Yuzhen Lou

Pascal Thebault
email: pascal.thebault@univ-rouen.fr

Fabrice Burel

Nasreddine Kebir
email: nasreddine.kebir@insa-rouen.fr

Antibacterial properties of metal and PDMS surfaces under weak electric fields

Keywords: Electrical field, Bacterial adhesion, PDMS, Metallic surface, Bactericidal effect

The adhesion of several species of bacteria on two parallel rectangular electrodes under weak electric fields was studied. The electrodes were based on native metal or PDMS coated Cu. After 2 hours of contact at a voltage of 0.2 to 1V without any current, the Zn cathode showed a bacterial repellent effect with a difference in bacterial adhesion of about 1.5 to 2 log CFU/cm2 on the anode. Al electrodes were inactive due to their passivation by the alumina layer. At 1V, both Zn and Al exhibited more than 80% mortality of suspended bacteria. The Cu electrodes showed a very high bactericidal effect even at 0V, and the bacterial adhesion on its surface was too weak to see a difference between the two electrodes. A similar study carried out on PDMS surfaces, covering Cu electrodes, revealed that a difference of 1 log CFU/cm2 of bacterial adhesion between the cathode and anode surfaces can be obtained by applying a voltage ranging from 10 to 30V. This cathodic repellent effect was specific to staphylococcus species, suggesting that in the presence of a PDMS coating, the electrostatic forces on the surface are too low to be the main factor governing bacterial adhesion.

Introduction

Microbial contamination of surfaces often leads to the formation of a biofilm, resulting in serious problems for human health (e.g. device-related infections, healthcare-related infections) as well as for industrial sectors (e.g. corrosion of metal surfaces, food contamination, deterioration of pipes...), leading to a negative socio-economic impact [START_REF] Doulkeraki | Methicillin-resistant food-related Staphylococcus aureus: a review of current knowledge and biofilm formation for future studies and applications[END_REF][START_REF] Romero | Bacterial determinants of the social behavior of Bacillus subtilis[END_REF][START_REF] Winiecka-Krusnell | Bacterial infections of free-living amoebae[END_REF].

However, it should be noted that bacteria in their biofilms require antibiotic concentrations 100 to 1000 times higher than those of planktonic bacteria to achieve effective eradication [START_REF] Salwiczek | Emerging rules for effective antimicrobial coatings[END_REF]. Therefore, the development of materials capable of preventing the adhesion of bacteria and/or the formation of biofilms on their surface is a key element to avoid contamination. Gottenbos et al. have reported that the initial attachment of bacteria depends on the result of non-specific interactions such as Van der Waals, electrostatic, acid-base interactions and Brownian motion forces [START_REF] Gottenbos | Pathogenesis and prevention of biomaterial centered infections[END_REF].

Dunne et al also repoted that once the bacteria approache surfaces at a critical distance (usually less than 1 nm), adhesion is determined by the net sum of attractive or repulsive forces, including electrostatic and hydrophobic interactions, steric hindrance, van der Waals forces etc [START_REF] Dunne | Bacterial Adhesion : Seen Any Good Biofilms Lately ? Bacterial Adhesion : Seen Any Good Biofilms Lately ?[END_REF].

Three types of antibacterial surfaces are described in the literature: anti-adhesive surfaces, contact-killing surfaces and release-based surfaces [START_REF] Cloutier | Antibacterial Coatings: Challenges, Perspectives, and Opportunities[END_REF].

Anti-adhesive surfaces are designed to repel bacteria by optimizing their physico-chemical properties such as the surface charge (negative charges), the wettability or the topography (micro/nano-patterned surfaces) [START_REF] Alves | Mini-review: Antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces[END_REF][START_REF] Liu | Understanding the Role of Polymer Surface Nanoscale Topography on Inhibiting Bacteria Adhesion and Growth[END_REF][START_REF] Satriano | Bacterial adhesion onto nanopatterned polymer surfaces[END_REF][START_REF] Hasan | Recent advances in engineering topography mediated antibacterial surfaces[END_REF]. However, they cannot affect the viability of pathogen that can be released to contaminate others. Furthermore, the use of physical surface modifications (particularly surface topography) as non-specific methods to prevent the bacterial adhesion is much more complex than we can imagine [START_REF] Ammar | Influence of surface roughness on the initial formation of biofilm[END_REF]. Contact-killing surfaces which are generally based on cationic biocidal agents covalently bonded to substrate surfaces that attract negatively charged bacteria and kill them by disrupting their cell membrane [START_REF] Foksowicz-Flaczyk | Multifunctional durable properties of textile materials modified by biocidal agents in the sol-gel process[END_REF][START_REF] Lewis | Surpassing nature: Rational design of sterile-surface materials[END_REF][START_REF] Kaur | Antibacterial surface design -Contact kill[END_REF]. However, these surfaces quickly become inactive after being buried under a layer of dead bacteria.

Released-based surfaces are designed to leach antibacterial agents that kill not only adhered bacteria but also planktonic bacteria [START_REF] Procaccini | Silver doped silica-methyl hybrid coatings. Structural evolution and antibacterial properties[END_REF][START_REF] Kao | Metal ion-dependent tailored antibacterial activity and biological properties of polydopamine-coated titanium implants[END_REF][START_REF] Campoccia | A review of the biomaterials technologies for infection-resistant surfaces[END_REF]. The disadvantage of this kind of surfaces is its limited activity owing to the limited amount of the loaded antibacterial compounds. However, excessive use of antibiotics may lead to the phenomenon of bacterial resistance [START_REF] Riosa | Alternatives to overcoming bacterial resistances: State-of-the-art[END_REF].

It is noteworthy that the use of an electrical method to prevent infection of medical devices, without traumatising the patient by removal of the device, is of great interest In addition, over time, bacteria should not adapt as well with electrical treatment as with antibiotics [START_REF] Freebairn | Electrical methods of controlling bacterial adhesion and biofilm on device surfaces[END_REF]. Direct and alternating currents [START_REF] Del Pozo | The electricidal effect: reduction of Staphylococcus and Pseudomonas biofilms by prolonged exposure to low-intensity electrical current[END_REF][START_REF] Del Pozo | The electricidal effect is active in an experimental model of Staphylococcus epidermidis chronic foreign body osteomyelitis[END_REF] as well as acoustic (by piezo-electric actuators) [START_REF] Hazan | Effective prevention of microbial biofilm formation on medical devices by low-energy surface acoustic waves[END_REF] and radio frequency wave [START_REF] Caubet | A radio frequency electric current enhances antibiotic efficacy against bacterial biofilms[END_REF] treatments have already proven to be effective techniques to prevent initial bacterial adhesion and growth, through in vitro and in vivo experiments. On the other hand, the electrical current has been found to increase the efficacy of antibiofilm agents in a synergistic action called 'the bioelectric effect' [START_REF] Del Pozo | Bioelectric effect and bacterial biofilms. A systematic review[END_REF][START_REF] Costerton | Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria[END_REF][START_REF] Del Pozo | Effect of electrical current on the activities of antimicrobial agents against Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis biofilms[END_REF][START_REF] Blenkinsopp | Electrical enhancement of biocide efficacy against Pseudomonas aeruginosa biofilms[END_REF][START_REF] Jass | The efficacy of antibiotics enhanced by electrical currents against Pseudomonas aeruginosa biofilms[END_REF], which can lead to a dramatic decrease in the concentration of the antibiofilm compounds used. Thus, the development of electrically stimulated surfaces that prevent bacterial adhesion and biofilm formation is an emergent and attractive pathway.

Busalmen et al. [START_REF] Busalmen | Adhesion of Pseudomonas fluorescens (ATCC 17552) to nonpolarized and polarized thin films of gold[END_REF] have studied, using an optical microscope, the influence of an electrical field on the adhesion of Pseudomonas fluorescens onto the surface of gold electrodes, under flow conditions at two different ionic strengths (0.01 and 0.1 M NaCl; pH 7). By applying negative electric potentials (-0.5 and -0.2V relative to a reference electrode [Ag/AgCl-KCl saturated solution]), they have evidenced a decrease in bacterial adhesion of one Log (CFU/mm 2 ) after 15 min of contact, compared to adhesion at a potential of 0.2V. In another work [START_REF] Busalmen | Electrochemical polarization-induced changes in the growth of individual cells and biofilms of Pseudomonas fluorescens (ATCC 17552)[END_REF], using the same system, they demonstrated the impact of this electrical field on the growth of planktonic cells and biofilms of Pseudomonas fluorescens (cell morphology, size at cell division, time to division, and biofilm structure). Gall et al. [START_REF] Gall | The effect of electric fields on bacterial attachment to conductive surfaces[END_REF] have studied, using Quartz Crystal Microbalance with Dissipation analysis (QCM-D), the effect of applying an electrical field, perpendicular to the flow of a Pseudomonas fluorescens suspension, on the adhesion of bacterial cells to the gold electrode. Surprisingly, they demonstrated that the tested bacteria were rigidly attached to the negatively charged surface, unlike to the positively charged one, suggesting that the applied electric potential could influence the conformation of the bacterial cell surface, allowing the cells to overcome the electrostatic energy barrier.

In this study, we investigated the adhesion of different bacterial species, in a static mode (without flow), on metallic and PDMS surfaces under an electrical field (without any current), generated by two parallel metal electrodes (as in a capacitor) immersed in an aqueous suspension of bacteria. The impact of the applied electrical field on bacterial survival has also been studied.

Materials and methods

Materials

Sylgard ® 184 was purchased from DOW chemical company (USA) and was used as the coating material. Copper, zinc and aluminum blades (10×100×1.5 mm 3 , Jeulin, France) were used as electrodes. Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 35984), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (PA14) and Escherichia coli (K12 MG1655) strains were stored as frozen aliquots in brain heart infusion broth (BHI, Bacto, France) and 30% of glycerol at -20°C. Ultrapure water, obtained from a Milli-Q system (Siemens, France), was used in all cases.

Preparation of PDMS-coated copper or zinc electrodes

15 g of the two parts of Sylgard ® 184, i.e. silicone elastomer base and curing agent, in a ratio of 10:1 (w/w) were mixed and casted into a low-density polyethylene petri dish square (120×120×17 mm 3 ). Then, the mixture was degassed under vacuum, until all air bubbles were removed, and cured at 70 °C for 3 h to form a first PDMS layer. Six blades were then put on top of this layer and covered with a second layer of PDMS (20 g) using the same process. The resulting PDMS-coated electrodes (12×3.5×40 mm 3 ) were cut out by using a scalpel. The thickness of the coating was around 1 mm.

Preparation of Bacterial suspensions

For each experiment, all bacterial species were pre-cultured in BHI at 37°C under shaking at 140 rpm for approximately16 h. Then, bacteria were harvested by centrifugation (Sigma® 3-16KL, rotor 19776, Germany) under 1600 g for 15 minutes at 20°C and resuspended at a concentration of 10 7 ~10 8 CFU/ml in Milli-Q water to avoid the influence of charged particles in BHI or Phosphate Buffered Saline (PBS, Gibco, UK).

Zeta potential measurements

Zeta potentials of bacteria were measured with a Zetasizer Nano-ZS system (Malvern Panalytical, Ltd., UK) at 25 °C in Milli-Q water solution at 10 7 ~10 8 CFU/ml. Three measurements were carried out for each bacterial suspension.

Assessment of bacteria attachment onto PDMS and metallic blade surfaces under an electric field

The process used to evaluate the attachment of bacteria to the surface of metallic or PDMS-coated electrodes is shown in Figure 1. The PDMS-coated electrodes were sterilized in 70% ethanol overnight and washed twice with Milli-Q water before use. Uncoated electrodes were sterilized in 70% ethanol for 20 min, followed by sonication for 30 min, and washed twice with Milli-Q water before use. The bacterial adhesion was studied in a system of parallel plates with a distance of 1 cm between the two electrodes connected to the potensiostat (0-30 V, Velleman ® ). The electrodes were immersed (1 cm) in 7 ml of the previous prepared bacterial suspension during 2 h. Then, to remove unattached bacteria, these electrodes were washed in 15 ml of sterile Milli-Q water under slight shaking while the potentiostat was still on. They were then detached from the potentiostat. To recover the bacteria fixed to the surface, metallic electrodes were directly immersed in 7 mL of PBS solution, under sonication for 3 min. For PDMS-coated electrodes, a sample of PDMS was taken with a circular punch (Ø=1 cm) and was immersed in 3 ml of PBS solution under sonication for 3 min. Decimal dilutions of the resulting bacterial suspensions, containing bacteria detached from the surface, as well as those where the experiments were conducted, containing unattached surviving bacteria, were spread (20 µl or 100 µl) on BHI agar plates and incubated at 37°C for 24 h before colony counting. All experiments were performed in at least triplicate.

Results and discussion

Bacterial adhesion onto a surface is often explained by the Derjaguin, Landau, Verwey and Overbeek (DLVO) theory of colloidal stability. It describes initially repulsive electrical double layer interactions (electrostatic repulsion) between bacteria and similarly charged surfaces. At the same time, bacteria are attracted by van der Waals forces and approach surface contact as soon as they cross the electrostatic energy barrier by decreasing the interfacial distance. [START_REF] Gottenbos | Pathogenesis and prevention of biomaterial centered infections[END_REF][START_REF] Dunne | Bacterial Adhesion : Seen Any Good Biofilms Lately ? Bacterial Adhesion : Seen Any Good Biofilms Lately ?[END_REF] In this work, we aimed to use an electrical field to increase electrostatic forces between bacteria and cathodic (negatively charged) surfaces, in order to decrease bacterial adhesion and colonization. Thus, we have used two parallel electrodes, as in a capacitor, immersed in an aqueous suspension of bacteria (Figure 2). Cu, Al and Zn were used as models for metallic electrodes; PDMS was used as representative electrodes for plastic-based biomaterials.

Bacterial adhesion to metal electrodes

The results of the enumeration of live S. aureus adhering to electrodes of Cu, Zn and Al, after immersion in bacterial suspensions for 2 hours at an electrical voltage of 0 to 1 V, are shown in Figure 3 (A1). Above 1 V, the presence of current and electrolysis reactions can be observed, making it unnecessary to study electrostatic interactions. The survival of suspended bacteria was also monitored before and after immersion of the electrodes (Figure 3 For copper electrodes at 0 V, the concentration of adhered S. aureus was only about 2 log of CFU/cm 2 (Figure 3 (A1)). This low adhesion can be explained by the well-known bactericidal properties of copper [START_REF] Grass | Metallic copper as an antimicrobial surface[END_REF][START_REF] Vincent | Contact killing and antimicrobial properties of copper[END_REF][START_REF] Da Silva | Corrosion resistance and antibacterial properties of copper coating deposited by cold gas spray[END_REF], which is confirmed by the percentage of bacterial surviving in solution (Figure 3 (A2)). Indeed, less than 20 % of bacteria in solution are still alive (or cultivable) after 2 h. However, a slight difference in bacterial adhesion between anode (positively charged) and cathode (negatively charged) at 0.2 and 0.4 V was observed. Furthermore, the increase of voltage to 1 V, suppressed the adhesion of S. aureus onto the surface and increased the suspended bacterial mortality to more than 95 %. These results suggest that the bactericidal effect of copper could be enhanced by the application of an electric field and are consistent with the "bioelectric effect" [START_REF] Del Pozo | Bioelectric effect and bacterial biofilms. A systematic review[END_REF][START_REF] Costerton | Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria[END_REF][START_REF] Del Pozo | Effect of electrical current on the activities of antimicrobial agents against Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis biofilms[END_REF][START_REF] Blenkinsopp | Electrical enhancement of biocide efficacy against Pseudomonas aeruginosa biofilms[END_REF][START_REF] Jass | The efficacy of antibiotics enhanced by electrical currents against Pseudomonas aeruginosa biofilms[END_REF].

For the two other electrodes, a bacterial adhesion of approximately 4.5 log of CFU/cm 2 was achieved at 0 V. In the case of Al, no difference in bacterial adhesion between cathode and anode was observed regardless of the applied voltage. This could be explained by the well-known passivation of the aluminum surface by oxidation leading to the formation of a thin layer of alumina, which is an ionic salt (Al2O3). The electrical properties of this layer are very different from those of its pure metal, and its electrochemical static polarization is more difficult to achieve. For Zn electrodes, at all applied voltages, there was a decrease in the adhesion of S. aureus on the cathode compared to bacterial adhesion at 0V. At the same time, a slight increase on the anode was observed inducing a difference of more or less 2 log of CFU/cm 2 between these two electrodes. This result suggests that electrostatic interactions are, in this case, quite predominant. Therefore, this result also indicates that bacterial adhesion could be modulated on the Zn electrodes by the use of an electric field.

Moreover, for a voltage ranging from 0 to 0.4V, an insignificant decrease, of about 10 to 40%, of live bacteria in suspension was observed for both Al and Zn electrodes. However, at 1 V, a decrease in the live bacteria population in solution of about 80 % was observed for both electrodes. Soumya et al. [START_REF] Pandit | Influence of Electric Fields on Biofouling of Carbonaceous Electrodes[END_REF] reported that potentials at about 0.9 V would affect the redox potential across the cell membrane and disrupt redox homeostasis, thereby accelerating the production of endogenous reactive oxygen species (ROS) and inhibiting bacterial growth. In summary, these results show that the nature of the electrode and the voltage values have an impact on the adhesion of S. aureus to the surface as well as on suspended bacterial mortality.

On the other hand, in order to determine whether the structure of the external membrane of bacteria cells has any influence on these results, we studied the adhesion of E. coli, as a model of Gram-negative bacteria, to zinc electrodes under voltage values ranging from 0 to 1 V. Figure 4 shows that E. coli behaves almost similarly to S. aureus in terms of adhesion to the Zn electrodes after 2 h of contact. Indeed, a difference in E. coli adhesion of about 1.5 to 2 log of CFU/cm 2 between the anode and cathode was observed. In addition, the percentage of killing of E. coli in suspension was also quite similar to that of S. aureus, i.e. low for a voltage of 0, 0.2 or 0.4 V, and higher than 80% at 1 V.

Experiments were then carried out with different types of bacteria to verify whether these results are universal or not on zinc electrodes. The bacteria chosen are the most representative species involved in the phenomenon of contamination in the hospital environment. S. epidermidis and E. faecalis are Gram-positive bacteria and mainly cause skin and endocardial infections, respectively [START_REF] Lee | Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis[END_REF][START_REF] Wells | Infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis[END_REF]. Gram-negative bacteria such as P. aeruginosa and E. coli account for more than 30 % of nosocomial infections [START_REF] Lee | Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis[END_REF]. P. aeruginosa causes several infections in human organs such as the urinary, blood, respiratory and gastrointestinal systems [START_REF] Mamoudou | Les infections à pseudomonas aeruginosa au service des maladies infectieuses du CHU YO, Burkina Faso: À propos deux cas[END_REF]. E. coli can also cause urinary or bloodstream infections for example [START_REF] Welch | Haemolysin contributes to virulence of extra-intestinal E. coli infections[END_REF]. These experiments were performed only at 0 and 0.2 V. As shown in Figure 5, at 0.2 V, a difference of bacterial adhesion was obtained between the cathode and the anode for each strain, with a lower adhesion on the cathode. These results suggest that bacterial adhesion could be controlled under an electric field whatever the bacterial species. Furthermore, no significant decrease of bacterial concentration in solution was observed for all tested species.

Bacterial adhesion to the surface of PDMS under an electrical field

In a second part, we studied the bacterial adhesion, under the effect of an electric field, on the surface of PDMS, which is a material widely used as biomaterial.

Previous copper electrodes were coated with a PDMS layer of a thickness around 1 mm and used as electrodes. The applied voltage ranged from 0 to 30 V, which is the maximum voltage provided by the potentiostat. The copper electrodes were chosen because they can easily reveal whether the metal surface was completely covered by The results of the adhesion of S. aureus to the surface of the PDMS under an electric field are shown in Figure 6. It can be seen that without an electrical voltage, the bacterial adhesion was about 3 log CFU/cm 2 , which is higher than its value on the surface of the uncoated copper (Figure 3). The difference in bacterial adhesion between cathode and anode at 1 and 5 V was not significant. On the other hand, at higher applied voltages (10 to 30 V), a lower bacterial adhesion on the cathode, around 2 log CFU/cm 2 , was observed, probably due to the electrostatic repulsion between the negative charges on the surface of the PDMS and the negatively charged bacterial cells. In addition, the percentage of live bacteria in suspension was approximately 90% regardless of the applied voltage, which is quite consistent with the percentage observed in milli-Q water. These results show that the PDMS coating on the copper electrodes leads to (i) the disappearance of the bactericidal effect of copper in the bacterial suspension, meaning that the electrodes were fully coated, and (ii) no significant bacterial stress at applied high voltage, probably due to the chemical and electrical insulating properties of PDMS. Consequently, the applied voltage must be increased above 30 V to achieve a greater anti-adhesion effect. The study of adhesion of different bacterial species onto PDMS surfaces was also carried out at 30 V (Figure 7). No copper bactericidal activity was detected for all species. However, S. epidermidis appears to be quite sensitive to the applied electric field and showed a reduction of about 50% in the concentration of live bacteria in suspension. For surface adhesion, only S. epidermidis showed the same behaviour as S. aureus with a difference of about 1 log CFU/cm 2 between cathode and anode. This result is in contradiction with the previous result on the zinc electrodes at 0.2V. To understand this specificity of the Staphylococcus genus, measurements of the zeta potential of the bacterial membrane were performed (Table 1). As expected, all bacterial membranes are negatively charged, but E. coli and P.

S . a u r e u s S . e p id e r m id is E . f a e c a li s E . c o li

S . a u r e u s S . e p id e r m id is E . f a e c a li s E . c o li P A 1 4 Alive bacteria in suspension (

S . a u r e u s S . e p id e r m i d is E . f a e c a l is P A 1 4 E . c o li

Log of CFU/cm

S . a u r e u s S . e p id e r m i d is E . f a e c a li s P A 1 4 E . c o li

aeruginosa bacteria have the lowest zeta potential and therefore the highest overall negative charge. Theoretically, with a higher negative charge, E. coli and P.

aeruginosa should undertake stronger repulsive electrostatic interactions with the PDMS surface, resulting in lower adhesion to the anode surface, which is in contradiction with our experimental results (Figure 7). Therefore, these observations suggest that, in the presence of a PDMS coating, the electrostatic interactions are too weak to be the predominant factor governing bacterial adhesion. There are two possible reasons why Staphylococcus species are more sensitive to the low surface polarization of PDMS: (1) they are Gram+ bacteria, which have only one external phospholipidic membrane, unlike Gram-bacteria, which have two;

(2) they are spherical in shape with a lower contact surface, while the other bacteria are bacillus-shaped.

Conclusion

In this work, we have studied the bacterial adhesion of Gram positive (S. aureus, S. epidermidis and E. faecalis) and Gram negative (E. coli and P. aeruginosa) bacteria to metal and PDMS electrode surfaces under an electric field. After 2h of contact with each of these bacteria, the Zn electrodes showed a difference of 1.5-2 log of UFC/cm 2 between cathode and anode at voltages of 0.2 and 0.4 V, without significant killing of bacteria in suspension. Under the same conditions, the Al electrodes were found to be inactive in limiting and directing bacterial adhesion, probably because of surface oxidation causing its passivation. However, at 1V, the Zn and Al electrodes became bactericidal by killing bacteria in suspension. On the other hand, the bactericidal effect of Cu electrodes was very high at 0 V and seems to be reinforced by the electrical filed. Nevertheless, the bacterial adhesion on its surface was too weak to see a difference between the cathode and the anode. Once coated with PDMS, the bactericidal effect of Cu disappeared and a difference of 1 log of UFC/cm 2 , specific to staphylococcus species, was observed between the two electrodes above 10 V. Finally, this study demonstrates that the electrostatic force is the predominant factor governing the bacterial adhesion to Zn surfaces but not to those of Al and PDMS. For future works, high voltage should be used in order to increase the electrostatic bacterial repellent effect onto PDMS surfaces. Furthermore, metal electrodes could be used as physical disinfecting agents at 1V without current.

Figure 1 .

 1 Figure 1. Process used to evaluate the adhesion of bacteria to the surface of metallic or PDMS-coated electrodes

Figure 2 .

 2 Figure 2. Schematic principle of antibacterial action of cathode.

Figure 3 .

 3 Figure 3. Adhesion of S. aureus onto the surface of Cu, Zn and Al after 2h of contact under 0, 0.2, 0.4 and 1V: (A1) live bacteria adhered to the surface; (A2) live bacteria in suspension; control: live bacteria in suspension without any electrode.

Figure 4 .

 4 Figure 4. Adhesion of E. coli to the surface of zinc electrodes after 2h of contact at voltage values between 0 and 1 V: (A3) live bacteria adhered to the surface; (A4) live bacteria in suspension; control: live bacteria in suspension without any electrode.

Figure 5 .

 5 Figure 5. Adhesion of different bacterial species to the surface of zinc after 2h of contact at voltage values of 0 and 0.2 V: (A5) live bacteria adhered to the surface; (A6) live bacteria in suspension, control: live bacteria in suspension without any electrode.

  the PDMS or not due to the disappearance of the bactericidal activity of the Cu in suspension.

Figure 6 .

 6 Figure 6. Adhesion of S. aureus to the PDMS-coated copper surface (1mm) after 2 hours of contact at voltage values between 0 and 30 V: (B1) live bacteria adhering to the surface; (B2) live bacteria in suspension; control: live bacteria in suspension without electrode.

Figure 7 .

 7 Figure 7. Adhesion of different bacterial species to the surface of PDMS coated copper (1mm) after 2 hours of contact at 30 V: (B3) live bacteria adhering to the surface; (B4) live bacteria in suspension; control: live bacteria in suspension without any electrode.

Table 1 . Zeta potential of bacteria in sterile milli-Q water at a concentration of 10 7 -10 8 CFU / ml.

 1

	Bacteria	Zeta potential (mV)	Percentage
	S. aureus (ATCC 29213)	-39.3± 0.9	100%
		-38.5±1.7	51.80%±0.02
	S. epidermidis (ATCC 35984)
		-26.8±1.2	48.20%±0.02
	E. faecalis (ATCC 29212)	-35.8±1.0	100%

Acknowledgments

This work was funded by China Scholarship Council (CSC).


Génesis Osorio-Toribio

Miriam De J Velásquez-Hernández

Paulo G M Mileo

J Antonio Zárate

Javier Aguila-Rosas

Gerardo Leyva-Gómez

Roberto Sánchez-Sánchez

Jonathan J Magaña

Mario Alberto Pérez-Díaz

Isabel Abánades Lázaro

Miriam De

J Velásquez-Hernández

Roberto Sánchez- Sánchez

Ross S Forgan

Guillaume Maurin
email: guillaume.maurin1@umontpellier.fr

Enrique Lima
email: lima@iim.unam.mx

Ilich A Ibarra

Controlled Transdermal Release of Antioxidant Ferulate by a Porous Sc(III) MOF

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

INTRODUCTION

Metal-organic frameworks (MOFs) are one of the most recent classes of porous materials integrating a unique chemical and topological richness with an almost infinite combination of metal ions and multidentate organic linkers. 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[START_REF] Chen | Synthesis, functionalization, and applications of metalorganic frameworks in biomedicine[END_REF][START_REF] Horcajada | Metal-organic frameworks in biomedicine[END_REF][START_REF] Simon-Yarza | Nanoparticles of metalorganic frameworks: on the road to in vivo efficacy in biomedicine[END_REF] Some of these hybrid materials combine outstanding adsorption/separation performances of highly challenging molecules with green synthesis and easy scale-up that make them highly attractive to address a large panel of social concerns. [START_REF] Chen | Synthesis, functionalization, and applications of metalorganic frameworks in biomedicine[END_REF][START_REF] Horcajada | Metal-organic frameworks in biomedicine[END_REF][START_REF] Simon-Yarza | Nanoparticles of metalorganic frameworks: on the road to in vivo efficacy in biomedicine[END_REF] In particular, MOFs show great potential for the design of modern drug delivery technologies. [START_REF] Chen | Synthesis, functionalization, and applications of metalorganic frameworks in biomedicine[END_REF][START_REF] Horcajada | Metal-organic frameworks in biomedicine[END_REF][START_REF] Jie | Metal-Organic Frameworks for Biomedical Applications[END_REF][START_REF] Simon-Yarza | Nanoparticles of metalorganic frameworks: on the road to in vivo efficacy in biomedicine[END_REF][START_REF] Tibbetts | Recent Bio-Advances in Metal-Organic Frameworks[END_REF] Unlike conventional drug carriers envisaged so far, e.g., micelles, liposomes, dendrimers, and mesoporous silica nanoparticles, [START_REF] Gillies | Dendrimers and dendritic polymers in drug delivery[END_REF][START_REF] Porter | Lipids and lipid-based formulations: Optimizing the oral delivery of lipophilic drugs[END_REF][START_REF] Senapati | Controlled drug delivery vehicles for cancer treatment and their performance[END_REF][START_REF] Zhang | Cyclodextrinbased supramolecular systems for drug delivery: Recent progress and future perspective[END_REF][START_REF] Zhang | Mesoporous silica nanoparticles for increasing the oral bioavailability and permeation of poorly water soluble drugs[END_REF] MOFs offer a unique opportunity to modulate the incorporated drug payload and release kinetics by a fine engineering of their pore dimension (size/shape) and a fine tuning of the nature/strength of the adsorption sites decorating their internal pore walls as well as of the functionalisation of their external surfaces. [START_REF] Liang | Enhanced Activity of Enzymes Encapsulated in Hydrophilic Metal-Organic Frameworks[END_REF] The high degree of variability targets an efficient encapsulation of a broad range of highly challenging active pharmaceutical ingredients (APIs), in order to enhance their bioavailability and "shelf life". (Chen et al., 2018;[START_REF] Giménez-Marqués | Nanostructured metal-organic frameworks and their biorelated applications[END_REF][START_REF] Horcajada | Porous metal-organic-framework nanoscale carriers as a potential platform for drug deliveryand imaging[END_REF][START_REF] Liu | Recent developments on zinc (ii) metal-organic framework nanocarriers for physiological pHresponsive drug delivery[END_REF][START_REF] Luo | Metal--organic framework (MOF)-based nanomaterials for biomedical applications[END_REF][START_REF] Teplensky | Temperature Treatment of Highly Porous Zirconium-Containing Metal-Organic Frameworks Extends Drug Delivery Release[END_REF][START_REF] Wang | Metal-Organic Framework Encapsulation for Biospecimen Preservation[END_REF][START_REF] Xiao-Gang | Facile synthesis of a micro-scale MOF host-guest with long-lasting phosphorescence and enhanced optoelectronic performance[END_REF][START_REF] Ya-Pan | Bi-Microporous Metal-Organic Frameworks with Cubane [M4(OH)4] (M=Ni, Co) Clusters and Pore-Space Partition for Electrocatalytic Methanol Oxidation Reaction[END_REF][START_REF] Ying | Tetraphenylethylene-Decorated Metal-Organic Frameworks as Energy-Transfer Platform for the Detection of Nitro-Antibiotics and White-Light Emission[END_REF] Cargo drug loading in MOFs can be accomplished either by an encapsulation during the synthesis, [START_REF] Doonan | Metal-Organic Frameworks at the Biointerface: Synthetic Strategies and Applications[END_REF][START_REF] Liang | Biomimetic mineralization of metal-organic frameworks as protective coatings for biomacromolecules[END_REF][START_REF] Liang | Enhanced Activity of Enzymes Encapsulated in Hydrophilic Metal-Organic Frameworks[END_REF] or by a post-synthetic infiltration in the porosity of already-formed architectures. (Chen et al., 2018;[START_REF] Horcajada | Porous metal-organic-framework nanoscale carriers as a potential platform for drug deliveryand imaging[END_REF][START_REF] Teplensky | Temperature Treatment of Highly Porous Zirconium-Containing Metal-Organic Frameworks Extends Drug Delivery Release[END_REF][START_REF] Wang | Metal-Organic Framework Encapsulation for Biospecimen Preservation[END_REF] This later strategy has been widely explored in the development of controlled drug delivery systems of diverse natures for a large number of therapeutic agents. However, when contemplating any nano-carrier materials for biomedical applications several criteria are desired: (i) achieving an optimal drug loading efficiency, [START_REF] Horcajada | Metal-organic frameworks in biomedicine[END_REF] (ii) allowing the protection of the drug to avoid any degradation under physiological media, (iii) ensuring a gradual release of the drug once administrated to circumvent a "burst release effect", [START_REF] Bellido | Understanding the colloidal stability of the mesoporous MIL-100(Fe) nanoparticles in physiological media[END_REF][START_REF] Li | New insights into the degradation mechanism of metal-organic frameworks drug carriers[END_REF][START_REF] Ruyra | Synthesis, culture medium stability, and in vitro and in vivo zebrafish embryo toxicity of metal-organic framework nanoparticles[END_REF][START_REF] Velásquez-Hernández | Degradation of ZIF-8 in phosphate buffered saline media[END_REF] and (iv) not having toxic effects on the body. In this context, a number of porous MOFs, built up from bio-compatible metal ions i.e., Ca(II), Mg(II), Zn(II), Zr(IV), Ti(III), and Fe(III), has been discovered. [START_REF] Chen | Synthesis, functionalization, and applications of metalorganic frameworks in biomedicine[END_REF]Chen et al., 2018;[START_REF] Doonan | Metal-Organic Frameworks at the Biointerface: Synthetic Strategies and Applications[END_REF][START_REF] Horcajada | Porous metal-organic-framework nanoscale carriers as a potential platform for drug deliveryand imaging[END_REF][START_REF] Horcajada | Metal-organic frameworks in biomedicine[END_REF][START_REF] Liang | Biomimetic mineralization of metal-organic frameworks as protective coatings for biomacromolecules[END_REF][START_REF] Liang | Enhanced Activity of Enzymes Encapsulated in Hydrophilic Metal-Organic Frameworks[END_REF][START_REF] Simon-Yarza | Nanoparticles of metalorganic frameworks: on the road to in vivo efficacy in biomedicine[END_REF][START_REF] Teplensky | Temperature Treatment of Highly Porous Zirconium-Containing Metal-Organic Frameworks Extends Drug Delivery Release[END_REF][START_REF] Wang | Metal-Organic Framework Encapsulation for Biospecimen Preservation[END_REF] However, only a small fraction of them encompass the adequate features to fulfil all requirements mentioned above. [START_REF] Chen | Synthesis, functionalization, and applications of metalorganic frameworks in biomedicine[END_REF][START_REF] Horcajada | Metal-organic frameworks in biomedicine[END_REF][START_REF] Simon-Yarza | Nanoparticles of metalorganic frameworks: on the road to in vivo efficacy in biomedicine[END_REF] Typically, most of these MOFs suffer from a fast degradation in presence of phosphates, essential components in body fluids, hampering their applications for controlled drug delivery. [START_REF] Bellido | Understanding the colloidal stability of the mesoporous MIL-100(Fe) nanoparticles in physiological media[END_REF][START_REF] Li | New insights into the degradation mechanism of metal-organic frameworks drug carriers[END_REF][START_REF] Ruyra | Synthesis, culture medium stability, and in vitro and in vivo zebrafish embryo toxicity of metal-organic framework nanoparticles[END_REF][START_REF] Velásquez-Hernández | Degradation of ZIF-8 in phosphate buffered saline media[END_REF] The collapse of the framework is induced by a highly favourable complexation of the phosphate species to most of the metal centres tested so far, e.g. Zr(IV), Fe(III), and Zn(II). [START_REF] Bellido | Understanding the colloidal stability of the mesoporous MIL-100(Fe) nanoparticles in physiological media[END_REF][START_REF] Li | New insights into the degradation mechanism of metal-organic frameworks drug carriers[END_REF][START_REF] Ruyra | Synthesis, culture medium stability, and in vitro and in vivo zebrafish embryo toxicity of metal-organic framework nanoparticles[END_REF][START_REF] Velásquez-Hernández | Degradation of ZIF-8 in phosphate buffered saline media[END_REF] In this context, Sc(III)-MOFs has never been investigated for drug delivery applications to date. This is likely mostly a result of the controversy on the bio-compatible nature of this metal. On one hand, Sc(III) was demonstrated to be reactive towards proteins owing to its ability to replace Ca(II) in many biochemical events causing negative effects in enzyme systems and cell metabolism. [START_REF] Ford-Hutchinson | The Binding of Scandium Ions to Transferrin in vivo and in vitro[END_REF][START_REF] Sánchez-González | Accumulation of scandium in plasma in patients with chronic renal failure[END_REF] On the other hand, Sc(III) is successfully used as a radioactive isotope in medical applications, [START_REF] Horovitz | Biochemistry of scandium and yttrium, Part 1: physical and chemical fundamentals[END_REF][START_REF] Szkliniarz | Production of medical Sc radioisotopes with an alpha particle beam[END_REF] and a low concentration Sc(III) was proven to positively enhance specific antibiotic overproduction and have beneficial antibacterial effect. [START_REF] Kawai | The rare earth, scandium, causes antibiotic overproduction in Streptomyces spp[END_REF] Therefore, this critically urges for a comprehensive study on Sc(III) MOFs for potential drug delivery applications to address these open questions.

The present work reports the applicability of MFM-300(Sc), also denoted NOTT-400, [START_REF] Ibarra | Highly porous and robust scandium-based metal-organic frameworks for hydrogen storage[END_REF] as drug carrier for transdermal administration of ferulic acid (FA). FA is a natural superior antioxidant since its phenolic nucleus and unsaturated side chain allow the formation of resonance-stabilised phenoxy radical acting as free radical scavenger. [START_REF] Antolovich | LC-MS Investigation of Oxidation Products of Phenolic Antioxidants[END_REF]Chen et al., 2010b;[START_REF] Zduńska | Antioxidant properties of ferulic acid and its possible application[END_REF] FA also exhibits anti-diabetic, anti-cardiovascular and anti-inflammatory properties. [START_REF] Antolovich | LC-MS Investigation of Oxidation Products of Phenolic Antioxidants[END_REF]Chen et al., 2010b;[START_REF] Zduńska | Antioxidant properties of ferulic acid and its possible application[END_REF] Interestingly, this molecule has a protective role for the main skin structures such as collagen, fibroblasts, keratinocytes and elastin. [START_REF] Zih-Yi | The potential application of spring Sargassum glaucescens extracts in the moisture-retention of keratinocytes and dermal fibroblast regeneration after UVAirradiation[END_REF] Consequently, this therapeutic agent has been widely used in skin care formulations as photoprotective agent, delayer of skin photoaging processes, and in the treatment of rosacea. [START_REF] Antolovich | LC-MS Investigation of Oxidation Products of Phenolic Antioxidants[END_REF]Chen et al., 2010b;[START_REF] Zduńska | Antioxidant properties of ferulic acid and its possible application[END_REF] Due to the importance of FA as active ingredient in a variety of cosmetic products, the transdermal delivery is the most common administration route. [START_REF] Zih-Yi | The potential application of spring Sargassum glaucescens extracts in the moisture-retention of keratinocytes and dermal fibroblast regeneration after UVAirradiation[END_REF] However, the premature exposure of FA to sunlight undergoes to oxidation reactions leading to the formation of quinones, dimers, and aldehydes. [START_REF] Antolovich | LC-MS Investigation of Oxidation Products of Phenolic Antioxidants[END_REF] The photodegradation of FA not only limits its shelf-life, but also reduces its effectiveness before it permeates the stratum corneum (SC), [START_REF] Antolovich | LC-MS Investigation of Oxidation Products of Phenolic Antioxidants[END_REF] which is the most superficial layer of the epidermis, and acts as skin barrier. [START_REF] Kalpana | Challenges and opportunities in dermal/transdermal delivery[END_REF][START_REF] Pegoraro | Transdermal drug delivery: From micro to nano[END_REF] In order to circumvent these limitations, herein, we propose to encapsulate FA in the pores of MFM-300(Sc) with the main idea to prevent the photodegradation process of FA and to allow its continuous and sustained release over time that does not require frequent dosing.

RESULTS AND DISCUSSION

MFM-300(Sc) as drug carrier of FA (FER -) MFM-300(Sc) of chemical formula [Sc2(BPTC)(OH)2], was synthesised and activated according to a previously reported procedure (Supplemental Information). [START_REF] Ibarra | Highly porous and robust scandium-based metal-organic frameworks for hydrogen storage[END_REF] A series of characterisation tools confirmed the phase purity of the material and the full activation of its porosity (see Supplemental Information). This MOF crystallises in the chiral tetragonal space group I4122, with each [Sc2(µ-OH)] (Figure S1) binuclear centre octahedrally coordinated to six O-donors, four from four different carboxylate groups of BPTC -4 ligand (BPTC -4 = biphenyl-3,3´,5,5-tetracarboxylate) and two from two different µ-OH groups, leading to a 3D framework with a channel of about 8.1 Å (Figure S1). [START_REF] Ibarra | Highly porous and robust scandium-based metal-organic frameworks for hydrogen storage[END_REF] To evaluate the capacity of MFM-300(Sc) as drug delivery carrier, ferulic acid was initially loaded as ferulate species (FER -) (vide infra) within the MOF by a simple impregnation process, i.e. aqueous solution of ferulic acid (FA) at pH = 9, (Supplemental Information). Since the pore opening of MFM-300(Sc) exceeds the dimension of the drug (10.0 x 7.1 x 1.8 Å 3 ), FER -is expected to be confined in its channels. UV-vis absorption spectroscopy was employed to monitor the concentration of FER -during the impregnation process (Figure S2). The maximum FER -payload (16.1 wt%) was achieved after 5 days of incubation, we determined this percentage by quantifying free FER -in the supernatants when the MOF dispersion was centrifuged (Supplemental Information). The drug content was further corroborated by TGA analysis (Figure S5). Powder X-ray diffraction analysis of the drug-loaded matrix FER@MFM-300(Sc) (ferulate is adsorbed as FER -and Na + ) shows that the impregnation process does not modify the crystalline structure of MFM-300(Sc) (Figure S3) while N2 adsorption measurements revealed a decrease of the BET area and pore volume with respect to the pristine solid (915 m 2 g -1 and 0.39 cm 3 g -1 vs 1300 m 2 g -1 and 0.56 cm 3 g -1 , respectively) (Figure S4). Grand Canonical Monte Carlo (GCMC) simulations (see Supplemental Information for details) further predicted a lower uptake of FER -confined in the pores (10.7 wt%) associated with a decrease of the theoretical N2accessible surface area from the empty to the FER@MFM-300(Sc) materials (1480 m 2 g -1 vs 1290 m 2 g -1 ). This strongly suggests that part of the experimental payload is not associated with a confinement of FER -in the pores of the MOF.

The drug-release kinetics of FER@MFM-300(Sc) was further assessed by in-vitro studies using a dialysis bag diffusion technique (Supplemental Information). Two distinct regimes can be distinguished in the drug-release profile (Figure 1). Initially, ca. 37.0 % of FER -is released within the first 6 hours; this fast initial release is associated with the wellknown burst effect, [START_REF] Bellido | Understanding the colloidal stability of the mesoporous MIL-100(Fe) nanoparticles in physiological media[END_REF][START_REF] Li | New insights into the degradation mechanism of metal-organic frameworks drug carriers[END_REF][START_REF] Ruyra | Synthesis, culture medium stability, and in vitro and in vivo zebrafish embryo toxicity of metal-organic framework nanoparticles[END_REF][START_REF] Velásquez-Hernández | Degradation of ZIF-8 in phosphate buffered saline media[END_REF] and we can exclude that it is related to the degradation of MFM-300(Sc), since we have evidenced that the structure maintains its integrity after the drug release experiments and under the simulated physiological conditions used for the cytotoxicity assays (Supplemental Information, Figure S3). Interestingly, this payload (6.4 wt%) corresponds roughly to the deviation between the total experimental uptake and the predicted value for FER -confined in the pores (5.4 wt%). This first regime is thus most probably assigned to the release of FER -initially adsorbed at the external surface of the MOF. In other words, this first FER -release does not come from the micropores of MFM-300(Sc). This most likely comes from the external surface of the MOF crystals. Conversely, in the second regime the release process is considerably slower, releasing ca. 53 % of the loaded drug during further ca. 94 hours, approximately 4 days. Release of 9.7 wt% (end of the experiment) is in good agreement with the 10.7 wt% predicted by our calculations, supporting that this corresponds to the delivery of FER -initially confined in the pores of the MOF material.

Microscopic understanding of the FER@MFM-300(Sc) interactions

Monte Carlo (MC) and Density Functional Theory (DFT) calculations were further combined to understand the microscopic origin of this slow release. Analysis of the MC configurations for the adsorption of FER -in the dry state, revealed that the drug molecule establishes relatively strong hydrogen bonds with the µ-OH functional groups of MFM-300(Sc) via their carboxylic groups with characteristic OCOO--Oµ-OH distance of ca. 3.0 Å as seen by the corresponding radial distribution functions (RDF) plotted in Figure S15a. Such an arrangement illustrated in Figure 2a leads to a high DFT-calculated binding energy (-43.5 kcal•mol -1 ) that needs to be overcome in order for the molecules to be expelled from the MOF framework. Our MC simulations performed in solution further evidenced that FER - species interact strongly with water via their carboxylic groups with characteristic short OCOO--OW distance of 2.5 Å as seen by the corresponding RDF plotted in Figure S15b. A fraction of these water molecules equally establishes strong hydrogen bonds with the µ-OH groups of the MOF pore wall with characteristic Ow -Oμ-OH distance of 2.7 Å (Figure S15b). This concerted FER -water and water-MOF interaction scenario offers an optimal scenario to stabilise FER -species in the confined environment, water playing a pivotal role by acting as a bridge between the MOF pore-wall and FER -to retain strongly the drug as can be seen in Figure 2b. One can also notice that besides forming a strong hydrogen bond network, as seen by the high intensity of the peak at 2.8 Å in the RDF corresponding to the Ow -Ow pairs (Figure S15b), the water molecules form side interactions with the other functional groups of FER -(Figure 2b and Figure S15b). DFT simulations further confirmed that such an arrangement of FER -is highly stabilised with a corresponding binding energy of -120 kcal•mol -1 , consistent with a slow release observed experimentally. In line with these computational simulations, the experimental 13 C CP MAS spectra (Figure S7) confirm the interaction of FER -within the MOF material. In the spectrum of FER@MFM-300(Sc), the NMR signals originating from FER -are considerably lower in comparison to those for MFM-300(Sc). Two signals are observed in the carboxylate region, the first one at 174.9 ppm is the most intense and it is attributable to COO -bonded to Sc +3 in MFM-300(Sc). The second one appears at 170.7 ppm and it is attributable to COO -coming from the FER - species. The signal of COO -FER -•••Na + appears at 173.1 ppm, i.e. that COO -shifted 2.4 ppm to stronger fields when adsorbed in MFM-300(Sc), (Figure S7).

Indeed, the drug adsorption profile accomplishes the desired drug-delivery kinetic for transdermal drug administration applications, where the drug carrier systems are designed normally to release the cargo within 1 to 7 days.

Cytotoxicity experiments

Accordingly, the cytotoxicity of MFM-300(Sc) and its components (biphenyl tetracarboxylate=BPTC -4 , Sc +3 ) were investigated separately, monitoring their impact in the cell viability and proliferation of human dermal fibroblasts (Supplemental Information). [START_REF] Kalpana | Challenges and opportunities in dermal/transdermal delivery[END_REF] Five different concentration solutions (5, 25, 100, 250 and 500 ppm) of BPTC -4 , Sc +3 and MFM-300(Sc) were tested (Supplemental Information). The collected data revealed that both MFM-300(Sc) and Sc +3 did not considerably affect the cell viability, in comparison with positive controls. More than 98 % of the cells remain viable after 24 hours of incubation at any of the concentrations employed (Figure 3 and Figure S9). This behaviour remained almost constant even after 120 hours. However, the culture well plates pre-treated with BPTC -4 presented a considerable reduction of cell viability in a dose-dependent way. Since some cells separate from the culture plate when they are not viable, the number of cells was also assessed. The cells treated with MFM-300(Sc) only showed an 80% reduction of the number of live cells at 500 ppm. Cells treated with Sc +3 showed a 120 % reduction at 25-500 ppm. Treatments with BPTC -4 decrease considerably the number of cells from 5.5 times at 5 ppm to 8.8 times at 500 ppm. Moreover, the cell images collected on an epifluorescence microscope showed that even when using only 5 ppm of BPTC -4 , in the incubation media, the cells adopt a rounded morphology. This last observation indicates that the fibroblast attachment was not appropriate on the culture wells pre-treated with BPTC -4 (Figure 3 and Figure S9). These results not only demonstrate that Sc +3 and MFM-300(Sc) exhibit an acceptable biocompatibility for topical drug administration, but also confirm that BPTC -4 shows a negative cytotoxic effect.

These findings encouraged us to investigate the transdermal permeability of FER -upon the topical administration of this antioxidant using FER@MFM-300(Sc) as a drug carrier. An active ingredient will exert its effect on its therapeutic target by releasing it from its pharmaceutical form and it will subsequently arrive at its site-of-action.

Evaluation of the antioxidant effect of FER -released from the MFM-300(Sc) matrix

The ex-vivo permeation assays were carried out on Franz diffusion cells using low back human tissue (Figure 4a and Figure S10). This tissue was obtained from a female donor, who underwent aesthetic surgery, and it was employed with the prior consent of the donor (Supplemental Information, Figure S10). The Franz chambers consist of two compartments (donor and receptor) separated by the skin membrane (Figure 4a). The skin was placed in the donor compartment, where the stratum corneum (SC) was exposed to a suspension of FER@MFM-300(Sc) (41.4 mg mL -1 ) (Figure 4b and Figure S10). The receptor compartment was filled with PBS (phosphate buffered saline) at 37 °C and kept under stirring (500 rpm) for 87 hours. Afterwards, the receptor medium was sampled to determine the amount of antioxidant diffused across the skin barrier (referred as systemic diffusion). As a control test, this permeation experiment was also performed using an aqueous suspension of FA (6.68 mg mL -1 ) (Figure 4c). Then, once completed the permeation assay, the skin samples were further analysed to determine the biodistribution of the antioxidant within the skin layers. The tape stripping technique was employed to remove the SC layer of each skin sample. [START_REF] Binder | The role of viscosity on skin penetration from cellulose ether-based hydrogels[END_REF][START_REF] Escobar-Chávez | The tapestripping technique as a method for drug quantification in skin[END_REF][START_REF] Esposito | Monoolein liquid crystalline phases for topical delivery of crocetin[END_REF][START_REF] Limcharoen | Increasing the percutaneous absorption and follicular penetration of retinal by topical application of proretinal nanoparticles[END_REF] Then, these tape stripping samples, and the remaining part of each skin sample were analysed to estimate the amount of the antioxidant, retained in the SC and in the inner layers of the skin. The data collected shows that when using the FER@MFM-300(Sc) system ca. 10.40 % of the cargo permeates through the skin to the systemic compartment, whereas in the system with an aqueous suspension of free FA only ca. 5.71 % of the drug reaches the receptor compartment (Figure 4b and4c), presumably due to oxidation and poor solubility of free FA in the skin. Interestingly, the amount of the antioxidant retained in the SC when using the FER@MFM-300(Sc) formulation and free FA is comparable ca. 0.20 % and almost negligible, respectively. The carrier (MFM-300(Sc)) was retained in the SC as demonstrated by the size of the crystals (Figure S8). In addition, the quantity of the drug retained in the dermis and epidermis layers is around 0.29 % when using FER@MFM-300(Sc), and 0.39 % in the case of free FA. The permeation fluxes determined for FER -and FER@MFM-300(Sc) were 12.1x10 -4 and 5.9x10 -4 (nmol•cm -2 •h -1 ), respectively. These findings demonstrate that our carrier material (MFM-300(Sc)) improves the permeation of the drug through the human skin, in comparison to the free FA. Such differences might be attributed to the promotion in the absorption of FER -inside FER@MFM-300(Sc) possibly by transcellular or paracellular routes. In addition, there is a higher availability of the drug when it is administrated as the ion FER -specie than when it is administrated directly as the protonated specie (FA).

Thus, the poor solubility of FA in the aqueous suspension, as well as its propensity to oxidize, reduces the concentration of the available molecular species retarding the permeation through the skin layers. The therapeutic reach of the permeation of FER -from FER@MFM-300(Sc) in the epidermis-dermis region, systemic, or deposition from the SC, also implies the subsequent prolonged release for up to 5 days of the cargo with the innocuous biodegradation of the carrier, a requirement of special interest for chronic clinical conditions.

Evidence of non-permeation of FER@MFM-300(Sc) was recorded by SEM studies before and after the tape stripping technique due to the particle size of FER@MFM-300(Sc) (5x13 µm), which corroborates the new system as only for modified release and not for permeation (Figure S11). It is worth to emphasise that for this model with real human skin, we did not perform more repetition experiments to achieve a certain level of statistics due to the limitations of achieving reproducible samples (same origin of the human skin: ideally same patient, same area of extraction, age of the donor, sex, ethnicity, etc.). Thus, more than focusing on the absolute permeation values of FER -, it is very significant the trends that we show.

Finally, to examine if the therapeutic drug preserved its antioxidant pharmacological effects after the long-term release process, the antioxidant efficacy, of FER -released from the microporous carrier (MFM-300(Sc)), was tested by monitoring the reactive oxygen species (ROS) produced in dermal skin fibroblasts upon being treated with H2O2. First, the release of the antioxidant was performed by soaking the MOF composite (FER@MFM-300(Sc)) in the cell culture media for 5 days. Then, the fibroblasts were pre-treated with this incubation media for 20 min. Subsequently, the cells were exposed to H2O2 for 20 min to induce the oxidative stress. The efficacy of the antioxidant in protecting skin fibroblasts from H2O2 induced ROS toxicity was determined using CellRox flow cytometry assay (see Supplemental Information, Figure S9). The results obtained demonstrated that the unstained condition marks 100 % of cells without ROS expression (CellRox+), dead cells (7-AAD+) or doubly marked (CellRox/7-AAD+). In the basal state (without H2O2 treatment), only 0.2 % of the cells remain unlabelled and 67.5 % show ROS expression. Whereas, as expected, the cells incubated with H2O2 present an increase in ROS production (88.7 %), the unlabelled population decreases (0.6 %), and the population with dead cells and ROS+ showed 10.7 %. However, when the cells were treated with FER@MFM-300(Sc) (100 ppm) the percentage of unstained cells (without ROS or dead cell staining) increase considerably (25.9 %), in comparison with the values observed in the basal state (0.6 %). This fact confirms the FER -released from FER@MFM-300(Sc) maintains its bio-functionality by scavenging ROS species. Another result shows that only the application of the MFM-300(Sc) to the cells increase the unlabelled cells from 0.6 % to 1.3%, suggesting that the MFM-300(Sc) exhibits an unexpected antioxidant capacity. These findings suggest that FER@MFM-300(Sc) not only allows the long-term sustained release of FER -, but also enhances the bio-preservation of dermal skin fibroblasts during the delivery process.

In summary, this work demonstrates that MFM-300(Sc) is an efficient drug carrier by virtue of its excellent biocompatibility in human skin cells, remarkable stability under physiological conditions, and adequate controlled release for topical applications. The incorporation of FER -into MFM-300(Sc), in comparison to other well-known or even commercial materials like hydrotalcites, [START_REF] Lima | Controlled release of ferulic acid from a hybrid hydrotalcite and its application as an antioxidant for human fibroblasts[END_REF] provided a longer-term controlled release through human skin. MFM-300(Sc) also demonstrated a higher chemical stability in comparison to typical MOF drug delivery systems like MIL-100 and MIL-101. [START_REF] Horcajada | Metal-Organic Frameworks as Efficient Materials for Drug Delivery[END_REF][START_REF] Li | New insights into the degradation mechanism of metal-organic frameworks drug carriers[END_REF]This particular chemical stability for MFM-300(Sc) provides a benchmark material for future investigations in order to compare to other MOF carries, particularly those constructed with Sc(III) metal centres. In addition, our study reveals that this material improves the systemic delivery of ferulate leading to the controlled and sustained long-term delivery of the cargo, which avoids both the degradation of ferulate, and the continuous re-application of the drug during, relatively, a long time. These findings pave the way towards the extended use of Sc(III)-based MOFs as drug carriers.

LIMITATIONS OF THE STUDY

In this study, the topical drug bioavailability was assessed by in-vitro experiments using a two-chamber Franz cell. The main disadvantage of this system is that it does not consider the in-vivo skin processes and it does not mimic accurately the living state of the skin. For instance, the skin normally functions under air from the environment. However, during the permeation studies, SC is typically exposed to wet conditions for prolonged periods. On the other hand, the current work was limited to the release of FER -. Future efforts should test other drugs, in order to drive the extended use of Sc(III)-based MOFs as drug carriers.

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ilich A. Ibarra (argel@unam.mx). ex-vivo permeation experiments of FER -using the FER@MFM-300(Sc) formulation, and amount of antioxidant (%) retained within the skin and released in the receptor chamber (systemic) and (c) ex-vivo permeation experiments using an aqueous suspension of free FA, and percentage of antioxidant retained within the skin and released in the receptor chamber (systemic). See also Figures S8,S10 and S11.

FIGURE CAPTIONS

Figure 1 .

 1 Figure 1. Controlled release of FER in FER@MFM-300(Sc)Comparison between the in-vitro release profile of free FER -(red circles) and the FER -delivered from the MFM-300(Sc) thorough the dialysis bag technique (blue circles). Five independent invitro experiments were performed to generate the error bars (statistics). Lines are shown to guide the eyes. A schematic representation of the release of FER -from a FER@MFM-300(Sc) composite is shown as inset. See also FigureS1and S3.S6.

Figure 2 .

 2 Figure 2. Microscopic illustration of FER -adsorption in the MFM-300(Sc) porosity. Representative MC snapshots of the FER -adsorption in MFM-300(Sc) (a) at the dry state, displaying the single-adduct between the FER -carboxylic groups and the µ-OH groups of the pore wall and (b) in solution, showing the FER -interactions with H2O molecules either forming (1) a single-adduct with the µ-OH groups of the pore wall or (2) clusters at the centre of the pore. Sc, O, C and H are respectively represented in yellow, red, grey and white. MFM-300(Sc) atoms are shown translucid for clarity. H-bonds are shown in dashed blue lines. Captions associated to the RDFs are described in the text. See also Figures S2, S7 and S13-S15.

Figure 3 .

 3 Figure 3. Cytotoxicity experiments of MFM-300(Sc), Sc +3 and BPTC -4 . (a) Shows viable (green/calcein positives) and dead (red/EthD-1 positives) cells seeded on culture well plates pre-treated with different concentrations of MFM-300(Sc), Sc(III) and BPTC -4 . b) and c) show total number of cells or cell percentage of viable and dead cells respectively. ANOVA test and Sidak´s multiple comparisons test. * p<0.05, ** p<0.005, *** p<0.0005 and **** p<0.00005. Scale bars correspond to 100 μm. See also Figures S9 and S12.

Figure 4 .

 4 Figure 4. Ex-vivo permeation experiments of free and entrapped FER -. (a) Structural design of a Franz diffusion cell and schematic representation of the skin layers; (b)ex-vivo permeation experiments of FER -using the FER@MFM-300(Sc) formulation, and amount of antioxidant (%) retained within the skin and released in the receptor chamber (systemic) and (c) ex-vivo permeation experiments using an aqueous suspension of free FA, and percentage of antioxidant retained within the skin and released in the receptor chamber (systemic). See alsoFigures S8, S10 and S11.

ACKNOWLEDGMENTS

The authors thank Dr. A. Tejeda-Cruz (powder X-ray; IIM-UNAM), and PAPIIT UNAM (IN202820), Mexico for financial support. E.L. thanks PAPIIT UNAM (IN205320), Mexico for financial support. G. L-G. thanks PAPIIT UNAM (TA200318) and CONACyT (CB-A1-S-15759), Mexico for financial support. R. S.-S. thanks CONACyT (B-S-40505), Mexico for financial support. M. J. V. H. thanks CONACyT (419210) for the postdoctoral scholarship, Mexico. Thanks to U. Winnberg (ITAM) for scientific discussions and G. Ibarra-Winnberg for conceptualising the design of this contribution.

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

All the data needed to evaluate the conclusions of this work are detailed in the main text and/or the Supplemental Information. Additional data related to this paper may be requested from Ilich A. Ibarra (argel@unam.mx).

METHODS

Experimental details about materials and methods are properly described Transparent Methods supplemental file.The human tissue employed for the ex-vivo experiments, was used prior approval of the letter of consent and following the guidelines of the protocol approved by research committee of the National Rehabilitation Institute (Mexico City, Mexico) # 26/13 and the declaration of Helsinki.

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi

AUTHOR CONTRIBUTIONS

Génesis Osorio-Toribio and Miriam de J. Velásquez-Hernández: Synthesis and charcaterisation of MFM-300(Sc), assistance of different experiments, data collecting and integration of the full manuscript. Paulo G. M. Mileo and J. Antonio Zárate: computational calculations. Javier Aguila-Rosas, Gerardo Leyva-Gómez and Jonathan J. Magaña: ex-vivo experiments. Roberto Sánchez-Sánchez and Mario Alberto Pérez-Díaz in-vitro experiments. Isabel Abánades Lázaro and Ross S. Forgan: Reactive oxygen species (ROS) experiments. Guillaume Maurin, Enrique Lima and Ilich A. Ibarra: Principal leader investigators who conducted the entire project from the bio-compatible (Prof. Lima), computational (Prof. Maurin) and material (Prof. Ibarra) perspectives.

DECLARATION OF INTERESTS

The authors declare no competing interests.