INJECTABLE HYDROGELS AND USES THEREOF

Abstract

The invention relates to a hydrogel, in particular degradable or non degradable, comprising monomers of formula (I) and organosilica particles or porous silicon particles covalently bound thereto, optionally with non covalently bound organosilica and/or silicon particles mixed therewith, in particular degradable organosilica nanoparticles or core-shell nanocapsules; pharmaceutical, veterinary or cosmetic compositions thereof; and uses thereof as a medicament. The present invention finds applications in the therapeutic and diagnostic medical technical fields and also in cosmetic and veterinary technical fields.

##STR00001##

Claims

1-29. (canceled)

30. (canceled)

31. A hydrogel comprising monomers of formula (I) ##STR00038## 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; each occurrence of L.sub.1 independently 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, for each occurrence, 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 allyl, 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 group of formula
*R.sub.7(R.sup.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.12, 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; wherein at least a subset of occurrences of Y in the hydrogel polymer bears or comprises at least one organosilica particle wherein the organosilica matrix may be porous (preferably mesoporous) and contains responsively cleavable bonds within the organosilica framework.

32. The hydrogel according to claim 31, wherein at least in a subset of bracketed structures n: L.sub.1 represents independently a responsively cleavable covalent bond selected from: ##STR00039## a light-induced breakable group or a photo-responsive group; or R.sup.1-L.sub.1-R.sup.2* independently 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: ##STR00040## wherein each occurrence of q independently represents an integer, for example 1-6; and D independently represents 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: ##STR00041## 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: ##STR00042## v) a moiety comprising a sugar derivative such as mannose, a hyaluronic acid derivative, collagene, an amino acid or a peptide moiety.

33. The hydrogel according to claim 31, 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.

34. The hydrogel according to claim 31, wherein in the group of formula *R.sub.7(R.sub.8)*, R.sup.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.

35. The hydrogel according to claim 31, wherein in the group of formula *R.sub.7(R.sub.8)*, R.sup.7 may be N and R.sup.8 may be independently selected from the group comprising: ##STR00043##

36. The hydrogel according to claim 31, wherein at least a subset of occurrences of Y in the 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) a disintegratable organosilica nanoparticle; or (ii) a disintegratable 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 organosilica nanoparticle or nanocapsule contains 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 disintegratable 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.pindependently represents H or C1-6alkyl; and wherein the nanoparticle or nanocapsule outer surface may comprise 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.

37. The hydrogel according to claim 31, 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.

38. The hydrogel according to 36, wherein at least a subset of nanocapsules bound to the hydrogel polymer are further crosslinked via one or more #R.sup.5R.sup.6 groups to another hydrogel polymer of formula I.

39. The h hydrogel according to claim 36, 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.

40. The hydrogel according to claim 36, wherein L.sub.2 represents independently a responsively cleavable covalent bond selected from: ##STR00044## CarbamoylThioketal 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: ##STR00045## wherein each occurrence of q independently represents an integer, for example 1-6; and D independently represents 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: ##STR00046## 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=q23; or iv) a responsively cleavable moiety selected from: ##STR00047##

41. The hydrogel according to claim 40, wherein the organosilica particles bound to the hydrogel polymer has a diameter between 25 nanometers and 500 nanometers.

42. A pharmaceutical or cosmetic composition comprising the hydrogel of claim 31, and a pharmaceutically or cosmetically acceptable carrier.

43. A method for preparing the hydrogel of claim 31, comprising steps of: a) dissolving in water or alcoholic solutions: a monomer precursor of formula (IV) ##STR00048## at least one molecular crosslinker precursor having the structure A-R.sup.1-L.sub.1-R.sup.2-A, disintegratable organosilica nanoparticles bearing amino-containing tether groups at the outer surface; or disintegratable organosilica core/shell nanocapsules 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) stirring the solution obtained in step a), at any appropriate temperature, thereby allowing the polymerization carried out to form the hydrogel, c) 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 amino acid 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 C1alkyl; 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: ##STR00049## 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.

44. The method of claim 43, wherein the monomer precursor is of formula (IVa) ##STR00050##

45. The method of claim 43, wherein the linker L.sub.1 and *R.sup.1-L.sub.1-R.sup.2* are as defined in claim 2.

46. The method of claim 43, wherein the molecular crosslinker precursor A-R.sup.1-L.sub.1-R.sup.2-A is of formula ##STR00051##

47. The method of claim 43, wherein the selected precursor of formula BR.sup.8 is of formula ##STR00052##

48-60. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWING

[0308] 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).

[0309] FIG. 2. Synthesis of an hydrogel according to the invention (dPAA), embedding BNCs (a); scheme of the network (b); FTIR trace of dPAA (c); SEM showing the porosity of the nanocomposite (d).

[0310] FIG. 3: FIG. 3a represents the swelling ratio of the hydrogels incubated with 10 M GSH solution or Phosphate Buffer Saline (PBS), showing the degradation of the network (ordinate:swelling ratio) over time (abscissa:hours). FIG. 3 b are image of scanning transmission electron microscope representing the fragmentation of the particles in presence of the reducing GSH after 72 hours (ii) and image of scanning transmission electron microscope representing the particles in presence of the reducing PBS after 72 hours (i). FIG. 3 c represents cumulative release of Cyt-C from an hydrogel according to the invention by measure of absorbance at 410 nm (ordinate) overt time in hours (abscissa)

[0311] FIG. 4. HDFa-mediated degradation as function of time of dPAA hydrogels, solid line, containing 2.510.sup.5 cells and control (acellular dPAA), dashed line (a); macroscopic pictures showing the cell-mediated degradation of the dPAA (b). Overlay images (i.e brightfield and DiD channel) of the HDFa growing onto the scaffold (24 h) and gradually transferring to the bottom well plastic as the dPAA gets degraded (48 and 96 h); scale bar is 100 m.

[0312] FIG. 5. SEM image of the explanted tissue and hydrogel, showing collagen fibers within the hydrogel matrix (a), scale bar is 100 m; endoscopy images of the dPAA stained with Methylene Blue formed in vivo (b) and close up (c) showing with fibrous formation within the hydrogel matrix.

[0313] Change in mucosal elevation as a function of time after the injection of NS or dPAA into a resected porcine stomach (d). Methylene blue was mixed as color agent. Height values (black bar) obtained for NS were 6.7 mm, 4.2 mm and 2.9 mm after 10 sec, 10 min and 1 h respectively; for dPAA were 8.3 mm, 6.4 mm, 5.8 mm after 10 sec, 10 min and 1 h respectively.

[0314] FIG. 6. Endoscopic views of the different steps of the ESD procedure performed using a hydrogel according to the invention (dPAA) stained with Methylene Blue. Setting of the lesion, approx. 3 cm in diameter (a); injection of the dPAA solution (b); formation of the SFC after gelation of the dPAA (c); circumferential cutting (d); complete resection with protective layer of dPAA that remained adhered to the muscolaris (e); wound left after ESD with layer of the dPAA (f).

[0315] FIG. 7. Mechanism of network degradation 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 scaffold occurs at the disulfide cleavage site by thiol-disulfide exchange.

[0316] FIG. 8. Swelling ratio of the hydrogels comprising nanoparticles containing different cystamine amounts, incubated with 10 M GSH solution, showing the degradation of the network (ordinate:swelling ratio) over time (abscissa:hours). Hydrogel containing 10 wt % of cystamine is displayed in green, the one with 40 wt % is in red; dPAA was added as a reference.

[0317] FIG. 9. Viability of HDFa onto the degradable nanocomposite measured with alamarBlue assay (a). The plot displays the percentage of reduced alamarBlue, which is proportional to the cell metabolic activity of the cells, as function of time. The result shows an increase in metabolic activity, thus indicates the proliferation of cells onto the scaffold. Proliferation of the cells in 3D (b); 3-channel visualization of the HDFa (c), scale bar is 100 m. Cells in (b) and (c) were stained with Vybrant DiD (grey) to facilitate the imaging.

[0318] FIG. 10. Injection of the dPAA solution stained with Methylene Blue via a surgical 23-gauge needle (a); formation of a mucosal elevation (b); gelation occurred in less than 10 minutes, achieving a solid and elastic hydrogel, adhered to the tissue.

[0319] FIG. 11. .sup.1H NMR spectrum of DCNS before and after light irradiation.

[0320] FIG. 12. Characterization of model spherical MSPs, illustrated in the Examples.

[0321] FIG. 13. Complete characterization of the hybrid light-sensitive spherical MSPs with light-induced cleavable linkers within the organosilica matrix, illustrated in the Examples.

[0322] FIG. 14. 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.

EQUIVALENTS

[0323] 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 a person of ordinary skill 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.

[0324] 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

[0325] 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: Hydrogels Comprising Particles: Synthesis and In-Vitro and In-Vivo Uses

Materials and Methods

Abbreviations

[0326] MBA N,N-methylenebisacrylamide [0327] GABA gamma-aminobutiric acid [0328] MeOH Methanol [0329] L-DOPA [0330] Dopamine [0331] ENA Ethylendiamine [0332] PEHA Pentaethylenhexamine O-(2-aminomethyl)-O-methylpolyethylene glycol, [0333] MPGA PM 5000 [0334] CYST Cystamine [0335] DMENA N,N-Dimethylethylenediamine [0336] NH2-CytC@BNPs cleavable core/shell nanocapsules [0337] 1 mg/ml NH2-MSPs Solution of NH2-MSPs as described herein

[0338] Redox-Cleavable Nanocapsules Synthesis

[0339] 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.

[0340] 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.

[0341] The procedure can be adapted for the encapsulation of different globular proteins.

[0342] Redox-Cleavable Nanocapsules Functionalization

[0343] 40 mg of redox-cleavable nanocapsules are suspended in 5 mL of ethanol.

[0344] 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.

[0345] The resulting NH.sub.2-functionalized redox-cleavable nanocapsules (NPs, also designated NH2-CytC@BNPs) are then washed five times with distilled water and dried.

[0346] Redox-Responsive Degradable Hydrogel Functionalized with Silica Nanoparticles 200 mg of methylenebisacrylamide (MBA), 65 mg of cystamine hydrochloride and 70 L of N,N-dimethylethylenediamine are mixed together with 1.0 mL of a 1 mg/mL solution of NH.sub.2-functionalized breakable nanocapsules.

[0347] After 48 h, the hydrogel is formed.

[0348] The procedure can be modified and other NH.sub.2-functionalized silica nanoparticles can be used, such as breakable or non-breakable mesoporous silica nanoparticles.

[0349] The experimental protocol described above was repeated with four additional different compositions of non degradable (no cleavable crosslinker in the PAAm polymer network) or degradable hydrogels (presence of cleavable crosslinker in the PAAm polymer network). The hydrogel compositions and starting materials are summarized below. Unless otherwise indicated in the table below, all hydrogels formed within 48 hours.

TABLE-US-00002 Hydrogel Hydrogel MONOMER 1 M1 MONOMER 2 M2 number Properties (M1) amount (M2) amount 1 Degradable MBA (mg) 200.00 DMENA (l) 86.00 2 Non MBA (mg) 200.00 GABA (mg) 66.00 Degradable 3 Non MBA (mg) 200.00 GABA (mg) 66.00 Degradable 4 Non MBA (mg) 200.00 GABA (mg) 66.00 Degradable S1 Hydrogel CROSSLINKER Other 1 SOLVENT amount number 1 (CL1) CL1 amount OTHER 1 amount 1 (S1) (mL) 1 CYST (mg) 32.00 NH2- 1 mg Water 1.50 CytC@BNPs (mL) 2 PEHA (l) 30.00 1 mg/ml 1.50 NH2-MSPs (ml) 3 PEHA (l) 60.00 1 mg/ml 1.50 NH2-MSPs (ml) 4 PEHA (l) 120.00 1 mg/ml 1.50 NH2-MSPs (ml)

[0350] NH2-CytC@BNPs refers to NH2-functionalized core/shell redox-cleavable nanocapsules described above.

[0351] NH2-MSPs refers to NH2-functionalized hybrid light-sensitive MSPs described in Example 1.3 below.

[0352] 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.

[0353] Once obtained, the disk-shaped hydrogels were freeze-dried and weighted. Dried 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.

[0354] 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.

Photo-Cleavable Nanoparticles Synthesis

1.1 2-nitro-5-(((3-(triethoxysilyl)propyl)carbamoyl)oxy)benzyl (3-(triethoxysilyl)propyl)carbamate, (DCNS)

[0355] 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)

##STR00032##

[0356] 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.

1.2 Breakability Test on DCNS

[0357] 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. 11).

1.3 Synthesis of Hybrid MSPs

[0358] Firstly, model spherical MSPs were synthesised. The model particles were synthesized according to a modified Stber synthesis, shown in Scheme 2

##STR00033##

[0359] The model particles obtained were spherical characterized by an average diameter of ca 200 nm (SEM, TEM and DLS analysis in FIG. 12). 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. 12)

[0360] 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)

##STR00034##

[0361] 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. 13). 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.

[0362] The hybrid light-sensitive MSPs may be further functionalized, as described for core/shell nanocapsules above, for covalent incorporation as crosslinkers into hydrogel networks. 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.

1.4 Light Breakability Experiments of Hybrid MSPs

[0363] 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. 13). 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.

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

[0364] ##STR00035##

[0365] 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.

##STR00036##

##STR00037##

[0366] pH-Degradable Hydrogels

[0367] 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.

[0368] The experimental protocol described above was repeated with additional different compositions of non degradable (no cleavable crosslinker in the PAAm polymer network) or degradable hydrogels (presence of cleavable crosslinker in the PAAm polymer network). The hydrogel compositions and starting materials are summarized below. Unless otherwise indicated in the table below, all hydrogels formed within 48 hours.

TABLE-US-00003 Gel- Hy- ifica- dro- Hydro- tion gel gel MONO- MONO- MONO- CROSS- SOL- S1 time num- Prop- MER M1 MER M2 MER M3 LINKER CL1 OTHER Other 1 VENT 1 amount (37 ber erties 1 (M1) amount 2 (M2) amount 3 (M3) amount 1 (CL1) amount 1 amount (S1) (mL) C.) 5 Adhe- MBA 200,00 GABA 34,00 PEHA 120,00 Water 1,50 sive (mg) (mg) (l) (mL) 6 Degrad- MBA 200,00 DMENA 38,00 CYST 130,00 Water 1,50 able (mg) (l) (mg) (mL) 7 Adhe- MBA 200,00 GABA 50,00 PEHA 120,00 PVP 100,000 Water 1,50 150 sive (mg) (mg) (l) 40,000 (mL) (RT) (mg) 8 Adhe- MBA 200,00 GABA 50,00 PEHA 120,00 PVP 25,00 Water 1,50 150 sive (mg) (mg) (l) 40,000 (mL) (RT) (mg) 9 Adhe- MBA 200,00 GABA 50,00 PEHA 120,00 PVP 50,00 Water 1,50 165 sive (mg) (mg) (l) 40,000 (mL) (RT) (mg) 10 Non MBA 200,00 GABA 50,00 PEHA 120,00 Water 1,50 Degrad- (mg) (mg) (l) (mL) able 11 Non MBA 200,00 GABA 50,00 PEHA 70,00 Water 1,50 Degrad- (mg) (mg) (l) (mL) able 12 Non MBA 200,00 GABA 50,00 PEHA 80,00 Water 1,50 Degrad- (mg) (mg) (l) (mL) able 13 Non MBA 200,00 GABA 50,00 PEHA 90,00 Water 1,50 110 Degrad- (mg) (mg) (l) (mL) able 14 Non MBA 200,00 GABA 50,00 PEHA 100,00 Water 1,50 88 Degrad- (mg) (mg) (l) (mL) able 15 Non MBA 200,00 GABA 65,00 ENA 35,00 Water 1,50 48 h Degrad- (mg) (mg) (l) (mL) able 16 Non MBA 200,00 GABA 65,00 ENA 40,00 Water 1,50 15 h Degrad- (mg) (mg) (l) (mL) able 17 Non MBA 200,00 GABA 65,00 ENA 50,900 Water 1,50 8 h 30 Degrad- (mg) (mg) (l) (mL) able 18 Non MBA) 200,00 GABA 65,00 ENA 60,00 Water 1,50 7 h Degrad- (mg) (mg) (l) (mL) able 19 Non MBA 200,00 GABA 65,00 PEHA 50,00 Water 1,50 Degrad- (mg) (mg) (l) (mL) able 20 Non MBA 200,00 GABA 65,00 PEHA 70,00 Water 1,50 200 Degrad- (mg) (mg) (l) (mL) able 21 Non MBA 200,00 GABA 65,00 PEHA 90,00 Water 1,50 4 h Degrad- (mg) (mg) (l) (mL) able 22 Non MBA 200,00 GABA 65,00 PEHA 110,00 Water 1,50 4 h Degrad- (mg) (mg) (l) (mL) able 23 Non MBA 200,00 GABA 65,00 PEHA 140,00 Water 1,50 2 h 10 Degrad- (mg) (mg) (l) (mL) able 24 Adhe- MBA 200,00 Dop- 66,00 PEHA 89,00 Ascorbic 10,00 Water 1,50 sive (mg) amine (l) acid (mL) (mg) (mg) 25 Adhe- MBA 200,00 GABA 66,00 PEHA 70,00 PVP 50,00 Water 1,50 sive (mg) (mg) (l) 40,000 (mL) (mg) 26 Adhe- MBA 200,00 GABA 66,00 PEHA 70,00 PVP 100,00 Water 1,50 150 sive (mg) (mg) (l) 40,000 (mL) (mg) 27 Adhe- MBA 200,00 GABA 66,00 PEHA 70,00 PVP 50,00 Water 1,50 sive (mg) (mg) (l) 40,000 (mL) (mg) 28 Adhe- MBA 200,00 GABA 66,00 PEHA 70,00 PVP 100,00 Water 1,50 120 sive (mg) (mg) (l) 40,000 (mL) (mg) 29 Lumin- MBA 200,00 GABA) 66,00 PEHA 70,00 MeOH 1,00 escent (mg) (mg) (l) (mL) 30 Lumin- MBA 200,00 GABA 66,00 PEHA 70,00 MeOH 2,00 escent (mg) (mg) (l) (mL) 31 Adhe- MBA 200,00 GABA 67,00 PEHA 70,00 Water 1,50 200 sive (mg) (mg) (l) (mL) 32 Degrad- MBA 200,00 DMENA 70,00 CYST 65,00 Water 1,50 able (mg) (l) (mg) (mL) 33 Degrad- MBA) 200,00 DMENA 86,00 CYST 32,00 Water 1,50 able (mg) (l) (mg) (mL)

[0369] Degradation Kinetic of Stimuli-Responsive Hydrogels For redox-responsive materials, a 1 mm thick hydrogel cylinder is lyophilized and its dry weight is recorded. The hydrogel is then placed in a vial and 5 mL of a 10 M solution of reduced GSH are added. The swelling of the 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.

[0370] The same procedure was applied for pH-responsive materials, using pH=4 citrate buffer for degradation and PBS as a reference.

[0371] Degradation of dPAA hydrogels was examined in the presence of reduced glutathione (GSH), a disulfide reducing agents.

[0372] Briefly, the lyophilized hydrogels samples were incubated at 37 C. in 2 mL of a PBS solution with a GSH concentration of 10 M. dPAA hydrogels were incubated in PBS alone as a control.

[0373] The degradation kinetics were then evaluated via swelling ratio (SR) measurements in time.

[0374] SR were measured by a gravimetric method. In brief, lyophilized hydrogel samples were immersed in PBS at 37 C. Then, the samples were 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 were measured on an analytical balance. The SR were then calculated as a ratio of weights of swollen hydrogel (Ws) to dried hydrogel (W), using the following equation:

[00001]$\mathrm{SR}=\frac{W_s-W_d}{W_d}$

[0375] Degradation time was 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 was determined when we could observe a limpid solution, without solid residues.

[0376] In Vitro Cell Culturing

[0377] Cryopreserved human dermal fibroblast, adult (HDFa) were purchased from Thermo Fisher and the culture was initiated as suggested on the protocol. HDFa were grown in Medium 106 supplemented with Low Serum Growth Supplement (LSGS, Thermo Fisher). Cells were 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 were grown until reaching 80 to 85% of confluence. Then, they were washed twice with PBS and treated with trypsin/EDTA solution to detach them from the flask surface. Cells were split every 2-3 days; the medium was changed every other day.

[0378] In Vitro Cell Culturing onto the Nanocomposite Hydrogels

[0379] The hydrogel scaffolds equilibrated by adding culture media at 37 C. HDFa were detached from the culture flask by trypsination and approximately 2.510.sup.5 cells were seeded onto the hydrogel scaffolds. Then, the samples were placed in the incubator (37 C., 5% CO.sub.2) for about 30 minutes and fresh media was cautiously added on the top of the hydrogels to supply cells with nutrients. This was done to allow anchorage of the cells onto the scaffolds.

[0380] Cell Staining and Viability Studies

[0381] Cell viability was assessed using alamarBlue assay. Briefly, the alamarBlue solution was added to the culture medium (1:10 dilution) of unstained cells growing onto hydrogel scaffolds. After 3 h incubation, 200 L of the media were 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 were recorded using a VICTOR X5 Multilabel Plate Reader (Perkin Elmer).

[0382] Each sample was tested in three replicates and the results were expressed as percentage of reduced alamarBlue.

[0383] The viability of cells after complete degradation of the dPAA was measured by with a TC20 (trade mark) Automated Cell Counter (Bio-Rad).

[0384] Where required (confocal fluorescence microscopy images), HDFa were stained with Vybrant DiD (Life Technologies, Thermo Fisher Scientific, Waltham, Mass., USA), following the reported protocol, prior to seeding them onto the scaffolds.

[0385] Cell-Mediated Degradation of Hydrogel

[0386] The hydrogel were freeze-dried and weighed (W). Then 2.510.sup.5 HDFa were seeded onto the samples (see above). The cell-laden samples were collected at pre-determined time points and were freeze-dried to obtain their dry weight after degradation (W).

[0387] The cell-mediated degradation of the hydrogels, D, was calculated using the following equation:

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

[0388] Acellular hydrogels were used as degradation control.

[0389] Evaluation of the Gelation and Formation of SFC Ex Vivo

[0390] Fresh porcine stomachs were used for the ex vivo tests. The hydrogel solution was injected into the submucosal layers of the pig stomach using a 23-gauge needle. The dose was 2 ml for each sample and the stomach was kept to a temperature of about 37 C. with a lamp to ensure simulation of in vivo conditions. Gelation of the dPAA samples was assessed by cutting open the tissue after the desired time. The experiment was repeated three times.

[0391] Creating Submucosal Cushion and Performing ESD in a Living Pig

[0392] The pig was fasted for 1 day before operation.

[0393] Endoscopy was performed by the surgeon.

[0394] A standard endoscope (Karl Storz, Tuttlingen, Germany) was used in the pig under general anesthesia. Both the dPAA solution and the NS used as control contained a small amount of Methylene Blue as a color agent in order to facilitate visualization of the SFC.

[0395] After setting appropriate lesion sizes of approx. 3 cm in diameter in the porcine stomach, 810 ml of dPAA solution and NS were injected in the stomach submucosa through the endoscope accessory channel using a 23-gauge injection needle.

[0396] The mucosal elevation due to the injected dPAA at the target site was observed endoscopically before starting the ESD. It was compared under direct view with the elevation caused by NS during the procedure.

[0397] After injection, the ESD was performed and a circumferential mucosal incision was accomplished using a Needle knife (Olympus, Tokyo, Japan)

[0398] Injection of dPAA and ESD were repeated three times.

[0399] The animal was euthanized after completion of experiments; the whole procedure was followed and recorded using a Silver Scope Video Gastroscope (Karl Storz, Tuttlingem, Germany).

[0400] The main outcome measures were (1) the rapid gelation of dPAA 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 dPAA to the muscolaris layer and thus the increase of protection during the procedure and after it.

Study Design

[0401] A degradable nanocomposite hydrogel, also called dPAA below, was synthetized and characterized. It is composed of a polyamidoamines-based network with embedded breakable silica hollow nanocapsules, BNCs.

[0402] Both, the BNCs and the polymeric backbone of the scaffold contain disulfide linkers that could be cleaved in presence of glutathione (GSH). The nanocomposite could be completely degraded even at a very low concentration of GSH (i.e. 10 M), which was chosen to mimic the extra-cellular environment. Degradation and release kinetics of model protein cytochrome, loaded into the particles, were evaluated.

[0403] Next, the cell-mediated degradation of dPAA in the presence of adult Human Dermal Fibroblasts (HDFa) proliferating onto the scaffolds was tested. The assay demonstrated the achievement of cell-controlled degradation of the material; complete dissolution of the scaffold was observed after 96 hours when 2.510.sup.5 cells were seeded onto the nanocomposite.

[0404] Then, the injection of the hydrogel solution (i.e. before complete gelation) was attained through a 23-gauge needle and the formation of the hydrogel was evaluated ex vivo. This allowed us to observe a fast gelation (<10 minutes), probably due to the increase of temperature and the interactions between the dPAA and the collagen present in the submucosal layer, where the injection was performed. Moreover, the dPAA was able to provide a stable and long-lasting mucosal elevation when tried as SFC.

[0405] Finally, the dPAA was tested as SFC for ESD procedures in vivo, on a porcine stomach.

[0406] The formation of the hydrogel and SFC in vivo was observed after 3 minutes and allowed the surgeon to perform the ESD procedure with a single injection. The adherence of part of the dPAA to the muscularis layer not only protected it during the procedure but also potentially offers several advantages in the phase following the surgery. The cell-mediated degradation of the nanocomposite indeed has shown to lead to the release of the active component loaded into the particles. This behavior could be exploited to release antibiotics or active factors to assist the healing of the wounded tissue and finally to achieve a complete clearance of the hydrogel form the body.

Synthesis of the Materials

Synthesis of Breakable Nanocapsules

[0407] 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.

[0408] An hydrogel comprising these BNCs to construct hydrogels comprising nanoparticles or nanocapsule able to release active molecules during the degradation of the material.

[0409] 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.

[0410] 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.

[0411] 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.

[0412] 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.

[0413] 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.

Synthesis of the Nanocomposite Hydrogel, dPAA

[0414] Advantageously the inventors designed the network of the dPAA to achieve a degradation that could be triggered by cells proliferating onto the material, without the need of any additional stimulus. Thus, a crosslinker, for example a disulfide, was incorporated in the polymeric network of the hydrogel, i.e. cystamine. Disulfide bonds are susceptible of thiol exchange in the presence of reducing agents, such as glutathione (GSH), which is a cell metabolite.

[0415] The inventors demonstrated that the reducing microenvironment given by the presence of GSH in the extra-cellular environment could trigger the cleavage of the disulfide bonds, therefore providing the dissolution of the scaffold.

[0416] Disulfide-modified polyamidoamines-based hydrogels containing BNCs were synthesized following the previously reported method with some modifications..sup.[17] In particular, amino groups on BNCs were reacted with the unsaturated moiety of methylenebisacrylamide (MBA) through a one-pot Michael poly-addition in water, at room temperature (FIG. 2a)

[0417] Briefly, a mixture of MBA, and N,N-dimethylethylenediamine, DMEN, was stirred in a BNCs water dispersion (1 mg/ml) at room temperature in presence of cystamine. A transparent liquid was obtained after 30 minutes, and then was left in static conditions to complete the gelation process in 2 days. FIGS. 2a,b show a schematic representation of the synthesis and structure of the nanocomposite network.

[0418] The synthesis afforded transparent hydrogels formed in water at room temperature using a catalyst-free Michael-type addition. Gelation was confirmed by the absence of gravitational flow when the test tubes containing the hydrogels were inverted, through the so called inverted test tube method.

[0419] The formation of a crosslinked network was further confirmed by Fourier transformed infrared spectroscopy (FTIR), showed in FIG. 2c. In particular, peaks at 1640 and 1530 cm.sup.1 are typical for the absorption of the amides carbonyl (st and v of CO), while the 3260 cm-.sup.1 band is attributed to the NH stretching (st NH.sub.2). Furthermore, the absorption band at 1039 cm.sup.1, which was ascribed to the vibration of CSSC bond, confirmed of the successful incorporation of the disulfide cross-linker in the polymeric network. Since the samples were lyophilized to allow a correct peak determination, the presence of adsorbed water cannot be seen from FTIR spectrum, apart from a weak residual shoulder at 3430 cm.sup.1, which is related to the OH angular deformation of water.

[0420] The morphological analysis of the obtained hydrogel scaffolds was assessed via scanning electron microscopy (SEM) of the lyophilized scaffolds. SEM showed a highly porous structure, with pores diameter in the range of 40 to 100 m, as can be seen in FIG. 2d.

Degradation and Release Kinetics

Degradation in Presence of GSH

[0421] For some medical application, hydrogels should maintain the required mucosal elevation for a determined time (i.e. 30 min to 1 hour), and then degrade into fragments, in order to have a complete clearance from the body.

[0422] The nanocomposite presented in this work was degradable upon exposure to GSH, via the incorporation of cystamine cross-links throughout the polymeric network and in the particle (BNCs) framework. The potential degradation mechanism of the network is shown in FIG. 7.

[0423] The degradation kinetics of the dPAA was examined by measuring swelling ratio variations as function of time in the presence of a low concentrated GSH solution (i.e. 10 M GSH solution in PBS), mimicking the extracellular environment. Hydrogels, incubated in PBS in the absence of GSH were used as control.

[0424] The swelling ratio curve of dPAA showed two different phases: an initial phase where clear increase in swelling was observed, followed by a rapid downward phase (FIG. 3a).

[0425] The imbibing of the solvent into the hydrogel caused the initial increasing phase. This was then quickly outweighed by the cleavage of the disulfide bonds, leading to the complete degradation of the hydrogel.

[0426] A clear point of reverse gelation, defined as the point where there are less than 2 crosslinks per polymer chain and the branched polymer chains dissolve,.sup.[22] was identified after 24 hours.

[0427] As disulfide bonds were cleaved, mass loss increases with time until there is no longer a sufficient number of crosslinks to maintain the 3D network. Finally, the equilibrium swelling value was found equal to zero after 3 days, due to complete disintegration of the hydrogel network.

[0428] The dPAA equilibrated in PBS showed instead a first phase of swelling followed by a plateau that was reached after 24 hours, demonstrating that the nanocomposite is stable in absence of reducing agent. The swelling was followed for 6 days.

[0429] The fast degradation rate observed, even with a low concentration of GSH, could be ascribed to the chemical environment of the hydrogel network surrounding the disulfide moieties, as shown in other studies..sup.[20b] It has been reported that the chemical environment of the disulfide units plays a key role in determining their degradation kinetics..sup.[23] Thus, the presence of electronegative groups in the adjacency of the disulfide bonds in the dPAA make them more susceptible to cleavage and hence, lead to a fast degradation.

Tunability of the Degradation Kinetic

[0430] To evaluate the effect of the disulfide crosslinker density on the degradation kinetics of the hydrogel nanocomposite, other two samples were synthetized. These scaffolds had the same composition of the dPAA, except for the amount of cystamine. In particular, they contained a lower and a higher amount of cystamine, 10-wt % and 40-wt %, compared to the dPAA hydrogel, which had 20 wt %.

[0431] In this way, a range of degradation profiles and times that could be achieved in response to GSH reducing microenvironments and that could be controlled by adjusting the molar ratio of the disulfide bond were established.

[0432] The degradation kinetics were evaluated by immersing the samples in the reducing solution ([GSH]=10 M) and by measuring the swelling ratio after precise time intervals, as already described above.

[0433] The degradation profiles of the three samples are reported in FIG. 8.

[0434] The degradation time was found to be proportional to the amount of disulfide crosslinker; in particular a decrease was observed with the scaffold containing 10 wt % of cystamine, which completely after 24 hours. Instead the sample crosslinked with an higher amount of cystamine (40 wt %) displayed a longer degradation profile, terminating after 6 days with the complete disintegration of the network.

[0435] The possibility of tuning the degradation kinetic of the nanocomposite scaffold developed by changing the ratio of the disulfide linker, demonstrate its potential for applications where a precise control of the breakability over time is required.

[0436] Release of Model Protein Cyt-C

[0437] As mentioned in the previous sections, the degradable hydrogel was decorated with breakable nanocapsules able to degrade with the same mechanism of the hydrogel, through the reduction of the disulfide bonds, and able to release their content.

[0438] The fragmentation of the BNCs in presence of the reducing GSH after 72 hours, was further confirmed by scanning transmission electron microscope, as shown in FIG. 3b.

[0439] Cytochrome-C, Cyt-C, was used as a model protein to study the release kinetics thanks to its strong absorption in the visible region, due to the presence of the eme group.

[0440] The BNCs were thus embedded into the dPAA and the Cyt-C released from the nanocomposite was investigated, by incubating the scaffold in the 10 M solution of GSH in PBS.

[0441] The cumulative release of Cyt-C from the dPAA is reported in FIG. 3c and shows a slow release in the first 6 hours, followed by an increase of detected Cyt-C. The release was observed by measuring the absorbance of the solution at 410 nm. The release of Cyt-C from the scaffold is highly augmented by the degradation of the hydrogel structure, which can be seen from the shape of the curve between 24 and 72 hours, showing a steep growth in absorption. The overall amount of protein release from the scaffold was estimated to be 67% of the initial loading.

[0442] Overall, as demonstrate the incorporation of cleavable groups, both in the embedded NPs and into the hydrogel network, which can degrade in response to endogenous stimuli, is an attractive strategy for in vivo procedures, such as ESD. The system is completely cleared from the body and release molecules of choice during the degradation process, such as a drug to assist the healing of the wound or in situ chemotherapy. The versatility of the synthesis allows the tailor-made nanocomposite hydrogel preparation in response to the needs of individual patients.

In Vitro and Ex-Vivo Analyses

Cell-Mediated Degradation

[0443] Since the degradation of hydrogel and the release of the model protein was achieved at low GSH concentration, such as the extra-cellular one, the degradation of the scaffold in the presence of cells was tested. In particular, Human Dermal Fibroblast (HDFa) were chosen for this study because fibroblasts residing within the extracellular matrix in the body are critical for matrix synthesis and repair. Upon injury or wound formation, these cells migrate to the wound site to repair the damaged tissue..sup.[24] Thus, to simulate the cell-mediated degrading conditions in vivo, we selected HDFa.

[0444] The dPAA hydrogels for this test were synthetized in a 8 mm diameter and 1 mm height disc shape and. 2.510.sup.5 HDFa were seeded onto the hydrogels and cultured in the corresponding growth medium; acellular hydrogels incubated in growth medium were used as control.

[0445] AlamarBlue assay indicated that the majority of the encapsulated cells were viable and proliferating onto the scaffold (FIG. 9a) up to 4 days. This is consistent to what has been observed for similar polyamidoamines-based scaffolds and it confirmed that hydrogel containing disulfide moieties supported cell encapsulation and viability. FIG. 9b displays an image of the 3D proliferation of the cells stained in red (Vibrant DiD stain) for better visualization, indicating that they permeate in the depth of the scaffold. A 3-channel visualization of the surface of the dPAA is reported in FIG. 9c and is indicative of the growth of the cells onto the scaffold.

[0446] The hydrogel underwent degradation responding to cell-secreted GSH, in the absence of any external stimulus. In addition, many cell surface molecules contain thiol groups and thus could contribute to the cleavage of the disulfide bonds of the network.

[0447] The scaffold resulted largely reduced in size and weight after 72 hours and the complete degradation was achieved after 96 hours.

[0448] FIG. 4a,b), demonstrating a good agreement with the GSH degradation tests.

[0449] It was observed that the degradation process resulted into a gradual movement of the cells from the nanocomposite to the bottom of the well containing the scaffold (FIG. 4c). The viability of HDFa measured after the degradation of the scaffold showed that 87% of the cells were viable, thus confirming that the degradation products were non-toxic. Previous studies on linear polyamidoamines systems have shown that the degradation products were completely non-cytotoxic, since the degradation produced oligomers..sup.[25]

[0450] The acellular hydrogels used as control displayed minimal degradation during the course of the studies (FIG. 4a, dashed line). This small degradation (17%) was probably due to the presence of fetal bovine serum in the culture medium, which contains various proteins and amino acids with thiol groups.

[0451] The obtained results clearly demonstrate that the dPAA hydrogels were prone to HDFa-mediated degradation through thiol reductive exchange, therefore showing potential for in vivo applications requiring degradation of the scaffold.

Hydrogel: Injectability and Formation of SFC

[0452] The hydrogel according to the invention is a material that could be delivered in vivo via injection, and then rapidly gel inside the body.

[0453] Polyamidoamine-based hydrogels have the great advantage of allowing network formation under physiological conditions.

[0454] Thus, taking advantage of the catalyst-free water-based reaction through which hydrogels could be obtained, and having observed a time window of several hours between the beginning of the polymerization and the complete gelation, the obtained scaffold for the injection procedure was tested.

[0455] When the polyaddition reaction between MBA and DMEN was carried out using the biodegradable cystamine crosslinker, we observed the formation of a clear solution after 30 minutes, indicating that the reagents were completely dissolved and have started the polyaddition reaction.

[0456] The ability of the hydrogel solution to flow through a disposable 23-gauge catheter injection needle was then examined. The dPAA solution was able to flow under hand pressure and the maximum needle injection pressure was found to be comparable to saline solution (FIG. 10a). Then, we observed that this viscous hydrogel solution reacted at room temperature in static conditions to form a self-standing hydrogel after 48 h.

[0457] Better results were obtained by raising the temperature to 37 C., when we observed the formation of a self-standing hydrogel in 18 hours.

[0458] However, knowing that intestinal submucosa is rich in type I collagen and that this presents amino and hydroxyl groups as side groups, the possible formation of hydrogen bonds that could lead to a faster gelation kinetic in vivo was tested.

[0459] Thus the dPAA solution was injected ex vivo in the submucosa of a porcine stomach. The injection was performed on the tissue at 37 C. and the gelation, with formation of a SFC, was immediately observed (FIG. 10b). There was no leaching of the solution away from the injected site, and the tissue was cut opened, revealing the formation of the dPAA after 8-10 min post injection ex vivo (FIG. 10c).

[0460] The nanocomposite hydrogel was found completely adhered to the submucosal layer and it had to be removed with scissors and tweezers. Moreover, it was confirmed that there had not been diffusion of the solution into the surrounding tissues.

[0461] The investigation of injectability and gelation time was then performed in vivo. The hydrogel (dPAA) showed a gelation time of approximately 3 minutes when injected in the submucosal layer in a living pig.

[0462] As demonstrated, unexpectedly and advantageously, physiological conditions of temperature (37 C.) and pH (7.4), contribute to the faster gelation than what observed in vitro, as already reported for similar systems..sup.[28]

[0463] The intermolecular interaction between the hydroxyl and amino groups of the collagen side chains with amide groups of dPAA lead to the formation of hydrogen bonds that further crosslink the polymer network.

[0464] Moreover, the formation of mechanical entanglements between the collagen fibers and the dPAA backbone advantageously may also contribute to the formation of an interpenetrated hydrogel network, favoring the faster formation of a stable and elastic hydrogel in situ. This behavior was observed via SEM of the explanted tissues, which showed interactions between the hydrogel scaffold and collagen fibers (FIG. 5a).

[0465] In vivo images of the dPAA formed in situ (FIG. 5b) and stained with Methylene Blue also displayed fibrous formation within the hydrogel matrix (FIG. 5c).

[0466] Thus, the formation and lasting of a SFC ex vivo by the hydrogel dPAA and a NS was examined. In particular, fresh resected porcine stomachs were used, and 2 ml of NS or DPAA solution were injected. Protrusions appeared at the injection site and the height changes in submucosal elevation were recorded after 10 seconds, 10 minutes and 1 hour, to cover the whole time of the ESD procedure, which is approximately 40 minutes (FIG. 5d).

[0467] Although both the examined solution and the hydrogel led to the mucosal elevation right after the injection, the hydrogel according to the invention comprising nanocomposite displayed higher mucosal lifting, 8.3 mm vs 6.7 mm, for the dPAA and the NS respectively, with the same amount of solution injected. This showed that already part of the NS solution was absorbed by the surrounding tissues after 10 seconds.

[0468] After the injection of the hydrogel solution, the formation of a solid SFC was detected, which showed only a slight change in size over 1 hour, i.e. from 8.3 mm to 5.8 mm. No significant change in shape or consistency of mucosal lifting was observed. This behavior was due to the formation of the dPAA hydrogel under the submucosa.

[0469] In contrast, the elevation created with NS gradually collapsed, showing a reduction of 37% in size after 10 minutes and of more than half after 1 hour (i.e. height from 6.7 mm to 2.9 mm).

[0470] In short, this example demonstrates the higher performance of the hydrogel of the invention nanocomposite in the formation of a higher and longer lasting mucosal elevation.

[0471] In Vivo ESD Procedure (Endoscopic Submucosal Dissection) A feasibility study to evaluate the in vivo efficacy of hydrogel according to the invention (dPAA) in a living pig was performed. We first set appropriate lesion sizes of approx. 3 cm in diameter in the porcine stomach and then 8-10 ml of the hydrogel solution were injected in the submucosa.

[0472] The ESD procedure was performed in triplicate in different areas of the same porcine stomach; NS solution was used as control.

[0473] In FIG. 6a is reported the endoscopic view of the stomach at time 0 before the injection of the pre-hydrogel solution. It shows the set of an appropriate lesion of approx. 3 cm in diameter.

[0474] In all the cases the injected hydrogel solutions according to the invention in the submucosa led to the gelation of the material in 3 minutes, which thus formed a clear and stable mucosal elevation (FIG. 6b,c).

[0475] A comparison with the normal saline solution generally used showed initially no significant difference compared to the SFC formed by hydrogel of the invention (dPAA). However, the elevation of the SFC formed by NS had obviously reduced after 15 min, due to quick diffusion of the NS at the target site and absorption of the liquid by the tissue, thus it was necessary to repeated the injections to keep the lifted submucosa and be able to finish the surgery.

[0476] In contrast, the mucosal lifting obtained with dPAA allowed the surgeon to perform the entire ESD procedure (40 min) without requiring a second injection, therefore significantly simplifying the procedure and avoiding large injection of liquids.

[0477] The presence of the hydrogel allowed the use of the common electrocautery settings and the long-lasting conservation of the mucosal elevation created with the dPAA enabled the surgeon to smoothly accomplish the circumferential submucosal resection (FIG. 6d). This demonstrate that no significant complications or perforation occurred during the procedure due to the reliable and long-lasting mucosal lifting achieved with the dPAA.

[0478] Then, the lesion was dissected en bloc without any sign of mucosal or muscularis damage, confirming that the dPAA hydrogel formed in situ was able to dissect the submucosal layer (FIG. 6e). Intact mucosal specimens were conveniently achieved via the performed ESD procedures. This is essential, as a definite resection provides accurate histological assessment and thus, can reduce the risk of neoplastic recurrence.

[0479] The same procedure was performed in the colon and in the esophagus with excellent results too.

[0480] Part of the hydrogel stayed under the resected mucosa was observed. In this way a protecting layer of hydrogel was obtained onto the newly formed cavity (FIG. 6f).

[0481] The possibility of releasing an active component from the dPAA during its degradation is highly beneficial at the end of such delicate procedure. Biomolecules, such as adrenaline, proton-pump inhibitors or antibiotics could potentially be efficiently delivered to assist the cauterization of the resected tissue or the prevention of inflammations.

[0482] Moreover, the design of the nanocomposite hydrogel enhances the versatility of the system, enabling the selection of different possible releasing factors, personalized in relation to the patient's requirements.

[0483] The reported non-survival animal study was conducted to examine the formation of SFC from the dPAA in vivo and the feasibility of ESD procedure with the novel material.

CONCLUSION

[0484] A hydrogel of the invention, in particular a degradable nanocomposite hydrogel was successfully developed by embedding breakable nanocapsules into a disulfide-containing polyamidoamines-based hydrogel.

[0485] In addition, a degradable nanocomposite hydrogel was successfully developed by embedding breakable nanocapsules into a disulfide-containing polyamidoamines-based hydrogel.

[0486] The example demonstrate that disulfide bonds of the embedded BNCs and of the network can be completely cleaved in 3 days when the hydrogel is incubated in a GSH solution mimicking the extra-cellular concentration (10 M).

[0487] Most importantly, an example of hydrogel according to the invention sustained the proliferation of HDFa and underwent complete degradation in response to cell-secreted molecules from HDFa seeded onto the scaffold without any external stimulus.

[0488] The degradation of the nanocomposite allowed the release of a model protein encapsulated into the BNCs.

[0489] Advantageously, the obtained hydrogel according to the invention can be delivered to the desired tissue, for example by facile injection through a 23-gauge needle. Its applicability in-vivo models was proved: the aqueous hydrogel solution was injected in the submucosa of a porcine stomach in vivo. It formed an elastic hydrogel in 3 minutes most likely due to temperature increase and interaction with collagen fibers present in the submucosal layer of the mammals.

[0490] Such an important result demonstrates that the hydrogel according to the invention is a novel injection agent, for example subcutaneous, for example for use in ESD.

[0491] The example also demonstrate that the hydrogel formed a reliable SFC in vivo, enabling a long-lasting mucosal elevation, which was superior to commonly used NS. This facilitated en bloc resection of the lesion, which was successfully accomplished with just a single injection.

[0492] No perforation, major bleeding or tissue damage were observed during ESD. Moreover, part of the in situ formed hydrogel adhered tightly to the muscolaris, under the resected mucosa, allowing protection of the membrane during the procedure and after it.

[0493] The results demonstrate a slow degradation kinetics and parallel release of the chosen active molecule in vitro throughout a period of 72 hours, triggered by the proliferation of cells into the scaffold.

[0494] Thus, a similar behavior is obtained in vivo, allowing the release of antibiotics, drugs or proteins such as that could assist the healing of the resected tissue during the degradation of the material, and finally the complete clearance of the scaffold.

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