Mathilde Champeau
email: mathilde.champeau@cea.fr

Dorothée Jary

Laurent Mortier

Serge Mordon

Séverine Vignoud

A facile fabrication of dissolving microneedles containing 5-aminolevulinic acid

Keywords: 5-aminolevulinic acid, hyaluronic acid, dissolving microneedles, solvent casting molding method, photodynamic therapy

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

Introduction

Transdermal drug delivery, usually ensured by using creams, lotion or patches, offers several important advantages over traditional drug delivery methods (oral administration or injection).

It indeed enables to control the amount of drug delivered at local areas, which allows to administrate lower doses [START_REF] Escobar-Chavez | Nanocarriers for transdermal drug delivery[END_REF]. A targeted drug administration also decreases undesirable side effects and increases therapeutic benefits [START_REF] Taveira | Topical administration of anticancer drugs for skin cancer treatment[END_REF]. Moreover the non-invasive nature of transdermal drug delivery allows to improve patient acceptability and compliance [START_REF] Lutton | Microneedle characterisation: The need for universal acceptance criteria and GMP specifications when moving towards commercialisation[END_REF]. This way of administration is commonly used for the treatment of skin cancers by photodynamic therapy (PDT) by using creams. PDT is a treatment relying on a combination of three parameters: drug content, oxygenation rate and light irradiation intensity. 5-aminolevulinic acid (5-ALA) is widely used as a PpIX photosensitive precursor and topically applied on the skin lesions. Light irradiation at PpIX exciting wavelength combined with the presence of oxygen promotes photochemical reaction leading to cell death. Treatment efficiency is linked to different factors including drug penetration. However, drug penetration is often hampered by the stratum corneum (SC), which is the outer first layer of epidermis that provides a barrier to external environment and difficult to penetrate for many drugs [START_REF] Lutton | Microneedle characterisation: The need for universal acceptance criteria and GMP specifications when moving towards commercialisation[END_REF]. Moreover, the physicochemical properties of 5-ALA such as its hydrophilicity or zwitterionic nature also limit its penetration, which prevents access to deep skin layers [START_REF] Kearney | Potential of microneedles in enhancing delivery of photosensitising agents for photodynamic therapy[END_REF][START_REF] Champeau | Photodynamic therapy for skin cancer: How to enhance drug penetration?[END_REF]. These limitations make PDT unsuitable for basal cell carcinoma (superficial, nodular or infiltrative) and invasive squamous cell carcinoma that have tumor cells in deep epidermis or dermis [START_REF] Donnelly | Hydrogel-Forming and Dissolving Microneedles for Enhanced Delivery of Photosensitizers and Precursors[END_REF][START_REF] Lin | Current evidence and applications of photodynamic therapy in dermatology[END_REF]. To overcome this lack of drug penetration, different (chemical or physical) strategies have been suggested [START_REF] Champeau | Photodynamic therapy for skin cancer: How to enhance drug penetration?[END_REF], among which the use of microneedles (MNs) appears to be a promising technique [START_REF] Donnelly | Microneedle-mediated intradermal delivery of 5-aminolevulinic acid: Potential for enhanced topical photodynamic therapy[END_REF][START_REF] Donnelly | Microneedle arrays permit enhanced intradermal delivery of a preformed photosensitizer[END_REF][START_REF] Lee | Photodynamic therapy with methyl 5aminolevulinate acid combined with microneedle treatment in patients with extensive alopecia areata: Correspondence[END_REF].

MNs are assembled together to form an array or patch that enables to bypass the SC by creating holes in it, resulting in enhanced transdermal drug delivery through the created microchannels [START_REF] Donnelly | Processing difficulties and instability of carbohydrate microneedle arrays[END_REF]. MNs have been developed with a height inferior to 900 µm in order to limit pain. Indeed, this height criterion improves patient compliance and safety since MN cannot penetrate deep vascular and nervous regions. At the same time, this height is adequate to successfully reach the dermal layers without inducing bleeding and touching active immune cells. Due to their microsize, MN cannot be seen by patients, which reduces needle phobia [START_REF] Gill | Effect of Microneedle Design on Pain in Human Volunteers[END_REF]. Although MN dimensions are in the microscale, their design with adequate materials confers them strong mechanical properties to pierce the SC, which allows the diffusion in skin tissues of hydrophilic or high molecular weight drugs [START_REF] Prausnitz | Transdermal drug delivery[END_REF].

Different types of MN-mediated drug delivery have been developed. Solid MNs can be inserted in the skin and then removed letting micron-scale pores in the skin surface, followed by drug formulation application. This passive diffusion of drugs requires a two-step application process, which is prone to errors in the delivered dose [START_REF] Iliescu | A review on transdermal drug delivery using microneedles: Current research and perspective[END_REF]. It also can be dangerous for the patient itself since materials used to produce solid MNs such as silicon have dubious biocompatibility and can break, which could lead to complications [START_REF] Donnelly | Hydrogel-Forming 29 and Dissolving Microneedles for Enhanced Delivery of Photosensitizers and Precursors[END_REF][START_REF] Liu | Transdermal delivery of relatively high molecular weight drugs using novel selfdissolving microneedle arrays fabricated from hyaluronic acid and their characteristics and safety after application to the skin[END_REF]. To avoid the two-step application, drugs have been coated on solid MNs. Nevertheless, the drugs were rapidly delivered in small quantity (≤ 2 mg) [START_REF] Donnelly | Processing difficulties and instability of carbohydrate microneedle arrays[END_REF][START_REF] Cormier | Transdermal delivery of desmopressin using a coated microneedle array patch system[END_REF][START_REF] Jain | 5-Aminolevulinic acid coated microneedles for photodynamic therapy of skin tumors[END_REF] and the risk of broken materials was still present. Hollow MNs are another type of MN whose principle is based on hypodermic needles. Such MNs could avoid the two-step process and deliver a large drug amount but due to their micro size they may become blocked by compressed dermal tissue when inserted into the skin [START_REF] Donnelly | Processing difficulties and instability of carbohydrate microneedle arrays[END_REF].

Recently, dissolving MNs have been attracting attention for drug delivery since they allow to get rid of the aforementioned issues. The principle is that a drug is encapsulated in the needle matrix that is biocompatible, soluble and mechanically robust. When inserted in the skin, this matrix is dissolved by body fluids and delivers the drug with a controlled kinetic profile depending on the chosen matrix. Materials used as matrix are often polymers, such as carbohydrates [START_REF] Miyano | Sugar micro needles as transdermic drug delivery system[END_REF][START_REF] Lee | Dissolving microneedles for transdermal drug delivery[END_REF] or other bio-sourced components [START_REF] Liu | Transdermal delivery of relatively high molecular weight drugs using novel selfdissolving microneedle arrays fabricated from hyaluronic acid and their characteristics and safety after application to the skin[END_REF]. Polymers such as polyvinylpyrrolidone [START_REF] Sullivan | Dissolving polymer microneedle patches for influenza vaccination[END_REF] or carboxymethylcellulose [START_REF] Lee | Dissolving microneedles for transdermal drug delivery[END_REF][START_REF] Lee | Dissolving Microneedle Patch for Transdermal Delivery of Human Growth Hormone[END_REF] allow a rapid dissolution ( ≤ 5 min or ≤ 20 min, respectively) whereas polylactic acid or poly(lactic-co-glycolic acid) are biodegraded in a few days [START_REF] Park | Polymer Microneedles for Controlled-Release Drug Delivery[END_REF][START_REF] Nayak | Potential of biodegradable microneedles as a transdermal delivery vehicle for lidocaine[END_REF].

Because the drug is embedded in the polymer matrix, the fabrication process must be soft to ensure drug stability and avoid as much as possible the use of organic solvents. Hyaluronic acid (HA) is a polysaccharide that can be shape-molded and stored at room temperature (RT). HA is a component naturally present in the human body, widely distributed in body tissues, intracellular fluids and well excreted by different organs ; it is a polymer fully biocompatible and already used in many medical fields [START_REF] Necas | Hyaluronic acid (hyaluronan): A review[END_REF]. Moreover, this is a water soluble polymer that can be dissolved after skin contact in a few minutes (≤ 10 min) [START_REF] Zhu | Rapidly Dissolvable Microneedle Patches for Transdermal Delivery of Exenatide[END_REF][START_REF] Mönkäre | IgGloaded hyaluronan-based dissolving microneedles for intradermal protein delivery[END_REF]. These characteristics make it a good candidate to produce dissolving MNs to transport 5-ALA.

In the present study, we aimed at exploring the processing and the properties of HA-MN with 5-ALA embedded in the polymer matrix. 5-ALA was embedded only in one easy step, in the whole patch including the basis without MN. The aim was to deliver 5-ALA from the top of the skin to the deeper skin lesions to allow PDT treatment on the whole damaged skin volume.

Since 5-ALA and HA were mixed together, interactions between the polymer and the drug was carefully studied and 5-ALA stability was investigated for different time periods. Furthermore, microneedles shape was designed as a "pencil-tip" to confer them strong mechanical properties to penetrate the cutaneous lesions. As all basal cell carcinomas do not extend to the same depth, different heights of needles were processed and characterized. Moreover MN-patch could be loaded with large different amounts of 5-ALA which offer different dosages. The characteristics of the MN-patch (compression, insertion tests, dissolution rate) have been conducted to evaluate whether this strategy is a promising approach to enhance PDT outcomes on deep skin cancers. All other chemicals were of analytical reagent grade.

Materials and methods

Fabrication of HA microneedles patches

Soluble HA microneedles were fabricated by solvent casting molding method. This technique consists of using an aluminium master structure to obtain a PDMS mold from which dissolvable polymer microneedle replicates are formed. The overall steps of the preparation of dissolving microneedles are displayed in scheme 1.

• Aluminum master structure

An array of 20 × 20 MN was machined on an aluminium surface of 25 × 25 mm 2 . MN design is similar to a pencil-tip: a conical tip sits on top of a cylinder body. Two types of MN were designed: a tall one (M N t ) and a little one (M N l ). For the tall microneedle patch, the height was 750 µm (height tip = 300 µm and height body = 450 µm), the base diameter was 300 µm and each MN was tip-spaced with 1200 µm. For the little microneedle patch, the height was 400 µm (height tip = 150 µm and height body = 250 µm), base diameter was 300 µm and each MN was tip-spaced with 1200 µm. • PDMS mold A PDMS solution (10% reticulated) was poured on the master structure tipped up, then vacuum was applied and PDMS was dried at (70 ± 1) • C for 24 h.

• Molding of HA microneedles

A HA solution (50 mg mL -1 ) was prepared by vigorous mixing in distilled water and different amounts of 5-ALA were added to the solution leading to final concentrations of 0, 10 or 50 mg mL -1 . 200 µL of HCl at 0.03 mol L -1 were added to the solution in order to be at pH of 5 and ensure 5-ALA stability. A solution amount of 2.0 mL was casted in the PDMS mold. The mold was centrifugated at 21 • C and 4000 rpm for 35 min to ensure a complete filling of the cavities. Then it was placed in an oven at (40 ± 1) • C for 24 h, after which the HA microneedle patch was gently removed from the mold. Each patch is expected to contain 100 mg of HA and 0 mg (MN-0), 20 mg (MN-20) or 100 mg (MN-100) of 5-ALA.

Characterization of 5-ALA-MN-patch

Microscopic analysis of HA microneedle arrays

A digital microscope (VHX 8000, Keyence) was used to observe the morphology (height, tip diameter, width of base) of microneedle patch under normal light. Microneedles heights were analyzed with the ImageJ software.

Mechanical properties

To determine the loss in height of MN when compressed at a precise force, a texturometer (TA.XT Plus, Stable micro systems, UK) was used. The MN array was placed on the metal platflorm and an axial compression force was applied with the probe. The MN array was pressed at a rate of 0.5 mm s -1 with a force ranging from 0 N per needle to 0.5 N per needle for 10 s.

Pre-test and post-test speed were of 5 mm s -1 , and the trigger force was set at 0.01 N. The patch was then observed under digital microscope and measurements were performed with the ImageJ software.

Water content in the microneedle patch

To evaluate the residual water content in the microneedle patch, thermogravimetric analysis (TGA) was performed at a heating rate of 5 • C/min on a TGA Q5000 from TA Instruments and TA universal analysis was the software used. To evaluate the amount of 5-ALA in the MN-patch, potassium phthalate monobasic (KHP) was added as an internal standard. A solution of KHP was prepared at 10 mg mL -1 in D 2 O.

For analysis, a small amount of the MN-patch (approximately 10 mg) was dissolved in 650 µL of D 2 O and 50 µL of the KHP solution was added. Then proton nuclear magnetic resonance spectra were recorded using a Bruker Spectrospin spectrometer operating at 300 MHz and 295 K. The TopSpin software was used to analyze the spectra. The content of 5-ALA was calculated using one of the signals of the internal KHP standard and one of the signals of the analyte as presented in equation 1, where x represents the analyte 5-ALA, N H KHP is the number of protons of KHP [START_REF] Taveira | Topical administration of anticancer drugs for skin cancer treatment[END_REF], N H x is the number of protons of 5-ALA (2), m KHP is the weighted amount of standard material potassium phthalate monobasic, M w x is the molar weight of 5-ALA (131.13 g mol -1 ), M w KHP is the molar weight of KHP (204.22 g mol -1 ), A x is the integral between 4.0 and 4.15 ppm and A KHP is the integral between 7.45 and 7.6 ppm.

m x = N H KHP × m KHP × M w x × A x N H x × M w KHP × A KHP (1)
The same equation 1 was also used to quantify the amount of the degradation product of 5-ALA, which is 2,5-pyrazinedipropionic acid (PY). When the analyte is PY, N H x is equal to 2, M w x is 224.21 g mol -1 and A x is the integral between 8.35 and 8.45 ppm.

The weight percentages of 5-ALA and PY were then obtained according to equations 2 and 3, respectively, where m 5-ALA and m P Y are calculated by equation 1 and m tot corresponds to the mass of the MN-patch (either MN-20 or MN-100) used for the analysis.

w 5-ALA = m 5-ALA m tot (2)
w P Y = m P Y m tot (3)
2.4.2. Interactions between drug and polymer followed by FTIR and 13 C NMR Molecular interactions between 5-ALA and HA in the MN-patch were studied with ATR-FTIR. FTIR spectra of the MN patch were performed using a spectrometer IRAffinity-1, MIracle-10 from Shimadzu in the wave number range from 500 to 5000 cm -1 with a resolution of 4 cm -1 .

Samples were directly placed on the crystal surface and scanned 32 times per analysis.

To determine the chemical structure of hyaluronic acid, analysis of the 13 C nucleus present in the compound was studied by 13 C NMR spectroscopy. Approximately ten milligrams of HA was dissolved in 700 µL of D 2 O. A DEPT-Q (Distorsionless enhancement by polarization transfer including the detection of quaternary nuclei) sequence was performed to complete carbon assignments using a Bruker Spectrospin spectrometer operating at 75 MHz, 355 K and using a 5 mm QNP probe.

2.5. Penetration and dissolution of the microneedles patch

Skin model

As an alternative to biological tissue, a modeling clay (Plastiline ®, composed of kaolin, sulfur and glycerine) was spread to obtain a thickness of 2 cm and used as a skin model regarding mechanical properties. Microneedle patches (MN-0, MN-20 and MN-100) were inserted in this phantom skin by thumb pressure that is estimated to be at 0.17 N/needle (measurement performed with the texturometer) and then was removed. The skin model was observed with a digital microscope to evaluate the penetration profile and depth.

Ex vivo rat skin

Ex vivo rat skin was streched and fixed with pins on a polystyrene plate. MN-patches were inserted in the ex vivo rat skin by thumb pressure. The pressure was kept constant for 10 sec.

Then the MN-patch was kept inserted in the skin for a precise amount of time (from 5 min to 45 min) with the help of an occlusive tape (Tegaderm™, 3M). After MN-patch removal, the skin was observed in top view and then it was embedded in tissue freezing medium (TFM, M-M FRANCE). As for the MN-patch, it was kept in a dry Petri dish until microscope analysis was carried out to quantify the dissolution kinetics.

Drug release measurements

Petri dishes (surface: 9.6 cm 2 ) were filled with 750 µL of water acidified with HCl to obtain a pH of 5. Therefore, a thin water layer (< 1 mm) was present in all the Petri dishes. Then MN-patches loaded with 5-ALA, either MN-20 or MN-100, were placed needles down into the Petri dishes and slightly agitated at 75 rpm. At predetermined time points, the solution was collected for analysis and replaced by a same volume of fresh water at pH5. The amount of solution removed was freeze dried for 72 h and then dispersed in D 2 O for quantifying released 5-ALA by 1 H NMR according to the previous equations hereinbelow. The experiments were conducted in triplicate.

In vitro cytotoxicity study

The cytotoxicity of MN-patches as well as individual products, 5-ALA, 5-ALA-free patches (MN-0) and product of 5-ALA biodegradation (PY), was assessed. To this end, the patches or pure chemicals were dissolved in cell culture media. After 24 h of preincubation of the cells (NIH3T3 murine fibroblats cells, 4 × 10 3 cells/well) in DMEM containing 10 % of new born calf serum and 1 % of Pen Strep, the medium was replaced by one containing the different tested products followed by a new incubation of 24 h at 37 • C. The WST-1 reagent was then added. After 2 h at 37 • C, absorbance at 450 nm was recorded using a microplate reader (Infinite M1000, Tecan). The absorbance was linked to the metabolic activity of cell mitochondria and the density of living cells (DLC) was calculated as mentioned in equation 4, where AS, APC and ANC respectively represent the absorbances of the sample, the positive control and the negative control, the negative control being cells growing in the culture media without any added product and the positive control being cells growing in the cell culture media containing

H 2 O 2 at 0.01 mol L -1 . DLC = AS -AP C AN C -AP C × 100 (4)

Results and discussion

Microneedle preparation and characterizations

Tall microneedle (M N t ) and little microneedle (M N l ) were successfully produced with expected dimensions using the solvent casting molding method as displayed in figure 1. The heights of the M N t and M N l microneedles were respectively 749 ± 6 µm and 390 ± 7 µm and the base diameter was 285 ± 10 µm which corresponds well to the original master structure dimensions.

The slight difference in microneedle height (respectively 0.13 % and 2.5 % for M N t and M N l ) and diameter (5 %) might be explained by the retraction of the HA matrix during the drying process.

Mönkäre et al. also reported a height reduction of 6.6 % when they developed immunoglobulinGloaded hyaluronan-based dissolving microneedles by solvent casting molding method and they explained it by the shrinking of the HA during drying process [START_REF] Mönkäre | IgGloaded hyaluronan-based dissolving microneedles for intradermal protein delivery[END_REF]. Similarly Zhao et al. fabricated 5-ALA-loaded dissolving microneedles made of HA and attribute the 9.3 % reduction in height to water evaporation during drying process [START_REF] Zhao | Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor[END_REF] which could consequently induce polymer shrinkage.

Drug distribution was expected to be homogeneous as the solution containing 5-ALA and HA was homogeneous before pouring. Indeed, a uniform pale yellow color could be observed from the tip to the base of the MN-patch containing 5-ALA (Figure 1, right). However, crystallization or precipitation of 5-ALA could have taken place during drying, leading to inhomogeneous drug distribution. Consequently analysis of 9 different parts of the patch was performed in order to quantify drug content in each part. Each piece was analyzed by 1 H NMR (Figure 2) and the percentage of 5-ALA was calculated according to 1 H NMR equations (Eq 1 and Eq 2).

The theoretical values for the 5-ALA relative weight percentage, considering only dry matters, are respectively equal to 17 %(w/w) and 50 %(w/w) for MN-20 and MN-100. Taking into account the residual water, a lower 5-ALA absolute weight percentage is expected. Indeed, the results presented in figure 3 show that the absolute percentage of 5-ALA was of 10.0 %(w/w) and 31.2 %(w/w) for MN-20 and MN-100, with low intra and inter patches standard deviations (< 2% (w/w)) demonstrating the homogeneous distribution of the drug. For the MN-20 patch, the content in water was evaluated at 14 %(w/w) by TGA corresponding to a drop of the 5-ALA weight percentage from 17 %(w/w) to 14 %(w/w), which is in good agreement with the obtained value of 10.0 %(w/w) taking into account all the experimental errors.

The process investigated in this study allows to produce reproducible MN-patches with precise amount of drug homogeneously distributed in the whole patch. Moreover, the amount of 5-ALA loaded in the MN-patch was a thousand times more important than previous studies lead by Zhao and not in the whole needle [START_REF] Zhao | Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor[END_REF][START_REF] Zhu | 5-Aminolevulinic Acid-Loaded Hyaluronic Acid Dissolving Microneedles for Effective Photodynamic Therapy of Superficial Tumors with Enhanced Long-Term Stability[END_REF]. Therefore they were forced to process the microneedles in several steps whereas performing a unique easy solvent casting method as presented in section 2.2 simplifies the process which would be an important asset for industrialization. Even when the active component is mixed with the polymer solution, several steps may be involved. It was the case for Mönkäre et al. that applied vacuum prior to centrifugation to produce IgGloaded hyaluronan-based dissolving microneedles [START_REF] Mönkäre | IgGloaded hyaluronan-based dissolving microneedles for intradermal protein delivery[END_REF]. For the MN-patches developed in this study, centrifugation was sufficient to ensure the complete filling of all microneedle tips as the poured solution at a low HA concentration (50 mg mL -1 ) had a moderate viscosity with a shear The others peaks between 2 and 6 ppm are characteristics either of 5-ALA or HA.

viscosity measured at 0.05 Pa s (shear rate: 50 s -1).

5-ALA stability in the MN-patch

To ensure a long drug stability, special care was taken by controlling the temperature, the 255 pH and the 5-ALA concentration because these three parameters are considered to be the most influencing for respecting 5-ALA stability [START_REF] Champeau | Photodynamic therapy for skin cancer: How to enhance drug penetration?[END_REF]. Indeed, 5-ALA is known to be unstable and dimerize in PY, constituting the major degradation product. This is usually a problematic aspect in the design of drug delivery systems intended for photodynamic therapy [START_REF] Mccarron | Analysis of pyrazine 2,5-dipropionic acid in 5-aminolevulinic acid-loaded urological and topical delivery vehicles: Methodology and assay validation[END_REF].

According to previous studies led by De Blois et al., the risk of 5-ALA to dimerize is reduced 260 when the temperature is decreased to 21 • C [START_REF] De Blois | Development of a stable solution of 5-aminolaevulinic acid for intracutaneous injection in photodynamic therapy[END_REF]. The authors modelled the degradation rate of 5-ALA as a second order reaction kinetics and an Arrhenius plot allowed to determine the half The second parameter that has been adjusted in order to optimize 5-ALA bioavaibility, was the pH, as it is known that 5-ALA is degraded at pH higher than 5 [START_REF] Bunke | Degradation mechanism and stability of 5-aminolevulinic acid[END_REF]. Since human skin pH is estimated on average at 5 [START_REF] Lambers | Natural skin surface pH is on average below 5, which is beneficial for its resident flora[END_REF], pH 5 has been defined as the optimal value for 5-ALA MN-patch preparation that should not induce skin disorders or irritation when the MN-patch will dissolve [START_REF] Baranda | Correlation between pH and irritant effect of cleansers marketed for dry skin[END_REF]. This pH value was adjusted by addition of HCl, with a final concentration of 3 mmol L -1 .

The third parameter to adjust is the 5-ALA concentration that is required to be kept as low as possible to ensure its stability [START_REF] Elfsson | Stability of 5aminolevulinic acid in aqueous solution[END_REF]. Nevertheless, due to the final application in PDT requiring high doses of drug 2 , two high contents of 5-ALA in MN-patches were evaluated (20 mg to 100 mg per patch).

First, the presence of 5-ALA in the MN-patch prepared with a pH solution at 5 and a drying 1 Half-life can be defined as the time where the 5-ALA concentration reduced to half of its original value. 2 The conventional PDT treatment using the prodrug in a Metvixia® cream advise to administrate ≈ 80 mg.

temperature of 40 • C was confirmed by FTIR. Figure 4 shows the IR spectra of MN-20 and MN-100 compared with those of HA and 5-ALA alone. On the four spectra, the bands observed between 3200-3600, 2850-3000 and 1550-1650 cm -1 were assigned to vibration of OH, CH 2 and primary NH 2 respectively. The bands at 1730 cm -1 and at 1600 cm -1 correspond respectively to the carbonyl groups of 5-ALA and the amid group of HA [START_REF] Chen | Infrared spectral comparison of 5-aminolevulinic acid and its hexyl ester[END_REF][START_REF] Reddy | Purification and characterization of hyaluronic acid produced by Streptococcus zooepidemicus strain 3523-7[END_REF]. These two bands appear in MN-20 spectra and are more intense in the MN-100 sample which evidenced the presence of the two compounds in the final material. The band relative to primary amino group is also present in the MN-20 and MN-100 spectra, which confirms the presence of 5-ALA in the patch. MN-20 and MN-100 spectra do not present new large peaks which might suggest that there is no covalent bonds between 5-ALA and HA. Nevertheless, this spectra cannot show that no chemical reaction occurred between the HA polymer and the 5-ALA prodrug. Even if unexpected, since the MNpatch preparation was performed at low temperature (40 • C) without the presence of a coupling agent [START_REF] Bulpitt | New strategy for chemical modification of hyaluronic acid: Preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels[END_REF], a reaction between the HA carboxyl group might occur with the amino group present on the 5-ALA to form an amido compound. Infrared spectroscopy correlation table indicates that the characteristic absorption peaks of the amido-group are between 3050-3500 cm -1 (2 large bands), 1630-1710 cm -1 for respectively the vibration of N-H and C=O. Therefore these bands are potentially covered by the HA signal and results of figure 4 cannot strictly confirm that no covalent groups were created.

To further verify that HA was not modified because of 5-ALA, 13 C NMR was performed. As presented in figure 5, all the characteristic peaks of HA were present in the HA that has been in contact with 5-ALA and correspond to the one reported in literature [START_REF] Napier | Effect of calcium on structure and function of a hyaluronic acid matrix: Carbon-13 nuclear magnetic resonance analysis and the diffusional behavior of small solutes[END_REF][START_REF] Izawa | Streptococcus thermophilus produces exopolysaccharides including hyaluronic acid[END_REF]. Some slight shifts (inferior to 1 ppm) may be observed but they have been previously described as conformational rearrangements due to hydrogen bonds [START_REF] Kvam | 1H-and 13C-NMR studies of solutions of hyaluronic acid esters and salts in methyl sulfoxide: Comparison of hydrogenbond patterns and conformational behaviour[END_REF][START_REF] Cowman | C-NMR Studies of Hyaluronan: Conformational Sensitivity to Varied Environments[END_REF]. Except at 28 ppm where a low intensity peak was observed due to an impurity, no other peaks were detectable. Consequently, this experiment demonstrate that no covalent bonding was created between 5-ALA and HA. Furthermore the filtrate of the MN-patch isolated with 10 kDa Amicon® Ultra Centrifugal Filters was analysed by 1 H NMR and only evidenced the presence of 5-ALA with a spectrum similar to pure 5-ALA.

Once no interaction between 5-ALA and HA was proven, the 5-ALA stability was studied.

Initially, 5-ALA and PY were separately dissolved in D 2 O and figure 6 summarizes the data derived from their 1 H NMR analysis. The chemical shifts presented were close to those reported in the literature [START_REF] Mccarron | Analysis of pyrazine 2,5-dipropionic acid in 5-aminolevulinic acid-loaded urological and topical delivery vehicles: Methodology and assay validation[END_REF][START_REF] Bunke | Degradation mechanism and stability of 5-aminolevulinic acid[END_REF][START_REF] Jaffe | Nuclear magnetic resonance studies of 5-aminolevulinate demonstrate multiple forms in aqueous solution[END_REF]. PY spectrum shows a singlet signal for the aromatic proton (a) at Indeed TGA experiments showed that the water content was 15 %(w/w) after 145 days whereas it was 14 %(w/w) at day 1. The adsorption of H 2 O molecules on the microneedles patch may increase the global mass of the patch (m tot ) and could affect the absolute weight percentage of 5-ALA according to equation 2 which could explain a 1 % slight decrease. Overall, this mean value of 7 %(w/w) stays in a satisfactory experimental range and evidences a very good stability of 5-ALA in the MN-20 patch over 5 months.

On the other hand, there was no degradation of 5-ALA in PY higher than 1 %(w/w) until the 145 th day. A very limited degradation below 1% during this period is considered as a very encouraging result. Further optimization of storing conditions could be undertaken in order to decrease this degradation level. Indeed, PY is a compound with unknown toxicity and scant information concerning its effect during PDT is available. Its presence should then be kept to a minimum [START_REF] Donnelly | Derivatives of 5-aminolevulinic acid for photodynamic therapy[END_REF] by avoiding 5-ALA degradation, which should guarantee a safe and successful PDT treatment [START_REF] Mccarron | Analysis of pyrazine 2,5-dipropionic acid in 5-aminolevulinic acid-loaded urological and topical delivery vehicles: Methodology and assay validation[END_REF]. Although, all the parameters responsible of 5-ALA stability were not optimized as much as possible, the stability results (figure 7) were satisfactory since they were higher than those expected regarding previous results reported in the literature [START_REF] Mccarron | Analysis of pyrazine 2,5-dipropionic acid in 5-aminolevulinic acid-loaded urological and topical delivery vehicles: Methodology and assay validation[END_REF][START_REF] De Blois | Development of a stable solution of 5-aminolaevulinic acid for intracutaneous injection in photodynamic therapy[END_REF][START_REF] Bunke | Degradation mechanism and stability of 5-aminolevulinic acid[END_REF].

MN-patch mechanical properties: compression and insertion

Morphology and compression mechanical properties

Prior to biological validations by inserting the patch in skin models or animals skin, it was necessary to control whether the MN-patch had sufficient mechanical properties to pierce the stratum corneum without microneedles fracture, bending or height reduction.

345

Mechanical compression tests were performed to evaluate the MN-patch mechanical strength and results for the M N l are displayed in figure 8. There was no significant deformation or fracture when the compression force applied was below 0.5 N/needle. The loading at 20 or 100 mg in the M N l -patch did not affect the mechanical behaviour. A slight height reduction (about 50 µm) could be observed for the M N l -0, M N l -20 or M N l -100 when the force compression applied was equal to 0.5 N/needle. It represents a loss in height of 12.5 %. Almost the same behaviour was observed for the M N t -0 and M N t -20 (results not shown) since the loss in height do not exceed 7 % even at 0.5 N/needle. For the M N t -100 at 0.3N/needle and 0.5 N/needle, the loss in height were higher and reached 17 % and 53 %. Nevertheless, the insertion force in the skin was measured with the texturometer and estimated at 0.17 N/needle which insure that insertion could occur without microneedle bending or breaking. The performances of all our needles except M N t -100 were comparable or even better than the previous reported one indicating a height reduction of ALA-loaded HA microneedle patch (initial height: 900 µm) of almost 200 µm for a compression force applied of 0.5 N/needle, which corresponds to a loss in height of 22 % [START_REF] Zhao | Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor[END_REF]. Furthermore, the important height reduction observed for the M N t -100 might be explained by Zhu et al. work, indicating that the decrease of polymer interactions is due to 5-ALA small molecules that insert between the polymer chains. Consequently, the Young's modulus is decreased and the microneedle become more deformable [START_REF] Zhu | 5-Aminolevulinic Acid-Loaded Hyaluronic Acid Dissolving Microneedles for Effective Photodynamic Therapy of Superficial Tumors with Enhanced Long-Term Stability[END_REF].

In order to gather all required properties for the intended application, the shape of microneedle has been designed in order to reach sufficient mechanical strength for skin insertion, and optimal height for therapeutic indication. To this end, the morphology of the MN-patch developed here consists in a "pencil-tip" shape (Figure 1) that presents an aspect ratio for the M N t and M N l respectively equal to 5:2 and 4:3 that should be sufficient to pierce and facilitate insertion in the skin [START_REF] Lee | Dissolving microneedles for transdermal drug delivery[END_REF][START_REF] Gittard | The effects of geometry on skin penetration and failure of polymer microneedles[END_REF]. Indeed, the aspect ratio recommended for clinical use by Gittard et al. was 2:1 as it exhibits high mechanical strength and no difficulty for penetration. Nevertheless, this value is only an arbitrary recommendation since they also showed that other aspect ratio such as 3:1 could be efficient too. The most important conclusion of their study showed that when the aspect ratio increased it was associated with lower failure force [START_REF] Gittard | The effects of geometry on skin penetration and failure of polymer microneedles[END_REF]. Secondly and still to ensure efficient mechanical properties, the base was cylindrical with a diameter fixed at 300 µm. It has been demonstrated that contrary to square or rectangular MN, circular MN can withstand higher forces (stress and bending force) [START_REF] Aggarwal | Geometrical effects in mechanical characterizing of microneedle for biomedical applications[END_REF]. Thirdly, for the intended application of skin cancer, it is necessary to reach the lower epidermis or the upper dermis without inducing pain or high tissue damages. Thus, depending on the state of the lesions, a low microneedle height of 400 µm or a high one of 750 µm would be the most appropriate. Moreover, these chosen heights are inferior to 900 µm which has been identified as a high value inducing breaking of the MNs [START_REF] Aggarwal | Geometrical effects in mechanical characterizing of microneedle for biomedical applications[END_REF][START_REF] Davis | Insertion of microneedles into skin: Measurement and prediction of insertion force and needle fracture force[END_REF].

Insertion tests in ex vivo skin and a skin model

Since mechanical strength of microneedles was measured as high enough compared to theoretical skin hardness, it was necessary to further examine their insertion ability. The MN-patch was applied to hair shaved ex vivo rat skin and removed. Subsequently, microholes were created on top of the skin and a penetration profile might be observed in cross section. Nevertheless, figure 9 shows that the insertion depth in ex vivo rat skin was quite low (less than 20 µm) which was far shorter than the microneedles height (750 µm). The main reason of this observation is due to the tissue embedding process with freezing medium which can deeply modify cutaneous 390 structures by shrinking. It was thus necessary to find a skin phantom model with similar mechanical properties and easy to cut without a prior embedding process in order to assess the puncture ability of our device.

Classically, gelatin based gels are used but as it is prepared in water, HA-based MN dissolved very rapidly during insertion assay. In order to overcome this difficulty, a Plastiline® material made of oils, mineral waxes and fillers has been chosen with a Young's modulus measured at 10 kPa very close from the skin's one. Therefore it was possible to observe the microchannels created after insertion.

After microneedles insertion, a slice of the skin phantom was manually cut under a binocular magnifier with a scalpel following a ruler. The cross-section observation suggests that the insertion depth was about 374 ± 10 µm and 594 ± 61 µm for respectively M N l and M N t as presented in figure 10. Larrañeta et al. worked on another alternative that consists in assembling multiple layers of Parafilm™. Authors then inserted the microneedles and evaluated the percentage of holes created in each Parafilm™layer [START_REF] Larrañeta | A proposed model membrane and test method for microneedle insertion studies[END_REF]. As Plastiline®, Parafilm™method allows to develop a rapid microneedle quality control. Similarly, Enfield et al. chose a Blu-Tack® material which is a pressure-sensitive clay-like adhesive known for its low elasticity and soft pliable consistency [START_REF] Enfield | In-vivo dynamic characterization of microneedle skin penetration using optical coherence tomography[END_REF].

The microneedle array with a needle height of 280 µm was inserted into this artificial material and the micropores created were observed under optical coherence tomography system. The depth penetration measured on Blu-Tack® material and forearm skin were respectively 254±7 µm and 179 ± 14 µm [START_REF] Enfield | In-vivo dynamic characterization of microneedle skin penetration using optical coherence tomography[END_REF]. Overall, materials mimicking tissue (Plastiline ®, Parafilm™or Blu-Tack®) appeared to be good alternatives to biological tissue for MN insertion studies however the depth penetration might be overvalued as shown by Enfield et al. [START_REF] Enfield | In-vivo dynamic characterization of microneedle skin penetration using optical coherence tomography[END_REF]. On the other hand, top view observation on ex vivo rat skin (figure 9, left) and on the membrane model (figure 10, left) evidenced that microneedles had perfored the skin barrier since microholes were observed. The interval between them was equal to 1200 µm which corresponds well to the original spacing on master structure. Pore diameters created were measured on the Plastiline membrane model at 269 ± 10 µm which is close to the microneedle base diameter set up at 300 µm.

In addition, the spacing between the microneedles has been demonstrated as a parameter influencing the insertion force required to make the microneedles penetrate through the skin.

Indeed, Olatunji et al. developed Gantrez® microneedles and showed that the insertion forces required for 330 µm tip-spaced MNs (16 MNs/array) and 900 µm tip-spaced MNs (4 MNs/array) were respectively equal to 0.030 N/needle and 0.028 N/needle (insertion speed of 0.5 N mm -1 ) [START_REF] Olatunji | Influence of Array Interspacing on the Force Required for Successful Microneedle Skin Penetration: Theoretical and Practical Approaches[END_REF]. Another study led by Kochhar et al. established that penetration percentage was more important when spacing between MNs tips increased. For instance, they revealed that microneedles (microneedle array: 1.44 cm 2 ) with a base diameter set at 300 µm and a tip-interspacing of 1800 µm had optimal characteristics to penetrate the skin with 64 % penetration [START_REF] Kochhar | Effect of Microneedle Geometry and Supporting Substrate on Microneedle Array Penetration into Skin[END_REF]. A large spacing also avoids to form a "bed of nails" described by the same authors as a too high density of microneedles that reduce penetration and efficiency of microneedle penetration [START_REF] Kochhar | Effect of Microneedle Geometry and Supporting Substrate on Microneedle Array Penetration into Skin[END_REF]. Moreover a high density of microneedles is also restrictive as demonstrated by Gill et al. who showed that a 10-fold increase in the number of microneedles is two-fold more painful [START_REF] Gill | Effect of Microneedle Design on Pain in Human Volunteers[END_REF]. In this study, in agreement with these previous research works, the spacing between microneedle tips was set at 1200 µm with 400 MNs/array. Such parameters enable easy manual MN-patch insertion as presented in figures 9 and 10, would be sufficient for high dose delivery while avoiding crowding, and should ensure a minimal pain.

3.4. Dissolution rate of the MN-patch after insertion in ex vivo rat skin, release kinetics of 5-ALA and MN-patch biocompatibility HA has been widely used the last decade as a component of artificial matrices and in bioengineering for tissue scaffolding or skin reparation. HA is known to be a highly water-soluble polysaccharide. Consequently when the HA-based MN-patch will be applied on the skin, which is approximately composed of 70 % of water, it is firstly expected that the MN-patch dissolves and releases 5-ALA [START_REF] Verdier-Sévrain | Skin hydration: A review on its molecular mechanisms[END_REF]. When the whole patch will be dissolved into the skin, HA will be further biodegraded since this polymer is already present in the human body and naturally degraded. In fact, the catabolism of HA has been widely described in the literature as a depolymerization process due to the enzymatic cleavage of glycosidic linkages leading to smaller molecule chains. These smaller molecules are naturally present in the human body and will join the natural elimination process consisting in a degradation metabolism in liver or lymph nodes thanks to endothelial cells. The half-life of HA was estimated between 1 and several days. Then the remainder enters in the bloodstream and is eliminated with a half-life between 2 and 5 min [START_REF] De Boulle | A Review of the Metabolism of 1,4-Butanediol Diglycidyl Ether-Crosslinked Hyaluronic Acid Dermal Fillers[END_REF][START_REF] Fraser | Turnover and metabolism of hyaluronan[END_REF][START_REF] Fraser | Hyaluronan: Its nature, distribution, functions and turnover[END_REF]. Therefore, HA is a good candidate to be dissolved by the skin, leading to 5-ALA release, and be furthermore rapidly biodegraded by a natural enzymatic pathway. Kinetics of MN-patch dissolution was estimated by removing the patch from the ex vivo skin at different times and results are displayed in figure 11. At 10 min, there was no obvious dissolution for the M N l whereas M N t dissolution was initiated. This fact could be explained by the M N t size that can reach deeper skin layer and particularly the lower dermis which is more hydrated and could enhance HA dissolution. A significant dissolution for both patches (M N l or M N t ) occurred after 45 min. After 60 min, the disintegration of the patch base was noticeable.

These experiments showed that when the MN-patch will be applied on the skin, a first drug release at 45 minutes time scale will take place due to microscale needles fast dissolution. Then a second slower drug release at 60 minutes time scale will occur due to the MN-patch base disintegration and could constitute a drug reservoir. However, the dissolution was not homogenous. Indeed, MN-patch surface is quite large (25 × 25 mm 2 ) and it is conceivable that the entire patch do not receive homogenous force which lead to partial microneedle penetration and inhomogenous dissolution. Different studies have They proved a successful insertion but there was no microscopical images of the microneedle disintegration [START_REF] Ripolin | Successful application of large microneedle patches by human volunteers[END_REF]. Overall, adhesive bandages may be an effective solution to ensure microneedle insertions and keeping them into the skin. Nevertheless, if the lesion is located on the nose a bandage may be unsuitable to maintain a pressure on the MN-patch and a thumb pressure may be more easier to perform. Therefore, the force that 8 people exerted spontaneously with their thumb, for 10 seconds using the texturometer was measured. We have observed that when the indication was "apply a pressure on the whole patch that could allow its insertion in the skin" the force was measured at 24.5 ± 7.5 N which represent 0.34 N/needle and is higher than the one needed for good needles insertion in the skin.

In the other hand, the dissolution rate can be controllable by the initial polymer solution i.e HA concentration and/or HA molecular mass. Indeed, a lower HA concentration will result in a faster dissolution. Similarly an upper HA molecular mass will lead to a lower dissolution due to stronger entanglement between polymeric chains (Preliminary experiments were realized on gelatin phantom skin and are not presented here). In fact, Leone et al. have also described these properties and they mentioned that a balance may be found between dissolution rate and mechanical properties since a lower molecular (4.9 kDa) mass may dissolve faster than upper one at 4.9 kDa but microneedles appeared less robust [START_REF] Leone | Hyaluronan molecular weight: Effects on dissolution time of dissolving microneedles in the skin and on immunogenicity of antigen[END_REF].

This first evaluation of MN-patch dissolution rate was comforting with the end-use application desired. Nevertheless, on ex vivo rat skin, it was not possible to evaluate the amount of 5-ALA released at predetermined time points because a long extraction protocol would have been necessary and might have degraded the 5-ALA. Therefore, an in vitro drug release experiment was set up in an attempt to mimic the dissolution rate observed in ex vivo rat skin. Indeed, after 80 min, macroscopic observation highlighted that the patch was almost completely dissolved as it was after 60 min in ex vivo rat skin. As displayed in figure 12, the amount of 5-ALA released was respectively equal to 77 % and 88 % for MN-20 and MN-100. Therefore, it may be assumed that these quantities may also be released during ex vivo or in vivo experiments which could be a sufficient quantity to perform a PDT treatment in a reasonable time-frame.

The 5-ALA release kinetics of MN-100 appears to be slightly faster than that of the MN-20 patch. Indeed, drug delivery is governed by diffusion through the polymer (Fick's law) and by the polymer dissolution (due to convection) [START_REF] Chen | Fundamental of Diffusion and Dissolution[END_REF]. Considering that MN-100 contains more 5-ALA, the prodrug diffusion through the microneedles might be more intense and explain this larger 5-ALA percentage released. Overall, the ability of the patch to deliver 5-ALA in a short time period (< 1h30) is impressive compared to previous studies [START_REF] Donnelly | Bioadhesive patch-based delivery of 5-aminolevulinic acid to the nail for photodynamic therapy of onychomycosis[END_REF][START_REF] Donnelly | Drug Delivery of Aminolevulinic Acid from Topical Formulations Intended for Photodynamic Therapy[END_REF][START_REF] Morrow | Influence of penetration enhancers on topical delivery of 5-aminolevulinic acid from bioadhesive patches[END_REF]. For example in 2006, McCarron et al. showed that a bioadhesive patch made of poly(methyl vinyl ether-co-maleic anhydride) and tripropyleneglycol methyl ether and containing either 19 mg cm -2 or 50 mg cm -2 of 5-ALA only released 57 % in 6 hours [START_REF] Mccarron | Design and evaluation of a water-soluble bioadhesive patch formulation for cutaneous delivery of 5-aminolevulinic acid to superficial neoplastic lesions[END_REF]. However, the in vitro measurements that the authors investigated were conducted with a Franz diffusion cell and they only dosed the amount that cross all the skin phantom. We may assume that 5-ALA was still contained in the skin itself.

Although 5-ALA is a key parameter at the origin of PpIX production, instead of dosing this prodrug precursor, it would also be interesting to evaluate the amount of PpIX itself by testing the patches in vivo on skin lesions in order to evaluate PpIX accumulation in the different skin layers [START_REF] Donnelly | Drug Delivery of Aminolevulinic Acid from Topical Formulations Intended for Photodynamic Therapy[END_REF].

Since dissolution was effective on ex vivo rat skin, and release of 5-ALA was effective, it is now important to control its cytotoxicity with a view to future use of this device in vivo. To evaluate the biocompatibility of the MN-20 and MN-100 patches, a WST-1 cytotoxicity assay was performed and results are presented in figure 13.

The density of living cells cultured in medium which were in contact with MN-0 was com- parable to the negative control indicating that MN-0 was not cytotoxic. It was expected since MN-0 was only composed of HA and this polymer is well known for its biocompatibility as a naturally occuring biopolymer. For example, in a 70 kg-human body roughly 15 g of HA is present and 50 % is concentrated in the skin [START_REF] Stern | Hyaluronan catabolism: A new metabolic pathway[END_REF]. Developing HA based microneedles was relevant and cytotoxicity experiments approved it.

Furthermore the density of the living cells cultured in medium which were in contact with MN-20 and MN-100 were also comparable to the negative control no matter the concentration. As the MN-p contained 5-ALA and PY, different conditions with these two molecules were also tested and results are displayed in figure 14. As presented in section 3.1, the weight percentages of 5-ALA in the MN-20 and MN-100 were respectively equal to 10.0 %(w/w) and 31.2 %(w/w). Therefore, when the cytotoxicity of the patch was tested at the higher concentration (40 µg mL -1 ), the 5-ALA concentration was at most equal to 4.0 µg mL -1 or 12.5 µg mL -1 and these concentrations appeared to be non cytotoxic for the cells (figure 14, left) which was comparable to the results obtained in the litterature by Li et al., Dan et al. [65,[START_REF] Dan | 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Promotes Phenotypic Switching from Dedifferentiated to Differentiated Phenotype via Reactive Oxygen Species and p38 Mitogen-Activated Protein Kinase in Vascular Smooth Muscle Cells[END_REF]. Moreover, the density of living cells after contact with PY remains about 100 % which was comforting as a 1 % degradation of 5-ALA in PY was observed in MN-patch.

Conclusion

Microneedle patches were fabricated by a single step of solvent casting molding method. The manufacture way very easy, low cost and relevant which would allow a facile translation of this technology. Two heights were developed: 400 µm and 750 µm. Each microneedle patch could contain 20 mg or 100 mg of 5-ALA consequently a single application is sufficient to deliver the amount required for PDT treatment. 5-ALA stability was followed to ensure that there was no dimerization in PY and exhibited a very good stability in the MN-patch even 5 months after manufacturing. Mechanical compression tests were performed and the different kinds of MNpatches do not present a significant deformation when the force applied was not superior to 0.3 N/needle. These results suggested that MN-patch had efficient strength to pierce the stratum corneum. Indeed, ex vivo rat skin was well perforated due to microneedle application since micropores were created. A clear penetration profile was distinguishable on phantom skin with depth penetration of 374 ± 10 µm and 594 ± 61 µm for respectively M N l and M N t . Dissolution rate was estimated on ex vivo rat skin demonstrating that the microneedles were dissolved within 45 min and the support started to dissolve after 60 min.

According to the previous results, MN-patches might be a promising technique to enhance 5-ALA penetration and produce PpIX in deeper skin lesions. In the very near future, in vivo tests on rats suffering from precancerous skin lesions will be carried out to show microneedle possible benefit during PDT treatment.

2. 1 . Chemicals 5 -

 15 ALA hydrochloride salt (GMP grade) was purchased from Biosynth Chemistry & Biology. Polydimethylsiloxane (PDMS) (Sylgard , Dow Corning), potassium phtalate monobasic (KHP, TraceCERT®), Dulbecco's modified eagle medium (DMEM), penicillin streptomycin solution (Pen Strep) and the cell proliferation reagent WST-1 were obtained from Sigma-Aldrich Chemical Co. HA with a molecular weight (MW) of (5.50 ± 0.04) × 10 4 g mol -1 was purchased from Contipro France. 2,5-pyrazinedipropionic acid (PY) was purchased from Carbosynth Product.

Scheme 1 :

 1 Scheme 1: Preparation of the MN-patch by solvent casting molding method. Three major steps are essential: aluminium master structure design, fabrication of PDMS mold and casting of the solution in the mold. Two heights were designed for the aluminium master structure: a tall one M Nt at 750 µm and a little one M N l at 400 µm.

2. 4 .

 4 Drug stability in the MN patch 2.4.1. Chemical stability of 5-ALA by quantitative proton nuclear magnetic resonance ( 1 H NMR)

Figure 1 :

 1 Figure 1: Digital microscopy images of MN-patch fabricated by solvent casting molding method. (Left: M N l -0, right: M Nt-20.) Scale bars represent 500 µm.

  et al. or Zhu et al. since both authors only added 5-ALA in the tips of the HA-microneedles

Figure 2 :

 2 Figure 2: 1 H NMR spectrum of a piece of MN-20. The integration value of the singlet of 5-ALA at 4.04 ppm was for 2 protons and the integration value of the multiplet of KHP between 7.4 and 7.5 ppm was for 0.64 protons.

Figure 3 :

 3 Figure 3: A: photograph of the M Nt-20 patch, the dotted lines represent the different parts of the patch to evaluate the percentage of 5-ALA in each samples numeroted from 1 to 9. B: table summarizing the weight percentages of 5-ALA in the MN-20 and MN-100 patches. Experiments were repeated three times. Results are presented as mean value ± standard deviation.

8. 4

 4 ppm and two triplet signals at 3.1 and 2.8 ppm characterising CH 2 groups (b and c) from the propionic acid chains. 5-ALA spectrum shows a singlet signal for the CH 2 (x) groups at 4.1

Figure 4 :

 4 Figure 4: IR spectra of 5-ALA, HA and the microneedle patches MN-20 and MN-100. Blue strips correspond to 5-ALA signature bands and orange strips to HA signature bands.

Figure 5 :

 5 Figure 5: A: 13 C NMR spectrum of MN-0 (HA only). B: HA from MN-20 was isolated with 10 kDa Amicon® Ultra Centrifugal Filters, analyzed by 13 C NMR and compared to the one above. C / CH 2 are down and CH / CH 3 are up on the spectrum. C: Structure of HA and summary of the 13 C chemical shifts (parts per million).

Figure 6 :Figure 7

 67 Figure 6: Structure of 2,5-pyrazinedipropionic acid and 5-aminolevulinic acid and summary of 1 H chemical shifts (parts per million)

  g h t p e r c e n t a g e ( % , w / w ) T i m e ( d a y s )

Figure 7 :

 7 Figure 7: Stability of 5-ALA in the MN-20 patch. Analyses were performed in triplicate. Results are presented as mean value ± standard deviation.

  340

Figure 8 :

 8 Figure 8: Mechanical behaviour of M N l after compression at different forces. Analyses were performed on three different patches with at least 10 height measurements on different microneedles from each patch. Results are presented as mean value ± standard deviation.

Figure 9 :

 9 Figure 9: Left: top view observation of rat skin after MN-patch removal. Right: cross section of cryoconserved rat skin after hematoxylin/eosin coloration. The blue circles show the penetration profile of microneedle in the skin.

Figure 10 :

 10 Figure 10: Left: top view observation of phantom skin after MN-patch removal. Middle: cross section of phantom skin after M N l removal. Right: cross section of phantom skin after M Nt removal. Experiments repeated 5 times. Scale bars represent 1000 µm.

Figure 11 :

 11 Figure 11: Microscopic images of microneedle patches after their application to ex vivo rat skin. Upper line corresponds to M N l and lower line to M Nt. From left to right: 10, 45 and 60 min. Scale bars represent 500 µm.

  u l a t i v e d r u g r e l e a s e ( % ) T i m e ( m i n )

Figure 12 :

 12 Figure 12: Cumulative amount of released 5-ALA in water at pH5 from MN-20 and MN-100 patches during 80 min.

Figure 13 :

 13 Figure 13: Density of living NIH3T3 fibroblasts after 24 h incubation with MN-0, MN-20, or MN-100. Negative control corresponds to cells incubated in medium only. Positive control corresponds to cells incubated in medium containing H 2 O 2 .

Figure 14 :

 14 Figure 14: Density of living NIH3T3 fibroblasts after 24 h incubation with 5-ALA or PY.

Acknowledgments

This project is supported by the European Union -European Regional Development Fund. LETI/DTBS is supported by the French National Research Agency in the framework of the "Investissement d'avenir" program Glyco@Alps (ANR-15-IDEX-02) and Arcane Labex (ANR-12-LABX-003). The authors would also like to thank Manuel Alessio for the design of the aluminium microneedle master structure. Mathilde Menneteau is also acknowledged for her help with cytotoxicity experiments. The authors are grateful to Nicolas Duraffourg for assistance with the NMR analysis.


Yannick Stephan
email: yannick.stephan@umontpellier.fr

Angelina R Sutin

Martina Luchetti

Brice Canada

Antonio Terracciano

Euromov

PERSONALITY AND HbA1c 1 RUNNING HEAD: Personality and HbA1c Personality and HbA1c: Findings from Six Samples

Keywords: Personality, diabetes, HbA1c

Personality traits are associated with risk of diabetes, but most research to date has relied on participants reported diagnosis rather than objective markers of glycaemia. The present study examined the association between the five major domains of personality (neuroticism, extraversion, openness, agreeableness and conscientiousness) and haemoglobin A1c (HbA1c). Participants (N> 26,000) were individuals aged from 16 to 104 years from six large community samples from the US, Europe, and Japan who had data on personality, demographic factors, body mass index (BMI), physical activity, and HbA1c. Of the five factors, only higher conscientiousness was related consistently to lower HbA1c level across most samples and in the meta-analysis. Conscientiousness was also related to lower risk of HbA1c ≥6.5% (OR = .85, 95%CI = 0.80-0.90). BMI and physical activity partially mediated the link between conscientiousness and HbA1c. There were not consistent associations for the other four traits across the six samples and no consistent associations between personality and likelihood of undiagnosed diabetes. The present study found replicable associations between conscientiousness and HbA1c in adulthood. Assessment of conscientiousness may improve the identification of individuals at risk of diabetes and guide personalized interventions for regulation of HbA1c level.

Introduction

Type 2 diabetes is one of the leading causes of disability and mortality worldwide [START_REF] James | Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017[END_REF]. The prevalence rate is estimated to range between 8 to 10% (NCD Risk Factor Collaboration, 2016;[START_REF] Menke | Prevalence of and trends in diabetes among adults in the United States, 1988-2012[END_REF], with higher prevalence among older adults [START_REF] Menke | Prevalence of and trends in diabetes among adults in the United States, 1988-2012[END_REF]. Lifestyle factors, such as physical inactivity and body mass index, play a crucial role in the incidence of Type 2 diabetes [START_REF] Abdullah | The magnitude of association between overweight and obesity and the risk of diabetes: a meta-analysis of prospective cohort studies[END_REF][START_REF] Joseph | Physical activity, sedentary behaviors and the incidence of type 2 diabetes mellitus: the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF]. In addition, personality traits, commonly defined as enduring patterns of thoughts, feelings, and behaviors, have been found to predict the risk of diabetes (Cukic et al., 2016;Cukic & Weiss, 2014;[START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF][START_REF] Weston | Personality traits predict the onset of disease[END_REF]. Among the Five Factor Model of personality [START_REF] Mccrae | An introduction to the five-factor model and its applications[END_REF], conscientiousness (the propensity to be self-disciplined and thoughtful) is associated consistently with a lower risk of diabetes [START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF][START_REF] Weston | Personality traits predict the onset of disease[END_REF]. This association is explained in part by the lower obesity risk and more frequent physical activity of conscientious individuals [START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF]Sutin et al., 2016;Sutin & Terracciano, 2016). The evidence for neuroticism (the tendency to experience distress and negative emotions) is more mixed: some studies found that higher neuroticism is predictive of lower risk of diabetes (Cukic & Weiss, 2014) whereas others reported that higher neuroticism is a risk factor (Cukic et al., 2016) or not associated [START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF].

Openness (the tendency to be creative and curious) has been related to a lower likelihood of diabetes in one study (Cukic et al., 2016) but not in others [START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF][START_REF] Weston | Personality traits predict the onset of disease[END_REF]. Extraversion (the propensity to be sociable and active) and agreeableness (the propensity to be altruistic and trusting) are generally not associated with diabetes (Cukic et al., 2016;Cukic & Weiss, 2014;[START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF][START_REF] Weston | Personality traits predict the onset of disease[END_REF].

This research has mostly examined the association between personality traits and selfreported diabetes, using questions on whether a medical doctor ever told the participant that they have diabetes or whether they have been treated for diabetes. Less is known about the link between personality traits and objective markers of glycaemia, specifically haemoglobin A1c (HbA1c). HbA1c is an indicator of individuals' glycemic status over the past two to three months and is used for the diagnosis of type 2 diabetes (clinical threshold of HbA1c ≥ 6.5%, American Diabetes Association, 2010). HbA1c can be used to identify individuals with undiagnosed diabetes (i.e., individuals with HbA1c levels past the clinical threshold but have not yet been diagnosed by a medical doctor). The few published studies on the association between personality and HbA1c are relatively inconsistent. Sutin and colleagues (2018), for example, found that lower conscientiousness was associated with higher HbA1c in the Health and Retirement Study (HRS), and Waller and colleagues (2013) found that lower conscientiousness and lower agreeableness were associated with higher HbA1c in a sample of young patients with type 1 diabetes. In contrast, Cukic and colleagues (2015) found that lower openness was associated with higher HbA1c in the Lothian Birth Cohort 1936 but that the other four traits were unrelated to it. Other studies have found no association between personality and HbA1c among individuals with diabetes [START_REF] Phillips | The five-factor model of personality and self-reported versus biomarker diabetic control[END_REF][START_REF] Skinner | Personality traits, self-care behaviours and glycaemic control in Type 2 diabetes: The Fremantle Diabetes Study Phase II[END_REF]. The current literature thus provides an unclear picture of the association between personality and HbA1c. To our knowledge, no large scale, analysis of multiple samples has been conducted to test for replicable associations between personality traits and HbA1c. Furthermore, no research has yet tested whether personality traits may be related to the HbA1c-threshold for diabetes-diagnosis and whether personality traits predicts undiagnosed diabetes.

Based upon six large samples of adults, the present study examined the association between personality and HbA1c in adulthood. Consistent with past research on self-reported diabetes [START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF], it was hypothesized that conscientiousness would be related to lower HbA1c. In addition, given the importance of BMI and physical activity to HbA1c and their association with personality, BMI and physical activity were tested as mediators. Finally, we also tested whether personality predicts undiagnosed diabetes and whether the association between personality and HbA1c varies depending upon self-reported diabetes. The UKHLS is a large, nationally representative panel study of UK households.

Method

Personality traits and demographic factors were obtained from the third wave (2011-2013).

HbA1c was available for some participants on Wave 2 (2010-2012) and for other participants on Wave 3. The analysis was conducted among a total of 8,417 individuals with complete data (56% women, Mean age: 51.34, SD: 16.54).

Measures

Personality.

Participants completed the Midlife Development Inventory (MIDI; [START_REF] Zimprich | Factorial structure and age-related psychometrics of the MIDUS personality adjective items across the lifespan[END_REF] in the MIDUS, MIDJA, HRS, ELSA and NSHAP. They were asked to indicate on a scale ranging from 1 (not at all) to 4 (a lot) how well adjectives assessing the five traits described themselves such as moody (e.g. neuroticism), outgoing (e.g. extraversion), curious (e.g. openness), warm (e.g. agreeableness), and organized (e.g. conscientiousness). A 26 version of the MIDI was used in the MIDUS, MIDJA, HRS, and ELSA, whereas a 21-item version was used in the NSHAP. A 15-item version of the Big Five Inventory [START_REF] Soto | Short and extra-short forms of the Big Five Inventory-2: The BFI-2-S and BFI-2-XS[END_REF] was used in the UKHLS. Following the sentence "I see myself as someone who…",

Participants were asked to rate items assessing neuroticism (e.g., nervous), extraversion (e.g., is talkative), openness (e.g., has an active imagination), agreeableness (e.g., is kind), and conscientiousness (e.g., is efficient) using a scale from 1 (does not apply to me at all) to 7 (applies to me perfectly).

HbA1c.

Blood samples were collected in the six samples. In the MIDUS, the HbA1c assay was performed using a Cobas Integra Systems instrument (Roche Diagnostics). In the MIDJA, HbA1c assay was performed at the Showa Lab in Tokyo, Japan using a latex agglutination assay (Showa Medical Service Co. LTD). In the HRS and NSHAP, HbA1c uses an automated ion-exchange high-performance liquid chromatography (IE-HPLC) system. In UKHLS, HbA1c is measured from whole blood using HPLC cation exchange on a Tosoh G8 analyser.

In ELSA, HbA1c was measured using a Tosoh G7 analyser (Tosoh, Tokyo, Japan). HbA1C

was expressed into percentage of the total hemoglobin in the MIDUS, MIDJA, HRS, and NSHAP whereas it was expressed into mmol/mol in ELSA and the UKHLS. These values were converted into % using the following formula: (Value in mmol/mol/10,929)+2.15.

Self-reported diabetes.

In each sample, participants were asked to indicate whether they ever had been diagnosed with diabetes by a doctor or a health professional or whether they have been treated

for diabetes. Individuals were categorized into either self-reported diabetes (either reported diagnosis and/or treatment for diabetes) or no self-reported diabetes. Of note, no distinction was made between type I and II diabetes.

Mediators.

Mediation analysis was conducted to examine whether the association between personality and HbA1c was mediated by BMI or physical activity. In the six samples, objective measurement of weight and height were used to compute BMI as kg/m 2 . Physical activity measures were available in five out of six samples (physical activity was not available at wave 3 in the UKHLS). In the MIDUS participants were asked to indicate the frequency of their vigorous and moderate leisure physical activity during both the summer months and the winter months on a scale from 1 (never) to 6 (several times a week or more). Items were averaged. In the HRS and ELSA, the mean of two items asking how often individuals participated in vigorous and moderate physical activity on a scale from 1 (hardly ever or never) to 4 (more than once a week) was computed. In the MIDJA, participants reported how frequently they followed exercise therapies such as yoga or thaï chi in the past 12 months on a scale from 1 (never) to 5 (a lot). In the NSHAP, participants were asked to report their frequency of vigorous physical activity on a scale ranging from 0 (never) to 5 (five or more times a week).

Covariates.

In each sample, age, gender (coded as 1 for male and 0 for female), and education were controlled for. Race was also controlled for in the HRS, MIDUS, NSHAP, ELSA, and UKHKS and was coded as 0 for white and 1 for other.

Data Analysis

In each sample, regression analysis was used to predict HbA1c from personality, controlling for the covariates. Each personality trait was analyzed separately. The PROCESS macro [START_REF] Hayes | An introduction to mediation, moderation, and conditional process analysis: A regression-based approach[END_REF], using 5,000 bootstrapped samples and 95% bias-corrected confidence intervals, was used to test whether BMI and physical activity mediated these associations, controlling for the covariates. Supplementary regression analyses were conducted to test the association between personality and HbA1c separately among individuals with and without self-reported diabetes.

Logistic regression was used to examine whether personality traits were associated with the likelihood of exceeding the clinical threshold indicative of diabetes (HbA1C ≥ 6.5%), controlling for demographic factors. Furthermore, the same analysis was conducted to test for the association between personality and undiagnosed diabetes, that is HbA1c ≥ 6.5% among individuals without a diagnosis of diabetes. This analysis controlled for the same covariates and excluded individuals with self-reported diabetes. To facilitate interpretation of the logistic regression analysis, each trait was standardized as z-scores (M = 0 and SD = 1). A random effects meta-analysis was conducted that combined the results from linear and logistic regressions with Comprehensive Meta-Analysis software. A meta-analysis of indirect and direct effects in the mediation analysis was also conducted.

Results

Supporting our main hypothesis, higher conscientiousness was related to lower HbA1c, controlling for demographic factors (see Table 2). This association was observed in every sample except for MIDUS. Higher extraversion was related to lower HbA1c in HRS, ELSA, and NSHAP and higher HbA1c in MIDUS. Agreeableness was associated with lower HbA1c in ELSA and higher HbA1c in MIDUS. Higher openness was related to lower HbA1c in MIDJA and NSHAP. No association was found between neuroticism and HbA1c across the six samples. The meta-analysis supported the association between conscientiousness and lower HbA1c and suggested a marginally significant relationship with openness (p=.045). No other associations were found (see Table 2).

The mediational analysis suggested that the association between conscientiousness and HbA1c was mediated by both BMI and physical activity in the HRS, NSHAP, ELSA and by BMI in the UKHLS (Table 3). The meta-analysis confirmed this pattern of mediation (see Table 3). The proportion of mediated effect by BMI and physical activity was, respectively, 18% and 21% in the HRS, 16% and 29% in the NSHAP, 19% and 17% in ELSA, and was 32% for BMI in the UKHLS. The association between extraversion and HbA1c was mediated by both BMI and physical activity in the HRS (proportion mediated: 27% and 39%, respectively) and ELSA (proportion mediated: 10% and 19%, respectively), and by physical activity in the NSHAP (proportion mediated: 20%). Physical activity mediated the association between openness and HbA1c in the NSHAP (proportion mediated: 30%) (Table 3). The meta-analysis supported the mediating role of physical activity in the relationship between both extraversion and openness and HbA1c. The pattern of associations was less consistent when examining individuals with and without self-reported diabetes separately (see supplementary analysis).

Analyses were conducted to examine whether personality traits were related to the clinical threshold in the total sample. The results revealed that higher conscientiousness was related to lower risk of exceeding the diabetes-diagnosis threshold (see Table 4). There was also a weak effect for agreeableness associated with a lower likelihood of exceeding the clinical cutoff. Finally, there was little evidence that personality was associated with undiagnosed diabetes. Conscientiousness was associated with a reduced risk of undiagnosed diabetes in the ELSA, and extraversion and agreeableness were related to higher risk of undiagnosed diabetes in the UKHLS and the NSHAP, respectively (Table 5). No associations were found in the MIDUS and the HRS. These analyses were not conducted in the MIDJA because of the small number of participants both without self-reported diabetes and clinical level of HbA1c (N= 6).

Discussion

Based upon six large samples of adults, the present study suggests that conscientiousness is related to HbA1c. That is, as expected, higher conscientiousness was related consistently to lower HbA1c, and this association was mediated partly by BMI and physical activity. Although less consistent, extraversion, openness and agreeableness were associated with lower HbA1C level in some samples. Personality traits were mostly unrelated to likelihood of undiagnosed diabetes. The present study provides robust evidence for the relation between conscientiousness and HbA1c.

Among the five traits, conscientiousness was the most consistent personality correlate of HbA1c. This result supports past findings on the association between conscientiousness and lower likelihood of incident self-reported diabetes [START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF]. While such comparisons should be done cautiously, it is of note that in the previous study conscientiousness predicted incident self-report diagnosis of diabetes with a meta-analytic OR = 0.87 (95%CI = 0.82-0.91) [START_REF] Jokela | Personality and risk of diabetes in adults: Pooled analysis of 5 cohort studies[END_REF], which is very similar to the meta-analytic OR = .85 (95%CI = 0.80-0.90) we found for the cross-sectional association of conscientiousness with HbA1c ≥ 6.5% (Table 4). These findings suggest that the self-report work is unlikely to overestimate the associations between personality and risk of diabetes.

However, we found little evidence of an association between personality and undiagnosed diabetes, that is a higher diagnostic level of HbA1c among individuals without a diagnosis of diabetes. Overall, the relationship between personality and objective measure of diabetes provides a relatively similar conclusion to the research based on self-reported measures. This study also supports the role of conscientiousness in more positive health outcomes [START_REF] Strickhouser | Does personality predict health and wellbeing? A metasynthesis[END_REF]. Several pathways may explain this association. As indicated by the mediation analysis, self-disciplined, organized and thoughtful individuals have lower level of HbA1c in part because they have lower adiposity and are more physically active. There are likely to be other pathways that also contribute to this association. For example, higher conscientiousness is related to higher adherence to doctors' orders and medication [START_REF] Hill | The role of adherence in the relationship between conscientiousness and perceived health[END_REF]) that may reduce the likelihood of elevated HbA1c [START_REF] Skinner | Personality traits, self-care behaviours and glycaemic control in Type 2 diabetes: The Fremantle Diabetes Study Phase II[END_REF][START_REF] Wheeler | Personality traits as predictors of adherence in adolescents with type I diabetes[END_REF]. Individuals higher in conscientiousness tend to consume less alcohol [START_REF] Hakulinen | Personality and alcohol consumption: Pooled analysis of 72,949 adults from eight cohort studies[END_REF][START_REF] Luchetti | Personality traits and facets linked with self-reported alcohol consumption and biomarkers of liver health[END_REF] and have better sleep quality [START_REF] Stephan | Personality and sleep quality: Evidence from four prospective studies[END_REF], which are both associated with lower HbA1c and protect against diabetes [START_REF] Baliunas | Alcohol as a risk factor for type 2 diabetes: A systematic review and meta-analysis[END_REF][START_REF] Knutson | Role of sleep duration and quality in the risk and severity of type 2 diabetes mellitus[END_REF]. Furthermore, conscientiousness is related to a healthy diet (Mottus et al., 2013;[START_REF] Weston | Personality traits predict dietary habits in middle-to-older adults[END_REF] and better fitness [START_REF] Terracciano | Personality, metabolic rate and aerobic capacity[END_REF] that may contribute to lower HbA1c. At the biological level, conscientiousness is associated with lower systemic inflammation [START_REF] Luchetti | Five-Factor Model personality traits and inflammatory markers: New data and a meta-analysis[END_REF]) that may also be implicated in the development of diabetes [START_REF] Wang | Inflammatory markers and risk of type 2 diabetes: a systematic review and meta-analysis[END_REF].

In contrast to conscientiousness, the associations with extraversion, openness and agreeableness were much less consistent. In addition, neuroticism was unrelated to HbA1c across the six samples and in the meta-analysis. This finding suggests that the propensity to experience distress and negative emotions is not implicated in individuals' glycemic status.

Perhaps the detrimental effects of negative emotions are canceled out by better glycemic control driven by health-related worries.

The present study adds to existing knowledge by providing the most comprehensive evidence to date of the association between personality traits and HbA1c across adulthood.

That is, replicable associations were found across samples with participants who ranged in age from 16 to 104. It also adds to existing models on personality and health. HbA1c could be a biological pathway that contributes to the association between personality and a range of health-related outcomes. Specifically, conscientiousness is related consistently to lower risk of dementia [START_REF] Terracciano | Personality traits and risk of cognitive impairment and dementia[END_REF] and mortality [START_REF] Graham | Personality predicts mortality risk: An integrative data analysis of 15 international longitudinal studies[END_REF]. Elevated HbA1c is a risk factor for dementia [START_REF] Ramirez | Elevated HbA1c is associated with increased risk of incident dementia in primary care patients[END_REF] and mortality [START_REF] Forbes | Mean HbA1c, HbA1c variability, and mortality in people with diabetes aged 70 years and older: a retrospective cohort study[END_REF]. Therefore, conscientiousness may be related to lower dementia and higher longevity in part through its association with lower levels of HbA1c. From a practical perspective, assessment of conscientiousness may improve the identification of individuals at risk of diabetes. In sum, the present study found replicable associations between personality and HbA1c in adulthood. In particular, higher conscientiousness was consistently related to lower level of HbA1c and reduced probability of diabetes diagnosis.

Furthermore, such assessment

  may guide personalized interventions directed toward the regulation of HbA1c level and ultimately the promotion of health among individuals low in conscientiousness.The present study has several strengths, particularly the inclusion of six large samples of adults from different cultures, the use of validated measures of all five major dimensions of personality, and the focus on HbA1c, an objectively assessed and clinically relevant outcome.There are also several limitations to consider. The observational design of the present study does not allow to establish causality. Reciprocal associations may exist, such that HbA1c may also be predictive of personality level and change. Longitudinal research designs are needed to test for these reciprocal associations. Moreover, further research using a facet-level measures of personality may provide a more detailed understanding of the personality-HbA1c association. Although the inclusion of an Asian sample increases the generalizability of our findings, future work should examine the conscientiousness-HbA1c association in other international samples, to test, for example, if this association replicates in countries with lower gross domestic product. Furthermore, the present study focused on a limited set of mediating variables. Future research may include additional potential mediators such as depressive symptoms and cognitive decline. Finally, the present study missed information about the extent to which participants had type 1 or type 2 diabetes.

Table 1 .

 1 Characteristics of the Samples MIDJA: N= 377; HRS: N= 9745; ELSA: N= 4656; NSHAP: N= 1888; UKHLS: N= 8417. Ns were lower for BMI and physical activity, due to missing data. a See method section for differences in measures used in each sample

	MIDUS	MIDJA	HRS	ELSA	NSHAP	UKHLS

Table 2 .

 2 Summary of Regression Analysis Predicting HbA1C from Personality Traits in the Six Samples

		Neuroticism	Extraversion	Openness	Agreeableness	Conscientiousness
		β	β	β	β	β
	MIDUS a	-0.02(-0.09;0.04)	0.10**(0.04;0.16)	0.05(-0.01;0.11)	0.07*(0.01;0.14)	-0.01(-0.07;0.05)	Note.
	MIDJA b	-0.01(-0.11;0.09)	-0.05(-0.15;0.04)	-0.10*(-0.20;-0.004)	-0.06(-0.15;0.04)	-0.10*(-0.20;-0.008)	MIDUS: N= 988;
	HRS a	0.01(-0.005; 0.03) -0.04***(-0.06; -0.02)	-0.02(-0.04;0.003)	-0.02*(-0.04;-0.0008)	-0.06***(-0.08;-0.04)	MIDJA: N= 377; HRS:
	ELSA a	0.02(-0.004;0.05) -0.09***(-0.12;-0.06)	-0.03(-0.06;0.003)	-0.03*(-0.06;-0.005)	-0.09***(-0.12;-0.06)	N= 9745;
	NSHAP a	-0.01(-0.06;0.03) -0.08***(-0.12;-0.03) -0.05*(-0.10;-0.006)	-0.03(-0.08;0.02)	-0.05*(-0.10;-0.007)	ELSA: N= 4656;
	UKHLS a	-0.01(-0.03;0.01)	0.01(-0.01;0.03)	-0.01(-0.03;0.01)	-0.01(-0.03;0.007)	-0.04***(-0.06;-0.02)	NSHAP: N= 1888;
	Random Effect	0.002	-0.027	-0.021*	-0.015	-0.06***	UKHLS:
		(-0.010;0.015)	(-0.072;0.018)	(-0.041;0.000)	(-0.035;0.005)	(-0.078;-0.037)	N= 8417; β =
	Tau Heterogeneity	0.000	0.051	0.017	0.016	0.017	Standardize coefficient d regression
	a Adjusted for age, sex, education, and race
	b Adjusted for age, sex, education
	* p < .05, ** p < .01, *** p <.001

Table 3 .

 3 Summary of Bootstrap Analysis Bootstrap estimates and 95% bias-corrected confidence interval for indirect effects of personality traits on HbA1c through BMI and physical activity, controlling for age, sex, education, and race (except for the MIDJA).

	Bootstrap Analysis b

b

Acknowledgment

The Midlife in the United States (MIDUS) is sponsored by the MacArthur Foundation Research Network on Successful Midlife Development, the National Institute on Aging (P01-AG020166 ; U19-AG051426), and grants from the General Clinical Research Centers Program (M01-RR023942, M01-RR00865) and the National Center for Advancing Translational Sciences (UL1TR000427). The Midlife in Japan study (MIDJA) was supported by a grant from the National Institute on Aging (5R37AG027343). The Health and Retirement Study (HRS) is sponsored by the National Institute on Aging (NIA-U01AG009740) and conducted by the University of Michigan. Funding for the English Longitudinal Study of Ageing is provided by the National Institute of Aging [grants 2RO1AG7644-01A1 and 2RO1AG017644] and a consortium of UK government departments coordinated by the Office for National Statistics. The National Health, Social Life and Aging Project (NSHAP) is supported by the National Institutes of Health, including the National Institute on Aging, the Office of Women's Health Research, the Office of AIDS Research, and the Office of Behavioral and Social Sciences Research (grants R01 AG021487, R37 AG030481, R01 AG033903, R01 AG043538, and R01 AG048511). UKHLS is primarily funded by the Economic and Social Research Council (ESRC).

Data Availability:

MIDUS data are publically available at http://midus.wisc.edu/index.php. MIDJA data are publically available at http://midus.wisc.edu/index.php. HRS data are publically available at http://hrsonline.isr.umich.edu/. ELSA data are available from the UK Data Service (UKDS, https://www.ukdataservice.ac.uk/). Information on how to access the NSHAP data can be found at: http://www.norc.org/Research/Projects/Pages/national-social-life-health-and-agingproject.aspx. Data of UKHLS are available at : https://www.understandingsociety.ac.uk/.

Declaration of Interest: None

Ethical Approval:

The six samples received approval from their respective Institutional Review Board. All participants in each sample provided informed consent. This study was based on publicly available de-identified datasets, and therefore was exempt from Institutional Review Board review.

c Direct effect of personality traits on HbA1C adjusted for mediators, age, sex, education, and race (except for the MIDJA); Coefficients are standardized regression coefficient