Hien Phan

Robert Cavanagh

Damien Destouches

Francis Vacherot

Blandine Brissault

Vincenzo Taresco

Jacques Penelle

Benoit Couturaud
email: benoit.couturaud@cnrs.fr

H2O2-Responsive Nanocarriers Prepared by RAFT-Mediated Polymerization-Induced Self-Assembly of N -(2-(Methylthio)ethyl)acrylamide for Biomedical Applications

Keywords: polymer-based nanomedicine, polymerization-induced self-assembly (PISA), reactive oxygen species (ROS), hydrogen peroxide nanoparticles, drug delivery

come

Introduction

It is becoming increasingly apparent that block copolymer nanocarriers are of great benefit to drug delivery designs and innovative biomaterials applications in a range of therapeutic areas. [START_REF] Rösler | Advanced Drug Delivery Devices via Self-Assembly of Amphiphilic Block Copolymers[END_REF][START_REF] Adams | MINIREVIEW Amphiphilic Block Copolymers for Drug Delivery[END_REF][START_REF] Taresco | Functionalized Block Co Polymer Pro Drug Nanoparticles with Anti Cancer Efficacy in 3D Spheroids and in an Orthotopic Triple Negative Breast Cancer Model[END_REF] The versatility of block chemistries enables their final supra-macromolecular architectures to be finely tuned, while their amphiphilic balances can be adjusted to exert appropriate stability and colloidal properties. [START_REF] Vasey | Amphiphilic Triand Tetra-Block Co-Polymers Combining Versatile Functionality with Facile Assembly into Cytocompatible Nanoparticles[END_REF][START_REF] Phan | Role of Self assembly Conditions and Amphiphilic Balance on Nanoparticle Formation of PEG PDLLA Copolymers in Aqueous Environments[END_REF] Block copolymer NPs are traditionally prepared by nanoprecipitation or thin film hydration methods which usually face several limitations such as low polymer concentrations used (< 1wt%) and multiple intermediate steps. [START_REF] Tyrrell | Fabrication of Micellar Nanoparticles for Drug Delivery through the Self-Assembly of Block Copolymers[END_REF] To address these challenges, a promising alternative known as polymerization-induced self-assembly (PISA) has been introduced, since the polymerization of block copolymers and their self-assemblies into NPs are directly obtained in one pot. [START_REF] Phan | Polymerisation-Induced Self-Assembly (PISA) as a Straightforward Formulation Strategy for Stimuli-Responsive Drug Delivery Systems and Biomaterials: Recent Advances[END_REF][START_REF] Canning | A Critical Appraisal of RAFT-Mediated Polymerization-Induced Self-Assembly[END_REF][START_REF] Lansalot | Polymerization-Induced Self-Assembly[END_REF][START_REF] Foster | Predicting Monomers for Use in Polymerization-Induced Self-Assembly[END_REF][START_REF] Couturaud | Pentafluorophenyl Methacrylate)-Based Nano-Objects Developed by Photo-PISA as Scaffolds for Post-Polymerization Functionalization[END_REF] In addition to such facile synthetic route, PISA elicits possibility of achieving morphological transitions from spherical micelles, cylinders to vesicles at very high concentration (to 50 wt%) during the polymerization process. [START_REF] D'agosto | RAFT Mediated Polymerization Induced Self Assembly[END_REF][START_REF] Phan | Polymerization-Induced Self-Assembly (PISA) for in Situ Drug Encapsulation or Drug Conjugation in Cancer Application[END_REF][START_REF] Zhao | Polymerization-Induced Self-Assembly (PISA) -Control over the Morphology of 19F-Containing Polymeric Nano-Objects for Cell Uptake and Tracking[END_REF][START_REF] Wang | New Insights into RAFT Dispersion Polymerization-Induced Self-Assembly: From Monomer Library, Morphological Control, and Stability to Driving Forces[END_REF][START_REF] Rieger | Guidelines for the Synthesis of Block Copolymer Particles of Various Morphologies by RAFT Dispersion Polymerization[END_REF][START_REF] Hunter | Synthesis of Diblock Copolymer Spheres, Worms and Vesicles: Via RAFT Aqueous Emulsion Polymerization of Hydroxybutyl Methacrylate[END_REF] Thereby, PISA can be a potential formulation alternative over conventional methods to accelerate the commercialization with low manufacture cost and high batch reproducibility.

Together with the emergence of many novel and promising drug molecules, increased concerns are being placed upon developing drug delivery systems that can facilitate high access of bioactive agents to diseased areas. This is because following systemic administration, therapeutic molecules are likely to face premature plasma degradation and non-specific biodistribution, thus limiting their access and accumulation at the targeted cells. This is particularly true for anticancer drugs, which are usually cytotoxic to both healthy and tumor cells, resulting in many severe off-target side effects. Therefore, to maximize treatment outcomes biomolecules should be protected and stabilized within drug delivery carriers during blood circulation, while being selectively released upon reaching targeted tissues. In this regard, the designed drug delivery systems should be able to sense the differences in their surrounding environments and specifically respond to pathological signals, thus programing their drug release profiles as appropriate. [START_REF] Taresco | Stimuli-Responsive Prodrug Chemistries for Drug Delivery[END_REF][START_REF] Sahle | Design Strategies for Programmable Oligonucleotide Nanotherapeutics[END_REF][START_REF] Steele | Stimuli-Sensitive and -Responsive Polymer Biomaterials[END_REF][START_REF] Cheng | Dual and Multi-Stimuli Responsive Polymeric Nanoparticles for Programmed Site-Specific Drug Delivery[END_REF][START_REF] Couvreur | Nanotechnology: Intelligent Design to Treat Complex Disease[END_REF] Meanwhile, disease cells are distinct from healthy cells due to the presence of certain abnormal signals or the imbalance between biological molecules, hence disturbing the physiological mechanisms of cell metabolism and cell survival. One widely known phenomenon is the abnormal increase of reactive oxygen species (ROS) that leads to oxidative stress and cell disruption, thus causing various diseases such as cancer, diabetes, and neurodegenerative diseases. [START_REF] Perillo | ROS in Cancer Therapy: The Bright Side of the Moon[END_REF][START_REF] Waris | Reactive Oxygen Species: Role in the Development of Cancer and Various Chronic Conditions[END_REF][START_REF] Liou | Reactive Oxygen Species in Cancer[END_REF] ROS are free-radical by-products of oxygen metabolism during cell regulation process, which plays a beneficial role in maintaining normal cellular activities at a concentration of 3-10 µM but promotes cellular dysregulation and diseases at 100 µM. [START_REF] Giorgio | Hydrogen Peroxide: A Metabolic by-Product or a Common Mediator of Ageing Signals?[END_REF] Such property has largely been exploited to design nano-vehicles encoded with functionalities sensitive to the increased ROS, inducing molecular structural transitions or disassociation into sub-units to release internal payloads at targeted sites. [START_REF] Sun | A ROS-Responsive Polymeric Micelle with a π-Conjugated Thioketal Moiety for Enhanced Drug Loading and Efficient Drug Delivery[END_REF][START_REF] Xu | ROS-Responsive Polyprodrug Nanoparticles for Triggered Drug Delivery and Effective Cancer Therapy[END_REF][START_REF] Wang | Facile Synthesis of a H2O2-Responsive Alternating Copolymer Bearing Thioether Side Groups for Drug Delivery and Controlled Release[END_REF][START_REF] Tang | Self-Accelerating H2O2-Responsive Plasmonic Nanovesicles for Synergistic Chemo/Starving Therapy of Tumors[END_REF][START_REF] Yang | Reactive Oxygen Species (ROS)-Based Nanomedicine[END_REF][START_REF] Ye | Recent Advances on Reactive Oxygen Species-Responsive Delivery and Diagnosis System[END_REF][START_REF] Kwon | Nanomedicines for Reactive Oxygen Species Mediated Approach: An Emerging Paradigm for Cancer Treatment[END_REF][START_REF] Lallana | Oxidation-Responsive Polymers: Which Groups to Use, How to Make Them, What to Expect from Them (Biomedical Applications)[END_REF][START_REF] El-Mohtadi | Oxidation-Responsive Materials: Biological Rationale, State of the Art, Multiple Responsiveness, and Open Issues[END_REF] In the context of ROS-responsive drug delivery systems, block copolymer NPs bearing thioether moieties have been widely studied in recent reports, since the hydrophobic thioether incorporated in the water-insoluble segments could be transformed into hydrophilic sulfoxides in the presence of oxidation species such as H 2 O 2 , promoting a solubility transition and disrupting the amphiphilic balance to ultimately disassociating block copolymers NPs to releasing their payloads. [START_REF] Yan | Facile Synthesis of ROS-Responsive Biodegradable Main Chain Poly(Carbonate-Thioether) Copolymers[END_REF][START_REF] Yoo | Protease-Activatable Cell-Penetrating Peptide Possessing ROS-Triggered Phase Transition for Enhanced Cancer Therapy[END_REF][START_REF] Piergentili | Thioanisole Ester Based Logic Gate Cascade to Control ROS-Triggered Micellar Degradation[END_REF][START_REF] Rajes | Oxidation-Sensitive Core-Multishell Nanocarriers for the Controlled Delivery of Hydrophobic Drugs[END_REF][START_REF] Qiao | Self-Assembled ROS-Sensitive Polymer-Peptide Therapeutics Incorporating Built-in Reporters for Evaluation of Treatment Efficacy[END_REF][START_REF] Luo | Self-Assembled Redox Dual-Responsive Prodrug-Nanosystem Formed by Single Thioether-Bridged Paclitaxel-Fatty Acid Conjugate for Cancer Chemotherapy[END_REF][START_REF] Van Der Vlies | Thioether-Based Polymeric Micelles with Fine-Tuned Oxidation Sensitivities for Chemotherapeutic Drug Delivery[END_REF][START_REF] Wang | Synthesis of an Oxidation-Sensitive Polyphosphoester Bearing Thioether Group for Triggered Drug Release[END_REF][START_REF] Dong | Copper-Coordination Induced Fabrication of Stimuli-Responsive Polymersomes from Amphiphilic Block Copolymer Containing Pendant Thioethers[END_REF][START_REF] Dong | Methionine-Based PH and Oxidation Dual-Responsive Block Copolymer: Synthesis and Fabrication of Protein Nanogels[END_REF][START_REF] Duro-Castano | One-Pot Synthesis of Oxidation-Sensitive Supramolecular Gels and Vesicles[END_REF][START_REF] Shu | Tumor Microenvironment Triple-Responsive Nanoparticles Enable Enhanced Tumor Penetration and Synergetic Chemo-Photodynamic Therapy[END_REF][START_REF] Song | Recent Studies on Hydrogels Based on H2O2-Responsive Moieties: Mechanism, Preparation and Application[END_REF] Taking advantage of the one-step PISA formulation technique while integrating the oxidationsensitive thioethers into PISA-made nano-assemblies for controlled drug release, we aim to firstly synthesize an acrylate-based H 2 O 2 -responsive thioether monomer, N-(2-(methylthio)ethyl)acrylamide, which can be polymerized by a poly(ethylene glycol) methacrylate macro-chain transfer agent (macro-CTA) via PISA-RAFT process to form NPs at 10 wt%. This study features the in situ preparation of H 2 O 2 -responsive NPs in one pot by PISA and investigates the stability of the obtained particle morphologies in bio-mimicking environments, both under physiological and tumor-associated elevated H 2 O 2 conditions. Nile Red dye was employed as a hydrophobic drug model to be loaded into the hydrophobic core of spherical micelles during PISA and the study of drug release in H 2 O 2 was undertaken. The impacts of particle shapes on their biocompatibilities and cell uptake were also assessed on MCF-7 breast cancer cells.

Materials and Methods

Materials

All chemicals were used as obtained without further purification unless otherwise stated.

Poly(ethylene glycol) methyl ether methacrylate M n = 500 g mol -1 (denoted as PEGMA500) (> 95 %, Aldrich) and poly(ethylene glycol) methacrylate M n = 360 g mol -1 (denoted as PEGMAOH) (> 95 %, Aldrich) were activated by aluminum oxide column before use. 4-Cyano-4-(((ethylthio)carbonothioyl)thio)pentanoic acid (CEPA) chain transfer agent (CTA) was synthesized according to the established protocol. [START_REF] Truong | Rapid Synthesis of Ultrahigh Molecular Weight and Low Polydispersity Polystyrene Diblock Copolymers by RAFT-Mediated Emulsion Polymerization[END_REF] Chemical has been sourced as followed : 2,2'-Azobis(2-methylpropionamidine) dihydrochloride azo initiator (V-50, MW = 271.19 g mol -1 , Aldrich), 2-(methylthio)ethylamine (95%, Thermo Scientific), 1,4-dioxane (anhydrous ≥ 99.9%, Aldrich), acryloyl chloride (≥ 97 %, Aldrich), triethylamine (TEA, 99 %, Aldrich), phosphate buffered saline (PBS, Aldrich), bovine serum albumin (BSA, Thermo Fisher Scientific), Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific), fetal calf serum (FBS, Sigma-Aldrich), RPMI (Invitrogen), Triton X-100 (Aldrich), diamidino-2-phenylindole (DAPI, Aldrich), lysotracker green DND-26 (Thermo-Fisher), Hoechst 33342 (Thermo-Fisher), PrestoBlue cell viability assay (Thermo Fisher Scientific), L-glutamine (Sigma-Aldrich), Sulfo-Cy5 NHS (Lumiprobe), nile red (≥97.0% (HPLC) from Sigma-Aldrich). MCF-7 human breast adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC; Manassas, Virginia) and used in a passage window of 10. DU145 and 22Rv1 human prostate cancer cells and PNT2 non-tumor human cells were obtained from LGC Promochem (Molsheim, France).

Methods

Synthesis of ROS-sensitive monomer N-(2-(methylthio)ethyl)acrylamide (MTEAM)

1.0 g (11.1 mmol, 1.0 mL) 2-(methylthio)ethylamine was added to a round bottom flask containing 50 mL dry THF and cooled to 0 °C. 1.1 g (11.1 mmol, 1.5 mL) triethylamine was then added to the solution and left to stir for 10 min. 0.1 g (11.1 mol, 0.9 mL) acryloyl chloride was added dropwise and the solution was allowed to warm to room temperature. After 12 h, the white precipitate was filtered and the THF was removed under reduced pressure. The crude mixture was dissolved in 50 mL of DCM and washed with saturated NaHCO 3 (2 x 50 mL) and brine (50 mL). The organic layer was dried over anhydrous MgSO 4 , filtered, and the solvent was evaporated in a rotavapor. The obtained oil was purified by column chromatography on silica gels using initially 100 % cyclohexane, then cyclohexane:dichloromethane = 50:50 (v/v) and slowly to dichloromethane:methanol = 98:2 (v/v), resulting in the colorless oil at 80.5 % yield.

Synthesis of hydrophilic P(PEGMA-co-PEGMAOH) macro-CTA

The chain transfer agent 2-cyano-5-((4-fluorophenyl)amino)-5-oxopentan-2-yl ethyl carbonotrithioate (CEPA CTA) was synthesized according to previous procedure. [START_REF] Truong | Rapid Synthesis of Ultrahigh Molecular Weight and Low Polydispersity Polystyrene Diblock Copolymers by RAFT-Mediated Emulsion Polymerization[END_REF] P(PEGMAco-PEGMAOH) macro-CTA (abbreviated as P) was prepared by RAFT polymerization with two monomers, PEGMA and PEGMAOH in the presence of CEPA CTA, initiated by AIBN at 80 °C in dioxane at a molar ratio of PEGMA:PEGMAOH:CEPA:AIBN = 90:10:1:0.2. The total concentration of two monomers in the solution was 0.5 mol L -1 . To begin with, 7.3 mg CEPA (27.8 µmol), 1.25 g PEGMA (2.5 mmol, 1.16 mL), 0.1 g PEGMAOH (278 µmol, 90.5 µL) and 912 µg AIBN (5.6 µmol) were added to an 8-mL glass vial and dissolved in 5.56 mL of dioxane.

The vial was sealed and degassed under argon for 30 min and placed in a pre-heated oil bath at 80 °C. 100 µL sample was withdrawn periodically at predetermined time to determine the monomer conversion (%) according to [START_REF] Rösler | Advanced Drug Delivery Devices via Self-Assembly of Amphiphilic Block Copolymers[END_REF] H NMR (CDCl 3 ) and molecular weight distribution and dispersity according to SEC (PMMA standard, DMF). On the 1 H NMR (CDCl 3 ) spectra, the integration of the peak belonging to the methyl groups (-CH 3 ) of PEGMA was fixed at 3 due to the 3 protons at different time points to observe changes of the proton peaks representing the methyl groups (-CH 2 ) of acrylate functions of PEGMA500 and PEGOH360 at 5.5 ppm or 6.1 ppm, with reference to such at t = 0 min. The monomer conversion is thus presented as:

Monomer conversion (%) = ! ! !!! ! ! ! !!!
100 in which I t and I 0 are integrals at time t and t = 0, respectively.

For SEC, 30 µL of the withdrawn samples were dried under the fume food overnight to evaporate the solvent dioxane and dissolved in 1.5 mL filtered dimethylformamide (DMF), followed by filtering through polytetrafluoroethylene (PTFE) membranes with pore sizes of 0.45 µm to eliminate impurities and bacteria, before subjecting to SEC with poly(methyl methacrylate) (PMMA) standard.

Polymerization-induced self-assembly (PISA) of MTEAM monomer and P(PEGMA-

co-PEGMAOH) macro-CTA

MTEAM monomer (denoted as M), P(PEGMA-co-PEGMAOH) macro-CTA (denoted as P), and V-50 were added into a glass vial at a molar ratio of M:P:V-50 = 300:1:0.2 in deionized water. 80 mg P (2.1 µmol), 93 mg M (641 µmol), and 116 µg V-50 (0.4 µmol) were added to an 8-mL vial and dissolved in 1.73 mL water to obtain a total polymer concentration of 0.1 g mL -1 (10 wt%). This vial was degassed under argon for 30 min and placed in a pre-heated oil bath at 60 °C. 100 µL sample was withdrawn at predetermined time intervals using a deoxygenated syringe to study the polymerization kinetics using 1 H NMR (DMSO), SEC (PMMA standard, DMF) and TEM.

Colloidal stability in biological media

The stability was undertaken by treating spherical and vesicle solutions with bovine serum albumin (BSA) 0.2 wt% and cell culture medium (DMEM) at 37 °C. The first step involved the preparation of stock solutions of NPs at 2 mg mL -1 and of BSA at 2 mg mL -1 . 500 µL NPs (2 mg mL -1 ) were added to a 1.5 mL-Eppendorf and mixed with the BSA solution prepared above (2 mg mL -1 ) to produce the final solution containing 1 mg mL -1 NPs and 1 mg mL -1 BSA. To another Eppendorf, 500 µL NPs (2 mg mL -1 ) was reacted with 500 µL DMEM. NPs at 1 mg mL -1 in PBS were used as a control. Three samples of spherical micelles (in PBS, BSA, DMEM) and three samples under similar conditions of vesicles were incubated in a water bath at 37 °C for 24 h.

Oxidation in H 2 O 2

The stock solution of H 2 O 2 was prepared at 0.1 M by diluting 10 µL H 2 O 2 9.8 M (the commercial product's original concentration) in 990 µL PBS. From this mother solution several H 2 O 2 concentrations were prepared at 20 mM, 2 mM, 0.2 mM and 20 µM in PBS. Stock solutions of spheres or vesicles were prepared at 2 mg mL -1 . 500 µL NPs (2 mg mL -1 ) were treated with 500 µL of different H 2 O 2 concentrations including 20 mM, 2 mM, 0.2 mM or 20 µM in different Eppendorfs, resulting in final mixture solutions of 1 mg mL -1 NPs with 10 mM, 1 mM, 0.1 mM or 10 µM H 2 O 2 , respectively. These samples were placed in a water bath at 37 °C for 48 h and their sizes and zeta potentials were assessed at predetermined times.

Encapsulation and release study of Nile Red

Nile Red (NR) dye was loaded into the hydrophobic core of the spherical micelles during PISA at a ratio of NR/MTEAM/ P(PEGMA-co-PEGMAOH)/V-50 = 0.05/45/1/0.4 with 10 wt% of total solid contents. Briefly, stock solution of NR was prepared at 1 mg mL -1 in chloroform. 18 µL of this solution was taken into a 1-mL vial and dried under reduced pressure to remove the chloroform. 5.24 µg MTEAM monomer was then added to the dried NR and mix well until a homogenous solution achieved. The mixture of NR and MTEAM monomer was added to another vial containing 0.03 g P(PEGMA-co-PEGMAOH) macro-CTA in 344 µL deionized H 2 O. 8.7 µL of V-50 was taken from a stock solution of V-50 of 0.01 g mL -1 and added to the PISA reaction mixture above. The sample was degassed for 30 min under argon and placed in a pre-heated oil bath at 60 °C for 4 h. The PISA was quenched by exposing the reaction mixture to air and assessed in [START_REF] Rösler | Advanced Drug Delivery Devices via Self-Assembly of Amphiphilic Block Copolymers[END_REF] H NMR and DLS. The reaction mixture was centrifuged at 60000 rpm for 4 h at room temperature, and that free NR in water tended to precipitate to the bottom of the vial and the supernatant containing NR-loaded micelles was collected for quantification of drug loading and drug release study. i) Quantification of drug loading (DL) and encapsulation efficiency (EE)

The solution of NR-loaded NP was freeze-dried overnight, and the obtained solid was dissolved in an appropriate volume of DMSO to measure the UV-Vis absorbance of NR at 550 nm. This data was compared with a known calibration curve of NR in DMSO to calculate the drug loading and encapsulation efficiency, as follows:

DL (%) = !"#$%&!!"!!"#$!!"#$%$ !"#$%&!!"!!"#$%!!"#$%#$& !!100 EE (%) = !"#$%&!!"!!"#$!!"#$%$ !"#$%&!!"!!"#$!!"!#!$%%&!!""#" !!100 ii) Drug release in H 2 O 2
40 µL of the NR-loaded NP dispersion was diluted with 460 µL deionized H 2 O in a 1-mL quartz cuvette, obtaining a NP concentration of 8 mg mL -1 . 500 µL of H 2 O 2 20 mM was added to this mixture, resulting in a particle concentration of 4 mg mL -1 and H 2 O 2 of 10 mM. 4 mg mL -1 of NR-loaded particles in PBS was used as a control. The cuvette was placed in a UV-Vis machine and scanned from 200 -800 nm every 1 h at 37 °C to observe changes in the spectra over the course of H 2 O 2 oxidation.

Attachment of Cy5

Both spheres (P-b-M 45 ) and vesicles (P-b-M 300 ) have the hydroxyl functions of PEGMAOH exposed on the surfaces that were used to conjugate with the activated N-hydroxysuccinimide (NHS) ester function of sulfo-cyanine5 NHS ester at a molar ratio of 1:0.1. 900 µL P-b-M 300 (1.1 µmol) and 86.5 µL sulfo-cyanine5 NHS ester (0.11 µmol) were added into a 5-mL vial and stirred under the fume hood for 24 h. The solution was then dialyzed against deionized water (MWCO 6 -8 kDa) for 48 h to remove free Cy5. The obtained mixture was lyophilized and then dissolved in an appropriate volume of deionized water to measure the absorbance in UV-Vis at 646 nm. Comparing the absorbance value recorded with the prepared calibration curve of Cy5 in H 2 O enabled the quantification of the attached Cy5 and the conjugation yield.

Cell culture

Cell viability and uptake of two NP dispersions of spheres and vesicles were assessed in cancer and normal cell lines, including MCF-7 human breast adenocarcinoma cells, DU145 and 22Rv1 human prostate cancer cells and PNT2 non-tumor human cells. MCF-7 cells were cultured in DMEM supplemented with 10 % (v/v) fetal calf serum (FBS) and 2 mM L-glutamine (complete DMEM). DU145, 22RV1, and PNT2 cells were cultured in RPMI supplemented with 10 % FBS.

Cells were incubated under humidified atmosphere at 37 °C with 5% CO 2 .

Cytocompatibility evaluation

The PrestoBlue cell viability assay was performed to assess nanoparticle cytotoxicity. MCF-7 cells were seeded at 1x10 4 cells per well in 96 well plates and cultured for 24 hours prior to assaying. Nanoparticles were exposed to cells for 24 hours and applied in 100 µL phenol red free DMEM containing 10 % (v/v) FBS and 2 mM L-glutamine. Triton X-100 applied at 1% (v/v) applied in phenol red free medium was used as a cell death (positive) control and a vehicle control containing no nanoparticles used as a negative control. Following exposure cells were washed twice with warm PBS and 100 µL 10% (v/v) PrestoBlue reagent diluted in phenol red free medium applied per well for 60 minutes. The resulting fluorescence was measured at 560/600 nm (λ ex /λ em ). Relative metabolic activity was calculated by setting values from the negative control as 100 % and positive control values as 0 % metabolic activity.

Cell viability was studied using MTT assay for spheres and vesicles in two human prostate cancer cell lines, DU145 and 22Rv1, as well as non-tumor human prostate cell line, PNT2. Cells were seeded into a 96-well plate at a concentration of 1 × 10 4 cells per well in 100 μL of the complete medium. The cells were then incubated for 24 h prior to analysis. For a typical test, the cells were incubated for 72 h in a fresh complete medium containing samples at designed concentrations: 0.005 -1 mg mL -1 (equivalent NP concentration). Three parallel wells were set for each concentration and three individual experiments were carried out. MTT (1 mg mL -1 ) was then added into each well and incubated further for 2 h. Cells were washed twice in PBS and the produced formazan was solubilized in 100 μL of isopropanol. The absorbance of MTT assay was measured at 595 nm using a microplate reader.

Fluorescent live cell microscopy

MCF-7 cells were seeded in CellView™ 35 mm diameter glass bottom cell culture dishes at a density of 2.5 x 10 5 cells per dish and cultured for 48 h. Sphere-Cy5 and vesicles-Cy5 (250 µg mL -1 ) were incubated with cells for 4 h at 37 °C with 5 % CO 2 . Following exposure, NP solutions were removed, and cells washed three times with ice cold PBS. Cells were then stained with 10 µg mL -1 Hoechst 33342 or 10 µg/mL Hoechst 33342 and 50 nM Lysotracker green DND-26 applied in Hank's Balanced Salt Solution (HBSS) for 30 min. Staining solution was removed, and cells washed twice with PBS. FluoroBrite DMEM was added to wells and cells were imaged on an inverted Nikon Eclipse TE 300 fluorescent microscope on DAPI, FITC and Cy5 filters. Images were processed using ImageJ software (1.52f) and the Coloc 2 plug in was used for calculation of Pearson's correlation coefficient (PCC) for co-localization studies.

Fluorometric-based uptake assessment

To further investigate cellular uptake, quantitative kinetic uptake experiments was performed. 50- 52 CF-7 breast cells were plated in 12 well plates at a seeding density of 1.2x10 5 cells per well and cultured for 48 h. Culture medium was then removed and 1 mL of 250 µg mL -1 spheres-Cy5 and vesicles-Cy5 applied in 10 % (v/v) FBS containing DMEM (no phenol red). Following exposure, NPs solutions were removed, and cells washed three times with ice cold PBS. 500 µL of 1 % (v/v) Triton X-100 solution applied in PBS was then added per well for 10 min at 37 °C.

To assess nanoparticle uptake, 100 µL of the resulting solution was collected per well and transferred to 96 well plates for the measurement of fluorescence at 640/680 nm (λ ex /λ em ).

Quantification of nanoparticles uptake was achieved via calibration curves of known nanoparticle concentrations diluted in 1% (v/v) Triton X-100 in PBS solution. Values were normalized to viable cell number per well determined by the trypan blue exclusion test and cell counting on a haemocytometer.

Results and Discussion

Synthesis of ROS-sensitive monomer N-(2-(methylthio)ethyl)acrylamide (MTEAM or M)

MTEAM monomer (M) was prepared through reacting 2-(methylthio)ethylamine with acryloyl chloride and trimethylamine for 12 h. Figure SI1A presents the 1 H NMR spectrum of MTEAM in CDCl 3 , showing singlet of 3 protons of methyl group (-CH 3 , a), triplets of 2 protons belonging to ethyl moieties (-CH 2 -CH 2 -, b, c), singlet of 1 proton of amine function (-NH, d), and proton peaks of acrylate group (-H, e, f, g), confirming the successful synthesis of the monomer.

Synthesis of hydrophilic P(PEGMA-co-PEGMAOH) macro-CTA (abbreviated as P)

The RAFT co-polymerization of PEGMA (M n = 500 g mol -1 ) and PEGMAOH (M n = 360 g mol - 1 ) was carried out in the presence of CEPA CTA and AIBN initiator at a molar ratio of PEGMA:PEGMAOH:CEPA:AIBN = 90:10:1:0.2 at 80 °C in dioxane, Figure 1A. The kinetics of the polymerization was thoroughly investigated using 1 H NMR (CDCl 3 ) and SEC (DMF, PMMA standard), seen Figure 1B and 1C, respectively. The semilogarithmic plot of monomer conversion as a function of time appears to increase linearly over a polymerization period of 270 min reaching 90 % conversion, Figure 1B. This inferred that the polymerization rate followed a first-order kinetics over the entire reaction and almost no terminations did occur. The SEC chromatograms display an increase in molecular weights over time with characteristic peaks and moderately narrow dispersity of 1.3 -1.5, suggesting that a reasonable control over the polymerization was obtained with the formation of a well-defined macro-CTA suitable for chain extension as depicted in Figure 1C. Indeed, it is not possible to assess the relative amount of each monomer, PEGMA or PEGOH in the final copolymers by NMR due to the high similarities in 1 H NMR signal patterns for the comonomer units. Given the very high structural resemblances between the two methacrylate monomers and the first-order polymerization kinetics, it can be reasonably expected that the copolymer composition at time t (min) would match those of the initial monomer feed at time t = 0 (min) as the structural difference between the two methacrylate esters is quite minimal (i.e., an OH group instead of a methoxy at the very end of the PEG chains) and is not expected to affect the kinetic copolymerization parameters. Both monomers, PEGMA500 and PEGMAOH360, were initially reacted at a molar ratio of 90:10, effectively yielding copolymer with the same composition. As the SEC results obtained for such copolymers cannot provide more than an estimate of the absolute molecular weights, an alternate approach based on the living character of the RAFT polymerization was used. The final degree of polymerization at full conversion can indeed be obtained from the [M] 0 /[RAFT agent] 0 ratio, and the degrees of polymerization at any conversion could be obtained by multiplying the above value by the conversion. To obtain the molecular weight, the degree of polymerization has additionally to take into account the composition of the copolymer and the molecular weights of the narrow-size comonomers. For example, if the reaction is quenched at t = 180 min, yielding a 77 % omonomer conversion, thereby, the respective molecular weight M n of P(PEGMA-co-PEGMAOH) (P) can be obtained as follows:

! ! = !!!!!(!""!!!!"!!"#!!") !"" = 37 kg mol -1
This value of 37 kg mol -1 was utilized for calculation in further experiments, but its accuracy has to be considered with caution, with an error that can certainly be expected to be in the 5-10 % range given the several assumptions that had to be made in the calculation.

Polymerization-induced self-assembly (PISA) of ROS-sensitive MTEAM monomer (M) and P(PEGMA-co-PEGMAOH) macro-CTA (P)

The macro-CTA P(PEGMA-co-PEGMAOH) (P) synthesized and characterized above was chain extended with the MTEAM monomer (M) in one-pot PISA using V-50 initiator in deionized water at 60 °C for 3 h (Figure 2A, Table SI1). Figure 2B exhibits three trends, (i) from 0 to 0.75 h, the monomer conversion increases from 5 % to 19 %, (ii) which rises up to 82 % at 1.5 h, and (iii) increases slowly to 100 % toward 3 h. SEC chromatograms show the molecular weight increase during the reaction, Figure 2C. DLS data also indicate the average particle size increases corresponding to the three different kinetics regimes, from 59 nm at 0.75 h, to 75 nm at 1 h, and 89 nm at 3 h with very low dispersity of 0.13 -0.24 in all three cases, Figure 2D. Not only do the particle sizes increase, but their morphologies are also transformed over the three stages of polymerization as indicated above. The morphological transition proceeds as follows:

(i) first, spherical micelles are formed at 45 min (Figure E1), (ii) these spheres then fused together to form a mixture of spheres and short fused micelles at 1 h (Figure E2), which later elongated to longer fused micelles at 1.25 h (Figure E3), and (iii) eventually evolve to vesicles at 3 h (Figure E4). Table 1 summarizes characteristics of the obtained morphologies. This transitional procedure is in accordance with the general structural changes observed in PISA polymerizations along with the increasing DPs. [START_REF] Zhao | Polymerization-Induced Self-Assembly (PISA) -Control over the Morphology of 19F-Containing Polymeric Nano-Objects for Cell Uptake and Tracking[END_REF][START_REF] Couturaud | Pentafluorophenyl Methacrylate)-Based Nano-Objects Developed by Photo-PISA as Scaffolds for Post-Polymerization Functionalization[END_REF][START_REF] Karagoz | Simultaneous Polymerization-Induced Self-Assembly (PISA) and Guest Molecule Encapsulation[END_REF]

Colloidal stability in biological media

The colloidal stability studies were undertaken with spherical micelles (Figure 2E1) and vesicles (Figure 2E4), due to their well-defined structures in TEM as compared to the fused micelles that did not provide a clear form of worm-like micelles. Hence, spherical particles (P-M 45 ) and vesicles (P-M 300 ) were incubated in bio-relevant environments, i.e., BSA 0.2 % and DMEM at 37 °C, and their sizes were determined by DLS after 24 h. Figure 3 exhibits that the size distributions of spheres and vesicles in BSA and DMEM remain similar to those of the control NPs in PBS and cause no precipitations. Zeta Potentials (ZPs) were recorded, showing the same value of around -10 mV in PBS (control), BSA 0.2 wt%, and DMEM over 24 h (Figure SI2).

These results indicated a high stability for PISA-made nano-objects under biological conditions.

Oxidation in H 2 O 2

Spheres (P-M 45 ) and vesicles (P-M 300 ) at 1 mg mL -1 were reacted with various concentrations of H 2 O 2 at 10 µM, 0.1 mM, 1 mM, and 10 mM at 37 °C.

Initially, spheres treated with 1 mM H 2 O 2 showed a decline in size and absolute ZPs over the course of 24 h, with multiple peak distributions starting to appear after 11 h (Figure 4A, Figure 4B), demonstrating that NPs disassociated into small and aggregated pieces upon oxidation. This disruptive process was accelerated when treating the same pristine spherical NPs with 10 mM H 2 O 2 , resulting in particles quickly crashing out after 2 h. Although the realistic level of H 2 O 2 in tumor cells is documented at 0.1 mM, the addition of H 2 O 2 0.1 mM to the particle solution induced disassembly relatively late after 48 h, observed from the oxidized signal on 1 H NMR (Figure 5). In addition to size and ZP, the particle disruption in H 2 O 2 can also be confirmed by observing the derived count rate (kcps) in DLS (Figure 4C), as it correlates with the size and/or concentration of particles in the measured solutions. The optimal count rates should be between 100 and 500 kcps, above which the particle solution is not appropriately diluted or very concentrated while below which particles cannot be detected by DLS's laser, probably due to very small or too diluted NPs. Before any treatment, particle solutions display a count rate of 420 -450 kcps which falls into the acceptable range (t = 0 h). However, the incubation of 10 mM or 1 mM H 2 O 2 caused the count rates to decrease exponentially to less than 100 kcps with no particles being detected after 6 h and 24 h, respectively. Figure 4D shows TEM images of intact spherical particles and aggregates produced from the oxidation after a 24 h treatment with 1 mM H 2 O 2 . Spherical NPs, after being treated with 1 mM H 2 O 2 for 24 h underwent a disruption, which resulted in the formation of smaller aggregates (41 nm), significantly larger PDI (0.4), and a much more diluted dispersion. The fractured small pieces of particles tended to scatter or form larger precipitation, leading to the heterogeneous mixture of small and large aggregates in the TEM image of Figure 4B.

Meanwhile, spheres incubated in 10 µM H 2 O 2 mimicking the normal ROS concentration had their sizes unchanged during the treatment period (Figure SI3). Together with the good particle stability in bio-relevant environments illustrated in section 3.4, these results suggest that spherical micelles are stable under physiological conditions, but sensitive to oxidation and prone to be disrupted in H 2 O 2 . On the other hand, vesicles (Figure 2E4) were found stable at all tested H 2 O 2 concentrations. The TEM image of Figure SI5 indicates that the vesicles are slightly swollen after incubating with H 2 O 2 , which corresponds to an increase in particle sizes from 90 nm to 130 nm (data not shown).

The swelling phenomenon is probably attributed to the partial oxidation of the hydrophobic core, and that NPs became slightly more hydrophilic, then water can easily penetrate, thus particles swell and increase in sizes. Despite being partly swollen, vesicles remained stable in H 2 O 2 , likely due to the strong hydrogen bonds potentially formed between the hydroxyl functions of PEG and water from two sides of the vesicle membranes, inside toward the hydrophilic core, and outside toward the external surrounding water. Such strong and rigid hydrogen interactions from both surfaces could probably reduce and prevent the penetration of H 2 O 2 , and that the vesicles were only marginally swollen and still stable. The particle size increase observed following the oxidation process also correlates with previously reported oxidation-sensitive nanomaterials. [START_REF] Piergentili | Thioanisole Ester Based Logic Gate Cascade to Control ROS-Triggered Micellar Degradation[END_REF][START_REF] Rajes | Oxidation-Sensitive Core-Multishell Nanocarriers for the Controlled Delivery of Hydrophobic Drugs[END_REF][START_REF] Van Der Vlies | Thioether-Based Polymeric Micelles with Fine-Tuned Oxidation Sensitivities for Chemotherapeutic Drug Delivery[END_REF]

Encapsulation and release of hydrophobic dye Nile Red

The hydrophobic drug model Nile Red (NR) was simultaneously entrapped into the spherical micelles during the PISA with drug loading of 0.03 wt% (compared to the initially theoretical value of 0.05 wt%), and encapsulation efficiency of 43 %. The NR-loaded NPs increased to 78 nm in diameter with PDI of 0.25, with a reference to the plain NPs of 59 nm and PDI of 0.1, indicating that NR was efficiently entrapped to make such change in size. This was followed by the drug release study of the NR-loaded micelles when exposed to 10 mM H 2 O 2 . NR is very hydrophobic, highly fluorescent in organic solvents and non-fluorescent in water. Especially H 2 O 2 does not induce fluorescence-quenching effect on free NR, which has been utilized to assess the drug release under oxidation conditions, and to eventually demonstrate that the fluorescence quenching of NR is merely caused by the disruption of NPs and not by H 2 O 2 . [START_REF] Yoo | Protease-Activatable Cell-Penetrating Peptide Possessing ROS-Triggered Phase Transition for Enhanced Cancer Therapy[END_REF][START_REF] Gracia Lux | Biocompatible Polymeric Nanoparticles Degrade and Release Cargo in Response to Biologically Relevant Levels of Hydrogen Peroxide[END_REF] Figure 6 shows the spectra of NR-loaded NPs at 10 mM H 2 O 2 , indicating a reduce in absorbance spectra over time. Although free NR is fluorescent limiting, the loaded NR in an aqueous dispersion can display fluorescence since it is protected in the non-polar core of the NPs. The maximal absorbance of 600 nm is observed at t = 0 h. However, when being exposed for a longer time in H 2 O 2, the particles tend to dissociate, free NR is released which shows very limited fluorescence or absorbance in water, thus the amounts of loaded NR decrease and accordingly, their absorbance peaks reduce gradually. In a meanwhile, the incubation of NR-loaded NPs in PBS at the same condition did not show any changes in the absorbance spectra, demonstrating the stability of NR in the normal aqueous environment (Figure SI6).

Attachment of Cy5

Spheres (P-M 45 ) and vesicles (P-M 300 ) were labelled with fluorescent sulfo-cyanine5 NHS ester to facilitate NPs imaging and tracking for biological assessment. This was achieved by functionalizing the hydroxyl end-groups of P(PEGMA-co-PEGMAOH) block with the highly reactive sulfo-NHS ester moieties of Cy5 dye. After a 48 h-dialysis and lyophilization, the obtained solids were resuspended in appropriate volumes of water, and their UV-Vis absorbances were then recorded at 646 nm and compared with the prepared calibration curve of Cy5 in water, resulting in the attached Cy5 concentrations of 48.2 µg mL -1 (55.4 % yield) and 69.7 µg mL -1 (79.5 % yield) for spheres and vesicles, respectively.

Cell culture

The Cy5-labelled spheres and vesicles were evaluated in in vitro cell culture of MCF-7 breast cancer, and DU145, 22Rv1 prostate cancer cells, and PNT2 non-cancerous cells to observe their effects on cytotoxicity and cell internalization (only implementing on MCF-7) over time.

Cytocompatibility evaluation

The biocompatibility of spheres (P-M 45 ) and vesicles (P-M 300 ) with Cy5 conjugation were assessed in MCF-7 breast cancer cells using the PrestoBlue viability assay (Figure 6A), and prostate cells namely DU145 and 22Rv1 (cancer cells) and PNT-2 (non-tumor cells) using the MTT assay (Figure SI7, SI8). The results display no cytotoxicity on the tested cell lines at concentrations up to 1 mg mL -1 with approximately 100 % of metabolic activity or cell viability upon incubation. This observation confirmed that the newly synthesized MTEAM monomer and thus its polymerization into the hydrophobic cores of NPs cause no detrimental effects on the eventual biocompatibility, as well as indicates the absence of toxic residues or solvent traces in the NP dispersions from the synthesis and modification procedures.

Cell uptake

Spheres and vesicles were labelled with Cy5 and incubated with MCF- In addition to observing the intracellular location of NPs, the fluorometric-based uptake kinetics indicates time-dependent uptake of two morphologies and the number of NPs internalized over time. Figure 7B shows that spherical and vesicle NPs display the fastest rate of uptake within the first 60 min. However, this follows different trends afterward in which the uptake of sphere NPs continues to increase rapidly, while vesicles exhibit a very slight increase in cell uptake until 240 min. The differing uptake rates and profiles observed suggests that the spherical and vesicle NPs are internalized via different mechanisms. [START_REF] Rennick | Key Principles and Methods for Studying the Endocytosis of Biological and Nanoparticle Therapeutics[END_REF] Moreover, a higher number of spherical micelles were taken up as compared to vesicles at all the timepoints tested. These results implied that 60 nm spherical micelles were internalized more efficiently and imparted higher level of uptake than the 90 nm vesicle NPs. This observation of NP size-dependent uptake reflects reports in the literature that demonstrate increased uptake with smaller NPs. [START_REF] Lu | Size Effect on Cell Uptake in Well-Suspended, Uniform Mesoporous Silica Nanoparticles[END_REF][START_REF] Khetan | Quantitative Analysis of the Correlation between Cell Size and Cellular Uptake of Particles[END_REF][START_REF] Wu | Size-Dependent Cellular Uptake and Localization Profiles of Silver Nanoparticles[END_REF][START_REF] Foroozandeh | Insight into Cellular Uptake and Intracellular Trafficking of Nanoparticles[END_REF] This phenomenon has been attributed to NP size-dependent utilization of distinct endocytosis pathways. For example, clathrin-mediated endocytosis may be available for both sized NPs to exploit, due to clathrincoated vesicle being in the range of 100-150 nm in diameter. [START_REF] Manzanares | Endocytosis: The Nanoparticle and Submicron Nanocompounds Gateway into the Cell[END_REF][START_REF] Ehrlich | Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits[END_REF] However, the caveolin-mediated route may be restricted to only the smaller 60 nm spherical NPs. Caveolin membrane invaginations and vesicles are reported to be < 80 nm in diameter, [START_REF] Manzanares | Endocytosis: The Nanoparticle and Submicron Nanocompounds Gateway into the Cell[END_REF][START_REF] Richter | High Resolution 3D Quantitative Analysis of Caveolar Ultrastructure and Caveola Cytoskeleton[END_REF] hence limiting internalization of the 90 nm vesicle NPs via this endocytosis pathway. Further study with endocytosis inhibitors would be required to confirm these hypotheses. Nevertheless, the increased uptake observed with spherical NPs indicates that these particles would be the most appropriate as vehicles for drug delivery. While the current study succeeded in preparing spherical micelles in situ, which were stable in physiological conditions yet disassociated in the increasing H 2 O 2 or ROS-rich environments, the remaining challenge is how to achieve vesicles that are also sensitive to high tumorigenic H 2 O 2 concentrations when using similar PISA procedure and materials. Therefore, future studies should be investigating the effects of hydrophilic and/or ROS-hydrophobic segments on the disassociation of particles in ROS-rich conditions. Drug molecules could be encapsulated to assess drug release kinetics. Finally, it is noteworthy of studying the behaviors of NPs on other cell lines related to ROS overexpression e.g., neurodegenerative diseases, and carrying out in vivo experiments.

Figure 1 .

 1 Figure 1. RAFT polymerization of PEGMA and PEGMAOH with CEPA CTA in dioxane at 80

Figure 2 .

 2 Figure 2. PISA polymerization kinetics of MTEAM monomer (M) by chain extension of

Figure 3 .

 3 Figure 3. DLS size distributions of (A) spheres and (B) vesicles in BSA and DMEM after 24 h.

Figure 4 .

 4 Figure 4. Characterization of spherical micelles of P-M 45 in 1 mM H 2 O 2 during 24 h. (A)

Figure 5 .

 5 Figure 5. (A) Illustration of oxidative reaction of P-M 45 block copolymer NPs with H 2 O 2 ; (B) 1 H

Figure 6 .

 6 Figure 6. UV-Vis spectra of the NR-loaded NPs as incubated in 10 mM H 2 O 2 for 6 h at 37 o C.

  7 cells for 4 h to assess cell internalization. The confocal microscopy demonstrates the intracellular accumulation and uptake for both NPs, Figure 7C. Spheres and vesicles are shown to co-localize with lysosomes with Pearson's Correlation Coefficient (PCC) values calculated at 0.59 and 0.51 for spheres and vesicles, respectively, indicating potential endocytosis of NPs and trafficking to lysosomal system, Figure 7D.

Figure 7 .

 7 Figure 7. (A) Cytocompatibility of NPs of spheres (P-M 45 ) and vesicles (P-M 300 ) on MCF-7

4 .

 4 Conclusions and PerspectivesH 2 O 2 -responsive NPs were readily prepared by one-pot PISA polymerization of the oxidationsensitive MTEAM monomer in the presence of P(PEGMA-co-PEGMAOH) macro-CTA in water, showing the increases of molecular weights and particle sizes, along with a morphological transition from spherical micelles, to fused micelles, and vesicles of less than 100 nm over the polymerization kinetics. The spherical micelles prepared from the P-M 45 block copolymer indicated sufficient stability in physiological environments (BSA 0.2 %wt and DMEM). However, they disassociated upon incubating with 0.1 -10 mM H 2 O 2 solutions, as the thioether functionalities in the hydrophobic core were oxidized into hydrophilic sulfoxides, thus disrupting the amphiphilic balance and ultimately the micellar structures. On the other hand, vesicles or polymersomes of P-M 300 appeared slightly swollen as being partially oxidized under all the tested H 2 O 2 conditions. The hydrophobic dye Nile Red was loaded during PISA at 43 % of encapsulation efficiency and indicated a controlled release of Nile Red over the course of H 2 O 2 incubation. Spheres and vesicles were well-tolerated in MCF-7 human breast cancer as well as prostate cells. Both NP shapes were demonstrated to accumulate inside lysosomes in MCF-7 cells which suggested the cell internalization was potentially realized via an endocytosismediated pathway, with higher number of spherical NPs being taken up compared to vesicles due to the smaller size.

Table 1 .

 1 Summary of structural features observed for the PISA-made P-M block copolymer NPs

		DP	M n	a (kg mol -1 )	M n	b (kg mol -1 )	Đ b	Size c (nm)	PDI c
	Sphere	45	43.9	62.2	1.8	58.9	0.1
	Fused micelle	210	67.9	77.7	1.9	80.2	0.1
	Vesicle	297	80.6	80.8	2.6	88.9	0.2

a Calculated from 1 H NMR; b SEC (PMMA standard, DMF); Đ: Polydispersity (M w /M n ); c DLS

  Meanwhile spherical micelles are sufficiently stable under normal conditions yet still demonstrate effective responsiveness to H 2 O 2 , since they only formed hydrogen bonds with the external layer of water, through which the H 2 O 2 solutions could permeate more easily to cause the oxidation and the nano-disassembly.

Acknowledgment

HP is financially supported by MESRI scholarship and UPEC for the academic years of 2019-2022. Benoit Couturaud is grateful for the support from DIM RESPORE.

Conflict of interest

The authors declare no conflict of interest.

Supporting Information

Supporting Information is available containing NMR spectra, DLS size distribution curves, and TEM images.

Author information

Corresponding Author

Benoit Couturaud: benoit.couturaud@cnrs.fr

Author Contributions

HP worked on the conceptualization, experimentation, and writing of the original draft. RC and DD contributed to the biological tests and discussion. BB read the manuscript. VT, JP and BC reviewed and edited the current work. All authors have given approval to the final version of the manuscript.

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