Synthesis of a biodegradable branched copolymer mPEG-b-PLGA-g-OCol and its pH-sensitive micelle

Journal Pre-proof Synthesis of a biodegradable branched copolymer mPEG-bPLGA-g-OCol and its pH-sensitive micelle

Yanwei Li, Xue Zhang, Jingpeng Zhang, Jing Ma, Lin Chi, Nannan Qiu, Yanhui Li PII:

S0928-4931(18)31036-1

DOI:

https://doi.org/10.1016/j.msec.2019.110455

Reference:

MSC 110455

To appear in:

Materials Science & Engineering C

Received date:

11 April 2018

Revised date:

2 November 2019

Accepted date:

16 November 2019

Please cite this article as: Y. Li, X. Zhang, J. Zhang, et al., Synthesis of a biodegradable branched copolymer mPEG-b-PLGA-g-OCol and its pH-sensitive micelle, Materials Science & Engineering C (2019), https://doi.org/10.1016/j.msec.2019.110455

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© 2019 Published by Elsevier.

Journal Pre-proof

Synthesis of a Biodegradable Branched Copolymer mPEG-b-PLGA-g-OCol and its pH-Sensitive Micelle Yanwei Li1, Xue Zhang1,2, Jingpeng Zhang1,2, Jing Ma1,2, Lin Chi1, Nannan Qiu1, Yanhui Li1* 1 School of Materials Science and Engineering, Changchun University of Science and Technology, Changchun 130022, China E-mail: [email protected]

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2 Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,

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Chinese Academy of Sciences, Changchun 130022, China Abstract

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An amphiphilic biodegradable branched copolymer, mPEG-b-PLGA-g-OCol, was

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synthesized by grafting copolymer (methoxy polyethylene glycol)-b-Poly (D,L-lactic-co-

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glycolic acid) (mPEG-b-PLGA) on oligomeric collagen (OCol), to form a branched structure with mPEG-b-PLGA as side chain and OCol as backbone. mPEG-b-PLGA and mPEG-b-

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PLGA-g-OCol were both amphipathic and can self-assemble into micelles in aqueous solution.

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The mPEG-b-PLGA-g-OCol micelles showed pH-sensitive behaviors and the particle size below 100 nm in slightly acidic environment such as tumor tissue milieu interieur to perform passive targeting. Observed by SEM, when the solution pH increased from 5 to 9, the morphology of mPEG-b-PLGA-g-OCol micelles changed from small spheres to larger ones to rings. For biodegradable mPEG-b-PLGA-g-OCol, the micelles will gradually degrade in body. Further, doxorubicin (DOX) was effectively loaded in the micelles with drug loading and encapsulation efficiency of 3.48% and 25.8%, respectively. To evaluate antineoplastic effect of DOX-laden micelles in vitro, MTT test, flow cytometry and CLSM were performed and found that DOX-laden micelles exhibited higher cellular proliferation inhibition against HeLa cells. These features indicated that the mPEG-b-PLGA-g-OCol micelles were potential drug carrier for cancer therapy. -1-

Journal Pre-proof Key words: Poly (D,L-lactic-co-glycolic acid), Collagen, Drug carrier, pH sensitive, Micelle pH=5

(b)

pH↓ LA

pH=7

(a)

200nm

ROP pH=9

mPEG OCol

(c)

200nm

pH↑

mPEG-b-PLGA-g-OCol

PLGA

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500nm

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1. Introduction

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Micelle is a kind of nanoparticle, most of which are made by amphiphilic block

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copolymer and has attracted much more attention. Many researchers have studied micelles as drug carriers because the hydrophilic shell of micelles can enhance solubility and maintain

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stability of hydrophobic drugs in aqueous solution to prolong their circulation in body,[1-3] and

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hydrophilic surface combined with nano size make micelles difficult to be recognized and captured by the reticuloendothelial systems (RES). [2] If the size of drug-loaded micelles is

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below100 nm, when used in antitumor therapy, the drug-loaded micelles will be accumulated around tumor by passive targeting via enhanced permeation and retention effect (EPR). [4-7] Poly (D,L-lactic-co-glycolic acid) (PLGA), an important biodegradable and biocompatible polymer, has been widely used as drug carriers.[8-10] As an aliphatic polyester, the hydrophilicity of PLGA is difficult to form micelles in water. When used as drug carriers and injected into body, lack of hydrophilic groups induces PLGA particles rapid clearing by RES. [11, 12]

So, it is necessary to modify PLGA molecules or PLGA based particles’ surface with

hydrophilic molecule, such as poly(ethylene glycol) (PEG), to yield hydrophilic shell to reduce rapid RES uptake.[13-15] Moreover, in order to achieve better tumor therapy, biodegradable, pH-responsive, and targeting PLGA based drug carriers have been developed. Such drug carriers not only perform selective drug release via a pH switch or stimulus -2-

Journal Pre-proof response groups but also degrade in body after therapy.[16-19] Tsai et al [20] synthesized redoxresponsive diselenide bond containing amphiphilic polymer, Bi(mPEG-PLGA)-Se2 from mPEG-PLGA and 3,3’-diselanediyldipropanoic acid to self-assemble into stable micelles in aqueous solution, then loaded DOX to form DOX-loaded micelles, which showed antitumor activities against HeLa cells and endowed redox stimuli triggered drug release in cytosol and nuclei of cancer cells. Zeng et al [21] synthesized a series of branched amphiphilic block copolymers consisting of cholic acid (CA) initiated poly(D,L-lactide-co-glycolide) (CA-

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PLGA) and water-soluble polyethyleneimine cross-linked polyethylene glycol (CA-PLGA-b-

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(PEI-PEG)), then complexed with insulin via electrostatic interaction to obtain nanoscale

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micelle/insulin complexes to be considered as a potential protein carrier. Xu et al [22] used

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monomethoxy (polyethylene glycol)-b-P(D,L-lactic-co-glycolic acid)-b-P(L-glutamic acid) polymer to prepare pH-sensitive nanoparticle with simultaneous encapsulation of curcumin

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and doxorubicin (CURDOX-NPs) with simultaneously targeting to differentiated tumor cells

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and cancer stem cells. Jia et al [23] prepared blank and DOX-loaded micelles formed by PHisb-PLGA-b-PEG-b-PLGA-b-PHis with temperature- and pH- sensitive properties. The

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copolymer micelles can respond to temperature and pH stimuli, indicating that they have potential usage in temperature- and pH- triggered drug delivery. In the field of drug delivery, collagen has been utilized for a long period [24-26]. Oligomeric collagen (OCol) is a polypeptide produced by further hydrolysis of denatured collagen. On collagen chains, the RGD sequence exists and plays important roles, such as cancer cell recognition and cell adhesion.

[27]

As one of tumor targeting molecules, RGD is an ideal

medium on drug carriers to perform positive targeting to tumor.[28-29] The biological properties of OCol are similar to collagen, the RGD sequence also exists on OCol. Furthermore, assembled NPs including micelles from monomers, oligomers, or polymers with amino or carboxyl groups can perform pH-sensitive and will be optimal drug carriers targeting to

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Journal Pre-proof tumor.[30-33] So, selecting OCol as modifier to modify PLGA or PLGA based NPs is an ideal way to achieve novel drug carriers with pH-sensitive and tumor targeting. Above all, this study used collagen as a raw material to degrade into OCol then to link with mPEG-b-PLGA by covalent bonds, the obtained copolymer named as mPEG-b-PLGA-gOCol. As designed, the copolymer had a branched structure with mPEG-b-PLGA as side chain and OCol as backbone. The micelles self-assembled by mPEG-b-PLGA-g-OCol were pH-sensitive and characterized by fluorescence spectroscopy and dynamic light scattering

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(DLS). When the solution pH increased from 5 to 9, for the mPEG-b-PLGA-g-OCol micelles,

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the morphology would change from small spheres to larger ones to rings observed by SEM.

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To use as drug carriers, the micelles had great advantage to control and release DOX in vitro.

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Moreover, negative carboxyl groups on mPEG-b-PLGA-g-OCol will compact with positive DOX to form stable micelles in neutral environment compared with mPEG-b-PLGA. When

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the micelles run in microacid environment, carboxyl groups will protonate and have the same

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charge with DOX to repel DOX release from the micelles, [33] which made mPEG-b-PLGA-g-

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OCol a promising material for drug delivery (Figure 1).

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Journal Pre-proof Figure 1. Formation of DOX-laden micelles assembled by mPEG-b-PLGA-g-OCol in aqueous solution, and when injecting into body, the micelles arrive and accumulate around tumor tissue by passive target and perform acid sensitive drug release.

2. Experimental Section 2.1. Material

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Monomethoxy-poly(ethylene glycol) (mPEG) (Mn=750, 2000 and 5000) was purchased

Institute

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Applied

Chemistry

and

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from Sigma-Aldrich. L-Lactide (LA) and glycolide (GA) were kindly provided by Changchun recrystallized

thrice

in

ethyl

acetate.

N-

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hydroxylsuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) were purchased from GL

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Biochem Ltd. (Shanghai, China). (4-Dimethyl-amino) pyridine (DMAP, 99%) was obtained

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from Acros. Hexane, methylene chloride, and chloroform were refluxed over CaH2 and distilled under a nitrogen atmosphere. Dimethyl sulfoxide (DMSO) was dried and distilled in

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the presence of sodium immediately before use. Collagen was extracted from bovine tendon.

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OCol with molecular weights ranging from 3,500 to 7,000 was obtained by collagen degradation in citric acid and purification through dialysis. 2.2. Synthesis of mPEG-b-PLGA and mPEG-b-PLGA-g-OCol Copolymers mPEG-b-PLGA (Mn = 10 kDa) were synthesized by LA and GA ringopening polymerization (ROP) with mPEG (0.75, 2, 5 kDa) as an initiator and stannous octoate as a catalyst.[33] mPEG was dissolved in toluene (40 mL) and distilled to remove residual water from mPEG at 140 °C for 5 h. LA and GA (WLA/WGA = 4:1), and stannous octoate (1 ‰ total mole number of LA and GA mixture) were added into mPEG toluene solution under the protection of argon and reacted at 120 °C for 72 h. Then the products were dissolved in chloroform and precipitated in excess cold diethyl ether three times to eliminate unreacted monomers, oligomers and catalyst. After vacuum filtration and drying at room -5-

Journal Pre-proof temperature for 24 h, hydroxyl-terminated mPEG-b-PLGA-OH was obtained. 1H NMR (400 MHz, CDCl): δ=3.4ppm (b: s, 4H; CH2), 3.7ppm(c: m, 3H; CH3), 1.6ppm (a: s, 3H; CH3), 5.2ppm (e: m, 1H; CH) , 4.7ppm (d: m, 2H; CH2). The mPEG-b-PLGA-g-OCol copolymer was synthesized in three steps: (1) The end group -OH of mPEG-b-PLGA-OH was converted by react with maleic anhydride into -COOH to yield mPEG-b-PLGA-COOH. mPEG-b-PLGA-OH diblock copolymer (1 mmol) and maleic anhydride (1.1 mmol) were dissolved in dry dioxane (70 mL) with magnetic stirring. Pyridine

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(1.1 mmol) and triethylamine (1.1 mmol) were added in the mixture and the solution was

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stirred at 70 °C for 48 h under a dry argon atmosphere. Crude products were obtained by

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precipitation in cold diethyl ether and then dissolved in dichloromethane (DCM) to precipitate

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again, and vacuum-dried at room temperature for 24 h. (2) mPEG-b-PLGA-COOH (0.6 mmol) and NHS (3 mmol) were dissolved in DCM (20 mL) with magnetic stirring. DCC (3

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mmol) was added in and then place in an ice-water bath. The reaction mixture was protected

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by argon and stirred at 0 °C for 1 h and then at room temperature for 24 h. The by-product 1,3-dicyclohexylurea (DCU) was precipitated from the solution and removed by filtration.

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Then the filtrate was precipitated by cold diethyl ether and vacuum-drying to obtain mPEG-bPLGA-NHS.[34] (3) OCol (0.4 mmol) was dissolved in anhydrous dimethyl sulfoxide (DMSO, 15 mL) at room temperature. Simultaneously, mPEG-b-PLGA-NHS was dissolved in 15 mL of DMSO, and slowly added into the OCol solution and stirred at room temperature for 3 h. After reaction, the mixture was dialyzed against the mixed solution of deionized water and DMSO with concentration of DMSO decreasing from 100% to 0% for 72 h ( Mn cut-off of the dialysis tube was Mn=7,000) at room temperature and then lyophilized to obtain mPEG-bPLGA-g-OCol copolymers. [35] FTIR (KBr): ν = 3334 cm-1 (m; br; NH), 1753 cm-1 (s; C=O), 1658 cm-1 (m; C=O), 1550 cm-1 (w; C-N), 1100 cm-1(s; νs(C-O-C)). 1H NMR (400 MHz, CDCl): δ=2.67ppm (f: s, 4H; CH2), 2.8ppm (g: s, 4H; CH2). 4.4ppm (i: q, 1H; CH), 8.06ppm(h: br, 1H; NH). -6-

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The synthesis procedure was shown in Scheme 1.

Scheme 1. Process of copolymer mPEG-b-PLGA-g-OCol synthesis: (1) mPEG-b-PLGA-OH was prepared by ring-opening polymerization using LA and GA as monomer, mPEG as an initiator; (2) End group -OH on mPEG-b-PLGA-OH was converted into -COOH by reacted with maleic anhydride; (3) mPEG-b-PLGA-COOH was activated with NHS; (4) mPEG-bPLGA-NHS was grafted on OCol to form a branch structure. (Red labels on atoms are used for 1H NMR designation in Figure S2) -7-

Journal Pre-proof 1

H NMR spectra were recorded in CDCl3 and DMSO-d6 at 25 °C on Bruker AV-400

NMR spectrometer. FT-IR spectra were recorded on a BIO-Rad FTS150 instrument. Gel permeation chromatography (GPC) measurements were conducted on a Waters 515 GPC instrument containing Waters UltrahydrogelTM columns (Ultrahydrogel Linear) calibrated by poly(ethylene glycol) standards with CHCl3 as the eluent (flow rate = 1 mL/min, at 35 °C). The molecular weights of the mPEG-b-PLGA-g-OCol copolymers were calculated by elemental N and C analysis. Elemental analysis calcd (%) for mPEG-b-PLGA: C 51.19, H

2.3. Preparation and characterization of micelles

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6.923, N 0.175; for mPEG-b-PLGA-g-OCol: C 50.98, H 7.274, N 1.7.

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mPEG-b-PLGA and mPEG-b-PLGA-g-OCol micelles were prepared as follows:

[36]

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Firstly, the copolymer (0.1 g) was dissolved in DMSO (4 mL in a 100 mL volumetric flask).

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Then 40 mL of double-distilled water was added dropwise into the solution with gentle agitation. Finally, DMSO was removed by dialysis (MWCO 3.5 KDa) for 48 h against

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deionized water exchanged every 6 h.

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The micelles were diluted with concentration range from 10-5 g/L to 1.0 g/L to confirm the critical micelle concentrations (CMCs) by a fluorescence technique (Perkin-Elmer LS50B luminescence spectrometer) with pyrene as the probe. For fluorescence exciation spectra, the detection wavelength was set at 390 nm with a scan rate of 240 nm /min. DLS was used to measure the size distribution of the micelles via a vertically polarized He-Ne laser (DAWN EOS, Wyatt Technology). The scattering angle was 90° and the measurements were carried out at 25 °C. The morphology of the micelles was observed by environmental scanning electron microscopy (ESEM Micrion FEI PHILIPS) and transmission electron microscope (JEOL JEM-2010, Japan). 2.4. pH-sensitive properties

-8-

Journal Pre-proof The micellization of mPEG-b-PLGA-g-OCol copolymer was investigated in different pH (pH = 5, 6, 7, 8, 9) solutions, while mPEG-b-PLGA micelles used as control. pH-driven changed in the sizes and morphologies of mPEG-b-PLGA-g-OCol and mPEG-b-PLGA micelles were detected by DLS and FESEM. 2.5. Drug loading and release in vitro mPEG-b-PLGA-g-OCol and mPEG-b-PLGA (10.0 mg) and doxorubicin hydrochloride

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(DOX·HCl) (4.0 mg) were dissolved in 1.0 mL of DMSO and incubated at room temperature

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for 1 h. Then, 1.0 mL of deionized water was added dropwise into the solution with stirring. After the mixture continued to stir at room temperature for 6 h, DMSO was removed by

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dialysis against deionized water for 24 h. The solution was filtered and freeze-dried to obtain

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DOX-loaded vesicles. The drug loading content (DLC %) and drug loading efficiency

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(DLE %) of the DOX-loaded vesicles were calculated as follows: DLC = (actual amount of DOX in micelles/amount of DOX-loaded micelles) × 100%

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DLE = (actual amount of DOX in micelles/input amount of DOX in micelles) × 100%

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The in vitro DOX-release behaviors of the DOX-laden micelles (including mPEG-bPLGA-g-OCol or mPEG-b-PLGA DOX-laden micelles) were investigated in PBS at pH 7.4. Freeze-dried DOX-laden micelles were suspended in 5 mL of PBS and put into a dialysis bag (MWCO 3.5 KDa) to dialyze against 50 mL of PBS at 37 °C with continuous stirring. At predetermined intervals, 2 mL of PBS was taken out and 2 mL of fresh PBS was replenished. The amount of released drug was assayed by UV-vis spectrophotometry (UV-2401) at 480 nm. 2.6. Cytotoxicity assay The relative cytotoxicities of blank micelles against HeLa cells were evaluated in vitro by MTT assay.[36] In brief, cells were cultured in 96-well plates with 8,000 cells per well in 200 µL Dulbecco's modified Eagle's medium (DMEM) for 24 h in a 5% CO2 incubator at 37 °C. Then after the cells were cultured with blank micelles for 48h, the cells were received -9-

Journal Pre-proof MTT assay measured at 490 nm on a BioRed microplate reader. The cell viability (%) was calculated according to the following Equation (1): Cell viability (%) = (Asample / Acontrol) × 100%

(1)

The Asample and Acontrol are the absorbance in the presence of sample and the blank in DMEM at 490 nm, respectively. The cell viability of DOX-laden micelles was also measured by the MTT assay. Analogously, cells were cultured in 96-well plates with 8,000 cells per well in 200 µL DMEM

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for 24 h. Afterwards, the DOX-laden micelles and free DOX were added in with different

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DOX final concentrations. After being cultured for 24 h and 48 h, the absorbance was

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detected on a Bio-Rad 680 microplate reader at 490 nm. The cytotoxicity was also calculated

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according to Equation (1).

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2.7. Flow cytometric analyses

HeLa cells were seeded into 6-well plates with 200,000 cells per well and cultured in

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DMEM for 24 h. Afterwards, DOX-laden mPEG-b-PLGA-g-OCol micelles was added at final

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DOX concentration of 3.0 µg/mL. After 1 h incubation, cells were washed three times with PBS and trypsinized. Next, the solutions were centrifuged for 5 min at 1000 rpm after 1.0 mL of PBS was mixed, and then the cells were suspended in 0.5 mL of PBS. Finally, the suspended cell solutions were measured by flow cytometry (Beckman, California, USA). 2.9. Confocal laser scanning microscopy (CLSM) The CLSM was used to detect the intracellular release of DOX qualitatively. The HeLa cells were incubated in 6-well plates (200,000 cells/well) in 2.0 mL DMEM for 24 h. Then the cells were cultured with the DOX-laden micelles for 1 h at 37 ℃. Afterwards, the cells were fixed with 4% paraformaldehyde for 30 min and washed with PBS for three times. Finally, the cells were cultured in 4,6-diamidino-2-phenylindole (DAPI, blue) for 5 min to stain the nuclei and observed by a confocal laser scanning microscopy (Olympus FluoView 1000). - 10 -

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3. Results and Discussion 3.1. Synthesis of mPEG-b-PLGA and mPEG-b-PLGA-g-OCol mPEG-b-PLGA copolymers were synthesized by LA and GA (WLA:WGA=8:2) ROP with mPEG (0.75, 2, 5 kDa) as initiator and stannous octanoate as catalyst. The molecular weight of mPEG-b-PLGA copolymers was estimated by GPC and calculated by 1H NMR listed in Table S1. The designed number average molecular weight (Mn) of three kinds of mPEG-b-

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PLGA copolymers were all set at 10 KDa, named as 0.75-P10 (Mn of mPEG was 750), 2-P10

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(Mn of mPEG was 2000) and 5-P10 (Mn of mPEG was 5000). When mPEG-b-PLGA

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copolymers were grafted onto OCol, the polymers named as 0.75-P10O, 2-P10O and 5-P10O, respectively. As can be seen from Table S1, only for 5-P10, proportional of LA and GA

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corresponded to the design with narrow molecular weight distribution.

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The FTIR spectra of mPEG-b-PLGA (5-P10), mPEG-b-PLGA-g-OCol (5-P10O), and OCol were shown in Figure S1. A strong sharp peak at 2889 cm-1 was attributed to -CH2

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stretching vibration, and two very strong sharp peaks at 1753 cm-1 and 1100 cm-1 assigned to

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C=O stretching vibrations and C-O-C symmetric vibrations in polyester, were both found in the mPEG-b-PLGA and mPEG-b-PLGA-g-OCol spectra. Compared with the mPEG-b-PLGA, new absorption peaks appeared in mPEG-b-PLGA-g-OCol spectrum: a broad peak at 3334 cm-1 assigned to N-H stretching vibrations belonging to amide A and amide II first frequency multiplication absorption, a medium sharp peak at 1658 cm-1 assigned to C=O stretching vibrations (amide I), and a weak peak at 1550 cm-1 assigned to C-N stretching and N-H bending (amide II) vibrations (Figure S1), all these peaks also exited in OCol as its characteristic peaks, while became weaken. For mPEG-b-PLGA-g-OCol, characteristic peaks of mPEG-b-PLGA and OCol emerged simultaneously, indicating that grafting polymerization should occur between OCol and mPEG-b-PLGA.

- 11 -

Journal Pre-proof Successful conversion from mPEG-b-PLGA to mPEG-b-PLGA-g-OCol was also confirmed by the 1H NMR as shown in Figure S2. 1H NMR (400 MHz, CDCl3, δ) of mPEGb-PLGA: 3.4 (b: s, 4H; CH2) and 3.7(c: m, 3H; CH3) attributed to mPEG block; 1.6 (a: s, 3H; CH3) and 5.2 (e: m, 1H; CH) attributed to PLA block; 4.7 (d: m, 2H; CH2) attributed to PGA block. The Mn of the PLA and PGA blocks can be calculated from the ratio of monomeric units in the copolymer listed in Table S1. 1H NMR (400 MHz, CDCl3, δ) of mPEG-b-PLGAg-COOH: 2.67 (f: s, 4H; CH2); mPEG-b-PLGA-NHS: 2.8 (g: s, 4H; CH2 on the five-

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membered ring of NHS) indicated the carboxyl group had been activated. For 1H NMR

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(400 MHz, DMSO-d6, δ) of OCol: 4.4 (i: q, 1H; CH) was assigned to protons on proline and

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hydroxyproline linked to a peptide bond, 8.06 (h: br, 1H; NH) was assigned to the proton

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signal of the CO-NH amino linkage, both exited in mPEG-b-PLGA-g-Col. Moreover, the resonances at 8.06, 4.32, 4.17, 3.7, 3.5, 2.7, 2.25 and 1.84 ppm appeared in OCol, also exited

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in mPEG-b-PLGA-g-Col, improved mPEG-b-PLGA had been grafted onto OCol successfully.

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The molecular weight and grafting ratio of mPEG-b-PLGA-g-OCol copolymer (5-P10O) were calculated by elemental N and C analysis because there was no element N in mPEG-b-

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PLGA copolymer listed in Table S2. As a result, there was 4~5 mPEG-b-PLGA copolymers grafted on OCol in one 5-P10O molecule, and final Mn of 5-P10O was calculated as 57700. In summary, the mPEG-b-PLGA-g-OCol copolymer was synthesized successfully with OCol as backbone and mPEG-b-PLGA as branches. 3.2. Micelle preparation Amphiphilic block copolymers will assemble into micelles in aqueous solution above the critical micelle concentration (CMC). When the concentration was below the CMC, amphiphilic block copolymers will disassemble and dissolve in water. The CMC was significantly affected by chain structure, block length, and relative molecular weight of the amphiphilic block polymer. - 12 -

Journal Pre-proof In this study, 0.75-P10, 2-P10, 5-P10 and 0.75-P10O, were all used to assemble into micelles in aqueous solution by the same method listed in table S3. As can be seen, 2-P10O, 5-P10, 5-P10O can self-assemble in water, but 2-P10O micelle was unstable. When 2-P10O micelle sustained in water for several days, some precipitate was emerged. While for 5-P10 and 5-P10O micelles, both were stable enough as the solution maintained semitransparent after one month, and chosen for the follow studies. The CMC was estimated to confirm the mPEG-b-PLGA and mPEG-b-PLGA-g-OCol

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micelle formation by using pyrene as a hydrophobic fluorescence probe. Figure 2(a) showed

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the excitation spectra of pyrene with various concentrations of 5-P10 block copolymer. A red

-p

shift from 333 nm to 335 nm was observed with 5-P10 concentration increasing, indicating

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micelle formation. Figure 2(b) showed the intensity ratio (I335/I333) of the pyrene excitation spectra changed with 5-P10 concentration increasing. The CMC was obtained from the

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intersection of two straight tangent lines: the base line and the rapidly rising line, the cross

(a) 600 400 200 0

1 0.5 0.25 0.1 0.05 0.025 0.01 0.005 0.0025 0.001 0.0001 mg/ml

1.08

(b) 1.04

I335 / I333

Fluorescence Intensity

800

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samples of 5-P10O.

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point was CMC of 5-P10. Figures 2(c) and 2(d) were the excitation spectra and CMC of the

1.00 0.96 0.00669mg/mL

0.92 310

320 330 340 Wavelength, nm

1E-4

350

- 13 -

1E-3

0.01 C, mg/mL

0.1

1

Journal Pre-proof 1 0.5 0.25 0.1 0.05 0.025 0.01 0.005 0.0025 0.001 0.0001 mg/ml

(c) 600 400 200

1.08

(d) 1.04

I335.2 / I333

Fluorescence Intensity

800

1.00 0.96

0

0.00353mg/mL

0.92 310

320

330 340 Wavelength, nm

350

1E-4

1E-3

0.01 C, mg/mL

0.1

1

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Figure 2. (a) Excitation spectra of 5-P10 block copolymer of various concentrations with

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pyrene as a probe; (b) plot of I335/I333 changed with 5-P10 concentration increasing to confirm CMC; (c) Excitation spectra of 5-P10O block copolymer of various concentrations with

-p

pyrene as a probe; (d) plot of I335.2/I333 changed with 5-P10O concentration increasing to

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confirm CMC.

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Copolymers 5-P10 and 5-P10O can self-assemble in aqueous solution to form stable micelles. The CMCs of 5-P10 and 5-P10O were 0.00669 mg/mL and 0.00353 mg/mL,

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respectively. It was an abnormal phenomenon that the increase of hydrophilic segment

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induced CMC decreasing. So there were two factors influence micelle formation. Firstly, the length of the hydrophilic segment (mPEG) played an important role in micelles formation, as longer mPEG block can improve the copolymer assembly. Secondly, if mPEG-b-PLGA copolymer grafted on OCol, the copolymer will form comb-like structure, which can enhance ability to assemble into micelles, but it’s not a decisive factor according to the results of Table S3. In theory, the lower the CMC is, the easier the polymers self-assemble into micelles in aqueous solution. Thus, compared with 5-P10, branched 5-P10O could self-assemble into micelles in aqueous solution more easily. 3.3. Size and morphology of micelles The distributions of micelles’ size were measured by DLS (concentration was 0.1 mg/mL). The results were shown in Figure 3 (a) and (b). Both particle sizes of 5-P10 and 5-P10O - 14 -

Journal Pre-proof micelles showed a single distribution in nanoscale. The mean diameter of 5-P10 micelles was 132 nm, while for 5-P10O micelles was 60.7 nm. The distribution of both micelles ranged from tens to one hundred nanometers according to DLS. As a result, after 5-P10 grafting on OCol, the forming micelles’ diameters showed a descending trend. 0.20

(a)

(b)

0.15 0.10

0

1

10

0.00

2

10

10

Diameter, nm

0.05

0

10

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0.00

0.10

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0.20

Fractional Intensity

Fractional Intensity

0.25

1

10 Diameter, nm

2

10

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Figure 3. Particle sizes measured by DLS: (a) 5-P10 and (b) 5-P10O. Morphology of (a) 5-

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P10 and (b) 5-P10O micelles observed by SEM inserted in every figure.

The morphologies of both 5-P10 and 5-P10O micelles observed by SEM (Figure 3

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inserted images) and TEM (Figure S3) were solid nanospheres. In the same scalebar, the size of 5-P10O micelles was smaller than 5-P10 micelles. Perhaps when 5-P10O self-assembled into micelles, the chains with a branched structure entangled each other and then compressed to get smaller size.

3.4. pH-sensitive properties of 5-P10O micelles The particle sizes of 5-P10O micelles responding to pH were characterized by DLS, 5-P10 micelles as control. Figure 4(a) showed the pH dependence of 5-P10O and 5-P10 micelle size. When the solution pH increased from 5 to 9, the average particle size of 5-P10 around 120 nm was almost no change. By contrast, the average particle size of 5-P10O increased from 57.3 nm to 102.8 nm as the pH increased from 5 to 9. Apparently, the structure of 5-P10O - 15 -

Journal Pre-proof micelles was depended on the solution pH. This was because OCol as a polypeptide had carboxyl groups and amino groups pendent on the chain, based on the reaction shown in Scheme1, most of the amino groups on OCol had reacted with carboxyl groups on 5-P10 to obtain 5-P10O, as a result, carboxyl groups residues were left on 5-P10O, which will perform pH sensitive to influence the structure of the micelles. As seen in Figure 4(a), the size of 5P10O micelles increased with pH increasing.

150

***

***

*

-p

100

5

6

7

pH value

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(b)

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re

50

0

**

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Size, nm

***

of

5-P10 5-P10O

(a)

8

(c)

200nm

200nm

9

(d)

500nm

Figure 4. The particle sizes of 5-P10O and 5-P10 micelle changing with pH increasing from 5 to 9 (interval = 1) (a). Morphologies of 5-P10O micelles in solutions (with pH 5, 7, and 9) change from small sphere (b) to bigger one (c) to ring (d). (*p < 0.05 was considered statistically significant, **p < 0.01 and ***p < 0.001 were considered extremely significant).

The morphologies of 5-P10O micelles at pH 5, pH 7 and pH 9 were observed by FESEM pictures and shown in Figure 4 (b), (c) and (d), which were changed from solid spheres to bigger ones to rings with pH varies from 5 to 9. In the inner of 5-P10O micelles, when - 16 -

Journal Pre-proof environment changed from acidic to alkaline, carboxyl groups will deprotonize to induce inner OCol blocks turning out from core to shell, then the compact core of micelles swelled and loosened resulting morphologies changing. So amphiphilic branched mPEG-b-PLGA-g-OCol copolymer with hydrophilic terminal blocks (mPEG) and hydrophobic middle blocks (PLGA) as arms, a hydrophilic main chain (OCol) as backbone (seen in Scheme1), can self-assemble into pH sensitive micelles with mPEG as corona, PLGA condensed OCol as a core in aqueous solution. In neutral and slightly

of

acidic environments, carboxyl groups of OCol will make the micelle more stable. It was why

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grafting with OCol allowed mPEG-b-PLGA to self-assemble into micelles more easily, and 5-

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P10O micelles had a smaller size than 5-P10 micelles. When the surrounding environment of

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the 5-P10O micelles changed from slightly acidic or neutral to alkaline, yielding a large amount of OH- emerged and invaded into the inner of the micelles, making the PLGA-

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encapsulated hydrophilic OCol core become loose, and the structure of the micelle gradually

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changed from solid spheres to vesicles with size increasing. At a certain alkaline pH, the OCol will reverse out from the micelle core to form a double-layered structure. This double-layer

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micelle structure formed ring shaped aggregates as Figure 4 (d). 3.5. Drug loading and release

In this study, an anthracycline anticancer drug DOX was loaded into 5-P10 and 5-P10O micelles to study their drug encapsulation capacity. Drug release behavior from micelles was obtained through dialysis. When 5-P10 and 5-P10O were used to prepare micelles, DOX was dissolved in co-solvent to encapsulate into micelles when hydrophobic blocks-PLGA shrank as micelles core. The unloaded DOX and co-solvent DMSO were removed by dialysis against distilled water. The drug loading contents (DC, %) of 5-P10 and 5-P10O micelles were 2.49% and 3.48%, and the drug loading efficiencies (DE, %) were 13.6% and 25.8%, respectively. From the DC and DE, branched copolymers 5-P10O tended to carrier more drugs in micelles’ - 17 -

Journal Pre-proof formation. The drug release behaviors of the micelles were observed at pH 7.4 for 50h as in Figure 5(a), at changeable pH also observed in Figure 5 (b).

50 40 30

5-P10 5-P10O

20 10 0

0

10

20

30

40

70 60 50

pH 7.4

40

pH 5

30

5-P10 5-P10O

20 10 0

50

0

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Time (h)

pH 7.4~5

(b)

80

of

(a)

60

90

DOX Realeased Percentage,%

DOX Realeased Percentage,%

70

5

10 15 Time (h)

20

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Figure 5. Release percentages of DOX from 5-P10 and 5-P10O micelles at pH7.4 (a), and

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DOX released from 5-P10 and 5-P10O micelles at changeable pH (b).

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From Figure 5(a), the release rate of DOX from 5-P10 micelles was faster than that of 5P10O micelles, and more than 50% of the loaded DOX released from 5-P10 micelles after

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24 h. By contrast, only 38% of the loaded DOX released from 5-P10O micelles in the same

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time. These results indicated that 5-P10O micelles loaded with DOX showing slow release at pH 7.4. The release rate of DOX from micelles may be affected by OCol grafting. Perhaps more negative carboxyl groups on 5-P10O will compact with more positive DOX to form stable drug-laden micelles in neutral environment, made 5-P10O micelles the best ones for DOX loading. Therefore, modulating DOX release from 5-P10O micelles can be achieved by adjusting the environment pH and the result was observed in Figure 5(b). When the environment pH changed from 7.4 to 5, for DOX-laden 5-P10O micelles, a burst release emerged and the final release percentages of DOX was up to 84.7% at pH 5. While for DOXladen 5-P10 micelles, a slightly increase was observed but no burst release. The results indicated 5-P10O micelles were sensitive to slightly acid environment. So, when the micelles were aggregated at microacid environment around tumor, carboxyl groups will be protonated - 18 -

Journal Pre-proof and have the same kind of discharge with DOX to let DOX release quickly from the micelles.[33] 3.6. In vitro cell viability For evaluating the cell viability of free DOX and DOX-laden micelles, HeLa cells were choose to measure the cell viability by MTT assay. 5-P10O micelles were observed to be less cytotoxic than 5-P10 micelles (above 85% viability) (Figure S3). The in vitro cell viability of

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DOX-laden 5-P10O and 5-P10 micelles against HeLa cells were also evaluated. As shown in

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Figure 6(a) and (b), in contrast to DOX-laden 5-P10 micelles, DOX-laden 5-P10O micelles exhibited inhibition efficiency to HeLa cells at 48 h, while at 24h, both micelles exhibited

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slimily cell growth inhibition. The results showed that the faster DOX releasing from DOX-

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laden 5-P10O micelles was triggered by the endosomal pH, leading to improved killability of

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cancer cells compared with the pH-insensitive DOX-laden 5-P10 micelle. Therefore, the 5-

40 20 0

Cell Viability (%)

80 60

100

DOX-Loaded 5-P10 DOX-Loaded 5-P10O

(a)

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Cell Viability (%)

100

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P10O micelles can be used as an efficient drug carrier to restrain cancer cells.

80 60

***

40 20 0

0.76 1.56 3.12 6.25 12.5 25.0 50.0 Concentration(μg/mL)

DOX-Loaded 5-P10 DOX-Loaded 5-P10O

(b)

0.76 1.56 3.12 6.25 12.5 25.0 50.0 Concentration(μg/mL)

Figure 6. The cell viability of DOX-Loaded 5-P10, DOX-loaded 5-P10O and free DOX toward HeLa cells after incubation for 24 h (a), 48 h (b) (*p < 0.05, **p < 0.01, ***p < 0.001). 3.7. Cellular uptake The cellular uptake behaviors of DOX-laden 5-P10O micelle in HeLa cells were detected by flow cytometry and CLSM. The DOX-laden 5-P10 and 5-P10O micelles were cultured - 19 -

Journal Pre-proof with HeLa cells for 1 h. As shown in Figure 7, the HeLa cells cultured with DOX-laden 5P10O micelles showed obviously higher fluorescence intensity compared with DOX-laden 5P10 micelles, that is to say, more 5-P10O micelles had been internalized. Or, the faster intracellular DOX release lead to the stronger fluorescence intensity region. Perhaps micelles with small size benefited to cell uptake resulting in more 5-P10O micelles aggregated in Hela cells, or more DOX released in the acid endosome, both will influence fluorescence intensity increasing. Moreover, the higher DOX fluorescence was observed in DOX-laden 5-P10O

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micelles compared with DOX-laden 5-P10 micelles, as seen in CLSM (Figure 8.) directly.

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Micelles made by the branched polymer 5-10O were more likely to induce endocytosis with

lP

Control 5-P10 5-P10O

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Count

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-p

higher cell uptake. As a result, 5-10O was a promising drug carrier.

102

103

104

105

Figure7. Cellular uptake tested by flow cytometry with HeLa cells incubated with PBS (as control), DOX-laden 5-P10 micelles, and DOX-laden 5- P10O micelles.

DOX

5-P10O

5-P10

DAPI

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Merge

Journal Pre-proof Figure8. The confocal microscope images of HeLa cells incubated with DOX-laden 5-P10O micelles and DOX-laden 5-P10 micelles for 1 h, cell nuclei stained by DAPI (blue), DOX fluorescence in cells (red), and overlays of the three images. Conclusion In this study, an amphiphilic biodegradable branched copolymer with OCol as the backbone and mPEG-b-PLGA as branched arms was synthesized and characterized

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successfully. Copolymers 5-P10 and 5-P10O can assemble into micelles in aqueous solution

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with PLGA as hydrophobic core and mPEG as hydrophilic corona, and for 5-P10O, OCol will be condensed into the micelle core. According to TEM observation, both of 5-P10 and 5-

-p

P10O micelle were solid nanospheres. 5-P10O micelles were sensitive to acid leads to particle

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size decreasing with pH decrease. And when the solution pH increased from 5 to 9, for the

lP

pH-respond 5-P10O micelles, the morphology changed from small spheres to larger ones to rings observed by SEM. To use as drug carriers, the 5-P10O micelles showed great advantage

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to carry DOX than 5-10P micelles, that is to say, negative carboxyl groups on 5-P10O

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compacted with positive DOX to form stable micelles in neutral environment. When the surrounding condition of micelles changed from neutral to microacid environment, carboxyl groups will protonate to let DOX release quickly from 5-P10O micelles. Furthemore, DOXladen 5-P10O micelles showed stronger cell inhibition than 5-P10 against Hela cells in vitro with higher cell uptake. Therefore, 5-P10O micelles were able to encapsulate antitumor drug to yield drug-laden vesicles and they have potential application as drug carrier for cancer therapy.

Acknowledgments This work was supported by the National Natural Science Foundation of China (50903009), Jilin science & technology department, science and technology development - 21 -

Journal Pre-proof project (20100115), Foundation of Department of Education of Jilin Province (No. 2016363, JJKH20170606KJ),

China

Postdoctoral

Science

Foundation

(No.

2014M561268,

2016M601356), Science and technology innovation fund of Changchun University of Science and Technology (No. XJJLG-2017-07), and Research Foundation of Education Department of Jilin Province (Grant No. JJKH20190582KJ).

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Journal Pre-proof

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Graphical abstract

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Journal Pre-proof Highlights 1. Branch copolymer mPEG-b-PLGA-g-OCol was synthesized by grafting mPEG-b-PLGA on OCol backbone. 2. The copolymer was biodegradable with low cytoxicity. 3. The copolymer was amphiphilic and can self-assemble into micelles in aqueous solution, and DOX can load in. 4. In neutral environment the micelles were stable, while when circumstance pH changed, the

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structure of the micelles will change sensitively to perform loaded DOX release.

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