms, and total scan time = 2 min and 18 sec.

In vitro and in vivo Ultrasound Imaging. To test the in vitro O2 generation, the PMHN-DA and AMN (0.06 mmol Kg-1 in terms of Mn) were respectively injected to 1 mL H2O2 (200 μM) in a sealed chamber, and then the US images were recorded. For in vivo US imaging, the tumor-bearing mice was intravenously administered with the PMHN-DA and AMN (0.06 mmol Kg-1 in terms of Mn), respectively. A Vevo 1100 micro-US imaging system (Visual Sonics MS-250D, 40 MHz, B-mode) was used to 15


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monitor the in vitro and in vivo US imaging. Vevo 2100 Workstation Software was used to measure the signal intensities of echo imaging.

Blood Circulation, Biodistribution, and Clearance. Balb/c mice grafted with 4T1 murine breast tumors (n = 3) were used for in vivo biodistribution evaluation, and normal Balb/c mice (n = 3) were used for in vivo blood circulation and clearance evaluation. The mice for the three experiments were all intravenously injected with PMHA-DA at a dose of 0.06 mmol Kg

-1

in terms of Mn. Blood, different tissues

including tumor, bone, muscle, skin, intestine, stomach, lung, heart, kidney, spleen and liver, and the urine and feces of mice were respectively collected at the indicated time point and weighted. All the blood, tissues, urine and feces samples were digested with HNO3/HCl (1:3) under heating, and the content of Mn in the solutions was analyzed with the ICP-MS.

In Vivo Photothermal Therapy. When the sizes of tumors were about 70 cm3, the Balb/c mice with orthotopic 4T1-bearing tumors were classified into four groups randomly (n = 5): (a) untreated control, (b) the mice treated with laser irradiation (808 nm, 2 W cm-2, 5 min), (c) the mice intravenously injected with PMHN-DA (3.25 mg Kg-1), and (d) the mice intravenously injected with PMHN-DA (3.25 mg Kg-1) and exposed to NIR laser. An IR thermal imaging camera was used to monitor the changes in tumor temperature of the mice. The therapeutic effect was estimated by measuring tumor sizes at the interval of one day, which were calculated as (V) = width2 × length/2. The relative tumor volume was analyzed as V/V0 (where V0 is the tumor volume before treatment), and their body weight were monitored.

Histological Assessment. The three groups of mice including the healthy mice without treatment, the mice with intravenous injection of PMHN-DA (3.25 mg kg-1) and laser treatment, and the group of healthy mice with injection of PMHN-DA (0.06 mmol kg1

Mn) but without laser treatment were sacrificed 60 days after treatment. The 4%

paraformaldehyde solution was used to fix the harvested organs including intestine, 16


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lung, heart, spleen, kidneys and liver, which were embedded into paraffin. H&E (Beyotime, Shanghai, China) was used to stain the slices of those organs, and they were subsequently observed through an optical microscope (BX51, Olympus, Tokyo, Japan).

Statistical Analysis. A Student’s t-test was used to carry out statistical analysis. The estimation of a statistically significant difference was performed as *p< 0.05, **p < 0.01, ***p < 0.001. The values were presented as the mean ± SD in the figure captions.

ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information The Supporting Information is available free of charge on the ACS Publication website. More details on the materials and methods, and additional tables (Table S1-S2), figures (Figure S1-S27).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Jianbin Tang: 0000-0003-4498-5705 Author Contributions Bing Xiao and Xiaoxuan Zhou contributed equally to this work.

ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (51522304, 21174128, 51390481), the National Basic Research Program (2014CB931900), the Zhejiang Provincial Natural Science Foundation of China (LR18E030002).

REFERENCES 17


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(1) Kunjachan, S.; Ehling, J.; Storm, G.; Kiessling, F.; Lammers, T. Noninvasive Imaging of Nanomedicines and Nanotheranostics: Principles, Progress, and Prospects. Chem. Rev. 2015, 115, 10907-10937. (2) Chen, G.; Roy, I.; Yang, C.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826-2885. (3) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939. (4) Fan, W.; Yung, B.; Huang, P.; Chen, X. Nanotechnology for Multimodal Synergistic Cancer Therapy. Chem. Rev. 2017, 117, 13566-13638. (5) Ni, D.; Zhang, J.; Wang, J.; Hu, P.; Jin, Y.; Tang, Z.; Yao, Z.; Bu, W.; Shi, J. Oxygen Vacancy Enables Markedly Enhanced Magnetic Resonance ImagingGuided Photothermal Therapy of a Gd(3+)-Doped Contrast Agent. ACS nano 2017, 11, 4256-4264. (6) Yan, L.; Amirshaghaghi, A.; Huang, D.; Miller, J.; Stein, J. M.; Busch, T. M.; Cheng, Z.; Tsourkas, A. Protoporphyrin IX (PpIX)-Coated Superparamagnetic Iron Oxide Nanoparticle (SPION) Nanoclusters for Magnetic Resonance Imaging and Photodynamic Therapy. Adv. Funct. Mater. 2018, 28, 1707030. (7) Wang, S.; Lin, J.; Wang, Z.; Zhou, Z.; Bai, R.; Lu, N.; Liu, Y.; Fu, X.; Jacobson, O.; Fan, W.; Qu, J.; Chen, S.; Wang, T.; Huang, P.; Chen, X. Core-Satellite Polydopamine-Gadolinium-Metallofullerene Nanotheranostics for Multimodal Imaging Guided Combination Cancer Therapy. Adv. Mater. 2017, 29, 1701013. (8) Chen, Q.; Feng, L.; Liu, J.; Zhu, W.; Dong, Z.; Wu, Y.; Liu, Z. Intelligent AlbuminMnO2 Nanoparticles as pH-/H2O2-Responsive Dissociable Nanocarriers to Modulate Tumor Hypoxia for Effective Combination Therapy. Adv. Mater. 2016, 28, 7129-7136. (9) Cai, X.; Gao, W.; Ma, M.; Wu, M.; Zhang, L.; Zheng, Y.; Chen, H.; Shi, J. A Prussian Blue-Based Core-Shell Hollow-Structured Mesoporous Nanoparticle as a Smart Theranostic Agent with Ultrahigh pH-Responsive Longitudinal Relaxivity. Adv. Mater. 2015, 27, 6382-6389. (10)Zhu, H.; Li, J.; Qi, X.; Chen, P.; Pu, K. Oxygenic Hybrid Semiconducting Nanoparticles for Enhanced Photodynamic Therapy. Nano Lett. 2018, 18, 586-594. (11)Zhang, W.; Li, S.; Liu, X.; Yang, C.; Hu, N.; Dou, L.; Zhao, B.; Zhang, Q.; Suo, Y.; Wang, J. Oxygen-Generating MnO2 Nanodots-Anchored Versatile Nanoplatform for Combined Chemo-Photodynamic Therapy in Hypoxic Cancer. Adv. Funct. Mater. 2018, 28, 1706375. (12)Liu, T.; Zhang, M.; Liu, W.; Zeng, X.; Song, X.; Yang, X.; Zhang, X.; Feng, J. Metal Ion/Tannic Acid Assembly as a Versatile Photothermal Platform in Engineering Multimodal Nanotheranostics for Advanced Applications. ACS nano 2018, 12, 3917-3927. (13)Ma, Z.; Jia, X.; Bai, J.; Ruan, Y.; Wang, C.; Li, J.; Zhang, M.; Jiang, X. MnO2 Gatekeeper: An Intelligent and O2-Evolving Shell for Preventing Premature Release of High Cargo Payload Core, Overcoming Tumor Hypoxia, and Acidic H2O2-Sensitive MRI. Adv. Funct. Mater. 2017, 27, 1604258. 18


Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(14)Sailor, M. J.; Park, J. H. Hybrid Nanoparticles for Detection and Treatment of Cancer. Adv. Mater. 2012, 24, 3779-3802. (15)Sharma, H.; Mishra, P. K.; Talegaonkar, S.; Vaidya, B. Metal Nanoparticles: a Theranostic Nanotool against Cancer. Drug Discov. Today 2015, 20, 1143-1151. (16)Xie, J.; Lee, S.; Chen, X. Nanoparticle-based Theranostic Agents. Adv. Drug Delivery Rev. 2010, 62, 1064-1079. (17)Xavier, P. L.; Chaudhari, K.; Baksi, A.; Pradeep, T. Protein-Protected Luminescent Noble Metal Quantum Clusters: An Emerging Trend in Atomic Cluster Nanoscience. Nano reviews 2012, 3, 14767. (18)An, F. F.; Zhang, X. H. Strategies for Preparing Albumin-based Nanoparticles for Multifunctional Bioimaging and Drug Delivery. Theranostics 2017, 7, 3667-3689. (19)Chen, Q.; Liu, Z. Albumin Carriers for Cancer Theranostics: A Conventional Platform with New Promise. Adv. Mater. 2016, 28, 10557-10566. (20)Yang, W.; Guo, W.; Chang, J.; Zhang, B. Protein/Peptide-Templated Biomimetic Synthesis of Inorganic Nanoparticles for Biomedical Applications. J. Mater. Chem. B 2017, 5, 401-417. (21)Pan, J.; Wang, Y.; Pan, H.; Zhang, C.; Zhang, X.; Fu, Y.-Y.; Zhang, X.; Yu, C.; Sun, S.-K.; Yan, X.-P. Mimicking Drug-Substrate Interaction: A Smart Bioinspired Technology for the Fabrication of Theranostic Nanoprobes. Adv. Funct. Mater. 2017, 27, 1603440. (22)Wang, Y.; Yang, T.; Ke, H.; Zhu, A.; Wang, Y.; Wang, J.; Shen, J.; Liu, G.; Chen, C.; Zhao, Y.; Chen, H. Smart Albumin-Biomineralized Nanocomposites for Multimodal Imaging and Photothermal Tumor Ablation. Adv. Mater. 2015, 27, 3874-3882. (23)Zhao, Y.; Peng, J.; Li, J.; Huang, L.; Yang, J.; Huang, K.; Li, H.; Jiang, N.; Zheng, S.; Zhang, X.; Niu, Y.; Han, G. Tumor-Targeted and Clearable Human ProteinBased MRI Nanoprobes. Nano Lett. 2017, 17, 4096-4100. (24)Jin, Q.; Liu, J.; Zhu, W.; Dong, Z.; Liu, Z.; Cheng, L. Albumin-Assisted Synthesis of Ultrasmall FeS2 Nanodots for Imaging-Guided Photothermal Enhanced Photodynamic Therapy. ACS Appl. Mater. Interfaces 2018, 10, 332-340. (25)Yang, W.; Guo, W.; Le, W.; Lv, G.; Zhang, F.; Shi, L.; Wang, X.; Wang, J.; Wang, S.; Chang, J.; Zhang, B. Albumin-Bioinspired Gd:CuS Nanotheranostic Agent for In Vivo Photoacoustic/Magnetic Resonance Imaging-Guided Tumor-Targeted Photothermal Therapy. ACS nano 2016, 10, 10245-10257. (26)Sheng, J.; Wang, L.; Han, Y.; Chen, W.; Liu, H.; Zhang, M.; Deng, L.; Liu, Y. N. Dual Roles of Protein as a Template and a Sulfur Provider: A General Approach to Metal Sulfides for Efficient Photothermal Therapy of Cancer. Small 2018, 14, 1702529. (27)Yang, T.; Tang, Y.; Liu, L.; Lv, X.; Wang, Q.; Ke, H.; Deng, Y.; Yang, H.; Yang, X.; Liu, G.; Zhao, Y.; Chen, H. Size-Dependent Ag2S Nanodots for Second NearInfrared Fluorescence/Photoacoustics Imaging and Simultaneous Photothermal Therapy. ACS nano 2017, 11, 1848-1857.

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ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

(28)Lin, T.; Zhao, X.; Zhao, S.; Yu, H.; Cao, W.; Chen, W.; Wei, H.; Guo, H. O2Generating MnO2 Nanoparticles for Enhanced Photodynamic Therapy of Bladder Cancer by Ameliorating Hypoxia. Theranostics 2018, 8, 990-1004. (29)Yang, Z.; He, W.; Zheng, H.; Wei, J.; Liu, P.; Zhu, W.; Lin, L.; Zhang, L.; Yi, C.; Xu, Z.; Ren, J. One-Pot Synthesis of Albumin-Gadolinium Stabilized Polypyrrole Nanotheranostic Agent for Magnetic Resonance Imaging Guided Photothermal Therapy. Biomaterials 2018, 161, 1-10. (30)Ma, R.; Bando, Y.; Zhang, L.; Sasaki, T. Layered MnO2 Nanobelts: Hydrothermal Synthesis and Electrochemical Measurements. Adv. Mater. 2004, 16, 918-922. (31)Liu, Y.; Ai, K.; Liu, J.; Deng, M.; He, Y.; Lu, L. Dopamine-Melanin Colloidal Nanospheres: An Efficient Near-Infrared Photothermal Therapeutic Agent for In Vivo Cancer Therapy. Adv. Mater. 2013, 25, 1353-1359. (32)Fan, Q.; Cheng, K.; Hu, X.; Ma, X.; Zhang, R.; Yang, M.; Lu, X.; Xing, L.; Huang, W.; Gambhir, S. S.; Cheng, Z. Transferring Biomarker into Molecular Probe: Melanin Nanoparticle as a Naturally Active Platform for Multimodality Imaging. J. Am. Chem. Soc. 2014, 136, 15185-15194. (33)Zhang, R.; Fan, Q.; Yang, M.; Cheng, K.; Lu, X.; Zhang, L.; Huang, W.; Cheng, Z. Engineering Melanin Nanoparticles as an Efficient Drug-Delivery System for Imaging-Guided Chemotherapy. Adv. Mater. 2015, 27, 5063-5069. (34)Yang, J.; Choi, J.; Bang, D.; Kim, E.; Lim, E. K.; Park, H.; Suh, J. S.; Lee, K.; Yoo, K. H.; Kim, E. K.; Huh, Y. M.; Haam, S. Convertible Organic Nanoparticles for Near-Infrared Photothermal Ablation of Cancer Cells. Angew. Chem., Int. Ed. 2011, 50, 441-444. (35)Lin, M.; Wang, D.; Li, S.; Tang, Q.; Liu, S.; Ge, R.; Liu, Y.; Zhang, D.; Sun, H.; Zhang, H.; Yang, B. Cu(II) Doped Polyaniline Nanoshuttles for Multimodal Tumor Diagnosis and Therapy. Biomaterials 2016, 104, 213-222. (36)Li, B.; Yuan, F.; He, G.; Han, X.; Wang, X.; Qin, J.; Guo, Z. X.; Lu, X.; Wang, Q.; Parkin, I. P.; Wu, C. Ultrasmall CuCo2S4 Nanocrystals: All-in-One Theragnosis Nanoplatform with Magnetic Resonance/Near-Infrared Imaging for Efficiently Photothermal Therapy of Tumors. Adv. Funct. Mater. 2017, 27, 1606218. (37)Li, Y.; He, L.; Dong, H.; Liu, Y.; Wang, K.; Li, A.; Ren, T.; Shi, D.; Li, Y. FeverInspired Immunotherapy Based on Photothermal CpG Nanotherapeutics: The Critical Role of Mild Heat in Regulating Tumor Microenvironment. Adv. Sci. 2018, 5, 1700805. (38)Ye, M.; Qian, Y.; Tang, J.; Hu, H.; Sui, M.; Shen, Y. Targeted Biodegradable Dendritic MRI Contrast Agent for Enhanced Tumor Imaging. J. Controlled Release 2013, 169, 239-245. (39)Chen, Y.; Ye, D.; Wu, M.; Chen, H.; Zhang, L.; Shi, J.; Wang, L. Break-Up of Two-Dimensional MnO2 Nanosheets Promotes Ultrasensitive pH-Triggered Theranostics of Cancer. Adv. Mater. 2014, 26, 7019-7026. (40)Song, M.; Liu, T.; Shi, C.; Zhang, X.; Chen, X. Bioconjugated Manganese Dioxide Nanoparticles Enhance Chemotherapy Response by Priming Tumor-Associated Macrophages toward M1-like Phenotype and Attenuating Tumor Hypoxia. ACS nano 2016, 10, 633-647. 20


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(41)Zhao, Z.; Fan, H.; Zhou, G.; Bai, H.; Liang, H.; Wang, R.; Zhang, X.; Tan, W. Activatable Fluorescence/MRI Bimodal Platform for Tumor Cell Imaging via MnO2 Nanosheet-Aptamer Nanoprobe. J. Am. Chem. Soc. 2014, 136, 11220-11223. (42)Yu, L.; Chen, Y.; Wu, M.; Cai, X.; Yao, H.; Zhang, L.; Chen, H.; Shi, J. "Manganese Extraction" Strategy Enables Tumor-Sensitive Biodegradability and Theranostics of Nanoparticles. J. Am. Chem. Soc. 2016, 138, 9881-9894. (43)Zhao, Z.; Bao, J.; Fu, C.; Lei, M.; Cheng, J. Controllable Synthesis of Manganese Oxide Nanostructures from 0-D to 3-D and Mechanistic Investigation of Internal Relation between Structure and T1 Relaxivity. Chem. Mater. 2017, 29, 1045510468. (44)Bai, C.; Jia, Z.; Song, L.; Zhang, W.; Chen, Y.; Zang, F.; Ma, M.; Gu, N.; Zhang, Y. Time-Dependent T1-T2 Switchable Magnetic Resonance Imaging Realized by c(RGDyK) Modified Ultrasmall Fe3O4 Nanoprobes. Adv. Funct. Mater. 2018, 28, 1802281. (45)Prasad, P.; Gordijo, C. R.; Abbasi, A. Z.; Maeda, A.; Ip, A.; Rauth, A. M.; DaCosta, R. S.; Wu, X. Y. Multifunctional Albumin–MnO2 Nanoparticles Modulate Solid Tumor Microenvironment by Attenuating Hypoxia, Acidosis, Vascular Endothelial Growth Factor and Enhance Radiation Response. ACS nano 2014, 8, 3202-3212. (46)Liu, Y.; Ai, K.; Ji, X.; Askhatova, D.; Du, R.; Lu, L.; Shi, J. Comprehensive Insights into the Multi-Antioxidative Mechanisms of Melanin Nanoparticles and Their Application To Protect Brain from Injury in Ischemic Stroke. J. Am. Chem. Soc. 2017, 139, 856-862.

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Scheme 1. Schematic illustration of the integration of polymerization and biomineralization to synthesize multifunctional nanotheranostic agents. DA: dopamine , EGC: epigallocatechin, PY: pyrrole, PD: diaminopyridine.

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Figure 1. (a) Hydrodynamic diameters of PMHNs with different monomers. (b) Typical TEM images of PMHN-DA. (c) HRTEM images of a single PMHN-DA (inset: single MnO2 nanodot). (d) FETEM images and the elemental mapping images (C, O, and Mn) of PMHN-DA. (e) XPS spectrum of MnO2 in PMHN-DA. (f) CD spectra of PMHN-DA and pure BSA. (g) ESR spectrum of PMHN-DA.

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Figure 2. (a) Photothermal heating profile of the PMHNs from different photothermiaderived monomers under laser irradiation (808 nm, 2.0 W cm−2, and 5 min). (b) Photothermal response of PMHN-DA aqueous solution treated with an NIR laser (808 nm, 2.0 W cm−2) for 500s, and subsequently the laser was shut off. (c) Linear time data versus -lnθ obtained from the cooling stage of (b). (d) Changes in temperature of the PMHN-DA aqueous solution upon laser irradiation (808 nm, 2.0 W cm−2, and 5 min) for four laser on/off cycles. (e) Relative viabilities of 4T1 cells after PMHN-DAinduced PTT for 48 h at various concentrations. (f) Confocal images of propidium iodide (red, dead cells) and calcein AM (green, live cells) costained cells after incubation with PMHN-DA and laser irradiation (808 nm, 2.0 W cm-2, and 5 min). Inset: the laser exposed areas were indicated in a and b, and the live cells and dead cells were displayed in c and d, respectively. (g) T1 relaxation rates vs Mn concentrations of 24


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different PMHNs. (h) T1-weighted MR images at various Mn concentrations of PMHNDA (No1-5 and 8) solutions. (i) Magnetization curve of PMHN-DA and AMN at 300 K.

Figure 3. In vivo multifunctional MRI. (a) T1-weighted MR images and (c) Signal to noise ratio (SNR) changes of the orthotopic 4T1-tumor bearing mice before and after intravenous administration of PMHN-DA solutions (0.06 mmol Kg-1 Mn). (b) T1weighted MR images and (d) SNR changes of liver-metastases-bearing mice before and after intravenous administration of PMHN-DA solutions (0.06 mmol Kg-1 Mn). (e) T1weighted MR images of the kidneys in normal mice before and after intravenous administration of PMHN-DA solutions (0.02 mmol Kg-1 Mn). (f) In vitro and (g) in vivo ultrasound imaging of PMHN-DA or AMN NPs-treated H2O2 solutions and tumors (0.06 mmol Kg-1 Mn). 25


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Figure 4. (a) Infrared thermal images and (b) tumor temperature profiles of orthotopic 4T1 tumor-bearing mice recorded in laser irradiation after intravenous injection of PMHN-DA (3.25 mg Kg-1) or PBS. (c) Tumor growth profiles, (d) body weight, and (e) survival curves of orthotropic 4T1 tumors in mice of various groups after different treatments (n=5). (f) H&E-stained images of the major organs from the control healthy mice, PMHN-DA-treated healthy mice 60 days after the injection (0.06 mmol Kg-1Mn), and PMHN-DA-treated tumor-bearing mice 60 days after the photothermal therapy (3.25 mg Kg-1). Scale bar: 200 μm. ToC Figure

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A biomineralization strategy is developed by integrating polymerization and biomineralization in albumin templates, which allows for facile synthesis of multifunctional nanotheranostic agents via a reproducible one-pot method.

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Integration of Polymerization and Biomineralization as a Strategy to

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