âBottom-Upâ Construction of Hyperbranched Poly(prodrug-co

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“Bottom-Up” Construction of Hyperbranched Poly(prodrugco-photosensitizer) Amphiphiles Unimolecular Micelles for Chemo-Photodynamic Dual Therapy Pei Sun, Nan Wang, Xin Jin, and Xinyuan Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13055 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 2, 2017

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ACS Applied Materials & Interfaces

“Bottom-Up” Construction of Hyperbranched Poly(prodrug-cophotosensitizer) Amphiphiles Unimolecular Micelles for ChemoPhotodynamic Dual Therapy Pei Sun, Nan Wang, Xin Jin*, Xinyuan Zhu School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China. Keywords: “bottom-up” strategy; chemo-photodynamic dual therapy; unimolecular micelles; drug delivery, redox-responsive

Abstract Despite of the great advantages of chemo-photodynamic combination therapy, tedious synthesis steps and laborious purification procedures make the fabrication of chemo-photodynamic combined therapeutic platforms rather difficult. In this study, we develop a facile “bottom-up” strategy to fabricate hyperbranched poly(prodrug-cophotosensitizer) amphiphiles, h-P(CPTMA-co-BYMAI)-b-POEGMA (hPCBE), for chemo-photodynamic dual therapy. The easily prepared hPCBE possess a bottom-upconstructed

hydrophobic

core

h-P(CPTMA-co-BYMAI)

(hPCB)

direct

copolymerized from reduction-responsive CPT prodrug monomer (CPTMA) and boron dipyrromethene-based photosensitizer monomer (BYMAI), as well as a biocompatible shell polymerized from hydrophilic monomers. Due to the covalently interconnected core-shell structure, hPCBE exists as unimolecular micelles in aqueous solution and exhibits excellent structural stability under dilution condition. The hPCBE micelles can effectively internalized by MCF-7 cells and release CPT 1/36

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triggered by the reductive milieu. In addition, photosensitizer moieties embedded in the hPCB core could generate singlet oxygen (1O2) effectively under irradiation, endowing hPCBE with the boosting of chemotherapeutic efficacy. Compared with the single chemotherapy of hyperbranched polyprodrug amphiphiles h-PCPTMA-bPOEGMA (hPCE) and photodynamic therapy of hyperbranched polyphotosensitizer amphiphiles h-PBYMAI-b-POEGMA (hPBE), hPCBE shows higher in vitro cytotoxicity. We expect that our approach will further boost research on designing of multifunctional drug delivery systems via the facile “bottom-up” strategy.

1. Introduction Because of the high efficiency, chemotherapy remains one of the major strategies for

most

cancer

cases.

After

continuous

treatment

with

an

individual

chemotherapeutic modality, however, considerable side effects and drug resistance would be induced.1,2 In order to overcome the limitations of single chemotherapy, attention has been directed toward the combination of multiple therapies,3-6 especially chemotherapy and photodynamic therapy (PDT). PDT utilizes photosensitizers (PS) that are activated by light to produce highly reactive singlet oxygen (1O2), which interact with adjacent biological macromolecules and thus damage cancer cells through different mechanisms from chemotherapy.7-9 In addition, PDT has the advantages in terms of minimally invasive nature, capability of repeated doses, and better selectivity from normal tissues.10 Up to now, many of the most commonly-used chemotherapy drugs have already been investigated in combination with PDT using polymeric drug delivery systems, and revealed higher therapeutic effect compared to the single therapy.11-19 Considering the pharmacokinetics in vivo, attachment of smallmolecular anticancer drugs and photosensitizers to polymeric carriers via covalent bonds exhibits much more stability and controllability than physical encapsulation.20-

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22

The most common method for this purpose is to conjugate small-molecular

anticancer drugs and photosensitizers to the synthesized polymers via “post-synthesis” approach, which often suffers from tedious reaction steps with limited control of the site and degree of drug loading. In this context, it is an urgent demand to develop a facile and control method for the preparation of chemo-photodynamic dual therapeutic platforms. A facile “bottom-up” strategy may possibly overcome the above drawbacks.23 With such “bottom-up” strategy, the therapeutic agents were first modified to prepare polymerizable monomers, and then directly incorporated during the synthesis of the polymers by the polymerization of the polymerizable monomers. Indeed, several pioneering applications in drug delivery with bottom-up-constructed therapeutic platforms have appeared.24-27 For instance, Liu and co-workers bottom-up-synthesized a polyprodrug amphiphiles PEG-b-PCPTM via the directly polymerization of reduction-cleavable camptothecin prodrug monomer.28 Another prominent example is the “bottom-up” synthesis of linear polyprodrug amphiphiles by the ring-opening polymerization of PEG and prodrug monomers consisting of a cyclic polymerizable group.29 Very recently, we have also pioneered the bottom-up construction of multiprodrug-arm hyperbranched copolymers for cancer therapy.30 However, these bottomup-constructed therapeutic platforms were involved in only single chemotherapy, and the bottom-up-constructed chemo-photodynamic dual therapeutic platform has never been reported so far. Inspired by the polymerizable prodrug monomers used in the “bottom-up” strategy, we envisioned that photosensitizer with the possibility of modification with polymerizable functional groups could also be incorporated into a therapeutic platform via the “bottom-up” strategy. Such novel multiple therapeutic

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system brings together facility of “bottom up” strategy and efficiency of chemophotodynamic dual therapy. Herein, we report the “bottom-up” fabrication of hyperbranched poly(prodrug-cophotosensitizer) amphiphiles hPCBE for chemo-photodynamic dual therapy (Scheme 1). For this purpose, anticancer drug CPT was selected to prepare polymerizable prodrug monomer CPTMA with a reduction-responsive linker, and dipyrromethenebased photosensitizer was chosen to prepare polymerizable photosensitizer monomer BYMAI.31-35 The hyperbranched poly(prodrug-co-photosensitizer) core hPCB was directly copolymerized form the two polymerizable monomers via the “bottom up” strategy. Then, hydrophilic shell POEGMA was polymerized on the surface of hydrophobic core hPCB from oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) monomers to prepare hyperbranched poly(prodrug-co-photosensitizer) amphiphiles hPCBE. Due to such covalently interconnected core-shell structure, hPCBE exist as structurally stable unimolecular micelles in aqueous solution and exhibit excellent stability under dilution condition. The hPCBE micelles can be effectively internalized by MCF-7 cells, and CPT release from hPCBE will be triggered by the reductive milieu, resulting in tumor cells death. In addition, photosensitizer moieties embedded in the hPCB core will generate singlet oxygen (1O2) under irritation, endowing hPCBE with the boosting of chemotherapeutic efficacy. Comparing with the single chemotherapy of hyperbranched polyprodrug amphiphiles hPCE and photodynamic therapy of hyperbranched polyphotosensitizer amphiphiles hPBE, hPCBE shows higher in vitro cytotoxicity, suggesting a potential therapeutic platform for chemo-photodynamic dual therapy.

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Polymerizable group Hyperbranched poly(prodrug-cophotosensitizer) core

Responsive linker S-S

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Anticancer drug

Prodrug monomer

“Bottom-up” fabrication

Hyperbranched poly(prodrug-cophotosensitizer) amphiphiles

OEGMA

RAFT polymerization Photosensitizer

Uptake by tumor cells

Light Photosensitizer monomer

3O

1O 1O

2

2

2

PDT Improved cytotoxicity Reductive release

Chemotherapy

Scheme 1. Schematic illustration of the “bottom-up” fabrication of hyperbranched poly(prodrug-co-photosensitizer) amphiphiles hPCBE, and their reduction-responsive drug release and 1O2 generation under irradiation for chemo-photodynamic dual therapy.

2. Materials and methods 2.1. Materials Oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA, Mn = 475 g/mol, mean degree of polymerization is 5-6) purchased from Aldrich was passed through a neutral alumina column to remove the inhibitor and then stored at 20 °C prior to use. 2,2'-Azobis(2-methylpropionitrile) (AIBN) was purchased from J&K Scientific Ltd. (China) and recrystallized twice from ethanol before used. FITCAnnexin V apoptosis detection kit was purchased from BD Biosciences. All other reagents, unless otherwise noted, were purchased from commercial sources and used without further purification. 5/36



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2.2. Characterization. 1H and

13

C nuclear magnetic resonance (NMR) spectra

were collected in deuterated chloroform (CDCl3) solution with a Varian MERCURY plus 400 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal standard. Fourier transform infrared (FTIR) spectra were measured on a PerkinElmer Paragon 1000 spectrophotometer by KBr sample holder method. High-resolution mass spectrometry (HRMS) data were obtained for each sample from 50 to 1000 Da with a 0.10 s scan time and a 0.01 s inter scan delay over a 10-min analysis time on a Waters Micromass Q-TOF Premier mass spectrometer. The molecular weights and polydispersity index (PDI) were determined by gel permeation chromatography (GPC). The Perkin-Elmer series 200 system (10 µm PL gel 300 × 7.5 mm mixed-B and mixed-C column, polystyrene calibration) equipped with a refractive index (RI) detector. N,N-dimethylformamide (DMF) containing 0.01 mol/L lithium bromide was used as the mobile phase at a flow rate of 1 mL/min at 30 °C. Ultraviolet-visible (UVvis) absorption of the sample solutions were measured at room temperature by using a Thermo Electron-EV300 UV-vis spectrophotometer. Fluorescent spectra were recorded on a PerkinElmer LS 50B fluorescence spectrometer. Dynamic Light Scattering (DLS) measurements were performed on a Malvern Zetasizer NanoZS apparatus, and all samples were measured at a scattering angel of 90°, laser operating at 633 nm. Transmission Electron Microscopy (TEM) studies were carried on a JEOL 2010 instrument operated at 200 kV. The samples were prepared by dropping the sample solution onto the carbon-coated copper grid and naturally air dried. 2.3. Synthesis of reduction-responsive prodrug monomer CPTMA. CPTMA was prepared according to the procedure described in the literature.28 Camptothecin (CPT, 2.0 g, 5.74 mmol) and DMAP (2.11 g, 17.3 mmol) were suspended in dry DCM

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(50 mL) under argon atmosphere. Triphosgene (0.567 g, 1.92 mmol) in 1 mL dry DCM was added and the mixture was stirred at room temperature for 30 min. HSEMA (1.40 g, 6.31 mmol, in 15 mL dry THF) was added dropwise, and the reaction mixture was stirred overnight during which a white precipitate was formed. After filtration and evaporating all the solvents, the residues were diluted with diethyl acetate and washed with water, 1.0 M HCl, and brine, respectively. The organic layer was collected and dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (ethyl acetate) to give CPTM as a pale solid powder (2.47 g, yield: 73%). 1H NMR, (400Hz, CDCl3), δ (ppm) = 8.41 (s, 1H), 8.23 (d, J= 11.3 Hz, 1H), 7.95 (d, J = 10.8 Hz, 1H), 7.85-7.83 (m, 1H), 7.70-7.68 (m, 1H), 7.35 (s, 1H), 6.08 (s, 1H), 5.73-5.69 (m, 2H), 5.42 (s, 1H), 5.30 (d, J = 23.6 Hz, 2H), 4.40-4.32 (m, 4H), 2.96-2.91 (m, 4H), 2.30-2.15 (m, 2H), 1.91(s, 3H), 1.030.99 (m, 3H).

13

C NMR, (400Hz, CDCl3), δ (ppm) =167.25, 167.05, 157.31, 153.46,

152.32, 148.92, 146.52, 145.62, 135.98, 131.17, 130.73, 129.70, 128.48, 128.20, 128.11, 125.97, 120.29, 95.97, 78.05, 67.09, 66.56, 62.42, 50.02, 37.29, 36.6, 31.91, 18.24, 7.64. ES I-MS m/z (M+H+) calcd. 597.1365, found 597.1365 (M+H+). 2.4. Synthesis of fluorescent molecule BODIPY. To a solution of p-hydroxy benzaldehyde (1 g, 8.19 mmol) and 2,4-dimethylpyrrole (1.85 mL, 18.02 mmol) in THF (150 mL) was added several drops of trifluoroacetic acid under a nitrogen atmosphere. The mixture was stirred at room temperature for 6 h, and the solution of 2,3-dichloride-5,6-dicyano-p-benzoquinone (DDQ, 2.05 g, 9.01 mmol) in THF (100 mL) was added. The resulting mixture was stirred continuously for another 5 h. After the addition of triethylamine (25 mL) and BF3·OEt2 (31 mL) dropwise to the reaction mixture with an ice-water bath, the mixture was kept stirring at room temperature overnight, then filtered through celite. The resulting mixture was filtrated and the

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solid was washed with CH2Cl2. The combined filtrate was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was re-dissolved in CH2Cl2 and the solution was washed with 15% NaHCO3 solution followed by water. The organic phase was dried over anhydrous Na2SO4, then filtered and evaporated. The crude product was purified by column chromatography using progressively more polar 50/1 to 9/1 (hexane/EtOAc) as eluent to afford BODIPY as an orange solid (1.1 g, yield 39%). 1H NMR (400 MHz, CDCl3) δ (ppm) = 7.12 (d, J = 12 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 5.98 (s, 6H), 2.55 (s, 2H), 1.44 (s, 6H). 2.5. Synthesis of BYMA. BODIPY (1.2 mmol, 475 mg) was dissolved anhydrous CH2Cl2 under nitrogen atmosphere, and DBU (2 equiv., 2.4mmol, 365mg) was slowly added with a syringe to the solution. Then methacryloyl chloride (1.5 equiv., 1.8 mmol, 190 mg) was added to the dark solution. After stirring at room temperature for 24 h, the mixture was concentrated and the residue purified by chromatography on silica gel (CH2Cl2/petroleum ether: 7/3) to afford the product BYMAI (432mg, 78%). 1

H NMR (400 MHz, CDCl3) δ (ppm): 7.33-7.26 (m, 4H), 6.39 (s, 1H), 5.99 (s, 2H),

5.81 (s, 1H), 2.56 (s, 6H), 2.09 (s, 3H), 1.44 (s, 6H). 2.6. Synthesis of BYMAI. At room temperature, BYMA (102 mg, 0.25 mmol) was dissolved in 15 mL anhydrous CH2Cl2, then N-iodosuccinimide (NIS, 140 mg, 0.62 mmol) was added into the solution. The mixture was stirred at room temperature for 24 h, and then washed with Na2S2O3 solution and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (CH2Cl2/petroleum ether: 1/2) to give the product BYMAI as a purple solid (60 mg, yield 36%). 1H NMR (400 MHz, CDCl3): δ (ppm) = 7. 33-7. 26 (m, 4H), 6.41 (s, 1H), 5.82 (s, 1H), 2.65 (s, 6H), 2.09 (s, 3H), 1.46 (s, 6H).

13

C NMR: δ (ppm) = 165.64,

157.20, 152.09, 145.54, 140.55, 135.75, 132.26, 131.55, 129.42, 129.22, 128.08,

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123.23, 122.95, 86.09, 30.86, 29.94, 25.71, 24.96, 18.61, 17.42, 16.30. HRMS: m/z calculated for [C23H21BF2 I2N2O2]+: 659.98; found: 659.94. 2.7.

Synthesis

of

hyperbranched

poly(prodrug-co-photosensitizer)

h-

P(CPTMA-co-BYMAI) core, hPCB. The hPCB core was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization technique. Typically, a glass ampoule was charged with s-(4-vinyl)benzyl S’-propyltrithiocarbonate (VBPT) (32 mg, 0.12 mmol), CPTMA (596 mg, 1.0 mmol), BYMAI (132 mg, 0.2 mmol), AIBN (3.9 mg, 0.024 mmol), and 3 mL 1,4-dioxane and dimethyl sulfoxide (DMSO) mixed solvents (1:1, v/v). It was then degassed by three freeze-thaw cycles. The polymerization was carried out at 70 °C for 24 h, and quenched by putting the glass ampoule into an ice-water bath. The crude product was obtained by precipitation into diethyl ether, and then purified by precipitated into diethyl ether from CH2Cl2 for three times. The final product was dried under vacuum at 35 °C for 24 h, yielding a purple red solid (566 mg, yield 77%). 2.8. Synthesis of hyperbranched poly(prodrug-co-photosensitizer) amphiphiles h-P(CPTMA-co-BYMAI)-b-POEGMA, hPCBE. hPCBE was synthesized by the RAFT

polymerization

of

OEGMA

using

hPCB

core

as

hyperbranched

macromolecular RAFT agent. Typically, hPCB (100 mg), OEGMA (500 mg, 1.67 mmol), AIBN (3.9 mg, 0.024 mmol) and 3 mL DMSO were placed in a glass ampoule. The resulting mixture was degassed via three freeze-pump-thaw cycles and then immersed into an oil bath thermostated at 70 °C to start the polymerization. After 24 h, the polymerization was terminated by ice-water bath. The crude product was obtained by precipitated into diethyl ether. The crude product was re-dissolved in CH2Cl2 and precipitated into diethyl ether for three times. The final product was dried under vacuum at 35 °C for 24 h, yielding highly viscous (480 mg, yield 80%).

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2.9. Synthesis of hyperbranched polyprodrug amphiphiles h-PCPTMA-bPOEGMA,

hPCE. In a similar procedure, photosensitizer BYMAI free

hyperbranched polyprodrug amphiphiles hPCE was synthesized. The hyperbranched polyprodrug core h-PCPTMA (hPC) was first synthesized. In brief, a glass ampoule was charged with VBPT (32 mg, 0.12 mmol), CPTMA (715.3 mg, 1.2 mmol), AIBN (3.9 mg, 0.024 mmol), and 3 mL 1,4-dioxane and DMSO mixed solvents (1:1, v/v). It was then degassed by three freeze-thaw cycles and sealed under vacuum. The polymerization was carried out at 70 °C for 24 h, and quenched by putting the glass ampoule into an ice-water bath. The crude product was obtained by precipitation into diethyl ether, and then purified by precipitated into diethyl ether from CH2Cl2 for three times. The final product was dried under vacuum at 35 °C for 24 h, yielding a purple red solid (613 mg, yield 82%). Then, the obtained hPC core was employed as hyperbranched macromolecular RAFT agent to polymerize OEGMA to prepare hPCE. hPC (100 mg), OEGMA (500 mg, 1.67 mmol), AIBN (3.9 mg, 0.024 mmol) and 3 mL DMSO were placed in a glass ampoule, then the mixture was degassed via three freeze-pump-thaw cycles. Subsequently, the glass ampoule was immersed into a 70 °C oil-bath. After reaction for 24 h, the polymerization was terminated by icewater bath. The crude product was obtained by precipitated into cold diethyl ether. The crude product was then dissolved in CH2Cl2 and precipitated into diethyl ether for three times. The final product was dried in vacuum at 35 °C for 24 h, yielding highly viscous (504 mg, yield 84%). 2.10. Synthesis of hyperbranched polyphotosensitizer amphiphiles h-PBYMAIb-POEGMA, hPBE. Briefly, a glass ampoule was charged with VBPT (16.1 mg, 0.06 mmol), BYMAI (396 mg, 0.6 mmol), AIBN (1.8 mg, 0.012 mmol), and 1.5 mL 1,4-dioxane and DMSO mixed solvents (1:1, v/v). The solution was degassed by three

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freeze-thaw cycles. The polymerization was carried out at 70 °C for 24 h, and quenched by putting the glass ampoule into an ice-water bath. The crude product was obtained by precipitation into diethyl ether, and then purified by precipitated into cold diethyl ether from CH2Cl2 for three times. The final product was dried under vacuum at 35 °C for 24 h, yielding a purple red solid (342 mg, yield 83%). Then, the obtained hPB was employed as hyperbranched macromolecular RAFT agent to further polymerize OEGMA. hPB (100 mg), OEGNA (500 mg, 1.67 mmol), AIBN (3.9 mg, 0.024 mmol) and 3 mL DMSO were placed in a glass ampoule, then the solution was degassed via three freeze-pump-thaw cycles. The reaction was carried out at 70 °C for 24 h. Then, the polymerization was terminated by ice-water bath, and the crude product was obtained by precipitated into cold diethyl ether. The obtained crude product was dissolved in CH2Cl2 and precipitated into diethyl ether for three times. The final product was dried under vacuum at 35 °C for 24 h, yielding highly viscous (468 mg, yield 78%). 2.11. Preparation of unimolecular micelles. hPCBE unimolecular micelles were prepared via the cosolvent approach and water dialysis displacing approach.25 Typically, hPCBE was firstly dissolved in DMF at a concentration of 20.0 mg/mL. Then, the solution was directly dialyzed (molecular weight cutoff, MWCO = 3,500 g/mol) against deionized water for 48 h, during which the deionized water was renewed every 4 h. Finally, the hPCBE unimolecular micellar dispersion was concentrated or diluted to the desired concentration for further experiments. hPCE and hPBE unimolecular micelles were prepared likewise. 2.12. Drug loading content and in vitro CPT release study of hPCBE and hPCE. The drug loading content (DLC) of hPCE, hPBE, or hPCBE (CPT and BYMAI) were measured as follows: hPCE, hPBE, and hPCBE were dissolved in

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DMSO, respectively. The UV absorbance of the solution at 360 nm (CPT) and 539 nm (BYMAI) was measured and the total loading of drugs was determined by using the linear standard curves, respectively. DLC was calculated according to the following formula: DLC (wt%) = (weight of drug/weight of polymer) × 100% The release profile of CPT from hPCBE or hPCE was evaluated using a dialysis method and performed as follows: 2 mL of hPCBE or hPCE solution (1.0 mg/mL) was transferred into a dialysis tubing (MWCO = 3,500 g/mol). The dialysis tubing was immersed into 50 mL pH = 7.4 PBS medium with different concentration of DTT (0 mM DTT, 2 µM DTT, 5 mM DTT or 10 mM DTT) at 37 °C with shaking. After varying time intervals, 2 mL of external medium was taken and replaced with 2 mL of fresh medium, and the amount of released CPT was determined by UV absorption at the wavelength of 360 nm. 2.13. Singlet oxygen generation (1O2) measurements in aqueous solution. To assess whether the photosensitizer embedded hyperbranched amphiphiles (hPCBE and hPBE) could generate cytotoxic 1O2 rapidly in aqueous solution. In vitro 1O2 generation measurement of hPCBE and hPBE in aqueous solution was carried out according to the reported method. P-Nitrosodimethylaniline (RNO) and imidazole were used as 1O2 scavengers. According to the previous reports, RNO does not react with 1O2 directly; however, it can react with the product formed by the reaction of 1O2 with imidazole, resulting in a decrease in absorption at 440 nm (the absorbance of RNO). Solution of RNO (30 µM) and imidazole (0.5 mM) in PBS (placed in a 96well plate) was added hPCBE or hPBE (final BYMA concentration of 5 µg/mL), which was then irradiated under green LED light (λ = 520-560 nm, 1.5 mW/cm2) for different periods of time, and the OD values at 440 nm were recorded on a BioTek

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Synergy H4 hybrid reader. For the control experiment, irradiation was also carried out on an RNO and imidazole solution in the absence of the photosensitizer, and Rose Bengal was used as the reference. 2.14. Cell culture. MCF-7 cells (a human breast adenocarcinoma cell line) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplied with 10 % fetal bovine serum (FBS) and antibiotics (50 units mL-1 penicillin and 50 units mL-1 streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. 2.15. Cellular uptake of micelles. The cellular uptake behaviors of micelles in MCF-7 cells were studied using both flow cytometry analysis and confocal laser scanning microscopy (CLSM). For flow cytometry study, MCF-7 cells were seeded in 6-well plates at a density of 5.0 × 105 cells per well in 2 mL of complete DMEM. After 24 h incubation, the cells were treated with Rhodamine B-labelled micelles (hPCBE, hPCE, or hPBE). Then, the cells were incubated at 37 °C for 15 min, 30 min, 1 h, 2 h, and 4 h. The untreated cells were used as control. Thereafter, culture medium was removed and cells were washed with PBS for three times and treated with trypsin. Subsequently, the cells were collected, centrifuged (1000 rpm, 5 min), and washed with PBS twice. Finally, the centrifuged cells were suspended in 1 mL PBS for analysis. The data for 1.0 × 104 gated events were collected and the analysis was performed by means of a BD LSRFortessa flow cytometer and CELL Quest software. For confocal laser scanning microscopy (CLSM) study, MCF-7 cells were plated in 6-well plates at 2.0 × 104 cells per well in 2 mL complete DMEM (a clean coverslip was put in each well) and incubated overnight. The culture medium was removed and cells were treated with rhodamine B-labelled micelles for 15 min, 2 h and 4 h at 37 °C. Then the medium was removed, cells were washed with Hank’s balanced salt solution (HBSS) for three times and fixed by 4 % formaldehyde for 30

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min at room temperature. After washed by cold HBSS for three times, the cells were stained with SYTOX® green for 15 minutes at room temperature and the slides were rinsed with HBSS. The resulting slides were mounted and imaged on a LEICA TCS SP8 fluorescence microscopy. 2.16. Cellular 1O2 detection upon irradiation. MCF-7 cells were seeded in 6-well plates at a density of 5.0 × 105 cells per well in 2 mL of complete DMEM and incubated overnight. The culture medium was removed and cells were treated with fresh DMEM medium (2 mL) containing hPCBE or hPCE micelles at the final BYMAI equivalent concentration of 5 µg/mL. The untreated cells were used as the blank control. After 4 h incubation, cells were further incubated with 20 µM DCFHDA for 20 min, then exposed to irradiation for 15 min or not. Subsequently, the fluorescence intensity of DCF inside the cells was detected by flow cytometry, which was representative of the level of 1O2 generation. 2.17. In vitro cytotoxicity study. The cytotoxicity of hPCBE against MCF-7 cells was evaluated by MTT assay. The hPCE, hPBE and free CPT were used as control. Briefly, cells were seeded in 96-well plates at a density of 1 × 104 cells per well and cultured overnight. The culture medium was removed and cells were treated with 200 µL of fresh DMEM medium containing serial dilutions of hPCBE, hPCE, hPBE or free CPT. For dark cytotoxicity, the cells were incubated for another 24 h. For light cytotoxicity, after 4 h co-incubation with drugs, the cells were exposed to green LED light for 15 min and then incubated for another 20 h. Untreated cells were used as the blank control. At the end of each incubation, 20 µL per well of MTT assay stock solution (5 mg mL-1 in PBS) was added. After 4-hour incubation, the medium was carefully removed, and the resulting blue formazan crystals were dissolved in 200 µL per well DMSO. Finally, the plates were shaken for 10 min, and the absorbance of

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formazan product at a wavelength of 490 nm was measured in a BioTek Synergy H4 hybrid reader to calculate the number of viable cells.

3. Results and discussions (a)

CPT

CPTMA

(b)

BODIPY

BYMA

BYMAI

(c) CPTMA

+

VBPT

OEGMA, AIBN

1,4-Dioxane/DMSO, AIBN , 70 oC

DMSO, 70 oC

BYMAI

h-P(CPTMA-co-BYMAI) -b-POEGMA (hPCBE)

h-P(CPTMA-co-BYMAI) (hPCB)

(d) OEGMA, AIBN

CPTMA

DMSO, 70 oC VBPT 1,4-Dioxane/DMSO, AIBN , 70 oC

h-PCPTMA-b-POEGMA h-PCPTMA (hPC)

(hPCE)

(e) BYMAI VBPT

OEGMA, AIBN

1,4-Dioxane/DMSO, AIBN , 70 oC

DMSO, 70 oC

h-PBYMAI (hPB)

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h-PBYMAI-b-POEGMA (hPBE)


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

Page 16 of 37

Scheme 2. Schematic illustration for the synthesis of (a) polymerizable reductionresponsive CPT prodrug monomer CPTMA and (b) polymerizable photosensitizer monomer BYMAI. (c) “Bottom-up” construction of hyperbranched poly(prodrug-cophotosensitizer) amphiphiles hPCBE, (d) hyperbranched polyprodrug amphiphiles hPCE, and (e) hyperbranched polyphotosensitizer amphiphiles hPBE. 3.1. Synthesis and characterization of hyperbranched poly(prodrug-cophotosensitizer) amphiphiles hPCBE. As one of the most efficient methods, the RAFT

technique

has been

widely employed

to fabricate the advanced

macromolecular architectures.36,37 In current study, hyperbranched poly(prodrug-cophotosensitizer) amphiphiles hPCBE was synthesized

via two-step RAFT

polymerization (Scheme 2). The first step involves the “bottom-up” construction of hyperbranched poly(prodrug-co-photosensitizer) core hPCB. For this purpose, anticancer drug CPT is used as a model drug, and the reduction-responsive prodrug monomer CPTMA was synthesized as shown in Scheme 2a.19 The polymerizable boron dipyrromethene-based photosensitizer monomer, BYMAI, was synthesized via three-step procedures (Scheme 2b), and 1H NMR (Figure S7), 13C NMR (Figure S8) and HRMS analyses (Figure S9) of BYMAI confirm its chemical structure. With the polymerizable monomers CPTMA and BYMAI in the hand, we then employed RAFT technique to copolymerize them using a inimer-type RAFT chain transfer agent VBPT, affording hPCB as hyperbranched poly(prodrug-co-photosensitizer) core (Scheme 2c). The 1H NMR spectrum and the attributions of hPCB are shown in Figure 1a. The multiple peaks range from 6.8-8.5 ppm (m, n, j, k, l, i, s) can be assigned to the characteristic protons from CPT moieties, and the signal appear at 2.58 ppm corresponding to the characteristic protons of methyl groups in the BYMAI moieties. With embedding CPT and BYMAI in the hyperbranched polymeric matrix, 16/36


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the UV-vis absorption spectrum (Figure 2a) of hPCB displays characteristic absorption peaks both of CPT and BYMAI at 359 and 539 nm, respectively. As expected, for the fluorescence spectrum of hPCB (Figure 2b), upon excitation at 365 nm, an intense emission peak at 430 nm corresponding to the emission of CPT can be observed; and there is also a weak emission peak at 577 nm attributing to the emission of BYMAI. When the exciting wavelength was tuned to 520 nm, the intense emission of BYMAI at 577 nm is observed. This result not only verifies the successful “bottom-up” construction of the hPCB with the anticancer drug CPT and photosensitizer BYI embedded into the network, but also imply the embedded CPT and BYMAI maintain an intact structure. The second step of the synthesis of hPCBE involves the RAFT polymerization of OEGMA on the surface of hPCB core to grow hydrophilic corona chains. The 1H NMR spectrum and the attributions of hPCBE are shown in Figure 1b. Comparing to the hPCB core, new signals at 3.39 (b), 3.66 and 4.09 (a) ppm characteristic of OEGMA units confirm the successful fabrication of hPCBE. In addition, comparing to the FTIR spectrum of hPCB, the new peak at 2869 cm−1 appearing in spectrum of hPCBE corresponds to the C-H stretching vibration of OEGMA units, and the absorption peak of C-O-C stretching vibration of OEGMA units is also clearly observed at 1110 cm-1. Moreover, hPCBE exhibits the same luminescent performance as the hPCB core from the spectra of UV-vis (Figure 2a) and fluorescence (Figure 2b), and the content of the anticancer drug CPT and photosensitizer BYMAI in hPCBE were determined to be about 12.4 and 4.5 wt% according to the standard curve of the UV-vis absorbance of free CPT and BYMAI, respectively. All this results demonstrate

that

hyperbranched


amphiphiles

hPCBE has been synthesized successfully. As comparison, photosensitizer BYMAI-

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free hyperbranched polyprodrug amphiphiles hPCE (Scheme 2d) and anticancer drug CPT-free hyperbranched polyphotosensitizer amphiphiles hPBE (Scheme 2e) were also synthesized and characterized following similar procedures.

(a)

(b)

Figure 1. 1H NMR spectra of (a) hyperbranched poly(prodrug-co-photosensitizer) core hPCB and (b) hyperbranched poly(prodrug-co-photosensitizer) amphiphiles hPCBE.

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Figure 2. (a) UV-vis and (b) fluorescence spectra of CPT, BYMAI, hPCB and hPCBE. 3.2. Characterization of hPCBE Micelles. The structure of hyperbranched amphiphiles hPCBE employs the hyperbranched poly(prodrug-co-photosensitizer) hPCB as hydrophobic core and polyOEGMA multi-arms as hydrophilic shell. According to the previous reports, such core-shell amphiphilic hyperbranched copolymers might exit as unimolecular micelles or multimolecular aggregates in aqueous media.38 In the current study, the aggregation behavior of hPCBE in aqueous media was explored according to the conventional cosolvent approach (THF and water).24 The hPCBE was first dissolved in THF (1.0 mg/mL), which is a common good solvent for both hPCB core and POEGMA shell, thus the intermolecular aggregation of hPCBE could be ruled out. As shown in Figure 3a, the size of hPCBE measured by DLS in THF is ~ 37.8 nm (PDI = 0.20). The size of hPCBE in water at the same concentration is then determined to be ~ 32.8 nm (PDI = 0.193), which is nearly identical to that in THF. The little size difference probably due to the shrinkage of the hydrophobic hPCB core in water.21 The TEM image of the hPCBE unimolecular micelles (Figure 3b) shows a monodisperse spherical morphology with average diameter of ~ 24 nm, which consists with the size determined by DLS. It’s well know that the amphiphilic hyerbranched copolymers with core-shell structure can exist as unimolecular micelles in aqueous solution. And with the increase of the concentration,

the

unimolecular

micelles

will

further

aggregate

forming

multimolecular aggregates. We then investigate the aggregation behavior of hPCBE by measuring the critical aggregation concentration (CAC). As shown in Figure S17, the CAC value of hPCBE is determined to be about 1.61 mg/mL. Base on the above results, we conclude that the hPCBE aggregate to multimolecular micelles in high

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concentration, and exist as stable covalently interconnected unimolecular micelles in a certain concentration range. Such covalently interconnected unimolecular micelles have been regarded as viechles with excellent stability comparing to the conventional micelles formed via the noncovalent interaction, especially against high-folds dilution and other microenvironment changes.39-45 To verify the structural stability of hPCBE unimolecular micelles, we then examined the concentration-dependent size using DLS. As displayed in Figure 3c, the hPCBE micelles exhibit robust stability and remain almost constant size (32-34 nm) when the aqueous solution is diluted from 1000 to 50 µg/mL. Moreover, the size of the hPCBE unimolecular micelles (Figure 3d) varies very little over the span of 30 days in aqueous solution, indicating the good stability probably due to their covalently interconnected core-shell structure. Employing similar methods, the aggregation behaviors of hPCE and hPBE unimolecular micelles were also measured. DLS results reveal that the hydrodynamic diameters of hPCE (Figure S18a) and hPBE (Figure S18b) are ~ 25.2 and 38.6 nm, which is in accordance with the diameters ~ 30.9 nm and 46.3 nm measured in THF. The two unimolecular micelles could be clearly observed under TEM, and appear well-dispersed and uniform spherical morphology with average diameter of ~ 18 for hPCE (Figure S18c) and 32 nm for hPBE (Figure S18d), respectively. The size of hPCE and hPBE in aqueous solution also exhibits robust stability against dilution (Figure S19) and good storage stability (Figure S20).

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(b)

(a)

(c)

Dh = 32.8 nm

Dh = 35.9 nm

PDI = 0.193

PDI = 0.20

(d)

Figure 3. (a) DLS curves of hPCBE in water and THF, respectively. (b) TEM images of hPCBE. (c) DLS curves of hPCBE unimolecular micelles in water at varying concentrations. (d) Influence of storage time on size of hPCBE unimolecular micelles. 3.3. In vitro drug release. A fast release of small-molecular anticancer drug from the drug delivery systems is a critical factor for the efficacy of cancer therapy. In current study, the prodrug monomer CPTMA contains a reduction-cleavable disulfide linker and a carbonate moiety, both of which render hPCBE with the self-immolative cleavage feature in the reductive environment. Thus, the in vitro release behavior of hPCBE micelles was evaluated by dialysis in PBS (pH 7.4) containing different concentrations of DTT (0 mM, 2µM, 5 mM, 10 mM) at 37 °C. As shown in Figure 4a, the release rate of CPT from hPCBE micelles shows a good relation to the concentration of DTT, and the general trend is that higher DTT levels lead to faster release of CPT from the micelles. In particular, less than 5% cumulative CPT release 21/36



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was detected in the absence of DTT or in the presence of 2 µM DTT after 96 h incubation, suggesting a minimal drug leakage in non-reductive condition. Upon incubation in the presence of 5 mM DTT, approximately 28% CPT was gradually released within 48 h due to the reduction-responsive cleavage of disulfide bonds and slow diffusion from the micellar cores. With the further increasing of DTT concentration to 10 mM, the cumulative CPT release is up to approximately 60% after 48 h incubation. As mentioned above, the CPT release is high dependent on reductive environment, but the 1O2 generated by photosensitizer BYMAI embedded in hPCBE possess strong oxidation, thus we then evaluate the influence of irradiation on the CPT release rate from hPCBE micelles. As displayed in Figure 4b, the CPT release behavior under irradiation shows a similar curve to that of in the dark, suggesting the CPT release is not affected by irradiation. In addition, after 48 h incubation, the CPT content of the hPCBE external dialysis medium were determined by LC-MS. As shown in Figure S21, in the presence of 5 mM DTT, the CPT peak area of hPCBE external dialysis medium in the dark and under irradiation is determined to be about 817 and 777, respectively. And upon incubation with 10 mM DTT, the CPT peak areas in the dark and under irradiation increase to 1339 and 1355, respectively. These results are in accord with the data of the cumulative release curves, and collectively confirm the effective release of active anticancer drug CPT from the hPCBE whether in the dark or under irradiation. In vitro CPT release experiment was also performed for hPCE, exhibiting reduction-responsive controlled release profiles. The cumulative release of CPT from hPCE (Figure S23) without (0 mM DTT) or with low DTT concentration (2 µM DTT) treatment is less than 5% after 48 h, while up to 62% with 10 mM DTT treatment. All of this information suggest that the reduction-responsive linkers between CPT and hyperbranched polymer matrix are stable under

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Page 23 of 37

physiological condition (0 mM and 2 µM DTT), while they can be readily cleaved under the existence of reduction-stimulus (5 mM and 10 mM DTT) and provide a

(a)

(b) Cumulative release (%)

desirable level of drug after the micelles were trapped into the reductive environment.

Cumulative 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


with irradiation

with irradiation

Figure 4. Cumulative release curve of CPT from hPCBE micelles (a) in the dark and (b) under irradiation. Error bars were based on three repetitions at each time point. 3.4. Generation of singlet oxygen In addition to the effective CPT release of hPCBE, high 1O2 generation rate is also an important parameter for the efficacy of the chemo-photodynamic dual therapeutic system. Singlet oxygen would induce the damage of cellular constituents and subsequent cell death, which can denote the phototoxicity of micelles. To this end, we therefore measured the 1O2 generation ability of the hPCBE and hPBE in aqueous media. The ability of hPCBE and hPBE to generate 1O2 was firstly proved by using pNitrosodimethylaniline (RNO) and imidazole as combination chemical probe, and the rate of 1O2 generation was expressed as the decrease of the OD value of RNO at 440 nm. Rose Bengal was used as the reference. As shown in Figure 5a, the OD values of hPCBE and hPBE at 440 nm show almost no decrease without the irradiation,46 indicating almost no generation of 1O2. Upon exposing to irradiation, there is a rapid decrease of the OD values of hPCBE and hPBE at 440 nm (Figure 5b), suggesting a

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rapidly generation of 1O2 with comparable generation rate to Rose Bengal. In addition, the photostability of hPCBE and hPBE was evaluated by the change of the OD value at 539 nm (UV-vis absorbance peak). Figure 5c shows only slightly decrease of OD value even after 3-hours irradiation, which suggests that the photosensitizer BYMAI embedded in the hPCBE and hPBE exhibits excellent stability against photobleaching. The cellular 1O2 generate ability of hPCBE and hPBE were also confirmed by the DCFH-DA staining method, and the result was expressed by the increase of cellular green fluorescent intensity. As shown in Figure 5d, for hPCBE and hPBE groups, irradiation treatment brought out 225 or 231 times increased fluorescent intensities comparing to none irradiation groups, indicating a significantly elevated levels of 1O2 in cell. Above results demonstrate that hPCBE and hPBE micelles could rapidly generate 1O2 under the irradiation and exhibit excellent photostability.

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Figure 5. 1O2 generation of hPCBE or hPBE in aqueous media monitored by OD value of RNO at 440 nm without (a) or with (b) irradiation. (c) Photostability of hPCBE or hPBE in aqueous solution monitored by OD value at 539 nm under irradiation. (d) Flow cytometry detection of cellular 1O2 generation with the DCFHDA staining method. 3.5. Cellular Uptake studies. For cancer therapy, the internalization of micelles by tumor cells affects the therapeutic efficacy. In this study, Rhodamin B was selected as a fluorescent probe and conjugated into hPCBE to prepare RB-labeled hPCBE, which was used to explore the cellular uptake behavior of the hPCBE micelles. The cell internalization of hPCBE micelles in MCF-7 cells were firstly studied by flow cytometry analysis. MCF-7 cells were treated with RB-labeled hPCBE micelles for predetermined time intervals and cellular uptake was expressed by the increase of cellular fluorescent intensity. As displayed in Figure 6a, the relative geometrical mean fluorescence intensity of the pretreated MCF-7 cells increases with the incubation time. After 15 min incubation with the RB-labeled hPCBE, the red fluorescence signal of Rhodamin B can be clearly observed in the cells. With the incubation time prolonging to 2 h and 4 h, the fluorescence intensity obviously increases, which can be attributed to the increased micelles uptake by MCF-7 cells. To further investigate the cellular uptake behaviors of hPCBE micelles, confocal laser scanning microscopy (CLSM) was performed. After culturing MCF-7 cells with RB-labeled micelles for predetermined time intervals, the nuclei were stained by SYTOX and then the cells were observed by CLSM. As depicted in Figure 6b, red fluorescence signal of RBlabeled micelles is clearly observed in the cytoplasm after 15 min incubation and rises greatly after 2 h incubation. When the incubation time was prolonged to 4 h, fluorescence signal further increases remarkably, suggesting the increased cellular 25/36



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uptake of the hPCBE micelles. These results are in accordance with those obtained using flow cytometry analysis, and collectively reveal the effectively internalization of hPCBE by the MCF-7 cells. The RB-labeled hPCE and hPBE were also prepared, and the effective MCF-7 cellular internalization of hPCE (Figure S25) and hPBE (Figure S26) micelles were studied by the similar method.

Figure 6. Cellular uptake of hPCBE micelles by MCF-7 cells. (a) Time-dependent profiles of RB-labelled hPCBE micelles fluorescence intensity in the MCF-7 cells by flow cytometry analysis (15 min, 30 min, 1 h, 2 h and 4 h). Insert: representative flow cytometry histogram profiles of MCF-7 cells treated with RB-labelled hPCBE 26/36


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micelles for 4 h, the untreated cells are used as a control. (b) CLSM photos of MCF-7 cells incubated with RB-labelled hPCBE micelles for 15 min, 2 h and 4 h. Cell nuclei are stained with SYTOX. 3.6. In vitro cytotoxicity studies. The proliferation inhibition of hPCBE micelles against MCF-7 cancer cells with or without irradiation was evaluated by MTT assay, comparing with free CPT, single chemotherapy of hPCE micelles, and single photodynamic therapy of hPBE micelles. The untreated cells were used as the control. The cytotoxicity of free CPT and single chemotherapeutic hPCE micelles was examined first. As displayed in Figure 7a, hPCE micelles exhibit comparable cytotoxicity to the free CPT, suggesting that the embedded CPT in hPCE micelles could escaped effectively triggered by the reductive conditions in cancer cells. Moreover, the irradiation showed no influence on the cytotoxicity of both free CPT and hPCE micelles. As expected, photosensitizer-embedded hPBE micelles shows almost no cytotoxicity in the dark, while the cytotoxicity enhances remarkably upon irradiation, which indicate that single photodynamic therapeutic hPBE micelles can generate 1O2 and cause cancer cell death effectively. For the anticancer drug CPT and photosensitizer BYMAI double embedded hPCBE micelles (Figure 7b), the dark cytotoxicity is nearly the same as that of hPCE and free CPT, while the cytotoxicity enhances remarkably upon irradiation, showing much better anticancer efficiency than all the other groups. The higher cytotoxicity suggests that the hPCBE micelles enter into tumor cells, and the released CPT and embedded photosensitizer BYMAI show a cooperative manner of chemo and photodynamic activities. To evaluate the apoptosis and necrosis of the MCF-7 cells after treatment with the hPCBE micelles, apoptosis assay involving the flow cytometry analysis of FITCAnnexin V/propidium iodide (PI) double staining was performed, and comparing with 27/36



free CPT, single chemotherapy of hPCE micelles and single photodynamic therapy of hPBE micelles. The untreated cells were used as the control. As shown in Figure 7c, irradiation used in this study alone did not affect cell viability (groups: PBS and PBS + light), and there is also almost no difference was observed between the light and dark apoptosis results of free CPT and hPCE micelles treated cells. Resulting from the embedded photosensitizer BYMAI, the ratio of apoptosis cells reduced by hPBE micelles in the dark or under irradiation is 6.7% or 41.7%, respectively. The ratio of apoptosis cells induced by hPCBE micelles in the dark is 26.6%, and increases to 65.7% while under irradiation. Comparing to the other two single therapeutic formulations (hPCE and hPBE), the hPCBE micelles promote a much higher apoptotic rate of MCF-7 cells with the same dose. The experimental results of the singlet oxygen detection test, MTT assay and apoptosis assay were consistent, revealing that the “bottom-up” hPCBE micelles may be a promising platform for chemo-photodynamic dual therapy.

(a)

(c)

(b)

CPT

PBS

hPCE

0.5%

2.6%

1.3%

6.3%

0.3%

94.5%

2.4%

75.8%

16.6%

83.5%

2.2%

0.6%

8.9%

0.5%

PI

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

Page 28 of 37

13.1%

1.5%

1.5%

11.2%

93.3%

3.8%

73.4%

13.9%

hPBE + light 3.9%

6.7%

6.0 58.3

93.3%

3.2%

71.6%

18.9%

77.1%

hPCBE

1.4%

hPCE + light

CPT + light

PBS + light 1.3%

hPBE 3.1%

18.5%

hPCBE + light 18.4%

14.5%

28.6%

16.6%

34.3%

22.6%

18.4 12.7

58.3%

ANNEXIN V-FITC

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Figure 7. In vitro cytotoxicity of (a) CPT and hPCE micelles, (b) hPBE and hPCBE micelles against MCF-7 cells with or without irradiation of the green light, respectively. The data are presented as average ± standard error (n = 6). (c) Flow cytometric analysis for apoptosis of MCF-7 cells treated with CPT, hPCBE, hPCE and hPBE micelles for 24 h. Lower left, living cells; Lower right, early apoptotic cells; upper right, late apoptotic cells; upper left, necrotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area. 4. Conclusion We have demonstrated in this work that hyperbranched poly(prodrug-cophotosensitizer) amphiphiles can be “bottom-up” designed and synthesized for chemo-photodynamic dual therapy. The easily prepared hPCBE possess a hydrophobic

hyperbranched


core

hPCB

copolymerized from polymerizable reduction-responsive prodrug monomers CPTMA and photosensitizer monomers BYMAI, as well as a hydrophilic shell polymerized from OEGMA monomers. Due to the covalently interconnected core-shell structure, hPCBE exists as structurally stable unimolecular micelles in aqueous solution with size of about 32.8 nm and shows excellent structural stability under dilution condition. The hPCBE micelles can effectively internalized by MCF-7 cells and be triggered by the reductive milieu to release CPT, resulting in effective chemo treatment to tumor. In addition, photosensitizer moieties embedded in the hPCB core will generate 1O2 under irritation, endowing hPCBE micelles with the boosting of chemotherapeutic

efficacy.

Comparing

with

the

single

chemotherapy

of

hyperbranched polyprodrug amphiphiles hPCE and single photodynamic therapy of hyperbranched polyphotosensitizer amphiphiles hPBE, hPCBE shows higher in vitro cytotoxicity, suggesting a potential drug delivery system for chemo-photodynamic 29/36



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dual therapy. We expect that this novel hyperbranched poly(prodrug-cophotosensitizer) amphiphiles constructed via the “bottom-up” strategy may provide a facile and effective way in preparing multifunction drug delivery systems. Associated content Supporting information Synthesis route of HSEMA, VBPT and Rhodamin B-labelled hPCBE, hPCE and hPBE; NMR data, FTIR and fluorescence characterization data, GPC curves; stability of the micelles; zeta potential distribution of micelles, and cellular uptake of micelles. This material is available free of charge via the Internet at http://pubs.acs.org. Author information Corresponding author [email protected]; [email protected] Author contributions X. Z., and X. J. contributed the original idea and supervised the project. P. S. performed all the experiments and participated in results analysis. All authors contributed to discussions regarding the research. P. S. wrote the manuscript with inputs from all authors. Notes The authors declare no competing financial interests. Acknowledgment This research was supported by the National Basic Research Program of China (2015CB931801), National Natural Science Foundation of China (51503122, 51690151) and Shanghai Rising-Star Program (17QC1401100). 30/36


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References 1.

Krishna, R.; Mayer, L. D. Multidrug Resistance (MDR) in Cancer: Mechanisms,

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Bioreducible

Unimolecular

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Based

on

Amphiphilic

Multiarm

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Graphical abstract: “Bottom-Up” Fabrication of Hyperbranched Poly(prodrug-co-photosensitizer) Amphiphiles Unimolecular Micelles for Chemo-Photodynamic Dual Therapy Pei Sun, Nan Wang, Xin Jin and Xinyuan Zhu Hyperbranched poly(prodrug-cophotosensitizer) core

S-S

Polymerizable prodrug monomer

“Bottom-up” fabrication

Polymerizable photosensitizer monomer

Hyperbranched poly(prodrug-cophotosensitizer) amphiphiles

OEGMA

Light

Uptake by tumor cells 3O

1O

1O 2

2

2

PDT

Improved cytotoxicity

Chemotherapy

We

reported

here

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first


Reductive release

“bottom-Up”

fabrication

amphiphiles

unimolecular

chemo-photodynamic dual therapy.


of

hyperbranched micelles

for

âBottom-Upâ Construction of Hyperbranched Poly(prodrug-co

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