Mitochondria and Nuclei Dual-Targeted Hollow Carbon Nanospheres

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Mitochondria and nuclei dual-targeted hollow carbon nanospheres for cancer chemo-photodynamic synergistic therapy Ruizhi Xie, Shu Lian, Huayi Peng, Changhe OuYang, Shuhui Li, Yusheng Lu, Xuning Cao, Chen Zhang, Jianhua Xu, and Lee Jia Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00259 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Molecular Pharmaceutics

80x60mm (200 x 200 DPI)

ACS Paragon Plus Environment

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

Mitochondria and nuclei dual-targeted hollow carbon nanospheres for cancer chemo-photodynamic synergistic therapy

Ruizhi Xie#1, Shu Lian#1, Huayi Peng#2, Changhe OuYang1, Shuhui Li1, Yusheng Lu1,3, Xuning Cao4, Chen Zhang3, Jianhua Xu2, Lee Jia1,3*

1Cancer

Metastasis Alert and Prevention Center, College of Chemistry; Fujian

Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou, Fujian 350116, China. 2College

of Pharmacy, Fujian Medical University. Fuzhou 350116, China

3Institute

of Oceanography, Minjiang University, Fuzhou, Fujian 350108,

China. 4State

Key Laboratory of Photocatalysis on Energy and Environment, College

of Chemistry, Fuzhou University Fuzhou 350002, China.

Correspondence should be addressed to L.J. ([email protected]; [email protected]) # These

authors contributed equally to this work.

1


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Abstract Dual-targeted nanoparticles are gaining increasing importance as a more effective anticancer strategy by attacking double key sites of tumor cells, especially in chemo-photodynamic therapy. To retain the nuclei inhibition effect and

enhance

DOX-induced

apoptosis

by

mitochondrial

pathways

simultaneously, we synthesized the novel nanocarrier (HKH) based on hollow carbon nitride nanosphere (HCNS) modified with hyaluronic acid (HA) and the mitochondrial localizing peptide

D[KLAKLAK]2

(KLA). DOX-loaded HKH

nanoparticles (HKHD) showed satisfactory drug loading efficiency, excellent solubility and very low hemolytic effect. HA/CD44 binding and electrostatic attraction between positively-charged KLA and A549 cells facilitated HKHD uptake via endocytosis mechanism. Acidic microenvironment, hyaluronidase and KLA-targeting altogether facilitate doxorubicin toward mitochondria and nuclei, resulting in apoptosis, DNA intercalating, cell-cycle arrested at S-phase and light-induced ROS production. Intravascular HKHD inhibited tumor growth in A549-implanted mice with good safety. The present study, for the first time, systemically reveals biostability, targetability, chemophotodynamics and safety of the functionalized novel HKHD. Keywords: Graphitic hollow carbon nitride nanosphere, Doxorubicin, Dualtargeted nanoparticles, Mitochondria, Photodynamic therapy

2



1. Introduction The combination of chemotherapy and photodynamic therapy has emerged as a promising strategy for cancer therapy due to its synergistic effects.1 Doxorubicin (DOX) is a broad-spectrum chemotherapy drug to treat tumors, but its limitations include low selectivity, systemic side effects, and drug resistance.2-4 Another therapeutic modality, photodynamic therapy (PDT) that utilizes photosensitizers (PSs) to produce cytotoxic singlet oxygen under irradiation has been widely integrated with fluorescence imaging for cancer therapeutics.5-6 However, clinical applications of PDT have been hindered by limited aqueous solubility and tumor specificity of PSs, and PDT has failed to treat all types of cancer.7-8 Theoretically, if DOX and PSs can be accurately delivered to the tumor cells, its therapeutic effect will be greatly improved.9-10 Furthermore, beyond conventional tumor targeting, a number of smart PDT agents and chemotherapy drugs have been designed to enable targeting of specific cell organelles such as mitochondria or nuclei that are vulnerable to reactive oxygen species (ROS), so as to further optimize the efficacy of PDT.7, 11

Nucleus is the most important cellular organelle, containing genomic materials (nuclear DNA, RNA, and chromosomes) and regulates important functions like transcription, cell cycle and cell division in healthy cells as well as in cancer cells.12-16 Mitochondrion also holds particularly great promise as a target, as it plays an essential role in supplying energy for cells and regulating cell apoptosis.17-22 Free doxorubicin (DOX) enters the nuclei of tumor cells rapidly without tarrying in the mitochondria, which weakens the potential of DOX effects on mitochondrial pathway.23 If DOX accumulates in mitochondria, it can increase the formation of ROS and damage of mitochondrial respiratory chain components, induce lipid peroxidation of mitochondrial membrane, and finally result in the shedding of cytochrome c from mitochondria,24 and activation of caspase cascade.3, 25-31 Therefore, it is imperative to develop a new drug carrier, 3


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which not only can deliver DOX to the nucleus of tumor cells but also can deliver DOX to mitochondria of tumor cells to enhance antitumor activity of doxorubicin 32-33.

Nanotechnology-based chemotherapies have ability of specifically and safely reaching tumor sites due to their unique physical and biological properties,

resulting

in

higher

efficacy

and

lower

toxicity

than

chemotherapies.34-36 We have developed various nanotechnology-based intelligent pharmaceuticals for targeted drug delivery for cancer metastasis treatment.37-40 As a new nanomaterial hollow g-C3N4 nanospheres (HCNS) has recently emerged owing to its controllable shell thickness as a light-harvesting platform for catalyzing hydrogen evolution under visible light irradiation.41 Due to the absence of metal elements, HCNS may be a highly biocompatible material for biomedical applications.42-43 Carbon nitrides exist in several allotropes with diverse properties, while the graphitic phase is regarded as the most

stable

under

conditions.44

ambient

As

effective

visible-light

photosensitizers for PDT, HCNS possess a uniform hollow structure45 and a porous shell with much enhanced charge collection and separation at the photocatalytic interface, and show a theoretically enhanced ROS generation properties.41,

46-47

The high-degree condensation of the tri-s-triazine ring

structure endows HCNS to be highly photoluminescent, which could enable long-term

in

vivo

particle

tracking

and

in

vitro

fluorescence

cell

labeling/imaging.48-50 D[KLAKLAK]2

(KLA) lysine units could facilitate cellular uptake of fabricated

delivery systems as the lysine interacts with tumor cell membranes via electrostatic

attraction

internalization.51-52

KLA

and

hydrogen

disrupts

the

bonding

to

promote

negatively-charged

cargo

mitochondrial

membrane and mediates cell death resulting from mitochondrial-dependent apoptosis.53 To facilitate the penetration of nanoparticles through the potential barrier of the mitochondrial intermembrane and improve the mitochondrial 4



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targeting efficiency, we select pro-apoptotic peptide KLA, a cation with a delocalized positive charge which can target the negative charge of mitochondria, serving as the targeting moiety.53-54 Highly-hydrophilic hyaluronic acid (HA)19, 23 a water-soluble mucopoly-saccharide and a negatively-charged polysaccharide,52 has excellent biodegradability, biocompatibility, and nonimmunogenicity. Moreover, it can also specifically bind the cluster determinant 44 (CD44) receptor, which is overexpressed on the surface of various tumor cells. HA forms a shell on the surface of nanoparticles after combination with HCN-KLA, and this shell could restrict the release of a loaded drug from the nanocarrier. Owing to HCNS’ unique 3D hollow structure with huge internal and external surface area, in the present study, we tried to explore HCNS pharmaceutical properties by functionalizing the novel HCNS with DOX and two targeting units HA and KLA via covalent bonding to produce HKHD (Scheme 1). We demonstrated

mitochondrion-

and

nucleus-targeting

ability

and

chemophotodynamic mechanisms and safety of HKHD in the in vitro and in vivo settings, and showed the high payload of the hollow HKHD for DOX in comparison with the non-hollow nanospheres. To the best of our knowledge, the system pharmaceutical study and evaluation of the graphitic hollow carbon nitride nanospheres have not been revealed before. 2. Materials and methods 2.1. Materials KLA peptide with a terminal cysteine (D[KLAKLAK]2-Cys) was obtained from Chinapeptides Co.,LTD (Shanghai, China). HA and Doxorubicin hydrochloride were purchased from Dalian Meilun Biotechnology (Dalian, China).

1-Ethyl-3-(3-dimethly-aminopropyl)

hydroxysuccinimide

(NHS)

and

carbodiimide

3-MaleiMidopropionic

(EDC),

N-

acid

N-

hydroxysucciniMide ester (MAL) were acquired from Aladdin. RPMI 1640 medium and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide 5


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(MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mito tracker Red was provided by KeyGEN BioTECH (Jiangsu, China). The rabbit antihuman Caspase-3, Caspase-9, Cytochrome c and β-actin antibodies were procured from Wanleibio Co., Ltd. (Shenyang, China). The secondary antibody goat anti-rabbit IgG horseradish peroxidase conjugate was obtained from Promega Co., Ltd. (Shanghai, China). A 430 nm laser (operating mode, CW; output power after fiber, 50W; LED display) purchased from Epileds (Taiwan, china). All the other reagents used in this study were of analytical grade. 2.2. Synthesis of HCN-KLA-HA 2.2.1. Synthesis of the SiO2 template The solid SiO2 template was synthesized according to a converted Stober method.36, 46, 55 Briefly, 3.50 g of aqueous ammonia (32 wt %), 58.5 g of ethanol, and 10 g of deionised water were added to a flask and stirred for 30 min at 30 °C, then 5.6 mL of TEOS was added under vigorous stirring and kept still for 2 h to obtain solid SiO2 nanoparticles. Next, C18TMOS (2.2 g) and TEOS (5.2 mL) were mixed and added into the reaction mixture under magnetic stirring. The mixed solution was then kept at ambient temperature for 3 h without stirring. The mixture was centrifuged, dried and calcined to obtain the final silica template. 2.2.2 Synthesis of HCNS HCNS was prepared by thermal polymerization according to the previous method.55 1 g of an HCl-treated silica template was mixed with 8 g of cyanamide in a flask. The mixture was kept vacuum for 4 h before subjected to sonication at 55 °C for 4 h. After this, the mixture was stirred at 60 °C overnight. The resultant mixture was centrifuged and dried in air to obtain solid that was transferred to a crucible and heated to 550 °C under flowing N2 for 4 h with a ramp rate of 275 °C h-1. The obtained yellow powder was ultrasonicated with 30 mL of water to form a yellow suspension and then centrifuged at 6000 rpm for 3 min, the resulting supernatant was treated with 4 M NH4HF2 solution for 6



12 h to remove the silica template, followed by centrifugation and washed three times with D. I. water and once with ethanol. The final yellow HCNS powders were obtained by drying at 80 °C in a vacuum oven overnight. 2.2.3. Synthesis of HCNS-HA (HH) and HCN-KLA-HA (HKH) HCNS-HA was synthesized by chemical grafting amidated HCN-NH2 to HA through amide formation, and the procedures were similar to that we reported previously.56 For the synthesis of HKH, HCNS (3 mg) was dissolved in 10 mL distilled water under sonication, DIPEA (20 µL) was added into the solution under stirring at 0 °C, MAL (6 mg) was added into the reaction flask. Next, KLA (9 mg) was added into the above mixture to obtain HCN-KLA. Subsequently, HA, cross-linking reagents EDC and NHS were added into the HCN-KLA solution and the procedures were similar to that described previously.51, 56-57 2.3. Preparation of HD, HHD and HKHD The methods were similar to what we reported.58 Briefly, the drug solution was prepared by DOX (8 mg) in deionized water, then mixed and stirred with HCNS (10 mg), HH (10 mg) or HKH (10 mg) for 2 days, and the mixture was centrifuged and washed to remove the unbound excess DOX. The precipitate was finally freeze dried to obtain the drug-loaded powders. All the washing solutions were collected, and the loading of the drug was evaluated by UV-Vis spectroscopy at 560 nm. The drug-loading capacity (DL) was calculated using the following formula: DL = (W1-W2) /W0 X100% Where W1 was the total weight of DOX, W2 was the weight of unencapsulated DOX. W0 was the total weight of nanoparticles. 2.4. General characterization Scanning electron microscope（SEM）image was acquired on Hitachi S4800 FE-SEM. The morphological properties of HCNS was assessed by transmission electron microscopy (TEM), which were recorded by field emission Hitachi HT7700 TEM (Hitachi, Japan). The morphology of the 7


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synthetic HKH and HKHD were detected by atomic force microscopy (AFM, Multimode 8, Bruker, USA). A Fourier transform infrared (FTIR) spectrometer (Intelligent, Nicolet 360, USA) was used to record the FTIR spectra. Fluorescence was recorded with a Hitachi F-7000 fluorescence spectrometer (Varian Ltd., USA). X-ray photoelectron spectra (XPS) survey scans were measured by an ESCALAB 250 X-ray photoelectron spectrometer (Thermo Scientific, USA). XRD measurements with CuK-α1 radiation as the X-ray source for excitation were used to examine sample structure and crystallite size. Thermogravimetric analysis (TGA) curves and differential scanning calorimetry (DSC) thermograms of nanocomposites were obtained by using a STA449C/6/G synchronous thermal analyzer (TA Instruments, Netzsch, GER), which was operated at 10°C min-1 over a temperature range of 35 to 1000°C in a dynamic atmosphere of N2 gas. The hydrodynamic size distribution and zeta potential of the nanocomposites were measured by using a Malvern Instruments Zetasizer HS III (Malvern, UK). 2.5. Stability study and hemolysis assay The stabilities of nanospheres in water for 5 weeks, or in different media were checked following the procedures that we reported previously.56 Briefly, at regular time points, the samples were collected to analyze the changes of particle sizes. Hemolysis assay experiments were carried out according to a previous report.59-60 Mouse blood stabilized by EDTA. RBCs were obtained by removing the serum from the blood by centrifugation and suction. After being washed with PBS solution five times; 0.2 mL of diluted RBS suspension was then mixed with 1.3 mL of PBS as a negative control, 1.3 mL of deionized water as a positive control, and 1.3 mL of HKHD suspensions at concentrations ranging from 25 to 200 ug mL-1. The mixtures were then vortexed and left to stand for 2 h at room temperature. After that time, the samples were centrifuged and the absorbance of the supernatants at 541 nm was measured by UV-vis spectroscopy. 8



2.6. In vitro DOX release study The methods were similar to what we described previously.35, 61 Briefly, 1.1 mg of drug conjugated nanoparticles were suspended in PBS buffer (5 mL, pH 7.4) or acetate buffer (5 mL, pH 5.5). Meanwhile, to examine the action of Hyal for DOX released, HKHD nanoparticles were dispersed with Hyal (2 mg mL−1) and sealed in a dialysis membrane (MW = 8 kDa). At a given time, a 300 μL portion of the aliquot was collected from the incubation medium and the amount of the released DOX was quantified at 488 nm. In vitro DOX release of HD, HHD and HKHD nanoparticles were monitored by microplate reader. 2.7. Cell culture and animals A549 cells were authenticated, and the methods of cell culture were developed based on what we reported before.35, 62-63 Thirty female Balb/c-nude mice (about 20 g weight, 4-6 weeks old) were purchased from Fuzhou Wushi Animal Center and maintained in cages in an SPF-grade animal room with access to food and water ad libitum. All animal experiments were performed in accordance with guidelines for the Institutional Animal Care and Use Committee of Fuzhou University and operated following the NSFC regulations concerning the care and use of experimental animals. 2.8. Cellular uptake To verify the targeting specificity, both A549 (CD44 receptor-positive) and HELF cells (CD44 receptor-negative) pre-seeded in six-well plates were incubated with HCNS and HKH for 4 hours. The methods were similar to what we reported.64 The cellular uptake of the nanoparticles by cells was evaluated using the BD FACSAriaIII (BD Biosciences), and the obtained data were analyzed with FlowJo software. Cellular uptake behavior of nanoparticles in A549 cells was also observed by flow cytometry. Cells were seeded and cultured in 6-well plates for 24 h. The cells were incubated with DOX, HD, HHD, HKHD and HKHD+Hyal (with 1mg ml-1 Hyal) at a drug concentration of 12.44 ug mL-1, respectively. After 9


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incubation for additional 4 h, the cells were washed twice with PBS to eliminate free nanoparticles. Intracellular DOX was also measured by fluorescence activated cell sorting (FACS). 2.9. HKHD endocytosis To determine if endocytosis is involved in HKHD traversing A549 cells, we cultured cells on a dish in the presence of endocytosis inhibitor Dynasore (50 uM, 30 min) at 37°C. The cells were washed with PBS followed by addition of HKHD to the cell culture for 30 min. After washing the cells with PBS three times, the HKHD signals generated from A549 cells were analyzed by flow cytometry. 2.10. Lysosome escape assay The internalization pathway of HKH was investigated. A549 cells were plated on confocal dishes and cultured overnight. 1 mL of a fresh cell medium containing 30 ug mL-1 of HKH was added to the dishes and incubated for 4 h. The samples (except the controls) were exposed under irradiation with a 430 nm lamp for 30 min and cultured for another 6 h, and the cells were then stained by LysoTracker Red (50 nM) for 15 min at 37°C. After washing three times with PBS, cell imaging was performed using CLSM. 2.11. Mitochondria and nuclei targeting capacity Intracellular mitochondria and nuclei targeting capacity of HKHD were determined. A549 cells were seeded on confocal dishes and cultured for 24 h. The cells were then treated with DOX, HHD and HKHD for 4 h, 6 h and 10 h, respectively. Then the cells in dishes were stained Mito Tracker Red (50 nM) and Hoechst 33258 (50 nM) for mitochondria and nuclei, respectively. After staining, the cells were washed three times with PBS and take images by CLSM. 2.12. Inhibition on intracellular ATP production The intracellular ATP levels were measured using Luminescence ATP Detection Assay by an ATPliteTM kit (PerkinElmer) after A549 cells were treated with different formulations. Cells at a density of 1 X104 cell well-1 were seeded and cultured in 96-well plates for 24 h, and were then incubated with 10



different drug-loaded nanospheres (12.44 ug mL-1 of DOX of HD, HHD and HKHD) for 4 h at 37 °C. 100 uL well -1ATPlite 1step was added, and incubated for 10 minutes in dark after plate shaking. A microplate reader was used to detect the absorbance spectra at Luminescence. 2.13. Intracellular ROS detection The ROS measurement during irradiation of HKHD was performed by using a 2, 7-dichlorofluorescin diacetate (DCFH-DA). Briefly, the A549 cells were treated with HD, HHD and HKHD (in equivalent drug concentration of 12.44 ug mL-1), and incubated for another 4 h. After this, the medium was washed several times with PBS to eliminate free nanoparticles and replaced by a fresh cell medium. 10 mM of DCFH-DA was added and incubated for additional 30 min in dark to ensure that DCFH-DA sufficiently covered the adherent cells. The cells were then irradiated using a 430 nm light-emitting lamp for 30 min. For the test group in the dark, we protected the wells from the light while irradiating the plate. The intracellular ROS generation was evaluated using flow cytometry. 2.14. Cell cycle analysis Analyses were carried out as we reported.56-57 Briefly, A549 cells were incubated with DOX, HD, and HKHD (in equivalent drug concentration of 12.44 ug mL-1) for 4 h and exposed under light irradiation for further 30 min, and then treated with nanoparticles for 20 h. The cells without irradiation treatment were used as the negative control. After the treatment, the cell cycle distribution was determined by flow cytometry. 2.15. Cell viability assay The cell viability of blank nanospheres were tested using a standard MTT assay, and the methods were similar to what we reported.65 Briefly, A549 and LO2 cells were seeded and cultured in 96-well plates at 37°C overnight, and were then incubated with different nanoparticles for 24 h (48 h, 72 h). Furthermore, we investigated intracellular PDT effect of drug-loaded 11


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nanoparticles on A549 cells. Then, various concentration of HKH (12.5, 25, 50, 100 ug mL-1), DOX and HKHD (all the test were carried out at equivalent DOX concentration of 5.18, 10.37, 20.74, 41.48 ug mL-1) were added to the cells were incubated for 4 h. After this, the cells was exposed under light irradiation for 30 min and cultured for further 20 h. At the end of the treatment period, viability was determined by a microplate reader at 488 nm. 2.16. Western blot analysis Analyses were carried out as we reported.35 Briefly, after 24 h of nanoparticle treatment, A549 cells were lysed and suspended in sample buffer. Lysates were electrophoresed and transferred to polyvinylidene difluoride (PVDF) membranes (BioRad). Membrane was then rinsed once with TBST and then incubated in the primary antibody (Caspase-3, Caspase-9, Cytochrome c and β-actin) solution, and probed with the secondary antibodies. The β-actin was used as an equal loading control. Chemiluminescent signals were detected using the ChemiDoc XRS system (Bio-Rad). 2.17. Tumor suppression A549 cells were subcutaneously implanted in the front right flank of female athymic nude mice (1 X 107 cells/100 uL/animal, 5 mice in each group). BALB/c nude female mice were randomly separated into seven groups (n = 5) and intravenously injected with 100 uL of solution at an equivalent DOX dose of 5.0 mg kg-1 (except the HKH group), when the tumor volume was approximately 100 mm3. One injections were administered every other day (from day 25 to 39). After that, the tumors were exposed under light irradiation. Mouse weight and the tumor volume were measured every three days. The tumor volume was measured using Vernier caliper and calculated as V = (length X width2)/2.66 At the end of the treatment, the tumor-bearing mice were sacrificed, and tumor tissue was stripped and weighted. The tumor inhibitory rate (TIR) was calculated with the formula: TIR = [(WC – We)/ We] × 100%. WC and We are the tumor weight of the control group and experimental group, respectively. The 12



major organs (heart, liver, spleen, lung, kidney and tumor) were collected, homogenized and measured by fluorescence spectrophotometry to determine the vivo biodistribution of the HKHD. Furthermore, in order to evaluate the toxicity and side effects of different formulations, blood samples were collected and tested by Sysmex.XS-800I in the Fuzhou general hospital of Nanjing military command (Fuzhou, China). Besides, the main organs and tumor tissues were stained with hematoxylin–eosin (H&E) to observe histopathological changes. 2.18. Statistical analysis For all studies, statistical analyses were conducted using the GraphPad Prism 5.0 (Graphpad software, San Diego, CA). In general, for two experimental comparisons, a two-tailed unpaired Student’s t-test was used unless otherwise indicated. For multiple comparisons, one-way ANOVAs were applied. When cells were used for experiments, three replicates per treatment were chosen as an initial sample size. All n values defined in the legends refer to biological replicates. Data were assessed as mean ± SD. The symbol *, ** and *** indicate no significant difference, P ＜ 0.05, P ＜ 0.01, and P ＜ 0.001, respectively. A probability (P) value of 0.05 or less was considered significant, P of 0.01 or less was considered highly significant, and P of 0.001 or less was considered extremely significant. 3. Results and discussion 3.1. Preparation and characterization of DOX-loaded HKH The HCNS was synthesized using the same silica template as we previously reported method46,

55

and described in Method 4.2. The synthetic

procedure for HCN-KLA-HA is illustrated in Figure.1a. The spherical morphology of HCNS (Figure.S1) was imaged by scanning electron microscopy (SEM), and the transmission electron microscopy (TEM) confirm this spherical morphology and indicate the corresponding individual hollow sphere (Figure.1b, Figure.S2). The characterization of HKH and HKHD was characterized by 13


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atomic force microscopy (AFM). As exhibited in Figure. 1c and 1d, the HKHD showed relatively spherical shape. As demonstrated in Figure.1e and 1f, mean diameter of the HKH and HKHD exhibited a uniform size distribution of 230 nm. This unique nanostructure, a rough surface, and a hollow cavity, is appropriate for adsorbing and delivering drug molecules or other active substances. The resulting HKH was characterized and verified by FTIR (Figure.1g). After HA and KLA coating, the new absorption at 1050 cm−1 could be assigned to the －C－O stretching of the －CH2OH groups in HA molecule. Compared to the FTIR spectrum of HA, the sharp band at 807 cm −1 originated from the breathing vibration of the tri-s-triazine units, and the skeletal vibrations at 1200 －1600 cm −1 were assigned to the aromatic CN heterocycles. Vibration at 2175 cm−1 was attributed to the C=N and N=C=N groups. The characteristic FTIR spectral peak verified that HKH was successfully synthesized. Besides, the large absorption at 2900–3600 cm−1 should be attributed to the absorbed H2O molecules and amino groups, which were consistent with the amide bond C=O stretching and N － H bending vibrations.46 X-ray photoelectron spectroscopy (XPS) show chemical elements C, N in HCNS. As displayed in the XPS spectra (Figure.S3-a), the main peaks at 282.1 eV, 284.7 eV and 287.8 eV could be assigned to the graphitic carbon (sp2 C － C bonds) and the sp2 -hybridized carbon respectively in the N-containing aromatic ring (N－C=N). For the N1s spectrum, the peak located at 395.8 eV was for sp2 -hybridized nitrogen in triazine rings (C － N=C), 398.7 eV for tertiary nitrogen N － (C)3 groups and 400.9 eV for amino functions caring hydrogen (C－N－H).46 Figure.31 (b, c) represents the X-ray diffraction (XRD) patterns of the HCNS and HKH. Nanoparticles show only one XRD peak at 27.14º attributed to the (002) reflection of CN-based materials,55 and the (002) peak of HKH became broader and gradually less intense because of KLA and HA modification. These above results demonstrate the structures of the nanospheres. As shown in Figure.1h, a strong fluorescence peak centered at 469 nm with a broad shoulder ranging 14



from 460 nm to 500 nm appeared when the particles were excited at 436 nm. After chemical modification of KLA and HA, the optical properties of HKH were as uniform as pristine HCNS, could be directly utilized for bioimaging without the need for modification of the fluorophores. Thermogravimetric analysis (TGA) was performed to provide an estimation of the approximate weights of the polymeric coating. As shown in Figure.1i, HCNS showed a weight loss of 9.62% at 100°C, which could represent the weight of the residual solvent and unwashed surfactant. We could observe that the polymeric coating (KLA, HA) were 26.70% and 22.91%, respectively. Besides, it could be found that we synthesized HCNS with thermal stability is 550°C. Differential scanning calorimetry (DSC) thermograms of HCNS, HCNMAL and HCN-KLA revealed that a distinct endothermic peak was associated at 700°C and 680°C (melting point) (Figure.1j). Zeta potential (Figure. S4) of the nanoparticles increased from −32.33 mV to +38 mV and then decreased to −17.7 mV, further proving that the HKH nanoparticles were successfully modified with amine-terminated KLA and HA. In addition, the negatively charged carbon surface could attract the positively charged DOX molecules. It should be noted that after the KLA and HA modification, the nanoparticles showed excellent stability in the RPMI-1640 medium without any visible precipitation for 24 hours (Figure. S5). 3.2. Physical stability of HKHD The HKHD was a kind of red uniform dispersed in solution, and exhibited smaller particle size and higher drug loading (Figure. 1k). The drug loading, average particle size and zeta potential of HKHD were 70.88%, 236.53 nm (PDI: 0.27) and -12.73 mV, respectively. The graphite plane of HKH can interact strongly with the DOX aromatic molecule through π−π stacking, resulting in the high loading capacity of DOX.67 Nanocarriers with particle sizes 10-500 nm could eventually accumulate at the tumor site due to the abnormalities of the tumor vasculature.58,

68

The moderate surface charge exhibited by the 15


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nanocarriers conferred their suitable physical and physiological stability in the system circulation. The successful intravenous injection of DOX-loaded HKHD in vivo must guarantee the excellent blood compatibility of the carriers, such as very low hemolytic effect. Herein, we describe an investigation of the hemolytic properties of HKHD with mammalian the red blood cells (RBCs). To the best of our knowledge, no prior study on the hemolytic interaction between HCNS and mammalian RBCs has been reported. The result of hemolysis assay in the supernatant is shown in Figure. 2a. Red blood cells are red due to the presence of hemoglobin in the cells. During the hemolysis assay experiments, hemoglobin will be released into the solution by hemolysis, and the resulting solution will become visually red (inset of Figure. 2a) in the case of strong hemolysis. No visible hemolysis effect can be observed visually at the increased concentrations of HKHD. The RBCs’ hemolysis effect was further determined by measuring the absorbance of the supernatant at 541 nm (hemoglobin) by UV-vis spectroscopy. The long-term stability of the DOXloaded nanoparticle was assessed by monitoring the changes in their sizes at different time intervals at 25°C in an aqueous environment. As shown in Figure. 2b, the particle size of HKHD was stable for four weeks and increased at 5th weeks. HKHD possessed a superior stability in H2O and cultural medium (Figure. S6). However, slight changes of particle size was observed in 20% FBS cultural medium. 3.3. Drug loading and release Owing to the porous nanostructure, large surface area in the shell and electrostatic interactions between the positively charged DOX molecules and the negatively charged surface of HKH, the nanoparticles had a sufficient capacity for loading drugs.67 According to UV-Vis absorption measurements, the amount of DOX molecules loaded to the hollow nanospheres (HCNS, HH and HKH) was 44.17% (441.7 mg g-1), 49.75% (497.5 mg g-1) and 70.88% (708.8 mg g-1), respectively. The high drug loading capacities of HKH could be 16



attributed to its biocompatible and monodisperse. Furthermore, capability of the nanocarriers to accommodate a high payload ensured delivery of ample amounts of DOX to the tumor site.69 Figure. 2c shows that compared with DOX solution, HKH-DOX showed a very low fluorescence intensity, suggesting that HCNS could efficiently quench the fluorescence of DOX, which could was recovered in the presence of the intracellular enzyme hyaluronidase (Hyal). These results collectively suggested that the intracellular Hyal could effectively degrade HA, resulting the release of DOX from HKH that traps DOX in side. Release kinetics of DOX from HD, HHD and HKHD were determined at different pH at 37°C. PBS at pH 7.4 was exploited to simulate the blood circulation while PBS at pH 5.5 was applied to simulate the acidic environment of tumor, respectively.61 As shown in Figure. 2d, the release rate of DOX at pH 5.5 from HKHD was about 48%, whereas from HHD, 30%; from HD, 22%. Meanwhile, 31%, 17%, and 13% of loaded DOX were released from HKHD, HHD and HD nanoparticles within 50 h in pH 7.4, respectively. The above data indicated that the release of DOX is pH-dependent. Additionally, HKHD exhibited a slow released profile, indicative of a sustained release. The pHsensitive and higher release of DOX from HKHD may provide, the maximum drug release in the acidic environments of the cancer cells, thus improving the antitumor efficacy.70-71 On the other hand, a considerable amount 65% of DOX was released in the presence of Hyal, suggesting that the intracellular Hyal plays a crucial part in triggering DOX release. This was probably because the hyaluronidase enzyme could cleave the HA, and acidic environment protonates the amino of DOX.46, 72 The interaction between positively-charged KLA and protons would also attenuate the adsorption of HCN-KLA to DOX, accelerating the release of DOX. 3.4. Cellular uptake and lysosome escape assay The HA-functionalized nanoparticles could target the tumor site.23 After CD44 receptor-mediated endocytosis, the nanosphere is embedded in 17


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lysosomal vesicles and HA could be specially degraded by Hyal,73 finally KLA peptide directed these nanoparticles into mitochondria. The involvement of the CD44 receptor in the uptake of HKH was studied by measuring the uptake of HCNS and HKH in A549 and HELF cells exhibiting high and low CD44 expressions. As shown in Figure. S7, the uptake of HKH was significantly higher than HCNS’ in A549 cells. HCNS were taken up by HELF cells lines but as expected, the uptake of HKH had no remarkable change in HELF cells. The results of flow cytometry confirmed that the uptake of HKH by A549 cells was facilitated by the specific interaction between CD44 receptor and HA. The cellular uptake of HKHD was studied by the DOX fluorescence expressed A549 cells. As shown in Figure. 2e, cells incubated with HKHD and HKHD+Hyal groups showed much stronger DOX fluorescence compared to DOX, HD and HHD groups. The quantitative analysis (Figure. 2f) also demonstrated that a much high cellular DOX concentration was found in this two groups. This result suggests that positively-charged KLA enhances cellular internalization in a tumor (acidic) microenvironment and eventually lead to more cytotoxicity. To further explore the mechanisms by which HKH is endocytosed as a single entity by A549 cells (Figure. 2g), we incubated A549 cells with HKHD in the presence and absence of endocytosis inhibitor Dynasore. Dynasore is a cell-permeable and commonly-used selective endocytosis inhibitor via inhibition of dynamin GTPase activity.74 Dynasore treatment significantly reduced intracellular HKHD by 2-fold in comparison with PBS treatment evidenced by flow cytometry (Figure. 2h). These results indicated that HKHD enters the cell through endocytosis. Confocal microscopy was employed to examine if HKH could escape from lysosome upon light irradiation. As shown in Figure. 2i (Dark), an obvious blue fluorescence (HKH) co-located with red fluorescence (lysosome) was observed in A549 cells, indicating that HKH was mainly distributed in the lysosomal compartments following the endosomal pathway. However, after light 18



irradiation, the blue HKH redistributed from lysosomes (Figure. 2i; Light), indicating the lysosomal release of HKH. These results confirmed that light could be utilized to regulate the lysosomal escape effect of the HKH for generating ROS and then breaking lysosomal membranes. 3.5. Mitochondria and nuclei targeting capacity To determine whether the enhanced anticancer effect of HKHD stemmed from the mitochondria-mediated apoptosis pathway. We stained mitochondria of A549 cells with Mito Tracker Red (green), which displayed mitochondria as green fluorescent signals under confocal laser scanning microscopy (CLSM). As illustrated in Figure. 3a, nearly no red fluorescent signals overlapped with green fluorescence of mitochondria in A549 cells treated with 4 h free DOX group and HHD; while the red fluorescent signals overlapped with the green fluorescent signals of mitochondria of A549 cells prominently after treatment with HKHD, illustrating that the targeting moiety KLA in HKHD could guide the delivery of DOX into mitochondria. Interestingly, we found that the red fluorescence signal distributed in both mitochondria and nuclei of A549 cells at 6 h. Moreover, the red fluorescence intensity of HKHD enhanced over time in mitochondria and nuclei, suggesting that internalized free doxorubicin redistribute into mitochondria and nucleus. HKHD would hopefully inhibit proliferation of A549 cells by attacking mitochondria and interfering the synthesis of DNA in nuclei simultaneously. In this way, HKHD could make full use of the dual inhibition effect of DOX against both mitochondria and nuclei. 3.6. Specific mitochondrial damage by HKHD After confirming the mitochondria targeting HKHD, we investigated the influence of HKHD on the intracellular ATP production in A549 cells. As shown in Figure. 3c, the cells showed a markedly decreased intracellular ATP levels after treatment with DOX, HD and HKHD, compared to the untreated cells. Moreover, HKHD showed a remarkable effect on inhibiting ATP production

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compared to HD. These data suggest that HKHD is able to interfere with the mitochondrial function to inhibit the intracellular ATP production. In general, apoptosis of many cancer cells induced by anticancer drugs is associated with ROS. Mitochondria is a major source of ROS.75 We used DCFH-DA to determine intracellular ROS levels generated by HD, HHD and HKHD. The non-fluorescent DCFHs could be converted into fluorescent dichlorofluorescein

(DCF)

in

the

presence

of

ROS.

DCF

had

excitation/emission maxima at 488 nm/525 nm, enabling detection using flow cytometry. As shown in Figure. 3d, ROS levels in A549 cells increased after treatment with HD, HHD and HKHD, respectively. In addition, compared with non-irradiation, the nanospheres with light irradiation had the ability to produce ROS with HKHD being the most significant (Figure. 3e). These results confirmed the effective light-activated ROS generation by HKHD to destruct mitochondria of A549 cells. 3.7. In vitro cytotoxicity by HKHD We first measured the cytotoxicity of blank nanospheres without light irradiation. As shown in Figure. 4a, the nanospheres (HCNS, HH and HKH) exhibited no significant cytotoxicity on A549 cells from 0 to 125 ug mL-1. However, the HKH showed slight cytotoxicity on LO2 cells (Figure. 4b) and A549 cells after 72 h treatment (Figure. 4c), indicating the pro-apoptotic peptide KLA could target the negatively charge mitochondria membrane and destroy their functions after a long time incubation. To evaluate the DNA damage by HKHD in A549 cells, the cell population at each cell cycle stage (G1, S or G2M) was examined using flow cytometry. We analyzed the cell cycle arrest by using propidium iodide (PI)-labeled DNA in A549 cells 24 h post-treatment. The flow cytometry analysis (Figure. 4d) showed that after HKHD treatment, cells in G1, S and G2-M phase accounted for 83.39%, 16.61%, and 0%, respectively (Figure. 4d-without light). The cell population in the S phase was slightly increased to 16.61%, owing to DOX-induced DNA damage. These results 20



proved that drug-loaded nanoparticles arrested the cells in S phase. Whereas, HKHD with light changed G1, S and G2-M population to 78.16%, 21.81%, and 0%, respectively (Figure. 4d-light). The cell population in the S phase was remarkably increased, from 8.69% to 21.16%. As showed in Figure. 4e, it was evident that light synergized A549 cells arrested in S phase. Next, we studied the synergism of HKHD with photodynamic therapy. As seen from Figure. 4f, HKHD showed the increased cytotoxicity compared to free DOX, suggesting HKHD possessed stronger inhibition ability of tumor cell proliferation than free DOX. The enhanced cytotoxicity might be attributed to the HKH-based improved drug mitochondria delivery and mitochondria damage. We further tested the effect of photodynamic destruction on A549 cells.23 As depicted in Figure. 4g, the cytotoxicity of free DOX was not synergized by light irradiation. Conversely, compared with dark controls, the cell viability decreased with an increase in the concentration of HKH, and this should be attributed to the high ROS induced after light irradiation. Strikingly, HKHD with light irradiation significantly enhanced cell viability inhibition in comparison with all other groups, indicating the synergism of HKHD with PDT. 3.8. A549 apoptosis by HKHD Cytochrome c released from mitochondria to cytosol could activate a cascade of caspase-9 and caspase-3 reactions, important indicators of cell apoptosis.76-78 We then examined the effect of nanoparticles on expressions of caspase and other proteins related to mitochondrial function (Figure. 4h). From the western blot images, it is clear that HKHD induced high expressions of cytochrome c, caspase-3 and caspase-9 in A549 cells compared to HD and HHD groups. These results collectively proved that HKHD manifested significant cytotoxicity effects on A549 cells. We also quantified the relative expression of proteins using Western blot analysis. Compared with dark controls, the cytochrome c, caspase-3 and caspase-9 levels in A549 are about 1.87-fold, and 1.36-fold and 1.17-fold, respectively in HKHD plus light irradiation 21


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(HKHD+L). These results clearly suggest that treatment with light result in the cleavage of caspase-3, caspase-9 and release of cytochrome c from mitochondria to the cytosol. More importantly, it is evident that the apoptosis of A549 induced by HKHD+L was mediated through the targeting mitochondria. 3.9. In vivo antitumor efficacy of HKHD The potential of HKHD+L to elevate antitumor efficiency was evaluated on the A549 tumor-bearing mice. Changes in body weight of mice were considered as an indicator of safety.79 As shown in Figure. 5a, all groups had no remarkable changes in body weight observed. In terms of tumor growth (Figure. 5b and Figure. S8), the average sizes of tumor in HD, HHD, HKHD and HKHD+L groups were not distinguishable from each other on days 25 to 33. However, the growth of tumor in HKHD and HKHD+L groups was delayed compared with the DOX, HD and HHD groups from days 33 to 39. The growth of tumor in HKHD+L group was better inhibited than HKHD group due to PDT. The final tumor weights were shown in Figure. 5c, and the highest tumor-inhibition efficiency was viewed with HKHD+L (86.19%), which was significantly higher than that of free HKHD (79.77%), HHD (70.26%), HD (65.14%) and DOX (59.31%). As displayed in Figure. 5e, the tumor tissue of HKHD+L group was looser and intercellular blank, compared with other five groups. Furthermore, Figure. 5d showed the distribution of HKHD nanoparticles in tumor-bearing nude mice. The fluorescence signal of DOX was primarily located in the tumor and lung, and only a few in the sections of other normal organs. Overall, owing to grafting HKH, HKHD not only showed high tumor accumulation but also possess potential to reduce side effects of DOX (cardiotoxicity and nephrotoxicity), mainly due to the enhanced permeability and retention (EPR) effect and CD44 targeting. The PDT and quicker drug release in mitochondria and nuclei made the free DOX produce better pharmacological action. The above results indicated that a combination therapy could make the higher tumor inhibition efficiency induced by HKHD. 22



It is worth noting that the side effects of DOX, such as cardiotoxicity, nephrotoxicity and chemotherapy-induced liver injury limited its clinical application and reduced the compliance of patient severely.23, 80-81 Therefore, the toxicity of HKHD was systemically investigated. Routine blood test showed that HKHD have no remarkable changes as compared to the saline group in Figure. 5f. DOX produced its significant blood toxicity as reported.82 Whereas, DOX entrapped in the hollow nanospheres produced much lesser blood toxicity. The H&E staining of major organs including heart, liver, spleen, lung and kidney was conducted to evaluate the safety of HKHD. As shown in Figure. 5g, free DOX, HD and HHD groups showed typical cardiomyopathy characterized as irregularly arranged muscle tissue fibers and some dissolved and broken muscle fiber. However, almost no damage or inflammation was observed in the major organs after injection of HKH and HKHD, indicating that nanoparticles are generally safe and biocompatible. The lack of appreciable blood toxicity and organ damage further suggested low toxicity of the nanoparticles, and HKHD could be as a promising drug delivery system for treatment of cancers. In the present study, we demonstrated that functionalization of HCNS with HA and KLA can serve as a powerful photosensitizer, and carry high payload of DOX for delivery of DOX into both mitochondria and nuclei of A549 cells. The DOX loaded-HKH showed suitable particle sizes, applicable zeta potentials, effective drug releasing and acceptable biostability. The mitochondria and nuclei dual-targeted ability of HKHD paved the way for DOX to destruct mitochondria and nuclei, thereby enhancing DOX anticancer activities. Direct ROS generation by HKHD under light irradiation maximizes its PDT efficiency, causing activation of caspase-3 and caspase-9, and release of cytochrome c, and initiating apoptosis cascade. Overall, these chemo-photodynamic synergistic effects of dual-targeted HKHD nanoparticles enhance the therapeutic efficacy superior to monotherapy.

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Acknowledgments This research was supported by the Ministry of Science and Technology of China (2015CB931804); the Natural Science Foundation of China (81773063; U1505225; 81273548; 81571802), the Natural Science Foundation of Fujian Province (2016J06020), and the Fujian Provincial Youth Top-notch Talent Support Program.

Author Information The authors declare no competing financial interest. Correspondence and requests for materials should be addressed to L.J. ([email protected])

Supporting Information SEM and TEM images of HCNS; XPS spectra of HCNS; XRD patterns of HCNS and HKH; zeta potential data; stability analyses of the nanoparticles in different media; the uptake of HKH in A549 and HELF cells; the picture of the A549 tumor-bearing mice and tumors (PDF)

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Scheme 1. Synthesis and chemophototherapeutic mechanisms of mitochondrion- and nucleus-targeting hollow graphitic carbon nitride nanospheres (HCNS) containing KLA, HA and DOX. (a) Covalent synthesis of HCNS, KLA and HA to form the conjugate HCNS-KLA-HA (HKH) that is then loaded with DOX to produce the functionalized chemophototherapeutic HKHD. (b) Antitumor mechanisms of HKHD. HA-mediated uptake of HKHD by A549 cells involves endocytosis, which can be inhibited by Dynasore. Light enhances release of endocytosed HKHD from lysosomes. Guided by KLA, HKHD targets mitochondria to induce sequential release of Cyt c, Caspase-9 and -3 to produce apoptosis. Nuclear-targeting HKHD can directly release DOX to nuclei under acidic condition causing DNA intercalating. Intravenous HKHD inhibits growth of A549 subcutaneously-implanted to nude mice with or without light irradiation.

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Figure. 1. Synthesis procedures, characterization and high-payload of HKHD. (a) The novel material HCNS reacts with maleimide that then reacts with SH-group of KLA followed by acetamide reaction with HA to form HKH. The left inset shows SEM imaging of HCNS. (b) TEM show the corresponding individual hollow sphere of HCNS. (c-f) Analysis of particle size of the hollow nanospheres. AFM images of HKH (c) and HKHD (d). Dynamic light scattering (DLS) measurements of HKH (e) and HKHD (f). (g) FTIR spectra of HA, HCN, 32



HH, and HKH. (h) Fluorescence emission spectra of HCN and HKH at an excitation wavelength of 436 nm. (i) Thermogravimetric analysis (TGA) curves of HCNS, HCN-MAL, HCN-KLA and HKH. (j) Differential scanning calorimetry (DSC) thermograms of HCNS, HCN-MAL, HCN-KLA and HKH. (k) Physicochemical properties and loading capacities of HD, HHD and HKHD. The hollow HKHD with huge interior and exterior surface area shows DOX payload 3.3-fold more than the non-hollow mesoporous silica nanoparticles (MSN)61 does compared on the mg g-1 basis.

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Figure. 2. In vitro stability, DOX-payload and release of HKHD and its cellular uptake by A549. (a) Hemolysis assay for HKHD, using PBS as a negative control and water as a positive control (left two tubes in the inset), and the HKHD was suspended at different concentrations (right five tubes in the inset). The mixtures were centrifuged to detect the presence of hemoglobin in the supernatant visually (inset pictures of the tubes). UV-vis absorption spectra to detect the presence of hemoglobin at 541nm. (b) Stability of HKHD in water at 25°C for 4 weeks. (c) Fluorescence quenched once DOX entrapped in HKH. Hyal, by hydrolyzing HA, released the entrapped DOX and resumed DOX fluorescence. (d) Dynamic release of DOX from the hollow nanospheres. Note, Hyal and pH 5.5 facilitated DOX release. (e) Flow cytometry scanning of cellular uptake by A549 cells treated with DOX, HD, HHD, HKHD, and HKHD+Hyal. (f) Fluorescent quantitation of DOX uptake by A549 cells. (g-h) Flow cytometry scanning (g) and quantitation (h) showing endocytosis of HKHD by A549 cells, and the endocytosis was inhibited by Dynasore. (i) Cellular internalization and distribution of HKH into lysosomes of A549 cells with or without light irradiation observed by confocal laser scanning microscopy (CLSM). Note, less HKH in lysosomes after irradiation (purple). 60× oil immersion objective and 10× ocular lens. Each data point represents the mean ± SEM (n=3).

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Figure. 3. Intracellular localization of hollow nanospheres and their effects on ATP and ROS production. (a-b) Confocal images show localization of HKHD in mitochondria (a; 4 h, green MitoTracker) and nuclei (blue Hoechst) of A549 cells in comparison with DOX and HHD. 60× oil immersion objective and 10× ocular lens. Dynamic distribution of HKHD into mitochondria (merged yellow) and nuclei (merged purple) increased at 4, 6 and 10 h incubation. (c) Intracellular ATP produced by A549 cells after 4 h-incubation with DOX, HD, and HKHD. The latter targets mitochondria to reduce ATP production. *, P< 0.05; **, P< 0.01; ***, P

Mitochondria and Nuclei Dual-Targeted Hollow Carbon Nanospheres

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