Florian Nolay

Emmanuel Sevin

Mathieu Létévé

Abed Bil

Fabien Gosselet

Karim El Kirat

Florence Djedaini-Pilard

Sandrine Morandat

Laurence Fenart

Cédric Przybylski

Véronique Bonnet
email: veronique.bonnet@u-picardie.fr

First step to the improvement of the blood brain barrier passage of atazanavir encapsulated in sustainable bioorganic vesicles

Keywords: Amphiphilic Cyclodextrin, vesicles, Vectorisation, Atazanavir, Blood Brain Barrier

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

Introduction

The blood-brain barrier (BBB) is a highly selec?ve physical barrier separating blood circulation from the central nervous system. It is formed by tight junctions between brain endothelial cells, which selectively prevent the diffusion of molecules [START_REF] Cecchelli | Modelling of the blood-brain barrier in drug discovery and development[END_REF]. Compounds may cross the BBB by two primary pathways: i) paracellular transport, in which compounds pass between the endothelial cells, and ii) transcellular transport via transcytosis thought endothelial cells [START_REF] Hersh | Evolving Drug Delivery Strategies to Overcome the Blood Brain Barrier[END_REF][START_REF] Cecchelli | Modelling of the blood-brain barrier in drug discovery and development[END_REF]. Selective transporters and efflux pumps are expressed by the endothelial cells comprising the BBB, which contribute to the regulating passage of molecules 2 [START_REF] Abdelwahed | Sulfated and non-sulfated amphiphilic β-cyclodextrins: Impact of their structural properties on the physicochemical properties of nanoparticles[END_REF]. As a result, the BBB prevents the majority of therapeutic drugs issued by intravenous or oral administration from passing into the brain [START_REF] Lahiani-Skiba | Optimization of entrapment of metronidazole in amphiphilic β-cylodextrin nanospheres[END_REF]. In particular, larger substances such as vesicles or liposomes are generally unable to penetrate the brain cells [START_REF] Lundy | Inducing a Transient Increase in Blood-Brain Barrier Permeability for Improved Liposomal Drug Therapy of Glioblastoma Multiforme[END_REF]. However, some promising examples of aforementioned assemblies act as carriers that are able to cross the BBB [START_REF] Hersh | Evolving Drug Delivery Strategies to Overcome the Blood Brain Barrier[END_REF][START_REF] Vieira | Getting into the brain: liposome-based strategies for effective drug delivery across the blood-brain barrier[END_REF][START_REF] Lundy | Inducing a Transient Increase in Blood-Brain Barrier Permeability for Improved Liposomal Drug Therapy of Glioblastoma Multiforme[END_REF].

In this context, cyclodextrin derivatives [START_REF] Coisne | Cyclodextrins as Emerging Therapeutic Tools in the Treatment of Cholesterol-Associated Vascular and Neurodegenerative Diseases[END_REF][START_REF] Vecsernyés | Cyclodextrins, Blood-Brain Barrier, and Treatment of Neurological Diseases[END_REF], especially amphiphilic cyclodextrins (CDs) are promising tools to serve as drug delivery systems to the central nervous system (CNS; [START_REF] Bonnet | Cyclodextrin nanoassemblies: a promising tool for drug delivery[END_REF]. β-cyclodextrin (β-CD) are cyclic oligosaccharides composed of seven α-(1 4) linked D-glucopyranosides obtained by the enzymatic degradation of starch. They possess a hydrophobic toroidal cavity which is well known to allow the encapsulation of a wide range of hydrophobic molecules. This property is widely exploited to enhance the solubilisation of poorly aqueous soluble drugs [START_REF] Dodziuk | Cyclodextrins and Their Complexes[END_REF]. Derivatives of CDs could be useful to form supramolecular assemblies such as nanoparticles, and they can also be associated with phospholipid to form mixed vesicles. [START_REF] Kauscher | Incorporation of Amphiphilic Cyclodextrins into Liposomes as Artificial Receptor Units[END_REF] The chemistry of amphiphilic cyclodextrins remains challenging, because it requires either per-or mono-substitution of CD to obtain well-defined structures which are suitable as drug carriers [START_REF] Sallas | Advances in synthesis and supramolecular chemistry[END_REF]. Numerous chemical modifications have been carried out on CDs by grafting alkyl chains of variable length to different positions in order to obtain amphiphilic derivatives. It was previously demonstrated that such derivatives were able to form supramolecular aggregates, which acted as potential drug carriers [START_REF] Bilensoy | Recent advances and future directions in amphiphilic cyclodextrin nanoparticles[END_REF]. For example, CDs have been modified with hydrophobic moieties (e.g., with cholesteryl, glycerolipidyl or phospholipidyl unit) to target membranes [START_REF] Auzely-Velty | Cholesteryl-cyclodextrins: synthesis and insertion into phospholipid membranes[END_REF][START_REF] Moutard | Novel glycolipids based on cyclodextrins[END_REF][START_REF] Furlan | Nanoparticles based on lipidyl-β-cyclodextrins: synthesis, characterization, and experimental and computational biophysical studies for encapsulation of atazanavir[END_REF]. To simplify synthesis, aliphatic chains were also grafted directly onto β-CDs to produce amphiphilic compounds that were able to be incorporated in lipid bilayers [START_REF] Gallois-Montbrun | Direct synthesis of novel amphiphilic cyclodextrins[END_REF][START_REF] Oliva | New Lipidyl-Cyclodextrins Obtained by Ring Opening of Methyl Oleate Epoxide Using Ball Milling[END_REF]. Nevertheless, in most cases reported in the literature, cyclodextrin-based nanoparticles are only obtained in pure water [START_REF] Choisnard | Nanoparticles of βcyclodextrin esters obtained by self-assembling of biotransesterified β-cyclodextrins[END_REF][START_REF] Mcnicholas | Amphiphilic N-glycosyl-thiocarbamoyl cyclodextrins: Synthesis, self-assembly, and fluorimetry of recognition by lens culinaris lectin[END_REF][START_REF] Abdelwahed | Sulfated and non-sulfated amphiphilic β-cyclodextrins: Impact of their structural properties on the physicochemical properties of nanoparticles[END_REF] or stabilized in biocompatible buffers in the presence of surfactants such as Pluronic PE/F68, Tween 80, Mantonox ® 80 or Miglyol ® 812 [START_REF] Lahiani-Skiba | Optimization of entrapment of metronidazole in amphiphilic β-cylodextrin nanospheres[END_REF][START_REF] Noomen | Emulsions of β-cyclodextrins grafted to silicone for the transport of antifungal drugs[END_REF][START_REF] Gèze | Colloidal systems made of biotransfered α, β and γ cyclodextrins grafted with C10 alkyls chains[END_REF]. Several years ago, we designed new amphiphilic permethylated β-CD bearing a phosphoramide moiety which linked a spacer arm and two oleyl chains to the cyclodextrin scaffold. The presence of phosphoramide considerably enhanced the polarity of the entire system, favourising the so-called hydrophilic/hydrophobic balance, which is a desired property for drug delivery in biological media. [START_REF] Gervaise | Synthesis of lipophosphoramidyl-cyclodextrins and their supramolecular properties[END_REF]. The introduction of these two lipid chains was achieved in one step by an Atherton-Todd coupling. These compounds appeared to be very promising for drug delivery with the efficient encapsulation of carboxyfluorescein and scopolamine in several physiological conditions. Nevertheless, the synthesis was not very versatile, and the quantities obtained were not sufficient for further biological studies. In this work, we investigated the interaction of vesicles, including lipophosphoramidyl permethylated β-CDs (Lip-β-CDs) encapsulating atazanavir (ATV), with the endothelial cells of the BBB. The drug candidate is a well-known protease inhibitor used as an antiretroviral agent against HIV. Nevertheless, the ATV transfer rate through the BBB is very low, resulting in a poor therapeutic effect in cerebrospinal fluid to efficiently protect against HIV replication (Ene et al., 2011, Varatharajan and[START_REF] Varatharajana | The transport of anti-HIV drugs across blood-CNS interfaces: Summary of current knowledge and recommendations for further research[END_REF]. In addition to greatly improving the synthesis of a library of Lip-β-CDs, several physico-chemical experiments to delineate the effect of chain length were performed. The results were analysed and discussed in terms of various parameters, such as nanoparticle size and stability, encapsulation capacity, loading efficiency and cellular toxicity and uptake using a human BBB model [START_REF] Heymans | Mimicking brain tissue binding in an in vitro model of the blood-brain barrier illustrates differences between in vitro and in vivo methods for assessing the rate of brain penetration[END_REF], Jahne et al., 2016[START_REF] Hachem | Efficient Docosahexaenoic Acid Uptake by the Brain from a Structured Phospholipid[END_REF].

Material and methods

Material and reagents

βand γ-cyclodextrins were purchased from Wacker Chemicals (Munich, Germany) whereas β-Alanine methyl ester hydrochloride was from TCI Europe (Zwijndrecht, Belgium). High purity fatty alcohols as most of the reagents were from Sigma-Aldrich (Saint-quentin Fallavier, France). Reyataz capsules, a generous gift from Amiens University Hospital, were treated by liquid liquid extractions to keep pure atazanavir (ATV) only. Fluka supplied solvents (Saint-quentin Fallavier, France). N,Ndimethylformamide (DMF) was distilled beforehand under argon atmosphere (on barium oxide) and stored on molecular sieves prior to use. Deuterated solvents were purchased from Eurisotop (Saint-Aubin, France). Atazanavir sulfate, [3H], 1-5 Ci (37-185 GBq)/mmol, were obtained from Hartmann Analytic (Braunschweig, Germany). Sucrose, [14C(U)], )/mmol were supplied from PerkinElmer (Les Ulis, France). Details of used devices for thin layer chromatography (TLC), Chromatography, mass spectrometry (MS), nuclear magnetic resonance (NMR), Dynamic light scattering (DLS) and Fluorimetry are available in SI.

Synthesis Procedures

Oleylphosphites 1-4 were already described in literature (Mevel et al., 2007 and2008). Characterization were in accordance with data in literature as well as 6 VI -amino-6 VI -deoxy-2 II ,3 IIIdi-O-methyl-hexakis(2,3,6-tri-O-methyl)cyclomaltoheptaose 13 [START_REF] Gervaise | Synthesis of lipophosphoramidyl-cyclodextrins and their supramolecular properties[END_REF]. Description of the synthesis of compounds 5-12 is available in SI.

6 VI -amido-β-alanyl-(6 VI -di(dodecyloxy)phosphoramidyl)-6 VI -deoxy-2 II ,3 III -di-O-methylhexakis(2,3,6-tri-O-methyl)-cyclomaltoheptaose 15 O,O-didodecyl-N-(2-carboxyethyl)phosphoramidate 9 (268 mg, 0.53 mmol, 1.5 eq) was dissolved in anhydrous N,N dimethylformamide (20 mL) under argon. Then, 1-hydroxybenzotriazole (472 mg, 3.5 mmol, 10 eq) and N,N-diisopropylcarbodiimide (545 µL, 3.5 mmol, 10 eq) were added. After stirring for 2h, the 6 VI -amino-6 VI -deoxy-2 II ,3 III -di-O-methyl-hexakis(2,3,6-tri-O-methyl)cyclomaltoheptaose 13 solution (495 mg, 0.35 mmol, 1 eq dissolved in 20 mL of chloroform with few drops of triethylamine) was added. The reaction mixture was stirred at room temperature for 24h. The solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography on silica gel DCM/MeOH (98/2 and 95/5) with 71% yield. TLC: Rf = 0.51 (DCM/MeOH 95:5 (v/v)) 1 H NMR (300 MHz, CDCl3), δ (ppm): 6.18 (m, 1H, NH), 5.13 (m, 7H, H1, H I ), 3.94 (m, 4H, H1'), 3.90-3.29 (m, 35H, H3, H III , H4, H IV , H5, H V , H6, H VI ) , 3.62 (s, 18H, O(6)CH3), 3.50 (s, 21H, O(3)CH3), 3.37 (s, 21H, O(2)CH3), 3.19 (m, 9H, H2, H II , H9), 2.37 (t, 2H, H8),1.62 (m, 4H, H2') , 1.25 (m, 36H, H3'-H11'), 0.87 (t, 6H, H12', JH12'-H11'= 6.49 Hz) 13 O-ditetradecyl-N-(2-carboxyethyl)phosphoramidate 10 (174 mg, 0.48 mmol, 2.3 eq) was dissolved in anhydrous N,N dimethylformamide (6.7 mL) and methylene chloride (25 mL) under argon. Then, 1hydroxybenzotriazole (430 mg, 3.182 mmol, 15 eq) and N,N-diisopropylcarbodiimide (500 µL, 0.318 mmol, 15 eq) were added. After stirring for 3h, the 6 VI -amino-6 V I-deoxy-2 II ,3 III -di-O-methylhexakis(2,3,6-tri-O-methyl)cyclomaltoheptaose 13 solution (300 mg, 0.212 mmol, 1 eq dissolved in 2.5 mL of methylene chloride with few drops of triethylamine) was added. The reaction mixture was stirred at room temperature for 24h. The solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography on silica gel DCM/MeOH (100/0, 95/5 and 8/2) with 68% yield. TLC Rf = 0.44 (DCM/MeOH 95:5 (v/v)) 1 H NMR (300 MHz, CDCl3) δ (ppm): 6.20 (s, 1H, NH), 5.16-5.07 (m, 7H, H1, H I ), 3.95 (t, 4H, H1'), 3.97-3.45 (m, 35H, H3 O-dihexadecyl-N-(2-carboxyethyl)phosphoramidate 11 (288 mg, 0.467 mmol, 1,5 eq) was dissolved in anhydrous N,N dimethylformamide (10 mL) and methylene chloride (40 mL) under argon. Then, 1hydroxybenzotriazole (420 mg, 3.11 mmol, 10 eq) and N,N-diisopropylcarbodiimide (720 µL, 4.61 mmol, 15 eq) were added. After stirring for 3h, the 6 VI -amino-6 VI -deoxy-2 II ,3 III -di-O-methylhexakis(2,3,6-tri-O-methyl)cyclomaltoheptaose 13 solution (440 mg, 0.31 mmol, 1 eq dissolved in 4 mL of methylene chloride with few drops of triethylamine) was added. The reaction mixture was stirred at room temperature for 24h. The solvent was removed by evaporation under reduced pressure and the residue was dissolved in methylene chloride and cooled at -20°C. 1hydroxybenzotriazole was precipitated and removed by filtration. Then, the residue was purified by column chromatography on silica gel DCM/MeOH (100/0, 95/5 and 8/2) with 78% yield. TLC: Rf = 0.44 (DCM/MeOH 95:5 (v/v)) 1 H NMR (300 MHz, CDCl3), δ (ppm): 6.15 (m, 1H, NH), 5.11 (m, 7H, H1, H I ), 3.90 (m, 4H, H1'), 3.80-3.45 (m, 35H, H3, H III , H4, H IV , H5, H V , H6, H VI ) , 3.63 (s, 18H, O(6)CH3), 3.50 (s, 21H, O(3)CH3), 3.38 (s, 21H, O(2)CH3), 3.14 (m, 9H, H2, H II , H9), 2.31 (t, 2H, H8),1.54 (m, 4H, H2') , 1.17 (m, 52H, H3'-H15'), 0.81 (t, 6H, H16', JH16'-H15'= 6.39 Hz) 13

VI -amido-β-alanyl-(VI -di(oleyloxy)phosphoramidyl)-VI -deoxy-2 II ,3 III -di-O-methylhexakis(2,3,6-tri-O-methyl)-cyclomaltoheptaose 18

O,O-dioleyl-N-(2-carboxyethyl)phosphoramidate 12 (283 mg, 0.42 mmol, 1.5 eq) was dissolved in anhydrous N,N dimethylformamide (15 mL) under argon. Then, 1-hydroxybenzotriazole (378 mg, 2.8 mmol, 10 eq) and N,N-diisopropylcarbodiimide (436 µL, 2.8 mmol, 10 eq) were added. After stirring for 1h, the 6 VI -amino-6 VI -deoxy-2 II ,3 III -di-O-methyl-hexakis(2,3,6-tri-O-methyl)cyclomaltoheptaose 13 solution (395 mg, 0.28 mmol, 1 eq dissolved in 10 mL of chloroform with few drops of triethylamine) was added. The reaction mixture was stirred at room temperature for 24h. The solvent was removed by evaporation under reduced pressure and the residue was dissolved in methylene chloride and cooled at -20°C. 1-hydroxybenzotriazole was precipated and removed by filtration. Then, the residue was purified by column chromatography on silica gel DCM/MeOH (98/2 and 96/4) with 85% yield. TLC: Rf = 0.42 (DCM/MeOH 95:5 (v/v)) 1 H NMR (300 MHz, CDCl3), δ (ppm): 6.17 (m, 1H, NH), 5.33 (m, 4H, H9', H10'), 5.12 (m, 7H, H1, H I ), 3.96 (m, 4H, H1'), 3.89-3.41 (m, 35H, H3, H III , H4, H IV , H5, H V , H6, H VI ) , 3.62 (s, 18H, O(6)CH3), 3.50 (s, 21H, O(3)CH3), 3.37 (s, 21H, O(2)CH3), 3.17 (m, 9H, H2, H II , H9), 2.36 (t, 2H, H8), 1.99 (m, 8H, H8', H11'), 1.64 (m, 4H, H2') , 1.26 (m, 44H, H3'-7', H12'-17'), 0.87 (t, 6H, H18', JH18'-H17' = 6.37 Hz) 13 6 VI -mono-amino-6 VI -deoxy-2 II ,3 III -di-O-methyl-heptakis(2,3,6-tri-O-methyl) cyclomaltooctaose 14 γ-CD (5.0 g, 3.85 mmol, 1 eq) was solubilized in pyridine (300 mL) under argon. After the solubilization (40 min), the 2,4,6-triisopropyl sulfonyl chloride (3.5 g, 11.56 mmol, 3 eq) was added. The reaction mixture has been stirred at room temperature under argon for 24h. Then, the solvent was removed by evaporation under reduced pressure until there is a minimum of solvent and acetone (500 mL) was added. The precipitation obtained was filtered and was washed with acetone. The white powder of 6 VI -(O-2,4,6-triisopropylbenzenesulfonyl)-cyclomaltooctaose was put in desiccator at 60°C under reduced pressure one night. Then, the 6 VI -(O-2,4,6-triisopropylbenzenesulfonyl)-cyclomaltooctaose (6.07 g, 3.89 mmol, 1 eq) was dissolved in DMF (130 mL) at 80°C. After solubilization, the sodium azide (0.51 g, 7.77 mmol, 2 eq) was added and the reaction mixture has been stirred at 80°C for 24h. Then, the solvent was removed by evaporation under reduced pressure, a little bit of water has been added to solubilize the product and acetone (200 mL) was added. The precipitation obtained was filtered and was washed with acetone. The white powder of 6 VI -azido-6 VI -deoxy-cyclomaltooctaose was put in desiccator at 60°C under reduced pressure one night. Then, the 6 VI -azido-6 VI -deoxy-cyclomaltooctaose (1.72 g, 1.30 mmol, 1 eq) was dissolved in dry DMF (70 mL). The solution was cooled down at 0°C in ice bath and sodium hydride (60%, m/m) (3.12 g, 78.1 mmol, 60 eq) was added. After solubilization, methyl iodide (15 mL, 240.9 mmol, 185 eq) was added. The reaction mixture has been stirred at room temperature and under inert atmosphere for 1 day. The addition of few drops of ethanol stopped the reaction. The reaction mixture is then filtered and the filtrate was evaporated under reduced pressure. The obtained oil was put in a minimum of water and extracted with dichloromethane (3x50 mL). The organic phase was washed with water (2x50 mL), dried on sodium sulfate, filtered and the solvent was removed by evaporation under reduced pressure. A yellow oil was obtained which is a mixture of the expected compound and the per(2,3,6-tri-O-methyl)cyclomaltooctaose, formed from the γ-CD regenerated during the synthesis of 6 VI -azido-6 VI -deoxy-cyclomaltooctaose. This mixture would be purified in the next step. Then, the 6 VI -mono-azido-6 VI -deoxy-2 II ,3 III -di-O-methyl-heptakis(2,3,6-tri-O-methyl) cyclomaltooctaose (3.041 g, 1.85 mmol, 1 eq) was dissolved in DMF (155 mL). After solubilization, the triphenyl phosphine (2.43 g, 9.25 mmol, 5 eq) was added and the reaction mixture has been stirred at room temperature for 2h. The solution was cooled down at 0°C in ice bath and ammoniac solution (85 mL, 38%) was added drop by drop. The reaction mixture has been stirred at room temperature for 1 night. Then, the residue was purified by column chromatography on silica gel DCM/MeOH (100/0, 95/5 and 8/2) with 5% global yield. TLC: Rf = 0.52 (DCM/MeOH 9/1 (v/v)) 1 O,O-didodecyl-N-(2-carboxyethyl)phosphoramidate (51 mg, 0.10 mmol, 1.5 eq) was dissolved in anhydrous N,N dimethylformamide (3 mL) under argon. Then, 1-hydroxybenzotriazole (90 mg, 0.67 mmol, 10 eq) and N,N-diisopropylcarbodiimide (100 µL, 0.67 mmol, 10 eq) were added. After stirring for 2h, the 6 VI -amino-6 VI -deoxy-2 II ,3 III -di-O-methyl-heptakis(2,3,6-tri-O-methyl)cyclomaltooctaose solution (108 mg, 0.067 mmol, 1 eq dissolved in 3 mL of chloroform with few drops of triethylamine) was added. The reaction mixture was stirred at room temperature for 24h. The solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography on silica gel DCM/MeOH (98/2 and 95/5) with 52%. TLC: Rf = 0.46 (DCM/MeOH 9/1 (v/v)) 1 H NMR (300 MHz, CDCl3), δ (ppm): 6.18 (m, 1H, NH), 5.13 (m, 8H, H1, H I ), 3.94 (m, 4H, H1'), 3.90-3.29 (m, 35H, H3, H III , H4, H IV , H5, H V , H6, H VI ) , 3.62 (s, 18H, O(6)CH3), 3.50 (s, 21H, O(3)CH3), 3.37 (s, 21H, O(2)CH3), 3.19 (m, 10H, H2, H II , H9), 2.37 (t, 2H, H8),1.62 (m, 4H, H2') , 1.25 (m, 36H, H3'-H11'), 0.87 (t, 6H, H12', JH12'H11' = 6.41 Hz) 13 O,O-ditetradecyl-N-(2-carboxyethyl)phosphoramidate (52mg, 0.093mmol, 1.5eq) was dissolved in anhydrous N,N dimethylformamide (3mL) under argon. Then, 1-hydroxybenzotriazole (83mg, 0.62mmol, 10eq) and N,N-diisopropylcarbodiimide (96µL, 0.62mmol, 10eq) were added. After stirring for 2h, the 6VI-amino-6VI-deoxy-2II,3III-di-O-methyl-heptakis(2,3,6-tri-Omethyl)cyclomaltooctaose solution (100mg, 0.062mmol, 1eq dissolved in 3mL of methylene chloride with few drops of triethylamine) was added. The reaction mixture was stirred at room temperature for 24h. The solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography on silica gel DCM/MeOH (98/2 and 95/5) with 54%. 1 H NMR (300 MHz, CDCl3), δ (ppm): 6.16 (m, 1H, NH), 5.26 (m, 8H, H1, H I ), 3.94 (m, 4H, H1'), 3.87-3.37 (m, 35H, H3, H III , H4, H IV , H5, H V , H6, H VI ) , 3.63 (s, 18H, O(6)CH3), 3.51 (s, 21H, O(3)CH3), 3.37 (s, 21H, O(2)CH3), 3.19 (m, 9H, H2, H II , H9), 2.37 (m, 2H, H8),1.64 (m, 4H, H2'), 1.25 (m, 48H, H3'-H13'), 0.87 (t, 6H, H12', JH14'-H13' = 6.41 Hz) 13 C NMR (75 MHz, CDCl3), δ (ppm): 98.07 (8C, C1, C I ), 82.14 (24C, C2, C II , C3, C III , C4, C IV ), 71.33, 70.93(15C, C6, C5, C V ), 66.69 (2C, C1'), 61.36 (7C, O(6)CH3), 59.17

Vesicles preparation and characterization

The nanoparticle preparation used is the lipidic film rehydration. Our compounds solubilized in ethanol and the 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) solubilized in hexane/ethanol (9:1 v/v) were mixed, with or without ATV (20µL of 5mM in EtOH). Then, the organic solvent was evaporated by nitrogen flux and the dry film was placed in desiccator for 2h to remove any trace of organic solvent. Then, the dry film is rehydrated by 2mL of Ringer-Hepes (RH) buffer pH 7.4 to reach a final concentration of 400 µM. The dry film was re-suspended by vigorous homogenization to produce MLVs. Then the solution is sonicated for 3 cycles at 2 min 30s (power: 33% of 500 W, probe diameter: 13 mm, Bioblock Scientific VibraCellTM 75115) followed by a filtration on 0.2 µm filters (Tuffryn ® by Acrodisc ® ) to eliminate titanium particles from the probe. The excess of ATV which was not encapsulated in the vesicles is eliminated by Size Exclusion Chromatography (SEC, Sephadex G50, length: 5 cm, diameter: 1.5 cm). The loaded vesicles are collected in the tubes 3 and 4, corresponding to an elution volume of ≈3 and ≈4 mL, respectively. In the case of 3 H-ATV, concentration was measured after SEC at 0.975 µM using Liquid Scintillation Analyser.

Calibration curve of Atazanavir fluorescence intensity in Ringer-Hepes buffer pH 7.4

This calibration curve was performed with a fluorescence spectrometer Synergy H1 (BioTek, Winooski, USA). A stock solution of ATV in DMSO (10 mM) is prepared. This compound is not soluble directly in the RH buffer, so, first, 5 µL of the ATV stock solution was putted in a small pillbox and then the RH was added (2 mL) to reach the concentration of 25 µM. An optimization of the measurement is performed to have the better S/N ratio in the RH. It is found that the better emission and excitation wavelengths are λex = 290 nm and λem = 338 nm, respectively. Then, from the stock solution (25 µM) a cascade dilution by 2 is performed and the fluorescence measurements were performed. The limit of detection is determined at 0.4 µM and the calibration curve obtained (see Figure S1).

Atazanavir in vitro drug release

100 μL of CD/POPC mixed vesicle and POPC vesicle charged with ATV in RH buffer pH 7.4, prepared following the procedure described above, were dialyzed using Single-Use Rapid Equilibrium Dialysis (Thermo Scientific™). NPs were placed in vertical cylinder of dialysis membrane with a molecularweight cutoff of 8 kDa. 300 μL of RH buffer pH 7.4 were placed in buffer chamber. The plate with dialysis membranes was placed at 37°C under vigorous shaking. ATV was quantified in both drug and buffer chambers by fluorescence spectroscopy.

In vitro model of blood brain barrier

Bovine Brain Capillary Endothelial Cells (BBCECs) were cultivated during 12 days with rat glial cell in Transwell™ Costar polycarbonate inserts, 3 μm pore size, coated with rat tail collagen. The resulting co-culture was maintained for at least 12 days under standard conditions: a humidified 5% CO2 atmosphere, with renewal of the culture medium (DMEM supplemented with 10% Horse Serum, 10% Calf Serum, 1% glutamine and 0.5% gentamycin) every second days. Under these conditions, BBCECs exhibit most of the characteristics of the BBB such as low permeability to non-permanent markers [START_REF] Schultz | Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models[END_REF][START_REF] Candela | Physiological pathway for low-density lipoproteins across the blood-brain barrier: transcytosis through brain capillary endothelial cells in vitro[END_REF][START_REF] Dehouck | Drug transfer across the blood-brain barrier: correlation between in vitro and in vivo models[END_REF][START_REF] Culot | An in vitro blood-brain barrier model for high throughput (HTS) toxicological screening[END_REF]. All experiments were performed in triplicate and at 37°C. On the day of the experiment, β-CDs were dissolved at the selected concentration in HEPES-buffered Ringer´s solution (NaCl 150 mM, KCl 5.2 mM, CaCl2 2.2 mM, MgCl2 0.2 mM, NaHCO3 6 mM, Glucose 2.8 mM, HEPES 5 mM, water for injection). At the start of the transport experiments, buffered 2.5 mL of Ringer´s solution was added to the receiver compartments without cells. Inserts with bovine brain endothelial cells were then placed in these wells and 1.5 mL compound's solution were added to the donor compartment.

Endothelial permeability coefficients (Pe) were calculated as described previously [START_REF] Dehouck | Drug transfer across the blood-brain barrier: correlation between in vitro and in vivo models[END_REF]. In this calculation, both filter without cell permeability (PSf = filter with collagen coating) and filter with cell permeability (PSt = filter with collagen with ECs) were taken into account. Pe = 1/PSe = 1/PSt + 1/PSf, where PS denote for the permeability over the surface area (in mL/min) which was the slope determined by linear regression of the average volume of compound cleared (Cl) versus time over the experiment. PSf was the collagen coated filter permeability previously determined. PSt was calculated using the volume of compound cleared (Cl in mL or cm 3 ) at each time interval following the equation: Cl = A / Cd. Where, A was the amount of compound in the basolateral compartment (in mol), Cd was the concentration in the luminal compartment (in mol/cm 3 ). The total cleared volume at each time point was calculated by summing Cl at each time interval. EC permeability coefficient (Pe, cm/min) was obtained by dividing PSe values by the surface area of the filter (cm²), as follow: Pe = PSe / S. Toxicity study of tested compounds was performed using radiolabelled sucrose ( 14 C sucrose) which is a paracellular marker to control the toxicity of products. This small hydrophilic molecule presents, in physiological conditions, a low cerebral penetration and its endothelial permeability coefficient reveals the endothelial cell monolayer integrity. The co-incubation of 14 C sucrose (final concentration 1.82KBq/mL) with the tested compound was performed in triplicates. Co-incubation of 14 C sucrose and the tested compound allows to assess drug toxicity by evaluating the permeability coefficient for 14 C sucrose. 14 C-sucrose was analyzed using Liquid Scintillation Analyser (Tri-carb 2100TR).

Uptake of 3 H-Atazanavir in endothelial cells

After 120 minutes of drug transport experiment, inserts ± BBCECs were washed 5 times with cold RH buffer pH 7.4. Cells were lysed with 500 μL of the triton X100 1% solution. Cell lysates and Filters were transferred to polyethylene scintillation vials with addition of scintillation cocktail (Ultima Gold MW). The amounts of 3 H-ATV were determined using Liquid Scintillation Analyser (Tri-carb 2100TR).

Preparation of POPC/ fluorescent probe (R18) vesicles

40 µL POPC solubilized in hexane/ethanol (9/1 v/v) mixture (5 mM) was put in a pillbox. The R18 probe is introduced at 6.3 mass% compared the mass of lipid, so 9.58 µL (1 mg/mL in ethanol) was added to the POPC in the pillbox. Then, the same procedure is applied as for the formation of the NPs 2.9. Fusogenicity studies To study fusogenicity, the fluorescence measurements were performed with a Cary Elipse fluorescence spectrometer using a quartz cell. The final concentration, in the cell, is fixed at 50 µM with a 1:4 molar ratio between the vesicles of amphiphilic CD/POPC and R18/POPC. So, a volume of 100 µL of each vesicle is added. Kinetics measurements were performed during 31 min. First, 800 µL of TRIS buffer pH 7.4 with 100 µL of R18/POPC was mixed and the measurement was performed during 1 min to obtain the value of "zero fusion". Then, the CD/POPC is added and the measurement is performed.

Results and discussion

Synthesis of lipophosphoramidyl permethylated CDs

An optimisation of the synthesis of Lip-β-CDs 15-18 was performed in order to scale up the synthesis which was previously described [START_REF] Gervaise | Synthesis of lipophosphoramidyl-cyclodextrins and their supramolecular properties[END_REF]. Fatty alcohols reacted with diphenylphosphite in a Kugelrohr apparatus, leading quantitatively to dialkylphosphite 1-4, depending on the chain length i.e. C12, C14 to C16 and C18:1 according to elsewhere described procedure (Mevel et al., 2007 and2008). Fatty phosphites 1-4 are then introduced in Todd-Atherton reaction with β-alanine methyl ester with rather good yield (Figure 1 and see SI for experimental details). After the saponification of methyl ester to 9-12 (see SI for experimental details), a peptidic coupling with permethylated 6-amino-6-deoxy-β-cyclodextrin 14 obtained by the conventional route [START_REF] Gervaise | Synthesis of lipophosphoramidyl-cyclodextrins and their supramolecular properties[END_REF] was performed. Five compounds were obtained in the gram scale with good yields (65-85%). The second advantage of this new process is the versatility of the synthesis in terms of tuning oligosaccharidic macrocycle or aliphatic chains. We demonstrated that this route could also be applied to permethylated 6-amino-6-deoxy-γcyclodextrin with C12 19 and C14 20 chain lengths (Figure 2) obtained with 52% and 54%, respectively (for the scheme of the chemical route see Figure S2). The difficulty is the preparation of the permethylated 6-amino-6-deoxy-γ-CD 14, which was obtained only at a 5% yield. The first monomodification step of γ-CD was performed using triisopropylsulfonyl chloride, as described in the literature [START_REF] Palin | Mono-6-(O-2,4,6triisopropylbenzenesulfonyl)-γ-cyclodextrin, a novel intermediate for the synthesis of monofunctionalised γ-cyclodextrins[END_REF]. The yield of this step was very low, impairing the scale-up of these compounds for further experiments.

Characterization of Lipophosphoramidyl permethylated β-CDs

Our goal is to achieve an in vitro test, in which a high purity of the compounds is a mandatory condition. We thus performed a very thorough characterization of the structure of our compounds by NMR (Figure 3 for 15 and see Figures S3-S8 for other compounds) and used mass spectrometry to decipher the structural backbone. The 1 H NMR spectrum of compound 15 shows a high resolution in a nonpolar solvent, meaning that the pure mono-substituted compound and NH of the amide group at δ = 6.18 ppm is characteristic of the linkage of the carboxylic acid of lipidic part to the amino group of the cyclodextrin.

Full MS spectra showed that ions were only detected under 1+ and 2+ charge states with common H + , NH4 + , Na + and K + cations. Whatever the compounds, sodiated forms are the prominent ions, singly and doubly charged, for compounds 15 (Figure 4), 16 and 18 (Figure S9 andS10). Exact masses were also confirmed by ESI-HRMS, for example, for [M+2Na] 2+ with experimental m/z 973.5291 (theoretical 973.5281; ∆m: 1.0 ppm), 1001.5603 (theoretical 1001.5594; ∆m: 0.9 ppm) and 1055.6066 (theoretical 1055.6062; ∆m: 0.2 ppm) for compounds 15, 16 and 18, respectively. For 17, ESI-HRMS measured m/z 2036.1959 for [M+Na] + (theoretical 2036.1928; ∆m: 1.5 ppm). Moreover, even if MS is not a quantitative approach, clear spectra with no noticeable impurities were obtained. Plotting the precursor abundance according to the HCD deposited energy allowed the well-known survival yield curves to be accessed and the CE50 values, that is, the energy required to decompose 50% of the initial precursor, to be obtained [START_REF] Przybylski | Probing the common alkali metal affinity of native and variously methylated β-cyclodextrins by combining electrospray-tandem mass spectrometry and molecular modeling[END_REF]. Moreover, as previously highlighted, the energy employed during HCD fragmentation can be directly provided in an eV scale. These values can be used to compare the stability of various molecular structures within a family of similar compounds. Here, deducted CE50 values were equal to 40.3 (73eV), 40.4 (76eV) and 40.1% (79eV) for doubly sodiated species of compounds 15, 16 and 18 respectively (see Figure S11). Comparison of CE50 values reveals that the gradual increase of acyl chains length leads to a slight strengthening of the molecular stability, that is, from 73 to 79 eV for C12 to C18:1, respectively. Careful examination of the MS/MS spectrum of the doubly sodiated ion of compound 15 obtained at its CE50 value (40%) may portray the main decomposition products, highlighting the most exposed moiety (Figure 4B). Hence, in addition to a usual charge reduction (the loss of one Na + ) at m/z 1924.0712, various fragments can be observed. The most intense fragment at m/z 765.3578 corresponds to the cleavage of the phosphoramide bond (N-P), leading to the release of a whole phosphoramidyl group. Such loss was confirmed by the same remaining 6-amido-β-alanyl-6-deoxy-TRIMEB as a monosodiated form at m/z 1507.7280. At least three other ions were also easily detected at m/z 1436.68, m/z 889.4350 and m/z 663.3079. The former corresponds to mono-amino-TRIMEB and the latter to a 6-amido-β-alanyl-6-deoxy-TRIMEB which has lost one methylated glucose (MeGlc). The third ion, at m/z 889.4350, is particularly interesting, since it indicates that one full chain remains between the two. Such results can be tentatively explained by a self-inclusion phenomenon (i.e., the insertion of one of 15's own lipid chains inside β-CD hydrophobic cavity), as previously observed by us and others especially for dioleoyl-glycerolipidyl-β-CD [START_REF] Furlan | Nanoparticles based on lipidyl-β-cyclodextrins: synthesis, characterization, and experimental and computational biophysical studies for encapsulation of atazanavir[END_REF]. Indeed, such self-inclusion of one lipid chain can partially prevent this chain from undergoing a fragmentation process.

To both track fragmentation trends and map the genealogies of product ions originating from consecutive losses, an energy-resolved mass spectrometry (ERMS) strategy was undertaken on compound 15. ERMS was, in particular, successfully used to characterize structural isomers of oligosaccharides [START_REF] Kurimoto | Analysis of Energy-Resolved Mass Spectra at MSn in a Pursuit To Characterize Structural Isomers of Oligosaccharides[END_REF], to capture glycan connectivity in glycopeptides [START_REF] Kolli | Energy-resolved collision-induced dissociation pathways of model Nlinked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment[END_REF] and to delineate Lipid A variants [START_REF] Crittenden | Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains[END_REF].

As an example, we investigated ERMS in an MS/MS energy-variable manner to characterize structural features, via dominant fragmentation pathways, corresponding to the doubly sodiated species of compound 15 (Figure S12).

As already described, from the low range of this HCD deposited (< 30%, < 55 eV), only one fragment appears to correspond to 6-amido-β-alanyl-6-deoxy-TRIMEB, starting from 23% (41 eV; Figure S12 A, full dark green trace). As the collision energy was increased from 30% (55 eV), the precursor ion was progressively and constantly fragmented, leading to the apparition of mono-amino-TRIMEB (Figure S12B, full blue trace) and compound 15 with one lipidyl chain lost (Figure S12C, violet trace). This last result is quite surprising, since amido-β-alanyl-6-deoxy-TRIMEB was logically expected to be the main fragment of a consecutive fragmentation of lipophosphoramidyl moieties. Nonetheless, such results can be assumed by a parallel fragmentation pathway originating from a different conformation of compound 15. Such an alternative supports the aforementioned hypothesis as regards a selfinclusion phenomenon and demonstrates the co-existence of two populations of compounds, that is, a first one with two free pending lipidic chains and a second conformation involving one fatty acid chain included in the cavity of the CD. Moreover, the use of collision-induced dissociation (CID) in place of HCD allowed for operation at a slower energy regime but with a higher number of collisions, leading to the formation of monolipidyl-phosphoramidyl-MeGlc (Figure S13). Fragmentation pathways were proposed according to these results in Figure S14. All these structural deciphering experiments showed that compounds were present both in high purity grade and with well-defined structures and thus could be confidently used for biological assessment.

The toxicity of these compounds was then tested on an in vitro model of the BBB. This model aimed to co-cultivate bovine brain capillary endothelial cells from rat glial cells. This model has been intensively characterized and is widely used to test drug toxicity. This parameter is checked by measuring the permeability of a small hydrophilic compound, the 14 C sucrose, across the cell monolayer. As shown by Figure 5, the labelled sucrose passage across the BBCEC monolayer was not altered in presence of concentrations below 5 µM of amphiphilic CDs (Figure 5). The permeability below 0.75.10 -3 cm.min -1 at 5µM for 15-17, which is lower than that of β-CD(C9)2OOMe, another amphiphilic cyclodextrin tested previously [START_REF] Oliva | New Lipidyl-Cyclodextrins Obtained by Ring Opening of Methyl Oleate Epoxide Using Ball Milling[END_REF]. At 50 µM, 15 increased 14 C Sucrose permeability, suggesting that the lowest concentrations must be used for further studies. The choice of mixed vesicles seems to be a good strategy to form vesicles both reducing the quantity of the amphiphilic CDs needed and preventing toxicity against the BBB [START_REF] Focus ; Binkowski-Machut | How cyclodextrins can mask their toxic effect on the blood-brain barrier[END_REF][START_REF] Vieira | Getting into the brain: liposome-based strategies for effective drug delivery across the blood-brain barrier[END_REF]. Our goal is to prepare mixed vesicles composed of phospholipid (POPC) and three amphiphilic cyclodextrins of the family and encapsulating ATV. To control the size of the vesicles, the film hydration technique was chosen to formulate the Lip-β-CDs with POPC in presence of the drug. The diameter of the mixed vesicles prepared with ATV was measured by DLS to be in the range 150-210 nm depending on the aliphatic chain. The polydispersity index (PdI) for 15/POPC is low, involving monodisperse vesicles, whereas Lip-β-CDs with longer chains exhibit a higher PdI, revealing quite polydisperse particles (Table 1). The size of these vesicles is consistent with an application for transcytose on the blood brain barrier. Recently, Lundy et al. [START_REF] Lundy | Inducing a Transient Increase in Blood-Brain Barrier Permeability for Improved Liposomal Drug Therapy of Glioblastoma Multiforme[END_REF] highlighted that a size in the 50-200 nm range is generally accepted as optimal. Our vesicles could be assimilated to niosomes. Indeed, in the literature, nano-niosomes were described as vesicles composed of cholesteryl derivatives and non-ionic surfactants, especially glycolipidic ones [START_REF] Moghassemi | Nano-niosomes as nanoscale drug delivery systems: An illustrated review[END_REF]. They can be prepared by the film hydration technique. Niosomes with β-CD have already been successfully employed to improve cellular uptake of drugs [START_REF] Oommen | Niosome Entrapped β-Cyclodextrin Methotrexate Complex as a Drug Delivery System[END_REF]. We studied the in vitro release of ATV from POPC liposomes or mixed vesicles of amphiphilic cyclodextrins/POPC compared to ATV alone (Figure 6). Atazanavir is poorly soluble in an aqueous buffer, so we work in diluted conditions. Moreover, the formulations led to colloidal stable suspension.

Preparation of vesicles containing Atazanavir

In four hours, 100% of ATV alone was released, whereas in the case of mixed vesicles, 40-50% of the ATV was released in approximately two hours. The same profile was observed regardless of the amphiphilic CD in the formulation (data not shown). However, during the same duration, we observed an ATV release of 80% with the liposomes of POPC alone. In this case, the curve obtained is close to that of ATV alone. We thus demonstrated the efficiency of amphiphilic CD in slowly releasing a drug such as ATV. This result is in good agreement with the encapsulation and release of methotrexate using noisome-based CDs [START_REF] Oommen | Niosome Entrapped β-Cyclodextrin Methotrexate Complex as a Drug Delivery System[END_REF].

After seven days, the release of ATV was not complete with amphiphilic CD/POPC vesicles. This could be due to a lack of detection sensitivity, because of chemical degradation of ATV. Indeed, ATV is known to be unstable in aqueous solutions and rather sensitive to heat and slightly basic conditions [START_REF] Dey | Development and validation of a stability-indicating RP-HPLC method for estimation of atazanavir sulfate in bulk[END_REF].

In vitro BBB model toxicity and transport experiments

We then checked the toxicity of vesicles alone on the BBB cells by measuring the permeability of the barrier in contact with different concentrations of vesicles (0 -400 µM). At concentrations below 50 µM of vesicles, no alteration of the permeability inferior to 0.8. 10 -3 cm/min of the monolayer was observed, much less than quaternary ammonium βCD (QAβCD) nanoparticles already used as carriers on the BBB model with no toxicity [START_REF] Gil | Quaternary Ammonium β-Cyclodextrin Nanoparticles for Enhancing Doxorubicin Permeability across the In Vitro Blood-Brain Barrier[END_REF].

Knowing that the integrity of the model was demonstrated, further experiments were carried out to improve the transport of 3 H-ATV through the BBB in vitro model. It was not technically possible to detect 3 H-ATV in the abluminal side of the model, but we showed a significant enhancement (Figure 7) of the cellular uptake of 3 H-ATV in the endothelial cells of the BBB. Mixed vesicles composed of 18/POPC were thus able to enhance the intracellular accumulation of 3 H-ATV by four-fold or higher, showing a clear endocytosis of vesicles by endothelial cells. Mixed vesicles containing amphiphilic CDs with shorter chains, 15 and 16, are less efficient as drug carriers but also enhanced uptake, by 1.1 and 1.6 fold, respectively. This result is consistent with features of efficient drug carriers reported in the literature. Comparatively, doxorubicin uptake in the BBB model was enhanced by 2.2 fold with QAβCD nanoparticles [START_REF] Gil | Quaternary Ammonium β-Cyclodextrin Nanoparticles for Enhancing Doxorubicin Permeability across the In Vitro Blood-Brain Barrier[END_REF], whereas the concentration of ribavirin, included in niosomes, was six-fold higher in liver cells than free drug [START_REF] Hashim | Preparation and characterization of niosomes containing ribavirin for liver targeting[END_REF]. The rationalisation of this clear impact of the chain length on cellular uptake requires a good understanding of the effect of Lip-β-CDs 15, 16 and 18/POPC vesicles on a membrane model. The fusogenicity of various vesicles was thus evaluated by fluorescence measurements. Two different vesicles were prepared, one with various molar ratio of Lip-β-CDs /POPC one using R18 as a fluorescent probe and three phosphatidylcholines (POPC:PLPC:SAPC (1:1:1)), sphingomyelin, cholesterol 4:3:1 as a biomimetic model of the plasmic membrane. One minute after mixing both of these vesicles, an increase of fluorescence was noted leading to larger vesicle formation involving dequenching of fluorescence due to fusogenic properties (Figure 8). These results indicate that a very strong fusogenicity of 15/POPC with a 20-50% molar ratio occurred, in contrast to when POPC alone is used as a control. A maximum fluorescence rate was reached around 70%, portraying good fusion with biomimetic SUVs. The behaviour of 15/POPC mixed vesicles is very similar to that described by Kros et al., who used lipopeptides incorporated in liposomes to produce the fusion of lipid membranes [START_REF] Marsden | Controlled liposome fusion mediated by SNARE protein mimics[END_REF]. Conversely, compound 16-based mixed vesicles exhibited a maximum fluorescence rate at approximatively 35% with biomimetic SUVs. That shows a less fusogenic character with the biomimetic membrane than 15. As regards 18/POPC mixed vesicles, we still observed a low fusogenicity, which is close to that obtained with POPC SUVs alone. Because of the fusogenicity, vesicles are probably diluted in the membranes of cells before acting as carriers through membrane cells and consequently preventing the endocytosis process.

Conclusion

In this study, we proposed an efficient synthetic route to prepare lipophosphoramidyl CDs at the gram scale. Obtained amounts of each compound were able to be assembled in vesicles with POPC encapsulating antiretroviral drugs, that is, ATV. Conversely to usual liposomes, a slow and controlled release was observed thanks to lipophosphoramidyl CDs vesicles. Moreover, two particularly important features were highlighted: (i) the absence of toxicity observed on an in vitro BBB model as a critical aspect for drug delivery applications and (ii) a high enhancement of cellular uptake of ATV in endothelial cells for oleylphosphoramidyl CDs (C18:1, compound 18). This clear impact of chain length can be explained by fusogenicity properties regarding the grafted chain length. Indeed, oleylphosphoramidyl CDs exhibit a very low fusogenicity, which prevents the dilution of vesicles in the membrane and presumably increases the drug delivery rate into the cell.

  C NMR (75 MHz, CDCl3), δ (ppm): 99.03 (7C, C1, C I ), 81.81-80.30 (21C, C2, C II , C3, C III , C4, C IV ), 70.92 (14C, C5, C V , C6, C VI ), 66.52 (2C, C1'), 61.44 (6C, O(6)CH3), 59.00-58.47 (14C, O(3)CH3, O(2)CH3), 37.81 (2C, C8, C9), 31.89-22.66 (28C, C2'-C15'), 14.09 (2C, C16') 31 P NMR (120 MHz, CDCl3), δ (ppm): 110.53 (s, 1P) and HRMS (ESI): m/z 2036.1959 [M+Na] + , (C97H181N2O38PNa requires 2036.1928).

  C NMR (75 MHz, CDCl3), δ (ppm): 130.13 (4C, C9', C10'), 99.20 (7C, C1, C I ), 82.21-80.47 (21C, C2, C II , C3, C III , C4, C IV ), 71.38-71.08 (13C, C5, C V , C6), 66.65 (2C, C1'), 61.61 (6C, O(6)CH3), 59.16-58.49 (14C, O(3)CH3, O(2)CH3), 39.96 (1C, C VI ), 37.94 (2C, C8, C9), 32.75-22.82 (28C, C2'-C8', C11'-C17'), 14.25 (2C, C18') 31 P NMR (120 MHz, CDCl3), δ (ppm): 110.55 (s, 1P) HRMS (ESI): m/z 1055.6066 [M+2Na] 2+ , (C101H185N2O38PNa2 requires 1055.6064).

  H NMR (300MHz, CDCl3), δ (ppm): 5.21 (m, 8H, H1, H I ), 3.91-3.30 (m, 49H, H2, HII, H3, HIII, H4, HIV, H5, HV, H6, HVI), 3.64 (s, 21H, O(6)CH3), 3.51 (s, 24H, O(3)CH3), 3.37 (s, 24H, O(2)CH3), 3.21 (m, 8H, H2, H II ). 13 C NMR (75MHz, CDCl3), δ (ppm): 98.05 (8C, C1, C I ), 82.00 (24C, C2, C II , C3, C III , C4, C IV ), 71.30 (7C, C6), 70.92 (8C, C5, C V ), 61.43 (8C, O(6)CH3), 59.18 (7C, O(3)CH3), 58.88 (7C, O(2)CH3). ZQ (ESI): m/z 1641.0 [M+Na] + (C71H127NO39Na requires 1640.80) 6 VI -amido-β-alanyl-(6 VI -di(dodecyloxy)phosphoramidyl)-6 VI -deoxy-2 II ,3 III -di-O-methylheptakis(2,3,6-tri-O-methyl)-cyclomaltooctaose 19

  C NMR (75 MHz, CDCl3), δ (ppm): 99.20 (8C, C1, C I ), 82.20-80.79 (24C, C2, C II , C3, C III , C4, C IV ), 71.38, 71.19 (15C, C6, C5, C V ), 66.67 (2C, C1'), 61.61 (7C, O(6)CH3), 59.16-58.76 (16C, O(3)CH3, O(2)CH3), 39.97 (1C, C VI ), 37.95 (2C, C8, C9), 32.05-22.82 (20C, C2'-C11'), 14.24 (2C, C12') 31 P NMR (120 MHz, CDCl3), δ (ppm): 110.46 (s, 1P) ZQ (ESI): m/z 2129.9 [M+Na] + (C98H181N2O43PNa requires 2128.18)

  (16C, O(3)CH3, O(2)CH3), 40.00 (1C, C VI ), 37.91 (2C, C8, C9), 32.06-22.84 (24C, C2'-C13'), 14.28 (2C, C12') ZQ (ESI): m/z 2185.3 [M+Na] + (C102H189N2O43PNa requires 2184.24)

Figure 1 :

 1 Figure 1: Chemical convergent route to lipophosphoramidyl-β-CDs 15-18.

Figure 2 :

 2 Figure 2: Lipophosphoramidyl-γ-CD 19 and 20 obtained by the same pathway.

Figure 3

 3 Figure 3: 1 H NMR of lipophosphoramidyl permethylated β-CD C12 15 in CDCl3 (298K, 400 MHz)

Figure 4 .

 4 Figure 4. ESI-HRMS and ESI-HRMS/MS of compound 15. A) Full MS spectrum and B) MS/MS spectrum obtained for fragmentation of [M+2Na] 2+ in HCD at 40% (73eV). The two insets show the comparison between experimental and theoretical m/z for singly and doubly sodiated species.

Figure 5 :

 5 Figure 5: Study of the permeability of 14 C sucrose on an in vitro BBB model in the presence of various amphiphilic cyclodextrins 15 (C12), 16 (C14), 17 (C16) and 18 (C18:1).

Figure 6 :

 6 Figure 6: In vitro release of ATV encapsulated in mixed vesicles of POPC/amphiphilic CD 15 (red ), POPC liposome (violet ) compared to ATV alone (green )

Figure 7 :

 7 Figure 7: Cellular accumulation of 3 H-ATV formulated in mixed vesicles amphiphilic CD 15, 16 and 18, respectively, with POPC (Normalization with respect to the internalization of 3 H-ATV alone in endothelial cells).**p < 0.01, ***p < 0.001.

Figure 8 :

 8 Figure 8: Fusogenicity effect of lipophosphoramidyl β-CDs/POPC mixed vesicles on R18/4:3:1 phosphatidylcholines (POPC :PLPC : SAPC (1:1:1)), sphingomyelin, cholesterol vesicles in a TRIS buffer (10 mM, NaCl 150 mM, pH 7,4). 15 (A), 16 (B) and 18 (C).

6 VI -amido-β-alanyl-(6 VI -di(tetradecyloxy)phosphoramidyl)-6 VI -deoxy-2 II ,3 III -di-O-methyl- hexakis(2,3,6-tri-O-methyl)-cyclomaltoheptaose 16 O,

	CDCl3), δ (ppm): 110.46 (s, 1P) HRMS (ESI): m/z 973.5291 [M+2Na] 2+ , (C89H165N2O38PNa2 requires
	973.5281, m/z 1924.0685 [M+Na] + , (C89H165N2O38PNa requires 1924.0670).
	C NMR (75 MHz, CDCl3), δ (ppm): 99.20 (7C, C1, C I ), 82.20-80.79 (21C, C2, C II , C3, C III , C4, C IV ),
	71.38, 71.19 (13C, C6, C5, C V ), 66.67 (2C, C1'), 61.61 (6C, O(6)CH3), 59.16-58.76 (7C, O(3)CH3, O(2)CH3),
	39.97 (1C, C VI ), 37.95 (2C, C8, C9), 32.05-22.82 (20C, C2'-C11'), 14.24 (2C, C12') 31 P NMR (120 MHz,

Table 1 :

 1 Measurements by DLS of the diameter of vesicles obtained for three formulations varying by chain length of lipidic moiety (n = 3).

	Vesicles formulation	Diameter (nm)	PdI
	15(C12)/POPC/ATV	171	0.192
	16(C14)/POPC/ATV	210	0.296
	18(C18 :1)/POPC/ATV	149	0.302

Acknowledgments

This work and the PhD grant (FN) was supported, as part of the "Agroressources" Research Program, by the Conseil Régional de Picardie under the name SYNVECT.

Graphical abstract

Highlights

• Lipophosphoramidyl cyclodextrins (Lip-β-CDs) were successfully synthesized.

• Controlled release of drug was shown with vesicles POPC/(Lip-β-CDs).

• No toxicity was observed on an in vitro BBB model.

• Oleylphosphoramidyl CDs enhanced cellular uptake of Atazanavir in BBB cells.

• The very low fusogenicity of oleylphosphoramidyl Lip-β-CDs increased the drug delivery rate.


Florence Tubach
email: <florence.tubach@bch.aphp.fr

Bruno Giraudeau

Philippe Ravaud

'é É

Keywords: Anti-Inflammatory Agents, Non-Steroidal, therapeutic use, Humans, Osteoarthritis, Hip, drug therapy, Osteoarthritis, Knee, drug therapy, Pain Measurement, Patient Satisfaction, Randomized Controlled Trials as Topic, methods, Research Design, Treatment Outcome Minimal Clinically important Difference, Patient Acceptable Symptom State, Improvement, Randomized Control Trial, Treatment Effect, Minimal Disease Activity

The variability in minimal clinically important difference and patient acceptable symptomatic state values did not have an impact on treatment effect estimates.

The variability in minimal clinically important difference and patient acceptable symptomatic state values did not have an impact on treatment effect estimates

Florence Tubach 1 2 * , Bruno Giraudeau 3 , Philippe Ravaud 1 2 Mod les et m thodes de l valuation th rapeutique des maladies chroniques 1 to help with the interpretation and communication of the results of clinical trials using patient reported outcomes measured by continuous variable (e.g. pain on a visual analogous scale -VAS). The MCID is the minimal difference representing a clinically important difference in the patient s perspective. Very close is the Minimal Clinically Important Improvement, MCII ( ) that addresses only the direction of ' 5 improvement, not worsening. In this paper we will use the term of MCID for these two very similar concepts. The PASS is the state beyond which patients consider their state as acceptable. Therapeutic success can thus be defined at the individual level (i.e. for each patient) as an improvement greater than the MCID, or as achieving a state acceptable at the end of the study. On a methodological point of view, MCID and PASS are then two different ways of dichotomising a continuous outcome into a binary variable of therapeutic success (the MCID being a threshold for the change in score and the PASS for the final score). At the end of the trial, they allow to report the proportion of patients benefiting from treatment (difference in proportion of improved patients or patients in an acceptable state between treatment arms). These concepts have been recognised useful ( ), in addition to the conventional way of reporting results of trials (e.g. 6 -8 difference in mean change), known to be usually a more powerful approach (

), but not meaningful to everyone ( ). MCID and 9 -11 12 PASS values have been estimated in different studies, in different diseases and in different countries ( , ) Especially for the 1 -5 13 -20 concept of important improvement, a large literature exists on the Minimal Clinically Important Difference (MCID) ( ). From these different estimates, to have a practical tool allowing to calculate a Number Needed to Treat, to compare studies or to pool them for a meta-analysis, for each outcome criterion, the medical community has to move towards a consensus on the MCID and PASS values for each outcome criteria so that every report would use the same cut-off values for comparison of therapeutic success. However, there is a variability among the different estimates from different studies (but estimates remain in an acceptable and clinically relevant range) and the choice of the value to be used subsequently is difficult. It should be data driven (based on the estimates) and expert based to have a good face validity. A key point in the discussion is whether the choice of the MCID and/or PASS cut-off values may have a great impact or not on the treatment effect estimates expressed as difference in success rate (proportion of patients benefiting from treatment). The aim of this study was to explore this point.

METHODS

This is an analytic calculation study. The calculations were performed using pre-specified mean and standard deviation values for the distribution of the endpoint score in the control group and considering a normal distribution. We based our example on a pain endpoint assessed through a 0 100mm Visual Analogous Scale (VAS), but this can be extrapolated to any 0 100 score. The control group --

were derived from a cohort of 1194 patients hip or knee osteoarthritis patients treated with a nonsteroid anti-inflammatory drug during 4 weeks ( ). Then, from the hypothesized effect sizes (0.25 and 0.50), we derived the mean score value in the experimental group 21

(the SD being supposed to be the same as in the control group). For different MCID and PASS values, we calculated (from normality assumption) the success rate for the control and experimental groups as the probability of having a value (change from baseline score/final score) lower than the threshold (MCID/PASS). This then allowed to derive the difference in success rates between the control and experimental groups (i.e. the proportion of patients benefiting from treatment). We also calculated the Number needed to treat (NNT) as the inverse of the absolute success rate increase caused by treatment, i.e. 1/difference in success rate) ( ). 22

For illustration purpose, we drawn figures plotting i) success rates in each group, ii) difference between these success rates as functions of the MCID or PASS thresholds and iii) derived NNT. Finally, we considered two effect sizes that can sensibly be observed (0.25 and 0.50) and made the MCID and the PASS vary across a range around relevant values according to results from previous published

6

RESULTS

shows the success rates in each treatment arm, the difference in success rates as function of the MCID values and the NNT. Figure 1 For instance, with an effect size of 0.25, mean change in score in control group is 20.2 (SD 24.5), mean change in score in experimental the difference in success rates, i.e. the proportion of patients benefiting from treatment (right vertical axis). In red, the NNT. In the green frame, the most relevant values of MCID in accordance to the OMERACT members. axis). In blue, the difference in success rates, i.e. the proportion of patients benefiting from treatment (right vertical axis). In red, the NNT. In the green frame, the most relevant values of PASS in accordance to the OMERACT members.

  , Universit Paris-Diderot -Paris VII é , Facult de m decine Paris 7 16, Rue Henri Huchard 75018 Paris,FR é é D partement d pid miologie, biostatistique et recherche clinique 2 é INTRODUCTION The Minimal Clinically Important Difference (MCID)( ) and the Patient Acceptable Symptom State (PASS)( ) are two concepts 1 -3 4

  . The ranges we used were 40 to 10 for MCID values, and 20 to 50 for PASS values, 4 scores. We particularly focused on the most relevant values for MCID and PASS in accordance to the OMERACT members -(experts in the field of outcome criteria in rheumatology): 15 to 25 for MCID values, and 30 to 40 for PASS values. ( )

Figure 1

 1 Figure 1 Impact of the MCID value on the difference in success rate between arms. Mean change in pain 20.2 in the control group; Standard =deviation of the change 24.5; ES 0.25 and 0.50, respectively. In black, the success rate in each treatment group (left vertical axis). In blue, = =

Figure 2

 2 Figure 2 Impact of the variability of the PASS value on the difference in success rate between arms. Mean final pain score 41.5 in the control group; = Standard deviation of the final score 22.8; ES 0.25 and 0.50, respectively. In black, the success rate in each treatment group (left vertical = =

group is 26.33 (SD 24.5), difference in mean score is 6.13. If the MCID value is 20, the success rate is 50.3 in the control group and -= -% 60.2 in the experimental group, thus the difference in success rates is 9.9 and the number needed to treat (NNT) is 10. For MCID % % values ranging from 40 to 10, the difference in success rate between arms ranges from 7.9 to 9.9 (NNT from 10 to 13) if the effect

size is 0.25, and from 15.9 to 19.7 (NNT from 5 to 6) if the effect size is 0.50. In the range of the most relevant values in accordance to % % the OMERACT members, the difference in success rate ranges from 9.4 to 9.9 (NNT from 10 to 11) if the effect size is 0.25; or 17.8 % % % to 19.7 (NNT from 5 to 6) if the effect size is 0.50.

%

shows the success rates in each treatment arm, the difference in success rates as function of the PASS values and the NNT. Figure 2 For instance, if the PASS value is 30 and the effect size 0.25, the success rate is 30. 7

DISCUSSION

This study shows that, in a range of relevant MCID and PASS values, the values chosen for MCID or PASS cut offs has a low impact on the difference in success rate between arms in a trial. In this calculation study, we assumed a normal distribution, which is reasonable for the patient-reported outcomes from our experience (data not shown). Thus our results can not be applied if an outcome criteria is not normally distributed.

No relevant difference in treatment effect estimates (assessed by the proportion of patients benefiting from treatment) was observed regardless of the cut-offs. The difference in success rate is particularly consistent in the range of the most relevant values in accordance to the OMERACT members: 15 to 25 for MCID values and 30 to 40 for PASS values. ( )

This is of help to choose, among a range of relevant values for a given endpoint, a cut off to be recommended as MCID or PASS, for use to report results of trials. However, this does not imply that any cut off can be recommended or that there is no need to agree on a single value (for each criteria) that would be use to report all trials. The aim of using MCID and PASS to report trial results in addition to the difference in mean change (which remains in general a more powerful approach) ( , , ), is to provide more meaningful 9 10 23 information to readers not very familiar with effect size or difference in mean change. Switching from the group level to the individual level (reflecting daily practice) is not natural to most people. Thus providing the difference between arms in terms of difference in proportion of improved patients, or of patient in an acceptable state at the end of the trial is a valuable complement to the conventional way of reporting results. Furthermore, it might be an interesting tool to improve the communication of trials results to the patients, and thus to enhance shared decision making.

To achieve these goals, MCID and PASS cut-offs must have a good face validity. For instance, if PASS for pain is higher than the inclusion criteria used to be candidate to enter a study (usually 40 mm on a 0 100 VAS measuring pain), there is a problem of face validity because it would mean that we enrol patients in therapeutic trials although they are doing well. Furthermore, the message is quite different when saying that 22 and 32 of patient experienced an important improvement in control and treatment arms respectively, or if these % %

proportions are 62 or 72 respectively. In both situations the difference in success rates between arms is 10, but the MCID is different.

% %

We believe that these issues should be taken into account for the choice of the MCID and PASS values in the range of those estimated from studies. This choice should be expert-based, and may involve physicians, trialists and patients used to the concerned outcome criteria in monitoring their treatments. Our findings show that there is a room to take these issues into account without influencing the difference in success rate.