Miktoarm Amphiphilic Block Copolymer with Singlet Oxygen-Labile

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Miktoarm Amphiphilic Block Copolymer with Singlet OxygenLabile Stereospecific #-aminoacrylate Junction: Synthesis, Self-assembly, and Photodynamically Triggered Drug Release Gurusamy Saravanakumar, Hyeongmok Park, Jinhwan Kim, Dongsik Park, Swapan Pramanick, Dae Heon Kim, and Won Jong Kim Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00290 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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Miktoarm Amphiphilic Block Copolymer with Singlet Oxygen-Labile Stereospecific Aminoacrylate Junction: Synthesis, Self-assembly, and Photodynamically Triggered Drug Release Gurusamy Saravanakumar1, Hyeongmok Park1, Jinhwan Kim2, Dongsik Park1, Swapan Pramanick1, Dae Heon Kim3,* and Won Jong Kim1,* 1

Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang

37673, Republic of Korea. 2

Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang, 37673,

Republic of Korea. 3

Department of Biology, Sunchon National University, Sunchon 57922, Republic of Korea.

KEYWORDS. stereospecific miktoarm block copolymer, cleavable block copolymer, shell sheddable micelles, singlet oxygen-responsive, triggered drug release.

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ABSTRACT. Incorporation of a desired stimuli-responsive unit in a stereospecific manner at the specific location within a non-linear block copolymer architecture is a challenging task in synthetic polymer chemistry. Herein, we report a facile and versatile method to synthesize AB2 miktoarm block copolymers bearing a singlet oxygen (1O2)-labile regio and stereospecific -aminoacrylate linkage with 100% E-configuration at the junction via a combination of amino-yne click chemistry and ring opening polymerization. Using this strategy, a series of 1O2-responsive AB2 amphiphilic miktoarm copolymers composed of hydrophilic polyethylene glycol (PEG) as the A constituent and hydrophobic polycaprolactone (PCL) as the B constituent (MA-PEG-b-PCL2) was synthesized by varying the block length of PCL. The self-assembly characteristics of these well-defined MAPEG-b-PCL2 copolymers in an aqueous condition were studied by solvent displacement and thinfilm rehydration method, and their morphologies were investigated using transmission electron microscopy. The copolymers formed spherical, cylindrical or lamella morphologies, depending on the chain length and preparation conditions. A hydrophobic photosensitizer chlorin e6 (Ce6) and anticancer drug doxorubicin (DOX) were efficiently encapsulated into the hydrophobic core of MA-PEG-b-PCL2 copolymer micelles. These coloaded micelles were taken up by human breast cancer (MDA-MB-231) cells. Upon red laser light irradiation, the 1O2-generated by the Ce6 induced photocleavage of the -aminoacrylate moiety, leading to the dissociation of the micellar structure and triggered intracellular drug release for effective therapy. Overall, rapid disassembly upon 1O2 generation and subsequent controlled intracellular drug release suggested that these micelles bearing -aminoacrylate linkage have a huge potential for on-demand drug delivery.


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INTRODUCTION Amphiphilic block copolymers have garnered significant research attention owing to their propensity to form myriad of self-assembled nanostructures, such as spherical micelles, cylindrical or worm-like micelles and polymeric vesicles.1-3 These nanostructures have found potential applications in diverse field ranging from material science to biomedical engineering; some of which includes use of these nanostructures as: templates for the preparation of functional inorganic nanoparticles4-6 and fabrication of nanoporous membrane,7-9 reactors for catalysis,10-12 and carriers for drug delivery.13-16 In particular, for some of these applications, the selective removal of specific constituent block at mild condition after self-assembly is required to achieve more desired properties. For example, in the fabrication of nanoporous membrane, the removal of minor constituent domain of the copolymer is considered to be crucial to create a highly ordered nanopores.17,

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Similarly, in the drug delivery applications, the shedding of the hydrophilic

polyethylene glycol (PEG) shell of drug carriers at the extracellular tumor site was reported to be capable of improving the cellular internalization while minimizing the nonspecific interactions during circulation.19,

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In some cases, the shedding of shell also induced dissociation of the

nanostructure at the intracellular compartments and facilitated triggered-drug release for more effective therapy.21 Thus, the design of block copolymers by judiciously incorporating the specific stimuli-responsive unit as trigger within the polymeric structure is crucial to control the PEG shedding in the extracellular and intracellular spaces. Sheddable or cleavable amphiphilic block copolymers with a stimuli-responsive moiety at the hydrophilic-hydrophobic junction are more interesting building blocks in the fabrication of smart nanocarriers for controlled and targeted drug delivery.22, 23 In general, there have been three strategies utilized for the synthesis of this type of cleavable block copolymers: (i) installation of


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an initiation site with cleavable moiety at the one chain end of the homopolymer through covalent conjugation chemistry, followed by the polymerization of monomers,21, 24 (ii) conjugation of two homopolymers through a cleavable moiety by organic coupling reactions,25,

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and (iii)

polymerization of two monomers sequentially or in a single pot using a cleavable small molecule heterobifunctional initiator via established similar or a different polymerization mechanisms. 27 Nonetheless, the recent progress in controlled and living polymerization techniques and facile click chemistry reaction has enabled the synthesis of well-defined block copolymers with a cleavable junction. By employing the aforementioned strategies, diverse cleavable block copolymer structures are synthesized with various stimuli-responsive groups, such as, a reducible disulfide bond,21,

28, 29

a pH-labile acetal group,30,

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and a photocleavable o-nitrobenzyl group,32-34 an

enzyme-cleavable peptide linkage,35 and reactive oxygen species (ROS) cleavable moieties36 including vinyldithioether37 and dialkoxyanthracene38 linkages at the junction. Compared to the traditional drug carriers, which exhibit slow drug release for a prolonged period of time via diffusion controlled mechanism, these sheddable nanocarriers using the cleavable block copolymers could rapidly release drug in response to the stimuli, resulting in a greatly enhanced therapy. Despite the promising characteristics of these sheddable self-assembled nanostructures, the cleavable block copolymers employed for these purpose are mainly limited to the linear architecture, which have been mostly utilized disulfide linkage among the others. It should also be emphasized that non-linear or branched copolymers have numerous advantages for use as drug carriers, including high drug loading capacity and the formation of more stable nanoassemblies with different interesting morphologies, compared to their linear structure.39-42 However, very little attention has been focused on the development of cleavable copolymers with branched architectures,43-45 which might be largely due to difficulties involved in the laborious synthetic


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process. Thus, the development of modular and facile approaches for the synthesis of novel sheddable non-linear block copolymer with other biologically relevant stimuli are needed. In this study, we described a versatile method to synthesize AB2-type miktoarm amphiphilic block copolymer comprising ROS singlet oxygen (1O2)-labile -aminoacrylate linkage with 100% E-configuration at the core-shell junction between the hydrophilic PEG as the A constituent and hydrophobic poly(caprolactone) (PCL) as the B constituent by a combination of amino-yne click chemistry and ring opening polymerization. PEG and hydrophobic aliphatic polyester PCL were chosen as constituents of copolymer owing to their biocompatibility, biodegradability, and low toxicity. In order to test the scope of this methodology to prepare copolymers with a wider range of molecular weights, a series of block copolymers were synthesized by varying the block length of PCL. The self-assembly characteristics of these well-defined miktoarm copolymers in an aqueous condition, and their red-light laser-induced 1O2-mediated disassembly of the resulting nanostructures, and accompanied drug release were investigated by co-encapsulating 1O2generating photosensitizer chlorin e6 (Ce6) and an anticancer drug doxorubicin (DOX) (Scheme 1). Furthermore, intracellular drug release and cellular toxicity of these Ce6/DOX co-loaded nanoassemblies under light-induced 1O2 condition were evaluated to demonstrate the potential of nanoassemblies as stimuli-responsive drug delivery carrier.


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Scheme 1. Synthesis of amphiphilic miktoarm PEG-b-PCL2 copolymer bearing 1O2-labile aminoacrylate via combination of amino-yne click chemistry and ring opening polymerization, and schematic illustration of self-assembly and red-laser induced 1O2-mediated dissociation of MA-PEG-b-PCL2 micelle. Chemical structures of Ce6 and DOX are shown below.

EXPERIMENTAL SECTION


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Materials and methods Monomethoxy poly(ethylene glycol) (PEG45-OH, Mn = 2000 g mol-1), propiolic acid (95%), p-Toluenesulfonic acid monohydrate (p-TsOH, 98.5%), -caprolactone (-CL, 97%), and diethanolamine (98.0%) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Tin (II) 2ethylhexanoate (Sn(Oct)2) was obtained from Alfa Aesar. Chlorine e6 (Ce6) and doxorubicin hydrochloride (DOX HCl) were supplied from Frontier Scientific Inc. (Logan, UT, USA), and Wako Pure Chemical Industries (Osaka, Japan), respectively. -CL was dried over calcium hydride (CaH2), and freshly distilled under reduced pressure. All other chemicals were of analytical grade and used as received without further purification unless otherwise specifically mentioned. 1

H nuclear magnetic resonance spectroscopy (1H NMR) spectra were recorded on Bruker

Avance 300 and 500 MHz FT-NMR with deuterium chloroform (CDCl3) as the internal standard solvent. The chemical shifts are reported in parts per million (ppm) and referenced to the CDCl3 singlet at 7.26 ppm. The splitting patterns are designated as s (singlet), d (doublet), t (triplet), m (multiplet) and br (broad), and coupling constants (J) are reported in Hertz (Hz). Diffusion ordered NMR spectroscopy (DOSY) experiments were performed on Bruker Avance III HD 800 MHz NMR spectrometer. Gel permeation chromatography (GPC) measurements were performed on a Waters Alliance e2695 GPC system, equipped with three consecutive Styragel® columns (HR1, HR2 and HR4). The detection was performed on a 2414 refractometer. THF was used as the eluent at a flow rate of 1 mL/min. The molecular weights were calibrated using polystyrene standards. The dynamic light scattering (DLS) measurements were conducted using a Zetasizer Nano S90 system (Malvern Instruments, Worcestershire, UK). UV-visible spectra were recorded on a UV 2550 spectrophotometer (Shimadzu), and fluorescence spectra were recorded on a RF-5301 PC


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spectrofluorophotometer (Shimadzu). The morphologies of the nanoassemblies were observed using transmission electron microscopy (TEM; JEM-1011, JEOL, Tokyo, Japan). The samples were prepared by dropping the colloidal suspensions of the nanoassemblies on the copper grid followed by staining with uranyl acetate. Synthesis of PEG45-(OH)2 macroinitiator First, PEG45-propiolate was synthesized by esterification of PEG45-OH with propiolic acid in the presence of p-TsOH as a catalyst. PEG45-OH (10 g; 5 mmol), propiolic acid (0.525 g; 7.5 mmol) and p-TsOH (0.19 g; 1 mmol) were taken in a round-bottom flask equipped with a Dean-Stark apparatus, and 100 ml dry toluene was added as a solvent. The resulting mixture was allowed to reflux for 36 h with continuous removal of the yielded water. After completion of the reaction (as monitored by 1H NMR), the reaction mixture was allowed to cool to room temperature, and concentrated using a rotary evaporator. The residue was dissolved in a minimum amount of CH2Cl2, and precipitated in diethyl ether. The crude product was purified by repeated precipitation in ether to afford desired PEG45-propiolate. (Yield: 8.62 g; 83.31%). 1H NMR (300 MHz, CDCl3): δ 4.34 (t, J = 6.0 Hz, 2H, -CH2-O-C(=O)-), 3.87 (t, J = 6.0 Hz, 1H, -COOCH2CH2-), 3.77-3.53 (CH2CH2O-), 3.38 (s, 3H, -OCH3), 2.96 (s, 1H, -C(=O)-C≡CH). 13C NMR (125 MHz, CDCl3): δ 152.73 (ester carbonyl carbon, -C(=O)-C≡CH), 75.56 (-C(=O)-C≡CH), 74.62 (-C(=O)-C≡CH), 70.65 (-CH2CH2O-), 68.63 (-COOCH2CH2-), 65.30 (-COOCH2CH2-), 59.12 (-OCH3). Second, the as-synthesized PEG45-propiolate (2.0 g; 0.967 mmol) and diethanolamine (0.122 g; 1.16 mmol) were dissolved in CH2Cl2, and the reaction was allowed to proceed at room temperature. After confirming the disappearance of alkyne peak monitored by 1H-NMR, the reaction was stopped, the solvent was evaporated and the product was precipitated in ether. The


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polymer was further purified by dissolving in minimal amount of CH2Cl2 and reprecipitated in ether. (Yield: 1.92 g; 91.20 %). 1H NMR (300 MHz, CDCl3): δ7.51 (d, J = 15.0 Hz, 1H, -N-CH= of E-isomer), 4.62 (d, J = 12.0 Hz, 1H, -C(=O)-CH= of E-isomer), 4.23 (t, J = 6.0 Hz, 2H, -CH2O-C(=O)-), 3.87 (t, J = 6.0 Hz, 2H, PEG-O-CH2-), 3.80 (t, J = 6.0 Hz, 4H, -CH2-OH), 3.71-3.53 (-CH2CH2O- of PEG), 3.42-3.39 (4H, -CH2-N-CH2-), 3.37 (s, 3H, -OCH3). 13C NMR (125 MHz, CDCl3): δ 169.41 (ester carbonyl carbon, -C(=O)-CH=CH-), 152.24 (-C(=O)-CH=CH-), 85.31 (C(=O)-CH=CH-), 72.09 (-CH2-O-C(=O)-), 70.72 (-CH2CH2O-), 69.88 (PEG-O-CH2-), 62.47 (CH2-OH), 61.87 (-CH2-CH2-OH), 59.18 (-OCH3). Mn (GPC) = 2410 g mol-1; Mw/Mn = 1.19. A small shoulder peak at the high molecular weight due to unknown impurities was observed in GPC trace. Typical synthesis of MA-PEG-b-PCL2 copolymers Miktoarm block copolymers MA-PEG-b-PCL2 were synthesized via ROP of -CL using the MA-PEG45-(OH)2 as a macroinitiator and Sn(Oct)2 as a catalyst. A typical procedure for ROP with [-CL0]/[I0] = 10 (coded as P1) is described below. MA-PEG45-(OH)2 macrointiator (0.3 g; 0.138 mmol), -CL (0.158 g; 1.38 mmol), Sn(Oct)2 (0.006 g; 0.014mmol) and 0.7 mL of toluene were charged into a flame dried Schlenk flask under dry nitrogen. After degassing the mixture, polymerization was carried out in a thermostated oil bath at 110 °C for 24h. After cooling to room temperature, the polymer was precipitated in ether. The polymer was redissolved in CH2Cl2 and precipitated again in ether. The precipitated polymer was filtered and dried under vacuum at room temperature. 1H NMR (300 MHz, CDCl3): δ 7.39 (d, J = 12.0 Hz, 1H, -N-CH= of E-isomer), 4.70 (d, J = 15.0 Hz, 1H, -C(=O)-CH= of E-isomer), 4.24-4.18 (6H, -CH2-O-C(=O)- ,–N-{CH2-CH2O-C(=O)}2-), 4.06 (t, J = 6.0 Hz, -CH2-OH of PCL), 3.87 (t, J = 6.0 Hz, 2H, PEG-O-CH2-), 3.64-


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3.53 (-CH2CH2O- of PEG), 3.44-3.39 (4H, -CH2-N-CH2-), 3.38 (s, 3H, -OCH3 of PEG), 2.30 (m, -C(=O)-CH2- of PCL), 1.64 (m, -C(=O)-CH2-CH2-CH2-CH2-CH2O of PCL), and 1.39 (m, -C(=O)CH2-CH2-CH2-CH2-CH2O of PCL). 13C NMR (125 MHz, CDCl3): δ 173.55 (-C(=O)-CH2CH2- of PCL), 169.09 (-C(=O)-CH=CH-), 151.78 (-C(=O)-CH=CH-), 86.05 (-C(=O)-CH=CH-), 70.55 (CH2CH2O-), 64.16 (-C(=O)-CH2-CH2-CH2-CH2-CH2O of PCL), 59.07 (-OCH3), 34.15 (-C(=O)CH2-CH2-CH2-CH2-CH2O of PCL), 28.38 (-C(=O)-CH2-CH2-CH2-CH2-CH2O of PCL), 25.56 (C(=O)-CH2-CH2-CH2-CH2-CH2O of PCL), 24.60 (-C(=O)-CH2-CH2-CH2-CH2-CH2O of PCL). Mn (NMR) = 3142 g mol-1, Mn (GPC) = 4.634 g mol-1, Mw/Mn = 1.22. A similar procedure was followed for the synthesis of other polymer compositions of MA-b-PCL2 with [-CL0]/[I0] = 20, 40, and 80, which are coded as P2, P3 and P4 respectively. Preparation of polymeric nanoassemblies The miktoarm block copolymer nanoassemblies were prepared using two different methods, solvent displacement and thin-film hydration methods. For the preparation of nanoassemblies via solvent displacement method, distilled water was added drop-wise into MA-PEG-b-PCL2 copolymers dissolved in THF at a concentration of 5 mg/mL, followed by extensive dialysis against distilled water to remove the organic solvent. Finally, the resulting colloidal nanossemblies were briefly sonicated for about 2 min, and filtered through 0.45 m syringe filter prior to characterization. For thin-film hydration method, P2 was dissolved in CH2Cl2 with a concentration of 5 mg/mL. Then, the solvent was evaporated using rotary evaporator to obtain a thin film, which was hydrated with distilled water under moderate stirring at 60 °C for 2h. The resulting colloidal nanoassemblies were sonicated for 15 min. After filtering through 0.45 m syringe filter, the size and morphologies of the nanoassemblies were recorded using TEM.


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Determination of critical aggregation concentration (CACs) CACs of the miktoarm block copolymers were determined by fluorescence spectroscopy method using pyrene as a probe. In brief, a series of polymer solutions with concentrations ranging from 1.25  10-5 to 0.5 mg/mL were prepared by diluting the polymer stock solutions. An aliquot of pyrene stock solution in acetone was transferred to a series of vials, and then acetone was completely evaporated under a stream of nitrogen. The polymer solutions were added to the vials to obtain a final pyrene concentrations of 6  10-7 M in each vial. After being equilibrated the solution overnight in the dark, the excitation spectrum was recorded using a spectrofluormeter with the emission wavelength fixed at 390 nm. The ratio of the fluorescence intensity of peak at 337 nm (I337) to the peak at 334 nm (I334) was determined and plotted against the logarithm of the polymer concentration. The CAC value was obtained as the crossover point of two tangents of the curve. Preparation of Ce6-loaded, DOX-loaded or Ce6/DOX co-loaded nanoassemblies The drug-loaded nanoassemblies were prepared following the thin-film hydration method as described above. In a typical experiment, block copolymer P2 (50 mg) was dissolved in 10 mL of CH2Cl2. To this appropriate feed amount of Ce6 dissolved in ethanol/acetone (3:1; v/v) was added and mixed well. Then, the solvent was evaporated under rotary evaporator to obtain a thin-film, which was hydrated with distilled water 100 mL under moderate shaking at 60 °C. The nonencapsulated drugs were removed by dialysis (membrane cutoff 3.5 kDa) for 24 h, and lyophilized to obtain Ce6-loaded nanoassemblies. The same method was employed for the preparation of DOX loaded and Ce6/DOX co-loaded nanoassemblies. For this, DOX was dissolved in CHCl3/MeOH mixture (1:1; v/v) and desalted using 3 equivalents of TEA before adding to polymer solution. The


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loading content (LC) and loading efficiency (LE) of Ce6 in the samples were determined using UV-Visible spectrophotometer at 664 nm. The LC and LE of DOX were determined by spectrofluorophotometer at 587 nm. In vitro drug release The in vitro release profile of DOX from the P2-Ce6-DOX micelles was evaluated using a dialysis method. For this experiment, the micelles were dispersed in PBS solution (pH 7.4) at a concentration of 1 mg/mL and placed in a dialysis membrane bags (MW cutoff = 3500). The dialysis bags were immersed in release media (PBS, pH 7.4), and each samples were gently shaken in a 37 °C water bath at 100 rpm. At predetermined time intervals, the release medium was refreshed and the DOX concentration was determined using spectrofluorophotometer at 586 nm. For light-triggered release of DOX, the micelles were irradiated with red light laser (50 or 100 mW/cm2; 660nm, 30 min) before transferring to the dialysis bag. In vitro cytotoxicity MDA-MB-231 cells (human breast adenocarcinoma cells) were cultured in a 5% CO2 humidified incubator under Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing fetal bovine serum (FBS, Hyclone), 100 U/mL of penicillin, and 100 μg/mL streptomycin. For cytotoxicity evaluation, cells were seeded on 96-well plate at an initial density of 6000 cells/well and incubated for 24 h. Fresh medium was treated with samples containing various concentration of DOX, and incubated further 18 h. A 660 nm laser was then subjected for 10 min at a power density of 50 mW/cm2, followed by further 24 h incubation. Cells were then washed and treated with 1 mg/mL of thiazolyl blue tetrazolium bromide (MTT) containing fresh medium and incubated for 4 h. After the removal of medium, dimethylsulfoxide (DMSO) was added and the


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absorbance at 570 nm was measured using a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, PerkinElmer). All statistical results were presented using Student’s t-test. Intracellular monitoring of drug release To visualize the intracellular light-responsive DOX release from P2-Ce6-DOX, MDA-MB231 cells were seeded on a coverslip at an initial density of 20000 cells/well and incubated for 24 h. Fresh medium was treated with P2-DOX or P2-Ce6-DOX (2 μM DOX, 4.84 μM Ce6) and incubated for 2.5 h. A 660 nm laser was then irradiated for 10 min at a power density of 50 mW/cm2, followed by fixation using 10% neutral buffered formalin (NBF). Cells on a coverslip were mounted in Vectashield antifade mounting medium with DAPI (Vector Laboratories) and observed using an Olympus FV-1000 confocal laser scanning microscope (CLSM) and analyzed with the Olympus Fluoview version 1.7 software.

Results and Discussion Synthesis of amphiphilic miktoarm block copolymer bearing 1O2-labile -aminoacrylate linkage Our approach to the synthesis of well-defined AB2-type miktoarm block copolymers with a stereospecifically placed 1O2-responsive moiety at the junction point of hydrophilic PEG and hydrophobic PCL blocks is illustrated in Scheme 1. First, a PEG macroinitiator (MA-PEG-(OH)2) bearing a 1O2-labile -aminoacrylate group and two hydroxyl group functionalized terminals was synthesized in two steps, that is, esterification of monomethoxy PEG-OH with propiolic acid via Fisher esterification, followed by a facile and catalyst free amino-yne click reaction of the resulting


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PEG-propiolate and a commercially available diethanolamine. In the past decade, several click reactions,46 such as copper-mediated or strain-promoted azide-alkyne cycloadditions,47, 48 thiolene or thiol-yne conjugation reactions,49,

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and Diels-Alder cycloadditions,51 have made

significant impact and greatly expanded the scope of polymer synthesis to accesses a wide range of functional polymers. However, some of these click reactions require metal catalysts, which necessitates further purification process and limits its applications. Interestingly, the amino-yne click reaction between the activated alkyne and secondary amine can readily proceeds at ambient conditions without the aid of any catalyst to obtain compounds in a high yield with a regio- and stereospecific -aminoacrylate linkage,52 which can be easily cleaved by the ROS 1O2.53 It should also be mentioned that the high stereospecificity demonstrated by this method, here at the branching point, may provide additional opportunities to structural engineer the polymeric backbone architecture. The structure of PEG45-propiolate and MA-PEG45-(OH)2 were confirmed using 1H-NMR 13C-NMR and FT-IR (Figure S3) . The terminal alkyne peak of the PEG-propiolate at  2.96 ppm (Figure 1a) completely disappeared and two new peaks representing the aminoacrylate linkage (-N-CH=CH-C(=O)O-) were observed at  7.51 ppm (-N-CH=) and  4.62 ppm (-C(=O)-CH=) (Figure 1b). The corresponding coupling constant of the –CH=CH- group in this linkage was found to be 15 Hz, confirming the presence of solely E-configuration.52 The block copolymer architecture, chain conformation and molar mass may have significant effect on the morphology of the resulting self-assembled aggregates. It is also worth mentioning the synthetic approach introduced in this study is quite versatile to prepare well-defined branched polymers with defined stereospecificity at the junction.


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Figure 1. 1H-NMR spectra of (a) PEG-propiolate and (b) miktoarm PEG macroinitiator bearing -aminoacrylate group (MA-PEG45-(OH)2) in CDCl3.

Second, the miktoarm MA-PEG-b-PCL2 copolymer was synthesized by ring-opening polymerization of -CL monomer using the as-synthesized MA-PEG-(OH)2 as a macroinitiator and Sn(Oct)2 as a catalyst. By varying the molar feed ratio of [-CL0]/[I0], a series of miktoarm block copolymers with different PCL lengths were obtained. The structure and chemical composition of the copolymers were determined by 1H NMR. Figure 2a represents a typical spectrum of miktoarm copolymer P2. All the resonance peaks corresponding to the PEG and PCL


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block repeating units were observed, individual peaks are assigned with alphabets in the structure and indicated in the spectrum. Then, the compositions of the copolymers were determined by comparing the integration ratios of the methylene peak at  3.53 (-CH2CH2O-) of PEG and at  1.39 (-C(=O)-CH2-CH2-CH2-CH2-CH2O) of PCL. The estimated compositions of the MA-PEGb-PCL2 copolymers are summarized in Table 1. The GPC traces of MA-b-PCL2 copolymers are showed in Figure 2b, which clearly exhibited monomodal distribution with narrow polydispersity (1.22-1.45). The GPC chromatograms shifted toward higher molecular weights by increasing the feed ratio of monomer to macroinitiator. The plots of [-CL0]/[I0] versus Mn(GPC) and Mn(NMR) (Figure S1) showed a linear trend for MA-PEG-b-PCL2 copolymer series, indicating that the polymerization of -CL monomer using MA-PEG-(OH)2 was well controlled. Therefore, MAPEG-b-PCL2 copolymers of any desired PCL lengths can be synthesized by varying the feed ratio of -CL monomer to MA-PEG-(OH)2 macroinitiator.


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Figure 2. (a) 1H-NMR spectrum of MA-PEG-b-(PCL8)2 copolymer (P2) in CDCl3 and (b) GPC traces of MA-PEG-b-PCL2 copolymers.


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Self-assembly characteristics of MA-PEG-b-PCL2 copolymers It is well-known that amphiphilic block copolymers can self-assemble into various morphologies such as micelles and polymersomes, in aqueous solution. In this study, the designed MA-PEG-b-PCL2 copolymers have other unique feature, that is, presence of stereospecific Econfiguration at the junction of hydrophilic PEG and hydrophobic PCL. The self-assemblies of MA-PEG-b-PCL2 (P1 to P4; with fPEG = 0.23 to 0.71) were prepared by two different methods, solvent displacement and thin-film hydration methods, with a concentration of 0.5 mg/mL, and their morphologies were characterized using TEM. As shown in Figure 3a, P1 copolymer with short PCL arms and fPEG of 0.71 formed a clear monodispersed spherical micelles with size about 50 nm. However, increasing the PCL arm block length resulted in the nanoassmblies with nonspherical morphologies. The copolymers P2 with fPEG = 0.56 and P3 with fPEG = 0.33 gave rise to fiber or cylindrical micelles (with varying thickness about 20-60 nm) along with some spheres. Further, increasing the core-forming PCL length of copolymer P4 (fPEG = 0.23) favored lamella/platelets structures. Previous studies on semicrystalline block copolymer self-assembly have also indicated increasing the length of crystalline core-forming block favors the formation of morphologies with low interfacial curvature, such as cylinders, lamellae and platelet micelles, whereas high curvature spherical structure become predominant as the core crystallinity decreases.54, 55 Interestingly, the self-assembly of all the four copolymers via thin-film hydration method resulted in the cylindrical morphologies with varying thickness and lengths (Figure 3b). The P1 copolymer (fPEG = 0.71) that formed uniform spherical micelles via solvent-displacement method also predominantly exhibited thin cylindrical morphology with only a few spherical micelles in thin-film hydration method. These results also indicate that, in addition to the composition or block ratio of the copolymer, several other physicochemical parameters, such as


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solvent, temperature and preparation conditions may greatly influence the resulting morphologies of the self-assembled structures.

Figure 3. TEM images of miktoarm block copolymer nanoassemblies prepared from P1 to P4 by (a) solvent-exchange method and (b) thin-film hydration method.

The CACs represent the concentration at which the block copolymers formed self-aggregates in aqueous solution, and it is also an important physicochemical parameters that indicates the thermodynamic stability of the self-assembled structures. The CACs of the amphiphilic MA-PEGb-PCL2 was determined by fluorescence spectroscopy method using pyrene as probe, which has been widely employed to study the self-aggregation characteristics of amphiphilic block copolymers and surfactants. Pyrene on its own exhibits little fluorescence intensity in aqueous environments due to its poor solubility. However, upon self-aggregation of copolymers, it preferably locate into the hydrophobic core, resulting in the increase of fluorescence intensity. As


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shown in the Figure 4, the CAC was estimated from the crossover point of the plot of I337/I334 versus logarithm concentrations, and the values were in the range of 1.33 to 6.46 mg L-1, which is comparable to the other PEG-polyester miktoarm block copolymers reported earlier.42 MA-PEGb-PCL2 copolymer with a long PCL arms had low CAC values, which indicated that an increase in the molecular weight of hydrophobic PCL facilitated formation of self-aggregates even at low concentrations. In general, miktoarm block copolymers with relatively low CAC values are highly advantageous for drug delivery, because their stability may remain unaffected even upon dilution with body fluids after intravenous injection. Furthermore, stable carriers with prolonged in vivo circulation may provide opportunity for tumor-targeting via enhanced permeation and retention effect.


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Figure 4. Determination of critical aggregation concentration of miktoarm block copolymer nanoassemblies. The fluorescence intensity ratio of I337/I334 of pyrene as a function of the block copolymer concentrations of (a) P1, (b) P2, (c) P3 and (d) P4.


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Red-light laser induced 1O2-mediated scission of block copolymer In the case of MA-PEG-b-PCL2 copolymers, 1O2-labile -aminoacrylate linkage is precisely placed between the miktoarm junction between PCL arms and PEG chain. Before exploring PEG shedding behavior of MA-PEG-b-PCL2 nanoassemblies, 1O2-mediated scission of the block copolymer was preliminary evaluated by exposing the representative copolymer P2 (with ~5 wt.% Ce6) with red-light laser irradiation of different power densities for 30 min. It should also be mentioned one important advantage of -aminoacrylate linkage is that it undergoes fast oxidative degradation without any toxic oxidation byproducts, thus, permitting to design a safe and efficient carrier.53 As shown in Figure 5b and 5c, upon laser irradiation with power densities of 50 and 100 mW/cm2, the olefinic peaks of the aminoacrylate at (–N-CH=CH-C(=O)O-) at  7.39 ppm and  4.70 ppm were decreased and a new peak at  8.06 ppm corresponding to the N-formyl appended PCL arm was appeared, indicating the light-induced 1O2-mediated degradation of -aminoacrylate linkage. However, the P2 containing Ce6 without laser irradiation did not show any degradation product formation (Figure 5a), suggesting that the polymer can be stored stable even with the presence of photosensitizer under dark condition for a long time. As the light can be easily focused into specific pathological areas such as tumor, therefore, it is possible to realize more precise drug delivery using MA-PEG-b-PCL2 based carriers. The light-induced 1O2-mediated scission of P2 was also further supported by the DOSY NMR spectra (Figure S4 and S5), which clearly showed a slow diffusion coefficient for the P2 under dark conditions than that one exposed to light, owing to the high molecular weight of intact P2. In addition, the signals corresponding to the PCL and PEG units were maintained at similar diffusion coefficient, suggesting the PEG and PCL are remained as a single polymer unit. However, P2 exposed to light irradiation of both 50 mW/cm2 and 100 mW/cm2, resulted in a high diffusion coefficients. In particular, the diffusion coefficient


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of PCL signals were clearly distinguishable from PEG, indicating the scission of P2 into two components. Thus, the 2D DOSY spectra results were well corroborated with 1D 1H-NMR, and clearly demonstrated the PEG and PCL remained as a single intact block copolymer unit as P2 in dark was cleaved into two constituent blocks under light-induced 1O2.The 1O2-mediated cleavage of the block copolymer was also further analyzed by the GPC (Figure S2), which indicated the shift of the block copolymer to low molecular weight and appearance of peak associated with the PEG macroinitiator although it was difficult to determine the cleavage of P2 quantitatively. Therefore, by comparing the 1H-NMR peak integration of  4.70 ppm of -C(=O)-CH= of aminoacrylate linkage and  4.06 ppm of -CH2-OH of PCL of P2 before and after laser irradiation, we quantitatively determined the degree of scission of P2. Interestingly, the results indicate about 73% and 84% cleavage of P2 after irradiation with 50 mW/cm2 and 100 mW/cm2, respectively. These results suggest that the -aminoacrylate linkage between the PEG and PCL arms can be efficiently cleaved under light-induced 1O2.


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Figure 5. 1H-NMR spectra of P2 with Ce6 (~5 wt%) in the (a) dark, and after exposing laser irradiation of power density (b) 50 mW/cm2 and (c) 100 mW/cm2 for 30 min, where the solvent is CDCl3.


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Preparation of DOX and Ce6 co-loaded nanoassemblies and laser-induced 1O2-mediated disassembly After confirming the red-light induced 1O2-mediated scission of the block copolymer, its potential as stimuli-responsive drug carrier was studied by physical encapsulation of 1O2generating photosensitizer Ce6 and anticancer drug DOX in to P2 copolymer with fPEG = 0.56 via thin-film hydration method. As the control samples, only Ce6-loaded and DOX-loaded nanoassemblies were also prepared following the same method. The loading content and loading efficiency of P2-Ce6-DOX, P2-Ce6 and P2-DOX nanoassemblies are summarized in Table 2. The loading efficiency of Ce6 was found to be higher than that of DOX. To prove red light-induced 1

O2-mediated disassembly of the nanoassemblies, the change in size distribution of P2-Ce6-DOX

with and without laser irradiation (50 and 100 mW/cm2) was followed at different time intervals using DLS. As shown in Figure 6a, P2-Ce6-DOX without any light irradiation exhibited unimodal size distribution, and did not show any change in particle size distribution as a function of time. But the one exposed to 50 and 100 mW/cm2 (Figure 6b and 6c) laser irradiations showed polydisperse size distribution even after 15 min of exposure time, which might be due to the 1O2mediated cleavage of -aminoacrylate linkage of the copolymer main chain and the subsequence disassembly of the nanostructures. We also monitored the light scattering intensity of the particles as a function time, which can be used to investigate the degradation behavior of self-assembled nanoparticles.56 As shown in Figure 6d, there is no significant change in scattering intensities was observed for P2-Ce6-DOX without light irradiation. However, P2-Ce6-DOX exposed to laser irradiation showed a dramatic decrease in relative scattering intensity. P2-Ce6-DOX exposed to 100 mW/cm2 laser irradiation showed more rapid decrease in scattering intensity than that of one


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exposed to 50 mW/cm2, which indicated that the disassembly of nanostructures, and associated the release rate of the loaded drugs, can be controlled by laser power density. To further investigate the light-induced 1O2-degradation of P2-Ce6-DOX, TEM images of the nanoassemblies were recorded before and after exposing to laser irradiation. P2-Ce6-DOX showed spherical morphology before irradiation (Figure 6e), and highly degraded irregular aggregates were observed for the one after laser irradiation (Figure 6f), which also supports the 1O2- mediated disassembly. It is also interesting to note that P2 copolymers transformed to spherical shape after drug loading, compared to cylindrical structure of blank nanaoassembly. In addition to the block copolymer compositions, as mentioned above, the morphology may also depend on a various other physicochemical parameter such as temperature, solvent-polymer interactions, additives, and preparation conditions. Nonetheless, the above results clearly confirm 1O2-mediated cleave of the block copolymer could induce disassembly of the nanostructures by disturbing the hydrophilicliphophilic balance. Overall, the results indicates that the hydrophobic Ce6 and DOX can be coloaded into the core of the nanostructures, and released into the desired targeted site in a lightinduced 1O2-controlled manner.


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Figure 6. Change of size distribution profile of P2-Ce6-DOX micelles at (a) dark, and after 660 nm laser irradiation of power density (b) 50 mW/cm2 and (c) 100 mW/cm2 as a function of time. (d) Time-dependent changes in light scattering of P2Ce6-DOX micelles at dark and after 660 nm laser irradiation as a function of time. TEM images of P2-Ce6-DOX (e) before and (f) after 660 nm laser irradiation.


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In vitro light triggered release of DOX The release of DOX from the P2-Ce6-DOX micelles was investigated using a dialysis method in PBS solution (pH 7.4) at 37 °C, with and without light irradiation of different power densities (50 mW/cm2 and 100 mW/cm2). The accumulative release profiles of DOX from the micelles are shown in Figure 7. The P2-Ce6-DOX micelles under dark condition showed a slow and minimal drug release, about only 26% of DOX was released within 24 h. However, as expected, the drug release from P2-Ce6-DOX micelles was accelerated upon laser light irradiation, and the amount of DOX released from micelles with light irradiation of 100 mW/cm2 reached about 68% within 24 h. These results clearly demonstrated that the -aminoacrylate linkages in the micelles can be readily cleaved under the generation of light-induced 1O2, which subsequently dissociates the micelle structure and accelerates the DOX release. In particular, micelles exposed to more intense laser light irradiation exhibited faster DOX release, indicating drug release rate can be controlled by varying the laser power density. Taken together, these results suggest that the 1O2-responsive miktoarm block copolymer micelles could serve as an efficient carrier for on demand targeted drug delivery.


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80

Cummulative Release (%)

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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60

40

20

MA-P2-Ce6-DOX; Dark MA-P2-Ce6-DOX; 50 mW/cm2 MA-P2-Ce6-DOX; 100 mW/cm2

0 0

5

10

15

20

25

30

Time (h)

Figure 7. Release profile of DOX from P2-Ce6-DOX micelles without and with laser irradiation of different power densities (50 mW/cm2 and 100 mW/cm2) in PBS (pH 7.4). The error bars in the graph represent standard deviations (n = 3).

In vitro cytotoxicity and intracellular drug release Our P2-Ce6-DOX possesses several beneficial aspects as a drug delivery carrier. This is firstly because of its high stability in biological environment governed by AB2 type miktoarm structure, which minimizes non-specific leakage of cargo molecule on undesired region.


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Additionally, by employing light-responsive bond cleavage, spatiotemporally controllable drug release would be achieved to maximize the therapeutic effect in the cellular environment. In order to examine the potential of AB2-type miktoarm amphiphilic block copolymer comprising ROSlabile -aminoacrylate linkage as a drug delivery carrier, intracellular therapeutic effect was evaluated on human breast cancer (MDA-MB-231) cell line (Figure 8a and S6). As expected, polymer itself did not exhibit any cytotoxicity. In addition, nanoassembly without Ce6 (P2-DOX) exhibited negligible cytotoxicity probably because of low leakage of DOX from highly stable nanoassembly comprising miktoarm block copolymers. Interestingly, P2-Ce6 itself did not exhibit high cytotoxicity upon light irradiation despite of ROS generation from Ce6, implying the amount of ROS generated from P2-Ce6 was not enough to induce cell death itself. Meanwhile, P2-Ce6DOX exhibited remarkable therapeutic effect when the cells were subjected with light, demonstrating the ROS-assisted bond cleavage induced the maximal therapeutic effect by specifically releasing the DOX into the cytosolic region. Accordingly, intracellular specific DOX release from P2-Ce6-DOX upon light irradiation was monitored using CLSM (Figure 8b). P2-DOX did not release DOX from stable nanoassembly regardless of light irradiation. As well, P2-Ce6-DOX without light irradiation exhibited negligible DOX release in the cytoplasm. In the case of P2-Ce6-DOX upon light irradiation, however, remarkable DOX release was monitored in the cytoplasmic region of MDA-MB-231 cells. This result further proves the postulation that high therapeutic effect of P2-Ce6-DOX induced by the ROS-responsive bond cleavage and subsequent DOX release from highly stable nanoassembly comprising miktoarm block copolymers.


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Figure 8. In vitro evaluation of nanoassembly. (a) Red-light laser induced cellular photo-toxicity of samples in MDA-MB-231 cell lines. Data represent mean  SD. (***P < 0.01) (b) Cellular uptake and intracellular DOX release from P2-DOX and P2-Ce6-DOX nanoassmblies with and without 660 nm laser irradiation observed by CLSM using MDA-MB-231 cell lines.


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CONCLUSIONS In summary, a series of amphiphilic cleavable MA-PEG-b-PCL2 copolymers of varying PCL block lengths bearing 1O2-labile -aminoacrylate group with only E-configuration at the block junction were facilely synthesized via combination of amino-yne click chemistry and ROP. The synthetic methodology disclosed here can be effectively employed to design and synthesis architecturally diverse block copolymers with defined stereospecificity. In aqueous condition, the MA-PEG-b-PCL2 copolymers with relatively low CAC can readily formed self-assembled structures with different morphologies, such as spherical, cylindrical or lamella, depending on the block length of PCL arms, and preparation conditions. After physical co-loading of photosensitizer Ce6 and anticancer drug DOX into the hydrophobic core, self-aggregates adopted a spherical morphology from the cylindrical geometry as was observed for the empty aggregates. Upon redlaser light irradiation, after cellular uptake, the 1O2 generated from the Ce6 induced scission of the

-aminoacrylate linkage at the core-shell junction, leading to the dissociation of the nanostructure and facilitated triggered intracellular release of DOX for effective therapy. The results from these studies indicate that the red-laser light induced 1O2-responsive sheddable micellar platform with concomitant drug release could be highly promising for on-demand delivery of therapeutic agents for potential clinical applications.

TABLES. Table 1.. Characteristics of MA-PEG-b-PCL2 copolymers bearing 1O2-labile -aminoacrylate.


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Sample Feed Code

a

Polymer

Molecular Weight

Mw/Mnb

fPEGc

CACd

( 103 g mol-1)

[-CL0]/[I0]

Mn (NMR)a

Mw (GPC)b

P1

10

MA-PEG45-b(PCL4)2

3.142

4.634

1.22

0.71

6.46

P2

20

MA-PEG45-b(PCL8)2

4.055

5.890

1.25

0.56

4.56

P3

40

MA-PEG45-b(PCL21)2

7.023

13.138

1.45

0.33

3.64

P4

80

MA-PEG45-b(PCL35)2

10.162

23.094

1.44

0.23

1.33

Determined by comparing the NMR peak integrated ratios of methylene (-O-CH2-CH2-O-) peak

of PEG and methylene ( -C(=O)-CH2-CH2-CH2-CH2-CH2O) peak of PCL in CDCl3. bDetermined by GPC using polystyrene as standard and THF as eluent. c Volume fraction of PEG was calculated using Mn (NMR) and the densities of bulk polymers: PEG = 1.13 g cm-3 and PCL = 1.25 gcm-3. d Critical aggregation concentration (CAC) was determined using pyrene as the fluorescent probe.


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Table 2. Physicochemical characteristics of Ce6/DOX loaded micelles. Sample

Feed Amount

Loading Content

Loading Efficiency

(LC) (wt.%)

(LE) (wt.%)

Ce6

DOX

Ce6a

20

20

15.540.61 5.840.17

77.693.04 29.200.83

P2-Ce6

20

-

16.411.28 -

82.066.42 -

P2-DOX

-

20

-

-

P2-Ce6-

DOXb

Ce6a

DOXb

DOX

a

3.240.11

16.180.56

determined by UV-Visible spectroscopy, and bdetermined by fluorescence spectroscopy.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. The plot of [-CL0]/[I0] vs Mn(NMR) and Mn(GPC), GPC trace of P2, 2D DOSY NMR spectra before and after laser irradiation, FT-IR spectra of polymers, and in vitro cytotoxicity evaluation of samples with different concentration of DOX (PDF)


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGMENT This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) (NRF-2017M3A9F5030930), and the Basic Science Research Program through the NRF (2017R1E1A1A01074088, 2017R12017R1A4A1015594, and 2017R1C1B2009362) funded by the Ministry of Science, ICT & Future Planning. REFERENCES 1. Kataoka, K.; Harada, A.; Nagasaki, Y., Block copolymer micelles for drug delivery: Design, characterization and biological significance. Adv. Drug Del. Rev. 2012, 64, 37-48. 2. Letchford, K.; Burt, H., A review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes. Eur. J. Pharm. Biopharm. 2007, 65, (3), 259-269. 3. Torchilin, V. P., Micellar nanocarriers: pharmaceutical perspectives. Pharm. Res. 2006, 24, (1), 1. 4. Hsueh, H.-Y.; Chen, H.-Y.; Hung, Y.-C.; Ling, Y.-C.; Gwo, S.; Ho, R.-M., Well-Defined Multibranched Gold with Surface plasmon resonance in near-infrared region from seeding growth approach using gyroid block copolymer template. Adv. Mater. 2013, 25, (12), 1780-1786. 5. Sasidharan, M.; Nakashima, K., Core–shell–corona polymeric micelles as a versatile template for synthesis of inorganic hollow nanospheres. Acc. Chem. Res. 2014, 47, (1), 157-167. 6. Hsueh, H.-Y.; Yao, C.-T.; Ho, R.-M., Well-ordered nanohybrids and nanoporous materials from gyroid block copolymer templates. Chem. Soc. Rev. 2015, 44, (7), 1974-2018. 7. Peinemann, K.-V.; Abetz, V.; Simon, P. F. W., Asymmetric superstructure formed in a block copolymer via phase separation. Nat. Mater. 2007, 6, 992. 8. Clodt, J. I.; Filiz, V.; Rangou, S.; Buhr, K.; Abetz, C.; Höche, D.; Hahn, J.; Jung, A.; Abetz, V., Double Stimuli-Responsive Isoporous Membranes via Post-Modification of pH-Sensitive selfassembled diblock copolymer membranes. Adv. Funct. Mater. 2013, 23, (6), 731-738. 9. Phillip, W. A.; O’Neill, B.; Rodwogin, M.; Hillmyer, M. A.; Cussler, E. L., Self-assembled block copolymer thin films as water filtration membranes. ACS Appl. Mater. Interfaces 2010, 2, (3), 847-853. 10. Bennett, R. D.; Xiong, G. Y.; Ren, Z. F.; Cohen, R. E., Using block copolymer micellar


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Scheme 1. Synthesis of amphiphilic miktoarm PEG-b-PCL2 copolymer bearing 1O2-labile β-aminoacrylate via combination of amino-yne click chemistry and ring opening polymerization, and schematic illustration of self-assembly and red-laser induced 1O2-mediated dissociation of MA-PEG-b-PCL2 micelle. Chemical structures of Ce6 and DOX are shown below 234x237mm (150 x 150 DPI)


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Figure 1. 1H-NMR spectra of (a) PEG-propiolate and (b) miktoarm PEG macroinitiator bearing βaminoacrylate group (MA-PEG45-(OH)2) in CDCl3 128x124mm (150 x 150 DPI)



Figure 2. (a) 1H-NMR spectrum of MA-PEG-b-(PCL8)2 copolymer (P2) in CDCl3 and (b) GPC traces of MAPEG-b-PCL2 copolymers 132x240mm (150 x 150 DPI)


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Figure 3. TEM images of miktoarm block copolymer nanoassemblies prepared from P1 to P4 by (a) solventexchange method and (b) thin-film hydration method 187x103mm (150 x 150 DPI)



Figure 4. Determination of critical aggregation concentration of miktoarm block copolymer nanoassemblies. The fluorescence intensity ratio of I337/I334 of pyrene as a function of the block copolymer concentrations of (a) P1, (b) P2, (c) P3 and (d) P4 173x179mm (150 x 150 DPI)


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Figure 5. 1H-NMR spectra of P2 with Ce6 (~5 wt%) in the (a) dark, and after exposing laser irradiation of power density (b) 50 mW/cm2 and (c) 100 mW/cm2 for 30 min, where the solvent is CDCl3 175x209mm (150 x 150 DPI)



Figure 6. Change of size distribution profile of P2-Ce6-DOX micelles at (a) dark, and after 660 nm laser irradiation of power density (b) 50 mW/cm2 and (c) 100 mW/cm2 as a function of time. (d) Time-dependent changes in light scattering of P2Ce6-DOX micelles at dark and after 660 nm laser irradiation as a function of time. TEM images of P2-Ce6-DOX (e) before and (f) after 660 nm laser irradiation 165x228mm (150 x 150 DPI)


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Figure 7. Release profile of DOX from P2-Ce6-DOX micelles without and with laser irradiation of different power densities (50 mW/cm2 and 100 mW/cm2) in PBS (pH 7.4). The error bars in the graph represent standard deviations (n = 3) 149x155mm (150 x 150 DPI)



Figure 8. In vitro evaluation of nanoassembly. (a) Red-light laser induced cellular photo-toxicity of samples in MDA-MB-231 cell lines. Data represent mean ± SD. (***P < 0.01) (b) Cellular uptake and intracellular DOX release from P2-DOX and P2-Ce6-DOX nanoassmblies with and without 660 nm laser irradiation observed by CLSM using MDA-MB-231 cell lines 168x251mm (150 x 150 DPI)


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Table of Content 187x113mm (150 x 150 DPI)


Miktoarm Amphiphilic Block Copolymer with Singlet Oxygen-Labile

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