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Enzymatic PEG-poly(amine-co-disulfide ester) Nanoparticles as pH and Redox-responsive Drug Nanocarriers for Efficient Antitumor Treatment Ya Chen, Meifei Su, Yingqin Li, Jinbiao Gao, Chao Zhang, Zhong Cao, Jiangbing Zhou, Jie Liu, and Zhaozhong Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10148 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017

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ACS Applied Materials & Interfaces

Enzymatic PEG-poly(amine-co-disulfide ester) Nanoparticles as pH and Redox-responsive Drug Nanocarriers for Efficient Antitumor Treatment

Ya Chena,†, Meifei Sua,†, Yingqin Lia, Jinbiao Gaoa, Chao Zhanga, Zhong Caoa, Jiangbing Zhoub, Jie Liua,*, Zhaozhong Jiangc,*

a

Department of Biomedical Engineering, School of Engineering, Sun Yat-sen

University, Guangzhou, Guangdong 510006, China b

Department of Neurosurgery and Department of Biomedical Engineering, Yale

University, New Haven, Connecticut 06511, United States c

Department of Biomedical Engineering, Molecular Innovations Center, Yale

University, 600 West Campus Drive, West Haven, Connecticut 06516, United States

†

These authors contribute equally to this work.

*E-mail: [email protected] [email protected]

1

ACS Paragon Plus Environment


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Abstract

We have designed and constructed novel multifunctional nanoparticle drug delivery systems that are stable under physiological conditions and responsive to tumor-relevant pH and intracellular reduction potential. The nanoparticles were fabricated

from

enzymatically

synthesized

PEG-poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionat e)

(PEG-PPMD)

and

PEG-poly(ε-caprolactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PCMD) block copolymers via self-assembly processes in aqueous solution. At acidic pH and in the presence of a reductant (e.g., D,L-dithiothreitol or glutathione), the nanosized micelle particles rapidly swell and disintegrate due to the protonation of amino groups and reductive cleavage of disulfide bonds in the micelle cores. Consistently, docetaxel (DTX)-loaded PEG-PPMD and PEG-PCMD micelles can be triggered synergistically by acidic endosomal pH and a high intracellular reduction potential to rapidly release the drug for efficient killing of cancer cells. The drug formulations based on PEG-PPMD and PEG-PCMD copolymers exhibited a substantially higher potency than free DTX in inhibiting tumor growth in mice while their therapeutic effects on important organ tissues were minimal. These results demonstrate that PEG-PPMD and PEG-PCMD nanoparticles have a great potential to serve as site specific, controlled drug delivery vehicles for safe and efficient antitumor treatment.

Keywords:

lipase,

poly(amine-co-disulfide

ester),

PEGylation,

nanoparticle,

pH-responsive, redox-responsive, drug delivery, anticancer treatment.

2


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1. Introduction

Nanoparticle-mediated drug delivery is one of the most prospective platforms for cancer chemotherapy primarily due to its capability of protecting drugs from premature biodegradation and of increasing drug accumulation in solid tumors via the enhanced permeability and retention (EPR) effect, thus reducing chemotherapeutic side effects 1-3. To achieve desirable treatment results, drug-loaded nanoparticles must remain stable under physiological conditions for a sufficient long period of time. Recently, substantial efforts have been made to improve the drug delivery efficiency of nanoparticle formulations by using functional nanoparticles that are responsive to rich stimuli (e.g., acidic pH and glutathione) present at tumor sites 4-7. Upon arrival at the disease sites after administration, the nanoparticles are triggered by the stimuli to rapidly disintegrate and unload encapsulated drugs for efficient killing of cancer cells. It is known that while the extracellular pH of the normal tissues and blood is slightly basic (pH of ~7.4), solid tumors are weakly acidic (pH of 5.7-7.0) as the result of lactic acid accumulation due to poor oxygen perfusion caused by their defective neovascular structures 8-11. Further, even more acidic conditions (pH of 4.0 to 6.0) are encountered in endosomes and lysosomes

12

. In addition to acidic pH, glutathione

(GSH, a tripeptide containing cysteine) is another stimulus trigger useful for designing functional nanoparticle drug delivery systems. GSH concentration is approximately 1 to 10 mM in normal cells, but is at least four times higher in many tumor cells

13,14

. In contrast, in a normal extracellular environment (e.g., plasma),

GSH concentration is only around 2 to 20 µM

13

. Among stimuli-responsive

nanoparticles reported,7,15-17 only a few18-23 are biodegradable drug nanocarriers capable of responding to tumor relevant pH and intracellular reduction potential and only two such nanocarrier systems21,23 have been shown to be effective in vivo. A number of stimuli-responsive nanoparticles have previously been formed from synthetic polymers that are produced via chemical polymerization processes 7,16. In the past several years, we have explored alternative, environmentally benign, enzyme-catalyzed

polymerization

procedures

for

preparation

of

functional 3



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biodegradable polyesters for drug and gene delivery applications

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24-34

. Because of the

high activity and selectivity of the enzyme catalysts and their extraordinary tolerance toward organic functional groups

35-40

, copolyesters with diverse chain structures and

functionalities were successfully synthesized typically in one step from readily available monomers

41- 47

. Further, the enzymatic polyesters possess high purity and

are metal-free, which render them particularly suitable for biomedical uses. We

have

recently

described

the

synthesis

of

PEG-poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-sebacate) (PEG-PPMS) copolymers that are responsive to acidic endosomal pH

48

, and

PEG-poly(ω-pentadecalactone-co-butylene-co-3,3'-dithiodipropionate) (PEG-PPBD) copolymers that are responsive to intracellular glutathione

49

. Consequently,

anticancer drug-loaded nanoparticles formed from PEG-PPMS and PEG-PPBD exhibited a potency that can be enhanced correspondingly by acid and glutathione. Here, we report successful synthesis of two new types of functional polyesters bearing both tertiary amino groups and disulfide groups in the polymer main chain: PEG-poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionat e)

(PEG-PPMD)

and

PEG-poly(ε-caprolactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PCMD). The amphiphilic block copolymers were produced in one step via lipase-catalyzed copolymerization of lactone (ω-pentadecalactone or ε-caprolactone), N-methyldiethanolamine (MDEA), dimethyl 3,3'-dithiodipropionate (DTDP) and poly(ethylene glycol) methyl ether (MeO-PEG-OH). These polymers contain dithio diester, amino diol and lactone repeat units, and are structurally different from previously reported pH and redox-responsive materials. PEG-PPMD and PEG-PCMD were designed to possess following structural components and functionalities: PEG for nanoparticle colloidal stability, lactone units for tuning micelle stability and improving cellular uptake,28 amino and disulfide functional groups for synergistic pH and redox-responsive properties. The PEG-PPMD and PEG-PCMD micelle nanoparticles were loaded with a representative anticancer drug docetaxel (DTX, a common commercial anti-mitotic chemotherapy medicine) for evaluation of their 4


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drug delivery efficiency. Scheme 1 illustrates key steps regarding anticipated therapeutic actions of the pH and redox-responsive nanoparticles in delivering the drug to tumor cells. In this article, we demonstrate the construction of stable nanoparticles with desirable sizes from PEG-PPMD and PEG-PCMD copolymers, their excellent biocompatibility and cellular uptake properties, swift responses of the particles to tumor relevant acidic pH and intracellular reduction potential, in vitro efficacy of the DTX-loaded nanoparticles in killing cancer cells, as well as their high in vivo antitumor efficiency. To the best of our knowledge, PEG-PPMD and PEG-PCMD represent the first examples of enzymatic polymers with pH and redox dual-responsive properties.

Scheme 1. Illustration of major steps involving DTX delivery by PEG-PPMD and PEG-PCMD nanoparticles to cancer cells.

2. Experimental section

5



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2.1. Materials. ω-Pentadecalactone

(PDL,

≥98%),

ε-caprolactone

(CL,

99%),

N-methyldiethanolamine (MDEA, 99%), poly(ethylene glycol) methyl ether (2000 Da, MeO-PEG2K-OH) and L-buthionine-(S,R)-sulfoximine (BSO) were purchased from Sigma-Aldrich Chemical Co. and were used as received. Immobilized CALB (Candida antarctica lipase B supported on acrylic resin) catalyst or Novozym 435 (from Aldrich) was dried at 40 °C under 2.0 mmHg for 20 h prior to use. Docetaxel (DTX) was purchased from Beijing Norzer Pharmaceutical Co., Ltd.; Duopafei® (commercial DTX injection) for in vivo treatment was manufactured by Qilu Pharm Co.,

Ltd

(Jinan,

China).

DiR

(1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) was purchased from Thermo Fisher Scientific Inc. Both HeLa cells and CT-26 cells were acquired from Shanghai cell bank of Chinese Academy of Science (Shanghai, China) and were maintained at 37 °C under 5% CO2 humidified atmosphere. DMEM and RPMI-1640 [from Gibco, both containing 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin] were used as the culture media for the HeLa cell line and CT-26 cell line, respectively.

2.2. Instrumental methods. NMR spectra were obtained on an Agilent 500 spectrometer using tetramethylsilane as the internal standard. The number and weight average molecular weights (Mn and Mw, respectively) of polymers were measured by gel permeation chromatography (GPC) using chloroform as the mobile phase and polystyrenes as the standards according to previously reported procedures.42 The size and zeta potential of PEG-PPMD and PEG-PCMD micelles were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments). Transmission electron microscope (TEM) was used to observe the morphology of micelle particles after staining with phosphotungstic acid.

2.3.

Synthesis

of 6


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PEG2K-poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-3,3′-dithiodip ropionate) (PEG2K-PPMD) copolymers. The

block

ω-pentadecalactone

copolymers (PDL),

were

prepared

via

copolymerization

N-methyldiethanolamine

(MDEA),

of

dimethyl

3,3′-dithiodipropionate (DTDP) with MeO-PEG2K-OH using Novozym 435 as the catalyst. The feed ratios (shown in Table 1) were selected to yield the block copolymers with 40 wt% PEG after complete polymerization reactions. Typically, PDL, MDEA, DTDP and MeO-PEG2K-OH in different ratios (Table 1) were mixed with Novozym 435 (10 wt% vs total substrate) and diphenyl ether (200 wt% vs total substrate). The resultant reaction mixtures were stirred at 90 °C under atmospheric nitrogen gas for 21 h and then under 1.8 mmHg vacuum for 70 h. The formed PEG2K-PPMD block copolymers were purified according to the procedures used for isolating PEG-PPBD copolymers as reported previously.49 Each PEG-PPMD copolymer is denoted as PEG-PPMD–x% PDL indicating molar percentage content of PDL units vs. (PDL + diester) units in the PPMD segments of the copolymer.

Table 1. Characterization of PEG2K-PPMD and PEG2K-PCMD Block Copolymers Lactone/DTDP/ Sample

a

MDEA/PEG2K (feed molar ratio)

Isolated Yield b

Lactone Unit Content (mol%) c

Mw (Da)

M w/ M n

PEG-PPMD-12% PDL

10:90:86:8

94%

12%

11000

1.7

PEG-PPMD-27% PDL

25:75:71:8

86%

27%

11300

1.7

PEG-PPMD-43% PDL

40:60:56:8

75%

43%

12500

1.7

PEG-PPMD-54% PDL

55:45:41:8

83%

54%

13300

1.8

PEG-PPMD-70% PDL

70:30:26:8

84%

70%

15200

1.7

PEG-PCMD-13% CL

10:90:86:8

89%

13%

8300

1.4

PEG-PCMD-26% CL

25:75:71:8

90%

26%

8100

1.5

PEG-PCMD-41% CL

40:60:56:8

91%

41%

8700

1.5

PEG-PCMD-53% CL

55:45:42:6

92%

53%

8700

1.5

PEG-PCMD-70% CL

70:30:27:6

85%

70%

9400

1.5

a. All PEG-PPMD and PEG-PCMD copolymers contain (40±1) wt% PEG. b. The lactone comonomer is PDL for PEG-PPMD or CL for PEG-PCMD. c. Mol% lactone units vs. (lactone + diester) units in the copolymer chains. 7



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PEG-PPMD block copolymer: 1H NMR (CDCl3; ppm) 1.26 (br.), 1.61 (m), 2.27-2.32 (m), 2.36 (s), 2.71-2.77 (m), 2.92 (t), 3.64 (s), 4.04-4.10 (m), 4.16-4.22 (m), plus a singlet at 3.38 ppm;

13

C NMR (CDCl3; ppm) 24.92, 25.02, 25.89, 25.93, 28.58,

28.65, 29.16, 29.26, 29.28, 29.47, 29.52, 29.58-29.63 (m), 33.01, 33.05, 33.15, 33.20, 34.06, 34.14, 34.27, 34.39, 42.84, 55.82, 55.91, 61.89, 62.42, 63.34, 63.89, 64.38, 64.99, 70.55, 171.60, 171.72, 173.78, 173.98; plus small peaks at 59.02, 69.03 and 71.92 ppm due to terminal –OCH3 and –COO-CH2-CH2–O– groups.

2.4.

Synthesis

of

PEG2K-poly(ε-caprolactone-co-N-methyldiethyleneamine-co-3,3′-dithiodipropio nate) (PEG2K-PCMD) copolymers. The PEG-PCMD copolymers were synthesized following procedures analogous to those used for the preparation of PEG-PPMD polymers above except that ε-caprolactone (CL) was employed instead of PDL as the lactone comonomer. Thus, CL, MDEA, DTDP and MeO-PEG2K-OH in different ratios (shown in Table 1) were blended with Novozym 435 (10 wt% vs total substrate) and diphenyl ether (200 wt% vs total substrate). The resultant mixtures were agitated at 80 °C under atmospheric nitrogen gas for 20 h and then under 2.0 mmHg vacuum for 71 h. The formed PEG-PCMD copolymers were purified using the same method as described above for isolation of the PEG-PPMD copolymers. Each PEG-PCMD copolymer is denoted as PEG-PCMD–x% CL indicating molar percentage content of CL units vs (CL + diester) units in the PCMD segments of the copolymer. PEG-PCMD block copolymer: 1H NMR (CDCl3; ppm) 1.39 (br.), 1.66 (m), 2.29-2.37 (m), 2.70-2.77 (m), 2.92 (t), 3.64 (s), 4.05-4.11 (m), 4.16-4.21 (m), plus a singlet at 3.38 ppm; 13C NMR (CDCl3; ppm) 24.48, 24.50, 24.53, 24.56, 25.46, 25.48, 25.51, 28.27, 28.33, 33.02, 33.04, 33.11, 33.13, 34.05, 34.09, 42.83, 55.79, 55.87, 61.96, 62.41, 64.11, 64.59, 70.56, 171.56, 171.63, 173.36, 173.48; plus small peaks at 8


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59.01, 69.02 and 71.92 ppm due to terminal –OCH3 and –COO-CH2-CH2–O– groups.

2.5. Preparation of blank and docetaxel (DTX)-loaded PEG-PPMD and PEG-PCMD micelles The blank and DTX-loaded micelles were fabricated using a dialysis method. PEG-PPMD or PEG-PCMD copolymers (38 mg) with or without DTX (2 mg) were dissolved in 1 mL of tetrahydrofuran (THF). The resultant solutions were continuously added into 5 ml of PBS (10 mM, pH 7.4) using a syringe to induce micellization. Subsequently, the micelle solutions were stirred for 30 min at room temperature and dialyzed against PBS (10 mM, pH 7.4) overnight using 3500 Da cutoff size dialysis bag. The dialyzed micelle solutions were then centrifuged for 20 min at 8000 rpm using MWCO 100 kDa ultrafiltration centrifuge tubes. Finally, an aliquot of the concentrated micelle solutions was lyophilized, and the yield of each micelle sample was calculated and recorded.

2.6. pH and redox-triggered disassembly of micelles The size change of the PEG-PPMD and PEG-PCMD micelles in response to acidic or reductive conditions in PBS solution was analyzed by dynamic light scattering (DLS). Typically, aliquots of blank micelle solutions were added into six different media: (i) PBS buffer (10 mM, pH 7.4), (ii) PBS buffer (10 mM, pH 7.4) containing 10 mM D,L-dithiothreitol (DTT), (iii) PBS buffer (10 mM, pH 7.4) containing 50 mM DTT, (iv) PBS buffer (10 mM, pH 5.0), (v) PBS buffer (10 mM, pH 5.0) containing 10 mM DTT, and (vi) PBS buffer (10 mM, pH 5.0) containing 50 mM DTT. After the resultant mixtures were incubated in a shaking bed at 37 °C and 100 rpm for 48 h, the micelle sizes, size distributions and count rates were determined by DLS.

2.7. Measurement of DTX contents in micelles The content of DTX encapsulated in PEG-PPMD or PEG-PCMD micelles was measured by high performance liquid chromatography (HPLC, Agilent 1260) 9



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equipped with an Eclipse XDB-C18 column. The mobile phase [a 1:1 (v/v) acetonitrile/water mixture] was used at 1 mL/min flow rate. The UV absorption at 230 nm was used for detection. In a typical procedure, an aliquot of micelle samples was dissolved in 950 µL THF, and the organic solutions were centrifuged (13000 rpm, 10 min) and filtered using 0.2 µm syringe filters. The filtered solutions (10 µL) were then injected to the HPLC for DTX content analysis. The DTX loading (DL) and the drug entrapment efficiency (EE) were calculated as following:

Drug loading (%) =

drug amount in micelles

× 100% mass of micelles drug amount in micelles Entrapment efficiency (%) = × 100% drug feeding

2.8. In vitro drug release The drug release behavior of DTX-loaded PEG-PPMD and PEG-PCMD micelles was studied using a dialysis method. Each DTX-loaded micelle sample was placed into four dialysis bags (MWCO 3500 Da) which respectively were immersed into four different PBS solutions (10 mM) containing 0.5% (w/v) Tween 80: (i) PBS buffer with pH 7.4, (ii) PBS buffer with pH 7.4 and 10 mM DTT (D,L-dithiothreitol), (iii) PBS buffer with pH 5.0, and (iv) PBS buffer with pH 5.0 and 10 mM DTT. The micelle samples were incubated at 37 °C in a shaking bed with a rotation speed of 100 rpm. At each time interval, 1 mL of the external PBS solutions was withdrawn and the same amount of fresh PBS was then added. The harvested PBS solutions were measured by HPLC and the amount of DTX released from the micelles were calculated. All experiments were perform in triplicate.

2.9. Cellular uptake and intracellular distribution To evaluate the cellular uptake capacities of PEG-PPMD and PEG-PCMD micelles, fluorescence probe molecule coumarin-6 (C6) was encapsulated in PEG-PPMD-12%, 43% and 70% PDL and PEG-PCMD-13%, 41% and 70% CL micelles according to the protocol employed for preparation of DTX-loaded micelles 10


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(Section 2.5) and the cellular uptake of the C6-loaded micelles was examined by flow cytometry using HeLa cells. Specifically, HeLa cells in 500 µl medium at a density of 4.0 × 105 cells/mL were seeded in a 24-well plate overnight at 37 °C under 5% CO2. Subsequently, free C6, C6-loaded PEG-PPMD micelles and C6-loaded PEG-PCMD micelles were respectively added to each well at a C6 concentration of 0.2 µg/mL. After incubation for 1 to 8 h, the culture media were removed and the cells were washed three times with cold PBS solution (10 mM, pH 7.4). The washed cells were detached with trypsin, harvested and centrifuged at 2000 rpm for 5 min to remove the supernatants. Upon resuspension of the cells in 500 µl PBS, the cellular internalization efficiency of the micelle samples was analyzed by FACSCalibur at an excitation wavelength of 488 nm and an emission wavelength of 585 nm (10000 cells per group). The intracellular location of C6-loaded micelles was visualized using confocal laser scanning microscopy (CLSM). HeLa cells (1 × 105 cells/well) were seeded on 15 mm glass-bottom dishes in a 6-well plate overnight. The cells were incubated with free C6, C6-loaded PEG-PPMD-12% PDL micelles or C6-loaded PEG-PCMD-13% CL particles at a C6 concentration of 0.2 µg/mL. After incubation for 2 and 6 h, the media were removed and the cells were washed three times with cold PBS solution. Thereafter, the cell lysosomes were stained with 75 nM Lysotracker-red and the cell nuclei were stained with 10 µg/mL Hoechst 33342. The cells were then rinsed, fixed with paraformaldehyde (PFA), washed by PBS and then observed by CLSM. The excitation wavelength for detecting Hoechst 33342, Lysotracker-red, and C6 was 405 nm, 577 nm and 467 nm, respectively.

2.10. In vitro cytotoxicity of micelles The cytotoxicity of both blank micelles and DTX-loaded micelles were evaluated against HeLa cells and CT-26 cells using MTT method according to our previous report.49 Briefly, the cells (3.0 × 103 cells/well) were seeded in 96-well plates and allowed to adhere overnight. Then the culture medium was removed and 200 µL of the fresh medium with different concentration of the blank or DTX-loaded micelles 11



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were added to each well. In order to investigate the stimuli-responsive properties of the DTX-loaded PEG-PPMD and PEG-PCMD micelles, the culture media with different pH (7.4 or 6.5) and different reduction potential (10 mM GSH or 0.2 mM BSO) were used to mimic different intracellular environments. After incubation with micelles under different medium environments for additional 48 h, the cell viabilities were measured using MTT assay.

2.11. In vivo biodistribution study CT-26 tumor-bearing mouse models were used to investigate the biodistribution of the DiR-loaded polymeric micelles. Fluorescence probe DiR was encapsulated in PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles according to the protocol employed for preparation of DTX-loaded micelles (Section 2.5). CT-26 cells (1×106 cells/0.1 mL) were injected subcutaneously in the right back of Balb/C mice (5 weeks, 16 g). When tumors reached 200-300 mm3 in volume, the DiR-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles (0.5 mg/kg DiR) were injected intravenously through the tail vein. In vivo fluorescence images were taken at 4, 12 and 24 h after injection using the IVIS Lumina XR imaging system (the excitation wavelength and emission wavelength of DiR are respectively 750 nm and 780 nm). The mice were sacrificed for further observation of organ accumulation of DiR after its administration for 24 h. The heart, lung, spleen, liver, kidney and tumor were harvested, washed with cold saline and photographed using the IVIS Lumina XR imaging system.

2.12. In vivo antitumor efficiency The antitumor efficiency of DTX-loaded PEG-PPMD-12% PDL micelles and PEG-PCMD-13% CL micelles was evaluated using Balb/C mice bearing mouse colon carcinoma CT-26 xenograft. CT-26 cells (1×106 cells) were injected subcutaneously in the right back of Balb/C mice (5 weeks, 16 g). When tumors reached 50－100 mm3 in volume, treatments were started and the initial treatment day was designated as day 0. On day 0, the mice were randomly assigned to one of the following 4 groups (at least 12


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4 mice in each group): 0.9% NaCl (control), free DTX (Duopafei®), DTX-loaded PEG-PPMD-12% PDL micelles and DTX-loaded PEG-PCMD-13% CL micelles. Mice were injected intravenously through the tail vein with free DTX, DTX-loaded PEG-PPMD-12% PDL micelles and PEG-PCMD-13% CL micelles (at 10 mg/kg DTX dose) every three days for four times. The control group of mice were administered via injection with 0.9% NaCl following the same procedure. The tumor volume was measured every other day using a vernier caliper and the body weight of the mice was recorded at the same time. At day 27, the mice were sacrificed to collect the tumors and important organs (heart, liver, spleen, lung and kidney), whose tissues were analyzed by hematoxylin-eosin (H&E) staining to determine the antitumor effects of the micelles.

3. Results and discussion

3.1. Synthesis and characterization of PEG-PPMD and PEG-PCMD block copolymers The amphiphilic block copolymers containing tertiary amino and disulfide functional groups were prepared via copolymerization of lactone (PDL or CL), N-methyldiethanolamine (MDEA), dimethyl 3,3'-dithiodipropionate (DTDP) with MeO-PEG2K-OH in a two stage process using CALB as the catalyst (Scheme 2). The comonomer feed ratios employed, and the composition and properties of the resultant polymer products are shown in Table 1. All purified PEG-PPMD and PEG-PCMD copolymers contain 40 (±1) wt% PEG (calculated from the proton NMR spectra) and

Scheme 2. Enzymatic Synthesis of PEG-PPMD and PEG-PCMD Block Copolymers

13



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were obtained in 75% to 94% yield. The Mw values range from 11000 to 15000 Da for PEG-PPMD copolymers and from 8100 to 9400 Da for PEG-PCMD copolymers. The results in Table 1 indicate that the monomer feed ratio can be adjusted to control the polymer composition. GPC analyses revealed mono-model molecular weight distributions for both PEG-PPMD and PEG-PCMD block copolymers, and no free PEG2K was found in the polymers. Because MeO-PEG-OH is a chain terminator, the PEG-PPMD and PEG-PCMD copolymers can contain PEG-polyester diblock chains and/or PEG-polyester-PEG triblock chains where polyester is PPMD or PCMD (Scheme 2). PEG-PPMD and PEG-PCMD copolymers possess both PEG segments and polyester segments consisting of lactone, MDEA and DTDP repeat units (Scheme 2). The molecular structure and composition of the copolymers were determined by 1H and

13

C NMR spectroscopy. The PEG and lactone contents in the block copolymers

were calculated from their proton NMR spectra (the results reported in Table 1). Detailed NMR analyses, including structural assignments for the proton resonances and major carbon-13 absorbances of the copolymers, are provided in the supporting information file (Figures S1 and S2). To determine repeat unit distributions in the polyester blocks of the copolymers, the carbonyl

13

C absorbances of the polymers

were quantitatively measured. Both PEG-PPMD and PEG-PCMD copolymers exhibit four

carbonyl

13

C

resonances

due

to

lactone*-lactone,

lactone*-MDEA, 14


Page 15 of 38

DTDP*-lactone and DTDP*-MDEA diads (Figure 1). For PEG-PPMD-43% PDL, PEG-PPMD-70% PDL, PEG-

DTDP*-PDL

DTDP*-MDEA

PDL*-MDEA

PDL*-PDL

15.0

10.0

5.0

0.0

ppm (t1)

174.00

173.50

173.00

172.50

172.00

171.50

DTDP*-CL DTDP*-MDEA

CL*-CL

(A)

CL*-MDEA

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


400 300 200 100 0

173.50 ppm (t1)

173.00

172.50

172.00

171.50

(B) Figure 1. Carbonyl C-13 resonance absorptions of different diads in (A) PEG2KPPMD-70%PDL, and (B) PEG2K-PCMD-70%CL (solvent: CDCl3).

PCMD-41% CL and PEG-PCMD-70% CL, the measured abundances of the four diads were compared to the values calculated respectively for random PPMD chains or PCMD chains at same compositions (Table 2). The results reveal that the repeat unit arrangements in the polyester chain blocks of the PEG-PPMD and PEG-PCMD copolymers are nearly random.

Table 2. Diad Distributions in the Polyester Segments of PEG-PPMD or PEG-PCMD Chains lactone*-lactone a

lactone*-MDEA a

DTDP*-lactone a

DTDP*-MDEA a

Meas. b

Calc. c

Meas. b

Calc. c

Meas. b

Calc. c

Meas. b

Calc. c

PEG-PPMD-43%PDL

0.08

0.08

0.20

0.20

0.20

0.20

0.52

0.53

PEG-PPMD-70%PDL

0.30

0.29

0.25

0.25

0.25

0.25

0.20

0.21

PEG-PCMD-41%CL

0.07

0.07

0.20

0.19

0.20

0.19

0.53

0.55

Sample

15



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PEG-PCMD-70%CL

0.29

0.29

0.25

0.25

0.25

Page 16 of 38

0.25

0.21

0.21

a. Repeat unit abbreviations: MDEA for N-methyldiethyleneamine unit; DTDP for 3,3′-dithiodipropionate unit. The lactone unit represents ω–pentadecalactone for PEG-PPMD or ε–caprolactone for PEG-PCMD. b. Measured by carbon-13 NMR spectroscopy. c. Calculated for random poly(lactone-co-MDEA-co-DTDP) chains. Abundance of lactone*-lactone diad = fL × fL; abundance of lactone*-MDEA = fL × (2 × fM); abundance of DTDP*-lactone = (2 × fD) × fL; abundance of DTDP*-MDEA = (2 × fD) × (2 × fM). The symbols fL, fM and fD represent respectively molar fractions of lactone, MDEA and DTDP units in the copolymer chains. 3.2. Fabrication and characterization of DTX-loaded micelles The PEG-PPMD and PEG-PCMD copolymers self-assembled readily to form micelle nanoparticles in aqueous medium. The formation of the micelle nanoparticles was monitored by fluorometry using pyrene as a fluorescent probe and the critical micelle concentration (CMC) values were calculated. Figure S3 displays the variations in fluorescence intensity ratio (I3/I1) of pyrene vs logarithm of polymer concentration for different PEG-PPMD and PEG-PCMD copolymers in PBS. With increasing polymer concentration from 0.0001 mg/mL to 1.0 mg/mL, the curves show cross points yielding the CMC values of the polymeric micelles. Above CMC values, stable micelles encapsulating an increased amount of pyrene are formed 50. The CMC values calculated for the PEG-PPMD copolymers with 12%, 43% and 70% PDL are respectively 10.9 µg/mL, 10.6 µg/mL and 8.5 µg/mL. A similar trend is found for PEG-PCMD micelle samples. The CMC values of the copolymers with 13%, 41% and 70% CL are 15.5 µg/mL, 14.7 µg/mL and 9.7 µg/mL, correspondingly. Thus, higher lactone content in the polymer chains improves the stability of the copolymer micelles in aqueous medium by increasing hydrophobicity in the micelle cores. It is also notable that PDL is a stronger hydrophobicity-enhancer than CL, leading to lower CMC values for PEG-PPMD vs PEG-PCMD at a same lactone content.

Table 3. Characterization Data of DTX-loaded PEG-PPMD and PEG-PCMD Micelles in PBS (10 mM, pH 7.4).

16


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Sample

Size(nm)

PDI

Zeta (mV)

DL(%)

EE(%)

PEG-PPMD-12% PDL

121.3±9.6

0.197±0.01

-7.7±3.8

3.27±0.15

65.4±5.1

PEG-PPMD-43% PDL

115.4±14.5

0.154±0.02

-6.8±0.5

3.33±0.36

66.6±11.2

PEG-PPMD-70% PDL

84.2±20.8

0.250±0.01

-5.9±0.2

3.29 ±0.24

65.8±8.2

PEG-PCMD-13% CL

202.8±30.0

0.252±0.04

-8.1±0.1

3.25±0.14

65.0±4.7

PEG-PCMD-41% CL

170.7±16.1

0.119±0.01

-6.6±0.7

3.59±0.03

71.8±1.0

PEG-PCMD-70% CL

140.4±28.3

0.188±0.01

-6.0±0.9

3.78±0.16

75.6±5.4

Docetaxel (DTX) is a hydrophobic chemotherapeutic agent, and its standard formulations (Taxotere® and Duopafei®) require Tween 80 and ethanol vehicle for parenteral administration

51

. Here, we use biodegradable PEG-PPMD and

PEG-PCMD copolymers for encapsulation of DTX to improve its solubility and cellular uptake. The average size, polydispersity index (PDI), and zeta potential of DTX-loaded PEG-PPMD and PEG-PCMD micelles measured by DLS are shown in Table 3. The drug-loaded PEG-PPMD micelles had an average size between 84 and 121 nm, which is smaller than the average sizes between 140 and 203 nm observed for the DTX-encapsulated PEG-PCMD micelles. This likely due to the fact that the presence of PDL units in PEG-PPMD chains render the copolymer micelle cores significantly more hydrophobic than those of the PEG-PCMD micelles, thus substantially reducing the water absorption in the nanoparticle cores

52,53

. The

morphologies of DTX-loaded micelles formed from PEG-PPMD-12% PDL and PEG-PCMD-13% CL copolymers were examined by TEM (Figure 2). The TEM images of both micelle samples show uniform, spherical shape with comparable average sizes and narrow size distributions. The sizes of the micelles

17



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Page 18 of 38

Figure 2. The TEM image and particle size distribution of DTX-loaded micelles: (A, B) PEG-PPMD-12% PDL and (C, D) PEG-PCMD-13% CL. The scale bar = 100 nm.

measured by TEM are smaller than those measured by DLS. Possibly, the removal of water from the micelle samples during the TEM analysis may shrink the micelle particles

54

. All DTX-loaded micelle samples were slightly negative-charged on

surface (Table 3). This is beneficial for in vivo drug delivery because previous studies indicate that nanoparticles with zeta potential values ranging from -10 to +10 mV can decrease serum protein binding and increase particle circulation time in the blood.55,56 Consistently, the average size of PEG-PPMD and PEG-PCMD micelles remained fairly constant upon incubation for 7 d in PBS (10 mM，pH 7.4) with 10% FBS (Figure S4 in supporting information). These results demonstrate that the PEG-PPMD and PEG-PCMD micelles are stable under the physiological conditions and the PEG shells in the micelle particles are efficient in preventing the nanoparticles from forming large agglomerates in PBS solution with 10 vol% FBS. The drug loading and entrapment efficiency for the DTX-loaded micelles were determined by HPLC analysis (Table 3). The DTX entrapment efficiencies (EE) are 65%-67% for the PEG-PPMD micelles and are in the range between 65% and 76% 18


Page 19 of 38

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for the PEG-PCMD micelles. The drug loading (DL) amounts in all micelle samples are comparable (3.3-3.8 wt%).

3.3. pH and redox dual-responsive properties of PEG-PPMD and PEG-PCMD nanoparticles Functional nanoparticles responsive to acidic pH and intracellular reduction potential are useful drug carriers that can selectively deliver and release a drug at controllable rates to specific disease sites (e.g., acidic tumors and tumor cells). Thus, the responsive behaviors of blank PEG-PPMD and PEG-PCMD micelles upon exposure to acidic and reductive conditions, either individually or synergistically, were investigated. The micelles were incubated for 48 h in different PBS buffers with pH of 7.4 or 5.0 containing various amount of D,L-dithiothreitol (DTT, 0 to 50 mM), and the size variations of the micelle particles were measured by DLS analysis. The DLS plots of six PEG-PPMD and PEG-PCMD micelle samples (PEG-PPMD with 12%, 43% and 70% PDL and PEG-PCMD with 13%, 41% and 70% CL) are shown in Figure S5 and the data for representative PEG-PPMD-12% PDL and PEG-PCMD-13% CL nanoparticles are summarized in Table 4. At pH of 7.4, the mean particle size of PEG-PPMD and PEG-PCMD micelle samples remains essentially constant (Figure S4). In general, upon decreasing the pH to 5.0, the sizes of the micelles increase significantly due to protonation of the tertiary amino groups in the micelle cores which become more hydrophilic to absorb extra water molecules from the media (Table 4). There is an exceptional case where the average size of PEG-PCMD-13% CL micelles was smaller in PBS (without DTT) at pH of 5.0 vs 7.4 (Table 4). Possibly, some of the large micelle particles become completely soluble in the medium after excessive swelling at pH of Table 4. The Size Variations of PEG-PPMD and PEG-PCMD Micelles Incubated for 48 h in PBS Solutions with different pH and DTT Contents Average size

Incubation condition

PEG-PPMD-12%

pH 7.4 + 0 mM DTT

220

0.33

332

PDL

pH 7.4 + 10 mM DTT

396

0.56

130

（nm）

PDI

Count rate

Sample

(kcps)

19



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Page 20 of 38

pH 7.4 + 50 mM DTT

459

1.00

40

pH 5.0 + 0 mM DTT

255

0.52

100

pH 5.0 + 10 mM DTT

345

0.89

56

pH 5.0 + 50 mM DTT

576

1.00

43

pH 7.4 + 0 mM DTT

223

0.20

224

pH 7.4 + 10 mM DTT

251

1.00

66

PEG-PCMD-13%

pH 7.4 + 50 mM DTT

349

1.00

37

CL

pH 5.0 + 0 mM DTT

187

0.16

157

pH 5.0 + 10 mM DTT

281

1.00

55

pH 5.0 + 50 mM DTT

453

1.00

47

5.0, thus reducing the average size of the whole sample. This is consistent with the count rate results showing that the count rate value is substantially lower for the PEG-PCMD-13% CL micelles upon decreasing the pH of PBS from 7.4 to 5.0 (Table 4). Similar phenomena were observed previously for other cationic polymers 48,57. It is also notable from Table 4 that during protonation, the size distribution of the micelles tends to be broader and the count rate (in proportion to micelle concentration) tends to drop, confirming that protonation decreases the stability of the PEG-PPMD and PEG-PCMD micelles in aqueous medium. When DTT concentration is increased from 0 to 50 mM in the PBS media at a constant pH, PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles also swell remarkably with average particle size increase between 150% and 230%, or respectively by up to 320 and 270 nm (Table 4). In addition to protonation, the presence of DTT further increases the micelle size, broadens the micelle PDL value and decreases the micelle count rate, which is ascribable to the cleavage of -S-Sbonds in the micelles forming more hydrophilic and looser micelle inner cores

58

.

Therefore, reductants such as DTT can effectively trigger disintegration of the PEG-PPMD and PEG-PCMD nanoparticles for controlled drug release and delivery applications. Importantly, the response of both PEG-PPMD and PEG-PCMD micelles to pH and reduction potential are markedly synergistic (Table 4), which should render these nanocarriers especially effective for drug delivery to target sites with acidic pH and high reduction potential such as tumors. 20


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3.4. In vitro drug release from DTX-loaded micelles The in vitro release of DTX from PEG-PPMD and PEG-PCMD micelles were investigated at 37 °C under following four different conditions: (i) in PBS with pH of 7.4, (ii) in PBS with pH of 7.4 and 10 mM DTT, (iii) in PBS with pH of 5.0, and (iv) in PBS with pH of 5.0 and 10 mM DTT. The drug release rate is dependent on the composition of the copolymers and is affected also by the medium pH and reduction potential. The accumulative release of DTX from the PEG-PPMD and PEG-PCMD micelles are depicted in Figure 3. Generally, the drug release rate of all micelles followed biphasic release kinetics. For example, at physiological pH of 7.4, the DTX-loaded PEG-PPMD micelles released 35-38% drug during the initial 12 h, then a gradual release of additional 25-32% drug was found for the subsequent 156 h (Figure 3, A-C). The accumulated DTX released from the micelles of PEG-PPMD-12% PDL, PEG-PPMD-43% PDL and PEG-PPMD-70% PDL copolymers were respectively 70%, 66% and 69% at the end of the incubation period (168 h). Upon decreasing the medium pH from 7.4 to 5.0, all micelle samples respond to the acidic pH to exhibit accelerated drug release rates (Figure 3). In particular for the PEG-PCMD-13% CL, PEG-PCMD-41% CL and PEG-PCMD-70% CL micelles over the 168 h incubation period, the total released drug is respectively 67%, 70% and 67% at pH 7.4, which increases correspondingly to 89%, 95% and 88% at pH 5.0 (Figure 3, D-F). This acid-triggered fast drug release is consistent with our previous results obtained from PEG-poly(PDL-co-MDEA-co-sebacate) (PEG-PPMS) drug delivery system 48 and is attributable to the swelling of the micelle particles due to protonation of the micelle cores. In addition to being responsive to pH, many of the PEG-PPMD and PEG-PCMD micelles (e.g., PEG-PPMD-12% PDL, PEG-PPMD-43% PDL, PEG-PCMD-41% CL, PEG-PCMD-70%

21



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Page 22 of 38

Figure 3. In vitro drug release from DTX-loaded micelles of following polymers incubated in PBS under different pH and redox conditions: (A) PEG-PPMD-12% PDL, (B) PEG-PPMD-43% PDL, (C) PEG-PPMD-70% PDL, (D) PEG-PCMD-13% CL, (E) PEG-PCMD-41% CL, and (F) PEG-PCMD-70% CL.

CL) also respond predictably to DTT that was added to the media, and are triggered by the reductant to release the drug at an accelerated rate. The minimal response to DTT for the PEG-PPMD-70% PDL micelles is presumably due to their highly hydrophobic, PDL-rich micelle cores that prohibit the diffusion of water-soluble DTT from the media to react with and cleave the low abundant disulfide bonds in the micelles (Figure 3C). The abnormal DTT-responsive drug release behaviors were also 22


Page 23 of 38

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observed for DTX-loaded PEG-PCMD-13% CL micelles (Figure 3D). This is likely due to the rapid reduction of the copolymer by DTT, causing precipitation of bulky polymer that traps DTX with a minimal drug release rate (Figure S6). Importantly, the drug release rate from the PEG-PPMD and PEG-PCMD micelles is responsive to and regulated by both medium pH and reductant DTT. Further, the accelerated drug-release effects triggered by pH and DTT are substantially synergistic, and the fastest DTX release rates occurred at acidic pH of 5.0 and in the presence of 10 mM DTT (Figure 3). Compared to the previously reported PEG-PPMS micelles that are solely pH-responsive

48

and PEG-PPBD micelles that are solely redox-responsive 49,

the current PEG-PPMD and PEG-PCMD nanoparticles with synergistic pH and redox-responsive properties are expected to be significantly more potent nanocarriers for intracellular delivery and release of chemotherapeutic agents to cancer cells since their drug delivery efficiency can be boosted by both acidic tumor or endosomal pH and the unusually high reduction potential (due to high GSH level) in cancer cells.

3.5. In vitro cellular uptake and intracellular distribution of PEG-PPMD and PEG-PCMD nanoparticles The cellular uptake efficiency of the DTX-loaded PEG-PPMD and PEG-PCMD micelles was evaluated with both flow cytometry and confocal laser scanning microscopy (CLSM). To facilitate the cellular uptake study, the DTX drug was replaced by fluorescent probe molecule coumarin-6 (C6) and C6-loaded micelles of PEG-PPMD and PEG-PCMD were used instead. Figure 4 shows the mean fluorescence intensity (MFI) values of HeLa cells incubated with free C6 or the C6-encapsulated micelle samples for up to 8 h. It is evident that the cellular uptake process is time-dependent with an increasing number of the micelles being internalized by the cells during the first 6 h. Over this period, C6-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles exhibit higher cellular uptake efficiency than other micelle samples, and are best performers in their own micelle sample groups (Figure 4). Among PEG-PPMD micelle samples, the cellular uptake efficiency decreases with increasing PDL content 23



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Page 24 of 38

Figure 4. Uptake of free C6 and C6-loaded PEG-PPMD and PEG-PCMD micelles by HeLa cells. The intracellular C6 MFI values were measured by flow cytometry after 1-8 h incubation. Data are given as mean ± SD (n = 3).

in the copolymer. The free C6 showed fastest cellular uptake due to its high hydrophobicity and quick diffusion through the cell membrane. To determine if the micelles can escape from endosomes and lysosomes, the locations of the C6-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles in HeLa cells relative to the positions of acidic endosomes/lysosomes (stained with Lysotracker Red) were visualized using CLSM (Figure 5). Clearly, the C6-loaded micelles were internalized by the cells and distributed over the whole cytoplasm after 2 h incubation. Importantly, the green fluorescence of both micelle samples exhibited minimal co-localization with the Lysotracker Red-stained organelles at 6 h of incubation, indicating their rapid endosomal escaping capability. We believe that the pH buffer capacity of the tertiary amino groups present in the PPMD and PCMD segments of the copolymer chains facilitates the endosomal escape of the internalized micelles due to proton sponge effects. Further, the intracellular GSH is anticipated to react with the disulfide bonds in the micelles to cause polymer chain cleavage and disintegration of the micelle particles, thus promoting C6 release in the cells. These results strongly support our assumption that the pH and redox-responsive PEG-PPMD and PEG-PCMD nanoparticles encapsulating anticancer drugs can be triggered by tumor-relevant acidic pH and intracellular GSH to rapidly release the drug molecules 24


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for efficient killing of cancer cells.

Figure 5. CLSM images of HeLa cells incubated with free C6, C6-loaded PEG-PPMD and PEG-PCMD micelles (green) for (A) 2 h and (B) 6 h. The nuclei were stained with Hoechst 33342 (blue) and the endosomes/lysosomes were stained with LysoTracker® Red (red). The scale bar = 15µm.

3.6. In vitro cytotoxicity The in vitro cytotoxicity of blank and DTX-loaded PEG-PPMD and PEG-PCMD micelles was evaluated on HeLa cells and CT-26 cells. All blank micelle samples exhibited minimal cytotoxicity and the viabilities of the cells incubated with the 25



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Page 26 of 38

micelles at various polymer concentrations up to 400 µg/mL were over 80% (Figure 6). Further, these PEG-PPMD and PEG-PCMD micelle samples are compatible with human blood, showing essentially no hemolytic activity ( 0.9% NaCl. To further evaluate the antitumor activity, tumor specimens

Figure 9. The measured tumor volume (A) and body weight (B) of the Balb/C mice treated with free DTX (Duopafei®), DTX-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelle formulations at 4 x 10 mg/kg DTX dose for 21 days. 30


Page 31 of 38

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Arrows indicate the dates when the formulations were administered.

from the mice receiving different treatments were collected for the hematoxylin and eosin (H&E) staining. From the H&E staining of all tumor tissue samples (Figure 10A), we observed cracked nuclear membranes and condensed chromatins that were marginalized and divided into blocks or apoptotic bodies, indicating obvious apoptosis of the tumor cells. The rate of necrosis of pieces is in the order: 0.9% NaCl (60-70%) > DTX-loaded PEG-PCMD-13% CL micelles (50-60%) > DTX-loaded PEG-PPMD-12% PDL micelles (40-50%) > free DTX (10-20%). The highest rate of necrosis of pieces obtained for 0.9 % NaCl is primarily due to the extremely big size of its treated tumors which causes self-necrosis. On the other hand, the lowest rate of necrosis of pieces obtained for the free DTX is possibly attributed to the instability and fast biodegradation

Figure 10. H&E staining of (A) tumor specimens from the mice receiving treatment of 0.9% NaCl, free DTX, DTX-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles for 27 days, (B) organs from the mice on day 27 after treatment of the PEG-PPMD and PEG-PCMD nanoparticle formulations at DTX dose of 4 x10 mg/kg. The scale bar = 50 µm. of the unprotected drug molecules. These experimental results demonstrate the advantages of using intracellular stimuli-responsive nanoparticles for controlled delivery and release of chemotherapeutic drugs to kill cancer cells. Finally, while 31



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Page 32 of 38

increasing efficacy of anticancer drug formulations is important, it is also essential to minimize their effects on normal organ tissues during treatment. Figure 10B depicts the H&E staining images of various organs including heart, liver, spleen, lung and kidney, which were harvested from the mice on day 27 after the treatment with DTX-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles at DTX dose of 4 x 10 mg/kg. Consistent with the minimal body weight loss (Figure 9B), no apparent histological damage in these organs was observed following the treatment courses. Therefore, the DTX formulations based on PEG-PPMD or PEG-PCMD micelles are not only therapeutically efficient, but also potentially safe for antitumor treatment in vivo.

4. Conclusions We have designed and constructed novel multifunctional nanoparticle drug delivery systems that are stable under physiological conditions and responsive to tumor-relevant pH and intracellular reduction potential. The nanoparticles were fabricated from new enzymatic PEG-PPMD and PEG-PCMD block copolymers via a self-assembly process in aqueous solution. At acidic pH and in the presence of a reductant (e.g., DTT or GSH), the nanosized micelle particles rapidly swell and disintegrate due to the protonation of amino groups and reductive cleavage of disulfide bonds in the micelle cores. Consistently, DTX-loaded PEG-PPMD and PEG-PCMD micelles can be triggered synergistically by both acidic endosomal pH and a high intracellular reduction potential to rapidly release the drug for efficient killing of cancer cells. The drug formulations based on PEG-PPMD and PEG-PCMD copolymers exhibited a substantially higher potency than free DTX in inhibiting tumor growth in mice while their therapeutic effects on important organ tissues were minimal. These results demonstrate that PEG-PPMD and PEG-PCMD nanoparticles are promising to serve as site specific, pH and redox-responsive drug nanocarriers for safe and efficient antitumor treatment.

32


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Supporting Information.

The supporting information is available free of charge on the ACS Publications website. Structural analysis of PEG-PPMD and PEG-PCMD block copolymers, critical micelle concentration (CMC) measurement, in vitro micelle stability test, DLS measurement on the size change of PEG-PPMD and PEG-PCMD micelles in response to different pH and reduction potential in the medium, visual inspection of PEG-PPMD and PEG-PCMD micelle responses to aqueous media with varied pH and DTT concentration, erythrocyte agglutination and hemolysis assay, and effects of pH and reduction potential on cytotoxicity of DTX-loaded PEG-PCMD nanoparticles.

Acknowledgments

This work was supported by the Natural Science Foundation of Guangdong Province (2016A030313315, 2014A030312018), the Science and Technology Planning Project of Guangdong Province (2015A050502024), the Tip-top Scientific and Technical Innovative Youth Talents of Guangdong Special Support Program (2014TQ01R651), and the Fundamental Research Funds for the Central Universities (161gzd05). We thank Yale University for supporting the biomaterial synthesis (project no. 1044076).

References

(1) Torchilin, V. Tumor Delivery of Macromolecular Drugs Based on the EPR Effect. Adv. Drug Delivery Rev. 2011, 63, 131-135. (2) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nanoparticle Therapeutics: An Emerging Treatment Modality for Cancer. Nat. Rev. Drug Discovery 2008, 7, 771-782. (3) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Polymeric Systems for Controlled Drug Release. Chem. Rev. 1999, 99, 3181-3198. (4) Du, J. Z.; Du, X. J.; Mao, C. Q.; Wang, J. Tailor-Made Dual pH-Sensitive Polymer-Doxorubicin Nanoparticles for Efficient Anticancer Drug Delivery. J. Am. Chem. Soc. 33



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2011, 133, 17560-17563. (5) Zugates, G. T.; Anderson, D. G.; Little, S. R.; Lawhorn, I. E. B.; Langer, R. Synthesis of Poly(beta-amino ester)s with Thiol-reactive Side Chains for DNA Delivery. J. Am. Chem. Soc. 2006, 128, 12726-12734. (6) Wu, W.; Driessen, W.; Jiang, X. Q. Oligo(ethylene glycol)-Based Thermosensitive Dendrimers and Their Tumor Accumulation and Penetration. J. Am. Chem. Soc. 2014, 136, 3145-3155. (7) Karimi, M.; Ghasemi, A.; Zangabad, P. S.; Rahighi, R.; Basri, S. M. M.; Mirshekari, H.; Amiri, M.; Pishabad, Z. S.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.; Aref, A. R.; Haghani, L.; Bahrami, S.; Hamblin, M. R. Smart Micro/nanoparticles in Stimulus-responsive Drug/gene Delivery Systems. Chem. Soc. Rev. 2016, 45, 1457-1501. (8) Potineni, A.; Lynn, D. M.; Langer, R.; Amiji, M. M. Poly(ethylene oxide)-modified Poly(beta-amino ester) Nanoparticles as a pH-sensitive Biodegradable System for Paclitaxel Delivery. J. Controlled Release 2003, 86, 223-234. (9) Yin, H.; Lee, E. S.; Kim, D.; Lee, K. H.; Oh, K. T.; Bae, Y. H. Physicochemical Characteristics of pH-sensitive Poly(L-histidine)-b-poly(ethylene glycol)/poly(L-lactide)-b-poly(ethylene glycol) Mixed Micelles. J. Controlled Release 2008, 126, 130-138. (10) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Design of Environment-sensitive Supramolecular Assemblies for Intracellular Drug Delivery: Polymeric Micelles That Are Responsive to Intracellular pH Change. Angew. Chem., Int. Ed. 2003, 42, 4640-4643. (11) Stubbs, M.; McSheehy, P. M. J.; Griffiths, J. R.; Bashford, C. L. Causes and Consequences of Tumour Acidity and Implications for Treatment. Mol. Med. Today 2000, 6, 15-19. (12) Yin, H. Q.; Bae, Y. H. Physicochemical Aspects of Doxorubicin-loaded pH-sensitive Polymeric Micelle Formulations from a Mixture of Poly(L-histidine)-b-poly(ethylene glycol)/poly(L-lactide)-b-poly(ethylene glycol) (vol 71, pg 223, 2009). Eur. J. Pharm. Biopharm. 2009, 72, 634-634. (13) Wu, G. Y.; Fang, Y. Z.; Yang, S.; Lupton, J. R.; Turner, N. D. Glutathione Metabolism and Its Implications for Health. J. Nutr. 2004, 134, 489-492. (14) Kuppusamy, P.; Li, H. Q.; Ilangovan, G.; Cardounel, A. J.; Zweier, J. L.; Yamada, K.; Krishna, M. C.; Mitchell, J. B. Noninvasive Imaging of Tumor Redox Status and Its Modification by Tissue Glutathione Levels. Cancer Res. 2002, 62, 307-312. (15) Bahadur, K. C. R.; Xu, P. S. Multicompartment Intracellular Self-Expanding Nanogel for Targeted Delivery of Drug Cocktail. Adv. Mater. 2012, 24, 6479-6483. (16) Cheng, R.; Meng, F.; Deng, C.; Klok, H. A.; Zhong, Z. Dual and Multi-stimuli Responsive Polymeric Nanoparticles for Programmed Site-specific Drug Delivery. Biomaterials 2013, 34, 3647-3657. (17) Chen, W.; Yuan, Y.; Cheng, D.; Chen, J.; Wang, L.; Shuai, X. Co-delivery of Doxorubicin and siRNA with Reduction and pH Dually Sensitive Nanocarrier for Synergistic Cancer Therapy. Small 2014, 10, 2678-2687. (18) Yoon, S.; Kim, W. J.; Yoo, H. S. Dual-responsive Breakdown of Nanostructures with High Doxorubicin Payload for Apoptotic Anticancer Therapy. Small 2013, 9, 284-293. (19) Cheng, W. R.; Kumar, J. N.; Zhang, Y.; Liu, Y. pH- and Redox-Responsive Poly(ethylene glycol) and Cholesterol-Conjugated Poly(amido amine)s Based Micelles for Controlled Drug 34


Page 35 of 38

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


Delivery. Macromol. Biosci. 2014, 14, 347-358. (20) Chen, J.; Qiu, X. Z.; Ouyang, J.; Kong, J. M.; Zhong, W.; Xing, M. M. Q. pH and Reduction Dual-Sensitive Copolymeric Micelles for Intracellular Doxorubicin Delivery. Biomacromolecules 2011, 12, 3601-3611. (21) Chen, F.; Zhang, J.; Wang, L.; Wang, Y.; Chen, M. Tumor pH(e)-triggered Charge-reversal and Redox-responsive Nanoparticles for Docetaxel Delivery in Hepatocellular Carcinoma Treatment. Nanoscale 2015, 7, 15763-15779. (22) Lu, Y.; Mo, R.; Tai, W. Y.; Sun, W. J.; Pacardo, D. B.; Qian, C. G.; Shen, Q. D.; Ligler, F. S.; Gu, Z. Self-folded Redox/Acid Dual-responsive Nanocarriers for Anticancer Drug Delivery. Chem. Commun. 2014, 50, 15105-15108. (23) Yi, H. Q.; Liu, P.; Sheng, N.; Gong, P.; Ma, Y. F.; Cai, L. T. In situ Crosslinked Smart Polypeptide Nanoparticles for Multistage Responsive Tumor-targeted Drug Delivery. Nanoscale 2016, 8, 5985-5995. (24) Jiang, Z. Z. Lipase-Catalyzed Synthesis of Poly(amine-co-esters) and Poly(lactone-co-beta-amino esters). ACS Symp. Ser. 2013, 1144, 29-42. (25) Zhou, J. B.; Liu, J.; Cheng, C. J.; Patel, T. R.; Weller, C. E.; Piepmeier, J. M.; Jiang, Z. Z.; Saltzman, W. M. Biodegradable Poly(amine-co-ester) Terpolymers for Targeted Gene Delivery. Nat. Mater. 2012, 11, 82-90. (26) Zhang, X. F.; Tang, W. X.; Yang, Z.; Luo, X. G.; Luo, H. Y.; Gao, D.; Chen, Y.; Jiang, Q.; Liu, J.; Jiang, Z. Z. PEGylated Poly(amine-co-ester) Micelles as Biodegradable Non-viral Gene Vectors with Enhanced Stability, Reduced Toxicity and Higher in vivo Transfection Efficacy. J. Mater. Chem. B 2014, 2, 4034-4044. (27) Yang, Z.; Jiang, Z. Z.; Cao, Z.; Zhang, C.; Gao, D.; Luo, X. G.; Zhang, X. F.; Luo, H. Y.; Jiang, Q.; Liu, J. Multifunctional Non-viral Gene Vectors with Enhanced Stability, Improved Cellular and Nuclear Uptake Capability, and Increased Transfection Efficiency. Nanoscale 2014, 6, 10193-10206. (28) Chen, Y.; Li, Y. Q.; Gao, J. B.; Cao, Z.; Jiang, Q.; Liu, J.; Jiang, Z. Z. Enzymatic PEGylated Poly(lactone-co-beta-amino ester) Nanoparticles as Biodegradable, Biocompatible and Stable Vectors for Gene Delivery. ACS Appl. Mater. Interfaces 2016, 8, 490-501. (29) Zhang, J.; Cui, J.; Deng, Y.; Jiang, Z.; Saltzman, W. M. Multifunctional Poly(amine-co-ester-co-orthoester) for Efficient and Safe Gene Delivery. ACS Biomater. Sci. Eng. 2016, 2, 2080-2089. (30) Mazzocchetti, L.; Scandola, M.; Jiang, Z. Z. Enzymatic Synthesis and Structural and Thermal Properties of Poly(omega-pentadecalactone-co-butylene-co-succinate). Macromolecules 2009, 42, 7811-7819. (31) Liu, J.; Jiang, Z. Z.; Zhang, S. M.; Saltzman, W. M. Poly(omega-pentadecalactone-co-butylene-co-succinate) Nanoparticles as Biodegradable Carriers for Camptothecin Delivery. Biomaterials 2009, 30, 5707-5719. (32) Liu, J.; Jiang, Z.; Zhang, S.; Liu, C.; Gross, R. A.; Kyriakides, T. R.; Saltzman, W. M. Biodegradation, Biocompatibility, and Drug Delivery in Poly(omega-pentadecalactone-co-p-dioxanone) Copolyesters. Biomaterials 2011, 32, 6646-6654. (33) Yang, Z.; Zhang, X.; Luo, X.; Jiang, Q.; Liu, J.; Jiang, Z. Enzymatic Synthesis of Poly(butylene-co-sebacate-co-glycolate) Copolyesters and Evaluation of the Copolymer Nanoparticles as Biodegradable Carriers for Doxorubicin Delivery. Macromolecules 2013, 46, 35



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 36 of 38

1743-1753. (34) Han, L.; Kong, D. K.; Zheng, M. Q.; Murikinati, S.; Ma, C.; Yuan, P.; Li, L. Y.; Tian, D. F.; Cai, Q.; Ye, C. L.; Holden, D.; Park, J. H.; Gao, X. B.; Thomas, J. L.; Grutzendler, J.; Carson, R. E.; Huang, Y. Y.; Piepmeier, J. M.; Zhou, J. B. Increased Nanoparticle Delivery to Brain Tumors by Autocatalytic Priming for Improved Treatment and Imaging. ACS Nano 2016, 10, 4209-4218. (35) Gross, R. A.; Ganesh, M.; Lu, W. H. Enzyme-catalysis Breathes New Life into Polyester Condensation Polymerizations. Trends Biotechnol. 2010, 28, 435-443. (36) Kobayashi, S.; Makino, A. Enzymatic Polymer Synthesis: An Opportunity for Green Polymer Chemistry. Chem. Rev. 2009, 109, 5288-5353. (37) Gross, R. A.; Kumar, A.; Kalra, B. Polymer Synthesis by in vitro Enzyme Catalysis. Chem. Rev. 2001, 101, 2097-2124. (38) Kobayashi, S.; Uyama, H.; Kimura, S. Enzymatic Polymerization. Chem. Rev. 2001, 101, 3793-3818. (39) Kumar, A.; Gross, R. A. Candida antarctica Lipase B-catalyzed Transesterification: New Synthetic Routes to Copolyesters. J. Am. Chem. Soc. 2000, 122, 11767-11770. (40) Jiang, Z.; Liu, C.; Gross, R. A. Lipase-catalyzed Synthesis of Aliphatic Poly(carbonate-co-esters). Macromolecules, 2008, 41, 4671-4680. (41) Jiang, Z. Z. Lipase-Catalyzed Synthesis of Aliphatic Polyesters via Copolymerization of Lactone, Dialkyl Diester, and Diol. Biomacromolecules 2008, 9, 3246-3251. (42) Jiang, Z. Z. Lipase-Catalyzed Synthesis of Poly(amine-co-esters) via Copolymerization of Diester with Amino-Substituted Diol. Biomacromolecules 2010, 11, 1089-1093. (43) Jiang, Z. Z. Lipase-Catalyzed Copolymerization of Dialkyl Carbonate with 1,4-Butanediol and omega-Pentadecalactone: Synthesis of Poly(omega-pentadecalactone-co-butylene-co-carbonate). Biomacromolecules 2011, 12, 1912-1919. (44) Martino, L.; Scandola, M.; Jiang, Z. Z. Enzymatic Synthesis, Thermal and Crystalline Properties of a Poly(beta-amino ester) and Poly(lactone-co-beta-amino ester) Copolymers. Polymer 2012, 53, 1839-1848. (45) Jiang, Z. Z.; Zhang, J. W. Lipase-catalyzed Synthesis of Aliphatic Polyesters via Copolymerization of Lactide with Diesters and Diols. Polymer 2013, 54, 6105-6113. (46) Feng, Y.; Knüfermann, J.; Klee, D.; Höcker, H. Enzyme-catalyzed Ring-opening Polymerization of 3(S)-Isopropylmorpholine-2,5-dione. Macromol. Rapid Commun. 1999, 20, 88-90. (47) Feng, Y.; Klee, D.; Höcker, H. Lipase-catalyzed Ring-opening Polymerization of 3(S)-sec-Butylmorpholine-2,5-dione. Macromol. Biosci. 2001, 1, 66-74. (48) Zhang, X. F.; Liu, B.; Yang, Z.; Zhang, C.; Li, H.; Luo, X.; Luo, H. Y.; Gao, D.; Jiang, Q.; Liu, J.; Jiang, Z. Z. Micelles of Enzymatically Synthesized PEG-poly(amine-co-ester) Block Copolymers as pH-responsive Nanocarriers for Docetaxel Delivery. Colloids Surf., B 2014, 115, 349-358. (49) Liu, B.; Zhang, X.; Chen, Y.; Yao, Z.; Yang, Z.; Gao, D.; Jiang, Q.; Liu, J.; Jiang, Z. Enzymatic Synthesis of Poly( ω -pentadecalactone-co-butylene-co-3,3 ′ -dithiodipropionate) Copolyesters and Self-assembly of the PEGylated Copolymer Micelles as Redox-responsive Nanocarriers for Doxorubicin Delivery. Polym. Chem. 2015, 6, 1997-2010. (50) Huh, K. M.; Lee, K. Y.; Kwon, I. C.; Kim, Y. H.; Kim, C.; Jeong, S. Y. Synthesis of 36


Page 37 of 38

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


Triarmed Poly(ethylene oxide)-Deoxycholic Acid Conjugate and Its Micellar Characteristics. Langmuir 2000, 16, 10566-10568. (51) Li, Y. F.; Yang, F. F.; Chen, W.; Liu, J. Y.; Huang, W.; Jin, M. J.; Gao, Z. G. A Novel Monomethoxy Polyethylene Glycol-Polylactic Acid Polymeric Micelles with Higher Loading Capacity for Docetaxel and Well-Reconstitution Characteristics and Its Anti-metastasis Study. Chem. Pharm. Bull. 2012, 60, 1146-1154. (52) Theerasilp, M.; Nasongkla, N. Comparative Studies of Poly(epsilon-caprolactone) and Poly(D,L-lactide) as Core Materials of Polymeric Micelles. J. Microencapsulation 2013, 30, 390-397. (53) Riley, T.; Stolnik, S.; Heald, C. R.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Physicochemical Evaluation of Nanoparticles Assembled from Poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) Block Copolymers as Drug Delivery Vehicles. Langmuir 2001, 17, 3168-3174. (54) Fontana, G.; Licciardi, M.; Mansueto, S.; Schillaci, D.; Giammona, G. Amoxicillin-loaded Polyethylcyanoacrylate Nanoparticles: Influence of PEG Coating on The Particle Size, Drug Release Rate and Phagocytic Uptake. Biomaterials 2001, 22, 2857-2865. (55) Li, S. D.; Huang, L. Pharmacokinetics and Biodistribution of Nanoparticles. Mol. Pharmaceutics 2008, 5, 496-504. (56) Levchenko, T. S.; Rammohan, R.; Lukyanov, A. N.; Whiteman, K. R.; Torchilin, V. P. Liposome Clearance in Mice: the Effect of A Separate and Combined Presence of Surface Charge and Polymer Coating. Int. J. Pharm. 2002, 240, 95-102. (57) Liu, Q. Q.; Yu, Z. Q.; Ni, P. H. Micellization and Applications of Narrow-distribution Poly[2-(dimethylamino)ethyl methacrylate]. Colloid Polym. Sci. 2004, 282, 387-393. (58) Zhang, Y.; He, J. L.; Cao, D. L.; Zhang, M. Z.; Ni, P. H. Galactosylated Reduction and pH Dual-responsive Triblock Terpolymer Gal-PEEP-a-PCL-ss-PDMAEMA: A Multifunctional Carrier for The Targeted and Simultaneous Delivery of Doxorubicin and DNA. Polym. Chem. 2014, 5, 5124-5138.

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