INJECTABLE HYBRID ALGINATE HYDROGELS AND USES THEREOF

Abstract

The invention relates to a hybrid hydrogel, in particular degradable or non-degradable, comprising a first hydrogel polymer of formula (I) in association with an alginate hydrogel polymer, and optionally organosilica particles in particular degradable or non-degradable nanoparticles, or porous silicon particles; pharmaceutical, veterinary and/or cosmetic compositions thereof; and uses thereof as a medicament. The invention notably relates to the use of such hybrid hydrogel in the treatment of fistulas and physiological leaks/leakages, notably in the gastrointestinal tract. The present invention finds applications in the therapeutic and diagnostic medical technical fields and also in cosmetic and veterinary technical fields.

Claims

1. A hybrid hydrogel comprising: A) A first hydrogel polymer comprising monomers of formula (I): ##STR00050## wherein n is an integer representing the number of monomers (I) in the hydrogel polymer; for each occurrence of the bracketed structure n, Y independently represents: a molecular crosslinker for connecting at least a monomer of formula (I) in the framework to at least another monomer of formula (I) in another framework through a linker having the following structure:
*-R.sup.1-L.sub.1-R.sup.2-*; wherein: each occurrence of *-R.sup.1-L.sub.1-R.sup.2-* independently represents a responsively cleavable moiety or a non-cleavable moiety; each occurrence of * denotes a point of attachment of the linker to a monomer of formula (I) in the hydrogel's framework; L.sub.1 represents a responsively cleavable covalent bond, a moiety containing a responsively cleavable covalent bond and/or a stable covalent bond; R.sup.1 and R.sup.2 independently represent an optionally substituted C1-20 alkylenyl moiety, an optionally substituted C1-20heteroalkylenyl moiety, an optionally substituted ethenylenyl moiety, CC or an optionally substituted phenyl moiety, wherein the C1-20 alkylenyl, C1-20 heteroalkylenyl or ethenylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6 alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO.sub.2, CN, isocyano, ORp, N(Rp)2 wherein each occurrence of Rp independently represents H or C1-6alkyl; wherein *-R.sup.1-L.sub.1-R.sup.2-* may independently comprise sugar derivatives such as mannose, hyaluronic acid derivatives, collagene, aminoacids or peptides; or a group of formula
*-R.sub.7(R.sub.8)-* wherein the * symbols denote the points of attachment of Y within the monomer backbone of formula (I); R.sup.7 represents N, R.sup.8 represents an optionally substituted C1-20 alkyl, C1-20alkenyl or C1-20alkynyl moiety, a C1-20 alkyl optionally substituted with carboxyl moiety, an optionally substituted C1-20heteroalkyl moiety, an optionally substituted C1-20alkylphenyl moiety or an optionally substituted phenyl moiety, wherein each of the foregoing C1-20 alkyl, C1-20alkenyl, C1-20alkynyl, C1-20heteroalkyl or C1-20alkylphenyl moieties may bear one or more substituents selected from halogen, OR, CO.sub.2R or N(Rp)2; where R may represent H or C1-6alkyl and each occurrence of Rp may independently represent H or C1-6alkyl; and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO.sub.2, CN, isocyano, ORp, N(Rp)2 wherein each occurrence of Rp independently represents H, C1-6alkyl or C1-6 alkoxy; wherein R.sup.8 may be optionally crosslinked to another monomer of formula (I) in another hydrogel polymer chain; or a hyaluronic acid, alginic acid, peptide, cellulose, amino acid, sugar such as glucose, lactose or mannose derivatives, or oligonucleotide moiety; for each occurrence of the bracketed structure n, R.sub.10 independently represents an optionally substituted C1-20 alkylenyl moiety, wherein the C1-20 alkylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl; for each occurrence of the bracketed structure n, R.sub.11 and R.sub.12 independently represent H, an optionally substituted C1-20 alkyl, C1-20alkenyl or C1-20alkynyl moiety, an optionally substituted C1-20heteroalkyl moiety, or an optionally substituted phenyl moiety, wherein each of the foregoing C1-20 alkyl, C1-20alkenyl, C1-20alkynyl or C1-20heteroalkyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO.sub.2, CN, isocyano, ORp, N(Rp)2 wherein each occurrence of Rp independently represents H or C1-6alkyl; for each occurrence of the bracketed structure n, X independently represents an optionally substituted C1-20 alkylenyl moiety, wherein the C1-20 alkylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl; and B) at least a polysaccharide-based hydrogel, preferably an alginate-based hydrogel, preferably a hydrogel based on an alginate polymer having formula (II): ##STR00051## wherein each occurrence of Z independently represents a counterion such as Ca, Mg, Na, K, Li, Rb and m, 1, p are independently integers.

2. Hybrid hydrogel according to claim 1, wherein at least in a subset of bracketed structures n: L.sub.1 represents independently a responsively cleavable covalent bond selected from: ##STR00052## a light-induced breakable group or a photo-responsive group; or *-R.sup.1-L.sub.2-R.sup.2-* represents: i) a pH-cleavable linker comprising to imine groups conjugated with an aromatic group such as phenyl, preferably a linker comprising a para di-imino phenyl group; ii) a pH-cleavable linker of formula: ##STR00053## wherein each occurrence of q independently represents an integer, for example 1-6; and D represents independently for each occurrence a C1-C3 alkylenyl moiety, or N(Rz) wherein Rz represents H or C1-6alkyl; iii) a light-induced cleavable linker having formula: ##STR00054## wherein q1 and q2 independently represent an integer from 1 to 6, preferably from 1 to 3. For example, q1 and q2 may both represent an integer from 1 to 6, preferably from 1 to 3, more preferably q1=q2=3; or iv) a responsively cleavable moiety selected from: ##STR00055## v) a moiety comprising a sugar derivative such as mannose, a hyaluronic acid derivative, collagene, an aminoacid or a peptide moiety.

3. Hybrid hydrogel according to claim 1, wherein in the linker having the structure *-R.sup.1-L.sub.1-R.sup.2-*, R.sup.1 and R.sup.2 are identical, and each represent CH.sub.2, (CH.sub.2).sub.2, (CH.sub.2).sub.3, (CH.sub.2).sub.4, or phenyl.

4. Hybrid hydrogel according to any one of claims 1 to 3 wherein in the group of formula
*-R.sub.7(R.sub.8)-*, R.sub.7 is N and R.sup.8 represents a C1-C6 alkyl substituted with a carboxyl moiety, a C1-C6 alkyl substituted with one or more hydroxyl groups, C1-C6 alkoxy, C1-C6 alkyl substituted with N(Rp)2 wherein each occurrence of Rp independently represents a C1-6alkyl.

5. Hybrid hydrogel according to any one of claims 1 to 4 wherein in the group of formula *-R.sub.7(R.sub.8)-*, R.sup.7 may be may be N and and R.sup.8 may be independently selected from the group comprising: ##STR00056##

6. Hybrid hydrogel according to any one of claims 1 to 5 wherein at least a subset of occurrences of Y in the first hydrogel polymer represents *-N(R.sup.8)-* wherein R.sup.8 represents a C1-20alkyl or C1-20heteroalkyl moiety, preferably C1-6alkyl or C1-6heteroalkyl, most preferably C1-6alkyl, bearing: (i) an organosilica nanoparticle; or (ii) an organosilica nanocapsule having a core/shell structure, and a molecule of interest or bioactive macromolecule or bioactive macromolecule cluster encapsulated within said nanocapsule, wherein the bioactive macromolecule(s) or macromolecule cluster(s) within the nanocapsule is/are preferably in an active conformation; wherein the organosilica matrix of the nanoparticle or nanocapsule may be disintegrable and may contain responsively cleavable bridges #-R.sup.3-L.sub.2-R.sup.4-# between Si atoms within the organosilica framework; preferably the organosilica matrix of the disintegrable organosilica nanoparticle or core/shell nanocapsule may be porous, most preferably mesoporous; wherein: each occurrence of # denotes a point of attachment to a Si atom in the organosilica material's framework; L.sub.2 represents a responsively cleavable covalent bond; and R.sup.3 and R.sup.4 independently represent an optionally substituted C1-20 alkylenyl moiety, an optionally substituted C1-20 heteroalkylenyl moiety, an optionally substituted ethenylenyl moiety, CC or an optionally substituted phenyl moiety, wherein the C1-20alkylenyl, C1-20 heteroalkylenyl or ethenylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO.sub.2, CN, isocyano, OR.sup.p, N(R.sup.p).sub.2 wherein each occurrence of R.sup.p independently represents H or C1-6alkyl; and wherein the nanoparticle or nanocapsule outer surface comprises one or more groups of formula
#-R.sup.5R.sup.6 wherein each occurrence of # denotes a point of attachment to a Si atom at the outer surface of the hybrid organosilica material's framework; each occurrence of R.sup.5 independently represents an optionally substituted C1-20alkylenyl moiety, an optionally substituted C1-20heteroalkylenyl moiety, an optionally substituted ethenylenyl moiety, CC or an optionally substituted phenyl moiety, wherein the C1-20alkylenyl, C1-20heteroalkylenyl or ethenylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO2, CN, isocyano, ORp, N(Rp)2 wherein each occurrence of Rp independently represents H or C1-6alkyl; and each occurrence of R.sup.6 independently represents OR, SR or N(Rf).sub.2; preferably N(Rf).sub.2; wherein each occurrence of R and Rf independently represents H or C1-6alkyl.

7. Hybrid hydrogel according to any one of claims 1 to 6 wherein R.sub.10 represents CH or CHCH.sub.2; and R.sub.11 and R.sub.12 independently represent H or C1-C6 alkyl.

8. Hybrid hydrogel according to 6 wherein at least a subset of nanocapsules bound to the first hydrogel polymer are further crosslinked via one or more #-R.sup.5R.sup.6 groups to another first hydrogel polymer of formula I.

9. Hybrid hydrogel according to claim 6 or 8 wherein the nanoencapsulated molecule is selected from proteins, enzymes, antibodies, peptides, DNA, RNA, PNA, gene fragments and small molecules with or without pharmaceutical activity; preferably proteins, enzymes, antibodies, peptides, DNA, RNA, PNA and gene fragments.

10. Hybrid hydrogel according to any one of claims 6 to 9, wherein L.sub.2 represents independently a responsively cleavable covalent bond selected from: ##STR00057## a light breakable group or a photo-responsive group, or #-R.sup.3-L.sub.2-R.sup.4-# represents: i) a pH-cleavable linker comprising to imine groups conjugated with an aromatic group such as phenyl, preferably a linker comprising a para di-imino phenyl group; ii) a pH-cleavable linker of formula: ##STR00058## wherein each occurrence of q independently represents an integer, for example 1-6; and D represents independently for each occurrence a C1-C3 alkylenyl moiety, or N(Rz) wherein Rz represents H or C1-6alkyl; iii) a light-induced cleavable linker having formula: ##STR00059## wherein q1 and q2 independently represent an integer from 1 to 6, preferably from 1 to 3. For example, q1 and q2 may both represent an integer from 1 to 6, preferably from 1 to 3, more preferably q1=q2=3; or iv) a responsively cleavable moiety selected from: ##STR00060##

11. Hybrid hydrogel of any one of claims 1 to 10, wherein the organosilica particles bound to the hydrogel polymer has a diameter between 25 nanometers and 500 nanometers.

12. Hybrid hydrogel of any one of claims 1 to 11, wherein the hydrogel is non covalently mixed with (i) an organosilica nanoparticle; and/or (ii) an organosilica nanocapsule having a core/shell structure, and a molecule of interest or bioactive macromolecule or bioactive macromolecule cluster encapsulated within said nanocapsule, wherein the bioactive macromolecule(s) or macromolecule cluster(s) within the nanocapsule is/are preferably in an active conformation; wherein the organosilica nanoparticle or nanocapsule is as defined in claim 6.

13. A pharmaceutical or cosmetic composition comprising a hydrogel of any one of claims 1 to 12, and a pharmaceutically or cosmetically acceptable carrier.

14. A method for preparing a hybrid hydrogel of any one of claims 1 to 12, comprising steps of: a) dissolving in water or alcoholic solutions: a monomer precursor of formula (IV) ##STR00061## at least one molecular crosslinker precursor having the structure A-R.sup.1-L.sub.1-R.sup.2-A, optionally organosilica nanoparticles optionally bearing amino-containing tether groups at the outer surface; or organosilica core/shell nanocapsules optionally bearing amino-containing tether groups at the outer surface and encapsulating a bioactive macromolecule or bioactive macromolecule cluster, and/or another molecule of interest that may or may not have biological activity and/or pharmaceutical or cosmetic activity; wherein the bioactive macromolecule or bioactive macromolecule cluster encapsulated within the nanocapsule is preferably in active conformation; and Optionally, a selected precursor of formula BR.sup.8; b) adding a solution of alginate, for example an aqueous solution of sodium alginate, which may be added concomitantly with step a) or separately from step a); c) Stirring the solution obtained in step b), at any appropriate temperature, thereby allowing the polymerization carried out to form the hydrogel, d) Optionally adding a suitable organic solvent, thereby precipitating the hydrogel: wherein: each occurrence of A independently represents a nucleophilic moiety, preferably N(Rf).sub.2 wherein each occurrence of Rf may represent H or C1-6alkyl; B independently represents a nucleophilic moiety, preferably N(Rf).sub.2 wherein each occurrence of Rf may represent H or C1-6alkyl; L.sub.1 independently represents a responsively cleavable covalent bond, a moiety containing a responsively cleavable covalent bond and/or a stable covalent bond; and R.sup.1 and R.sup.2 independently represent an optionally substituted C1-20 alkylenyl moiety, an optionally substituted C1-20heteroalkylenyl moiety, an optionally substituted ethenylenyl moiety, CC or an optionally substituted phenyl moiety, wherein the C1-20 alkylenyl, C1-20 heteroalkylenyl or ethenylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6 alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO.sub.2, CN, isocyano, ORp, N(Rp)2 wherein each occurrence of Rp independently represents H or C1-6alkyl; wherein *-R.sup.1-L.sub.1-R.sup.2-* may independently comprise a sugar derivative such as mannose, a hyaluronic acid derivative, collagene, an aminoacid or a peptide moiety; R.sub.10 independently represents an optionally substituted C1-20 alkylenyl moiety, wherein the C1-20 alkylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl; R.sub.11 and R.sub.12 independently represent an optionally substituted C1-20 alkyl, C1-20alkenyl or C1-20alkynyl moiety, an optionally substituted C1-20heteroalkyl moiety, or an optionally substituted phenyl moiety, wherein each of the foregoing C1-20 alkyl, C1-20alkenyl, C1-20alkynyl or C1-20heteroalkyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl, and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO.sub.2, CN, isocyano, ORp, N(Rp)2 wherein each occurrence of Rp independently represents H or C1-6alkyl; X independently represents an optionally substituted C1-20 alkylenyl moiety, wherein the C1-20 alkylenyl moiety may bear one or more substituents selected from halogen or OR where R may represent H or C1-6alkyl; and R.sup.8 may independently represent: an optionally substituted C1-20 alkyl moiety, a C1-20 alkyl optionally substituted with carboxyl moiety, an optionally substituted C1-20heteroalkyl moiety, an optionally substituted C1-20alkylphenyl moiety or an optionally substituted phenyl moiety, wherein each of the foregoing C1-20 alkyl, C1-20heteroalkyl or C1-20alkylphenyl moieties may bear one or more substituents selected from halogen, OR, CO.sub.2R or N(Rp)2 where R may represent H or C1-6alkyl, and each occurrence of Rp may independently represent H or C1-6alkyl; and the phenyl moiety may bear one or more substituents independently selected from halogen, C1-6alkyl, NO2, CN, isocyano, ORp, N(Rp)2 wherein each occurrence of Rp independently represents H, C1-6alkyl or C1-6 alkoxy; the residue of the corresponding amino acid H.sub.2NR.sup.8; a C1-C6 alkyl substituted with a carboxyl moiety, a C1-C6 alkyl substituted with one or more hydroxyl groups, C1-C6 alkoxy, C1-C6 alkyl substituted with N(Rp)2 wherein each occurrence of Rp independently represents a C1-6alkyl; a C1-C6 alkyl substituted with N(Rp)2 wherein each occurrence of Rp independently represents a C1-6alkyl; a C2 alkyl substituted with-N(Rp)2 wherein each occurrence of Rp independently represents a C1 alkyl; a C1-20alkylphenyl moiety optionally substituted with one or more OR wherein R may represent H or C1-6alkyl; a group of any one of the following formulae: ##STR00062## a hyaluronic acid, alginic acid, peptide, cellulose, amino acid, sugar (for example glucose, lactose or mannose derivatives) or oligonucleotide moiety; or a C1-20alkyl or C1-20heteroalkyl moiety, preferably C1-6alkyl or C1-6heteroalkyl, most preferably C1-6alkyl, bearing an organosilica particle, preferably organosilica nanoparticles or core-shell nanocapsules, preferably the organosilica matrix may be porous, most preferably mesoporous, and may contain responsively cleavable bonds L.sub.2 or responsively cleavable linkers #-R.sup.3-L.sub.2-R.sup.4-# within the organosilica framework as defined in claim 6.

15. The method of claim 14, wherein the monomer precursor is of formula (IVa) ##STR00063##

16. The method of claim 14 or 15, wherein the linker L.sub.1 and *-R.sup.1-L.sub.1-R.sub.2-* are as defined in claim 2.

17. The method of any one of claims 14 to 16 wherein the molecular crosslinker precursor A-R.sup.1-L.sub.1-R.sup.2-A is of formula ##STR00064##

18. The method of any one of claims 14 to 17 wherein the selected precursor of formula BR.sup.8 is of formula ##STR00065##

19. A hybrid hydrogel covalently non-covalently mixed with, or covalently conjugated to, organosilica nanoparticles or organosilica nanocapsules having a core/shell structure, obtainable by a method of any one of claims 14 to 18; wherein the organosilica matrix of the organosilica nanoparticles or core/shell nanocapsules may preferably be porous, most preferably mesoporous, and wherein the organosilica matrix of the nanoparticles or nanocapsules may be disintegrable and may contain responsively cleavable bridges #-R.sup.3-L.sub.2-R.sup.4-# between Si atoms within the organosilica framework as defined in claim 6.

20. A hybrid hydrogel of any one of claims 1 to 12 or a pharmaceutical composition of claim 13, for use as medicament.

21. Hybrid hydrogel according to claim 20 for use in sealing a wound, for enhancing tissue regeneration, as fillers for example for submucosal fluid cushion for surgery, tissue reconstitution in a subject-in-need thereof, for the treatment of diabetes, for the treatment of spinal cord injury.

22. Hybrid hydrogel according to claim 20 for use as a medicament for the treatment of cancer, preferably tumor, more preferably for the resection of solid tumors.

23. A method for sealing acute and/or chronic wounds and/or perforation in a subject-in-need thereof, the method comprising administering to the subject a therapeutically effective amount of a hybrid hydrogel of any one of claims 1 to 12 or a pharmaceutical composition of claim 13, thereby sealing the wound and/or perforation.

24. A method for treating a disease, preferably cancer tumor, in a subject-in-need thereof, the method comprising administering to the subject a therapeutically effective amount of a hybrid hydrogel of any one of claims 1 to 12 or a pharmaceutical composition of claim 13, thereby treating the disease in the subject.

25. Use of a hybrid hydrogel of any one of claims 1 to 12, in a cosmetic composition.

26. Use of a hybrid hydrogel of any one of claims 1 to 12 or a cosmetic composition of claim 25, for delivering a cosmetically bioactive macromolecule to the skin.

27. Use according to claim 25 or 26, wherein the cosmetically bioactive macromolecule is collagen, keratin, elastin, calcitonin, hyaluronic acid, aminoacids, retinol, antioxidants, vitamins or silk proteins.

28. A method for systemically delivering a drug, or a bioactive macromolecule in a biologically active form, to a subject in need thereof, the method comprising, administering to the subject a therapeutically effective amount of a hybrid hydrogel of any one of claims 1 to 12 or a pharmaceutical composition of claim 13.

29. The method of claim 28, wherein said bioactive macromolecule is selected from proteins, oligonucleotides, antibodies, peptides, PNA, DNA, RNA, gene fragments, a hormone, a growth factor, a protease, an extra-cellular matrix protein, an enzyme, an infectious viral protein, an antisense oligonucleotide, a dsRNA, a ribozyme, a DNAzyme, antibiotics, antinflammatory, steroids, chemiotherapeutics.

30. A unit dosage form for local delivery of a molecule to a tissue of a subject, the unit dosage form comprising, a therapeutically effective amount of a hybrid hydrogel of any one of claims 1 to 12 or a pharmaceutical composition of claim 13, wherein said macromolecule is selected from proteins, oligonucleotides, antibodies, peptides, PNA, DNA, RNA, gene fragments, a hormone, a growth factor, a protease, an extra-cellular matrix protein, an enzyme, an infectious viral protein, an antisense oligonucleotide, a dsRNA, a ribozyme and a DNAzyme.

31. A delivery system for enhancing wound healing, tissue regeneration and/or tissue regeneration in vivo, said system comprising a hybrid hydrogel of any one of claims 1 to 12 or a pharmaceutical composition of claim 13.

32. A hybrid hydrogel according to any one of claims 1 to 12, for use in the treatment of fistula.

33. Hybrid hydrogel for use according to claim 32, wherein the hybrid hydrogel is non-covalently mixed with, or covalently conjugated to, organosilica nanoparticles or organosilica nanocapsules having a core/shell structure; wherein the organosilica matrix of the organosilica nanoparticles or core/shell nanocapsules may preferably be porous, most preferably mesoporous, and wherein the organosilica matrix of the nanoparticles or nanocapsules may be disintegrable and may contain responsively cleavable bridges #-R.sup.3-L.sub.2-R.sup.4-# between Si atoms within the organosilica framework as defined in claim 6.

34. Hybrid hydrogel for use according to claims 32 or 33, in the treatment of acute or chronic fistula.

35. A method for treating fistula in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a hybrid hydrogel as defined any one of claims 1 to 12, or a composition according to claim 13 comprising a pharmaceutically acceptable carrier.

36. Method according to claim 35 for the treatment of acute or chronic fistula.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0340] FIG. 1. Scheme of the synthesis and functionalization of BNCs containing disulfide moiety in the framework and loaded with Cyt-C inside the silica capsule (a); SEM image of the monodispersed functionalized BNCs, in the insert SEM picture of a naked nanoparticle (b); scheme of degradation after GSH exposure and release of Cyt-C (c).

[0341] FIG. 2. Synthesis of a PAAm hydrogel covalently conjugated to redox-responsive degradable organosilica nanocapsules prepared in Example 3.1 (a); scheme of the network (b); FTIR trace of dPAA (c); SEM showing the porosity of the nanocomposite (d).

[0342] FIG. 3. Mechanism of network degradation of a PAAm polymer used as first hydrogel polymer of formula (I) in the hybrid hydrogels of the present invention, upon exposure to GSH, schematic representation and pictures of the hydrogel network before and after degradation. The yellow lines represent the disulfide units; the degradation of the hydrogel

[0343] FIG. 4. Injection of a solution of PAAM and alginate (hybrid hydrogel of the present invention) stained with Methylene Blue via a surgical 23-gauge needle (a); formation of a mucosal elevation (b); gelation occurs in less than 10 minutes, achieving a solid and elastic hydrogel, adhered to the tissue.

[0344] FIG. 5. .sup.1H NMR spectrum of DCNS before and after light irradiation (cf. Example 1.3).

[0345] FIG. 6. Characterization of model spherical MSPs, illustrated in Example 1.3.

[0346] FIG. 7. Complete characterization of the hybrid light-sensitive spherical MSPs with light-induced cleavable linkers within the organosilica matrix, illustrated in Example 1.3.

[0347] FIG. 8. Schematic representation of the light-induced cleavability experiments and SEM images of the investigated organosilica particles comprising light-induced cleavable linkers within the organosilica matrix.

[0348] FIG. 9. FIG. 9A represents a schematic representation of the application of hybrid hydrogel according to the invention onto the duodenal mucosa, in order to interfere with nutrients adsorption and, particularly, with glucose metabolism, which is particularly active at the level of the foregut. The endoscope is advanced in the duodenum and the hybrid hydrogel is sprayed in order to cover the duodenal mucosa, while moving backwards. FIG. 9B represents endoscopic injection of a hybrid hydrogel according to the invention at the level of the lower esophageal sphincter (LES) to obtain a sphincter augmentation (increased closure strength to prevent reflux episodes).

[0349] FIG. 10 represents a general scheme for the treatment of fistula using a hybrid hydrogel according to the invention.

[0350] FIG. 11 represents CT scans performed in Example 6. FIG. 11A represents comparative CT scans of water, Iomeron 400, 2% sodium alginate solution, hybrid hydrogel prepared in Example 6 (with Iomeron as solvent), and hybrid hydrogel prepared according to Example 2.2 (water as solvent). FIG. 11A represents a CT scan of the fistula after injection of the hybrid hydrogel.

[0351] FIG. 12 represents comparative uniaxial compression test of 2% sodium alginate crosslinked with calcium chloride, 2% alginate/calcium/PAAm and pure PAAm hydrogel.

EQUIVALENTS

[0352] The representative examples that follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art.

[0353] The following examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and the equivalents thereof.

Exemplification

[0354] The present invention and its applications can be understood further by the examples that illustrate some of the embodiments by which the inventive product and medical use may be reduced to practice. It will be appreciated, however, that these examples do not limit the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the present invention as described herein and as hereinafter claimed.

EXAMPLES

Example 1: Synthesis of Organosilica Particles

1.1. Redox-Cleavable Core/Shell Nanocapsules Synthesis

[0355] The nanocapsules used are those disclosed in E. A. Prasetyanto, A. Bertucci, D. Septiadi, R. Corradini, P. Castro-Hartmann, L. De Cola, Angew. Chem. Int. Ed. 2016, 55, 3323. [21]. This platform is composed of a silica shell able to encapsulate functional proteins in their active folding and it is engineered to degrade upon contact with a reducing agent, such as GSH present in the biological environment with a complete release of the loading.

[0356] Cytochrome C (Cyt-C) was chosen as model cargo, since its strong absorption in the visible region allowed us to investigate the release kinetics during the hydrogel degradation.

[0357] The synthesis of the BNCs, in order to prevent denaturation of the loaded active molecule, was performed following the reported reverse nano-emulsion procedure. In particular, the silica precursor, tetraethyl orthosilicate (TEOS) was added to bis[3-(triethoxysilyl)propyl]disulfide in a ratio 7:3 TEOS:bispropyldisulfide derivative, in order to introduce the redox-sensitive moiety. Well-defined and monodispersed spherical nanocapsules with a diameter of around 6010 nm were obtained.

[0358] Then, the obtained pristine BNCs were functionalized with 3-aminopropyltriethoxysilane, to be able to covalently link the BNCs to the polymeric hydrogel network. A scheme of the synthesis and functionalization, as well as the SEM of the pristine and functionalized BNCs and of the degradation via GSH is displayed in FIG. 1.

[0359] The surface functionalization was confirmed by the shift from negative to positive values of the -potential, from 10.5 mV of the pristine nanocapsules to +2.2 mV.

[0360] Then, the functionalized BNCs (1 mg/ml) were used to synthetize the dPAA nanocomposite hydrogel through surface-grafting of the aminated BNCs to the polyamidoamine backbone of the scaffold.

[0361] Briefly, the protocol was as follows:

[0362] Triton X-100 (7.08 mL) and n-hexanol (7.20 mL) were dissolved in Cyclohexane (30 mL). Separately, 1.20 mL of a 5 mg/mL aqueous solution of Cytochrome C from equine heart were mixed with 0.16 L of tetraethyl orthosilicate and 0.24 mL of bis[3 (triethoxysilyl)propyl] disulfide.

[0363] After shaking, this mixture was added to the former organic medium. Eventually, 200 L of 30% ammonia aqueous solution were added and the water-oil emulsion was stirred overnight at room temperature. After that, 80 mL of pure acetone were added to precipitate the NPs and the material was recovered by means of centrifugation, washing five times with water and one with ethanol.

[0364] The procedure can be adapted for the encapsulation of different globular proteins.

1.2. Redox-Cleavable Core/Shell Nanocapsules Functionalization

[0365] 40 mg of breakable nanocapsules prepared in Example 1.1. are suspended in 5 mL of ethanol. 44 L of 3-aminopropyltriethoxysilane (PM=221.37, d=0.946, 0.094 mmol) and 20 L of triethylamine (TEA) are added to the suspension, that is stirred at R.T. for 18 hours.

[0366] The resulting core/shell nanocapsules bearing aminopropyl tether moieties at the outer surface (NPs, also designated NH2-CytC@BNPs) are then washed five times with distilled water and dried.

1.3. Photo-Cleavable Nanoparticles Synthesis

1.3.1 2-nitro-5-(((3-(triethoxysilyl)propyl)carbamoyl)oxy)benzyl (3-(triethoxysilyl)propyl)carbamate, (DCNS)

[0367] This photosensible molecule was synthesized by the reaction of the alcohol groups of the 5-hydroxy-2-nitrophenyl alcohol and Iscocyanopropyltriethoxysilane by the presence of triethylamine as catalyst (see scheme 1)

##STR00044##

[0368] The reaction product could be obtained in a 53% of yield and had been characterized by .sup.1H-NMR and .sup.13C-NMR, FTIR spectroscopy and ESI-mass spectrometry. Furthermore the absorption spectra had been recorded for further light breakability experiments of the linker itself.

1.3.2. Breakability Test on DCNS

[0369] The light-induced breakability of the DCNS compound had been performed by irradiating the compound with light produced by a Hg lamp. To this purpose, the compound was dissolved in DMSO-d.sub.6 in a NMR tube. In this way the photo degradation could be followed by recording .sup.1H-NMR spectra over a certain period of time. Indeed the photogradation reaction could be observed and it is indicated by the signal derived from the aldehyde proton at 10.92 ppm (FIG. 5).

1.3.3. Synthesis of Hybrid MSPs

[0370] Firstly, model spherical MSPs were synthesised. The model particles were synthesized according to a modified Stber synthesis, shown in Scheme 2

##STR00045##

[0371] The model particles obtained were spherical characterized by an average diameter of ca 200 nm (SEM, TEM and DLS analysis in FIG. 6). Furthermore these model particles possess a hexagonal mesostructure with an estimated average pore size of 2.5 nm (see SAXS and pore size distribution in FIG. 6)

[0372] Once the standard synthesis protocol had been established, hybrid silica particles were synthesised by the co-condensation of DCNS into the silica structure. (see scheme 3)

##STR00046##

[0373] The hybrid silica particles obtained by this synthetic approach were spherical and characterized by satisfactorily monodispersity and diameter of ca. 200 nm and 20 wt. % of organic material as determined by TGA. The incorporation of the DCNS linker was proven by XPS analysis. The deconvolution of high resolution scans of the C(1s) and N(1S) indicated the presence of peaks characteristic for the functional groups present in the linker (FIG. 7).

[0374] A pH measurement of the reaction mixture before and after the addition of DCNS linker confirmed the hypothesis that the hydrolysis of the carbamate occurs in these conditions. In fact, the pH value changes from 11 to 7. The hydrolysis product could be extracted from the aqueous mixture and the recorded .sup.1H NMR spectrum showed the presence of a carbamic acid derivative.

[0375] The hybrid light-sensitive MSPs may be further functionalized, as described for core/shell nanocapsules above, for covalent incorporation as crosslinkers into hydrogel networks.

[0376] For example, 40 mg of hybrid light-sensitive MSPs are suspended in 5 mL of ethanol. 44 L of 3-aminopropyltriethoxysilane (PM=221.37, d=0.946, 0.094 mmol) and 20 L of triethylamine (TEA) are added to the suspension, that is stirred at R.T. for 18 hours. The resulting NH.sub.2-functionalized hybrid light-sensitive MSPs (NPs, also designated NH2-MSPs herein) are then washed five times with distilled water and dried.

1.3.4. Light Breakability Experiments of Hybrid MSPs

[0377] In order to evaluate the light-induced breakability of hybrid light breakable silica particles a suspension of these particles in ethanol was irradiated with a Hg lamp (FIG. 7). As a control experiment, particles were kept in the dark. The SEM and STEM analyses showed that only the particles exposed to light break partially apart, whereas the non-irradiated particles showed no change in morphology. The partial breaking of particles could be explained by the fact, that during the particle formation some of the photolabile linker was hydrolized, while the remaining non-hydrolized linker was incorporated to a certain extent in the hybrid particles.

1.3.5. Synthesis of triethoxy(3-(4-nitro-3-((3-(triethoxysilyl)propoxy)methyl)phenoxy)propyl)silane

[0378] ##STR00047##

[0379] The diether compound can be prepared from 5-hydroxy-2-nitrobenzylalchol through allylation and subsequent hydrosilylation reaction, as depicted in Scheme 4. The synthetic steps are described in detail in Scheme 5.

##STR00048##

##STR00049##

1.4. Photo-Cleavable Organosilica Nanoparticles Functionalization

[0380] 40 mg of photo-cleavable organosilica nanoparticles prepared in Example 1.3. are suspended in 5 mL of ethanol.

[0381] 44 L of 3-aminopropyldimethoxysilane (PM=221.37, d=0.946, 0.094 mmol) and 20 L of triethylamine (TEA) are added to the suspension, that is stirred at R.T. for 18 hours.

[0382] The resulting nanoparticles bearing aminopropyl tether moieties at the outer surface are then washed five times with distilled water and dried.

Example 2: Synthesis of Hybrid PAAm-Alginate Hydrogels

2.1. pH-Degradable PAAm Hydrogels

[0383] 1 g of MBA and 250 mg of GABA were weighted in a 50-ml round bottom flask. 0.85 g of diimPEHA were dissolved in 7.5 mL of distilled water and the solution was added to the flask at 45 C. under magnetic stirring until the suspension become clear. The fluid is placed in a glass vial and the temperature is then raised to 60 C.

2.2. pH-Degradable Hybrid Alginate-PAAm Hydrogel

[0384] The pH-degradable PAAm hydrogel prepared in Example 2.1 is mixed with a solution of sodium alginate in water. A solution of calcium chloride or any other suitable calcium salt solution in water is added and the mixture is hand-shaken until it is completely solid.

TABLE-US-00002 Sodium PAAm/ Calcium salt alginate Alginate Conc. M Gelation PAAm hydrogel Conc. % w/v, weight volume time (quantity) mL volume added ratio added (second) 5 mL 1% in H.sub.2O, 1 4.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL 10 mL 1% in H.sub.2O, 1 .sup.9:1 0.5M CaCl.sub.2 15 mL in H.sub.2O, 0.5 mL 5 mL 2% in H.sub.2O, 1 .sup.4:1 0.5M CaCl.sub.2 5 mL in H.sub.2O, 0.5 mL 10 mL 2% in H.sub.2O, 1 8.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL

Example 3: Synthesis of Hybrid PAAm-Alginate Hydrogels Functionalized with Organosilica Particles

3.1. Preparation of PAAm Hydrogel Covalently Conjugated to Redox-Responsive Degradable Organosilica Nanocapsules

[0385] 200 mg of methylenbisacrylamide (MBA), 65 mg of cystamine hydrochloride and 70 L of N,N-dimethylethylendiamine are mixed together with 1 mL of a 1 mg/mL solution of NH.sub.2-functionalized redox-cleavable organosilica core/shell nanocapsules prepared in Example 1.2. After 48 h, the hydrogel is formed.

[0386] The procedure can be modified and other NH.sub.2-functionalized silica nanoparticles can be used, such as responsively cleavable or non-responsively cleavable mesoporous organosilica nanoparticles. The protocol can be reproduced using the amino-functionalized photo-cleavable organosilica nanoparticles prepared in Example 1.4.

[0387] When the hydrogels were needed for in vitro experiments (i.e. GSH degradation and cellular viability and degradation), the obtained solution was transferred to glass vials (500 l per vial) and allowed to react in static conditions at r.t. Glass vials with inner diameter of 8 mm were used as molds. The hydrogels were obtained after 48 hours.

[0388] Once obtained, the disk-shaped hydrogels were freeze-dryed and weighted. Dryed hydrogels were used to study the swelling ratio at different pH and the degradation kinetics with different concentrations of GSH. This step allowed us as well to sterilized the materials for in vitro experiments.

[0389] Sterile and ultrafiltered water was used during hydrogel preparation for in vivo tests; the synthesis was carried out in closed sterile vials and protected from bacteria contamination, the final product was assumed to be free of bacterial contamination.

3.2. Hybrid Alginate-PAAm Hydrogel Covalently Conjugated to Redox-Cleavable Core/Shell Organosilica Nanocapsules (Method 1)

[0390] The PAAm hydrogel covalently conjugated to organosilica nanocapsules, prepared in Example 3.1., is mixed with a solution of sodium alginate in water. A solution of calcium chloride or any other suitable calcium salt solution in water is added and the mixture is hand-shaken until it is completely solid.

TABLE-US-00003 Sodium PAAm/ Calcium salt NP-conjugated alginate Alginate Conc. M, Gelation PAAm hydrogel Conc. % w/v, weight volume time (quantity) mL volume added ratio added (min.) 5 mL 1% in H.sub.2O, 1 4.5:1 0.5M CaCl.sub.2 7 mL in H.sub.2O, 0.5 mL 10 mL 1% in H.sub.2O, 1 .sup.9:1 0.5M CaCl.sub.2 13 mL in H.sub.2O, 0.5 mL 5 mL 2% in H.sub.2O, 1 .sup.4:1 0.5M CaCl.sub.2 5 mL in H.sub.2O, 0.5 mL 10 mL 2% in H.sub.2O, 1 8.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL

3.3. Hybrid Alginate-PAAm Hydrogel Covalently Conjugated to Redox-Cleavable Core/Shell Organosilica Nanocapsules (Method 2)

[0391] Hybrid PAAm-alginate hydrogels covalently conjugated to organosilica nanocapsules are prepared as previously reported (cf. Example 3.1.) using a solution of 1 mg/mL of redox-cleavable core/shell nanocapsules in sodium alginate. As such, 200 mg of methylenbisacrylamide (MBA), 65 mg of cystamine hydrochloride and 70 L of N,N-diethylethylendiamine are mixed together with 1 mL of a 1 mg/mL solution of NH.sub.2-functionalized redox-cleavable organosilica core/shell nanocapsules prepared in Example 1.2. in sodium alginate. When the pre-gel solution become homogeneous, a water solution of calcium chloride is added to trigger gelation.

TABLE-US-00004 Sodium PAAm/ Calcium salt alginate Alginate Conc. M Gelation PAAm hydrogel Conc. % w/v, weight volume time (quantity) mL volume added ratio added (second) 5 mL 1% in H.sub.2O, 1 4.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL 10 mL 1% in H.sub.2O, 1 .sup.9:1 0.5M CaCl.sub.2 15 mL in H.sub.2O, 0.5 mL 5 mL 2% in H.sub.2O, 1 .sup.4:1 0.5M CaCl.sub.2 5 mL in H.sub.2O, 0.5 mL 10 mL 2% in H.sub.2O, 1 8.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL

Example 4: Hydrogels Characterization and Uses in Non Invasive Surgery Procedures

Degradation Kinetic of Stimuli-Responsive Hybrid Hydrogels

[0392] For redox-responsive materials, a 1 mm thick hydrogel cylinders is lyophilized and its dry weight is recorded. The hybrid hydrogel is then placed in a vial and 5 mL of a 10 M solution of reduced GSH are added. The swelling of the hybrid hydrogel is recorded at the appropriate time-points. The experiment is repeated in triplicated and then with a solution of GSH 10 mM and with a solution of PBS as a reference.

[0393] The same procedure can be applied for pH-responsive hybrid hydrogels, using pH=4 citrate buffer for degradation and PBS as a reference.

[0394] Degradation of hybrid hydrogels covalently conjugated to organosilica particles can be examined in the presence of reduced glutathione (GSH), a disulfide reducing agents. Briefly, the lyophilized hybrid hydrogel samples are incubated at 37 C. in 2 mL of a PBS solution with a GSH concentration of 10 M. Hybrid hydrogels without organosilica particles are incubated in PBS alone as a control.

[0395] The degradation kinetics can then be evaluated via swelling ratio (SR) measurements in time.

[0396] SR are measured by a gravimetric method. In brief, lyophilized hybrid hydrogel samples are immersed in PBS at 37 C. Then, the samples are removed from PBS at set time points (after 1 h, 6 h, 12 h, 24 h, 48 h, 72 h, 144 h), blotted free of surface water using filter paper and their swollen weights are measured on an analytical balance. The SR are then calculated as a ratio of weights of swollen hybrid hydrogel (Ws) to dried hybrid hydrogel (W), using the following equation:

[00001]$S.Math.R=\frac{W_s-W_d}{W_d}$

[0397] Degradation time is defined as the time where there were no longer sufficient crosslinks to maintain the 3D network and the material was completely disintegrated. Experimentally, complete degradation can be determined when a limpid solution can be observed, without solid residues.

[0398] In Vitro Cell Culturing

[0399] Cryopreserved human dermal fibroblast, adult (HDFa) are purchased from Thermo Fisher and the culture is initiated as suggested on the protocol. HDFa are grown in Medium 106 supplemented with Low Serum Growth Supplement (LSGS, Thermo Fisher). Cells are kept in 75 cm.sup.2 culture flasks (Corning Inc., NY, USA) at 37 C. with a controlled atmosphere of 5% CO.sub.2 and are grown until reaching 80 to 85% of confluence. Then, they are washed twice with PBS and treated with trypsin/EDTA solution to detach them from the flask surface. Cells are split every 2-3 days; the medium is changed every other day.

[0400] In Vitro Cell Culturing onto Hybrid Hydrogels

[0401] The hybrid hydrogel scaffolds are equilibrated by adding culture media at 37 C. HDFa are detached from the culture flask by trypsination and approximately 2.510.sup.5 cells are seeded onto the hybrid hydrogel scaffolds. Then, the samples are placed in the incubator (37 C., 5% CO.sub.2) for about 30 minutes and fresh media is cautiously added on the top of the hybrid hydrogel to supply cells with nutrients. This is done to allow anchorage of the cells onto the scaffolds.

[0402] Cell Staining and Viability Studies

[0403] Cell viability is assessed using alamarBlue assay. Briefly, the alamarBlue solution is added to the culture medium (1:10 dilution) of unstained cells growing onto hybrid hydrogel scaffolds. After 3 h incubation, 200 L of the media are transferred to a 96-well plate and absorbance signals generated from the dye resazurin (dark blue) being reduced to resorufin (pink) by metabolically active cells are recorded using a VICTOR X5 Multilabel Plate Reader (Perkin Elmer).

[0404] Each sample is tested in three replicates and the results are expressed as percentage of reduced alamarBlue.

[0405] The viability of cells after complete degradation of the hybrid hydrogel was measured by with a TC20 (trade mark) Automated Cell Counter (Bio-Rad).

[0406] Where required (confocal fluorescence microscopy images), HDFa are stained with Vybrant DiD (Life Technologies, Thermo Fisher Scientific, Waltham, Mass., USA), following the reported protocol, prior to seeding them onto the scaffolds.

[0407] Cell-Mediated Degradation of Hydrogel

[0408] The hybrid hydrogels are freeze-dried and weighed (W). Then 2.510.sup.5 HDFa are seeded onto the samples (see above). The cell-laden samples are collected at pre-determined time points and were freeze-dried to obtain their dry weight after degradation (W).

[0409] The cell-mediated degradation of the hybrid hydrogels, D, is calculated using the following equation:

[00002]$D\left(\%\right)=\frac{W_i-W_f}{W_i}100$

[0410] A cellular hydrogels are used as degradation control.

[0411] Evaluation of the Gelation and Formation of SFC Ex Vivo

[0412] Fresh porcine stomachs are used for the ex vivo tests. The hybrid hydrogels solution is injected into the submucosal layers of the pig stomach using a 23-gauge needle. The dose can be 2 ml for each sample and the stomach is kept to a temperature of about 37 C. with a lamp to ensure simulation of in vivo conditions. Gelation of the hybrid hydrogels samples is assessed by cutting open the tissue after the desired time. The experiment may be repeated three times.

[0413] Creating Submucosal Cushion and Performing ESD in a Living Pig

[0414] The pig is fasted for 1 day before operation.

[0415] Endoscopy is performed by the surgeon.

[0416] A standard endoscope (Karl Storz, Tuttlingen, Germany) is used in the pig under general anesthesia. Both the hybrid hydrogels solution and the NS used as control contains a small amount of Methylene Blue as a color agent in order to facilitate visualization of the SFC.

[0417] After setting appropriate lesion sizes of approx. 3 cm in diameter in the porcine stomach, 810 ml of hybrid hydrogels solution and NS are injected in the stomach submucosa through the endoscope accessory channel using a 23-gauge injection needle.

[0418] The mucosal elevation due to the injected hybrid hydrogels at the target site is observed endoscopically before starting the ESD. It is compared under direct view with the elevation caused by NS during the procedure.

[0419] After injection, the ESD is performed and a circumferential mucosal incision is accomplished using a Needle knife (Olympus, Tokyo, Japan)

[0420] Injection of hybrid hydrogels and ESD may be repeated three times.

[0421] The animal is euthanized after completion of experiments; the whole procedure is followed and recorded using a Silver Scope Video Gastroscope (Karl Storz, Tuttlingem, Germany).

[0422] The main outcome measures are (1) the rapid gelation of hybrid hydrogels when injected into the submucosa and (2) the long-lasting SFC formed; (3) the feasibility of the dissection procedure during ESD; (3) the adhesion of hybrid hydrogels to the muscolaris layer and thus the increase of protection during the procedure and after it.

Example 5: Treatment of Fistula and Leaks

[0423] Digestive leaks and fistulas are mostly the result of inflammatory bowel diseases or surgical manipulation of the gastrointestinal (GI) tract and their management remains challenging. Despite recent progress in interventional endoscopy that provides a minimally invasive alternative to surgery, complex acute leaks and chronic fistula remain the most difficult to treat: the healing rate is still insufficient, in particular for complex fistulas or large anastomotic leaks.

[0424] In order to validate the use of hybrid hydrogels of the invention to treat digestive leaks and chronic fistula experimental models in a large animal were created.

[0425] On-lay based application of the hybrid hydrogel can be used to treat gastrointestinal perforations and to create a chemical film barrier to bypass areas of the gut responsible for metabolic diseases (FIG. 9A).

[0426] Injection based hydrogel therapy can be used as filling agent to restore, heal and treat mechanical, functional and metabolic diseases: gastro-esophageal reflux disease (GERD) by restoring the lower esophageal sphincter pressure (FIG. 9B), GI fistulas by occluding the fistula tract and insulin-resistance/metabolic syndrome, by creating a physical barrier to the absorption of nutrients in crucial segments of the small bowel.

5.1. Treatment of Chronic Fistula

[0427] The first step of the procedure was the dissection of the lateral side of the neck of the animal to be treated (e.g. a pig).

[0428] A 5 cm skin incision was made on the neck. After dissection layer by layer the esophagus was identified and a convenient spot on the cervical esophagus 30 cm from the dental arches was chosen by transillumination using a light of the gastroscope.

[0429] A large bore needle was introduced into the esophageal lumen under endoscopic view and a guide wire fed into the needle in the esophageal lumen and retrieved by the endoscope.

[0430] A9-Fr T-tube was inserted over the guide and retrieved from the cervicotomy with the distal T part sitting into the esophagus. The catheter was then tunneled subcutaneously and secured to the skin. The same procedure was performed on the opposite side.

[0431] The T-tubes are left in place 4 weeks in order to create permanent communication between the esophageal lumen and the skin.

[0432] The following procedure was followed to treat digestive fistulas by hybrid hydrogel filling, in the above animal model of upper gastrointestinal tract fistulas. A) A drain is placed in the upper esophagus, through a cervicotomy approach, as previously described. B) The fistula path is obtained after 30 days survival. C) The fistula tract is filled with the hydrogel.

5.2. Treatment of Acute Fistula

[0433] In-vivo acute digestive gastro-jejunal fistula tracts were created by tubulisation of a segment of small bowel (3 cm long and 4 mm in diameter) which was then attached to the gastric wall. The small bowel cylinders were then closed at their distal end with a surgical suture.

[0434] A gastroscopy was performed by using a standard single channel endoscope to access the fistulas endoscopically.

[0435] In-vivo injection of the components of a hybrid hydrogel according to the invention was performed in 2 steps using a plug through the scope 2.8 mm plastic delivery catheter connected to a three-way valve. The hybrid hydrogel components were sodium Alginate 2%, and PAAm hydrogel (hydrogel polymer of formula (I), as described generally herein), which were injected concomitantly with Ca.sup.2+ to effect gelation. The PAAm hydrogel of Example 2.1 was used as hydrogel polymer of formula (I). The hybrid hydrogel gelled in vivo in a few minutes (<10 min.), thereby efficiently filling the fistula tract (and treating the fistula).

[0436] Step 1. The endoscopic delivery catheter was placed inside the proximal orifice of the fistula during the injection and removed after 2 minutes.

[0437] Step 2. A second injection was done by means of an extraction biliary catheter equipped with an inflatable balloon at its tip. The balloon was inflated in correspondence of the proximal opening of the fistula after the injection procedure and kept inflated for 2 minutes.

[0438] This allowed the components to have sufficient time to react and avoided the percolation of the solution in its liquid phase. The balloon was then deflated and the device extracted from the fistula. A careful endoscopic look was performed to confirm the presence of the gel inside the fistula.

[0439] A gastrectomy was then performed to examine the internal orifice of the fistula. The hydrogel was formed and solid and could only be removed by milking forcefully the fistula tract, which demonstrated the successful treatment of the fistula.

5.3. Examples

[0440] A huge challenge with existing hydrogels is the inadequate gelation time (too long, >10 minutes) which is not adapted for non-invasive surgical procedures, notably for the treatment of fistulas, because it hinders the possibility of a simple endoscopic injection of the material, considering that the hydrogel would just percolate outside the fistula.

[0441] The use of a hybrid hydrogel according to the invention, which is able to solidify in an extremely short time (less than five second), allowed to address the problem.

5.3.1. Comparative Example: Mixing of PAAm Hydrogel with Plasma and then Coagulation with Fibrinogen

[0442] In-vivo tests were performed to check the gelation properties of a bi-component hydrogel system made of blood-containing PAAm hydrogel of Example 2.1 and thromboplastin from rabbit (sigma). Thromboplastin was reconstituted as recommended by the producer in 10 mM CaCl.sub.2.

[0443] A section of about 1 cm of length of the small bowel of a pig was sealed with surgical thread at the extremities. 1.3 mL of PAAm hydrogel of Example 2.1, 1 mL of porcine blood and 1 mL of reconstituted thromboplastin were mixed together and immediately injected in the bowel section. No leakage from the injection site or from the sealing was observed. After 10 minutes the section was opened to check hydrogel gelation and adhesion. The hydrogel was not formed and only small blood clots were observed.

5.3.2. Hybrid Hydrogel According to the Invention and Gelation with Ca.SUP.2+

[0444] Ex-vivo tests were conducted on porcine small bowel. The bowel was explanted the day before, carefully washed, frozen for the night, de-frozen just before the tests and washed again. The bowel then divided in 1 cm long subsection with surgical thread, and reverted to have the mucosa in the external part and the mucosa inside the lumen. Hybrid hydrogels of the invention (mixture of PAAm hydrogel of Example 2.1/sodium alginate with different compositions) were injected into each section, followed by injection of the solution of Ca.sup.2+. Good gelation and adhesion was observed.

TABLE-US-00005 Sodium PAAm/ Calcium salt alginate Alginate Conc. M Gelation PAAm hydrogel Conc. % w/v, weight volume time (quantity) mL volume added ratio added (second) 5 mL 1% in H.sub.2O, 1 4.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL 10 mL 1% in H.sub.2O, 1 8.5:1 0.5M CaCl.sub.2 15 mL in H.sub.2O, 0.5 mL 5 mL 2% in H.sub.2O, 1 .sup.4:1 0.5M CaCl.sub.2 5 mL in H.sub.2O, 0.5 mL 10 mL 2% in H.sub.2O, 1 .sup.8:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL

[0445] In contrast, No gelation was observed with injection of only PAAm hydrogel. With only alginate/Calcium gelation is observed, but no adhesion.

5.3.3. Injection of Pre-Gel (PAAm+Alginate) and Gelation with Ca.SUP.2+

[0446] In-vivo tests were conducted on two fistula models obtained linking two 3 cm long sections of the intestine to the stomach of a pig. The fistulas were then accessed endoscopically.

[0447] The fistula model was prepared as described above and the distal extremity was closed. The stomach was then cut and the proximal opening of the fistula exposed. With a three-way valve, a mixture of PAAm hydrogel of Example 2.1 and 1% sodium alginate was injected inside the fistula. Then 0.1 M Ca.sup.2+ were injected inside the fistula. Exemplary amounts of PAAm hydrogel, sodium alginate and Ca.sup.2+ used in this Example are detailed in the Table in section 5.3.2 above. After one minute, hydrogel formation was checked by observing the possible percolation of fluids. The hybrid hydrogel was formed and solid. The stomach is the removed from the animal and the hydrogel is removed from the fistula applying pression to the closed extremity.

5.3.4. Sequential Injection of Alginate, PAAm and Gelation with Ca.SUP.2+

In Vitro

[0448] In the following experiment, in-vitro injection of the components of the hybrid hydrogel (Sodium Alginate 2%, PAAm hydrogel of Example 2.1) was performed through a 2.8 mm standard endoscope-compatible plastic endoscopic sheath. The procedure was to inject the solution of alginate first, then the hydrogel to clean the sheath from the alginate, and then injection of the Ca.sup.2+ solution was done to effect gelation of the hybrid hydrogel. The sheath was then washed with water to avoid gelation. Results were excellent, with no blocking observed and a fast in-vial gelation. Exemplary amounts of PAAm hydrogel, sodium alginate and Ca.sup.2+ used in this Example are detailed in the Table in section 5.3.2 above.

In Vivo

[0449] This approach was tested in-vivo through endoscopy. A model of fistula was prepared as described above, and then Alginate, PAAm hydrogel of Example 2.1 and Ca.sup.2+ were sequentially injected using the procedure tested in-vial. The results were good and we observed gel formation and no blocking of the catheter.

TABLE-US-00006 Sodium PAAm/ Calcium salt alginate Alginate Conc. M Gelation PAAm hydrogel Conc. % w/v, weight volume time (quantity) mL volume added ratio added (second) 5 mL 1% in H.sub.2O, 1 4.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL 10 mL 1% in H.sub.2O, 1 .sup.9:1 0.5M CaCl.sub.2 15 mL in H.sub.2O, 0.5 mL 5 mL 2% in H.sub.2O, 1 .sup.4:1 0.5M CaCl.sub.2 5 mL in H.sub.2O, 0.5 mL 10 mL 2% in H.sub.2O, 1 8.5:1 0.5M CaCl.sub.2 8 mL in H.sub.2O, 0.5 mL

Example 6: Radiopacity Tests

[0450] A hybrid hydrogel according to the invention containing a contrast solution was also used to check the visualization via CT scan.

[0451] Having a material that is radiopaque is of great interest: this allows the surgeons to check if the fistula is completely filled with the material and to follow in time the degradation of the hydrogel.

[0452] A conventional contrast agent, Iomeron (iodium-based contrast agent), was used as a solvent for the synthesis of the hybrid hydrogel, which was prepared according to Example 2.2, replacing water with Iomeron. The resulting pre-gel was injected inside a fistula (model fistula described above), followed by CaCl.sub.2 for gelation. The hybrid hydrogel formed showed good contrast compatible with the real application, as evidenced in FIG. 11B.

TABLE-US-00007 Sodium PAAm/ Calcium salt alginate Alginate Conc. M Gelation PAAm hydrogel Conc. % w/v, weight volume time (quantity) mL volume added ratio added (second) 5 mL 1% in H.sub.2O, 1 4.5:1 0.5M CaCl.sub.2 7 mL in H.sub.2O, 0.5 mL 10 mL 1% in H.sub.2O, 1 .sup.9:1 0.5M CaCl.sub.2 15 mL in H.sub.2O, 0.5 mL 5 mL 2% in H.sub.2O, 1 .sup.4:1 0.5M CaCl.sub.2 5 mL in H.sub.2O, 0.5 mL 10 mL 2% in H.sub.2O, 1 8.5:1 0.5M CaCl.sub.2 8 mL in H2O, 0.5 mL

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