Crosslinkable polypeptide and hyaluronic acid coatings
11602578 · 2023-03-14
Assignee
- Université de Strasbourg (Strasbourg, FR)
- Institut National De La Sante Et De La Recherche Medicale (Inserm) (Paris, FR)
- Protip Medical (Strasbourg, FR)
Inventors
- Philippe Lavalle (Wintzenheim, FR)
- Pierre SCHAAF (Molsheim, FR)
- Nihal Engin Vrana (Strasbourg, FR)
- Angela Mutschler (Strasbourg, FR)
- Cynthia Calligaro (Strasbourg, FR)
- Lorène Tallet (Strasbourg, FR)
Cpc classification
A61L31/16
HUMAN NECESSITIES
A61L2300/252
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
A61L2300/404
HUMAN NECESSITIES
International classification
A61L31/16
HUMAN NECESSITIES
Abstract
The present invention concerns a polyelectrolyte coating comprising at least one polycationic layer consisting of at least one polycation consisting of n repetitive units having the formula (1) and at least one polyanionic layer consisting of hyaluronic acid. The polyelectrolyte coating has a biocidal activity and the invention thus further refers to the use of said polyelectrolyte coating for producing a device, in particular a bacteriostatic medical device, more particularly an implantable device, comprising said polyelectrolyte coating, and a method for preparing said device and a kit.
Claims
1. A polyelectrolyte coating, comprising: (a) from 18 to 60 polycationic layers consisting of at least one polycation consisting of n repetitive units having the formula (1), ##STR00011## wherein n is an integer comprised between 2 and 100, and each R group, identical or different, is selected from the group consisting of —NH.sub.2, —CH.sub.2—NH.sub.2 and —NH—C(NH)—NH.sub.2, and (b) from 18 to 60 polyanionic layers consisting of hyaluronic acid, wherein at least a portion of the hyaluronic acid has a molecular weight of between 400 kDa and 3000 kDa, or a derivative thereof wherein said polyelectrolyte coating has antimicrobial activity.
2. The polyelectrolyte coating according to claim 1, wherein at least one polyanionic layer consists of hyaluronic acid wherein at least a portion of the hyaluronic acid has a molecular weight between 800 kDa and 3000 kDa, or a derivative thereof.
3. The polyelectrolyte coating according to claim 1, wherein the polycationic layers and the polyanionic layers are cross-linked or wherein the polyanionic layers are cross-linked at a level of cross-linking such that at least one polycation in the polycationic layers keeps a mobility of at least 70%.
4. The polyelectrolyte coating according to claim 1, wherein the polycationic layers consist of n repetitive units having the formula (1), ##STR00012## wherein n is an integer comprised between 2 and 100, and R is chosen from —NH.sub.2, —CH.sub.2—NH.sub.2 and —NH—C(NH)—NH.sub.2.
5. The polyelectrolyte coating according to claim 1, wherein R of formula (1) is —NH—C(NH)—NH.sub.2.
6. The polyelectrolyte coating according to claim 1, which comprises 18 to 50 polycationic layers, and/or 18 to 50 polyanionic layers.
7. A device comprising a polyelectrolyte coating according to claim 1.
8. The device of claim 7, wherein the polyelectrolyte coating covers at least a portion of the surface of said device.
9. The device of claim 8, wherein said device is an implantable device.
10. The implantable device according to claim 8, wherein the implantable device is selected from the group comprising catheters, arteriovenous shunts, breast implants, cardiac and other monitors, cochlear implants, defibrillators, dental implants, maxillofacial implants, middle ear implants, neurostimulators, orthopedic devices, pacemaker and leads, penile implants, prosthetic devices, replacement joints, spinal implants, voice prosthesis, artificial hearts, contact lenses, fracture fixation device, infusion pumps, intracranial pressure device, intraocular lenses, intrauterine devices, joint prosthesis, mechanical heart valves, orthopedic devices, suture materials, urinary stents, vascular assist device, vascular grafts, vascular shunts and vascular stents, and artificial vessels of permanent or transient types.
11. The device according to claim 7, wherein said device is an implantable device comprising a pharmaceutically active drug.
12. A method for preparing a device comprising the polyelectrolyte coating according to claim 1, the method comprising: (a) providing a device; (b1) depositing on the surface of said device (i) from 18 to 60 polycationic layers consisting of at least one polycation consisting of n repetitive units having the formula (1), ##STR00013## wherein n is an integer comprised between 2 and 100, and each R group, identical or different, is selected from the group consisting of —NH.sub.2, —CH.sub.2—NH.sub.2 and —NH—C(NH)—NH).sub.2, and then ii) from 18 to 60 polyanionic layers consisting of hyaluronic acid, wherein at least a portion of the hyaluronic acid has a molecular weight of between 400 kDa and 3000 kDa or a derivative thereof, or (b2) depositing on the surface of said device ii) and then i) as defined above, and optionally repeating step b1) and/or b2).
13. The method according to claim 11, further comprising a step of cross-linking the at least one polycationic layer and the at least one polyanionic layer or cross-linking the at least one polyanionic layer at a level of cross-linking such that the at least one polycation in the at least one polycationic layer keeps a mobility of at least 70%.
14. A method comprising using a polyelectrolyte coating according to claim 1 for producing a device.
15. A kit comprising the polyelectrolyte coating according to claim 1.
16. The method of claim 14, wherein the device is an implantable device.
17. The method of claim 16, wherein the implantable device is selected from the group comprising catheters, arteriovenous shunts, breast implants, cardiac and other monitors, cochlear implants, defibrillators, dental implants, maxillofacial implants, middle ear implants, neurostimulators, orthopedic devices, pacemaker and leads, penile implants, prosthetic devices, replacement joints, spinal implants, voice prosthesis, artificial hearts, contact lenses, fracture fixation device, infusion pumps, intracranial pressure device, intraocular lenses, intrauterine devices, joint prosthesis, mechanical heart valves, orthopedic devices, suture materials, urinary stents, vascular assist device, vascular grafts, vascular shunts and vascular stents, and artificial vessels of permanent or transient types.
18. The device according to claim 14, wherein said device is an implantable device comprising a pharmaceutical active drug.
19. The polyelectrolyte coating of claim 1, wherein said polyelectrolyte coating has anti-inflammatory activity.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
1. Example 1
(30) 1.1 Material
(31) Polyelectrolyte multilayer coatings have been built up with the following polyelectrolytes. Polycations of poly(I-arginine hydrochloride) (PAR) were purchased from Alamanda Polymers, USA. Different PAR polymers used differ from the numbers of arginine per chain: PAR10 10 arginine (R), Mw=2.1 kDa, PDI=1); PAR30 (30 R, Mw=6.4 kDa, PDI, =1.01), PAR100 (100 R, Mw=20.6 kDa, PDI=1.05), and PAR200 (200 R, Mw=40.8 kDa, PDI=1.06). Poly(L-ornithine hydrochloride) (PLO) was purchased from Alamanda Polymers, USA. Different PLO polymers used differ from the numbers of ornithine per chain: PLO30 (30 R, Mw=5.9 kDa, PDI, =1.03), PLO100 (100 R, Mw=18.5 kDa, PDI=1.03), and PLO250 (250 R, Mw=44.7 kDa, PDI=1.02). Poly(L-lysine hydrochloride) (PLL) such as PLL30 (30 R, Mw=5.4 kDa, PDI, =1.02) was purchased from Alamanda Polymers, USA.
(32) Hyaluronic acid (HA, Mw=150 kDa) used as the polyanion was from Lifecore Biomed, USA.
(33) 1.2 Methods
(34) Monitoring build-up of multilayer coatings: Coating or film build-up was followed using an in situ quartz crystal microbalance (QCM-D, E1, Q-Sense, Sweden). The quartz crystal is excited at its fundamental frequency (about 5 MHz), as well as at the third, fifth, and seventh overtones (denoted by v=3, v=5, v=7 corresponding respectively to 15, 25, and 35 MHz). Changes in the resonance frequencies (Δf) are measured at these four frequencies. An increase of Δf/v is usually associated to an increase of the mass coupled with the quartz. PAR (i.e. PAR10, PAR30, PAR50, PAR100 or PAR200) and HA were dissolved at 0.5 mg.Math.mL.sup.−1 in sterilized buffer containing 150 mM NaCl and 10 mM of tris(hydroxymethyl)-aminomethan (TRIS, Merck, Germany) at pH 7.4. The polyelectrolyte solutions were successively injected into the QCM cell containing the quartz crystal and PAR was the first deposited polyelectrolyte. They were adsorbed for 8 min and then, a rinsing step of 5 min with NaCl-Tris buffer was performed.
(35) Buildup of (PAR/HA).sub.24 with dipping robot: For the construction of 24 bilayers of PAR/HA ((PAR30/HA).sub.24) an automated dipping robot was used (Riegler & Kirstein GmbH, Berlin, Germany). Glass slides (12 mm in diameter) were first washed with Hellmanex® II solution at 2%, H.sub.2O, and ethanol and dried with air flow. The solutions of polyelectrolytes were prepared as described above for QCM experiments. Glass slides were dipped alternatively in polycation and polyanion solutions and extensively rinsed in NaCl-Tris buffer between each step. After construction, the coatings were dried with air flow and then immerged in NaCl-Tris buffer and stored at 4° C. before use. Thicknesses of obtained coatings were evaluated by deposition of 100 μL of PLL-FITC (poly-L-lysine labelled with fluorescein isothyocyanate, a green fluorescent probe) (0.5 mg.Math.mL.sup.−1 in Tris-NaCl buffer) on top of the PAR/HA multilayer coatings. After 5 minutes and diffusion of PLL-FITC through the whole coating, a rinsing step was performed with Tris-NaCl buffer. Observations of the coatings were carried out with a confocal microscope Zeiss LSM 710 microscope (Heidelberg, Germany) using a 20× objective (Zeiss, Plan Apochromat).
(36) 24 bilayers of PLUHA (PLL30/HA).sub.24 and 24 bilayers of PLO/HA (i.e. (PLO30/HA).sub.24, (PLO100/HA).sub.24 and (PLO250/HA).sub.24) were prepared in analogy, wherein PLO or PLL was dissolved at 0.5 mg.Math.mL.sup.−1 in sterilized buffer containing 150 mM NaCl and 10 mM of tris(hydroxymethyl)-aminomethan (TRIS, Merck, Germany) at pH 7.4.
(37) Other polyelectrolyte coatings, for example polyelectrolyte coatings comprising 48 bilayers of PAR/HA such as (PAR30/HA).sub.48 and (PAR10/HA).sub.48 were prepared in analogy.
(38) Antibacterial Assays:
(39) Staphylococcus aureus (S. aureus, ATCC 25923) strains were used to assess the antibacterial properties of the test samples. Bacterial strain was cultured aerobically at 37° C. in a Mueller Hinton Broth (MHB) medium (Merck, Germany), pH 7.4. One colony was transferred to 10 mL of MHB medium and incubated at 37° C. for 20 h, to provide a final density of 10.sup.6 CFU.Math.mL.sup.−1. To obtain bacteria in the mid logarithmic phase of growth, the absorbance at 620 nm of overnight culture was adjusted of 0.001.
(40) Glass slides coated with (PAR/HA).sub.24 with PAR10, PAR30, PAR100, are sterilized by using UV-light during 15 minutes, then washed with NaCl-Tris buffer. After washing, each glass slides were deposited in 24-well plates with 300 μl of S. aureus, A.sub.620=0.001, and incubated during 24 hours at 37° C.
(41) For negative control, uncoated glass slides were directly incubated with S. aureus using a similar method.
(42) For positive control, Tetracycline (10 μg.Math.mL.sup.−1) and Cefotaxime (0.1 μg.Math.mL.sup.−1) were added in S. aureus solution in contact with uncoated glass slides.
(43) To quantify bacteria growth or inhibition after 24 h, the absorbance of the supernatant at 620 nm was measured.
(44) The assay was performed similarly for Glass slides coated with (PLL30/HA).sub.24, (PLO30/HA).sub.24, (PLO100/HA).sub.24 and (PLO250/HA).sub.24.
(45) The antibacterial assay for M. Luteus, E. Coli and P. aeruginosa were performed in analogy to the bacterial assay described for Staphylococcus aureus described above.
(46) Bacteria Live Dead Assay: To evaluate the health of bacteria which are on the surface, the BacLight™ RedoxSensor™ CTC Vitality Kit (ThermoFischer Scientific Inc., France) was used. This kit provides effective reagents for evaluating bacterial health and vitality. The kit contains 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which has been used to evaluate the respiratory activity of S. aureus. Indeed, healthy bacteria will absorb and reduce CTC into an insoluble, red fluorescent formazan product. Bacteria which are dead or with a slow respiratory activity will not reduce CTC and consequently will not produce red fluorescent product. Finally this kit gives a semi-quantitative estimate of healthy vs unhealthy bacteria. SYTO® 24 green-fluorescent nucleic acid stain (ThermoFischer Scientific Inc., France) is used for counting all bacteria. A solution of 50 mM CTC and 0.001 mM Syto 24 in pure water is prepared. Each glass slides were washed with phosphate-buffered saline buffer, pH=7.4 (PBS) then 270 μl of PBS and 30 μL of CTC/Syto 24 solution were added. The plate were incubated 30 minutes at 37° C., away from light. Each surfaces were observed with confocal microscopy (Zeiss LSM 710 microscope, Heidelberg, Germany), using a 63× objective, immerged in oil. Excitation/Emission wavelength of stains was 450/630 nm for CTC and 490/515 nm for Syto 24.
(47) Biocompatibility test: Human fibroblast (CRL-2522 from ATCC/LGC Standards, France) was cultured at 37° C. in Eagle's Minimum Essential Medium (EMEM, ACC/LGC) with 10% of Fetal Bovin Serum (FBS, Gibco/ThermoFicher Scientific Inc., France) and 1% of penicillin streptomycin (Pen Strep, Life Technologies/ThermoFicher Scientific Inc., France). 50 000 cells were incubated in each well of a 24 well-plates during 24 h. Glass slides coated with (PAR/HA).sub.24 were incubated simultaneously in a 6 well-plates with 1 mL of medium. After 24 h, the medium of the wells containing cells was removed and replaced by the supernatant that was in contact with the multilayers for 24 h. Human fibroblasts were incubated during 24 h at 37° C. Then, the supernatant was removed and incubated with 10% of AlamarBlue (ThermoFischer Scientific Inc., France) during 2 h. The cell viability was determined by measuring the fluorescence of produced resofurin (Excitation/Emission wavelength=560/590 nm). Cells were washed twice with PBS and fixed with PFA 4% solution during 10 minutes, and then again washed twice with PBS. A solution of Phalloidin was prepared in PBS buffer with 1% of bovin serum albumin (BSA). The staining solution were placed on the fixed cells for 30 minutes at room temperature and washed two times with PBS buffer. A solution of DAPI was prepared and placed on the cells at the same conditions as previously. Fluorescence images were captured using Nikon Elipse Ti-S with 63×PL APO (1.4 NA) objective equipped with Nikon Digital Camera (DS-Q11MC with NIS-Elements software, Nikon, France), and processed with ImageJ (http://rsb.info.nih.gov/ij/). Excitation/Emission wavelength for Rhodamine Phalloidin was 540/565 nm and for DAPI 350/470 nm.
(48) Time-lapse microscopy: Glass slides coated with (PAR/HA).sub.24 were sterilized by using UV-light during 15 minutes, then washed with NaCl-Tris buffer. After washing, each glass slides were mounted in a Ludin Chamber (Life Imaging Services, Switzerland) at 37° C., 5% CO.sub.2, with 1 mL μl of S. aureus (used as described previously, with A.sub.620=0.001), stained with Syto 24 during the culture. The time-lapse sequence was performed during 24 h with a Nikon TIE microscope equipped with a 60×PL Apo oil (1.4 NA) objective and an Andor Zyla sCMOS camera (Andor Technology LtD. United Kingdom), was used with Nikon NIS-Elements Ar software (Nikon, France). Phase contrast and fluorescence images were acquired every 5 min for 24 h. Images were processed with ImageJ.
(49) Circular Dichroism: Circular dichroism (CD) spectra were recorded using a Jasco J-810 spectropolarimeter (Jasco Corporation, UK) as an average of 3 scans obtained using a 0.1 mm path length quartz cuvette at 22° C. from 180 to 300 nm with data pitch of 0.1 nm and a scan speed of 20 nm/min. All spectra were corrected by subtraction of the buffer spectra. Spectra for each PAR were obtained at a concentration of 2 mg.Math.mL.sup.−1 in NaCl-Tris Buffer. All CD data were expressed as mean residue ellipticity.
(50) Fluorescent labelling of PAR: For labeling PAR chains, PAR (15 mg.Math.mL.sup.−1 in 100 mM NaHCO.sub.3 pH 8.3 buffer) was incubated with fluorescein isothiocyanate (FITC, Sigma Aldrich, France) at 1:2 molar ratio of PAR/FITC at room temperature for 3 h. This solution was dialyzed against 1 L of water at 4° C. with a Slide-A-Lyser Dialysis Cassette (Thermo Fischer Scientific Inc, USA), cut-off=3500 MWCO. PAR-FITC was then produced and stored in aliquots of 2 mL (0.5 mg.Math.mL.sup.−1 in NaCl-Tris buffer).
(51) Release Experiments: For the first experiment, a multilayer coating (PAR30/HA).sub.24 was built by using PAR-FITC. Release experiments were performed at 37° C. during 24 h in presence of MHB medium or a S. aureus/MHB solution (A.sub.620=0.001). 300 μL of mineral oil were added on the top of the supernatant to prevent any evaporation during the monitoring. The release of PAR-FITC in solution was performed by measuring the fluorescence of the supernatant over time with a spectrofluorimeter (SAFAS Genius XC spectrofluorimeter, Monaco) with excitation/emission wavelength of 488/517 nm. Three samples were studied for each conditions.
(52) For the second experiment, a multilayer film (PAR30/HA)24 was incubated at 37° C. with two conditions: A) with 300 μl of S. aureus solution A620=0.001 and B) 300 μl of MHB only. After 24 h, the supernatant in contact with the LbL was taken and incubated with a new S. aureus solution to have a final A620=0.001. After 24 h at 37° C., the absorbance at 620 nm was measured. Three samples were studied for each condition.
(53) Fluorescence Recovery After Photobleaching (FRAP) experiments: The diffusion coefficient, D, and the proportion of mobile molecules, p, was measured for (PAR/HA).sub.24 multilayers containing PAR-FITC by performing photobleaching experiments (FRAP, Fluorescence Recovery After Photobleaching).
(54) A glass slide coated with the multilayer was introduced in a home-made sample holder and immerged in 200 μl of Tris-NaCl buffer. One circular regions (4.4 μm in radius and referred as “R4” in an image of 35 μm×35 μm or 10.6 μm in radius and referred as “R10” in an image of 85 μm×85 μm) were exposed for 700 msec to the laser light set at its maximum power (λ=488 nm). Then, the recovery of the fluorescence in the bleached area was observed over time. Observations were carried out with a Zeiss LSM 710 microscope (Heidelberg, Germany) using a 20× objective (Zeiss, Plan Apochromat).
(55) Cross-linking of (PAR30/HA)24: Crosslinking was performed by immersing the (PAR30/HA)24 films in a solution containing EDC (100 mM) and N-hydroxysuccinimide (10 mM) in NaCl (0.15 M) during 15 h at 4° C. Films were rinsed 2 times with a NaCl (0.15 M) solution. The films were immerged in a solution of ethanolamine (1M) during 40 minutes at 4° C. to neutralize all carboxylates functions that have not react. The films were rinsed with NaCl solution and the NaCl-Tris buffer solution was used for the last rinsing step.
(56) 1.3 Results
(57) In order to test the buildup of the PAR/HA coatings, quartz crystal microbalance (QCM) was used.
(58) The inventors also estimated the thicknesses of the films by using the model of Voinova et al. (Phys. Scripta 1999, 59, 391-396). After 8 deposited pair of layers (or “bi-layers”) the thicknesses of the films built up with PAR10, PAR30, PAR100 or PAR200 as polycations equals 70, 130, 200 or 450 nm respectively (
(59) An opposite behavior was previously demonstrated for multilayer coatings buildup with chitosan/HA with various MW of chitosan (Richert 2004, Langmuir). An exponential growth of the coatings was observed for all the MW of chitosan used, however the coating buildup was more rapid when the mass of the chitosan chains was smaller. This behavior was related to the diffusive properties of the chitosan chains in the coatings: shorter chains should diffuse more through the coating which should lead to a higher increase in the mass increment after each layer deposition. However in the present study, experimental conditions are different as an homopolypeptide was selected as the polycation instead of a polysaccharide and the range of their chain length was smaller.
(60) Then, coatings with 24 bilayers ((PAR/HA).sub.24) were observed with confocal microscopy. In order to label fluorescently the coatings, poly(lysine)-FITC was added as the last layer on top of the coatings. Cross-section images of coatings build up with PAR of different residue numbers depict thick bands with a green labeling through the whole coating section (
(61) Next, the antimicrobial properties of (PAR/HA)24 multilayers for PAR of different number of residues was evaluated. The films were tested against a gram positive bacteria, S. aureus, a strain well known to be associated with nosocomial infections and more particularly with implant-related infections. For example in the case of orthopaedic implants, S. aureus with S. epidermis is involved in 70% of infections (biomaterials 84, 2016, 301). S. aureus were incubated for 24 h at 37° C. in the presence of MHB medium on the (PAR/HA)24 coatings. The bacteria were incubated at high density on surfaces for 24 h at 37° C. in the presence of MHB medium. The normalized growth of pathogens (%) was estimated by comparing absorbance at 620 nm in the presence of multilayer films in comparison with the positive control (without multilayer films and in presence of antibiotics in the medium) and the negative control (without multilayer films and in the absence of antibiotics in the medium). No significant inhibition was observed for films built with PAR10, PAR50, PAR100 and PAR200. However, for PAR30 (30 residues), more than 95% of bacterial growth inhibition was observed after 24 hours. This suggests that PAR30 strongly impact viability of S. aureus. It must be pointed out that the molecular weight effect is extremely striking and up to now such an effect on the multilayer film functionality, whatever this function, was never observed (see
(62) To evaluate more precisely the health of bacteria in contact with the surfaces, the respiratory activity of S. aureus using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was monitored. Healthy bacteria will absorb and reduce CTC into an insoluble, red fluorescent formazan product and bacteria which are dead will not reduce CTC and consequently will not produce fluorescent product (Data not shown). A total inhibition of bacteria on (PAR30/HA) surfaces was clearly observed and it was extremely rare to find an area with few bacteria (Data not shown). Comparatively, PAR10, PAR100 or PAR200 surfaces did not prevent bacterial adhesion and growth, a similar density of healthy bacteria as on non-treated surfaces was found. This outstanding result is in full correlation with growth inhibition in supernatant described above where PAR30 was also the only coating strongly effective against bacteria.
(63) In order to elucidate if the bacterial growth inhibition of the (PAR/HA).sub.24 coatings is only limited to S. aureus, bacterial growth inhibition of (PAR30/HA).sub.24 was further tested against other gram positive and gram negative bacteria. Accordingly, the antimicrobial properties of (PAR30/HA).sub.24 multilayer was evaluated for methilin resitant S. aureus strain, M. Luteus, E. coli and P. aeruginosa. The result shown in
(64) In this context, the inventors were interested if the antibacterial activity is limited to the herein described (PAR/HA) multilayer coatings or if a coating comprising as polycation another polypeptide would also demonstrate the same antibacterial activity. The inventors of the present invention further evaluated (PLUHA).sub.24 and (PLO/HA).sub.24 multilayers for PLL and PLO of different number of residues. Surprisingly, as it can be concluded from
(65) In the following the inventors of the present invention wanted to further elucidate the underlying mechanism that confer to the coatings of the invention the strong inhibitory property on the surface and in the supernatant. As an example the (PAR30/HA).sub.24 coating was further investigated. Accordingly, the minimal inhibitory concentration (MIC) of PAR in solution was determined using bacterial assay as described in Experimental Section. For concentrations up to 0.04 mg.Math.mL.sup.−1, all PAR (PAR10, PAR30, PAR100 or PAR200) totally inhibited S. aureus growth (
(66) However, PAR chains of different number of residues were not markedly different in their activity in solution and thus the origin of the PAR30 activity observed with PAR30/HA films is not related to its higher activity in solution.
(67) To address the conformations of PAR chains and to check if PAR30 chains have a specific secondary structure that could explain their inhibitory properties compare to longer or shorter chains, circular dichroism (CD) experiments were performed. Firstly, secondary structures of PAR chains in NaCl/TRIS buffer solution (150 mM NaCl, 10 mM Tris, pH 7.4) (Data not shown). All CD spectra of PAR chains (PAR10, PAR30, PAR100 and PAR200) show a unique negative minimum at about 200 nm characteristic of a random coil conformation in solution. In a second step, PAR conformation in PAR/HA multilayer coatings was monitored (Data not shown). Surprisingly, spectra of the coatings depict totally different profiles: no more minima at 200 nm were observed, however one minima at about 10 and another one at about 222 nm were monitored, except for (PAR10/HA)24, which present a unique negative minimum at 200 nm (random coil). They can probably not be attributed to HA chains as it is known that in solution at pH 7.4 they have an unordered conformation (Zahouani, ACS Applied Materials, 2016, 8, 14958-14965). Moreover the PAR/HA spectrum correspond more probably to chains in α-helix conformations characterized by these two minimums. This indicates that PAR chains should change from a coil conformation in solution to an α-helix in the coating. Similar behavior were previously observed for LbL build up with polylysine and poly(glutamic acid) (Boulmedais F. et al. Langmuir, 2002, 18, 4523). Interestingly, unordered antimicrobial peptides are known to adopt an α-helix conformations when they interact with the bacterial membrane and this mechanism is a key point in their mechanism of action (Porcellini F. et al. 2008, Biochemistry, 47, 5565 and Lugardon K. et al., 2001, J Biol Chem., 276(38): 35875). Here in polyelectrolyte multilayer coatings, PAR chains already adopt an α-helix conformation most probably due to the interactions between PAR and HA and local high concentration of PAR. This mechanism can be helpful to fight faster and in a more efficiency way against invading bacteria.
(68) But, because the films built with different PAR chain lengths present similar spectra, the secondary structure of PAR chains cannot explain the striking molecular weight effect on the bactericidal property of the PAR30/HA films.
(69) In view of the absence of specific properties of PAR30 in solution compared to shorter or longer PAR chains, the antimicrobial abilities of PAR30/HA films should be related to the film property by itself. In this context, we investigated if the bactericidal property of the film is due to the release of PAR30 chains from the multilayer into the solution or if bacteria need to come in contact with the film to be killed. For this purpose two types of experiments were performed. Using fluorescently labelled PAR30 chains we first determined the release of PAR30 chains into the solution containing solely MH medium with and without S. aureus.
(70) Finally, we also performed an experiment where bacteria were brought in contact with a (PAR30/HA)24 film for 24 hours. Bacteria growth was totally inhibited. The supernatant of this experiment was removed and brought it in contact with a fresh suspension of bacteria. Here again, the bacteria growth was no further inhibited (Data not shown). These results demonstrate that the release of the PAR30 chains from the film in the supernatant cannot be at the origin of the bactericidal effect. Finally we can hypothesize that the bactericidal effect is directly related to the contact of the bacteria with the PAR30/HA film which acts as a contact-killing multilayer.
(71) Then the inventors investigated if the bactericidal activity of the PAR30/HA multilayer is related to the mobility of these chains in the films. Indeed, it is known that the exponential character of a multilayer is related to the diffusion ability of at least one of its constituents in and out of the film during each deposition step. They first determined the mobility of the different chains, PAR10, PAR30, PAR100 and PAR200 in the (PAR/HA)24 multilayer by using FRAP method.
(72) From these curves we can deduce the percentage of mobile chains over the timescale of the experiments. It clearly appears that PAR10 and PAR30 chains are more mobile (between 85 to 90% of mobile fraction) than the PAR100 (only 63% of mobile fraction). The largest chains PAR200 correspond to the slowest with about 12% of the population which is mobile (
(73) To confirm the dependence of mobility on the bactericidal effect of the film, the inventors cross-linked the PAR30/HA multilayers using a standard EDC-NHS cross-linking method which creates a covalent link between amine groups of PAR and carboxylic groups of HA. The proportion of mobile chains measured by FRAP method decreases significantly from 88% for the non-crosslinked film to 20% for the cross-linked one (Data not shown). When the cross-linked film was brought in contact with S. aureus, only about 40% of inhibition of the bacterial growth was observed after 24 h of contact (
(74) These results suggest that the percentage of mobile PAR chains in the multilayer is an important parameter controlling its bactericidal property of the film (
(75) Finally, using confocal microscopy, the inventors also investigated the structure of the film after 24 hours of contact with bacteria (Data not shown). For this purpose, films were constructed by incorporating fluorescently labelled PAR30 chains. It was found that after 24 hours of contact, the film is no longer homogeneous but that non-fluorescent areas appear. Because these areas have a smooth shape, they suggest a reorganization of the film. Such a behavior is not observed in the absence of bacteria where the films remain homogeneous. Such a reorganization could be consecutive to de decrease of the PAR chains in the film and suggest the following bactericidal mechanism: When bacteria come in contact with the multilayer, the negative bacteria membranes act as strong attractive surfaces for the PAR chains. Mobile PAR chains are thus soaked out of the film by the bacteria membranes and destabilize them. Chains of large molecular weight have a stronger membrane destabilization power than smaller molecular weight ones as it comes out from the MICs determined in solution. PAR10 chains are 10 times less active than PAR100 or PAR200 chains but PAR30 chains are only 2 times less active than PAR100 or PAR200 chains. Yet, in a film one can assume that the concentration of arginine monomers is fairly independent of the molecular weight of the PAR chains. Thus, the concentration of chains decreases when the molecular weight of the polyelectrolytes increases. For example the concentration PAR30 chains in the film should be 3 times higher than that of PAR100. In addition there are of the order of 90% of PAR10 chains that are mobile whereas only 70% of PAR100 chains are mobile. This leads to 4 times more PAR10 chains than PAR100 in the film. There could be other factors explaining the higher propensity of films PAR/HA films built with PAR30 to be strongly bactericidal but the chain mobility is without doubt an important one.
(76) The inventors clearly demonstrate that the property of the coating is related to the length of the PAR polyelectrolyte chain. The concentration of the mobile PAR chains is a key-factor in the antimicrobial effectiveness and thus the films having 24 layers and being buildup with PAR containing 30 residues of arginine seem optimal for such bioactivity.
(77) PAR containing 10 residues per chain seems not active in (PAR/HA).sub.24 films despite its high mobility in the films and its MIC which is close to that of PAR30. The inventors considered that this can be attributed to the film buildup which is about two times thinner with PAR10 compared to PAR30. After 24 bilayers, the amount of free PAR10 chains able to inhibit for 24 h bacterial growth could be too low.
(78) To verify this hypothesis, the inventors performed therefore additional experiments with PAR10/HA films containing a higher number of bilayer (48 instead of 24) (
(79) This indicates that a sufficient number of free PAR chains should be available to confer antimicrobial properties to the films. For PAR10 or PAR30, this number is reached with 48 or 24 bi-layers respectively.
(80) The inventors consider that, concerning the mechanism of action of PAR30 (or PAR10 for thicker films), it should be related to diffusion of PAR30 or PAR10 chains out of the film enhanced by the attractive electrostatic interactions between the positively charged PAR and the negatively charged bacterial membrane. This interaction should occur as soon as the bacteria come in contact with the PAR/HA film. Time-lapse microscopy experiments have clearly shown that the bacteria are killed when they touch the PAR30/HA surface (data not shown). As PAR chains are mobile, they can diffuse and stick to the membrane. Then the mechanism should be closed to the mechanism of action of antimicrobial peptides, which are positively charged peptides that interact with bacterial membrane.
(81) In order to investigate the biocompatibility of the PAR/HA coatings, the inventors seeded human primary fibroblasts from skin with medium that was in contact for 24 h with (PAR30/HA).sub.24 glass slides. After 24 h of seeding, no sign of toxicity was observed, the viability was equivalent to control conditions, i.e. glass surfaces (data not shown). This preliminary test demonstrates that the PAR released in the presence of medium in the supernatant shows no apparent sign of toxicity for the primary cells used. This is a positive point in the perspective of the application of PAR/HA films as coatings of implanted medical devices.
(82) The inventors further investigated the biocidal activity of the coatings of the invention, in particular of (PAR30/HA).sub.24 coatings, over more than 24 hours. Therefore, a (PAR30/HA).sub.24 multilayer coating was put in contact with a fresh suspension of S. aureus after each 24 h and the (PAR30/HA).sub.24 shows after 24 and 48 h of incubation a total inhibition of bacteria and a bacterial growth that is strongly reduced (by 70%) after 72 h. This demonstrates the biocidal activity of the coatings of the invention over 3 days and three successive contaminations (
(83) To summarize, the inventors of the present invention have found that multilayer coatings comprising PAR, PLL or PLO, in particular PAR30 and PAR10, as polycation and HA as polyanion present a strong anti-microbial effect against S. aureus, M. Luteus, E. coli and P. aeruginosa. The inventors demonstrated in context of PAR30 coatings that this effect strikingly depends on the molecular weight of the polypeptide chains. This effect is explained by the concentration of mobile in the multilayers and their power to kill bacteria as a function of the molecular weight. This mechanism can be transferred to PAR10, because PAR30 as well as PAR30 both demonstrate high mobility.
(84) However, under the conditions tested PAR10 films require more than 24 bilayers in order to be active. As demonstrated by the inventors since PAR10 and PAR30 have several capabilities in common the activity seems to depend on the amount of PAR10 or PAR30 present in the films and PAR10 biofilms require more PAR10 layers to obtain the same biocidal activity as PAR30 films.
(85) These results open the route to new type of applications of polyelectrolyte multilayers, in particular of antibacterial multilayers, where the function can be tuned by the molecular weight of the polyelectrolytes.
2. Example 2
(86) 2.1 Material
(87) Polyelectrolyte multilayer coatings have been built up using the polyelectrolytes described herein above in section 1.1. The following polyelectrolytes were used in addition: Polycations of poly(I-arginine hydrochloride) (PAR) were purchased from Alamanda Polymers, USA. Different PAR polymers used differ from the numbers of arginine per chain: PAR50 (50 arginine (R), Mw=9.6 kDa, PDI=1.03); PAR70 (70 arginine (R), Mw=13.4 kDa, PDI, =1.01), PAR150 (150 arginine (R), Mw=29 kDa, PDI=1.04).
(88) 2.2 Methods
(89) The methods used are as described herein above in the corresponding section under paragraph 1.2.
(90) 2.3 Results
(91) 2.3.1 Effect of Number of Arginine Residues on PAR/HA Antimicrobial Activity: Coatings with PAR30, PAR50, PAR70, PAR100 and PAR150 after 24 Hours of Incubation
(92) PAR with 30, 50, 100 and 150 residues have been tested in order to confirm previous results and to obtain complimentary results. Measurements were performed with a glass slide coated with (PAR/HA).sub.24 (i.e. 24 layers of PAR alternating with 24 layers of HA) and placed in a 24 well-plate, as previously described (Chem. Mater. 2016, 28, 8700). 300 μL of S. aureus at a concentration of 8.105 CFU.Math.mL.sup.−1 was deposited in each well and incubated for 24 h at 37° C. Then the absorbance of the supernatant at 620 nm was measured.
(93) PAR50/HA Versus PAR30/HA with 24 Bilayers
(94) PAR50/HA films built up with 24 bilayers show a complete bactericide effect on bacteria (
(95) PAR50/HA Versus PAR30/HA with 48 Bilayers
(96) Increasing the number of bilayer from 24 to 48 show similar results: inhibition of S. aureus growth is total, for coatings based either on PAR50 or on PAR30 (
(97) PAR100/HA and PAR150/HA Versus PAR30/HA with 24 Bilayers
(98) PAR100/HA coatings built with 24 bilayers depict a total antimicrobial activity (
(99) PAR10/HA Versus PAR30/HA with 24 Bilayers
(100) PAR10/HA coatings with 24 bilayers totally inhibit bacteria in the supernatant (
(101) PAR70/HA Versus PAR30/HA with 24 Bilayers
(102) Bacteria in the supernatant where a PAR70/HA coating is placed are totally inhibited (
(103) 2.3.2. Long Term Antimicrobial Activity of PAR/HA Coatings: PAR10, PAR30, PAR50, PAR100 and POR30 after 24/48 or 72 h of Incubation
(104) PAR30/HA Versus POR30/HA with 24 Bilayers after 24/48 or 72 h of Incubation
(105) No bacteria were monitored after 24, 48 or 72 h in the supernatant when (PAR30/HA).sub.24 or (POR30/HA).sub.24 coatings were used (
(106) PAR10/HA, PAR50/HA and PAR100/HA with 24 Bilayers after 24/48 or 72 h of Incubation
(107) Similar experiments were performed with PAR10/HA, PAR50/HA and PAR100/HA coatings. After 24 h, no bacteria were measured (
(108) 2.3.3 Storage and Sterilization of PAR/HA Coatings
(109) Storage of PAR/HA Coatings
(110) In order to check if drying procedures and storage of PAR/HA coatings allow to maintain or not the antimicrobial activity, a (PAR30/HA).sub.24 coating was tested after drying it (rinsing with pure water and drying at ambient temperature) and storage at 4° C. for 1 or 7 days (
(111) Sterilization of PAR/HA Coatings
(112) Activity of (PAR30/HA).sub.24 coatings have been tested after a drying procedure and an autoclave sterilization following regular cycles used for sterilization of medical devices (30 minutes with cycles at 121° C.) (
(113) 2.4. Conclusions
(114) Finally several conclusions can be drawn from these studies: (PAR/HA).sub.24 coatings built with PAR10, PAR30, PAR50, PAR70, PAR100 show a total antimicrobial activity against S. aureus after 24 h of inoculation. However, in our previous preliminary studies, PAR10 and PAR100 were not always active with 24 h. This is probably because PAR10 and PAR100 correspond to chain lengths at the limit of values which are effective. On the contrary, PAR30 and PAR50 always show a total antimicrobial activity in our experiments (at least more than 10 individual experiments for both have been realized). (PAR150/HA).sub.24 coatings never show some antimicrobial activities after 24 h of incubation of S. aureus. (PAR/HA).sub.24 coatings built with PAR30, PAR50, PAR100 and POR30 after 24/48 or 72 h of incubation show a total inhibition of bacteria which demonstrate their efficiency over 3 days and three successive contaminations. On the contrary, PAR10 is no more active after the third inoculation (72 h). (PAR/HA).sub.24 coatings can be stored at 4° C. for several days after drying without any loss in their activity. Moreover application of standard sterilization protocol used for medical devices can be apply to (PAR/HA).sub.24 coatings, the antimicrobial properties of the coating activity will be maintained.
3. Example 3
(115) Variation of molecular weight of HA in (PAR/HA) multilayers
(116) 3.1. Material
(117) Polyelectrolyte multilayer coatings have been built up with the following polyelectrolytes. Poly-L-arginine (PAR) such as PAR10 (10 arginine (R), Mw=2.1 kDa, PDI=1); PAR30 (30 R, Mw=6.4 kDa, PDI, =1.01), PAR100 (100 R, Mw=20.6 kDa, PDI=1.05), and PAR200 (200 R, Mw=40.8 kDa, PDI=1.06) were purchased from Alamanda Polymers. Hyaluronic acid used as the polyanion was from Lifecore Biomed, USA. The molecular weight (Mw) of hyaluronic acid represents an average of all the molecules in the population and thus represents the molecular Mass Average (Molecular Weight Average). For this experiment, different molecular weights were used: Mw=150 kDa (HA.sup.150), Mw=823 kDa (HA.sup.800), Mw=2 670 kDa (HA.sup.2700).
(118) 3.2. Methods
(119) 3.2.1. Preparation of Polyelectrolytes Solutions
(120) PAR and HA were dissolved at 0.5 mg.Math.mL.sup.−1 in sterilized buffer containing 150 mM NaCl and 10 mM TRIS at pH 7.4. Concerning the solution of HA, one type of molecular weight could be selected but two or more molecular weights of HA could also be mixed to form one solution of HA, denoted HA.sup.X,Y; with x,y=molecular weights of the mixture of HA.sup.X+HA.sup.y.
(121) 3.2.2. Buildup of (PAR/HA) Films
(122) For the construction of n bilayers of PAR/HA (denoted as (PAR/HA).sub.n,), an automated dipping robot was used (Riegler & Kirstein GmbH, Germany). Glass slides (12 mm in diameter) were first washed with Hellmanex II solution at 2%, 1 M HCl, H.sub.2O, and ethanol and dried with an air flow. Glass slides were dipped alternately in the PAR and HA solutions, with PAR as first deposited layer, and extensively rinsed in NaCl-TRIS buffer between each step. During the process, there is a possibility of changing the nature of the building blocks that are deposited during each step. In this context, HA solution could be changed during the construction to form a film with different buildings blocks, such as (PAR/HA.sup.X).sub.n(PAR/HA.sup.Y).sub.m(PAR/HA.sup.Z).sub.o, etc.; with x, y, z=molecular weight of HA (kDa) and n, m, o=number of bilayers. After construction, the films were dried with an air flow, then immersed in NaCl-TRIS buffer, and stored at 4° C. before use.
(123) Observations of the films were carried out with a confocal Zeiss LSM 710 microscope (Heidelberg, Germany) using a 20× Plan Apo (0.8 NA) objective (Zeiss). The microscopic observations of the obtained films were evaluated by deposition of 100 μL of PAR-FITC (PAR labeled with fluorescein isothiocyanate, 0.5 mg.Math.mL.sup.−1 in NaCl-TRIS buffer) on top of the (PAR/HA) multilayer films.
(124) After 5 min of deposition and diffusion of PAR-FITC through the whole film, a rinsing step was performed with NaCl-TRIS buffer.
(125) 3.2.3. Antibacterial Assay
(126) Gram-positive bacteria, such as S. aureus, or M. luteus, and Gram-negative bacteria, such as E. coli or P. aeruginosa were used to assess the antibacterial properties of the samples. The bacterial strain was cultured aerobically at 37° C. in a Mueller Hinton Broth (MHB) medium (Merck, Germany), pH 7.4. One colony was transferred to 10 mL of MHB medium and incubated at 37° C. for 20 h. To obtain bacteria in the mid-logarithmic phase of growth, the absorbance at 620 nm of overnight culture was adjusted to 0.001, corresponding to a final density of 8×10.sup.5 CFU.Math.mL.sup.−1.
(127) Glass slides coated with (PAR/HA), films were sterilized by using UV light during 15 min and then washed with NaCl-TRIS buffer. After washing, all glass slides were deposited in 24-well plates with 300 μL of S. aureus, A.sub.620=0.001, and incubated during 24 h at 37° C. For negative control, uncoated glass slides were directly incubated with S. aureus using a similar method. To quantify bacteria growth or inhibition after 24 h, the absorbance of the supernatant at 620 nm was measured.
(128) The BacLight RedoxSensor CTC Vitality Kit (Thermo Fisher Scientific Inc., France) was used for evaluation of the health of bacteria present on the surface. This kit gives a semi quantitative estimate of healthy vs unhealthy bacteria. SYTO 24 green-fluorescent nucleic acid stain (Thermo Fisher Scientific Inc., France) was used for counting all bacteria. A solution of 50 mM CTC and 1 μM SYTO 24 in pure water was prepared. All glass slides were washed with phosphate-buffered saline buffer (PBS), pH=7.4. Then 270 μL of PBS and 30 μL of CTC/SYTO 24 solution were added. The plates were incubated for 30 min at 37° C., away from light. Each surface was observed by confocal microscopy (Zeiss LSM 710 microscope, Heidelberg, Germany), using 63× Plan Apo (1.4 NA) objective immersed in oil. Excitation/emission wavelengths of stains were 450 nm/630 nm for CTC and 490 nm/515 nm for SYTO 24.
(129) 3.2.4. Anti-Inflammatory Tests
(130) THP-1 Cells
(131) For cell experiments in 2D (cells seeded on top of the film), the studies were performed with THP-1 cells (human monocytic cell line; ATCC). The THP-1 cells were cultured in RPMI 1640 GlutaMAX (Gibco Life Technologies) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 0.2% fungizone, and 0.05 nM 2-mercaptoethanol. Films were UV-treated for 15 min. For each cell experiment, 50 000 cells were deposited on top of the films, the system was first put at 37° C. for 15 min for adhesion, and after that the medium was added. The plate was then put into the incubator at 37° C.
(132) After fixation with PFA, the THP-1 cells were washed twice with the Tween 20 solution (0.2% in PBS) for 5 min. The samples were incubated for 30 min with the BSA (3% v/v) and glycine (1%) solution in PBS. Then, two rising steps with 5% goat serum in PBS for 5 min each were performed. The samples were incubated with diluted primary antibodies and incubated at room temperature for 1 h. The primary antibodies were (i) mouse anti-human CD80 primary Ab (Thermo scientific) at a dilution of 1/200 in 5% (v/v) goat serum in PBS and (ii) rabbit anti-human CD206 primary Ab (Abcam) at a dilution of 1/176 in 5% (v/v) goat serum in PBS (final concentration=1 μg mL.sup.−1). The samples were rinsed 3 times with 0.2% Tween 20 for 5 min. The diluted secondary antibodies were incubated at room temperature for 1 h in the dark. The secondary antibodies were (i) Alexa Fluor-568 goat anti-mouse IgG (H+L) (Thermo scientific) for mouse anti-CD80 primary antibody (M1 phenotype marker) at a dilution of 1/250 in 5% (v/v) goat serum in PBS (final concentration=8 μg mL.sup.−1) and (ii) Alexa Fluor-488 goat anti-rabbit IgG (H+L) (Thermo scientific) for Rabbit anti-CD206 primary antibody (M2 phenotype marker), 2 drops mL.sup.−1 of solution (in 5% v/v goat serum in PBS). The samples were rinsed three times with 0.2% Tween 20 for 5 min each time. Finally, the nuclei were labeled with DAPI (1 mg mL.sup.−1; Promokine) at a dilution of 1/100 in PBS and two rinsing steps were performed. Real-time reverse transcription qPCR (real-time RT-qPCR) was used for quantifying biologically relevant changes in the mRNA levels of THP-1-encapsulated cells. The expression levels of CD86, TNF-α, STAT1, CD163L1, ID10, and IL1RA were measured by real-time qPCR using 96-well Prime PCR custom plates (BIORAD). CD86, IL-6, STAT1, and TNF-α are M1 markers and IL-10, CD206, CD163L1 and IL-1RA correspond to M2 markers. Reactions were carried out for 50 cycles in a CFX-Connect (BIORAD). GAPDH was used as a reference gene for all of the RT-qPCR obtained results.
PBMCs
(133) Buffy coats were obtained from the National Blood Service (U.K.) following Ethics committee approval. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinised blood by Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation. Monocytes were isolated from PBMCs using the MACS magnetic cell separation system (positive selection with CD14 MicroBeads and LS columns, Miltenyi Biotec). This method routinely yielded 95% pure monocytes as determined by flow cytometric analysis of CD14 expression.
(134) Purified monocytes were cultured at 1×10.sup.6 cells/mL/well in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U.Math.mL.sup.−1 penicillin, and 100 μg.Math.mL.sup.−1 streptomycin (all from Sigma-Aldrich) in 12-well tissue culture-treated plates containing the coated films. Samples and controls were incubated at 37° C., 5% CO.sub.2 for 1, 3 and 6 days. Scheme 1 shows the time line of the experiments and the readouts made. controls.
(135) Blue Assay
(136) The AlamarBlue assay kit (Thermo Fisher Scientific, USA) was used to determine the cell viability as per the manufacturer's instructions. Controls were monocytes plated in 12-well tissue culture-treated plates in the same medium in the absence of any cytokines (TC control).
(137) Cytokine Analysis
(138) Supernatants were collected and assayed for the cytokines TNF-α, IL-12, IL-1β, CCL18 and IL-1 RA by ELISA as per the manufacturer's instructions (Table 1).
(139) TABLE-US-00001 TABLE 1 ELISA kits and standard/sample dilutions Cytokine ELISA Kit supplier IL-4 PeproTech 900-K14 IL-1β Antibody Solutions AS56-P, AS57-B (Capture and detection antibodies) Peprotech 200-01B (Standard protein) IL-1RA PeproTech 900-K474 IL-12 PeproTech 900-K96 CCL18 R&D Systems DY394 TNFα Peprotech 900-K25
4. Example 4
(140) Cross-Linking of (PAR/HA) Multilayers with EDC/NHS Method
(141) Interaction between PAR and HA via: electrostatic interactions where the cations are the guanidinium groups of PAR and the anions are the carboxylate groups of HA. covalent interactions due to the formation amide bond —CO—NH— between reactive carboxylate group of HA (thanks to the presence of EDC and NHS) and guanidinium groups of PAR.
4.1. Material
(142) Polyelectrolyte multilayer coatings have been built up with the following polyelectrolytes. Poly-L-arginine (PAR) such as PAR10 (10 arginine (R), Mw=2.1 kDa, PDI=1); PAR30 (30 R, Mw=6.4 kDa, PDI, =1.01), PAR100 (100 R, Mw=20.6 kDa, PDI=1.05), and PAR200 (200 R, Mw=40.8 kDa, PDI=1.06) were purchased from Alamanda Polymers. Hyaluronic acid used as the polyanion was from Lifecore Biomed, USA. The molecular weight (Mw) of hyaluronic acid represents an average of all the molecules in the population and thus represents the molecular Mass Average (Molecular Weight Average). For this experiment, different molecular weights were used: Mw=150 kDa (HA.sup.150), Mw=823 kDa (HA.sup.800), Mw=2 670 kDa (HA.sup.2700). N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC, Mw=155 Da) and N-hydrosuccinimide (NHS, Mw=115 Da) were purchased from Sigma Aldrich.
(143) 4.2. Methods
(144) 4.2.1. Preparation of Polyelectrolytes Solutions
(145) The polyelectrolytes solutions were prepared as disclosed in example 3 at point 3.2.1.
(146) 4.2.2. Buildup of (PAR/HA) Films
(147) The (PAR/HA/films were built up as disclosed in example 3 at point 3.2.2.
(148) 4.2.3. Cross-Linking of (PAR/HA) Films
(149) Cross-linking was performed by immersing the (PAR/HA) films in a solution containing EDC and NHS in NaCl (0.15 M) during 15 h at 4° C. Concentrations of EDC and NHS could be selected from 0.2 to 200 mM. Indeed, the mobility rate of polyelectrolyte into the films is dependent of the concentration of EDC/NHS used for cross-linking (see Francius et al. (2006) Microscopy Research and Technique 69:84-92). Films were rinsed two times with a NaCl (0.15 M) solution and then immersed in a solution of ethanolamine (1M) during 40 min at 4° C. to neutralize all carboxylate functions that have not reacted. Then the films were rinsed with NaCl (0.15 M) solution, and the NaCl-TRIS buffer solution was used for the last rinsing step. Crosslinked (PAR/HA) films could be covered with (PAR/HA) multilayers that is uncrosslinked so that the inventors can obtain a film with different buildings blocks such as ((PAR/HA.sup.X).sub.n crosslinked/(PAR/HA.sup.Y).sub.m uncross-linked) with n, m=number or bilayers; X, Y=molecular weight of HA in kDa.
(150) 4.2.4. Fluorescence Recovery after Photobleaching Experiments.
(151) The proportion of mobile molecules, p, was measured for (PAR/HA) films containing PAR30-FITC (HA with fluorescein isothiocyanate as a fluorescent dye) and HA-Rho (HA with rhodamine as a fluorescent dye) by performing fluorescence recovery after photobleaching (FRAP) experiments. A glass slide coated with a PAR/HA film was introduced in a homemade sample holder and covered with 200 μL of NaCl-TRIS buffer. One circular region (4.4 μm in radius in an image of 35 μm×35 μm) was exposed for 700 ms to the light of a laser set at its maximum power (λ=488 nm for PAR-FITC and λ=541 nm for HA-Rho). Then, the recovery of fluorescence in the bleached area was followed over time. Observations were carried out with a Zeiss LSM 710 microscope (Heidelberg, Germany) using a 20× Plan Apo (0.8 NA) objective (Zeiss). At the same time, three equally sized circular reference areas outside of the bleached area were monitored.
(152) The intensities in these areas are used to normalize the intensity in the bleached area so that bleaching due to image acquisition was accounted for.
(153) Under the hypothesis that (i) the recovery is due to the Brownian diffusion of the mobile labeled molecules, (ii) all moving labeled molecules have the same diffusion coefficient, and (iii) the initial intensity profile (i.e., immediately after the bleaching) has a sharp edge (a circle in the present case), the time evolution of the normalized intensity can be derived theoretically. It depends on the diffusion coefficient, D, and the proportion of mobile labeled molecules, p, as well as the radius, R, of the initially bleached area. Note that, according to the aforementioned theory, the area observed during the recovery must have the same radius and the same center as the bleached area. Bleached areas were observed for at least 6 min.
(154) 4.2.5. Antibacterial Assay.
(155) Antibacterial assays were performed as disclosed in example 3 at point 3.2.3.
(156) 4.2.6. Anti-Inflammatory Tests
(157) Anti-inflammatory tests were performed as disclosed in example 3 at point 3.2.4.
5. Example 5
(158) Cross-Linking of (PAR/HA) Multilayers with HA-Aldehyde
(159) Interaction with PAR and HA-Aldehyde via: electrostatic interactions where the cations are the guanidinium groups of PAR and the anions are the carboxylate groups of HA. covalent interactions due to the formation imine bond —CH═N— between aldehyde of HA and amine terminal and guanidinium groups of PAR (amine terminal is most rapid to react than guanidinium).
(160) 5.1. Material
(161) Material was as disclosed in example 3 at point 3.1.
(162) 5.2. Methods
(163) 5.2.1. Synthesis of HA-Aldehyde
(164) For the preparation of 1 g of HA-Aldehyde (sodium formyl hyaluronate or sodium 6(GlcNAc)-oxo hyaluronate), 1.1 g of HA is dissolved in 100 mL of demineralized water containing 5 equiv. of disodium hydrogen phosphate (Lach-Ner Ltd., Czech Republic). The catalyst (TEMPO) (0.01 equiv.) was added and the mixture was stirred. The following step was the addition of 0.5 equiv. of sodium hypochlorite at 5° C. The reaction solution was then stirred for 2 h at this temperature. The final product was isolated by dialysis (cut off 12 kDa) against demineralized water and by freeze drying procedure, as disclosed in Knopf-Marques et al. (2016) Biomacromolecules 17:2189-2198). By playing with parameters (reaction time, concentrations), the degree of substitution of Aldehyde can be tuned and finally the degree of crosslinking of the PAR/HA-Aldehyde film will be modulated.
(165) 5.2.2. Preparation of Polyelectrolytes Solutions
(166) PAR and (HA or HA-Aldehyde) were dissolved at 0.5 mg.Math.mL.sup.−1 in sterilized buffer containing 150 mM NaCl and 10 mM TRIS at pH 7.4. Concerning the solution of HA, only one type of molecular weight could be selected but two or more molecular weights of HA could also be mixed to form one solution of HA, denoted HA.sup.X,Y; with x,y=molecular weight of mixed HA. A mixture of HA.sup.x,y/HA-Aldehyde.sup.x,y can also be prepared. A device of the invention is prepared by successive deposition of PAR30 and HA solutions on it. PAR30 was the first deposited polyelectrolyte. Each polyelectrolyte was adsorbed for 5 min, and then a rinsing step with NaCl-TRIS buffer was performed for 5 min.
(167) 5.2.3. Buildup of (PAR/HA) Films
(168) For the construction of n bilayers of PAR/Polyanions (Polyanions=HA or HA-Aldehyde), denoted as (PAR/HA).sub.n, an automated dipping robot was used (Riegler & Kirstein GmbH, Germany). Glass slides (12 mm in diameter) were first washed with Hellmanex II solution at 2%, 1 M HCl, H.sub.2O, and ethanol and dried with an air flow. Glass slides were dipped alternately in the PAR and HA solutions, with PAR as first deposited layer, and extensively rinsed in NaCl-TRIS buffer between each step. HA solution could be changed during the construction to form a film with a successive repetition of various types of multilayers, such as (PAR/HA-Aldehyde.sup.X).sub.n(PAR/HA.sup.Y).sub.m(PAR/HA-Aldehyde.sup.Z).sub.o, etc.; with x, y, z corresponding to molecular weight of HA in kDa and n, m, o corresponding to number of bilayers. After construction, the films were dried with an air flow, then immersed in NaCl-TRIS buffer, and stored at 4° C. before use.
(169) Observations of the films were carried out with a confocal Zeiss LSM 710 microscope (Heidelberg, Germany) using a 20× Plan Apo (0.8 NA) objective (Zeiss). The microscopic observations of the obtained films were evaluated by deposition of 100 μL of PAR30-FITC (PAR labeled with fluorescein isothiocyanate, 0.5 mg.Math.mL.sup.−1 in NaCl-TRIS buffer) on top of the (PAR30/HA) multilayer films.
(170) After 5 min of deposition and diffusion of PAR-FITC through the whole film, a rinsing step was performed with NaCl-TRIS buffer.
(171) 5.2.4. Fluorescence Recovery after Photobleaching Experiments.
(172) FRAP assays were performed as disclosed in Example 4 at point 4.2.4.
(173) 5.2.5. Antibacterial Assay.
(174) Antibacterial assays were performed as disclosed in example 3 at point 3.2.3.
(175) 5.2.6. Anti-Inflammatory Tests Anti-inflammatory tests were performed as disclosed in example 3 at point 3.2.4.
6. Example 6
(176) Other Methods for Cross-Linking HA in (PAR/HA) Multilayers
(177) Interaction with PAR and HA via electrostatic interactions Cross-linking of HA chains via a modification of HA chemical structure.
(178) 6.1. Photocrosslinking HA is modified with a vinylbenzyl groups (VB). The VB-modified HA incorporated into the films of (PAR/HA) can be crosslinked upon UV irradiation as disclosed in Pozos-Vazquez et al. (2009) Langmuir 25:3556-3563. HA is modified with a methacrylate group. The methacrylated HA incorporated into the films of (PAR/HA) can be crosslinked upon UV irradiation and the presence of a photoinitiator as disclosed in Yamanlar et al. (2011) Biomaterials 32:5590-5599.
(179) 6.2. Use of HA-Tyramine
(180) HA-Tyr conjugate is synthesized by amide bond formation between carboxyl groups of HA and amine groups of tyramine. Then, (PAR/HA-Tyr) cross-linked films are prepared by radical cross-linking reaction using horse radish peroxydase and H.sub.2O.sub.2, as disclosed in Kim et al. (2011) Acta Biomateriala 7:666-674.
(181) 6.3. Use of HA-Aldehyde and HA-NH.sub.2
(182) In building block of (PAR/HA), HA is modified into HA-NH.sub.2 and, in another building blocks of (PAR/HA), HA is modified into HA-Aldehyde. In the presence of genipin, the two HA can be cross-linked and form a cross-linked film (PAR/HA-NH.sub.2)(PAR/HA-Aldehyde), as disclosed in Khunmanee et al. (2017) Journal of Tissue Engineering 8:1-8.
(183) 6.4. Use of HA-Catechol
(184) HA is modified with catechol using a chemical linkage of dopamine to the carboxyl group of HA by the EDC coupling reaction. Then, cross-linked films were prepared by using PAR and HA-catechol, as disclosed in Halake et al. (2017) Journal of Industrial and Engineering Chemistry 54:44-51.
7. Example 7
(185) Evaluation of the Cross-Linking Ratio
(186) The inventors are monitoring through FRAP experiments the percentage of diffusing molecules through labelling of PAR with FITC or HA with Rhodamine (see example 4, point 4.2.4). 100% means that all chains are mobile, and 0% that absolutely no chains are able to diffuse over the experimental duration (typically, 1 hour). A decrease of the % of mobile chains is expected when the cross-linking is performed. However this will depend of the cross-linking method used. For example, when HA-tyramine will be used in PAR/HA films, once cross-linked, only HA chains will show a loss of mobility, PAR should keep a ratio of mobile chains about 90% (for PAR30 and HA with a MW of 150 000, see Mutschler et al. (2016) Chem. Mater. 28:8700-8709).
8. Example 8
(187) Biocidal Effects Obtained Using HA of High Molecular Weight
(188) Materials and Methods
(189) Materials
(190) The polyelectrolyte multilayer films have been built up with the following polymers. The polycation was poly(L-arginine hydrochloride) whose chains consisted in 30 residues (PAR30, 30R, Mw=6.4 kDa, PDI=1.01) and was purchased from Alamanda Polymers, USA. Hyaluronic acid (HA, Mw=29, 108, 823 and 2670 kDa) used as a polyanion was produced by Lifecore Biomed, USA. Tris(hydroxymethyl)-aminomethane (TRIS) was purchased from Merck, Germany.
(191) Buildup of PAR/HA Films
(192) PAR and HA were dissolved at 0.5 mg.Math.mL.sup.−1 in sterilized buffer containing 150 mM NaCl and 10 mM of TRIS at pH 7.4.
(193) For the construction of 24 bilayers of PAR30/polyanion (denoted as (PAR30/polyanion).sub.24), an automated dipping robot was used (Riegler & Kirstein GmbH, Germany). Glass slides (12 mm in diameter) were first washed with Hellmanex® II solution at 2%, HCl 1M, H.sub.2O, and ethanol and dried with an air flow. Fresh solutions of polyelectrolytes were used. Glass slides were dipped alternately in the polycation and polyanion solutions and extensively rinsed in NaCl-TRIS buffer between each step. After construction, the films were dried with an air flow and then immersed in NaCl-TRIS buffer and stored at 4° C. before use.
(194) Fluorescent Labelling of Films
(195) Observations of the films were carried out with a confocal Zeiss LSM 710 microscope (Heidelberg, Germany) using a 20× Plan Apo (0.8 NA) objective (Zeiss). The microscopic observations of the obtained films were evaluated by deposition of 100 μL of PAR-FITC (PAR labeled with fluorescein isothiocyanate, 0.5 mg.Math.mL.sup.−1 in NaCl-TRIS buffer) on top of the PAR30/polyanion multilayer films for 15 min. Fluorescent labeling of PAR was as described in Mutschler et al. (2016) Chem. Matter 28:8700-8709. After 5 min of deposition and diffusion of PAR-FITC through the whole film, a rinsing step was performed with 500 μL of NaCl-TRIS buffer.
(196) Antibacterial Assay
(197) Staphylococcus aureus (S. aureus, ATCC 25923) strain was used to assess the antibacterial properties of the samples. Bacterial strain was cultured aerobically at 37° C. in a Mueller Hinton Broth (MHB) medium (Merck, Germany), pH 7.4. One colony was transferred to 10 mL of MHB medium and incubated at 37° C. for 20 h. To obtain bacteria in the mid logarithmic phase of growth, the absorbance at 620 nm of overnight culture was adjusted to 0.001, corresponding to a final density of 8.10.sup.5 CFU.Math.mL.sup.−1.
(198) Glass slides coated with (PAR30/HA).sub.24 films were sterilized by using UV-light during 15 min, then washed with NaCl-TRIS buffer. After washing, all glass slides were deposited in 24-well plates with 300 μL of S. aureus, A.sub.620=0.001, and incubated during 24 h at 37° C. in the presence of MHB medium. Then, the normalized growth of pathogens was estimated by monitoring the absorbance at 620 nm in the presence of multilayer films in comparison with the positive control (without films and in the presence of antibiotics in the medium) and the negative control (without films and in the absence of antibiotics in the medium).
Results
Antimicrobial Activity
(199) The growth of pathogens S. aureus was estimated after 24 h by monitoring the absorbance at 620 nm in the presence of (PAR30/HA).sub.24 multilayer films built with HA of MW=29 or 108 or 823 or 2670 kDa. Comparison of this absorbance with the positive control (without films and in presence of antibiotics in the medium) and the negative control (without films and in the absence of antibiotics in the medium) was done to normalize the results. The results obtained are shown in Table 1 below.
(200) TABLE-US-00002 TABLE 1 Normalized pathogen growth (%) Condition Mean Standard deviation Glass + (PAR30/HA29).sub.24 −0.6 0.4 Glass + (PAR30/HA108).sub.24 −0.9 0.2 Glass + (PAR30/HA823).sub.24 −0.9 0.2 Glass + (PAR30/HA2670).sub.24 −0.6 0.4 Glass 100 2.3 Glass + antibiotics 0.0 0.4 Medium 0.1 0.6
(201) Total inhibition of proliferation was monitored for films built with HA of MW=29 or 108 or 823 or 2670 kDa. This indicates that whatever the MW of HA used, the PAR30/HA films prevent the growth of S. aureus. Only glass surfaces without coating exhibits high S. aureus proliferation. Moreover, through labelling of the respiratory activity of bacteria with the surfaces, the inventors observed with the fluorescent microscope that absolutely no bacteria were on the surface of the PAR30/HA films whatever the MW of HA used.
(202) Films made with PAR50 and various MW of HA were also tested. Antimicrobial activity was measured in solution with coatings based on (PAR50/HA.sup.108).sub.24, (PAR50/HA.sup.823).sub.24 and (PAR50/HA.sup.2670).sub.24 where no bacteria were detected through absorbance measurements. The results obtained are shown in Table 2 below.
(203) TABLE-US-00003 TABLE 2 Normalized pathogen growth (%) Condition Mean Standard deviation Glass + (PAR50/HA108).sub.24 −0.4 0.7 Glass + (PAR50/HA823).sub.24 −0.2 0.3 Glass + (PAR50/HA2670).sub.24 0.3 0.9 Glass 100.0 2.5 Glass + antibiotics 0.0 0.3 Medium −0.6 0.2
(204) These results confirm that a biocidal activity is obtained even when HA of high molecular weight is used in the coatings of the invention.
9. Example 9
(205) Determination of Anti-Inflammatory Properties of Coatings Using HA of High Molecular Weight
(206) Materials and Methods
(207) Materials
(208) The polyelectrolyte multilayer films have been built up with the following polymers. The polycation was poly(L-arginine hydrochloride) whose chains consisted in 30 residues (PAR30, 30R, Mw=6.4 kDa, PDI=1.01) and was purchased from Alamanda Polymers, USA. Hyaluronic acid (HA, Mw=29, 108, 823 and 2670 kDa) used as a polyanion was produced by Lifecore Biomed, USA. Tris(hydroxymethyl)-aminomethane (TRIS) was purchased from Merck, Germany.
(209) Buildup of PAR/HA Films
(210) PAR and HA were dissolved at 0.5 mg.Math.mL.sup.−1 in sterilized buffer containing 150 mM NaCl and 10 mM of TRIS at pH 7.4.
(211) For the construction of 24 bilayers of PAR30/polyanion (denoted as (PAR30/polyanion).sub.24), an automated dipping robot was used (Riegler & Kirstein GmbH, Germany). Glass slides (12 mm in diameter) were first washed with Hellmanex® II solution at 2%, HCl 1M, H.sub.2O, and ethanol and dried with an air flow. Fresh solutions of polyelectrolytes were used. Glass slides were dipped alternately in the polycation and polyanion solutions and extensively rinsed in NaCl-TRIS buffer between each step. After construction, the films were dried with an air flow and then immersed in NaCl-TRIS buffer and stored at 4° C. before use.
(212) Anti-Inflammatory Assays
(213) Monocyte Isolation: isolation and cultivation of monocytes were performed as described in Kzhyshkowska et al. (2006) J. Cell. Mol. Med. 10:635. Monocytes were cultured on the films at a concentration of 1×10.sup.6 cells mL.sup.−1 in macrophage SFM (Invitrogen, Darmstadt, Germany) supplemented with 5×10.sup.−3 M glucose (Sigma-Aldrich, Steinheim, Germany) and dexamethasone (Sigma-Aldrich, Steinheim, Germany) at concentration of 10.sup.−8 M. Monocytes were stimulated with cytokines IL-4 (10 ng mL.sup.−1) (Peprotech, Hamburg, Germany) or IFNγ (100 ng mL.sup.−1) (Peprotech, Hamburg, Germany) as indicated.
(214) ELISA: Concentration of human TNF-α and CCL18 (R&D Systems) levels in cell culture supernatants, collected after first, third, fifth, and sixth day of incubation, were analyzed according to the manufacturer's instructions. Four individual donors were analyzed. All samples were analyzed in duplicates. Measurements were performed with Tecan Infinite 200 microplate reader (Tecan, Mannedorf, Switzerland) at 450 nm/570 nm wavelengths.
Results
(215) This protocol enables demonstrating anti-inflammatory properties of polyelectrolyte coatings using HA of high molecular weight.
10. Example 10
(216) Biocidal Effects Obtained with Coating Comprising Cross-Linked Layers Optionally Covered by Non-Cross-Linked Layers and Optionally Loaded with PAR30
(217) Materials and Methods
(218) Materials
(219) The polyelectrolyte multilayer films have been built up with the following polymers. The polycation was poly(L-arginine hydrochloride) whose chains consisted in 30 residues (PAR30, 30R, Mw=6.4 kDa, PDI=1.01) and was purchased from Alamanda Polymers, USA. Hyaluronic acid (HA, Mw=108 kDa) used as a polyanion was produced by Lifecore Biomed, USA. Tris(hydroxymethyl)-aminomethane (TRIS) was purchased from Merck, Germany. 1.4-butanediol diglycidyl ether (BDDE) was purchased from Sigma-Aldrich, USA.
(220) Buildup of PAR/HA Films
(221) PAR and HA were dissolved at 0.5 mg.Math.mL.sup.−1 in sterilized buffer containing 150 mM NaCl and 10 mM of TRIS at pH 7.4.
(222) For the construction of 24 bilayers of PAR30/HA (denoted as (PAR30/HA).sub.24), an automated dipping robot was used (Riegler & Kirstein GmbH, Germany). Glass slides (12 mm in diameter) were first washed with Hellmanex® II solution at 2%, HCl 1M, H.sub.2O, and ethanol and dried with an air flow. Fresh solutions of polyelectrolytes were used. Glass slides were dipped alternately in the polycation and polyanion solutions and extensively rinsed in NaCl-TRIS buffer between each step. After construction, the films were dried with an air flow and then immersed in NaCl-TRIS buffer and stored at 4° C. before use.
(223) Crosslinking of PAR/HA Films
(224) BDDE solution was prepared at a concentration of 0.01, 0.1, 1 or 10% in a NaOH 0.1 M solution. PAR/HA films were dipped in this solution either 4 h at 50° C. and then rinsed two times with NaCl-TRIS buffer.
(225) Addition of Supplemental Layers
(226) Immediately after the cross-linking step, 5 PAR30/HA layers were added according to the same protocol as above.
(227) Loading of the Films with PAR30
(228) Immediately after cross-linking or after the addition of supplemental layers, PAR30 or PAR30-FITC was added at 0.5 mg/mL in Tris-NaCl for 5 min.
(229) The films were then washed in Tris-NaCl buffer and kept in Tris-NaCl buffer at 4° C. until used.
(230) Fluorescent Labelling of Films
(231) Observations of the films were carried out with a confocal Zeiss LSM 710 microscope (Heidelberg, Germany) using a 20× Plan Apo (0.8 NA) objective (Zeiss). The microscopic observations of the obtained films were evaluated by deposition of 100 μL of PAR-FITC (PAR labeled with fluorescein isothiocyanate, 0.5 mg.Math.mL.sup.−1 in NaCl-TRIS buffer) on top of the PAR30/polyanion multilayer films for 15 min. Fluorescent labeling of PAR was as described in Mutschler et al. (2016) Chem. Matter 28:8700-8709. After 5 min of deposition and diffusion of PAR-FITC through the whole film, a rinsing step was performed with 500 μL of NaCl-TRIS buffer.
(232) Antibacterial Assay
(233) Staphylococcus aureus (S. aureus, ATCC 25923) strain was used to assess the antibacterial properties of the samples. Bacterial strain was cultured aerobically at 37° C. in a Mueller Hinton Broth (MHB) medium (Merck, Germany), pH 7.4. One colony was transferred to 10 mL of MHB medium and incubated at 37° C. for 20 h. To obtain bacteria in the mid logarithmic phase of growth, the absorbance at 620 nm of overnight culture was adjusted to 0.001, corresponding to a final density of 8.10.sup.5 CFU.Math.mL.sup.−1.
(234) Glass slides coated with (PAR30/HA).sub.24 films were sterilized by using UV-light during 15 min, then washed with NaCl-TRIS buffer. After washing, all glass slides were deposited in 24-well plates with 300 μL of S. aureus, A.sub.620=0.001, and incubated during 24 h at 37° C. in the presence of MHB medium. Then, the normalized growth of pathogens was estimated by monitoring the absorbance at 620 nm in the presence of multilayer films in comparison with the positive control (without films and in the presence of antibiotics in the medium) and the negative control (without films and in the absence of antibiotics in the medium).
Results
(235) The growth of pathogens S. aureus was estimated after 24 h by monitoring the absorbance at 620 nm in the presence of (PAR30/HA).sub.24 multilayer films (built with HA of MW=108 kDa) and cross-linked with BDDE at a concentration of 1% or 10%. Comparison of this absorbance with the positive control (without films and in presence of antibiotics in the medium) and the negative control (without films and in the absence of antibiotics in the medium) was done to normalize the results.
(236) No inhibition of proliferation was monitored for films built with BDDE at a concentration of 1% or 10%. This indicates that PAR30/HA cross-linked films obtained using the above cross-linking procedure were not able to prevent the growth of S. aureus anymore. The inventors explained this observation by the fact that PAR chains are probably immobilized in the film structure as demonstrated by FRAP analyses: no recovery of fluorescence of PAR.sup.FITC chains after photobleaching is observed. Through crosslinking of HA with such concentrations of BDDE, porosity is probably lower and probably not sufficient to allow PAR30 mobility.
(237) Accordingly, the inventors built again these cross-linked films but they added a PAR30 layer. Similar experiments of growth of pathogen S. aureus 24 h on these new coatings were performed.
(238) The results obtained are shown in Table 3 below.
(239) TABLE-US-00004 TABLE 3 Normalized pathogen growth (%) Condition Mean Standard deviation Glass + (PAR30/HA108).sub.24 [Tris −0.8 0.3 NaCl] + PAR30 Glass + (PAR30/HA108).sub.24 [NaOH −0.2 0.4 0.1M] + PAR30 Glass + (PAR30/HA108).sub.24 [BDDE −1.2 0.3 10%] + PAR30 Glass 100.0 2.4 Glass + antibiotics 0.0 0.6 Medium 1.7 2.0
(240) Films cross-linked with BDDE 10% are fully antimicrobial and inhibit 100% of bacteria. Adding a PAR30 layer finally allows to recover the antimicrobial activity, at least when a cross-linking with 10% BDDE is performed. FRAP analyses show that most of the last PAR30 chains are able to diffuse in films built with BDDE.
(241) Addition of (PAR30/HA).sub.5/PAR30 layers on cross-linked (PAR30/HA).sub.24 films (built with HA of MW=108 kDa) shows that for a low BDDE (0.01%, 0.1% or 1%) or a high BDDE concentration (10%), total bacterial inhibitions are obtained.
(242) The results obtained are displayed in Table 4 below.
(243) TABLE-US-00005 TABLE 4 Normalized pathogen growth (%) Condition Mean Standard deviation Glass + (PAR30/HA108).sub.24 [NaOH 2.2 1.5 0.1M] + (PAR30/HA108).sub.5 + PAR30 Glass + (PAR30/HA108).sub.24 [BDDE −0.8 0.5 0.01%] + (PAR30/HA108).sub.5 + PAR30 Glass + (PAR30/HA108).sub.24 [BDDE −1.5 0.5 0.1%] + (PAR30/HA108).sub.5 + PAR30 Glass + (PAR30/HA108).sub.24 [BDDE −1.4 0.9 1%] + (PAR30/HA108).sub.5 + PAR30 Glass + (PAR30/HA108).sub.24 [BDDE −1.0 1.2 10%] + (PAR30/HA108).sub.5 + PAR30 Glass 100.0 3.5 Glass + antibiotics 0.0 0.3 Medium 0.1 0.7
(244) These results confirm that biocidal activity can be observed with polyelectrolyte coatings comprising cross-linked layers.