Specific Cancer Cytosolic Drug Delivery Triggered by Reactive

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Specific cancer cytosolic drug delivery triggered by reactive oxygen species-responsive micelles Lu-Yi Yu, Geng-Min Su, Chi-Kang Chen, Yi-Ting Chiang, and Chun-Liang Lo Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00916 • Publication Date (Web): 18 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016

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Specific cancer cytosolic drug delivery triggered by reactive oxygen species-responsive micelles Lu-Yi Yu †, Geng-Min Su †, Chi-Kang Chen †, Yi-Ting Chiang ‡, Chun-Liang Lo †, §, * †

Department of Biomedical Engineering, National Yang Ming University, Taipei, Taiwan 112,

ROC ‡

School of Pharmacy, China Medical University, Taichung 40402, Taiwan, ROC

§ Biophotonics & Molecular Imaging Research Center (BMIRC) and Biomedical Engineering Research Center, National Yang Ming University, Taipei, Taiwan 112, ROC [*] To whom correspondence and reprint requests should be addressed. Prof. C.L. Lo E-mail: [email protected] Fax: + 886-2-2821-0847 KEYWORDS: reactive oxygen species-responsive, micelles, endosomal escape, cytosolic drug delivery, endocytosis.

ACS Paragon Plus Environment

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ABSTRACT:

Cytosolic drug delivery, a major route in cancer therapy, is limited by the lack of efficient and safe endosomal escape techniques. Herein, we demonstrate a reactive oxygen species (ROS)responsive micelle composed of methoxy polyethylene glycol-b-poly(diethyl sulfide) (mPEGPS) copolymers which can induce specific endosome escape in cancer cells by changes in the hydrophobicity of copolymers. Owing to the more ROS levels in cancer cells than normal cells, the copolymers can be converted into more hydrophilic and insert into and destabilize the cancer intracellular endosome membrane after cellular uptake. More importantly, we show that acidintolerant drugs successfully maintain their bioactivity and cause selective cytotoxicity for cancer cells over normal cells. Our results suggest that the endosomal escape induced by hydrophobic-hydrophilic exchange of copolymers has great potential to locally and efficiently deliver biological agents (e.g., proteins and genes) in the cancer cell cytosol.


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■ INTRODUCTION

The endocytic internalization pathway is the major uptake mechanism of cells used in nanoparticle delivery. Most nanoparticle-encapsulated biological agents, such as drugs, genes, oligonucleotides, and proteins, are unstable in acidic endocytic compartments; therefore, the endosomal escape of nanoparticles or their encapsulation agents becomes the determining factor for improving therapeutic efficacy.1-3 Several methods, such as the proton sponge effect, endosomal membrane fusion, and photochemical internalization can induce the endosomal escape of nanoparticles or biological agents.1-4 In the proton sponge technique, nanoparticles should have the ability to absorb protons at low pH.1,2 However, such nanoparticle types typically have excess tertiary amines, yielding strong positive charges and causing severe cell cytotoxicity in either normal or tumorigenic cells.3,5 For photochemical internalization, photosensitizers should be combined with nanoparticles to induce reactive singlet oxygen for destroying the endosomal–lysosomal membranes.1,4,6 However, this method requires irradiation for stimulating the photosensitizers and is used in extremely shallow tissues. Otherwise, endosomal membrane fusion is limited to some viruses and mimicked viral particles. To implement this pathway, mimicked viral particles should be additionally modified using some specific virus peptides such as HA27,8 or GLAL9,10. To overcome the aforementioned defects and limitations, we developed a reactive oxygen species (ROS)-responsive micelle, which was self-assembled from methoxy polyethylene glycolb-poly(diethyl sulfide) (mPEG-PS) copolymers, to precisely induce endosomal escape in cancer cells, as illustrated in Scheme 1. The diblock copolymers were polymerized through condensation polymerization from bis(4-nitrophenyl) diethyl sulfide, thiobis(ethylamine), and


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mPEG. It is known that the levels of ROS in cancer cells are 100 times higher (approximately 100 µM) than those in normal cells.11-13 Therefore, the first step for achieving cytosolic drug delivery was cancer-specific oxidation of polysulfide via high levels of H2O2 (H2O2, a major ROS species in cancer cells

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) to convert sulfides into sulfoxides and sulfones. Under H2O2

oxidation, the hydrophobic core of micelles gradually becomes hydrophilic and the payloads released into endosomes. The following second step was that the dissociated polymers subsequently inserted into endosomal membranes and destabilized them to release the payloads into the cytosol. Herein, we used α-tocopheryl succinate (α-TOS), a vitamin E derivative, as the payload because it has been confirmed to induce cell apoptosis in various cancer cells and exhibit low toxicity in normal cells.14-16 However, the ester bond of α-TOS hydrolyzes to become α-tocopherol (α-TOH) under acidic conditions and loses its apoptotic ability.17 Encapsulating αTOS in micelles could determine whether the micelles specifically respond in cancer cells and precisely release α-TOS into the cytosol. Although the ROS-responsive micelles had been widely used in drug delivery18-24, this study presents a first example of a potential endosomal escape method from the hydrophobic-hydrophilic exchange of copolymers, which could not only specifically deliver biological agents into the cancer cell cytosol but also apply this idea to other types of responsive materials.


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■ MATERIALS AND METHODS

Materials mPEG (MW, 5000), α-TOS, DAPI, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), Dulbecco modified Eagle medium, McCoy’s 5A, and other organic solvents were purchased from Sigma-Aldrich Scientific. 2,2′-Thiodiethanol was purchased from Ferak Berlin. H2O2 and ethyl ethanoate were purchased from MP Biomedicals. Foetal bovine serum and penicillin/streptomycin were purchased from Invitrogen. Cy5.5-NHS ester was purchased from GE Healthcare Life Science. LysoTracker®Red DND-99 and CellMask™ Orange were purchased from Thermo Fisher Scientific. The Spectra/Por® 1 dialysis membrane (MW cut-off [MWCO], 6000-8000) was purchased from Spectrum Labs. Synthesis of mPEG-nitrophenyl carbonate mPEG (1 mmol) and triethylamine (1.1 mmol) were dissolved in dichloromethane (DCM) under nitrogen; 4-nitrophenylchloroformate (1.1mmol) was added dropwise and reacted at 0 °C for 24 h. After the reaction, the product was precipitated from diethyl ether three times and dried in vacuum for 1 day. The product was subsequently analyzed through 1H-NMR (Bruker NMR 400 MHz) and Fourier transform infrared spectroscopy (FTIR, SHIMADZU IRAffinity-1), as shown in Supporting information Figs. S1 and S2. The purity of mPEG-nitrophenyl carbonate was approximately 89% through 1H-NMR. mPEG-nitrophenyl carbonate: (DMSO-d6): δ 3.0-3.1 (m, -CH3 from mPEG), δ 3.3-3.4 (s, -O-CH2CH2-O- from mPEG),δ 7.6,8.3 (d, -CH from nitrophenyl). mPEG-nitrophenyl carbonate (KBr): 1176 cm-1 (C-O-C stretching), 1258 cm-1 (OC stretching), 1732 cm-1 (C=O stretching), 2700-2900 cm-1 (C-H stretching).


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Synthesis of bis(4-Nitrophenyl) diethyl sulfide. Thiodiethanol (1 mmol) and triethylamine (3 mmol) were dissolved in DCM under nitrogen. 4-Nitrophenylchloroformate (3 mmol) was subsequently added into the mixture at 0 °C for 24 h. The mixture was extracted with an NaCl saturated solution three times and recrystallized in ethyl acetate to obtain the final product. The product was analyzed through 1HNMR and FTIR, as shown in Supporting information Figs. S3 and S4.The purity of bis(4nitrophenyl) diethyl sulfide was approximately 100%, as assessed through 1H-NMR. bis(4nitrophenyl) diethyl sulfide (DMSO-d6): δ 2.9, 4.4 (t, CH2CH2 from 2.2’-thiodiethanol), δ 7.6, 8.3 (d, CH from nitrophenyl). bis(4-nitrophenyl) diethyl sulfide (KBr): 1176 cm-1 (C-O-C stretching), 1732 cm-1 (C=O stretching), 3300 cm-1 (-OH stretching). Synthesis of ROS-responsive block copolymers. The block copolymers were synthesized through condensation polymerization. Typically, mPEG-nitrophenyl carbonate (1 mmol) with different molar ratios of bis(4-nitrophenyl)diethyl sulfide (27, 45, and 54 mmol), 2,2’-thiobis(ethylamine) (30, 50, and 60 mmol), and triethylamine (27, 45, and 54mmol) were dissolved in dimethyl sulfoxide (DMSO) and reacted at 50 °C for 5 days under nitrogen. The reaction mixture was precipitated from diethyl ether three times, dialyzed against DMSO, and subsequently freeze-dried to obtain mPEG-PS copolymers. The pure product was analyzed through 1H-NMR, GPC (SHIMADZU), and FTIR, as shown in Supporting information Figs. S5-S7. mPEG-b-poly(diethyl sulfide)-NH2 (DMSO-d6): δ 2.9, 4.4 (m, CH2CH2 from 2.2’-thiodiethanol), δ 3.0-3.1 (m, -CH3 from mPEG) , δ 3.3-3.4 (s, -OCH2CH2-O- from mPEG). mPEG-b-poly(diethyl sulfide)-NH2 (KBr): 1176 cm-1 (C-O-C


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stretching), 1258 cm-1 (C-O stretching), 1611 cm-1 (N-H bending), 1732 cm-1 (C=O stretching), 2850 cm-1 (C-H stretching), 3335 cm-1 (N-H stretching). Preparation and characterization of micelles. The mPEG-PS copolymers (2 mg) were dissolved in DMSO (8 mL) and placed into dialysis bags (MWCO, 6000-8000) for preparing micelles through dialysis. The particle sizes and size distributions of micelles were determined through DLS (Zetasizer 3000 HSA, Malvern). The particle sizes were analyzed using the CONTIN method. In addition, surface charges of the micelles were analyzed using a zeta nanoparticle analyzer (NanoPlus, Particulate Systems) by combining a heterodyne system and the photon correlation method. The micelle morphology was observed through TEM (JEM-2000EXII, JEOL) at an accelerating voltage of 100 k by using 2% of uranyl acetate for micellar positive stain. Stability and ROS-responsive of micelles. The stability test was conducted by incubating the micelles at pH5.5 and pH7.4 phosphatebuffered saline (PBS) and 37°C for 3 days. In addition, the ROS-responsive tests were conducted by mixing the micelles with 133 µM and 1.33 µM H2O2 at 37°C for 3 days. After 1, 3, 6, 12, 24, 48, and 72 h, the micelles were measured through DLS to obtain particle sizes and size distributions. The morphologies were also observed through TEM at 1, 3, and 6 h by using 2% of uranyl acetate for positive staining. Preparation of α-TOS-loaded micelles. The mPEG-PS copolymers (2 mg) and α-TOS (2 mg) dissolved in DMSO were dialyzed against ddH2O to fabricate α-TOS-loaded micelles. Theα-TOS-loaded micelles were freeze-


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dried and subsequently analyzed through UV-Vis spectrometry (Lambda 35, PerkinElmer) at 291 nm to determine the drug content and loading efficiency. Characteristics of the copolymers after oxidation. First, the ROS-responsive tests were conducted by treating the copolymers in pH 7.4 PBS with 133 µM and 1.33 µM H2O2 at 37 °C. After 0, 3, 12, and 24 h, the copolymers were dialyzed against ddH2O to obtain oxidized copolymers and subsequently analyzed using elemental analyzer (FLASH 2000 CHNS/O Analyzers, Thermo Fisher Scientific) to understand the degree of oxidation. The copolymers were also analyzed using FT-IR to understand the chemical structure change of copolymers. Second, the copolymers were treated in pH7.4 PBS with 133 µM H2O2 at 37 °C. After 0, 3, 6, 9, 12, and 24 h, the copolymers were dialyzed against ddH2O to obtain oxidized copolymers, which were dissolved in methanol and analyzed through GPC to determine the changes of molecular weight of copolymers. Third, the copolymers were treated in pH7.4 PBS with 133 µM H2O2 at 37 °C. After 0, 3, 6, 9, 12, and 24 h, the copolymers were dialyzed against ddH2O to collect oxidized copolymers, which were dissolved in ddH2O for analyzing the zeta potential of these copolymers by using a zeta nanoparticle analyzer. And, the copolymers were dissolved in pH 2.0 ddH2O and titrated with 0.1 N NaOH for analyzing the buffering capacity. Finally, the copolymers were treated in pH 7.4 PBS with 133 µM H2O2 at 37 °C. After 0, 3, 6, 9, 12, and 24 h, the copolymers were dialyzed against ddH2O to collect oxidized copolymers. Different concentrations of copolymers were subsequently prepared for a CMC study. The CMC of each copolymer was determined using a fluorescence technique by using pyrene as the hydrophobic fluorescence probe. A saturated aqueous solution of pyrene (6 × 10−7 mol/L) was used for these experiments. Fluorescence intensities of the pyrene-encapsulated micelle core were determined using a multimode microplate reader (Tecan Infinite 200). The


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excitation wavelength for the emission spectra was 332 nm, and the excitation spectra were recorded at 373 and 383 nm. Hemolysis and the interaction of the copolymers with RBCs. First, blood was collected from healthy New Zealand rabbit. The serum was then removed by centrifugation at 1200 rpm for 5 min to obtain RBCs, and the RBCs were washed three times with a sterile isotonic 0.9% NaCl solution. Otherwise, the copolymers were oxidized in pH7.4 PBS with 133 µM H2O2 at 37 °C. After 0, 3, 6, 9, 12, and 24 h, they were dialyzed against ddH2O to collect oxidation copolymers; these copolymers (10 mg) were then dissolved in ddH2O (1mL) with sonication. The copolymers (10 mg) in ddH2O (1.2 mL) were then mixed with RBC suspensions (200 µL). Positive and negative controls were also prepared by mixing 200 µL of RBC suspensions with 1.2 mL of ddH2O and 0.9% NaCl, respectively. Following incubation for 6 and 12 h, the samples were centrifuged for 5 min at 4000 rpm. The absorbance of the supernatant was measured through UV-Vis spectroscopy; the absorbance is directly proportional to the amount of hemoglobin released. Cy 5.5-NHS ester was subsequently reacted with oxidation copolymers for 1 day and dialyzed against ddH2O to remove extra Cy5.5. The RBCs were labelled with the CellMask™ Orange Plasma Membrane stain for 1 day and were washed three times with sterile isotonic 0.9% NaCl solution through centrifugation at 1200 rpm for 5 min. The Cy5.5-labelled copolymers were co-cultured with RBCs under coverslides at 37 °C for 0, 3, 6, 9, 12, and 24 h and observed through confocal laser scanning microscopy (CLSM; Zeiss LSM 880). CLSM was performed to observe the internalization by using excitation wavelengths of 405, 559, and 635 nm and emission wavelengths of 567 and 694 nm for CellMask™ Orange Plasma Membrane stain (Thermo Fisher Scientific) and Cy 5.5 (GE Healthcare), respectively.


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The endosomal escape ability in HCT116 and L929 cells. To obtain fluorescent dye-labelled α-TOS-loaded micelles, Cy5.5-NHS ester was reacted with mPEG-PS copolymers for 1 day and subsequently mixed with α-TOS to prepare micelles through dialysis. The accumulation of micelles and endosomal escape behavior in HCT116 and L929 cells were determined through CLSM (Olympus FV1000). HCT116 and L929 cells were seeded on coverslides for 12 h at 37 °C and subsequently treated with α-TOS loaded micelles for 3, 6, 9, and 24 h. The cells were washed twice with PBS to remove α-TOS-loaded micelles and were subsequently cultured in a pH 7.4 medium. After an interval, the cells were washed twice with PBS and mounted on a slide with 4% paraformaldehyde for CLSM. CLSM was performed to observe the internalization by using excitation wavelengths of 543 and 633 nm and emission wavelengths of 405, 559, and 635 nm as well as 461, 590, and 694 nm for DAPI, LysoTracker®Red DND-99 (Thermo Fisher Scientific) and Cy 5.5 (GE Healthcare), respectively. Drug release behaviors. α-TOS-loaded micelles were collected in dialysis bags (MWCO, 6000–8000) and coincubated in pH7.4 and pH5.5 with 133 µM H2O2 and 1.33 µM H2O2 at 37 °C to determine αTOS-releasing behaviors. At time intervals, the α-TOS concentration was determined through UV-Vis spectrometry at an absorption wavelength of 291 nm. Cytotoxicity and α-TOS bioactivity evaluation. The cytotoxicity of α-TOS, empty micelles, and α-TOS-loaded micelles was evaluated using the MTT assay for L929 and HCT116 cells. α-TOS, empty micelles, and α-TOS-loaded micelles were incubated with the cells (1 ×104 cells) for 24 and 48 h. Following the incubation,


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the medium in each well was removed and washed with PBS twice. The MTT assay was subsequently performed to evaluate the cell viability by using an ELISA reader. The cell cycles were also analyzed. HCT116 and L929 cells (1 ×105 cells) were incubated with α-TOS (50 µM) and α-TOS-loaded micelles (containing 50µM of α-TOS). Following 3-, 6-, and 24-h incubation, α-TOS and α-TOS-loaded micelles were removed. The cells were washed with PBS twice and collected and fixed with iced ethanol for 30 min. The cells were subsequently collected through centrifugation, washed with PBS twice, stained with propidium iodide solutions for 30 min, and analyzed through flow cytometry (BD FACSCalibur). The cell cycle phases were determined using ModFit LT, and α-TOS hydrolysis was analyzed. HCT116 and L929 cells (1 ×105 cells) were incubated with α-TOS (100 µM), α-TOH, α-TOS-loaded micelles (containing 100 µM of αTOS). Following 24-h incubation, α-TOS (100 µM) and α-TOS-loaded micelles were removed and lysed through sonication for 5 min. Following centrifugation at 7000 rpm for 10 min, the supernatant was freeze-dried and dissolved in ethanol. The α-TOS levels in these solutions were analyzed through HPLC (SHIMADZU). Statistical analysis. All data were presented with an average values and its standard deviation, shown as mean ± S.D. Comparison between groups was analysis with the two-tailed Student's t-test (Excel, 2007). Differences were considered statistically significant when the p values were less than 0.05 (p < 0.05).


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■ RESULTS AND DISCUSSION

Micelle preparation and characterization. The mPEG-PS diblock copolymers were synthesized using three compositions of sulfide 10, 14, and 21 repeating units. The detailed characterizations are shown in Supporting information (Supporting information, Figure S1-7 and Scheme S1-3). The micelles were prepared by assembling the copolymers through dialysis. We observed that the copolymers with 14 repeating units of sulfide had more desirable particle size and size distribution, approximately 144 nm and 0.132, respectively (Table 1). The morphology of micelles with 14 repeating units of sulfide was analyzed through transmission electron microscopy (TEM, Supporting information, Figure S8). The micelles were found to be spherical, but the particle sizes of micelles around 100 nm. The sizes by TEM observations were smaller than their hydrodynamic diameter, perhaps because the drying process for TEM observations shrunk the structure of micelles. To obtain α-TOS-loaded micelles, the same gram ratio of the 14 repeating units of sulfide-containing copolymers and αTOS was mixed, followed by dialysis against ddH2O. The size of the drug-loaded micelles increased to 225 nm, and the size distribution was 0.03 (Table 1). TEM indicated a spherical morphology (Supporting information, Figure S8). The drug loading capacity and encapsulation efficiency of micelles measured through 42.2±3.3 wt% and 32.3±1.9 wt%, respectively. The zeta potential value of micelles determined using a zeta nanoparticle analyzer was -9.66 mV. ROS-responsive studies. To determine the characteristics of the copolymers, we first oxidized them in different concentrations of H2O2. The element contents of copolymers during 24 h were monitored using


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elemental analyzer. Figure 1a demonstrates that the degree of oxidation (oxygen contents) of the copolymers in high levels of H2O2 (133 µM) significantly increased with time and were higher than that in low levels of H2O2 (1.33 µM). The oxygen-to-sulfur ratio of the copolymers revealed that the oxygen atoms increased 1.76-fold after 24 h oxidation (Supporting information, Figure S9). Additionally, the wavenumber for sulfone groups at 1396 cm-1 was clearly observed from FT-IR, indicating that the copolymers could be oxidized to form not only sulfoxide groups, but sulfone groups (Supporting information, Figure S10). Studies have reported that polyurethane can be degraded at high H2O2 levels.25,26 Therefore, gel permeation chromatography (GPC) was used to measure the changes in the molecular weight (MW) of the copolymers at 133 µM H2O2. We observed no degradation of the copolymers after 24 h (Figure 1b). Moreover, the critical micelle concentrations (CMCs) of the copolymers were determined to understand the hydrophobic-hydrophilic exchanges among the copolymers. The CMC of the copolymers after oxidation changed with oxidation time: 0.035 mg/mL for 3 h oxidation; 0.099 mg/mL for 9 h; and 0.145 mg/mL for 24 h (Supporting information, Figure S11); this proved that the copolymers became more hydrophilic, which can induce their dissociation from micelles.27,28 We also measured the zeta potential of the copolymers after oxidation to understand the surface charge changes. The results revealed that copolymers maintain neutral charges under acidic conditions (Figure 1c), indicating that copolymers cannot electrostatically interact with the cancer endosomal membrane. The buffering capacity of copolymers also revealed that copolymers cannot absorb protons to induce proton sponge effect (Supporting information, Figure S12). The stability of the micelles was then evaluated at pH 7.4 and 5.5. We observed that the micelles were stable at both pH conditions for 24 h (Supporting information, Figure S13). After 3 days, the particle size of the micelles at pH 5.5 slightly increased by approximately 20%. These


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results indicated that pH is not the key factor affecting copolymer dissociation in the initial 24 h. Subsequently, the micelles were treated with different H2O2 concentrations and pH. The micelles exhibited significant size, size distribution, kilo counts per second (Kcps), and morphological changes under high H2O2 conditions at pH 5.5 but not at pH 7.4 (Figure 1d-1f). TEM revealed micelle disruption under high H2O2 and low pH conditions after 3 h (Figure 2). These experimental results emphasize that H2O2 is the major induced response factor for micelles, whereas pH is a cofactor enhancing micellar dissociation. Interaction of the copolymers with RBCs. Because oxidized copolymers are not completely dissolved in water, the oxidized copolymer cluster can be used to replace micelles in a simulated endosomal escape study. Red blood cells (RBCs) are the standard model for simulating endosomes.29,30 First, through UV-Vis spectrometry, we determined the hemolysis of RBCs co-cultured with copolymers having different oxidation degrees to investigate which oxidation degree and co-culture time point cause RBC disruption. The results indicate that the copolymers oxidized for 9 h and co-cultured with cells for 12 h showed a higher hemoglobin release (approximately 18%) than did the other copolymers (Figure 3a and Supporting information, Figure S14). After copolymer oxidation for 9 h, the oxidation degree inversely correlated with the observed hemolysis. This was because a higher oxidation of copolymers renders them too hydrophilic for insertion into the cell membrane. Second, the oxidized copolymers were labelled with Cy5.5 at the end of the PS segments, and the RBCs were stained with CellMask Organe fluorescent dyes. The insertion behaviors of the copolymers with 9 h oxidation on the RBCs were directly monitored through confocal microscopy. We observed that the superimposition of the copolymers and cell membrane increased with time (Figure 3b and Supporting information, Figure S15), showing that


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the copolymers gradually inserted into the RBCs. At 9 h co-incubation, a portion of lysed RBCs were observed. On the basis of confocal imaging and copolymer characteristics, we summarized that high H2O2 levels can change micelle hydrophobicity and cause micelle dissociation, leading to copolymer insertion into the RBC membrane and thereby induce membrane destabilization. The endosomal escape in HCT116 and L929 cells. The next step was to determine the endosomal escape ability at the cellular level; HCT116 colon cancer cells representing high levels of H2O2 and L929 mouse fibroblast cells representing low levels of H2O2 were co-cultured with α-TOS-loaded micelles. The copolymers were labelled with Cy5.5 before micelle preparation. The cells were treated with LysoTracker Red DND-99 and DAPI for staining endosomes and nucleus, respectively. In L929 cells, we observed that the fluorescent intensity of the copolymers overlapped with that of the LysoTracker dye and was quenched because the copolymers were tightly assembled into the micelles at 9 h (Figure 4). At 12 h, the related high fluorescence of the copolymers out of the micelles appeared in endosomes, indicating the poor endosomal escape ability of the micelles under low H2O2 conditions. However, for HCT116 cells, copolymer fluorescence was high and overlapped in the endosomes after 6 h, demonstrating that copolymers were oxidized and dissociated from the micelles. From 9 to 12 h, fluorescent intensity from the copolymers out of the endosomes was significant, revealing that the copolymers were inserted into the endosomal membrane and resulted in its disruption. Most studies have indicated that endosomal escape of nanoparticles or biological agents initiated at 6 h.30,31 Our study demonstrated that this ROS-responsive copolymer induce endosome disruption at 6-9 h, which was not inferior to that reported in previous studies. Cytotoxicity and drug bioactivity evaluation.


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To further evidence the specific cytosolic drug delivery of biological agents in cancer, we first evaluated the drug release behavior of α-TOS-loaded micelles at different H2O2 and pH. The release behaviors of α-TOS from micelles were pH independent and showed significant differences at different H2O2 concentrations. The α-TOS release rates of the micelles at high H2O2 but different pH conditions were similar; almost 100 wt% of α-TOS was released after 3 days (Figure 5a). However, the micelles released only 40 wt% of α-TOS at low H2O2. The results indicated that micelles have favorable response ability to ROS. Through confocal microscopy, we used the same cells to investigate the cytotoxicity of empty and α-TOS-loaded micelles. We observed that empty micelles had favorable biocompatibility, but α-TOS-loaded micelles had higher cytotoxicity to cancer cells (Figure 5b). The IC50 values of α-TOS-loaded micelles and free α-TOS for HCT116 cells were approximately 60 and 40 µM after 24 and 48 h co-incubation, respectively. Almost all cancer cells were killed by 100 µM of α-TOS-loaded micelles following the 48 h treatment. However, more than 60% of L929 cells survived in 100 µM of α-TOS-loaded micelles for 48 h (Figure 5b). Notably, the α-TOS has been confirmed to induce cell apoptosis in various cancer cells and exhibit low toxicity in normal cells.14-16 Free α-TOS showed less effective toward the normal cells is expected. Additionally, free α-TOS could penetrate into cell by diffusion, while micelles could be uptaken into cells not by diffusion but by endocytosis. Therefore, free α-TOS molecules could keep their bioactivity and showed high effective toward HCT116 cells. In contrast, the released α-TOS from micelles was hydrolyzed in the acidic endosomal surroundings. mPEG-PS micelles in this study could induce faster endosomal escape to avoid α-TOS hydrolysis. Thus, the cytotoxicity of HCT116 cells treated by α-TOS-loaded micelle was similar to that by free α-TOS treatment. We also investigated the effect of α-TOSloaded micelles on the cell cycle. The experimental results clearly indicated that α-TOS-loaded


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micelles (50 µM, less than the IC50 value) arrested the cell cycle of HCT116 cells at the S phase for 24 h (Figure 5c), which was in concordance with previous studies.14-17,32 However, the bioactivity of free α-TOS on the cell cycle under the same concentration was negligible for 24 h, perhaps because the free α-TOS concentration was insufficient to display significant differences. In L929 cells, the patterns of all treatments were similar because α-TOS-loaded micelles were insensitive in low H2O2 cells (Figure 5c). Finally, we measured the concentration of α-TOS in cancer and fibroblast cells through high-performance liquid chromatography (HPLC) after incubating α-TOS-loaded micelles with the cells for 24 h. We observed a large proportion of αTOS (94 wt%) in cancer cells, indicating that micelles provided high efficiency to protect payload and most α-TOS molecules were not degraded owing to their successful escape from endosomes (Figure 5d). Nevertheless, a large amount of α-TOS (almost 100 wt%) was converted to α-TOH in L929 cells because of hydrolysis at low pH endosomes or secondary lysosomes.


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■ CONCLUSIONS

In conclusion, we developed a safe, specific, and efficient platform, unlike the most conventional methods, for endosomal escape based on hydrophobic-hydrophilic exchange of ROS-responsive copolymers for specific drug delivery in the cancer cell cytosol. Our experimental results indicate that ROS-responsive micelles not only preserved the bioactivity of the payload but also exhibited selective cytotoxicity in cancer cells over normal cells. In light of the endosomal escape performance, the further design of the hydrophobic-hydrophilic exchanged copolymers will open new opportunities for protein or gene therapy in cancer.


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■ ASSOCIATED CONTENT

Supporting information This supporting information is available free of charge via the Internet at http://pubs.acs.org. Experimental material, Synthesis scheme, NMR data, FT-IR data, GPC data, DLS data, EA data, CMC data, Zeta data, Hemolysis data, Confocal data, including Scheme S1-S3, Figures S1S15.


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■ AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]. Fax: +886-2-2821-0847. Author Contributions C.-L. Lo and L.-Y. Yu designed the experiments. L.-Y. Yu, G.-M. Su, C.-K. Chen and Y.-T. Chiang performed the experiments. L.-Y.Yu and C.-L.Lo analyzed results and wrote the manuscript.


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■ ACKNOWLEDGMENT The authors would like to thank the National Health Research Institutes (NHRI) of the Republic of China, for financially supporting this work (NHRI-EX105-10527EI). TEM images and flow cytometry were supported in part by the Electron Microscopy Facility and Flow cytometry Core Facility in NYMU. Confocal images were supported in part by Imaging Core Facility of Nanotechnology of the UST-YMU. Additionally, we thank the Instrumentation Center at National Taiwan University for technical support with the EA experiment.


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■ REFERENCES

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10. Nakase, I.; Kogure, K.; Harashima, H.; Futaki, S. Methods Mol Biol. 2011, 683, 525-533. 11. Liou, G.-Y.; Storz, P. Free Radic Res. 2010, 44, 479-496. 12. Giorgio, M.; Trinei, M.; Migliaccio, E.; Pelicci, P. G. Nat Rev Mol Cell Biol. 2007, 8, 722728. 13. Szatrowski, T. P.; Nathan, C. F. Cancer Res. 1991, 51, 794-798. 14. Lee, K. Y.; Chiang, Y. T.; Hsu, N. Y.; Yang, C. Y.; Lo, C. L.; Ku, C. A. Acta Biomater. 2015, 24, 286-296. 15. Neuzil, J.; Weber, T.; Gellert, N.; Weber, C. Br J Cancer. 2001, 84, 87-89. 16. Duhem, N.; Danhier, F.; Préat, V. J Control Release. 2014, 182, 33-44. 17. Dong, L. F.; Grant, G.; Massa, H.; Zobalova, R.; Akporiaye, E.; Neuzil, J. Int. J. Cancer. 2012, 131, 1052–1058. 18. Napoli, A.; Tirelli, N.; Kilcher, G.; Hubbell, J. A. Macromolecules 2001, 34, 8913-8917. 19. Napoli, A.; Tirelli, N.; Wehrli, E.; Hubbell, J. A. Langmuir 2002, 18, 8324-8329. 20. Valentini, M.; Napoli, A.; Tirelli, N.; Hubbell, J. A. Langmuir 2003, 19, 4852-4855. 21. Napoli, A.; Valentini, M.; Tirelli, N.; Müller, M.; Hubbell, J. A. Nat Mater. 2004, 3, 183189. 22. Gupta, M. K.; Meyer, T. A.; Nelson, C. E.; Duvall, C. L. J. Control Release 2012, 162, 591598.


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23. Chiang, Y. T.; Yen, Y. W.; Lo, C. L. Biomaterials. 2015, 61, 150-161. 24. Saravanakumar, G.; Kim, J.; Kim, W. J. Adv. Sci. 2016, 1600124. 25. Yildirimer, L.; Buanz, A.; Gaisford, S.; Malins, E. L.; Remzi Becer, C.; Moiemen, N.; Reynolds, G. M.; Seifalian, A. M. Sci Rep. 2015, 5, 15040. 26. Christenson, E. M.; Anderson, J. M.; Hiltner, A. Corros Eng Sci Techn. 2007, 42, 312-323. 27. Glavas, L.; Odelius, K.; Albertsson, A.-C. Soft Matter. 2014, 10, 4028-4036. 28. LaRue, I.; Adam, M.; Zhulina, E. B.; Rubinstein, M.; Pitsikalis, M.; Hadjichristidis, N.; Ivanov, D. A.; Gearba, R. I.; Anokhin, D. V.; Sheiko, S. S. Macromolecules. 2008, 41, 6555– 6563. 29. Evans, B. C.; Nelson, C. E.; Yu, S. S.; Beavers, K. R.; Kim, A. J.; Li, H.; Nelson, H. M.; Giorgio, T. D.; Duvall, C. L. J Vis Exp. 2013, 73, 50166. 30. Yang, H.; Li, Y.; Li, T.; Xu, M.; Chen, Y.; Wu, C.; Dang, X.; Liu, Y. Sci Rep. 2014, 4, 7072. 31. Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K. Seifert, S.; Andree, C.; Stöter, M.; Epstein-Barash, H.; Zhang, L.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M. Nature Biotechnology 2013, 31, 638–646. 32. Weber, T.; Lu, M.; Andera, L.; Lahm, H.; Gellert, N.; Fariss, M. W.; Korinek, V.; Sattler, W.; Ucker, D. S.; Terman, A.; Schröder, A.; Erl, W.; Brunk, U. T.; Coffey, R. J.; Weber, C.; Neuzil, J. Vivo. Clin Cancer Res. 2002, 8, 863-869.


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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 ACS Paragon Plus Environment

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■ FIGURE LEGENDS

Scheme 1. The research concept. The micelles composed of amphiphilic copolymers were uptaken by high H2O2 cancer cells through the endocytosis process. Following copolymer oxidation by H2O2, the copolymers became more hydrophilic; they dissociated and inserted into the endosomal membrane to induce membrane breakage and release drugs into the cytosol. mPEG-PS represents methoxy polyethylene glycol-b-poly(diethyl sulfide) copolymers. mPEGPSSO represents methoxy polyethylene glycol-b-poly(diethyl sulfide-co-diethyl sulfoxide) copolymers. The morphology of α-TOS-loaded micelles was analyzed through TEM. The micelles were stained with uranyl acetate (2 wt%). The scale bar is 100 nm. Figure 1. Characteristics of mPEG-PS copolymers and micelles. a) The oxidation degree for the copolymers with 14 repeating units of sulfide after oxidation by 133 µM and 1.33 µM H2O2, as analyzed using elemental analyzer. b) The retention times of the copolymers oxidized by 133 µM H2O2 through GPC. c) The zeta potential of copolymers at different pH values. d-f) The particle size, size distribution, and Kcps of micelles at different H2O2 concentrations and pH, as analyzed through DLS. Asterisk indicate statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.005). Figure 2. The morphological changes in the micelles at different H2O2 concentrations and pH solutions for 1, 3, and 6 h. The micelles were also stained with uranyl acetate (2 wt%). The scale bar was 100 nm. Figure 3. Interaction of copolymer clusters with red blood cells. a) The hemolysis percentages of RBCs co-cultured with oxidized copolymers. b) The confocal images of the copolymers after 9-h


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oxidation by 133µM H2O2 co-cultured with RBCs monitored, as through Zeiss 880 confocal microscopy. The upper plot shows the three-dimensional image. The lower plot shows the orthogonal image. Green represents Cy5.5-conjugated copolymers, and orange represents CellMask plasma membrane dyes. The scale bar was 5 µm. Asterisk indicate statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.005). Figure 4. Endosomal escape behavior of micelles in HCT116 and L929 cells. a) The micelles were co-cultured with L929 cells. b) The micelles were co-cultured with HCT116 cells. Blue represents the cell nucleus stained with DAPI. Green represents Cy5.5-conjugated copolymers. Red represents LysoTracker dyes, which could stain acidic endosomes and secondary lysosomes. The scale bar was 10 µm. Figure 5. Bioactivity of α-TOS released from micelles in cancer and fibroblast cells. a) The αTOS release behavior of α-TOS-loaded micelles at different H2O2 concentrations and pH, as measured through UV-Vis spectrometry at 291 nm. b) The cytotoxicity of empty micelles, αTOS-loaded micelles, and free α-TOS in cancer and fibroblast cells for 1 and 2 days, as assessed using the MTT assay. The left and right plots represent the cell viabilities of HCT116 and L929 cells, respectively. c) The cell cycle assay through flow cytometry for HCT116 (left) and L929 (right) cells treated with empty micelles, α-TOS-loaded micelles, and free α-TOS for 1 day. d) The α-TOS concentration in cancer (light blue line) and normal (green line) cells after treatment with α-TOS-loaded micelles for 1 day, as assessed through HPLC. The upper left plot shows the retention times of free α-TOS (red line) and α-TOH (blue line) for references. Asterisk indicate statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.005).


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■ TABLE.

Table 1. The characteristics of ROS-responsive copolymers, empty micelles and drugloaded micelles. Code

% Molar ratio [a] of mPEG:DES

Empty micelles [b]

Drug-loaded micelles [b]

Size (nm)

PDI

Size (nm)

PDI

PS10

1:10

230.3±11.5

0.09±0.00

424.4±32.1

0.35±0.21

PS14

1:14

144.1±12.5

0.13±0.05

225.4±14.6

0.03±0.02

PS20

1:20

219.9±10.9

0.09±0.01

376±46.2

0.41±0.23

[a] The composition of copolymers was measured by 1H NMR. (DES: diethyl sulfide). [b] The size and size distribution (PDI) was measured by DLS.


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Scheme 1.

mPEG-PS copolymer Oxidized copolymer α-TOS Oxidized hydrophobic region H2O2 High H2O2 cancer cell Internalization

Membrane disruption (Endosomal escape)

Copolymer oxidation

Copolymer insertion

Copolymer dissociation


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Figure 1. b)

5k

***

60

C

45 30

*

15

Zeta potential (mV)

75

Oxidation 3h Oxidation 6h Oxidation 9h Oxidation 12h Oxidation 24h

6k

***

H2O2 1.33µM

c)

4k 3k 2k

0 0

5

10

15

20

6

Time (h)

7

8

e) ***

9

*** ******

non-oxidation oxidation 9h

7.0

6.5

6.0

5.5

pH value

f)

480

PDI Change (%)

pH7.4, 1.33µM pH7.4, 133µM pH5.5, 1.33µM pH5.5, 133µM

-8

Time (min)

d) 1600 1400 1200 1000 800 600 400 200 0

-4

-16 7.5

0

25

0

-12

1k

420 360 300 240

pH7.4, 1.33µM pH7.4, 133µM pH5.5, 1.33µM pH5.5, 133µM

*** *** ******

***

180 120

120

Kcps Change (%)

H2O2 133µM

90

Signal (mV)

Degree of Oxidation (%)

a)

Size Change (%)

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

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100 80 60 40 20

60

0

***

*** pH7.4, 1.33µ M *** pH7.4, 133µM ****** pH5.5, 1.33µM pH5.5, 133µM

0 1 3 6 12 24 48 72

0 1 3 6 12 24 48 72

0 1 3 6 12 24 48 72

Time (h)

Time (h)

Time (h)


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Figure 2.

1.33 µM H2O2

133 µM H2O2

1.33 µM H2O2

(pH 7.4)

(pH 7.4)

(pH 5.5)

(pH 5.5)

3h

1h

133 µM H2O2

6h

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

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Figure 3. a)

b) 3h

Hemolysis (%)

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

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20 18 16 14 12 10 8 6 4

6h

9h

*** ***

*** *** ******

Co-colture 6 h Co-colture 12 h

0

5

10

15

20

25

Oxidation time (h)


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Figure 4. DAPI

Cy5.5

Lysotracker

Merge

9h

6h

3h

a)

12 h

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

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DAPI

Cy5.5

Lysotracker

Merge

9h

6h

3h

b)

12 h

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

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Figure 5.

b) 120 pH7.4, 1.33µ M pH7.4, 133 µM pH5.5, 1.33µ M pH5.5, 133 µM

80 60

***

40

*

20 0

***

0

*** *

*** *** *** *** ***

100

*

80 60 40 20 0 -20

empty micelles 24h α -TOS loaded micelles Free α -TOS 24h

-20

10 20 30 40 50 60 70 80

0

20

40

48h 24 h 48h

60

48 h

120

*** *** *** ***

100 80 60 40 20 0 -20

80 100

empty micelles 24h α -TOS loaded micelles Free α -TOS 24h

-20

Drug concentration ( µ M )

Time (h)

c)

0

20

40

48h 24h 48h

60

48h

80 100

Drug concentration ( µ M )

d) **

**

50

Free α-TOS α-TOS-loaded micelles empty micelles

*

*

40 *

30 20 10 0

Free α-TOS α-TOS-loaded micelles empty micelles

60

L929 Cell Count (%)

60

50 40

*

*

*

30

*

*

20

α -TOS α -TOH

Signal (mV)

Drug Release (%)

100

***

HCT116 Cell Viability (%)

120

L929 Cell Viability (%)

a)

HCT116 Cell Count (%)

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

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HCT116 L929

10 0

G0/G1

S

G2/M

10 11 12 13 14 15 16

G0/G1

S

G2/M

10

12 14 Time (min)

16


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For Table of Contents Only Specific cancer cytosolic drug delivery triggered by reactive oxygen species-responsive micelles Lu-Yi Yu, Geng-Min Su, Chi-Kang Chen, Yi-Ting Chiang, Chun-Liang Lo

Copolymer oxidation

Endosomal escape

Red blood cells

HCT116 cells

Copolymer dissociation

Copolymer insertion


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Specific Cancer Cytosolic Drug Delivery Triggered by Reactive

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