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Engineered polymeric micelles for combinational oxidation anticancer therapy through concurrent HO-1 inhibition and ROS generation Joungyoun Noh, Eunkyeong Jung, Jeonghun Lee, Hyejin Hyun, Seri Hong, and Dongwon Lee Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019
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Engineered polymeric micelles for combinational oxidation anticancer therapy through concurrent HO-1 inhibition and ROS generation Joungyoun Noh a, Eunkyeong Jung b, Jeonghun Lee b, Hyejin Hyun b, Seri Hong b, Dongwon Lee a,b,*
of PolymerNano Science and Technology, Chonbuk National University,
Baekjedaero 567, Jeonju, Chonbuk, 54896, Republic of Korea
of BIN Convergence Technology, Chonbuk National University, Baekjedaero
Cancer cells have a large amount of ROS (reactive oxygen species) because of disturbed ROS homeostasis. Cancer cells therefore undertake redox adaptation to drive proliferation in tumor environments and even survive during anticancer treatment by upregulating endogenous antioxidants. As one of antioxidant defense systems, heme oxygenase-1 (HO-1) acts as essential roles in tumor development by offering antioxidant bilirubin to protect cancer cells under stress conditions. It can be therefore reasoned that the combination of ROS generation and HO-1 inhibition would exert synergistic anticancer effects through the amplification of oxidative stress and provide a new opportunity for targeted anticancer therapy. To establish targeted anticancer therapy based on amplified oxidative stress, we developed molecularly engineered polymer, termed CZP, which incorporates ROS generating CA (cinnamaldehyde) and HO-1 inhibiting ZnPP (zinc protoporphyrin) in
its backbone and could form stable micelles in aqueous solutions. CZP micelles not only elevated oxidative stress but also suppressed the expression of antioxidant HO-1, leading to apoptotic cell death. CZP micelles could also significantly suppress the tumor growth without body weight loss, tumor recurrence and noticeable toxicity in organs. This study demonstrates that combination of ROS generation and HO-1inhibition synergistically magnifies oxidative stress to kill cancer cells and oxidative stress amplifying CZP micelles may provide a promising strategy in anticancer treatment.
Keywords: oxidative stress; oxidation anticancer therapy; cancer; micelles; heme oxygenase-1 1. Introduction In spite of substantial improvement in the drug discovery, molecular biology and oncology over the past decade, cancer is as one of the major causes of death worldwide as ever.1-3 One of the straightforward and powerful approaches for cancer treatment is chemotherapy using various
anticancer drugs, but most anticancer drugs have some limitations for instance, deficient bioavailability, lack of target specificity, the possibility to induce multidrug resistance and adverse side effects.2,4 To get over these shortcomings, extensive efforts have been dedicated to developing effective anticancer therapeutic strategy. “Oxidation therapy” is one of the recently developed anticancer therapies and has gained increasing attractions.5-7 Oxidation therapy exploits the redox imbalance in cancer cells. In comparison with normal cells, cancer cells have an elevated level of ROS such as singlet oxygen, hydrogen peroxide and hydroxyl radicals and therefore are under oxidative stress.7-8 Although the intracellular level of ROS in cancer cells are not exactly determined, almost all cancer cells are known to have excessive ROS such as prostate cancer cells and colorectal cancer cells.9-11 Cancer cells are hence more easily affected by therapeutic agents including arsenic trioxide (As2O3), cisplatin, glucose oxidase and cinnamaldehyde that stimulate ROS generation to kill cancer cells preferentially.5,12-13 On the other hand, quinone methide (QM), -phenylethyl isocyanate and buthionone sulfoximine were reported to deplete antioxidant defense systems and exacerbate the redox imbalance in cancer cells, giving rise to cell death.14-18 One of promising and powerful approaches to maximize oxidative stress in anticancer treatment would be the combination of ROS generation and antioxidant depletion.6,12,15,19 Concurrent delivery of ROS generators and antioxidant inhibitors to cancer cells could drastically amplify the oxidative stress to kill cancer cells and could be referred to as “combinational oxidation anticancer therapy”. Various combinations of ROS-generating compounds and antioxidant-inhibiting compounds could be made to achieve synergistic effects in the amplification of oxidative stress, targeting selective destruction of cancer cells.4,15 In this work, cinnamaldehyde (CA) and zinc
protoporphyrin (ZnPP) were selected as a ROS-generating compound and antioxidant-inhibiting agent, respectively. CA is a major ingredient in cinnamon which has been extensively used in foods including beverage, ice cream and candies and herbal medicine.20-21 Accumulative evidence has demonstrated that CA and its derivatives trigger apoptosis of cancer cells through mitochondrial membrane disruption and caspase activation.22-24 On the other hand, ZnPP is known to elevate oxidative stress by depleting antioxidant heme oxygenase-1 (HO-1), which is a heme degrading enzyme. HO-1 is present at low concentration under normal conditions, but plays a vital role in tumor development by protecting cancer cells under stress conditions such as heat shock, hypoxia, high-energy radiation, ROS and heavy metals.15,25-26 It has been reported that cancer cells exploit the cytoprotective functions of HO-1 as a shield from chemo- and radiotherapy. Moreover, Maeda et al. have recently reported that ZnPP exerts potent anticancer activity by inhibiting HO-1 and also moderates the therapeutic responses of cancer cells to ROS generating anticancer drugs.27-28 Although a number of researchers have documented the therapeutic potential of CA and ZnPP in cancer treatment, it is necessary to develop methodologies to deliver CA and ZnPP concurrently to targeted cancer cells to maximize synergistic therapeutic effects and overcome their intrinsic limitations such as poor solubility, low bioavailability and lack of targetability.15,29-33 One of the promising strategies to address these challenges encountered in combinational oxidation anticancer therapy would be the covalent incorporation of both ROS generators and antioxidant inhibitors in the chain of stimulus-responsive biodegradable polymer.30,34 This strategy could realize the concurrent delivery of both therapeutic agents and high drug loading capacity and also enhance their bioavailability. On the basis of great therapeutic potential of CA and ZnPP and
the advantages of polymeric prodrugs, we developed CZP (CA and ZnPP-incorporated polymer) as a combinational oxidation anticancer therapeutic agent.
Scheme 1. A schematic diagram showing the construction of CZP micelles with their working mechanism of combinational oxidation anticancer therapy.
As illustrated in Scheme 1, CA is incorporated covalently in the poly(-amino ester) through the formation of acid-sensitive acetal linkages. ZnPP is also grafted to side chains of the polymer. CZP could form thermodynamically stable micelles through self-assembly in aqueous environments and be activated by acidic pH in cancer microenvironments to
liberate CA and ZnPP. Therefore, the hypothesis behind the therapeutic actions of CZP micelles is that ZnPP-mediated inhibition of antioxidant HO-1 renders cancer cells highly vulnerable to CA-mediated ROS insults and therefore the elevated oxidative stress could induce preferential cancer cell death. In addition, CZP micelles with a mean diameter of <200 nm could target tumor passively through probably the enhanced permeability and retention (EPR) effect. Herein, we report the feasibility of CZP micelles as a combinational oxidation anticancer drug.
2. Materials and Methods 2.1 Materials. Cinnamaldehyde, poly(ethylene glycol) (PEG) methyl ether (Mn ~2,000), 2hydroxylethyl acrylate, hydroquinone, p-toluenesulfonic acid, 4,4′-trimethylenedipiperidine, protoporphyrin IX zinc (II) and ethanol amine were acquired from Sigma-Aldrich (St. Louis, MO, USA). Tetrahydrofuran, trimethylamine and dichloromethane were gained from Samchun (Korea). All chemicals and reagents used were of analytical grade. 2.2 Synthesis 2.2.1 Synthesis of compound 1. 2-Hydroxyl ethyl acrylate (41.62 mmol), trans-cinnamaldehyde (13.87 mmol), hydroquinone (5.173 mmol) and p-toluenesulfonic acid (0.07 mmol) were dissolved in 200 ml dry benzene under stirring in a round bottom flask. The reaction was allowed at 95C
overnight. After being cooled to room temperature, the reaction mixture was given trimethylamine (1 mL). After removing benzene under reduced pressure, Compound 1 was obtained by column chromatography with a mixture of hexane/ethyl acetate (2:8 v/v) and dried under vacuum. The chemical structure of compound 1 was confirmed by NMR. 2.2.2 Synthesis of compound 2. Compound 1 (2.88 mmol), 4.4’-trimethylene-dipiperidine (2.88 mmol), ethanolamine (0.288 mmol) and methoxy PEG monoacrylate (0.288 mmol) were dissolved in dry tetrahydrofuran under stirring in an round bottom flask at room temperature. Polymerization reaction was allowed for 96 h and the product was poured into cold hexane to terminate polymerization. Compound 2 was dried under vacuum. The chemical structure of compound 2 was proved by NMR and its molecular weight was verified by a gel permeation chromatography (Alliance HPLC, Waters, Milford, MA). 2.2.3 Synthesis of CZP. Compound 2, (1-ehtyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlorede) (EDC) and 4-(dimethylamino)pyridine were dissolved in dimethyl sulfoxide, to which ZnPP was added. The reaction mixture was stirred at room temperature and reaction was allowed for 48 h. The product was extracted by dichloromethane and water and the organic layer was removed to obtain red colored CZP (yield 82%). Conjugation of ZnPP was confirmed by FTIR (Spectrum GX, Perkin Elemer). The molecular weight of CZP was verified by a gel permeation chromatography. 2.3 Preparation and Characterization of CZP micelles. CZP (1 mg) dissolved in anhydrous tetrahydrofuran (100 µL) was added into 10 mL of phosphate buffer saline (PBS) of pH 7.4. After evaporation of tetrahydrofuran using a rotary evaporator, CZP micelles were
acquired at a concentration of 100 µg/mL. The hydrodynamic size of CZP micelles was measured using a particle size analyzer (Brookhaven Instrument Corp., Holtsville, NY). To observe the shape of the CZP micelles, a drop of CZP micelles was laid down on a grid. CZP micelles were analyzed using a transmission electron microscope. The critical micelle concentration (CMC) of CZP micelles in pH 7.4 PBS was determined employing pyrene. The fluorescence emission was monitored with an excitation wavelength of 334 nm using a fluorospectrometer (FP-6500, JASCO Corp., Japan). The ratio of I373/I384 was plotted against the CZP concentrations. UV-Vis absorption of the CZP micelles was analyzed with a UV-Vis spectrophotometer (S-3100, Scinco, Korea). The fluorescence images of CZP were acquired using a fluorescence imaging system (FOBI, Neoscience, Korea). 2.4 Cellular uptake of CZP micelles. Cells were grown in RPMI medium supplemented with 10 % fetal bovine serum at 37 ℃ in an incubator with 5% CO2. A549 cells were cultured in a glass bottom dish (MatTek Corp., Ashland, MA) and were treated with CZP micelles for 2 h or 6 h. After gentle washing with fresh medium, cells were observed under a confocal laser scanning microscope (LSM 510 510 Meta, Carl Zeiss, Inc. Germany). 2.5 Measurement of ROS generation. A549 cells were treated with various agents for 12 h. For the purpose of intracellular ROS measurement, cells were treated with dichlorodihydrofluorescein diacetate (DCFH-DA) for 15 min. The stained cells were observed under a confocal laser scanning microscope and the quantitatively analyzed using a flow cytometer (FACS Caliber Becton
Dickinson, San Jose, CA, USA). For mitochondrial membrane potential depolarization assay, cells were treated with various concentrations of therapeutic agents. After being transferred to a culture tube, cells were stained with JC-1 dye and then were quantitatively analyzed by flow cytometry. 2.6 Cytotoxicity of CZP micelles. A549 cells (5 105) were treated with various concentrations of CA, ZnPP of CZP for 24 h. Cells were given 100 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetarazolium bromide) solution and then incubated for 2 h. Dimethyl sulfoxide (900 µL) was added to each well. The absorbance at 570 nm was measured using a microplate reader (Biotek Instruments, Winooski, VT, USA). The cell viability was determined by comparing with the absorbance of untreated control cells. For apoptosis assay, cells were treated with Annexin VFITC and propidium iodide and incubated at room temperature in the dark for 15 min. Cells were then given 400 µL of 1 binding buffer. The level of apoptosis was analyzed using a flow cytometer. 2.7 Western blotting. After treatment with various agent, cells were lyzed using a lysis buffer on ice. Total proteins were extracted from the lysates and then separated by performing electrophoresis on polyacrylamide gel. The separated proteins were transferred to PVDF membranes using electric current. The membranes were probed for the proteins of HO-1, Bcl-2, Bax, caspase-3, PARP or β-actin. The amount of protein staining was determined by the chemiluminescent detection method. 2.8 Imaging of tumors with CZP micelles. Nude mice (BALB/c, 6 weeks old) were purchased from the Orient Bio, Korea. Tumors were established by injecting A549 cells (2 106 cells) subcutaneously in their left flank. When tumors developed to have a volume of ~250 mm3, CZP micelles (10 mg/kg) were individually injected intravenously through a tail vein. At 12 h and 24 h
after treatments, the fluorescence images of tumors were captured using a fluorescence imaging system (FOBI, Neoscience, Korea). 2.9 Anticancer therapy using CZP micelles. The tumor-bearing nude mice were randomly divided into five group (n = 4 each group). When tumor volumes reached ~200 mm3, CA (1.4 mg/kg), ZnPP (0.6 mg/kg), co-treatment of CZ with ZnPP or, CZP micelles (10 mg/kg) were injected intravenously through a tail vein. Administration was repeated every 3 days and the dimensions of tumors were measured. Tumors and organs were excised for histological examination. After formalin fixation, excised tumor and organs specimens were embedded in paraffin. The tissue blocks were sectioned with a thickness of ~5 µm. Histological analysis was performed with tissue sections stained with H&E (hematoxylin and eosin) and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Animal experiments were performed under the guidance of the Institutional Animal Care and Use Committee (IACUC) of Chonbuk National University.
2.10 Statistical analyses. One-way ANOVA using a program of GraphPad Prism 5.0 was performed to make comparison between groups. Probability (p) values of <0.05 were considered statistically significant.
3.1 Design and Synthesis of CZP. For the synthesis of CZP, considering excellent structural tunability, mild reaction condition, biocompatibility and ease of preparation as the most important criteria in the design of polymeric prodrugs, poly(β-amino ester) was explored as a framework.35 We first synthesized CA-diacrylate (Figure S1, compound 1) to covalently incorporate CA into the main chain of poly(β-amino ester). From the polymerization reaction of CA-diacrylate, trimethylene-dipiperidine, ethanolamine and PEG acrylate as shown in Figure S2, the amphiphilic CA-incorporated polymer (compound 2) was obtained. CZP was developed from the conjugation of ZnPP to the side hydroxyl group of compound 2. The content of ZnPP was optimized after careful consideration of conjugation capacity of poly(β-amino ester), HO-1 inhibiting activity of ZnPP and toxicity of ZnPP (5 M).28 As shown in Figure S3, the hydroxyl group of compound 2 was observed to disappear after conjugation of ZnPP. The chemical structure of CZP was further confirmed by FT-IR spectroscopy (Figure S4). Gel permeation chromatography revealed that the average molecular weight of CZP was ~8,300 Da with polydispersity of 1.25 (Figure S5). It was found that the contents of CA and ZnPP in CZP were ~20 wt% and ~8 wt%, respectively.
Figure 1. Synthesis and degradation of dual acid-sensitive amphiphilic CZP for combinational oxidation anticancer therapy.
CZP was amphiphilic because of a hydrophilic PEG block and a hydrophobic poly(β-amino ester) backbone that possesses acid-sensitive acetal linkages and protonable tertiary amine groups (Figure 1). Due to the amphiphilic nature and dual acid-responsiveness in the backbone, CZP could
self-assemble in aqueous solutions to form micelles and release CA and ZnPP in the mildly acidic tumor environments and endosomal compartments.4,12,31
3.2 Characterization of CZP micelles. Under aqueous conditions, amphiphilic CZP could form monodispersed micelles through self-assembly in the aqueous environment. The mean hydrodynamic diameter was ~160 nm (Figure 2a). As shown in Figure 2b, CZP micelles were uniformly round and were smaller than compound 2 micelles. The reason is that when hydrophobic ZnPP was conjugated in the backbone of polymer, the amphiphilic nature of ZnPP increased, allowing it to tightly pack and form more stable micelles. Hydrophobic fluorescent pyrene was used to determine the CMC of CZP micelles. From Figure 2c illustrating the index of micelle hydrophobicity (I384/I373) with CZP concentrations, the CMC was determined to be 10 µg/ml. We also investigated the pH-responsive behaviors of CZP micelles. The value of I384/I373 decreased with decreasing pH (Figure 2d). Under acidic conditions (pH< 5.5), the amine groups incorporated in CZP could be rapidly protonated and the acetal linkages could be also cleaved. As a result, the micelles lost the structural integrity and dissociated.12 The stability of CZP micelles was also studied by monitoring the change in their diameter. CZP micelle showed no noticeable
diameter change in neutral pH solution containing 10 wt% serum protein for 4 days (Figure 2e). However, upon the reduction of pH to 5.4 by the addition of HCl, their mean diameter abruptly increased because the amine groups incorporated in CZP underwent rapid hydrophobichydrophilic transition at acidic pH and CZP micelles disrupted. The pH-dependent morphology of CZP micelles was verified by transmission electron microscopy (Figure 2f).
Figure 2. Physicochemical characterization of CZP micelles. (a) Size distribution of CZP micelles. (b) Transmission electron micrograph of compound 2 micelles and CZP micelles. (c) Determination of CMC of CZP micelles. (d) pH-dependent stability of CZP micelles. (e) The change in the diameter of CZP micelles with or without 10 % FBS. Values are mean ± SD (n=4). (f) Transmission electron micrograph of CZP micelles after
incubation at pH 5.4. (g) 1H NMR spectra of CZP after hydrolytic degradation at pH 5.4. (h) UV-vis absorption of CZP micelles and compound 2. (i) Florescence images of CZP micelles at different concentrations.
We also carried out 1H-NMR to study the acid-triggered release of CA from CZP micelles during hydrolysis in D3COOD/D2O (pD 5.4). CZP could release noticeable amounts of CA from 12 h incubation at acidic pH 5.4 (Figure S6), demonstrated by the disappearance of acetal protons (~5.2 ppm) and the appearance of aldehyde protons at 9.5 ppm (Figure 2g). It is consistent with the release kinetics of CA from compound 2 in our previous study.4 Therefore, these observations clearly demonstrate that in an acidic environment, CZP micelles undergo sequential two step acidtriggered changes. Hydrophobic tertiary amine groups become hydrophilic due to protonation and then the acetal linkages are cleaved to release CA and ZnPP. The optical properties of CZP were analyzed by UV-vis spectroscopy. ZnPP in organic solvent has two distinct absorbance peaks, 300-450 nm and 500-600 nm.28,36 Consistent with the previous studies, CZP micelles exhibited strong absorbance at 300-450 nm and 500-600 nm due to the conjugation of ZnPP (Figure 2h). We also found that CZP micelles exhibit fluorescence emission, in a concentration-dependent manner (Figure 2i). It may be noted from these findings that ZnPP was successfully conjugated and the photophysical properties of CZP micelles may be useful for fluorescence imaging of tumor.
3.3 CZP micelles-induced ROS amplification and HO-1 expression. As CZP micelles were hypothesized to exert anticancer effects in the cells, the intracellular uptake of CZP micelles was verified using a confocal laser scanning microscope. From 3 h after incubation, strong and widespread fluorescence was shown in the cytoplasm of cells (Figure S7), supporting that CZP micelles are internalized into cells probably through endocytosis.37
We first investigated the effects of CZP micelles on the expression of HO-1 in cancer cells as the present work was intended to establish combinational oxidation anticancer therapy by regulating antioxidant HO-1. As shown in Figure 3a, after the treatment with CA for 12 h, the level of HO-1 in A549 cells increased to some extent. On the other hand, HO-1 inhibitor, ZnPP, significantly dropped the level of HO-1. These observations support our rationales that cells upregulate cytoprotective and antioxidant HO-1 in response to external stress such as CA-mediated elevation of oxidative stress and ZnPP reduces the cytoprotective effect in cancer cells by inhibiting HO-1 expression. The co-treatment of CA 50 µM and ZnPP 5 µM also caused significant reduction in the expression of HO-1, suggesting that the level of HO-1 is regulated by HO-1 inhibiting ZnPP rather than ROS generating CA. As expected, CZP micelles dramatically downregulated HO-1, in a concentration dependent manner. Theoretically, 50 µg/ml of CZP micelles could release 50 M of CA and 5 M of ZnPP. CZP micelles (50 µg/ml) suppressed the HO-1 expression more effectively than the mixture of equivalent CA (50 M) and
ZnPP (5 M). These findings demonstrate that CZP micelles are readily internalized into cells and deliver ZnPP to inhibit the cytoprotective HO-1. In addition, this supports the previous studies that ZnPP derivatives such as pegylated ZnPP inhibits the expression of HO-1, like free ZnPP.28 Interestingly, CZP micelles diminished the expression of HO-1 more effectively at 50 µg/mL than at 100 µg/mL. It can be reasoned that more ROS generation compromises the suppression on ZnPP-mediated HO-1 inhibition. CA causes the generation of ROS to kill cell death.20-21 We, therefore investigated whether CZP micelles could generate ROS in cancer cells using a ROS probe DCFH-DA. As shown in Figure 3b and Figure S8, moderate green fluorescence was observed with cells treated with CA and ZnPP for 12 h, indicating that the generation of intracellular ROS. When treated with both CA and ZnPP simultaneously, cells showed significantly increased fluorescence intensity compared with treatment of each. CZP micelles also induced remarkable ROS generation in cells, concentration-dependently. In particular, CZP micelles induced a larger generation of ROS than the mixture of equivalent CA and ZnPP. It can be explained by the rationale that free CA readily undergoes oxidation of aldehyde in cell culture medium prior to intracellular uptake, leading to conversion into cinnamic acid which does not generate ROS.33,38 In contrast, internalized CZP micelles release CA in cells and therefore induce ROS generation more effectively. To further certify the intracellular ROS generation, cancer cells were pre-treated with hydrogen peroxide scavenging catalase. As expected, catalase significantly inhibited CZP micelles-induced ROS generation. These findings indicate that CZP micelles release ZnPP and CA, which in turn synergize to enhance the generation of ROS mainly hydrogen peroxide. CZP micelles-induced elevation of intracellular ROS generation was further substantiated by flow cytometry (Figure 3c). The results of flow cytometric analysis were consistent with those of fluorescence imaging.
Therefore, we could reason that CZP micelles are a combinational oxidation anticancer therapeutic agent that magnifies the generation of intracellular ROS. One of the unique characteristics of ROS-mediated apoptosis of cancer cells is the mitochondrial damages which provoke mitochondrial membrane potential disruption.23,39 In order to substantiate whether the combinational oxidation anticancer therapy induces the mitochondrial damages, CZP micelles-treated cells were analyzed using a mitochondrial membrane potential probe, JC-1.40 At 16 h post treatment, CA, ZnPP and their combination enhanced mitochondrial depolarization, evidenced by the increased the green/red intensity ratio. As expected, CZP micelles also induced drastic mitochondrial membrane disruption (Figure 3d). These results demonstrate that CZP micelles could disrupt mitochondrial membrane potential and induce apoptosis of cancer cells.
Figure 3. CZP micelles-induced elevation of oxidative stress in A549 cells. (a) Modulation of HO-1 by CA, ZnPP and CZP micelles. (b) Confocal microscopic images of cells generating ROS. (c) The level of intracellular ROS in calls. (d) Changes in mitochondria membrane potential of cells. CAT stands for catalase.
3.4 Anticancer activity of CZP micelles in vitro. Motivated by the promising results that CZP micelles significantly elevate the intracellular ROS generation in cancer cells, the MTT assay was carried out to assess their cytotoxic effects on A549 cells. At 24 h post treatment, 50 µM CA and 5 µM ZnPP showed the marginal effect on the cell viability despite the elevation of ROS generation and mitochondrial membrane potential disruption. However, CZP micelles significantly decreased the cell viability, dose-dependently. The mixture of CA and ZnPP also showed moderate synergistic anticancer effects, but less extent than equivalent CZP micelles. As hydrogen peroxide, one of the most abundant ROS is degraded by catalase, the cytotoxic effect of CZP micelles was significantly diminished by the treatment of catalase (Figure 4a). These results support that anticancer activity of CZP micelles results mainly from ROS-mediated oxidative stress amplification
and CZP micelles serve as oxidation anticancer therapeutic agents. As shown in Figure S9, CZP micelles also displayed cytotoxicity against DU145 cells, but less extent than A549, probably because DU145 cells have a higher level of antioxidant defense systems. We therefore used A549 cells to elucidate the mechanism of anticancer activity of CZP micelles for the remaining of experiments.
Figure 4. CZP micelles-induced cell death. (a) Cytotoxicity of CZP micelles to A549 cells. **p<0.01, ***p<0.001 relative to the untreated group. (b) The effects of CZP micelles on
the expression of apoptosis-related proteins. (c) Flow cytometric analysis for apoptotic cell death. Western blot assay was performed to investigate the effects of CZP micelles on the expression of apoptosis-related genes including Bcl-2, Bax, procaspase-3 and PARP-1.41 As shown in Figure 4b, CA and ZnPP suppressed the expression of apoptosis-related proteins, to some extent. However, CZP micelles significantly downregulated the anti-apoptotic Bcl-2 and upregulated proapoptotic Bax. In addition, CZP micelles remarkably induced the cleavage of procacpase-3 and PARP-1. These results suggest that CZP micelles induce apoptosis through ROS- mediated mitochondria pathway and caspase activation. We further evaluated the apoptosis-inducing capabilities of CZP micelles by the flow cytometric assay using an Annexin V-FITC/propidium iodine method. After 24 h of treatment, CA and ZnPP induced apoptosis to some extent and co-treatment of CA and ZnPP caused more cell death than treatment of each. However, CZP micelles induced more apoptosis than co-treatment of equivalent CA and ZnPP, demonstrated by the larger population in the right upper and lower quadrant corresponding to apoptotic death (Figure 4c). As expected from Figure 4a, catalase remarkably suppressed CZP micelle medicated-apoptosis, indicating that CZP micelles cause ROS-mediated apoptosis.
3.5 Therapeutic anticancer activity of CZP micelles. The promising results of in vitro studies prompted us to study the antitumor therapeutic efficacy of CZP micelles in vivo. Based on the previous studies reporting that ZnPP has fluorescence emission at ~ 650 nm, we first investigated
the propensity of CZP micelles to accumulate in tumor using a fluorescence imaging system. Figure 5a shows the fluorescence images of tumor-bearing mice 12 h and 24 h after intravenous administration of CZP micelles. Although the fluorescence intensity of CZP micelles is low because of the limited tissue penetration resulting from the short emission wavelength (650 nm),15,28 tumors of CZP micelles-treated mice showed distinct fluorescence signal compared to background. The tumor preferential accumulation of CZP micelles can be realized by EPR effects because mainly of their small size and hydrophilic PEG corona.42
Figure 5. Therapeutic anticancer activity of CZP micelles. (a) Florescence images of tumor-bearing mice after administration of CZP micelles. (b) Images of tumor-bearing mice after treatment with various formulations. (c) Changes in tumor volume during the
We next investigated the in vivo anticancer activity of CZP micelles for 30 days of observation (Figure 5b-c). CA (1.4 mg/kg), ZnPP (0.6 mg/kg) and CZP micelles (10 mg/kg) were then administrated to mice bearing tumors of 200 mm3 through a tail vein injection every 3 days. Untreated mice exhibited continuous and gradual tumor growth. Both CA and ZnPP exhibited marginal antitumor effects. Co-treatment of CA and ZnPP exerted the higher antitumor activity than each alone. However, CZP micelles markedly suppressed tumor growth and their anticancer effects were significantly higher that than co-treatment of equivalent CA and ZnPP. It can be explained by the rationales that CA and ZnPP are not expected to target the same cancer cells concurrently due to the different pharmacokinetics, but CZP could deliver the both complementary pharmacophores (CA and ZnPP) into the same site simultaneously to realize synergistic and combined therapeutic effects. These findings also strongly demonstrate the importance of simultaneous delivery of both CA and ZnPP through their covalent incorporation in acid-activatable polymers. To further gain insight into the anticancer efficacy of CZP micelles, pathological examination of tumors was carried out (Figure 5d). A large number of dead cells which have disrupted membrane and significant DNA degradation in tumor sections were observed with a CZP micelles-
treated group. CZP micelles-treated groups also displayed a significantly large number of TUNEL positive cells, demonstrating that CZP micelles effectively kill tumor cells in through apoptosis and suppress the proliferation of tumor cells. The body weight of tumor-bearing mice was evaluated for 30 days of treatment since it reflects the general toxicity of anticancer agents. Mice showed no noticeable change in their body weight during the treatment (Figure S10). No obvious systemic toxicity of CZP micelles was also demonstrated by H&E staining, TUNEL staining and DHE staining (Figure S11). CA, ZnPP and CZP micelles induced negligible damages to organs. These observations suggest that CZP micelles have excellent safety profiles at therapeutic doses. 4. Conclusions CZP micelles were developed as a new family of combinational oxidation anticancer therapeutic agent. In design, amphiphilic CZP incorporates both ROS-generating CA and antioxidant HO-1 inhibiting ZnPP in its hydrophobic backbone and also has hydrophilic segment. CZP could form thermodynamically stable micelles through self-assembly under aqueous conditions. CZP micelles disrupted preferentially at acidic pH and release CA and ZnPP in acid-triggered manners. Additionally, CZP micelles suppressed antioxidant HO-1 and generated ROS to synergistically elevate oxidative stress, giving rise to apoptotic cell death. CZP micelles exerted strong antitumor effects in a mouse xenograft model without conspicuous adverse effects. We anticipate that oxidative stress augmenting CZP micelles hold tremendous potential as anticancer therapeutic agents.
Supporting Information The Supporting information is available free of charge. Synthesis and characterization of CZP. Results of biological experiments.
Acknowledgements This work was supported by a grant of Korean Health Industry Development Institute (HI15C1619), Ministry of Health & Welfare and Mid-career Research Program through National Research Foundation (2016R1A2B4008489), Ministry of Science, ICT and Future Planning, Republic of Korea.
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