Esra Aydinlioglu

Mona Abdelghani

Gaëlle Le Fer

Jan C M Van Hest

Olivier Sandre
email: olivier.sandre@enscbp.fr

Sébastien Lecommandoux
email: sebastien.lecommandoux@u-bordeaux.fr

Robust Polyion Complex Vesicles (PICsomes) based on PEO-b-Poly(Amino Acid) Copolymers Combining Electrostatic and Hydrophobic Interaction: Formation, siRNA Loading and Intracellular Delivery

Keywords: polypeptides, PEO-b-poly(amino acids) block copolymers, polyion complexes, ring opening polymerization, small interfering RNA

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In recent years, polymeric vesicles have gained much interest due to their unique hollow architecture with tunable membrane properties. Vesicular compartments, which are segregated from the outer aqueous medium by a membrane, can protect bioactive materials such as proteins or nucleic acids, making them potentially useful for versatile biological applications. [START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF][START_REF] Tanner | Polymeric Vesicles: From Drug Carriers to Nanoreactors and Artificial Organelles[END_REF][START_REF] Lee | Polymersomes for Drug Delivery: Design, Formation and Characterization[END_REF][START_REF] Hu | Stimuli-Responsive Polymersomes for Biomedical Applications[END_REF] However, for conventional polymeric vesicles made from amphiphilic block copolymers such as poly(acrylic acid)-block-polystyrene (PAA-b-PS) or poly(ethylene oxide)-block-polybutadiene (PEO-b-PB), the inertness and impermeability of their membrane can hinder their use for the active delivery of water soluble bioactive molecules via a pure diffusive mechanism. [START_REF] Spulber | Photoreaction of a Hydroxyalkyphenone with the Membrane of Polymersomes: A Versatile Method to Generate Semipermeable Nanoreactors[END_REF][START_REF] Wang | Concurrent Block Copolymer Polymersome Stabilization and Bilayer Permeabilization by Stimuli-Regulated "Traceless" Crosslinking[END_REF][START_REF] Wang | Regulating Vesicle Bilayer Permeability and Selectivity via Stimuli-Triggered Polymersome-to-PICsome Transition[END_REF] To overcome this drawback, Kataoka et coll. developed PICsomes, standing for "PolyIon Complex vesicles" that are prepared mostly from PEG-b-polypeptide block copolymers and have a unique three-layered structure with a semi-permeability character. [START_REF] Koide | Semipermeable Polymer Vesicle (PICsome) Self-Assembled in Aqueous Medium from a Pair of Oppositely Charged Block Copolymers: Physiologically Stable Micro-/Nanocontainers of Water-Soluble Macromolecules[END_REF] In an early study, they reported uniform size PICsomes, which were formed by mixing cationic PEG46-b-poly(aspartamide)100 and PEG46-b-poly(aspartate)100. [START_REF] Chuanoi | Structural Factors Directing Nanosized Polyion Complex Vesicles (Nano-PICsomes) to Form a Pair of Block Aniomer/Homo Catiomers: Studies on the Aniomer Segment Length and the Catiomer Side-Chain Structure[END_REF] Among structural factors that drive formation of nanoscale sized PICsomes (instead of PIC micelles or other PIC nanoparticles), [START_REF] Hammond | Building Biomedical Materials Layer-by-Layer[END_REF][START_REF] Insua | Polyion Complex (PIC) Particles: Preparation and Biomedical Applications[END_REF] a main control parameter was shown to be to limit the PEG weight fraction (f-PEG) % in the block copolymers. [START_REF] Dong | Monodispersed Polymeric Nanocapsules: Spontaneous Evolution and Morphology Transition from Reducible Hetero-PEG PICmicelles by Controlled Degradation[END_REF] They demonstrated that too high PEG fraction prevents the growth of PICsomes and mostly directs towards micelle formation because of the immiscibility of the two (PEG and PIC) parts, as well as large steric hindrance exerted by the PEG strands. Chemical architecture of polymers is also another parameter that needs to be taken into account for PICsome formation: Kataoka et al. also stated that for a more stable PIC formation, longer aliphatic side chains of the polyelectrolytes are needed to favor the formation of vesicles. [START_REF] Chuanoi | Structural Factors Directing Nanosized Polyion Complex Vesicles (Nano-PICsomes) to Form a Pair of Block Aniomer/Homo Catiomers: Studies on the Aniomer Segment Length and the Catiomer Side-Chain Structure[END_REF] They also were shown stable even in harsh (i.e. high salinity and protein content) in vivo conditions and efficient to deliver water soluble 4 bioactive molecules such as nucleic acids, proteins [START_REF] Kishimura | Encapsulation of Myoglobin in PEGylated Polyion Complex Vesicles Made from a Pair of Oppositely Charged Block Ionomers: A Physiologically Available Oxygen Carrier[END_REF] and enzymes. [START_REF] Koide | Semipermeable Polymer Vesicle (PICsome) Self-Assembled in Aqueous Medium from a Pair of Oppositely Charged Block Copolymers: Physiologically Stable Micro-/Nanocontainers of Water-Soluble Macromolecules[END_REF][START_REF] Sueyoshi | Enzyme-Loaded Polyion Complex Vesicles as in Vivo Nanoreactors Working Sustainably under the Blood Circulation: Characterization and Functional Evaluation[END_REF][START_REF] Anraku | Living Unimodal Growth of Polyion Complex Vesicles via Two-Dimensional Supramolecular Polymerization[END_REF][START_REF] Anraku | Systemically Injectable Enzyme-Loaded Polyion Complex Vesicles as in Vivo Nanoreactors Functioning in Tumors[END_REF][START_REF] Anraku | Spontaneous Formation of Nanosized Unilamellar Polyion Complex Vesicles with Tunable Size and Properties[END_REF][START_REF] Hori | Robust Polyion Complex Vesicles (PICsomes) under Physiological Conditions Reinforced by Multiple Hydrogen Bond Formation Derived by Guanidinium Groups[END_REF] PICsomes can be obtained at the nanoscale from few tens to several hundred nanometers with tunable permeability based on the covalent cross-linking degree of the vesicular membrane through amide bond formation between the -COOH and -NH2 side groups of the peptides, which makes them ideal nano/microreactors for the delivery of biomacromolecules. [START_REF] Kishimura | Development of Polyion Complex Vesicles (PICsomes) from Block Copolymers for Biomedical Applications[END_REF][START_REF] Bennett | MR Imaging Techniques for Nano-Pathophysiology and Theranostics[END_REF] Thanks to their facile preparation for the encapsulation of water-soluble drugs and considering that they do not need any organic solvent to be self-assembled, PICsomes are highly suitable for biomedical studies. Moreover, the PEG chains tethered to the polypeptide blocks forming the PICsome membrane serve as inner and outer shell repulsive layers prolonging their lifetime in the blood stream. [START_REF] Hamley | PEG-Peptide Conjugates[END_REF][START_REF] Anraku | Size-Controlled Long-Circulating PICsome as a Ruler to Measure Critical Cut-off Disposition Size into Normal and Tumor Tissues[END_REF] Originally discovered in plant biology, small interfering RNA (siRNA) is a trigger of RNA interference (RNAi), which is a post-transcriptional gene expression mechanism that gained enormous interest since 2 decades due to its potential therapeutic use, especially for antiviral and anticancer therapies, but also for treatment of cardiovascular, neurological, or infectious diseases. [START_REF] Elbashir | Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells[END_REF] RNAi has potential therapeutic applications based on selective degradation of a target messenger RNA (mRNA) in mammalian cell cytoplasm. [START_REF] Elbashir | Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells[END_REF][START_REF] Rana | Illuminating the Silence: Understanding the Structure and Function of Small RNAs[END_REF] But due to its fragility and high negative charge density, its bioavailability is limited as ascribed to rapid degradation by nuclease enzymes, causing inefficient cellular uptake. [START_REF] Kaczmarek | Advances in the Delivery of RNA Therapeutics: From Concept to Clinical Reality[END_REF][START_REF] Kim | Recent Progress in Development of SiRNA Delivery Vehicles for Cancer Therapy[END_REF][START_REF] Wittrup | Knocking down Disease: A Progress Report on SiRNA Therapeutics[END_REF] Thus delivery vehicles are needed for an efficient and targeted delivery of siRNA to the cell cytoplasm. One possibility offered by the highly negatively charged repeating phosphate groups of siRNAs consists in neutralizing it through polyion complexation with oppositely charged macromolecules. Efficient candidates to do so are block copolymers combining polycationic and neutral (usually PEG) blocks, coassembling with siRNA and providing a neutral protective layer around it. Among various block or stat copolymer architectures proposed so far in literature to form PIC assemblies with siRNA or mRNA as potential nanomedicines [START_REF] Sharma | Nanoparticulate RNA Delivery Systems in Cancer[END_REF] -for example, polyaspartamide derivatives PAsp(DET), PAsp(DPT), and PAsp(TEP), [START_REF] Suma | Enhanced Stability and Gene Silencing Ability of SiRNA-Loaded Polyion Complexes Formulated from Polyaspartamide Derivatives with a Repetitive Array of Amino Groups in the Side Chain[END_REF][START_REF] Kim | SiRNA Delivery from Triblock Copolymer Micelles with Spatially-Ordered Compartments of PEG Shell, SiRNA-Loaded Intermediate Layer, and Hydrophobic Core[END_REF] oligosaccharide (chitosan)-b-PEO, [START_REF] Delas | Effects of Chain Length of Chitosan Oligosaccharides on Solution Properties and Complexation with SiRNA[END_REF] poly(alkyl-L-glutamine) copolymers, [START_REF] Klemm | Efficient Gene Delivery of Tailored Amphiphilic Polypeptides by Polyplex Surfing[END_REF] diblock elastin like polypeptides (ELPs) [START_REF] Abdelghani | Self-Assembly or Coassembly of Multiresponsive Histidine-Containing Elastin-Like Polypeptide Block Copolymers[END_REF][START_REF] Bravo-Anaya | Nucleic Acids Complexation with Cationic Elastin-like Polypeptides: Stoichiometry and Stability of Nano-Assemblies[END_REF] , linear [START_REF] Zhou | Targeted Polyelectrolyte Complex Micelles Treat Vascular Complications in Vivo[END_REF] or branched [START_REF] Chaya | Dynamic Stabilization of Unit Polyion Complexes Incorporating Small Interfering RNA by Fine-Tuning of Cationic Block Length in Two-Branched Poly(Ethylene Glycol)-b-Poly(l -Lysine)[END_REF] PEG-b-poly(Llysine) -PICsomes offer the additional advantage of possible co-encapsulation with other soluble cargos, such vesicular nanocarriers being named "siRNAsomes". [START_REF] Kim | Self-Assembly of SiRNA/PEG-b-Catiomer at Integer Molar Ratio into 100 Nm-Sized Vesicular Polyion Complexes (SiRNAsomes) for RNAi and Codelivery of Cargo Macromolecules[END_REF] Another feature to consider about siRNA is that it has a rigid cylindrical structure with a length around 6 nm. [START_REF] Rana | Illuminating the Silence: Understanding the Structure and Function of Small RNAs[END_REF][START_REF] Hyeon | Shape, and Flexibility of RNA Structures[END_REF] Kataoka et al. investigated the mechanisms of self-assembly by polyionic complexation between PEG-b-PLys and either single stranded (ssRNA) or double stranded siRNA. Multimolecular selfassembly (i.e. micellization) was strongly affected as ascribed to the rigidity of siRNA compared to ssRNA. As a consequence, siRNA and PEG-b-PLys form bimolecular PICs rather than larger multimolecular assemblies, at a wide range of concentrations. Thus to form multimolecular assemblies, additional attractive forces such as hydrophobic interactions and hydrogen bonds are needed within the polycationic segments to improve the driving force of complexation and reach superior transfection capability for the delivery of siRNA. [START_REF] Kim | Multifunctional Polyion Complex Micelle Featuring Enhanced Stability, Targetability, and Endosome Escapability for Systemic SiRNA Delivery to Subcutaneous Model of Lung Cancer[END_REF][START_REF] Kataoka | Block Copolymer Micelles for Drug Delivery: Design, Characterization and Biological Significance[END_REF] As an example, Kataoka et al.

developed A-B-C type triblock copolymers featuring shell-forming A-segment, nucleic acidloading B-segment, and stable core forming C-segment, directing toward construction of a threelayered polymeric micelle that can act as siRNA vehicle. These triblock copolymers were composed of hydrophilic and neutral poly(ethylene glycol) (PEG), polycationic poly(L-lysine) (PLys), and poly(N-(N-(2-aminoethyl)-2-aminoethyl) aspartamide) (PAsp(DET)) bearing a hydrophobic dimethoxy nitrobenzyl ester (DN) moiety in the side group. Such triblock copolymer with hydrophobic moieties showed stable encapsulation of siRNA in serumcontaining medium with strong sequence-specific gene silencing effect in cultured cancer cells. [START_REF] Kim | SiRNA Delivery from Triblock Copolymer Micelles with Spatially-Ordered Compartments of PEG Shell, SiRNA-Loaded Intermediate Layer, and Hydrophobic Core[END_REF] In another study, phenylboronic acid (PBA) functionalized cationic PIC micelles were used for intracellular ATP-triggered release of siRNAs. The strength of the ribose-PBA binding force and the hydrophobicity of PBA, both critical determinants of complex stability, as well as the ATP sensitivity, were shown to be controllable on the basis of the substituent group structures. [START_REF] Naito | A Phenylboronate-Functionalized Polyion Complex Micelle for ATP-Triggered Release of SiRNA[END_REF] As a last example, PEG114-poly(L-Glu10-co-L-Phe10) and PEG114-poly(L-Lys9-co-L-Phe10) copolymers with oppositely charged random co-polypeptide blocks were combined to form PIC micelles exhibiting a "charge conversion mechanism" for the pH-induced delivery of doxorubicin hydrochloride (DOX•HCl). These PIC micelles have shown superior activity and reduced side toxicity compared to free DOX in A549 tumor bearing nude mice. [START_REF] Lv | Charge-Conversional Peg-Polypeptide Polyionic Complex Nanoparticles from Simple Blending of a Pair of Oppositely Charged Block Copolymers as an Intelligent Vehicle for Efficient Antitumor Drug Delivery[END_REF] Considering this literature context, it appears still challenging to prepare non-toxic and stable PIC micelles in physiological conditions with the ability to deliver nucleic acid derivatives efficiently. In this work, we synthetized PEO-poly(amino acids) copolymers composed of PEOnpoly(L-Glux-co-L-Phey) and PEOn-poly(L-Lysx-co-L-Phey) with varying neutral hydrophilic fraction f-PEO (wt. %) and hydrophobic fraction f-L-Phe (wt. %). The self-assembly properties of each polymer were investigated individually, then the complexation of each pair of identical degrees of polymerization of PEO (n), of charged unit Glu or Lys (x) and of Phe (y) was studied at different [NH3 + /COO -] charge ratios and f-PEO (wt. %) repulsive ratios. The resulting polyion complex particles were studied by mono-angle dynamic light scattering (DLS), multi-angle light scattering (MALS) and transmission electron microscopy (TEM), consistently showing PICsomes structures. We also demonstrated stability of PICsomes in presence of high salt concentrations, even without any added crosslinking agent. In literature, there are many examples of poly(L-lysine) based siRNA loaded polyionic complexes but only few examples with tripartite particles. [START_REF] Boudier | Tripartite SiRNA Micelles as Controlled Delivery Systems for Primary Dendritic Cells[END_REF] Furthermore, these complexes were further used for the encapsulation of siRNA as possible gene transfection agent. Their gene silencing activity was studied with the firefly luciferase activity assay on mutant bioluminescent Luc HeLa cells and compared with the classically used polycationic transfecting agent lipofectamine.

Experimental section

Materials

Methyl-and amino-terminated poly(ethylene oxide)s (mPEO-NH2, (Mn) PEO, without spacer = 2,000 and 5,000 g•mol -1 ) were purchased from Rapp Polymere GmbH (Tübingen, Germany) and used without further purification. γ-Benzyl-L-glutamate-N-carboxyanhydride (γ-BLG NCA), εtrifluoroacetyl-L-lysine N-carboxyanhydride (L-Lys(TFA) NCA) and L-Phenylalanine-Ncarboxyanhydride (Phe NCA) were purchased from Isochem (Vert-le-Petit, France). Dry DMF was obtained from a Solvent Purification System and freshly used for the polymerization.

Trifluoroacetic acid (TFA, 99%), methanesulfonic acid (MSA), and potassium carbonate (K2CO3) were purchased from Alfa Aesar, Thermo Fisher GmbH (Karlsruhe, Germany). Diethyl ether, DMSO, anisole and methanol were used as received from Sigma Aldrich (L'Isle d'Abeau, France). Dialysis was conducted using a Spectra/Por ® 6 with molecular weight cut-off (MWCO) of 3.5 kDa membrane (Merck Millipore, Darmstadt, Germany). Dulbecco's modified eagle medium (DMEM), HEPES buffer (pH 7.4), and phosphate buffered saline (PBS) were purchased from ThermoFisher Scientific. Small interfering RNAs (siRNA) were obtained from Eurogentec (Angers, France): Two tubes of 21-mer siRNA duplexes targeting the pGL3 firefly luciferase gene (siRNA pGL3 luciferase control, also called siRNA duplex -firefly, antisense strand: dTdT 3'overhang, and sense strand: dTdT 3' overhang, 5 nmol of duplex per tube) were used in our experiments. For the determination of efficacy of siRNA encapsulation, Quant-iT™ microRNA assay kit (Molecular Probes, Thermo Fisher) was used as described in manufacturer's protocol.

CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega) and Luciferase Assay System (Promega) were purchased for toxicity and for gene silencing activity, respectively. HeLa EGFP-Luciferase bioluminescent cell line was provided by Utrecht University. operating at 400 MHz and equipped with a Bruker multinuclear z-gradient direct probe head capable of producing gradients in the z direction with 53.5 G⋅cm -1 strength. [START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF] H NMR spectra were recorded with an acquisition time of 4 sec, a relaxation delay of 1-2 sec and 64 scans.

Size exclusion chromatography (SEC) was used to determine the apparent number-average molar masses (Mn) and dispersities (Đ) using dimethylformamide (DMF + lithium bromide (LiBr) 1g•L -1 ) as eluent. Measurements in DMF were performed on an Ultimate 3000 system from Thermo Fisher Scientific equipped with a diode array detector (DAD) and a differential refractive index (dRI) detector (Optilab, Wyatt Technology, Santa Barbara CA, USA).

Polymers were separated on two KD-803 Shodex™ gel columns (300 × 8 mm) (exclusion limits from 1000 Da to 50 000 Da) at a flowrate of 0.8 mL•min -1 and column temperature held at 50°C. A series of polystyrene standards was used as the calibration. For certain copolymers synthesized from PEO46-NH2 macroinitiator, the SEC measurements were performed using dimethysulfoxide (DMSO + 0.1% LiBr) as eluent instead. In that case polymers were separated on Tosoh TSK G3000HHR and G2000HHR (7.8×300) columns (exclusion limits from 200 Da to 60 000 Da) at a flowrate of 0.5 mL•min -1 and column temperature held at 80°C. A dextran series was used as the calibration standard.

Mono-angle dynamic light scattering (DLS) measurements were performed on a NanoZS90

Zetasizer™ (ZEN3690, Malvern Instruments, Malvern, UK) operating with a He-Ne laser source at the wavelength λ=633 nm and scattering angle θ=90° at a constant central position in the cuvette (constant scattering volume). The provider's software was used to obtain Z-average hydrodynamic radii or diameters and polydispersity indexes (PDI) by analyzing the autocorrelation functions 𝑔𝑔 1 (𝑡𝑡) in terms of relaxation time distribution P(τ) (Equation 1) and using the 2 nd order cumulant method. [START_REF] Johnson | Laser Light Scattering[END_REF] 𝑔𝑔 1 (𝑡𝑡) = ∫ P(𝜏𝜏) exp (

-𝑡𝑡 𝜏𝜏 )𝑑𝑑𝜏𝜏 = 𝑒𝑒 -〈𝛤𝛤〉𝑡𝑡 �1 + 𝜇𝜇 2 2! 𝑡𝑡 2 + ⋯ � (1)
Hydrodynamic radius (Rh) was determined from the Stokes-Einstein relation (Equation 2).

𝑅𝑅 ℎ = 𝑘𝑘 B 𝑇𝑇 6π𝜂𝜂 s 𝐷𝐷 0 ⁄ obtained from 〈𝛤𝛤〉 = 𝐷𝐷 0 𝑞𝑞 2 with 𝑞𝑞 = 4𝜋𝜋𝜋𝜋 sin(𝜃𝜃 2 ⁄ ) 𝜆𝜆 ⁄ (2a) PDI = 𝜇𝜇 2 〈𝛤𝛤〉 2 ⁄ ( 2b
)
where D0 is the diffusion coefficient of the particles, PDI is the polydispersity index, <Г>=1/τ is the main decay rate and µ2 the second order coefficient of the cumulant series both obtained by fitting the 𝑔𝑔 1 (𝑡𝑡) autocorrelation function, θ is the scattering angle, λ the light wavelength and q the scattering vector, ηs is the viscosity and n the refraction index of the solvent, T is the absolute temperature and kB the Boltzmann constant.

Multi-angle light scattering (MALS) measurements were performed using an ALV/CG-6-8F compact goniometer, with a 35 mW red He-Ne linearly polarized laser (λ = 632.8 nm), an ALV/LSE-5004 multiple tau digital correlator and using a thermostat bath controller (20 °C).

The data were acquired with the ALV correlator software, the counting time being typically 15 sec for each scattering angle. Angles ranged from 20° to 150° corresponding to scattering vectors q from 6.85 × 10 -3 nm -1 to 2.55 × 10 -2 nm -1 . The autocorrelation functions (g1(t)) were analyzed in terms of a multi-modal relaxation time distribution P(τ) (Equation 1) and the hydrodynamic 10 radius (Rh) was determined from the Stokes-Einstein relation (Equation 2), the translational diffusion constant D0 being determined from the slope of the q 2 dependence of decay rate (<Г>=1/τ=D0•q 2 , τ being the main relaxation time deduced from the fitting of g1(τ)). Static light scattering (SLS) data were reduced using standard procedures. The solvent intensity was subtracted from that of the sample. Absolute calibration was performed against toluene. The static light scattering data allowed to calculate the radius of gyration (Rg) determined from a

Guinier plot according to Equation 3:

ln�𝐼𝐼(𝑞𝑞)� ≅ ln(𝐼𝐼 0 ) -𝑅𝑅 g 2 𝑞𝑞 2 3 ⁄ (3

Synthesis of poly(ethylene oxide)n-b-poly(L-glutamic acidx-co-L-phenylalaniney)

Copolymers were synthesized by ring-opening polymerization of BLG-NCA and Phe-NCA monomers with mPEO-NH2 as macroinitiator. Deprotection of γ-benzyl L-glutamate (BLG) moieties of copolymers into L-glutamic acid (Glu) was performed subsequently as described in literature. [START_REF] Lv | Charge-Conversional Peg-Polypeptide Polyionic Complex Nanoparticles from Simple Blending of a Pair of Oppositely Charged Block Copolymers as an Intelligent Vehicle for Efficient Antitumor Drug Delivery[END_REF][START_REF] Lv | Doxorubicin-Loaded Amphiphilic Polypeptide-Based Nanoparticles as an Efficient Drug Delivery System for Cancer Therapy[END_REF] In a typical polymerization, PEO-NH2 (0.11 g, 0.05 mmol, Mn=2,200 g⋅mol -1 ) was dissolved in dry DMF (1 mL) after an azeotropic dehydration process with toluene, then Phe-NCA (0.422 g, 2.2 mmol) and BLG-NCA (0.868 g, 3. Deprotection of TFA-L-lysine units of copolymers into L-lysine (Lys) was performed subsequently as described in literature. [START_REF] Sun | Conformation-Directed Formation of Self-Healing Diblock Copolypeptide Hydrogels via Polyion Complexation[END_REF][START_REF] Rodríguez-Hernández | Highly Branched Poly(L-Lysine)[END_REF] In a typical polymerization, mPEO-NH2 (0.

Formation of PICsomes and siRNA loading

For the formation of polyionic complexes, each polymer was firstly dissolved at 1 mg•mL -1 concentration in HEPES buffer at pH 7.4 and then mixed with their counterpart at desired molar charge ratios Z. The mixtures were vortexed 3 min, as described in literature. [START_REF] Chuanoi | Fabrication of Polyion Complex Vesicles with Enhanced Salt and Temperature Resistance and Their Potential Applications as Enzymatic Nanoreactors[END_REF] For efficient complexation, overnight incubation at 300 rpm using an orbital shaker at room temperature was used. After formation, all nanoparticles were characterized by dynamic (DLS) and multi-angle light scattering (MALS), and neutralization curves versus molar charge ratio Z were determined by zeta potential measurements.

Loading of siRNA in the polymeric particles was performed by incubation of siRNA (20 µM in serum medium (Opti-MEM) with the preformed polymer complexes (also in Opti-MEM) at 14 the designated [Amine]/[Phosphate+Carboxylate] ratios for 1-2 h at room temperature. More precisely, the charge ratios were defined as following:

𝑍𝑍 = [-NH 3 + ] PEO 𝑛𝑛 -poly(Lys 𝑥𝑥 -𝑐𝑐𝑐𝑐-Phe 𝑦𝑦 ) [-PO 4 -] siRNA + [-CO 2 -] PEO 𝑛𝑛 -poly(Glu 𝑥𝑥 -𝑐𝑐𝑐𝑐-Phe 𝑦𝑦 ) (4)
SiRNA loaded particles were further characterized by DLS in an ultralow volume quartz cuvette (Ultra-Micro Cell 105.251-QS, Hellma Analytics).

Gel retardation assay

Gel electrophoresis was performed using 0.8% agarose gel at 95 V constant voltage for 40 min. Then the gel was visualized with SYBR Safe DNA gel stain (Invitrogen) with a GE ImageQuant 350 Imager.

Determination of the Effect of Polyionic Complexes on Cell Viability by MTS Assay

MTS assay (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS, Promega ® ) was performed to confirm the cell viability after exposure to the PIC nanoparticles. Wild-type (WT) HeLa cells were seeded in DMEM medium with 10% fetal bovine serum (FBS) and placed into a 96-well plate to a final confluency of 10,000 cells/mL, and were incubated overnight at 37°C with 5% CO2. Next day, cells were exposed to different concentrations of PIC nanoparticles. The cells were incubated for 48 h at 37° C with 5% CO2. Afterward, the MTS assay was performed 24 h and 48 h post-exposure to the nanoparticles.

Ultraviolet-visible (UV/Vis) absorbance was recorded on a Jasco V-650 UV/Vis spectrometer at 490 nm and 293 K for the MTS assay.

In vitro luciferase gene silencing in cultured HeLa cells

Human cervical cancer cells stably expressing luciferase (HeLa-Luciferase) were seeded in 35 mm petri dishes (25,000 cells/dish) in DMEM medium containing 10% FBS and allowed to attach overnight. Then, the medium was removed and replaced with loaded PICsomes (at either 200 nM or 400 nM siRNA with charge ratios Z= [Amine]/[PO3 -] +[COO -] =1 or 10). For each analysis, control samples were prepared by addition of medium diluted with pure Opti-MEM instead of PICsome suspension. The added volumes of luciferin (luciferase substrate) and nanoparticles were 80 µL per 2 mL of medium. The cells were incubated with the nanoparticles for 24 h, then the nanoparticles were removed and replaced by complete DMEM medium and incubated for another 48 hr. Gene knock-down was evaluated using the luciferase bioluminescence assay using a Spark ® multimode microplate reader (Tecan, Männedorf, Switzerland).

Results and Discussion

Synthesis of oppositely charged PEO-b-poly(amino acid) copolymers

N-carboxy-α-amino-anhydride (NCA) ring-opening polymerization (ROP) is the most straightforward controlled polymerization technique enabling to obtain high molar masses polypeptides with no detectable racemization of the chiral centers. Polypeptides and hybrid materials exhibiting secondary structure and multi-functionalities have been obtained through the (co)polymerization of diverse NCA monomers. [START_REF] Hadjichristidis | Synthesis of Well-Defined Polypeptide-Based Materials via the Ring-Opening Polymerization of α-Amino Acid N-Carboxyanhydrides[END_REF][START_REF] Deming | Polypeptide and Polypeptide Hybrid Copolymer Synthesis via NCA Polymerization[END_REF][START_REF] Tian | Biodegradable Synthetic Polymers: Preparation, Functionalization and Biomedical Application[END_REF] For this study, PEO-b-poly(L-Glu-co-L-Phe)

and PEO-b-poly(L-Lys-co-L-Phe) block copolymers were synthesized via the ROP of BLG-NCA or Lys(TFA)-NCA with Phe-NCA as co-monomer using mPEO-NH2 as macroinitiator, followed by removal of the protecting groups (Scheme 1 and Scheme 2). The structures of the copolymers were determined by 1 H NMR and their apparent molar mass (Mn) and dispersity (Ð) by SEC. For the formation of PICsomes, we chose oppositely charged PEO-b-poly(amino acid) copolymers of slightly different f-PEO and f-L-Phe ratios yet with nearly equal degrees of polymerization (DP) of the charged amino acids L-Glu and L-Lys, namely DP around 60 and 100, as seen in Table 1.

The typical 1 H NMR spectra of the resulting copolymers (after deprotection) in deuterated DMSO-d6 (for PEO-b-poly(L-Glu-co-L-Phe)) or in CDCl3 + TFA (PEO-b-poly(L-Lys-co-L-Phe))

are shown in Figures S1 andS3, respectively, where all peaks were assigned. The degrees of polymerization and other molecular characterizations are summarized in Table 1. The Mn values of all synthesized copolymers were calculated by [START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF] H NMR spectroscopy (Figures S1 andS3). In most polymerizations, the targeted DPs were reached experimentally. For example, the conditions described in Methods (targeted DPs of 44 for L-Phe and 66 for BLG) led to the copolymer formula: PEO46-b-poly(L-BLG62-co-L-Phe42). Corresponding SEC analyses (Figures S2 andS4) show that these copolymers exhibit narrow molar mass distribution (Ð = 1.09 to 1.17). This narrow distribution may be attributed to the choice of starting PEO-NH2 with also narrow molar mass distribution (Ð = 1.13) and by the controlled polymerization character that was accomplished by the ROP mechanism of the NCA monomers. Copolymer

L-Phe-NCA L-BLG

f-PEO (%) a f-L-Phe (%) b (Mn) 1 H-NMR (g•mol -1 ) c (Mn) SEC (g•mol -1 ) d Ð d PEO114-b-poly(L-Glu60-co-L-Phe36) 28 29

Formation and physicochemical characterizations of PICsomes

We first examined formation of PICsomes by DLS at 90° at different targeted molar ratio Z between NH3 + and COO -moieties for the copolymers based on PEO46. Zeta potential of each sample was checked at different charge ratios. Once Z was increased above one (up to 1.4), almost perfectly neutral particles were obtained, with a tendency to make large aggregates, suggesting neutralization among polymer chains and particle growth, presumably by Ostwald ripening mechanism as reported previously for PICsomes with an excess of added polycations. [START_REF] Liu | Inducible Dynamic Behavior of Polyion Complex Vesicles by Disrupting Charge Balance[END_REF] From these DLS and zetametry results, we observed that neutralization was obtained slightly above 1:1 charge ratio, more precisely around Z=1.4, that might be due to a difference in the protonation state of the charged residues among the chains, PGlu and PLys being both weak electrolytes (Figure 1.a and Table 2). For the PICsome formation from PEO114-based copolymers, DLS measurements were also performed at different charge ratio Z: Results revealed more aggregation and larger polydispersity indexes (PDI) than with PEO46-based copolymers. To produce more homogeneous nanoparticles, extrusion was applied to the samples, which is a classical method in liposome research to obtain better controlled sizes and unilamellarity. [START_REF] Olson | Preparation of Liposomes of Defined Size Distribution by Extrusion through Polycarbonate Membranes[END_REF] For this purpose, a polycarbonate filter membrane with 200 nm well-defined, cylindrical pore channels was used. After 11 steps of extrusion, slightly better defined particles were observed for PEO114-based copolymer complexes at Z=1 (Figure S5 and table S1), while the derived count rate was almost constant (meaning that negligible amount of polymers remained on the extrusion membrane). We already mentioned that f-PEO (%) ratio limiting is very important to design nanosized PICsomes. More detailed characterizations were performed with multi-angle light scattering (MALS) for each condition of polyionic complexation (Figure S6 and S7). MALS experiments were performed with a scattering angle varying from 20° to 150°.

The linear variation of the main relaxation frequency versus the squared scattering vector q 2 and its passing through the origin confirm the presence of well-defined and spherical objects. MALS results also indicate possible formation of vesicles from the ratio of the radius of gyration to the hydrodynamic radius, [START_REF] Kunz | Dynamic Light Scattering from Spherical Particles[END_REF] with value Rg/Rh=1.1 for PEO46-based complexes and Rg/Rh=1.0 for PEO114-based complexes, confirming their vesicular structure. facilitate their visualization by TEM. [START_REF] Chuanoi | Fabrication of Polyion Complex Vesicles with Enhanced Salt and Temperature Resistance and Their Potential Applications as Enzymatic Nanoreactors[END_REF][START_REF] Goto | Facile Preparation of Delivery Platform of Water-Soluble Low-Molecular-Weight Drugs Based on Polyion Complex Vesicle (PICsome) Encapsulating Mesoporous Silica Nanoparticle[END_REF] An average diameter of 38.5 nm (standard deviation of 9 nm) was estimated by analysis of N=83 particles (Figure 2). The smaller size compared to DLS is consistent with a number-weighted average diameter obtained by TEM, differing from the intensity-weighted average value determined by DLS that is more sensitive to larger sizes, reflecting the rather broad size dispersity of PICsomes (PDI around 0.3). Since the PEO46 based copolymer pair with a lower f-PEO (%) ratio was more favorable for complexation, we moved on to more detailed characterization with this complex. It is important to verify the stability of polyionic complexes at physiological ionic strength and osmolarity (0.15 mM NaCl; 300 mOsm•L -1 ) and temperature (37 °C), in order to establish if they potentially can be used for biomedical applications. [START_REF] Anraku | Spontaneous Formation of Nanosized Unilamellar Polyion Complex Vesicles with Tunable Size and Properties[END_REF][START_REF] Kokuryo | SPIO-PICsome: Development of a Highly Sensitive and Stealth-Capable MRI Nano-Agent for Tumor Detection Using SPIO-Loaded Unilamellar Polyion Complex Vesicles (PICsomes)[END_REF] For this purpose, DLS experiments were performed at different salt concentrations, and a slight increase of the size of polyion complexes was observed, with no significant change in their PDI. Even after 1-day incubation at 37 °C with 0.15 M NaCl (9 g•L -1 solution i.e. isotonic with blood), the nanoparticles were neither degraded nor disassembled. At higher salt concentrations, larger sizes and PDI values were observed as ascribed to the screening of the electrostatic repulsion (Figure 3). These results indicate that the π-π interactions between L-Phe moieties provide enough increased stability of the PICsomes thanks to hydrophobic and aromatic interactions. The pH-dependent stability profiles of PICsomes were further characterized by DLS. The size distribution of PICsomes was clearly dependent on pH value, which may indicate the lowered association force of the PIC membranes in response to a decrease in pH, causing progressive increase in the protonation degree of the COO -groups of glutamic acid units in the chains (Figure S8). The PEO46-based copolymer pair was chosen to load siRNA in its complexes since they bear around 100 charged residues in their polypeptide blocks with a lower f-PEO. For this pair, both siRNA encapsulation and gene silencing activity of the complexes were studied in HeLa cells.

Formation of siRNA loaded PICsomes

The duplex -firefly double stranded oligonucleotide chosen as model siRNA was encapsulated by simple mixing with PEO46-b-poly(L-Lys100-co-L-Phe65) and PEO46-b-poly(L-Glu99-co-L-Phe65) copolymers. Complexation was proceeded firstly by mixing siRNA and PEO46-b-poly(L-Glu99co-L-Phe65), then this mixture of polyanions was subsequently complexed with a solution of PEO46-b-poly(L-Lys100-co-L-Phe65) polycations. This complexation process was performed at various molar charge ratios Z=[N]/([P]+[COO -]) in Opti-MEM (reduced serum medium), and each complex was analyzed by DLS and zetametry. The formation of empty PICsomes in serum media was also made and showed narrower size distributions than loaded PICsomes, besides a small secondary peak of large size aggregates (Figure S9). The siRNA loading was confirmed by gel electrophoresis for each molar ratio. The hydrodynamic size and ζ-potential of complexes were larger at higher Z ratios as observed by DLS and PALS, respectively (Figure 4). These results suggest that negatively charged siRNA strands were successfully bound to oppositely charged poly(L-lysine) segments in the PICsomes and that there was no macroscopic aggregation nor phase separation (coacervation), as observed when complexing with poly(L-Glu) polyanions only, revealing nanoscale well defined assemblies instead. Actually, charge reversal when increasing the Z ratio caused even a decrease of the hydrodynamic size of the complexes (Table 3). The complexation efficacy was studied with Quant-iT™ RiboGreen™ RNA Assay Kit using a fluorescent dye for quantification of oligonucleotides and DNA in solution. 58 While siRNA integrated inside the particles cannot interact with the dye, only negligible amount of naked siRNA could be detected by this fluorescent labelling (Table 3). For each complex, similar levels of siRNA were targeted like in standard curves (Figure S10). Therefore, the encapsulation rates were at least 99% of the total amount of siRNA. The siRNA loading was also determined qualitatively for each ratio via a gel retardation assay (Figure 4d). The complete disappearance of the free siRNA band at the four studied ratios indicates that all siRNA molecules were complexed through electrostatic interactions and thus could not migrate in the agarose gel under the applied electric field. TEM images of siRNA loaded PICsomes show formation of uniform spherical nanostructures, which implies that siRNA is sandwiched between PIC membranes (Figure 5). In particular for the Z=1 charge ratio, the complexes are much monodisperse in sizeaverage diameter of 26±6 nm counted on N=722 particles -matching their number-average diameter Dh∼30-40 nm measured by DLS (Figure 4a). S10).

Cellular delivery of siRNA-loaded PICsomes

As a preliminary study, the gene silencing activity of siRNA loaded PICsomes was investigated in cultured human cervical cancer cells stably expressing luciferase (HeLa-Luc). An siRNA sequence against the firefly luciferase gene was used to selectively inhibit the expression of luciferase. The commercially available Lipofectamine RNAimax was used as positive control transfection agent. The reduction in protein expression level was determined by measuring the bioluminescence using the Promega luciferase assay kit. We chose the highest and lowest

[N]/([P]+[COO -]) ratios with two different final siRNA concentrations in solution. For this purpose, the effect of PICsomes at different concentrations on cell viability was firstly determined via MTS assay, allowing for further gene silencing experiments. Although there was ∼40% reduction of cell mitochondrial enzyme activity as measured by the MTS assay at different concentrations of cell incubation with PEO46-based copolymer complexes (Figure 6), this effect did not appear to be dose-dependent, and no significant toxicity was finally observed. The luciferase-based bioluminescence intensity from the cells treated with each PICsome was measured as an indicator of gene silencing. The highest siRNA concentration among PICsomes (400 nM) was also complexed with Lipofectamine 2000 to obtain positive control complexes that were also incubated with the cells for 24h, before gene knock-down was evaluated. These cell transfection results proved the efficiency of the siRNA loaded PICsomes (Figure 7). For both ratios Z=1 and Z=10, PICsomes with the highest siRNA concentration (400 nM) showed that they could deliver siRNA and down-regulate the luciferin protein expression down to 20% of activity, as compared to 30% with lipofectamine.

Conclusion

In this study, amphiphilic copolymers with a neutral PEO block and a charged copolypeptide

Example of characterization of poly(ethylene oxide)n-b-poly(L-glutamic acidx-co-L-phenylalaniney) with 1 H NMR and SEC

As an example, the [START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF]

1

  Robust polyion complex vesicles (PICsomes) based on PEO-b-poly(amino acid) copolymers combining electrostatic and hydrophobic interaction: formation, siRNA loading and intracellular delivery Esra Aydinlioglu 1 , Mona Abdelghani 2 , Gaëlle Le Fer 1,3 , Jan C. M. van Hest 2 , Olivier Sandre 1, * and Sébastien Lecommandoux 1, *

Instrumentation 1 H

 1 NMR experiments were performed in deuterated trifluoroacetic acid (TFA-d) at 25 °C on a Bruker Avance I NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany)

)

  Zeta potential (ζ) of nanoobjects was determined on the NanoZS90 instrument by phase analysis light scattering (PALS) using the Smoluchowski approximation in different buffer solutions containing saline up to 0.15 M NaCl to mimic physiological conditions. All the measurements were at least the average of triplicate values and performed at 25 °C, in pure DI water or in saline at different NaCl concentrations.Transmission electron microscopy (TEM) images were recorded on a Hitachi H7650 microscope working at 80 kV equipped with a GATAN Orius 11 Megapixel camera. Samples were prepared by dropping 5 µL of a 1 mg•mL -1 colloidal suspension onto a copper grid (200 mesh coated with an ultrathin lacey/continue carbon film (30/5-10 nm), Cu-200LD, Pacific Grid Tech, San Francisco CA, USA) enabling observation with natural contrast. Certain suspensions were analyzed with positive contrasting using either uranyl acetate (2% w/v) or phosphotungstic acid (0.75% w/v) as staining agents.

12 (

 12 3 mmol) which were weighted in a glovebox under pure argon, introduced in a flame-dried Schlenk tube, and dissolved in dry DMF (6 mL) were added under nitrogen using a syringe. After 3 days of reaction, poly(ethylene oxide)-b-poly(γ-benzyl-L-glutamate-co-L-phenylalanine) (PEO-b-poly(L-BLG-co-L-Phe)) block copolymer was purified by repeated precipitation from DMF into an excess amount of cold diethyl ether. The copolymer was dried under vacuum, dialyzed against water and freeze-dried to obtain a white powder.[START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF] H NMR (400 MHz, in d-TFA assigned chemical shifts δ (ppm): 2.26 and 2.68 (-CH2-CH2-CO-), 3.13 (C6H5-CH2-of polyphenylalanine), 3.99 (PEO chain), 4.83 (CH of poly-γ-benzyl glutamate and polyphenylalanine), (C6H5-CH2-), 6.82-7.01 (C6H5-) (FiguresS1-S2 in Supplementary Info). In a second step, deprotection of BLG units was achieved in mild acidic conditions to afford L-glutamic acid without racemization.[START_REF] Bonduelle | Smart Metallopoly(l-Glutamic Acid) Polymers: Reversible Helix-to-Coil Transition at Neutral PH[END_REF][START_REF] Sun | Conformation-Directed Formation of Self-Healing Diblock Copolypeptide Hydrogels via Polyion Complexation[END_REF] Poly(ethylene oxide)114-bpoly(L-BLGx-co-L-phenylalaniney) (500 mg, 2.28 mmol of BLG units) was dissolved in trifluoroacetic acid (TFA) (5 mL) and the reaction mixture was stirred at 0°C (ice bath). Then anisole (1 mL) and methanesulfonic acid (MSA) (5 mL) were added to the solution and the reaction mixture was stirred during 20 min at 0°C followed by stirring during 50 min at room temperature and maximum speed. The copolymer was finally precipitated in Et2O and collected by centrifugation. The resulting mixture was dissolved in saturated sodium bicarbonate (NaHCO3) solution to deprotonate the glutamic acid groups, dialyzed against distilled water (MWCO 3.5 kDa), and finally lyophilized to give the debenzylated polymer as a white solid fluffy polymer (typical yield 80%).[START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF] H NMR (400 MHz, TFA-d, chemical shifts δ (ppm): 3.40 PEO chain), 6.82-7.11(C6H5-CH2-), 2.58 (C6H5-CH2-of polyphenylalanine), 4.38 (CH of poly(glutamic acid) and polyphenylalanine), 1.75-2.01(-CH2-CH2-COOH), 2.31(-CH2-CH2-COOH) (Figure S1B). Synthesis of poly(ethylene oxide)n-b-poly(L-lysinex-co-L-phenylalaniney) In a first step, copolymers were synthesized by ring-opening polymerization (ROP) of εtrifluoroacetyl-L-lysine N-carboxyanhydride (L-Lys(TFA) NCA) and L-phenylalanine Ncarboxyanhydride (L-Phe NCA) monomers using mPEO-NH2 as macroinitiator. Various [monomers]/[macroinitiator] and [L-Lys(TFA) NCA]/[L-Phe NCA] molar ratios were used to obtain copolypeptides with different block lengths and hydrophilic/lipophilic balances.

3 (

 3 11 g, 0.05 mmol, Mn=2,200 g⋅mol -1 with the spacer) was dissolved in dry DMF (1 mL) after an azeotropic dehydration process with toluene, then L-Phe-NCA (0.460 g, 4 mmol) and L-Lys(TFA) NCA (0.965 g, 6 mmol) which were previously kept in a glovebox under pure argon, introduced in a flame-dried Schlenk tube, and dissolved in dry DMF (6 mL) were added via a syringe under nitrogen. After 3 days of reaction at room temperature, poly(ethylene oxide)-block-poly(Llysine(TFA)-co-L-phenylalanine) block copolymer was purified by repeated precipitation from DMF into an excess of cold diethyl ether. The copolymer was dried under vacuum, dialyzed against water and freeze-dried to obtain a white powder.[START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF] H NMR (400 MHz, TFA-d, chemical shifts δ (ppm): 6.91-7.40 (m, NH-COCF3, C6H5-CH2 and NH from peptide bonds), 4.25-4.68 (m, CO-CH-NH), 3,66 (s, O-CH2-CH2 PEO block), 3.3 (s, CH3-O-CH2 PEO block), 3.25 (m, CH2-NH-COCF3), 2.84 (m, C6H5-CH2), 1.25-1.63 (m, CH2-CH2-CH2) (Figure S3B).13In a second step, deprotection of L-lysine(TFA) units was achieved. Poly(ethylene oxide)block-poly(L-lysine(TFA)-co-L-phenylalanine) was dissolved in a 9:1 methanol: water mixture (10 mg•mL -1 ) and K2CO3 (2 eq per lysine residue) was added. The reaction was stirred for 8 h at 50°C, and methanol was then removed under vacuum. The resulting solution (10% of original volume) was then diluted in water (3 times the remaining volume), transferred to a 5,000 MWCO dialysis membrane, and then dialyzed against 0.10 M aqueous NaCl solution at pH HCl) for 24 h, followed by DI water for 48 hours with water bath changes twice per day. The content of the dialysis membrane was then freeze-dried to afford a white powder.[START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF] H NMR (400 MHz, TFA-d, chemical shifts δ (ppm): 6.30-7.30 (m, C6H5-CH2 and NH from peptide bonds), 3.70-4.25 (m, CO-CH-NH), 3,70 (s, O-CH2-CH2 PEO block), 2.80 (s, CH3-O-CH2 PEO block), 2.70 (m, CH2-NH3 + ), 2.40-2.50 (m, C6H5-CH2), 0.5-1.40 (m, CH2-CH2-CH2) (Figure S2).

Scheme 1 . 2 Scheme 2 .

 122 Scheme 1. Synthesis route of poly(ethylene oxide)n-b-poly(L-glutamic acidx-co-Lphenylalaniney)

  Hydrophilic weight fraction based on PEO content f-PEO (%)=(Mn PEO /Mn tot )×100 b Hydrophobic weight fraction based on L-Phe content f-L-Phe (%)=(Mn PPhe /Mn tot )×100 c Based on number of monomer units calculated by 1 H NMR d Obtained by SEC with RI detector in DMF + 0.1% LiBr via polystyrene calibration. e Obtained by SEC with RI detector in DMSO + 0.1% LiBr via dextran calibration (all polymers were characterized by SEC before their deprotection).

44

 44

Figure 1 .Figure 2

 12 Figure 1. a) Size distribution profiles of PICsomes formed by PEO46-based copolymers at different charge ratios Z determined from DLS measurements (Malvern, 90°). b) DLS g1(τ) autocorrelation functions and their fitting with 2 nd order Cumulant model.

Figure 2 .

 2 Figure 2. TEM images of PICsomes obtained from PEO46-based copolymers in 10 mM PB buffer (without saline) at pH 7.4 (positively stained with 1.5% uranyl acetate solution).

Figure 3 .

 3 Figure 3. (a) Size distribution profiles for the stability of complexes after 1-day incubation at 37°C in 150 mM NaCl; (b) Stability against different salt concentrations at Z=1:1 charge ratio from DLS measurements (Malvern, 90°)

Figure 4 .

 4 Figure 4. a) Size distribution profiles and b) g1(τ) autocorrelograms of siRNA loaded PICsomes formed from PEO46 based copolymers at different charge ratios 𝑍𝑍 = [N] ([P] + [C]) ⁄ (where N=NH2 + , P=PO3 -, C=COO -) determined by DLS measurements in Opti-MEM at 37°C (Malvern, 90°); c) Zeta potential measurements; d) Gel retardation assay at different charge ratios Z=1, 3, 5 and 10.

Figure 5 .

 5 Figure 5.TEM images of siRNA loaded PICsomes: a) at [N]/([P]+[COO -])=10 ratio (inset is a more focused image) (scale bar:100 nm); b) at [N]/([P]+[COO -])=1 ratio (scale bar: 200 nm)

Figure 6 .

 6 Figure 6. Percentage of cell viability of HeLa cell line after incubation with empty PEO46-based PICsomes for 24 and 48 h obtained with MTS assay at several incubation concentrations.

Figure 7 .

 7 Figure 7. Relative luminescence intensity of HeLa-Luc cells incubated for 24 h with PICsomes loaded at two siRNA concentrations (100 and 400 nM) at two 𝑍𝑍 = [N] ([P] + [C]) ⁄ ratios (Z=10 or 1) or with siRNA complexed with lipofectamine (at 400 nM). The bioluminescence assay was performed after 48 h. Relative luminescence intensities were obtained by normalizing to those from non-treated control cells. Results are expressed as the mean ± S.D (n=6).

  block of either negative, PEO-b-poly(L-Glux-co-L-Phey), or positive charge, PEO-b-poly(L-Lysxco-L-Phey), were synthesized and studied for possible PICsome formation. Different characterization methods were used to demonstrate PICsome formation at different molar charge ratios for each complex. PEO46-based amphiphilic copolymers showed to be more favorable for the formation of PICsomes, in agreement with literature that advises to use lower molar mass PEO blocks. Moreover, stability of PICsomes in saline at 37°C was improved thanks to possible hydrophobic π-π interactions among the polypeptide chains. PICsome formulations were further extended to be loaded with siRNA. The results revealed that PICsomes can efficiently complex siRNA through electrostatic interactions. The complexation at the highest (Z=10) and lowest(Z=1) charge ratios 𝑍𝑍 = [N] ([P] + [C])⁄ (where N=NH2 + , P=PO3 -, C=COO -) with 400 nM siRNA showed more effective gene silencing activity compared to the positive control using siRNA-lipofectamine complex, with no significant toxicity. There is still a need to understand different aspects such as intracellular delivery and trafficking of these siRNA loaded PICsomes aka "siRNAsomes" to evaluate them as potential gene delivery agents, but these preliminary results are already very encouraging. (58) Kuhlman, P.; Duff, H. L.; Galant, A. A Fluorescence-Based Assay for Multisubunit DNA-Dependent RNA Polymerases. Anal. Biochem. 2004, 324 (2), 183-190. https://doi.org/10.1016/j.ab.2003.08.038. Graphical abstract A series of pH-sensitive PEO-b-poly(L-Glux-co-L-Phey) and PEO-b-poly(L-Lysx-co-L-Phey) PEO-copolypeptides are synthesized. The hydrophobic L-Phe co-monomer influences secondary structure and shifts effective pKa. Co-assembly of oppositely charged PEOcopolypeptides with similar amino acid content forms PICsomes stable in isotonic NaCl thanks to π─π stacking of L-Phe. Duplex-firefly siRNA encapsulation shows superior and less toxic luciferase gene silencing in HeLa cells than lipofectamine polyplex.

  Figure S1A and S1B respectively, and fully assigned. The degrees of polymerization (DP) of L-BLG and

Figure S3. A) 1 H

 1 Figure S3. A) 1 H NMR spectrum of PEO114-b-P(L-Lys(TFA)52-co-L-Phe20) in TFA-d (before deprotection). B) 1 H NMR spectrum of PEO114-b-P(L-Lys52-co-L-Phe20) in CDCl3+TFA (20 %) (after

Figure S5 .

 S5 Figure S5. Size distribution profiles of PICsomes formed by PEO114-based copolymers at different

Figure S6 .

 S6 Figure S6. Multi-angle light scattering analysis of PICsomes at 1:1 charge ratio prepared from the lower

Figure S7 .

 S7 Figure S7. Multi-angle light scattering analysis of PICsomes at 1:1 charge ratio prepared from the

Figure S8 .

 S8 Figure S8. Size distribution profiles for the pH responsiveness of PICsomes formed from PEO46-based

Table 1 .

 1 Molecular characteristics of the different copolymers obtained by ROP of NCA monomers at different hydrophilic weight fractions used for PICsome formations.

Table 2 .

 2 Summary of results at different charge ratios after formation of polyionic complexes for PEO46 polymers determined by DLS. Broadness of the size distribution determined by DLS estimated by 𝐷𝐷 h × √PDI.

	Z ratio [NH3 + /COO -]	Dh by intensity (nm)	PDI	PDI width (nm)*	ζ potential (mV)
	0.1:1	210	0.30	115	-29±5
	0.5:1	170	0.30	93	-17±3
	1:1	194	0.34	113	-12±7
	1.4:1	2300	0.29	1240	-0.2±4

*

Table 3 .

 3 siRNA complexations at different ratios studied by DLS and their encapsulation efficacy determined by Quant-iT™ RiboGreen™ RNA Assay Kit

	Z ratio [N/(P+COO -)] N=NH2 + P=PO3 -	Dh by (nm) a intensity	PDI a	EE % b
	10	240	0.23	99
	5	277	0.31	99
	3	267	0.29	99
	1	216	0.25	98
	a from DLS measurements (Malvern, 90°) b Encapsulation efficiency of siRNA loaded
	PICsomes determined by Quant-iT™ RiboGreen™ RNA Assay by comparison with the standard
	curve (Figure

Funding: This work was supported by the European Union's Horizon 2020 research and innovation programme Marie Sklodowska-Curie Innovative Training Networks (ITN) Nanomed, under grant No. 676137 Acknowledgments: The authors are grateful to Dr Anne-Laure Wirotius for 1 H NMR training, Amelie Wax for GPC analysis, Dr Christophe Schatz and Quentin Larger for help with light scattering experiments and discussions. Transmission Electron Microscopy (TEM) was performed at the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging; authors are grateful to Sabrina Lacomme for the TEM training sessions.

Author Contributions: E.A.: experiments, methodology, data collection, analysis, and writing; M.A.: experiments, data collection, and analysis; E.A, G.L.F, J. C. M. v. H, O.S. and S.L conceptualization, methodology, analysis, writing, and supervision. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

Supplementary data for:

Robust polyion complex vesicles (PICsomes) based on PEO-b-poly(amino acid) copolymers combining electrostatic and hydrophobic interaction: formation, siRNA loading and intracellular delivery Esra Aydinlioglu 1 , Mona Abdelghani 2 , Gaëlle Le Fer [START_REF] Christian | Polymersome Carriers: From Self-Assembly to SiRNA and Protein Therapeutics[END_REF][START_REF] Lee | Polymersomes for Drug Delivery: Design, Formation and Characterization[END_REF] ) with an RI detector and dextran used as a calibration standard.

Characterization of poly(ethylene oxide)n-b-poly(L-glutamic acidx-co-L-phenylalaniney) by size exclusion chromatography

PEO46-NH2

Flow marker Copolymer

Characterization of poly(ethylene oxide)n-b-poly(L-Lysinex-co-L phenylalaniney) with 1 H NMR and

Formation of PEO114-based ionic complexations


Spolia in se at the fair, 1867-1889: a channel for Islamic reuse in nineteenth-century

From 1867 onward, Universal Expositions became gigantic feasts of spolia in re, to borrow Brilliant's classical phrase defining stylistic reuses of the past in contemporary design.

National pavilions took the form of replicas and creative interpretations of the historical architecture of the countries they represented. The furnishings displayed within their premises or on sale in the corresponding commercial galleries followed similar formulas. Much less known is the fact that spolia in se, that is material reuse, was also a fixture of many a pavilion at Universal Expositions. A case in point is Egypt which featured as early as 1867, and in subsequent fairs, authentic furnishings, as well as décors and specimens repurposing historic fragments into modern frames. When the event was over, Egypt's pavilions were dismantled and fragments reused elsewhere, as happened with the reception hall presented in 1873. A fashion for building and decorating with Islamic antiques developed in parallel in Cairo.

Through selected examples, the paper proposes to uncover the range of agencies and dynamics that shaped the spread of this characteristic architectural genre, among which Egyptian displays at World's fairs and French precedents in material medievalism.

Ultimately, it pleas for a proper historiographical recognition of its outcomes.

Bio

Mercedes Volait is CNRS Research professor and member of InVisu, a research unit devoted to visual and material history, based at Institut national d'histoire de l'art, in Paris. She has published numerous volumes on the history of architecture and heritage in modern Egypt, including 5 monographs and 8 collections of essays. Her current research focuses on the global outreach and consumption of architecture and craft from

Text

In 1881, Bohemian architect František Schmoranz (1845-1892) and his partner Jan Machytka (1844-1887) worked out designs for an "Arab Room" to be installed at the Museum of Art and Industry in Vienna (currently the MAK). 1 (Fig. 1) Whatever were the terms of the commission, Schmoranz received in April 1881 the permission to reuse, for the purpose, remnants from the Egyptian structures that he had designed for the Vienna World's Fair in 1873. 2 At the end of the fair, as was common practice, the Egyptian section had been dismantled and portions had been acquired and stored by the Austrian administration in charge of historic buildings, the Burghauptmannschaft. As products of Austro-Hungarian workmanship and design evidencing ability with Egyptian Islamic décor, the spoils were understandably worth preserving. They kept indeed the record of a memorable ephemeralia.

The Egyptian ruler, Khedive Ismā'īl (r. 1863-1879), had devised grandiose plans for the country's presence at the Viennese fair. Its material expression had to be "splendid and magnificent" as reported by the general commissioner in charge, Egyptologist Heinrich Brugsch (1827-1894). 3 Accuracy was to be at the core of the endeavour. At an extremity of the main building, the replica of a funerary mosque was meant to be as faithful as possible to the aspect of comparable religious buildings and was to be furnished with original elements, such as a minbar (pulpit) and a dikka (lectern). The portable objects were to be provided by the Minister of Religious Endowments (awqāf) in Cairo, depending on availabilities. 4 Egypt's tangible heritage was also to be represented by an adequate collection of Islamic artworks from the age of the Fatimids and the Mamluks (tenth to the fifteenth century). 5 The initial plan experienced bifurcations, as funding and time constraints altered myriad features in the course of implementation, but the scenery achieved still enthused viewers. 6

1 Recent publications on the room include Němeček, "František Schmoranz le Jeune (1845-1892)," in Le Caire dessiné et photographié au XIX e siècle, ed. Mercedes Volait (Paris: Picard, 2013), 117-38; Pflugradt-Abdel Aziz, "Exhibiting Islamic Architecture in the Second Half of the Nineteenth Century: The Architect Carl von Diebitsch and His Successor Franz Schmoranz Jr in the Service of Egyptian Viceroy Ismaʿil Pasha," Art in Translation 11:2 (2019): 200-22; and Ofner, "Das »Arabische Zimmer« (1883-1931) von Franz Schmoranz und Johann Machytka aus dem ehem. k. k. Österreichischen Museum für Kunst und Industrie," in Gezimmertes Morgenland Orientalische und orientalisierende Holzinterieurs in Mitteleuropa im späten 19. Jahrhundert. Phänomenalität, Materialität, Historizität, ed. Julia Rüdiger and Maximilian Hartmuth (Vienna: Bohlau, 2021), 153-68. 2 Ofner, "Das »Arabische Zimmer« (1883-1931),"164. 3 Brugsch, Mein Leben und mein Wandern [My Life And My Travels] (Waban, Mass. : Heinrich George Brugsch, 1992), Reprint of the second edition, Berlin, 1894, ed. George Laughead Jr. and

Repurposing remnants from the 1873 Exposition

As the Arab room was in turn dismantled in 1931 and again only partially preserved, it is not easy to retrieve the scope and provenance of what Schmoranz and Machytka repurposed. In all likelihood, they produced a mixture of sorts recycling spoils from the mandara (reception hall) installed in the Egyptian section, possibly supplemented with additional material. The mandara is primarily known through written depictions, and a detailed material comparison with the room is thus out of scope but Schmoranz is reported to have invested in its shape and fixtures the intimate knowledge he had acquired of Cairo's domestic architecture, from surveying a number of examples in 1870-1872,7 aided by Jan Machykta who joined effort in 1870-1871. 8 The Arab room seems to have combined replicas of originals from Cairo such as the stained glasses and the salsabīl belonging to the sabīl-kuttāb built by Al-Ashraf Qā'itbāy in 1479 by the mosque of al-Azhar, both of which possibly used in 1873, with items made up by Austro-Hungarian craftsmanship on Schmoranz's drawings (six doors, twelve walled cupboards and latticework). The epigraphic frieze that was to run on top of the cupboards as per Schmoranz and Machytka's designs was pull apart at some point, as it does not appear on the photographs of the room taken in 1897 but do feature in an engraving from the time of the room's inauguration. (Fig. 1) Its components have recently surfaced from storage and have been inspected. [START_REF]Auf den Spuren von Franz Schmoranz junior durch die MAK Sammlung[END_REF] The historic tiles feature white calligraphy on plain cobalt blue ground and may have belonged to a larger inscription for the existing text is not fully legible, as if pieces were missing. The loose tiles, many of them cracked, are said to have been acquired by Schmoranz in 1870 from the Mekhitarist Congregation in Vienna, [START_REF] Ofner | Das »Arabische Zimmer[END_REF] and were possibly reused in 1873, or meant to be reused for that occasion. Their provenance is unclear, but a sixteenthcentury or later Ottoman or Persian building is a likely candidate. [START_REF] Blessing | The Blue-and-White Tiles of the Muradiye in Edirne: Architectural Decoration between Tabriz, Damascus, and Cairo[END_REF] As Armenians, the Mekhitarists had privileged contacts with the Middle East, where fellow denizens were soon to become actively involved in the global trade of antiquities. [START_REF]Examples and bibliography provided in Volait, Antique Dealing and Creative Reuse in Cairo and Damascus[END_REF] In contrast, the ware displayed on the shelves and cupboards of the Arab room were ceramics gifted to the MAK at a later stage, allegedly by the Ottoman government. 13 In other words, the chamber was both a reduction of the initial mandara and more; in any event, it was purely eclectic and artificial.

Yet, its watercolours masterfully retained a sense of Cairene architecture and craft thanks to Schmoranz and Machytka's expertise and exquisite design.

While a style room, rather than a proper period room, 14 the MAK re-installation brings to attention a two-fold by-product of Universal expositions that has remained off the radar to this day, at least in the case of Middle Eastern pavilions: the reuse of antiques in their architecture, and their subsequent afterlife after the fair. Ephemeral architecture at international fairs has generally been viewed through the prism of stylistic reuse of historic forms in contemporary design. In other words, it has been studied as "spolia in re," rather than as "spolia in se," or material reuse, to refer to Richard Brilliant's classical distinction of the two main types of aesthetic engagements with the past in contemporary design. 15 The literal reuse hereby discussed concerns Mamluk and Ottoman fragments that were set up in modern frames at the scale of architecture, décor or furniture, in Egypt and elsewhere, during the long nineteenth century. In such instances, spolia in se and spolia in re were commonly combined, generating in the process composite structures and artworks that defy the usual categorisations of Islamic art historiography, and indeed art historical ones at large. The resulting artistic forms can be characterised as hybrid, but the label does little justice to the full range and substance of their expressions, as I have argued elsewhere. 16 In the following, I contend two things: firstly, that nineteenth-century Islamic reuse in Egypt and elsewhere bore connections to French material medievalism, and secondly, that the Egyptian participation in Universal Expositions, and in particular to that of 1867 in Paris, actively promoted the genre.

Creative reuse in architecture and furniture in khedivial Cairo

Creative reuse of Islamic antiques in nineteenth-century architecture in Cairo is best exemplified by commissions due to French expatriates in the 1870s, some being implemented on designs authored by French architect Ambroise Baudry (1838-1906), who was active in 13 Ofner,156. 14 In theory, a period room refers to an existing historic room translocated to a museum to illustrate a given architectural style of interior decoration; in practice, the scheme experienced diverse declinations, The Period Rooms, ed. Sandra Costa, Dominique Poulot, Mercedes Volait (Bologna: Bononia University press, 2016). 15 Richard Brilliant, "I piedistalli del giardino di Boboli: spolia in se, spolia in re," Prospettiva 31 (Oct. 1982): 2-17. 16 Volait, Antique Dealing and Creative Reuse, op. cit. Chapter 4 passim.

Egypt from 1871 to 1886. 17 An icon in the group is the hôtel particulier of aristocrat Gaston de Saint-Maurice (1831-1905), who acted as grand equerry of the Egyptian ruler from 1868 to 1878. The grand mansion, built in the heart of suburban Cairo from 1872 to 1879, brought together historic fragments such as marble fountains (two basins and a mural one), rows of Iznik and Damascene tiles used to frame doors and decorate panels, as well as doors inlaid with Mamluk carved and chequered plaques. 18 (fig.

2) The masterpiece of the house was a double-height cruciform grand parlour featuring dados and floors in opus sectile of coloured marble; it was covered by carved and painted ceilings, underlined by calligraphic friezes, of which one held biblical epigraphy in Arabic. The room was lighted by a large mashrabiyya closing one of its recesses, while a central shukhshaykha (wooden hexagonal skylight) provided zenithal luminosity filtered through coloured glass. The authentic fragments were combined with plaster replicas of historic ornament, and with new stucco work inspired by existing examples, for instance a stuccoed niche in the mosque of al-Mūʾayyad Shaykh (r. 1415-21). (Fig. 3 and4). The house was ultimately furnished with local fittings, in the guise of tabouret-tables, a canopied vernacular bed, Indo-Portuguese chests, candlesticks, caskets, and all kinds of brass ware, of which Saint-Maurice gathered many precious specimens, a selection of which entered the South Kensington Museum (current Victoria and Albert Museum, London) in 1884 after having been exhibited in the show L'Égypte des Khalifes that he curated at the Paris Exposition Universelle in 1878. 19 In the very words of its patron, the intention had been to "build a house along an ancient plan, with materials coming from the most beautiful dwellings of the fourteenth, fifteenth and sixteenth-century, [with] sculpted ceilings, painted and gilded, mosaics of marble and motherof-pearl, doors in bronze and ivory, Persian and Cairene tiles, mashrabiyyas, inscriptions, plaster casts, etc. etc." 20 The outcome was deemed a "master piece in Arab restoration" by a French visitor, 21 the word defining then a very distinct praxis than the one it has come to 17 Crosnier Leconte and Volait, L'Égypte d'un architecte : Ambroise Baudry (1838-1906) (Paris : Somogy, 1998). 18 embody today, and one significantly more interventionist. In the French architectural jargon of the time, "restoration," also termed "restitution," primarily defined the art of completing missing parts in the rendering of an ancient building based on the extended classical culture acquired at the École des Beaux-arts in Paris, and for a happy few, while on residence at the Villa Medicis in Rome. [START_REF] Pinon | Les envois de Rome (1778-1968[END_REF] This was the antiquarian culture invested in the creation of the Saint-Maurice house.

A photograph keeps the record of the type of second-hand material that the French aristocrat collected for potential reuse, in this instance the base of a large mashrabiyya in need of heavy repair (Fig 5). The salvage, as such material was commonly termed in English at the time, exemplifies the poor condition of what was available. There is no direct evidence on how Gaston de Saint-Maurice procured the pieces repurposed in his house. A much later source contends that he legally salvaged architectural fragments on site, thanks to the official permission he had been granted to this end, at a time when Cairo was being modernised by way of cutting up new streets through its historic urban fabric. [START_REF] De | La légation de France a changé de quartier[END_REF] Truly enough, the opening of the main new thoroughfare in the city, the Citadel street, cut through 398 building plots, [START_REF] ʿalī Mubārak | jadīda li-Miṣr al-Qāhira wa-mudunihā wa-bilādihā al-qadīma wa-lshaḥīra (Cairo: al-Maṭba'[END_REF] and the clearing and resale of the ensuing rubble in stone and timber employed sixteen officials alone in 1872. [START_REF]List of appointees in the Administration of parks and gardens and other services, 1872-75, Muhāfaza Misr, Mahāfiḍ 1872[END_REF] However, announcements published in the Egyptian official gazette (al-Waqā'i al-Miṣriyya, irregularly released from 1828) delineate a broader context for salvage picking in Cairo. The journal recurrently advertised public auctions of rubble (anqāḍ, pl. of nuqḍ) from derelict structures (khirba or khariba), of which the city contained countless examples. Most were endowed properties (waqf) that had reached advanced ruin, either because of fire or recurrent seism (3 major ones hit Cairo on 7 August 1847, 12 October 1856, and in 1863), but moreover for poor building maintenance over long stretches of time. During the late 1860s, announcements for rubble auctioning appeared at least twice a year in the gazette. 26 None of the corresponding locations had any link with urban embellishment. Dilapidation was a more general condition of architecture in the city, and one that possessed a long track record. 27 The abundant topographical photography of Cairo corroborates the derelict condition of much of its historic buildings in the 1870s. (Fig. 6) Private transactions with property owners wishing to dispose of obsolete fixtures cannot be disregarded either. 28 Another significant example of modern architecture featuring repurposed historic décors in Cairo was the house that Ambroise Baudry erected for himself in 1875-1876. He too had managed to get a position at the khedivial court, as chief architect of Gīza Palace, and promptly acquired enough means to start building his own residence in suburban Cairo. The composite interiors he conceived incorporated historic woodwork (ceilings and door leafs) as well as a rich sample collection of Iznik and Damascene tiles, together with plaster replicas, and woodwork after his own designs. (Fig 7) A keen collector of Islamic art, with a good eye for rare collectibles, Baudry did amass during his stay in Egypt several hundred of Islamic antiques, including about three hundred tiles and myriad thirteenth-to fifteenth-century carved and chequered plaques that he had an indoor carpenter, the Maltese Peppe Gliveu, repurposing for him into panels, door leaves and cabinets. (Fig. 8) The one hundred and twenty-six loose plaques that entered the collection of the Metropolitan Museum in New York after his death evidence a specific collecting behaviour aimed at securing a second life to medieval fragments. 29 (Fig. 9)

Mixed-period furniture from Cairo praised at Universal Expositions

Cabinet-making was indeed an area where mixed-period and mixed-culture artworks did thrive, way beyond Baudry's circle. Not all specimens in the so-called Arabesque style so praised in Cairo were an amalgamation of old and new, but a significant number were, until the exhaustion of stock, one suspects. Some of these composite furnishings were crafted by a celebrated Italian cabinet-maker, Giuseppe Parvis (1832-1909), who had settled in 1859 in Cairo, where he promptly developed an extremely successful line of historicist furniture, both in Islamic and (Ancient) Egyptian style. He presented his productions at all Universal Expositions since 1867 and won awards at about each of them in the following decades. 30 His 27 Loiseau, Reconstruire la maison du sultan, 1350-1450 : ruine et recomposition de l'ordre urbain au Caire (Cairo : Publications de l'Institut français d'archéologie orientale, 2010). 28 Contrasting attitudes to the commodification of historic architecture within Egyptian society are discussed in Volait, Antique Dealing, op. cit., Chapter 3 passim. 29 The objects from the estate of Ambroise Baudry were purchased in 1907, thanks to the Rogers fund, from Cairene dealer Panayotis Kyticas by the merchant Albert M. Lythgoe, for the MET. All can be viewed on the MET online collection and can be retrieved by searching "A. Beaudry" because of a typo in the spelling of the collector's name. 30 Volait, "Revival, Replica and Reuse : Fashioning 'Arabesque' furniture in Khedival Cairo," The Arabist, Budapest Studies in Arabic 41 (2020): 229-42. exhibits in Vienna included the double-leaf door inlaid with carved plaques in ebony that he was commissioned for the khedivial mandara, as well as three large cupboards, one of which was part of a style room intended for the conspicuous palace-cum-botanical gardens being built for the affluent Italian merchant Pietro Ciccolani, in the outskirts of Cairo at Shubra. [START_REF] Signor | L'Exposisizione Universale di Vienna Illustrata[END_REF] (Fig. 10) Other cabinet-makers were to follow Parvis's track. In 1891, a commercial gazette listed four other manufacturers specialising in Islamic-style furnishings in Cairo: the Furino Brothers, Giuseppe and Nicola Jacovelli, Elias Hatoun, and Gaspare Giuliana. [START_REF]Annuaire égyptien administratif et commercial[END_REF] Typically, the last two were also dealers in Islamic antiques, while a major part of the Jacovelli brothers's business was in restoring Mamluk woodwork, metalwork and marble-mosaic in Cairo. [START_REF] Balboni | The Jacovellis were also keen collectors of Islamic art ; Paribeni, "Dall'Egitto all'Italia: la collezione Jacovelli di arte islamica del Museo Nazionale di Palermo[END_REF] Historicist cabinet-making, creative reuse, repair and the trade in antiques were clearly intertwined activities at the time.

The oddest piece in the mixed-period genre was presented in 1876 at the Centennial Exhibition in Philadelphia, aside furnishings and cupboards exhibited by Parvis. The item was an amalgam of woodwork from various periods: it was described at the time as "a door of the sanctuary of a mosque in Cairo carved and inlaid with ebony and ivory." (Fig 11) The work was valued $14,000a considerable amount for those years. Interestingly enough, its composite nature passed completely unnoticed then. The guide to the fair just added that "it was made in the fourteenth century" and had been secured by the newly-born Boston Art Museum, [START_REF]The Centennial Exhibition of 1876, What We Saw and How We Saw It[END_REF] as an example of good design and skilled workmanship that could inspire American art students. [START_REF] Weinstein | Silk and Gold, Islamic Art from the Museum of Fine Arts[END_REF] No close inspection is required to realise that the assemblage is completely made up, for no other function than display. The carved polygons are not inserted into a wooden structure as usual, but are lazed on a wooden board; some carving does not fit the design in which it is placed. The whole is obviously a reunion of disparate parts, with significant recent interventions. Its attribution to the fourteenth century solely rests on the inscription placed at the bottom of the door. The ebony panel celebrates Sultan al-Zāhir Barqūq (r. 1382-1389), and would have been salvaged from one of his buildings, possibly his funerary complex erected in 1384-1386 in the heart of Cairo. Nail holes and cavities on the brother, and had been visiting in Cairo in 1875 and 1876. Paul Baudry was duly impressed by the achievement, and communicated back his verdict in explicit terms: Ambroise's house is a gem. We would be rich if the building stood in the surroundings of boulevard Saint-Germain, or simply at the Batignolles; the doors and ceilings, the marbles and tiles, come from sixteenth-century palaces, it is an Arab Cluny. 39 The Cluny mention refers to the refurbishment and refurnishing, from 1839 onwards, of the Abbey of Cluny, one of the very few medieval mansions that had been left in Paris. It was the initiative of passionate collector and antiquarian Alexandre Du Sommerard (1779-1842).

Until his death in 1842, Sommerard worked at restoring, at times heavily, the medieval objects he had collected during his lifetime, and more importantly, at rearranging them in rooms along a systematic ordering, combined with an attention to everyday life, that pretended to enliven the reading of historical texts. He could test many arrangements before fixing one. The aim was to awake a sense of the past through the "unmediated perception of objects," to quote Stephen Bann in his must-read piece on Cluny. 40 In other words, Cluny was history in kind, rather than in writing. Sommerard's methodical collection became in 1843 the Musée de Cluny, and was continued by his son Edmond, but very little survives today of their evocative historical display, apart from some remnants in storage and a couple of images.

The idea, however, prospered. Varied declinations of Sommerard's vision materialised in the following decades. It came to give its name to an artisanal technique -"à la clunisienne"which consisted in producing new period furniture from a single recycled fragment. For instance, a sofa could be made out of an old armchair by cutting the latter in two halves and adding a central part between them. It was the market response to a demand for antiques that exceeded the offer by far. A Parisian master in the genre, woodworker Ernest Eymonaud (1847-1933), made a fortune in a trade advertised as the "reconstruction with old fragments of antique furniture for amateurs." 41 He mobilised for the purpose the traditional skills of the charron, the art of repairing cart wooden wheels, that has been his family's craft for 39 My translation and my emphasis. The quote in French reads : "La maison d'Ambroise est un bijou. Nous serions riches si l'immeuble était situé dans les environs de boulevard St Germain, ou simplement aux Batignolles, les portes et les plafonds, les marbres et les faïences viennent des palais du 16 ème siècle, c'est un Cluny arabe." Letter of Paul Baudry to Louise Garnier, 22 December 1876, Correspondence between Charles and Louise Garnier, and Paul Baudry, 2d part, 1873-1885, Mss 742, item 97, Bibliothèque de l'École des Beauxarts, Paris. 40 Bann, "Historical Text and Historical Object: the Poetics of the Musée de Cluny." History and Theory 17, no. 3 (1978): 251-66. 41 My translation from "la reconstruction avec de vieux fragments de meubles anciens pour amateurs"; Charpy, "Le théâtre des objets. Espaces privés, culture matérielle et identité bourgeoise, Paris, 1830-1914," PhD Tours University, 2010, p. 536-38. generations, but has been made obsolete by the progress of motorisation, thus exemplifying both a professional metamorphosis instigated by the new infatuation with the past in French society, and the deep entanglement of old and new that sustained creative reuse. 42 Paul Baudry was far from being the only one perceiving "Arab style" architecture in Egypt as a local declination of the Cluny idea. The Saint-Maurice's mansion did also prompt an equal connection,43 as did most Orientalist interiors then, whether built in Egypt or in Europe. 44 While undoubtly Eurocentric, the evocative label consecrates the centrality of Cluny as point of reference to any appraisal and revival of past architecture, be it European or Middle Eastern. It inscribes Islamic style architecture and décor in a broader intertemporal and intercultural framework. More importantly here, I argue that "Arab Clunys" identified in the designs of French architects and patrons in Cairo were not unprecedented in Egyptian architectural culture itself, whether in its promotion abroad or its genuineness at home.

The Egyptian promotion of reuse on the global stage

Earlier instances of creative reuse had taken place at the Universal Exposition of 1867 in Paris. It was the first time that nations were represented by stand-alone architecture. The Egyptian ruler had arranged for a lavish presence. Egypt had four buildings erected in the park, and substantial commercial space rented in the main premises of the fair. While change also occurred during construction, it was not on the scale of what will happen in Vienna, and the ostentatious Egyptian participation earnt sensational success. 45 Abundant visual material exists on the Egyptian pavilions, but the focus is placed here on a plate that has escaped attention so far. Its caption reads "Marqueterie ancienne employée dans un meuble moderne" (Historic inlay reused in a modern furnishing). (Fig 12) The piece is ascribed to the khedivial kiosk and thanks to a rare photograph of its interiors (Fig. 13), one can identify precisely the composite furniture in which the panel was reused. It formed the back of a console presenting a Mamluk Koran and arms belonging to the Khedive, just a few steps behind his bust sculpted by French artist Charles Cordier (1827-1905). The arrangement composed an evocative double amalgamation of inherited and appropriated visual and material cultures. The roundels decorating the doors' tops in the same pavilion were possibly spolia too. They can be paired with cut-wood work used to decorate the bases of late Ottoman mashrabiyyas (Fig. 5); at the time, cut-wood was also employed to adorn ceilings.

Further salvage ready for reuse was displayed for sale in the Egyptian commercial alley located in the vast circular galleries built in the Champ-de-Mars. Some of the elements on offer are known in some details because one collection was bulk-purchased by the South Kensington Museum before the fair even ended, from an Egyptian high-rank official, architect-collector Husayn Fahmī (c. 1827-1891). 46 One fragment of historic marquetry offered on sale closely relates to the inlaid panel reused in the khedivial kiosk. (Fig. 14) Other carved plaques, said to be coming from the fourteenth-century mosque of al-Māridānī, were set in what can be reconstructed, from a compilation of textual descriptions, as a muchremanufactured octagonal tabouret-table, with parts allegedly completed with French wood.

The tentative identification cannot be checked as the object has been deaccessioned in 1939,47 but the mention of "modern French" elements in its description suggests that the repurposing may have taken place in France, where the business of reviving, or rather "resurrecting," old pieces at any cost was blooming, as already mentioned. A set of thirty-five plaques from the Lājīn minbar (1296) that could be reassembled to form a complete rosette, was also on offer at the same fair, and is now at the MAK in Vienna. 48 These few cases demonstrate that before French Arab Clunys flourished in Cairo, equivalent composite artworks had been part of Egyptian official and commercial exhibits in Paris. It implies that the circulation of creative reuse did not only work from Paris to Cairo, but functioned the other way around too, and more to the point, even predated the materialisation of the Cluny idea by French agency in Egypt.

The trail of creative reuse of Islamic antiques can be followed up in later fairs, involving this time mostly French Islamic art collectors. Spectacular composite doors (now in the V&A) were exhibited for instance by Gaston de Saint-Maurice at the show he curated for the 1878 Exposition Universelle in Paris. The famous "Rue du Caire" assembled for the 1889 World's Fair, again in Paris, did contain many authentic pieces incorporated in its constructions: the mashrabiyyas in particular were original specimens brought from Cairo, most attributed to the 46 Volait,Antique Dealing,[START_REF]The Centennial Exhibition of 1876, What We Saw and How We Saw It[END_REF][START_REF] Weinstein | Silk and Gold, Islamic Art from the Museum of Fine Arts[END_REF][36][37][38][39][40][41][42][43][44][45][46][47][48][START_REF] De | L'Architecture arabe des khalifes d'Égypte à l'[END_REF][START_REF] Volait | Fous du Caire : Excentriques, architectes and amateurs d'art en Égypte, 1867-1914[END_REF][51][START_REF] Fiori | Paris déplacé du XVIII e siècle à nos jours : Architecture, fontaines, statues, décors[END_REF][START_REF] Emery | Meubles: The Ever Mobile Middle Ages[END_REF] Accession number 1049-1869 ; List of objects recommended for sale by Boards of Survey, 20 January 1939 (39/446) and 15 April 1939 (39/1712), MA/50/2/35, Victoria and Albert Archive, London. 48 The Future of Tradition-the Tradition of Future, ed. Dercon, Krempel and Shalem (London and New York: Prestel, 2010), 93-4. The accession number of the set is Or. 3405.

seventeenth century, and it was also the case of the shop fronts displayed, an eleventh-century inscribed panel, and all the tiles presented. [START_REF] De | L'Architecture arabe des khalifes d'Égypte à l'[END_REF]

At the intersection of two cultures of the Antique

Reuse also prevailed for the pavilions themselves. One of the Egyptian buildings at the 1867 Exposition, the okel, was purchased by Charles Cordier and was reconstructed as his studio in the outskirts of Paris. Another one was reinstated as a porter's lodge in an estate by the city of Le Havre. [START_REF] Volait | Fous du Caire : Excentriques, architectes and amateurs d'art en Égypte, 1867-1914[END_REF] Doors from the khedivial kiosk were retrieved at the end of the fair by Husayn Fahmī. The patron of the Cairo Street at the 1889 Exposition recycled some of the mashrabiyyas brought to Paris in a summer cottage he had built by the Atlantic in Normandy. 51 Whether French or Egyptian, the buyers were all art lovers deeply attached to the recreation of Islamic architecture and craft from Egypt.

Yet, Egyptian pavilions were far from being the only ones being dismantled, relocated and reconstructed for new functions. In France, most ephemeral architecture, and indeed enduring ones, [START_REF] Fiori | Paris déplacé du XVIII e siècle à nos jours : Architecture, fontaines, statues, décors[END_REF] could experience new lives at a time when "restoration," "renovation," "recycling," and "reconstruction" were still unfixed and labile concepts encapsulating very distinct activities from what they mean today. All were notions primary of a rhetorical nature, as has been shown for the reappraisal of medieval furniture in post-revolutionary France. And each did fabricate pure fantasies while serving competing agendas, depending on understandings of the Medieval Ages as times of cross-cultural sharing or ones seen through the chauvinist lens of French primacy. [START_REF] Emery | Meubles: The Ever Mobile Middle Ages[END_REF] In the case of Egypt, the remanufacturing of the spoils of the past encompassed extrameaning, because it was connected to a centuries-old tradition of "upcycling," to use contemporary parlance. Thanks to a dry climate sustaining the natural conservation of almost any material over ages, and because of limited resources encouraging the continuous reuse of existing structures, Egypt was in a way the land of reuse.

It is no wonder then that Cluny-type recreations of Islamic architecture, commonly termed "restorations" at the time, found in Egypt a perfect terrain on which to blossom. They could benefit in this context from the articulation of two cultures of the Antique: on the one hand, the lasting tradition of Egyptian reuse, nurtured by the abundance of raw historic material, and on the other, the French understanding of "restoration," as an illusionist reconstruction of bygone times. The two cultures eventually came into fusion, and World's fairs did have their share in connecting them.

Offering modern creative reuse a place in historiography and the museum

There is a need, consequently, to expand Islamic Art historiography to offer modern Islamic creative reuse a proper place in art history. One museum storing mixed-period and mixedculture furnishings and interiors from the region has been contributing its share. In the context of the refurbishment of its legendary Cast courts, achieved in 2018, the Victoria and Albert Museum has offered visibility to the plaster casts of Islamic ornament connected to reuse in modern Cairo, after extensive research on the global phenomenon of "Copy culture" on the one hand, 54 and sustained investigation in its early Islamic collections on the other. 55 Thanks to both, a replica of an arabesque ornament from the wikala (caravanserai) of Al-Ashraf Qā'itbāy, built in 1477 in the vicinity of the mosque of al-Azhar, is now on view in room 46, with detailed explanations on its provenance and use: Gaston de Saint-Maurice had exhibited it in L'Égypte des Khalifes show at the Exposition Universelle of 1878 in Paris, and it was part of the many pieces from this show that the V&A had purchased. 56 The overmantel designed by Ambroise Baudry in 1875-1876 also found its way to museum display, after another episode of removal and reuse. It had been acquired by a member of the khedivial dynasty, Prince Yūsuf Kamāl (1882-1967), after the French architect's house was dismantled in 1936, and was transported to its palace in Matariyya, a suburb of Cairo, to be reinstalled in an "Arab Room." 57 At the death of the prince, the piece entered the national collections as part of his bequest. It had most probably already lost its provenance. It is currently on view in the Mamluk galleries of the Islamic Art Museum in Cairo (room 8) because of the precious fourteenth-century carved plaques that adorn it, but no reference is 54 In the context of the 150th anniversary of the Convention for Promoting Universally Reproductions of Works of Art for the Benefit of Museums of all Countries; Copy Culture. Sharing in the Age of Digital Reproduction, ed. Cormier (London: V&A Publishing, 2018). 55 Moya Carey and the author have been studying early Islamic art collecting at the V&A from the 1860s to the 1890s. 56 The accession number of the piece is 1022 :7-1884, Carey and Volait, "Framing Islamic Art". 57 Volait, "Revival, Replica, and Reuse," 240 and fig. 8. made to the peculiar story, and journey, of the furniture. 58 Despite growing interest in nineteenth-and twentieth-century tangible heritage in Egyptian and international academia alike, the rediscovery and re-appraisal of a rich moment in the late art and architecture of the city of Cairo is proceeding at its own pace in the museum sphere. When I shared the history of the piece with the then director of the museum, Ahmad al-Shoky, in the late 2010s, he concluded that a gallery devoted to the nineteenth century would be a most welcome addition to the current display (initially designed with expertise from the Louvre Museum), and all the more because there was much related material in storage. Since then, the museum has had several temporary displays focusing on nineteenth-century pieces selected from its holdings. Casting a place for intertemporal and intercultural pieces in art history and the museum is certainly no easy game, for it requires subverting established taxonomies and delineating new ones. However, the pieces of the puzzle are on the table; it is a matter now of working at (re)assembling them. In a world of increased connectedness and with climate change demanding recycling by any means, the challenge should be seen as timely and motivating.

  Volait, Maisons de France au Caire : le remploi de grands décors mamelouks et ottomans dans une architecture moderne (Cairo : Publications de l'Institut français d'archéologie orientale, 2012), Chapter 1 passim. High definition photographs of the house can be viewed online at http://facchinelli.huma-num.fr/ 19 Carey and Volait, "Framing 'Islamic Art' for Aesthetic Interiors: Revisiting the 1878 Paris Exhibition," International Journal of Islamic Architecture 9, no. 1 (1 March 2020): 31-59. 20 My translation from the original : "Il s'agit d'une maison que j'ai fait construire au Caire sur un plan ancien, avec des matériaux provenant des plus belles habitations des XIV, XV et XVI° siècles. Plafonds sculptés peints et dorés, mosaïques de marbre et de nacre, portes de bronze et d'ivoire, faïences de Perse et du Caire, moucharabiehs, inscriptions moulages et matériaux, etc, etc.", extract from a letter of Gaston de Saint-Maurice to Sir Philip Cunliffe Owen, director of the South Kensington Museum, 24 October 1882, Saint-Maurice nominal file, MA1/S180, Victoria and Albert Archive, London. 21 Ch. Gabriel (pseudonym of Gabriel Charmes), "Cinq mois au Caire, IV," Journal des débats politiques et littéraires, 26 August 1879, 2.

  Similarly, the new National Museum of Egyptian civilisation, which opened in Cairo in 2017, has been exhibiting examples of nineteenth-century creative reuse in woodwork, albeit without labelling. Meanwhile, Islamic revival furniture from the khedivial palaces' collections is being reinstalled in the Gayer-Anderson Museum in Cairo.

Wilhelm Lauser, "Die Ka'ah," Allgemeine Kunst-Chronik. Zeitschrift fur Kunst, Kunstgewerbe und Literatur 7, no. 25 (June 23, 1883): 317-18; Němeček, "František Schmoranz, » op. cit.; Brugsch, op. cit. Awaiting proper publication, the drawings and watercolours kept at the Schmoranz archive in Chrudim offer ample testimony of an extended acquaintance with Islamic architecture in Cairo.

His Cairo's drawings were exhibited in 1876, together with those made by Schmoranz and other Austrian artists; Schmoranz, "Katalog der historischen Ausstellung des islamitischen Orientes, umfassend Darstellungen

Prel-Berard, "Un compagnon du Tour de France," La lettre du cercle généalogique de la Drôme, no. 96 (Fall 2018): 16-8.

"La maison arabe de M. de Saint-Maurice est aussi remarquable dans son genre que l'est dans le sien le musée de Cluny," "Une fête chez le ministre de France au Caire," Le Monde illustré, 9 May 1885.

Volait, "Les intérieurs orientalistes du comte de Saint-Maurice et d'Albert Goupil: des 'Cluny arabe' au Caire et à Paris à la fin du XIX e siècle," in The Period Rooms. Allestimenti storici tra arte, collezionismo e museologia, eds. Costa, Poulot, Volait (Bologne : Bononia University Press, 2017), 103-14.

A recent synthesis of historic accounts is given by Nour, "Egyptian-French Cultural Encounters at The Paris Exposition Universelle of 1867," MDCCC/1800 6 (July 2017):35-49.

Boston door suggest that the frame may have been part of a minbar. The Barqūq complex did lack its original pulpit since the fifteenth century and it is plausible that pieces from it would have been found somewhere in the 1870s. 36 The curious door is an archetypical by-product of the creative reuse so prevalent in late nineteenth-century Cairo.

No information is given on its maker. Its current attribution to Ambroise Baudry is not substantiated by solid evidence. 37 The Egyptian activity of the French architect is well documented in his extended correspondence kept by descendants; clues would have existed if he had had any hand in the endeavour, and had won the above-mentioned sum. More importantly, the type of woodwork that Baudry designed could not differ more from the recreated door, as an eye can easily perceive when contrasting it with the overmantel made for his house. (Fig. 8) Truly enough, Baudry's furniture bears two inscriptions replicating, in light-coloured wood, the one adorning the Boston door and its four carved ebony colonettes do possess some semblance with their equivalents in the door. But that's about it. The Baudry's piece holds a much larger proportion of modern woodwork; it is ostensibly modern design beautified with historic fragments, rather than one faking a historic object. What can be safely assumed is that parts of both pieces may have shared a common provenance in some derelict structure.

The connection made to French material medievalism

Artworks playing with old and new do not easily fit conventional nomenclatures. More so when they relate to the "age of empire," and stem from the interaction of global and vernacular cultures brought about by ever-growing European expansion overseas. 38 Mainstream art historical narratives, as well as established taxonomies of cultural heritage, tend to allocate material culture to single localities, ethnicities and temporalities.

Conventional historiography is not at ease with multi-layered matters and pluri-cultural contexts.

The reception of composite artworks at the time can help critically locate them. The appraisal of Baudry's house by a prominent French artist of the Second Empire provides an illuminating, if unexpected, insight. The celebrated figure happened to be Ambroise's elder 36 Behrens-Abouseif, Cairo of the Mamluks : A History of the Architecture and its Culture (Cairo : The American University in Cairo Press, 2007), 229. 37 Weinstein, Ink, Silk and Gold, 49. 38 Hobsbawm, The Age of Empire, 1875-1914(New York : Vintage, 1989). The internationalisation of vernacular cultures provoked by imperialism is notably discussed in Bhabha, The Location of Culture (London : Routledge, 1994).