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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*,†,‡,§ †
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, People's Republic of China Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province, People's Republic of China § Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou, China ‡
S Supporting Information *
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 Ca 2+ nanogenerator (DECaNG), which can induce elevation of intracellular Ca2+ through the following three 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 808 nm laser) can accelerate Ca2+ generation from the basic Ca2+ nanogenerator by disturbing the crystal lattice of hollow mesoporous copper sulfide via NIR-induced heat. Curcumin can facilitate Ca2+ release from the endoplasmic reticulum to cytoplasm and inhibit expelling of Ca2+ in cytoplasm through the 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 had an outstanding photothermal effect. Interestingly, it was found that DECaNG exerted a 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 a 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 treatment through the 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
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reast cancer is one of the most severe diseases with a 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 © 2018 American Chemical Society
Received: March 18, 2018 Accepted: July 2, 2018 Published: July 2, 2018 6806
DOI: 10.1021/acsnano.8b02034 ACS Nano 2018, 12, 6806−6818
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Cite This: ACS Nano 2018, 12, 6806−6818
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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.
overloading of mitochondrial Ca2+ has been a promising approach to kill tumors. 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 stimuli such as receptors and cytotoxicity agents.21,25,26 However, application of these methods was limited due to the protective mechanism of cells via Ca2+ excretion. So an effective method to generate Ca2+ directly in the cells is needed. To date, there are few multifunctional drug delivery systems 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 nanocarrier 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 More impor-
As a promising approach to cause mitochondrial dysfunction, mitochondrial Ca2+ overload has 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 the 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 the 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, 6807
DOI: 10.1021/acsnano.8b02034 ACS Nano 2018, 12, 6806−6818
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ACS Nano
Figure 2. Specific Ca2+ and CUR release. (A) Pictures of centrifugated CaNG at different pHs for 1 h. (B) TEM images of CaNG at pH 7.4, 6.5, and 5.0. Scale bar: 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).
tantly, CaP nanoparticles can release abundant Ca2+ in an acid pH environment.31 In spite of the 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 a combination of photothermal therapy and photodynamic therapy.32 Considering the above problems of CaP, copper sulfide (CuS) can be used to hybridize with CaP. First, hollow mesoporous CuS (HMCuS) has a good hydrophilicity and high drug loading capacity.33 Second, under near-infrared light radiation (NIR, such as 808 nm laser), CuS nanoparticles had an excellent photothermal effect, showing promise for photothermal therapy (PTT) of tumors.34 In addition, NIR can disturb the crystal lattice of HMCuS and facilitate disintegradation of CaP and release Ca2+ by photothermal effect, elevating Ca2+ levels.35 All of these advantages make it a 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. First, 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, and utilization of NIR could accelerate disintegration of nanocarriers and Ca2+ release. In addition, the system would have 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 a prominent photothermal effect, which could be enhanced in a tumor acid environment. A 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 cross-linker. 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. To characterize the successful synthesis of ECaNG, transmission electron microscopy (TEM) and ultraviolet− visible (UV−vis) absorption spectrum were conducted. It can be seen from Figure 1B that it was obviously hollow and mesoporous in HMCuS with the diameter of about 100 nm, while CaNG was about 150 nm with a more obvious 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 has not only the characteristic peak of CUR at 429 nm but also a broad peak in the 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 a 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 a 0.047 of polydispersity index (PDI), in accordance with TEM results. Atomic force microscopy (AFM) image of CaNG also 6808
DOI: 10.1021/acsnano.8b02034 ACS Nano 2018, 12, 6806−6818
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ACS Nano
solutions at different pHs 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 pH 5.0, almost 80% of the Ca element in CaNG (Figure S3A), which was obviously higher than that at pH 5.0 for disintegration of 30 min. The results revealed that Ca2+ could be persistently released from CaNG until complete disintegration of CaNG. All of the 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
