NEW HYDROGELS HAVING A SILYLATED STRUCTURE, AND METHOD FOR OBTAINING SAME

Abstract

The present invention relates to hydrogels prepared using silylated organic molecules (such as silylated biomolecules), a process for obtaining the same, and uses thereof.

Claims

1.-7. (canceled)

8. A process for producing a hydrogel comprising the steps of: a) sol-gel polymerization of at least one molecule of formula (I): ##STR00006## wherein: n is an integer greater than or equal to 2; A is a structural organic polymer, preferentially of synthetic origin which may be, for example, selected from proteins, peptides such as collagen derivatives, in particular the sequences comprising Pro-Hyp-Gly or Pro-Pro-Gly or Asp-Pro-Gly or Pro-Lys-Gly tripeptide repeats, self-assembly peptide sequences such as Arg-Ala-Asp-Ala (SEQ ID 4), oligoprolines, oligoalanines, polysaccharides, such as hyaluronic acid and derivatives thereof, oligonucleotides, C.sub.1-C.sub.6-alkylene-glycol polymers, or polyvinylpyrrolidone; Xa is a chemical bond or a spacer group preferentially represented by a divalent radical derived from a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 10 carbon atoms, optionally intercalated with one or more structural linkers selected from arylene or fragments —O—, —S—, —C(═O)—, SO.sub.2 or —N(R.sub.1)—, wherein said chain is unsubstituted or is substituted by one or more radicals selected from halogen atoms, a hydroxyl group, a C.sub.1-C.sub.4 alkyl group, a benzyl group and/or a phenethyl group; R.sub.1 represents a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, a benzyl or a phenethyl; Y.sub.1, Y.sub.2, Y.sub.3, which may be identical or different, each independently represents a hydrogen atom, a halogen atom, an —OR.sub.2 group, an aryl or a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 6 carbon atoms optionally substituted by a halogen atom, an aryl group or a hydroxyl group; R.sub.2 represents a hydrogen atom, an aryl group or a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 6 carbon atoms; wherein at least two Xa groups as defined above are linked to different attachment points on A; b) mixing with water, optionally at the same time as step a); and c) recovering the hydrogel.

9. The process for producing a hydrogel according to claim 8, wherein said process comprises the addition, at the same time as or subsequent to step a), of at least one type of molecule of formula (II): ##STR00007## wherein: m is an integer greater than or equal to 1, preferentially equal to 1; B is an active ingredient, preferentially a biomolecule or a fluorophore, which may be, for example, selected from a peptide, an oligopeptide, a protein, such as collagen, a deoxyribonucleic acid, a ribonucleic acid, a polysaccharide, such as a pectin, a chitosan, a hyaluronic acid, a polyarabinose and polygalactose polysaccharide, and a glycolipid; Xb is a chemical bond or a spacer group preferentially represented by a divalent radical derived from a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 10 carbon atoms, optionally intercalated with one or more structural linkers selected from arylene or fragments —O—, —S—, —C(═O)—, SO.sub.2 or —N(R.sub.3)—, wherein said chain is unsubstituted or is substituted by one or more radicals selected from halogen atoms, a hydroxyl group, a C.sub.1-C.sub.4 alkyl group, a benzyl group and/or a phenethyl group; R.sub.3 represents a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, a benzyl or a phenethyl; Z.sub.1, Z.sub.2, Z.sub.3, which may be identical or different, each independently represents a hydrogen atom, a halogen atom, an —OR.sub.4 group, an aryl or a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 6 carbon atoms optionally substituted by a halogen atom, an aryl group or a hydroxyl group; R.sub.4 represents a hydrogen atom, an aryl group or a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 6 carbon atoms; and wherein preferentially only one of the Z.sub.1, Z.sub.2, or Z.sub.3 groups is a halogen atom or an OR.sub.4 group.

10. The process for producing a hydrogel according to claim 8, wherein the sol-gel polymerization process is carried out at physiological pH or in that the hydrogel is formed in the presence of a sufficient amount of water so that the water content of the hydrogel is at least 50 wt. % relative to the total weight of the hydrogel formed.

11. The process for producing a hydrogel according to claim 8, wherein said hydrogel is polymerized on or in at least a first hydrogel as a support, thus resulting in a multi-layer hydrogel.

12. A hydrogel that can be obtained by the process according to claim 8.

13. Therapeutic and/or surgical method comprising the use of hydrogel according to claim 12 in a patient in need thereof.

14. In vitro tissue engineering method comprising the use of a hydrogel according to claim 12.

15. The process for producing a hydrogel according to claim 9, wherein the sol-gel polymerization process is carried out at physiological pH or in that the hydrogel is formed in the presence of a sufficient amount of water so that the water content of the hydrogel is at least 50 wt. % relative to the total weight of the hydrogel formed.

16. The process for producing a hydrogel according to claim 9, wherein said hydrogel is polymerized on or in at least a first hydrogel as a support, thus resulting in a multi-layer hydrogel.

17. The process according to claim 8, wherein the molecule of formula (I) is prepared in homogeneous conditions.

18. The process according to claim 9, wherein the molecule of formula (II) is prepared in homogeneous conditions.

19. The process according to claim 8, wherein the polymerization is carried out in situ in a living organism.

20. The process according to claim 9, wherein the polymerization is carried out in situ in a living organism.

Description

FIGURES

[0172] FIG. 1: this figure relates to cytotoxicity tests on L929 fibroblasts. The “TC-PS” control (“low control” column in the figure) represents cell viability on TC-PS (i.e., without hydrogel). The “lysed cells” control (“high control” column in the figure) corresponds to complete lysis of the cells and thus to maximum toxicity. The “PLA-50” control makes it possible to confirm cell viability in the presence of poly(lactic acid). The “2.5% NaF silylated PEG hydrogel” sample concerns cells incubated 24 h in the presence of a hydrogel containing 10% silylated PEG by mass obtained with 2.5% NaF by weight. The “0.3% NaF silylated PEG hydrogel” sample concerns cells incubated 24 h in the presence of a hydrogel containing 10% silylated PEG by mass obtained with 0.3% NaF by weight.

[0173] FIG. 2: this figure concerns tests to quantify the release of fluorescein from various hydrogels according to the invention. The hydrogels grafted with fluorescein by covalent bond show much lower release rates than the hydrogel containing fluorescein simply enclosed with no covalent bond.

[0174] FIG. 3: this figure concerns tests of cell adhesion on the hydrogels according to the present invention. Two controls were selected for comparison: one indicates the measured fluorescence values of the culture medium in the absence of cells and the other is the fluorescence measured for the cells deposited directly on TC-PS. On this comparative basis, three series of tests were carried out with silylated PEG hydrogels according to the invention with 10% bare silylated PEG by mass, then with 7.5% molar or 15% molar (relative to the number of moles of silylated PEG) of a cell adhesion peptide (“RGD”) linked by covalent bond to said hydrogel.

[0175] Caption for FIG. 3: [0176] Columns (of the histogram) in white (i.e., the first columns starting from the left of the histogram): culture medium in the absence of cells; [0177] Columns in light gray (i.e., the second columns starting from the left of the histogram): cells deposited on bare PEG hydrogel; [0178] Columns in medium gray (i.e., the third columns starting from the left of the histogram): cells deposited on PEG hydrogel containing 7.5% RGD hybrid peptide; [0179] Columns in dark gray (i.e., the fourth columns starting from the left of the histogram): cells deposited on PEG hydrogel containing 15% RGD hybrid peptide; [0180] Columns in black (i.e., the fifth columns starting from the left of the histogram): cells deposited on TCPS.

[0181] FIG. 4: this figure concerns antibacterial tests with hydrogels according to the present invention. The bacteria which were the subject of the test are E. coli, S. aureus and P. aeruginosa. Two control tests were carried out: one being an evaluation of the number of bacterial colonies on simple agar gel, the other on bare silylated PEG hydrogel according to the present invention (i.e., a silylated PEG hydrogel with 10% silylated PEG by mass). An antibacterial peptide was grafted onto this same gel (i.e., bare PEG hydrogel) by covalent bonds in various proportions (7.5 mol. % of antibacterial peptide and 15 mol. % of antibacterial peptide relative to the number of moles of silylated PEG). The bacterial colonies which were formed in contact with the gets are counted after 24 h of incubation at 37° C.

[0182] Caption for FIG. 4: [0183] Columns (of the histogram) in black (i.e., the first columns starting from the left of the histogram): colonies seeded on agar; [0184] Columns in white (i.e., the second columns starting from the left of the histogram): colonies seeded on bare PEG hydrogel; [0185] Columns in light gray (i.e., the third columns starting from the left of the histogram): colonies seeded on PEG hydrogel with 7.5% antibacterial hybrid peptide; [0186] Columns in dark gray (i.e., the fourth columns starting from the left of the histogram): colonies seeded on PEG hydrogel with 15% antibacterial hybrid peptide.

[0187] FIG. 5: illustrates a Cryo-SEM view of the hydrogel containing the bi-silylated peptide prepared.

[0188] FIG. 6: illustrates the adhesion of murine mesenchymal stem cells (mMSC) on the hydrogel according to the invention, on collagen foams and on TC-PS. The dark gray histograms represent the TC-PS support. The histograms with black dots represent the hydrogel support according to the invention. The histograms with black diagonal lines represent the collagen-type commercial support.

[0189] FIG. 7: illustrates the proliferation of mMSC on the hydrogel according to the invention, on the collagen foam and on TC-PS. The caption for the histograms is the same as for FIG. 6.

EXAMPLES

[0190] The examples below in no way limit the scope of the protection sought and are provided by way of illustration according to the present invention.

Abbreviations

[0191] ACN, acetonitrile; Ahx, ε-aminohexanoic acid; Boc, t-Butyloxycarbonyl; DCM, dichloromethane; DIEA, diisopropylethylamine; DMF, N—N′-dimethylformamide; DPBS, Dulbecco's phosphate buffered saline; ESI-MS, electrospray ionization mass spectrometry; Fmoc, fluorenylmethoxycarbonyl; HBTU, —N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; LC/MS, mass spectrometry coupled with liquid chromatography; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PEG, polyethylene glycol; pip, piperidine; PS, polystyrene; NMR, nuclear magnetic resonance; RT, room temperature, i.e., ranging between 20 and 25° C.; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TIS, triisopropylsilane.

[0192] Materials and Methods

[0193] Analytical HPLCs were carried out on an Agilent Infinity 1260 apparatus equipped with a diode array and a Kinetex C.sub.18 reversed-phase column, 2.6 μm, 50×4.6 mm with a gradient of 0% to 90% (% by volume) of B in 5 min with eluent A: water/0.1% TFA and eluent B: ACN/0.1% TFA with a flow rate of 2.5 mL/min.

[0194] Purifications by preparative HPLC were carried out on a Waters HPLC 4000 apparatus, equipped with a UV 486 detector and a Waters DeltaPak C.sub.18 reversed-phase column, 40×100 mm, 100 Å, 15 μm, with a flow rate of 50 mL/min. The solvents used are H.sub.2O/0.1% TFA and ACN/0.1% TFA.

[0195] Samples for LC/MS analyses were prepared in a water/ACN mixture (50:50, v/v) containing 0.1% TFA. The LC/MS device consists of a Waters Alliance 2695 HPLC, coupled to a Water Micromass ZQ spectrometer (electrospray ionization, positive mode). Analyses were carried out with a Phenomenex Onyx reversed-phase column, 25×4.6 mm with a flow rate of 3 mL/min with a gradient of 0% to 100% (vol. %) of B in 2.5 min with eluent A: water/0.1% HCO.sub.2H; eluent B: ACN/0.1% HCO.sub.2H. UV detection was set at 214 nm. Mass spectra were acquired with a solvent flow rate of 200 μL/min. Nitrogen is used as the nebulizing and drying gas. Data are obtained by scanning m/z from 100 to 1000 in 0.75 seconds or from 200 to 1600 in 0.9 seconds. High-resolution mass spectrometry analyses were carried out in positive mode on a time-of-flight (TOF) spectrometer equipped with an electrospray ionization source.

[0196] .sup.1H, .sup.13C and .sup.29Si NMR spectra were recorded at room temperature (RT) in deuterated solvents on a spectrometer at 400, 101 and 79 MHz, respectively. Chemical shifts (δ) are given in parts per million using the residual non-deuterated solvents as references (CHCl.sub.3 in CDCl.sub.3, δH=7.26 ppm; DMSO-d6, δH=2.50 ppm). Signals are designated s (singlet), d (doublet), t (triplet), q (quadruplet), dt (doublet of triplets), m (multiplet), etc. Coupling constants are measured in hertz.

[0197] Attachment of an Amino Acid to the 2-Chlorotrityl Chloride Resin:

[0198] The 2-chlorotrityl chloride resin (1.44 mmol Cl/g, 1 eq) is placed in a solid-phase peptide synthesis reactor equipped with a sintered glass. The protected amino acid Fmoc-AA-OH (3 eq) is coupled to the resin in the presence of DIEA (5 eq) in DMF overnight. After standard washings (3×DMF, 1×MeOH and 1×DCM), the Fmoc-AA-Cltrityl resin is dried under vacuum for 12 h. Resin load is determined by 299 nm detection of the piperidine-dibenzofulvene adduct which is formed in the pip/DMF (20:80 v/v) deprotection solution.

[0199] Fmoc Deprotection:

[0200] The Fmoc-Rink Amide AM PS resin or Fmoc-peptidyl-resin is placed in a solid-phase reactor equipped with a sintered glass. The Fmoc group is removed by two successive treatments with a DMF-piperidine solution (80:20; v/v, 2×20 min). Between the two treatments, the solution is filtered and replaced with fresh solution. The conventional washing steps are carried out at the conclusion of the deprotection (3×DMF, 1×MeOH and 1×DCM).

[0201] Coupling of an Acid Amino:

[0202] The amino acid N-ter protected with an Fmoc group (3 eq) is dissolved in DMF (10 mL per g of resin) in the presence of HBTU (3 eq) and DIEA (3 eq) for 10 min. This solution is added to the peptidyl-resin the N-ter of which is free. The resin is stirred at RT for 1 h 30 min then washed (3×DMF, 1×MeOH and 1×DCM).

Example 1: Silylation of PEG

[0203] Polyethylene glycol with an average molecular mass of 2000 g/mol (2.00 g, 1.00 mmol) is dried under vacuum at 80° C. overnight then dissolved in anhydrous THF (12 mL) under argon. Triethylamine (1.66 mL, 12 mmol, 12 eq) and isocyanatopropyl-triethoxysilane (744 μL, 3 mmol, 3 eq) are added. The mixture is refluxed for 48 h then concentrated under reduced pressure. The bi-silylated PEG is then precipitated in hexane. After centrifugation, it is washed 3 times with hexane and dried under vacuum. It is obtained in the form of a white powder stored at 4° C. under argon. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.00 (sl, 2H, NH), 4.18 (t, J=4.7 Hz, 4H, H-6), 3.79 (q, J=7.0 Hz, 12H, H-2), 3.62 (s, 177H, CH.sub.2 PEG), 3.14 (dd, J=13.2, 6.7 Hz, 4H, H-5), 1.58 (qu, J=7.7 Hz, 4H, H-4), 1.20 (t, J=7.0 Hz, 18H, H-1), 0.64-0.54 (m, 4H, H-3). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 156.72 (C), 70.55 (CH.sub.2), 63.76 (CH.sub.2), 58.26 (CH.sub.2), 43.29 (CH.sub.2), 23.12 (CH.sub.2), 18.38 (CH.sub.3), 9.12 (CH.sub.2). .sup.29Si NMR (79 MHz, CDCl.sub.3) δ −45.73 (s).

Example 2: Synthesis of a Peptide Derived from Collagen and Bi-Silylated

[0204] Preparation of the Tripeptide: Fmoc-Pro-Hyp-Gly-OBzl

[0205] Coupling of Boc-Hyp-OH:

[0206] Into a 5000-mL single-neck round-bottom flask are introduced H-Gly-OBzl.HCl (11.42 g, 56.6 mmol, 1 eq) dissolved in AcN, and DIEA (37.44 mL, 226.5 mmol, 4 eq). In a beaker, Boc-Hyp-OH (13.3 g, 56.6 mmol) is dissolved in AcN. DIEA and pyBOP (29.3 g, 56.63 mmol, 1 eq) are added to this solution. The two amino acids are contacted and stirred at RT for 4 h. The reaction is monitored by analytical HPLC.

[0207] Once the reaction ends, AcN is evaporated and the orange oil obtained is solubilized in ethyl acetate. This solution is washed with aqueous solutions of KHSO.sub.4, NaHCO.sub.3 and NaCl. The organic phase is then dried with MgSO.sub.4 and the solvent evaporated under reduced pressure.

[0208] N-Ter Deprotection of the Dipeptide:

[0209] The product is then dissolved in 150 mL of TFA for 40 min until gas evolution has completely ceased. The TFA is then evaporated under reduced pressure and the dipeptide is precipitated in diethyl ether then freeze-dried.

[0210] Coupling of Fmoc-Pro-OH:

[0211] Fmoc-Pro-OH is coupled to H-Hyp-Gly-OBzl according to the same protocol as the coupling of Boc-Hyp-OH described above. The reaction lasts 2 h. At the conclusion of the washings, the tripeptide is purified on silica gel (Biotage apparatus, SNAP 340 g column, gradient of 0% MeOH in DCM to 10% MeOH in DCM, product eluted at 50% of the gradient, 71% yield).

[0212] C-Ter Deprotection:

[0213] Into a 250-mL single-neck round-bottom flask are introduced Fmoc-Pro-Hyp-Gly-OBzl (20.4 g, 40.2 mmol) dissolved in EtOH and 200 mg of Pd/C. The whole is placed under hydrogen bubbling for 6 h at 60° C. The reaction is monitored by analytical HPLC. Once the reaction ends, the solution is filtered through Celite then concentrated under reduced pressure. The solid is taken up in H.sub.2O/AcN 50:50 v/v and freeze-dried (Yield: 86%).

[0214] Synthesis of Peptide Ac-Lys-(Pro-Hyp-Gly).sub.3-Lys-NH.sub.2 (Seq ID 5) on a Support Starting with the Tripeptide Block

[0215] Synthesis is carried out in a syringe equipped with a sintered glass on a Rink Amide PS resin the load of which is 0.94 mmol/g with a 1-mmol synthesis scale. The resin is swollen in DCM then washed with DMF. It is deprotected by means of two treatments with pip/DMF deprotection solution (15 mL for 5 min, 3 washings with DMF, 15 mL for 20 min then washings (3×DMF, 3×DCM, 1×DMF)). The usual coupling conditions are slightly modified. The couplings are extended to 2 h and are carried out with 1.5 eq of Fmoc-Lys(Boc)-OH or Fmoc-(Pro-Hyp-Gly)-OH, 5 eq of DIEA and 1.5 eq of HATU which replaces HBTU. At the end of the coupling, the resin is washed with the following solvents: (3×DMF, 3×DCM, 1×DMF). The last coupling is also followed by deprotection of the Fmoc group. Next, the peptidyl-resin is acetylated with acetic acid (2 eq) in DCM (10 mL) in the presence of BOP (2 eq) and DIEA (4 eq) for 1 h 30 min. The resin is washed then cleaved in a TFA/TIS/H.sub.2O mixture (95:2.5:2.5 v/v/v, 50 ml). The “cleavage” solution is concentrated under reduced pressure and the peptide is precipitated with ether. After centrifugation and removal of the supernatant, the crude peptide is taken up in a water/ACN mixture and freeze-dried. Finally, it is purified by preparative HPLC on a Luna C.sub.18 reversed-phase column (15 μm, 250×50 mm) with a flow rate of 120 mL/min with a gradient of 0% to 6% of B in 6 min, of 6% to 10% of B in 8 min and of 10% to 18% in 24 min with eluent A: H.sub.2O/0.1% TFA and eluent B: ACN/0.1% TFA. Yield: 49%; purity >99%. LC/MS (ESI.sup.+): t.sub.R=0.73 min, 1118 ([M+H].sup.+, 5%), 559 ([M+2H].sup.2+, 100).

[0216] Silylation

[0217] Ac-Lys-(Pro-Hyp-Gly).sub.3-Lys-NH.sub.2 (20 mg, 14.9 μmol) is dissolved in anhydrous dimethylformamide (300 μL) under argon. Diisopropylethylamine (12.4 μL, 71.3 μmol, 4.8 eq) and then 3-isocyanatopropyltriethoxysilane (9.7 μL, 39.3 μmol, 2.6 eq) are added to the nonapeptide solution. The reaction mixture is left 50 min under stirring. The end of the reaction is monitored by LC/MS. The solvent is evaporated under reduced pressure. Next, the silylated nonapeptide is precipitated with diethyl ether. After centrifugation, the hybrid peptide is washed 3 times with diethyl ether then the powder obtained is dried under vacuum. LC/MS (ESI.sup.+): t.sub.R=0.82 min, 704 ([M+2H-2H.sub.2O].sup.2+, 100%), 695 ([M+2H-3H.sub.2O]].sup.2+, 80) and t.sub.R=0.87 min (conformer), 704 ([M+2H-2H.sub.20].sup.2+, 70%), 695 ([M+2H-3H.sub.20]].sup.2+, 100).

Example 3: Preparation of a Hydrogel Composed of a Molecule of Formula (I)

[0218] A molecule of formula (I), for example the bi-silylated PEG prepared in example 1 or the collagen-derived bi-silylated peptide synthesized in example 2, is dissolved in pH 7.4 phosphate buffer (DPBS Dulbecco's phosphate buffered saline), preferably at a concentration of 10% by mass, in the presence of sodium fluoride (3 mg of NaF per mL of DPBS). The non-viscous solution is incubated at 37° C.; a get then forms. The gelation time depends on the nature and the concentration of the molecule of formula (I).

Example 4: Synthesis of Silylated Peptide (EtO).SUB.3.Si—(CH.SUB.2.).SUB.3.—NHCO-(βAla).SUB.4.-Gly-Ara-Gly-Asp-Ser-Pro-OH (Seq ID 6)

[0219] H-(βAla).sub.4-Gly-Arg-Gly-Asp-Ser-Pro-OH (Seq ID 6) is synthesized on a 2-chlorotrityl chloride resin (load: 1.44 mmol/g, synthesis scale: 0.5 mmol) using an Fmoc/tBu strategy. The amino acids used are successively Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp-(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH and Fmoc-βAla-OH four times. Each amino acid coupling is followed by N-ter deprotection of the Fmoc group. “Cleavage” of the peptide and deprotection of the side chains are carried out in a TFA/TIS/H.sub.2O mixture, 95:2.5:2.5 v/v/v, for 4 h. After precipitation, the peptide is purified by preparative HPLC on a C.sub.18 column. It is obtained after freeze-drying with 63% yield. It is then silylated by reaction with 3-isocyanatopropyltriethoxysilane (1.1 eq) in DMF at a concentration of 30 mM in the presence of DIEA (3 eq). The reaction is monitored by analytical HPLC. The DMF is then evaporated under reduced pressure and the silylated peptide is precipitated in diethyl ether. It is recovered by centrifugation at the conclusion of 3 washings in diethyl ether.

[0220] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.61-8.40 (m, 2H, NH Asp and Gly), 8.21-8.04 (m, 2H, NH Gly N-ter and Arg), 7.99-7.78 (m, 3H, NH βAla)), 7.58-7.42 (m, 1H, NH Ser), 7.34-6.98 (m, 3H, OH Ser, COOH Asp and C-ter), 5.97 (t, J=5.6 Hz, 1H, NH urea), 5.78 (t, J=5.6 Hz, 1H, NH urea βAla), 4.56 (q, J=6.9 Hz, 1H, Hα Ser), 4.49-4.38 (m, 1H, Hα Asp), 4.38-4.26 (m, 1H, Hα Arg), 4.22 (dd, J=8.7, 4.1 Hz, 1H, Hα Pro), 4.02-3.75 (m, 4H, Hα Gly), 3.73 (q, J=6.9 Hz, 6H, CH.sub.2 ethoxy), 3.67-3.46 (m, 4H, Hδ Pro and Hβ Ser), 3.46-3.28 (m, 2H, Hδ Arg), 3.28-3.11 (m, 8H, Hβ BAla), 2.92 (q, J=6.1 Hz, 2H, H-3), 2.60-2.48 (m, 2H, Hβ Asp), 2.29 (t, J=7.1 Hz, 2H, Hα βAla), 2.26-2.09 (m, 6H, Hα βAla), 1.87 (dd, J=13.3, 6.5 Hz, 2H, Hγ Pro), 1.60-1.56 (m, 2H, Hβ Arg), 1.55-1.42 (m, 2H, Hδ Arg), 1.38 (m, 2H, H-2), 1.19-1.09 (t, J=7.2 Hz, 9H, CH.sub.3 ethoxy), 0.55-0.42 (m, 2H, H-1). .sup.13C NMR (101 MHz, DMSO-d6) δ 174.27 (C), 172.82 (C), 171.38 (C), 171.27 (C), 171.14 (C), 170.86 (C), 170.80 (C), 169.58 (C), 169.43 (C), 169.02 (C), 168.77 (C), 158.41 (C), 157.52 (C), 62.14 (CH.sub.2), 59.54 (CH), 58.14 (CH.sub.2), 53.51 (CH), 52.60 (CH), 49.78 (CH), 47.02 (CH.sub.2), 42.46 (CH.sub.2), 42.36 (CH.sub.2), 40.89 (CH.sub.2), 36.68 (CH.sub.2), 36.30 (CH.sub.2), 35.88 (CH.sub.2), 35.76 (CH.sub.2), 30.10 (CH.sub.2), 29.13 (CH.sub.2), 25.39 (CH.sub.2), 24.91 (CH.sub.2), 24.01 (CH.sub.2), 18.68 (CH.sub.3), 7.72 (CH.sub.2). .sup.29Si NMR (79 MHz, DMSO-d6) δ −45.10. LC/MS (ESI.sup.+): Only the hydrolysis products of the ethoxysilane groups to silanols are detected. t.sub.R=0.62 min, 1035 ([M+H].sup.+, 50%), 509 ([M+H—OH].sup.2+, 60), 500 ([M-2OH].sup.2+, 100). HRMS: 1119.5481. C.sub.45H.sub.74N.sub.18O.sub.14Si implies [M+H].sup.+, 1119.5479.

Example 5: Synthesis of Silylated Peptide HO(CH.SUB.3.).SUB.2.Si—(CH.SUB.2.).SUB.3.—NHCO-Ahx-Arg-Ara-NH.SUB.2

[0221] Antibacterial peptide H-Ahx-Arg-Arg-NH.sub.2 is synthesized on a Rink Amide resin (load 0.94 mmol/g, synthesis scale: 3 mmol) using an Fmoc/tBu strategy. The amino acids used are successively Fmoc-Arg(Pbf)-OH twice and Fmoc-ε-aminohexanoic acid. Each coupling is followed by N-ter deprotection of the Fmoc group. The tripeptide is then silylated on a support in DMF (10 mL/g resin) by using 3-isocyanatopropyldimethylchlorosilane (3 eq) in the presence of DIEA (3 eq). The silylation reaction is left under stirring overnight then the resin is washed (3×DMF, 1×MeOH and 1×DCM) and cleaved in TFA for 5 h. The “cleavage” solution is concentrated under reduced pressure then the silylated antibacterial peptide is precipitated in diethyl ether and finally purified by preparative HPLC on a C.sub.18 column (Eluent A: H.sub.2O/0.1% TFA, eluent B: ACN/0.1% TFA, gradient: 0% to 7% of B in 2 min then 7% to 30% of B of 23 min, the product eluted at 16% of B). After freeze-drying, the silylated antibacterial peptide is obtained in the form of a white powder (Yield: 68%, purity >98%). .sup.1H NMR (400 MHz, D.sub.2O) δ 4.19 (ddd, J=14.4, 8.6, 5.7 Hz, 2H, Hα Arg), 3.09 (t, J=6.9 Hz, 4H, Hδ Arg), 2.96 (td, J=6.8, 1.9 Hz, 4H, H-3 and H-4), 2.17 (t, J=7.3 Hz, 2H, H-8), 1.82-1.59 (m, 4H, Hβ Arg), 1.59-1.44 (m, 6H, H-7 and Hγ Arg), 1.41-1.31 (m, 4H, H-2 and H-5), 1.24-1.12 (m, 2H, H-6), 0.53-0.41 (m, 2H, H-1), 0.00 (s, 6H, Si(CH.sub.3).sub.2). .sup.13C NMR (101 MHz, DMSO-d6) δ 172.40 (C), 171.76 (C), 170.69 (C), 157.38 (C), 156.00 (C), 51.38 (CH), 51.02 (CH), 41.54 (CH.sub.2), 34.28 (CH.sub.2), 29.01 (CH.sub.2), 28.34 (CH.sub.2), 28.08 (CH.sub.2), 25.24 (CH.sub.2), 24.24 (CH.sub.2), 23.16 (CH.sub.2), 14.24 (CH.sub.2), −0.51 (CH.sub.3). .sup.29Si NMR (79 MHz, DMSO-d6) δ 7.97 (dimer). LC/MS (ESI.sup.+): t.sub.R=0.71 min, 602 ([M+H].sup.+, 10%), 302 ([M+2H].sup.2+, 100), 293 ([M+2H—NH.sub.3].sup.2+, 30). HRMS: 602.3926. C.sub.24H.sub.51N.sub.11O.sub.5Si implies [M+H].sup.+, 602.3922.

Example 6: Synthesis of a Hydroxydimethylsilyl Fluorescein

[0222] ##STR00004##

[0223] N-Boc-1,3-propanediamine (56.4 mg, 0.324 mmol, 1.05 eq) is added to a solution of fluorescein isothiocyanate (FITC, 120 mg, 0.308 mmol) in anhydrous DMF (3 mL) in the presence of DIEA (100 μL). The reaction mixture is stirred for 1 h at RT under argon. Next, the DMF is evaporated under reduced pressure. The Boc-amino fluorescein is precipitated and washed in diethyl ether then dried. It is then solubilized in TFA (4 mL) and this solution is stirred for 1 h. The reaction mixture is concentrated and precipitated in diethyl ether. After centrifugation, the supernatant is removed. The fluorescein amine is purified by preparative HPLC on a C.sub.18 column (Eluent A: H.sub.2O/0.1% TFA, eluent B: ACN/0.1% TFA, gradient: 0% to 15% of eluent B in 3 min then 15% to 40% of eluent B in 25 min, the product eluted at 22% of eluent B) and obtained in the form of TFA salt (180 mg, 100%, purity=95%).

[0224] 3-Isocyanatopropylchlorodimethylsilane (16.2 μL, 0.0910 mmol, 1.05 eq) is added to a solution of fluorescein amine purified beforehand (50.0 mg, 0.0867 mmol) in anhydrous DMF (2 mL) in the presence of DIEA (45.2 μL, 0.260 mmol, 3 eq). The reaction mixture is stirred under argon for 1 h. The solvent is evaporated under reduced pressure and the crude product is obtained by precipitation in diethyl ether. The hydroxydimethylsilyl fluorescein is purified by preparative HPLC on a C.sub.18 column (Eluent A: H.sub.2O/0.1% TFA, eluent B: ACN/0.1% TFA, gradient: 0% to 20% of eluent B in 4 min, 20% to 26% of eluent B in 6 min then 26% to 46% of eluent B in 30 min, the product eluted at 32% of eluent B, yield: 42%, purity: 97%) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.01 (sl, 1H, COOH), 9.97 (s, 1H, NH thiourea from FITC), 8.20 (s, 1H, H-4), 8.12 (s, 1H, NH thiourea), 7.71 (d, J=7.6 Hz, 1H, H-6), 7.14 (d, J=8.3 Hz, 1H, H-5), 6.65 (d, J=2.2 Hz, 2H, H-9 and H-10), 6.59-6.51 (m, 4H, H-7, H-8, H-11 and H-12), 5.88 (s br, 2H, NH urea), 3.43-3.38 (m, 2H, H-3), 3.03 (t, J=6.5 Hz, 2H, H-1), 2.92 (t, J=6.9 Hz, 2H, H-3′), 1.62 (qu, J=6.5 Hz, 2H, H-2), 1.37-1.29 (m, 2H, H-2′), 0.47-0.36 (m, 2H, H-1′), 0.00 and −0.04 (2 s, 6H, H-4′ of the dimer and of the monomer, respectively). .sup.13C NMR (101 MHz, DMSO-d6) δ 179.51 (C), 167.65 (C), 158.62 (C), 157.51 (C), 151.02 (C), 140.47 (C), 128.75 (CH), 128.19 (CH), 125.68 (C), 123.18 (CH), 116.66 (C), 115.79 (CH), 114.24 (C), 111.71 (CH), 108.87 (C), 101.36 (CH), 41.55 (CH.sub.2), 40.52 (CH.sub.2), 35.85 (CH.sub.2), 28.80 (CH.sub.2), 23.13 (CH.sub.2), 14.20 (CH.sub.2), −0.51 (CH.sub.3), −0.75 (CH.sub.3). .sup.29Si NMR (79 MHz, DMSO-d6) δ 11.24 (monomer), 7.99 (dimer). LC/MS (ESI.sup.+): t.sub.R=1.33 min, 623 ([M+H].sup.+, 60%), 390 ([M+H—NH.sub.2(CH.sub.2).sub.3NH—CONH(CH.sub.2).sub.3Si(CH.sub.3).sub.2OH].sup.+, 15), 312 ([M+2H].sup.2+, 60), 303 ([M+2H—H.sub.2O].sup.2+, 100. HRMS: 623.1996. C.sub.30H.sub.34N.sub.4O.sub.7SSi implies [M+H].sup.+, 623.19%.

Example 7: Preparation of Hydrogels Comprising a Molecule of Formula (I) and a Molecule of Formula (II)

[0225] A molecule of formula (I), for example the bi-silylated PEG prepared in example 1 or the collagen-derived bi-silylated peptide synthesized in example 2, is dissolved in pH 7.4 phosphate buffer (DPBS Dulbecco's phosphate buffered saline), preferably at a concentration of 10% by mass, in the presence of sodium fluoride (3 mg of NaF per mL of DPBS). A molecule of formula (II), for example the silylated peptide containing sequence Arg-Gly-Asp synthesized in example 4 or the antibacterial silylated peptide prepared in example 5 or the silylated fluorescein described in example 6, is added to the solution of the molecule of formula (I) at a concentration ranging between 1% and 15 mol. % relative to the molecule of formula (I). The non-viscous solution is incubated at 37° C.; a get then forms. The gelation time depends on the nature and the concentration of the selected molecules.

Example 8: Examples of Applications of a Hydrogel According to the Invention

[0226] Optimization of Synthesis

[0227] The bifunctional unit (EtO).sub.3—Si—(CH.sub.2).sub.3—NHCO-(PEG2000)-OCONH—(CH.sub.2).sub.3—Si—(OEt).sub.3 (i.e., “bi-silylated hybrid PEG block”) was synthesized by reacting polyethylene glycol (MW=2000 Da) with 3-isocyanatopropyltriethoxysilane. Next, the bi-silylated hybrid PEG block was engaged in the sol-get process, consisting of hydrolysis of ethoxysilyls to silanols and the condensation thereof to form siloxane bonds. This process was carried out at 37° C., at pH 7.2-7.4 in phosphate buffer (DPBS). Sodium fluoride (NaF) was used as nucleophilic catalyst to accelerate the condensation reactions. Various concentrations of bi-silylated hybrid PEG and of sodium fluoride were tested, thus showing their influence on gelation time (table 1):

TABLE-US-00001 TABLE 1 Gelation time of solutions of bi-silylated hybrid PEG in DPBS at 37° C. and viscoelastic moduli of the hydrogels obtained Composition of the gel Bi-silylated hybrid Gelation PEG block NaF time G′ G″ (% by mass) (% by mass) (min) (Pa) (Pa) 20 5.0 10 n.d. n.d. 20 2.5 15 77750 204 10 5.0 20 n.d. n.d. 10 2.5 35 18960  49 10 0.3 120 9947   61 5 5.0 50 n.d. n.d. 5 2.5 220 5413   39

[0228] The effect of the PEG/water ratio and of the NaF concentration on the mechanical properties of the hydrogels was also studied. The viscoelastic response of the hydrogels was measured in oscillation mode using an AR 2000 rheometer (TA Instruments, Inc.) with a parallel geometry of 20 mm diameter (normal force=2 N). Changes in the storage (G′) and loss (G″) moduli were measured as a function of oscillation frequency within the linear viscoelastic range (0.1% deformation, from 0.01 Hz to 10 Hz) for gels of different compositions. The moduli values for a 10 Hz frequency are presented in table 1. All of the samples exhibit the properties of a solid with G′ greater than G″. The storage moduli were used as measurement of the elasticity of the hydrogels. They remained stable for one week. Extending from 5000 to 80000 Pa depending on the composition of the gel, they encompass a wide range of stiffness. Very few variations were observed on the loss moduli for all samples. Thus, according to the application concerned, the stiffness of the gets can be adjusted by varying the bi-silylated hybrid PEG and/or NaF concentrations.

[0229] Cytotoxicity Tests of the Bare Hydrogel

[0230] Cytotoxicity tests were carried out on two of the hydrogels presented above each containing 10% bi-silylated hybrid PEG by mass relative to the mass of solvent and 0.3% or 2.5% NaF by mass, respectively.

[0231] Line L929 murine fibroblasts were seeded in tissue culture-treated polystyrene wells. After 24 h of proliferation, these cells were incubated with the hydrogels for a further 24 h. Cytotoxicity was then measured using a test showing the release of lactate dehydrogenase by the cells. As expected, the hydrogels containing the highest NaF concentration proved toxic. However, more than 80% cell viability was observed for an NaF concentration of 3 mg/mL, which means that the latter hydrogel is not toxic to the cells (FIG. 1).

[0232] These results were confirmed by microscopic observations which showed healthy spindle-shaped cells.

[0233] Verification of the Functionalization of the Hydrogel

[0234] In order to show the covalent incorporation of a (bio)molecule into the gel and the absence of release over time of the grafted molecules, fluorescein derivatives were selected to be chemically linked to the hydrogels in accordance with the present invention (see FIG. 2 for the exact formulae). Thus, if there were to be a release of the grafted molecules, the use of fluorescein would make it possible to easily detect it.

[0235] Fluorescein isothiocyanate (FITC) was used to prepare two types of fluorescein derivatives giving covalent bonds (triethoxysilane and dimethylhydroxysilane), as well as a non-silylated molecule used as control. Each fluorescein derivative was dissolved at a concentration of 5.2 mM in a solution of bi-silylated hybrid PEG, itself at a concentration of 10% by mass relative to the mass of DPBS used as solvent. Sodium fluoride was added, and the solutions were homogenized. Fluorescent hybrid hydrogels were obtained at 37° C. in 30 minutes. The various hydrogels were placed in phosphate buffer (10 mL) and fluorescein release was monitored by HPLC. In the case of non-covalent enclosure, complete release of fluorescein was observed within 72 h. As expected, fluorescein release is limited (relative to the control) in the case of the covalent derivatives, and reaches a plateau after 72 hours. This indicates the stability of the hybrid covalent bonds. A maximum release of 9% and 20% (relative to the total amount of fluorescein introduced) was observed for the hydrogels obtained with “triethoxysilylfluorescein” and “dimethythydroxyfluorescein”, respectively (FIG. 2). This release may be attributed to the hybrid molecules trapped non-covalently, which could not have reacted during the gelation process. This is consistent with the fact that the triethoxysilyl derivative should be more reactive than dimethylhydroxysilyl during the condensation reaction.

[0236] Cell Adhesion Test

[0237] A peptide containing the RGD sequence was selected to promote cell adherence. It is sequence GRGDSP (SEQ ID 7).

[0238] The deprotected peptide H-GRGDSP-OH (Seq ID 7) was first prepared by peptide synthesis on a solid support using an Fmoc/tBu strategy and functionalized with a triethoxysilyl group using 3-isocyanatopropyttriethoxysilane (ICPTES). A solution of bi-silylated hybrid PEG at 10% by mass relative to the mass of DPBS containing 0.3% NaF by mass was prepared and the silylated GRGDSP hybrid peptide was added to this solution. The relative concentration of silylated GRGDSP in the mixture before reaction was set at 7.5% (first solution) and 15% (second solution) in moles relative to the number of moles of bi-silylated hybrid PEG. These two solutions were placed at 37° C. overnight, to provide two “silylated-PEG/RGD” hydrogels.

[0239] L929 fibroblasts were seeded on the surface of the silylated-PEG/RGD hydrogels and on a non-functionalized hybrid PEG hydrogel (“bare hydrogel”). Adherent cells after 30 min, 1 h and 2 h of incubation were detected and assayed using the PrestoBlue Cell Viability Reagent® (FIG. 3). No cell adhesion was observed on the bare hydrogel. On the other hand, cell adhesion was very effective in the case of the hydrogel containing 15% molar silylated RGD (solution 2). Indeed, adhesion on the latter after 30 min of incubation is better than on the tissue culture-treated polystyrene (TC-PS), and this for adhesion surfaces of identical size.

[0240] Antibacterial Tests

[0241] Likewise, hydrogels with antibacterial properties were prepared by using the peptide sequence H-Ahx-Arg-Arg-NH.sub.2 suitably silylated on the N-terminal side. To that end, the peptide H-Ahx-Arg(Pbf)-Arg(Pbf)-NH— on Rink amide resin was functionalized at the N-terminal end with a dimethylhydroxysilyl group before cleavage of the resin and deprotection of the side chains. The resulting hybrid peptide was added to solutions of bi-silylated hybrid PEG according to the protocol described above for the “silylated PEG-RGD” hydrogel. The antibacterial activity of the hydrogels was evaluated against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The hydrogels were surface-inoculated with bacteria and covered with trypticase soy agar. After 24 h of incubation at 37° C., the bacterial colonies were counted (FIG. 4). The “bare” PEG hydrogels seem to inhibit the growth of P. aeruginosa even in the absence of peptide. For E. coli and S. aureus, the antibacterial effect was provided by the grafted peptide. Indeed, 15 mol. % antibacterial peptide induced complete inhibition of S. aureus growth and reduced E. coli growth by 80%.

[0242] Adhesion and Cell Proliferation Test

[0243] A hydrogel as obtained in Example 3 above, containing a bi-silylated hybrid peptide illustrated below, was prepared:

##STR00005##

[0244] It was able to be shown that this hydrogel has an alveolar internal structure which may be favorable to cell proliferation. Cryo-scanning electron microscopy (SEM) analyses show a multimicrometric alveolar system.

[0245] This is indeed quite visible in FIG. 5, which shows a Cryo-SEM view of the hydrogel containing the bi-silylated peptide prepared.

[0246] Cell Adhesion

[0247] It was also shown that the hydrogel obtained allows adhesion of murine mesenchymal stem cells (mMSC) more effectively than the tissue culture-treated polystyrene and after 4 h as effectively as a commercial collagen foam.

[0248] 50 μL of a cell suspension at 60,000 cells per mL was deposited on various materials, including the hydrogel according to the invention as described in this section. Cell culture-treated polystyrene (TC-PS) and a commercial foam of purified and cross-linked bovine type I collagen were used as controls.

[0249] Various adhesion times at 37° C. were studied before removing the medium, rinsing with DPBS then counting the cells by means of a CellTiter-Glo viability test. The results are reported in FIG. 6.

[0250] FIG. 6 shows the adhesion of mMSC on the hydrogel according to the invention, on collagen foams and on TC-PS.

[0251] Cell Proliferation

[0252] The hydrogel is as good a support as the collagen foams for cell proliferation.

[0253] 1000 mMSC were deposited on various materials, including the hydrogel according to the invention. Cell culture-treated polystyrene (TC-PS) and a commercial foam of purified and cross-linked bovine type I collagen were used as controls.

[0254] Each day for 3 days, the culture medium of a sample series is replaced, and the cells are counted by means of a CellTiter-Glo viability test. The results are reported in FIG. 7.

[0255] FIG. 7 indeed shows the proliferation of murine mesenchymal stem cells (mMSC) on the hydrogel according to the invention, on collagen foam and on TC-PS.

[0256] Cells Survival

[0257] The hydrogel allows satisfactory survival of enclosed cells for at least 25 hours.

[0258] A major advantage of the process described in the present patent is the possibility of adding cells to the still-liquid mixture of silylated precursors. Thus, the gel forms without addition of additional chemical reagent, while enclosing the cells. Murine mesenchymal stem cells were thus encapsulated for 25 hours with excellent viability, comparable to the positive control of cells cultured in 2D on a TC-PS surface.

[0259] The encapsulation protocol is as follows. A solution of hybrid hydrogel is prepared by solubilization of 30 mg of the hybrid peptide of example 3, the structure of which is indicated above, in 250 μL of DMEM culture medium containing 4.5 g/L glucose and 0.12 mg/mL NaF. The solution is incubated at 37° C. for 17 h 15 min. At that time, the solution is still liquid, but its viscosity has increased. 50 μL of a suspension of mMSC at 500,000 cells per mL in DMEM medium is added. The concentration of bi-silylated hybrid peptide is then 10% by mass. The hybrid solution is homogenized and 30 μL of this solution is deposited in the wells of a 96-well cell culture plate. The gel gradually forms at 37° C. 25 hours later, a solution of Calcein-AM and Ethidium homodimer III in DPBS is added to the gels and the latter are analyzed by confocal microscopy.

[0260] It is noted that most of the cells enclosed within the hydrogel according to the invention were stained with calcein and are thus alive. The viability of the cells enclosed within the gel is comparable to the viability of the cells deposited on TC-PS.

CONCLUSION

[0261] The ease of synthesis and of use of the hydrogels according to the present invention was shown. These hydrogels were usefully employed with molecules of diverse chemical structures, proving the versatility of the process according to the present invention. Hydrogels exhibiting satisfactory rheological, biological and/or physicochemical properties were thus able to be prepared with very little variation (indeed no variation) of the operating conditions, apart from the nature of the grafted molecules.