Gold Cube-in-Cube Based Oxygen Nanogenerator: A Theranostic

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Gold Cube-in-Cube Based Oxygen Nanogenerator: A Theranostic Nanoplatform for Modulating Tumor Microenvironment for Precise Chemo-Phototherapy and Multimodal Imaging Xing Zhang, Zhongqian Xi, Jeremiah Ong’achwa Machuki, Jianjun Luo, Dongzhi Yang, Jingjing Li, Weibing Cai, Yun Yang, Lijie Zhang, Jiangwei Tian, Kaijin Guo, Yanyan Yu, and Fenglei Gao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09786 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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Gold Cube-in-Cube Based Oxygen Nanogenerator: A Theranostic Nanoplatform for Modulating Tumor Microenvironment for Precise Chemo-Phototherapy and Multimodal Imaging Xing Zhang1,2, Zhongqian Xi1, Jeremiah Ong’achwa Machuki1, Jianjun Luo1,2, Dongzhi Yang1, Jingjing Li1, Weibing Cai1, Yun Yang3, Lijie Zhang3, Jiangwei Tian4, Kaijin Guo1,2,*, Yanyan Yu1 and Fenglei Gao1,* 1. Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy,

Xuzhou

Medical

University, Jiangsu 221002, P. R. China. 2. Institute of Orthopedics, Department of Orthopedics, Affiliated Hospital of Xuzhou Medical University, Jiangsu 221002, P. R. China. 3. Nanomaterials and Chemistry Key Laboratory, Wenzhou University, Zhejiang 325027, P. R. China 4. School of Traditional Chinese Pharmacy, China Pharmaceutical University, Jiangsu 211198, P. R. China Email address: [email protected] (F. Gao), [email protected] (K. Guo).

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ABSTRACT Engineering a versatile oncotherapy nanoplatform integrating both diagnostic and therapeutic functions has always been an intractable challenge in targeted cancer treatment. Herein, to actualize the theme of precise medicine, a nanoplatform is developed by anchoring Mn-Cdots to doxorubicin (DOX)-loaded mesoporous silica-coated gold cube-in-cubes core/shell nanocomposites and further conjugating them to a Arg-Gly-Asp (RGD) peptide (denoted as RGD-CCmMC/DOX) to achieve active-targeting effect. Under a 635 nm irradiation, the nanoplatform acts as oxygen nanogenerator that produces O2 in situ and amplifies the content of singlet oxygen (1O2) in the hypoxic tumor microenvironment (TME), which has been demonstrated to attenuate tumor hypoxia and synchronously enhance photodynamic efficacy. Moreover, the gold cube-in-cube core in this work has proven as a photothermal agent for hyperthermia, which exhibits a favorable photothermal effect with a 65.6% calculated photothermal conversion efficiency under 808 nm irradiation. In addition, the nanoplatform achieves heat/pH-sensitive drug realease with precise control to specific-tumor sites, executing combined chemo-phototherapy functions. Besides, it functions as a multimodal bioimaging agent of photothermal, fluorescence and magnetic resonance imaging for accurate diagnosis and guidance of therapy. As validated by in vivo and vitro assays, the TME-responsive nanoplatform is highly biocompatible and effectively obliterates 4T1 tumor xenograft on nude mice after triple-synergetic treatment. This work presents a rational design of versatile nanoplatforms which modulate the TME to enable high therapeutic performance and multiplexed imaging, which provides an innovative paradigm for targeted tumor therapy. KEYWORDS: cancer theranostics; oxygen nanogenerator; hypoxia alleviation; targeting; chemo-phototherapy; multimodal imaging; 2


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Over the past years, malignant tumors have been considered to be among the most dreadful diseases that jeopardize human health.1-3 Despite considerable progress in enriching anticancer treatment modalities, numerous types of carcinomas cannot be completely eliminated due to the poor tumor perfusion and high metastatic nature.4-6 Furthermore, it has been recognized that the curative effect of traditional cancer treatments is unsatisfactory owing to their non-specific targeting characteristic and adverse side effects, which hinders their clinic application.7,8 Faced with such predicament, many scientists are attempting to develop a versatile nanoplatform in which medical diagnostics, drug delivery, and oncotherapy are integrated into a single nanosystem.9,10 Such oncotherapy platforms that impart both diagnostic and therapeutic functions have received extensive scientific attention in the field of precision medicines. To date, burgeoning phototherapy methods, such as photodynamic therapy (PDT) and photothermal therapy (PTT), have been integrated with chemotherapy and/or multimodal imaging to generate theranostic nanoplatforms, which is expected to achieve better therapeutic performance relative to monotherapeutic modes.7,11,12 However, the designing and engineering of such high-performance nanoplatforms that cotransport therapeutic and imaging agents remains an intractable challenge. PTT, a newly emerging therapeutic mode, exploits PTT agents to convert energy from laser into heat, causing the elevation of local temperature and hyperthermia (>41 °C) for irreversible tumor damage.13,14,72 This damage includes protein denaturation and nucleic acids damnification which inhibits tumor growth and facilitates tumor ablation.15,16 Currently, gold-based nanomaterials, especially gold nanocubes or nanorods, have been extensively investigated and exploited in PTT owing to their surface plasmon resonance. Despite their usefulness, low photostability and high scattering loss limits their application.17-20 To date, application of gold cube-in-cubes which have a 3


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special interior nanogap as photothermal agents is yet to be investigated or reported in PTT field.21,22 In this work, gold cube-in-cubes exhibiting superb photothermal effect, long-term photostability, and 65.6% photothermal conversion efficiency have been fabricated by our team and employed as an advantageous PTT agent. Although gold cube-in-cube has favorable photothermal conversion efficiency and stability, the therapeutic efficacy of sole PTT is not as satisfactory compared to synergistic therapeutic modes owing to the heterogeneous distribution of resulting heat. Thus, strategies that combine PTT with other therapeutic techniques, such as photodynamic therapy and so on, should be actively pursued to provide high overall therapeutic performance for oncologic treatments. As another non-invasive therapeutic strategy, photodynamic therapy relies on the fact that photosensitizers are activated under appropriate laser irradiation to generate large amounts of cytotoxic reactive oxygen species (ROS) to kill cancer cells.23-25,71 However, the efficiency of PDT is severely hampered by the inherent hypoxic TME.1,26,27 Tumor hypoxia and acidic nature, which are distinct hallmarks of TME, not only induce irreversible tumor metastasis and angiogenesis,28,29 but also inflict hypoxia-associated resistance thereby compromising the therapeutic efficiency of several cancer therapies that demand oxygen.30,31 So far, many intriguing strategies aimed at transporting oxygen to the hypoxic regions or in situ generation of oxygen inside the solid tumor for efficient tumor oxygenation, such as MnO2,32,33 perfluorocarbon,34 or carbon nitride35 mediated oxygen delivery, have been explored. Very recently, the application of Mn-Carbon dots (Mn-Cdots), which have catalase-like activity that triggers endogenous H2O2 decomposition to produce O2 and to amplify singlet oxygen (1O2) generation, has led to the recognition that Mn-Cdots are highly effective in regulating hypoxic TME for tumor hypoxia attenuation.36 Thus, leveraging 4


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on the sensitive pH-/H2O2-responsive properties of Mn-Cdots, some Mn-Cdots were anchored onto gold cube-in cube@mSiO2 nanocomposite to form a CCmMC nanosystem, which was served as an oxygen nanogenerator, for four major reasons: (i) Mn-Cdots are smart nanophotosensitizers that amplify the formation of ROS or 1O2; (ii) Mn-Cdots with enzyme-like activities can be reduced into Mn2+, and simultaneously trigger the decomposition of endogenous H2O2 under acidic TME of solid tumors, thereby increasing in situ oxygen concentrations in the hypoxic regions—an effect that improves the oxygen-dependent-featured PDT; (iii) the CCmMC nanosystem enables the co-loading of Mn-Cdots and DOX provide efficient transport of therapeutic agents into the tumor sites; (iv) Mn-Cdots offer contrasts for T1-weighted magnetic resonance imaging (MRI) and FL imaging, whereas the gold cube-in-cube core endows the nanosystem with photothermal imaging, executing multimodal imaging for precise tumor diagnosis. Importantly, the mild hyperthermia mediated by CCmMC during PTT can speed up the intratumoral blood flow so as to enhance oxygen distribution and oxygenation effect, and eventually improve the PDT efficacy with a synergistic effect. Today, coalescing the phototherapy and chemotherapy to target multiple essential pathways required for tumor growth has become a vigorous trend since it affords multiple advantages in terms of enhancing cancer therapeutic efficiency.8,37-40,70 The approach of using mesoporous silica-coated gold cube-in-cubes as cargo reservoirs to enhance drug loading and the pH/heat-responsive Mn-Cdots as gatekeepers to realize drug release with precise and flexible control within the TME will achieve better chemotherapeutic efficacy. In addition, the heat generated from PTT can remotely stimulate the release of drug from the CCmMC nanovehicles simultaneously along with the pH-sensitive drug release. Recently, numerous empirical studies have revealed that alleviation 5


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of hypoxia in tumor tissues by increasing oxygen levels augments the sensitivity of tumor cells to DOX, thereby suppressing tumor progression.41,42 Once the hypoxic levels in tumor tissues are reversed, targeted delivery of anticarcinogen agents to the tumor sites would enormously improve the overall therapeutic effects.43,44 In healthy tissues, the employment of synergistic therapies with untargeted nanosystems is often accompanied with some undesired phototoxicity and anticarcinogen toxicity. Also, due to the passive targeting via the enhanced permeability retention (EPR), only a small amount of anticarcinogen agents can access the tumor tissues, which compromises the therapeutic efficacy of the current nanosystems.45-47 Therefore, to address this non-targeting limitation, an Arg-Gly-Asp (RGD) tumor-homing peptide, which specifically binds to neuropilin-1 and αvβ3 integrin receptor overexpressed on the surface of cancer cells, was introduced and decorated on the CCmMC NPs to accomplish the task of active targeting effect.48,49 This allows these nanoparticles to achieve deep intratumoral permeability and retention thereby enhancing therapeutic agents accumulation at the targeted tumor tissues.50-52 More importantly, the great photoluminescence characteristic of Mn-Cdots, the beneficial photothermal property of gold cube-in-cubes, and enhanced T1-magnetic resonance signal of Mn2+ can be utilized for triple-modal fluorescence/photothermal/magnetic resonance (FL/PTI/MRI) imaging, which allows the visualization and tracking of tumor progression as well as guiding anticancer treatments. In this work, a TME-responsive and O2 self-supplement theranostic nanoplatform based on gold cube-in-cube was developed for triple-collaborative targeted tumor therapy (Scheme 1). In the design, the nanoplatform was successfully fabricated by anchoring Mn-Cdots to doxorubicin-loaded mesoporous silica-coated gold cube-in-cubes core/shell nanocomposites followed by conjugation with RGD targeting ligands (denoted as RGD-CCmMC/DOX) to achieve the active-targeting effect. 6


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When the nanoplatforms accumulated at the tumor sites, the Mn-Cdots efficiently consumed endogenous H2O2 produced by tumors thereby elevating in situ oxygen concentrations for efficient tumor hypoxia attenuation and improved PDT efficacy. This eliminated the concomitant chemotherapeutic drug-resistance caused by hypoxia with an auxiliary effect. Moreover, the gold cube-in-cube core in the nanoplatform has proven to be an advantageous photothermal agent, which exhibits a favorable photothermal effect with a 65.6% photothermal conversion efficiency under 808 nm irradiation. In addition, Mn-Cdots acted as gatekeepers to precisely release the encapsulated DOX in a heat-/pH-sensitive manner at specific-tumor sites. Meanwhile, the nanoplatform served as a FL/PTI/MRI multimodal bioimaging agent, furnishing complementary information for accurate diagnosis and imaging-guided cancer therapy. As expected, the versatile nanoplatform was highly biocompatible and showed enhanced therapeutic effect after triple-synergetic treatments in vivo and vitro. In conclusion, this work successively designed and engineered a versatile nanoplatform with the TME-modulating effect which not only resulted in a high-performance synergism that combined PTT/PDT/chemotherapy for targeted cancer therapy, but also forms the basis for the design of possible nanosystems for tumor treatment.

RESULTS AND DISCUSSION Characterization of Versatile Nanoplatforms. The synthesis process and applications of the versatile nanoplatforms are illustrated in Scheme 1. In this work, ROS was produced by Mn-Cdots (photosensitizers), local hyperthermia was generated from the gold cube-in-cube core upon laser irradiation and the precise tumor targeting capability of the CCmMC nanoplatform was achieved via RGD targeting ligands. The morphology and structure of CCmMC NPs were characterized by transmission electron microscopy (TEM), as depicted in Figure 1. Gold nanocubes with uniform 7


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cubical morphology and monodispersed size were synthesized using seed-growth procedure (Figure 1A1~A2). The cornerstone of the CCmMC NPs was the gold cube-in-cubes (Au CCs) with a special interior nanogap, which were fabricated via an ion replacement method (Figure 1B1~B2). A layer of cubic mesoporous silica was coated on the gold cube-in-cubes to form Au CCs@mSiO2 via an optimized sol-gel approach (Figure 1C1~C2), which acted as a precursor to accelerate the formation of CCmMC NPs. Subsequently, CCmMC NPs were synthesized based on the strong electrostatic interactions between negatively charged Mn-Cdots and positively charged NH2-Au CCs@mSiO2, which was confirmed by the zeta potential (Figure S1b). After introducing amino groups, the zeta potential of CCm-NH2 still remained positive. A decrease in zeta potential from 24.5±1.6 mV to 5.6±0.5 mV confirmed the successful attachment of Mn-Cdots to amine-modified Au CCs@mSiO2. TEM (Figure 1D1~D2) showed that the CCmMC NPs possessed mondispersity and cubical morphology, and the Mn-Cdots were coated around the Au CCs@mSiO2 nanocomposites. Careful observation revealed that the tiny Mn-Cdots uniformly covered the surface of the Au CCs@mSiO2 or scattered around Au CCs@mSiO2. Also, the hydrodynamic size of CCmMC measured by dynamic light scattering (DLS) was about 116.5 nm (Figure S1a). Meanwhile, the Mn-Cdot were highly crystalline with an interlayer spacing of 0.21 nm and an average diameter of 4.1±0.5 nm, which was confirmed by HRTEM image and DLS (Figure S2a, S2b and S2d). Corresponding atomic force microscopy (AFM) image and X-ray photoelectron spectroscopy (XPS) were also performed and the results are presented in Figure S2c and S2e. To further validate the elemental composition of CCmMC NPs, the scanning transmission electron microscopy (STEM) and elemental mapping images were obtained. It was noticed that the gold elements were highly concentrated at the core, while the silicon and oxygen signals were observed 8


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at the outer zone, and the signals from manganese and carbon were recorded at the outermost region, displaying a homogeneous distribution of Au, Si, O, Mn and C elements. This confirmed the successful integration of three independent components. Furthermore, the structure and crystalline phase of the samples was verified by X-ray Diffraction (XRD), which are provided in Figure 1F. The obvious diffraction peaks at 2θ degree of 38.2°, 44.4° and 64.7° were assigned to (111), (200) and (220) crystal planes of gold cube-in-cubes. Moreover, XRD spectra of CCmMC analysis revealed the characteristic peaks at 24.7°, 34.9°, 58.8°, which can be ascribed to (002), (111) and (220) of the crystalline Mn-Cdots. The XRD pattern of RGD-CCmMC/DOX NPs displayed reduced peak intensities in comparison with the CCmMC NPs, which confirm the immobilization of RGD and DOX on the surface of CCmMC. XPS spectra was applied to further explore the composition of CCmMC NPs as shown in Figure S3. The spectrum of CCmMC revealed the characteristic peaks associated with Mn 2p, C 1s, O 1s, Au 4d and Au 4f. In the Mn 2p XPS spectra, two peaks 641.8 and 653.4 eV derived from Mn 2p3/2 and Mn 2p1/2 were recorded, providing evidence for the existence of Mn2+. After confirmation of the synthesis and composition of nanocomposites, the UV-vis absorption characteristics, which is an extremely significant parameter for characterizing PTT agents,61,62 was investigated. The absorbance of gold cube-in-cubes, which contained two characteristic absorbance peaks, was much higher than that of gold nanocubes in this work (Figure S4). The UV-vis absorption spectra of gold cube-in cubes, CCmMC and RGD-CCmMC/DOX NPs were examined to evaluate their optical properties as indicated in Figure 1G. Gold cube-in-cubes possessed two characteristic absorbance peaks in the range of 580-680 nm and 720-880 nm, which were ascribed to the different sizes of gold cubes. UV-vis absorbance of the CCmMC nanoparticles still retained two unchanged characteristic peaks with slightly broader 9


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spectra. Notably, absorption peaks of CCmMC NPs and RGD-CCmMC/DOX NPs within the optimal visible and near-infrared (NIR) window were not significantly attenuated, which confirms their potential to be exploited as combination PTT/PDT nanoagents. N2 physisorption and pore distribution results of the CCm NPs and RGD-CCmMC/DOX NPs are shown in Figure 1H and Figure S5. The isotherm of CCm NPs displayed a typical type IV feature, demonstrating that CCm NPs maintained a mesoporous architecture, which was beneficial to its effective anticarcinogen loading. The specific surface area of the CCm was determined to be 316 m2 g−1 according to the Brunauer-Emmett-Teller method and a pore distribution of about 2.9±0.3 nm was obtained via N2 adsorption-desorption isotherm analysis. Compared to the CCm NPs, the amount of N2 adsorbed by RGD-CCmMC/DOX NPs decreased significantly, as well as the Brunauer-Emmett-Teller surface area, which might be ascribed to DOX loading in the cubic mesoporous channels. Photothermal Effect in Vitro, Pharmacokinetics and Cellular Internalization. Enhancing NIR absorption could give rise to efficient photothermal effect, which is the foundation of photothermal therapy. Therefore, we systematically evaluated and investigated the photothermal performance of CCmMC nanovehicles. As shown in Figure 2A and Figure 2B, the temperature elevation of CCmMC suspension displayed a concentration and irradiation duration-dependent pattern. Moreover, the temperature of CCmMC suspension (400 μg/mL) increased by about 33 ℃ under 808 nm laser irradiation as soon as 5 min. In contrast, slight temperature elevation was observed in the physiological saline solution under the same treatment conditions, indicating that CCmMC NPs possessed high NIR laser-induced photothermal performance. Some previous experimental studies have reported that cancer cells can be killed within 4-6 min at temperatures above 50 °C.14,55,56 In this study, the temperature of the CCmMC suspension rapidly increased 10


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within 5 min as the laser power density increased from 0.3 to 1.2 W/cm2 (Figure 2C), and the temperature elevations were dependent on power density. After irradiation for 5 min with 1.2 W/cm2 irradiation, the temperature of the CCmMC suspension increased by nearly 40 °C compared with 0.3 W/cm2 irradiation which produced an increase of 7 °C, implying that the CCmMC NPs were highly efficient at converting laser energy into thermal energy. In PTT field, the curative efficacy of photothermal ablation relies on the photothermal conversion efficiency and stability of nanomaterials, effectively restricting the clinical application of numerous PTT agents.57,58 As illustrated in Figure 2D, CCmMC nanoparticles exhibited higher photothermal conversion efficiency and photothermal stability (10 min laser on and 10 min laser off) compared to Au cubes. The photothermal conversion efficiency of CCmMC was calculated to be about 65.6% under irradiation by an 808 nm laser, which was much higher than that of gold nanocube (37.2%) (Figures S6). In addition, the temperature change of Au cube-in-cube at a duration of 1200 s was extremely similar to the photothermal behaviors of CCmMC, as given in the Figures S7. To further explore the cyclic photothermal transduction stability of CCmMC, the CCmMC suspension was subjected to 4 repeated irradiation cycles (Figure 2E). The result revealed that the temperature of CCmMC suspension elevated rapidly and reached a plateau upon irradiation for 10 min, which then naturally cooled close to the ambient temperature within 15 min. The rapid heating and cooling pattern demonstrated significant photothermal conversion stability and reproducibility of CCmMC nanoplatforms. According to these results, it can be inferred that CCmMC nanoplatforms can be employed as efficient NIR-mediated PTT agents for precise tumor phototherapy. To investigate the effect of pH and temperature as dual-stimuli on the pharmacokinetic release behavior of CCmMC/DOX as drug nanovehicles, DOX was first loaded into the mesoporous 11


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structure of nanovehicles. The malignant proliferation of cancer cells leads to excessive accumulation of metabolic products, which decreases the pH of the tumor microenvironments.59,60 Therefore, we selected three pH values (5, 6.6, and 7.4) to evaluate the drug metabolism of the DOX-loaded nanovehicles. After 24 h of continuous agitation in darkness, DOX in the pore channels of CCmMC nanovehicles were completely sealed with Mn-Cdots through electrostatic interaction, and UV-vis spectrophotometer analysis showed that the DOX loading efficiency was about 78%. Afterwards, the in vitro release behavior of CCmMC/DOX NPs were conducted in PBS buffer of different pH values at 37 °C or 50 °C. As depicted in Figure 3A, the cumulative DOX release rate of 37 °C was about 15.2% at pH 7.4, 34.5% at pH 6.6, and 50.6% at pH 5.0 during 96 h, respectively. Obviously, CCmMC/DOX nanovehicle displayed a sustained release with stepwise increases from 15.2% to 50.6% when the pH values were adjusted from 7.4 to 5.0, which ideally mimic intracellular and extracellular physiological states, indicating that the release behavior of nanovehicles followed a pH-dependent pattern. The higher DOX-release rate at acidic conditions may be explained by the fact that as the pH dropped, substantial Mn-Cdots-COO- became protonated, leading to the dissociation of Mn-Cdots from the surface of Au CCs@mSiO2, the opening of gated channels, and release of the encapsulated cargo.63 Another reason is that the acidic condition increased the solubility of DOX which in turn accelerated the release of the drug from the mesoporous pores of the nanocarriers. Besides, the DOX cumulative release rate was significantly higher at 50 °C than at 37 °C in PBS at all pH conditions in Figure 3A or Figure S8, demonstrating that temperature played a pivotal role in boosting the release of targeted molecules. This is ascribed to the fact that the increase in temperature enervated the electrostatic interaction between Mn-Cdots-COO- and cationic NH3+ groups on the Au CCs@mSiO2 surface, thereby facilitating 12


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DOX release. Moreover, it is worthy to note that 62% of DOX was released within 24 h when the pH value was 5.0, whereas only 6% of the DOX was slowly released from the nanoparticles in the subsequent 72 h. Thus, these results illustrate that the pH and temperature may act as a remote switch to rationally control the release of DOX very effectively from CCmMC/DOX NP, thereby optimizing antineoplastic effects in vitro and in vivo. Afterwards, to enhance biocompatibility and strengthen tumor selectivity of CCmMC, a pS-PEG2000-RGD were introduced into CCmMC to form RGD conjugated CCmMC nanoparticles (RGD-CCmMC) due to the formation of hydrogen-bonded complexes and/or amidation reaction.68,69 To confirm the cellular uptake characteristics, the endocytosis mechanism of RGD-CCmMC/DOX NPs and intracellular release behaviors of the encapsulated drugs were investigated on 4T1 cells using confocal laser scanning microscopy (CLSM). The CLSM imaging was performed on 4T1 cells incubated with the RGD-CCmMC/DOX NPs (30 μg/mL) for various durations (1 h, 2 h, and 4 h). It is widely acknowledged that endolysosomal compartments are closely associated with the cellular endocytosis of nanosystems.64 In this work, lysotracker green, a commercial lysosome binding dye, was utilized to ascertain the localization of the nanosystems and lysosomes. As shown in Figure 3B, strong fluorescence of DOX and Lysotracker were observed, demonstrating that the RGD-CCmMC/DOX nanosystems had been internalized by 4T1 cells via an endolysosomal pathway.64 Besides, a time-dependent increase in red fluorescence was observed in the cells, which was ascribed to the release of DOX from the pore channels due to the “on” state of the “gatekeeper” Mn-Cdots under this condition. The merge images of DAPI, DOX, and lysotracker green indicated that the nanosystems were mainly distributed in the cytoplasm after 1 h of incubation. When the incubation time reached 4 h, DOX was gradually released from 13


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RGD-CCmMC/DOX located in the endocytic compartments into the nucleus, which was consistent with the featured fluorescence of the nucleus region after 4 h of incubation. Flow cytometry analysis was further performed to quantitatively investigate the effect of RGD conjugation on the cell internalization mechanism, and the results are shown in Figure 3C. The cellular fluorescence intensity of RGD-conjugated CCmMC NPs was significantly higher than that of the nontargeted CCmMC NPs at all time-points, signifying that cellular internalization of RGD-CCmMC/DOX was strengthened due to the synergistic effect produced by tumor-targeted molecule recognition and the Nrp-1 receptor. This finding not only indicated the successful conjugation of functional RGD onto the nanoplatform, but also confirmed the availability of active-targeting peptide for enhanced delivery of the theranostic nanoplatform. The nanoplatform decorated with targeting moiety of RGD displayed improved cell internalization efficiency and enhanced tumor targeting delivery of encapsulated cargos, which provided better therapeutic performance. As shown in Figure 3D, the 4T1 cells displayed strong green fluorescence signals and left a clear nucleus without green fluorescence, confirming that large amounts of Mn-Cdots had been internalized into the cells and only distributed in the cytoplasm. Moreover, the cellular green fluorescence intensity of the RGD-CCmMC/DOX-treated cells was much higher compared with CCmMC/DOX NPs, which coincided with results of flow cytometry analysis. After endocytosis, the intracellular localization and distribution of RGD-CCmMC/DOX NPs in 4T1 cells were directly confirmed by cellular TEM imaging (Figure 3E). Further analysis of the TEM images showed the local distribution of nanoplatforms in the cytoplasm in the form of aggregations. Taken together, we conclude that RGD-modified CCmMC NPs had improved cellular uptake and delivery of tumor-targeted anticarcinogen agents, making them suitable for targeting strategy. 14


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Oxygen Generation, Extracellular and Intracellular ROS Production, and Hypoxia Alleviation. According to a previous study, Mn2+ can be oxidized by H2O2 to MnO2 which then acts as a nanoagent to trigger oxygenation under acidic conditions. The catalytic decomposition of H2O2 to generate oxygen can be explained by the following reaction equations.36,62 Mn2+ + H2O2 MnO2 + H2O2 + 2H+

2H+ +MnO2

(1)

Mn2+ + 2H2O + O2

(2)

Therefore, to examine the catalytic power of the CCmMC NPs to generate oxygen, we measured the amount of dissolved oxygen in the mixed solution and the degree of decomposition of H2O2 in real time using an oxygen probe and hydrogen peroxide reagent kit. The pH of H2O2 solution was adjusted to 6.6, mimicking the acidity level of tumor microenvironments. As shown in Figure 4A, when CCmMC and acidic H2O2 coexisted, the oxygen level in the solution increased rapidly accompanied with the generation of large amounts of gas bubbles, demonstrating the strong catalytic capability of CCmMC NPs to generate O2 and to trigger the decomposition of H2O2. In comparison, no oxygen was produced in the presence of only H2O2. Figure 4B shows that the concentration of H2O2 dropped to about 20% within 2 h, further elucidating the high efficiency of CCmMC NPs to catalyze the production of oxygen in a weak acidic PBS solution. Moreover, so as to simulate in vivo physiological environment in which H2O2 is continuously produced by cancer cells, the O2 generated by the CCmMC was measured while continuously adding exogenous H2O2 into the reaction system every 120 min. As indicated in Figure 4C, CCmMC NPs displayed the catalytic power to reproducibly induce oxygen production in a re-usable manner. After repeated addition of H2O2 thrice, the concentration of H2O2 decreased sharply during a single cycle, but the catalytic efficiency of CCmMC did not reduce with the number of cycles, confirming that the 15


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as-prepared CCmMC NPs were catalytically stable and effective in decomposing H2O2 to generate O2. The intracellular O2 levels in 4T1 cells treated with different formulations were further measured using an O2 sensing probe [Ru(dpp)3]Cl2 (RDPP). As expected, the cellular red fluorescence was very low and negligible after treating the cells with CCmMC NPs and H2O2 under 635 nm laser irradiation (Figure S9a). In contrast, saline or CCmMC group displayed intense red fluorescence characteristics. This confirmed that CCmMC NPs could effectively catalyze the decomposition of H2O2 to produce intracellular O2 in living cells and relieve tumor hypoxia. Similarly, in Figure S9b, the lowest fluorescence intensity in CCmMC+ H2O2 group demonstrated the generation of cellular O2. As depicted in Figure S9c, the red fluorescence intensity decreased gradually as a function of treatment time. Obviously, 4T1 cells could hardly display any red fluorescence at 3 h, indicating the total amount of O2 generated is highest at this time, which triggers the production of large amounts of ROS for effective PDT. Considering the high stability and the strong capacity of CCmMC NPs to decompose H2O2 for O2-evolving process, we further tested the feasibility of applying CCmMC NP as a PDT agent. Several studies have demonstrated that ROS-generation capacity of nanomaterials is greatly enhanced when the hypoxic state in cancer cells is suppressed. Thus, we determined the capacity of CCmMC NPs to generate singlet oxygen (1O2). Numerous studies have reported the relatively high content of H2O2 in TME (concentration range from 100 µM to 1 mM).S1-S3 The production of extracellular ROS was detected using singlet oxygen sensor green (SOSG) probe, which was analyzed by a fluorescence spectrophotometer. SOSG probe can rapidly and irreversibly react with 1O

2

to produce SOSG endoperoxides, emitting strong green fluorescence. As indicated in Figure 16


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4D, when mixed with CCmMC NPs, the fluorescence intensity of the solution containing CCmMC and SOSG was gradually increased as a function of irradiation time, suggesting a sustained generation of cytotoxic 1O2. In view of the extracellular 1O2 generation results, we were compelled to further determine the intracellular ROS levels generated by CCmMC NPs. A 2', 7'-dichlorofluorescein diacetate (DCFH-DA) probe was utilized to investigate the intracellular ROS levels in different formulations during the PDT process (Figure 4E). The ROS fluorescence intensity triggered by CCmMC NPs was very low and negligible without laser radiation, which was not conspicuously different compared to saline. However, the fluorescence intensity of ROS produced by CCmMC under 635 nm laser irradiation for 5 min was increased significantly, suggesting that laser irradiation was a prerequisite for the success of PDT. It should be noted that the green fluorescence of CCmMC was much stronger after the addition of the acidic H2O2 buffer compared with CCmMC without H2O2 under 635 nm laser irradiation. This is ascribed to the fact that CCmMC may react with the added H2O2 to produce a certain amount of O2 and the rapid elevation in oxygen concentration may lead to the increase of singlet oxygen triggered by the Mn-Cdots. Indeed, as indicated in Figure S10, the fluorescence intensity of singlet oxygen in CCmMC NPs and H2O2 treated group was significantly higher than that of other groups based on flow cytometry analysis, further confirming that the PDT effect of CCmMC NPs was sufficiently amplified by hydrogen peroxide, which is consistent with the above results. As shown in Figure 4F, the RGD-CCmMC group induced stronger fluorescence intensity in 4T1 cells when compared to that of CCmMC based on CLSM observation, confirming that the RGD-CCmMC significantly increased the intracellular ROS levels. The differences in ROS generation may account for the increased cellular internalization of CCmMC NPs. The RGD-conjugated CCmMC NPs can be 17


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easily internalized into 4T1 cells via the Nrp-1 receptor-mediated internalization pathway, thereby producing higher ROS levels. Therefore, it is reasonable to conclude that CCmMC NPs are potential PDT agents which can be delivered into intracellular sites to alleviate TME-mediated hypoxia. In Vitro Cytotoxicity Assay and Antitumor Performance. Biocompatibility is regarded as a prerequisite of nanoagents used for biomedical applications. Therefore, we further evaluated the cytotoxicity of RGD-CCmMC NPs against 4T1 and L929 cell lines using the Cell Counting Kit-8 (CCK-8) assay. As shown in Figure 5A, after incubation with nanoparticles for 24 h, RGD-CCmMC NPs did not induce obvious toxicity in 4T1 and L929 cells as evidenced by the cell viability of more than 85% even when the NPs concentration was up to 400 µg/mL. Furthermore, the cell viability was not significantly different between 4T1 and L929 cells following treatment with varying concentrations of the NPs. Also, there is no obvious difference in cell viability between CCmMC and RGD-CCmMC under the same concentration in Figure S11, indicating favorable biocompatibility of CCmMC and RGD-CCmMC NPs. Moreover, to assess the hemocompatibility of the RGD-CCmMC NPs, we determined hemolysis of red blood cells (RBCs) using deionized water (positive) and PBS (negative) as the control groups, and digital photographs are presented in Figure 5B. No visual red color was observed as the concentration of NPs increased, which indicated that NPs did not cause any noticeable hemolysis at all indicated concentrations. Additionally, there was a non-significant increase in hemolytic efficiency with the concentration. The highest degree of hemolysis reached only 6%, showing that the level of hemolysis caused by NPs was negligible. These results revealed that RGD-CCmMC NPs were highly biocompatible and can be utilized as nanocarriers for efficient phototherapy. 18


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Enhanced photothermal effect and improved ROS generation induced by the CCmMC NPs, which incented us to investigate their phototherapeutic effect on 4T1 cells in vitro. Figure 5C shows the viability of 4T1 cells after incubation with different concentrations of CCmMC NPs with or without laser irradiation (1 W/cm2) based on the standard CCK-8 assays. The cell viability of all formulation groups decreased with the increase in CCmMC NPs concentration, indicating a concentration-dependent therapeutic effect. Compared with CCmMC+L635 (PDT group) or CCmMC+L808 (PTT group), the CCmMC+L635+L808 group showed a much higher cytotoxicity effect at an equivalent concentration, which demonstrated that the therapeutic synergism between PTT and PDT was more efficacious in inducing a high number of apoptotic/necrotic cancer cells. The viability of cells treated with RGD-CCmMC/DOXs (400 µg/mL) and irradiated with 635 nm and 808 nm laser had the lowest cell survival ratio, of about 4±2.12%, which was attributed to the efficient superadditive killing effect of PTT/PDT and thermo/pH-responsive DOX release behavior. Cytotoxicity of various therapeutic formulation was further examined by fluorescence costaining of live/dead cells using Calcein-AM and PI, visualizing the live cells (green) and dead cells (red) (Figure 5D). In the PTT group alone or PDT group alone, half of the cancer cells displayed green fluorescence, illustrating that both had moderate inhibitory effects. When the cells were incubated with RGD-CCmMC/DOX NPs and exposed to 635 nm plus 808 nm laser, nearly all cells died since all cells in a field view showed red fluorescence. This strongly revealed that the triple-combination therapy had obvious superiority over other groups. Besides, apoptosis/necrosis assay was conducted by flow cytometry with Annexin V-FITC/ PI fluorescence staining to further verify the level of cell death (Figure 5E). The cell survival ratio was close to 100% and negligible apoptosis was detected in the control group. Notably, RGD-CCmMC/DOX treatment significantly induced cell 19


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apoptosis/necrosis, compared with 61.7% of PTT, or 54.2% of PDT and 75.9% of synergetic PTT and PDT. This sufficiently evidenced that RGD-CCmMC/DOX-mediated triple therapy had the highest total apoptotic/necrosis ratio compared to other groups, confirming that the enhancement of ROS, hyperthermia effect and anticarcinogen eruption resulted in improved therapeutic efficacy. This result was in agreement with the above results of the in vitro cytotoxicity assay and live/dead staining assay. Taken together, CCmMC/DOX NP can be exploited as a potential phototherapy agent for efficient tumor ablation based on the combination of chemo-phototherapy. In Vivo Fluorescence/Photothermal/Magnetic Resonance Multimodal Imaging. Motivated by the superior performance of RGD-CCmMC in cellular experiments in vitro, we further evaluated the intratumor levels and penetration behaviors of the RGD-CCmMC nanovehicles in vivo to determine their potential for fluorescence imaging-guided targeted therapy. The RGD-CCmMC NPs (0.5 mg/kg) were intravenously administered into 4T1 tumor-bearing BALB/c mice. In vivo biodistribution of RGD-CCmMCs was traced by whole-body fluorescence imaging using the NightOWL II LB983 in vivo imaging system. As shown in Figure 6A, fluorescence was observed at the tumor site as early as 3 h postinjection and the increase in the fluorescence signal of the tumor showed a duration-dependent pattern, which reflected rapid accumulation, high penetration and targeting selectivity. The fluorescence signals were then progressively strengthened and reached the peak intensity at 12 h postinjection, which suggested an efficient enrichment of nanovehicles at the targeted tumor region and an optimal phototherapy at that time point in vivo. The following three reasons may account for the improved tumor-specific accumulation and targeting selectivity: (i) an enhanced permeability and retention (EPR) effect; (ii) the RGD homing peptide facilitated tumor permeability by activating neuropilin-1 receptors; (iii) MAPK pathway and ERK1/2 pathway were 20


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activated by phosphorylation of through phosphorylation of endothelial cell-specific receptor tyrosine kinase.66,67 Subsequently, the fluorescence signal of the tumor decreased progressively during the following 12 h, which indicated that CCmMCs were gradually metabolized. Mice were sacrificed at 24 h postinjection, organs and tumors were then dissected and imaged to evaluate the fluorescence signal intensities, providing strong evidence for tracing the in vivo biodistribution of nanoparticles. As presented in Figure 6B, high fluorescence level was observed within the tumor tissue, whereas limited fluorescence was observed in the liver, spleen, lung, and kidney, which suggested that accumulation and biodistribution of RGD-CCmMC NPs to other main organs was inevitable. The liver and lung tissues displayed a much higher fluorescence signal intensity than the spleen and kidney, but the strongest fluorescence was observed in the tumor region (Figure 6C). This result further confirmed the high selectivity of RGD-CCmMCs for the targeted tumor tissues. The targeted accumulation of the NPs in the tumor and great fluorescence imaging property enabled the utilization of RGD-CCmMC to locate the tumor and its margins thereby providing a precise imaging-guided cancer phototherapy nanoplatform for precision medicine. Figure 6D shows the photothermal images of the nude mice at different time intervals under the continuous 808 nm laser irradiation (1 W/cm2). Avoidance of the overheating effect is a question worthy of careful consideration.S4-S6 After 5 min of laser irradiation, the tumor temperature of RGD-CCmMC-injected group was rapidly elevated to 51.6 ± 1.1 °C and reached a constant temperature eventually, which exceeded the damage threshold for inducing irreversible tumor ablation. The rapid increase in temperature in a short time was ascribed to the fact that the RGD targeting ligands were able to specifically bind to αvβ3 integrin receptors overexpressed on the 4T1 cells, thus promoting targeted amassment of the NPs in the tumor. In comparison, the tumor 21


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temperature of CCmMC-treated mice following irradiation for 5 min reached 46.4±0.9 °C, which is much lower than that of RGD-CCmMC NPs. This indicates the low tumor accumulation and non-selective targeting property of CCmMC NPs. There was only a slight tumor temperature rise (~4.8 °C) for the PBS group. Corresponding 3D IRTM photothermal images of PBS, CCmMC, and RGD-CCmMC are displayed in Figure 6E, which provided a three-dimensional analysis for tumor diagnosis. Besides, the temperature variation from the central zone of the tumor to the boundary was observed intuitively. As shown in Figure 6F, there was no obvious temperature elevation at the tumor site after injection of PBS within 7 min of laser irradiation in the control group, demonstrating no obvious damage to the tumor. In the PTT field, controlled localized temperature increments (>50 °C) have been shown to cause apoptosis/necrosis of cancer cells, which is sufficient to kill cancer cells.65 These results revealed that RGD-CCmMC nanovehicles provided strong PTI capability and an enhanced PTT effect, which matched with the temperature increase data from in vitro experiments. High penetration and enhanced accumulation of RGD-CCmMC nanovehicles inside the solid tumor were further affirmed by the T1-weighted MRI imaging in vivo. The MRI images of various concentrations of the RGD-CCmMC NPs exhibited a clear concentration-dependent brightening effect, as shown in Figure 7A. As indicated in Figure 7B, a strong MRI signal was observed in the targeted-tumor site at 12 h postinjection, which persisted at 24 h later. At 12 h postinjection of RGD-CCmMC NPs, the T1-MRI signal intensity displayed 4.2-folds positive enhancement quantitatively at the tumor region (Figure 7C), indicating the ability of magnetic resonance contrast strengthening the effect of RGD-CCmMC nanocomposite. Moreover, this revealed a high retention and accumulation of RGD-CCmMC NPs in the tumor sites. This was achieved via the active 22


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targeting effect of RGD-homing peptide and the passive enhanced EPR effect, which are consistent with the result of the aforementioned fluorescence imaging. Thus, magnetic resonance, fluorescent and photothermal imaging results collectively evidenced that the RGD-conjugated CCmMC NPs are suitable for future clinical use and can be utilized as promising imaging agents in guiding and monitoring cancer therapy in vivo. In Vivo Anticancer Efficacy of Triple-collaborative Tumor Therapy. Inspired by the high phototherapeutic efficacy of CCmMC NPs in vitro, we then performed in vivo experiments to further explore the anticancer efficacy of CCmMC NPs for combination PDT/PTT/chemotherapy in vivo. 4T1 tumor-bearing mice were randomly divided into eight groups (n = 5 in each group): (i) PBS only; (ii) 635 nm and 808 nm laser; (iii) CCmMC only; (iv) CCmMC/DOX; (v) CCmMC+ 808 nm laser (PTT); (vi) CCmMC+635 nm laser (PDT); (vii) CCmMC+635 nm+808 nm laser (PDT+PTT); (viii) CCmMC/DOX+635 nm+808 nm laser (PTT+PDT+chemotherapy); (ix) RGD-CCmMC/DOX+635

nm+808

nm

laser

(PTT+PDT+chemotherapy+active-targeting).

CCmMC NPs, RGD-CCmMC NPs (200 μg/mL) or PBS (100 µL) were administrated via intravenous (i.v.) injection, PTT was given via 808 nm laser irradiation (L808, 1 W cm−2, 10 min), and PDT was given via 635 nm laser irradiation (L635, 50 mW cm−2, 10 min). The tumor volumes and tumor weight were monitored in real time every 3 days after receiving various treatments. As shown in Figure 8A, no any inhibitory effect on tumor growth was detected in the L635+L808 and CCmMC groups over the course of 21 days treatment. The treatment with CCmMC/DOX exhibited negligible antitumor effects, indicating the individual chemotherapy may trigger hypoxia-associated drug resistance without obvious tumor damage. Furthermore, individual PDT, PTT or PTT+PDT groups moderately slowed the growth of tumors without distinct differences. Notably, for the 23


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CCmMC/DOX+Laser treated group, there was a significant tumor suppression in tumor volume relative to the initial. Compared with these groups, we observed an optimal tumor inhibitory effect in the RGD-CCmMC/DOX+Laser group, which achieved nearly almost thorough tumor ablation. This gratifying therapeutic effect was ascribed to the accomplishment of triple-collaborative PTT/PDT/chemotherapy for eliminating tumors. Besides, body weight displayed no obvious change in all groups (Figure 8B), indicating negligible systemic biotoxicity of various nanoparticles. Tumors were collected, and the digital graphs of excised tumors (Figure 7D) and the average tumor weight (Figure 8C) were visually displayed. The mice treated with RGD-CCmMC/DOX plus laser displayed the smallest tumor size and weight, once again confirming that the triple-collaborative therapy has the most desirable therapeutic efficacy. Besides, survival curves of mice (Figure 8D) further ascertained that RGD-CCmMC/DOX group had the longest survival time and highest survival rate during the treatment. This improved survival rate was ascribed to a combination of Mn-Cdot-mediated enhanced PDT, gold cube-in-cube-mediated PTT and DOX chemotherapy. The hematoxylin and eosin (H&E) staining was also performed on tumor slices after various treatments on the 21st day. Analysis of the H&E stained tumor sections further demonstrated the occurrence of severe histological damages and typical pathological changes such as severe karyopyknosis, apoptosis or necrosis of tumor cells in the RGD-CCmMC/DOX group, which revealed excellent tumor ablating capability. Conversely, mice in other treatment groups only displayed partial tumor destruction and small necrotic regions interspersed with infiltrating tumor cells to different extents, achieving limited therapeutic benefit (Figure 8E). In addition, to detect whether RGD-CCmMC NPs can be effectively eliminated from the bodies of mice, ICP-MS was utilized to quantitatively detect the gold content in feces and blood at various time points, as presented in Figure S12. Au 24


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content in feces reached the maximum value at 3-day post-injection. Besides, Au content in blood decreased dramatically in the early stages (first 24 h) and the levels of Au in blood continued to decrease gradually in the following periods after postinjection. At day 7 post-injection, the levels of Au in blood were negligible, demonstrating that NPs could generally be cleared out from the bodies of mice after undergoing a long-circulation, which may be ascribed to the results of liver, spleen processing and mononuclear phagocytic system.S7-S8 Therefore, these results demonstrated that triple-collaborative treatment mediated by the RGD-CCmMC/DOX nanoplatform provided optimal therapeutic outcomes in vivo solid tumors. The Long-Term Biotoxicity of the RGD-CCmMC Nanoparticle. To evaluate the future bio-safety of the RGD-CCmMC nanoparticles, we also performed a systematic investigation on their in vivo toxicity. The main mice organs including the tumor, heart, liver, spleen, kidney and lung were collected from various groups at 30 days postinjection and then they were subjected to histological analysis. As shown in Figure 9, neither noticeable organ impairment nor obvious inflammation or necrosis was observed for all treatment groups with or without laser irradiation based on H&E staining. These results demonstrated that nearly no detrimental effects caused by the nanovehicles to major organs and their superior bio-safety. Moreover, blood biochemistry and blood panel analysis (n=3) were also carried out at 1 day, 1 week and 1 month post-treatment, and the results are presented in Figure 10. After the injection of RGD-CCmMC nanoparticles, the levels of these metrics only slightly fluctuate at the initial days, and the levels recovered rapidly over time. Liver function index (alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST)), the kidney function index (the serum urea nitrogen (BUN), creatinine (Cr), Catalase (CAT), Glutathione Peroxidase (GSH-PX), Superoxide Dismutase (SOD)), as well as 25


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Hematology parameter (blood glucose (GLU), blood fat (CHOL) and and Mean Corpuscular Volume (MCV)), were found to be within normal reference range and not significantly different from the control group levels, confirming negligible hepatotoxicity and nephrotoxicity. This suggested that RGD-CCmMC nanoparticles did not induce obvious hepatic toxicity, kidney disorders or other adverse side effects in vivo during the treatments. On the basis of the foregoing data, we evidenced that the targeted chemo-phototherapy nanoplatforms designed in this study exhibited favorable biocompatibility and promising prospects for in vivo applications.

CONCLUSIONS In this paper, we have successfully designed and synthesized RGD-CCmMC/DOX NP to serve as a versatile nanoplatform with a TME-modulating effect for triple-collaborative targeted cancer therapy and mutilmodal imaging. The resulting RGD-CCmMC/DOX nanoplatforms efficiently consumed endogenous H2O2 inside TME thereby elevating in situ oxygen concentrations under 635 nm irradiation, which distinctly attenuated tumor hypoxia and simultaneously enhanced PDT. Moreover, the nanoplatform displayed a considerable PTT effect and 65.6% photothermal conversion efficiency due to its gold cube-in-cubes core. In addition, Mn-Cdots served as “gatekeepers” which block the premature release of entrapped anticarcinogen, enabling accurate heat/pH-sensitive release of anticarcinogen at the tumor sites. Incorporation of RGD targeting ligands endowed the nanoplatform with active-targeting capability. Furthermore, in vivo and vitro assays revealed that the nanoplatform showed high biocompatiblity and desirable therapeutic effect after cooperative treatment. Thus, this work underscored the utility of a paradigm of a versatile theranostic nanoplatform designed to achieve superadditive PTT/PDT/chemotherapy antitumor efficacy, which provides the basis for the design of possible nanosystems for tumor treatment. 26


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EXPERIMENTAL SECTION Synthesis of Au Nanocubes (AuNCs). The previously reported seed-growth method53,54 was employed to synthesize Au nanocubes. The as-prepared Au seed solution was incubated at 30 ℃ for at least 1 h. To synthesize Au growth solution, 8 mL of the 100 mM CTAB solution was mixed with 40 mL double distilled water (DDW) in 100 mL round bottom flask. 1mL of the 10 mM HAuCl4 solution and 4.75 mL of a 100 mM ascorbic acid solution were sequentially added to the mixture solution. After adding 4.75 mL ascorbic acid solution, the rose red color of the mixture solution became transparent, demonstrating a reduction of Au3+ to Au1+. Then, the seed liquid was diluted 10 times, 25 μL of the diluted seed liquid was added dropwise, and then mixed uniformly. After overnight incubation, two centrifugations (6000 rpm, 15 min) were used to separate the precipitated nanoparticles. The Au nanocubes were collected and re-dispersed in 9 mL DDW after discarding the supernatant. Synthesis of Au Cube-in-Cubes (Au CCs). 4 mL of the 50 pM Au nanocubes was mixed with 400 μL of 2~5 mM AgNO3 solution on a vortex mixer which reacted to form Au@Ag core-shell nanocubes. In this step, ascorbic acid served as the reducing agent and CTAC served as the capping agent. To synthesize Au CCs, 4 mL of 0.01 mM HAuCl4 solution was injected into the Au@Ag core-shell nanocubes suspension solution using a micro-injection pump at a speed of 20 μL/min at 30℃ in the presence of 4 mL of 100 mM CTAB and 870 μL of 100 mM ascorbic acid. Then the mixed solution was incubated for 20 min and centrifuged twice at 6000 rpm for 10 min. Finally, the precipitated Au CCs were dispersed in 4 mL DDW for further use. Synthesis and Chemical Modification of the Au CCs@mSiO2 (CCm) Surface. A layer of mesoporous silica was coated on the Au CCs to form Au CCs@mSiO2 via the optimized sol-gel 27


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approach, in which CTAB served as a hard template. 1 mL CTAB (100 mM) was added dropwise into 4 mL Au CCs suspension, and stirred for at least 1 h. Next, ammonia was added to the above mixture solution, and the pH value was adjusted to about 10. TEOS was added in three, each time adding 50 μL, 70 μL and 90 μL at the interval of 30 min after the completion of the pH adjustment. The mixed solution was reacted at room temperature for 10 h. The supernatant was discarded via centrifugation twice at 10000 rpm for 15 min to obtain Au CCs@mSiO2 precipitate, which was mainly used to completely remove CTAB. The precipitate was then dispersed in 4 mL of ethanol, and the pH was adjusted to less than 1 with HCl and allowed to stand for 2 h. Finally, after centrifugation thrice with ethanol, the obtained Au CCs@mSiO2 was dispersed in 10 mL PBS. For amino-functionalized CCm, 0.5 mL of APTES was added into the above solution. The mixture was reacted at 90 °C in an oil bath for 24 h to achieve amino-group modification. The CCm-NH2 was collected through centrifugation of the solution followed by washing with ethanol several times to ensure complete removal of the residual APS. Synthesis of Au CCs@mSiO2@Mn-Cdots (CCmMC). Mn-dots were synthesized substantially by solvothermal treatment using manganese (II) phthalocyanine (Mn-Pc) as the raw material. Mn-PC was dissolved in absolute ethylalcohol, and then heated to 180~200 ℃ for 24 h. After cooling to room temperature, the resultant Mn-Cdots were filtered using millipore 0.22 µm membranes then dialyzed against ethylalcohol several times to remove the free Mn-Pc, and then re-dispersed in ethylalcohol. To cap Mn-Cdots onto CCm-NH2, the CCm-NH2 was dispersed in 10 mL PBS with sonication for 1 h to achieve uniform distribution. Mn-Cdots solution was added into the solution under violent stirring for 12 h to form Mn-Cdots capped CCm (CCmMC), which was ascribed to the strong coulombic interaction. The CCmMC were collected via centrifugation-rinsing 28


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cycles and stored at 4 °C for further use. Surface Modification of pS-PEG2000-RGD on CCmMC. 2 mg CCmMCs were re-dispersed in 1 mL DDW followed by addition of 0.2 mg of pS-PEG2000-RGD, and the mixture solution was stirred at 4 °C for 2~4 h. The excess pS-PEG2000-RGD was removed through centrifugation and washing with deionized water several times in order to obtain the functionalized end-product of CCmMC-PEG-RGD (RGD-CCmMC). DOX Loading and Heat/pH-Triggered Release Kinetic Behaviors. 5 mg of as-synthesized gold CCm-NH2 were dispersed in 4 mL DOX solution (1 mg/mL) and the mixture was stirred at 600 rpm for 24 h at room temperature in the dark to form DOX-loaded Au CCs@mSiO2. To remove DOX anchored on the surface of nanoparticles, the product was centrifuged and washed with PBS at least five times. The drug loading capacity was calculated by measuring the absorbance of DOX in the supernatant at 494 nm. Afterwards, Mn-Cdots stock solution reacted with the DOX load nanoparticles to produce DOX-loaded Au CCs@mSiO2@Mn-Cdots in PBS, which were designated as CCmMC/DOX. To investigate the temperature and pH-triggered release kinetic behaviors of DOX from the CCmMCnanoplatform, the release profile of DOX was evaluated in PBS solution of different pH (5.0, 6.6 and 7.4) at 37 °C (physiological temperature) and 50 °C (tumor site temperature) by determining the cumulative amount of DOX in the supernatant at predetermined time intervals. In Vitro Cellular Uptake Studies. To investigate the cellular endocytosis characteristics of CCmMC/DOX, we seeded 5×104 4T1 cells into a 35 mm confocal dish and incubated overnight to allow cells to adhere firmly to the bottom of the dish. Subsequently, the cells were washed thrice with PBS, and 1.5 mL of CCmMC/DOX suspension (50 μg/mL in DMEM) was added to the dish 29


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and incubated at 37 °C for various times (1 h, 2 h, and 4 h). After washing thrice with PBS to remove residual nanoparticles and dead cells, the cells containing DOX and Mn-Cdots signals were recorded real-time via confocal laser scanning microscope (CLSM, Olympus, FV10i). Furthermore, nanoplatform-mediated cellular uptake process was monitored using TEM imaging. Measurement of Photothermal Performance and Stability in Vitro. To measure the photothermal effect of the synthesized CCmMC NPs, 1 mL aqueous dispersion of CCmMC NPs with different concentrations (12.5, 25, 50, 100, 200 and 400 μg/mL) were irradiated under an 808 nm laser with different power intensities (0.3, 0.6, 0.9, and 1.2 W/cm2) for 5 min. An IR thermal camera (Fotric 225) was employed to monitor the temperature variation of the CCmMC aqueous solution and photothermal imaging at the tumor sites. Then, to further investigate the photothermal stability of CCmMC nanoplatforms, CCmMC solution (400 µg/mL) was cyclically irradiated with an 808 nm laser (1 W/cm2) for 600 s (laser on), and then naturally cooled to room temperature without irradiation for 900 s (laser off), and this cycle was repeated four times. Cell culture and Measurement of Cytotoxicity of CCmMC NPs. 4T1 and L929 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and incubated at 37 ℃ in a humidified atmosphere with 5% CO2. The 4T1 and L929 cells were grown in DMEM or α-MEM medium with 10% fetal bovine serum and 1% penicillin-streptomycin. CCK-8 assay based on L929 and 4T1 cells was employed to assess the in vitro cytotoxicity of CCmMC NPs. The cells were seeded in 96-well plates (1 × 104/well) and incubated overnight, followed by the addition of various concentrations CCmMC NPs (12.5, 25, 50, 100, 200 and 400 μg/mL). Then the medium was removed and 10 µL CCK-8 solutions were added into each well. 30


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Finally, the optical density (OD) values at 490 nm were measured using a Bio-Rad 680 microplate reader to determine the relative cell viability, which was regarded as a cytotoxicity index. Cytotoxicity of Various Therapeutic Methods. To explore the cytotoxicity of different formulations, 4T1 cells were incubated with CCmMC at different concentrations (12.5, 25, 50, 100, 200 and 400 μg/mL in cell culture medium). After incubation for 6 h, 4T1 cells were treated with only

PBS,

CCmMC/DOX,

CCmMC+L808

(PTT

group),

CCmMC+L635

(PDT

group),

CCmMC+L808+L635 (PDT+PTT group), CCmMC/DOX+L808+L635 (PDT+PTT+chemo group) and RGD-CCmMC/DOX+L808+L635 (PDT+PTT+chemo+active-targeting group), and then irradiated with 808 nm or 635 nm laser for 5 min. Finally, the cells were visualized after co-staining with calcein-AM and PI for 30 min with a CLSM to determine the live and dead cell. Detection of Extracellular and Intracellular ROS levels. The extracellular ROS levels were detected using singlet oxygen sensor green (SOSG) probe. Briefly, 100 μg SOSG was dissolved in 330 μL of methanol to prepare a 500 μM stock solution. Next, the stock solution was diluted 250 times (2 μm) and then added into 2 mL of CCmMC suspension (200 µg/mL) followed by irradiation with a 635 nm laser at different intervals. Finally, the supernatant was centrifuged, collected and the amount of ROS generated was qualitatively measured using a fluorescence microplate reader. A ROS-sensitive probe, DCFH-DA, which is converted into DCFH by intracellular esterase was used to examine the intracellular ROS levels by CLSM. The 4T1 cells were seeded into 35 mm confocal dishes (1×105 cells/well) and treated with CCmMC (200 µg/mL) overnight at 37 °C. Cells were further incubated with 20 µM DCFH-DA for 40 min and irradiated with a 635 nm laser with an output power of 500 mW/cm2 for 10 min. The cells were incubated for an additional 4 h after 31


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Page 32 of 57

irradiation. The green fluorescence signals stemming from intracellular ROS were acquired with a confocal laser scanning microscope (CLSM, ex/em = 488/520). Measurement of Extracellular H2O2 and O2 levels. After addition of 100 μg CCmMC NPs to 10 mL H2O2 solution (100 μM), ammonium molybdate spectrophotometric method was utilized to detect the H2O2 concentration at different time-points. The in vitro production of O2 from H2O2 solutions upon CCmMC addition at pH 6.6 was evaluated by an oxygen probe (JPBJ-608 portable dissolved oxygen meters, Shanghai REX Instrument Factory). The amount of oxygen generated by the CCmMC NPs in H2O2 (100 μM) solution was recorded and compared to that of H2O2 solution without CCmMC NPs. In vivo Photothermal Imaging. The 4T1 cell suspension was subcutaneously inoculated into the right shoulder or leg of female Balb/c mice. When the tumor size reached about 60 mm3, the mice bearing tumor were injected with RGD-CCmMC suspension intravenously (100 µL, 500 μg/mL). An IR thermal camera (Fotric 225) was utilized to perform photothermal imaging at different times when the tumor sites were exposed to 808 nm laser (1.0 W/cm2). Before irradiating with NIR laser, Balb/c mice given different pretreatments were divided into three groups for photothermal imaging process as follows: (i) those that received PBS group only; (ii) those that received i.v. injection of 500 μg/mL CCmMC; (iii) those that received i.v. injection of 500 μg/mL RGD-CCmMC. In vivo Fluorescence Imaging. When the volumes of 4T1 tumors reached 60 mm3, the nude mice were intravenously injected with RGD-CCmMC NPs (100 μL, 2.5 mg/kg) via the tail vein. The fluorescence imaging (Ex: 640 nm, Em: 720 nm) was carried out at 3, 6, 9, 12, 24 and 48 h post-injection using an in vivo imaging system (NightOWL II LB983, Germany). The mice were euthanized and tumor and major organs (heart, liver, spleen, kidneys, and lung), were harvested at 32


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the end of the last FL scans at 48 h p.i. The organs were imaged via FL imaging system to quantify the residual amount of the nanovehicles in vivo. In Vitro and In Vivo Magnetic Resonance Imaging. The CCmMC NPs with various Mn concentrations were placed in 2 mL of tubes. The T1-weighted MRI imaging in vitro was carried out using a 1.2 T MRI instrument (Shanghai Niumai Corporation Ration NM120-Analyst). The in vivo MRI experiments were conducted by a 3.0 T Siemens Magnetom Trio medical MRI system (Affiliated Hospital of Xuzhou Medical University, Radiology Department of MRI, Xuzhou, China). 100 µL aqueous solution of RGD-CCmMC NPs (500 μg/mL) was injected intravenously into tumor-bearing nude mice via tail vein and then MRI was recorded before and at 12 h, 24 h postinjection. In vivo Synergistic Antitumor Therapy. In vivo phototherapy was performed on tumor-bearing mice. A total of 45 mice were randomly divided into eight groups to receive different antitumor treatments: (i) only received PBS group; (ii) irradiation with 635 nm and 808 nm laser; (iii) i.v. injection with CCmMC; (iv) injection with CCmMC/DOX; (v) injection with CCmMC and irradiation with L808 (PTT group); (vi) injection with CCmMC and irradiation with L635 (PDT group); (vii) injection with CCmMC and irradiation with L635+L808 (PTT+PDT group); (viii) injection with CCmMC/DOX and irradiation with L635+L808 (PTT+PDT+chemotherapy group); (ix)

injection

with

RGD-CCmMC/DOX

and

irradiation

with

L635+L808

(PTT+PDT+chemotherapy+active-targeting group). All treatments based on PTT were conducted under appropriate irradiation with a laser (1 W/cm2, 10 min) at 24 h post-injection, and tumor volumes, sizes, and body weights were measured every 2 days after the treatment. Histopathological Examination. After 21 days of treatment, mice from all the groups were 33


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euthanized and their tumors and tissues (heart, liver, spleen, lung, and kidney) were excised from all groups and fixed in 4% neutral buffered formalin for histological analysis. Afterwards, the tumors and organs were gradient-dehydrated with different concentrations of ethanol and xylene. The samples were then embedded in liquid paraffin and sliced for staining with hematoxylin and eosin (H&E). Finally, the morphological characteristics of each organ were examined by a microscope (VS120, Olympus, Japan). Long Term Toxicity Assessment In Vivo. Healthy Balb/c mice were intravenously injected with RGD-CCmMC NPs suspension at a dose of 30 mg/kg and sacrificed at various time points after injection. An approximately 1 mL blood sample from each mouse was collected at 1 day, 1 week, and 1 month in a sodium EDTA anticoagulant tube for the blood biochemistry examination and complete blood panel analysis. Liver function was evaluated by determining the serum levels of ALT, ALP and AST. Kidney function was determined by measuring BUN, Cr, CAT, GSH-PX, SOD levels. Other parameters such as GLU, CHOL and MCV were also measured to verify the long-term toxicity in vivo. Statistical Analysis. All data are presented as mean value ± standard deviations. Student’s test and

two-way ANOVA are applied to evaluate the significance among two groups or multiple

groups. p < 0.05 was considered as the minimal level of significance and is indicated by a single asterisk (*), and P < 0.01 is indicated by a double asterisk (**).

ASSOCIATED CONTENT Supporting Information Supplementary data regarding this article are available free of charge via the Internet at http://pubs.acs.org. 34


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The Supplementary data contain Abbreviation identification, Reagents and materials, Instruments, Hemolysis assay, Animals tumor model, Detection of Intracellular O2 Generation, DLS and zeta potential, Characterization of Mn-Cdots, XPS spectra, UV-vis absorption spectra, Pore size distribution profile, Plot of cooling time, The temperature change of Au cube-in-cube, Cumulative DOX, Detection of intracellular O2 generation, Flow cytometry detection of ROS generation, Cell viability, Detection of Au content in feces and blood and Reference. Conflict of Interest The authors declare no competing financial interest.

AUTHOR INFORMATION Corresponding Author *Email address: [email protected] (F. Gao), Tel.: +86-516-83262138. *Email address: [email protected] (K. Guo), Tel.: +86-516-85805386.

ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the Natural Scientific Foundation of China (NNSFC) (21565002), Research Project of Jiangsu Provincial Health Department (H201528), Natural Science Foundation of Jiangsu Province (BK20171174), the Key Program of Science and Technique Development Foundation in Jiangsu Province (BE2015627), Jiangsu Postdoctoral Science Foundation (1701045C) and China Postdoctoral Science Foundation Funded Project (2016M591929). The Authors appreciate Prof. Pengpeng Chen from College of Chemistry and Chemical Engineering of Anhui University for N2 adsorption-desorption measurements.

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(61) Leng, C.; Zhang, X.; Xu, F.; Yuan, Y.; Pei, H.; Sun, Z.; Li, L.; Bao, Z. Engineering Gold Nanorod-Copper Sulfide Heterostructures with Enhanced Photothermal Conversion Efficiency and Photostability. Small 2018, 7, 169-183. (62) Son, B. H. D.; Mai, V. Q.; Du, D. X.; Phong, N. H.; Cuong, N. D.; Khieu, D. Q. Catalytic Wet Peroxide Oxidation of Phenol Solution over FeMn Binary Oxides Diatomite Composite. J. Porous Mater. 2016, 24, 601-611. (63) Zhou, L.; Li, Z.; Liu, Z.; Ren, J.; Qu, X. Luminescent Carbon Dot-Gated Nanovehicles for pH-Triggered Intracellular Controlled Release and Imaging. Langmuir 2013, 29, 6396-6403. (64) Gao, H.; Xiong, Y.; Zhang, S.; Yang, Z.; Cao, S.; Jiang, X. RGD and Interleukin-13 Peptide Functionalized Nanoparticles for Enhanced Glioblastoma Cells and Neovasculature Dual Targeting Delivery and Elevated Tumor Penetration. Mol Pharm 2014, 11, 1042-1052. (65) Jaque, D.; Maestro, L. M.; Rosal, B.; Gonzalez, P.; Benayas, A.; Plaza, J. L.; Rodriguez, E.; Sole, J. M. Nanoparticles for Photothermal Therapies. Nanoscale 2014, 6, 9494-9530. (66) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Girard, O. M.; Hanahan, D.; Mattrey, R. F.; Ruoslahti, E. Tissue-Penetrating Delivery of Compounds and Nanoparticles into Tumors. Cancer cell 2009, 16, 510-520. (67) Becker, P. M.; Waltenberger, J.; Yachechko, R.; Mirzapoiazova, T.; Sham, J. S.; Lee, C. G.; Elias, J. A.; Verin, A. D. Neuropilin-1 Regulates Vascular Endothelial Growth Factor-Mediated Endothelial Permeability. Circ Res 2005, 96, 1257-1265. (68) Murashov, V. V.; Leszczynski, J. Adsorption of the Phosphate Groups on Silica Hydroxyls: An AB Initio Study. J. Phys. Chem. A 1999, 103, 1228-1238. (69) Zhou, X.; Zhang, Q.; Chen, L.; Nie, W.; Wang, W.; Wang, H.; Mo, X.; He, C. A Versatile Nanocarrier Based on Functionalized Mesoporous Silica Nanoparticles to Co-deliver Osteogenic 44


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Gene and Drug for Enhanced Osteo-differentiation. ACS Biomater. Sci. Eng. 2019, 5, 710-723. (70) Li, Y.; Li, N.; Pan, W.; Yu, Z.; Yang, L.; Tang, B. Hollow Mesoporous Silica Nanoparticles with Tunable Structures for Controlled Drug Delivery. ACS Appl. Mater. Interfaces 2017, 9, 2123-2129. (71) Yu, Z.; Sun, Q.; Pan, W.; Li, N.; Tang, B. A Near-Infrared Triggered Nanophotosensitizer Inducing Domino Effect on Mitochondrial Reactive Oxygen Species Burst for Cancer Therapy. ACS Nano 2015, 9, 11064-11074. (72) Yu, Z.; Wang, M.; Pan, W.; Wang, H.; Li, N.; Tang, B. Tumor Microenvironment Triggered Fabrication of Gold Nanomachines for Tumor-Specific Photoacoustic Imaging and Photothermal Therapy. Chem. Sci. 2017, 8, 4896-4903.

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Scheme 1. (A) Schematic illustration of the synthesis process for the versatile RGD-CCmMC/DOX nanovehicles. (B) Schematic illustration of the therapeutic mechanism of the RGD-CCmMC/DOX nanoplatforms to enhance the overall anticancer efficiency of triple-combination photodynamic/photothermal/chemo-therapy in a solid tumor.

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Figure 1. Characterization of CCmMC NPs. TEM images of gold cubes (A1~A2), gold cube-in-cubes (B1~B2), Au CCs@mSiO2 (C1~C2) and CCmMC NPs at different magnifications. The insets showed solution state photographs of (A) gold cubes, (B) gold cube-in-cubes, (C) Au CCs@mSiO2 and (D) CCmMC. (E) STEM images and corresponding element mapping of CCmMC nanovehicles. (F) XRD patterns of gold cube-in-cubes, CCmMC and RGD-CCmMC/DOX NPs. (G) The UV-vis absorption spectra of gold cube-in-cubes, CCmMC and RGD-CCmMC/DOX NPs. (H) N2 adsorption-desorption isotherms of CCm and RGD-CCmMC/DOX NPs, the inset was their corresponding pore size distribution profile.

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Figure 2. (A) Thermographic images of CCmMC NPs aqueous solutions irradiated with 808 nm laser at a power intensity of 1 W/cm2 from 0-5 min at various concentrations. (B) Photothermal conversion characterizations of CCmMC aqueous solution of various concentrations under 1 W cm−2 808 nm laser irradiation for 5 min, saline was used as a control. (C) Temperature elevation profiles of 400 µg mL−1 CCmMC solutions under various laser power densities. (D) The temperature change in CCmMC and Au Cube aqueous solution (400 µg mL−1) in response to NIR laser on and off at a duration of 1200 s. (E) Temperature curves of CCmMC under continuous NIR laser irradiation for 4 cycles (808 nm, 1 W/cm2, 600 s with laser and 900 s without laser for each cycle).

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Figure 3. Drug release characterization of RGD-CCmMC/DOX NPs. (A) Cumulative DOX release from RGD-CCmMC/DOX nanovehicles under different pH values (5.0, 6.6, 7.4 ) at 37 ℃ and 50 ℃. Cellular uptake behaviors of RGD-CCmMC/DOX NPs in 4T1 cells. (B) Confocal microscopy images of 4T1 cells stained with DAPI and Lysotracker Green after 1 h, 2 h and 4 h incubation with RGD-CCmMC/DOX NPs at a concentration of 30 μg/mL. Scale bars: 30 µm. (C) Quantitative detection of intracellular Mn-Cdot fluorescence in cells incubated with CCmMC/DOX or RGD-CCmMC at different times (1, 2 and 4 h) via flow cytometer. (D) Confocal FL images of 4T1 cells incubated with the CCmMC/DOX or RGD-CCmMC. Scale bar: 20 µm. (E) TEM images of the RGD-CCmMC/DOX NPs uptake by 4T1 cells and the aggregations of nanovehicles in the cytoplasm. 49


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Figure 4. (A) O2 generation after treatment with CCmMC NPs in PBS solution at pH of 6.6 with increasing photoirradiation time. (B) Decomposition of H2O2 by CCmMC NPs in PBS solution at pH of 6.6 in 30 min. (C) Repetitive catalytic ability of CCmMC NPs with repetitive addition of H2O2 at pre-determined time-points. (D) Absorption spectra of SOSG solution containing CCmMC NPs under various irradiation times with a 635 nm laser, saline was used as the control. (E) CLSM images of 4T1 cells treated with different formulations under laser irradiation. The production of intracellular ROS and O2 generation were measured by the green fluorescence intensity of DCF (laser: 635 nm, 50 mW cm−2, 10 min, scale bar = 20 µm). (F) Confocal FL images of DAPI and DCF stained 4T1 cells with CCmMC or RGD-CCmMC in presence of H2O2. 50


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Figure 5. (A) Cell viability after treated with various concentrations of RGD-CCmMC determined by CCK-8 assay of 4T1 and L929 cells. Each value represents the mean ± SD of three replicates. (B) The hemolytic percentage of RGD-CCmMC NPs to human red blood cells (PBS as a negative control and deionized water as a positive control). (C) Relative cell viability of various formulation groups after incubation with different concentrations NPs with or without laser irradiation by standard CCK-8 assays. (D) Fluorescence microscopy images of 4T1 cells with various treatments using Calcein AM/PI staining. Green and red colors represented live and dead cells, respectively. Scale bar=100 μm. (E) Flow cytometric analysis of apoptosis/necrosis of 4T1 cells by staining with Annexin V-FITC and PI after various treatments. 51


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Figure 6. (A) In vivo fluorescence of RGD-CCmMC NPs by tail vein injection at different times and the organs fluorescence within 48 h. Ex vivo fluorescent signals (B) and quantitative detected (C) in the tumors and major organs induced by laser irradiation after intravenous injection for 24 h. (D) In vivo representative photothermal images of tumor sites after intravenous injection of PBS, CCmMC NPs or RGD-CCmMC NPs as a function of the irradiation time under 808 nm irradiation (1 W/cm2). (E) Corresponding 3D IRTM photothermal images of tumor sites after receiving PBS, CCmMC and RGD-CCmMC treatments at the fifth minute. (F) Corresponding real-time temperature changes in the tumor sites during NIR laser irradiation.

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Figure 7. The magnetic property of RGD-CCmMC and the in vivo MRI. (A) T1-weighted MRI images of RGD-CCmMC in aqueous solution at various Mn concentration. (B) In vivo T1-weighted MRI images of 4T1 tumor-bearing mouse after tail intravenous injection of RGD-CCmMC at preinjection and 12 h, 24 h postinjection. (C) Relative T1-weighted MRI signals of tumors at 0 h, 12 h, and 24 h p. i of RGD-CCmMC. (D) Photographs of excised tumors from tumor-bearing mice after received different treatments on the 21st day.

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Figure 8. Therapeutic efficacy in the 4T1 cell tumor model. (A) Relative tumor volume, (B) body weights, (C) average tumor weight, (D) survival curves of 4T1 tumor-bearing mice in different groups after receiving various treatments during the procedure. Data are presented as mean ± SD, *p < 0.05, **p < 0.01. (E) H&E staining of tumor sections collected from various treatment groups on day 21. Scale bar=100 µm.

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Figure 9. Hematoxylin and eosin staining of tumors and major organs (heart, liver, spleen, lung and kidney) of 4T1 tumor-bearing mice after received different treatments with or without laser irradiation. Scale bar=100 μm.

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Figure 10. Hematology and blood biochemical assay in vivo. Blood indexes of ALP, ALT, AST, BUN, Cr, CAT, GSH-PX, SOD, GLU, CHOL and MCV from the mice treated with RGD-CCmMC at a dose of 30 mg/kg or PBS at various time points (1 day, 1 week and 1 month). Data are presented as mean ± SD.

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Table of contents graphic Gold Cube-in-Cube Based Oxygen Nanogenerator: A Theranostic Nanoplatform For Modulating Tumor Microenvironment for Precise Chemo-Phototherapy and Multimodal Imaging

Xing Zhang, Zhongqian Xi, Jeremiah Ong’achwa Machuki, Jianjun Luo, Dongzhi Yang, Jingjing Li, Weibing Cai, Yun Yang, Lijie Zhang, Jiangwei Tian, Kaijin Guo*, Yanyan Yu and Fenglei Gao*

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Gold Cube-in-Cube Based Oxygen Nanogenerator: A Theranostic

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