ROS-Activated Ratiometric Fluorescent Polymeric Nanoparticles for

Feb 9, 2018 - Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Depa...
0 downloads 16 Views 1MB Size
Subscriber access provided by READING UNIV

Article

ROS-Activated Ratiometric Fluorescent Polymeric Nanoparticles for Self-Reporting Drug Delivery Mei Zhang, Cheng-Cheng Song, Shan Su, Fu-Sheng Du, and Zi-Chen Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18438 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

ROS-Activated Ratiometric Fluorescent Polymeric Nanoparticles for Self-Reporting Drug Delivery Mei Zhang, Cheng-Cheng Song, Shan Su, Fu-Sheng Du,* and Zi-Chen Li*

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Department of Polymer Science & Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 2 of 27

ABSTRACT. Reactive oxygen species (ROS)-responsive theranostic nanomedicines have attracted wide interest in recent years because ROS stress is implicated in some pathological disorders such as inflammatory diseases and cancers. In this article, we report a kind of innovative ROS-responsive theranostic polymeric nanoparticles that are able to load hydrophobic drugs and to fluorescently self-report the in vitro or intracellular drug release under ROS triggering. The fluorescent nanoparticles were formed by amphiphilic block copolymers consisting of a PEG segment and an oxidation-responsive hydrophobic block. The copolymers with different hydrophobic block lengths were synthesized by the atom transfer radical polymerization (ATRP) of a phenylboronic ester-containing acrylic monomer with small fraction of a ROS-activatable 1,8-naphthalimide-based fluorescent monomer, using PEG-Br as the macroinitiator. The copolymer nanoparticles were stable in neutral phosphate buffer but degraded upon H2O2 triggering, with the degradation rate depending on the hydrophobic block length and the concentration of H2O2. The degradation of nanoparticles was accompanied by a colorimetric change of the fluorophore from blue to green, which affords the nanoparticles the ability to detect H2O2 by a ratiometric fluorescent approach. Moreover, the nanoparticles could encapsulate doxorubicin (DOX) and the H2O2-triggered DOX release was well associated with the change in ratiometric fluorescence. CLSM results reveal that the fluorescent nanoparticles were internalized into A549 cells through the endocytosis pathway. The ROS-stimulated degradation of the nanoparticles and intracellular DOX release, and the fate of the degraded polymers could be monitored by ratiometric fluorescent imaging. Finally, the naked nanoparticles and the degradation products are cytocompatible, while the DOX-loaded ones exhibit concentration dependent cytotoxicity. Of importance, the stimulation with exogenous H2O2 or LPS enhanced obviously cell-killing capability of the DOX-loaded nanoparticles due to the ROS-enhanced intracellular DOX release.

Key

word:

oxidation-responsive;

ratiometric

fluorescence;

theranostic

polymer;

self-reporting; drug delivery

2

ACS Paragon Plus Environment

Page 3 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

INTRODUCTION Theranostic nanomedicine that integrates imaging and therapeutic functionalities in one entity is an emerging and rapidly growing branch of medicines. It represents a promising modality that is beneficial for the temporal and spatial visualization of the therapeutic active agents, intracellularly or in vivo, and the evaluation of therapeutic efficacy. This may improve the therapy index of diseases such as cancers, facilitate the clinical translation of various drug delivery systems under research, and contribute to the personalized medicine.1-5 Among various imaging modalities, fluorescent optical imaging (FI) is highly attractive because of its advantages such as the ease of use, the versatility, high sensitivity and the ability to monitor multiple processes at the same time. FI technique has been widely applied for the understanding of various physiological and pathological processes, the preclinical researches of nanomedicines, and the clinical uses such as intraoperative imaging and fluorescent mammography.6-9 Small molecule fluorophores have been extensively studied and commonly used for the detection of various chemical species and the exploration of biological mechanism on cellular level, whereas the polymeric fluorescent nanoparticles with varied dyes, covalently attached or physically encapsulated, are more promising for preclinical researches and future clinical applications owing to their intrinsic benefit of nanosize, the improved water solubility and biocompatibility of the dyes, and the ability of versatile functionalization.10-13 Particularly, the stimuli-responsive polymeric nanoparticles with activatable fluorescence characteristics have drawn great attention in nanomedicines because of the improved detection sensitivity and selectivity or image quality as compared to the termed “always-on” counterparts. 14-21 Reactive oxygen and nitrogen species (ROS and RNS), existing homeostatically in human body, play important roles in normal physiological processes.22-24 However, ever-increasing evidences indicate that ROS/RNS stress (overproduction of ROS/RNS) in cells is closely correlated with various pathologies, including inflammation diseases and cancers.25-28 Therefore, ROS or RNS have been deemed as pathological markers for diagnosis of the oxidative stress-associated disorders ROS-overproduced

diseases.32-37

This

29-31

or as therapeutic targets in curing the

stimulates

the

development

of

various

ROS/RNS-activatable chemical or biological sensors, imaging agents and prodrugs, and a 3

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 4 of 27

number of oxidation-responsive polymers.38-49 While most of these ROS-sensitive systems focus on one function, that is detection/imaging or therapy/drug delivery, little of them has the theranostic feature. Recently, the ROS-activatable small molecule theranostic prodrugs have been reported, showing potential for the monitoring fluorescently intracellular drug release or the image-guided tumor therapy.50-53 Moreover, some ROS-responsive polymer nanoparticles or polymeric prodrugs possessing theranostic characteristics have been developed.54-58 These polymers were prepared by the step-growth polymerization method, which limits the controllability of the polymer structure, in particular molecular weight and its dispersity. Considering the validity of ROS stress as a biomarker of various pathologies, continuous efforts are necessary for the development of the ROS-responsive polymer-based theranostic nanomedicines. Herein, we report a type of innovative ROS-activatable theranostic polymer nanoparticles that are capable of loading hydrophobic drugs and self-reporting the payload release upon ROS stimulation. The nanoparticles are formed by the amphiphilic block copolymers consisting of PEG and an oxidation-responsive hydrophobic block that contains pendent phenylboronic pinacol ester groups and a small fraction of 1,8-naphthalimide dye (Scheme 1). The present theranostic system possesses the following unique features: 1) the copolymers are prepared by ATRP which enables the fine tuning of the polymer structure including composition and block length;59 2) ROS triggering leads to a ratiometric colorimetric change of the naphthalimide dye, which could improve the sensitivity and accuracy of fluorescent detection;60 3) the payload release is approximately linearly correlated with the fluorescence ratiometric change; 4) covalently conjugation of the dye would prevent its leakage, making it easy to visualize the intracellular release of the fluorescent drug and to trace the polymer carrier within cells;61-63 and finally, the block copolymers and their oxidative degradation products are biocompatible, which is essential for the potential in vivo applications.

4

ACS Paragon Plus Environment

Page 5 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

O O

R

O

N

O HN

O B

O

O

O

R=

O O

N

O O

O

N

O

MF

F O

M

HO

O

O O B O

B

O

O

(ii)

(i)

+

O

R

O

O

O

n O

m O

O

O

HN

O

O 113

O

O B

O

NH2

O

O

Scheme 1. Synthesis of MF block copolymers and H2O2-induced dissociation of the copolymer nanoparticles. (i) mPEG113-Br, CuBr, Me6TREN, anisole, 60 oC, 15 h; (ii) H2O2, PB solution (pH 7.4, 50 mM).

EXPERIMENTAL SECTION Materials.

The

oxidation-sensitive

monomer,

4-(4,4,5,5-Tetramethyl-1,3,2-

dioxaborolan-2-yl)benzyl acrylate (M), and p-hydroxymethylphenylboronic pinacol ester were synthesized following the published procedures.59 The macroinitiator mPEG113-Br was prepared from poly(ethylene oxide) monomethyl ether (mPEG113, Mw = 5000 Da, Fluka). Tris(2-dimethylaminoethyl) amine (Me6TREN) was prepared in our lab. CuBr (Acros) was treated with acetic acid, methanol and ether sequentially. Toluene was refluxed over sodium for 15 h prior to distillation. Triethylamine (TEA) and DMSO-d6 were treated with KOH and CaH2, respectively. NaOD (40 wt % in D2O) and deuterated D3PO4 (85 wt% in D2O) purchased from Alfa Aesar were used to prepare deuterated phosphate buffer (PB), to which was added sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS, J&K Chemical Ltd) as an internal standard (0.03 wt%). Doxorubicin hydrochloride (DOX•HCl, Alfa Aesar), catalase (CAT, from bovine liver, Sigma), lipopolysaccharide (LPS, Sigma), acrylic anhydride (Alfa Aesar) were used as received. 4-Amino-1,8-naphthalic anhydride, 2-(2-aminoethoxy)ethanol, 4-(N,N-dimethyl amino)pyridine (DMAP), triphosgene, CH2Cl2 (99.8%, SuperDry), N,N-dimethylformamide (DMF, SuperDry), and pyridine (SuperDry) were purchased from J&K

Chemical

Ltd

and

used

as

received.

DRAQ-5

(1,5-bis((2-

(dimethylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione), Lyso Tracker Red, cell counting kit-8 (CCK-8) were received from Invitrogen Co. Regenerated cellulose dialysis membrane (MWCO: 100 KDa and 10 KDa) were obtained from Beijing Huamei Scientific Co. Hydrogen peroxide (H2O2, 30 wt%) and other reagents were obtained from Beijing Chemical Reagent Co. and applied as received. 5

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 6 of 27

Syntheses of Fluorescent Monomer and Block Copolymers. The fluorescent monomer (F) was synthesized following a route as shown in Scheme S1. The detailed preparation protocols of monomer F and block copolymers (MF series) are presented in the Supporting Information. Preparation of Block Copolymer Nanoparticles. The solutions of the block copolymer nanoparticles in PB (pH 7.4, 50 mM) were prepared by a solvent-evaporation approach using acetone as the organic solvent. Briefly, copolymer (1.0 mg) was dissolved in acetone (1.0 mL) and this solution was added dropwise to 10 mL PB (pH 7.4, 50 mM) under stirring at room temperature. After another 12 h under stirring at 40 oC, acetone was removed thoroughly by evaporation to afford the nanoparticle solution (0.1 mg/mL) which was applied for the measurements of LLS, TEM, and UV-Vis absorption and fluorescence spectra. For the H2O2– initiated degradation monitored by 1H NMR, the copolymer nanoparticles were formulated by the same protocol but in deuterated PB solution (50 mM, pH 7.4). The concentration of polymer was 5 mg/mL. 1

H NMR, GPC, TEM and LLS Measurements. The detailed experimental procedures

are given in the Supporting Information. UV-Vis Absorption and Fluorescence Measurements. The MF1-MF4 nanoparticle solutions were equilibrated for 30 min at 37 oC prior to the measurements. A Lambda 35 UV-Vis spectrometer equipped with a temperature controller was used to record the absorption spectra of the nanoparticle solutions in a 1 cm quartz cell with a fast scanning rate. Blank PB solution was applied as a reference. The absorption spectra were measured from 300 to 700 nm. Both emission and excitation slit widths were 2 nm. A440/A370 is defined as the ratio of absorbance at 440 nm to that at 370 nm. For fluorescence measurements, a Hitachi F-7000 spectrometer with a temperature controller was used with a scanning rate of 2400 nm/min and an excitation wavelength of 430 nm. The fluorescence spectra were measured from 440 to 700 nm. Both excitation and emission slit widths were 5 nm. F535/F495 is defined as the ratio of emission intensity at 535 nm to that at 495 nm. Loading and Release of DOX. Doxorubicin hydrochloride was dissolved in acetone with 5-fold excessive TEA, the final concentration of DOXHCl was 2.0 mg/mL. This DOX 6

ACS Paragon Plus Environment

Page 7 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

solution was added to the solution of MF nanoparticles (2.0 mg/mL) in 50 mM PB (pH 7.4). After stirring overnight at 37 oC, the obtained solution was moved into a dialysis membrane (MWCO: 100 kDa) and dialyzed in PB (pH 7.4, 50 mM) at 37 oC for 24 h under dark conditions. In this process, the medium was displaced five times. Then, the nanoparticle solution after dialysis was diluted to the polymer concentration of 1.0 mg/mL. To measure the loading efficiency and capacity, 5 µL H2O2 (30 wt%) was added into 1.0 mL of the DOX-encapsulated nanoparticle solution (NPs@DOX). After stirring for 24 h, the nanoparticles were dissociated completely. The solution was put into a dialysis membrane (MWCO: 10 kDa) and dialyzed in 50 mM phosphate buffer (pH 7.4) at 37 oC for 24 h in the dark. The dialysis medium was collected and the DOX content in this medium was measured on a Shimadzu 2450 UV-Vis spectrometer at 485 nm. The calibration curve of DOX was obtained from a series of solutions containing different concentrations of DOX in PB (pH 7.4, 50 mM). The measurements were carried out in triplicate under dark conditions. Loading efficiency and loading capacity were defined as DOX in nanoparticle/DOX in feed (wt %) and DOX in nanoparticle/copolymer nanoparticle (wt %), respectively. In vitro DOX release from the nanoparticles was conducted by a dialysis ptotocol. 1.0 mL of NPs@DOX solution was transferred into a dialysis tubing (MWCO: 10 KDa) quickly. The tubing was immersed in 10 mL phosphate buffer (pH 7.4, 50 mM) with H2O2 (1.25 mM) under stirring at 37 oC in the dark. At different time points, 1.0 mL of the dialysis solution was taken out for UV-Vis measurement (485 nm) and 1.0 mL fresh medium was replenished. Meanwhile, the solution inside the tubing was withdrawn for recording the fluorescence spectra. After each measurement, the solution was added back into the tubing. All the measurements were conducted thrice in the dark. The block copolymer nanoparticles or their oxidation degraded polymeric products (PEG-b-PAA) could not leak out from the dialysis tubing (MWCO: 10KDa) based on the alternative control experiments. Intracellular Trafficking Observed by CLSM. A549 cells (5 × 104 cells/mL) were cultured for 12 h in a 20 mm confocal microscope dish with DMEM medium. The conditions of humidified atmosphere, 5% CO2, 37 °C were used for cell culture and attachment. The medium was supplemented with an antibiotic-antimycotic mixture and 10% fetal bovine serum. Subsequently, to each well 10 µL of MF1 nanoparticle solution with a polymer 7

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 8 of 27

concentration of 5 mg/mL was added. The final concentration of the nanoparticles in the cell culture medium was 0.5 mg/mL. The cells were cultured for another 1, 3, 6, 12, and 24 h, respectively, and then washed with PBS thrice. For colocalization measurements, the cells were co-cultured with Lyso-Tracker Red (100 nM) for additional 30 min at 37 oC prior to the confocal laser scanning microscope (CLSM) observations. After discarding the medium and washing with PBS thrice, cells were imaged on a Nikon AIR PLUS confocal laser scanning microscope with a water 60× objective lens. For the Z-stacked images, a series of optical sections was Z-stacked by moving the focal plane of the instrument step-by-step through the depth of the cell. ROS-Triggered Colorimetric Change in Cell. The A549 cells were seeded and grown in a 20 mm confocal microscope dish (5 × 104 cells/mL) following the aforementioned procedure, and incubated with 0.5 mg/mL MF1 nanoparticle solution for 12 h. After washing with PBS thrice, 200 µM H2O2 in the cell culture medium was added. The cells were incubated for additional 6, 12, and 24 h, respectively, and washed with PBS three times. Lysosome was counterstained with Lyso-Tracker Red for 30 min at 37 oC prior to the CLSM observations using the same microscope mentioned above. For the intracellular colorimetric change experiments triggered by different stimuli, A549 cells were treated with 0.5 mg/mL of MF1 nanoparticle for 12 h and washed with PBS thrice. Then, 200 µM H2O2 or 2 µg/mL LPS or a mixture of 200 µM H2O2 and 1000 U/mL catalase was added to the specific dish. All the cells were cultured for another 24 h and washed with PBS thrice. The cells were fixed with paraformaldehyde and counterstained with the nucleic dye (DRAQ-5) for 15 min at 37 oC. After replacement of the cell culture medium, fluorescent images were taken with the aforementioned microscope with an water 60× objective lens. ROS-Activated Intracellular DOX Release. For the detection of intracellular DOX release triggered by H2O2, A549 cells were treated with 5 µg /mL free DOX and 0.5 mg/mL of NPs@DOX (1.2 wt% DOX loaded MF1 nanoparticle) for 12 h, then 200 µM H2O2 was addied to some specific dishes. The cells were incubated for another 12 h to ensure the released DOX to migrate into the cell nuclei. Afterwards, the cells were imaged immediately by the aforementioned microscope with an water 60× objective lens. 8

ACS Paragon Plus Environment

Page 9 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

In Vitro Cytotoxicity Assay. To assess the cytotoxicity of copolymer nanoparticles and their oxidative degradation products in A549, Hela, and 293T cells, CCK-8 assays were applied. Cells were seeded in the 96-well plates (6000 cells per well) and cultured in a 5% CO2 humidified atmosphere at 37 oC for 12 h. Each sample was added to the wells at concentrations ranging from 10 to 500 µg/mL and the cells were cultured for 12 h. Afterwards, the cells were washed with PBS thrice and the wells were replenished with DMEM culture medium. To some specific wells 200 µM H2O2 or 2 µg/mL LPS was added. The cells were incubated for additional 24 h, washed with PBS thrice, and subjected to CCK-8 analysis. The absorbance at 450 nm of the solution in each well was determined on a Tecan infinite M200 microplate reader. Cell viability (%) was denoted as (Asample/Acontrol) × 100. Measurements were carried out in triplicate. The sample for the cytotoxicity assay of the degradation products of MF1 nanoparticle was prepared by the following procedure. 5 mg MF1 nanoparticle was dispersed in 1 mL phosphate buffer solution (pH 7.4, 10 mM), to which 15 µL H2O2 (10 M) was added. After 12 h at 25 oC, the solution was mixed with MnO2 and incubated for additional 12 h. Then, MnO2 was removed by centrifugation, the upper aqueous solution was used for cytotoxicity assay. The empty MF1 nanoparticle and the DOX-loaded MF1 nanoparticle were also dispersed in 10 mM PB solution at 37 oC prior to the cytotoxicity assay experiments. The negative control was mPEG113 while DOX dissolved in phosphate buffer solution (pH 7.4, 10 mM) was used as the positive control. RESULTS AND DISCUSSION Synthesis of Block Copolymers and Formation of Their Nanoparticles. The non-fluorescent monomer (M) was synthesized following the published method.59 The ROS-activatable ratiometric fluorescent monomer (F) was prepared following the synthetic route as shown in Scheme S1. Four amphiphilic block copolymers (MF1-MF4) were synthesized by ATR copolymerization of M and F using mPEG113-Br as a macroinitiator (Table 1, Figure S4 and S5). They are different in the hydrophobic block length but with the similar contents of the fluorescent unit (~2% in molar percentage). Each of the copolymers shows a unimodal distribution of the GPC traces. 9

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 10 of 27

All the block copolymers could not dissolve directly in water, however, they formed spherical nanoparticles in aqueous PB solution (pH 7.4, 50 mM) by the solvent evaporation method. According to the results of LLS and TEM measurements, the dimeters of the nanoparticles are in the range 40-120 nm. The nanoparticle sizes increase with increasing the hydrophobic block length from MF1 to MF4 (Table 1, Figure S6 and S7). In the 1H NMR spectra of the nanoparticles in deuterated PB solution, we could see the proton signals of PEG but not those of the hydrophobic segment, which may indicate a micelle-like morphology of the nanoparticles (Figure S8). Table 1. Characterization of the block copolymers and their nanoparticles

MF1 MF2 MF3 MF4

a

M/F b

DP b

Mn b

Mn c

Ðc

Rh d (nm)

Rg d (nm)

Rg/Rh d

97.8/2.2 97.7/2.3 97.9/2.1 97.6/2.4

36 51 72 82

15600 20100 26100 29300

24500 30600 38900 43400

1.31 1.26 1.23 1.25

24 35 45 58

19 29 39 53

0.78 0.82 0.88 0.91

a

Polymerization condition: [M]:[F] = 99:1; [mPEG113-Br]:[CuBr]:[M6TREN] = 1:2:2; ([M]+[F])/ [mPEG113-Br] = 50, 105, 135, and 165, respectively; 60 oC. b Molar ratio of M to F in copolymers MF1-MF4, degree of polymerization and number-averaged molecular weight determined by 1H NMR. c Measured by GPC using polystyrenes as the standards and THF as the eluent. d Measured in 50 mM PB (pH 7.4) with a polymer concentration of 0.1 mg/mL at 37 oC by LLS.

H2O2-Triggered Dissociation of the Copolymer Nanoparticles. As reported previously, the pendent phenylboronic ester/acid units enable the MF block copolymers sensitive to H2O2. 59

In the absence of H2O2, the copolymer nanoparticles were stable against hydrolysis of the

ester bonds in PB solution (pH 7.4), which was supported by the 1H NMR and LLS measurements. Neither 1H NMR spectrum nor the scattered light intensity of the nanoparticle solutions changed in 48 h at 37 oC. Moreover, Rg and Rh of the nanoparticles did not show obvious change in the same period (Figure S8-S10). In contrast, upon exposure to H2O2, the phenylboronic acid/ester could be oxidized to its phenol derivative which further decomposed into the carboxylic acid and p-quinone methide (QM) via 1,6-elimination (Scheme S2). The highly reactive QM was transformed quickly to 4-hydroxymethylphenol. The H2O2-triggered decomposition of the pendent phenylboronic ester caused a significant increase in water solubility of the block copolymers, leading to a gradual dissociation of nanoparticles. 10

ACS Paragon Plus Environment

Page 11 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Meanwhile, the amine-caged chromophore was also oxidatively de-protected through the similar mechanism, which would induce a colorimetric switch from blue emission to green emission.64

Figure 1. (A) Time-dependent 1H NMR spectra of MF3 nanoparticle (5 mg/mL) in deuterated PB solution (pH 7.4, 50 mM) with 62.5 mM of H2O2 at 37 oC. (B, C) Oxidative decomposition kinetic curves of (B) MF1-MF3 nanoparticles (5 mg/mL) with (solid symbols) or without (empty symbols) 62.5 mM of H2O2 and (C) MF3 nanoparticle (5 mg/mL) at different concentrations of H2O2, 37 oC.

The aforementioned oxidative dissociation process of the MF nanoparticles was clearly demonstrated by 1H NMR. We could see the gradual increase in peak intensity of the signals that were assigned to 4-hydroxymethylphenol (plus its phosphate precursor, e’+e”) and poly(acrylic acid) backbone (a’, b’) (Figure 1A). By comparing the peak intensities of 11

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 12 of 27

protons e’+e’’ with that of the internal standard (DSS), the kinetic curves of oxidative decomposition of the pendent phenylboronic ester were obtained (Figure 1B and 1C). It was found that the decomposition rate followed an order of MF1 > MF2 > MF3, decreasing with the increase in the hydrophobic block length. Furthermore, the decomposition rate was accelerated with increasing H2O2 concentration, but not following a linear relationship, which implied that the decomposition kinetics was greatly influenced by the diffusion process of H2O2 and H2O into the copolymer nanoparticles.59 We further studied the oxidative dissociation profiles of the MF nanoparticles by LLS. The changes in excess scattered light intensity, Rh, Rg and Rg/Rh as a function of time are summarized in Figure 2. In the presence of 1.25 mM H2O2, all the four copolymer nanoparticles underwent H2O2-triggered dissociation as proved by the gradually decreased light intensity. The dissociation rate of the nanoparticles decreased from MF1 to MF4, which is well consistent with the 1H NMR results. The dissociation process could be divided into three stages. Take MF2 nanoparticle as an example. At the initial stage (0-710 min), the scattered light intensity decreased drastically whereas Rh, Rg and Rg/Rh remained constant. In this period, the decomposition of the phenylboronic ester/acid groups initiated by H2O2 and the consequential peeling off of the degradation products occurred mainly at the outer layer of the nanoparticle, but accompanied by a slight swelling of the inner part of the nanoparticle. In the second stage, from 710 min to ~1100 min, more H2O2 and H2O diffused into the inner part and further decomposition of the phenylboronic ester/acid groups occurred, which resulted in the significant swelling of the nanoparticle. This was supported by the increase in Rh, Rg, as well as Rg/Rh ratio. At the same time, the scattered light intensity continued to decrease to almost the lowest level. In the final stage, the swollen nanoparticles decomposed further and dissociated into free single polymer chains, probably with the coexistence of some loose associates of the water soluble polymers (Figure S11).65-66 Moreover, the oxidative dissociation of MF3 nanoparticle was carried by triggering of H2O2 with different concentrations, following the similar mechanism (Figure S12 and S13). Again, the LLS results demonstrated a positive relationship between the dissociation rate and H2O2 concentration. 12

ACS Paragon Plus Environment

(B)

100

60 40

250

MF1 MF2 MF3 MF4

200

MF1 MF2 MF3 MF4

80

Rh (nm)

(A)

150 100

20 50

0 0

800

1600

2400

0

3200

0

Time (min)

600

1200

1800

2400

3000

Time (min)

(D)

(C) 180

MF1 MF2 MF3 MF4

120

MF1 MF2 MF3 MF4

1.4

Rg/Rh

150

Rg (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

% Initial intensity (a. u.)

Page 13 of 27

90

1.2 1.0

60 0.8

30 0 0

600

1200

1800

Time (min)

2400

3000

0

600

1200

1800

2400

3000

Time (min)

Figure 2. Time dependence of (A) excess scattered light intensity, (B) Rh, (C) Rg, and (D) Rg/Rh of MF1-MF4 nanoparticles (0.1 mg/mL) in PB solution (50 mM, pH 7.4) with 1.25 mM H2O2 at 37 oC.

H2O2-Induced Ratiometric Colorimetric Change. Both fluorescence and UV-Vis absorption spectra were used to clarify the H2O2-triggered colorimetric changes of the MF nanoparticles. In the absence of H2O2, both UV-Vis absorption and fluoresence spectra of the nanoparticle solutions (in PB, pH 7.4) remained unchanged in 24 h at 37 oC, indicating the stability of the chromophore in nanoparticles against hydrolysis. However, significant colorimetric changes of both fluorescence and absorption were observed upon the addition of H2O2 (Figure 3A and 3B). The initial emission maximum (508 nm, blue) and absorption band at 370 nm red-shifted to 535 nm (green) and 445 nm, respectively, upon incubation with 1.25 mM H2O2 for 24 h, which is attributed to the H2O2-triggered transformation of the carbamate group to the more electron-donating amino group of the choromophore.64 The fluorescence ratio (F535/F495) or absorption ratio (A440/A370) of the MF3 nanoparticle solution at various concentrations of H2O2 was plotted against time (Figure 3C and 3D). It was found that both ratios increased initially and then leveled off after reaching some values, which were dependent on the concentrations of H2O2. A higher concentration of H2O2 resulted in a faster increase of the ratio. By plotting the final fluorescence ratio or absorption ratio (after incubation for 48 h) against H2O2 concentration (Figure 3E and 3F), we could see that both ratios increased almost linearly with H2O2 concentration until they reached a saturation point 13

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 14 of 27

at 0.25 mM of H2O2, wherein all of the pnenylboronic ester/acid groups were oxidized in the solution.

Figure 3. (A, B) Time-dependent changes in (A) fluorescence and (B) absorption spectra of MF2 nanoparticle (0.1 mg/mL) in PB solution (50 mM, pH 7.4) in the presence of 1.25 mM H2O2 at 37 oC. (C, D) Plots of (C) F535/F495 (λex =430 nm) and (D) A440/A370 of MF3 nanoparticle vs time with different concentrations of H2O2. (E, F) The final ratio of (E) F535 /F495 and (F) A440/A370 of MF3 nanoparticle as a function of H2O2 concentration. The data were obtained after incubation for 48 h. (G, H) Plots of (G) F535/F495 and (H) A440/A370 of MF1-MF4 nanoparticles vs time with (solid symbols) or without (empty symbols) H2O2 (1.25 mM).

We also monitored the colorametric changes of the MF1-MF4 nanoparticle solutions at a fixed concentration of H2O2 (1.25 mM) against time (Figure 3G and 3H). The changing rate of the ratio (fluorescence or absorption) decreased with increasing the hydrophobic block 14

ACS Paragon Plus Environment

Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

length of the copolymers, being well consistent with the results of 1H NMR and LLS measurements. This may imply that the chromophore units were distributed uniformly in the nanoparticles. DOX Loading and H2O2-Triggered Release. The hydrophobic anticancer drug DOX could be loaded into MF copolymer nanoparticles by the solvent evaporation approach using acetone as organic solvent (Table 2). From MF1 to MF3, the loading efficacy increased slightly and the size of the DOX-loaded nanoparticles (NPs@DOX) increased from ~120 nm for MF1 to ~200 nm for MF3 (Figure S15). Moreover, the DOX-loaded nanoparticle was larger than the empty nanoparticle formed by the same copolymer (Table 2), which could be attributed the hydrophobicity of the deprotonated DOX. It should be noticed that the fluorescence of the chromophore was quenched significantly by the loaded DOX (Figure S16). Table 2. Characterization results of NPs@DOX and the empty nanoparticles

NPs@DOX

Empty nanoparticle

LC a (wt%)

LE b (wt%)

Rh c (nm)

Rg c (nm)

Rg/Rh

Rh c (nm)

Rg c (nm)

Rg/Rh

MF1

6.4±0.14

43±1.0

59

51

0.86

39

30

0.78

MF2 MF3

6.7±0.38 7.0±0.20

45±2.5 47±1.3

76 88

68 81

0.89 0.91

49 61

41 54

0.83 0.89

a

Loading capacity (LC) defined as DOX in nanoparticle/polymer nanoparticle (x 100%). b Loading efficiency (LE) defined as DOX in nanoparticle/DOX in feed (x 100%). DOX/polymer in feed: 15 wt%. c Measured by LLS in 50 mM PB (pH 7.4) at 37 oC, with a polymer concentration of 1.0 mg/mL.

The in vitro colorimetric changes of NPs@DOX and the DOX release profiles were investigated by the dialysis method with or without H2O2, and the results are summarized in Figure 4. In the absence of H2O2, the fluorescent spectra of NPs@DOX did not change with negligible fluorescence at the wavelength of ~500 nm in 48 h, confirming the stability of the pendent naphthalimide chromophore again (Figure S16). The cumulative DOX release was less than 30% in 48 h (Figure 4C). In contrast, upon triggering of 1.25 mM H2O2, the green fluorescence at ~540 nm and also the ratio of F535/F475 increased gradually (Figure 4A, 4B). At the same time, the DOX release was accelerated greatly, with the rates depending on the hydrophobic block length. Nearly 100% DOX was released in 24 h from MF1 nanoparticle and in 43 h from MF2 and MF3 nanoparticles, which are consistent with the oxidative 15

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

dissociation profiles of the MF nanoparticles. To assess whether in vitro DOX release could be monitored by the colorimetric change, the cumulative release was plotted versus F535/F475, showing approximately a linear relationship (Figure 4D). This indicates that the ratiometric fluorescence of NPs@DOX can be applied for in situ reporting the DOX release profile.

Figure 4. (A) Time-dependent changes in fluorescence spectra of the DOX-loaded MF2 nanoparticle (1.0 mg/mL) in PB solution (50 mM, pH 7.4) in the presence of 1.25 mM H2O2 at 37 oC. (B) Changes in F535/F475 (λex =430 nm) of NPs@DOX (1.0 mg/mL) as a function of time with (solid symbols) or without (empty symbols) 1.25 mM H2O2. (C) Cumulative release of DOX from the MF nanoparticles in PB solution (pH 7.4, 50 mM) in the absence (empty symbols) or presence (solid symbols) of 1.25 mM H2O2 at 37 oC. (D) The plots of cumulative DOX release vs F535/F475.

Intracellular Trafficking of MF1 Nanoparticle. To clarify the cellular internalization process and the intracellular trafficking of MF1 nanoparticle, colocalization experiments were conducted in A549 cells using CLSM (Figure 5). The MF1 nanoparticle and Lyso-Tracker Red are represented by blue color and orange color, respectively. It was found that negligible amount of MF1 nanoparticles was internalized by the cells within 1 h. The obvious cellular uptake of the nanoparticles was observed only after incubation for 6 h. At this time point the majority of the nanoparticles were colocalized with lysosome, which indicated that the nanoparticles entered into the cells via an endocytosis pathway.57 After 12 h, some nanoparticles escaped from endosome or lysosome. However, significant amount of the nanoparticles were still trapped in the lysosomes even after 24 h. The slow cellular 16

ACS Paragon Plus Environment

Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

internalization rate and the low efficacy of endosome/lysosome escaping of the MF1 nanoparticles could be ascribed to the shielding effect of the PEG shell.67-68 It was noticed that, in the whole process, we did not observe green fluorescence of the MF1 nanoparticle, demonstrating that the ROS level in the pristine A549 cells was not high enough to activate efficiently the colorimetric change of the chromophore inside the nanoparticle.

Figure 5. (A) CLSM images of A549 cells after incubation with MF1 nanoparticle for different times. (B) The Z-stacked cellular fluorescence images of MF1 nanoparticle-treated A549 cells (12 h). The Z-stacked images clearly reveal that the nanoparticles are localized within lysosome with a dot-like shape. Blue: nanoparticle (Ex: 405 nm, Em: 450-500 nm); green: nanoparticle (Ex: 488 nm, Em: 550-570 nm); orange: Lyso-Tracker Red (Ex: 561 nm, Em: 570-620 nm).

ROS-Triggered Intracellular Colorimetric Change. Since the endogenous ROS of A549 cells was too low to trigger the colorimetric change, the exogenous H2O2 was added to elevate the intracellular ROS level. As shown in Figure S18, after treating the cells with H2O2, the intensity of blue emission decreased while that of green emission increased gradually with time. The obvious overlapping of green color and orange color after 24 h indicates the colocalization of some oxidatively activated nanoparticles with lysosomes. Moreover, the 17

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 18 of 27

changing magnitude in the intensity ratio (green/blue) was much greater than that of the intensity itself. This is helpful to enhance the detection sensitivity of intracellular ROS level. To further elucidate the function of exogenous stimuli on colorimetric activation of the chromophore, the MF1 nanoparticle-internalized A549 cells were treated individually with H2O2 or lipopolysaccharide (LPS) or a mixture of H2O2 and catalase for 24 h, and counterstained with the nuclear dye DRAQ-5 (Figure 6). It is clear that both the exogenous H2O2 and LPS could stimulate colorimetric change from blue to green, and in contrast, the untreated cells or the cells treated with H2O2/catalase retained blue color. Quantitative analyses revealed that the emission intensity ratio of green to blue increased from 1.06 (without stimulus) to 3.06 (stimulated by H2O2) or 2.61 (stimulated by LPS). On the other hand, catalase that catalyzes the decomposition of H2O2 caused a reduction of the intensity ratio from 3.06 to 1.17. Again, the ratiometric approach was proved to be more sensitive to monitor the intracellular ROS (Figure 6B-6D). Furthermore, the colocalization experimental results indicated that neither pristine MF1 nanoparticles nor their polymeric degradation products entered the nuclei (stained in red), they were distributed mainly in the cytoplasm.

Figure 6. (A) CLSM images of MF1 nanoparticle-treated A549 cells upon incubation with H2O2 or LPS or a mixture of H2O2 and catalase for 24 h. The cells were stained with DRAQ-5. Blue: nanoparticle (Ex: 405 nm, Em: 450-500 nm); green: nanoparticle after stimulation (Ex: 488 nm, Em: 550-570 nm); red: DRAQ-5 18

ACS Paragon Plus Environment

Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

(Ex: 640 nm, Em: 663-738 nm). (B, C) Fluorescence intensity and (D) intensity ratio (green to blue) of the cells. Results are presented as the mean ± SD in triplicate. Asterisk (*) denoted statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

ROS-Triggered Intracellular DOX Release. As mentioned above, the H2O2-triggered in vitro DOX release was positively correlated with the ratiometric colorimetric change. Herein, we evaluated the intracellular DOX release profile from MF1 nanoparticles qualitatively by CLSM (Figure 7). DOX was internalized by A549 cells and entered into the nuclei quickly (within 1 h). In contrast, the NPs@DOX was internalized with a much slower rate, being similar to the empty nanoparticles (Figure 5). Without exogenous H2O2, the intracellular blue color was very weak due to fluorescence quenching of the naphthalimide chromophore by the loaded DOX. The merged images demonstrated the colocalization of DOX and the chromophore (pink color), and little amount of DOX in the nuclei (24 h). It indicated that the intracellular DOX release was very slow under the endogenous ROS triggering. Upon treating with exogenous H2O2 for 12 h, the green color increased significantly because of the H2O2-induced colorimetric change and the concurrent dissociation of the nanoparticles, which resulted in an obvious acceleration of DOX release. We further stained the nuclei with DRAQ-5. The obvious overlapping of red color (DOX) and purple color (DRAQ-5) indicates that almost all DOX entered the nuclei while the polymeric degradation products were located in cytoplasm (Figure S19). On the whole, the ROS-triggered intracellular drug release can be reported well by the concurrent ratiometric colorimetric change based on the MF nanoparticles.

Figure 7. (A, B) CLSM images of A549 cells incubated with (A) free DOX and (B) DOX-loaded (1.2 wt%) MF1 nanoparticle for 12 h. (C) CLSM images of A549 cells treated with the DOX-loaded MF1 19

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 20 of 27

nanoparticles for 12 h and then incubated with H2O2 for additional 12 h in fresh medium. Blue: nanoparticle (Ex: 405 nm, Em: 450-500 nm); green: nanoparticle after stimulation (Ex: 488 nm, Em: 550-570 nm); red: DOX (Ex: 561 nm, Em: 610-640 nm). The concentration of DOX in the culture medium is 6 µg/mL.

In Vitro Cytotoxicity. The in vitro cytotoxicity assays of MF1 nanoparticles with or without DOX were carried out in A549 cells under varied conditions in order to evaluate the cytocompatibilty of the empty nanoparticles and their degradation products, and to see how the exogenous stimuli influence the cell killing capability of the NPs@DOX (Figure 8). MF1 nanoparticles and its oxidative degradation products did not display obvious cytotoxicity even at a concentration up to 500 µg/mL, revealing the good cytocompatibility. However, MF1 NPs@DOX showed the concentration dependent cytotoxicity to A549 cells with IC50 > 32 µg/mL DOX. Upon treating the cells with H2O2 or LPS, the cytotoxicity of the NPs@DOX was enhanced obviously. The IC50 was reduced to 22 µg/mL for H2O2 and 26 µg/mL for LPS, respectively. Under the same condition, the incubation with H2O2 or LPS showed a negligible effect on cytotoxicity of the empty MF1 nanoparticle. Therefore, the exogenous stimuli-enhanced cytotoxicity of the NPs@DOX is most probably due to the ROS-induced enhancement of the intracellular DOX release, which is agreed well with the CLSM results as shown in Figure 7. The similar phenomenon was observed for the cytotoxicity assays in Hela cell and in the non-cancerous 293T cell (Figure S20). Since the sensitivity of the MF nanoparticles was not high enough to respond, with a moderate rate, to the intracellular H2O2 level even for the cancerous cells, the NPs@DOX may not be an ideal nanomedicine for the targeted tumor therapy. It is well known that there are various ROS-generating agents that could temporally enhance the ROS levels within cells or in tumor tissues.36, 69-70 By combining the MF nanoparticles and a suitable ROS-generating agent, we may expect a more practical nanoparticulate delivery system for the therapies of tumors or inflammatory diseases.

20

ACS Paragon Plus Environment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Cell Viability (%)

Page 21 of 27

140

PEG NPs NPs-degrade DOX NPs+H2O2 NPs+LPS NPs@DOX

120

NPs@DOX+H2O2

NPs@DOX+LPS

**** **** ***

100 80

**** ***

60 40 20 0

10

50

100

150

200

500

Concentration of Polymers (µg/mL)

Figure 8. Cell viability of A549 cells measured by CCK-8 assay at 37 oC. The DOX-loading content in MF1 NPs@DOX is 6.4 wt%. The concentrations of DOX are 0.64, 3.2, 6.4, 9.6, 12.8, 32.0 µg/mL, respectively. Asterisk (*) denotes statistical significance: *** P < 0.001, **** P < 0.0001.

CONCLUSION We have demonstrated the novel theranostic amphiphilic block copolymers consisting of a PEG segment and an oxidation-responsive hydrophobic block containing the pendent phenylboronic pinacol esters and ~2% naphthalimide fluorophore that shows the ROS-activatable colorimetric change. The copolymers could form stable micelle-like nanoparticles that are capable of loading DOX. ROS could trigger the degradation of nanoparticles, enhance the payload release, and concurrently induce a change in fluorescence from blue to green. By using the ratiometric fluorescent approach, we could monitor in vitro or intracellular DOX release, and of importance, the fluorescence ratiometric change is approximately linearly correlated with the in vitro payload release. Since the naphthalimide dye was covalently conjugated onto the polymer, we could trace fluorescently the polymeric carriers within cells and found that they did not enter the nuclei, regardless prior to and after oxidative degradation. Both the copolymer nanoparticles and their degradation products are cytocompatible. On the other hand, the DOX-loaded nanoparticles exhibited concentration dependent cytotoxicity, which was further enhanced by ROS stimulation.

ASSOCIATED CONTENT Supporting Information Additional 1H NMR spectra, GPC curves, LLS results, TEM images, and more results of 21

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 22 of 27

cytotoxicity assay and CLSM observation. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Phone: +86-10-62757155; E-mail : [email protected] (F. Du) * Phone: +86-10-62755543; E-mail : [email protected] (Z. Li) ORCID Fu-Sheng Du: 0000-0003-3174-6107 Zi-Chen Li: 0000-0002-0746-9050

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21474001 and 21534001) and National Key Research and Development Program of China (No. 2016YFA0201400). We thank Prof. Wenbin Zhang (CCME of Peking University), Dr. Hao Wang and Dr. Zengying Qiao (CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety) for their help in cytotoxicity assay and CLSM experiments. REFERENCES 1.

Chen, H.; Zhang, W.; Zhu, G.; Xie, J.; Chen, X. Rethinking Cancer Nanotheranostics. Nat. Rev. Mater.

2017, 2, 17024. 2.

Janib, S. M.; Moses, A. S.; MacKay, J. A. Imaging and Drug Delivery Using Theranostic Nanoparticles.

Adv. Drug Delivery Rev. 2010, 62, 1052-1063. 3.

Jo, S. D.; Ku, S. H.; Won, Y.-Y.; Kim, S. H.; Kwon, I. C. Targeted Nanotheranostics for Future Personalized

Medicine: Recent Progress in Cancer Therapy. Theranostics 2016, 6, 1362-1377. 4.

Kievit, F. M.; Zhang, M. Cancer Nanotheranostics: Improving Imaging and Therapy by Targeted Delivery

Across Biological Barriers. Adv. Mater. 2011, 23, H217-H247. 5.

Muthu, M. S.; Leong, D. T.; Mei, L.; Feng, S.-S. Nanotheranostics-Application and Further Development

of Nanomedicine Strategies for Advanced Theranostics. Theranostics 2014, 4, 660-677. 6.

Zhao, T.; Huang, G.; Li, Y.; Yang, S.; Ramezani, S.; Lin, Z.; Wang, Y.; Ma, X.; Zeng, Z.; Luo, M.; de Boer, 22

ACS Paragon Plus Environment

Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

E.; Xie, X.-J.; Thibodeaux, J.; Brekken, R. A.; Sun, X.; Sumer, B. D.; Gao, J. A Transistor-Like pH Nanoprobe for Tumour Detection and Image-Guided Surgery. Nat. Biomed. Eng. 2016, 1. 0006. 7.

Kunjachan, S.; Ehling, J.; Storm, G.; Kiessling, F.; Lammers, T. Noninvasive Imaging of Nanomedicines

and Nanotheranostics: Principles, Progress, and Prospects. Chem. Rev. 2015, 115, 10907-10937. 8.

Chen, G.; Roy, I.; Yang, C.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-Based

Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826-2885. 9.

Etrych, T.; Lucas, H.; Janoušková, O.; Chytil, P.; Mueller, T.; Mäder, K. Fluorescence Optical Imaging in

Anticancer Drug Delivery. J. Controlled Release 2016, 226, 168-181. 10. Zhao, J.; Song, S.; Zhong, M.; Li, C. Dual-Modal Tumor Imaging via Long-Circulating Biodegradable Core-Cross-Linked Polymeric Micelles. ACS Macro Lett. 2012, 1, 150-153. 11. Lin, W.; Li, Y.; Zhang, W.; Liu, S.; Xie, Z.; Jing, X. Near-Infrared Polymeric Nanoparticles with High Content of Cyanine for Bimodal Imaging and Photothermal Therapy. ACS Appl. Mater. Interfaces 2016, 8, 24426-24432. 12. Elsabahy, M.; Heo, G. S.; Lim, S.-M.; Sun, G.; Wooley, K. L. Polymeric Nanostructures for Imaging and Therapy. Chem. Rev. 2015, 115, 10967-11011. 13. Thapaliya, E. R.; Zhang, Y.; Dhakal, P.; Brown, A. S.; Wilson, J. N.; Collins, K. M.; Raymo, F. M. Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores. Bioconjugate Chem. 2017, 28, 1519-1528. 14. Wang, Y.; Zhou, K.; Huang, G.; Hensley, C.; Huang, X.; Ma, X.; Zhao, T.; Sumer, B. D.; DeBerardinis, R. J.; Gao, J. A Nanoparticle-Based Strategy for the Imaging of a Broad Range of Tumours by Nonlinear Amplification of Microenvironment Signals. Nat. Mater. 2014, 13, 204-212. 15. Viger, M. L.; Collet, G.; Lux, J.; Nguyen Huu, V. A.; Guma, M.; Foucault-Collet, A.; Olejniczak, J.; Joshi-Barr, S.; Firestein, G. S.; Almutairi, A. Distinct ON/OFF Fluorescence Signals from Dual-Responsive Activatable Nanoprobes Allows Detection of Inflammation with Improved Contrast. Biomaterials 2017, 133, 119-131. 16. Hu, X.; Li, Y.; Liu, T.; Zhang, G.; Liu, S. Intracellular Cascade FRET for Temperature Imaging of Living Cells with Polymeric Ratiometric Fluorescent Thermometers. ACS Appl. Mater. Interfaces 2015, 7, 15551-15560. 17. Li, C.; Liu, S. Polymeric Assemblies and Nanoparticles with Stimuli-Responsive Fluorescence Emission Characteristics. Chem. Commun. 2012, 48, 3262-3278. 18. Luby, B. M.; Charron, D. M.; MacLaughlin, C. M.; Zheng, G. Activatable Fluorescence: From Small Molecule to Nanoparticle. Adv. Drug Delivery Rev. 2017, 113, 97-121. 19. Wang, H.; Di, J.; Sun, Y.; Fu, J.; Wei, Z.; Matsui, H.; Alonso, A. C.; Zhou, S. Biocompatible PEG-Chitosan@Carbon Dots Hybrid Nanogels for Two-Photon Fluorescence Imaging, Near-Infrared Light/pH Dual-Responsive Drug Carrier, and Synergistic Therapy. Adv. Funct. Mater. 2015, 25, 5537-5547. 20. Zhu, C.; Yang, Q.; Liu, L.; Wang, S. Rapid, Simple, and High-Throughput Antimicrobial Susceptibility Testing and Antibiotics Screening. Angew. Chem. Int. Ed. 2011, 50, 9607-9610. 21. Song, Z.; Mao, D.; Sung, S. H. P.; Kwok, R. T. K.; Lam, J. W. Y.; Kong, D.; Ding, D.; Tang, B. Z. Activatable Fluorescent Nanoprobe with Aggregation-Induced Emission Characteristics for Selective In Vivo Imaging of Elevated Peroxynitrite Generation. Adv. Mater. 2016, 28, 7249-7256. 22. Niethammer, P.; Grabher, C.; Look, A. T.; Mitchison, T. J. A Tissue-Scale Gradient of Hydrogen Peroxide Mediates Rapid Wound Detection in Zebrafish. Nature 2009, 459, 996-999. 23. Winterbourn, C. C. Reconciling the Chemistry and Biology of Reactive Oxygen Species. Nat. Chem. Biol. 2008, 4, 278-286. 23

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 24 of 27

24. Schieber, M.; Chandel, N. S. ROS Function in Redox Signaling and Oxidative Stress. Curr. Biol. 2014, 24, R453-R462. 25. Lisanti, M. P.; Martinez-Outschoorn, U. E.; Lin, Z.; Pavlides, S.; Whitaker-Menezes, D.; Pestell, R. G.; Howell, A.; Sotgia, F. Hydrogen Peroxide Fuels Aging, Inflammation, Cancer Metabolism and Metastasis. The Seed and Soil Also Needs "Fertilizer". Cell Cycle 2011, 10, 2440-2449. 26. Gupta, S. C.; Hevia, D.; Patchva, S.; Park, B.; Koh, W.; Aggarwal, B. B. Upsides and Downsides of Reactive Oxygen Species for Cancer: The Roles of Reactive Oxygen Species in Tumorigenesis, Prevention, and Therapy. Antioxid. Redox Sign. 2012, 16, 1295-1322. 27. Zhou, R.; Yazdi, A. S.; Menu, P.; Tschopp, J. A Role for Mitochondria in NLRP3 Inflammasome Activation. Nature 2011, 469, 221-225. 28. Li, Y.; Hu, K.; Yu, Y.; Rotenberg, S. A.; Amatore, C.; Mirkin, M. V. Direct Electrochemical Measurements of Reactive Oxygen and Nitrogen Species in Nontransformed and Metastatic Human Breast Cells. J. Am. Chem. Soc. 2017, 139, 13055-13062. 29. Van de Bittner, G. C.; Dubikovskaya, E. A.; Bertozzi, C. R.; Chang, C. J. In Vivo Imaging of Hydrogen Peroxide Production in a Murine Tumor Model with a Chemoselective Bioluminescent Reporter. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 21316-21321. 30. Chan, H. P.; Lewis, C.; Thomas, P. S. Exhaled Breath Analysis: Novel Approach for Early Detection of Lung Cancer. Lung Cancer 2009, 63, 164-168. 31. Aran, K.; Parades, J.; Rafi, M.; Yau, J. F.; Acharya, A. P.; Zibinsky, M.; Liepmann, D.; Murthy, N. Stimuli-Responsive Electrodes Detect Oxidative Stress and Liver Injury. Adv. Mater. 2015, 27, 1433-1436. 32. Dixon, S. J.; Stockwell, B. R. The Role of Iron and Reactive Oxygen Species in Cell Death. Nat. Chem. Biol. 2014, 10, 9-17. 33. Pelicano, H.; Carney, D.; Huang, P. ROS Stress in Cancer Cells and Therapeutic Implications. Drug Resistance Updates 2004, 7, 97-110. 34. Gorrini, C.; Harris, I. S.; Mak, T. W. Modulation of Oxidative Stress as An Anticancer Strategy. Nat. Rev. Drug Discov. 2013, 12, 931-947. 35. Dharmaraja, A. T. Role of Reactive Oxygen Species (ROS) in Therapeutics and Drug Resistance in Cancer and Bacteria. J. Med. Chem. 2017, 60, 3221-3240. 36. Yin, W.; Li, J.; Ke, W.; Zha, Z.; Ge, Z. Integrated Nanoparticles To Synergistically Elevate Tumor Oxidative Stress and Suppress Antioxidative Capability for Amplified Oxidation Therapy. ACS Appl. Mater. Interfaces 2017, 9, 29538-29546. 37. Kwon, J.; Kim, J.; Park, S.; Khang, G.; Kang, P. M.; Lee, D. Inflammation-Responsive Antioxidant Nanoparticles Based on a Polymeric Prodrug of Vanillin. Biomacromolecules 2013, 14, 1618-1626. 38. Song, C. C.; Du, F. S.; Li, Z. C. Oxidation-Responsive Polymers for Biomedical Applications. J. Mater. Chem. B 2014, 2, 3413-3426. 39. Chen, X.; Wang, F.; Hyun, J. Y.; Wei, T.; Qiang, J.; Ren, X.; Shin, I.; Yoon, J. Recent Progress in the Development of Fluorescent, Luminescent and Colorimetric Probes for Detection of Reactive Oxygen and Nitrogen Species. Chem. Soc. Rev. 2016, 45, 2976-3016. 40. Chu, T.-S.; Lü, R.; Liu, B.-T. Reversibly Monitoring Oxidation and Reduction Events in Living Biological Systems: Recent Development of Redox-Responsive Reversible NIR Biosensors and Their Applications in in Vitro/in Vivo Fluorescence Imaging. Biosens. Bioelectron. 2016, 86, 643-655. 41. Major Jourden, J. L.; Cohen, S. M. Hydrogen Peroxide Activated Matrix Metalloproteinase Inhibitors: A Prodrug Approach. Angew. Chem. Int. Ed. 2010, 49, 6795-6797. 42. Wang, J.; Sun, X.; Mao, W.; Sun, W.; Tang, J.; Sui, M.; Shen, Y.; Gu, Z. Tumor Redox 24

ACS Paragon Plus Environment

Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Heterogeneity-Responsive Prodrug Nanocapsules for Cancer Chemotherapy. Adv. Mater. 2013, 25, 3670-3676. 43. Liu, B.; Wang, D.; Liu, Y.; Zhang, Q.; Meng, L.; Chi, H.; Shi, J.; Li, G.; Li, J.; Zhu, X. Hydrogen Peroxide-Responsive Anticancer Hyperbranched Polymer Micelles for Enhanced Cell Apoptosis. Polym. Chem. 2015, 6, 3460-3471. 44. Kuang, Y.; Balakrishnan, K.; Gandhi, V.; Peng, X. Hydrogen Peroxide Inducible DNA Cross-Linking Agents: Targeted Anticancer Prodrugs. J. Am. Chem. Soc. 2011, 133, 19278-19281. 45. Lee, S. H.; Gupta, M. K.; Bang, J. B.; Bae, H.; Sung, H.-J. Current Progress in Reactive Oxygen Species (ROS)-Responsive Materials for Biomedical Applications. Adv. Healthc. Mater. 2013, 2, 908-915. 46. d'Arcy, R.; Tirelli, N. Fishing for Fire: Strategies for Biological Targeting and Criteria for Material Design in Anti-Inflammatory Therapies. Polym. Adv. Technol. 2014, 25, 478-498. 47. Tapeinos, C.; Pandit, A. Physical, Chemical, and Biological Structures based on ROS-Sensitive Moieties That Are Able to Respond to Oxidative Microenvironments. Adv. Mater. 2016, 28, 5553-5585. 48. Xu, Q.; He, C.; Xiao, C.; Chen, X. Reactive Oxygen Species (ROS) Responsive Polymers for Biomedical Applications. Macromol. Biosci. 2016, 16, 635-646. 49. Deng, Z.; Hu, J.; Liu, S. Reactive Oxygen, Nitrogen, and Sulfur Species (RONSS)-Responsive Polymersomes for Triggered Drug Release. Macromol. Rapid Commun. 2017, 38, 1600685. 50. Yuan, Y.; Zhang, C.-J.; Xu, S.; Liu, B. A Self-Reporting AIE Probe with A Built-in Singlet Oxygen Sensor for Targeted Photodynamic Ablation of Cancer Cells. Chem. Sci. 2016, 7, 1862-1866. 51. Liu, L.-H.; Qiu, W.-X.; Li, B.; Zhang, C.; Sun, L.-F.; Wan, S.-S.; Rong, L.; Zhang, X.-Z. A Red Light Activatable Multifunctional Prodrug for Image-Guided Photodynamic Therapy and Cascaded Chemotherapy. Adv. Funct. Mater. 2016, 26, 6257-6269. 52. Kumar, R.; Han, J.; Lim, H.-J.; Ren, W. X.; Lim, J.-Y.; Kim, J.-H.; Kim, J. S. Mitochondrial Induced and Self-Monitored Intrinsic Apoptosis by Antitumor Theranostic Prodrug: In Vivo Imaging and Precise Cancer Treatment. J. Am. Chem. Soc. 2014, 136, 17836-17843. 53. Kim, E.-J.; Bhuniya, S.; Lee, H.; Kim, H. M.; Cheong, C.; Maiti, S.; Hong, K. S.; Kim, J. S. An Activatable Prodrug for the Treatment of Metastatic Tumors. J. Am. Chem. Soc. 2014, 136, 13888-13894. 54. Pu, H.-L.; Chiang, W.-L.; Maiti, B.; Liao, Z.-X.; Ho, Y.-C.; Shim, M. S.; Chuang, E.-Y.; Xia, Y.; Sung, H.-W. Nanoparticles with Dual Responses to Oxidative Stress and Reduced pH for Drug Release and Anti-Inflammatory Applications. ACS Nano 2014, 8, 1213-1221. 55. Yuan, Y.; Liu, J.; Liu, B. Conjugated-Polyelectrolyte-Based Polyprodrug: Targeted and Image-Guided Photodynamic and Chemotherapy with On-Demand Drug Release upon Irradiation with a Single Light Source. Angew. Chem. Int. Ed. 2014, 53, 7163-7168. 56. Li, M.; Li, S.; Chen, H.; Hu, R.; Liu, L.; Lv, F.; Wang, S. Preparation of Conjugated Polymer Grafted with H2O2-Sensitive Prodrug for Cell Imaging and Tumor Cell Killing. ACS Appl. Mater. Interfaces 2016, 8, 42-46. 57. Qiao, Z.-Y.; Zhao, W.-J.; Cong, Y.; Zhang, D.; Hu, Z.; Duan, Z.-Y.; Wang, H. Self-Assembled ROS-Sensitive Polymer–Peptide Therapeutics Incorporating Built-in Reporters for Evaluation of Treatment Efficacy. Biomacromolecules 2016, 17, 1643-1652. 58. Xu, X.; Saw, P. E.; Tao, W.; Li, Y.; Ji, X.; Bhasin, S.; Liu, Y.; Ayyash, D.; Rasmussen, J.; Huo, M.; Shi, J.; Farokhzad, O. C. ROS-Responsive Polyprodrug Nanoparticles for Triggered Drug Delivery and Effective Cancer Therapy. Adv. Mater. 2017, 29, 1700141. 59. Song, C. C.; Ji, R.; Du, F. S.; Liang, D. H.; Li, Z. C. Oxidation-Accelerated Hydrolysis of the Ortho Ester-Containing Acid-Labile Polymers. ACS Macro Lett. 2013, 2, 273-277. 60. Chan, J.; Dodani, S. C.; Chang, C. J. Reaction-Based Small-Molecule Fluorescent Probes for Chemoselective Bioimaging. Nat. Chem. 2012, 4, 973-984. 25

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 26 of 27

61. Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. Cell-Specific, Activatable, and Theranostic Prodrug for Dual-Targeted Cancer Imaging and Therapy. J. Am. Chem. Soc. 2011, 133, 16680-16688. 62. Krüger, H. R.; Schütz, I.; Justies, A.; Licha, K.; Welker, P.; Haucke, V.; Calderón, M. Imaging of Doxorubicin Release from Theranostic Macromolecular Prodrugs via Fluorescence Resonance Energy Transfer. J. Controlled Release 2014, 194, 189-196. 63. Chen, K.-J.; Chiu, Y.-L.; Chen, Y.-M.; Ho, Y.-C.; Sung, H.-W. Intracellularly Monitoring/Imaging The Release of Doxorubicin from pH-Responsive Nanoparticles Using Förster Resonance Energy Transfer. Biomaterials 2011, 32, 2586-2592. 64. Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J. An ICT-Based Approach to Ratiometric Fluorescence Imaging of Hydrogen Peroxide Produced in Living Cells. J. Am. Chem. Soc. 2008, 130, 4596-4597. 65. Yan, J. J.; Ji, W. X.; Chen, E. Q.; Li, Z. C.; Liang, D. H. Association and Aggregation Behavior of Poly(ethylene oxide)-b-Poly (N-isopropylacrylamide) in Aqueous Solution. Macromolecules 2008, 41, 4908-4913. 66. Huang, X. N.; Du, F. S.; Cheng, J.; Dong, Y. Q.; Liang, D. H.; Ji, S. P.; Lin, S. S.; Li, Z. C. Acid-Sensitive Polymeric Micelles Based on Thermoresponsive Block Copolymers with Pendent Cyclic Orthoester Groups. Macromolecules 2009, 42, 783-790. 67. Du, F. S.; Wang, Y.; Zhang, R.; Li, Z. C. Intelligent Nucleic Acid Delivery Systems Based on Stimuli-Responsive Polymers. Soft Matter 2010, 6, 835-848. 68. Sun, Q.; Zhou, Z.; Qiu, N.; Shen, Y. Rational Design of Cancer Nanomedicine: Nanoproperty Integration and Synchronization. Adv. Mater. 2017, 29, 1606628.

69. Noh, J.; Kwon, B.; Han, E.; Park, M.; Yang, W.; Cho, W.; Yoo, W.; Khang, G.; Lee, D. Amplification of Oxidative Stress by A Dual Stimuli-Responsive Hybrid Drug Enhances Cancer Cell Death. Nat. Commun. 2015, 6, 6907. 70. Su, Z.; Chen, M.; Xiao, Y.; Sun, M.; Zong, L.; Asghar, S.; Dong, M.; Li, H.; Ping, Q.; Zhang, C. ROS-Triggered and Regenerating Anticancer Nanosystem: An Effective Strategy to Subdue Tumor's Multidrug Resistance. J. Controlled Release 2014, 196, 370-383.

26

ACS Paragon Plus Environment

Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Graphics for TOC

27

ACS Paragon Plus Environment