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Enhanced Intracellular Ca2+ Nanogenerator for Tumor Specific Synergistic Therapy via Disruption of Mitochondrial Ca2+ Homeostasis and Photothermal Therapy Lihua Xu, Guihua Tong, Qiaoli Song, Chunyu Zhu, Hongling Zhang, Jinjin Shi, and Zhenzhong Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02034 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Figure 1. (A) Schematic illustration of the synthesis processes of ECaNG. Characterization of the nanoplatform. (B) TEM pictures of HMCuS, CaNG and ECaNG. (C) Ultravoilet-visible absorption spectrum of the nanoplatform. (D) Dynamic light scattering analysis of CaNG. (E) XPS characterization of CaNG. (F) TEM mapping of CaNG. 228x228mm (300 x 300 DPI)

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Figure 2. Specific Ca2+ and CUR release. (A) Pictures of centrifugated CaNG at different pH for 1 h. (B) TEM images of CaNG at pH7.4, pH 6.5 and pH 5.0. Scale ruler: 200 nm. (C) The effect of pH, NIR and disintegration time on release of Ca2+ from CaNG. (n=3) (D) Release profile of CUR from ECaNG at different pH. (n=3) 141x79mm (300 x 300 DPI)


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Figure 3. Thermal images of CaNG at different pH with irradiation by 808nm laser (2W/cm2) for different time. 89x110mm (300 x 300 DPI)


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Figure 4. Ca2+ production by DECaNG. CLSM images and coincubation ratio analysis of biodistribution of (A) CUR and (B) ECaNG in lysosomes for different time treatment to MCF-7 cells. Scale bar: 10 µm. (C) CLSM images and mean fluorescence intensity analysis of Intracellular Ca2+ production after treatment with CaNG and CaNG+NIR for 6 h. NIR was used at 4 h of incubation for 1 min. Scale bar: 7.5 µm. (n=6) 239x268mm (300 x 300 DPI)


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Figure 5. (A) CLSM images and Colocation ratio analysis of biodistribution of Ca2+ released from CaNG in mitochondria after treatment with CaNG in MCF-7 cells for different time. Scale bar: 7.5 µm. Mechanisms study of cell apoptosis. (n=3) (B) Mitochondria membrane potential changes of MCF-7 cells treated with different preparations by fluorescence microscope. Scale bar: 100 µm. (C) Western blot assays of intracellular Caspase-3, Bcl-2 and Cyto C. (D) Intracellular ATP content analysis. (n=3) 199x175mm (300 x 300 DPI)


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Figure 6. Cell inhibition rate of different treatment on MCF-7 cells for 24 h. (n=6) 86x65mm (300 x 300 DPI)


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Figure 7. (A) In vivo optical images of tumor bearing mice at different times after injection with IR783 and IR783/CaNG in vein. (B) Thermal images of mice treated by DECaNG for different time. NIR: 808 nm laser with power density of 2 W/cm2. 99x52mm (300 x 300 DPI)


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Figure 8. (A) Body weight changes of different groups treated. (n=5) (B) Relative tumor volume changes of tumor bearing mice with different treatment. (n=5) (C) Ca2+ content in tumor tissues after treatment with DECaNG. (n=3) (E) H&E staining and TUNEL staining of tumor tissues exfoliated from different groups. Scale bar: 25µm in H&E staining and 100 µm in TUNEL staining. 146x85mm (300 x 300 DPI)


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Figure 9. ICP-MS analysis of Cu amounts in the organs of mice collected at 8, 24 and 48 h after injection of HMCuS and CaNG. ID/g: injected dosage per gram tissues. (n=3) 116x54mm (300 x 300 DPI)


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Enhanced Intracellular Ca2+ Nanogenerator for Tumor Specific Synergistic Therapy via Disruption of Mitochondrial Ca2+ Homeostasis and Photothermal Therapy Lihua Xu a,b,c,1, Guihua Tong a,1, Qiaoli Song a, Chunyu Zhu a, Hongling Zhang a,b,c, , Jinjin Shi a,b,c,*, Zhenzhong Zhang a,b,c,* a School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, PR.China b Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province, PR. China c Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou, PR. China *Corresponding authors: Prof. Zhenzhong Zhang and Dr. Jinjin Shi. Email: [email protected], [email protected].

ABSTRACT: Breast cancer therapy has always been a hard but urgent issue. Disruption of mitochondrial Ca2+ homeostasis has been reported as an effective antitumor strategy, while how to contribute to mitochondrial Ca2+ overload effectively is a critical issue. To solve this issue, we designed and engineered a dual enhanced Ca2+ nanogenerator (DECaNG), which can induce elevation of intracellular Ca2+ through the following 3 ways. Calcium phosphate (CaP) doped hollow mesoporous copper sulfide was the basic Ca2+ nanogenerator to generate Ca2+ directly and persistently in the lysosomes (low pH). Near-infrared light radiation (NIR, such as 808nm laser) can accelerate Ca2+ generation from the basic Ca2+ nanogenerator by disturbing the crystal lattice of hollow mesporous copper sulfide via NIR induced heat. Curcumin can facilitate Ca2+ release from endoplasmic reticulum to cytoplasm and inhibit expelling of Ca2+ in cytoplasm through cytoplasmic membrane. The in vitro study showed that DECaNG could produce a large amount of Ca2+ directly and persistently to flow to mitochondria, leading to upregulation of Caspase-3, cytochrome C and downregulation of Bcl-2 and ATP followed by cell apoptosis. In addition, DECaNG owned outstanding photothermal effect. Interestingly, it was found that DECaNG exerted stronger photothermal effect at lower pH due to the super small nanoparticles effect, thus enhancing photothermal therapy. In the in vivo study, the nanoplatform had good tumor targeting and treatment efficacy via combination of disruption of mitochondrial Ca2+ homeostasis and photothermal therapy. The metabolism of CaNG was sped up through disintegration of CaNG into smaller nanoparticles, reducing the retention time of the nanoplatform in vivo. Therefore, DECaNG can be a promising drug delivery system for breast cancer therapy. KEYWORDS: Ca2+ nanogenerator; mitochondrial Ca2+ homeostasis; tumor specific synergistic therapy; enhanced photothermal effect.


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Breast cancer is one of the most severe diseases with high incidence rate. There are 1.5–1.7 million new diagnoses of breast cancer worldwide each year.1 Breast cancer therapy has been an urgent worldwide issue to be solved due to low treatment efficiency and severe adverse effects of chemotherapeutics, such as cardiotoxity of doxorubicin. As is known to all, mitochondria are a primary factory to supply energy to cells to keep working and it is a critical tie among many signal pathways, involved in cell apoptosis, calcium homeostasis, lipids and amino acids metabolism and so on.2-5 Therefore, persistent mitochondrial dysfunction has been an effective method to induce cell death. Many drug delivery systems have been constructed for tumor treatment through mitochondria apoptotic pathway.6-8 During treatment of tumor cells, some of signal pathways are disturbed, followed by damage of outer mitochondrial membrane and decrease of mitochondrial membrane potential, which can activate caspase dependent cell apoptosis.9-11 In addition, once mitochondrial dysfunction constantly occurs, cells may lead to death due to the deficiency of ATP.12 As a promising approach to make mitochondrial dysfunction, mitochondrial Ca2+ overload has been gotten more attention due to the antitumor effect of Ca2+.13, 14 When temporary imbalance of intracellular Ca2+ occurs, mitochondria as sensors and regulators will take in Ca2+ from cytoplasm to regulate intracellular calcium homeostasis.15 However, excessive Ca2+ in mitochondria triggers cell apoptosis.16 It has been reported that compared with normal cells, intracellular Ca2+ homeostasis is altered because of remodeling of Ca2+ transport in tumor cells.17 Alteration of Ca2+ signal pathways in tumor cells leads to different effects of antitumor drugs. For example, curcumin (CUR) has been widely used for cancer therapy.18-20 In tumor cells, CUR can persistently facilitate Ca2+ release from endoplasmic reticulum to cytoplasm to activate caspase and inhibit expelling of Ca2+ in cytoplasm through cytoplasmic membrane, while it is ineffective in normal cells, indicating that tumor cells are more sensitive to the persistent Ca2+ overload than normal cells.21-24 Therefore, overloading of mitochondrial Ca2+ has been a promising approach to kill tumor. Recently, many researchers have been focusing on how to induce influx of Ca2+ into cells and facilitate release of Ca2+ from endoplasmic reticulum to cytoplasm especially to mitochondria by external stimulus such as receptors and cytotoxicity agents.21, 25, 26 However, application of these methods was limited due to protective mechanism of cells via Ca2+ excretion. So it is needed to seek for an effective method to generate Ca2+ directly in the cells. To date, there is few multifunctional drug delivery system that can lead to persistent enrichment of excessive Ca2+ in mitochondria via both nanocarriers and drugs in tumor tissues for tumor therapy. As a kind of nanocarriers for Ca2+ release, calcium phosphate (CaP) is a good choice. Due to its good biocompatibility, bioactivity and biodegradability, CaP has been used for bone tissue engineering and tumor treatment.27-30


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More importantly, CaP nanoparticles can release abundant Ca2+ at acid pH environment.31 In spite of above advantages of CaP, strong hydrophobicity and low drug loading efficiency limit its application in cancer therapy. Moreover, monotherapy of excessive mitochondrial Ca2+ has not completely met the needs of tumor therapy. Recently, many kinds of synergistic therapy have been used and achieved great antitumor effect. For example, the platform that Yang et al. designed had high in vivo and in vitro antitumor efficiency by combination of photothermal therapy and photodynamic therapy.32 Considering above problems of CaP, copper sulfide (CuS) can be used to hybridize with CaP. Firstly, hollow mesoporous CuS (HMCuS) own good hydrophilcity and high drug loading capacity.33 Secondly, under near-infrared light radiation (NIR, such as 808nm laser), CuS nanoparticles owned excellent photothermal effect, showing promise for photothermal therapy (PTT) of tumor.34 In addition, near-infrared light radiation can disturb the crystal lattice of HMCuS and facilitate disintegradation of CaP and release elevation of Ca2+ by photothermal effect.35 All these advantages made it potential for tumor therapy of HMCuS. Herein, a dual enhanced intracellular Ca2+ nanogenerator (DECaNG) was constructed in this work, which was involved in nanocarriers, CUR and NIR. Firstly, the system could produce Ca2+ in tumor cells effectively and sensitively. As a kind of Ca2+ nanogenerator, CaP doped HMCuS could escape from lysosomes through consuming lysosomal H+, meanwhile being disintegrated and releasing Ca2+; CUR could induce endoplasmic reticulum Ca2+ release; Utilization of NIR could accelerate disintegradation of nanocarriers and Ca2+ release. In addition, the system would own excellent tumor specificity due to pH ultrasensitivity of CaNG. CaNG in tumor sites could be disintegrated and release primary Ca2+. Moreover, the system would have prominent photothermal effect, which could be enhanced at tumor acid environment. Combination of photothermal therapy and imbalance of mitochondrial Ca2+ homeostasis could treat tumor synergistically. The above viewpoints were verified in MCF-7 cells and MCF-7 cells bearing nude mice. RESULTS AND DISCUSSION Synthesis and Characterization of ECaNG In the present work, CUR loaded CaNG was prepared as enhanced Ca2+ nanogenerator (ECaNG), according to a gentle coprecipitation method based on the preparation of HMCuS (Figure 1A). In brief, Cu2O nanoparticles were formed with CuCl2.2H2O as Cu sources, NaOH as pH regulator, hydrazine hydrate (N2H4.H2O) as reductant and PVP as crosslinker. As sources of CaP, CaCl2 and Na2HPO4 were added quickly when Cu2O nanoparticles were being formed followed by sulfuration of Cu2O with Na2S to obtain CaNG. Subsequently, CUR was loaded into the interior of CaNG by electrostatic absorption. At last, poloxamer F68 was capped onto CaNG as a biocompatible reagent and gatekeeper. Thus ECaNG was constructed.


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To characterize the successful synthesis of ECaNG, transmission electron microscopy (TEM) and ultraviolet -visible absorption spectrum were conducted. It can be seen from Figure 1B that there was obviously hollow and mesoporous in HMCuS with the diameter of about 100 nm, while CaNG was about 150 nm with more obviously hollow structure and thinner shell. There were some morphologically inhomogeneous agglomerates in the hollow structure of ECaNG compared with CaNG, indicating that CUR was successfully loaded into CaNG. As shown in Figure 1C, ECaNG had not only the characteristic peak of CUR at 429 nm, but a broad peak in near-infrared region (NIR, λ= 700–1000 nm) of HMCuS as reported,36 confirming the successful synthesis of ECaNG. It has been reported that CUR can induce elevation of intracellular Ca2+,21 so successful loading of CUR might be an enhanced approach to intracellular Ca2+ generation. Photothermal effect of HMCuS was attributed to strong absorbance in NIR and surface plasmon resonance (SPR) effect, indicating that CaNG had similar ability for photothermal therapy in spite of doping of CaP. Additionally, dynamic light scattering analysis (Figure 1D) showed that the size of CaNG was 164±5 nm with 0.047 of polydispersity index (PDI), in accordance with TEM results. Atomic Force Microscopy (AFM) image of CaNG also confirmed the spherical structure and size distribution, and the zeta potential of CaNG was -22±0.6 mV (Figure S1A,B in the supporting information). To further verify the components and structure, X-ray photoelectron spectroscopy (XPS) and TEM mapping were used. XPS results revealed that CaNG contained five elements (Cu, S, Ca, P, and O) and TEM mapping visualization showed that above five elements were almost distributed into the shell of CaNG, indicating that CaP was successfully doped into the shell of CaNG (Figure 1E,F). Valence state of various elements in CaNG was demonstrated in Figure S1C. The peaks in Ca 2p appeared at the binding energies of 350.58 eV and 346.98 eV were attributed Ca 2p1/2 and Ca 2p3/2, and the peaks in P 2p appeared at the binding energies of 133.48 eV and 132.58 eV were assigned to P 2p1/2 and P 2p3/2, indicating existence of CaP. For Cu and S elements, Cu 2p1/2, Cu 2p3/2 and S-Cu were located at the binding energies of 951.78 eV, 931.78 eV and 161.78 eV, indicating existence of CuS. To further analysis phase structure of CaNG, XRD was conducted. As was shown in Figure S1D, a diffraction pattern of the CuS nanostructures were consistent with the hexagonal CuS (JCPDS card 06–0464). The main diffractions peaks at 27.2°, 28.9°, 31.2°, 47.9°, and 52.8° were assigned respectively to the (101), (102), (113), (110), and (108) planes. In addition, the diffraction peaks of CaP were identical with calcium phosphate (JCPDS card 16-0728). Diffraction peaks located at 31°and 45.6° were assigned to CaP. It indicated that CaNG was consist of CaP and CuS. Doping of CaP into the shell revealed that CaP was interacted with CuS for forming of shell of CaNG. Near-infrared laser irradiation can disturb the crystal lattice of CuS, facilitating disintegration of CaP into Ca2+. Nitrogen adsorption isotherm of CaNG was studied as depicted in Fig S2A and exhibited a type of V


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iso-therm, one of the typical curves for mesoporous nanoparticles. A considerably large surface area up to 123.1 m2/g was calculated, which endowed CaNG high drug loading efficacy. As seen in Fig S2B, when weight ratio of CaNG and CUR was 1:2, drug loading efficacy could reach up to 88%, confirming the high drug loading efficacy of CaNG. Specific Ca2+ and CUR release at acidic environments Because of successful doping of CaP into the shell of HMCuS, ECaNG was supposed to disintegrate and release Ca2+ and CUR at low pH environment. Firstly, we studied collapse of CaNG at different pH in 1 h. As seen in Figure 2A, after centrifugation, supernatant color was deepened and precipitation amount was reduced with decrease of solution pH from 7.4 to 5.0. To visualize the morphology changes of CaNG after treatment with different pH solutions, the three samples were characterized by TEM (Figure 2B). At pH7.4, all nanoparticles remained stable, while nanoparticles were partly destroyed at pH6.5. However, almost all nanoparticles were disintegrated into smaller nanoparticles in the whole field at pH5.0. The results of TEM confirmed the collapse of CaNG at lower pH gradually and complete Ca2+ release after interaction for 1 h, indicating persistent Ca2+ and CUR release. As we all know, tumor environment is acidic at pH6.5, while lysosomes are at pH5.0. Considering disintegration conditions and tumor environment, we thought that Ca2+ release was tumor specific because CaNG was only disintegrated at acidic environment followed by release of Ca2+ and CUR. To further evaluate influence factors and efficiency of Ca2+ release from CaNG after collapse, CaNG was dispensed into solutions at different pH and interacted for different time. NIR (808 nm laser, 2 W/cm2, 1 min) was also used. Concentration of Ca2+ in the supernatant was measured by ICP-MS (Figure 2C). It can be seen that after interaction for 30 min, concentration of Ca2+ rose from 3.98 to 202 µg/mL with decrease of solution pH, suggesting that Ca2+ generation was effective and pH sensitive, consistent with the results of CaNG collapse. In addition, after irradiation, concentration of Ca2+ further rose by 40 µg/mL. This might be attributed to disturbance of the crystal lattice of HMCuS and structural instability by NIR induced photothermal effect.37, 38 Therefore, NIR could accelerate Ca2+ generation. What is more, after 1 h of disintegration, concentration of Ca2+ reached to 285 µg/mL at pH5.0, almost 80% of Ca element in CaNG (Figure S3A), which was obviously higher than that at pH5.0 for disintegration of 30 min. The results revealed that Ca2+ could be persistently released from CaNG until complete disintegration of CaNG. All above results demonstrated that Ca2+ generation was tumor specific, effective, persistent and NIR enhanced. With disintegration of CaNG at lower pH, CUR release was supposed to be pH sensitive. In figure 2D, CUR was only released less than 20% after 12 h in pH7.4, indicating that poloxamer F68 was a good gatekeeper. In contrast, within 1 h, the cumulative release rate of CUR at pH5.0 and pH6.5 exceeded 60% and 35%, respectively. Therefore, ECaNG could also release abundant


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CUR persistently like Ca2+ to induce more intracellular Ca2+ generation during disintegration of ECaNG. Tumor-acid specific enhanced PTT and PTT induced acceleration of Ca2+ generation It has been reported that HMCuS has outstanding photothermal effect due to its strong absorption at near infrared region (808 nm), in which HMCuS can convert energy from light to heat effectively. Because of similar absorption at NIR region to HMCuS, CaNG was supposed to own excellent light-thermal conversion property. As displayed in Figure 3, temperature of solution at pH7.4 was increased by 10.8℃ in one minute. With the increase of irradiation time, solution temperature was elevated. After 5 min，the temperature of solution was increased to 61.8℃ at pH7.4. This confirmed excellent photothermal effect of CaNG, in accordance with the results of utravoilet-visible absorption spectrum. Due to tumor acidic environment, we further evaluated photothermal effect of CaNG at pH6.5 and pH5.0. It can be seen that, temperature elevation of solution at lower pH was irradiation time dependent as well. More importantly, ascending temperature speed of solution apparently increased with the decrease of solution pH. It may be attributed to the disintegration of CaNG into smaller CuS nanoparticles, which had stronger photothermal effect than bigger nanoparticles.39 So, collapse of CaNG at lower pH enhanced thermal therapy. In addition, molar extinction coefficient of CaNG was measured. As shown in Figure S3B, with increasing the concentration of CaNG, the corresponding absorbance was linearly enhanced. The molar extinction coefficient of CaNG గ

at 808 nm was calculated by using equ: ε = (A ݀ଷ ߩܰ஺) /(‫ܥܮ‬௪௧ ) , where A, d, ଺ ρ, NA, L and Cwt are the absorbance at 808 nm, the average diameter of nanospheres, the density of nanospheres, Avogadro’s constant, the pathlength through the sample and the weight concentration of the nanospheres, respectively. After calculation, molar extinction coefficient of CaNG was ca. 2.3 × 109 cm−1 M−1 at 808 nm. To further evaluate photothermal performance, we investigated photothermal effect of pure water and CaNG with different concentrations under 808 nm laser irradiation, as well as photostability of CaNG. According to Figure S3C, temperature of samples increased with increased concentration of CaNG with continuous irradiation of 808 nm laser, and the temperature of pure water increased weakly in 3 min. What is more, temperature elevation rate of CaNG samples was a little faster than that in the first 3 min when exposed to 808 nm laser again, which was attributed to partial disintegration induced by laser irradiation. These phenomena indicated that photothermal conversion of CaNG was concentration dependent and photostable. As mentioned in Figure 2C, NIR could accelerate Ca2+ generation. This showed that there was a mutual promoting relationship between thermal therapy and Ca2+ generation, which provided evidences for tumor synergistic


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treatment of DECaNG. Ca2+ generation by DECaNG in cells As mentioned before, Ca2+ generation was performed at lower pH (such as lysosomal environment). So we wondered if ECaNG was distributed in lysosomes. In the experiment, biodistribution of ECaNG and CUR in lysosomes were observed by confocal laser scanning microscope (CLSM) first. CUR with green fluorescence was used to indicate the intracellular behavior of CUR in ECaNG, while LysoRed probe with red fluorescence was used for indication of lysosomes and Hoechst33342 with blue fluorescence for cellular nucleus. As shown in the Figure 4A, when cells were treated with free CUR for different time, the colocation rate of CUR and lysosomes was always high until CUR entered into cell nucleus, indicating that CUR had no ability to escape from lysosomes. However, it can be seen from Figure 4B that ECaNG just partly entered into lysosomes after 2 h, and the colocation rate was less than 40%. With increase of incubation time, the colocation rate could reach to 85%. But at 6 h, it was found that CUR was partly in the cell nucleus and the colocation rate decreased to 45%. The results revealed that ECaNG could escape from lysosomes. As confirmed before, ECaNG could be disintegrated at low pH. Hence ECaNG might consume H+ in lysosomes, leading to lysosomal rupture and release of CUR and Ca2+. Due to collapse of ECaNG in lysosomes, abundant Ca2+ was supposed to be released from ECaNG. It has been reported that CUR can induce elevation of intracellular Ca2+ concentration, so we just explored the effect of CaNG and NIR on intracellular Ca2+ content in this work. Fluo-3 AM probe was used as intracellular Ca2+ indicator. CLSM was used to observe green fluorescence of Ca2+ indicator. As depicted in Figure 4C, compared with the control group, intracellular Ca2+ fluorescence intensity increased from 40 to 90 when cells were treated with CaNG and from 40 to 130 with CaNG+NIR, consistent with the the results of CaNG at pH5.0 in Figure 2C. This confirmed abundant Ca2+ generation of DECaNG in cells. Disruption of mitochondrial Ca2+ homeostasis by DECaNG Given that collapse of ECaNG and abundant Ca2+ generation of DECaNG, we speculated that elevated Ca2+ might flow into mitochondria, disrupting mitochondrial Ca2+ homeostasis. Biodistribution of Ca2+ in mitochondria was observed by CLSM and colocation rate was analyzed (Figure 5A). In the assay, MitoRed probe with red fluorescence was used as a mitochondria indicator, and Hoechst33342 with blue fluorescence as a cellular nucleus indicator. Distribution of intracellular Ca2+ was marked by Fluo-3 AM probe. After 2 h of coincubation, colocation rate of Ca2+ and mitochondria was only about 30%. According to CLSM images of ECaNG distribution in lysosomes (Figure 4B), at this moment, only part of CaNG was in lysosomes and being broken up. So less Ca2+ was released and flowed into mitochondria. However, the colocation rate raised to 80% after incubation for 6 h when ECaNG had been disintegrated and released plenty of Ca2+. All these results revealed that


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abundant Ca2+ produced by DECaNG flowed into mitochondria effectively, most probably disrupting mitochondria Ca2+ homeostasis. Abundant Ca2+ generation and flowing into mitochondria observed in this work encouraged us to study disruption of mitochondrial Ca2+ homeostasis associated cell apoptosis mechanism by DECaNG. The change of mitochondrial membrane potential (MMP) was measured through JC-1 staining. With the high mitochondrial membrane potential, JC-1 probes are supposed to be enriched in mitochondrial matrix, forming J-aggregates emitting red fluorescence. And when the mitochondrial membrane potential is low, JC-1 probes exist almost in monomer emitting green fluorescence. Thus, it is very convenient to detect the change of mitochondrial membrane potential by measuring relative proportions of green and red fluorescence intensity by fluorescence microscope. As shown in Figure 5B, in the control group, cells almost presented red fluorescence, indicating relatively high MMP. When cells were incubated with CaNG and CUR, green fluorescence signal in cells increased. However, in the ECaNG group, cells owned more green fluorescence due to combination of CUR and CaNG. With NIR irradiation, cells owned most green fluorescence, suggesting that DECaNG contributed to enormous decrease of mitochondrial membrane potential in cells with triple effect of CaNG, CUR and NIR. Subsequently, we explored the effect of DECaNG on Caspase-3, Cytochrome C and Bcl-2 by western blot. It is well-known that most apoptosis pathways are initiated by Caspase-3 activation.40, 41 During cell apoptosis, Cytochrome C will be released from mitochondria to cytoplasm, and Bcl-2 protein acting as an inhibitor of apoptosis will decrease.42 As shown in Figure 5C, after treatment with different formulations, Caspase-3 in cells was activated in all the experimental groups, most obvious in the DECaNG group. In addition, Figure 5C showed that after treatment with different formulations, the amount of cytosol cytochrome C increased, and anti-apoptotic protein Bcl-2 was down-regulated, indicating the opening of mitochondria mediated apoptosis pathway. With the structural damage and dysfunction of mitochondrial, ATP content might decrease in cells. It can be seen that in Figure 5D, ATP content decreased in the experimental groups to various degrees, compared with the control group. Especially in the DECaNG group, the relative ATP content decreased substantially, reaching to 15%, which revealed that DECaNG sharply cut off the energy supply to cells. Given that ATP content decreased significantly when tumor cells were treated by DECaNG, we deemed that abundant Ca2+ would also enhance PTT by downregulating HSP family. To verify our assumption, western blot analysis was conducted to investigate expression of HSP70 and HSP90. It can be seen in Figure S4 that under irradiation, expression of intracellular HSP70 and HSP90 significantly increased in the HMCuS group. However, in the CaNG group, the expression of HSP70 and HSP90 was downregulated, which was


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similar with that in the control group. From the results, we speculate that Ca2+ would contribute to downregulation of HSP family, which could further enhance PTT. Cytotoxicity induced by disruption of mitochondrial Ca2+ homeostasis Due to mitochondrial Ca2+ overload induced by DECaNG and its prominent photothermal effect, the nanoplatform had the promising potential for breast cancer therapy. To explore cytotoxicity of DECaNG, CCK-8 assay was performed to assess the lethality of DECaNG at different amount of CUR. In Figure 6, cell viability rate decreased with the increase of CUR amount embedded into CaNG. CaNG had similar antitumor ability with CUR. Compared with CUR at the same concentration, ECaNG contributed to more dead cells due to Ca2+ release induced by CUR. Besides Ca2+ production by CUR and CaNG, laser irradiation intensified cell death by heat production and Ca2+ elevation. For example, with 10 µg/mL of CUR, the CaNG group had about 25% dead cells, while the ECaNG group and DECaNG group had 40% and 60%, respectively. Nevertheless there was no significant difference between the CaNG group and ECaNG with the concentration of CUR increased from 1 µg/mL to 5 µg/mL, demonstrating that low Ca2+ played insignificant role in cell death. Furthermore, there was significant difference among the three groups respectively when the concentration of CUR varied from 5 µg/mL to 40 µg/mL, indicating synergistic of chemotherapy, thermotherapy and imbalance of mitochondrial Ca2+ homeostasis was effective for tumor treatment. Due to difference of Ca2+ signal pathways between tumor cells and normal cells, cytotoxicity of CaNG to HS578Bst cells was also tested. As shown in Figure S5, survival rate of HS578Bst cells treated with different concentration of CaNG (0.1 to 50 µg/mL) was always over 80%, indicating specific cytotoxicity of CaNG to tumor. In order to investigate the biodistribution behavior of CaNG, a near-infrared dye (IR783) was used to mark CaNG. As shown in Figure 7A, in the free IR783 group, after injection of IR783 for 1 h, the fluorescence signal was mainly in liver while the partial signal was in tumor and bladder at 3 h, indicating that IR783 was cleared by the body after injection for 3 h. After injection for 48 h, there was few fluorescence signal in the mice. Meanwhile, in the IR783/CaNG group, after injection for 1 h, the signal of IR783/CaNG was weaker compared with free IR783, while fluorescence signal occurred in tumor site, and increased from 3 to 8 h after injection. Compared fluorescence intensity at 8 h, the signal of IR783/CaNG in tumor at 36 and 48 h decreased, but still had a high level, while there was few fluorescence signal in normal tissues. The biodistribution of IR783/CaNG revealed that CaNG could significantly accumulate in the tumor site via enhanced penetration and retention effect, suggesting CaNG had a great potential in drug delivery for tumor targeting therapy. After 8 h of injection, more fluorescence signal of IR783/CaNG was observed in tumor than other time points, so in the following in vivo antitumor activity studies, the NIR laser irradiation time was set at 8 h


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after injection. The high distribution of CaNG in tumor and low distribution in normal tissues endowed CaNG a great advantage for antitumor drug delivery. To ensure the exposure time of NIR, photothermal effect of DECaNG in vivo was investigated. As seen from Figure 7B, temperature of tumor tissues treated by DECaNG increased by 17.7℃ in 30 s, compared with the control group of 2.1℃, indicating great photothermal effect of ECaNG. However, with continuous irradiation after 30s, temperature of brain and peripheral tissues of tumor increased besides tumor tissues, which was beyond tolerance of mice. Given that photothermal effect of ECaNG and tolerance of mice, we chose 30 s as the optimal exposure time of NIR to mice. Antitumor effect of DECaNG To investigate in vivo treatment efﬁcacy of DECaNG, comparative efﬁcacy studies were conducted. The MCF-7 tumor-bearing mice were divided into 5 groups and were treated with different formulations. Body weight changes and relative tumor volume as a function of time were plotted in Fig 8A and B. After treatment for 2 weeks, body weight of all the mice was slightly higher, indicating no significant toxicity to survival (Fig 8A). As depicted in Fig 8B, compared with the control group, CaNG and CUR showed certain therapeutic effect, resulting in the relative tumor volume (V/V0) of 3.68±0.45 (**p

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