Spatiotemporally Light-Activatable Platinum Nanocomplexes for

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Spatiotemporally Light-Activatable Platinum Nanocomplexes for Selective and Cooperative Cancer Therapy Hao Zhao, Jiabao Xu, Wenjing Huang, Guiting Zhan, Yanbing Zhao, Huabing Chen, and Xiangliang Yang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b00972 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Spatiotemporally

Light-Activatable

Platinum

Nanocomplexes for Selective and Cooperative Cancer Therapy Hao Zhao,† Jiabao Xu,† Wenjing Huang,† Guiting Zhan,† Yanbing Zhao,*,† Huabing Chen,*,‡ and Xiangliang Yang*,† †National

Engineering Research Center for Nanomedicine, College of Life Science and

Technology, Huazhong University of Science and Technology, Wuhan 430074, China ‡State

Key Laboratory of Radiation Medicine and Protection, Jiangsu Key Laboratory of

Neuropsychiatric Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China

ACS Paragon Plus Environment

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ABSTRACT. Highly efficient nanoarchitectures are of great interest for achieving precise chemotherapy with minimized adverse side effect in cancer therapy. However, a major challenge remains in exploring a rational approach to synthesize spatiotemporally selective vehicles for precise cancer chemotherapy. Here, we demonstrate a rational design of bifunctional lightactivatable platinum nanocomplexes (PtNCs) that produce dually cooperative cancer therapy through spatiotemporally selective thermo-chemotherapy. The Pt4+-coordinated polycarboxylic nanogel is explored as the nanoreactor template, which is exploited to synthesize bifunctional PtNCs consisting of zero-valent Pt0 core and surrounding bivalent Pt2+ shell with tunable ratios through a facile and controllable reduction. Without light exposure, chemotherapeutic Pt2+ ions are tightly bound on the surface of PtNCs, efficiently reducing undesirable drug leakage and nonselective damages on normal tissues/cells. Upon light exposure, PtNCs generate lots of heat via photothermal conversion from Pt0 core, and simultaneously trigger a rapid release of chemotherapeutic Pt2+ ions, thereby leading to spatiotemporally light-activatable synergistic effect of thermo-chemotherapy. Moreover, PtNCs show the enhanced tumor accumulation through the heat-triggered hydrophilicity-hydrophobicity transition upon an immediate light exposure after injection, dramatically facilitating in vivo tumor regression through their cooperative anticancer efficiency. This rational design of spatiotemporally activatable nanoparticles provides an insightful tool for precise cancer therapy.

KEYWORDS. bifunctional platinum nanocomplex, nanoreactor, photothermal therapy, activatable chemotherapy, cooperative cancer therapy


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Cancer is a serious hazard to human health. For instance, hepatocellular carcinoma (HCC) is the second leading cause of global cancer-related deaths, and single-modality treatment such as chemotherapy alone usually suffers from many limitations such as toxicity, drug tolerance, recurrence and metastasis.1-4 Recently, multifunctional nanoparticles have emerged as promising targeted delivery platforms of chemotherapeutic compounds, exhibiting a favorable synergy between chemotherapy and other therapeutic modalities.5-7 The tailor-made design of nanostructures shows great promise to overcome multiple barriers in cancer-targeted drug delivery such as blood circulation, vessel extravasation, intratumoral penetration, and intracellular drug release, together with minimized adverse side effects, thereby increasing the accessibility of therapeutic compounds to subcellular targets in organelles.8,9 In particular, a variety of smart nanoparticles have been extensively explored to deliver the payloads (such as chemotherapeutics, siRNA, photosensitizers, and gasotransmitters) for satisfying their intracellular delivery in response to intrinsic stimuli such as lysosomal pH, redox, and reactive oxygen species, being responsible for tumor-specific drug delivery.10-15 To further achieve precise drug release, some external stimuli (e.g. light, heat, ultrasound, and magnetism) have also been utilized to induce spatiotemporally responsive drug release through some turn-on/off approaches, together with reduced undesired release in normal tissues.16-22 However, most of these nanoparticles are still encountered with unsatisfied tumor suppression, owing to several drawbacks including premature drug release in normal tissues, insufficient tumor selectivity, and moderate stimuli-responsiveness at tumor microenvironment.23-27 Thus, rational design of multifunctional nanoparticles with precisely spatiotemporal release is highly desired for achieving potent cancer therapy with minimized adverse side effect.


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Near-infrared (NIR) light as an external stimulus has multiple advantages such as remote manipulation, good spatiotemporal resolution, and deep tissue penetration, showing great promise in biomedical applications including selective cancer therapy, bioimaging, and optogenetics.28-30 To date, versatile NIR light-responsive nanoparticles have been extensively explored to realize spatiotemporal drug releases for enhanced intracellular delivery of anticancer compounds.31,32 For instance, light-triggered hyperthermia of the nanoparticles was applied to induce distinct drug release through temperature-mediated phase transition, polymer dissociation, or de-stabilization of hydrogen bonding/π-π interaction,18,33-36 thereby resulting in preferable anticancer efficiency under light exposure.37,38 However, there are still encountered with several limitations such as sophisticated synthetic process and composition, undesirable drug release and poor targeted delivery,16,24,26,39,40 resulting in an urgent demand for fabricating the nanoparticles with highly selective drug release at tumor in a facile and mild approach toward precise cancer therapy.41,42 In our previous work,43,44 temperature-sensitive nanogels were developed for enhancing tumor accumulation and cellular uptake, by their heat-induced hydrophilicity-hydrophobicity transition. Here, we report a rational design of NIR light-activatable bifunctional platinum nanocomplexes (PtNCs) that can produce efficient cooperative cancer therapy through spatiotemporally selective thermo-chemotherapy (Scheme 1). The nanogels of Pt4+-coordinated polycarboxylic acid (Pt4+PNA nanogels) are emloyed as a nanoreactor template to effectively synthesize the bifunctional PtNCs through a facile and controllable reduction reaction, which are comprised of zero-valent Pt0 core and surrounding bivalent Pt2+ shell in a tunable composition. Upon NIR light exposure, PtNCs display a significant hyperthermia via effective photothermal conversion from Pt0 core, and meanwhile trigger an immediate release of chemotherapeutic Pt2+ ions from the surrounding Pt2+ shell through hyperthermia-triggered destabilization of metal-metal interaction, together without


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distinct Pt2+ release in the absence of light exposure, thereby leading to a selective and synergistic effect of thermo-chemotherapy. Moreover, PtNCs showed a light-tirggered tumor accumulation enhancement resulting from the hydrophilicity-hydrophobicity transition of the temperaturesensitive nanogels, consequently leading to a significant cooperative thermochemotherapy efficacy (Scheme 1B).

Scheme 1. (A) Synthetic scheme of bifunctional thermochemotherapy-activatable platinum nanocomplexes (PtNCs) for spatiotemporally selective cancer therapy. (B) Schematic illustration of light-activatable tumor accumulation through hyperthermia-mediated phase transition and light-activatable spatiotemporal drug release. The gray and green octahedrons represent the uncoordinated and coordinated Pt4+ ions in Pt4+-PNA nanogel, respectively. The gray rhombi represent uncoordinated Pt2+ ions, and green rhombi represent coordinated Pt2+ ions or released Pt2+ ions upon light irradiation. Grey spheres represent Pt0 atoms. Yellow dotted lines indicate metal-metal interaction between Pt2+ ions and Pt0 atoms.

RESULTS AND DISCUSSION


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Synthesis and Characterization of PtNCs. To construct bifunctional PtNCs with tunable compositions, the Pt4+-PNA nanogels were exploited as a nanoreactor template as depicted in Scheme 1A. Briefly, H2PtCl6 solution was added dropwise to the solution of poly(Nisopropylacrylamide-b-acrylic acid (PNA) at the carboxyl/Pt4+ molecular ratio of 1.4. The Pt4+PNA nanogels were fabricated through the coordination effect between Pt4+ ions and the carboxyl groups of linear PNA chains (Figure S1, Table S1), showing a nanoscale structure according to the TEM images and strong tyndall effect (Figure S2). Afterwards, sodium borohydride was introduced to reduce free uncoordinated Pt4+ and coordinated Pt4+ into Pt0 core and Pt2+ shell, respectively, and finally resulted in the formation of bifunctional PtNCs with the Pt0-Pt2+ coreshell structure, thus showing an effective approach to synthesize dual-valent Pt nanocomplexes through high affinity of electrostatic and metal-metal interactions.45-52 To elucidate the controlled synthesis of bifunctional PtNCs with core-shell nanostructure, we employed the absorption spectra to monitor the synthesis kinetics of PtNCs, which were synthesized using three Pt4+-PNA nanogels at the feeding carboxyl/Pt4+ ratios of 0.3, 1.4, and 2.7 as the nanoreactor template, respectively. Firstly, Pt4+ ions in these Pt4+-PNA nanogels were rapidly reduced to Pt2+ ions regardless of coordinated or uncoordinated Pt4+ ions, since the characteristic peak of Pt4+ ions (λ = 266 nm) was immediately disappeared after the addition of sodium borohydride.17 Subsequently, the Pt2+ ions that were uncoordinated with PNA suffered from a further reduction to Pt0 atoms, as confirmed by both slow disappearance of the characteristic peak of Pt2+ ions (λ = 212 nm) and corresponding occurrence of Pt0 atom peak (λ = 808 nm) (Figure 1A and Figure S3).53,54 However, the PNA-coordinated Pt2+ ions were hardly reduced to Pt0 atoms, which was verified by the fact that the characteristic peak of


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Figure 1. Structural characterization of PtNCs with the Pt2+/Pt0 ratio of 1.2. (A) Absorption spectra of PtNCs synthesized at different reaction time. Inset plot represented the corresponding enlarged absorption spectra in the range of 750-850 nm. (B) Valence distribution analysis of Pt in PtNCs through X-ray photoelectron spectroscopy (XPS). (C) TEM image of PtNCs (Inset is the high-resolution TEM image of PtNCs). (D) Highangle annular dark field-scanning transmission electron micrograph (HAADF-STEM) of PtNCs. (E) Linear scanning microanalysis of Pt, C and O elements in PtNCs. (F) Size distribution of PtNCs using dynamic light scattering (DLS). (G) X-ray diffraction (XRD) patterns of PtNCs and PNA. (H) Comparison of XPS spectra of O 1s between PtNCs and PNA. (I) Scheme of the microstructure of PtNCs. Gray and green rhombi represent uncoordinated Pt2+ ions and coordinated Pt2+ ions in PtNCs, respectively. Grey spheres represent Pt0 atoms. Yellow dotted lines indicate metal-metal interaction between Pt2+ ions and Pt0 atoms.


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Pt2+ ions (λ = 212 nm) in PtNCs was strengthened as the carboxyl/Pt4+ ratio was decreased in Pt4+PNA nanogels. Apparently, the carboxyl groups of PNA were able to inhibit the reduction reaction from Pt2+ ions to Pt0 atoms (Figure S4), thereby resulting in the formation of bifunctional PtNCs with the Pt2+/Pt0 ratios of 0.3, 1.2, and 2.3 when the feeding carboxyl/Pt4+ ratios were respectively 0.3, 1.4, and 2.7 in the nanoreactor of Pt4+-PNA nanogels, according to the peak-fitting process of Pt 4f orbits in their XPS spectra (Figure 1B and Figure S5A-C). In contrast, Pt4+ ions were fully reduced to zero-valent Pt0 atoms in the absence of PNA (the carboxyl/Pt4+ ratio of 0), consequently leading to the formation of bare Pt0 nanoclusters without the presence of Pt2+ (Pt2+/Pt0 ratio was 0), suggesting that PNA plays a key role in selective reduction of Pt4+ ions into Pt2+ or Pt0 (Figure S5A). Distinctly, there was also a linear correlation between Pt2+/Pt0 ratio in PtNCs and feeding carboxyl/Pt4+ ratio in the nanoreactor of Pt4+-PNA nanogels (Figure S5D), suggesting that the Pt2+/Pt0 ratio in PtNCs can be easily controlled by the feeding carboxyl/Pt4+ ratios in the nanogels. Thus, the nanogels as a template nanoreactor can be effectively applied to mediate the synthesis of PtNCs with tunable Pt2+/Pt0 compositions through regulating feeding carboxyl/Pt4+ ratios. To further verify the microstructures of PtNCs with various Pt2+/Pt0 ratios, their morphology was observed using TEM imaging. At the Pt2+/Pt0 ratios of 0.3, 1.2, and 2.3, PtNCs were found to possess the average diameters of 4.6 ± 0.4 nm, 3.1 ± 0.3 nm, and 2.0 ± 0.2 nm, respectively (Figure 1C and Figure S6 and S7), showing the reduced average diameters as compared to bare Pt0 nanoclusters (5.9 ± 0.4 nm). However, the high-angle annular dark field-scanning transmission electron (HAADF-STEM) imaging showed that higher carboxyl/Pt4+ ratios resulted in the enhanced thickness of PNA shell around the bright core of Pt0 atom and Pt2+ ion aggregates (Figure 1D,E and Figure S6). Reasonably, PtNCs exhibited a distinct nanostructure of Pt0-core and Pt2+shell, and favorable colloidal stability during the experimental periods (Figure S8). Their


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hydrodynamic diameters were 32.1 nm, 48.8 nm, and 75.2 nm with the Pt2+/Pt0 ratios of 0.3, 1.2, and 2.3 respectively (Figure 1F and Table S1). In the experiments of high-resolution TEM imaging, selected area electron diffraction (SAED) and X-ray diffraction (XRD) pattern, a distinct facecentered cubic lattice (d111, 2.24 ± 0.03 Å) was found for bare Pt0 nanoclusters without the presence of Pt2+ (Pt2+/Pt0 ratio was 0), but gradually disappeared as the Pt2+/Pt0 ratio of PtNCs was increased from 0 to 0.3, 1.2, and 2.3 (Figure 1G and Figure S6 and S9A).53 It indicates that amorphous Pt2+PNA shell were tightly bound on crystalline Pt0 core through so-called metal-metal interaction (Figure 1C and Figure S6).45-52 Compared to free PNA, PtNCs showed an O1s binding energy shift of ca. 0.4 eV in their XPS spectra (Figure 1H and Figure S9B), indicating that Pt2+-coordinated PNA were anchored on Pt0 core. This anchoring was also confirmed by the FT-IR spectra, that is, the characteristic peaks of νC=O (1711 cm-1) and νC-O (1174 cm-1) in PtNCs were weakened compared to those of uncoordinated PNA (Figure S9C). As a result, PtNCs show a core-shell nanostructure with Pt0 nanoclusters surrounded by Pt2+-coordinated PNA layer (Figure 1I). Photothermal Effect, Photothermal Conversion Efficiency, and Photostability of PtNCs. To evaluate the photothermal conversion of PtNCs, the temperature elevations (ΔT) were measured using a thermal imager under light irradiation. The ΔT values were increased with the concentration of PtNCs and NIR power density (Figure 2A and Figure S10), indicating a favorable capacity to generate heat. As compared to those widely used organic dyes and inorganic nanoparticles,55 PtNCs showed a good photostability and structural stability in five light exposure/cooling cycles, as confirmed by the comparison of their UV-Vis spectra, TEM and DLS before and after five times of NIR irradiation (Figure 2B and Figure S11). The influence of coreshell valence compositions of PtNCs on the photothermal effect was further investigated.


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Figure 2. Photothermal conversion and hyperthermia-activatable Pt2+ release of PtNCs. (A) Temperature elevation of PtNCs with the Pt2+/Pt0 ratio of 1.2 under 808 nm light exposure (1.0 W cm-2). (B) Temperature elevation of PtNCs with the Pt2+/Pt0 ratio of 1.2 at concentration of 0.5 mmol L-1 Pt under five 808 nm light exposure/cooling cycles (1.0 W cm-2) for their photostability. Inset pictures were TEM micrographs of PtNCs before and after five times of 808 nm light exposure (1.0 W cm-2). (C) Temperature elevation of PtNCs (0.5 mmol L-1 Pt) with various Pt2+/Pt0 ratios under 808 nm light exposure (1.0 W cm-2). (D) Infrared thermography of PtNCs with various Pt2+/Pt0 ratios after 10 min of 808 nm light exposure (1.0 W cm-2). (E) Hyperthermiaactivated release profile of Pt2+ ions from PtNCs at the different Pt2+/Pt0 ratios at the concentration of 0.5 mmol L-1 Pt under five light exposures of 5 min at 1.0 W cm-2 at the interval of 1 h, respectively. (F) Hyperthermiaactivated release profile of Pt2+ ions from PtNCs with the Pt2+/Pt0 ratio of 1.2 at the concentration o 0.5 mmol L1Pt

under five light exposures of 5 min at 0, 0.4, 1.0 and 1.5 W cm-2 at the interval of 1 h, respectively. (G) Long-


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term release profile of Pt2+ ions from PtNCs at the Pt2+/Pt0 ratio of 1.2 (0.5 mmol L-1 Pt) under six light exposures of 5 min at 1.0 W cm-2 at the interval of 2 days. (H) Release profile of Pt2+ ions from PtNCs at the Pt2+/Pt0 ratio of 1.2 (0.5 mmol L-1 Pt) under incubation at 37, 40, 60 and 80 oC in the dark. (I) Temperature elevation and simultaneous Pt2+ release from PtNCs with the Pt2+/Pt0 ratio of 1.2 at the concentration of 0.5 mmol L-1 Pt under continuous 5 min light exposure at 1.0 W cm-2, respectively. The released Pt2+ ions were immediately collected through ultrafiltration under light exposure of each minute during 5 min. Pt2+ ions release experiments were performed at 37 oC.

Under 10 min light exposure (808 nm, 1.0 W cm-2), PtNCs with Pt2+/Pt0 ratios of 0.3, 1.2, and 2.3 showed the temperature elevations of 43.9 °C, 34.6 °C, and 22.8 °C at 0.5 mmol L-1 Pt, while bare Pt0 nanoclusters (the Pt2+/Pt0 ratio was 0) as the control showed a temperature elevation of 47.6 °C (Figure 2C,2D). Obviously, the photothermal effect depends on the content of Pt0 core in PtNCs, instead of Pt2+ in the shell. The molar extinction coefficients of PtNCs were measured as 1.78 9.78 L g-1 cm-1(Table S2), which are smaller than organic photothermal dyes. In spite of low absorbance, PtNCs still realized distinct photothermal effect owing to their high photothermal conversion efficiency (η = 41-44%, Figure S12), which are much higher than those of the existing photothermal agents such as Au nanorods (21%) and CuS nanomaterials (25-40%).56,57 The Pt2+/Pt0 ratios had no influence on the photothermal conversion efficeincies of PtNCs. However, the Pt2+/Pt0 ratios within PtNCs had a significant influence on the extinction coefficient, and thus resulted in a valence-dependent photothermal effects (Table S2). Distinctly, all these PtNCs show a good photothermal conversion capacity for producing effective hyperthermia. In Vitro Photothermal-Induced Pulsatile Release of Cytotoxic Pt2+ Ions. Hyperthermiatriggered release of Pt2+ ions from PtNCs were investigated under several light exposure cycles. PtNCs with the Pt2+/Pt0 ratios of 0.3, 1.2 and 2.3 exhibited light-triggered pulsatile release


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behaviors, which include a sudden release of Pt2+ ions under light exposure and no release in absence of light exposure (Figure 2E). In particular, PtNCs with Pt2+/Pt0 ratio of 1.2 exhibited the highest release amount of 35.6% after five cycles of light exposure, while bare Pt0 nanoclusters (the Pt2+/Pt0 ratio was 0) caused a negligible release of Pt2+ ions (Figure 2E). Compared to PtNCs with Pt2+/Pt0 ratio of 1.2, PtNCs with Pt2+/Pt0 ratios of 0.3 and 2.3 showed the reduced release of Pt2+ ions (17.8% and 27.4%) under the same light exposure, being explained by their lower contents of Pt2+ ions and Pt0 atoms respectively in PtNCs. Therefore, PtNCs with Pt2+/Pt0 ratio of 1.2, which consist of 53.5% Pt2+ ions and 46.5% Pt0 atoms, possess the optimized hyperthermiatriggered pulsatile Pt2+ release. In addition, PtNCs showed light power density-dependent release of Pt2+ ions in the range of 0-1.5 W cm-2 (Figure 2F). To simulate clinical application, the longterm release profile of Pt2+ ions from PtNCs at the Pt2+/Pt0 ratio of 1.2 under repetitive NIR light exposures (1.0 W cm-2, 5 min) at the interval of 2 days was also investigated. PtNCs always show a pulsatile release behavior with light exposure and the release amount was up to 43.6% after six times of light exposure. While there were only negligible Pt2+ ions release without light exposure (Figure 2G). Furthermore, to ensure that Pt2+ release was triggered by thermal effect, the Pt2+ release profile were measured when PtNCs at the Pt2+/Pt0 ratio of 1.2 incubated at different temperatures (37, 40, 60, and 80 oC) for 10 min in the dark. Figure 2H showed that Pt2+ ions release showed a temperature-dependent behavior. There was no Pt2+ ions release when PtNCs incubated at 37 and 40 oC for 10 min, while it increased to 2.5% and 6.6% when PtNCs incubated at 60 oC and 80 oC, respectively, indicating thermal effect triggered Pt2+ ions release. Moreover, the temperature elevations were linearly increased within 5 min light exposure (Figure 2I), and the release amount of Pt2+ ions were linearly increased under the same light irradiation as well, thus displaying a linear correlation between temperature elevation and the release amount of Pt2+ ions


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under NIR light exposure. It means that the hyperthermia-triggered release of Pt2+ ions might follow the zero-order release kinectics. Therefore, the release amount of Pt2+ ions is able to be easily regulated by the light power density and exposure. The zero-order release characteristics of PtNCs can be reasonably explained by the metal-metal interaction of Pt2+ and Pt0, and thus provide a controllable release of Pt2+ ions from PtNCs, together without undesired leakage of Pt2+ ions under no light exposure.45,51 In Vitro Hyperthermia-Activated Chemotherapy upon NIR Light Exposure. To evaluate the light-activatable thermo-chemotherapeutic cytotoxicity of PtNCs under light exposure, PtNCs with various Pt2+/Pt0 ratios were incubated with murine heptoma H22 tumor cells for 24 h, followed by 808 nm light exposure at 1.0 W cm-2. Moreover, to further confirm the chemotherapeutic cell damage of Pt2+ shell or photothermal injury of Pt0 core in PtNCs, the Pt2+coordinated PNA nanogels (Pt2+-Nanogels) and PNA-stabilized bare Pt0 nanoclusters (Pt0-NCs, the Pt2+/Pt0 ratio was 0) were also synthesized as the controls to evaluate their cytotoxicity, respectively (Figure S13). All the nanoparticles showed no cytotoxicity without light exposure, even up to the dose of 1.0 mmol L-1 Pt (Figure S14A), indicating that PtNCs act as non-toxic agent in the absence of light exposure. Under 5 min light exposure at 1.0 W cm-2, PtNCs with Pt2+/Pt0 ratios of 0, 0.3, and 2.3 showed the IC50 values of 0.20, 0.18, and 0.17 mmol L-1 Pt, respectively, thus displaying Pt2+/Pt0-ratio-dependent cytotoxicity (Figure 3A).58,59 The light-induced cytotoxicity of PtNCs was explained well by the light-activable release of Pt2+ ions and enhanced cellular uptake (Figure S14B), producing a selective cytotoxicity in a precise manner. As shown in Figure 3B, Pt2+-Nanogels and Pt0-NCs showed the IC50 values of 0.16 and 0.22 mmol L-1 Pt, respectively, indicating that Pt2+ ions can effectively produce chemotherapeutic cell damage


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regardless of light exposure, and only Pt0 clusters are also able to generate distinct cell injury under light exposure (Figure 3B). In particular, PtNCs with the Pt2+/Pt0 ratio of 1.2 as a

Figure 3. In vitro light-activatable thermo-chemotherapeutic damage of PtNCs at various Pt2+/Pt0 ratios under 808 nm light exposure in H22 cells. (A) Relative viability of H22 cells after incubation with PtNCs at the different Pt2+/Pt0 ratios under 808 nm light exposure at 1.0 W cm-2 for 5 min. (B) Relative viability of H22 cells after incubation with Pt0-NCs, Pt2+-Nanogels and PtNCs with the Pt2+/Pt0 ratio of 1.2 without or with 808 nm


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light exposure at 1.0 W cm-2 for 5 min. (C) Viability of H22 cells treated with PtNCs, thermo-chemotherapeutic concurrence (Chemo-Thermo), chemotherapy and subsequent photothermal therapy (Chemo+Thermo), as well as photothermal therapy and subsequent chemotherapy (Thermo+Chemo), respectively. For chemotherapy, the released Pt2+ ions (0.025 mmol L-1) from PtNCs were used to incubate with the cells for 12 h, while Pt0-NCs (0.1 mmol L-1) were employed to generate photothermal therapy through the incubation with the cells for 12 h, followed by 5 min light exposure at 1.0 W cm-2. (D) PI and Calcein-AM staining and (E) their relative fluorescent intensity (PI/Calcein-AM) of H22 cells treated with PtNCs, Chemo-Thermo, Chemo+Thermo, and Thermo+Chemo using CLSM imaging, respectively. Scale bar: 200 μm. (F) Apoptoic level of H22 cells treated with PtNCs, Chemo-Thermo, Chemo+Thermo, and Thermo+Chemo using Annexin V-FITC/PI staining. Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001.

light-activable thermo-chemotherapeutic agent displayed a preferable cytotoxicity of 0.13 mmol L-1 IC50 as compared to chemotherapeutic Pt2+-Nanogels or photothermal Pt0-NCs alone (Figure 3B). Subsequently, the synergistic effect of PtNCs with different Pt2+/Pt0 ratios between Pt0mediated photothermal therapy and Pt2+-mediated chemotherapy was calculated using the cooperative index (CI). The CI values of PtNCs were 0.86, 0.75, and 0.98 with the Pt2+/Pt0 ratios of 0.3, 1.2, and 2.3 (Figure 3A,B), respectively. Distinctly, PtNCs have the lowest of CI value with the Pt2+/Pt0 ratio of 1.2, thus accounting for their strongest photoactive cytotoxicity, and also according with light-triggered Pt2+ release behavior as depicted in Figure 2D. This strongest cytotoxicity was also confirmed by the confocal laser scanning microscopy (CLSM) images of Calcein-AM/PI staining live/dead cells treated with PtNCs with the Pt2+/Pt0 ratio of 1.2 (Figure S15A). In addition, PtNCs act as non-toxic agent without cytotoxicity in the absence of light exposure due to their light-activatable Pt2+ release behavior, while Pt2+-Nanogels displayed distinct cell damage regardless of light exposure. And PtNCs showed excellent spatial selectivity where only the cells under light exposure zone died, while the un-irradiated cells were still alive (Figure


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S15B). It means that PtNCs possess both chemotherapeutic and photothermal cell injuries with a cooperative effect, both of which are selectively activated by NIR light. Reasonably, PtNCs with the Pt2+/Pt0 ratio of 1.2 were selected as the typical example for subsequent in vivo studies. The influence of the sequential treatments with chemotherapy and photothermal therapy on H22 tumor cells was investigated to confirm the advantage of both light-activatable chemotherapeutic and simultaneous photothermal cell injuries from PtNCs. H22 tumor cells were firstly incubated with the released Pt2+ ions from PtNCs via ultrafiltration, and subsequently treated with Pt0-NCs under light exposure for photothermal therapy (Chemo+Thermo), or were firstly treated with Pt0-NCs under light exposure for photothermal therapy and subsequently incubated with the released Pt2+ ions from PtNCs (Thermo+Chemo). In contrast, H22 tumor cells were also treated with both Pt0-NCs and the released Pt2+ ions under light exposure, indicating a simultaneous treatment of thermo-chemotherapy (Chemo-Thermo). The cytotoxicity of PtNCs under one time of light exposure was compared with those of the treating groups (Chemo+Thermo, Thermo+Chemo, and Chemo-Thermo). There are higher cytotoxicity against H22 tumor cells with the both treatments of Chemo-Thermo and PtNCs than those of Chemo+Thermo and Thermo+Chemo (Figure 3C). Moreover, PtNCs exhibited the highest cytotoxicity on the cells among all treatments under NIR irradiation, indicating that the spatiotemporal synchronization of thermotherapy and chemotherapy plays an important role on enhancing antitumor efficacy of PtNCs. Calcein-AM/PI staining was further applied to confirm the synergistic effect of lightactivatable thermo-chemotherapy using CLSM imaging. The treating groups of PtNCs and Chemo-Thermo showed the higher ratio of red/green fluorescence intensities as compared to the groups of Chemo+Thermo and Thermo+Chemo (Figure 3D,E). The flow cytometry results also indicated the apoptosis of H22 cells treated by PtNCs was up to 69.79% (Figure 3F), much higher


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than those by Thermo+Chemo (28.87%), Chemo+Thermo (43.10%) and Chemo-Thermo (55.62%). These in vitro data indicated PtNCs showed the strongest antitumor efficacy among all treatments through the synergistic effect of thermotherapy from Pt0 atoms and light-activable chemotherapy from spatiotemporal release of Pt2+ ions. In Vivo Pharmacokinetics, and Tumor-Specified Accumulation of PtNCs Directed by NIR Light Based on Hydrophilicity-Hydrophobicity Transition. To investigate in vivo blood circulation of PtNCs with the Pt2+/Pt0 ratio of 1.2, the protein adsorption and macrophage uptake of PtNCs were compared with those of PEG-modified Pt0 nanoclusters (PEG-PtNCs), which were also synthesized as a control (Figure S16). PtNCs and PEG-PtNCs showed a negligible difference in protein adsorption and macrophage uptake, suggesting a similar surficial chemistry between PNA and PEG (Figure S17A and B). Furthermore, PtNCs were intravenously administrated into the mice at the dose of 15.0 μmol Pt kg-1 as a control. It indicates that PtNCs had the half-life time (t1/2β) of 7.5 h, showing a comparable circulation time as compared to that of PEG-PtNCs (t1/2β , 7.9 h) (Figure S17C, Table S3). It is possibly attributed to the fact that PtNCs have a similar hydrophilic property of PNA shell at 37 oC to that of PEG-PtNCs. To improve the in vivo tumor accumulation, PtNCs were intravenously administrated into the mice bearing H22 tumors at a single dose of 15.0 μmol Pt kg-1, and subsequently NIR light (808 nm, 0.5 W cm-2, 15 min, Light on) was immediately applied on the tumor site to cause the temporal tumor hyperthermia after injection, which was expected to enhance the tumor accumulation of PtNCs at 24 h post-injection (Figure 4A). In the absence of immediate light exposure after injection (Light off), PtNCs resulted in the temperature elevation of only 7.6 °C at 24 h post-injection upon light exposure (1.0 W cm2,

5 min) (Figure 4B), while they showed the temperature increase of 16.1 °C at 24 h post-injection,

suggesting an enhanced tumor hyperthermia from PtNCs at tumor site. The similar results were


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also obtained for zero-valent Pt0-NCs (Figure S18A,B). Apparently, the immediate 15 min light exposure (0.5 W cm-2, Light on) on tumor site after injection can effectively improve tumor accumulation of PtNCs at 24 h post-injection, accounting for this increased hyperthermia at tumor. Afterwards, we further monitored the accumulation and

Figure 4. Light-activatable tumor accumulation of PtNCs with the Pt2+/Pt0 ratio of 1.2 at a dose of 15.0 μmol kg-1 Pt based on the hyperthermia-triggered hydrophilicity-hydrophobicity transition upon an immediately spatiotemporal 808 nm light exposure (Light on, 0.5 W cm-2, 15 min) post-injection. (A) Schematic illustration of the experiment schedule of light-activated tumor accumulation of PtNCs through the hydrophilicityhydrophobicity transition under an immediate light exposure after injection. (B) In vivo infrared thermograph of


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the tumors of the mice treated with PtNCs at 24 h post-injection under light irradiation (1.0 W cm-2, 5 min), which have already suffered from immediate 15 min light exposure (Light on) after injection or not (Light off). (C) Accumulation of PtNCs at the tumors from the mice at various time, which have already suffered from an immediate 15 min light exposure (Light on) after injection or not (Light off). The inset indicated the tumor accumulation of PtNCs during 15 min post-injection. (D) Ex vivo near-infrared fluorescence (NIRF) imaging and (E) NIRF intensity of the tumors from the mice injected with Cy5.5-labeled PtNCs and Cy5.5-labeled PEGPtNCs at 15 min post-injection, which have already suffered from an immediate 15 min light exposure (Light on) after injection or not (Light off). Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001.

retention of PtNCs in the tumors during 21 days, which suffered from immediate 15 min light exposure (Light on, 0.5 W cm-2) after injection. PtNCs showed the highest tumor accumulation of about 22.9% ID g-1 at 24 h post-injection (Figure 4C), while only 7.3% ID g-1 was observed for PtNCs without the immediate 15 min light exposure after injection (Light off). As a control, PEGPtNCs showed no significant difference in their tumor accumulations regardless of the immediate 15 min light exposure after injection (Figure S19). It was further confirmed by in vivo and ex vivo near-infrared fluorescence (NIRF) cancer imaging (Figure 4D,E and Figure S20). Clearly, the light-triggered tumor accumulation enhancement was attributed to the hyperthermia from PtNCs (Figure 4C-E). Since PtNCs had the temperature-sensitive PNAs on the surface, and thus showed the temperature-sensitive hydrophilicity-hydrophobicity transition, together with a lower critical solution temperature (LCST) of ca. 38.5 °C according to the light transmittance and hydrodynamic diameter measurements (Figure S21). Reasonably, PtNCs possess a hydrophilic surface at body temperature (< LCST), but might turn into a hydrophobic surface owing to the hydrophilicityhydrophobicity transition casued from hyperthermia under immediate 15 min light exposure on tumor site after injection.22,60-62 Hence, PtNCs display a light-triggered enhancement of tumor accumulation through their hyperthermia-triggered phase transition at tumor site.


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In Vivo Antitumor Efficiency and Histological Staining. To investigate the antitumor efficiency of light-activatable thermo-chemotherapy, PtNCs were intravenously injected into the Balb/c mice bearing H22 tumors. After light-triggered tumor accumulation enhancement by immediate NIR light exposure (Light on) on tumor after injection, six times of NIR light exposures (808 nm, 1.0 W/cm2, 5.0 min) were subsequently applied on the H22-bearing mice at 2, 4, 6, 8, 10, and 12 day post-injection, respectively (Figure 5A). Pt2+-Nanogels with chemotherapeutic cell damage and Pt0-NCs with photothermal injury were also injected as the controls, respectively, followed by the similar light exposures. The mice treated with Pt2+-Nanogels showed about 4-fold increase in their tumor volumes at 14 days post-injection whether the light exposures were applied on or not (Figure 5B). It indicates that Pt2+-Nanogels has a moderate effect on tumor inhibitation, resulting from the slow release of Pt2+ ions from Pt2+-Nanogels regardless of light exposure. However, Pt0-NCs showed a 3.4-fold increase in their volumes under light exposures, while they displayed about 10-fold increase of tumor volume in the absence of light exposure that was similar to that of Saline (~12-fold increase). Clearly, Pt0-NCs possess a light-activatable photothermal damage against tumors, together with no significant dark-cytotoxicity. Interestingly, in the absence of six light exposures, PtNCs showed a 6.9-fold increase in their tumor volume during 14 days post-injection, indicating a moderate anticancer efficiency. Reasonably, this is due to the effective chemotherapy from a part of Pt2+ that were released in vivo during the immediate 15 min light exposure (Light on) after injection for hyperthermia-tirggered tumor accumulation enhancement, although six light treatments were no applied to the tumors. To confirm this reason, this anticancer efficiency of PtNCs was also evaluated in the absence of both immediate light exposure (Light on) for hyperthermia-triggered tumor accumulation enhancement and six light exposures for treatment. It shows that no significant anticancer efficiency was observed for PtNCs (Figure S22).


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Reasonably, PtNCs are able to be also considered as a non-toxic agent in the absence of light exposure. Importantly, PtNCs were found to have the distinct tumor regression with 0.84-fold increase in their tumor.

Figure 5. In vivo antitumor efficacy on H22 tumor-bearing BALB/c mice treated with PtNCs with the Pt2+/Pt0 ratio of 1.2 at the dose of 15.0 μmol kg-1 Pt. (A) Schematic illustration of the experiment schedule of spatiotemporally light-activatable synergistic thermo-chemotherapy. (B) Tumor growth profile, (C) tumor weight, and (D) tumor photo of the mice treated with Pt0-NCs, Pt2+-Nanogels, and PtNCs under immediate light exposure (Light on, 0.5 W cm-2, 15 min) for hyperthermia-triggered tumor accumulation, and subsequent light exposures (1.0 W cm-2) of 5 min at 2, 4, 6, 8, 10, and 12 day post-injection, respectively. The tumors were collected for weight measurement and photogarph at the end of the experiment (14 days post-injection). (E) TUNEL/DAPI staining at 9 days post-injection, and H&E staining at 14 days post-injection of the tumors from


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the mice treated with Pt0-NCs, Pt2+-Nanogels, and PtNCs under immediate light exposure for hyperthermiamediated tumor accumulation and subsequent light treatments of 5 min at 2, 4, 6, 8, 10, and 12 day post-injection, respectively. Scale bar: 100 μm. (F) Relative fluorescence intensity of the tumor sessions stained using the TUNEL/DAPI in (E). Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001.

volumes under six light exposures during 14 days post-injection. Reasonably, this effective tumor regression of PtNCs might result from their synergistic effect between photothermal damage of Pt0 core and simultaneous chemotherapy of Pt2+ shell through light-activatable spatiotemporal delivery. The effective anticancer efficacy of PtNCs was also confirmed by the comparison of tumor weights and tumor mass at the end of this experiment (Figure 5C,D). In addition, no significant difference of the body weights of the mice was observed for PtNCs regardless of light exposure, implying a negligible systemic toxicity of PtNCs (Figure S23A). Afterwards, the TUNEL/DAPI and H&E stainings were aslo applied to verify the anticancer efficacy of PtNCs against the tumors, indicating that PtNCs resulted in the strongest cell apoptosis and subsequent tumor destruction under light exposure after 14 days post-injection (Figure 5E,F). Thus, PtNCs possess the light-activatable thermo-chemotherapy through spatiotemporally selective treatment against the tumors, together without dark cytotoxicity. Recently, some multifunctional nanoparticles (gold nanocages, gold nanorods, graphene) were constructed by sophisticated engineering approaches.63 By intracellular degradation of hyaluronic acid, for instance, gold nanocages@hyaluronic acid (AuNCs-HA) were developed for pinpointed intracellular drug release.64 A Pt nanocluster assembly (Pt-NA) were engineered using a pH-sensitive polymer and tumor-targeting peptide, disassembling into small Pt nanoclusters in acidic subcellular compartment.59 Eventually Pt-NA induced selective damage on tumor cells by pH-responsive release of Pt2+ ions. Compared to these nanoparticles, PtNCs avoided the premature leakage of


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Pt2+ ions under no NIR irradiation, and maximized their therapeutic outcome by spatiotemporally light-activatable synergy effect of thermo-chemotherapy. Moreover, owing to several advantages such as simple synthesis, adjustable structure, high photostability and favorable metallophilic interaction, PtNCs has been hopefully developed as a multi-valent nanoplatform for many metalbased drugs (e.g. Au, Ag, Pd, Ru, Rh, and Ir). Biocompatibility, Long-Term Biodistribution and Elimination. The biocompatibility of PtNCs was investigated using cytotoxicity, hemolysis and histopathology. Without light exposure, PtNCs showed no cytotoxicity against NIH3T3 mouse fibroblasts even up to 1.0 mmol L-1 Pt. When the cells were incubated with the released Pt2+ ions from PtNCs, however, the cell viability reduced to only 65% at 0.025 mmol L-1 Pt and 13.3% at 0.2 mmol L-1 Pt, respectively (Figure S23B). Moreover, PtNCs also showed a better blood-compatibility (only 0.5-0.6% of hemolysis rate) than cisplatin (2.1 ± 0.1%) in the concentration of 0.5 mmol L-1 Pt (Figure S23C). The H&E staining slices showed that PtNCs had no significant damage on heart, liver, spleen, kindey and lung, whether NIR light was applied or not. However, Pt2+-Nanogels showed some renal toxicity, that is, some glomeruli were swollen (red arrows in Figure S23D). In addition, the pharmacokinetic studies showed that PtNCs were rapidly eliminated because of their ultrasmall size (Figure S23E),65,66 and had no distinct influence on the hepatorenal function and hematological indexes even after 21 days post-injection (Figure S24, S25).67,68

CONCLUSION In summary, light-activatable bifunctional platinum nanocomplexes (PtNCs) have been successfully constructed using Pt4+-coordinated polycarboxylic nanogels as a nanoreactor,


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showing a well-defined nanostructure with zero-valent Pt0 core and bivalent Pt2+ shell. The ratio of Pt0 core and Pt2+ shell in PtNCs was easily tailored by the feeding amount of hexachloroplatinic acid and PNA, improving spatiotemporally synergistic effect of thermo-chemotherapy. Owing to the metallophilic interaction of Pt2+ ions and Pt0 atoms, the premature leakage of Pt2+ ions was efficiently inhibited in the absence of NIR irradiation, thus avoiding their damages on normal tissue/cells. Upon expose under NIR irradiation, PtNCs achieved a rapid release of Pt2+ ions due to the photothermal effect, thereby leading to light-activatable synergistic thermochemotherapeutic damage on tumor cells. Moreover, PtNCs also exhibited light-directed tumor accumulation as a specific light exposure was implemented on tumor tissue after injection, further promoting the spatiotemporal specificity and precision of light-activatable antitumor effect. With this spatiotemporally light-activatable releasing behavior, the multi-valent nano-platform represents an effective approach for improve the synergy of multimodal cancer therapy.

MATERIALS AND METHODS Materials. N-isopropylacrylamide (NIPAM, purity > 98.0%, Tokyo Chemical Industry, Tokyo, Japan) was purified by recrystallization from n-hexane for further use. CuCl (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) was purified using diluted hydrochloric acid for further use. Tert-Butyl acrylate (tBA, purity > 99.0%), Hexachloroplatinic acid hexahydrate (H2PtCl6.6H2O, Pt content: 38-40%), Platinum black (purity: 99.95%) were purchased from J&K Scientific Ltd, Beijing, China. Tris(2-dimethyl-aminoethyl)amine (Me6TREN, purity > 99.0%) was purchased from Alfa Aesar, Shanghai, China. Bis[2-(2′-broMoisobutyryloxy)-ethyl]disulfide (BiBOEDS, purity > 97.0%), Methyl-β-cyclodextrin (MβCD), 5-(N-ethyl-N-isopropyl) amiloride


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(EIPA), Chlorpromazine (CPZ) were purchased from Sigma-Aldrich, St., Louis, USA. Acetone (purity > 99.0%), Diethyl ether (purity > 99.0%), Tetrahydrofuran (THF, purity > 99.9%), Dichloromethane (purity > 99.5%), Concentrated hydrochloric acid (HCl, purity: 36%-38%), Concentrated nitric acid (HNO3, purity: 65.0%-68.0%), Dimethylsulfoxide (DMSO, purity > 99.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd, Shanghai, China. Triphenylphosphine (purity > 99.0%), Trifluoroacetic acid (TFA, purity > 99.0%), Platinum standard solution (1000 μg mL-1) were purchased from Aladdin Reagent Co., Ltd, China. Perchloric acid (HClO4, purity: 70.0%-72.0%), Cis-diammineplatinum dichloride (cisplatin, Pt content > 65.0%), Sodium borohydride (NaBH4, purity > 98.0%) were purchased from Macklin Biochemical Co., Ltd, Shanghai, China. Cy5.5 NHS ester (purity > 95%) was purchased from Famous Pharmaceutical Technology Co., Ltd, Beijing, China. Cy5.5-PEG-SH (MW 5000: 5000 Da, purity > 95%) was purchased from Hunan huateng pharmaceutical Co., Ltd., Changsha, China. DMEM medium, RPMI 1640 medium and Fetal bovine serum (FBS) were purchased from Gibco BRL/Life

Technologies,

Grand

Island,

NY,

USA.

3-(4,5-dimethyl-2-thiazolyl)-2,5-

diphenyltetrazolium bromide (MTT), Cell Counting Kit-8 (CCK-8), Calcein-AM/PI Double Stain Kit (500 T), Annexin V-FITC/PI Apoptosis Detection Kit (100 T) and Enhanced BCA Protein Assay Kit (5000 T) were purchased from Yeasen Biotechnology Co., Ltd., Shanghai, China. Except for NIPAM and CuCl, all of them were used as received. Milli-Q Ultrapure water (DI water) (18.2 MΩ) was used in all of the experiments. The glassware used in this work was douched with “DI water : aqua regia = 1 : 1” solution, and then washed with acetone and DI water thrice respectively. Preparation of Temperature Sensitive pAA100-b-pNIPAM200-b-pAA100 (PNA) Triblock Polymers. In order to form the Pt4+-PNA nanogels, temperature sensitive PNA triblock polymers


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(Mn = 49.6 kDa) were firstly synthesized according to our previous work by classical atom transfer radical polymerization (ATRP).61,62 Briefly, a Schlenk tube containing NIPAM (1.13 g, 10 mmol), BiBOEDS (15.3 µL, 0.05 mmol) and Me6TREN (27 μL, 0.1 mmol) dissolved in 5 mL of the actone/water (4/1, v/v) mixed solvent was sealed. After two cycles of liquid-nitrogenrefrigeration/vacuum/argon-filling, CuCl (10 mg, 0.1 mmol) were added into the reaction system to catalyze the polymerization. ATRP reaction was performed at 0 oC within ethanol bath after another cycle of liquid-nitrogen-refrigeration/vacuum/argon-filling. The first polymeric block pNIPAM200 (an viscous solution) was obtained after 48 h. Immediately, The second ATRP reaction was initiated after degassed tBA (1.5 mL, 10 mmol) was injected into the pNIPAM200 solutions using a syringe filled with argon, and three block polymer (ptBA100-b-pNIPAM200-b-ptBA100, PNtB) was obtained for another 48 h polymerization. The crude PNtB was precipitated from water and vacuum drying at 80 oC for 24 h. Then 2 g of dried PNtB was dissolved in 40mL dichloromethane in a 100 mL round-bottom flask under stirring at 30 oC oil-bath, and 4 mL TFA was added to trigger the hydrolysis of PNtB in order to acquire PNA. 24 h later, coarse PNA was prepared and transferred into dialysis bag (MWCO: 3500 Da) against DI water for 5 d. PNA was refined by freeze-drying the dialysis solution. 1 g PNA was added into 20 mL DI water under stirring, then using 1M NaOH to adjust the pH to 8.0, finally PNA water solution (5.0 wt%) was obtained. Preparation of Pt4+-PNA Nanogels. A series volumes (0.1, 0.5 and 1 mL) of PNA solutions (5.0 wt%) and 1.0 mL H2PtCl6 (100 mmol L-1) were mixed together and diluted to 15 mL with DI water, and then their pH values were adjusted to 8.0. After stirring for 24 h at 30 oC, Pt4+-PNA nanogels were obtained due to the coordination between Pt4+ and carboxylic groups. The resultant Pt4+-PNA nanogels were ultrafiltrated to remove free Pt4+ ions and un-coordinated PNA chains.


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Synthesis of PtNCs. To synthesize PtNCs with different Pt2+/Pt0 ratios, NaBH4 served as reductant to reduce Pt4+ ions to Pt2+ ions and Pt0 atoms. In brief, 11.3 mg NaBH4 was dissolved in 5 mL DI water and then injected into Pt4+-nanogels with various carboxyl/Pt4+ ratios using microinfusion pump (WZ50C6, Smiths Medical Instrument Co., Ltd, Beijing, China) at a speed of 20 mL h-1, respectively. At various time points, 0.5 mL of samples was sucked out and diluted to 3.0 mL using DI water. The growing kinetics of PtNCs was studied by UV-Vis absorption spectra. After the reactions finished (ca. 48 h), PtNCs were purified by centrifugation (8000 rpm × 10 min) using ultrafiltration tubes (MWCO: 100 kDa) for three times to remove the uncombined PNA and Pt2+ ions. Eventually, the resultant PtNC dispersions were concentrated to 2.0 mL for further use. PNA-stabilized Pt0 nanoclusters (Pt0-NCs) were synthesized as the control of zero-valent platinum. NaBH4 (5 mL, 2.26 mg mL-1) was injected into 1 mL of H2PtCl6 solusions (100 mmol L-1). After reacted under stirring for 60 min, the resultant Pt0 nanocluster was further co-incubated with PNA solution (5.0 wt%, 0.5 mL). Due to the coordination interaction between zero-valent platinum and carboxyl groups, zero-valent Pt0-NCs were formed in 48 h of co-incubation with PNA. Pt2+coordinated PNA nanogels (Pt2+-Nanogels) were synthesized by the reduction of Pt4+-PNA nanogel (the feeding carboxyl/Pt4+ ratio was 5.4) as the control of bivalent platinum. PEG-PtNCs, as the positive control for evaluating the long-circulation ability of PtNCs, were synthesized through mixing Pt0 nanocluster and Cy5.5-PEG-SH according to the same protocol of synthesis of Pt0-NCs.69 The growing kinetics of PtNCs was studied by UV-Vis absorption spectra. Electrochemical Measurements. Electrochemical measurements were used to investigate the influence of PNA on the redox potentials of Pt4+ ions. Firstly, working electrodes were prepared by the addition of 15 μL of free Pt4+ ions and Pt4+-PNA nanogels (carboxyl/Pt4+ ratio of 0.3, 1.4 and 2.7) onto the glassy carbon electrode (ca. 0.196 cm2). The working electrode was covered with


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0.05wt% Nafion solution (15 μL) after dried in air, and vacuum-dried for 30 min. Electrochemical testing were operated using a CHI 630D workstation (Shanghai Chenhua Apparatus Co., China) with a conventional three-electrode system, which were composed of a platinum wire as the counter electrode and a saturated calomel electrode (SCE) as the reference. In brief, cyclic voltammetry (CV) tests were carried out in N2-saturated 5% NaCl solutions (pH 8.0) at a sweep rate of 50 mV s-1. Characterization. The morphology and selected area electron diffractions (SAED) patterns of PtNCs were observed by transmission electron microscopy (TEM, Tecnai G2 20, FEI Co., Netherlands). And the microstructure, high-angle annular dark field-scanning transmission electron (HAADF-STEM) micrographs and linear scanning microanalysis for PtNCs were performed using high-resolution TEM (HRTEM, FEI Technai G2 F30, FEI Co., Netherlands). Wide-angle powder X-ray diffraction (XRD) measurements (Empyrean, PANalytical B.V. Co., Netherlands) were used to analyse lattice of PtNCs. Thermogravimetric analysis (TGA) of PtNCs were performed using thermal gravimetric analyzer (Pyris1 TGA, PerkinElmer Instruments Co., Ltd., Shanghai, China). Functional groups analysis was measured using Fourier transform infrared spectroscopy (FT-IR, VERTEX 70, Bruker Co., Germany). Platinum valence composition in the PtNCs were analysized by X-ray photoelectron spectroscopy (XPS, AXIS-ULTRA DLD-600 W, Shimadzu-Kratos Co., Japan). Platinum black and Pt2+-Nanogels were selected as the standards of Pt0 atoms and Pt2+ ions, respectively. The hydrodynamic diameters, zeta potentials and volume phase transition temperatures (VPTTs) of PtNCs were performed using dynamic light scattering instrument (DLS, Zetasizer Nano ZS90, Malvern Instruments Co., Ltd., Malvern, UK). The UVVis absorbance spectra were measured using UV-Vis spectrophotometer (Lambda 35, PerkinElmer Instruments Co., Ltd., Shanghai, China). Platinum content was measured using an


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inductively coupled plasma optical emission spectrometer (ICP-OES, PerkinElmer Ltd., Co., USA). The colloidal stability of PtNCs in various mediums (DI water, PBS buffer and RPMI 1640 medium containing 10% FBS) was investigated by measuring their hydrodynamic diameters respectively at 0 d, 7 d and 14 d. The concentration of PtNCs was diluted to 0.5 mmol L-1. Photothermal Effect, Photothermal Conversion Efficiency, and Photostability of PtNCs. 0.5 mL of PtNCs at various platinum concentrations (0.125, 0.25, 0.375, 0.5 mmol L-1) were irradiated using NIR light (wavelength is 808 nm) with various powers (0, 0.4, 0.7, 1.0 and 1.5 W cm-2) for 10 min. And real-time temperature was recored using a NIR thermal imager (FLIRE64501, FLIR Systems Inc., USA) every 30 s. To estimate the photothermal stability, 0.5mL of PtNCs (Pt concentration was 0.5 mmol L-1) were heated by NIR light (1.0 W cm-2) for 10 min, and then let them cooled to room temperature. The heating-cooling cycles was repeated five times, and the temperature was recored at an interval of 30 s. The UV-Vis absorbance spectra and TEM micrographs of PtNCs were compared before and after five times of heating-cooling cycles. the photothermal conversion efficiency (η) of PtNCs was calculated following equation (1).



hS Tmax  Tsurr   Qdis



I 1  10 A808



100%

(1)

Here, h, S were the heat transfer coefficient and surface area of the cuvette cell, respectively. Tmax and Tsurr were equilibrium temperature of solutions and ambient temperature, respectively. Qdis was measured using a quartz cuvette cell containing DI water. I was incident laser power (1.0 W cm-2), A808 was the UV/Vis absorbance values of four PtNCs (0.5 mmol L-1 Pt) at the wavelength of 808 nm, respectively. In Vitro Photothermal-Induced Pulsatile Release of Pt2+ Ions. In order to investigate the release of Pt2+ ions from PtNCs under NIR light exposure, 0.5 mL of PtNC dispersions (pH 5.0,


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Pt content was ca. 0.5 mmol L-1) were added in 1.5 mL EP tubes and incubated in an incubator at 37 oC. At various time points (1.0, 2.0, 3.0, 4.0 and 5.0 h), PtNC dispersions were irradiated with NIR light (1.0 W cm-2 × 5.0 min), and then taken out into a 15 mL ultrafiltration tube (MWCO: 100 kDa). After centrifuged (6000 rpm × 5 min) these PtNC dispersions, their ultrafiltrate was collected for measuring Pt content. Moreover, to evaluate the long-term photothermal-induced pulsatile release of Pt2+ ions form PtNCs at the Pt2+/Pt0 ratio of 1.2, the NIR light (1.0 W cm-2 × 5.0 min) exposure time interval was extended to 48 h. The procedure was the same as mentioned above. To study the influence of NIR light power density on the release of Pt2+ ions, 0.5 mL of PtNC disperisions (0.5 mmol L-1 of Pt content) were added in 1.5 mL EP tube, and then was irradiated using NIR light with various power densities (0, 0.4, 1.0 and 1.5 W cm-2) at various time points (1.0, 2.0, 3.0, 4.0 and 5.0 h). In addition, the Pt2+ ions release behaviors from PtNCs at the Pt2+/Pt0 ratio of 1.2 incubated at different temperatures (37 oC, 40 oC, 60 oC, 80 oC) in the dark for 10 min were also investigated to determine Pt2+ ions release was triggered by thermal effect. The accumulative releasing amount of Pt2+ ions (AR at the m times of NIR light exposure) as calculated as followed equation (2):

 AR 

m

CV

i 1 i i

(2)

C0V0

Here, Ci and Vi are the concentration and volume of Pt2+ ions in the collected supernatant at No. i of NIR light exposure, respectively. C0 is total platinum concentration in PtNCs, and V0 is the volume of PtNCs. Cytotoxicity of PtNCs on NIH3T3 Mouse Fibroblasts Without NIR Light Exposure. The MTT assay was used to evaluate the cytocompatibility of PtNCs on NIH3T3 cells. For cytotoxicity test, in brief, NIH3T3 cells were cultured in 96-well plates at the cell density of 1 × 104 per well


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in 200 μL complete DMEM media. After incubated under 5%/95% CO2/O2 atmosphere at 37 °C for 12 h, the media were replaced by four PtNC dispersions (Pt content is 0.3, 0.6, 0.9 and 1.2 mmol L-1 respectively in 100 μL serum-free DMEM media). After cultured for another 24 h, the media were sucked out and the cells were washed with cold PBS for 3 times. 200 μL MTT containing 20 μL mother liquor (5 mg mL−1) and 180 μL serum-free DMEM media was added in every wells, and the optical densities (ODs) at 492 nm were measured 15 minutes later. The Pt contents of cisplatin and free Pt2+ ions were 0.025, 0.05, 0.1, 0.15 and 0.2 mmol L-1 in 200 μL serum-free DMEM media. The blank and negative controls were serum-free DMEM media and the mixture solution of serum-free DMEM media and NIH3T3 cells, respectively. The cell viability (CV) was calculated following equation (3).

CV 

ODs  ODb 100% ODn  ODb

(3)

Here, ODs, ODb and ODn were the OD values of samples, blank control and negative control, respectively. Cellular Uptake of PtNCs Without and With NIR Light Exposure. To study the influence of NIR light exposure on cellular uptake, PtNCs (0.15 mmol L-1 Pt) were incubated with H22 cells (1 × 106 cells per well) in the 6-well plates without and with NIR light exposure (1.0 W cm-2, 15 min). Subsequently, the cells were gathered for cell counting and digesting. Afterward, Pt amounts were analyzed by using ICP-OES. Hemolysis Assay of PtNCs Without NIR Light Exposure. Hemolysis was used to evaluate the blood compatibility of PtNCs. To investigate the hemolysis, the whole blood of BALB/c mice (2 mL) was mixed with 2 mL of normal saline in a 10 mL centrifuge tube containing 5 μL 2% heparin sodium under stirring slowly. Afterwards, the mixture solution was centrifuged (1200 rpm,


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20 min) twice to collect concentrated red cell suspension (RCS). 2% of RCS diluent was prepared by adding 500 μL concentrated RCS into 24.5 mL normal saline. The RCS diluent (150 μL) was added into 150 μL of PtNC dispersions (0.5 mmol L-1 of Pt content), and cultured at 37 oC for 1.0 h. The mixtures were centrifuged (1500 rpm, 10 min) and collected the supernatant for ultrafiltration (8000 rpm, 10 min) in order to separate the hemoglobin (filtrate) and PtNCs (supernatant). The optical densities (OD value) of the supernatant were measured at the wavelength of 540 nm. Normal saline and DI water was used as negative and positive controls respectively, and the hemolysis ratio (Hr) was calculated following equation (4):

Hr 

ODs  ODn 100% OD p  ODn

(4)

Here, ODs, ODp and ODn are the OD values of the samples, the positive control and the negative control, respectively. In Vitro Hyperthermia-Activated Chemotherapy Upon NIR Light Exposure. Classical CCK-8 assay was used to evaluate the cytotoxicity of PtNCs on H22 cells. H22 cells were cultured in 96-well plates as mentioned above, the media were substituted with various PtNCs (0.3, 0.6, 0.9 and 1.2 mmol L-1 of Pt content, 200 μL of serum-free RPMI 1640 media), respectively. The cisplatin and Pt2+ ions with the Pt content of 0.025, 0.05, 0.1, 0.15 and 0.2 mmol L-1 as control. The blank and negative controls were serum-free DMEM media and the mixture solution of serumfree DMEM media and H22 cells, respectively. After 24 h of incubation, the 96-well plates were centrifuged (1200 rpm × 5 min) for collecting H22 cells, and washed with fresh PBS buffer for 3 times. Cell viability (CV) was measured as mentioned above and calculated following equation (3). The combination index (CI) of PtNCs was calculated from the half inhibition concentration (IC50) of Pt0-NCs, Pt2+-Nanogels and PtNCs under NIR light exposure.70 In brief, H22 cells were


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seeded in 96-well plates as mentioned above. Then, the media were replaced respectively with the serum-free RPMI 1640 media containing Pt0-NCs, Pt2+-Nanogels and PtNCs at Pt content of 0.025, 0.05, 0.1, 0.2, 0.4 and 0.6 mmol L-1. All H22 cells were irradiated by NIR light (1.0 W cm-1, 5 min). After cultured for another 24 h, the cell vialibities were measured using CCK-8 as mentioned above. And the CI of PtNCs was calculated by the followed equation (5):

CI 

DPt0 ( D0) Pt0



DPt 2 

(5)

( D0) Pt 2 

Here, (D0)Pt0 and (D0)Pt2+ represented the half inhibition concentration (IC50) of Pt0-NCs and Pt2+-Nanogels, respectively. D Pt0 and D Pt2+ represented the corresponding IC50 of zero-valent Pt0 and bivalent Pt2+ in PtNCs with the Pt2+/Pt0 ratio of 1.2, respectively. Photocytotoxicity Assay of PtNCs. The photocytotoxicity of PtNCs was evaluated by the same procedure of CCK-8 assay, as described above. After incubation for 24 h, H22 cells were stained with Calcein-AM/PI or stained with Annex V-FITC/PI for analyzing cell viability by confocal laser scanning microscope (CLSM, Olympus IX81, Japan) or flow cytometer (CytoFLEX, Beckman Coulter, USA) at the excitation wavelengths of 488 nm (Calcein-AM or Annex V-FITC) and 559 nm (PI) according to the protocols. It is impossible to use suspension H22 cell to examine the spatial selectivity of photocytotoxicity of PtNCs. Therefore, adherent HepG2 hepatoma cells were used to determine this property of PtNCs. In brief, 3 × 105 of HepG2 cells were seed in confocal dishes, and PtNCs at the Pt2+/Pt0 ratio of 1.2 at different concentrations were added into confocal dishes when HepG2 cells grew up to 80% of dish area. Then, the HepG2 cells were irradiated by NIR light (1.0 W cm-1, 5 min). After cultured for another 24 h, HepG2 cells were stained with Calcein-AM/PI for analyzing cell viability by CLSM.


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The Influence of Treatment Sequence on Antitumor Efficacy. To confirm the advantage of both light-activatable chemotherapeutic and simultaneous photothermal cell injuries of PtNCs, the influence of treat sequence on antitumor efficacy was further investigated on H22 cells. In order to ensure the same amount of Pt2+ ions in each group for chemotherapy. The administration dosage of Pt2+ ions was set to the released amount of Pt2+ ions (ca. 0.025 mmol L-1) from PtNCs under one time of NIR irradiation. In brief, H22 cells were seeded in 96-well plates as mentioned above. After cultured overnight, the cells were divided in three treat groups: (1) Chemo+Thermo group: the cell firstly incubated with the solution of the released Pt2+ ions for chemotherapy (0.025 mmol L-1 of Pt concentration, 50 μL) for 12 h, and subsequently NIR light (1.0 W cm-2, 5 min) were applied after Pt0-NCs (0.1 mmol L-1 of Pt content) was added into the culture. (2) Thermo+Chemo group: NIR light (1.0 W cm-2, 5 min) were firstly applied along with the addition of Pt0-NCs (0.1 mmol L-1 of Pt content) into the cells. Waiting for 10 min, the released Pt2+ ions (0.025 mmol L-1 of Pt concentration) was added to cells and co-incubated for 12 h. (3) ChemoThermo group: the released Pt2+ ions (0.025 mmol L-1 of Pt concentration) and Pt0-NCs (0.1 mmol L-1 of Pt concentration) were added together into the cells, and meanwhile NIR light (1.0 W cm-2, 5 min) were applied. (4) PtNCs group: PtNCs at the Pt2+/Pt0 ratio of 1.2 (0.213 mmol L-1 of Pt concentration) were added together into the cells, and meanwhile NIR light (1.0 W cm-2, 5 min) were applied. After another 12 h of incubation at 37 oC, the cells were washed with PBS buffer for three times, and a standard CCK-8 test procedure was carried on for the measurement of cell viability. The confocal laser scanning microscope and flow cytometer were also performed, as mentioned above. Protein Adsorption. To evaluate the protein adsorption capacity of PtNCs, PEG-PtNCs (negative control) and PtNCs with the Pt2+/Pt0 ratio of 1.2 (0.15 mmol L-1 of Pt content) were


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incubated in PBS (containing 10% FBS) for 4 h at 37 oC respectively, as reported before.71 these PtNCs were collected by centrifugation (20000 rpm, 30 min) using an ultracentrifuger (Optima MAX-TL, Beckman Coulter Co., Ltd., USA) and washed with cold PBS for 3 times. Finally, using enhanced BCA protein assay kit to measure the protein content according to the protocols. The background values of PEG-PtNCs and PtNCs with the Pt2+/Pt0 ratio of 1.2 were deducted in the calculation. Phagocytosis by Macrophage Raw264.7 Cells. To evaluate the phagocytosis by macrophages of PtNCs, RAW264.7 cells were seeded in a 6-well plate at the density of 5 × 105 cells per well, as reported before.71. After incubated in 5%/95% CO2/O2 atmosphere at 37 oC for 12 h, the cells were treated with complete DMEM medium (10% FBS) containing PEG-PtNCs (negative control) and PtNCs with the Pt2+/Pt0 ratio of 1.2 (0.15 mmol L-1 of Pt concentration) for 2 h at 37 oC, respectively. And the cells were washed with cold PBS for 3 times and detached by trypsin, gathered through centrifugation, re-suspended in PBS, counted with image analysis and digested to measure the platinum concentrations using ICP-OES. In Vivo Pharmacokinetics, Long-Term Biodistribution and Elimination. BALB/c mice (SPF, male, 16-18 g) were purchased from the center for disease control and prevention in Hubei province (Wuhan, China) and raised in SPF laboratory animal room. To investigate the pharmacokinetic behavior and long-term circulation of PtNCs, PEG-PtNCs was synthesized as positive control. In brief, PtNCs with the Pt2+/Pt0 ratio of 1.2 and PEG-PtNCs were injected into healthy BALB/c mice intravenously at 15 μmol kg-1 of Pt dose. Afterwards, blood samples were extracted at 10 min, 30 min, 2 h, 4 h, 8 h, 12 h and 24 h post-injection and Pt content was measured using ICP-OES after digestion. To demonstrate in vivo long-term biodistribution, PtNCs with the Pt2+/Pt0 ratio of 1.2 (15 μmol kg-1) was injected into healthy BALB/c mice via tail vein at a single


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injection, which were sacrificed at 15 min, 1 d, 3 d, 7 d, 14 d, 21 d post-injection for extraction of major tissues and tumor. Then, these tissues were digested for calculation of the biodistribution. Meanwhile, urine and faeces were also collected for calculation of the elimination. Pt contents were measured using ICP-OES as mentioned above. Preparation and Characterization of Cy5.5-Labeled PtNCs. Cy5.5-labeled PtNCs were prepared by coupling Cy5.5 NHS on PtNCs through ester linkage. Firstly, amino PNA was synthesized through azide reaction and azide groups were reduced by triphenylphosphine. In brief, 1.2 g of PNA was dissolved in 40 mL of DMF. Then, 65 mg of NaN3 dissolved in 4.5 mL of DMF was added into this reaction system (the molar ratio of alkyl bromide and azide groups was 1.0 / 1.1). After reacted at 25 oC with gentle stirring for 24 h, the obtained azide PNA was purified by dialysis (MWCO 14 kDa) and lyophilization. Afterwards, the obtained azide PNA was dissolved in 80 mL of THF and 22 mg of triphenylphosphine was added into this reaction system (the molar ratio of azide groups and triphenylphosphine was 1.0 / 1.5). After reacted at 25 oC for 24 h, the THF was removed by vacuum rotary evaporation. When it was nearly dried, 60 mL of water was added into it and the pH was adjusted to 9.0, and then the botained amino PNA was purified by dialysis (MWCO 14 kDa) and lyophilization. Then, Cy5.5-labeled PNA was prepared by coupling amino PNA with Cy5.5 NHS ester. Briefly, 1 g amino PNA was dissolved in 20 mL of DMF/DI water (1/1, v/v) mixed solution and the pH was adjusted to 8.0 using triethylamine. Then, 40 mg of Cy5.5 NHS ester (the molar ratio of amino groups and Cy5.5 NHS ester was 1.0 / 1.5) was added into this system. After reacted at 25 oC for 24 h in the dark, the obtained Cy5.5-labeled PNA was purified by dialysis (MWCO 14 kDa) and lyophilization. And the Cy5.5-labeled PtNCs were synthesized by using Cy5.5-labeled PNA as template. The UV/Vis and fluorescent spectra of Cy5.5-labeled PtNCs were measured by using using UV-Vis spectrophotometer and Time-


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Resolved Spectrofluorometer (Fluoromax-Plus, Horiba Jobin Yvon, Paris, France) before and after NIR light exposure (1.0 W cm-2, 15 min) to detect the signal stability of Cy5.5. Tumor-Specified Accumulation of PtNCs Directed by NIR Light. The H22 subcutaneous tumour model in BALB/c mice (male, SPF, 16-18g) were established by inoculation in the flank (2 × 106 H22 cells per mouse). To study tumor-specified accumulation property of PtNCs directed by NIR light, 100 μL Saline, PNA solution (2.0 wt%), Pt0-NCs (1.4 mmol L-1 of Pt concentration), Pt2+-Nanogels (1.6 mmol L-1 of Pt concentration) and PtNCs with the Pt2+/Pt0 ratio of 1.2 (3.0 mmol L-1 of Pt concentration) were intravenously injected into the tumor-bearing mice, respectively. Immediately, the tumors were exposed in NIR light (808 nm, 0.5 W cm-2, 15 min) for carrying out tumor-specified accumulation. Afterwards, the tumors were irradiated by NIR light (808 nm, 1.0 W cm-2, 5 min) at 24 h post-injection for launching photothermal therapy. The real-time temperature images in tumor were recorded by NIR thermal imager at an interval of 30 s. And the major tissues and tumor were harvested and digested for platinum measurement using ICP-OES at 15 min and 24 h after NIR light exposure on tumors or not. PEG-PtNCs were negative control. The influence of NIR light-directed accumulation on antitumor effect of PtNCs with the Pt2+/Pt0 ratio of 1.2 was also evaluated. In brief, 100 μL of Saline, and PtNCs (3.0 mmol L-1 of Pt content) were intravenously injected into the tumor-bearing mice, respectively. Immediately, the tumors were irradiated without /with NIR light (808 nm, 0.5 W cm-2) for 15 min. At 24 h postinjection, the tumors were irradiated by NIR light (808 nm, 1.0 W cm-2) for 5 min every 2 days. The tumor volume was measured every 2 days in 14 days of experimental periods, and calculated as following V = (L) × (W)2 / 2. In Vivo Antitumor Efficiency and Histological Staining. To evaluated antitumor activities of PtNCs, H22-tumor-bearing BALB/c mice model were established according to mentioned


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above. The mice were selected as the tumors grew up to about 125 mm3 of volume, and divided into 8 groups randomly (5 mice per group): (1) Saline, (2) PNA solution (2.0 wt%), (3)/(4) Pt0NCs -/+ Light (1.4 mmol L-1 Pt), (5)/(6) Pt2+-Nanogels -/+ Light (1.6 mmol L-1 Pt), (7)/(8) PtNCs with the Pt2+/Pt0 ratio of 1.2 -/+ Lgiht (3.0 mmol L-1 of Pt concentration). All of the groups were immediately irradiated with NIR light after injecting with corresponding materials. At 24 h postinjection, the tumors of the mice in Group (4), (6) and (8) were irradiated with NIR light (1.0 W cm-2) for 5 min every other day. The tumors volume were also measured every other day. To evaluate the cytotoxicity of PtNCs without NIR light exposure, H22-tumor-bearing BALB/c mice were divided into 5 groups randomly (3 mice per group) after the volume of tumors reached to about 200 mm3: (a) Saline; (b) the physical mixture of Pt2+-Nanogels (1.6 mmol L-1 of Pt content) and Pt0-NCs (1.4 mmol L-1 of Pt content) without NIR light exposure; (c) the physical mixture of Pt2+-Nanogels (1.6 mmol L-1 of Pt content) and Pt0-NCs (1.4 mmol L-1 of Pt content) with NIR light exposure (1.0 W cm-2) exposure for 5 min every other day; (d) PtNCs (3.0 mmol L-1 of Pt content) without NIR light exposure; (e) PtNCs (3.0 mmol L-1 of Pt content) with NIR light exposure (1.0 W cm-2) for 5 min every other day. All of the materials were injected through intratumoral (i.t.) administration to ensure the Pt amount in tumors were equal. The tumors volume were measured for 9 days, together with the tumors weighed to evaluate the influence of precision synchronization on the antitumor efficiency. Finally, all of the mice were sacrificed at the end of the experiments and the major tissues and tumor were collected for H&E staining and TUNEL/DAPI immunofluorescence staining. Serum Biochemistry assay. PtNCs (15 μmol kg-1) were injected into healthy BALB/c mice via tail vein, which were sacrificed at 3 d, 7 d, 14 d, 21 d post-injection for extraction of blood (0.8 mL) from the mice for blood biochemistry determinations. In brief, the collected bloods were


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stored at 4 oC overnight and then centrifuged (3000 rpm, 15 min) for obtaining the serum. 100 μL of serum were taken out and added to 200 μL PBS for blood biochemistry examination. Hematological Assessment. PtNCs (15 μmol kg-1) were injected into healthy BALB/c mice via tail vein, which were sacrificed at 3 d, 7 d, 14 d, 21 d post-injection for extraction of blood (0.5 mL, with 40 μL of 15 g L-1 EDTA for anticoagulation) from the mice for hematological assessments. The blood panels including white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean horpuscular hemoglobin concentration and platelets were measured with Automatic five-classification blood cell analyzer (XT-1800i, Sysmex Shanghai Ltd., Shanghai, China).67,68 Statistical Analysis. The data were expressed as the mean ± SD. The measurement data were analyzed statistically by the Student's t-test and double-factor variance analysis, while the enumerated data were treated with the chi-square (χ2) test and Fisher’s exact probability test. *P < 0.05 was considered as a statistically significant difference, and ***P < 0.001 was considered highly statistically significant. ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information The Supporting Information is available free of charge on the ACS Publications website. XPS, TEM images and hydrodynamic diameters of Pt4+-PNA nanogels, growth kinetic behavior by monitoring UV-Vis spectra, Cyclic voltammograms (CVs) of Pt4+-PNA nanogels, Valence state analysis, TEM, SAED and HAADF-STEM, colloidal stability, XRD, XPS and FT-IR spectra,


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temperature elevating profile, photothermal stability and photothermal conversion efficiency, cell viability of PtNCs treated H22 cells without light irradiation, pharmacokinetic and biodistribution analyses, tumor temperature variation, in vivo NIRF images, temperature sensitivity evaluation, H&E staining images of major tissues, hepatorenal function analysis of PtNCs treated Balb/c mice. AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected] *[email protected] ORCID Hao Zhao: 0000-0002-5848-9457 Jiabao Xu: 0000-0002-3802-8311 Yanbing Zhao: 0000-0002-0675-6680 Huabing Chen: 0000-0003-1637-2872 Author Contributions H.Z., J.X. and Y.Z. conceived the project. H.Z. and J.X. designed the experiments. H.Z., J.X., W.H. and G.Z carried out the experiments and analysed the data. H.Z. and J.X. wrote the manuscript. H.Z., Y.Z., H.C. and X.Y. contributed to the revision. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT


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This work was supported by the National Basic Research Program of China (973 Program, grant No. 2015CB931802 to Xiangliang Yang), the National Natural Science Foundation of China (grant No. 81673016 to Yanbing Zhao, 81627901 to Xiangliang Yang, and 51473109 to Huabing Chen), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) to Huabing Chen. We thank the Analytical and Testing Center of HUST and the Research Core Facilities for Life Science (HUST) for the related analysis. REFERENCES 1. Llovet, J. M.; Zucman-Rossi, J.; Pikarsky, E.; Sangro, B.; Schwartz, M.; Sherman, M.; Gores, G. Hepatocellular Carcinoma. Nat. Rev. Dis. Primers 2016, 2, 16018. 2. Stanaway, J. D.; Flaxman, A. D.; Naghavi, M.; Fitzmaurice, C.; Vos, T.; Abubakar, I.; AbuRaddad, L. J.; Assadi, R.; Bhala, N.; Cowie, B.; Forouzanfour, M. H.; Groeger, J.; Hanafiah, K. M.; Jacobsen, K. H.; James, S. L.; MacLachlan, J.; Malekzadeh, R.; Martin, N. K.; Mokdad, A. A.; Mokdad, A. H. et al. The Global Burden of Viral Hepatitis From 1990 To 2013: Findings From the Global Burden of Disease Study 2013. Lancet 2016, 388, 10811088. 3. Forner, A.; Reig, M.; Bruix, J. Hepatocellular Carcinoma. Lancet 2018, 391, 1301-1314. 4. Worns, M. A.; Galle, P. R. Hepatocellular Carcinoma in 2017: Two Large Steps Forward, One Small Step Back. Nat. Rev. Gastroenterol. Hepatol. 2018, 15, 74-76. 5. Chen, G.; Roy, I.; Yang, C.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826-2885. 6. Sherman, S. E.; Xiao, Q.; Percec, V. Mimicking Complex Biological Membranes and Their Programmable Glycan Ligands with Dendrimersomes and Glycodendrimersomes. Chem. Rev. 2017, 117, 6538-6631.


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7. Tregubov, A. A.; Nikitin, P. I.; Nikitin, M. P. Advanced Smart Nanomaterials with Integrated Logic-Gating and Biocomputing: Dawn of Theranostic Nanorobots. Chem. Rev. 2018, 118, 10294-10348. 8. Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20-37. 9. Bourzac, K. News Feature: Cancer Nanomedicine, Reengineered. Proc. Natl. Acad. Sci. USA 2016, 113, 12600-12603. 10. Li, S.; Jiang, Q.; Liu, S.; Zhang, Y.; Tian, Y.; Song, C.; Wang, J.; Zou, Y.; Anderson, G. J.; Han, J.; Chang, Y.; Liu, Y.; Zhang, C.; Chen, L.; Zhou, G.; Nie, G.; Yan, H.; Ding, B.; Zhao, Y. A DNA Nanorobot Functions As a Cancer Therapeutic In Response To a Molecular Trigger in Vivo. Nat. Biotechnol. 2018, 36, 258-264. 11. Chan, L.; Gao, P.; Zhou, W.; Mei, C.; Huang, Y.; Yu, X.; Chu, P. K.; Chen, T. Sequentially Triggered Delivery System of Black Phosphorus Quantum Dots with Surface ChargeSwitching Ability for Precise Tumor Radiosensitization. ACS Nano 2018, 12, 12401-12415. 12. Li, Y.; Li, Y.; Ji, W.; Lu, Z.; Liu, L.; Shi, Y.; Ma, G.; Zhang, X. Positively Charged Polyprodrug Amphiphiles with Enhanced Drug Loading and Reactive Oxygen SpeciesResponsive Release Ability for Traceable Synergistic Therapy. J. Am. Chem. Soc. 2018, 140, 4164-4171. 13. Sun, B.; Luo, C.; Yu, H.; Zhang, X.; Chen, Q.; Yang, W.; Wang, M.; Kan, Q.; Zhang, H.; Wang, Y.; He, Z.; Sun, J. Disulfide Bond-Driven Oxidation- and Reduction-Responsive Prodrug Nanoassemblies for Cancer Therapy. Nano Lett. 2018, 18, 3643-3650. 14. Deng, Z.; Qian, Y.; Yu, Y.; Liu, G.; Hu, J.; Zhang, G.; Liu, S. Engineering Intracellular Delivery Nanocarriers and Nanoreactors from Oxidation-Responsive Polymersomes via


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