Alysia Cox

Daniele Vinciguerra

Francesca Re
email: francesca.re1@unimib.it

Roberta Dal Magro

Simona Mura

Massimo Masserini

Patrick Couvreur

Julien Nicolas
email: julien.nicolas@u-psud.fr

Protein-Functionalized Nanoparticles Derived from End-Functional Polymers and Polymer Prodrugs for Crossing the Blood-Brain Barrier

Keywords: Polymer, prodrug, nanoparticle, blood-brain barrier, proteins, drug delivery

Nanoparticles may provide a viable way for neuroprotective drugs to cross the blood-brain barrier (BBB), which limits the passage of most drugs from the peripheral circulation to the brain.

Heterotelechelic polymer prodrugs comprising a neuroprotective model drug (adenosine) and a maleimide functionality were synthesized by the "drug-initiated" approach and subsequent nitroxide exchange reaction. Nanoparticles were obtained by nanoprecipitation and exhibited high colloidal stability with diameters in the 162-185 nm range and narrow size distributions.

Nanoparticles were then covalently surface-conjugated to different proteins (albumin, 2macroglobulin and fetuin A) to test their capability of enhancing BBB translocation. Their performances in terms of endothelial permeability and cellular uptake in an in vitro BBB model were compared to that of similar nanoparticles with surface-adsorbed proteins, functionalized or not with the drug. It was shown that bare NPs (i.e., NPs not surface-functionalized with proteins) without the drug exhibited significant permeability and cellular uptake, which were further enhanced by NP surface functionalization with 2-macroglobulin. However, the presence of the drug at the polymer chain-end prevented efficient passage of all types of NPs through the BBB model, likely due to a decrease in the hydrophobicity of the nanoparticle surface and alteration of the protein binding/coupling, respectively. These results established a new and facile synthetic approach for the surface-functionalization of polymer nanoparticles for brain delivery purposes.

Introduction

The blood-brain barrier (BBB) remains one of the major limiting obstacles preventing pharmacological advancement in the treatment of neurological, neurodegenerative, and psychiatric disorders [START_REF] Pardridge | The blood-brain barrier: Bottleneck in brain drug development[END_REF]. Although nanoparticles (NP) offer a promising approach to cross this barrier, surfacemodification is necessary to increase drug delivery to the central nervous system (CNS). With this in mind, synthetic and physiological peptides and proteins have extensively been used to coat or functionalize NPs for BBB targeting [START_REF] Saraiva | Nanoparticlemediated brain drug delivery: Overcoming blood-brain barrier to treat neurodegenerative diseases[END_REF][START_REF] Magro | The ability of liposomes, tailored for blood-brain barrier targeting, to reach the brain is dramatically affected by the disease state[END_REF][START_REF] Lalani | Protein functionalized tramadol-loaded PLGA nanoparticles: preparation, optimization, stability and pharmacodynamic studies[END_REF]. Many techniques have been utilized to identify novel synthetic peptides or previously untested physiological proteins, including peptide phage display and computer-assisted design [START_REF] Gao | Progress and perspectives on targeting nanoparticles for brain drug delivery[END_REF]. A wide array of these ligands has been successfully used to target receptor-mediated endocytosis and encourage passage of NPs through the BBB [START_REF] Krol | Challenges in drug delivery to the brain: nature is against us[END_REF], commonly through transferrin receptor [START_REF] Liu | B6 peptide-modified PEG-PLA nanoparticles for enhanced brain delivery of neuroprotective peptide[END_REF][START_REF] Salvati | Liposomes functionalized to overcome the blood-brain barrier and to target amyloid-β peptide: the chemical design affects the permeability across an in vitro model[END_REF][START_REF] Mourtas | Multifunctional nanoliposomes with curcumin-lipid derivative and brain targeting functionality with potential applications for Alzheimer disease[END_REF], lactoferrin receptor [START_REF] Yu | Self-assembled polymersomes conjugated with lactoferrin as novel drug carrier for brain delivery[END_REF][START_REF] Hu | Lactoferrin-conjugated PEG-PLA nanoparticles with improved brain delivery: in vitro and in vivo evaluations[END_REF], and low-density lipoprotein receptor (LDLr) [START_REF] Re | Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model[END_REF][START_REF] Bana | Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: Implications for therapy of Alzheimer disease[END_REF]. In particular, a modified fragment of apolipoprotein E, named mApoE, successfully enhanced brain delivery of liposomes designed for the treatment of Alzheimer's disease via transcytosis targeting LDLr [START_REF] Re | Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model[END_REF][START_REF] Bana | Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: Implications for therapy of Alzheimer disease[END_REF].

We recently analyzed the protein composition of NP corona after passage through an in vitro BBB model and found that specific proteins were able to cross endothelial BBB cells in relatively high abundance [START_REF] Cox | Evolution of Nanoparticle Protein Corona across the Blood-Brain Barrier[END_REF]. Among these proteins, we have selected three to test their capability to improve brain targeting of polymeric NP: albumin, α 2 -macroglobulin (α2M) and Fetuin A (Fet A).

Albumin, the most abundant serum protein, crosses the BBB in vivo in healthy conditions [START_REF] Kanoh | Serum matrix metalloproteinase-2 levels indicate blood-CSF barrier damage in patients with infectious meningitis[END_REF], and in higher quantities after traumatic brain injury [START_REF] Blyth | Validation of serum markers for blood-brain barrier disruption in traumatic brain injury[END_REF], in Huntington disease [START_REF] Pardo | Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease[END_REF], diabetes and dementia [START_REF] Janelidze | Increased blood-brain barrier permeability is associated with dementia and diabetes but not amyloid pathology or APOE genotype[END_REF]. α2M is a broad spectrum anti-protease that also crosses the healthy BBB [START_REF] Asano | Evaluation of blood-brain barrier function by quotient alpha2 macroglobulin and its relationship with interleukin-6 and complement component 3 levels in neuropsychiatric systemic lupus erythematosus[END_REF] and is a biomarker for multiple sclerosis [START_REF] Jensen | Significantly increased fractions of transformed to total α2-macroglobulin concentrations in plasma from patients with multiple sclerosis[END_REF]. It has anti-inflammatory effects which seem particularly promising in treating neurological conditions that are coupled with increased local or generalized inflammation [START_REF] Cucullo | Blood-brain barrier damage induces release of α2-macroglobulin[END_REF]. Fet A, also known as α 2 -HS-glycoprotein, is predominantly found in fetal serum where it prevents tissue calcification [START_REF] Häusler | The physiologic development of fetuin-a serum concentrations in children[END_REF]. It can cross the BBB during ischemic injury [START_REF] Laughlin | Fetuin-A, a new vascular biomarker of cognitive decline in older adults[END_REF].

Although albumin has previously been used to synthesize NPs that can cross the BBB [START_REF] Lin | Bloodbrain-barrier-penetrating albumin nanoparticles for biomimetic drug delivery via albuminbinding protein pathways for antiglioma therapy[END_REF], the use of α2M or Fet A for functionalizing NPs to specifically target the brain has never been investigated.

Among the different families of materials suitable for the design of targeted drug delivery systems, polymer NPs have attracted much attention because of their numerous benefits. Polymer synthesis methods indeed offer great flexibility in terms of control, macromolecular architecture, physico-chemical properties, and enable easy access to functional materials. For instance, different polymer NPs have been engineered to allow surface modification for CNS drug delivery with various ligands to enhance their permeability through the BBB [START_REF] Li | Targeting the brain with PEG-PLGA nanoparticles modified with phage-displayed peptides[END_REF][START_REF] Nicolas | Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymerbased nanocarriers for drug delivery[END_REF][START_REF] Kreuter | Drug delivery to the central nervous system by polymeric nanoparticles: what do we know?[END_REF]. NP coating by simple adsorption of targeting peptides has been extensively investigated [START_REF] Kreuter | Influence of the surface properties on nanoparticle-mediated transport of drugs to the brain[END_REF][START_REF] Göppert | Polysorbate-stabilized solid lipid nanoparticles as colloidal carriers for intravenous targeting of drugs to the brain: comparison of plasma protein adsorption patterns[END_REF][START_REF] Di Pietro | Immobilization of neurotrophin peptides on gold nanoparticles by direct and lipid-mediated interaction: a new multipotential therapeutic nanoplatform for CNS disorders[END_REF], but surface functionalization via covalent linkage remains the method of choice as it prevents protein desorption and loss of targeting, though it requires more elaborate synthetic strategies. Developing polymer prodrug nanocarriers, where the drug is linked to the polymer, usually comes with important benefits compared to traditional drug-loaded systems [START_REF] Delplace | Recent trends in the design of anticancer polymer prodrug nanocarriers[END_REF], including: (i) absence of "burst release" (i.e., uncontrolled release of drugs post-administration); (ii) the possibility to obtain higher drug loading and (iii) enhancement of the drug compatibility with the polymer matrix/core, allowing a broader range of drugs to be transported.

Heterotelechelic polymers are gaining great interest in the biomedical field [START_REF] Vinciguerra | Telechelic polymers from reversible-deactivation radical polymerization for biomedical applications[END_REF], as they offer an opportunity to embed two different biologically active moieties positioned at both extremities (e.g., drug, targeting ligands, imaging probes). In this context, we proposed the synthesis of heterotelechelic polymers bearing a drug of interest in the -position and a reactive group for further conjugation with BBB-crossing proteins in the -position.

As a proof of concept, adenosine (Ade) was chosen as a model drug for this drug delivery system. Its neuroprotective characteristics have already been demonstrated in vivo after stroke when delivered to BBB endothelial cells as a squalene-Ade molecular prodrug [START_REF] Gaudin | Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury[END_REF].

Here, we either adsorb or conjugate bovine serum albumin (BSA), α2M or Fet A at the surface of heterobifunctional polymer prodrug NPs (Figure 1) and report on the proteins effect on NP ability to cross the in vitro BBB, possibly enhancing Ade transport and therefore its neuroprotective activity. Moreover, targeting is vital to avoid side reactions, given the widespread expression of Ade receptors throughout the body [START_REF] Fredholm | International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors[END_REF][START_REF] Londos | Subclasses of external adenosine receptors[END_REF], and peripheral degradation [START_REF] Haskó | Adenosine: an endogenous regulator of innate immunity[END_REF]. Though investigation of the pharmacological activity of the prodrug NPs is outside the scope of this article, we investigate here the possibility to embed a relevant drug for the treatment of CNS-related diseases and study its impact on the nanoparticle interaction/functionalization with proteins, and their subsequent cellular uptake and permeability through a well-established and widely used in vitro transwell BBB model [START_REF] Magro | The ability of liposomes, tailored for blood-brain barrier targeting, to reach the brain is dramatically affected by the disease state[END_REF]. Tetrabutylammonium fluoride (TBAF) was purchased from Alfa Aesar (Kandel, Germany). N-tertbutyl-N-(1-diethylphosphono-2,2-dimethylpropyl) nitroxide (SG1, 85%) was kindly supplied by Arkema. AMA-SG1 alkoxyamine [START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF], Rho-TEMPO [START_REF] Vinciguerra | A facile route to heterotelechelic polymer prodrug nanoparticles for imaging, drug delivery and combination therapy[END_REF] and tris-O-silylated adenosine [START_REF] Erion | Design, synthesis, and characterization of a series of cytochrome P450 3A-activated prodrugs (hepdirect prodrugs) useful for targeting phosph (on) ate-based drugs to the liver §[END_REF] were prepared as reported previously. All other reactants were purchased from Sigma-Aldrich (Lyon, France) at the highest available purity and used as received. Deuterated chloroform (CDCl 3 ) was obtained from Eurisotop (France). All other solvents were purchased from Carlo-Erba (France) at the highest grade. EBM-2 basal medium was from Lonza (Switzerland). Nanosep centrifugal devices (Omega membrane, 300K) were from VWR International (Fontenay-sous-Bois, France).

Materials and Methods

Materials

Adenosine

BCA assay kit was from Thermo Fisher Scientific (Illkirch, France). hCMEC/D3 cells were provided by Pierre-Olivier Couraud (Université Paris Descartes (Paris, France). FITC-dextran 40 kDa was purchased from Sigma-Aldrich (Milan, Italy).

Analytical methods

Nuclear Magnetic Resonance Spectroscopy (NMR). NMR spectroscopy was performed in 5 mm diameter tubes in CDCl 3 at 25 °C. 1 H and 13 C NMR spectroscopy was performed on a Bruker

Avance 300 spectrometer at 300 MHz ( 1 H) or 75 MHz ( 13 C). The chemical shift scale was calibrated based on the internal solvent signals. To characterize nitroxide derivatives, pentafluorophenylhydrazine was added in situ and allowed to react before the analysis [START_REF] Benoit | Development of a universal alkoxyamine for "living" free radical polymerizations[END_REF][START_REF] Benoit | Accurate structural control and block formation in the living polymerization of 1, 3-dienes by nitroxide-mediated procedures[END_REF].

Mass spectrometry. Mass spectra were recorded with a Bruker Esquire-LC instrument. Highresolution mass spectra (ESI) were recorded on an ESI/TOF (LCT, Waters) LC-spectrometer.

Size Exclusion Chromatography (SEC). SEC was performed

Synthesis

Synthesis of PI. AMA-SG1 (120 mg, 0.327 mmol) was placed in a 15 mL-capacity pressure tube (Ace Glass 8648-164) fitted with a plunger valve and thermowell. After addition of isoprene (6.5 mL, 6.53 mmol) and dry 1,4-dioxane (6.5 mL), the tube underwent three cycles of freeze-thaw degassing and was then backfilled with argon. The tube was placed in a preheated oil bath at 115 °C for 16 h (PI1) and then cooled to r.t. by placing it under cold water. The residue was concentrated under reduced pressure and precipitated in cold methanol to give PI as a colorless viscous oil. Two other polymerizations were performed for 16 h with [isoprene] 0 /[AMA-SG1] 0 = 500/1 (PI2) and 400/1 (PI3). All polymers were characterized by SEC and 1 H NMR. PI1: M n,SEC = 2860 g.mol -1 , Đ = 1.13. PI2: M n,SEC = 4940 g.mol -1 , Đ = 1.18. PI3: M n,SEC = 4320 g.mol -1 , Đ = 1.16.

Synthesis of PI-Rho. Briefly, PI1 (300 mg, 0.105 mmol, 1 eq) and TEMPO-Rho (72 mg, 0.105 mmol, 1 eq) were placed in a 7 mL-vial and dissolved in dry pyridine (1 mL). After 20 min of degassing under argon, the vial was placed in a preheated oil bath at 110 °C and stirred for 16 h. PI-Rho was then precipitated two times in cold methanol and dried under reduced pressure until constant weight. Functionalization was confirmed by SEC equipped with a fluorescent detector (λ ex = 570 nm, λ em = 595 nm). M n,SEC = 2690 g.mol -1 , Đ = 1.13.

Synthesis of PI-OH.

Briefly, P2 (0.500 g, 0.101 mmol, 1 eq) and 4-hydroxy-TEMPO (0.017 g, 0.101 mmol, 1 eq) were placed in a 7 mL-vial and dissolved in 1 mL of dry pyridine. After 20 min of degassing under argon, the vial was placed in a preheated oil bath at 110 °C and stirred for 16 h.

PI-OH was then precipitated two times in cold methanol and dried under reduced pressure until constant weight. The polymer was characterized by SEC and 1 H NMR. M n,SEC = 5030 g.mol -1 , Đ = 1.15.

Synthesis of N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide. N-[2-

(2-Hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide was prepared according to a modified published procedure [START_REF] Sankaran | Ring-opening metathesis polymers for biodetection and signal amplification: synthesis and self-assembly[END_REF]. Exo-

Synthesis of PI-mal-furan.

A solution of succinic-mal-furan (0.043 g, 0,121 mmol, 2 eq), EDC•HCl (0.023 g, 0.121 mmol, 2 eq) and DMAP (cat) in dry DCM (1 mL) was stirred for 15 min and added dropwise to a solution of PI-OH (0. 300 g, 0.061 mmol, 1 eq) in DCM (1 mL). The mixture was stirred overnight under argon at r.t., before being quenched with water and extracted with DCM. The organic phases were then washed with brine and dried with MgSO 4 . After evaporation of the solvent, two precipitation in cold methanol were performed to obtain PI-malfuran as a colorless oil. The polymer was characterized by SEC and 1 H NMR. M n,SEC = 5200 g.mol - 1 , Đ = 1.15.

Synthesis of PI-mal. PI-mal-furan (0.200 g, 0.038 mmol) was dissolved in dry toluene (1 mL).

After 20 min of degassing under argon, the vial was placed in a preheated oil bath at 100 °C and stirred for 16 h. PI-mal was then precipitated two times in cold methanol and dried under reduced pressure until constant weight. The polymer was characterized by SEC and 1 H NMR, which also confirmed the disappearance of the maleimide protecting group. M n,SEC = 5650 g.mol -1 , Đ = 1.17.

Synthesis of Ade-PI.

The TBDMS-protected TBDMS-Ade-PI2 (0.300 g, 0.050 mmol) was dissolved in 1.5 mL THF and TBAF (1 M in THF, 150 μL) was added. The solution was stirred for 30 min and then purified by two consecutive precipitation in cold methanol to give Ade-PI as a colorless viscous oil. The polymer was characterized by SEC and 1 H NMR, which confirmed also the disappearance of TBDMS protecting groups. M n,NMR was calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA-SG1 -MW TBDMS with DP n,NMR calculated from ratio of areas under the peak at 8.69, 8.31 and 5.98 ppm (aromatic and anomeric proton of Ade) and 5.0-5.5

ppm (vinylic H in isoprene repeat unit (1,4-addition), corresponding to ~81% of total isoprene units [START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF]). M n,SEC = 6260 g.mol -1 , M n,NMR = 6280 g.mol -1 , Đ = 1.15.

Synthesis of tris-O-silylated adenosine-AMA-SG1 (TBDMS-Ade-AMA-SG1

Synthesis of TBDMS-

Synthesis of TBDMS-Ade-PI-OH.

Briefly, TBDMS-Ade-PI1 (0.500 g, 0.098 mmol, 1 eq) and 4hydroxy-TEMPO (0.017 g, 0.098 mmol, 1 eq) were placed in a 7 mL-vial and dissolved in 1 mL of dry pyridine. After 20 min of degassing under argon, the vial was placed in a preheated oil bath at 110 °C and stirred for 16 h. TBDMS-Ade-PI-OH was then precipitated two times in cold methanol and dried under reduced pressure until constant weight. The polymer was characterized by SEC and 1 H NMR. M n,NMR was calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA- SG1 -MW SG1 + MW TEMPO-OH with DP n,NMR calculated from ratio of areas under the peak at 8.69, 8.31 and 5.98 ppm (aromatic and anomeric proton of Ade) and 5.0-5.5 ppm (vinylic H in isoprene repeat unit (1,4-addition), corresponding to ~81% of total isoprene units [START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF]). M n,SEC = 5330 g.mol - 1 , M n,NMR = 7590 g.mol -1 , Đ = 1.15.

Synthesis of TBDMS-Ade-PI-mal-furan.

A solution of succinic-mal-furan (0.041 g, 0.117 mmol, 2 eq), EDC•HCl (0.022 g, 0.117 mol, 2 eq) and DMAP (cat) in dry DCM (1 mL) was stirred for 15 min and added dropwise to a solution of TBDMS-Ade-PI-OH (0.300 g, 0.059 mmol, 1 eq) in dry

DCM (1 ml). The mixture was stirred overnight under argon at r.t., before being quenched with water and extracted with DCM. The organic phases were then washed with brine and dried with ~81% of total isoprene units [START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF]). M n,SEC = 5370 g.mol -1 , M n,NMR = 9120 g.mol -1 , Đ = 1.15. unit (1,4-addition), corresponding to ~81% of total isoprene units [START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF]). M n,SEC = 5390 g.mol -1 , M n,NMR = 7230 g.mol -1 , Đ = 1.37. mal/Ade = 0.63

Synthesis of

Synthesis of Ade-PI-Rho. Briefly, Ade-PI (0.300 g, 0.048 mmol, 1 eq) and TEMPO-Rho (0.038 g, 0.048 mmol, 1 eq) were placed in a 7 mL-vial and dissolved in 1 mL of dry pyridine. After 20 min of degassing under argon, the vial was placed in a preheated oil bath at 110 °C and stirred for 16 h. NPs were stored at 4 °C and allowed to reach r.t. before each measurement. To establish the best conditions for protein functionalization, PI and Ade-PI NP colloidal stability was tested following incubation at r.t. overnight and at 37 °C for 30 min, in either PBS or hCMEC/D3 cell medium without serum (which is important to avoid coating with serum proteins).

Ade

hCMEC/D3 cell culture

Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) cells were used as a well-established model of the BBB that forms tight junctions (TJs), the major mechanism that prevents passage of most biomolecules and pharmacological agents from the blood to the brain [START_REF] Magro | The ability of liposomes, tailored for blood-brain barrier targeting, to reach the brain is dramatically affected by the disease state[END_REF][START_REF] Cox | Evolution of Nanoparticle Protein Corona Across the Blood-Brain Barrier[END_REF][START_REF] Tagit | Transport mechanisms of squalenoyl-adenosine nanoparticles across the blood-brain barrier[END_REF][START_REF] Weksler | The hCMEC/D3 cell line as a model of the human blood brain barrier[END_REF]. Briefly, hCMEC/D3 cells were seeded (60.000 cells.cm -2 ) between passages 25 and 35 on the apical side of transwell filters coated with rat-tail collagen type I (4 µg.cm -2 ). Cells were grown for 14 days in EBM-2 medium supplemented with fetal bovine serum (5%), hydrocortisone (1.4 µM), HEPES (10 mM), penicillin-streptomycin (1%), basic fibroblast growth factor (1 ng.mL -1 ), ascorbic acid (5 µg.mL -1 ), and lipid concentrate (1/100). Medium was changed every 2-3 days (500 µl in the apical compartment, 1 ml in the basolateral compartment). Trans-endothelial electrical resistance (TEER) was measured every day to monitor the formation of tight junctions (EVOM2, World Precision Instruments), and was also measured in the presence of NPs (0.1 mg.mL -1 added to the apical compartment for 3 h at 37 °C) and medium with no serum. Integrity of tight junctions was assessed by measuring the paracellular permeability of lucifer yellow (LY) across hCMEC/D3 monolayer. Briefly, LY (50 µM in PBS) was added to the apical compartment of the transwell system for 60 min. Every 30 min, samples were collected from the basolateral compartment.

Quantification of fluorescence (λ ex = 425 nm, λ em = 530 nm) was carried out using a spectrofluorimeter (Perkin Elmer, LS50 B). The apparent permeability coefficient (Papp) for LY was calculated as previously described [START_REF] Cecchelli | In vitro model for evaluating drug transport across the blood-brain barrier[END_REF]. Permeability of FITC-dextran (40 kDa) was assessed after incubating cells in transwells with medium supplemented with either 0 % or 5 % serum for 24 h. All medium was removed, and FITC-dextran was added to the apical compartment (1 mg.mL -1 in PBS), with 1 mL serum-depleted medium in the basolateral compartment. After 3 h incubation, fluorescence spectroscopy was used to quantify FITC-dextran in both compartments, and endothelial permeability was calculated [START_REF] Cecchelli | In vitro model for evaluating drug transport across the blood-brain barrier[END_REF].

Cell viability in the presence of polymer NPs

The viability of hCMEC/D3 cells after exposure to medium with no serum or NPs for 3 h at 37 °C was assessed using the MTT assay, as previously described [START_REF] Gregori | Novel antitransferrin receptor antibodies improve the blood-brain barrier crossing efficacy of immunoliposomes[END_REF]. Four types of bare NPs were used, each labelled with 5% wt. Rho: PI (PI), Ade-PI (Ade-PI), PI-mal (PI-mal) and Ade-PI-mal (Ade-PI-mal). Assessment of protein covalent binding. To establish whether proteins can covalently bind to the maleimide moieties present on the polymeric NPs, Ellman's protocol was used to determine free thiol groups [START_REF] Ma | Modular assembly of proteins on nanoparticles[END_REF][START_REF] Moser | Ellman's and aldrithiol assay as versatile and complementary tools for the quantification of thiol groups and ligands on nanomaterials[END_REF]. PI, Ade-PI, PI-mal and Ade-PI-mal NPs (50 g) were incubated with each protein (150 g) in 500 l serum-free medium overnight at r.t., and free protein was removed by ultrafiltration as before. 5 L of Ellman's reagent (2 mM DTNB in 50 mM sodium acetate) was added to NP-protein samples diluted 1:15 in PBS (final volume 150 L) in 96 well plates. Plate was shaken and incubated at r.t. for 5 min. Optical absorbance was measured at 412 nm. A standard curve was created using known amounts of N-acetyl-L-cysteine, and nmol of free cysteine per mg protein was calculated. All samples were measured in triplicate and experiments were repeated three times.

NP functionalization with physiological proteins and protein corona formation

Endothelial permeability and cellular uptake of NPs in an in vitro model of the healthy BBB

On the 14 th day after cell seeding in the transwell system, 0.1 mg.mL -1 of bare, protein-adsorbed or protein-conjugated NPs (determined to be non-toxic by MTT assay) suspended in 500 µL serumfree medium were added to the apical "blood" compartment of the transwell for 3 h at 37 °C. Note that the use of serum-free cell medium has already been utilized for similar NP permeability studies using this cell model [START_REF] Sancini | Functionalization with TAT-Peptide Enhances Blood-Brain Barrier Crossing In vitro of Nanoliposomes Carrying a Curcumin-Derivative to Bind Amyloid-b Peptide[END_REF][START_REF] Grossen | Functionalized Solid-Sphere PEG-b-PCL Nanoparticles to Target Brain Capillary Endothelial Cells In Vitro[END_REF]. Also, cell viability (MTT), permeability (TEER and EP after incubation with FITC-dextran, M W = 40 kDa) were tested using serum-free medium and there was no effect on hCMEC/D3 permeability and viability after 3 h incubation. The basolateral "brain" compartment contained 1 mL of serum-free medium. At different time points for up to 3 h, an aliquot from the basolateral compartment was collected and fluorescence of Rho was measured using a spectrofluorimeter (Perkin Elmer, LS50 B). Excitation was set at 555 nm, and emission at 578 nm for PI and PI-mal NPs or 587 nm for Ade-PI and Ade-PI-mal NPs. For each experiment, a standard curve was established using bare NPs of a known concentration that were not subjected to ultrafiltration. Each experiment used triplicate wells and was repeated at least three times.

Transwell filters without cells were used as a control to calculate the endothelial permeability (EP)

of NPs across the filter. EP takes into account passage of NPs through the transwell filter alone to give a value representative of the rate of passage through the cell monolayer, and was calculated as previously described [START_REF] Cecchelli | In vitro model for evaluating drug transport across the blood-brain barrier[END_REF].

To measure cellular uptake of NPs after 3 h incubation, cells were washed twice with Dulbeccos's PBS (DPBS) and incubated with 0.25% trypsin-EDTA for 5 min at 37 °C. Control wells without cells were used to rule out NPs adherence to the filter. Trypsinized cells were collected and centrifuged at 2,000 rpm (1500 g) for 5 min. Supernatant was discarded and the pellet of cells was resuspended in 750 µL cold lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% v/v Triton X-100, 0.1% SDS, 1 mM DTT). Cells were incubated for 30 min at r.t.

while rotating in the dark and then centrifuged at 10,000 rpm (7500 g) for 5 min. The supernatant was collected and fluorescence was measured as above described. Cellular uptake was expressed as a percentage of the initial amount of NPs added to the apical compartment, subtracting the fluorescence measurements obtained from wells without cells. Fluorescent microscopy images were acquired using the Operetta CLS High-Content Analysis System (PerkinElmer) equipped with 40x water objective. Excitation of NPs was set to 530-560 nm, emission at 570-650 nm.

Visualization of cellular uptake of

Results and Discussion

Overall synthetic strategy

Heterotelechelic polymers were synthesized by the "drug-initiated" method [START_REF] Nicolas | Drug-Initiated Synthesis of Polymer Prodrugs: Combining Simplicity and Efficacy in Drug Delivery[END_REF]. It relies on the controlled growth of a polymer chain from a drug covalently linked to an "initiator" using reversible-deactivation radical polymerization (RDRP) techniques, especially nitroxide-mediated polymerization (NMP) [START_REF] Harrisson | Nanoparticles with in vivo anticancer activity from polymer prodrug amphiphiles prepared by living radical polymerization[END_REF][START_REF] Guégain | Degradable polymer prodrugs with adjustable activity from drug-initiated radical ring-opening copolymerization[END_REF][START_REF] Bao | Simple Synthesis of Cladribine-Based Anticancer Polymer Prodrug Nanoparticles with Tunable Drug Delivery Properties[END_REF][START_REF] Bao | Fluorescent polymer prodrug nanoparticles with aggregation-induced emission (AIE) properties from nitroxide-mediated polymerization[END_REF][START_REF] Bao | Self-stabilized, hydrophobic or PEGylated paclitaxel polymer prodrug nanoparticles for cancer therapy[END_REF] and reversible addition-fragmentation chain transfer polymerization (RAFT) [START_REF] Bao | Self-stabilized, hydrophobic or PEGylated paclitaxel polymer prodrug nanoparticles for cancer therapy[END_REF][START_REF] Bui | Polymer prodrug nanoparticles based on naturally occurring isoprenoid for anticancer therapy[END_REF][START_REF] Maksimenko | Significant tumor growth inhibition from naturally occurring lipid-containing polymer prodrug nanoparticles obtained by the drug-initiated method[END_REF][START_REF] Bao | Structure-cytotoxicity relationship of drug-initiated polymer prodrug nanoparticles[END_REF][START_REF] Louage | Well-Defined Polymer-Paclitaxel Prodrugs by a Grafting-from-Drug Approach[END_REF][START_REF] Louage | Micellar Paclitaxel-Initiated RAFT Polymer Conjugates with Acid-Sensitive Behavior[END_REF]. This synthetic approach has key advantages: (i) purification of the drug-polymer prodrug is simplified as only unreacted monomer has to be removed; (ii) the conjugation of the drug is quantitative, leading to well-defined conjugates with one drug molecule attached to each polymer chain and (iii) the drug loading (DL) can be fine-tuned by varying the polymer chain length; the lower the chain-length, the higher the DL. Polyisoprene (PI) was selected as the polymer for its biocompatibility [START_REF] Yang | Low temperature heat shrinkable polymer material[END_REF] and structural similarity to natural terpenoids, hence representing a potential material for drug delivery applications [START_REF] Vinciguerra | A facile route to heterotelechelic polymer prodrug nanoparticles for imaging, drug delivery and combination therapy[END_REF][START_REF] Harrisson | Nanoparticles with in vivo anticancer activity from polymer prodrug amphiphiles prepared by living radical polymerization[END_REF][START_REF] Bao | Simple Synthesis of Cladribine-Based Anticancer Polymer Prodrug Nanoparticles with Tunable Drug Delivery Properties[END_REF][START_REF] Bao | Self-stabilized, hydrophobic or PEGylated paclitaxel polymer prodrug nanoparticles for cancer therapy[END_REF]].

To functionalize the other polymer chain-end and obtain heterotelechelic polymer prodrugs, we recently developed a general and robust methodology consisting of applying a nitroxideexchange reaction to "drug-initiated" synthesized polymers obtained by NMP [START_REF] Vinciguerra | A facile route to heterotelechelic polymer prodrug nanoparticles for imaging, drug delivery and combination therapy[END_REF][START_REF] Vinciguerra | Heterotelechelic polymer prodrug nanoparticles: Adaptability to different drug combinations and influence of the dual functionalization on the cytotoxicity[END_REF]. The terminal SG1 nitroxide was quantitatively replaced by a functional TEMPO nitroxide bearing the molecule of interest, which was either a fluorescent probe for imaging applications or a second drug for combination therapy. This methodology has important benefits such as nearly quantitative postfunctionalization yields and easy purification given the absence of any catalyst or reactant other than the free nitroxide and the polymer. Also, TEMPO can be functionalized by a variety of different moieties at the 4-position.

To allow NP surface functionalization with cysteine-containing proteins, the TEMPO nitroxide was derivatized by a protected maleimide group [START_REF] Heredia | Synthesis of protein-polymer conjugates[END_REF][START_REF] Nicolas | Living radical polymerization as a tool for the synthesis of polymer-protein/peptide bioconjugates[END_REF][START_REF] Gauthier | Peptide/protein-polymer conjugates: synthetic strategies and design concepts[END_REF]. TEMPO was also functionalized with a Rho moiety to endow NPs with fluorescence properties by a simple co-nanoprecipitation approach. Rho was chosen as a fluorescent probe because of its favorable characteristics such as good photostability, high quantum yield, high extinction coefficient and higher emission wavelengths than those coming from cell autofluorescence [START_REF] Benson | Cellular autofluorescence--is it due to flavins?[END_REF]. Rho was also previously modified with a piperazine ring to avoid intramolecular cyclization and allow good stability with pH modifications [START_REF] Nguyen | Practical synthetic route to functionalized rhodamine dyes[END_REF]. The covalent linkage of Rho to the polymer, as opposed to simple encapsulation, is intended to avoid potential leakage of the dye, which could result in the detection of artifact coming from the free dye by fluorescence spectroscopy [START_REF] Vinciguerra | A facile route to heterotelechelic polymer prodrug nanoparticles for imaging, drug delivery and combination therapy[END_REF][START_REF] Bao | Fluorescent polymer prodrug nanoparticles with aggregation-induced emission (AIE) properties from nitroxide-mediated polymerization[END_REF].

Synthesis of heterotelechelic polymer prodrug

PI was synthesized from AMA-SG1 alkoxyamine initiator (Scheme 1a) with M n ranging from 2860 to 4940 g.mol -1 and low dispersity (1.13-1.18, Table 1). PI-Rho was obtained by nitroxide exchange reaction from PI and TEMPO-Rho (Scheme 1b and Table 1). Unfortunately, it was not possible to calculate the post-functionalization yield by 1H NMR because of the lack of a suitable proton in α position to be compared with those of Rho, but previous demonstration by electron spin resonance [START_REF] Vinciguerra | A facile route to heterotelechelic polymer prodrug nanoparticles for imaging, drug delivery and combination therapy[END_REF] and characterization of heterotelechelic polymers showed below, led us to assume it was quasi-quantitative. As for Ade-PI (Scheme 1c), Ade hydroxyl groups were first protected by TBDMS and the resulting TBDMS-Ade was covalently linked to AMA-SG1 alkoxyamine initiator by HATU coupling to give TBDMS-Ade-AMA-SG1 (62% overall yield). By varying the experimental conditions, it then served as an initiator for the NMP of isoprene to yield well-defined TBDMS-Ade-PI (see experimental part) with M n,SEC ~5500 g.mol -1 and Đ = 1.16. After deprotection in the presence of TBAF, Ade-PI was obtained. The effectiveness of the reaction was confirmed by 1 H NMR, which showed the disappearance of the silylated protecting groups (Figure S1) and a slight decrease of the M n,NMR because of TBDMS group removal (see experimental part and Table 1).

Post-functionalization by nitroxide exchange reaction was performed on Ade-PI in presence of 1 eq. of TEMPO-Rho to yield Ade-PI-Rho as a fluorescent heterotelechelic polymer prodrug (Scheme 1d and Table 1). Ade-PI-Rho was characterized by 1 H NMR spectroscopy and gave the expected structure. Rho aromatic protons were detected in the 6.5-8 ppm region, together with those from the piperazine ring in the 4-4.5 ppm region (Figure S2). Complete disappearance of SG1 protons in the 3.2-3.4 ppm region was also observed. Integration of aromatic proton signals of Ade and Rho gave a nearly quantitative post-functionalization yield as the Rho/Ade molar ratio was as high as 94 mol.% (Table 1). SEC equipped with a fluorescent detector revealed comparable molar mass distribution profiles for both RI and fluorescence traces, supporting a uniform end-capping process by TEMPO-Rho (Figure S3a). Moreover, RI traces between Ade-PI and Ade-PI-Rho were almost perfectly overlaid, indicating negligible irreversible termination by recombination (Figure S3b).

To allow NP surface functionalization with BBB-targeting proteins by covalent linkage, a maleimide moiety was installed at the -chain end of PI and Ade-PI through the nitroxide exchange reaction. However, direct nitroxide exchange from a TEMPO bearing a furan-protected maleimide group led to extensive occurrence of termination reactions and doubling of the starting M n (Figure S4), likely because of in situ deprotection of the maleimide group at elevated temperature and radical addition onto it. The furan-protected maleimide was therefore introduced in a two-step process, by first performing the nitroxide exchange reaction with 4-hydroxy-TEMPO, followed by coupling with a succinate furan-protected maleimide (succinic-mal-furan, Scheme 2a).

PI-mal was also prepared by subjecting PI to nitroxide exchange in the presence of 4-hydroxyl-TEMPO to give PI-OH. After coupling with succinic-mal-furan under EDC assistance to give PImal-furan, PI-mal was recovered after maleimide deprotection (see experimental part, Scheme 2b

and Table 1). SEC analysis did not show noticeable changes in molar mass distributions and 1 H NMR confirmed the presence of maleimide and disappearance of SG1 protons. Even though absence of a suitable proton in α-position prevented determination of the coupling yield, it was expected to be very similar to Ade-PI-mal. A furan-protected maleimide derivative was reacted with succinic anhydride to give succinic-malfuran with a 96% yield (Scheme 2a), meanwhile the nitroxide exchange reaction was performed on TBDMS-Ade-PI with 4-hydroxy-TEMPO to give TBDMS-Ade-PI-OH (Scheme 2b and Figure S5). Even though absence of visible protons peaks on TEMPO prevented calculation of the nitroxide exchange yield by 1 H NMR, disappearance of SG1 protons (Figure S6) and the previous successful use of this reaction [START_REF] Vinciguerra | A facile route to heterotelechelic polymer prodrug nanoparticles for imaging, drug delivery and combination therapy[END_REF] indicates that it was quantitative. Subsequent coupling between TBDMS-Ade-PI-OH and a furan-protected maleimide assisted by EDC yielded the expected TBDMS-Ade-PI-mal-furan. The post-functionalization yield was calculated by comparing Ade aromatic and anomeric protons ( = 6.53, 4.22, 2.90 ppm) and the maleimide proton ( = 2.68 ppm), and reached 60% (Figure S5, experimental part). Ade was then deprotected with TBAF followed by maleimide deprotection in toluene at 100 °C to give Ade-PI-mal (Table 1). Complete disappearance of both protecting groups, appearance of a signal corresponding to maleimide protons at 7.14 ppm, and shift of Ade anomeric proton from 6.10 to 6.24 ppm (likely due to a change of the chemical environment), confirmed the effectiveness of the deprotections (Figure S6).

All polymers were also characterized by SEC and showed good conservation of the macromolecular characteristics throughout the different steps (Figure S7, experimental part).

In summary, six different polymers were synthesized (Table 1): (i) two Ade prodrug polymers, bearing a maleimide group (Ade-PI-mal) or not (Ade-PI); (ii) two Ade-free polymers, bearing a maleimide group (PI-mal) or not (PI) and (iii) two fluorescent polymers, bearing Ade (Ade-PI-Rho) or not (PI-Rho).

NP formulation and colloidal characteristics

The previously synthesized polymers were formulated into NPs in order to compare bare (i.e., protein-free) NPs to NPs functionalized with BBB-targeting proteins, either by adsorption or conjugation, and to investigate the influence of the drug on NP colloidal properties. Drug loading of the different polymer prodrugs ranged from 3.5 to 5 wt.%, depending on the M n of the polymer (Table 1). It is worth noting that higher drug loading values could be easily obtained by targeting lower molar mass polymers. Here, a relatively high M n was targeted to increase NP surface hydrophobicity, which is known to increase protein affinity towards the NP surface [START_REF] Gessner | Nanoparticles with decreasing surface hydrophobicities: influence on plasma protein adsorption[END_REF][START_REF] Walkey | Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake[END_REF].

Four different types of NPs were formulated by the nanoprecipitation technique at a concentration of 2.5 mg.mL -1 : drug-free NPs (PI) and polymer prodrug NPs (Ade-PI) for protein adsorption or drug-free NPs containing maleimide groups (PI-mal) and polymer prodrug NPs containing maleimide groups (Ade-PI-mal) for protein conjugation (Table 2). All NPs were also fluorescently-tagged by co-nanoprecipitation with PI-Rho or Ade-PI-Rho (5 wt.% Rho) to confer fluorescent properties and allow tracing during biological evaluations. NPs were very similar in terms of average size (D z = 162-185 nm) and had low particle size distribution (PSD) values varying from 0.10 to 0.21. Whereas zeta potential of PI-based prodrug NPs is usually strongly negative [START_REF] Harrisson | Nanoparticles with in vivo anticancer activity from polymer prodrug amphiphiles prepared by living radical polymerization[END_REF][START_REF] Bao | Simple Synthesis of Cladribine-Based Anticancer Polymer Prodrug Nanoparticles with Tunable Drug Delivery Properties[END_REF], all NPs here had a strong positive zeta potential value, between +40 and +50 mV. This is due to the presence of positive charges on the Rho molecule, as already observed for other PI NPs labeled with this fluorophore [START_REF] Vinciguerra | A facile route to heterotelechelic polymer prodrug nanoparticles for imaging, drug delivery and combination therapy[END_REF]. In addition to the nanoparticle surface hydrophobicity provided by the polymer, the positive charge from Rho could also increase the NP affinity towards the proteins used for functionalization [START_REF] Nandhakumar | Influence of surface charge on the in vitro protein adsorption and cell cytotoxicity of paclitaxel loaded poly(ε-caprolactone) nanoparticles[END_REF][START_REF] Deng | Plasma protein binding of positively and negatively charged polymer-coated gold nanoparticles elicits different biological responses[END_REF][START_REF] Yang | Stability of bovine serum albumin labelled by rhodamine B isothiocyanate[END_REF][START_REF] Sinigaglia | Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles[END_REF][START_REF] Patil | Protein adsorption and cellular uptake of cerium oxide nanoparticles as a function of zeta potential[END_REF]. Note that Rho has been extensively used for NP and peptide labelling in cell uptake/permeability studies, but the positive charge of the Rho alone has not been enough to dictate the cell-NP interactions [START_REF] Li | A dipeptide with enhanced anion binding affinity enables cell uptake and protein delivery[END_REF].

NP colloidal stability was assessed in water and they were all stable for at least 14 days (Figure 2a). To optimize protein functionalization conditions, PI and Ade-PI colloidal stability was also tested in PBS and hCMEC/D3 cell culture medium without serum at r.t., at 4 °C overnight, or at 37 °C for 30 min (Figure 2b and2c). The hCMEC/D3 immortalized human brain endothelial cell line is a widely used model of the BBB, forming the characteristic tight junctions, architectural structure, and protein expression found in the human barrier [START_REF] Weksler | Blood-brain barrier-specific properties of a human adult brain endothelial cell line[END_REF]. NPs were stable in cell medium but aggregated in PBS, therefore the former was used in further experiments.

Coating and functionalization of NPs with BBB crossing proteins

We recently found that specific proteins are enriched in the protein corona of gold NPs following passage through a transwell model of the BBB [START_REF] Cox | Evolution of Nanoparticle Protein Corona Across the Blood-Brain Barrier[END_REF], emphasizing their potential ability to reach the brain after in vivo administration. It was postulated that the formation of an artificial corona consisting of one of these specific proteins could enhance the passage of NPs through the BBB.

The presence of maleimide on the polymer should allow covalent binding of NPs with proteins that have a free cysteine residue. BSA [START_REF] Rombouts | Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation[END_REF], α2M [START_REF] Ghetie | Covalent binding of human α2-macroglobulin to deglycosylated ricin A chain and its immunotoxins[END_REF] and Fet A [START_REF] Dziegielewska | The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily[END_REF] have cysteine residues at different oxidation states that could be theoretically available for covalent binding to PImal and Ade-PI-mal NPs. Moreover, given the strongly positive NP surface charge (Table 2), simple adsorption of the proteins was also investigated, as it has been extensively shown that a wide array of proteins are easily adsorbed via strong electrostatic interactions with the surface of NPs [START_REF] Sathyamoorthy | Effect of surface modification on the In vitro protein adsorption and cell cytotoxicity of vinorelbine nanoparticles[END_REF]. However, there are several advantages of choosing covalent binding over the simple adsorption of proteins on the surface of NPs, including improved selectivity of protein binding.

Furthermore, as covalent bonds are stronger than electrostatic forces, this may possibly prevent the displacement of functionalized proteins by serum proteins upon in vivo administration, or their removal in the purification process [START_REF] Ivanova | A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly (tert-butyl methacrylate)[END_REF].

NPs were incubated overnight at r. To confirm that proteins could covalently bind to maleimide moieties present on the NPs and to discriminate between adsorbed and covalently linked proteins, Ellman's protocol was used to determine free thiol groups [START_REF] Ma | Modular assembly of proteins on nanoparticles[END_REF][START_REF] Moser | Ellman's and aldrithiol assay as versatile and complementary tools for the quantification of thiol groups and ligands on nanomaterials[END_REF]. If a NP is covalently bound to a protein, the number of free thiols present on the protein should decrease, as the thiol groups are attached to the maleimide moiety. Ellman's reagent was used to quantify free cysteine on BSA, α2M or Fet A following incubation with either PI, PI-mal, Ade-PI or Ade-PI-mal NPs (Figure S8). For all proteins, there was significantly less free cysteine following incubation with PI-mal and Ade-PI-mal NPs compared to incubation with PI and Ade-PI NPs respectively, indicating that covalent binding occurred in the presence of maleimide. Even though there is likely a mix of adsorbed/linked proteins at the surface of PI-mal and Ade-PI-mal NPs, their total amount was not significantly different to that of only adsorbed proteins at the surface of PI and Ade-PI NPs.

Further confirmation of successful protein functionalization was obtained by measuring the hydrodynamic diameter of NPs. It has been previously shown that NPs increase in size upon protein functionalization and protein corona formation [START_REF] Konduru | Protein corona: implications for nanoparticle interactions with pulmonary cells[END_REF]. Therefore, the size of NPs with or without protein functionalization was also measured after incubation in serum to form a corona, and subsequent ultrafiltration to remove excess unbound serum proteins. For all NPs, both protein functionalization and protein corona formation resulted in a significant increase in size, with the latter causing a more dramatic increase (Figure 4). The size increase upon corona formation is likely due to the adherence of multiple protein layers, containing proteins of diverse size bound directly to the NP surface and to each other. In the case of almost every NP-protein combination, prior protein functionalization prevented corona-associated size increases, and the size was comparable or identical to protein-functionalized NPs not exposed to serum. This is indicative of functionalization stability. Notably the ultrafiltration process itself caused a slight increase in PI (+36 nm), PI-mal (+23 nm), and Ade-PI-mal (+9 nm) NPs without proteins, but PSD values remained comparable before and after ultrafiltration (Figure S9). Indeed, the PSD values for most nanoparticle-protein or nanoparticle-serum complexes did not significantly change compared to bare NPs. This indicated that size changes are not due to aggregation of NPs upon protein exposure, and is in agreement with studies showing increases in NP size upon protein binding [START_REF] Ma | Modular assembly of proteins on nanoparticles[END_REF][START_REF] Konduru | Protein corona: implications for nanoparticle interactions with pulmonary cells[END_REF][START_REF] Van Hong Nguyen | Protein corona: a new approach for nanomedicine design[END_REF][START_REF] Dutta | Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials[END_REF]. Remarkably, bare PI NPs, whose safety has already been demonstrated in vivo [START_REF] Harrisson | Nanoparticles with in vivo anticancer activity from polymer prodrug amphiphiles prepared by living radical polymerization[END_REF][START_REF] Bao | Simple Synthesis of Cladribine-Based Anticancer Polymer Prodrug Nanoparticles with Tunable Drug Delivery Properties[END_REF], had high cellular uptake (5.3 ± 0.8%) and endothelial permeability (EP) (9.5 x 10 -4 cm.min -1 ) (Figure 5b and6). Compared to previously described bi-functionalized liposomes that can cross the BBB in vitro (EP = 2.5 x 10 -5 cm.min -1 ) and in vivo [START_REF] Bana | Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: Implications for therapy of Alzheimer disease[END_REF], EP of PI NPs was ~4 times higher, which is likely due, at least partially, to the high PI hydrophobicity, known to increase cellular uptake [START_REF] Bao | Simple Synthesis of Cladribine-Based Anticancer Polymer Prodrug Nanoparticles with Tunable Drug Delivery Properties[END_REF][START_REF] Monopoli | Biomolecular coronas provide the biological identity of nanosized materials[END_REF]. Importantly, the decrease in cell uptake observed upon protein functionalization of PI NPs (Figure 5b) did not match with a decrease in permeability (Figure 6). This indicates that the presence of proteins prevents the sequestering of NPs in the cell but may allow them to pass through the in vitro BBB at a similar or higher rate than bare PI NPs.

When considering PI and PI-mal NPs, the presence of maleimide moieties led to higher cellular uptake but a lower EP compared to PI NPs. However, covalently bound 2M at the surface of PI-mal NPs caused a significant increase in both cellular uptake and EP (Figure 5b and6).

Therefore, this protein may be promising as a functionalization tool to promote passage of certain NPs through the BBB. Conversely, BSA and Fet A did not significantly affect the cellular uptake neither the EP of any of the PI-based NPs. This observation may be explained by differences in the packing/orientation of the proteins or alterations in protein conformation on the surface of NPs that may change protein-receptor interactions and subsequent cellular uptake [START_REF] Ivanova | A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly (tert-butyl methacrylate)[END_REF][START_REF] Petrash | Changes in protein adsorption on self-assembled monolayers with monolayer order: comparison of human serum albumin and human gamma globulin[END_REF][START_REF] Vijayendran | A quantitative assessment of heterogeneity for surfaceimmobilized proteins[END_REF].

Interestingly, the presence of Ade significantly decreased the endothelial permeability (EP) of NPs by ~4-8 fold when comparing PI and Ade-PI NPs, regardless of protein functionalization (p < 0.01) (Figure 6). Given the relatively high water-solubility of Ade, many Ade moieties may be displayed at the surface of NPs, decreasing the NP surface hydrophobicity and thus modifying their interaction with proteins compared to PI NPs. This hypothesis is supported by the increase of the predicted HLB numbers for Ade-containing model prodrugs compared to Ade-free counterparts (Table S1). Similarly, Ade-PI-mal NPs had a lower cellular uptake than PI-mal NPs (5.3 ± 2.0 % vs 9.5 ± 1.4 %, p < 0.05), potentially due to a lower surface amount of reactive maleimide moieties caused by competition with hydrophilic Ade groups (Figure 5b). This may explain why 2M did not significantly affect the EP of NPs with Ade.

Conclusion

Here we described a robust procedure for engineering well-defined, PI-based heterotelechelic polymer prodrug NPs for drug delivery and brain targeting. Heterotelechelic polymer prodrugs were prepared by the "drug-initiated" strategy followed by their post-functionalization via the nitroxide exchange reaction to position maleimide groups for conjugation of BBB-crossing proteins (BSA, 2M or Fet A) at the NP surface, as confirmed by Ellman's test and DLS measurements. All NPs were stable, with average diameters in the 162-185 nm range, narrow particle size distributions, and long-term colloidal stability in water and cell culture medium.

We showed that non-BBB-targeted PI-NPs already exhibit higher permeability and cellular uptake than targeted systems described in the literature, rendering this novel nanodevice highly attractive as a drug delivery system for brain-directed drugs or contrast agents. We also demonstrated that surface functionalization of these NPs with 2M enhanced their passage through an in vitro BBB model. This could be a promising BBB-targeting strategy in vivo that would warrant further investigation. Also, our results suggested that in case polymer-prodrugs are envisioned, the nature/solubility of the drug and/or its positioning within the NPs are crucial to achieve efficient surface functionalization with BBB-targeting proteins to enhance passage through the BBB.

Figure 1 .

 1 Figure 1. General strategies for the preparation of (a) polymer nanoparticles and (b) polymer prodrug nanoparticles surface-adsorbed or surface-functionalized with proteins for the crossing of hCMEC/D3 human brain endothelial cells as a blood-brain barrier (BBB) model. BSA = albumin, 2M = 2-macroglobulin and Fet A = fetuin A.

  , tert-Butyldimethylsilyl chloride (TBDMSCl), imidazole, N,N-diisopropylethylamine (DIPEA), isoprene, 4-(dimethylamino)pyridine (DMAP), succinic anhydride, 4-hydroxy-TEMPO, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), rhodamine B base (Rho), N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC•HCl), exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride, 2-(2aminoethoxy)-ethanol as well as dry solvents, corning transwells (polystyrene membrane, 12 well plates, 0.4 µm pores), bovine α2M, bovine fetuin (Fet A), bovine serum albumin (BSA) and cell medium components were purchased from Sigma-Aldrich (Lyon, France) and used as received.

  at 30 °C with two columns from Polymer Laboratories (PL-gel MIXED-D; 300 × 7.5 mm; bead diameter 5 mm; linear part 400 to 4 × 10 5 g.mol -1 ), a differential refractive index detector (Spectra System RI-150 from Thermo Electron Corp.) and a scanning fluorescence detector (Waters 474). The eluent was chloroform at a flow rate of 1 mL.min -1 (Waters 515 pump) and toluene was used as a flow-rate marker. The calibration curve was based on polystyrene (PS) standards (peak molar masses, M p = 162-523 000 g.mol -1 ) from Polymer Laboratories. A polyisoprene (PI) calibration curve was constructed by converting the PS standard peak molecular weights (M PS ) to PI molecular weights (M PI ) using Mark-Houwink-Sakurada (MHS) constants determined for both polymers in CCl 4 at 25 °C. For PI, the MHS constants used were K PI = 2.44 × 10 4 and α PI = 0.712. For PS, K PS = 7.1 × 10 4 and α PS = 0.54 (M w < 16 700 g.mol -1 ) or K PS = 1.44 × 10 4 and α PS = 0.713 (M w > 16 700 g.mol -1 )[START_REF] Harrisson | Nanoparticles with in vivo anticancer activity from polymer prodrug amphiphiles prepared by living radical polymerization[END_REF]. This technique allowed the number-average molar mass (M n ), the weight-average molar mass (M w ) and the dispersity (M w /M n , Ð) to be determined.DynamicLight Scattering (DLS) and zeta potential. Nanoparticle diameters (Dz) and zeta potentials (ζ) were measured by dynamic light scattering (DLS) with a Nano ZS from Malvern (173° scattering angle) at a temperature of 25 °C. The surface charge of the NPs was investigated by ζ-potential (mV) measurement at 25 °C after dilution with 1 mM NaCl, using the Smoluchowski equation.

  Ade-PI. TBDMS-Ade-AMA-SG1 (110 mg, 0.115 mmol) was placed in a 15 mL-capacity pressure tube (Ace Glass 8648-164) fitted with plunger valves and thermowells. After addition of isoprene (5.7 mL, 57.2 mmol) and dry 1,4-dioxane (5.7 mL), the tube underwent three cycles of freeze-thaw degassing and was then backfilled with argon. The tube was then placed in a preheated oil bath at 115 °C for 16 h (TBDMS-Ade-PI1) and cooled to r.t. by placing under cold water. The residue was concentrated under reduced pressure and precipitated in cold methanol to give TBDMS-Ade-PI as a colorless viscous oil. Another polymerization was performed for 16 h with [isoprene] 0 /[TBDMS-Ade-AMA-SG1] 0 = 780/1 (TBDMS-Ade-PI2). All polymers were characterized by SEC and 1 H NMR. M n,NMR was calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA-SG1 with DP n,NMR calculated from the ratio of areas under the peak at 8.69, 8.31 and 5.98 ppm (aromatic and anomeric proton of Ade) and 5.0-5.5 ppm (vinylic H in isoprene repeat unit(1,4-addition), corresponding to ~81% of total isoprene units[START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF]). TBDMS-Ade-PI1: M n,SEC = 6000 g.mol -1 , M n,NMR = 6450 g.mol -1 and Đ = 1.16. TBDMS-Ade-PI2: M n,SEC = 5100 g.mol -1 , M n,NMR = 5050 g.mol -1 , and Đ = 1.16.

MgSO 4 .

 4 After evaporation of the solvent, two precipitations in cold methanol were performed to obtain TBDMS-Ade-PI-mal-furan as a colorless oil. The polymer was characterized by SEC and 1 H NMR. The post-functionalization yield was calculated by 1 H NMR using the chemical shifts of protons of mal ( = 6.53, 4.22, 2.90 and 2.68 ppm) and the chemical shifts of aromatic protons and anomeric proton of Ade ( = 8.69, 8.31 and 5.98 ppm). M n,NMR was calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA-SG1 -MW SG1 + MW TEMPO-mal-furan with DP n,NMR calculated from ratio of areas under the peak at 8.69, 8.31 and 5.98 ppm (aromatic and anomeric proton of Ade) and 5.0-5.5 ppm (vinylic H in isoprene repeat unit(1,4-addition), corresponding to

  Ade-PI-mal-furan. The TBDMS-protected TBDMS-Ade-PI-mal-furan (0.300 g, 0.056 mmol) was dissolved in 1.5 mL THF and TBAF (1 M in THF, 150 μL) was added. The solution was stirred for 30 min and then purified by two consecutive precipitations in cold methanol to give Ade-PI-mal-furan as a colorless viscous oil. It was then directly used for the next reaction.Synthesis of Ade-PI-mal.Ade-PI-mal-furan (0.200 g, 0.036 mmol) was dissolved in dry toluene (1 mL). After 20 min of degassing under argon, the vial was placed in a preheated oil bath at 100 °C and stirred for 16 h. Ade-PI-mal was then precipitated two times in cold methanol and dried under reduced pressure until constant weight. The polymer was characterized by SEC and 1 H NMR, which also confirmed the disappearance of maleimide protecting group. The post-functionalization yield was calculated by1 H NMR from ratio of areas under the peak of protons of mal at 6.53, 4.22, 2.90 and 2.68 ppm and aromatic and anomeric protons of Ade at 8.69, 8.31 and 5.98 ppm. M n,NMR was calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA-SG1 -MW SG1 -MW TBDMS + MW TEMPO-mal with DP n,NMR calculated from ratio of areas under the peak at 8.69, 8.31 and 5.98 ppm (aromatic and anomeric proton of Ade) and 5.0-5.5 ppm (vinylic H in isoprene repeat

94 2. 4

 944 -PI-Rho was then precipitated two times in cold methanol and dried under reduced pressure until constant weight. The post-functionalization yield was calculated by 1 H NMR using the chemical shifts of aromatic protons of Rho ( = 7.70, 7.55, 7.08 and 6.93-6.67 ppm) and the chemical shifts of aromatic protons and anomeric proton of Ade ( = 8.69, 8.31 and 5.98 ppm).Functionalization was confirmed by SEC equipped with a fluorescent detector (λ ex = 570 nm, λ em = 595 nm). M n,NMR was calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA- SG1 -MW SG1 -MW TBDMS + MW TEMPO-Rho with DP n,NMR calculated from ratio of areas under the peak at 8.69, 8.31 and 5.98 ppm (aromatic and anomeric proton of Ade) and 5.0-5.5 ppm (vinylic H in isoprene repeat unit (1,4-addition), corresponding to ~81% of total isoprene units[START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF]). M n,SEC = 7540 g.mol -1 , M n,NMR = 6120 g.mol -1 , Đ = 1.32. Rho/Ade = 0.Nanoparticle preparation PI (PI3), Ade-PI, PI-mal and Ade-PI-mal NPs were prepared by the nanoprecipitation technique[START_REF] Fessi | Nanocapsule formation by interfacial polymer deposition following solvent displacement[END_REF]. All NPs were obtained by co-nanoprecipitation of the desired polymer with a Rho-containing PI to reach 5 wt.% of Rho moieties with respect to the overall weight of NPs, as follows: Ade-PI-Rho/Ade-PI, Ade-PI-Rho/Ade-PI-mal, PI-Rho/PI and PI-Rho/PI-mal. This percentage gave the best compromise in terms of fluorescence signal and NP colloidal characteristics (e.g., average size, stability). Briefly, 2.5 mg of the corresponding polymers were dissolved in 0.5 mL of THF and the solution was quickly added to 1 mL MilliQ water. THF was evaporated at r.t. using a Rotavapor.Intensity-averaged diameter (D z ) in MilliQ water and zeta potential measurements in 1 mM NaCl were carried out in triplicate by DLS. Their colloidal stability was assessed in water for 14 days.

  Protein functionalization. 100 L of PI and Ade-PI NPs (for adsorption) or PI-mal and Ade-PImal NPs (for covalent conjugation) at 2.5 mg.mL -1 were incubated with 400 L of one of the proteins (MW Fet A = 49 kDa, MW α2M = 725 kDa and MW BSA = 66 kDa; 1.25 mg.mL -1 ) in hCMEC/D3 serum-free cell medium overnight at r.t., under which conditions the NPs were stable, as determined by previous stability tests. Free proteins were separated from NP-protein complexes by ultrafiltration (MWCO 300 kDA) at r.t. for 3 min at 14,000 g. NPs were resuspended in serumfree medium, and BCA assay (Thermo Fischer Scientific) was used to determine protein binding.Protein corona formation. To test potential size changes upon protein corona formation, 25 L bare, protein-adsorbed or protein-conjugated NPs (62.5 g) were incubated in 200 L cell medium containing 5% FBS for 30 min at 37 C. Unbound proteins were removed by ultrafiltration (MWCO 300 kDA) for 6 min at 14,000 g. NPs were resuspended in MilliQ water and size was measured by DLS.

  NPs by hCMEC/D3 cells using fluorescence microscopyhCMEC/D3 cells were seeded on rat collagen type I-coated Cell Carrier Ultra 96 well plates (10,000 cells/well) and grown for two days. When cells were confluent, they were incubated with 100 µL bare PI, Ade-PI, PI-mal and Ade-PI-mal NPs in serum-free cell medium (0.1 mg.mL -1 ) for 3 h at 37 °C. NPs were then removed from the wells and cells were washed twice with DPBS. Cells were fixed with 10% formalin solution (100 µL) at r.t. for 20 min, followed by three 10 min washes with DPBS. Cells were permeabilized with 100 µL Triton (0.2 % v/v) for 15 min at r.t. and washed once with DPBS. The actin cytoskeleton was stained with Phalloidin Alexafluor 633 (1:100 in DPBS) for 1 h at r.t., and then washed 3 times with DPBS. Hoescht (1:5,000 in DPBS) was incubated for 15 min at r.t. to stain nuclei. Cells were washed three times in DPBS and all wells were filled with MilliQ water (100 µL). Plates were stored in the dark at 4 °C until visualization.

Scheme 1 .

 1 Scheme 1. (a) Synthesis of polyisoprene (PI) by nitroxide-mediated polymerization (NMP) initiated by the AMA-SG1 alkoxyamine. (b) Synthesis of PI-rhodamine (PI-Rho) by the nitroxide exchange reaction with TEMPO-rhodamine (TEMPO-Rho) on PI. (c) Synthesis of adenosine-polyisoprene (Ade-PI) by NMP of isoprene from tris-O-silylated-adenosine-AMA-SG1 alkoxyamine (TBDMS-Ade-AMA-SG1), followed by deprotection of Ade. (d) Synthesis of adenosine-polyisoprenerhodamine (Ade-PI-Rho) by the nitroxide exchange reaction with TEMPO-rhodamine (TEMPO-Rho) on Ade-PI.

Scheme 2 .

 2 Scheme 2. (a) Synthesis of adenosine-polyisoprene-maleimide (Ade-PI-mal) by nitroxide-mediated polymerization (NMP) of isoprene from tris-O-silylated-adenosine-AMA-SG1 alkoxyamine (TBDMS-Ade-AMA-SG1), followed by the nitroxide exchange with 4-hydroxy-TEMPO on TBDMS-Ade-AMA-SG1, EDC-assisted functionalization by succinic-mal-furan and subsequent deprotections of Ade and maleimide. (b) Structure of PI-mal obtained by the nitroxide exchange with 4-hydroxy-TEMPO on PI, EDC-assisted functionalization by succinic-mal-furan and subsequent deprotection of maleimide.

Figure 2 .

 2 Figure 2. (a) Evolution of the intensity-averaged diameters (D z ) and particle size distributions (PSD) in water of Ade-PI, PI, Ade-PI-mal and PI-mal NPs blended with PI-Rho or Ade-PI-Rho (5 wt.%. Rho) measured by DLS over 14 days. Stability in milliQ water, PBS and cell culture medium without serum incubated overnight (O.N.) at room temperature (r.t.), O.N. at 4 °C, or for 30 min at 37 °C of (b) PI or (c) Ade-PI. NPs at 0.1 mg.mL -1 in order to determine the best conditions for protein functionalization (n = 3, error bars represent SEM).

  t. with each protein suspended in hCMEC/D3 cell medium without serum, to avoid serum protein interferences, then ultrafiltrated to remove unbound protein. BCA quantification indicated that the highest amount of protein binding occurred with α2M on all NPs (1.8-2.4 µg protein/µg NP) and the lowest with Fet A (0.5-1.1 µg protein/µg NP), possibly due to differences in MW as MW α2M = 725 kDa and MW Fet A = 49 kDa (Figure3). The presence of Ade significantly increased BSA binding in the presence of mal (1.4 µg protein/µg NPs on Ade-PI-mal vs 0.9 µg protein/µg NPs on PI-mal) but did not significantly affect α2M or Fet A binding.

Figure 3 .

 3 Figure 3. Amount of protein bound to NPs (g protein/g NP), as measured by BCA assay after incubation of NPs with protein in serum-free cell medium overnight at r.t., followed by ultrafiltration to remove unbound protein (n = 3, error bars represent SEM. Student's t test * p < 0.05, ** p < 0.01).

Figure 4 .

 4 Figure 4. Size of NPs upon protein functionalization and protein corona formation in serum, as measured by DLS after ultrafiltration to remove unbound proteins. Functionalization or coating of NPs with: (a) BSA, (b) α2M and (c) Fet A, with or without serum-derived protein corona (n = 3, error bars represent SEM. Statistical analysis (Student's t test) to compare size with bare NPs: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5 .

 5 Figure 5. Cellular uptake of PI, Ade-PI, PI-mal and Ade-PI-mal NPs in an in vitro BBB model. (a) Fluorescent microscopy images show localization of NPs in the perinuclear region of hCMEC/D3 endothelial cells after 3 h incubation at 37 °C. Orange staining is actin cytoskeleton, blue is cell nuclei, red is rhodamine labelled NPs; (b) Uptake of NPs by hCMEC/D3 cells, expressed as percentage of original NPs added to the apical side of the transwell (n = 3, error bars represent SEM, Student's t test *p < 0.05).

Figure 6 .

 6 Figure 6. Endothelial permeability (EP) of PI, Ade-PI, PI-mal and Ade-PI-mal NPs in an in vitro BBB model after 3 h at 37 °C (n ≥ 3, error bars represent SEM, Student's t test *p < 0.05, **p < 0.01).

Synthesis of succinic-mal-furan (N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6- tetrahydrophthalimide-4-oxobutanoic acid). Succinic

	modified published procedure [44]. Briefly, a solution of DMAP (0.290 g, 2.37 mmol) and succinic
	anhydride (0.237 g, 2.37 mmol) in dry DCM (10 mL) was added dropwise at 0 °C and under an
	argon atmosphere, to a solution of N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-
	tetrahydrophthalimide (0.200 g, 0.833 mmol) in dry DCM (2 mL). After stirring overnight at r.t.,
	the solution was quenched with 1 M HCl. The product was extracted three times with DCM. The
	combined organic layers were washed with brine and dried over MgSO 4 . After evaporation of the
	solvent, succinic-mal-furan was obtained as a white solid. Yield: 96%. 1 H NMR (CDCl 3 , 300
	MHz): δ 6.53 (s, 2H), 5.30 (d, 2H), 4.27 -4.15 (m, 2H), 3.66 (dq, 6H), 2.90 (s, 2H), 2.68 (s, 4H).
	13 C NMR (75 MHz, CDCl 3 ) δ 176.39, 171.95, 136.54, 80.77, 68.50, 67.05, 63.81, 47.49, 38.24,
	29.05, 28.88. MS (ESI-): m/z = 352.2 (M) -. Calc. for C 16 H 19 NO 8 : 353.1.
	3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride (0.50 g,
	3.01 mmol) was dissolved in 2-(2-aminoethoxy)-ethanol (1.58 g, 15.0 mmol) and stirred at 80 °C
	for 7 h. The reaction mixture was cooled to r.t. before addition of DCM. The solution was washed
	with brine, dried over MgSO 4 and concentrated to give a colorless oil. Yield: 39 % (0.318 g.). 1 H
	NMR (300 MHz, CDCl 3 ) δ 6.51 (s, 2H), 5.29 (s, 2H), 3.76-3.50 (m, 8H), 2.87 (s, 2H), 2.59 (b, 1H)
	ppm. 13 C NMR (75 MHz, CDCl 3 ) δ 176.38, 136.30, 81.02, 72.25, 67.06, 61.63, 47.40, 38.58.
	-mal-furan was prepared according to a

  C NMR (75 MHz, CDCl 3 ): δ 169.75, 152.42, 152.06, 149.66, 141.76, 88.39, 85.51, 85.19, 78.82, 78.69, 75.58, 72.02, 71.72, 70.04, 68.21, 62.63, 62.44, 61.82, 60.65, 35.41, 30.54, 28.24, 26.08, 25.85, 25.68, 18.50, 18.08, 17.83, 16.50, 16.23, -4.40, -4.71, -5.05, -5.39 ppm. MS (ESI+): m/z = 981.6 (M+Na) + . Calc. for C 29 H 48 N 3 O 7 P: 958.6.

). Tris-O-silylated adenosine (TBDMS-Ade, 2.0 g, 3.28 mmol) was dissolved in 10 mL of dry DMF. HATU (2.49 g, 6.56 mmol) and AMA-SG1 (2.37 g, 6.56 mmol) were dissolved in 10 mL of dry DMF in another round-bottomed flask, DIPEA (2.12 g, 16.4 mmol) was added dropwise by syringe. The mixture was stirred for 1 h and added by syringe to the first solution. The solution was stirred under argon atmosphere for 96 h at room temperature (r.t.), then diluted with 100 mL EtOAc.The organic phase was washed with 1 M HCl, sat. NaHCO 3 aqueous solution, and brine before being dried over MgSO 4 . The residue was concentrated under reduced pressure and purified by flash chromatography (SiO 2 , gradient from PetrolEther/EtOAc 9/1 v/v to PetrolEther/EtOAc 5/5 as eluent) to give 1.95 g of TBDMS-Ade-AMA-SG1 as a white-yellow solid. Yield: 62%.

1

H NMR (CDCl 3 , 300 MHz): δ 11.01 (d, 1H), 8.76 (s, 1H), 8.31 (d, 1H), 6.09 (m, 1H), 4.93-4.82 (d, 1H), 4.69 (dt, 1H), 4.37-4.29 (m, 2H), 4.23-3.98 (m, 5H), 3.81 (dt, 1H), 3.49 (d, 1H), 1.62 (d, 3H), 1.32 (t, 6H), 1.22-1.20 (m, 18H), 0.95 (d, 18H), 0.81 (d, 9H), 0.12 (d, 12H), -0.03 (d, 3H), -0.24 (d, 3H)

ppm.

13

Table 1 .

 1 Characterization of Polyisoprene (PI), Polyisoprene-Rhodamine (PI-Rho), Adenosine-Polyisoprene (Ade-PI), Adenosine-Polyisoprene-Rhodamine (Ade-PI-Rho), Adenosine-Polyisoprene-Maleimide (Ade-PI-mal) and Polyisoprene-Maleimide (PI-mal). Determined by SEC, calibrated with PS standards and converted into PI by using Mark-Houwink-Sakurada parameters. b Calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA-SG1 -MW TBDMS . c Calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA-SG1 -MW SG1 -MW TBDMS + MW TEMPO-Rho . d Calculated according to M n,NMR = (DP n,NMR x MW isoprene ) + MW TBDMS-Ade-AMA-SG1 -MW SG1 -MW TBDMS + MW TEMPO-mal . e Calculated from ratio of areas under the peak at 8.69, 8.31 and 5.98 ppm (aromatic and anomeric proton of Ade) and 5.0-5.5 ppm (vinylic H in isoprene repeat unit (1,4-addition), corresponding to ~81% of total isoprene units[START_REF] Harrisson | SG1 nitroxide-mediated polymerization of isoprene: alkoxyamine structure/control relationship and α, ω-chain-end functionalization[END_REF]). f Calculated from ratio of areas under the peak of aromatic protons of Rho at 7.70, 7.55, 7.08 (or of mal at 6.53, 4.22, 2.90 and 2.68 ppm) and 6.93-6.67 ppm and aromatic and anomeric protons of Ade at 8.69, 8.31 and 5.98 ppm.

	Prodrug	M n,SEC	a	Ð a	M n,NMR	DP n,NMR	e	Rho or mal	Drug loading
		(g.mol -1 )		(g.mol -1 )			/Ade f	(wt.%) g
	PI	2860		1.13	-	-		-	-
	PI-Rho	2690		1.13	-	-		-	-
	Ade-PI	6260		1.15	6280 b	83		-	4.3
	Ade-PI-Rho	7540		1.32	6120 c	74		0.94	3.5
	Ade-PI-mal	5390		1.37	7230 d	95		0.63	5.0
	PI-mal	5650		1.17	-	-		-	-

a g Calculated according to MW Ade /M n,SEC .

Table 2 .

 2 Colloidal Characteristics of NPs Prepared by Co-nanoprecipitation of PI, Ade-PI, PI-mal or Ade-PI-mal with PI-Rho or Ade-PI-Rho (5 wt.% Rho). Measured by dynamic light scattering (DLS) as an average of three different measures. b Co-nanoprecipitated with PI-Rho (5 wt.% Rho). c Co-nanoprecipitated with Ade-PI-Rho (5 wt.% Rho).

	Sample	D z (nm) a	PSD a	 a (mV)
	PI b	185 ± 1	0.13 ± 0.001	49 ± 2
	Ade-PI c	179 ± 3	0.11 ± 0.03	45 ± 1
	PI-mal b	164 ± 14	0.21 ± 0.01	41 ± 15
	Ade-PI-mal c	162 ± 3	0.10 ± 0.02	50 ± 4

a

Acknowledgements

This work was supported by the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska Curie grant agreement no. 642028 (NABBA). The authors thank Pierre-Olivier Couraud, Université Rene Descartes, for providing the hCMEC/D3 cells, and Stéphanie Nicolaÿ for mass spectrometry analyses (Service d'Analyses des Médicaments et Métabolites (Institut Paris-Sud d'Innovation Therapeutique (IPSIT)), Université Paris-Sud).

Arkema is acknowledged for kindly providing the SG1 nitroxide. CNRS and Université Paris-Sud are also acknowledged for financial support.

Cellular uptake and permeability of functionalized NPs through a transwell BBB model

The next step was to evaluate the influence of the nanoparticle surface coating/functionalization on the cellular uptake and permeability using an in vitro BBB model. A simple but well characterized and widely used model of the BBB was established by seeding hCMEC/D3 cells on transwell inserts separating an apical "blood" side from a basolateral "brain" side [START_REF] Magro | The ability of liposomes, tailored for blood-brain barrier targeting, to reach the brain is dramatically affected by the disease state[END_REF][START_REF] Cox | Evolution of Nanoparticle Protein Corona Across the Blood-Brain Barrier[END_REF][START_REF] Tagit | Transport mechanisms of squalenoyl-adenosine nanoparticles across the blood-brain barrier[END_REF][START_REF] Weksler | The hCMEC/D3 cell line as a model of the human blood brain barrier[END_REF]. Average TEER values peaked at 149 ± 3 .cm -2 on the 14 th day after seeding and the paracellular permeability of LY, a probe used to evaluate the integrity of in vitro tight junctions, was 1.51 ± 0.03 × 10 -3 cm.min - 1 , indicating the formation of tight junctions [START_REF] Cox | Evolution of Nanoparticle Protein Corona Across the Blood-Brain Barrier[END_REF][START_REF] Cecchelli | In vitro model for evaluating drug transport across the blood-brain barrier[END_REF]. MTT assay showed that none of the NPs affected hCMEC/D3 cell viability up to 0.1 mg.mL -1 after 3 h incubation (Figure S10).

Furthermore, TEER values remained unchanged upon incubation of hCMEC/D3 cells with PI, PImal, Ade-PI, or Ade-PI-mal NPs for at least 3 h (Figure S11) during which time the NPs were stable, despite an increase in diameter (Table S2).

All four bare PI-based NPs were internalized to some extent by hCMEC/D3 cells, as visualized by fluorescent microscopy (Figure 5a) and quantified in the transwell model (Figure 5b). Fluorescent microscopy showed that the NPs gathered in the perinuclear region of hCMEC/D3 cells after 3 h incubation, a pattern seen with other NPs and cell types [START_REF] Schütz | Lysosomal dysfunction caused by cellular accumulation of silica nanoparticles[END_REF][START_REF] Barua | Synergistic Targeting of Cell Membrane, Cytoplasm, and Nucleus of Cancer Cells Using Rod-Shaped Nanoparticles[END_REF].


Marc De Backer

Elise Berrier

Yann Martinon

Nicolas Dujardin

Florence Dufour

Jean-Paul Peres

Christophe Jehoulet

Peres Jean Paul

Infrared spectroelectrochemical studies of divided

come

Electrical energy produced by renewable sources such as wind power or photovoltaic devices is fluctuant by nature. In order to use it reliably, it is necessary to have a means of storing electricity. These storage devices are also of prime importance in systems where surges of power are frequent; e.g. applications such as transportation and regenerative braking.

Storage of electrical energy can be accomplished in an elegant way through the use of lightweight capacitors. Recently, devices based on the use of the electrochemical double-layer capacitor ͑EDLC͒ have been developed: the supercapacitors. [START_REF] Conway | Electrochemical Supercapacitors: Scientific Fundamentals and Technological Applications[END_REF] Supercapacitors are intermediate systems between electrochemical batteries, which can store high energy associated with medium power values, and dielectric capacitors, which can deliver very high power during times of the order of a few milliseconds. Supercapacitors are used for energy storage over time periods ranging from seconds to minutes; their peak power is up to 10-20 times higher than that of batteries, but their energy density is 20-50 times lower.

Their principle relies on the use of high specific area electrodes placed in an electrolyte and separated by a thin inert microporous membrane. This work focuses on organic electrolyte capacitors with electrodes manufactured using carbon dispersed in an aqueous solvent. To be useful, these devices must be able to remain charged in open-circuit conditions for great lengths of time. This implies that no irreversible reactions induced by the variation of the potential of each electrode should occur; thus, it is important to understand the mechanisms occurring during charge and spontaneous discharges.

During the charge of the EDLC, several phenomena can occur. 2 An increase in the number of ions forming the double layer can be caused by the application of a voltage. A local increase in ionic concentration near the carbon surface, but not directly connected with the double layer, can also be produced if a current flows through the capacitor at or above the electrolyte potential range.

Self-discharge processes are complex and involve mechanisms occurring on a short or a long time scale. When the supercapacitor is fully charged, its removal from the power supply induces a fast relaxation phenomenon. This process is reversible at short times. An irreversible process corresponding to faradaic reactions can also be observed on the same time scale. Another reason for self-discharge are the faradaic reactions of various groups at the surface of the carbon during ca. the first 500 h of use of the device. This phenomenon is irreversible. Another cause for voltage drop is the presence of an ''electrochemical shuttle'' due to electroactive redox couples involving impurities in the carbon layer.

To gain some insight into self discharge phenomena, a knowledge of the variations of composition of the electrolyte in the immediate vicinity of the electrode or even modifications of the electrode itself as the potential is swept is of prime importance. In situ spectroelectrochemical measurements are well suited to fulfill this goal. Vibrational spectroscopy yields structural information on all the species present in the solution or on the electrode. Therefore, it appears to be a good method to couple with electrochemistry. Raman spectroscopy associated with confocal microscopy seems ideal because of the spatial resolution that enables one to select which part of the system is studied: electrode or solvent. 3 However, Raman spectroscopy is not very sensitive to the presence of minor components, and the previous studies were not able to detect new species, although variations of the intensities of the major peaks during potential scans were recorded. Rapid scan Fourier transform infrared ͑FTIR͒ spectroscopy associated with electrochemistry in situ was used in this work. Preparation of new electrodes allowed us to keep enough IR light to obtain high-quality spectra.

Experimental

The spectroelectrochemical cell and its installation in an IR spectrometer have been previously described. 4 The cell was designed to accommodate various electrodes. The distance between the electrode and the optical window could be adjusted by a micrometric screw so that the cell path length was small enough ͑ca. 100 m͒ to avoid complete absorption of radiation. The window was made of CaF 2 , and the working range was from 1100 to 4000 cm Ϫ1 . The electrolyte was a 1 M solution of tetraethylammonium tetrafluoroborate (Et 4 NBF 4 ) in dry acetonitrile ͑Merck Supercapacitor Electrolyte LES 100; H 2 O: 25 ppm; HF: 68 ppm as given by the supplier͒ used as provided and kept inside the glove box. The other salts, NaBF 4 , Et 4 NClO 4 were dried at 120°C under vacuum (10 Ϫ5 Torr͒ for 24 h. A solution of the adduct CH 3 CN•BF 3 in CH 3 CN was prepared by letting BF 3 stay in contact with CH 3 CN in a greaseless, mercury-free, vacuum line. BF 3 was prepared according to published methods. [START_REF] Kwasnik | Handbook of Preparative Inorganic Chemistry[END_REF] Electrodes such as those used in industrial supercapacitors absorb all incident radiation from UV to IR. The major technological innovation in this work was the capability to manufacture ''finely divided carbon electrodes'' that would reflect enough light to record high-quality IR spectra. The electrodes were aluminum cylinders 12 mm o.d. ͑grade 1050͒ whose bases were finely polished. Heatshrinkable poly͑tetrafluoroethylene͒ ͑Adtech Polymer Eng. Ltd, Stroud, U.K.͒ was used to insulate the sides of the cylinder. A thin layer of ''ink'' made of divided carbon together with a binder c was spread on the electrode using a spin-coating technique. The layer's thickness was not measured but adjusted by trial and error so that enough IR energy was reflected from the surface. These electrodes were cured at 130°C under vacuum (10 Ϫ5 Torr͒ for 24 h. Afterward, all handlings were performed in a glove box ͑Jacomex͒ filled with z E-mail: marc.debacker@hei.fr c Activated carbon, synthetic origin, Brunauer, Emmett, and Teller method area 1600 m 2 /g; carboxymethyl cellulose and styrene butadiene rubber as binder in aqueous dispersion.

dry, oxygen-free, purified argon. The potential of the working electrode was controlled with a three-electrode setup; the counter electrode was a platinum loop and a silver wire acted as a pseudoreference. The potential sweep was provided by a potentiostat ͑PG30 Autolab͒. The FTIR spectrophotometer used was either a Mag-naRAM 860 or an Avatar ͑Nicolet-Thermo-Optek͒

Results

The electrochemical behavior of a carbon spin-coated aluminum electrode during a potential cycle 0, ϩ2 V, 0 V ͑vs. Ag͒ is shown in Fig. 1. ͑A similar curve is obtained during cycles toward negative potentials.͒ It is always observed that the first curve is markedly different from the following ones. This current vs. voltage curve is characteristic of a capacitive system associated with a series resistance as is observed in capacitors for potentials below 1.5 V. For potentials higher than this value, faradaic processes are probably involved, coming from either carbon surface groups or impurities linked to the carbon manufacturing process. The slight curvature observed on the negative going potential sweep observed between 0.6 and 0 V is possibly due to a reduction of a small quantity of species that were produced in the forward scan. Because there are no redox species in the solution, no peaks are observed or expected. Uncoated aluminum or platinum electrodes have only a resistive behavior and much weaker currents under these conditions.

To detect spectral modifications during a potential scan, the first spectrum was subtracted from the following ones. In this configuration, production of new species show as positive signals. As shown in Fig. 2, during an oxidative scan, the intensities of the major peaks decrease ͑solvent and support electrolyte: 1050-1150, 1360-1500, 2250-2350, 2850-3150 cm Ϫ1 ͒. These variations follow linearly the potential increase and are reversible. The disappearance of species in regions where the original spectrum shows very small absorbance is observed at 1600 and 1640 cm Ϫ1 . The intensities of these peaks follow linearly the potential from 0 to 1.1 V and then remain constant. This process is reversed on the return scan. New sharp peaks are formed in the region around 2360 and 1200 cm Ϫ1 where no absorbing species were present in the starting spectrum, and a broader band grows between 3200 and 3500 cm Ϫ1 . Evolution of the new peaks around 1220 and 2360 cm Ϫ1 while the potential was scanned from 0 to 2 V, are shown in Fig. 3a andb, respectively. These modifications begin at potentials higher than 1.25 V and they are not reversible.

To determine the origin of these new peaks, Et 4 NBF 4 was replaced with either Et 4 NClO 4 or NaBF 4 . No spectral modifications were detected with Et 4 NClO 4 , but the same behavior was observed with NaBF 4 , thus implying the role of the anion BF 4

Ϫ . Another origin of these peaks could be the formation of CO 2 in the acetoni-trile solution because a peak in this spectral region was previously reported. [START_REF] Pons | [END_REF] To answer these questions, two key experiments were performed. In one of them, CO 2 gas was bubbled through the solution, and in the other one CH 3 CN•BF 3 dissolved in acetonitrile was added into the cell. IR spectra were recorded with the same electrodes as used previously. The addition of CO 2 to the solution produces a new peak at 2340 cm Ϫ1 and almost no variation in the range of 1200 cm Ϫ1 . However, the addition of BF 3 produces a peak at 2360 cm Ϫ1 and a broad peak at 1190 and at 1220 cm Ϫ1 . Similar peaks were observed for the adduct CH 3 CN•BF 3 in the solid state. 7,8 Potential scans toward the negative potentials did not produce any new absorbing species. Control experiments using bright platinum or aluminum working electrodes were also performed; the current flowing through the cell was much lower, and no new peaks were detected in the region of 1200 or 2360 cm Ϫ1 , even in long duration experiments.

Discussion

FTIR spectroelectrochemistry is a well-established technique for the study of processes occurring next to an electrode. 9 However, it is the first time that it was used to investigate electrochemical processes occurring in the vicinity of a dispersed carbon electrode.

During positive potential scans, the difference absorption spectra are complex. Although the thickness of the solution layer used in our experiments is huge compared to that of the double layer, it is in this last layer that all the variations of concentration occur. Near the positive electrode, BF 4 Ϫ becomes trapped in the diffuse layer or in pores of the carbon coating, thus decreasing the concentration in the bulk. According to the electroneutrality of the solution, the concentration of the cation also decreases. As expected, the variation of concentration follows linearly the potential and is reversible. The same behavior and the possible mechanisms were described in a previous spectroelectrochemical study of a supercapacitor using Raman spectroscopy. 3 The intensity of the acetonitrile peak at 2250 cm Ϫ1 decreases in a linear and reversible way when the potential increases. This means that in the path of the light, solvent molecules are replaced by anions that are attracted to the positively charged electrode. This variation was not observed in a previous study using the same electrolytic solution but with bright platinum electrodes. 10 The explanation for this difference could be that our divided electrodes have a much larger area than the polished platinum and thus they amplify the phenomenon. Sharp negative peaks observed at 1608 and 1643 cm Ϫ1 are attributed to water and suggest that this molecule is de sorbed from the double layer or from the electrode as the potential becomes positive. The water bending mode is observed at 1600 cm Ϫ1 in the vapor phase. The bands observed in this work are shifted to higher energies, indicating several kinds of interactions with the components of the solution. This is correlated with the appearance of broad peaks in the 3200-3500 cm Ϫ1 region corresponding to extensively hydrogen-bonded water associated with anions.

New absorption peaks produced in the regions of 1200 and 2360 cm Ϫ1 indicate the formation of stable species, rather than a simple adsorption on the electrode, because they do not vanish when the potential returns to its initial value. They appear only when the anion BF 4

Ϫ is present in the solution. It is then logical to look for new compounds originating from the major components of the system CH 3 CN and Et 4 NBF 4 , the most obvious being the formation of the well-known addition complex CH 3 CN•BF 3 .

The spectroscopic evidence strongly supports the hypothesis of adduct formation. The new peak appearing at 2360 cm Ϫ1 is located in the range of the vibration frequency 2 of the triple bond CwN ͑occurring at 2266 cm Ϫ1 in pure acetonitrile͒. Modifications of the force constants of this vibrator when adducts were formed has first been published by Purcell and Drago. 11 Theoretical calculations on model molecules indicated that this frequency increase was due to an electronic transfer of the lone pair of the nitrogen toward the receptor molecule. Subsequent Raman and IR studies 7 have enabled a complete indexing of all the lines. The IR spectrum of the adduct in solid argon matrix has also been reported. 8 The geometry of CH 3 CN•BF 3 is known in the gas phase 12 and in the solid state. 7,13 Other peaks were also detected in the 1250 cm Ϫ1 range; they were assigned to the 13 mode corresponding mainly to BF 3 stretching. 14 Similar conclusions were reached by various investigators who used similar electrolytes but on different electrode materials. 10,15,16 It is important to investigate the possible pathways leading to the formation of this adduct under an oxidizing potential. From this work, it is known that a divided carbon electrode and the presence of BF 4

Ϫ are required and that, as far as the IR spectrum indicates the composition of the solutions in the vicinity of such an electrode does not evolve with time in the absence of an applied potential. This observation rules out the possibility of a catalytic decomposition of the constituents of the solution.

Tetrafluoroborate anion has been extensively used as an electrolyte for the anodic oxidation of organic compounds in aprotic solvents, and it is generally assumed that these anions are stable. However, fluorination of some organic molecules that are electrochemically oxidized in the presence of this anion was reported some time ago in the literature. 17 Although there are no substances undergoing oxidization in our solution, the possibility of decomposition of BF 4

Ϫ cannot be overlooked.

We have postulated, based on spectroscopic results, that the formation of a stable neutral compound such as the adduct CH 3 CN•BF 3 could explain the new peaks observed. However, in BF 3 or BF 4 Ϫ , the boron atoms are in the same oxidation state: a simple electrochemical process cannot lead to the formation of BF 3 .

Considering the minute amount of material present on our electrode, it is unlikely that oxidation of trace impurities contained in the carbon alone would lead to the observed spectral modification through a reaction with BF 4 Ϫ . However, the possibility that active sites are generated that could catalyze this reaction cannot be completely overruled.

One must then look for electrochemical reactions of other molecules present in the solution or adsorbed on the electrode material, keeping in mind that its area is very large. The carbon-coated electrode was dried under high vacuum but only at a temperature of 130°C; the presence of water trapped in the pores is thus possible. The spectroscopic evidence shows that water is desorbed under oxidizing potentials. 10 Furthermore, in the electrolyte solution, H 2 O and HF are present at a concentration in the order of 10 Ϫ3 M. Oxidation of fluoride anion leading to fluorine is very unlikely while that of water is possible in the potential range being studied. It can lead to the formation of H ϩ and oxygen.

It can then be tentatively proposed that the following reaction, independent of the potential, possibly catalyzed by the carbon material, could be triggered: BF 4 Ϫ ϩ H ϩ → HF ϩ BF 3 . As soon as produced, BF 3 would form the adduct CH 3 CN•BF 3 that we have spectroscopically identified. One can argue that, to strengthen our hypothesis, a deliberate addition of water to the system could be made to see if the growth of new peaks would be modified. This was not done for several reasons. First, as we have seen, there is already water adsorbed on the electrode, and a further addition would completely modify the nature of the interface. Second, this would be very different from the conditions of the real supercapacitor, and the results would be of little utility.

Conclusion

We have shown that FTIR can be used to probe the variation of concentration of species in the vicinity of a high specific area electrode covered with divided carbon. The magnitude of the area of the electrode thus obtained allows a relatively large current to flow, hence producing large chemical effects that can be detected. The variation of the concentration of ions in the double layer under a potential sweep was put into evidence. The desorption of water from the electrode occurred at positive potentials. However, all these phenomena were reversible and did not lead to permanent modifications.

It has been established that a new species identified as CH 3 CN•BF 3 was formed when the electrode was subjected to an oxidizing potential. This new species is not reduced during the discharge cycle and is stable. However the influence of the presence of this complex on the performances of supercapacitors has not yet been investigated.

CNRS assisted in meeting the publication costs of this article.

Figure 1 .

 1 Figure 1. Cyclic voltammogram of finely divided carbon electrode prepared by spin-coating. Scan rate: 5 mV s Ϫ1 ; Et 4 NBF 4 ͑1 M͒ in acetonitrile. Solid line: first sweep; dotted line: following cycles.

Figure 2 .

 2 Figure 2. Difference absorption spectrum during scan from 0 to ϩ2 V ͑vs. Ag͒. Scan rate 2 mV s Ϫ1 , 64 spectra recorded, Et 4 NBF 4 ͑1 M͒ in acetonitrile. Bottom part: original spectrum.

Figure 3 .

 3 Figure 3. Spectral modifications observed at ͑a͒ 1220 cm Ϫ1 and ͑b͒ 2360 cm Ϫ1 as function of the applied potential. Bottom spectrum is at ϩ1 V; topmost at ϩ2 V. Each spectrum is separated by 32 mV.