L Simon

V Lapinte

L Lionnard

N Marcotte

M Morille

A Aouacheria

K Kissa

J M Devoisselle

S Bégu

Polyoxazolines Based Lipid Nanocapsules for Topical Delivery of Antioxidants

Keywords: Antioxidant, Lipid nanocapsules, PEG free, Polyoxazolines, Skin protection, Topical delivery Conceptualization, formal analysis, methodology, validation, investigation, writing Conceptualization, methodology, writing review & editing, visualization, supervision, project Conceptualization, methodology, writing review & editing, visualization, supervision, project

Nano-sized lipid formulations offer a great potential for topical delivery of active compounds to treat and prevent human skin damages. Of particular importance is the high loading of hydrophobic molecules, the long-term stability and the auspicious penetration capacity especially reached when using lipid nanocapsules (LNC). Unfortunately, their formation currently relies on a phase inversion process that only operates when using a poly(ethylene glycol) (PEG) based surfactant belonging to the controversial PEG family that was subject of clinical awareness. The present study proposes an alternative to this overused polymer in formulations by designing LNC made of harmless amphiphilic polyoxazolines (POx). Implementing a short sonication step in the process allowed well-defined spherical nanoparticles of ~30 nm to be obtained. The structure of the so called LNC POx was composed of an oily core surrounded by a rigid shell of phospholipids and POx, which ensures a high stability over time, temperature, centrifugation and freezing. Encapsulation of the natural quercetin antioxidant led to a drug loading three times higher than for LNC constituted of PEG (LNC PEG). The antioxidant activity of loaded LNC POx was tested on mice fibroblasts and human keratinocytes after exposure to free radicals from peroxides and UVB irradiation, respectively. The radical scavenging capacity of quercetin loaded in the LNC POx was preserved and even slightly enhanced compared to LNC PEG, highlighting the POx value in nanoformulations.

Introduction

Environmental factors such as UV radiations, pollution or tobacco have a negative impact on human skin by accelerating skin aging and potentially leading to skin cancer [START_REF] Krutmann | The skin aging exposome[END_REF]. Human skin acts as a barrier against this hostile environment thanks to its unique structure and composition. The main protective layer, stratum corneum (SC), ensures stiffness and permeability to the skin due to corneocyte cells embedded in a lipid matrix [START_REF] Elias | Epidermal Lipids, Barrier Function, and Desquamation[END_REF]. The vital barrier function of the SC then becomes a hurdle to overcome when delivery of active compound (AC) to the deepest layer of the skin is concerned (e.g. the imiquimod with antimitotics properties). Indeed, only small (less than 500 Da) uncharged lipophilic (logP 1-3) molecules are able to go through the SC [START_REF] Roberts | Topical and cutaneous delivery using nanosystems[END_REF], whereas the largest or charged ones need to be formulated for a successful delivery through the skin. Topical formulations of lipid nano-sized vectors are one of the preferred solutions since their size allows for enhanced penetration [START_REF] Hatahet | Quercetin topical application, from conventional dosage forms to nanodosage forms[END_REF]. As a matter of fact, solid or polymeric nanoparticles and colloidal lipid nanocarriers loaded with AC were shown to induce therapeutic effect, crossing through the SC to reach their site of action [START_REF] Roberts | Topical and cutaneous delivery using nanosystems[END_REF]. Formulating the nanovector with lipid-based colloids increases the epidermis penetration thanks to a fluidization mechanism of the SC lipid barrier. Among the colloidal formulations developed, solid lipid nanoparticle (SLN), nanostructured lipid carrier (NLC), liposome and lipid nanocapsule (LNC) are increasingly exploited due to their enhanced efficiency [START_REF] Roberts | Topical and cutaneous delivery using nanosystems[END_REF]. It has to be mentioned that adding chemical penetration enhancers (CPE) to the topical formulations facilitates the passive diffusion of the AC once into the deepest layer of the skin [START_REF] Dragicevic | Chemical Penetration Enhancers: Classification and Mode of Action[END_REF].

The structure of the main lipid-based formulations used for topical delivery of AC is schematically depicted in Table 1. Also listed are the composition of the lipid formulations, some physical parameters and the advantages and drawbacks of the corresponding nanovector. Liposomes, the oldest known lipid-based formulation systems, consist of a lipid bilayer of phospholipids delimiting an inner aqueous core. Such structure allows liposomes to encapsulate either hydrophilic or hydrophobic active pharmaceutical ingredients that can diffuse through the skin layers, after the liposomes have adsorbed on the skin surface and merged with the lipid matrix [START_REF] Sala | Lipid nanocarriers as skin drug delivery systems: Properties, mechanisms of skin interactions and medical applications[END_REF]. Contrary to liposomes, SLN, NLC and LNC possess an oily core. SLN are composed of a solid lipid core at room temperature that is stabilized by a polymer shell, whereas the NLC core is made of a mixture of liquid and solid lipids surrounded by a surfactant layer [START_REF] Schäfer-Korting | Lipid nanoparticles for improved topical application of drugs for skin diseases[END_REF]. SLN and NLC interact with the SC and create a lipid rearrangement facilitating drug penetration [START_REF] Garcês | Formulations based on solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for cutaneous use: A review[END_REF]. Only LNC possesses an oily liquid core surrounded by a rigid surfactant and phospholipid membrane [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF].

Table 1: Properties and features of NLC, SLN, LNC and liposome formulations Abbreviations: HPH: High pressure homogenizer, PIT: Phase inversion process, DL: Drug loading Among all those lipid based nanoformulations, LNC demonstrates higher performances for topical delivery of a model compound as AC. Indeed, they allow similar permeation as SLN and NLC with a reduced intradermal drug accumulation and exhibit a better stability and a higher loading efficiency [START_REF] Abdel-Mottaleb | Lipid nanocapsules for dermal application: A comparative study of lipid-based versus polymer-based nanocarriers[END_REF]. LNC are usually formed by a low energy process using the phase inversion method, which was initially developed by [START_REF] Heurtault | A Novel Phase Inversion-Based Process for the Preparation of Lipid Nanocarriers[END_REF]. It relies on the properties of the nonionic surfactant polyethylene glycol (15)-hydroxystearate, which possesses a temperature-dependent hydrophilic-lipophilic balance (HLB) allowing emulsions to switch from oil in water to water in oil [START_REF] Anton | Nano-emulsions and nanocapsules by the PIT method: An investigation on the role of the temperature cycling on the emulsion phase inversion[END_REF]. After three heating and cooling cycles, the induced phase inversion is cooled down and diluted leading to LNC with a size of 20-100 nm [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF]. The in vitro and in vivo skin penetration study of the LNC prepared with ropivacaine as an AC showed an apparent morphology change of the SC, proving the transdermal delivery potency of this type of formulations [START_REF] Zhai | Lipid nanocapsules for transdermal delivery of ropivacaine: in vitro and in vivo evaluation[END_REF]. Moreover, studies for the topical delivery of quercetin showed superior penetration capacity of LNC compared to liposomes and smart crystals formulations, and the possibility to deliver the antioxidant to the viable epidermis upon application to human skin in vivo [START_REF] Hatahet | Liposomes, lipid nanocapsules and smartCrystals®: A comparative study for an effective quercetin delivery to the skin[END_REF]. This makes LNC the most promising lipid formulation for skin penetration and delivery to the epidermis.

Despite all the advantages of LNC for topical delivery, this lipid-based formulation suffers from its exclusive reliance on polyethylene glycol (PEG) surfactant. Indeed, PEG was proved to generate an immune response [START_REF] Zhang | Anti-PEG antibodies in the clinic: Current issues and beyond PEGylation[END_REF], [START_REF] Lubich | The Mystery of Antibodies Against Polyethylene Glycol (PEG) -What do we Know?[END_REF] and accumulate in body tissues [START_REF] Rudmann | High Molecular Weight Polyethylene Glycol Cellular Distribution and PEG-associated Cytoplasmic Vacuolation Is Molecular Weight Dependent and Does Not Require Conjugation to Proteins[END_REF], [START_REF] Viegas | 6 -Poly(oxazolines)[END_REF]. This represents a major drawback in the development of LNC for many applications ranging from nanomedicine to cosmetics. The clinical awareness on PEG overuse makes it particularly important to design new biocompatible surfactants as an alternative to the controversial PEG. In that context, we aimed at developing LNC devoid of PEG.

We proposed to use poly(2-R-2-oxazoline) (POx), a class of polymers with a peptidomimetic structure belonging to the polyamide family, as a surfactant for LNC formation. Indeed POx offers interesting properties such as its cytocompatibility and hemocompatibility [START_REF] Lorson | Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update[END_REF], in addition, it possesses a stealth behavior [START_REF] Yang | Time Resolved UV-Vis Absorption Spectra of Quercetin Reacting with Various Concentrations of Sodium Hydroxide[END_REF] and its HLB is easily tunable through synthesis [START_REF] Guillerm | How to Modulate the Chemical Structure of Polyoxazolines by Appropriate Functionalization[END_REF]. It also holds great promise as a platform polymer for drug delivery [START_REF] Moreadith | Clinical development of a poly(2oxazoline) (POZ) polymer therapeutic for the treatment of Parkinson's disease -Proof of concept of POZ as a versatile polymer platform for drug development in multiple therapeutic indications[END_REF]. As recently reviewed by Luxenhofer [START_REF] Lorson | Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update[END_REF], POx-based formulations with solid dispersions (Paclitaxel, curcumin, Doxorubicin) and drug formulations have already been described as well as theranostic drug delivery systems incorporating proteins and gene complexes and using partially hydrolyzed POx [START_REF] Dargaville | Poly(2-oxazoline) Hydrogels: State-of-the-Art and Emerging Applications[END_REF]. Recently, we also demonstrated the ability of POx to formulate stable mixed-micelles loaded with quercetin while maintaining its antioxidant activity [START_REF] Simon | Polyoxazolines based mixed micelles as PEG free formulations for an effective quercetin antioxidant topical delivery[END_REF].

In the present work, we evaluated the potency of POx to stabilize lipid nanocapsules, called LNC POx, for topical delivery. A new process allowing stable LNC POx to be obtained was developed. LNC POx was loaded with a natural flavonoid antioxidant, the quercetin (Q), which was previously studied for topical delivery in various formulations [START_REF] Nagula | Recent advances in topical delivery of flavonoids: A review[END_REF]. The model compound was chosen due to its readily assessable, reported antioxidant activities [START_REF] Hatahet | Quercetin topical application, from conventional dosage forms to nanodosage forms[END_REF] and used to prove LNC POx efficacy as a topical platform to protect and maintain bioactivity. The physico-chemical and mechanical properties of LNC POx were evaluated, and their effect on the cell viability of mice fibroblasts was assessed. The antioxidant activity of quercetin loaded in LNC POx was evaluated. Finally, a comparison study of the radical scavenging capacity of Q-LNC POx and Q-LNC PEG on mice fibroblasts and human keratinocytes was performed to evaluate the ability of Q-LNC POx to protect cells from an excess of radical species.

Materials and methods

Materials

Quercetin (95% HPLC), DPPH (2.2-diphenyl-1-picrylhydrazyl), phenazine methosulfate (PMS), tert-butyl hydroperoxide solution (TBHP) 70% in water, acetonitrile, diethyl ether, methanol, acetone, phosphoric acid and 2,7-dichlorofluorescein diacetate (DCFDA) were purchased from Sigma Aldrich (Germany). Tween 80 ® (polysorbate 80) was purchased from BASF (Germany) and 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was from Promega (USA). Lipophilic Labrafac ® (caprylic acid triglycerides) was brought from Gattefossé (Saint-Priest, France) and Lipoid S75 ® (fatfree soybean phospholipids with 70% phosphatidylcholine) was kindly provided by Lipoid (Ludwigshafen, Germany). Sodium chloride (NaCl) was bought from VWR. MilliQ water was obtained from Milli-Q Gradient A10 (Merck Millipore, Germany) apparatus. Chloroform (for HPLC, stabilized with ethanol) was bought from Carlo-Erba (Carlo Erba Reagent, Spain). All the reagents were used without further purification. Spectra/Por 6 dialysis membranes pre-wetted RC tubing with 0.5-1 kDa MWCO were purchased from Spectrum Labs (USA).

Methods

LNC POx formulation

The lipid nanocapsule (LNC POx) were obtained by mixing Labrafac®, Lipoid® S75, Milli-Q water, NaCl and amphiphilic polyoxazoline (POx, C 16 POx 15 ). C 16 POx 15 was synthesized as described in [START_REF] Simon | Polyoxazolines based mixed micelles as PEG free formulations for an effective quercetin antioxidant topical delivery[END_REF].

Three heating and cooling cycles (85 -30°C) of the mixture were performed under magnetic stirring. The solution was placed in an ice bath and sonicated for 4 minutes at 30% amplitude with a Digital Sonifier 250 sonication probe (Branson Ultrasonics Corporation, USA) using a Microtip 64-247A. Then quercetin was added as a powder to the solution and additional heating and cooling cycle was carried out. A last heating to 80 °C was then performed before addition of 2.5 mL of MilliQ water at 4 °C. The quercetin loaded LNC (Q-LNC POx) obtained was cooled down under magnetic stirring. The unloaded quercetin was separated by filtration through 0.2 µm syringe filter (Whatman). Blank-LNC (B-LNC POx) was prepared by the same method, except that quercetin was removed from the process.

The composition was optimized after doing a ternary phase diagram (POx, Labrafac ® oil, water) for which the concentration of Lipoid ® S75 and NaCl were fixed at 1.5% and 3% (w/w) respectively. The composition (w/w) containing 20% of POx, 15% Labrafac ® , 65% water (w/w) and 3% of quercetin was selected for its stability and high drug loading (section 2.6).

All the analyses described bellow were performed on freshly prepared LNC POx preparations. The LNC stabilized by PEG, called LNC PEG, were prepared according to the protocol for quercetin LNC 20 nm developed by [START_REF] Hatahet | Dermal quercetin lipid nanocapsules: Influence of the formulation on antioxidant activity and cellular protection against hydrogen peroxide[END_REF].

Dynamic light scattering (DLS) and electrophoretic mobility measurement

Zetasizer NanoZS apparatus (Malvern Instruments, UK) equipped with a He-Ne laser (632.8 nm) was used to evaluate the hydrodynamic diameter and polydispersity index (PDI) of LNC POx (20 µL in 1980 µL of MilliQ water) at 20°C at scattering angle of 173°. Zeta potential was measured on 1000 µL of diluted LNC solutions in disposable capillary cell (Malvern Instruments, UK). All the results were average of three independent measurements.

Stability of LNC

After preparation, the stability study was conducted at 4, 25 and 37°C for the B-LNC POx and at 4°C for the Q-LNC. The stability of the LNCs was assessed by measurements of the hydrodynamic diameter and PDI (see section 2.2). Side studies of LNC stability were conducted using centrifugation at 15000 rpm for 15 minutes and freezing at -22°C in a freezer. The hydrodynamic diameter and PDI were measured at described in section 2.2.

The stability of Q-LNC POx at 25°C was also evaluated by studying the in vitro release of quercetin diffusing through a nitrocellulose dialysis membrane (12 -14 MWFC Spectra/Pore dialysis membrane from Spectrum laboratories, INC USA). The receptor medium was composed of 40 mL PBS at pH 7.4 with 2 wt% Tween ® 80.

The quercetin concentration released after 1, 2, 3, 4, 5, 6 and 7 hours was measured by HPLC (section 2.7).

Transmission electron microscopy (TEM)

Transmission electron microscopy was performed on a TEM Jeol 1400 PLUS apparatus (Jeol. Ltbd, Tokyo, Japan) equipped with a Jeol 2K/2K camera. The samples were preliminarily diluted 1000 times before deposition on grids (type Cu formvar carbon) and were negatively colored with an aqueous uranyl acetate solution at 1.5 wt% and pH 5.

Atomic force microscopy (AFM)

Atomic force microscopy was performed on Nanoman (Bruker Instrument) and monitored by Nanoscope V software. The LNC samples were diluted 100 times in MilliQ water and 5 µL of the solution was deposited on a silicon wafer. The tapping mode and harmonix mode were used after drying of the drop at 37°C. For the tapping mode, PPP NCL tips (Nanosensors) were used at the resonance frequency of 157 kHz. Various amplitude setpoints were tested (from 500 to 250 mV) to modify the resulting force applied onto the sample. The harmonix mode was carried out using HMX-S tips (Bruker Instruments) by applying a force of 10 to 20 nN with a vertical resonance frequency of 48 kHz and horizontal of 791 kHz. No morphology modification of the LNC was observed.

Encapsulation efficacy (EE) and drug loading capacity (DL)

Drug loading (DL) and encapsulation efficacy (EE) were respectively calculated using the following equations:

(Eq.1)

Where total mass represents the mass of POx, Labrafac and quercetin.

(Eq.2)

The quercetin loaded in Q-LNC POx was quantified by HPLC (section 2.7) after filtration of the solution through a 0.2 µm filter (Whatman) to remove the residual unloaded quercetin.

HPLC analysis

High Pressure Liquid Chromatography (HPLC) analysis of quercetin was performed on LC6-2012HT apparatus (Shimadzu, Kyoto, Japan) using a Prontosil C18 column (120-5-C18 H5.0 µm, 250x4.0 mm). The detection was achieved using a UV-vis detector (Shimadzu, Kyoto, Japan) at 368 nm [START_REF] Yang | Time Resolved UV-Vis Absorption Spectra of Quercetin Reacting with Various Concentrations of Sodium Hydroxide[END_REF].

Acetonitrile/phosphoric acid at 0.2 wt% and pH=1.9 (40/60 %v) was used as mobile phase. The calibration curve was performed with solutions of quercetin in methanol from 1 to 100 µg/mL with a good linearity (r² = 0.9999).

The flow rate was 1 mL.min -1 . To determine the concentration of quercetin released to the aqueous medium receptor (see stability study of LNC, section 2.3) a calibration curve realized in PBS buffer at pH 7.4 and Tween ® 80 (2% wt) from 0.1 to 4 µg/mL (r² = 0.9996) was used.

In vitro antioxidant activity

The antioxidant activity of Q-LNC POx was determined from the reactivity of quercetin towards the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH°) [START_REF] Blois | Antioxidant Determinations by the Use of a Stable Free Radical[END_REF]. It was calculated from the decrease of the DPPH° radical absorbance at 517 nm using the following equation (Eq.3):

(Eq.3)
where Absorbance of control corresponds to the absorbance of a 100 µM DPPH° solution. Note that the concentration of quercetin loaded in Q-LNC POx (2.5, 5, 7.5, 10, 12.5, 15 µM) was lower than that of DPPH°.

Cell culture

Mice fibroblasts cell lines (NIH3T3) were purchased from American Type Cell Culture organization (USA).

Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Life Technologies), 1% L-glutamine and 1% penicillin/streptomycin equivalent to a final concentration of 2 nM for glutamine, 100 U.mL -1 for penicillin and 100 µg.mL -1 for streptomycin. They were incubated at 37°C in humidified 5% CO 2 atmosphere.

Normal Human Epidermal Keratinocytes (NHEK) were purchased from Promocell (C-123003) and cultured at 37°C in keratinocyte basal medium (C-20216 Promocell) supplemented with the supplement mix keratinocyte from Promocell (C-39016) under a humidified atmosphere containing 5% CO 2 . Cells were seeded to reach approximately 80% confluency at the time of treatment or irradiation. UVB irradiation was performed using a BS-02 UV irradiation chamber (Dr. Gröbel UV-Elektronik GmbH, Ettlingen, Germany). The lamp emits UVB irradiation with a peak at 311-312 nm and partially excludes shorter wavelengths, such as UVA.

2.10.

Cell viability NIH3T3 cells were seeded at 20,000 cells/cm² in 96 well plate and incubated for 24 hours at 37°C, 5% CO 2 to allow cell adhesion. Cells were treated or not (control) with quercetin, POx (B-LNC POx, Q-LNC POx) and PEG (B-LNC PEG, Q-LNC PEG) preparation. After 24 hours of exposure, a CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, USA) was used to evaluate the cell viability following the manufacturer's instructions. The absorbance was recorded at 490 nm using Multiskan TM GO Microplate Spectrophotometer (Thermo Scientific TM , Waltham, Massachusetts, USA). Absorbance of the basal media (with no cells and no treatment) was subtracted to the recorded absorbance for all conditions. Values of treated cells were normalized to non-treated cells (100% intensity).

Antioxidant effect of quercetin on UVB irradiated NHEK cells

NHEK cells were seeded at 30,000 cells per well in a 24-well plate. They were allowed to settle for 24 hours before treatment for 2 more hours with 5 μg/ml of the crude quercetin, Q-LNC POx or Q-LNC PEG preparations. Cells were then incubated with 20 µM DCFDA in a serum free medium for 30 minutes before a 100 mJ/cm 2 UVB irradiation was triggered. The cells were harvested and cellular fluorescence was assessed by flow cytometry (Canto, Becton Dickinson) directly after irradiation [START_REF] Masaki | Reactive Oxygen Species in HaCaT Keratinocytes After UVB Irradiation Are Triggered by Intracellular Ca2+ Levels[END_REF]. Data were processed using FlowJo software. Values of treated cells were normalized to non-treated cells (representing the cells with 100% ROS intensity).

Statistical analysis

The statistical analysis of the data resulting from the cell viability and the antioxidant effect on cells was conducted with Origin Pro software 8.1 (OriginLab, USA). A one-sample t-test with equal variance was performed to compare cell viability and antioxidant effect with formulations to viability of untreated cells (100%). A two-sample t-test with unequal variance was carried out to compare cell viability of formulations two by two. The P value reflects the significance with * =P <0.05, ** =P <0.01 and *** =P < 0.001.

Results and discussion

LNC formulation and characterization

The amphiphilic nonionic POx polymer (C 16 POx 15 ) used in this study is highly soluble in water and has a molecular weight of 1520 g/mol. It possesses a low viscosity (e.g. PEtOx 50 kDa [η] = 0.23 dL/g) and a high stability towards degradation [START_REF] Lorson | Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update[END_REF]. Its solubility in water remains constant with temperature (no LCST characteristic for this poly(-2-methyl-2-oxazoline) POx derivative) [START_REF] Glassner | Poly(2-oxazoline)s: A comprehensive overview of polymer structures and their physical properties[END_REF]), impeding the use of the phase inversion process developed by Heurtault et al [START_REF] Heurtault | A Novel Phase Inversion-Based Process for the Preparation of Lipid Nanocarriers[END_REF] to produce LNC. As a consequence, the protocol to form POx based LNC was redefined as represented in Scheme 1. Heating and cooling cycles were first performed to solubilize and homogenize the system by melting and mixing the solid lipids (Lipoid ® S75) with the liquid lipids (Labrafac ® ). Then, a short sonication step supplied the energy necessary to disperse the preparation [START_REF] Cohen | Interactions of engineered nanomaterials in physiological media and implications for in vitro dosimetry[END_REF] and reduce its size from 300 nm to 30 nm. Quercetin was added to the mixture to produce antioxidant LNC after this stage, thus avoiding its degradation from ultrasonic energy [START_REF] Qiao | Sonochemical effects on 14 flavonoids common in citrus: relation to stability[END_REF]. One of the advantages of this process is thus to preserve active compound (AC) sensitive to many temperature cycles and ultrasound treatments by post-insertion into the LNC. One last heating and cooling cycle was performed to favor quercetin loading. Then, the mixture was heated at 85°C and cold water (4°C) was rapidly introduced to anchor the system and generate LNC stabilized by polyoxazoline (LNC POx).

Using this process, a pre-formulation work was conducted to determine the suitable composition leading to the most stable nanosized LNC, with the lowest amount of POx and resulting in the highest drug loading. The POx concentration range explored varied from 10 to 20 mass percent, the Labrafac ® oily phase from 10 to 25 mass percent and water from 55 to 80 mass percent, whereas the phospholipids Lipoid ® S75 and NaCl were respectively fixed at 1.5 mass percent and 3 mass percent. The stability was evaluated from DLS measurement (data not shown).

Scheme 1 : Process of lipid nanocapsules formulation

As a result, POx was introduced at 20 mass percent, Labrafac ® oil at 15 mass percent and water 65 mass percent.

Other components such as NaCl and Lipoid ® S75 were added at 3 mass percent and 1.5 mass percent. The physicochemical and morphological properties of blank LNC (B-LNC POx) and LNC loaded with quercetin (Q-LNC POx) were fully characterized; their main properties are gathered in Table 2. The hydrodynamic diameter of B-LNC was measured by DLS at 30.5 ± 1.5 nm with a polydispersity index (PDI) of 0.16. Loading with quercetin (Q-LNC POx) resulted in almost similar hydrodynamic diameter (26.4 ± 0.6 nm with a PDI of 0.18 ± 0.01, Table 2). The zeta potential for both LNC was close to neutral, indicating that POx chains at the surface of the LNC stabilize the formulation by steric repulsions. LNC PEG, free and loaded with quercetin were prepared using Hatahet et al. protocol (Hatahet et al., 2017) for comparative purposes. The hydrodynamic diameter of B-LNC PEG and Q-LNC PEG were measured at 34.7 nm and 33.5 nm with a PDI of 0.03 and 0.05, respectively.

As for LNC POx, the presence of quercetin did not change the LNC size.

Table 2 : Characteristics of LNC POx

The morphology of LNC was observed by TEM after coloration with uranyl acetate solution. Well-defined spherical particles of similar size (< 70 nm) were observed for both B-LNC POx and Q-LNC POx. A typical image obtained with Q-LNC POx is presented on Figure S1. The spherical shape was confirmed by AFM (Figure 1). It also allowed determination of the nanoparticle stiffness using the tapping mode to evaluate the contact force and more especially the repulsive force of the nanoparticle by decreasing the amplitude (setpoint).

The topography of the LNC POx showed a positive phase indicating a repulsive force and mechanical properties.

The latter were quantified using the harmoniX mode for determination of the indentation modulus (DMT). The DMT profile of Q-LNC POx was measured at 1-2 GPa (Figure S2) and the associated topography profiles resulted in no permanent morphology deformation (Figures S3 andS4) up to a constraint of about 10-20 nN.

This evidences the mechanical properties of the LNC POx composed of a rigid capsule embedding on oily core just like the ones designed by Heurtault et al. [START_REF] Heurtault | A Novel Phase Inversion-Based Process for the Preparation of Lipid Nanocarriers[END_REF] for which a contact force > 10nN was applied.

Figure 1 : LNC POx topography from AFM measurement

It is reasonable to assume that the stiffness of the POx-based nanoparticles results from the rigid capsule made of phospholipids from the Lipoid® S75 and POx surrounding the oily core of Labrafac® as for the LNC produced with polyethylene glycol (15)-hydroxystearate as a surfactant [START_REF] Heurtault | A Novel Phase Inversion-Based Process for the Preparation of Lipid Nanocarriers[END_REF]. For LNC POx, the amphiphilic non ionic POx stabilized the hydrophobic part of the formulation into spherical nano-objects covered with the POx chains as previously described for similar amphiphilic POx (Korchia et al., 2015). An illustrative representation of the assembly is depicted in Scheme 2.

Scheme 2 : Inner assembly of LNC POx LNC formulations are well known for their high stability to dilution, centrifugation and over the long term [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF]. These properties were also investigated for the newly developed POx based LNC. The LNC POx stability to dilution by 1000 (data not shown) and to high speed centrifugation at 15 000 rpm for 15 minutes at 25°C was evaluated. The size and the PDI remained the same as reported in Table 2. Interestingly, after 5 days at -22°C the LNC POx hydrodynamic diameter did not change. The long term stability of B-LNC

POx was assessed at 4, 25 and 37°C by measuring the hydrodynamic diameter and PDI over a period of one month (Figure 2). At each temperature, the size of the particles slightly increased in the first 7 days (from 30 to 38 nm at 4°C, 42 nm at 25°C and 49 nm at 37°C), after 7 days the PDI remained almost identical at a low value of ~ 0.1, indicating stable monodisperse preparations. In any case, after a month, the size of the B-LNC POx remained well below 100 nm, which is fully compatible for topical delivery applications. It is noteworthy to mention that after 2 months at 25°C, the size of the B-LNC POx had only marginally increased; it reached 57 nm whereas it was 52 nm one month before (PDI < 0.1). A cell viability test was first conducted on mice fibroblasts (NIH3T3) to ensure that the platform was non toxic by itself. B-LNC POx and B-LNC PEG were both tested from 1 to 50 µg/mL to evaluate the IC 50 (cell viability)

(Figure S5). The IC 50 (cell viability) value was 43± 8 µg/mL for B-LNC PEG and 45± 8 µg/mL for B-LNC POx and the statistical analysis did not reveal any difference in term of cell viability. Thus, the cell viability in presence of B-LNC POx seems to be well-suited for topical delivery of AC as it is similar to that of B-LNC PEG.

Quercetin loaded LNC and scavenging capacity

The LNC POx was then loaded with an AC model to evaluate the encapsulation capacity and protection of bioactivity. The natural antioxidant quercetin was chosen for its hydrophobic nature, poor solubility and sensitivity to oxidation. The post-insertion method successfully led to a high encapsulation efficiency (EE) of 93 ± 1% corresponding to a drug loading (DL) of 7.9 ± 0.1%. Addition of ethanol (from 100 µL to 500µL) had no impact on DL and rather tended to destabilize the LNC by creating two phases, contrary to Q-LNC PEG synthesized by Hatahet et al. adapted from Heurtault protocol (Hatahet et al., 2017). Compared to others LNC [START_REF] Barras | Formulation and characterization of polyphenol-loaded lipid nanocapsules[END_REF] and NLC [START_REF] Chen-Yu | Development of a Quercetin-loaded nanostructured lipid carrier formulation for topical delivery[END_REF][START_REF] Pivetta | Topical formulation of quercetin encapsulated in natural lipid nanocarriers: Evaluation of biological properties and phototoxic effect[END_REF] developed to encapsulate quercetin, it has to be mentioned that Q-LNC POx exhibits the highest drug loading, which exceed that obtained with Q-LNC PEG [START_REF] Hatahet | Dermal quercetin lipid nanocapsules: Influence of the formulation on antioxidant activity and cellular protection against hydrogen peroxide[END_REF] using Cremophor and ethanol to reach a drug loading of 2.6 ± 0.1% with an encapsulation efficiency of 96.4 ± 1.2%. Moreover, Q-LNC POx preparation also improved quercetin loading compared to the POx stabilized mixed-micelles we recently developed [START_REF] Simon | Polyoxazolines based mixed micelles as PEG free formulations for an effective quercetin antioxidant topical delivery[END_REF] (from 3.6 to 7.9 %).

The stability of Q-LNC POx with time was investigated at 4 °C, a temperature relevant in the presence of quercetin that is sensitive to thermal degradation [START_REF] Wang | Degradation kinetics of fisetin and quercetin in solutions affected by medium pH, temperature and co-existing proteins[END_REF]. As for the blank LNC POx, the size of quercetin loaded nanocapsules slightly evolved over one month, from 26 nm to 40 nm with a PDI staying under 0.2 (Figure 3). As for B-LNC POx, Q-LNC POx was also stable to dilution and centrifugation (Table 2). The stability study of Q-LNC POx was completed by evaluating the quercetin leakage from the nanocapsules in aqueous media (Figure S6). After 7 hours, only 1.5% of quercetin was released from Q-LNC POx and it reached a low value of 5% after 48 hours. This result confirmed that quercetin is localized within the core of the LNC.

Interestingly, the LNC POx formulation reduced by four times the quercetin leakage in aqueous media compared to the mixed-micelles formulation [START_REF] Simon | Polyoxazolines based mixed micelles as PEG free formulations for an effective quercetin antioxidant topical delivery[END_REF]. The Q-LNC POx also seemed to better retain quercetin than Q-LNC PEG that showed a leakage of quercetin of almost 15% after 24 hours using the same experimental conditions [START_REF] Hatahet | Dermal quercetin lipid nanocapsules: Influence of the formulation on antioxidant activity and cellular protection against hydrogen peroxide[END_REF]. This result might be due to the rigid capsule surrounding the oily core acting as a sealed reservoir and ensuring a high stability for the particles.

Antioxidant tests were performed in order to evaluate the ability of the LNC POx platform to protect and maintain the bioactivity of the encapsulated compound. The radical scavenging capacity was assessed in vitro using a chemical assay (DPPH). It was also evaluated on mice fibroblasts and human keratinocytes by generating reactive oxygen species (ROS) respectively using organic peroxide and UVB irradiation.

The quercetin antioxidant is able to react with the 2,2-diphenyl-1-picrylhydrazyl (DPPH°) molecule by its free radical on the hydrazine position leading to DPPHH. The radical scavenging capacity of quercetin encapsulated in the Q-LNC POx was assessed by looking at its inhibitive interaction with DPPH°. The IC 50 (DPPH° inhibition) value deduced from the DPPH° decrease shows higher antioxidant activity for Q-LNC POx (IC 50 (DPPH° inhibition) = 2 µg/mL) than for crude quercetin, which exhibited a twice higher IC 50 (Figure S7). The higher antioxidant activity of Q-LNC POx can be due to a better protection of the antioxidant to oxidation, a greater conformation or organization in the nanocapsule compared to crude quercetin.

Quercetin loaded antioxidant activity

To go further in the evaluation of the antioxidant effect of Q-LNC POx with respect to classical PEG based LNC, both POx and PEG based Q-LNC were loaded at the same quercetin concentration and tested on mice fibroblasts and human keratinocytes. A cell viability test was performed on NIH3T3 to determine the maximum non toxic quercetin concentration to be used for the antioxidant tests. The IC 50 (cell viability) values were determined for crude quercetin (17.5 µg/mL), Q-LNC POx (23 µg/mL) and Q-LNC PEG (16 µg/mL) (Figure S8). The cell viability of blank LNCs was also evaluated B-LNC POx (43 µg/mL) and B-LNC PEG (45 µg/mL).

As for the uncharged POx and PEG-based LNC, the IC 50 (cell viability) value is not affected by the nature of the stabilizing polymer.

To perform the antioxidant study, the concentration of quercetin was selected such as to avoid formulation toxicity from the cell viability results; it was set at 5 µg/mL. The radical scavenging capacity of Q-LNC POx was first tested on NIH3T3 and compared to Q-LNC PEG and crude quercetin (Figure 4). Cells were treated with the formulations for 24 hours, then an oxidative stress was induced by tert-butyl hydroperoxide (TBHP).

The quantity of ROS generated was determined by fluorescence spectroscopy using the DCFDA probe. The results were normalized to untreated cells with TBHP exposure (non treated). The crude quercetin was able to reduce excess ROS generation to 69 ± 6% (P < 0.01) and similarly Q-LNC POx decreased surplus ROS to 70 ± 6% (P < 0.01). Q-LNC PEG was less able to counter ROS generation with a reduction to 81 ± 9% (P < 0.01).

The antioxidant activity of quercetin once loaded in LNC POx was thus maintained and even slightly enhanced compared to Q-LNC PEG. B-LNC POx was also tested and the cells resisted to the peroxides generated reflecting the innocuousness of POx upon oxidation. Another antioxidant test was performed on human keratinocytes with UV irradiation as a source of ROS.

Therefore, the antioxidant effect observed on mice fibroblasts can be transposed to human cells with ROS simulated from an environmental factor.

We first evaluated the scavenging activity of ROS naturally generated by the cells (Table 3, "No UV irradiation"). The fluorescence intensity was normalized to untreated cells (no formulation and no UV irradiation). UV irradiation was then carried out to evaluate the antioxidant capacity on overproduction of ROS (Table 3, "UV irradiation" column). The results were normalized to untreated cells. As shown in Table 3, the crude quercetin significantly reduced the ROS naturally produced by the cells to 59 ± 19% (P < 0.01) which is not the case for Q-LNC POx and Q-LNC PEG maintaining a quantity of ROS of 91 ± 16% and 94 ± 13%

respectively. On the contrary, once the cells irradiated, the crude quercetin was the one reducing most of the ROS over generated to 65 ± 6% (P < 0.01) whereas Q-LNC POx was able to reduce ROS at 83 ± 4% (P < 0.01) and Q-LNC PEG at 91 ± 15%. The statistical analysis revealed that crude quercetin and Q-LNC POx significantly decreased the ROS over generated. Therefore, Q-LNC POx was able to maintain the antioxidant activity of quercetin alike Q-LNC PEG. It has to be noticed that, even if the crude quercetin reduced most of the ROS, its too powerful activity could scavenge the ROS essential for cell survival.

Conclusion

This work demonstrates the ability of amphiphilic non ionic polyoxazolines (POx) to act as a surfactant to form lipid based nanoformulation suitable for topical delivery of quercetin. The optimized nanoformulation allows to shape well-defined monodisperse spherical lipid nanocapsules (LNC) of 30 nm diameter size. They are composed of a Labrafac ® oily core stabilized by a solid phospholipid and POx surfactant shell, whose stiffness ensured a high stability over time, centrifugation, freezing and dilution. Most importantly, LNC POx are free from the PEG-dependant inversion process universally used to date to design LNC. The process developed here associates a sonication step to heating/cooling cycles allowing loading the natural antioxidant quercetin at a high drug loading of 7.9 ± 0.1% without inducing any morphological change in the particles nor altering their stability. Interestingly, the LNC POx leads to a higher encapsulation and a slightly better radical scavenging capacity on mice fibroblasts and human keratinocytes than LNC PEG. This evidences the crucial role amphiphilic polyoxazolines might take in the future as PEG free topical delivery platform for topical and intravenous administration for applications in nanomedecine, dermatology and cosmetics. Lipid bilayer enclosing an aqueous core [START_REF] Zhai | Lipid nanocapsules for transdermal delivery of ropivacaine: in vitro and in vivo evaluation[END_REF] Liquid lipid core surrounded by a solid membrane from lecithin and surfactant [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF] Size 40-1000 nm [START_REF] Sala | Lipid nanocarriers as skin drug delivery systems: Properties, mechanisms of skin interactions and medical applications[END_REF] 40-1000 nm [START_REF] Sala | Lipid nanocarriers as skin drug delivery systems: Properties, mechanisms of skin interactions and medical applications[END_REF] 20-3000 nm [START_REF] Sala | Lipid nanocarriers as skin drug delivery systems: Properties, mechanisms of skin interactions and medical applications[END_REF] 20-100 nm [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF]

Type of lipid formulation

Preparation method

Hot or cold HPH [START_REF] Schäfer-Korting | Lipid nanoparticles for improved topical application of drugs for skin diseases[END_REF]

Disavantages

High degree of order leading to low DL [START_REF] Zhai | Lipid nanocapsules for transdermal delivery of ropivacaine: in vitro and in vivo evaluation[END_REF] Lower skin permeation than LNC [START_REF] Zhai | Lipid nanocapsules for transdermal delivery of ropivacaine: in vitro and in vivo evaluation[END_REF] Low capacity to encapsulate lipophilic drug; presence of traces of organic solvent; unstable in biological fluid [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF] Low DL of hydrophilic AC [START_REF] Huynh | Lipid nanocapsules: A new platform for nanomedicine[END_REF] Table 1 Scheme 1

2. 11 .

 11 Antioxidant effect of quercetin on TBHP treated NIH3T3 cellsNIH3T3 cells were seeded at 20,000 cells/cm² in a 96 well black plate with clear bottom (Corning ® Massachusetts, USA) and incubated 24 hours at 37°C, 5% CO 2 to allow cell adhesion. Then B-LNC (POx and PEG), crude quercetin, or Q-LNC (POx and PEG) possessing an equivalent quercetin concentration of 5 µg/mL were added and incubated for another 24 hours in supplemented DMEM. Cells were washed twice with PBS before addition of 200 µL of 2,7-dichlorofluorescein diacetate (DCFDA) reagent (20 µM in DMEM without phenol red). The serum free medium used provides reliable data by avoiding deacetylation of the DCFDA into non fluorescent compound that could later be oxidized by reactive oxygen species (ROS) into dichlorofluorescein (DCF). After 30 minutes of incubation at 37°C, 5% CO 2 , cells were rinsed with PBS and placed in 200 µL DMEM without phenol red. A solution of tert-butyl hydroperoxide (TBHP) (500 µM in PBS) was then added and the cells were incubated for additional 30 minutes. The fluorescence signal of DCF produced by the reaction of DCFDA reagent with ROS was then measured (λexc 485 nm, λem 535 nm) using a Tristar LB941 Spectrofluorimeter (Berthold Technologies, Germany). Values of treated cells were normalized to nontreated cells (100% intensity).

Figure 2 :

 2 Figure 2 : Hydrodynamic diameter (column) and PDI (symbol) of B-LNC POx at 4, 25 and 37 °C over time (n=3)

Figure 3 :

 3 Figure 3 : Hydrodynamic diameter (column) and PDI (symbol) of Q-LNC POx at 4 °C with time (n=3)

Figure 4 :

 4 Figure 4 : ROS relative intensity for crude quercetin, Q-LNC POx and Q-LNC PEG at a quercetin concentration of 5 µg/mL (n=3) on NIH3T3 cells. Fluorescence intensity was normalized to TBHP treated cells without formulation. A one sample t-test relative to untreated cells was performed (*: P < 0.05, **: P < 0.01 and ***: P < 0.001)

Table 3 :

 3 ROS relative intensity for crude quercetin, Q-LNC POx and Q-LNC PEG at a quercetin concentration

of 5 µg/mL (n=3) on human keratinocytes. Fluorescence intensity was normalized to irradiated cells (not treated with formulation). A one sample t-test relative to untreated cells was performed (*: P < 0.05, **: P < 0.01 and ***: P < 0.001).

Table 2

 2

Acknowledgements

The authors thank Michel Ramonda (CTM, Univ Montpellier, France) for help on AFM measurements, Christophe Dorandeu (ICGM, Univ Montpellier, France) for assistance on HPLC analysis and Thomas Cacciaguerra (ICGM, Univ Montpellier, France) for assistance on TEM experiments. We acknowledge the imaging facility MRI, member of the national infrastructure France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»)" for Cytometry Analysis.

Graphical abstract:

*Graphical Abstract (for review)

Declaration of interests

x The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The shape of the LNC was observed with the topography profile (Figure S3), indicating that the nanoparticles remained spherical with no deformation. The modulus profile (Figure S4) showed that the constraint applied on the LNC was about 1-2 GPa.


Elodie Perdreau
email: elodie.perdreau@chu-lyon.fr

Zakaria Jalal

Richard D Walton

Jérôme Naulin

Julie Magat

Bruno Quesson

Hubert Cochet

Olivier Bernus

Jean-Benoît Thambo

Assessment of the healing process after percutaneous implantation of a cardiovascular device: a systematic review

Keywords:

The healing process, occurring after intra-cardiac and intra-vascular device implantation, starts with fibrin condensation and attraction of inflammatory cells, followed by the formation of fibrous tissue that slowly covers the device. The duration of this process is variable and may be incomplete, which can lead to thrombus formation, dislodgement of the device or stenosis. To better understand this process and the neotissue formation, animal models were developed: small (rats and rabbits) and large (sheep, pigs, dogs and baboons) animal models for intra-vascular device implantation; sheep and pigs for intracardiac device implantation. After intra-vascular and intra-cardiac device implantation in these animal models, in vitro techniques, i.e. histology, which is the gold standard and scanning electron microscopy, were used to assess the 1,2,3 1,2,3,

device coverage, characterize the cell constitution and detect complications such as thrombosis. In humans, optical coherence tomography and intravascular ultrasounds are both invasive modalities used after stent implantation to assess the structure of the vessels, atheroma plaque and complications. Noninvasive techniques (computed tomography and magnetic resonance imaging) are in development in humans and animal models for tissue characterization (fibrosis), device remodeling evaluation and device implantation complications (thrombosis and stenosis). This review aims to [START_REF] Dahan | Dynamic autologous reendothelialization of small-caliber arterial extracellular matrix: a preclinical large animal study[END_REF] present the experimental models used to study this process on cardiac devices; [START_REF] Liu | Fluid upstream shear stress of rabbit aortic stenosis inhibits neointimal hyperplasia by promoting endothelization after balloon injury[END_REF] focus on the in vitro techniques and invasive modalities used currently in humans for intra-vascular and intra-cardiac devices and (3) assess the future developments of non-invasive techniques in animal models and humans for intra-cardiac devices.

Introduction

The healing process is an important stage that follows intra-cardiac and intravascular device implantation and leads to the formation of a neotissue covering the device [START_REF] Dahan | Dynamic autologous reendothelialization of small-caliber arterial extracellular matrix: a preclinical large animal study[END_REF]. It usually starts with fibrin condensation and an intense inflammatory reaction, involving cells that migrate from neighboring regions (host fibroblast-like cells, macrophages, lymphocytes, endothelial mature cells), extra-cellular matrix and thrombotic material. Circulating endothelial progenitors are also attracted, as well as angiogenic growth factors. It is followed by the formation of a poorly vascularized granulating tissue, slowly covering the device and evolving towards fibrosis, predominantly made of collagen and small vessels [START_REF] Liu | Fluid upstream shear stress of rabbit aortic stenosis inhibits neointimal hyperplasia by promoting endothelization after balloon injury[END_REF][START_REF] Foth | Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion devices[END_REF][START_REF] Sigler | Biocompatibility of septal defect closure devices[END_REF]. Chronic inflammatory reactions with multinucleated cells and lymphocyte infiltration may also be observed [START_REF] Sigler | Biocompatibility of septal defect closure devices[END_REF].

Ideally, device coverage should occur over a short time-course, be uniform and complete after implantation [START_REF] Dahan | Dynamic autologous reendothelialization of small-caliber arterial extracellular matrix: a preclinical large animal study[END_REF][START_REF] Pislaru | Magnetic forces enable rapid endothelialization of synthetic vascular grafts[END_REF]. The duration of this process is highly variable across different studies, depending on the environment (in vivo or in vitro) and device, its location in the heart and its material composition [START_REF] Liu | Fluid upstream shear stress of rabbit aortic stenosis inhibits neointimal hyperplasia by promoting endothelization after balloon injury[END_REF][START_REF] Syedain | 6-month aortic valve implantation of an off-the-shelf tissue-engineered valve in sheep[END_REF]. In animal models, the healing process occurs between 28 and 90 days [START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF]8]; in humans, between 6 and 18 months [START_REF] Virmani | Drug eluting stents: are human and animal studies comparable?[END_REF]. However, this process may be much longer, as some devices still remain uncovered 2 years after implantation, such as firstgeneration sirolimus-eluting stents [START_REF] Takano | Long-term follow-up evaluation after sirolimus-eluting stent implantation by optical coherence tomography[END_REF], atrio-septal device defect occlusion devices or left atrial appendage occluders [START_REF] Chen | Incomplete endothelialization and late dislocation after implantation of an Amplatzer septal occluder device[END_REF][START_REF] Nguyen | Endocarditis and incomplete endothelialization 12 years after Amplatzer septal occluder deployment[END_REF].

The thickness of the neotissue that develops on the inner surface of stents presents a great variability, from a few microns to a few millimiters, depending on multiple factors such as the type of device, implantation time, age of human or animal [START_REF] Foth | Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion devices[END_REF][START_REF] Farb | Morphological predictors of restenosis after coronary stenting in humans[END_REF][START_REF] Deutsch | In vitro endothelialization of expanded polytetrafluoroethylene grafts: a clinical case report after 41 months of implantation[END_REF].

With the recent development of numerous percutaneous devices, this process is more studied. Intra-vascular devices such as stents and vascular prosthesis, and intra-cardiac devices, such as atrio/ventricular septal defect occluders and left atrial appendage occluders, are concerned by this natural process and its potential short-and long-term complications. Indeed, in case of an incomplete or late device tissue coverage, thrombus formation, endocarditis, stent stenosis or dislodgement of the device can complicate the issue after implantation [START_REF] Chen | Incomplete endothelialization and late dislocation after implantation of an Amplatzer septal occluder device[END_REF][START_REF] Nguyen | Endocarditis and incomplete endothelialization 12 years after Amplatzer septal occluder deployment[END_REF][START_REF] Pendyala | The firstgeneration drug-eluting stents and coronary endothelial dysfunction[END_REF][START_REF] Li | Evaluation of long-term follow-up with neointimal coverage and stent apposition after sirolimus-eluting stent implantation by optical coherence tomography[END_REF]. Thereby, strut coverage of a coronary stent was demonstrated to be a powerful histological predictor of stent thrombosis [START_REF] Gonzalo | Incomplete stent apposition and delayed tissue coverage are more frequent in drug-eluting stents implanted during primary percutaneous coronary intervention for ST-segment elevation myocardial infarction than in drug-eluting stents implanted for stable/unstable angina[END_REF][START_REF] Musick | Sensor to detect endothelialization on an active coronary stent[END_REF]. Several factors were suspected to lead to late stent stenosis : the anti-proliferative drug of the device that not only prevented smooth muscle cell proliferation but also endothelialization; and the polymer of the prosthesis by its pro-inflammatory effect, all this resulting in endothelial denudation and risk of very late stent stenosis [START_REF] Foin | Incomplete stent apposition causes high shear flow disturbances and delay in neointimal coverage as a function of strut to wall detachment e[END_REF]. As for intra-cardiac devices, the surgical explantation showed incomplete device coverage and presence of thrombus in some cases, sometimes years after implantation [START_REF] Foth | Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion devices[END_REF].

Objectives

This review aims to (1) present the experimental models used to study this process on cardiac devices; (2) focus on the in vitro techniques and invasive modalities used currently in humans for intra-vascular and intra-cardiac devices and (3) assess the future developments of non-invasive techniques in animal models and humans for intra-cardiac devices.

Experimental models to study the healing process after intravascular and intra-cardiac device implantation Animal models were developed to study the healing process after device implantation, especially after stent positioning. An ideal animal model should reflect the evolution of healing processes in humans, allowing understanding of the genesis of complications, such as stenosis, and guidance for postimplantation treatment [START_REF] Chan | Evaluation of synthetic vascular grafts in a mouse carotid grafting model[END_REF]. This process appears to be similar in animals and humans in terms of the healing response, but the duration is much longer in humans. However, the selection of the animal is not benign as several factors can interfere with the healing process: species in terms of duration of the healing process; senescence of the animal as a longer duration of healing process exists for older animals; anatomical dimensions and graft surface/vessel caliber that will interfere with blood flow conditions [START_REF] Zilla | Prosthetic vascular grafts: wrong models, wrong questions and no healing[END_REF][START_REF] Byrom | Animal models for the assessment of novel vascular conduits[END_REF]. Cost-effectiveness, accessibility, time frame of the healing process, post-operative risk of death and cellular imaging tools available are other factors that will be useful to decide which model should be retained [START_REF] Chan | Evaluation of synthetic vascular grafts in a mouse carotid grafting model[END_REF].

Animal models for intra-vascular devices

Intra-vascular devices include stents and vascular prosthesis. Stents have been widely studied starting with bare-metal stents, then followed by drug-eluting stents. Drug-eluting stents allow localized elution of neointimal inhibiting drugs (sirolimus, paclitaxel, everolimus…) and prevent in-stent restenosis. Absorbable drug reservoirs and platforms have been then developed followed by bioengineered stents coated with antibodies specific to CD 34 to capture circulating endothelial progenitor cells [START_REF] Pendyala | The firstgeneration drug-eluting stents and coronary endothelial dysfunction[END_REF]. Intra-vascular device healing processes has been extensively studied.

Animal models are therefore well developed and cross-compared, from small e.Proofing https://eproofing.springer.com/journals_v2/printpage.php?token=jvT3xal... animals such as rats [START_REF] Chan | Evaluation of synthetic vascular grafts in a mouse carotid grafting model[END_REF][START_REF] Mirza | Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall[END_REF][START_REF] Wang | Atorvastatin accelerates both neointimal coverage and re-endothelialization after sirolimus-eluting stent implantation in a porcine model[END_REF][START_REF] Liu | Rapamycin inhibits re-endothelialization after percutaneous coronary intervention by impeding the proliferation and migration of endothelial cells and inducing apoptosis of endothelial progenitor cells[END_REF] and rabbits [START_REF] Liu | Fluid upstream shear stress of rabbit aortic stenosis inhibits neointimal hyperplasia by promoting endothelization after balloon injury[END_REF][START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF]8,[START_REF] Prati | Randomized comparison between 3-month Cre8 DES vs. 1-month Vision/Multilink8 BMS neointimal coverage assessed by OCT evaluation: The DEMONSTRATE study[END_REF][START_REF] Ma | Delayed re-endothelialization with rapamycin-coated stents is rescued by the addition of a glycogen synthase kinase-3β inhibitor[END_REF][START_REF] Tang | The impact of vascular endothelial growth factor-transfected human endothelial cells on endothelialization and restenosis of stainless steel stents[END_REF][START_REF] Toma | Vascular endoluminal delivery of mesenchymal stem cells using acoustic radiation force[END_REF][START_REF] Larsen | Capture of circulatory endothelial progenitor cells and accelerated reendothelialization of a bio-engineered stent in human ex vivo shunt and rabbit denudation model[END_REF] to large animals such as sheep [START_REF] Mertens | USPIO-labeled textile materials for non-invasive MR imaging of tissueengineered vascular grafts[END_REF][START_REF] Koobatian | Successful endothelialization and remodeling of a cell-free small-diameter arterial graft in a large animal model[END_REF][START_REF] Fukunishi | Tissue-engineered small diameter arterial vascular grafts from cell-free nanofiber PCL/Chitosan scaffolds in a sheep model[END_REF], pigs [START_REF] Dahan | Dynamic autologous reendothelialization of small-caliber arterial extracellular matrix: a preclinical large animal study[END_REF][START_REF] Pislaru | Magnetic forces enable rapid endothelialization of synthetic vascular grafts[END_REF][START_REF] Wang | Atorvastatin accelerates both neointimal coverage and re-endothelialization after sirolimus-eluting stent implantation in a porcine model[END_REF][START_REF] Attizzani | Serial evaluation of vascular response after implantation of a new sirolimus-eluting stent with bioabsorbable polymer [MISTENT]: an optical coherence tomography and histopathological study[END_REF][START_REF] Ruiter | Long-term effect of stents eluting 6-mercaptopurine in porcine coronary arteries[END_REF][START_REF] Wittchow | Bioresorbable drug-eluting magnesium-alloy scaffold: design and feasibility in a porcine coronary model[END_REF][START_REF] Zhang | Polymerfree dual drug-eluting stents evaluated in a porcine model[END_REF][START_REF] Koppara | Histopathological comparison of biodegradable polymer and permanent polymer based sirolimus eluting stents in a porcine model of coronary stent implantation[END_REF][START_REF] Rotmans | In vivo cell seeding with anti-CD34 antibodies successfully accelerates endothelialization but stimulates intimal hyperplasia in porcine arteriovenous expanded polytetrafluoroethylene grafts[END_REF][START_REF] Farooq | Intracoronary optical coherence tomography and histology of overlapping everolimus-eluting bioresorbable vascular scaffolds in a porcine coronary artery model[END_REF], baboons [START_REF] Jordan | The effect of a recombinant elastin-mimetic coating of an ePTFE prosthesis on acute thrombogenicity in a baboon arteriovenous shunt[END_REF] and dogs [START_REF] Liu | Rapamycin inhibits re-endothelialization after percutaneous coronary intervention by impeding the proliferation and migration of endothelial cells and inducing apoptosis of endothelial progenitor cells[END_REF]. Mouse models can serve to assess very small conduit (less than 2 mm) healing processes with serial examinations, small synthetic tubular vascular grafts made of multicellular spheroids [START_REF] Itoh | Scaffold-free tubular tissues created by a Bio-3D printer undergo remodeling and endothelialization when implanted in rat aortae[END_REF] or decellularized extracellular matrix [START_REF] Aubin | Customized interface biofunctionalization of decellularized extracellular matrix: toward enhanced endothelialization[END_REF], and to study the molecular mechanisms of graft failure in pre-clinical assessment [START_REF] Chan | Evaluation of synthetic vascular grafts in a mouse carotid grafting model[END_REF]. Rabbits are recommended for small vascular grafts (1-4 mm). Studies involving the closest anatomic and physiologic relatives to humans, the primates, are few. But some studies were conducted to assess the healing process after vascular graft and stent implantation, showing 60% of ePTFE artery conduits fully endothelialized after 12 months [START_REF] Clowes | Mechanisms of arterial graft failure. Role of cellular proliferation in early healing of PTFE prostheses[END_REF] and demonstrating the reduction of intimal hyperplasia with heparin-coated balloon-expandable stents compared with non-coated stents [START_REF] Lin | Carotid stenting using heparin-coated balloon-expandable stent reduces intimal hyperplasia in a baboon model[END_REF]. However, due to the cost and protected status of primates, alternative large animal models grew momentum. The use of dogs was assessed for vascular graft studies. However, because of a marked difference with the human hemostatic system and a lack of relevance to study graft patency and endothelialization, sheep and pigs were finally preferred [START_REF] Byrom | Animal models for the assessment of novel vascular conduits[END_REF]. Their availability, low cost, size/anatomy and blood/coagulation systems were found to better complement humans, especially for 4-6 mm grafts. Sheep are closer matches to human results for vascular graft studies and pigs for coronary stents. Sheep present a coagulation system closer to humans than pigs, with few post-operative complications; therefore an appropriate model to assess patency and anastomotic intimal hyperplasia after graft implantation [START_REF] Byrom | Animal models for the assessment of novel vascular conduits[END_REF]. Pigs are suitable to assess stent patency, calcification and endothelialization albeit with faster healing kinetics than in humans. The influence of ageing and growth on device efficacy and longevity is an important parameter for infantile and adolescent patients. Pigs present an appropriate model with most common strains having indeterminate growth.

Animal models for intra-cardiac devices

Intra-cardiac devices include bioprosthetic heart valves and cardiac defect occluders. Bioprosthetic valves are usually created from animal valve leaflets mounted on a polymer material stent. Atrial septal defect occluders are mostly made of nickel-titanium skeleton and biostable membranes such as PET and e-PTFE membrane. Biodegradable materials are in development. Intra-cardiac device investigations have been primarily studied in sheep and pig models. The sheep is one of the most widely used animal models for bioprosthetic heart valve and tissue-engineered valve investigations [START_REF] Syedain | 6-month aortic valve implantation of an off-the-shelf tissue-engineered valve in sheep[END_REF][START_REF] Vincentelli | In vivo autologous recellularization of a tissue-engineered heart valve: Are bone marrow mesenchymal stem cells the best candidates?[END_REF][START_REF] Schmitt | Percutaneous pulmonary valve replacement using completely tissueengineered off-the-shelf heart valves: six-month in vivo functionality and matrix remodelling in sheep[END_REF][START_REF] Hof | Challenges in developing a reseeded, tissue-engineered aortic valve prosthesis[END_REF]. Such models have enabled pre-clinical evaluations comprising of leaflet motion; effective orifice area; mechanical properties; remodeling of the extracellular matrix; endothelialization and calcification. As for septal defect occluders, experimental models were necessary to assess the healing process [START_REF] Milewski | Temporal healing patterns and coverage dynamics after new polish transcatheter PFO occluder implantation in a swine[END_REF] and biocompatibility after implantation [START_REF] Sigler | Biocompatibility of septal defect closure devices[END_REF]. Before placing the device, a defect must be created in the fossa ovalis, by transeptal puncture. Pig and sheep models were selected thanks to their close anatomy of the fossa ovalis and dimensions representative of humans, with a special interest in pigs for assessment of device longevity during growth.

AQ2

Bioengineering and in vitro models

Bio-engineered small-diameter vascular grafts consist of a biodegradable scaffold on which endothelial cells are cultured [START_REF] Whited | Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium[END_REF]. Customizing the cell populations and neotissue formations in vitro is one of the major goals of this technique. Instead of using a static system to enhance cell culture of the scaffold, a tubular perfusion system bioreactor was developed, allowing a greater maturation and differentiation of cells [START_REF] Melchiorri | In vitro endothelialization of biodegradable vascular grafts via endothelial progenitor cell seeding and maturation in a tubular perfusion system bioreactor[END_REF].

The influence of stent struts on local blood flow and the migration of endothelial cells were also studied on in vitro models, using a flow chamber that mimic the geometry of stent struts. It was shown that unidirectional flow leads to a unified forward migration of cells in the flow direction, whereas in the presence of bidirectional flow, complex non-uniform distributions were observed [START_REF] Hsiao | Endothelial repair in stented arteries is accelerated by inhibition of Rhoassociated protein kinase[END_REF].

Human studies

Explanted grafts and devices directly from humans have enabled the study of neotissue formation after implantation. Farb et al. provided a detailed histological study of 116 coronary stents, identifying a spectrum of risk factors for stenosis [START_REF] Farb | Morphological predictors of restenosis after coronary stenting in humans[END_REF]. A central segment of in vitro femoropopliteal bypass was analyzed 41 months after implantation by Deutsch et al., showing the presence of a mature endothelium covering the graft [START_REF] Deutsch | In vitro endothelialization of expanded polytetrafluoroethylene grafts: a clinical case report after 41 months of implantation[END_REF]. Experiments on human homograft valves were also used to investigate the influence of valve-preservation e.Proofing https://eproofing.springer.com/journals_v2/printpage.php?token=jvT3xal... techniques on endothelial cell growth [START_REF] Eberl | Experimental in vitro endothelialization of cardiac valve leaflets[END_REF]. Tissue-engineered pulmonary valves could also be studied after explantation from humans, allowing a cross-sectional study of magnetic resonance imaging (MRI) and histology to better understand graft failure mechanisms [START_REF] Voges | Adverse results of a decellularized tissue-engineered pulmonary valve in humans assessed with magnetic resonance imaging[END_REF]. Atrial and ventricular septal defect occluders were studied after surgical explantation by Foth et al., providing crucial information about the neotissue formation from five days to four years post-implantation [START_REF] Foth | Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion devices[END_REF].

In vitro techniques to assess the healing process on devices Histology, the gold standard Histology is the gold standard to evaluate the healing process, as it allows the most accurate characterization of the tissue covering the device [START_REF] Attizzani | Serial evaluation of vascular response after implantation of a new sirolimus-eluting stent with bioabsorbable polymer [MISTENT]: an optical coherence tomography and histopathological study[END_REF]. As the device is metallic, the choice of embedding process before histological and immunohistochemical analysis is important. The hard resin inclusion should be preferred to paraffine inclusion in order to preserve the interface tissue-implant and allow assessment of local processes [START_REF] Foth | Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion devices[END_REF]. Standard histology includes hematoxylin and eosin staining and Richardson blue staining to distinguish cells and scaffold material; Lawson van Gieson, Masson trichrome and Movat pentachrome stainings to detect collagen fibers [START_REF] Ruiter | Long-term effect of stents eluting 6-mercaptopurine in porcine coronary arteries[END_REF][START_REF] Voges | Adverse results of a decellularized tissue-engineered pulmonary valve in humans assessed with magnetic resonance imaging[END_REF]. Immunohistochemistry has also been applied to characterize specific cell types: von Willebrand factor, CD31 and CD34 for endothelial cells [START_REF] Pislaru | Magnetic forces enable rapid endothelialization of synthetic vascular grafts[END_REF][START_REF] Ruiter | Long-term effect of stents eluting 6-mercaptopurine in porcine coronary arteries[END_REF][START_REF] Itoh | Scaffold-free tubular tissues created by a Bio-3D printer undergo remodeling and endothelialization when implanted in rat aortae[END_REF]; CD 68 for macrophages, CD 79 for B-and CD 3 for T-cells, CD 45 for leukocytes, CD 34 for endothelial cells progenitors [START_REF] Foth | Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion devices[END_REF][START_REF] Voges | Adverse results of a decellularized tissue-engineered pulmonary valve in humans assessed with magnetic resonance imaging[END_REF][START_REF] Aoki | Endothelial progenitor cell capture by stents coated with antibody e[END_REF]. Inflammation and endothelialization scores involving the extent and severity of inflammation, thrombus/fibrin formation and recellularization of graft tissue can help to better evaluate the healing process [START_REF] Ruiter | Long-term effect of stents eluting 6-mercaptopurine in porcine coronary arteries[END_REF][START_REF] Voges | Adverse results of a decellularized tissue-engineered pulmonary valve in humans assessed with magnetic resonance imaging[END_REF][START_REF] Kar | Impact of Watchman and Amplatzer devices on left atrial appendage adjacent structures and healing response in a canine model[END_REF]. Specific scores concern intimal fibrin content for stent struts [START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF][START_REF] Wittchow | Bioresorbable drug-eluting magnesium-alloy scaffold: design and feasibility in a porcine coronary model[END_REF] and endothelialization extension [START_REF] Wittchow | Bioresorbable drug-eluting magnesium-alloy scaffold: design and feasibility in a porcine coronary model[END_REF]. Serial examinations on animal and human models have further enabled descriptions of the time frame of the healing process [3, 7, 8] (Fig. 1). Fig. 1 Histology example. Overview of an Amplatzer atrial septal defect-occlusion device 24 months after implantation. Micrographs show representative staining with Richardson blue (cellular components, blue; metal wires, black). Overview of an Amplatzer atrial septal defect-occlusion device 24 months after implantation. a Representative image of pattern of neotissue within the implant with irregular e.Proofing https://eproofing.springer.com/journals_v2/printpage.php?token=jvT3xal... orientation of cells (detail). b Representative image of pattern of cells of pseudointima with longitudinal orientation (parallel to the neoendothelium, detail)

Microscopy techniques

Microscopy techniques are used to characterize the coverage of tissue on cardiac devices. While light microscopy is still important to magnify histologic slides, scanning electron microscopy is largely used in vascular graft engineering [START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF][START_REF] Deutsch | In vitro endothelialization of expanded polytetrafluoroethylene grafts: a clinical case report after 41 months of implantation[END_REF][START_REF] Chan | Evaluation of synthetic vascular grafts in a mouse carotid grafting model[END_REF]. This high-resolution electron-based technique can provide 3D images of the surface covering a stent or a scaffold [START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF][START_REF] Chan | Evaluation of synthetic vascular grafts in a mouse carotid grafting model[END_REF][START_REF] Mertens | USPIO-labeled textile materials for non-invasive MR imaging of tissueengineered vascular grafts[END_REF] (Fig. 2). Local organization and the interactions of neotissue with devices can also be investigated [START_REF] Mertens | USPIO-labeled textile materials for non-invasive MR imaging of tissueengineered vascular grafts[END_REF]. Quantifying endothelialized area [START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF]8,[START_REF] Zhang | Polymerfree dual drug-eluting stents evaluated in a porcine model[END_REF] and characterizing the time-frame of the healing process [START_REF] Mirza | Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall[END_REF] can also be performed by studying stent struts coverage by smooth muscle cells, collagen and endothelium after stent implantation.

Moreover, incomplete endothelialized protruding parts of the metal framework after atrial septal defect-occlusion device implantation can be identified [START_REF] Sigler | Biocompatibility of septal defect closure devices[END_REF][START_REF] Deutsch | In vitro endothelialization of expanded polytetrafluoroethylene grafts: a clinical case report after 41 months of implantation[END_REF][START_REF] Chan | Evaluation of synthetic vascular grafts in a mouse carotid grafting model[END_REF][START_REF] Wang | Atorvastatin accelerates both neointimal coverage and re-endothelialization after sirolimus-eluting stent implantation in a porcine model[END_REF]. Regions of devices lacking coverage typically show adherence of platelets and macrophages [START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF].

Fig. 2

Scanning electron microscopy example. Magnified scanning electron microscopy views of overlapping Absorb (Everolimus-eluting bioresorbable vascular scaffold). Magnified scanning electron microscopy (SEM) view of the overlapping Absorb at 28 days (upper), demonstrating multiple uncovered Absorb struts primarily secondary to the direct overlay configuration of the "stacked inner" struts (white asterisks) to the corresponding "stacked outer" struts located abluminally. At 90 days (lower), all "stacked inner" struts (white asterisks) are covered. Transmission microscopy is another microscopic technique with a nanometric resolution, higher than scanning electron microscopy. It enables to appreciate the ultrastructure of the components of the neotissue and characterize more precisely the cells [START_REF] Deutsch | In vitro endothelialization of expanded polytetrafluoroethylene grafts: a clinical case report after 41 months of implantation[END_REF].

Two-photon microscopy or multi-photon microscopy, is a fluorescent imaging approach that combines a deep penetration and high resolution [START_REF] Dahan | Dynamic autologous reendothelialization of small-caliber arterial extracellular matrix: a preclinical large animal study[END_REF][START_REF] Whited | Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium[END_REF]. Collagen, elastin and endothelial cells can be distinguished, as well as their localization in the tissue [START_REF] Dahan | Dynamic autologous reendothelialization of small-caliber arterial extracellular matrix: a preclinical large animal study[END_REF].

Fiber optic-based imaging

An vitro imaging technique, described by Whited and al [START_REF] Whited | Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium[END_REF], consists of mapping fluorescently labelled endothelial cells placed in a half-vessel scaffold.

A micro-imaging channel is embedded in the wall of a scaffold through which the excitation fiber optic is inserted. The endothelial cell fluorescence may then be captured by a photosensitive detector to map the cell distributions within the scaffold. This allows for non-destructive, dynamic imaging of the endothelium of the vascular graft with no impact on the adjacent structures.

Invasive techniques available in humans

Histology and microscopy techniques can only be used on explanted devices. Intra-vascular ultrasounds (IVUS) and optical coherence tomography (OCT) were developed for intra-vascular grafts, allowing an invasive evaluation of the healing process on stents.

Intra-vascular ultrasounds

This invasive, catheter-based technique relies on the use of ultrasound impulses, emitted from a miniaturized transducer, mounted on the tip of a catheter [START_REF] Hoang | The role of intracoronary plaque imaging with intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in patients with coronary artery disease[END_REF]. It provides real-time, cross-sectional tomographic images of the internal surface of the blood vessel [START_REF] Vasquez | Impact of Intravascular Ultrasound in Clinical Practice[END_REF][START_REF] Erglis | Intravascular Ultrasound-based Imaging Modalities for Tissue Characterisation[END_REF]. The axial resolution is around 50 µm, depending on the catheter type. High ultrasound reflection between variable tissue interfaces enables differentiation of the layers of the vessel wall [START_REF] Hoang | The role of intracoronary plaque imaging with intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in patients with coronary artery disease[END_REF].

It can be used to evaluate a vessel in terms of lesion quantification and morphology assessment: before or after stent implantation or vascular grafting with higher sensitivity in assessing coronary artery lumen morphology [START_REF] Schiele | Impact of intravascular ultrasound guidance in stent deployment on 6-month restenosis rate: a multicenter, randomized study comparing two strategies-with and without intravascular ultrasound guidance[END_REF] (Fig. 3), after cardiac transplantation to detect coronary transplant vasculopathy and for evaluation of atherosclerotic plaque formation [START_REF] Cheneau | Time course of stent endothelialization after intravascular radiation therapy in rabbit iliac arteries[END_REF][START_REF] Hoang | The role of intracoronary plaque imaging with intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in patients with coronary artery disease[END_REF][START_REF] Vasquez | Impact of Intravascular Ultrasound in Clinical Practice[END_REF].

Fig. 3 OCT and IVUS example. OCT evidence of uncovered struts in drug-eluting stent late stent thrombosis. Representative angiographic (a), intravascular ultrasound (bd), and optical coherence tomography (e-g) cross-sectional images from a patient with very late stent thrombosis at 1836 days in a single sirolimus-eluting stent implanted in proximal left anterior descending artery. After thrombus aspiration, uncovered struts are detected by OCT, with remaining intraluminal thrombus e.Proofing https://eproofing.springer.com/journals_v2/printpage.php?token=jvT3xal... adherent to some strut. Positive remodeling was not observed by IVUS.

To customize the technique, the near-infrared spectroscopy imaging system was incorporated in the IVUS catheter and used to analyze the atherosclerotic plaque composition [START_REF] Hoang | The role of intracoronary plaque imaging with intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in patients with coronary artery disease[END_REF]. The reflected signal is converted in a chemogram that represents a color map of the location of lipid core plaques to characterize the composition of the plaque and select patients at high-risk of adverse outcome [START_REF] Hoang | The role of intracoronary plaque imaging with intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in patients with coronary artery disease[END_REF].

IVUS has not been extended to other devices, especially intra-cardiac devices such as occluders.

Intravascular optical coherence tomography

OCT is a near-infrared light-based modality that provides high resolution imaging (10 µm for axial resolution and 20-40 µm for lateral resolution) of the microstructure of the tissue and blood vessel wall in vivo [START_REF] Prati | Randomized comparison between 3-month Cre8 DES vs. 1-month Vision/Multilink8 BMS neointimal coverage assessed by OCT evaluation: The DEMONSTRATE study[END_REF][START_REF] Attizzani | Serial evaluation of vascular response after implantation of a new sirolimus-eluting stent with bioabsorbable polymer [MISTENT]: an optical coherence tomography and histopathological study[END_REF][START_REF] Tearney | Consensus standards for acquisition, measurement, and reporting of intravascular optical coherence tomography studies[END_REF].

In the interventional cardiology field, this invasive technique is used to evaluate the microstructure of the vessels, atheroma plaque and stents [START_REF] Attizzani | Serial evaluation of vascular response after implantation of a new sirolimus-eluting stent with bioabsorbable polymer [MISTENT]: an optical coherence tomography and histopathological study[END_REF][START_REF] Barlis | An optical coherence tomography study of a biodegradable vs. durable polymer-coated limus-eluting stent: a LEADERS trial sub-study[END_REF] (Fig. 3). The OCT catheter is placed at the distal part of the stent and the entire length of the region is scanned using the integrated automated pullback device [START_REF] Prati | Randomized comparison between 3-month Cre8 DES vs. 1-month Vision/Multilink8 BMS neointimal coverage assessed by OCT evaluation: The DEMONSTRATE study[END_REF][START_REF] Attizzani | Serial evaluation of vascular response after implantation of a new sirolimus-eluting stent with bioabsorbable polymer [MISTENT]: an optical coherence tomography and histopathological study[END_REF][START_REF] Hoang | The role of intracoronary plaque imaging with intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in patients with coronary artery disease[END_REF].

OCT enables distinction of the three layers of the vessels, to identify lesions such as unstable atherosclerotic plaques, macrophage accumulations and thrombi [START_REF] Tearney | Consensus standards for acquisition, measurement, and reporting of intravascular optical coherence tomography studies[END_REF]. For stented arteries, neointimal hyperplasia can be detected [START_REF] Murase | The relationship between re-endothelialization and endothelial function after DES implantation: Comparison between paclitaxcel eluting stent and zotarolims eluting stent[END_REF][START_REF] Lehtinen | Early vascular healing after endothelial progenitor cell capturing stent implantation[END_REF] and a morphometric analysis of the stent strut coverage can be performed [START_REF] Murase | The relationship between re-endothelialization and endothelial function after DES implantation: Comparison between paclitaxcel eluting stent and zotarolims eluting stent[END_REF][START_REF] Bonnema | An automatic algorithm for detecting stent endothelialization from volumetric optical coherence tomography datasets[END_REF]. Dissection of stents, noncoverage, calcification and misalignment of struts may also be detected [START_REF] Gonzalo | Incomplete stent apposition and delayed tissue coverage are more frequent in drug-eluting stents implanted during primary percutaneous coronary intervention for ST-segment elevation myocardial infarction than in drug-eluting stents implanted for stable/unstable angina[END_REF][START_REF] Prati | Randomized comparison between 3-month Cre8 DES vs. 1-month Vision/Multilink8 BMS neointimal coverage assessed by OCT evaluation: The DEMONSTRATE study[END_REF][START_REF] Attizzani | Serial evaluation of vascular response after implantation of a new sirolimus-eluting stent with bioabsorbable polymer [MISTENT]: an optical coherence tomography and histopathological study[END_REF][START_REF] Hoang | The role of intracoronary plaque imaging with intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in patients with coronary artery disease[END_REF][START_REF] Tearney | Consensus standards for acquisition, measurement, and reporting of intravascular optical coherence tomography studies[END_REF][START_REF] Kobayashi | Impact of target lesion coronary calcification on stent expansion[END_REF]. Such information can thus far only be obtained in vivo using OCT, thanks to its 10-fold higher resolution than IVUS [START_REF] Guagliumi | Examination of the in vivo mechanisms of late drug-eluting stent thrombosis[END_REF], which has successfully been applied to guide pharmalogical therapy for the prevention of late stent thrombosis [START_REF] Prati | Randomized comparison between 3-month Cre8 DES vs. 1-month Vision/Multilink8 BMS neointimal coverage assessed by OCT evaluation: The DEMONSTRATE study[END_REF]. It is also used in tissue-bioengineering, but to date, has not been evaluated in the assessment of intra-cardiac device coverage [START_REF] Gurjarpadhye | Dynamic assessment of the endothelialization of tissue-engineered blood vessels using an optical coherence tomography catheter-based fluorescence imaging system[END_REF]. One of the drawbacks of this technique is the inability to detect the endothelial layers that measure less than 10 µm and complications due to the invasiveness of the procedure [START_REF] Gurjarpadhye | Dynamic assessment of the endothelialization of tissue-engineered blood vessels using an optical coherence tomography catheter-based fluorescence imaging system[END_REF].

Future developments of non-invasive techniques in animal models and humans MRI

In cardiovascular tissue engineering, MRI is used as a non-invasive technique to monitor the remodeling of vascular grafts and their patency, to follow the cells implanted in vitro in the graft and to evaluate inflammation and thrombosis after implantation [START_REF] Mertens | USPIO-labeled textile materials for non-invasive MR imaging of tissueengineered vascular grafts[END_REF][START_REF] Fayol | Design of biomimetic vascular grafts with magnetic endothelial patterning[END_REF]. This is made possible by the adjunction of ultra-small superparamagnetic iron oxide biocompatible particles in the scaffold to render it visible and provide morphological information of the graft [START_REF] Mertens | USPIO-labeled textile materials for non-invasive MR imaging of tissueengineered vascular grafts[END_REF][START_REF] Fayol | Design of biomimetic vascular grafts with magnetic endothelial patterning[END_REF].

In humans, even if most of the modalities have been developed for intra-vascular devices, imaging the tissue covering intra-cardiac devices, such as septal occluders or left atrial appendage occlusion devices, is also challenging and clinically relevant.

Thanks to its high spatial and temporal resolutions, significant focus has been applied to tissue characterization using MRI [START_REF] Bucciarelli-Ducci | Cardiac MRI anatomy and function as a substrate for arrhythmias[END_REF][START_REF] Haaf | Cardiac T1 mapping and extracellular volume in clinical practice: a comprehensive review[END_REF][START_REF] Captur | Cardiac MRI evaluation of myocardial disease[END_REF], especially to detect myocardial fibrosis [START_REF] Gyöngyösi | Myocardial fibrosis: biomedical research from bench to bedside: myocardial fibrosis: biomedical research from bench to bedside[END_REF], myocardial edema and microvascular obstruction [START_REF] Bucciarelli-Ducci | Cardiac MRI anatomy and function as a substrate for arrhythmias[END_REF]. Fibrosis can be identified by late gadolinium enhancement, favored for visualization of myocardial focal fibrotic scar [START_REF] Berliner | Cardiac computed tomographic imaging to evaluate myocardial scarring/fibrosis in patients with hypertrophic cardiomyopathy: a comparison with cardiac magnetic resonance imaging[END_REF], as seen in ischemic and nonischemic cardiomyopathy. T1 and T2 MRI provides has further been used to enhance images of fibrotic, inflammatory and edematous tissue [START_REF] Ghosn | Important advances in technology and unique applications related to cardiac magnetic resonance imaging[END_REF][START_REF] Graham-Brown | Imaging of myocardial fibrosis in patients with endstage renal disease: current limitations and future possibilities[END_REF][START_REF] Vo | T1 Mapping in characterizing myocardial disease: a comprehensive review[END_REF].

Intra-cardiac imaging devices, has been mostly used to detect device position and cardiac remodeling after implantation [START_REF] Lapierre | Longterm follow-up of large atrial septal occluder (Amplatzer device) with cardiac MRI in a pediatric population[END_REF]. Yet, work is still to be done to optimize MRI for neo-tissue detection due to challenges associated with the required resolution and to alleviate image artefacts, particularly due to the metal framework of the prosthesis, especially occlude screw hubs.

Computed tomography

Computed tomography [CT] is known to detect focal myocardial fibrotic scars [START_REF] Berliner | Cardiac computed tomographic imaging to evaluate myocardial scarring/fibrosis in patients with hypertrophic cardiomyopathy: a comparison with cardiac magnetic resonance imaging[END_REF], providing an infra-millimetric resolution (0.5 mm compared to 1-2 mm for MRI) [START_REF] Maron | The remarkable 50 years of imaging in HCM and how it has changed diagnosis and management[END_REF][START_REF] Mahida | Cardiac imaging in patients with ventricular tachycardia[END_REF].

Multidetector computed tomography angiography and ultra-high-resolution computed tomography angiography are now in development to detect coronary stent stenosis. Thanks to its higher temporal and spatial resolution than 4 and 16slice CT, 64-slice multidetector computed tomography angiography presents a good correlation with angiography and IVUS for detection of diameter and area stent stenosis for > 3 mm stents. The motion and blooming artefacts are less important but the temporal resolution is not as good as IVUS [START_REF] Kwon | In-stent area stenosis on 64-slice multi-detector computed tomography coronary angiography: optimal cutoff value for minimum lumen cross-sectional area of coronary stents compared with intravascular ultrasound[END_REF]. For calcified lesions, ultra-high-resolution CT presents fewer artefacts and a better spatial resolution for in-stent lumen assessment > 2.5 mm [START_REF] Motoyama | Ultra-high-resolution computed tomography angiography for assessment of coronary artery stenosis[END_REF].

Regarding intra-cardiac devices, cardiac CT is mostly used for the follow-up of patients after percutaneous left atrial appendage occlusion to assess the presence of device thrombosis and peri-device leaks and to guide post-procedural antithrombotic therapy durations [START_REF] Cochet | Left atrial appendage patency and device-related thrombus after percutaneous left atrial appendage occlusion: a computed tomography study[END_REF][START_REF] Marini | Midterm results of percutaneous closure of very large atrial septal defects in children: role of multislice computed tomography[END_REF]. However, for some cases, cardiac CT cannot discriminate between laminated thrombus (reinforcement of antithrombotic treatment recommended) and locally prominent device covering (no treatment needed).

Thus, dedicated CT sequences to precisely identify the fibrotic tissue covering intra-cardiac devices could provide improved tailored patient management.

Sensors to detect endothelialization on coronary stents

Musick and al. [START_REF] Musick | Sensor to detect endothelialization on an active coronary stent[END_REF]. described the use of a piezoelectric microcantilever placed in the strut of an active stent to detect endothelialization of the struts. When cells attach to the cantilever, they cause an increase in mass and decrease in the resonant frequencies of the cantilever. The goal of this non-invasive process is to e.Proofing https://eproofing.springer.com/journals_v2/printpage.php?token=jvT3xal... monitor the healing process after stent implantation, but without the possibility to differentiate the types of covering tissues (endothelium versus fibrin). There is potential future clinical application of such sensors to provide non-invasive follow-up of healing status and determine the patient's anti-platelet therapy. An approach beneficial to intra-vascular devices but also intra-cardiac applications.

High frame rate ultrasound imaging

Noninvasive high frame rate ultrasound imaging may be a promising technique to detect neotissue formation. It was recently experienced in different fields of cardiology, notably to visualize the epicardial and intramural coronary vasculature to assess coronary microcirculation and coronary blood flow in normal and pathologic situations. It offers a visualization of the pre-arteriolar coronary vessel structure which size is 100 to 500 microns, in animals and humans [START_REF] Maresca | Noninvasive imaging of the coronary vasculature using ultrafast ultrasound[END_REF]. The shear wave imaging that relies on the mapping of the propagation velocities of shear waves related to tissue viscoelastic properties is another technique in development [START_REF] Strachinaru | Cardiac shear wave elastography using a clinical ultrasound system[END_REF][START_REF] Brekke | Ultra-high frame rate tissue Doppler imaging[END_REF].

Conclusion

Imaging neotissue covering a cardiac device is a necessary step yet remains challenging. In vitro techniques, such as histology, remain the gold standard even if several invasive techniques (OCT, IVUS) have been developed to detect vessel structure and coronary stent restenosis. Most of these techniques were developed for intra-vascular devices. With the evolution of MRI and CT imaging of cardiac fibrosis, we hope that such techniques will provide insight in to neotissue intracardiac device coverage in the near future.

AQ3

  Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. devices, Figure n°1, Copyright 2009, with permission from Wolters Kluwer Health, Inc. Fig. 2: reprinted from JACC: Cardiovascular Interventions 2013;6[5]:523-32, by Farooq et al., Intracoronary optical coherence tomography and histology of overlapping everolimus-eluting bioresorbable vascular scaffolds in a porcine coronary artery model the potential implications for clinical practice, Figure n°7, Copyright 2013, with permission from Elsevier Inc. Fig. 3: reprinted from JACC: Cardiovascular Interventions 2012;5[1]:12-20, by Guagliumi et al., Examination of the in vivo mechanisms of late drug-eluting stent thrombosis-Findings from optical coherence tomography and intravascular ultrasound imaging, Figure n°1, Copyright 2012, with permission from Elsevier Inc.

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Acknowledgements

The figures were reprinted: Fig. 1: reprinted from Circulation: Cardiovascular Interventions 2009;2:90-96, by Foth et al., Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion e.Proofing https://eproofing.springer.com/journals_v2/printpage.php?token=jvT3xal...

Funding

This study received financial support from the French Government as part of the "Investments of the Future" program managed by the National Research Agency (ANR), Grant reference ANR-10-IAHU-04.

Compliance with Ethical Standards

Ethical approval The manuscript does not contain clinical studies or patient data.