HYALURONIC ACID HYDROGELS WITH PROLONGED ANTIMICROBIAL ACTIVITY

Abstract

The present invention concerns a hydrogel comprising hyaluronic acid (HA) or a derivative thereof, loaded with at least one positively charged antimicrobial peptide, wherein said HA or derivative thereof is cross-linked with a cross-linking agent at the level of its hydroxyl moieties while the carboxyl moieties of HA or derivative thereof remain free and said HA or derivative thereof remains negatively charged; and a method for preparing said loaded hydrogel.

Claims

1. A hydrogel comprising hyaluronic acid (HA) or a derivative thereof, loaded with at least one positively charged antimicrobial peptide, wherein said HA or derivative thereof is cross-linked with a cross-linking agent at the level of its hydroxyl moieties while the carboxyl moieties of HA or derivative thereof remain free and said HA or derivative thereof remains negatively charged.

2. The hydrogel according to claim 1, wherein said positively charged antimicrobial peptide is selected from the group consisting of polyarginine, polyornithine and polylysine.

3. The hydrogel according to claim 1, wherein said positively charged antimicrobial peptide is polyarginine.

4. The hydrogel according to claim 3, wherein said polyarginine is of the following formula (1) ##STR00008## wherein n is an integer comprising between 2 and 250.

5. The hydrogel according to claim 4, wherein said polyarginine is of the following formula (1) ##STR00009## wherein n is 30.

6. The hydrogel according to claim 1, wherein said hyaluronic acid is hyaluronic acid having a molecular weight of between 800 and 850 kDa.

7. The hydrogel according to claim 1, wherein said cross-linking agent is butanediol diglycidyl ether (BDDE).

8. A method for preparing the hydrogel according to claim 1, wherein said method comprises the following steps: (a) mixing, in basic conditions, hyaluronic acid (HA) or a derivative thereof with a cross-linking agent which cross-links HA at the level of its hydroxyl moieties while the carboxyl moieties of HA or derivative thereof remain free and said HA or derivative thereof remains negatively charged, (b) depositing the mixture on a support and incubating it for 48 h to 72 h at room temperature to obtain a hydrogel, (c) recovering the hydrogel formed at step (b), (d) incubating said hydrogel in an aqueous buffer in conditions enabling the withdrawal of cross-linking agent residues and the hydrogel to swell, (e) loading the hydrogel obtained at step (d) with at least one positively charged antimicrobial peptide, and (f) recovering the loaded hydrogel obtained at step (e).

9. The method according to claim 8, wherein the mixture of step (a) comprises from 2 to 3% (w/v) of HA or derivative thereof, and at least 10% (v/v) of cross-linking agent.

10. The method according to claim 8, wherein said cross-linking agent is butanediol diglycidyl ether (BDDE).

11. The method according to claim 8, wherein said positively charged antimicrobial peptide is polyarginine.

12. The method according to claim 8, wherein said positively charged antimicrobial peptide is loaded at step (e) at a concentration of 0.05 to 1 mg/ml.

13. (canceled)

14. A medical device comprising the hydrogel according to claim 1.

15. The medical device according to claim 14, wherein said medical device is a wound dressing or a mesh prosthesis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0168] FIG. 1: Schematic representation of hydrogel preparation process.

[0169] FIG. 2: Construction between parafilm and glass slide.

[0170] FIG. 3: Bacterial growth on PAR pre-loaded and post-loaded HA films. Bacterial growth was evaluated after 24 h by optical density (OD) measurement at 620 nm. PARO corresponds to HA film without PAR. HA 2.5%+BDDE 20% films were added with 1 mg/mL PAR after cross-linking (=post-loaded films).

[0171] FIG. 4: Production of free-standing HA hydrogels. Schematic protocol for hydrogel discs preparation (A) to obtain HA hydrogel discs of different sizes (B).

[0172] FIG. 5: Loading of PAR into HA hydrogel discs. (A) Schematic presentation of PAR-FITC loading. (B) CLSM images of resulting hydrogel discs (loaded with 0.5 mg/mL PAR30-FITC): 3D reconstruction (left) and cross-cuts in Z (right).

[0173] FIG. 6: Loading of PAR30-FITC into HA hydrogel discs at different concentrations for 3 h and 24 h. (A) CLSM images (B) Quantification of PAR concentration in the center of the discs.

[0174] FIG. 7: Loading of PARs-FITC into HA hydrogel discs. CSLM images of HA hydrogel discs loaded with 0.5 mg/mL of three PARs labelled with FITC (on the left) and fluorescence profiles of the resulting discs (on the right).

[0175] FIG. 8: PAR mobility inside HA hydrogels. HA discs were loaded with 0.5 mg/mL of three different FITC-conjugated PARs for 24 h and rinsed. Then, FRAP experiments were conducted. (A) Comparison of the fluorescence recovery of three PARs. (B) Determination of the diffusion coefficients D and percentage of mobile molecules p for three PARs.

[0176] FIG. 9: Antibacterial activity of HA hydrogels loaded with PAR10, PAR30, PAR200: repetitive culture. Repetitive culture: after 24 h of bacterial culture, the samples were rinsed and seeded with fresh bacteria.

[0177] FIGS. 10-12: Antibacterial activity of HA hydrogels loaded with PAR10, PAR30, PAR200: repetitive culture. After 24 h of bacterial culture, the samples were rinsed and seeded with fresh bacteria. The graphs show bacterial growth in presence of hydrogel discs loaded with different concentrations of PAR10 (FIG. 10), PAR30 (FIG. 11) and PAR200 (FIG. 12).

[0178] FIG. 13: Cytotoxicity assay on PAR10 and PAR30-loaded HA hydrogels. (A). Balb/3T3 cells after 24 h show detachment/deformation under HA+PAR10 and HA+PAR30 discs (loaded at 0.05 mg/mL), while remaining in good health around the discs. (B) MTT test confirmed good cell viability. (C) Such reactivity (grade 2) is considered as mild reactivity without cytotoxic effect, according to ISO 10993.

[0179] FIG. 14: Cell viability evaluation by MTT test. Balb/3T3 cells were seeded in 24-well plate and put in contact with HA hydrogel discs loaded or not with PARs for 24 h. HA discs correspond to BDDE-crosslinked HA hydrogel discs without PAR, and HA+PARs correspond to HA hydrogels loaded with different PAR concentrations (mg/mL). (A) Cell images after 24 h of direct in vitro cytotoxicity test. (B) Cell viability measured by MTT test. Dashed line corresponds to 70% viability (cytotoxicity limit).

[0180] FIG. 15: Antibacterial activity of HA hydrogels loaded with PAR10, PAR30, PAR200: repetitive culture. Every 24 h of bacterial culture, the samples were seeded with fresh bacteria. The graphs show bacterial growth in presence of hydrogel discs loaded with PAR10, PAR30 and PAR200 (loading concentration is indicated).

[0181] FIG. 16: Antibacterial activity of HA hydrogel discs and hydrogel-coated meshes loaded with PAR. The graphs show bacterial growth in presence of hydrogel discs or hydrogel-coated meshes loaded with 0.05 mg/mL of PAR10 or PAR30 (loading concentration is indicated, mg/mL). The bacteria were incubated with materials at 37° C. for 6 days, then optical density was measured to evaluate bacterial growth.

[0182] FIG. 17: Antibacterial activity of HA hydrogels after autoclaving. The graphs show bacterial growth in presence of hydrogel-coated polypropylene (PP) meshes loaded with 0.05 and 0.1 mg/mL of PAR30 and sterilized by autoclaving, compared to non-autoclaved samples. The bacteria were incubated with materials at 37° C. for 24 h, then optical density was measured to evaluate bacterial growth.

[0183] FIG. 18: Antibacterial activity of HA hydrogels cross-linked with different % of BDDE. The graphs show bacterial growth in presence of HA hydrogel discs loaded with 0.05 and 0.1 mg/mL of PAR30 and sterilized by autoclaving, compared to non-autoclaved samples. The bacteria were incubated with materials at 37° C. for 24 h, then optical density was measured to evaluate bacterial growth.

[0184] FIG. 19: Antibacterial activity of HA hydrogels loaded with positively charged antibacterial polypeptides. The graphs show bacterial growth in presence of HA hydrogel discs loaded with 0.05 and 0.1 mg/mL of polyarginine PAR30, polyornithine PLO30 and polylysine PLL30. The bacteria were incubated with materials at 37° C. for 24 h, then optical density was measured to evaluate bacterial growth.

[0185] FIGS. 20-24: Release of PARs in NaCl and in culture media. HA discs were incubated with 0.5 mg.Math.mL.sup.−1 of three different FITC-conjugated PAR for 24 h. The discs were rinsed, then incubated for 72 h in NaCl 1 M, MH or DMEM.

[0186] FIG. 20: Confocal microscopy observations, percentage of PAR remaining after 72 h is indicated.

[0187] FIG. 21: Quantification by spectrofluorimetry of PAR release after 72 h in NaCl 1 M. The graphs represent averages from 3 independent experiments, and error bars represent standard deviations.

[0188] FIG. 22: Confocal microscopy images of the discs before and after incubation with MH and DMEM.

[0189] FIG. 23: Percentage of released PARs in MH, where 100% represent fluorescence intensity of the discs in Tris/NaCl.

[0190] FIG. 24: Percentage of released PARs in DMEM, where 100% represent fluorescence intensity of the discs in Tris/NaCl.

EXAMPLES

Example 1: HA Cross-Linking and Hydrogel Deposition

[0191] As a first step of HA hydrogel development, the inventors evaluated several substrates and deposition techniques. The goal was to obtain 10-100 μm thick and homogeneous HA layers.

[0192] To cross-link the films, the inventors chose butanediol diglycidyl ether (BDDE), which is used in the majority of market-leading HA hydrogels.

HA Crosslinking with BDDE

[0193] A preliminary experiment was done by cross-linking through mixing 823 kDa HA 2,5% (m/v), dissolved in 0.1 M NaOH by overnight stirring, with 5 and 10% (v/v) BDDE in glass jars. The reaction was conducted for 48 h until no more increase in solution viscosity was observed. HA solutions were observed before and after the cross-linking.

[0194] Before cross-linking, all three solutions were liquid. After 24 h, gelation of HA solution containing 10% BDDE was observed, and after 48 h, both 5% and 10% BDDE-containing solutions were cross-linked, while HA without BDDE remained liquid.

HA Hydrogels Produced by Drop Deposition, HA Pre-Mixed with BDDE

[0195] The inventors selected HA 823 kDa, which seemed to absorb more PAR30-rhodamine than other HAs.

[0196] They tested the method which consists in dissolving 5% HA 823 kDa in NaOH 0.25 M and pre-mixing it with BDDE 10% or 20%.

[0197] Deposition was tested on 12 mm diameter glass slides.

[0198] The glass slides were first washed in Hellmanex 2% solution, then in HCl 1 M (both steps followed by rinsing in demineralized water), then rinsed in ethanol 70% and dried. To improve HA adhesion, a layer of polyethyleneimine (PEI) was deposited by immersion of the glass slides in 0.5 mg/ml PEI solution in water for 30 minutes.

[0199] The mix was deposited on the prepared glass slides, and the plates containing the slides were sealed to avoid film drying (FIG. 1). The cross-linking reaction was conducted for 48 h.

[0200] HA dissolving in NaOH allows increasing the HA concentration up to 5% without the solution being too viscous. Pre-mixing BDDE with HA allows using smaller quantities of the cross-linker.

[0201] Layers obtained by deposition of 25 μL HA 5%, BDDE 10% were about 800 μm thick and homogeneous. When 10 μL HA 5%-BDDE 10% were deposited and spread on the slide, the film thicknesses decreased to approximately 250 μm. Finally, when 5 μL HA 5%+BDDE 20% were deposited between two glass slides, 50 μm film thicknesses were obtained.

[0202] Thus, deposition of HA pre-mixed with BDDE allows obtaining films of different thicknesses, depending on the deposited volume and deposition method.

[0203] At the end, the inventors selected HA 2.5%+20% BDDE (pre-mixed), 10 μL deposition between parafilm and glass slide (FIG. 2) which gave about 100 μm homogeneous films.

PAR Pre-Loading Vs. Post-Loading

[0204] Next, the inventors tested PAR-charged HA hydrogels for antibacterial activity. They assessed post-loading of PAR (adding 1 mg/mL solution of PAR onto cross-linked HA films). PAR loaded were of 4 different lengths: 10, 30, 150 and 200 residues, further referred to as PAR10, PAR30, PAR150 and PAR200. We also followed PAR release after 24 h from post-loaded films.

[0205] The antibacterial activity was tested using S. aureus culture (400 μL of bacterial suspension with an initial optical density OD=0.001 per well of a 24-well plate containing or not HA-covered, PAR charged or not glass slides). After 24 h, OD at 620 nm was measured and bacterial viability on the surfaces was assessed using BacLight Redox Sensor CTC Vitality kit (Molecular Probes) as a fluorescent marker.

[0206] The results showed inhibition of bacterial growth on the films post-loaded with PAR (all lengths).

[0207] The inventors identified the PAR post-loading method as the most efficient.

Example 2: Production and Characterization of Free-Standing HA Hydrogels

[0208] In the first time, the inventors set up a protocol to produce thin hydrogel films by mixing 823 kDa HA 2.5% (w/v) and 1.4-butanediol diglycidyl ether (BDDE) 20% (v/v) in NaOH 0.25 M and depositing the solution between parafilm and 12-mm diameter glass slide.

[0209] This approach gave about 100 μm thin films, but required using of parafilm, which is temperature-sensitive and can affect hydrogel formation. To avoid the lack of reproducibility, the inventors developed a new approach which allowed to produce free-standing hydrogels of different sizes, resistant and easy to manipulate.

Construction of Free-Standing HA Hydrogels

[0210] To prepare such hydrogels, 1.5 mL of 2.5% HA and 20% BDDE well-mixed solution in NaOH 0.25 M was poured into a 35-mm diameter Petri dish and allowed to cross-link at room temperature for 72 h.

[0211] The hydrogel was further cut into the discs of required size using a circle cutter, e.g. for the experiments in 24-well plates, 4 mm diameter discs were used.

[0212] The hydrogel discs were further rinsed in Tris 10 mM/NaCl 0.15 M buffer (pH=7.4) and could be kept at 4° C. for several weeks.

[0213] The schematic protocol for hydrogel disc preparation and the resulting discs of different sizes are shown in FIG. 4. The resulting hydrogels can be easily manipulated with a pincer or spatula.

Example 3: PAR Loading and Release Characterization

[0214] Loading of PAR into HA Hydrogel Discs

[0215] To load PAR into the hydrogels, the discs were immersed in PAR solution and incubated at room temperature. For 4 mm discs in 24-well plate, 0.5 mL of PAR solution was used. Then the discs were rinsed with Tris/NaCl buffer two times: one short and one long (at least 1 hour) rinsing.

[0216] The procedure, as well as an example of a resulting PAR30-FITC (PAR having 30 arginine residues and conjugated to FITC) loaded disc, are shown in FIG. 5.

[0217] The inventors compared loading of PAR30 for 3 h vs. 24 h. The results showed that after 24 h, PAR30 diffused more into the discs center (FIG. 6). Hence, 24 h loading was selected for further experiments.

[0218] They next studied loading of three different PARs: PAR10, corresponding to chains having 10 arginine residues; PAR30, corresponding to chains having 30 arginine residues and PAR200, corresponding to chains having 200 arginine residues.

[0219] To visualize PAR loading and diffusion inside the hydrogels, they again used fluorescently-labelled PARs (PAR10-FITC, PAR30-FITC, PAR200-FITC).

[0220] Fluorescent profiles showed more homogeneous distribution for PAR10 and PAR30 (FIG. 7).

[0221] Next, the inventors studied PARs mobility inside the hydrogels (FIG. 8) using FRAP (fluorescence recovery after photobleaching) technique. Qualitatively, PAR10 was the most mobile and PAR200 was the least mobile (FIG. 8A). Quantitative parameters such as diffusion coefficient were also determined (FIG. 8B).

PAR-Loaded Hydrogel Stability in Tris/NaCl Buffer

[0222] HA discs loaded with three different PARs (PAR10, corresponding to chains having 10 arginine residues; PAR30, corresponding to chains having 30 arginine residues 30 and PAR200 corresponding to chains having 200 arginine residues) were incubated for 48 h at room temperature or at 37° C. The results showed no PAR-FITC release in any of the conditions, suggesting that antibacterial HA-PAR discs remain stable in Tris/NaCl buffer even at higher temperature, which is good for discs manipulation and transportation.

[0223] More specifically, total amounts of PAR contained in the hydrogels were estimated by incubation of hydrogels loaded with fluorescently labelled PAR in concentrated NaCl to promote PAR release. After 72 h at 37° C. in NaCl 1M, the release was almost complete for PAR10 and close to 80% for PAR30 and PAR200, according to the confocal microscopy images (FIG. 20). The percentage of PAR remaining in the hydrogel discs after 72 h of incubation was measured with image processing; 100% corresponds to fluorescence intensity before release. Then, percentage of remaining PAR after NaCl 1 M incubation was determined to obtain a value of released PAR: about 97%, 78% and 78% for PAR10, PAR30 and PAR200, respectively. Incomplete release of PAR30 and PAR200 correlates with lower mobility demonstrated by FRAP experiments. Then, amounts of PAR-FITC released were quantified by measuring fluorescence intensity of the supernatant by spectrofluorimetry and referring to a calibration curve.

[0224] The results show that the discs incubated in 0.5 mg.Math.mL.sup.−1 PAR solutions released about 212 μg of PAR10-FITC, 157 μg of PAR30-FITC and 91 μg of PAR200-FITC after 72 h in NaCl 1 M (FIG. 21). When correcting the released quantities to 100%, it gives 218 μg, 201 μg and 117 μg of loaded PAR10-FITC, PAR30-FITC and PAR200-FITC, respectively. Discs volume is approximately 30 μL, so the discs contain about 7.3 mg.Math.mL.sup.−1 of PAR10, 6.7 mg.Math.mL.sup.−1 of PAR30 and 3.9 mg.Math.mL.sup.−1 of PAR200, respectively.

PAR Release in Bacterial and Cell Culture Media

[0225] Using FITC-labeled PARs of 10 units, 30 units and 200, the inventors evaluated their release at 37° C. from HA hydrogels in two different media.

[0226] They performed discs imaging after 24 h of incubation at 37° C. in MH (Mueller Hinton broth, bacterial culture medium) and in DMEM (Dulbecco's modified Eagle's medium+10% FBS+antibiotics, cell culture medium). The results showed that PAR release patterns were slightly different in MH and DMEM/FBS. In the latter, release of PAR30 and PAR200 was higher than in MH.

[0227] More specifically, PAR release from the hydrogels was followed during 72 hours in microbiological growth medium (MH) or cell culture medium (DMEM). PAR-FITC loaded hydrogels were placed into these media and incubated at 37° C., and PAR release was observed by confocal microscopy (FIG. 22). The release was faster for PAR10 in MH, compared to PAR30 and PAR200, which were released more gradually (FIG. 23). In DMEM, all three PAR had more or less similar release profiles and were completely released after 48 h (FIG. 24).

Example 4: Antibacterial Effect Vs. In Vitro Cytotoxicity

[0228] In the preliminary experiments, the inventors demonstrated antibacterial activity of PAR10, PAR30 and PAR200 loaded into HA hydrogels at 1 mg/mL.

[0229] To study antibacterial properties of PAR-loaded hydrogels in more details, they performed repetitive culture (FIG. 9) of bacteria with hydrogel discs loaded with different concentrations of PAR10, PAR30 and PAR200.

[0230] PAR30 had the most prolonged antibacterial effect, remaining efficient at 0.5 and 1 mg/mL (loaded) after 8 days of repetitive culture. PAR10 remained efficient for 8 days at 1 mg/mL, and PAR200 remained efficient for 7 days at 1 mg/mL (FIGS. 10-12).

Cytotoxicity Tests

[0231] Direct in vitro cytotoxicity test consists in putting material in contact with the cells for 24 h and performing MTT test to evaluate cell viability. According to ISO 10993, the tested material should cover approximately 1/10 of cell layer surface, which corresponds to ˜5 mm hydrogel discs per well of a 24-well plate.

[0232] The inventors used 4 mm diameter discs which swelled and became ˜6 mm diameter when they were placed in Tris-NaCl buffer.

[0233] After 24 h at 37° C., hydrogel discs were removed and MTT test was performed in order to measure cell metabolic activity, which often serves to estimate cell viability. Yellow water-soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid) is metabolically reduced in viable cells to a blue-violet insoluble formazan. The number of viable cells correlates to the colour intensity determined by photometric measurements after dissolving the formazan in alcohol (from ISO 10993).

[0234] So the discs, loaded or not with PAR, were sterilized and placed onto ˜80% confluent layer of Balb/3T3 cells. For MTT assay, the cells were incubated for 2 h in 0.2 mg/mL MTT-containing cell culture medium. The medium was then removed and formazan was dissolved in DMSO. Absorbance of resulting solutions was measured at 570 nm using spectrophotometer and images were taken around and under the discs to evaluate cell morphology.

[0235] In first experiments, hydrogels loaded with 0.05 mg/mL PAR10 and PAR30 (lowest loading concentration showing antibacterial effect) appeared to be non-cytotoxic by ISO 10993 norms, according to quantitative MTT test results (good viability) and qualitative reactivity gradation (reactivity limited to area under specimen) (FIG. 13).

[0236] The inventors confirmed these results and, in addition, performed cytotoxicity assay on PAR10 and PAR30 0.1 mg/mL-loaded hydrogels, which also appeared to be non-cytotoxic (FIG. 14). As previously, reactivity zone was limited to the area under the discs (data not shown) and cell viability was good (FIG. 20B). PAR200-loaded hydrogels, however, were classed as cytotoxic.

[0237] Of note, PAR10 and PAR30 0.1 mg/mL-loaded hydrogels showed 2 and 4 days of antibacterial effect in repetitive culture (=fresh bacteria added every day). In an additional test (FIG. 15), these numbers were even higher (3 and 5 days).

Example 5: Deposition of HA Hydrogels on Mesh Materials

[0238] In addition to setting up a protocol for free-standing hydrogel disc production, the inventors attempted hydrogel deposition onto two materials used for clinical applications: a non-woven fabric used for wounds desinfection, with high absorption power (Medicomp®) and polypropylene mesh for hernia repair.

[0239] HA-BDDE solution (50 or 100 μL) was deposited on 12-mm diameter fabric or mesh pieces and allowed to cross-link. Medicomp® absorbed and retained 100 μL of hydrogel solution, while 50 μL amount was more suitable for polypropylene meshes which are not absorbent. However, both materials were able to retain the hydrogels after cross-linking, and easy to manipulate.

[0240] Confocal images of PAR30-rhodamine loaded HA hydrogels deposited onto Medicomp® fabric and polypropylene meshes were obtained. In these images, fabric or mesh fibers are surrounded by PAR30-rhodamine labeled hydrogels. Some PAR30-rhodamine is also adsorbed on the fibers.

[0241] In terms of antibacterial activity, hydrogel-coated mesh materials were compared to hydrogel discs and showed similar bacterial growth inhibition at low concentration (FIG. 16) for 6 days.

Example 6: Storage and Sterilization

[0242] The hydrogels (free-standing, as well as deposited onto mesh materials) can be kept for several days at 4° C., dried, frozen and sterilized by autoclaving (FIG. 17) without losing antibacterial activity.

Example 7: Cross-Linking Percentage

[0243] Hydrogels with lower or higher cross-linking degrees (10% and 30% BDDE v/v) loaded with PAR30 showed similar antibacterial activity after 24 h, as compared to 20% BDDE (FIG. 18). However, they were more difficult to handle: 10% BDDE hydrogels were very soft and elastic, while 30% BDDE hydrogels were fragile and broke easily.

Example 8: Use of Other Positively Charged Antibacterial Polypeptides

[0244] After 24 h, HA hydrogels loaded with 0.05 and 0.1 mg/mL of polyornithine PLO30 and polylysine PLL30 showed similar antibacterial activity, as compared to PAR30-loaded hydrogels (FIG. 19).

Example 9: In Vivo Biocompatibility

[0245] Ten 8-week-old male Wistar rats (300-400 g in weight), provided by a certified breeding centre (Charles River, France) were used for this study.

[0246] The animals were received at the CREFRE (US 006/CREFRE-Inserm/UPS/ENVT) animal supplier (No. A31555010 issued Dec. 17, 2015). Protocols were submitted to the CREFRE ethics committee with approval, in accordance with the European directive (DE 86/609/CEE; modified DE 2003/65/CE) for conducting animal experiments. One week of acclimatization was respected. The animals were housed in ventilated cages with a double level (two animals per cage according to European standards). The animals were carefully monitored (behavior and food intake) and were weighed weekly throughout the experiment. The 10 rats received each 2 round implants (diameter of 1 cm), one implant on left side and one implant on right side. In total, there were 5 implantations of dried and autoclaved hydrogels deposited onto mesh materials for each of the following conditions: i) HA-only hydrogels; ii) HA hydrogels loaded with PAR10 at 0.1 mg.Math.mL.sup.−1; iii) HA hydrogels loaded with PAR30 at 0.05 mg.Math.mL.sup.−1; iv) HA hydrogels loaded with PAR30 at 0.1 mg.Math.mL.sup.−1.

[0247] The rats were induced by isoflurane 4% and maintenance of 2%. Each rat was placed in a prone position on a heated pad. After shaving and scrubbing with betadine, two 20 mm dorsal incisions were made over the thoracolumbar area, one on the right side and one on the left side. One scaffold was inserted at both sides into subcutaneous pockets. All the incisions were closed with Vicryl® 3-0. All rats received buprenorphine (0.6 mg/kg) injected subcutaneously twice per day for 5 days. All animals survived the duration of the study with no adverse effects. Euthanasia were performed after 14 days. The animals were first anesthetized with isoflurane device and mask and then slowly injected with an overdose of pentobarbital (150 mg/kg) in intraperitoneal route. After the expiration of the animal death, the implants with surrounding tissue were explanted and collected to perform histology.

[0248] For histological analysis, the samples were fixed in 4% formalin. Macroscopic sections were embedded in paraffin. Five-μm thick sections were stained with hematoxylin-eosin-saffron (HES). For each sample, microscopic optical analysis was realized with the software NDP.view2 (Hamamatsu, Massy, France) after slides scanning (NanoZoomer, Hamamatsu) with the following criteria: semi-quantitative assessment of acute inflammation, chronic inflammation, fibroblastic reaction, edema, fibrosis, angiogenesis and periprosthetic histiocytic reaction.

Results

[0249] Preliminary in vivo experiments were conducted on rats (10 animals). Each rat received 2 implants of hydrogel-coated meshes (d=1 cm), one implant on left side and one implant on right side. All animals survived the duration of the study with no adverse effects and all animals gained weight in a normal way.

[0250] After 14 days, the implants with surrounding tissue were explanted and collected to perform histological analysis. The results of the analysis showed the presence of inflammation in the tissues surrounding the implants. However, there was no difference between HA-only hydrogels and HA-PAR hydrogels, suggesting that PAR addition does not promote or increase inflammatory response.

CONCLUSION

[0251] In summary, the inventors developed hyaluronic acid (HA) hydrogels that can be loaded with polyarginine (PAR) and provide a long lasting antibacterial effect. This effect is dependent on the concentration and length of loaded PAR. PAR30 was identified as the most efficient in providing a prolonged antibacterial effect, which increases with PAR concentration.

[0252] The antibacterial hydrogels can be deposited onto wound dressings and mesh prosthesis and may help to prevent infections, thus improving tissue regeneration and/or implant integration.