Laura Etchenausia

Eva Villar-Alvarez

Jacqueline Forcada

Maud Save
email: maud.save@univ-pau.fr

Pablo Taboada
email: pablo.taboada@usc.es

Prof Pablo Taboada

Evaluation of cationic core-shell thermoresponsive poly(Nvinylcaprolactam)-based microgels as potential drug delivery nanocarriers

Keywords: Cationic thermoresponsive microgels, in-vitro drug delivery, cell internalization pathway

The present work investigates the potentiality of poly(N-vinyl caprolactam) (PVCL)-based thermoresponsive microgels decorated with cationic polymer brushes as drug delivery carriers. The effect of physico-chemical features of the colloids on cell viability response have to be carefully investigated to establish the range of suitable hydrodynamic diameters, crosslinking densities, lengths and ratios of the cationic polyelectrolyte shell which allow their efficient and effective use for cargo loading, transport and delivery. The colloidal stability of all cationic thermoresponsive microgels is maintained over several days of incubation at 37 °C in biological mimicking medium (Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum). The thin cationic polymer shell covalently anchored does not hinder the all range of microgels to be biocompatible while the higher cytotoxicity of the doxorubicin-loaded microgels on HeLa cells proves their antitumor activity. The core-shell PVCL drug delivery nanocarriers allow a sustained release of doxorubicin with a slightly higher viability of HeLa cells incubated in the presence of DOXOloaded microgels compared to the free DOXO. The nature of the endocytosis pathway is investigated through a quantification of the extent of the cellular survival rate in the presence of various cellular uptake inhibitors. A clathrin-dependent internalization was observed.

Introduction

In the field of human disease therapies and, more specifically, cancer therapies, pharmaceutical molecules (drugs, proteins, nucleic acids) are usually administered intravenously, which generally leads to short residence time, limited targeting and penetration of tumor tissues, toxic reactions in healthy tissues, and limitations to reach optimal effective doses. [START_REF] Wang | Recent advances of thermally responsive nanogels for cancer therapy[END_REF] Therefore, a major challenge consists in designing drug carriers in order to improve the safety and efficacy of therapeutic agents. Drug delivery systems (DDS) are ideally suitable to protect the active compounds from the surrounding environment (preventing their early degradation), consequently increasing their in-vivo circulation to deliver the drug concentration required for an optimal therapeutic efficacy, without damaging the healthy cells or tissues. [START_REF] Ramos | Cationic polymer nanoparticles and nanogels: from synthesis to biotechnological applications[END_REF][START_REF] Mura | Stimuli-responsive nanocarriers for drug delivery[END_REF] Thus, a robust delivery platform needs to satisfy some requirements. First of all, the size of the carrier is an important parameter to consider since it plays a key role in its degradation, its cellular uptake and its blood circulation life, among other functional aspects. [START_REF] Shang | Engineered nanoparticles interacting with cells: size matters[END_REF][START_REF] Wilczewska | Nanoparticles as drug delivery systems[END_REF][START_REF] Dhand | Role of size of drug delivery carriers for pulmonary and intravenous administration with emphasis on cancer therapeutics and lung-targeted drug delivery[END_REF] For instance, the size of the vehicle should be large enough to prevent rapid leakage in blood capillaries but small enough to escape the capture of macrophages in the reticuloendothelial system (RES), i.e., sizes ranging from a few tenths to a few hundreds of nanometers. Biocompatibility and non-toxicity are also imperative criteria for a suitable delivery system. [START_REF] Petros | Strategies in the design of nanoparticles for therapeutic applications[END_REF][START_REF] Dobrovolskaia | Immunological properties of engineered nanomaterials[END_REF] Among the different systems that have been envisaged as nanocarriers for therapeutics, submicronic polymeric nanogels or microgels are very promising candidates due to their versatile properties such as easy production and functionalization, large surface area, porous network, high loading capacity and potential responsiveness to environmental factors of some varieties, which allows a triggered release of the therapeutics. [START_REF] Oh | The development of microgels/nanogels for drug delivery applications[END_REF][START_REF] Vinogradov | Nanogels in the race for drug delivery[END_REF][START_REF] Saunders | Microgels: From responsive polymer colloids to biomaterials[END_REF][START_REF] Lu | Polyacrylamide hybrid nanogels for targeted cancer chemotherapy via co-delivery of gold nanoparticles and MTX[END_REF][START_REF] Baipaywad | Fabrication of Nanogels for Delivery of Molecules[END_REF][START_REF] Karimi | Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems[END_REF][START_REF] Mauri | Modulation of electrostatic interactions to improve controlled drug delivery from nanogels[END_REF][START_REF] Yi | Study on beta-cyclodextrincomplexed nanogels with improved thermal response for anticancer drug delivery[END_REF][START_REF] Ma | Advancement of multifunctional hybrid nanogel systems: Construction and application in drug co-delivery and imaging technique[END_REF] Thermoresponsive nano/microgels that undergo a volume phase transition in aqueous media upon subtle temperature changes have attracted interest as DDS within the last past years in order to control the sustained drug release and uptake performance when collapsing. [START_REF] Wang | Recent advances of thermally responsive nanogels for cancer therapy[END_REF][START_REF] Ramos | Cationic polymer nanoparticles and nanogels: from synthesis to biotechnological applications[END_REF][START_REF] Lyon | Thermoresponsive microgel-based materials[END_REF][START_REF] Wong | Dual-stimuli responsive PNiPAM microgel achieved via layer-by-layer assembly: Magnetic and thermoresponsive[END_REF][START_REF] Smeets | Designing responsive microgels for drug delivery applications[END_REF][START_REF] Sung | Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery[END_REF][START_REF] Olalde | Multifunctional bioactive glass scaffolds coated with layers of poly(D,L-lactide-co-glycolide) and poly(nisopropylacrylamide-co-acrylic acid) microgels loaded with vancomycin[END_REF][START_REF] Bardajee | Synthesis of a novel thermo/pH sensitive nanogel based on salep modified graphene oxide for drug release[END_REF] Among the different classes of thermoresponsive polymers, poly(N-vinylcaprolactam) (PVCL) has proven to be very attractive to design nano/microgels for biomedical applications thanks to its biocompatibility and lower critical solution temperature in the range of the physiological temperature. [START_REF] Ramos | Temperature-sensitive nanogels: poly(N-vinylcaprolactam) versus poly(N-isopropylacrylamide)[END_REF][START_REF] Liu | Poly(N-vinylcaprolactam): a thermoresponsive macromolecule with promising future in biomedical field[END_REF][START_REF] Aguirre | Biocompatible Stimuli-Responsive Nanogels for Controlled Antitumor Drug Delivery[END_REF][START_REF] Peng | A facile approach for thermal and reduction dual-responsive prodrug nanogels for intracellular doxorubicin delivery[END_REF][START_REF] Sun | Gadolinium-Loaded Poly(N-vinylcaprolactam) Nanogels: Synthesis, Characterization, and Application for Enhanced Tumor MR Imaging[END_REF][START_REF] Sonzogni | Effect of Delivery Platforms Structure on the Epidermal Antigen Transport for Topical Vaccination[END_REF] Polymeric cationic vehicles are of special interest due to their ability to interact with the negatively charged cell membrane facilitating their cellular uptake. [START_REF] Ramos | Cationic polymer nanoparticles and nanogels: from synthesis to biotechnological applications[END_REF] Moreover, they can strongly interact with negatively charged relevant biological molecules such as DNA and siRNA (whose charge and size make them non-permeable to cell membranes) facilitating their delivery into cells. [START_REF] Dincer | Intelligent polymers as nonviral vectors[END_REF][START_REF] Yue | Progress and perspectives in developing polymeric vectors for in vitro gene delivery[END_REF][START_REF] Yin | Non-viral vectors for gene-based therapy[END_REF][START_REF] Bui | Encapsidation of RNA-Polyelectrolyte Complexes with Amphiphilic Block Copolymers: Toward a New Self-Assembly Route[END_REF][START_REF] Ho | Innovative well-defined primary aminebased polyacrylates for plasmid DNA complexation[END_REF] Cationic thermoresponsive microgels have also been conjugated with anionic dyes for bio-imaging applications. [START_REF] Kodlekere | Design of functional cationic microgels as conjugation scaffolds[END_REF] The present work aims to investigate the in-vitro bioactivity of the original core-shell microgels as drug delivery nanocarriers to combine the advantage of the highly accessible covalently anchored cationic outer shell with the ones of a crosslinked thermoresponsive core with encapsulation capacity. We have previously reported the synthesis in aqueous dispersed media of this type of microgels using a strategy to covalently anchor the cationic polyelectrolyte exhibiting permanent cationic charges with the PVCL chains forming the crosslinked microgels. [START_REF] Etchenausia | Cationic Thermoresponsive Poly(Nvinylcaprolactam) Microgels Synthesized by Emulsion Polymerization Using a Reactive Cationic Macro-RAFT Agent[END_REF] The potential use of this cationic PVCL-based microgels for biomedical applications is conditioned by their stability in different biological mimicking media and their invitro cytotoxicity towards different cell lines. These properties are studied in the present work for a series of microgels with various structural features (crosslinking degree, length and ratio of the cationic polyelectrolyte and microgel swelling ratio) before to investigate the cellular uptake of the cationic PVCL-based microgels by confocal microscopy and more precisely their internalization pathway into HeLa cells by means of inhibition uptake experiments (Scheme 1). To complete the study, a model anticancer drug (doxorubicin, DOXO) is loaded into the PVCL-based microgels in order to test their potential use as chemotherapeutic drug delivery systems for cancer treatment.

Attention is thus paid to the drug release profiles of selected microgels and to the cytotoxicity of the DOXO-loaded microgels on HeLa and RAW cell lines.

Cell cytotoxicity assay

Loading and release of doxorubicin hydrochloride

Materials and Methods

Materials

Reagents and cell lines S1 and Table S2). The codes of the microgels displays the different experimental parameters used for their synthesis, like for instance SC1-PX76 4-E4. SC represents the initial solids content, the number following indicates the wt-% of VCL based on water, PXn is the reactive cationic polymeric stabilizer with n the degree of polymerization, the number following indicates the wt-% of stabilizer based on VCL and E corresponds to EGDMA crosslinker, the number following indicates the wt-% of EGDMA based on VCL.

Methods

Cell culture. The cervical HeLa cancer cell line was grown in a medium composed of DMEM, 1 v/v% penicillin-streptomycin, 1 v/v% sodium pyruvate, 1 v/v% amino acid and 10 v/v% of FBS.

The same medium was used for the growth of RAW cell line but DMEM/F-12 was used instead of DMEM. Both cell lines were grown in an 5% CO2-95% air atmosphere at 37°C. After attaining 80-90% confluence, cells were harvested using trypsin (0.25%)-EDTA. Cell count was conducted using an automated cell counter (Multisizer 3 Beckman Coulter Cell Counter).

Colloidal stability of bare PVCL-based microgels in biological mimicking media. The colloidal stability of bare PVCL-based microgels was tested at 37°C under slow stirring for 13 days, by dilution of the samples (0.05 mg.mL -1 ) in different biological mimicking media: cell culture medium (DMEM) supplemented with 10% FBS (pH 8.4), PBS (pH 7.4) and PBS supplemented with 10% FBS (pH 7.4). Stability of the microgels was also assessed in water, following the same procedure. The evolution of the hydrodynamic diameter (Dh) of the microgels along the incubation time was monitored by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Instruments). Samples were allowed to equilibrate at 37°C for 2 min before analysis.

Doxorubicin loading and release. Microgel dispersions at 2 mg.mL -1 in water and a DOXO solution at 1 mg.mL -1 in water were prepared and heated to 37°C using an oil-bath. Then, 500 µL of the microgel dispersion and 100 µL of the DOXO solution were mixed together at 37°C for 5 min and then the mixture was kept at 4°C for 24 h. Subsequently, microgel particles were separated from the aqueous solution containing free DOXO through centrifugation for 1 h at 15 000 rpm and × 100 Equation 2The in-vitro release of DOXO from selected microgels (1 mg.mL -1 ) was assessed by a dialysis method. Release of free DOXO was also performed as a control in order to take into consideration the simple diffusion of the free drug through the semipermeable membrane. DOXO-loaded SC1-PX76 4-E4, SC1-PX76 8-E8 and SC1-PX9 4-E4 microgel dispersions were dialyzed at 37 °C against two different serum-containing (10 % (v/v) FBS) buffer solutions: sodium acetate/acetic acid (SA) buffer solution (pH 5.5) and phosphate buffer saline (PBS) solution (pH 7.4). The DOXO release concentration in the release medium was determined spectrophotometrically at 488 nm. All experiments were performed in triplicate.

In vitro cell cytotoxicity assays. Cytotoxicity of bare PVCL-based microgels was evaluated on cervical HeLa and RAW 264.7 cell lines utilizing the Cell Counting Kit-8 (CCK-8) cytotoxicity assay. [START_REF] Chen | pH-responsive mechanism of a deoxycholic acid and folate comodified chitosan micelle under cancerous environment, The journal of physical chemistry[END_REF][START_REF] Jiang | DNA origami as a carrier for circumvention of drug resistance[END_REF] Both cell lines were seeded in 96 well-plates at a density of 5000 cells per well in 100 µL of growth medium (DMEM for HeLa cells and DMEM/F-12 for RAW cells, respectively) and allowed to grow for 24 h at 37°C. Then, the medium was removed and cells were exposed to 100 µL of various concentrations of cationic poly(N-vinylcaprolactam)-based microgels (Test 1:

[Microgel] = 0.005; 0.05; 0.1; 0.25; 0.5 and 1 mg.mL -1 -Test 2: [Microgel] = 0.5; 1; 2; 3.5; 5 and 10 mg.mL -1 ) dispersed in growing media for further incubation at 37°C in a 5% CO2 atmosphere for 24 and 48 h. After incubation, 10% CCK-8 solution per well was added and incubated for 1 h for RAW cell line and 90 min for HeLa cell line. The absorbance of viable cells was subsequently measured at 450 nm using an UV-Vis microplate absorbance reader (Microplate Manager 5.0, Bio-Rad Laboratories, USA). The non-treated cells served as a 100% cell viability control (blank).

Survival percentage or cell viability was calculated as follows:

% 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 𝐴𝑏𝑠 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 -𝐴𝑏𝑠 𝑚𝑖𝑐𝑟𝑜𝑔𝑒𝑙 𝐴𝑏𝑠 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 × 100 Equation 3
where Abstreated cells is the absorbance at 450 nm of cells incubated in the presence of microgels, and Absuntreated cells is the absorbance at 450 nm of control cells in the absence of microgel particles. The absorbance of microgels at 450 nm was substracted to the absorbance of treated cells for each microgel concentration.

The same procedure was followed to study the DOXO-loaded microgel cytotoxicity on RAW and HeLa cell lines after 24 h of incubation. Cells were exposed to various concentrations of DOXOloaded microgels: [DOXO-loaded microgel] = 2; 1; 0.5; 0.25 and 0.1 mg.mL -1 .

For each microgel, the concentration of DOXO (Table S4) was calculated according to equation 4 by using the microgel weight concentrations and the entrapment efficiency (see Table S2).

[𝐷𝑂𝑋𝑂](µ𝑀) = (

[𝑀𝑖𝑐𝑟𝑜𝑔𝑒𝑙] × 𝐸.𝐸 × 10 -3 𝑀 𝐷𝑂𝑋𝑂 ) × 10 6 Equation 4where E.E is the entrapment efficiency ( Equation 2) and MDOXO is the DOXO molar mass (MDOXO = 579.98 g.mol -1 ).

Cellular uptake by fluorescence microscopy. Prior to the cellular uptake experiments, the microgels were labeled with rhodamine 6G (λexc = 530 nm) according to the following procedure. Microgel dispersions at 1 mg.mL -1 in water and a solution of rhodamine 6G at 10 mg.mL -1 also in water were prepared and heated to 37°C in an oil-bath. Then, 1 mL of the microgel dispersion and 400 µL of the rhodamine solution were mixed together at 37°C and the mixture was kept at 4°C for 24 h.

Subsequently, microgel particles were separated from the aqueous solution containing free R6G through four successive centrifugations for 1 h at 15 000 rpm and 15°C. After centrifugations, the resulting pellet was re-dispersed in cell culture medium at the desired concentration. As a concentrated solution of R6G in water (10 mg.mL -1 ) was used, several centrifugations were required to separate the R6G-loaded microgels from free R6G. The cellular uptake of R6G-loaded microgels was followed by confocal fluorescence microscopy. HeLa cells were seeded on poly-(Llysine) coated glass coverslips (12 × 12 mm) placed inside 6 well-plates (15 × 10 4 cells/well with 3 mL of DMEM), and allowed to grow for 24 h at standard culture conditions. The growth medium was then removed, and 500 µL of a dispersion of R6G-loaded microgels in DMEM at a concentration of 0.5 mg.mL -1 and 2.5 mL in DMEM were added to each well. Cells were then incubated for 3 and 24 h, respectively. For energy-dependent uptake experiments at 4°C, cells were pre-incubated at 4°C with serum-free DMEM for 1 h, followed by incubation with 500 µL of R6Gloaded microgel dispersions in serum-free DMEM (at a concentration of 0.5 mg.mL -1 ) at the same temperature for 3 h. To inhibit a specific uptake mechanism of microgels into cells, these were preincubated with the following inhibitors individually for 1 h at 37°C: 400 mM hypertonic sucrose, 30 µM chlorpromazine, 30 µM cytochalasin and 30 µM nystatin, respectively. After the preincubation time, the inhibitor solutions were removed and the freshly prepared R6G-loaded microgel dispersed (at a concentration of 0.5 mg.mL -1 ) in media containing inhibitor at the same concentrations were added and further incubated for 3 h at 37°C. In this study, cells incubated for 3 h at 37°C in the presence of R6G-loaded microgel and in the absence of inhibitor were used as controls. For all the experiments described above, after the desired incubation times cell culture media were removed and the microgel-containing cells were washed three times with PBS (pH 7.4), fixed for 10 minutes with a solution of paraformaldehyde at 4 w/v% in PBS, washed with PBS, treated for 10 minutes with a solution of Triton X-100 (permeabilizer) at 0.25 (w/v)% in PBS and, finally, washed again with PBS. Coverslips were mounted on glass slides and cell nuclei were stained with 4'-6-diamidino-2-phenylindole dihydrochloride (DAPI, blue). The samples were cured at -20°C for 24 h before being visualized with an 63X oil-immersion objective using an inverted wide field fluorescence microscope (Leica DMI6000B, Leica Microsystems, Germany)

with the blue channel for DAPI-stained cell nuclei (λexc = 355 nm), the red channel for R6G (λexc = 590 nm) and the transmitted light channel in differential interference contrast (DIC) mode.

Results and discussion

1.

Colloidal stability of bare cationic PVCL microgels.

The stability in biological mimicking media of a series of poly(N-vinylcaprolactam) (PVCL) microgels surrounded by covalently anchored poly[2-(acryloyloxy)ethyl]trimethylammonium chloride (P(AETAC-X) cationic polymer shell is investigated for various colloids (Scheme 1). All these microgels dispersed in water will be named "bare microgels" in the following work. Their diameters at 37 °C range between 100 -200 nm (Table S2). Different physico-chemical parameters of the series of SC-PX-E microgels (Scheme 1) are controlled by their synthesis [START_REF] Etchenausia | Cationic Thermoresponsive Poly(Nvinylcaprolactam) Microgels Synthesized by Emulsion Polymerization Using a Reactive Cationic Macro-RAFT Agent[END_REF]: 1) the length of the cationic brushes, 2) the ratio of the cationic P(AETAC) versus PVCL, 3) the crosslinking density which can be tuned by either the initial crosslinker amount versus VCL or by the initial solids content of emulsion polymerization that influence the mesh size of the network. [START_REF] Etchenausia | Cationic Thermoresponsive Poly(Nvinylcaprolactam) Microgels Synthesized by Emulsion Polymerization Using a Reactive Cationic Macro-RAFT Agent[END_REF] For biomedical applications, the stability of the particles in biological media is of high importance since their particle size can affect their biodistribution and cell internalization and fate. [START_REF] Shang | Engineered nanoparticles interacting with cells: size matters[END_REF][START_REF] Hillaireau | Nanocarriers' entry into the cell: relevance to drug delivery[END_REF] The hydrodynamic diameters of the bare microgels were monitored for 13 days of incubation at 37°C to investigate their colloidal stability (Figure 1 and Figure S1 in Supplementary materials).

Microgels were dispersed (at 0.05 mg.mL -1 ) either in water (pH 6. In water, all microgels remained stable since their hydrodynamic diameters were almost constant during the whole incubation (13 days at 37°C). In contrast, an obvious increase of the microgel hydrodynamic diameter at the beginning of incubation was systematically observed in PBS buffer revealing a certain lack of microgel stability. Since the ionic strength of PBS buffer is around 150 mM, the increase of the microgel hydrodynamic diameters could be explained by the formation of aggregates due to the ionic screening of the particle electrical charges. Indeed, the reduction of the particle surface charges might lead to the microgel destabilization by decreasing the electrostatic repulsions between particles. The salt effect on microgel stability was also studied for SC5-PX76

4-E4 microgel in aqueous solution at different concentrations of KBr ranging from 10 mM to 100

mM. An increase of the microgel hydrodynamic diameters was observed above the microgel VPTT, suggesting the formation of aggregates (Figure S2 in Supplementary materials). Such colloidal instability and aggregation of positively charged carriers in fluids at high salt concentration is a well-known process, being one of the main drawbacks for their use, for example, as gene delivery systems. [START_REF] Yin | Non-viral vectors for gene-based therapy[END_REF][START_REF] Wiethoff | Barriers to nonviral gene delivery[END_REF] It should be noted that the aggregation of the cationic PVCL-based microgels is observed at their collapsed state (T > 30°C) but a similar value of Dh was measured at low temperature (swollen state) whatever the salt concentration (Figure S2 in Supplementary materials). The screened cationic polyelectrolyte does not provide then a sufficient protective layer against the hydrophobic interaction of the collapsed microgels.

In PBS supplemented with FBS, microgels are more stable than in the presence of pure PBS, at least at short incubation times (< 6 days, see for example, SC5-PX9 4-E4, SC1-PX9 4-E4; SC1-PX16 4-E4 and SC1-PX76 4-E4). In DMEM supplemented with FBS, the hydrodynamic diameters of SC1-PX76 8-E4, SC1-PX16 12-E4, SC1-PX76 4-E4, SC5-PX76 4-E4 and SC10-PX76 4-E4 microgels did not suffer any substantial changes for 6 days of incubation; interestingly, SC1-PX9 4-E4, SC5-PX9 4-E4, SC5-PX76 8-E4, SC1-PX76 8-E4 and SC1-PX16 4-E4 microgels do not undergo significant size change along the whole 13 days incubation period.

The origin of microgel destabilization in FBS supplemented media (either PBS or DMEM) could be the formation of aggregates of microgel particles as a consequence of the absorption of proteins onto the particle surfaces and the subsequent exchange kinetics between low affinity-highly abundant serum proteins and high affinity-less abundant ones. Indeed, in FBS the main constitutive plasma protein is bovine serum albumin (BSA), which is negatively charged at pH 7.4 (isoelectric point of BSA = 4.7). [START_REF] Van Tomme | Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching[END_REF] Since the microgel particles used in this work are positively charged, BSA could adsorb firstly and quick onto microgel surfaces by electrostatic attraction, increasing their size. [START_REF] Belmadi | Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration[END_REF] Moreover, the reduction of the microgel surface charge by adsorption of the protein molecules could lead to the formation of aggregates of microgel particles. [START_REF] Nuhn | Aggregation behavior of cationic nanohydrogel particles in human blood serum[END_REF][START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF] In addition to the adsorption of BSA through electrostatic attractions, hydrophobic interactions should also be considered in this process provided that it has been previously demonstrated that for poly(Nisopropylacrylamide)-based microgels protein adsorption was enhanced above the microgel VPTT as a result of the latter type of interactions between the protein and the hydrophobic collapsed microgel. [START_REF] Kawaguchi | Hydrogel microspheres III. Temperature-dependent adsorption of proteins on poly-N-isopropylacrylamide hydrogel microspheres[END_REF][START_REF] Trongsatitkul | Temperature dependence of serum protein adsorption in PEGylated PNIPAm microgels[END_REF] The higher stability of the microgels in FBS supplemented media (either PBS or DMEM) than in serum free one (PBS) might arise from salt aggregating effects being initially screened by the formation of a protein corona at the microgel surfaces. This process known as opsonization enables protein adsorption and recognition of foreign elements by macrophages and the reticulo-endothelial system (RES), allowing their clearance from the body. [START_REF] Belmadi | Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration[END_REF] Upon incubation, this protein corona might be destabilized through exchange dynamics with smaller ions or even other less abundant proteins possessing larger affinities, leading to a reduction of the microgel surface charge and, thus, to the particle aggregation/clustering.

On the other hand, it should be noted that there are not very clear specific trends between the colloidal stability of microgels and their structural features, but some qualitative observations can be made. For example, an increasingly amount of solids content in the microgel formulation seems to alter the colloidal stability, particularly in media supplemented with FBS at relatively long incubation times as observed when comparing microgels SC1-PX76 4-E4, SC5-PX76 4-E4 and SC10-PX76 4-E4 (Figure 1). Also, microgels bearing larger ratios of the polycation stabilizer undergo larger increments in their hydrodynamic sizes at 6-8 days of incubation pointing to a certain progressive loss of their colloidal stability, especially noted in the presence of FBSsupplemented media as observed when compared SC1-PX76 4-E4 and SC1-PX76 8-E4 or for SC1-PX16 4-E4 and SC1-PX16 12-E4 microgels (see Figure 1 and Figure S1). Conversely, no trends could be elucidated neither in terms of the stabilizer length nor the crosslinking ratio.

In summary, the incubation time should be carefully considered for an implementation of the cationic PVCL microgels in blood serum conditions. In serum supplemented media, the time along which these microgels remain stable is much longer than that required for routinely in-vitro studies (maximum 72h) [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF][START_REF] Standardization | Tests for in-vitro cytotoxicity[END_REF][START_REF] Xiong | Anti-tumor drug delivery system based on cyclodextrin-containing pH-responsive star polymer: in vitro and in vivo evaluation[END_REF][START_REF] Wang | Poly(vinylcaprolactam)-based biodegradable multiresponsive microgels for drug delivery[END_REF] or for potential in-vivo applications. In the present work, for experiments of microgel in-vitro cell cytotoxicity assays or internalization experiments microgels were used after dispersion in DMEM cell culture medium supplemented with FBS for a maximum of two days. Since in this medium the microgel hydrodynamic diameters remained constant for up to 6 days, microgel destabilization should not play a significant effect on the obtained results.

In vitro cell cytotoxicity assays of bare PVCL-based microgels

The cytotoxicity of bare PVCL-based microgels at different concentrations (from 0.005 to 10 mg.mL -1 ) was assessed on HeLa and RAW cell lines after 24 h and 48 h of incubation, using the Cell Counting Kit-8 (CCK-8) cytotoxicity assay. The cell viabilities for the different microgels (expressed as cell viability percentage) are reported as a function of the microgel concentration for HeLa and RAW cell lines at the different incubation times (Figure 2 and Figure S3 in Supplementary materials). The series of microgels achieve significant cell viabilities in both types of cells with a dosedependent behavior. A similar dose-dependent cytotoxicity was also observed for other PVCLbased nano/microgels [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF] or PVCL-based copolymers. [START_REF] Prabaharan | Thermosensitive micelles based on folateconjugated poly(N-vinylcaprolactam)-block-poly(ethylene glycol) for tumor-targeted drug delivery[END_REF][START_REF] Shah | Synthesis and characterization of thermo-responsive copolymeric nanoparticles of poly(methyl methacrylate-co-N-vinylcaprolactam)[END_REF] After 24 h of incubation, the growth inhibition observed in both HeLa and RAW cells lines is almost negligible for microgel concentrations up to 1 mg.mL -1 , and even for macrophages some additional proliferation compared to the control cells is noted. Moreover, cell viabilities for both cell lines were above 70% for microgel concentrations between 0.005 and 2 mg.mL -1 indicating that almost all of the microgels have a good biocompatibility. [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF][START_REF] Cavet | Comparison of the effect of multipurpose contact lens solutions on the viability of cultured corneal epithelial cells, Contact lens & anterior eye : the journal of the British Contact Lens[END_REF] After 48 h of incubation, a slight additional reduction of cell viability for microgels incubated with HeLa cells occurs, and this decrease is larger in the case of RAW macrophages. The observed larger cytotoxicity of microgels at concentrations > 2 mg.mL -1 and particularly, at the largest one 10 mg mL -1 , can be originated from an excess of microgel particles producing a damage to cells leading to their death. [START_REF] Slowing | Mesoporous silica nanoparticles for reducing hemolytic activity towards mammalian red blood cells[END_REF] Indeed, this overpopulation might involve membrane-cell disruption due to the high amount of nanoparticles in the medium interacting with the cells and/or large amounts of particles being internalized into cells, hence disrupting the cell cytoplasm (for example, by the formation of a high number of vacuoles), altering the cell metabolic activity and, thus, activating the programed cell death cascade pathways. In this regard, it is worth mentioning that cationic particles are usually more toxic to cells than neutral or negatively charged ones due to the stronger electrostatic interactions with the cell surface, hence, causing larger cellular membrane disruption effects (alteration of the anionic lipidic and protein domains). Herein, for microgel concentrations above 2 mg.mL -1 the cell viability is also lower for RAW cells than for HeLa ones, particularly after 48 h of incubation. This result was consistent with the fact that RAW macrophages uptake and internalize very efficiently any foreign element in the body. Thus, for nanosystems whithout a specific targeting, assessing the cytotoxicity on such cell line gives the ability of microgels to be uptaken by the reticulo-endothelial system. It is wellknown that the toxicity of a particle is size-dependent [START_REF] Podila | Toxicity of engineered nanomaterials: a physicochemical perspective[END_REF][START_REF] Naahidi | Biocompatibility of engineered nanoparticles for drug delivery[END_REF] and, for cationic systems, toxicity also depends on cation content. [START_REF] Garnett | Gene-Delivery Systems Using Cationic Polymers, Critical Reviews™ in Therapeutic Drug Carrier Systems[END_REF][START_REF] Mcallister | Polymeric Nanogels Produced via Inverse Microemulsion Polymerization as Potential Gene and Antisense Delivery Agents[END_REF] In this work, whatever the fraction of the cationic stabilizer in the microgel (4 to 12 wt%) or their initial hydrodynamic diameter at 37°C (see Table S2), all microgels are biocompatible (cell viabilities above 50%) in the range up to 2 mg.mL -1 , except for RAW cells after 48 h of incubation as a consequence of their extensive uptake. The incubation time-dependent cytotoxicity has already been observed for other PVCL-based microgels [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF] and PVCL-based polymers [START_REF] Vihola | Cytotoxicity of thermosensitive polymers poly(N-isopropylacrylamide), poly(N-vinylcaprolactam) and amphiphilically modified poly(Nvinylcaprolactam)[END_REF] This might be due to further microgel accumulation around/within cells promoting their death. In addition, the chain length of the cationic polymer used as stabilizer for the PVCL microgel synthesis has no relevant impact on the microgel cytotoxicity as similar percentages of cell viability were obtained after incubation of cells with SC1-PX76 4-E4 and SC1-PX9 4-E4 microgels (Figure 2 and Figure S3 in Supplementary materials), and even for RAW264.7 cells a slight enhancement can be observed at low microgel concentrations (0.005-0.5 mg mL -1 ) after 24 h of incubation for the microgel with the largest cationic stabilizer. This is in contrast to previous results showing that the cytotoxicity of the nanosystems increased with the chain length of the cationic polymer. [START_REF] Zhang | Differential expression of syndecan-1 mediates cationic nanoparticle toxicity in undifferentiated versus differentiated normal human bronchial epithelial cells[END_REF][START_REF] Cai | Effect of Chain Length on Cytotoxicity and Endocytosis of Cationic Polymers[END_REF][START_REF] Sahariah | Impact of chain length on antibacterial activity and hemocompatibility of quaternary N-alkyl and n,n-dialkyl chitosan derivatives[END_REF] This can be explained by the fact that, whatever the chain length of the P(AETAC-X) used as stabilizer for the synthesis of PVCL-based microgels, the final microgels presented similar values of the electrophoretic mobility (SC1-PX76 4-E4, electrophoretic mobility = 0.42  10 -8 m 2 /Vs in cationic buffer at pH 3 and 10 mM ionic strength, and SC1-PX9 4-E4, electrophoretic mobility = 0.44  10 -8 m 2 /Vs in cationic buffer at pH 3 and 10 mM ionic strength). Finally, just to mention that the extent of microgel crosslinking does not have influence on microgel cell toxicity except at the highest concentrations (> 5 mg mL -1 ) at which the most cross-linked microgels (SC1-PX76 8-E4 and SC1-PX76 8-E8, Figure S3) induce larger toxicities maybe related to a lower particle elasticity as reported elsewhere. [START_REF] Anselmo | Elasticity of Nanopartides Influences Their Blood Circulation, Phagocytosis, Endocytosis, and Targeting[END_REF] 3. Monitoring cellular uptake of microgels and uptake pathway.

Prior to their cellular uptake characterization by means of fluorescence microscopy, the microgels were labeled with the rhodamine 6G cationic dye. Figure 3 shows fluorescence images of the R6Gloaded microgel cellular uptake in HeLa cells after 3 h of incubation at 37°C. For most of the tested microgels, the DAPI-stained cell nuclei (blue) were surrounded by a diffuse rhodamine fluorescence (red) pattern suggesting a successful cell internalization of the microgels at short time and the subsequent sustained dye release inside the cell cytoplasm. [START_REF] Hoppert | Microscopic Techniques in Biotechnology[END_REF] The cationic dye was truly located at the cell cytoplasm and/or in some intracellular compartments, especially endoplasmic reticulum and/or mitochondria, for which R6G is a specific marker. In addition, some few observed pink colored nuclei would indicate overlapping of the fluorescence signal corresponding to microgels/dye located over the nuclei, as observed in the 2D projections (the dye used is not permeable to the nucleus membrane). The presence of microgel clusters was also visualized via the presence of red dotted spots in the images as a result of microgel aggregation when in contact to the biological medium as a consequence of their positive charges.

At longer incubation times (24 h, Figure S4 in Supplementary materials), rather similar fluorescence patterns as at 3 h were observed, that is, the DAPI-stained cell nuclei were surrounded by rhodamine fluorescence, suggesting the successful internalization and residence of the microgels/cargo inside cells. The fluorescence intensity of the dye was maintained constant after 24 h of incubation in agreement with a sustainable release of rhodamine 6G from the microgels along time, although for some microgels a very small decrease in fluorescence intensity, indicating a certain expulsion of the dye from the cell. However, this point would deserve further studies to rule out any possible contribution from dye quenching and/or degradation. In addition, from a qualitative analysis no relevant differences in uptake levels were noted for the different microgels in terms of their cross-linking ratio, stabilizer chain length and/or initial solids content of their synthesis. On the other hand, it should be highlighted that for high microgel accumulation levels inside cells, some few with damaged membranes could clearly be observed confirming the aforementioned statement (Figure S5 in Supplementary materials). Such loss of cell membrane integrity was already observed for cells in contact with other highly cationic nanomaterials. [START_REF] Zhang | Differential expression of syndecan-1 mediates cationic nanoparticle toxicity in undifferentiated versus differentiated normal human bronchial epithelial cells[END_REF] In the present work, much less cells with damaged membrane were observed at 3 h of incubation rather than at 24 h. This confirms that the microgel accumulation process into cells is timedependent, as already been observed with other PVCL-based microgels. [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF][START_REF] Wang | Poly(vinylcaprolactam)-based biodegradable multiresponsive microgels for drug delivery[END_REF][START_REF] Wang | Acid degradable poly(vinylcaprolactam)-based nanogels with ketal linkages for drug delivery[END_REF] In order to elucidate how microgels can be internalized into cancerous cells, inhibition experiments of different cell uptake pathways were performed. Endocytosis (the vesicular uptake of extracellular macromolecules) has been established as the main mechanism for the internalization of non-viral vectors into cells. [START_REF] Oliveira | Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases[END_REF] Endocytosis can be classified into two broad categories:

phagocytosis (for specialized mammalian cells) and pinocytosis (all cells). Among pinocytic pathways four main mechanisms can be distinguished: clathrin-mediated endocytosis, caveolae, macropinocytosis and clathrin/caveolae-independent endocytosis (Figure S6 in Supplementary materials). Internalization via endocytosis can be inhibited using different cell treatments. A general procedure for hindering endocytic pathways consists in lowering the temperature as endocytosis is an energy-dependent mechanism. The microgel internalization was studied at 4°C in order to assess if the internalization mechanism is a chemical-mechanical energy dependent-or a simple diffusion process. The fluorescence images of the R6G-loaded microgel cellular uptake in HeLa cells after 3 h of incubation at 4°C are depicted in Figure 4. For the three selected PVCL microgels, the internalization process took place satisfactorily at 4º C, disregarding an energy-dependent uptake pathway for the dye-loaded microgels. Indeed, the R6G release occurred into the cell cytoplasm for all the microgels. Moreover, in some cases, it seems that some few microgel clusters might be surrounding the cell nuclei, as observed by the reconstructed fluorescence image of SC1-PX76 4-E4 microgel in HeLa cell line after 3 h of incubation at 4°C (Figure S7 in Supplementary materials). Nevertheless, further experiments would be required to definitively confirm this point. At 4°C, R6G was also released at the cell surroundings (for SC1-PX9 4-E4 microgel for instance, Figure 4c). For these experiments, it is worth also mentioning the cell density was observed to be slightly lower than for uptake experiments performed at 37°C, probably as a consequence of some cellular death and their subsequent detachment from the well surface during the washing process. In order to understand which specific protein/mechanism is other involved in the particle incorporation within the cells, it is possible to inhibit specifically a certain endocytic pathway by adding a specific inhibitor for such route. [START_REF] Khalil | Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery[END_REF] Here, the cellular uptake mechanism of several microgels was investigated using four different inhibitors: two different clathrin-mediated endocytosis specific inhibitors (hypertonic sucrose and chlorpromazine (CPZ)), an inhibitor of macropinicytosis/phagocytosis (cytochalasin D), and an inhibitor of caveolae (nystatin). These inhibitors act dissociating the clathrin lattice, actin and/or caveolae, respectively, impeding the corresponding endocytic pathways. Figure 5 and Two other additional internalization routes, macropinocytosis/phagocytosis and caveolae, were additionally analyzed by fluorescence microscopy using the SC1-PX76 8-E8 microgel. Both cytochalasin D, a polymerization inhibitor of actin which is a protein largely involved in the formation of cell skeleton and mobility, and nystatin, a sterol-binding agent which disassembles caveolae, were used. In these cases, a good level of internalization of the microgels within cells could be observed suggesting that the inhibition of these both routes do not impede the incorporation of the microgel into cells (see Figure S8 and Figure S9 in Supplementary materials).

Finally, a quantification of the extent of internalization inhibition for microgels SC1-PX76 4-E4, SC1-PX76 8-E8 and SC1-PX9 4-E4 loaded with the anticancer DOXO at a concentration of 20 M using the different inhibiting compounds was performed using HeLa cells by evaluating the cell viability by the CCK-8 assay (see part 4 for details of DOXO encapsulation and related efficiency).

A maximum cell survival is observed when the clathrin-dependent endocytic pathway is inhibited, either with sucrose or chlorpromazine (CPZ) (Figure 7), which confirms the previous fluorescence microscopy analysis. For cytochalasin D, some increase in viability is observed compared to the control experiment thanks to a certain involvement of cytochalasin D in the clathrin-dependentpathway. [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF] For nystatin, cell viabilities were similar as the control experiments, i.e. in the absence of any inhibitor, demonstrating that the macropinicytosis/phagocytosis and caveolae are not the main internalization routes of the present cationic thermoresponsive PVCL microgels.

In vitro cell cytotoxicity assays of DOXO-loaded microgels

The loading of doxorubicin was performed for some selected microgels, SC1-PX76 4-E4, SC1-PX76 8-E4, SC5-PX76 4-E4, SC1-PX76 8-E8, SC1-PX9 4-E4 and SC1-PX16 4-E4 (Table S1). The chosen drug loading method (see Materials and Methods section) takes advantages of the sponge like nature of the microgels, allowing solute molecules to partition into the porous particle network. [START_REF] Qiu | Environment-sensitive hydrogels for drug delivery[END_REF] DOXO can be incorporated into microgels through electrostatic and/or hydrophobic interactions. [START_REF] Shi | Intracellular microenvironment responsive PEGylated polypeptide nanogels with ionizable cores for efficient doxorubicin loading and triggered release[END_REF] The drug loading (D.L.) and entrapment efficiency (E.E.) were calculated according to Equation 1 and Equation 2, respectively, and the values obtained for the different microgels are presented in Table S3 (Supplementary materials). The values of drug loading and entrapment efficiency are relatively low compared to the values obtained for other PVCL-based nano/microgels, [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF][START_REF] Wang | Poly(vinylcaprolactam)-based biodegradable multiresponsive microgels for drug delivery[END_REF][START_REF] Wang | Acid degradable poly(vinylcaprolactam)-based nanogels with ketal linkages for drug delivery[END_REF][START_REF] Lou | Temperature/pH dual responsive microgels of crosslinked poly(N-vinylcaprolactam-co-undecenoic acid) as biocompatible materials for controlled release of doxorubicin[END_REF]. Since the drug loading process was carried out at pH 6.5 for which DOXO is positively charged (pKa = 8.3), [START_REF] Sanson | A simple method to achieve high doxorubicin loading in biodegradable polymersomes[END_REF] electrostatic repulsions between the drug and the cationic PVCL-based microgels might limit the drug encapsulation. Moreover, electrostatic repulsions among DOXO molecules could also reduce the entrapment efficiency. Also, as EGDMA crosslinker is relatively hydrophobic compared to others, it might hinder the penetration of DOXO molecules into the microgel network (which is used in its water soluble hydrochloride salt form). [START_REF] Imaz | N-vinylcaprolactam-based microgels for biomedical applications[END_REF] Aguirre et al. [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF] studied the loading of DOXO into poly(2diethylaminoethyl)methacrylate (PDEAEMA)-based microgels and PDEAEMA-based core and PVCL-based shell microgels at acidic pH for which both microgels and DOXO were positively charged. As they used a dextran-based macro-crosslinker, the main driving force to encapsulate DOXO into the microgels was the interaction by H-bonding between the -OH groups of DOXO and the -OH groups of the dextran chains. Thus, a high encapsulation efficiency of the drug could be reached (i.e. ranging from 80 to 90%), even in the presence of electrostatic repulsions between the microgels and the drug. In the present work, at the DOXO concentration used (1 mg.mL -1 ), the levels of entrapment efficiency and drug loading are in a similar range for all the different microgels. Therefore, the structure of the different particles tested does not seem to influence the amount of DOXO encapsulated into the network.

The cumulative DOXO release of the microgels was also studied. DOXO-loaded SC1-PX76 4-E4, SC1-PX76 8-E8 and SC1-PX9 4-E4 microgel dispersions were dialyzed at 37 °C against different serum-containing (10% (v/v) FBS) solutions. Sodium acetate/acetic acid (SA) buffer solution (pH 5.5) was chosen in order to simulate the extracellular environment of tumoral cancer cells, and phosphate buffer saline (PBS) solution (pH 7.4) was selected to mimic blood serum conditions. Note that at 37°C the microgels are collapsed (T > VPTT, see Table S2). The in-vitro cumulative DOXO release at 37°C under both pHs is depicted in Figure 8. It is well-known that the drug release rate from microgels is theoretically affected by different factors such as the drug diffusion through the microgel particles and the extent of swelling of microgels. In the present case, at pH 7.4 the three selected SC1-PX76 4-E4, SC1-PX76 8-E8 and SC1-PX9 4-E4 microgels displayed a clear burst release phase within the first 7 h of incubation where ca. 29, 24 and 19% of the drug is released, followed by a short pseudo-plateau region between ca. 7 and 10 h. After this incubation period, the release is slightly accelerated to depict a very sustained release pattern at longer incubation times (> 15h), with total cargo releases between 30-40% (depending on the microgel) within the time framework of the present experiments (ca. 37 h). In contrast, under acidic conditions rather similar release patterns with stair-like profiles can be observed and with larger drug releases than at pH 7.4. A burst phase is again observed at short incubation times followed by a slowing of the release rate for a short period (more than a true pseudo-plateau region as observed at physiological pH). After ca. 9-10 h of incubation an additional abrupt increase in the cumulative release is noted up to ca. 18 h at which ca. 75-80% of the cargo is released; thereafter, a plateau region is reached along in which ca. 90% of the drug is already expelled from the microgels. The presence of an initial burst phase in the release profiles of the present microgels might be related to the adsorption of drug molecules located in the outer layer of the particles, which are released at first; meanwhile, the more sustainable release phase would be controlled by the rate of DOXO release from the microgel core thanks to the hindered diffusion from the highly cross-linked inner part of the microgels. The presence of a slight pseudoplateau/reduction of the release rate between 7-10 h of incubation in Figure 8 might be related to the difference of crosslinking density within the microgel, with a more densely crosslinked core. [START_REF] Etchenausia | Cationic Thermoresponsive Poly(Nvinylcaprolactam) Microgels Synthesized by Emulsion Polymerization Using a Reactive Cationic Macro-RAFT Agent[END_REF] In addition, important differences in the release extents between acidic and basic physiological conditions can be observed. At this point, it is important to note that the cationic stabilizer used for the synthesis of the microgels is permanently charged and, thus, insensitive to pH changes. Therefore, the larger cumulative releases observed at pH 5.5 might be attributed to the fact that, at acidic conditions, amine groups of DOXO molecules are protonated (i.e. positively charged). As the hydrophilicity of DOXO molecules is enhanced at acidic pH, the affinity of the drug with the microgel hydrophobic core might be reduced, enhancing the diffusion out of the particle. [START_REF] Cambon | Doxorubicin-loaded micelles of reverse poly(butylene oxide)poly(ethylene oxide)-poly(butylene oxide) block copolymers as efficient "active" chemotherapeutic agents[END_REF] Finally, no remarkable differences in DOXO release kinetics were observed between the three selected microgels. This would hence suggest that neither the extent of polymer stabilizer nor the amount of crosslinker would have a strong impact on the release profiles, at least in the range within the tested particles. As the interactions were mainly non-covalent, it was expected that the delivery would be governed by diffusion, which is affected by the swelling capability of the microgels and their interactions with the drug (especially electrostatic repulsion at acidic conditions). Since the used microgels showed very similar swelling-deswelling behavior, an analogous DOXO release kinetics was observed in all cases.

Cell viability of DOXO-loaded microgels was then studied at different microgel concentrations (from 0.1 to 2 mg.mL -1 ), thus at different DOXO concentrations, in both HeLa and RAW cell lines after 24 h of incubation (Figure 9). For each microgel, the concentration of DOXO was calculated from the microgel weight concentration and the entrapment efficiency (see Table S3) according to Equation 4. The results are given in Table S4. For HeLa cells, the IC50 (i.e, the concentration of drug required to reduce cell growth by 50%) of free DOXO after 24 h of incubation was 5.5 µM (see Supplementary materials). Therefore, for all microgels at concentrations above 0.1 mg.mL -1 , the DOXO concentrations loaded inside are above the minimum DOXO concentration providing cytotoxicity activities in vitro. The bare microgels were biocompatible and did not inhibit growth of both types of cell lines in the range of the studied microgel concentrations (0.1 to 2 mg.mL -1 ) as mentioned previously. In contrast, DOXO-loaded microgels exhibit higher cytotoxicity due to the effect of the drug (Figure 9). As expected, DOXO-loaded microgel cytotoxicity is dose-dependent as cell viability decreases by increasing the concentration of DOXO-loaded microgels and, hence, by increasing the concentration of DOXO. For the highest DOXO concentration used, the decrease of cell viability in the presence of DOXO-loaded microgels was more pronounced in the case of HeLa cells than for RAW ones. This result could be explained by the fact that RAW cells proliferate quicker than HeLa cells leading to a higher percentage of viable cells at the same percentage of dead cells. Additionally, HeLa cells are more sensitive to DOXO-loaded microgels as a result of their higher metabolic activity, which may lead to larger particle uptakes (confirmed by the cellular uptake experiments presented previously), and, hence, allowing a larger DOXO concentration inside the cells. For a given DOXO concentration, the level of cytotoxicity is in the same range for all the series of microgels, without relevant apparent influence of their structure and/or compositons in agreement with previous observation, and with an expected increase of cytotoxicity by increasing the DOXO concentration. It should be noted that the viability of HeLa cells is higher when incubated in the presence of DOXO-loaded microgels than with free DOXO as a consequence of the much more sustained release of DOXO from the microgels. Indeed, and as observed for other PVCL-based microgels, the release of the drug from the microgels to the cells through diffusion is a timedependent and sustained process. [START_REF] Aguirre | Biocompatible stimuliresponsive nanogels for controlled antitumor drug delivery[END_REF][START_REF] Wang | Poly(vinylcaprolactam)-based biodegradable multiresponsive microgels for drug delivery[END_REF][START_REF] Lou | Temperature/pH dual responsive microgels of crosslinked poly(N-vinylcaprolactam-co-undecenoic acid) as biocompatible materials for controlled release of doxorubicin[END_REF]

Conclusions

In summary, a series of biocompatible thermo-responsive microgels with covalently anchored cationic shells were synthesized directly in water by emulsion polymerization. The stability of the bare PVCL-based microgels was studied after incubation for 13 days at 37°C in different biological mimicking media revealing that the incubation time before aggregation should be carefully considered. Cytotoxicity assays of the bare PVCL-based microgels on HeLa and RAW cells indicated that all the microgel particles were biocompatible for concentration ranging from 0.005 to 2 mg.mL -1 . The inhibition of cell's growth was both a time and a dose-dependent process. The successful cellular uptake of the microgels is time-dependent as well as the release of R6G into the cell cytoplasm, with an apparently sustained release profile of the dye from the microgels into the cells. The mechanism of internalization of microgels and the interactions between microgels and HeLa cells was investigated through blocking different endocytic uptake pathways. It could be observed that inhibition of clathrins gave rise to a reduction in the extent of internalization of R6Gloaded microgels demonstrating that this pathway was one of the main mechanisms responsible for microgel internalization. Finally, doxorubicin anticancer model drug was loaded into some cationic core-shell thermoresponsive PVCL microgels. The DOXO-loaded microgels exhibited higher cytotoxicities on HeLa cells than bare PVCL microgels due to the effect of the drug. At similar initial DOXO concentrations and for incubation at 37°C for 24 h, the viability of HeLa cells was slightly higher when incubated in the presence of DOXO-loaded microgels than with free DOXO as a consequence of the sustained release of DOXO from the microgels, confirming the suitability of the cationic PVCL-based microgels as chemotherapeutic drug delivery nanocarriers.

Scheme 1 .

 1 Scheme 1. Structures of thermoresponsive PVCL-based microgels decorated with covalently

  [START_REF] Wilczewska | Nanoparticles as drug delivery systems[END_REF] or in three different biological mimicking media: i) bare PBS (pH 7.4), ii) PBS supplemented with 10% FBS (pH 7.4), iii) cell culture medium (DMEM, pH 8.4) supplemented with 10% FBS. PBS and PBS supplemented with FBS mimicks somehow the physiological serum conditions of increasing complexity whereas DMEM supplemented with FBS corresponds to the cell culture medium used for the in-vitro studies (cytotoxicity and cell internalization).

Figure 1 .

 1 Figure 1. Temporal evolution of the hydrodynamic diameter of bare selected microgels at 37°C,

Figure 2 .

 2 Figure 2. Cell viability of: () HeLa and () RAW cell lines after incubation at 37°C with different concentrations of microgels. Full line: 24 h of incubation; Dotted line: 48 h of incubation.

Figure 3 .

 3 Figure 3. Fluorescence microscopy images of R6G-loaded microgels in HeLa cells after 3 h of incubation at 37°C: (a) SC1-PX76 4-E4, (b) SC1-PX76 8-E4, (c) SC5-PX76 4-E4, (d) SC1-PX76 8-E8, (e) SC1-PX16 4-E4, (f) SC1-PX9 4-E4 microgels in HeLa cells stained with DAPI after 3 h of incubation at 37°C. Differential interference contrast (DIC) images (upper left side), DAPI-stained

Figure 4 .

 4 Figure 4. Fluorescence microscopy images of R6G-loaded microgels: (a) SC1-PX76 4-E4, (b) SC1-PX76 8-E8, (c) SC1-PX9 4-E4 microgels in HeLa cells stained with DAPI after 3 h of incubation at 4°C.

Figure 6

 6 Figure 6 show the fluorescence images of the R6G-loaded microgel cellular uptake in HeLa cells

Figure 5 .

 5 Figure 5. Fluorescence microscopy images of R6G-loaded microgels: (a) SC1-PX76 4-E4, (b) SC1-PX76 8-E4, (c) SC1-PX76 8-E8, (d) SC1-PX9 4-E4 microgels in HeLa cells stained with DAPI after 3 h of incubation at 37°C with hypertonic sucrose medium.

Figure 6 .

 6 Figure 6. Fluorescence microscopy images of R6G-loaded microgels: (a) SC1-PX76 4-E4, (b) SC1-

Figure 7 .

 7 Figure 7. Cellular survival rate after inhibition of different uptake pathways (Maximum uncertainty within  12 %).

Figure 8 .

 8 Figure 8. Cumulative DOXO release of () SC1-PX76 4-E4, () SC1-PX76 8-E8 and () SC1-

Figure

  S10 reports the cell viability of HeLa cells after incubation for 24 h with free DOXO and the different DOXO-loaded microgels in the same range of initial loaded DOXO concentrations (from 3 to 30 µM).

Figure 9 .

 9 Figure 9. Cell viability of: () HeLa and () RAW cell lines as a function of DOXO concentration after 24 h of incubation at 37°C.

  Cervical cancer HeLa cell line was obtained from Cell Biolabs and RAW 264.7 cell line from ATCC, Manassas, Virginia.

	N-Vinylcaprolactam (VCL, Sigma Aldrich, 98%), ethylene glycol dimethacrylate (EGDMA,
	Synthesis of PVCL-based microgels.
	Sigma Aldrich, 98%), potassium persulfate (KPS, Sigma Aldrich, 99%), sodium bicarbonate
	(NaHCO3, Sigma-Aldrich, 99.7%), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride
	(ADIBA or VA-044, Wako, 99%), 4,4'-azobis(4-cyano)pentanoic acid (ACPA, Fluka, 98 %),
	cetyltrimethylammonium	bromide	(CTAB,	Sigma	Aldrich,	99%),

tris(hydroxymethyl)aminomethane hydrochloride (TRIZMA, Sigma Aldrich, 99%) and 1,3,5trioxane (Sigma Aldrich, 99%) were used as received.

[2-(acryloyloxy)

ethyl]trimethylammonium chloride solution (AETAC, Arkema, 98 wt-% in water) was passed under inhibitors remover (Sigma Aldrich, 0.1 g in order to purify 50 mL of monomer) before use. Double deionized (DDI) water was used for the microgel synthesis. Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F-12), sodium pyruvate solution (100 mM) and non-essential amino acid solution were purchased from Life Sciences. Fetal Bovine Serum (FBS), Cell Counting Kit-8 (CCK-8), chlorpromazine hydrochloride (98%), phosphate buffered saline (PBS, 10× concentrate, BioPerformance Certified, suitable for cell culture, NaCl 0.137 M, KCl 0.0027 M, Na2HPO4 0.01 M, KH2PO4 0.0018 M), triton X-100, doxorubicin hydrochloride (98-102%, Scheme 1) and fluorescent dye rhodamine 6G (R6G, red, 99%, Scheme 1) were obtained from Sigma Aldrich. Trypsin-EDTA (0.25%), phenol red and penicillin-streptomycin (10,000 U/mL) were supplied by Gibco, ThermoFisher Scientific. D (+)-Sucrose (99%, RNAse and DNAse free) was obtained from Acros Organics. Paraformaldehyde was acquired from Fisher Scientific. 4'-6-diamidino-2-phenylindole dihydrochloride, ProLong Gold antifade (DAPI, blue) was supplied by Invitrogen.

PVCL-based microgels were synthesized following the previously reported

[START_REF] Etchenausia | Cationic Thermoresponsive Poly(Nvinylcaprolactam) Microgels Synthesized by Emulsion Polymerization Using a Reactive Cationic Macro-RAFT Agent[END_REF]

by batch emulsion copolymerization of N-vinylcaprolactam (VCL) monomer and ethylene glycol dimethacrylate (EGDMA) crosslinker stabilized by a reactive poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) P(AETAC-X) which was synthesized by controlled radical polymerization (see details of microgel synthesis and characterization in Electronic Supplementary Materials, Table

). Drug loading (D.L.) and entrapment efficiency (E.E.) were calculated using the following equations:

Acknowledgements


Luca Petrizza

Mickael Le Bechec

Hind Emile Decompte

El Hadri

Sylvie Lacombe
email: sylvie.lacombe@univ-pau.fr

Maud Save
email: maud.save@univ-pau.fr

Tuning photosensitized singlet oxygen production from microgels synthesized by polymerization in aqueous dispersed media

Table of content entry Miniemulsion copolymerization of vinyl acetate, N-vinylcaprolactam, vinyl benzyl Rose Bengal and divinyl adipate to synthesize switchable photosensitizer-grafted polymer colloids for interfacial photooxygenation reactions.

Latent colloids Irradiation  LED = 515 nm Photoactive microgels Quantum yield of singlet oxygen ( 1 O 2 ) production   EtOH

Introduction

Singlet oxygen production by photosensitization is a green chemistry process that takes advantage of oxygen from air as reactant and of visible light to excite a photosensitizer (PS).

Singlet oxygen is a powerful selective oxidant, used for Photodynamic Therapy (PDT), 1-3 for the treatment/disinfection of environmental pollutants, 4,5 or for fine chemical synthesis. 6 The covalent grafting of PS onto polymer substrates has been extensively investigated as a strategy to transpose poorly soluble photosensitizers in solvent (or in water) while enhancing their stability and recycling. A large part of the literature is devoted to the field of PDT with studies describing multi-step syntheses of either star-like or block copolymers, [7][8][9][10][11][12][13] and the design of hydrogels. [14][15][16] It should be mentioned that the block copolymer self-assembly into micelles as nanoparticles dispersed in water required the systematic use of an organic co-solvent. On the other hand, photosensitizing polymer beads, mainly based on polystyrene and/or poly(vinyl benzyl chloride) polymers, with large diameters above 5 µm, have been extensively studied as support for interfacial production of singlet oxygen to promote photooxygenation reactions. 17- 26 Polymer microbeads of 3-27 µm diameter were also prepared by thiol-ene photopolymerization and capped with C60 photosensitizer. 27 The singlet oxygen production mainly occurs at the surface of such large beads which are prone to sedimentation.

In the growing area of continuous-flow photochemistry implemented in microreactors for fine chemical oxidation, [28][29][30][31] a potential barrier to the use of the large photosensitizer-grafted polymer beads is their fast sedimentation rate and low specific surface area. It is thus important to develop a straightforward synthesis of stable submicronic polymer particles covalently grafted by an efficient organic photosensitizer.

Herein, we focus our attention on the covalent bonding of the Rose Bengal photosensitizer with the polymer to limit any catalyst leaching and to design robust organic photocatalysts suitable for photo-oxygenation of fine chemicals. Singlet oxygen is an elusive species with lifetime depending on its surrounding medium (16 µs and 3.3 µs in ethanol and water respectively), so its diffusion length is highly dependent on the solvent. 6,[START_REF] Boix-Garriga | Singlet Oxygen: Applications in Biosciences and Nanosciences[END_REF] At solid/liquid interface, singlet oxygen diffusion length is limited to several tens of nanometers. [START_REF] Ogilby | [END_REF]34 Consequently, the reagents to be oxidized have to be in the vicinity of the photosensitizer to enhance the efficiency of the photosensitized oxygenation. Moreover, due to solubility issues, relevant photo-oxygenation reactions of fine chemicals such as -terpinene or furfural have to be performed in organic solvents, among which alcoholic solvents are the less hazardous for any application. 6,35 To address these challenges, we describe here for the first time the synthesis of submicronic photosensitizing crosslinked colloidal particles by one pot miniemulsion copolymerization.

These PS-grafted microgels are designed to exhibit swelling capacity in ethanol and should present several advantages: enhancement of reactant diffusion during the photooxygenation process, increase of the exchange surface due to their sub-micronic size, colloidal stability in the absence of sedimentation. Polymerizations in aqueous dispersed media (emulsion, miniemulsion, dispersion) are environmentally friendly processes of interest to synthesize stable dispersions of submicronic polymer colloids in one step in the absence of solvents. 36,37 Although polymer particles can be directly synthesized in alcoholic media by dispersion polymerization, this implies the choice of polymers that precipitate in alcohol preventing their swelling in ethanol. To the best of our knowledge, the development of polymer microgels with covalent anchoring of organic photosensitizer is very scarce. In parallel with the synthesis of crosslinked nanocapsules from sacrificial silica core embedding thionine photosensitizer, Shiraishi et al. described the synthesis of poly(N-isopropylacrylamide-co-vinyl benzyl chloride) microgels by precipitation polymerization, post-derivatized by thionine. 38 Sensitized oxygenation activity via the turnover number was estimated with the transformation of phenol to p-benzoquinone. 38 Lyon et al. synthesized Rose Bengal-grafted PNIPAM-based microgels with hydrodynamic diameter ranging between 90-600 nm, but the singlet oxygen production of these colloids was not investigated at all. 39 Polyacrylamide hydrogel nanoparticles of 50-60 nm grafted with methylene blue were reported by Kopelman et al. for PDT purpose but the synthesis was performed by inverse microemulsion with 300 wt-% of surfactant versus monomers and hexane as organic continuous phase. 40 In the present work, the commercially available Rose Bengal (RB) was selected as photosensitizer due to both its high value of singlet oxygen quantum yield in water ( = 0.75) and ethanol ( = 0.68-0.80) 41 and its reactivity towards vinyl benzyl chloride (VBC) to produce a polymerizable vinyl benzyl Rose Bengal (VBRB) monomer. 42,43 Statistical copolymerization of VBRB with N-vinylcaprolactam (VCL), vinyl acetate (VAc), and divinyl adipate (DVA) crosslinker is implemented to covalently anchor RB to the crosslinked latex particles during miniemulsion polymerization. The choice of P(VAc-co-VCL) copolymer to form the sensitizing colloidal particles was driven by its solubility in ethanol, thus conferring a swelling ability to the crosslinked particles compared with the more conventional polystyrene latex particles that do not swell in such solvent. Several studies have reported self-quenching of the photosensitizer induced by aggregation of the photosensitizer. 19,20,25,[44][45][46][47][48][49] This will be taken as an advantage in the present work to design ON/OFF photosensitizing colloidal system by simple switching from water to ethanol. Here, we aim to provide a detailed study on singlet oxygen production via the measurement of the singlet oxygen quantum yield (∅ ∆ ). This allows an accurate comparison of the singlet oxygen production between the different systems (free rose Bengal, linear and crosslinked P(VAc-co-VCL-co-VBRB) polymer), independently from the RB concentration.

Experimental section

Materials.

4-vinylbenzyl chloride (VBC, 90 %), Rose Bengal (RB, 95 %), N,N-Dimethylformamide (DMF, 99.8 %), acetone (99.9 %), chloroform (99 %), methanol (99.8 %), diethyl ether (99.8 %), vinyl acetate (VAc, 99 %), N-vinylcaprolactam (VCL, 98 %), 2,2-azobis(2methylpropionitrile) (AIBN, 98 %), 1,3,5-trioxane (TR, 99 %), furfuryl alcohol (FFA, 99 %), ethanol (for UV spectroscopy, 96 %), 1,3-diphenylisobenzofuran (DPBF, 97 %), 1,3,5-trioxane and hexadecane (HD, 99 %) were purchased from Sigma-Aldrich Co. (St Louis, USA). Divinyl adipate (DVA, ≥99 %) was purchased from TCI Europe N.V. (Paris, France). Dimethyl sulfoxide-d6 (99.8 %) was purchased from Euriso-Top Sas (Saint-Aubin, France). Sodium dodecyl sulfate (SDS, 99 %) was purchased from ABCR GmbH (Karlsruhe, Germany). Sodium bicarbonate (SbC, NaHCO3, 99 %) was purchased from VWR International S.A.S. All the purchased chemicals were used as received except furfuryl alcohol, freshly distilled (boiling point 170°C) before use. ALUGRAM® aluminium sheets SIL G/UV254 for TLC were purchased from MACHEREY-NAGEL GmbH & Co (Düren, Germany). Dialysis was performed against water at room temperature under gentle stirring with Spectra/Por® Dialysis membrane (Spectrum Laboratories, Inc., Rancho Dominquez, USA, mol wt cutoff >3.5 kDa, average diameter 33 mm).

Characterization methods.

NMR spectra were recorded using a Brüker 400 MHz spectrometer at 25 °C. 1 H measurements were performed at 400 MHz and 13 C measurements at 100 MHz.

The hydrodynamic diameter (Dh) of the microgels were measured by Dynamic Light Scattering (DLS) using a Malvern Nano ZS instrument equipped with a He-Ne mW power laser operating at a wavelength of 633 nm. Dialyzed samples were housed in disposable polystyrene cuvettes of 1 cm optical path length, using double deionized water or ethanol as solvent, at 25° C and at a concentration of 0.05 g L -1 . The width of the hydrodynamic diameter distribution is provided by the Cumulant analysis and reported as the Polydispersity Index (PDI). Dh values reported in Table 1 were calculated from the intensity of light scattered by particles (10 measurements per sample) from a multiple narrow mode (non-negative least squares (NNLS) analysis) to obtain the particle size of each population.

The MG and VBRB@MG microgels were also analyzed by asymmetrical flow field-flow fractionation (AF4) running with aqueous eluent. An Eclipse 3+ AF4 instrument (Wyatt Technology, Dernbach, USA) equipped with a 1200 series high-performance liquid chromatography pump (Agilent Technologies, Les Ulis, France) was used as a separation system. All injections were performed with an Agilent Technologies 1260 ALS series autosampler. The detection system was formed by a 1200 series UV-vis absorbance detector (Agilent Technologies) and a multi-angle laser light scattering (MALLS) detector (DAWN HELEOS, Wyatt Technology). The AF4 channel thickness was fixed by a 250-µm Mylar spacer (with dimensions of 26.5 cm length and narrowing width from 2.1 to 0.6 cm). The accumulation wall was defined by a 10-kDa polyethersulfone (PES) membrane purchased from Wyatt Technology. The AF4 conditions used in this study were: an elution flow rate of 0.5 mL min -1 , an injection flow rate fixed at 0.2 mL min -1 , a focus flow rate of 0.5 mL min -1 and a cross flow rate (decreasing exponential) of 2e -0.27t mL min -1 (with t the time). 50,51 The mobile phase was prepared with sodium nitrate (NaNO3) diluted in deionized water (to reach a concentration of 0.5 mmol L -1 ) and filtered at 0.1 µm. The overlay of the UV-visible trace recorded at 559 nm, characteristic absorption wavelength of Rose Bengal derivatives, with light scattering trace confirms the presence of VBRB inside the microgels. The gyration radius (Rg) was measured from the plot of scattered light intensity versus the wave vector (q = (4/) sin/2) using the sphere model with ASTRA software (version 6, Wyatt technology). The polymer concentration was set at 5 mg L -1 . TEM images were recorded on a TEM JEOL JEM 1400 with a tungsten filament working at 120KeV, an objective diaphragm to improve the contrast and a Gatan Orius 1000 camera. The samples dispersed in water were casted onto TEM grids.

Photophysical measurements.

UV-VIS absorption spectra were recorded at 5, 10 and 25 °C by means of Perkin-Elmer Lambda 850 spectrophotometer. Corrected steady-state emission and excitation spectra were recorded with a photon counting Edinburgh FLS920 fluorescence spectrometer equipped with Xe lamp.

The concentrations of all compounds were adjusted to give an absorbance around 0.1 at the absorption maximum. Quartz cuvettes (Hellma) with optical path length of 1 cm were used for both absorbance and emission measurements. The detailed irradiation experiments and equations used for singlet oxygen quantum yield determination, photobleaching and cycling photooxygenation measurements are described in Electronic Supplementary Information.

General procedure for the synthesis of crosslinked particles and linear polymer by miniemulsion polymerization. The synthesis of the VBRB monomer is described in Electronic Supplementary Information (Fig. S1 †) and the corresponding 1 H, 13 C NMR and UV spectra are displayed in Fig. S2 †, Fig. S3 † and Fig. S4 †. The microgels were synthesized by miniemulsion polymerization carried out at 10 wt% of initial solid content. The organic phase containing VAc, VCL, TR, AIBN, HD, DVA and VBRB was stirred at 0°C for 15 min. The aqueous phase consisted of SDS and SbC, dissolved in double deionized water (DW) and was stirred at room temperature for 30 min and thus sonicated with an ultra-probe VIbraCell 72408, Bioblock Scientific in a continuous mode (power 30 % for 6 min). The organic phase was added to the aqueous phase and stirred at 0°C for 15 min. The reaction mixture was introduced in a 100 mL round bottom flask equipped with a stirring bar set at 300 rpm and purged with a gentle flow of nitrogen at 0°C for 30 min. The round bottom flask was placed into an oil-bath previously heated to 65 °C and the reaction mixture was stirred for 3 h, then cooled to room temperature and oxygen was introduced in the reaction mixture. The recipes for the microgel syntheses are reported in Table S1. The final monomer conversion calculated by 1 H NMR (See Fig. S5 † and S6 †) ranged from 50 to 90 %. The final microgels were dialyzed in DW at room temperature for 48 h. The codes MG-w correspond to the initial weight fraction (w) of DVA crosslinker used in the synthesis of microgels (MG), calculated on the basis of the mass of crosslinker and VAc/VCL monomers: w = mDVA/(mDVA + mVCL + mVAc). In order to assess the ability of the photoactive microgels to be separated from the reaction medium while being still photoactive, the VBRB@MG-14 microgels were centrifuged at 4°C, 10 000 g on a Beckman Coulter Aventi J30I centrifuge.

Results and discussion

Synthesis and colloidal characterization of photosensitizing polymer colloids.

Due to the aromatic structure of RB, the derived VBRB vinyl monomer is poorly soluble in water. Contrary to emulsion polymerization which requires monomer diffusion through the aqueous continuous phase, miniemulsion is advantageous to disperse highly hydrophobic species as the particle nucleation proceeds in the monomer droplets formed by shearing the initial dispersed phase. 36 The choice of the VCL and VAc monomers was driven by different parameters that were not achievable by using more conventional styrene or alkyl (meth)acrylate monomers, widely used in miniemulsion polymerization. First, the mixture of VAc and VCL was able to solubilize VBRB, otherwise almost insoluble in n-butyl acrylate or methyl acrylate for instance. The best organic solvent for RB derivatives is dimethyl formamide (DMF) and VCL is an amide, liquid at temperature above 35-38°C, with good solubilisation capacity towards VBRB. Secondly, VAc and VCL are sufficiently hydrophobic (([VCL]limit, water, 25°C = 41 g L -1 , [VAc]limit, water, 20°C = 23 g L -1 ) [START_REF] Barandiaran | Polymer Reaction Engineering[END_REF][START_REF] Basf | Safety Data Sheet Vinyl caprolactam Kerobit[END_REF] to promote the formation of monomer droplet at 10 wt-% (ie 100 g.L -1 ) of initial monomer content in water. Vinyl acetate enables the solubilisation of the solid VCL to form the initial monomer droplets at room temperature while being efficiently copolymerized with VCL, [START_REF] Etchenausia | [END_REF][55][56] which avoids the use of an organic co-solvent that should be removed at the end of polymerization as reported by Crespy et al.. 57 In the present work, the P(VAc-co-VCL)-based particles were synthesized by miniemulsion polymerization in the presence of hydrophobic hexadecane limiting the Ostwald Ripening, as depicted in Scheme 1. 36 Scheme 1 : Reaction scheme for the synthesis of photosensitizing microgels by miniemulsion copolymerization.

A series of four microgels were synthesized (Figure 1): i) two model photosensitizer-free microgels crosslinked with 8 or 14 mol % of the divinyl adipate (DVA) crosslinker (MG-8, MG-14 in Table 1), and ii) two homologue microgels containing Rose Bengal photosensitizer synthesized by miniemulsion copolymerization of 0.6 mol-% of VBRB (ie 5.6 wt %, initial loading 50 µmol g -1 ) based on the total number of moles of VAc, VCL and DVA (VBRB@MG- 8, VBRB@MG-14 in Table 1). In order to discriminate the effect of RB grafting onto P(VAcco-VCL) copolymer from the effect of the microgel environment on photoactivity, a linear P(VAc-co-VCL-co-VBRB) was synthesized by miniemulsion copolymerization in the absence of DVA crosslinker (VBRB@P in Table 1). The P(VAc-co-VCL) microgels were synthesized by miniemulsion polymerization up to complete monomer conversion within 3 hours. Stable microgels dispersed in the aqueous phase b) a)

exhibit a narrow particle size distribution (PDI < 0.1, see MG-8 and MG-14 in Table 1 and Fig.

S7 †). Miniemulsion process requires the addition of a hydrophobic molecule (hexadecane in

the present work) in order to limit the monomer diffusion through the water phase which would results in larger droplets at equilibrium. 36,58 The higher Laplace pressure of small droplets should be counterbalanced by the non-favorable increase of the osmotic pressure in case of increasing hexadecane concentration through monomer exit. The RB-free microgel crosslinked with 8 mol-% of DVA (MG-8) displays an average hydrodynamic diameter (Dh) of 190 nm, slightly higher than the microgels crosslinked with 14 mol-% of DVA (Dh = 171 nm for MG-14 in Table 1). A similar trend was observed for the RB-grafted microgels as 10 % of Dh decrease was observed by increasing the initial crosslinker ratio from 8 mol-% to 14 mol-% (see VBRB@MG-8, and VBRB@MG-14 in Table 1). It should be mentioned that all microgels dispersed in water are stable over months in the absence of sedimentation. The copolymerization of VBRB induced an increase of Dh measured in water (see MG-8 vs VBRB@MG-8 and MG-14 vs VBRB@MG-14 in Table 1). The VBRB monomer introduces ionic moieties inside the polymer network (see scheme 1). The ionic contribution to the total osmotic pressure is known to enhance microgel swelling. 59 The characterization of the VBRB@MG-8 microgels by TEM revealed a different contrast in the VBRB@MG microgels compared to the RB-free counterparts (Figure 2), which probably highlight the presence of the heavy iodide atoms in RB moiety. The dialyzed microgels were also analysed by Asymetrical Flow Field-Flow Fractionation (AF4) equipped with UV-visible detector set at a wavelength of 559 nm, a characteristic wavelength of RB moiety compared to the polymer, and a MALLS detector. As depicted in Figure 3, the absorbance traces of VBRB@P and VBRB@MG-8 samples overlay with the light scattering (LS) traces which supports the presence of VBRB within the colloids. It should be noted that the colloidal dispersion scatters light which induces an extra-absorbance at 559 nm due to the slight upper shift of the baseline of UV-visible spectra for all the microgels (see Fig. S8 †). However, at a concentration of 5 ppm, the ratio between the areas of the LS signal over the UV-visible signal is 5 times higher for VBRB@MG-8 microgel than for the MG-8 microgel (Table S2). This confirms that the UV-visible trace at 559 nm arises mainly from the VBRB units. Overlay of the light scattering (LS, orange) and UV-visible ( = 559 nm, blue) traces.

The microgels were dried to be further dispersed in ethanol, a solvent suitable for photooxygenation reactions of organic molecules of interest such as -terpinene or furfural. 6,35 Interestingly, both MG and VBRB@MG crosslinked microgels had the ability to swell in ethanol as shown by the higher Dh value measured in ethanol than in water (Table 1). The lower swelling ratio of the VBRB@MG compared to MG is due to the higher diameter in water as

Characterization of the photophysical properties of the polymer colloids.

In order to correlate the swelling ability of the microgels to the yield of singlet oxygen production, the singlet oxygen quantum yield ( was monitored in both water and ethanol.

The singlet oxygen quantum yield, corresponding to the number of moles of singlet oxygen produced per photon adsorbed by the photosensitizer, will be measured using two different quenchers, either furfuryl alcohol (FFA) in water or diphenylisobenzofuran (DPBF) in ethanol (Scheme 2). The oxidation of FFA to 6-hydroxy-(2H)-pyranone is monitored by HPLC, while DPBF concentration is calculated from the decrease of its absorbance spectra at 410 nm. The oxidation of FFA follows a pseudo first order kinetics (see Eq. 4 in Electronic Supplementary Information), while the decrease of DPBF followed a more general kinetic scheme (see Eq.5 in Electronic Supplementary Information).

Scheme 2. Singlet oxygen quenching by FA in water and by DPBF in ethanol

Prior to measurement of the quantum yield of singlet oxygen production, it is important to carefully analyze the UV-visible absorbance and emission spectra of the different materials.

Both absorption and emission spectra of the synthesized VBRB monomer were recorded in ethanol (see Fig. S4 †). The maximum absorbance and emission wavelengths (abs= 559 nm, em= 573 nm) as well as the shoulder peak at 514 nm were slightly red-shifted relative to free RB (abs,max = 548 nm, em,max = 569 nm) as already reported for other aromatic derivatives of RB. 60 This confirms the successful nucleophilic substitution of Rose Bengal with vinyl benzyl chloride to produce a pure styrenic monomer. The absorbance and emission spectra of both the RB-grafted microgels (VBRB@MG) and the non-crosslinked copolymer (VBRB@P) dispersed in water are displayed Figure 4. First, we can notice that the adsorption bands of the VBRB@P linear polymer are broader than those of free RB with a red-shifted maximum at 565 nm, an intense shoulder at 535 nm and a weak band at 428 nm. It has been previously reported that dimers of RB ethyl esters in water were characterized by two absorption bands at 526 and 572 nm. 61 The absorbance spectrum of the linear polymer VBRB@P (Figure 4), with a noticeable decrease of the I565/ I535 intensity ratio relative to the I548/ I514 ratio in Rose Bengal, and the broadening of the absorption bands, 62 both support the assumption of slight aggregation of VBRB moiety in the VBRB@P polymer dispersed in water. This is in agreement with our previous results showing that amphiphilic P(VAc-co-VCL) statistical copolymer chains with 50/50 composition in VAc/VCL slightly self-assembled via hydrophobic interactions, even below the phase transition temperature (PTT  17-20 °C) of these copolymers in water. [START_REF] Barandiaran | Polymer Reaction Engineering[END_REF][START_REF] Basf | Safety Data Sheet Vinyl caprolactam Kerobit[END_REF] The UV spectra of the linear VBRB@P polymer in water exhibit the characteristic traces of RB aggregation even at low temperature (5 °C and 10 °C) (Fig. S9 †). The same P(VAc-co-VCL) copolymers crosslinked with the hydrophobic DVA are likely in a more collapsed state within the colloidal particles as the microgels do not exhibit any swelling-to-collapse transition in water over the all temperature range from 10 to 50 °C (Fig. S10 †). This collapsed state induces an intense distortion of the absorption spectra of the grafted RB moieties in the VBRB@MG microgels (Figure 4). Figure 4 displays absorption spectra in which the drift of the baseline arising from the light scattered by the dense particles has been corrected (see raw data in Fig.

S8 †).

A very broad band is observed for both VBRB@MG-8 and VBRB@MG-14 microgels, with a maximum absorbance at 544 nm and prominent shoulders on the high energy side at 518 nm and 436 nm (this latter noticeably more intense than in VBRB@P) and on the low energy side at 576 nm. The spectra of these microgels are more difficult to rationalize since their shapes are indicative of optically thick samples strongly modified by diffusion effects. Higher dimer/monomer ratio in the cross-linked microgel can however be assumed.

Figure 4: a) Normalized absorption spectra b) emission spectra (ex 530 nm) in H2O of the soluble polymer VBRB@P (blue lines), microgels VBRB@MG-8 (continuous red lines), VBRB@MG-14 (dotted red lines), polymer concentration in H20 [13.0 mg L -1 ] together with RB spectra in H2O [1.0 µM] (black lines).

The emission spectrum of VBRB@P (em = 569 nm) (Figure 4b) is similar to that of free RB but exhibits a broader signal compared to the emission spectrum of free RB, as expected for aggregated species. On the contrary, the emission spectra of VBRB@MG-8 and VBRB@MG-14 microgels in water show a bathochromic shift (em 595 nm) relative to free RB (em 569 nm) and a growing shoulder at longer wavelengths typical of inner filter effects, 63 with reabsorption of the 0-0 emission band, indicative of strong aggregation in this optically thick sample. Additionally the fluorescence excitation spectra are expected to be different from the absorption spectrum for a solution containing monomeric photosensitizer and its aggregates absorbing in the same region. 64 This is actually observed for the VBRB@P linear copolymer and VBRB@MG-8, VBRB@MG-14 microgels in water (Fig. S11 †). To summarize these spectral data, aggregation of RB units is observed for the different RB-grafted polymers dispersed in water and this aggregation is much more significant in the crosslinked microgels than in the corresponding linear soluble polymer.

It is worth noting that the aggregation of RB moieties in the linear or crosslinked P(VAc-co-VCL-co-VBRB) dispersed in water is correlated to very low values of singlet oxygen quantum yields (0.01 <  < 0.05, Table 2). Thus, the synthesis of VBRB@MG microgels by polymerization in aqueous dispersed media (miniemulsion polymerization) produces "latent" microgels which are not photo-active in water under light irradiation at 515 nm due to the aggregated state of RB moieties in the collapsed polymer. In relationship with their respective solubility and swelling ability in ethanol, the photosensitized linear copolymer and microgels were further dispersed in ethanol to monitor their propensity to produce singlet oxygen. Note that VBRB, which is soluble in ethanol up to 0.05 mol L -1 , is as efficient as free RB to produce singlet oxygen in ethanol (see Table 2,  = 0.72). In ethanol, the absorbance and emission spectra of the RB-grafted copolymers are very close to that of the free VBRB monomer in the same solvent (Figure 5) and strikingly different from their spectra in water (Figure 4a andb).

Wavelength (nm)

VBRB VBRB VBRB@P VBRB@P VBRB@MG-8 VBRB@MG-8 VBRB@MG-14

Moreover, the extent of crosslinking had no influence on the spectra, as the patterns of VBRB@P, VBRB@MG-8 and VBRB@MG-14 perfectly overlaid. It can be also noticed that the absorption and excitation spectra of RB-based soluble linear polymer and dispersed microgels perfectly matched (Fig. S12 †). All these spectral features tend to show that the RB moieties do not aggregate inside the swollen microgels in ethanol, contrary to the strong aggregation observed in the collapsed state of the microgels dispersed in water. The three types of RB-grafted polymeric materials proved to be photoactive in ethanol producing singlet oxygen under irradiation. Indeed, the singlet oxygen quantum yields () ranged between 0.47 and 0.32 for VBRB@P and VBRB@MG (Table 2), the highest value being obtained with the soluble VBRB@P linear polymer. These values are rather high for supported Rose Bengal. 62 The efficiency of the VBRB@MG in producing singlet oxygen in ethanol can thus be ascribed to the absence of any aggregation of the covalently grafted RB in the swollen microgels. We can conclude that the latent non-photoactive VBRB@MG polymer colloids dispersed in water were turned ON into very active systems in ethanol. It is well known from the literature that the singlet oxygen production is drastically reduced by the photosensitizer aggregation, which enhances self-quenching of the excited state, 44,25 as demonstrated for instance with lipophilic phthalocyanines included in polymer hydrogels of various lipophilicity. 65 Aggregation effects, were also reported to be responsible for the decrease of singlet oxygen quantum yield of either RB esters insoluble in non-polar solvents 66 or RB-grafted to poly(styrene-covinylbenzylchloride) beads. 67 It should be noticed that VBRB@MG-14 microgels were centrifuged to assess the recovery of the supported photosensitizer separately from the photo-oxidized molecules. This recycling step is expected to minimize the tricky distillation steps usually applied to recover from the solvent the oxidized product free of photosensitizer. Centrifuged VBRB@MG-14 microgels were mixed with DPBF in ethanol to be further irradiated by the LED at 515 nm. The VBRB@MG-14 were still photoactive after the centrifugation cycle with a singlet oxygen quantum yield in a similar range ( = 0.32). irradiance of 3 mW cm -2 ); b Singlet oxygen quantum yields measured in ethanol with DPBF quencher (Xe-Hg Lamp Monochromator 547 nm, irradiance of 0.2 mW cm -2 ); c Extracted from reference 41 .

The reusability of VBRB@MG14 microgels was investigated through a repeated use of the photosensitized colloids in five photooxygenation cycles. The overlay of the DPBF conversion versus time for each cycle reveals that the high efficiency is maintained over the different cycles (Figure 6a) while no DPBF conversion could be detected in the dark. It is worth noting that under the conditions used for singlet oxygen measurement in ethanol at low irradiance (0.2 mW cm -2 ), neither RB nor VBRB@MG photobleached (constant absorbance at  569 nm). In order to highlight the interest of RB grafting, free RB and VBRB@MG-14 were irradiated in ethanol with a more powerful Xenon lamp (irradiance 55 mW cm -2 ) to allow the comparison of their photobleaching yield (see Eq. 1 in the experimental part). The VBRB@MG-14 microgels were more photo-stable (36 % photobleaching) than free RB (53 % photobleaching) over 6 hours of intense irradiation (Figure 6b). It should be noted that the photo-stability of VBRB@MG14 particles is maintained over more than 12 months with very close value of singlet oxygen quantum yield (Table S3).

Figure 6 : a) Five photooxygenation cycles of DPBF from VBRB@MG-14 (Xe-Hg Lamp Monochromator 547 nm, irradiance of 0.2 mW cm -2 ); b) Comparison of the rate of photobleaching of RB and VBRB@MG-14 in ethanol (300 W Xe Lamp, Irradiance 55 mW cm -2 between 400 and 800 nm).

Figure 1 .

 1 Figure 1. Pictures of the final polymer dispersions in water obtained by miniemulsion copolymerization of VAc and VCL (Table 1): a) MG-8; b) VBRB@MG8.

Figure 2 .

 2 Figure 2. TEM images of the dialyzed microgels: MG-8 (left), VBRB@MG-8 (right).

Figure 3 .

 3 Figure 3. A4F fractograms in NaNO3/H2O eluent of: a) VBRB@P and b) VBRB@MG-8.Overlay of the light scattering (LS, orange) and UV-visible ( = 559 nm, blue) traces.

  The swelling ability of microgels is of high interest to increase the diffusion of oxygen in the vicinity of the grafted photosensitizer.

Figure 5 .

 5 Figure 5. Normalized absorption (full lines) and emission (dotted lines, ex 530 nm) spectra in ethanol of VBRB ([VBRB] = 0.98 × 10 -6 M) and VBRB@P, VBRB@MG-8, VBRB@MG-14 at polymer concentrations ranging from 0.003 to 0.007 g L -1 .

  Photobleaching (norm. on λ Abs, max

Table 1 .

 1 Characterization

	Expt	Dh, H20 (nm) a (PDI) Dh, EtOH (nm) a (PDI)	SR b	Rg,H20 c
						(nm)
	MG-8	190 (0.05)	247 (0.06)		2.2	90
	MG-14	171 (0.06)	205 (0.03)		1.7	80
	VBRB@MG-8	262 (0.16)	285 (0.44)		1.3	100
	VBRB@MG-14	241 (0.17)	260 (0.31)		1.3	100
	a Hydrodynamic diameter (Dh) values and polydispersity index measured in water or ethanol
	for [microgel] =0.05 g L -1 ; b Swelling ratio, SR = Rh 3	EtOH / Rh 3	H20, where Rh is the
	hydrodynamic radius at 25° C;

of the microgels: RB loading, hydrodynamic diameter Dh in water and alcohol, gyration radius Rg, singlet oxygen quantum yields  in water and alcohols. c Gyration radius measured by MALLS.

Table 2 .

 2 Singlet oxygen quantum yields  of free RB, VBRB and dialyzed polymers.

	Expt	 (H2O) a	 (EtOH) b
	Free RB	0.75	0.68-0.80 c
			0.75
	VBRB	-	0.72
	VBRB@P	0.05	0.47
	VBRB@MG-8	0.03	0.27
	VBRB@MG-14	0.01	0.35

a Singlet oxygen quantum yields measured in water with FFA quencher (LED at 515 nm,

Acknowledgements

The authors would like to thank Agence Nationale de la Recherche (ANR) for funding PICPOSS project (ANR-15-CE07-0008-03), CNRS and UPPA for financial support. The authors would like to thank Abdel Khoukh, Elise Deniau and Bruno Grassl (UPPA) for their

Conclusions

In conclusion, novel photosensitizer-grafted polymer colloids of submicronic diameter were designed as efficient tool to trigger the photoactivity of supported Rose Bengal. Interestingly, the Rose-Bengal microgels were synthesized by a one-step miniemulsion copolymerization of vinyl benzyl rose Bengal, vinyl acetate, N-vinyl caprolactam and divinyl adipate crosslinker carried out in aqueous dispersed media. We designed a switchable system via the synthesis of non-photoactive latent polymer colloids in water which can be triggered into photoactive particles in ethanol with a good level of singlet oxygen quantum yield ( = 0.32-0.47) via Rose Bengal disaggregation through simple swelling of these microgels. Their photo-activity is maintained after 5 repeated use of the photosensitized microgels in photooxygenation cycles but also after their centrifugation, which opens perspectives towards an efficient separation of the supported sensitizer from the reaction medium. The reusability and recyclability of the organic photocatalyst limiting any distillation step is will increase the sustainability of the catalytic photooxygenation green process. The switchable photo-activity represents an advantage as latent photoactivity in water during storage should enhance the photostability while efficient photoactivity in ethanol is of particular interest to trigger photo-oxygenation reactions. These original sub-micronic microgels open the route towards further implementation of fine chemicals photooxygenation in flow photoreactors, in the absence of substrate sedimentation.

Conflicts of interest

There are no conflicts to declare.