Mitochondria-Specific Anticancer Drug Delivery Based on Reduction

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Mitochondria-Specific Anticancer Drug Delivery Based on Reductive-Activated Polyprodrug for Enhancing Therapeutic Effect of Breast Cancer Chemotherapy Yajun Wang, Tian Zhang, Cuilan Hou, Menghang Zu, Yi Lu, Xianbin Ma, Die Jia, Peng Xue, Yuejun Kang, and Zhigang Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10211 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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ACS Applied Materials & Interfaces

Mitochondria-Specific Anticancer Drug Delivery Based on Reductive-Activated

Polyprodrug

for

Enhancing

Therapeutic Effect of Breast Cancer Chemotherapy Yajun Wanga, b, Tian Zhanga, b, Cuilan Houc, Menghang Zua, b, Yi Lua, b, Xianbin Maa, b,

aKey

Die Jiaa, b, Peng Xuea, b, Yuejun Kang*a, b and Zhigang Xu*a, b

Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest

University), Ministry of Education, School of Materials and Energy, Southwest University, Chongqing, 400715, P. R. China bChongqing

Engineering Research Center for Micro-Nano Biomedical Materials and

Devices, Southwest University, Chongqing 400715, P. R. China c

Department of Cardiology, Shanghai Children’s Hospital, Shanghai Jiaotong University, No. 355 Luding Road, Shanghai, 200062, P.R. China

Email: Z. Xu ([email protected]); Y. Kang ([email protected]).

1

ACS Paragon Plus Environment

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Abstract: Mitochondria-targeted cancer therapies have achieved unprecedented advances attributed to their superior ability for improving drug delivery efficiency and producing an enhanced therapeutic effect. Herein, we report a mitochondria-targeted camptothecin (CPT) polyprodrug system (MCPS) covalently decorated with a high-proportioned CPT content, which can realize drug release specifically responsive to a tumor microenvironment (TME). The nonlinear structure of MCPS can form water-soluble unimolecular micelles with high micellar stability and improved drug accumulation in tumoral cells/tissues. Furthermore, a classical mitochondria-targeted agent, triphenylphosphonium bromide (TPP), was tethered in this prodrug system, which causes mitochondrial membrane potential depolarization and mediates the transport of CPT into mitochondria. The disulfide bond in MCPS can be cleaved by intracellular reductant such as glutathione, leading to enhanced destruction of mitochondria DNA and cell apoptosis induced by a high level of reactive oxygen species (ROS). The systematic analyses both in vitro and in vivo indicated the excellent tumor inhibition effect and biosafety of MCPS, which is believed to be an advantageous nanoplatform for subcellular organelle-specific chemotherapy of cancer.

Key words: Mitochondria-targeted; reduction-activated; polyprodrug; chemotherapy; cancer therapy

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1. Introduction Nanoparticle-based theranostics is an enabling tool in current cancer treatments. However, dose-dependence, multidrug resistance, rapid blood clearance and lack of specificity are still among the major challenges in drug delivery for cancer therapy.1 The treatment efficiency of nanotheranostics can be regulated by various drug delivery systems, such as micelles,2,3 polymers,4,5 dendrimers,6,7 liposomes,8 inorganic nanoparticles9 etc., which have shown highly promising features in promoting the biosafety of chemotherapy and reducing the toxicity to normal cells. Amphiphilic stimuli-responsive polyprodrug is believed to be a preferential strategy to overcome the limitations of many nanomedicine. Specifically, anticancer agents can be conjugated to amphiphilic polymers through a stimuli-responsive linkage, such as hydrazine bond,10,11 disulfide bond12 or thioketal bond.13 The obtained amphiphilic polyprodrug can self-assemble into nano-sized micelles in water with high drug loading rate, high stability, uniform micellar size and prolonged blood circulation half-life.14,15

When

the

polyprodrug

micelles

are

stimulated

by

tumor

microenvironmental (TME) cues, such as lower pH condition,16,17 higher level of H2O2,18 GSH19 or enzymes,20 the carried drug will be released due to the cleavage of TME-responsive linker. However, conventional chemotherapy relies heavily on nonspecific or random diffusion of drug molecules into tumor cells. For many small molecule drugs, including the DNA toxin camptothecin (CPT), the drug function is usually limited in intracellular organelles, such as mitochondria and nuclei, which are the energy producers or hosts of critical genetic information of the cells.21,22 On the other hand, drugs in subcellular space are not easy to escape in comparison with those in cytoplasm. Therefore, it is a more rational strategy to construct intracellular organelle-targeted nanotheranostics. The double membrane-structured mitochondrion is a crucial subcellular organelle in regulating cell metabolism by multiple paths (e.g., altering redox state and inducing cell apoptosis).23,24 Compared to those in normal cells, mitochondria in cancer cells express a higher level of reactive oxygen species (ROS) and reductant 3



(e.g., GSH),25-27 whereas the excessive ROS may damage lipids and DNA.28,29 Mitochondrial oxidative stress is a potential cancer therapeutic method, which works by consuming the intracellular reductants, resulting in a burst of ROS and thereby inducing cell death.30-32 Recently, various mitochondria-specific nanotheranostics, including those targeting on mitochondrial metabolism33 or correcting abnormal mitochondrial genes,34,35 have been developed for cancer therapy. In particular, molecules with lipophilicity and positive charge have been found to traverse the double-membrane more easily for delivery of therapeutic agents into mitochondria. As a typical mitochondria-targeted agent, triphrnylphosphonium bromide (TPP) has a deionized positive charge center and a hydrophobic surface area, which can improve the mitochondrial uptake of drug molecules.36,37 Moreover, TPP can induce the release of cytochrome C from the mitochondrial intermembrane, resulting in an up-regulated cell apoptosis pathway.38,39 Therefore, mitochondria-targeted drug delivery systems are expected to enhance the effect of chemotherapy. As discussed above, TME-activatable nanotheranostics could be a desirable strategy for targeted drug delivery with enhanced tumor permeation and therapeutic effects, while maintaining a high level of in vivo biosafety. Herein, we synthesized a mitochondria-targeted camptothecin (CPT) polyprodrug system (MCPS) for tumor-specific drug delivery (Scheme 1), which consist of an amphiphilic polyprodrug of dextran-P (OEGMA-co-CPT-co-TPP) (DCT). Compared with other mitochondria-targeted polyprodrug system,40,41 the present MCPS systems integrated biocompatible dextran (DEX), reduction-sensitive camptothecin polyprodrug and mitochondrial targeting into one system, endowing MCPS with unimolecular micelle feature, high drug loading capacity, remarkable in vivo biosafety and enhanced chemotherapy. Specifically, the CPT molecules in MCPS can not only damage DNA topoisomerase,42 but also induce enhanced mitochondrial apoptosis43 in comparison with non-targeted prodrug, which further produce a ROS enhancement and thus accelerate the apoptosis of cancer cells. The results obtained from in vitro and in vivo experiments provided a solid evidence of MCPS as an enabling therapeutic for inhibiting tumor growth and promoting cancer cell apoptosis. 4


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2. Methods and Experiments 2.1 Materials All chemicals and cell culture agents were supplied by Sigma-Aldrich Pte. Ltd. (USA) and Life Technologies (USA), unless otherwise noted, and used without further purification. Deionized (DI) water was supplied by Milli-Q Synthesis A10 System (Millipore). 2.2 Characterizations of polyprodrug micelles The chemical structure of polyprodrug and its intermediates were verified by a with a Bruker 600 MHz nuclear magnetic resonance (NMR) instrument. The morphology, micellar size and surface charge were determined by a JEM-1230EX transmission electron microscopy (TEM) and a Malvern Nano ZS90 size analyzer, respectively. The optical results were conducted on a Shimadzu UV-1800 Ultraviolet (UV)-vis spectroscopy and Shimadzu RF-5301PC spectrofluorometer. The cellular viability and drug loading were tested by Tecan SPARK-10 M plate reader. The cellular uptake and imaging were conducted on a NovoCyte 2060R flow cytometry and a Zeiss 800 confocal laser scanning microscopy. Blood analysis was acquired by Mindray BC-2600 Vet hematology analyzer. NIR fluorescence images were collected by IVIS Lumina Kinetic Series Ⅲ systesm (PerkinElmer). 2.3 Synthesis of PEGTPP PEGTPP was synthesized through an esterification of TPP and MAPEG. Briefly, (2-carboxyethyl) triphenylphosphonium bromide (TPP, 834.67 mg, 2 mmol) and N, N’-dicyclohexylcarbodimid (DCC, 498.29 mg, 2.4 mmol) were dissolved in 4 mL dichloromethane (DCM) under the protection of argon atmosphere and were allowed to react at 4 C for 0.5 h. Then, poly(ethylene glycol) methacrylate (MAPEG, Mn=500, 1.50 g, 3 mmol) and 4-(Dimethylamino) pridine (DMAP, 109.95 mg, 0.9 mmol) dissolved in 4 mL DCM was added as one portion. After 0.5 h stirring, the mixture was transferred into room temperature and stirred for 12 h. The solution was dialyzed for 8 h and then the solvent of the solution in dialysis bag was removed under vacuum to obtain the product. 2.4 Synthesis of DEX-P (OEGMA-co-CPT) (DC) polyprodrug 5



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Firstly, the camptothecin (CPT) monomer (MABHDCPT) with a disulfide bond was obtained by a previously reported method.44 Second, DEX-Br (23 mg, 0.105 mmol), oligo (ethylene glycol) methyl ether methacrylate (OEGMA, 131.25 mg, 0.2625 mmol), MABHDCPT (31.5 mg, 0.0525 mmol) was dissolved in a mixed solvent with 1 mL N, N-dimethylformamide (DMF) and 1 mL dimethyl sulfoxide (DMSO), following the mixed solution was added into a 25 mL tube under an argon atmosphere. Then, copper (I) bromide (CuBr, 15.2 mg, 0.105 mmol) was added. After three

freeze-pump-thaw

cycles

using

liquid

nitrogen,

tris

(2-dimethylaminoethyl)-amine (Me6TREN, 35 μL, 0.127 mmol) was injected into the mixture. The polymerization was carried out for 24 h at 25 C. The final product was placed in a dialysis bag (molecular weight cutoff, MWCO: 14000) and purified by THF at room temperature for 2 days. The obtained product was a viscous yellow liquid and denoted as DEX-P (OEGMA-co-CPT) (DC). 2.5 Synthesis of DEX-P (OEGMA-co-CPT-co-TPP) (DCT) prodrug Typically, Dextran-Br (DEX-Br, 23 mg, 0.105 mmol), OEGMA (420 mg, 0.84 mmol), PEGTPP (105 mg, 0.21 mmol), MABHDCPT (126 mg, 0.21 mmol) was dissolved in a mixed solvent with 2 mL DMF and 2 mL DMSO, after which the solution was transferred into a 25 mL reaction tube under an argon atmosphere. Then, CuBr (15.2 mg, 0.105 mmol) and Me6TREN (16 mg, 0.055 mmol) was added. After three freeze-pump-thaw cycles treatment, the polymerization was continued for 24 h at 25 C. The final product was dialyzed against tetrahydrofuran (THF), resulting in a viscous solid (denoted as DEX-P (OEGMA-co-CPT-co-TPP) (DCT)). 2.6 Preparation of polyprodrug micelles To prepare polyprodrug micelles, THF solution containing 5 mg polyprodrug was mixed with 5 mL DI water under magnetical stirring for 0.5 h, and then dialyzed against DI water for 2 days using a dialysis bag (MWCO: 3500). The final CPT concentration of polyprodrug micelles was diluted to 600 μg/mL for the following experiments. 2.7 TME-responsive release kinetics The drug release assay of polyprodrug micelles was performed by incubating 1 mL 6


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polyprodrug micelles in 80 mL PBS (pH 7.4) with various DL-dithiotheritol (DTT) concentration (0, 2 μM, 2 mM) using a dialysis method (MWCO: 3500), and then the bag was slightly shaken at 37 C. At fixed time intervals, 1 mL releasing solution was collected and 1 mL PBS with corresponding media was added. The released and uploaded CPT was determined according to a standard curve of CPT. 2.8 Cell culture Human breast cancer cells (MCF-7), 4T1 murine mammary carcinoma cells and human cervical cancer cells (HeLa) were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (China). These cells were cultured in DMEM (Dulbecco’s modified Eagle’s Medium) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin at 37 C under 5% CO2. 2.9 In vitro cytotoxicity assay of polyprodrug micelles MTT and calcein AM/propidium iodide (PI) double assays were used to assess the in vitro cytotoxicity. HeLa, MCF-7 or 4T1 cells were seeded in a 96-well plate at a density of 10000 cells/well. After a 16 h culture, the DMEM was replaced with fresh DMEM medium containing free CPT, DC or DCT micelles. After incubation for 48 h or 72 h, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and calcein AM/PI assays were carried out after a washing process using PBS. The results were analyzed by a Tecan microplate reader and a fluorescence microscope, respectively. 2.10 Intracellular drug distribution and uptake assay The intracellular drug distribution and uptake assays were analyzed by CLSM and flow cytometry. For drug distribution, HeLa (2×104) cells were seeded into an 8-well plate. After incubating for different time under different treatments, the cells were washed with PBS three times and treated by a mixture of formalin, bovine serum albumin and triton X-100 solutions. Finally, 400 μL DRAQ 5 (SYTO Deep Red Nucleic Acid Stain) solution was stained and the results were recorded by confocal laser scanning microscopy (CLSM). Nile red-labeled DC or DCT were used to trace the time-dependent cellular uptake

7



of polyprodrug micelles. HeLa cells seeded in 24-well plate at a density of 1×104 per well and cultured for 16 h. These cells were cultured with different prodrug groups for 0.5 h, 1 h, 2 h and 4 h. After that, cells were harvested and the results were analyzed using flow cytometry. 2.11 Mitochondria and lysosomes co-localization assay To investigate the lysosome escape and mitochondria target abilities of polyprodrug micelles, MCF-7 (2 × 104) cells were seeded into a 8-well plate and subject to different treatments. Then, the cells were washed with PBS and incubated with Lyso Tracker Red for 1 h or Mito Tracker Green for 45 min. Finally, the cells were washed with PBS three times and cultured in 400 μL DMEM for CLSM assay. 2.12 Cellular mitochondrial membrane potential and ROS level JC-1 fluorescence probe was used to assess the effect of polyprodrug micelles on (2×104) cells were seeded into a 8-well

mitochondria membrane potential. HeLa

plate and incubated for 16 h, followed by incubating with fresh medium containing polyprodrug micelles for 4 h. The untreated cells is served as control group. Then, cells were washed three times with PBS and stained with JC-1 at 37 C for 20 min. The fluorescence signal was detected by CLSM. HeLa (2 × 104) cells were seeded into a 8-well plate. After treatment with polyprodrug micelles for 4 h, the cells were washed with PBS and the cellular ROS level was detected using a ROS fluorescence probe, 2’, 7’-dichlorofluorescein diacetate (DCFH-DA, 10 μM). The fluorescence intensity was observed using CLSM. 2.13 In vitro spheroids solid tumor penetration effect To assess the tumor penetration ability of the polyprodrug micelles, 1% agarose gel was first prepared by dissolving agarose in 1×TAE (tris-acetic acid and EDTA) solution and was quickly added into a 96-well plate at a volume of 50 μL/well. After sterilization using ultraviolet light, 200 μL MCF-7 cells with a density of 103 per well were cultured in a 96-well plate for three days. Then, the obtained multi-cell spheroids (MCS) were subject to different treatments for 6 h. The penetrations ability of micelles were analyzed using CLSM. 2.14 Hemolysis assay 8


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Mouse peripheral blood were obtained from the eyelid posterior sinus, and centrifuged at 3000 rpm for 15 min. After washing with PBS, the erythrocytes were diluted to a concentration of 2 % using PBS and were continually incubated with micelles of different concentrations at 37 C for 1 h. Then, the samples were centrifuged and the leaked hemoglobin in supernatant was measured by recording the optical absorbance at 570 nm. 2.15 Animal models for in vivo studies All animal experiment protocols were in strict compliance with the National Guide for Care and Use of Laboratory Animals, and approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University. Kunming mice and BABL/C mice were supplied by Chongqing Teng-Xin Biological Technology Co. Ltd. The 4T1 Murine breast cancer model were constructed using female BABL/C mice subcutaneously injected with 5×106 cells into the right back. 2.16 In vivo biocompatibility assay To assess the in vivo biocompatibility of polyprodrug micelles, Kunming mice (n=4, 6 weeks old) were randomized into four groups and were injected through tail vein with saline, free CPT, DC micelles and DCT micelles with a CPT dosage of 5 mg/kg. At day 1 and day 7, 0.5 mL of blood was collected from the eye socket of mice and routine blood assay were conducted on a hematology analyzer (Mindray BC-2600 Vet). 2.17 In vivo fluorescence imaging and bio-distribution Cy7-labeled polyprodrug micelles were used to investigate the in vivo bio-distribution of micelles. BABL/C mice bearing 4T1 tumors were intravenously injected with free Cy7, DC/Cy7 micelles or DCT/Cy7 micelles with a Cy7 dosage of 1 mg/kg. After 24 h, mice were sacrificed and the fluorescence of entire body, major organs (heart, liver, spleen, lung and kidney) and tumors were captured by a NIR imaging system. 2.18 In vivo anti-tumor therapeutic assay The BALB/C mice bearing 4T1 tumors were administered with saline, free CPT, DC micelles or DCT micelles via tail vein injection (n=5). The tumor volumes were 9



measured using a digital caliper and the size of tumors was calculated based on the equation: Length×Width2/2. After the treatment, the body weight and tumor volume were measured every two days for 2 weeks. 2.19 Histological studies and immunofluorescence imaging After treatment and observations, mice were sacrificed and the tumor and main organs were excised, fixed, frozen, cut into a 10 μm thick slices for H&E staining. The tumor slices were also treated with terminal deoxynucleotidyl transferase dUTP nick (TUNEL) staining. All slices were imaged using a fluorescence microscope. 2.20 Data analysis The statistical significance of results were evaluated by t-test and analysis of variance. All data were demonstrated as mean ± SD.

Scheme. 1 Scheme illustration of camptothecin (CPT) polyprodrug-based dextran for enhanced chemotherapy. (A) The structure and micelles formation of DCT polyprodrug; (B) The DCT micelles showed a high accumulation process in tumor by EPR effect, were activated by reduction microenvironment and produced the mitochondria-specific and reductive-activated drug releases for tumor cell death.

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3. Results and discussion 3.1 Synthesis of mitochondria-targeted camptothecin (CPT) polyprodrug systems Dextran (DEX), a dendritic natural polysaccharide with good biocompatibility and advantageous polyhydroxyl chemical structure,45 can work as a preferable candidate for synthesis of block copolymers. Following the previously reported protocols, we synthesized DEX-Br macroinitiator46 that was characterized by 1H NMR result as shown in Fig. S1. The synthetic routes of PEGTPP, MABHDCPT, DC and DCT were shown in Fig. S2. For PEGTPP, the typical signal of TPP for aromatic protons could be observed at 7.67-7.90 ppm, while the proton signal of CH2 of MAPEG appeared at 4.23 ppm (Fig. S3). Besides, in the normalized UV-vis absorption spectra (Fig. 1A), PEGTPP showed both characteristic absorbance of OEGMA and TPP at 269 nm and 275 nm, respectively, indicating the successful reaction of OEGMA and TPP. In the 1H NMR spectrum of MABHDCPT (Fig. S4), the characteristic peaks at 2.86 ppm (peak m+l) of methylene and 7.70-8.42 ppm (peak a, b, c, d, e) of aromatic group could be observed. In the 1H NMR spectrum of DCT, the typical signal of aromatic protons from TPP was located at 8.1-8.2 ppm. Meanwhile, the characteristic peaks of MABHDCPT at 2.86 ppm and 4.1-4.23 ppm, OEGMA at 3.65 ppm also appeared in the 1H NMR spectra of DC and DCT. Furthermore, both polyprodrug micelles exhibited typical CPT peaks at 365 nm and 432 nm in the UV-vis absorption spectrum (Fig. 1C) and fluorescence spectrum (Fig. 1D), respectively. All these results verified that the polyprodrug was successfully synthesized. Next, the molecular weights of two polyprodrugs were measured using Agilent 1260 gel permeation chromatography (GPC), which showed monomodal peaks of both polyprodrugs obtained (Fig. 1B). The molecular weights of DC and DCT were estimated as 23600 g/mol and 16400 g/mol, respectively. The corresponding polydispersity index (PDI) were 1.52 and 1.39, respectively, indicating a controlled polymerization process. 3.2 Size, morphology and hemolysis analysis assays The technologies of TEM and DLS were performed to characterize the particle 11



morphology and size distribution of two polyprodrug micelles. As shown in Fig. 1E and 1F, both polyprodrug micelles exhibited a near spherical morphology (TEM images) with average diameters of 21.3±3.5 nm and 28.7±4.1 nm for DC micelles and DCT micelles, respectively. The corresponding zeta potentials were -21.9 ± 2.3 mV and -23.3 ± 0.8 mV (Fig. 1G), respectively. The hydrodynamic diameter detected by DLS were 22.52 nm and 33.63 nm for DC and DCT micelles, respectively. The slight difference between the measured TEM and DLS results might be due to the different hydration status of the micelles during the measurements. The nano-size micelles provide obvious benefits for prolonging the blood circulation half-life and improving the drug penetration in tumoral tissues under the enhanced permeability and retention (EPR) effect.47,48 A blood hemolysis assay was used to further investigate the in vitro hemocompatibility of the polyprodrug micelles. As shown in Fig. 1H, the supernatants of the negative control group and the drug-treated group were clear with the concentration of leaked hemoglobin less than 5%. In the UV-vis absorption spectrum (Fig. 1I), the absorbance at 540 nm and 570 nm of the positive control group was over 25 folds higher than the negative control group. All these results suggested that the polyprodrug has a good hemocompatibility for peripheral blood circulation.

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Fig. 1 Characterizations and hemolysis assay of mitochondria-targeted polyprodrug. (A) UV-vis spectra of TPP, OEGMA and OEGMA-TPP. The GPC traces (B), UV-vis spectra (C), fluorescence spectra (D) of DC and DCT. The TEM images and hydrodynamic size distribution of DC micelles (E) and DCT micelles (F). (G) Zeta potentials of DC and DCT. (H) Photographs of hemolysis analysis and the hemolytic rates after treatment with DC and DCT under different equivalent CPT concentrations. (I) Absorbance spectra of the blood supernatants after treatment with 0.1% Triton X-100 (positive controls) and PBS (negative controls).

3.3 Drug release and cell viability The CPT release in vitro behavior of DC micelles and DCT micelles was studied under various concentrations of reductant DTT (0, 2 μM or 2 mM). As shown in Fig. 2A and B, CPT release from two drug-loaded polyprodrug micelles was faster in the presence of 2 mM DTT than in other conditions. Specifically, 67 % and 73 % of CPT was released from DC and DCT, respectively, in 7 h under 2 mM DTT concentration. The relatively lower release amount might be restrained by the high-density POEGMA 13



chains in polyprodrugs.2 In contrast, less than 20 % of drug was released from DC and

DCT after 6 h in PBS free from DTT or with 2 μM DTT. The reduction-triggered CPT release is due to the cleavage of disulfide bond by reductant agent DTT. Therefore, the polyprodrug micelles can maintain minimal release during blood circulation, while activate rapid drug release when exposed to a highly reductive microenvironment in tumor cells. MTT assay was used to measure the cytotoxicity of polyprodrug micelles to HeLa, MCF-7 and 4T1 cells (Fig. 2C, D and E). For these three cancer cell lines, DCT micelles exhibited a higher cytotoxicity than DC micelles, suggesting the enhanced anti-tumor effect of mitochondria-targeted drug delivery. It was obvious that DC produced a lower cytotoxicity than DCT, which could be due to the controlled release kinetics of CPT from the micelles. The relatively lower cytotoxicity in comparison with free CPT might be induced by the slower transportation of endocytosis for prodrug groups.49,50 Further, Annexin V-FITC and

propidium iodide (PI) double staining assay also demonstrated the TPP-targeted enhanced cytotoxicity (Fig. 2F). Similar to the results by MTT assays, samples treated with DCT micelles exhibited higher ratio of dead cells than those treated with DC micelles, suggesting the effective inhibition of cancer cells by mitochondria-targeted DCT micelles.

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Fig. 2 In vitro release behaviors, cell viability and apoptosis. CPT release curves of DC (A) and DCT (B) in the PBS solution with different DTT concentrations (0, 2 μM, 2 mM). Data are presented as mean ± SD (n = 3).The cell viability of HeLa cells (C), MCF-7 cells (D) and 4T1 cells (E) incubated with free CPT, DC micelles or DCT micelles at different concentrations for 72 h. Data are presented as mean ± SD (n = 3). (F) Live and dead staining of HeLa cells incubated with free CPT, DC micelles and DCT micelles with CPT concentration of 30 μg/mL for 24 h at 37C. The untreated cells were used as control group. Scale bars: 200 μm.

3.4 In vitro cell uptake assay CLSM was employed to investigate the internalization and intracellular localization of free CPT and polyprodrug micelles. Fig. 3A displayed the fluorescence of CPT (blue), F-actin (Alexa Fluor 488 Phalloidin, green) and nuclei (DARQ5 Fluorescent Probe, red). The fluorescence signals of CPT in intracellular 15



compartments showed a time-depended enhanced process from polyprodrug micelles. The line profile further indicated that the CPT fluorescence intensity of DCT micelles in the cytoplasm was constantly enhanced from 1 h to 4 h, while there was no significant increase in the nuclei. Such observations implied the TPP-mediated mitochondria targeting effect of the micelles. Flow cytometry was used to quantify of the cellular uptaking rate of polyprodrug micelles. After incubation with HeLa cells for 4 h, the cellular uptaking rates of DC and DCT micelles labeled with Nile red reach 97.14 % and 99.90 %, respectively (Fig. 3B). The fluorescence intensity of intracellular DCT micelles was generally higher than that of DC micelles, suggesting the higher cellular uptaking rate of DCT micelles (Fig. 3C).

Fig. 3 Cellular uptake of polyprodrug micelles. (A) Confocal images and fluorescence intensity profiles (left) of HeLa cells incubated with polyprodrug micelles or free CPT for 1 h, 2 h and 4 h. The scale bars are 20 μm (enlarged images) or 50 μm (original 16


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images). (B) The time-dependent cellular uptaking by flow cytometric analysis. (C) The mean fluorescence intensity of intracellular DC micelles and DCT micelles by flow cytometric analysis.

3.5 Mitochondrial damage and ROS enhancement The mitochondria damage can directly activate the mitochondrial apoptotic pathway.51,52 As described above, reductant GSH can trigger the drug release of polyprodrug micelles. CLSM imaging was employed to study the intracellular fate of the released CPT. After incubation with cells for 4 h, DCT micelles (blue fluorescence) were found colocalized with lysosome (red fluorescence) (Fig. 4A) and mitochondria (green fluorescence) (Fig. 4B ), which suggesting that the passive cellular upatake mechanism of polyprodrugs. The ROS level was evaluated using 2’, 7’-dichlorofluorescein diacetate (DCFH-DA), which could be rapidly oxidized by ROS to generate dichlorofluorescein (DCF) with green fluorescence. In Fig. 4C, notable green fluorescence was detected in DCT micelle-treated group after 4 h incubation, while there was much weaker green fluorescence in DC micelle-treated or control group. Finally, JC-1 staining was used to investigate the DCT-induced mitochondrial dysfunction (Fig. 4D). The damaged mitochondrial membrane has relatively higher permeability, making JC-1 diffuse away from mitochondria and disperse in cytoplasmic matrix in the form of monomers. Meanwhile, the corresponding fluorescence of JC-1 changes from red (aggregates) to green (monomers), suggesting the depolarization of mitochondrial membrane and damage of mitochondria. Compared to the control group and DC micelle-treated group, the DCT micelle-treated cells exhibited a much stronger green fluorescence. The overexpressed antiapoptotic Bcl-2 gene would improve drug resistence to cancer chemotherapy.53,54 As revealed by western blotting and relative quantitative analysis (Fig. S8A and S8B), the MCPS treated group significantly downregulated the expression of Bcl-2, implying that the damage of mitochondria by DCT was involved in

the

regulation

of

Bcl-2

expression.

These

results

indicated

that

mitochondria-targeted drug release could not only destroy mitochondrial DNA, but 17



also disrupt mitochondrial membrane potential, resulting in a higher oxidative stress due to the produced ROS and thereby the synergistically enhanced chemotherapeutic effect.

Fig. 4 Confocal microscopic analysis of cellular co-localization and the DCT-induced mitochondria membrane depolarization. Co-localization images of MCF-7 cells stained with (A) lysotracker red and (B) mitochondria-green after treatment with prodrugs for 4 h. (C) HeLa cells treated with polyprodrugs for 4 h and stained with DCFH-DA (Green) showing the intracellular ROS. (D) HeLa cells treated with prodrugs for 4 h and stained with JC-1 showing the mitochondrial membrane depolarization. The red fluorescence shows the aggregates of JC-1, while green fluorescence shows the monomers of JC-1.

3.6 The penetration and biodistribution of DCT micelles Multicellular spheroids (MCS) of MCF-7 cells were established to investigate the penetration ability of polyprodrug micelles into solid tumoral tissue. As shown in Fig. 18


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5A, MCS were first incubated with free CPT, DC micelles and DCT micelles for 6 h, the blue fluorescence of CPT was detected by recording the confocal images scanning at different depth. Similar to those treated with free CPT, polyprodrugs also reached the interior of the MCS (~ 80 μm) with constant intensity, indicating the good permeability of polyprodrug micelles into solid tumors. Tumor bearing mice were further used to demonstrate the accumulation of polyprodrug micelles in vivo (Fig. 5B). An NIR fluorescence imaging system was used to analyze the distribution of free Cy7, DC/Cy7 micelles and DCT/Cy7 micelles. Compared to the weak fluorescence of DC/Cy7-treated group, DCT/Cy7-treated group exhibited a much stronger fluorescence intensity near tumor site at 24 h post-injection. The CPT released from non-targeted DC micelles tends to randomly diffuse into the surrounding tissues and rapidly metabolized. Meanwhile, the mitochondria-targeted DCT micelles can assist transport of CPT into mitochondria and thereby better maintain the drug concentration in tumoral tissues. The major organs of treated mice excised and observed in vitro to verify the organ distribution of polyprodrug micelles (Fig. 5C). The fluorescence signals in tumors from DCT/Cy7 micelle-treated mice were remarkably improved, while those in heart, spleen, lung and kidney were weakened as compared to other treated mice. More specifically, the fluorescence intensity in the tumor site of DCT/Cy7 micelle-treated mice was about 2.2 or 1.3 fold higher than those in the mice treated with free Cy7 or DC/Cy7 micelles, respectively (Fig. 5D). The excised tumors were further sectioned and frosted for microscopic analysis by CLSM, which showed clearly the enhanced drug accumulation in tumors mediated by DCT/Cy7 micelles. These results were consistent with those above by other characterizations, indicating that DCT micelles could accumulate preferentially in tumor sites and reduce off-targeting side effects.

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Fig. 5 Deep tumor tissue penetration of polyprodrug micelles. (A) Confocal images of MCF-7 multicellular spheroids treated with free CPT or polyprodrug micelles at 37 C for 6 h. (B) Fluorescence imaging of 4T1 tumor-bearing BABL/C mice after injection with free Cy7, DC/Cy7 micelles or DCT/Cy7 micelles (Cy7 concentration: 1 mg/kg) via tail veins. The white circles denote the tumor site. (C) Ex vivo biodistribution of Cy7 and polyprodrug micelles in major organs (tumor, heart, liver, spleen, lung and kidney). (D) Quantitative comparison of Cy7 signal intensity in different organs. (E) CLSM images of frosted tumor slices showing the fluorescence of CPT and Cy7. Scale bars: 100 μm.

3.7 In vivo anticancer activity 20


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Encouraged by the promising results from the above investigations, we established animal models to evaluate the efficacy of mitochondria-targeted chemotherapy mediated by DCT micelles in vivo. In Fig. 6A, the DCT-treated group showed the optimal tumor suppression effect compared to those treated with free CPT or DC. Specifically, a significant decrease in tumor weight (Fig.6C) and tumor cell apoptosis (Fig. 6D and E) were observed after the treatment with DCT. Meanwhile, the mice in free CPT-treated group showed slight reduction of body weight particularly in the later stage (Fig. 6B), while there was no apparent effect on the groups treated with DC or DCT (such as eating, drinking, appearance, body weight, activity or neurological status) . Additionally, there were no obvious pathological changes in the major organs in DCT micelle-treated group, suggesting the systemic safety of the prodrug micelles. After 14 days of treatment, mice were sacrificed and the tumor and major organs were excised for histopathological analysis by TUNEL (Fig. 6D) and H&E staining (Fig. 6E). There was no obvious histological damage found in the major organs. On the other hand, the tumor sections from DCT-treated groups showed more obvious damage than those from other treatment groups (PBS, free CPT and DC), indicating the remarkable tumor suppressive ability of DCT micelles in vivo. These results provided a clear evidence for the unique advantages of DCT micelles, including improved mitochondria-targeted effect, enhanced subcellular uptake and accumulation effect, and the intracellular GSH-triggered on-demand CPT release.

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Fig. 6 Tumor suppression effect of mitochondria-targeted DCT micelles in vivo based on breast tumor-bearing mouse model. Variation of tumor volume (A) and mouse body weight (B) in two weeks after treatment. (C) The weight of tumors excised from mice subject to different treatments at day 14. *P

Mitochondria-Specific Anticancer Drug Delivery Based on Reduction

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