US 20100028434A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0028434 A1 Chenite et al. (43) Pub. Date: Feb. 4, 2010

(54) TEMPERATURE CONTROLLED AND PH Publication Classification DEPENDENT SELF GELLING (51) Int. Cl. BIOPOLYMERICAQUEOUS SOLUTION A6II 35/12 (2006.01) A6II 47/26 (2006.01) (75) Inventors: Abdellatif Chenite, Kirkland (CA); A6II 47/08 (2006.01) Cyril Chaput, Montreal (CA); A6IR 9/00 (2006.01) DongWang, Montreal (CA); A6II 35/32 (2006.01) Amine Selmani, Laval (CA) A6II 35/34 (2006.01) A6II 35/36 (2006.01) Correspondence Address: A6II 35/56 (2006.01) DAVID S. RESNICK A6II 35/66 (2006.01) NIXON PEABODY LLP, 100 SUMMER STREET A636/00 (2006.01) BOSTON, MA 02110-2131 (US) (52) U.S. Cl...... 424/485; 514/777: 514/774; 424/93.1; (73) Assignee: BIOSYNTECH CANADA, INC., 424.793.7 Laval (CA) (57) ABSTRACT (21) Appl. No.: 12/576,354 The present invention relates a biopolymeric liquid aqueous composition for producing self-gelling systems and gels, (22) Filed: Oct. 9, 2009 which comprises: an acidic water-based medium, 0.1 to 10% by weight of a pH-gelling acid-soluble biopolymer; and 0.1 to Related U.S. Application Data 10% by weight of a water-soluble molecule having a basic character and a pKa between 6.0 and 8.4, or a water-soluble (63) Continuation of application No. 10/130,316, filed on residue or sequence of the molecule having a basic character Aug. 27, 2002, now abandoned, filed as application and a pKa between 6.0 and 8.4. The liquid composition has a No. PCT/CA00/01341 on Nov. 10, 2000. final pH ranging from 5.8 and 7.4, and forms a stable solid and homogeneous gel within a temperature range from 10 to 70° (60) Provisional application No. 60/165,641, filed on Nov. C. The present invention also relates to a method for preparing 15, 1999. the composition and uses thereof. Patent Application Publication Feb. 4, 2010 Sheet 1 of 4 US 2010/0028434 A1

Heating

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NB2% + BES2% at room temperature 450

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TEMPERATURE CONTROLLED AND PH 0013. A further aim of the present invention is to provide DEPENDENT SELF GELLING gels that would retain its solid or gel State at the physiological BIOPOLYMERICAQUEOUS SOLUTION temperature or 37° C. 0014 Still one aim of the present invention is to provide a BACKGROUND OF THE INVENTION method for the preparation of Such gels. 0001 (a) Field of the Invention 0015. In accordance with the present invention, there is 0002 The present invention relates to the composition of provided a biopolymeric liquid aqueous composition for pro molecular assemblies in liquid Solution that enables tempera ducing self-gelling systems and gels, which comprises: ture-controlled pH-dependant formation of biopolymeric 0016 a) an acidic water-based medium; and gels, such as polysaccharide-based, and methods of prepara 0017 b) 0.1 to 10% by weight of a pH-gelling acid tion thereof. soluble biopolymer; and 0003) (b) Description of Prior Art (0.018 c) 0.1 to 10% by weight of a water-soluble mol 0004 Biopolymers and macromolecules are attractive ecule having a basic character and a pKa between 6.0 materials for the preparation and design of self-gelling and/or and 8.4, or a water-soluble residue or sequence of the auto-assembling systems. Numerous attempts tend to molecule having a basic character and a pKa between develop such systems on the basis of polysaccharides and 6.0 and 8.4: polypeptides. wherein the liquid composition has a final pH ranging from 0005. In situ formed gels were also proposed with ionic 5.8 and 7.4, and forms a stable solid and homogeneous gel polysaccharides. A composition can be used as a medical within a temperature range from 10 to 70° C. device for drug delivery, the application of a diagnostic agent, 0019. The composition can be prepared from organic and/ or the prevention of post-operative adhesions, and is com or inorganic acids, such as hydrochloric acid, citric acid, posed of an aqueous liquid vehicle which is capable of being ascorbic acid, lactic acid, lactobionic acid, acetic acid, Sali gelled in situ. It includes at least one ionic polysaccharide, at cylic acid, formic acid, glutamic acid, phosphoric acid, ortho least one film forming polymer, and a medicament or phar phosphoric acid, or glycerophosphoric acid, or a mixture maceutical agent, water, and optionally, a counter-ion capable thereof. of gelating the ionic polysaccharide. However, the gelation is 0020. The biopolymer preferably comprises a pH-gelling reached by interaction between the ionic polysaccharide and acid-soluble polysaccharide, polypeptidic or poly(amino the film-forming polymer, or by counter-ion induced cross acids), or synthetic polymer, such as a solution of chitosan, linking of the ionic polysaccharide. Other in situ forming gels modified chitosan or chitosan derivative, the solution of chi are based upon polyoxyalkylene composition or polyoxy tosan being cationic and bearing amino groups. alkylene/polysaccharide mixture or alginate/cation mixture 0021. The molecule, residue or sequence may be an in situ. organic salt selected from the group consisting of mono 0006. It would be highly desirable to be provided with a phosphate salt, mono-Sulfonate salt, mono-sulfate Salt and biopolymeric gel that is formed while excluding any organic mono-carboxylate salt. Solvent, any organic monomers, any ionic or covalent cross 0022. Alternatively, the molecule, residue or sequence linking that may be potentially toxic or induce a reduced may be a salt of polyol selected from the group consisting of biological compatibility. mono-phosphate dibasic salt, mono-Sulfonate salt, mono-sul 0007. It would be highly desirable to be provided with a fate salt and mono-carboxylate salt of polyol, said polyol biopolymeric gel that is formed by stimulus-induced free being selected from the group consisting of glycerol, histidi interactions between biologically acceptable and well-recog nol, acetol, diethylstil-bestrol, indole-glycerol, sorbitol, ribi nized molecules. tol. Xylitol, arabinitol, erythritol, inositol, mannitol, glucitol, 0008. It would be highly desirable to be provided with, a palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, and temperature-controlled pH-dependant formed biopolymeric arachidonoyl-glycerol, or a mixture thereof. gels that could be used to encapsulate cells and cellular mate 0023 The glycerol may also be selected from the group rial while retaining their biological activity. consisting of glycerol-2-phosphate, sn-glycerol 3-phosphate 0009. It would be highly desirable to be provided with Such gels, which would retain its Solid or gel state at the and L-glycerol-3-phosphate salt, or a mixture thereof. physiological temperature or 37°C. 0024. In a further embodiment, the molecule, residue or sequence is a salt of a Sugar selected from the group consist ing of mono-phosphate dibasic salt, mono-Sulfonate salt, SUMMARY OF THE INVENTION mono-Sulfate salt and mono-carboxylate salt of a Sugar, said 0010. One aim of the present invention is to provide a way Sugar being selected from the group consisting of fructose, allowing the preparation of a neutral clear liquid solution of a galactose, ribose, glucose, Xylose, rhamnulose, Sorbose, pH-controlled acid-soluble biopolymer while avoiding any erythrulose, deoxy-ribose, ketose, mannose, arabinose, fucu unwanted precipitation or heterogeneous gelation. lose, fructopyranose, ketoglucose, Sedoheptulose, trehalose, 0011. A second major aim of the present invention is to tagatose. Sucrose, allose, threose, Xylulose, hexose, meth provide a neutral clear liquid solution of a pH-controlled ylthio-ribose, and methylthio-deoxy-ribulose, or a mixture acid-soluble biopolymer that will thermally form solid homo thereof. geneous gels at a temperature close to the physiological tem 0025. The molecule, residue or sequence may be selected perature. from the group consisting of sodium, magnesium or iron salt 0012 Another aim is to provide temperature-controlled of glycerol-2-phosphate, sn-glycerol-3-phosphate and pH-dependant formed gels, which could be used to encapsu L-glycerol-3-phosphate, glucose-1-phosphate, glucose-6- late cells and cellular material while retaining their biological phosphate, fructose-1-phosphate and fructose-6-phosphate, activity. or a mixture thereof. US 2010/0028434 A1 Feb. 4, 2010

0026. The molecule, residue or sequence is preferably a a) dissolving a pH-gelling acid-soluble biopolymer within an Sodium, magnesium or iron salt selected from the group con aqueous acidic solution of a pH from about 1.0 to about 5.0 to sisting of N-carbamoylmethyl-2-aminoethane Sulfonate obtain an aqueous biopolymer composition having a concen (ACES), N.N-bis(2-hydroxyethyl-2-aminoethane sulfonate tration of 0.1 to 5% by weight of the biopolymer; (BES), 3-N,N-bis(2-hydroxyethyl)amino-2-hydroxypro b) dissolving 0.1 to 10% by weight of a water-soluble mol panesulfonate (DIPSO), N-(2-hydroxyethylpiperazine-N'- ecule having a basic character and a pKa between 6.0 and 8.4. 3-propane-sulfonate (E PPS), N-2-hydroxyethylpipera ora water-soluble residue or sequence of the molecule having Zine-N'-4-butane-sulfonate (HE PBS), N-2-hydroxyethyl a basic character and pKa between 6.0 and 8.4, within the piperazine-N'-3-propanesulfonate (HEPES), N-2- aqueous biopolymer composition to obtain a clear liquid for hydroxyethylpiperazine-N'-2-hydroxypropanesulfonate mulation with a pH ranging between 5.8 and 7.4: (HEPSO), 2-N-morpholinolethanesulfonate (MES), 4-N- c) heating the liquid formulation at a temperature above 30° morpholinobutanesulfonate (MOBS), 3-N-morpholinobu C. to obtain a solid gel, the gel having a pH from about 5.8 to tanesulfonate (MOPS), 3-N-morpholino-2-hydroxypro about 7.4. panesulfonate (MOPSO), piperazine-N,N'-bis(2- 0035. The composition of the present invention may be ethanesulfonate (PIPES), piperazine-N,N'-bis(2- used in cosmetics, pharmacology, medicine and/or Surgery, hydroxypropanesulfonate (POPSO), 3-N-tris into an implantable device or an implant for repair, recon (hydroxymethyl)methylamino-2-hydroxypropanesulfonate struction and/or replacement of tissues and/or organs, as an (TAPSO), and N-tris hydroxymethylmethyl-2-minoethane implantable, transdermal or dermatological drug delivery sulfonate(TES), and derivatives or mixtures thereof. system, as an opthalmological implant or a drug delivery 0027. The molecule, residue or sequence is preferably system, or in cells-loaded artificial matrices for engineering selected from the group consisting of N,N-bishydroxyethyl and culture of bioengineered hybrid materials and tissue glycine (BICINE), bis2-hydroxyethyliminotris hydroxym equivalents. ethylmethane (BIS-TRIS), Glycyl-glycine (GLY-GLY), Tri 0036. The composition may be loaded with cells selected ethanolamine (TEA), N-tris hydroxymethylmethylglycine from the group consisting of chondrocytes (articular carti (TRICINE), and Tris hydroxymethylaminomethane lage), fibrochondrocytes (meniscus), ligament fibroblasts (TRIZMA), and derivatives or mixtures thereof. (ligament), skin fibroblasts (skin), tenocytes (tendons), myo 0028. Still in another embodiment, the molecule, residue fibroblasts (muscle), mesenchymal stem cells and kerati or sequence has either one acid group and at least one amino nocytes (skin). Such composition may be used in culturing group, or more amino groups than acid groups. The molecule, and engineering of artificial articular cartilage and cartilagi residue or sequence may also be an amino-acid residue, an nous tissues and organs, either for Surgical or laboratory amino-acid sequence or a poly(amino acids) having a basic testing applications. character and a pKa between 6.0 and 8.4. 0037. The composition of the present invention may also 0029. Examples of amino acid residue can be histidine be used in processing and engineering of living artificial (HIS), arginine (ARG), lysine (LYS), asparagine (ASP), and Substitutes for ligaments, tendons, skin, bone muscles and glutamine (GLN), or a mixture thereof. The amino acid resi any metabolic organs, either for Surgical or laboratory testing due may further be modified with a radical acetyl, t-butyl, applications, in living Substitutes for the replacement of benzyl, benzoyl, ethyl, formyl, or methyl. articular cartilages, fibrocartilages, cartilaginous organs, 0030 The molecule, residue or sequence is alternatively a ligaments, tendons, bone tissues or skin, to induce an ectopic sequence, derivative or polymer of at least one amino acid formation offibrocartilage-like or cartilage-like tissues, as an selected from the group consisting of alanine (ALA), histi injectable or implantable gel biomaterial which acts as Sup dine (HIS), arginine (ARG), lysine (LYS), aspartic acid ports, carriers, reconstructive devices or substitutes for the (ASP), glutamine (GLN), glycine (GLY), hydroxyproline formation in situ of bone-like, fibrocartilage-like or cartilage (HYP), isoleucine (ILE), leucine (LEU), norleucine (NLE), like tissues, and/or in cosmetics, pharmacology, medicine phenylalanine (PHE), proline (PRO), serine (SER), threonine and/or Surgery. (THR), tyrosine (TYR), and valine (VAL). 0038. For the purpose of the present invention the follow 0031 Preferably, the composition further comprises at ing terms and expressions are defined below. least one other water-soluble polymer, such as collagen, 0039. The term “gelating temperature' is intended to methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cel mean any temperature ranging from about 20°C. to about 80° lulose, hydroxyethyl propylcellulose, hydroxymethyl propyl C., preferably between 30° C. and 60° C., and more prefer cellulose, polyethylene oxide, polypropylene oxide, poly ably at about the physiological temperature or 37° C. (ethylene oxide-co-propylene oxide) copolymers, poly(eth 0040. The term “pH-controlled acid-soluble biopolymer ylene oxide-co-propylene oxide-co-ethylene oxide) copoly refers to a biological polymer that is solubilized in an acidic mers, polyvinyl alcohol, or polycaprolactone diols, and aqueous medium, and precipitates or gels heterogeneously derivatives or mixtures thereof. when the pH is increased. For example, chitosan is dissolved 0032. The composition of the present invention may fur in acid/water Solution at pH about 4.0, and precipitates or gels ther comprises a solid particulate or a water-soluble additive, heterogeneously when the chitosan Solution is neutralized at Such as a drug or a pharmaceutical agent, microorganisms, pHs above 6.2. plant cells, animal cells or human cells dispersed therein. 0041. The expression “three-dimensional refers hereinto 0033. The composition of the present invention may be the fact that the polymeric solution is simultaneously gelated used as a carrier for delivering a pharmaceutical agent in situ. and shaped by the mold wherein the solution was initially 0034 Still in accordance with the present invention, there poured. Gels can be produced in glass or plastic bechers, is provided a method for preparing a composition as defined dishes, tubes or between two plates So as to obtain any above. The method comprises the steps of: expected shapes. US 2010/0028434 A1 Feb. 4, 2010

0042. The expression “in situ gelation” refers hereinto the 0054 FIG. 2 illustrates a plot of the elastic modulus G' formation of gels by injecting the liquid solution within spe (Pa) vs. Temperature (Celsius) illustrating the thermal gel cific sites of mammalian or human environments, e.g. any ling/ungelling of chitosan (2% w/v. deacetylation 85%) solu tissues (muscles, bone, ligaments, cartilages) and organs. tion with MOPSO (3.0% w/v) upon cooling/heating: Gelation in situ allows complete and precise filling of tissue 0055 FIG. 3 illustrates a plot of the Elastic Modulus G' defects or body cavities. The gelation of biopolymer mixture (Pa) vs. Temperature (Celsius) illustrating the thermal gel is induced by the physiological temperature. ling/ungelling of chitosan (2% w/v. deacetylation 85%) solu 0043. The expression “endothermal gelation” refers tion with BIS-TRIS (3.0% w/v) upon cooling/heating: herein to the thermal mechanism of the solution, which 0056 FIG. 4 illustrates a plot of the Elastic Modulus G' enables the solution to gelate upon standing at the desired (Pa) vs. Temperature (Celsius) illustrating the thermal gel temperature. Induction of Sol to gel transitions of systems ling/ungelling of chitosan (2% w/v. deacetylation 85%) solu requires energy via, for example, the temperature. tion with MES (8.0% w/v) upon cooling/heating: 0044) The expression “residue' refers herein to a series of 0057 FIG. 5 illustrates a plot of the Elastic Modulus (Pa) biochemical molecules having a common specific chemical VS. Temperature (Celsius) illustrating the thermal gelling/ function. Example: the amino acid residues. ungelling of chitosan (2% w/v. deacetylation 85%) solution 0045. The expression “sequence” refers hereinto the asso with BES (2.0% w/v) upon cooling/heating: ciation of two or several molecules or residues. Example: a 0058 FIG. 6 illustrates a plot of the Turbidimetry (NTU) sequence of amino acid residues (LYS-ASP-PRO-GLY vs. Time illustrating the thermal gelling of chitosan (2% w/v. LYS). deacetylation 85%) solution with GP (8.0% w/v. pH=7.2) and 0046. The expression “basic character” refers hereinto the without GP (pH=5.4) at 37° C.; ability of a chemical molecule in aqueous Solution to capture 0059 FIG. 7 illustrates a plot of the Turbidimetry (NTU) protons (H), thus leading to an increase in pH. vs. Time illustrating the thermal gelling of chitosan (2% w/v. 0047. The expression “cells or cellular matters' refers deacetylation 85%) solution with BES (2.0% w/v) and with herein to living biologicals, such as isolated cells, cellular out BES (pH=5.4) at 37° C.; and dispersion, cell aggregates, cell spheroids or cells adhered to 0060 FIG. 8 illustrates a plot of the Turbidimetry (NTU) Solid microspheres particles, that are encapsulated within the vs. Time illustrating the thermal gelling of chitosan (2% w/v. gels. deacetylation 85%) solution with BIS-TRIS, at different BIS 0048. It is assumed herein that any pH-gelling acid TRIS content, from 2.0 to 4.0% w/v, at 37° C. soluble biopolymers or polymers would behave similarly. As a consequence, the term “biopolymer in the present inven DETAILED DESCRIPTION OF THE INVENTION tion may be replaced by the term “polymer”, “polypeptide', "poly(amino acids)'. 0061. In accordance with the present invention there is 0049. The present invention include method of forming proposed a new gelation mechanism that combines hydrogen different gelated materials, those materials being either bonding, electrostatic interactions and hydrophilic/hydro molded (customized shapes, tubes, membranes, films ...) or phobic interactions. It can only be achieved through complex formed in situ within biological environments (filling of tis interactions between biological macromolecules or synthetic Sue defects). polymers, water molecules and specific biochemical mol 0050. In a preferred embodiment, the self-gelling biopoly ecules having special actions. meraqueous solution has a pH above that for normal precipi 0062. In accordance with the present invention, the con tation, and turn into Solid gel upon thermal stimulation. This cerned biopolymer should be insoluble in water under neutral biopolymer gel can be used as a carrier for drugs or as a conditions pH-7. non-living therapeutics delivery systems, as Substituting 0063 A method is disclosed for preparing a composition materials for tissues and organs and as encapsulants for living which comprises the steps a) of dissolving a pH-gelling acid cells or microorganisms. Gel matrices are rapidly formed at soluble biopolymer within an aqueous acidic Solution of a pH temperatures between 30 to 60° C. Such aqueous systems are from about 1.0 to about 5.0 to obtain an aqueous biopolymer used as injectable filling materials, injected and gelated in situ composition having a concentration of 0.1 to 5% by weight of for filling and repairing tissue defects. said biopolymer, and b) dissolving 0.1 to 10% by weight of a 0051. In a second preferred embodiment, pH-dependant water-soluble molecule having a moderate basic character, or gelling acid water-soluble biopolymers and derivatives hav any water-soluble sequence of said molecule, within said ing a sufficient hydrophilicity are selected for preparing tem aqueous biopolymer composition to obtain a clear liquid for perature-stimulated gels. mulation with a pH ranging between 6.5 and 7.4. The final 0052 Biopolymeric gels can be applied to surgical recon step is the heating of liquid formulation at a temperature structive and regeneration uses and drug delivery purposes. above 30° C. to obtain a solid gel, wherein said gel has a They provide thermally reversible or irreversible bioerodible concentration of 0.1 to 5.0% by weight of said biopolymer, polymeric gels with biologically well-known and compatible and a concentration of 0.1 to 10% by weight of said molecule, components for a broad range of medical/biotechnological and has a pH from about 6.4 to about 7.4. applications. 0064. The aqueous acidic solution is prepared from organic or inorganic acids that are selected from the group BRIEF DESCRIPTION OF THE DRAWINGS consisting of acetic acid, ascorbic acid, glutamic acid, lactic acid, lactobionic acid, Salicylic acid, phosphoric acid, hydro 0053 FIG. 1 illustrates a plot of the Elastic Modulus G' chloric acid, propionic acid, formic acid, and a mixture (Pa) vs. Temperature (Celsius) illustrating the thermal gel thereof. Solubilization of pH-controlled acid-soluble ling/ungelling of chitosan (2% w/v. deacetylation 85%) solu biopolymers in aqueous Solution requires acidic aqueous tion with MOPS (2.0% w/v) upon cooling/heating: Solutions having a pH ranging from 1.0 to 5.0. US 2010/0028434 A1 Feb. 4, 2010

0065. The selected biopolymer is a pH-gelling acid used as tonicity enhancing agents. Glycerol and polyethylene soluble polysaccharide, polypeptidic or poly(amino acids), or glycol are proposed as plasticizers. Polyols (-ol: glycerol, synthetic polymer. Sorbitol . . . ) and Sugars (-ose: fructose, glucose, galactose. . 0066. The preferred polysaccharide is selected from a ... ) were used as thermal stabilizing agents for proteins in group comprising chitosan, modified chitosan or chitosan Solutions Depending on the selected molecules, they were derivative, said chitosan biopolymer being cationic and bear found to make or break structuring of water, create hydrogen ing amino groups. bonding, electrostatic or hydrophobic interacting, and present 0067. The acid-soluble polypeptide is selected from col endothermic transitions Polyols and Sugars stabilize proteins lageneous proteins, preferentially collagen. to heat denaturation through their structuring effect on water 0068 A second polymer, selected from a groups compris and the strengthen of hydrophobic interactions. ing collagen, methyl cellulose, hydroxyethyl cellulose, 0075. The molecule, residue or sequence is preferably a hydroxypropyl cellulose, hydroxyethyl propylcellulose, salt selected in a group comprising N-carbamoylmethyl-2- hydroxymethyl propyl cellulose, polyethylene oxide, aminoethane sulfonate (ACES), N.N-bis(2-hydroxyethyl-2- polypropylene oxide, poly(ethylene oxide-co-propylene aminoethane sulfonate (BES), 3-N,N-bis(2-hydroxy-ethyl) oxide) copolymers, poly(ethylene oxide-co-propylene oxide amino-2-hydroxypropanesulfonate (DIPSO), N-2- co-ethylene oxide) copolymers, polyvinyl alcohol, polyca hydroxyethylpiperazine-N'-3-propanesulfonate (HEPES), prolactone diols, and derivatives, and any mixture thereof, 2-N-morpholinoethanesulfonate (MES), 4-N-morpholino can be incorporated within the biopolymeric solution. butanesulfonate (MOBS), 3-N-morpholinobutanesulfonate 0069. The admixed molecule is required to play a double (MOPS), 3-N-morpholino-2-hydroxypropanesulfonate role: 1) to increase the pH within the biopolymeric solution up (MOPSO), 3-N-tris(hydroxymethyl)methylamino-2-hy the physiological conditions, and 2) to prevent the immediate droxypropanesulfonate (TAPSO), N-tris hydroxymethyl gelation or aggregation. The required molecule, preferen methyl-2-minoethanesulfonate(TES), bis2-hydroxy-ethyl tially selected from organic salts and amino-acids, should iminotris hydroxymethylmethane (BIS-TRIS), have a moderate basic character and a pKa between 6.0 and 3-morpholino 1-1-propanediol, and derivatives, and any mix 7.6. Typically, the selected molecule should have a great ture thereof. sensitivity in terms of hydrophilicity/hydrophobicity (hydro 0076. The molecule, residue or sequence is preferably phobic hydration and dehydration) and thermal sensitivity. selected from amino-acid residues, amino-acid sequences or Such effects are based upon a competition for hydration poly(amino acids) having a basic character and a pKa between apolar and polar groups of said molecule, which between 6.0 and 7.6, preferentially histidine (His). enables the design of molecular machines by free energy 0077. The molecule, residue or sequence is preferably a conversion. sequence, derivative or oligomer of amino acids including 0070. Other preferred molecules, residues or sequences alanine, (ALA), histidine (HIS), arginine (ARG), lysine are organic salts selected from mono-phosphate salts, mono (LYS), aspartic acid (ASP), glutamine (GLN), glycine Sulfonate salts, mono-Sulfate salts or mono-carboxylate salts; (GLY), hydroxyproline (HYP), isoleucine (ILE), leucine said organic salts being water-soluble and having a basic (LEU), norleucine (NLE), phenylalanine (PHE), proline character and a pKa between 6.0 and 7.6. (PRO), serine (SER), threonine (THR), tyrosine (TYR), and 0071. The organic salt is preferably a salt of polyol or valine (VAL). Sugar selected from mono-phosphate dibasic salts, mono 0078. A pharmaceutical or bioactive agent can be added to Sulfonate salts, mono-sulfate salts or mono-carboxylate salts the liquid biopolymer containing solution of step a) or b). It of polyol, said polyol being selected from the group consist can be highly soluble, sparingly soluble or non-soluble in the ing of glycerol, comprising glycerol-2-phosphate, sn-glyc aqueous formulation. Solid particulate additives such as non erol 3-phosphate and L-glycerol-3-phosphate salts, and any polymeric microspheres or nanospheres, mineral or ceramic mixture thereof. Salt of polyol or Sugar are known to greatly granules or powders, can be added to the biopolymer Solution modify the behavior of biopolymeric acidic aqueous solu of step a) or b). tions. 007.9 The mixture can be dispensed for gelation into a 0072 The salt of polyol is preferably selected from mono desired receiver, either in a mold or within a tissue, organ or phosphate dibasic salts, mono-Sulfonate salts, mono-sulfate body cavity. It can be kept in a stable ungelled liquid format salts or mono-carboxylate salts of polyol, said polyol being a temperature ranging from about 0°C. to about 20°C. The selected from a group of polyols comprising histidinol, solidifying temperature is ranging from about 37°C. to about acetol, diethylstil-bestrol, indole-glycerol, sorbitol, ribitol, 60° C., and preferably about 37° C. Xylitol, arabinitol, erythritol, inositol, mannitol, glucitol, 0080 Practically, the mixture is introduced within an ani palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, mal or human body by injection or endoscopic administra arachidonoyl-glycerol, and any mixture thereof. tion, and gelled in situ at a temperature of about 37°C. 0073. The salt of sugar is preferably selected from mono I0081 Table 1 below provides composition of some pre phosphate dibasic salts, mono-Sulfonate salts, mono-sulfate ferred examples for buffering/gelling agents with a 2% w/v. salts or mono-carboxylate salts of Sugar, said Sugar being chitosan solution (deacetylation 85%). selected from a group of Sugars consisting of fructose, galac tose, ribose, glucose, Xylose, rhamnulose, Sorbose, erythru TABLE 1 lose, deoxy-ribose, ketose, mannose, arabinose, fuculose, Concentration Intrinsic Gelling fructopyranose, ketoglucose, Sedoheptulose, trehalose, taga Agent pKa (mM) pH Temperature (C.) tose, Sucrose, allose, threose, Xylulose, hexose, methylthio BES 7.1 85.03 7.1 38.5 ribose, methylthio-deoxy-ribulose, and any mixture thereof. MOPS 7.2 86.5 7.2 32 0074 Polyols are frequently added to compositions for MOPSO 6.9 121.35 7.2 32.2 improving gel properties. Sorbitol and mannitol are currently US 2010/0028434 A1 Feb. 4, 2010

soluble additive is incorporated prior to the gelation, can be TABLE 1-continued administrated topically, directly to the body site to be treated or diagnosed. Anti-bacterial, anti-fungal, Steroidal or non Concentration Intrinsic Gelling steroidal anti-inflammatory, anti-cancer, anti-fibrosis, anti Agent pKa (mM) pH Temperature (C.) viral, anti-glucoma, miotic and anti-cholinergies, anti-psy BIS-TRIS 6.5 1912O 7.15 25.5 chotic, anti-histaminic and decongestant, anesthetic and anti MES 6.1 361.O 7.2 35.5 parasitic agents may be incorporated within the composition and gel. In a similar fashion, non-living pharmaceutical agents may be incorporated within the composition or gel for Formation of Biopolymeric Gels restorative, re-constructive or regenerative purposes. 0086 Living microorganisms, plant cells, animal cells or 0082. A selected biopolymer in powder form is dissolved human cells may be entrapped identically within the biopoly in an aqueous acidic solution until the occurrence of a clear mergel by introduction prior to the gelation. The cells or solution is obtained. The proportion of biopolymer varies micro-organisms loaded gels may be applied to biotechno from 0.5 to 10.0% w/v., preferentially from 1.0 to 3.0% w/v. logical purposes in medicine or in other industrial areas. The pH of the aqueous biopolymer solution ranges from 4.0 Biopolymer in situ forming gels can be formed Sub-cutane to 5.5. Aqueous biopolymer solutions can be sterilized either ously, intramuscularly, intra-peritoneally or within biological by autoclaving or filtration with in-line sterile filters (0.22 connective tissues, bone defects, fractures, articular cavities, micrometer). Freshly-prepared aqueous biopolymer Solu body conduits or cavities, eye cul-de-sac, Solid tumor vascu tions are stored preferably at low positive temperature (4°C.). latures, etc. . . . The added molecule with a moderate basic character is dis I0087. The present invention will be more readily under Solved in water, then admixed to the aqueous biopolymer stood by referring to the following examples, which are given Solutionata temperatures ranging from 4 to 15°C., preferably 10° C. When a clear homogeneous aqueous solution with a to illustrate the invention rather than to limit its scope. pH ranging from 5.8 to 7.0 is attained, the said solution is Example I poured into the desired receiver, and hold to appropriate tem perature to gel. I0088. This example shows typical experiments of acidic 0083. The nature of the acid that is used for the acidic biopolymer Solutions neutralized with an organo-phosphate, biopolymer solutions does not influence fundamentally the preferentially glycerophosphate (GP), and transformed to gel sol to gel transition of the system. The final pH within the upon standing at 37° C. solution is dependent upon the pH of the water/acid solution as well as the biopolymer and molecule concentrations. As Experiment 1: Gelation of Chitosan/GP the biopolymer and molecule are two basic components, they I0089. In typical experiment, 200 mg of chitosan 85% tend to increase the pH of the acidic solution wherein they are deacetylated was dissolved in 8.5 mL of aqueous HCl solu dissolved. Concentrations in this biopolymer and molecule tion (0.1M). The chitosan solution, so obtained had a pH of can be balanced to reach the appropriate pH of the solution, about 5.0, was cooled down to around 4°C. Then 800 mg of while taking into consideration the solubility limit of both B-glycerophosphate disodium salt pentahydrate dissolved in components, and particularly the one of biopolymer. 1.5 mL of water were added slowly to the chitosan solution, while maintaining the cold temperature. The pH of the result In Situ Formation of Gels ing homogeneous and clear liquid mixture become 7.1. This 0084. The selected molecule tested to be incorporated in mixture was disposed in a glass Scintillation vial in the incu the polymeric solution was histidine, but similar results were bator at 37° C. for 2 hours, enough time to achieve bulk obtained with other amino acids or synthetic molecules hav gelation process. ing similar functions and basic character. In situ gelation of 0090 Similar results were obtained when the f-glycero the biopolymer Solution can be conducted by dispensing the phosphate disodium salt was replaced by the C-glycerophos Solution from a hypodermic syringe. If needed, the Solution phate disodium salt. may be pre-gelated (initiate the thermal gelation) by keeping the Syringe and biopolymer Solution at desired temperature, Experiment 2: Gelation of Collagen/GP ideally 37° C., until the first signs of gelation appear. The 0091 Collagen was isolated from knee joint cartilage of ready-to-gel biopolymer mixture is then administrated so as calf, and was made mainly of type II collagen. An aqueous to fill tissue defects or cavities and complete in situ the gela solution of collagen (2% w/v) was prepared by dissolving 0.2 tion process (at 37° C.). A needle having a gauge of 20 and g of collagen in 8.5 ml of an acetic acid solution with a pH below is ideal material for injection of such gel solution. Body about 3.6. Once a clear solution was obtained, it was cooled cavities and tissue defects act as recipients for the solution, down to about 4°C., and then a cold solution of 800 mg of but the liquid materials remain in an open aqueous environ B-glycerophosphate disodium salt pentahydrate in 1.5 mL of ment. The conformability and diffusability of the biopolymer water was added under continuous stirring. When the result Solutions is dependent upon the Solution and material prop ing neutral Solution (pH 7.2) appeared quite homogeneous erties. Increased viscosity results information in situ of more and clear, it was poured in a Petridish and placed at 37°C. A compact and less conformable gels. homogeneous uniform gel formed within 1 hour. Therapeutic Use and Other Uses of Biopolymeric Gels Experiment 3: Gelation of Chitosan-Collagen/GP 0085. Such a biopolymeric gel as previously described is 0092 Collagen (100 mg) of the same origin (Example 1, an ideal material for drug delivery system. Such a in situ Experiment 2) was first dissolved in 10 ml of an acetic acid gel-like forming vehicle, wherein a solid particulate or water solution (0.1M). Then 100 mg of Chitosan was added to the US 2010/0028434 A1 Feb. 4, 2010

resulting Solution and stirred until all chitosan was com tion was cooled down to around 4°C., after which 400 mg of pletely dissolved. After the whole system was cooled downto BIS-TRIS dissolved in 1.5 mL of cold water was added around 4°C., and 800 mg of 3-glycerophosphate disodium slowly to the cold chitosan Solution under vigorous stirring. salt, dissolved in 1.5 ml of water, was added under continuous The pH of the resulting homogeneous and clear Solution stirring. Once the resulting neutral solution (pH=7.2) was increases to about 7.15. This solution was disposed in a glass perfectly homogeneous and clear, it was poured in a Petridish scintillation vial in the incubator at 37° C. Bulk gelation and placed at 37°C. The gel formed within 1 hour. occurs within 10 minutes. 0099 While the invention has been described in connec Example 2 tion with specific embodiments thereof, it will be understood 0093. This example shows the typical experiments of that it is capable of further modifications and this application acidic biopolymer Solutions neutralized with organo-Sul is intended to cover any variations, uses, or adaptations of the fonate, preferentially N,N-bis(2-hydroxyethyl-2-aminoet invention following, in general, the principles of the invention and including Such departures from the present disclosure as hane Sulfonate (BES), and transformed to gel upon standing come within known or customary practice within the art to at 37° C. which the invention pertains and as may be applied to the Experiment 1: Gelation of Chitosan/BES essential features hereinbefore set forth, and as follows in the Scope of the appended claims. 0094. In this experiment, 200 mg of chitosan 85% What is claimed is: deacetylated was dissolved in 8.5 mL of aqueous HCl solu 1. A polymeric liquid aqueous composition for producing tion (0.1M). The chitosan solution, so obtained had a pH of self-gelling systems and gels, which comprises: about 5.0, was cooled down to around 4°C. Then 200 mg of BES in form of sodium salt was dissolved in 1.5 mL of cold a) an acidic water-based medium; and water and added slowly to the cold chitosan solution under b) 0.1 to 10% by weight of a pH-gelling acid-soluble poly vigorous stirring. The pH of the resulting homogeneous and mer, with the proviso that said polymer is free of chito San and chitosan derivatives; and clear solution increases to about 7.17. This solution was dis c) 0.1 to 10% by weight of a water-soluble molecule having posed in a glass scintillation vial in the incubator at 37° C. a basic character and a pKa between 6.0 and 8.4: Bulk gelation occurs in 10 minutes. wherein said liquid composition has a final pH ranging Experiment 2: Gelation of Collagen/BES from 5.8 and 7.4, and forms a stable solid and homoge neous gel when heated within a temperature range from 0095 Collagen was of the same origin (Example 1, 10 to 70° C. Experiment 2). An aqueous solution of collagen (2% w/v) 2. A composition according to claim 1, wherein said com was prepared by dissolving 200 mg of collagen in 8.5 ml of an position is prepared from organic and/or inorganic acid. acetic acid solution with a pH about 3.6. Once a clear solution 3. A composition according to claim 2, wherein the organic was obtained, it was cooled down to about 4°C., and then a and/or inorganic acid is selected from the group consisting of cold solution of 200 mg of BES in 1.5 mL of water was added hydrochloric acid, citric acid, ascorbic acid, lactic acid, lac under continuous stirring. When the resulting neutral Solution tobionic acid, acetic acid, Salicylic acid, formic acid, glutamic (pH ~7.2) appeared quite homogeneous and clear, it was acid, phosphoric acid, orthophosphoric acid, and glycero poured in a Petri dish and placed at 37° C. A homogeneous phosphoric acid, or a mixture thereof. uniform gel formed within 15 minutes. 4. A composition according to claim 1, wherein said poly mer comprises a pH-gelling acid-soluble polysaccharide, Experiment 3: Gelation of Chitosan-Collagen/BES polypeptidic or poly(amino acids), or synthetic polymer. 0096 Collagen (100 mg) of the same origin (Example 1, 5. A composition according to claim 1, wherein said mol Experiment 2) was first dissolved in 10 ml of an acetic acid ecule is an organic salt selected from the group consisting of solution (0.1M). Then 100 mg of Chitosan was added to the mono-phosphate salt, mono-Sulfonate salt, mono-sulfate salt resulting solution and stirred until all Chitosan was com and mono-carboxylate salt. pletely dissolved. After the whole system was cooled downto 6. A composition according to claim 1, wherein said mol around 4° C., and 200 mg of BES in form of sodium salt ecule is a salt of polyol selected from the group consisting of dissolved in 1.5 ml of coldwater, was added undercontinuous mono-phosphate dibasic salt, mono-Sulfonate salt, mono-sul stirring. Once the Solution was perfectly homogeneous and fate salt and mono-carboxylate salt of polyol, said polyol clear, the liquid mixture was poured in a Petridish and placed being selected from the group consisting of glycerol, histidi at 37°C. The gel formed within 5 minutes. nol, acetol, diethylstil-bestrol, indole-glycerol, sorbitol, ribi tol. Xylitol, arabinitol, erythritol, inositol, mannitol, glucitol, Example 3 palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, and arachidonoyl-glycerol, or a mixture thereof. 0097. This example shows the typical experiments of 7. A composition according to claim 6, wherein the salt of acidic biopolymer solutions neutralized with tertiary glycerol is selected from the group consisting of glycerol-2- hydroxyalkylamine, preferentially bis-2-hydroxyethylimi phosphate, sn-glycerol 3-phosphate and L-glycerol-3-phos notris hydroxymethylmethane (BIS-TRIS), and trans phate salt, or a mixture thereof. formed to gel upon standing at 37° C. 8. A composition according to claim 1, wherein said mol ecule is a salt of a Sugar selected from the group consisting of Experiment 1: Gelation of Chitosan/BIS-TRIS mono-phosphate dibasic salt, mono-Sulfonate salt, mono-sul 0098 Chitosan solution with a pH around 5.0 was pre fate salt and mono-carboxylate Salt of a Sugar, said Sugar pared by dissolving 200 mg of chitosan 85% deacetylated in being selected from the group consisting of fructose, galac 8.5 mL of aqueous HCl solution (0.1M). This chitosan solu tose, ribose, glucose, Xylose, rhamnulose, Sorbose, erythru US 2010/0028434 A1 Feb. 4, 2010

lose, deoxy-ribose, ketose, mannose, arabinose, fuculose, 17. A composition according to claim 1, wherein said com fructopyranose, ketoglucose, Sedoheptulose, trehalose, taga position further comprises at least one other water-soluble tose, Sucrose, allose, threose, Xylulose, hexose, methylthio polymer other than chitosan. ribose, and methylthio-deoxy-ribulose, or a mixture thereof. 18. A composition according to claim 1, wherein said at least one other polymer is selected from the group consisting 9. A composition according to claim 1, wherein said mol of collagen, methyl cellulose, hydroxyethyl cellulose, ecule is selected from the group consisting of sodium, mag hydroxypropyl cellulose, hydroxyethyl propylcellulose, nesium or iron salt of glycerol-2-phosphate, sn-glycerol-3- hydroxymethyl propyl cellulose, polyethylene oxide, phosphate and L-glycerol-3-phosphate, glucose-1- polypropylene oxide, poly(ethylene oxide-co-propylene phosphate, glucose-6-phosphate, fructose-1-phosphate and oxide) copolymers, poly(ethylene oxide-co-propylene oxide fructose-6-phosphate, or a mixture thereof. co-ethylene oxide) copolymers, polyvinyl alcohol, and poly 10. A composition according to claim 1, wherein said mol caprolactone diols, and derivatives or mixtures thereof. ecule is selected from the group consisting of N-carbamoyl 19. A composition according to claim 1, further comprising methyl-2-aminoethane sulfonate (ACES), N,N-bis(2-hy a solid particulate or a water-soluble additive. droxyethyl-2-aminoethane sulfonate (BES), 3-N,N-bis(2- 20. A composition according to claim 1, further comprising hydroxy-ethyl)amino-2-hydroxypropanesulfonate a drug or a pharmaceutical agent. (DIPSO), N-2-hydroxyethylpiperazine-N'-3-propane-sul 21. A composition according to claim 1, further comprising fonate (EPPS), N-(2-hydroxyethylpiperazine-N'-4-butane microorganisms, plant cells, animal cells or human cells dis sulfonate (HEPBS), N-2-hydroxyethylpiperazine-N'-2- persed therein. ethanesulfonate (HEPES), N-(2-hydroxyethylpiperazine 22. A composition according to claim 1, for use as a carrier N'-2-hydroxypropanesulfonate (HEPSO), 2-N-morpholino for delivering a pharmaceutical agent in situ. ethanesulfonate (MES), 4-N-morpholinobutanesulfonate 23. A method for preparing a composition according to (MOBS), 3-N-morpholinopropanesulfonate (MOPS), claim 1, which comprises the steps of 3-N-morpholino-2-hydroxypropanesulfonate (MOPSO), a) dissolving a pH-gelling acid-soluble polymer within an piperazine-N,N'-bis(2-ethanesulfonate (PIPES), piperazine aqueous acidic solution of a pH from about 1.0 to about N,N'-bis(2-hydroxypropanesulfonate (POPSO), 3-N-tris 5.0 to obtain an aqueous polymer composition having a (hydroxymethyl)methylamino-2-hydroxypropanesulfonate concentration of 0.1 to 10% by weight of said polymer, (TAPSO), and N-tris hydroxymethylmethyl-2-aminoet said polymer being free of chitosan and chitosan deriva hanesulfonate (TES), and derivatives or mixtures thereof. tives; 11. A composition according to claim 1, wherein said mol b) dissolving 0.1 to 10% by weight of a water-soluble ecule is selected from the group consisting of N,N-bishy molecule having a basic character and a pKa between droxyethylglycine (BICINE), bis(2-hydroxyethyliminotris 6.0 and 8.4 within said aqueous polymer composition to hydroxymethylmethane (BIS-TRIS), Glycyl-glycine obtain a clear liquid formulation with a pH ranging (GLY-GLY), Triethanolamine (TEA), N-tris hydroxym between 5.8 and 7.4: ethylmethylglycine (TRICINE), and Tris hydroxymethyl c) heating said liquid formulation at a temperature above aminomethane (TRIZMA), and derivatives or mixtures 30° C. to obtain a solid gel, said gel having a pH from thereof. about 5.8 to about 7.4. 24. The method of claim 23, wherein said aqueous acidic 12. A composition according to claim 1, wherein said mol Solution is prepared from an organic or inorganic acid ecule has either one acid group and at least one amino group, selected from the group consisting of acetic acid, ascorbic or more amino groups than acid groups. acid, glutamic acid, lactic acid, lactobionic acid, salicylic 13. A composition according to claim 1, wherein said mol acid, phosphoric acid, hydrochloric acid, propionic acid, and ecule is an amino-acid residue, an amino-acid sequence or a formic acid, or a mixture thereof. poly(amino acids) having a basic character and a pKa 25. The method of claim 23, wherein said molecule is an between 6.0 and 8.4. organic salt selected from the group consisting of a mono 14. A composition according to claim 13, wherein said phosphate salt, a mono-sulfonate salt, a mono-Sulfate salt and amino acid residue is selected from the group consisting of a mono-carboxylate salt. histidine (HIS), arginine (ARG), lysine (LYS), asparagine 26. The method of claim 23, wherein said molecule is a salt (ASN), and glutamine (GLN), or a mixture thereof. of polyol selected from the group consisting of a mono 15. A composition according to claim 1, wherein said mol phosphate dibasic salt, a mono-Sulfonate salt, a mono-Sulfate ecule is an amino acid residue or amino acid derivative salt and a mono-carboxylate Salt of polyol, said polyol being selected from the group consisting of histidine (HIS), argin selected from the group consisting of glycerol, histidinol, ine (ARG), lysine (LYS), aspartic acid (ASP), and glutamine acetol, diethylstil-bestrol, indole-glycerol, sorbitol, ribitol, (GLN), said amino acid residue or amino acid derivative Xylitol, arabinitol, erythritol, inositol, mannitol, glucitol, being modified with a radical acetyl, t-butyl, benzyl, benzoyl, palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, and ethyl, formyl, or methyl. arachidonoyl-glycerol, or a mixture thereof. 16. A composition according to claim 1, wherein said mol 27. The method of claim 26, wherein said salt of glycerol is ecule is a sequence, derivative or polymer of at least one selected from the group consisting of glycerol-2-phosphate, amino acid selected from the group consisting of alanine sn-glycerol 3-phosphate and L-glycerol-3-phosphate salts, or (ALA), histidine (HIS), arginine (ARG), lysine (LYS), aspar a mixture thereof. tic acid (ASP), glutamine (GLN), glycine (GLY), hydrox 28. The method of claim 23, wherein said molecule is a salt yproline (HYP), isoleucine (ILE), leucine (LEU), norleucine of Sugar selected from the group selected from a mono-phos (NLE), phenylalanine (PHE), proline (PRO), serine (SER), phate dibasic salt, a mono-Sulfonate salt, a mono-Sulfate salt threonine (THR), tyrosine (TYR), and valine (VAL). and a mono-carboxylate salt of Sugar, said Sugar being US 2010/0028434 A1 Feb. 4, 2010 selected from the group consisting of fructose, galactose, 38. The method of claim 23, wherein said aqueous polymer ribose, glucose, Xylose, rhamnulose, Sorbose, erythrulose, composition turns into a gel at a temperature above 37°C. deoxy-ribose, ketose, mannose, arabinose, fuculose, fructo 39. The method of claim 23, wherein said polymer solution pyranose, ketoglucose, Sedoheptulose, trehalose, tagatose, further comprises a Solid particulate additive, said solid par Sucrose, allose, threose, Xylulose, hexose, methylthio-ribose, ticulate additive being added to the polymer solution of step and methylthio-deoxy-ribulose, or a mixture thereof. a) or b). 29. The method of claim 23, wherein said molecule is a 40. The method of claim 23, further comprising another Sodium, magnesium or iron salt of glycerol-2-phosphate, water-soluble polymer added to the polymer solution of step sn-glycerol-3-phosphate and L-glycerol-3-phosphate, glu a) or b), said other water-soluble polymer being free of chi cose-1-phosphate, glucose-6-phosphate, fructose-1-phos tosan and chitosan derivatives. phate or fructose-6-phosphate, or a mixture thereof. 41. The method of claim 40, wherein said other polymer is 30. The method of claim 23, wherein said molecule is selected from the group consisting of collagen, methyl cellu selected from the group consisting of N-carbamoylmethyl lose, hydroxyethyl cellulose, hydroxypropyl cellulose, 2-aminoethane sulfonate (ACES), N,N-bis(2-hydroxyethyl hydroxyethyl propylcellulose, hydroxymethyl propyl cellu 2-aminoethane sulfonate (BES), 3-N,N-bis(2-hydroxy lose, polyethylene oxide, polypropylene oxide, poly(ethylene ethyl)amino-2-hydroxypropanesulfonate (DIPSO), N-(2- oxide-co-propylene oxide) copolymers, poly(ethylene oxide hydroxyethylpiperazine-N'-3-propane-sulfonate (EPPS), co-propylene oxide-co-ethylene oxide) copolymers, polyvi N-2-hydroxyethylpiperazine-N'-4-butane-sulfonate nyl alcohol, and polycaprolactone diols, or derivatives (HEPBS), N-2-hydroxyethylpiperazine-N'-2-ethane thereof. sulfonate (HEPES), N-(2-hydroxyethylpiperazine-N'-2-hy 42. The composition of claim 1, wherein said composition droxypropanesulfonate (HEPSO), 2-N-morpholinoethane is loaded with cells selected from the group consisting of sulfonate (MES), 4-N-morpholinobutanesulfonate chondrocytes (articular cartilage), fibrochondrocytes (menis (MOBS), 3-N-morpholinopropanesulfonate (MOPS), cus), ligament fibroblasts (ligament), skin fibroblasts (skin), 3-N-morpholino-2-hydroxypropanesulfonate (MOPSO), tenocytes (tendons), myofibroblasts (muscle), mesenchymal piperazine-N,N'-bis(2-ethanesulfonate (PIPES), piperazine stem cells and keratinocytes (skin). N,N'-bis(2-hydroxypropanesulfonate (POPSO), 3-N-tris 43. A polymeric liquid aqueous composition for producing (hydroxymethyl)methylamino-2-hydroxypropanesulfonate self-gelling systems and gels, which comprises: (TAPSO), and N-tris hydroxymethylmethyl-2-aminoet a) an acidic water-based medium; and hanesulfonate (TES), and derivatives or mixtures thereof. b) 0.1 to 10% by weight of a Ph-gelling acid-soluble poly 31. The method of claim 23, wherein said molecule is mer, and selected from the group consisting of N,N-bishydroxyethyl c) 0.1 to 10% by weight of a water-soluble molecule having glycine (BICINE), bis2-hydroxyethyliminotris hy a basic character and a pKa between 6.0 and 8.4, said droxymethylmethane (BIS-TRIS), Glycyl-glycine (GLY water-soluble molecule being free of salt of polyol and GLY), Triethanolamine (TEA), N-tris hydroxymethyl Sugar, methylglycine (TRICINE), and Tris hydroxymethyl wherein said liquid composition has a final Ph ranging aminomethane (TRIZMA), and derivatives or mixtures from 5.8 and 7.4, and forms a stable solid and homoge thereof. neous gel when heated within a temperature range from 32. The method of claim 23, wherein said molecule has 10 to 70° C. either one acid group and at least one amino group, or more 44. A composition according to claim 43, wherein said amino groups than acid groups. composition is prepared from organic and/or inorganic acid. 45. A composition according to claim 44, wherein the 33. The method of claim 23, wherein said molecule is an organic and/or inorganic acid is selected from the group con amino-acid residue, an amino-acid sequence or a poly(amino sisting of hydrochloric acid, citric acid, ascorbic acid, lactic acids) having a basic character and a pKa between 6.0 and 8.4. acid, lactobionic acid, acetic acid, Salicylic acid, formic acid, 34. The method of claim 33, wherein said amino acid glutamic acid, phosphoric acid, orthophosphoric acid, and residue is selected from the group consisting of histidine glycerophosphoric acid, or a mixture thereof. (HIS), arginine (ARG), lysine (LYS), asparagine (ASN), 46. A composition according to claim 43, wherein said glutamine (GLN), or a mixture thereof. polymer comprises a Ph-gelling acid-soluble polysaccharide, 35. The method of claim 33, wherein said molecule is an polypeptidic or poly(amino acids), or synthetic polymer. amino acid residue oramino acid derivative selected from the 47. A composition according to claim 43, wherein said group consisting of histidine (HIS), arginine (ARG), lysine polymer comprises a solution of chitosan, modified chitosan (LYS), aspartic acid (ASP), and glutamine (GLN) modified orchitosan derivative, said solution of chitosan being cationic with a radical acetyl, t-butyl, benzyl, benzoyl, ethyl, formyl, and bearing amino groups. or methyl. 48. A composition according to claim 43, wherein said 36. The method of claim 23, wherein said molecule is a molecule is an organic salt selected from the group consisting sequence, derivative or polymer of at least one amino acid of mono-phosphate salt, mono-Sulfonate salt, mono-Sulfate selected from the group consisting of alanine (ALA), histi salt and mono-carboxylate salt. dine (HIS), arginine (ARG), lysine (LYS), aspartic acid 49. A composition according to claim 43, wherein said (ASP), glutamine (GLN), glycine (GLY), hydroxyproline molecule is selected from the group consisting of N-carbam (HYP), isoleucine (ILE), leucine (LEU), norleucine (NLE), oylmethyl-2-aminoethane sulfonate (ACES), N,N-bis(2-hy phenylalanine (PHE), proline (PRO), serine (SER), threonine droxyethyl-2-aminoethane sulfonate (BES), 3-N,N-bis(2- (THR), tyrosine (TYR), and valine (VAL). hydroxy-ethyl)amino-2-hydroxypropanesulfonate 37. The method of claim 23, wherein said aqueous polymer (DIPSO), N-2-hydroxyethylpiperazine-N'-3-propane-sul composition turns into a gel at a temperature above 10°C. fonate (EPPS), N-(2-hydroxyethylpiperazine-N'-4-butane US 2010/0028434 A1 Feb. 4, 2010 sulfonate (HEPBS), N-2-hydroxyethylpiperazine-N'-2- 61. A method for preparing a composition according to ethanesulfonate (HEPES), N-(2-hydroxyethylpiperazine claim 43, which comprises the steps of: N'-2-hydroxypropanesulfonate (HEPSO), 2-N-morpholino a) dissolving a pH-gelling acid-soluble polymer within an ethanesulfonate (MES), 4-N-morpholinobutanesulfonate aqueous acidic solution of a pH from about 1.0 to about (MOBS), 3-N-morpholinopropanesulfonate (MOPS), 5.0 to obtain an aqueous polymer composition having a 3-N-morpholino-2-hydroxypropanesulfonate (MOPSO), concentration of 0.1 to 10% by weight of said polymer; piperazine-N,N'-bis(2-ethanesulfonate (PIPES), piperazine b) dissolving 0.1 to 10% by weight of a water-soluble N,N'-bis(2-hydroxypropanesulfonate (POPSO), 3-N-tris molecule having a basic character and a pKa between (hydroxymethyl)methylamino-2-hydroxypropanesulfonate 6.0 and 8.4 within said aqueous polymer composition to (TAPSO), and N-tris hydroxymethylmethyl-2-aminoet obtain a clear liquid formulation with a pH ranging hanesulfonate (TES), and derivatives or mixtures thereof. between 5.8 and 7.4, said water-soluble molecule being 50. A composition according to claim 43, wherein said free of salt of polyol and Sugar, molecule is selected from the group consisting of N,N-bis c) heating said liquid formulation at a temperature above hydroxyethylglycine (BICINE), bis(2-hydroxyethylimi 30° C. to obtain a solid gel, said gel having a pH from notris hydroxymethylmethane (BIS-TRIS), Glycyl-glycine about 5.8 to about 7.4. (GLY-GLY), Triethanolamine (TEA), N-tris hydroxym 62. The method of claim 61, wherein said aqueous acidic ethylmethylglycine (TRICINE), and Tris hydroxymethyl Solution is prepared from an organic or inorganic acid aminomethane (TRIZMA), and derivatives or mixtures selected from the group consisting of acetic acid, ascorbic thereof. acid, glutamic acid, lactic acid, lactobionic acid, salicylic 51. A composition according to claim 43, wherein said acid, phosphoric acid, hydrochloric acid, propionic acid, and molecule has either one acid group and at least one amino formic acid, or a mixture thereof. group, or more amino groups than acid groups. 63. The method of claim 61, wherein said polymer is a 52. A composition according to claim 43, wherein said chitosan or a chitosan derivative, with a degree of deacetyla molecule is an amino-acid residue, an amino-acid sequence tion ranging from 35 to 99%. or a poly(amino acids) having a basic character and a pKa 64. The method of claim 61, wherein said molecule is an between 6.0 and 8.4. organic salt selected from the group consisting of a mono 53. A composition according to claim 52, wherein said phosphate salt, a mono-sulfonate salt, a mono-Sulfate salt and amino acid residue is selected from the group consisting of a mono-carboxylate salt. histidine (HIS), arginine (ARG), lysine (LYS), asparagine 65. The method of claim 61, wherein said molecule is (ASN), and glutamine (GLN), or a mixture thereof. selected from the group consisting of N-carbamoylmethyl 54. A composition according to claim 43, wherein said 2-aminoethane sulfonate (ACES), N,N-bis(2-hydroxyethyl molecule is an amino acid residue or amino acid derivative 2-aminoethane sulfonate (BES), 3-N,N-bis(2-hydroxy selected from the group consisting of histidine (HIS), argin ethyl)amino-2-hydroxypropanesulfonate (DIPSO), N-(2- ine (ARG), lysine (LYS), aspartic acid (ASP), and glutamine hydroxyethylpiperazine-N'-3-propane-sulfonate (EPPS), (GLN), said amino acid residue or amino acid derivative N-2-hydroxyethylpiperazine-N'-4-butane-sulfonate being modified with a radical acetyl, t-butyl, benzyl, benzoyl, (HEPBS), N-2-hydroxyethylpiperazine-N'-2-ethane ethyl, formyl, or methyl. sulfonate (HEPES), N-(2-hydroxyethylpiperazine-N'-2-hy 55. A composition according to claim 43, wherein said droxypropanesulfonate (HEPSO), 2-N-morpholinoethane molecule is a sequence, derivative or polymer of at least one sulfonate (MES), 4-N-morpholinobutanesulfonate amino acid selected from the group consisting of alanine (MOBS), 3-N-morpholinopropanesulfonate (MOPS), (ALA), histidine (HIS), arginine (ARG), lysine (LYS), aspar 3-N-morpholino-2-hydroxypropanesulfonate (MOPSO), tic acid (ASP), glutamine (GLN), glycine (GLY), hydrox piperazine-N,N'-bis(2-ethanesulfonate (PIPES), piperazine yproline (HYP), isoleucine (ILE), leucine (LEU), norleucine N,N'-bis(2-hydroxypropanesulfonate (POPSO), 3-N-tris (NLE), phenylalanine (PHE), proline (PRO), serine (SER), (hydroxymethyl)methylamino-2-hydroxypropanesulfonate threonine (THR), tyrosine (TYR), and valine (VAL). (TAPSO), and N-tris hydroxymethylmethyl-2-aminoet 56. A composition according to claim 43, wherein said hanesulfonate (TES), and derivatives or mixtures thereof. composition further comprises at least one other water 66. The method of claim 61, wherein said molecule is soluble polymer. selected from the group consisting of N,N-bishydroxyethyl 57. A composition according to claim 43, wherein said at glycine (BICINE), bis2-hydroxyethyliminotris hy least one other polymer is selected from the group consisting droxymethylmethane (BIS-TRIS), Glycyl-glycine (GLY of collagen, methyl cellulose, hydroxyethyl cellulose, GLY), Triethanolamine (TEA), N-tris hydroxymethyl hydroxypropyl cellulose, hydroxyethyl propylcellulose, methylglycine (TRICINE), and Tris hydroxymethyl hydroxymethyl propyl cellulose, polyethylene oxide, aminomethane (TRIZMA), and derivatives or mixtures polypropylene oxide, poly(ethylene oxide-co-propylene thereof. oxide) copolymers, poly(ethylene oxide-co-propylene oxide 67. The method of claim 61, wherein said molecule has co-ethylene oxide) copolymers, polyvinyl alcohol, and poly either one acid group and at least one amino group, or more caprolactone diols, and derivatives or mixtures thereof. amino groups than acid groups. 58. A composition according to claim 43, further compris 68. The method of claim 61, wherein said molecule is an ing a solid particulate or a water-soluble additive. amino-acid residue, an amino-acid sequence or a poly(amino 59. A composition according to claim 43, further compris acids) having a basic character and a pKa between 6.0 and 8.4. ing a drug or a pharmaceutical agent. 69. The method of claim 68, wherein said amino acid 60. A composition according to claim 43, further compris residue is selected from the group consisting of histidine ing microorganisms, plant cells, animal cells or human cells (HIS), arginine (ARG), lysine (LYS), asparagine (ASN), dispersed therein. glutamine (GLN), or a mixture thereof. US 2010/0028434 A1 Feb. 4, 2010

70. The method of claim 68, wherein said molecule is an 75. The method of claim 61, further comprising another amino acid residue oramino acid derivative selected from the water-soluble polymer added to the polymer solution of step group consisting of histidine (HIS), arginine (ARG), lysine a) or b). (LYS), aspartic acid (ASP), and glutamine (GLN) modified with a radical acetyl, t-butyl, benzyl, benzoyl, ethyl, formyl, 76. The method of claim 75, wherein said other polymer is or methyl. selected from the group consisting of collagen, methyl cellu 71. The method of claim 61, wherein said molecule is a lose, hydroxyethyl cellulose, hydroxypropyl cellulose, sequence, derivative or polymer of at least one amino acid hydroxyethyl propylcellulose, hydroxymethyl propyl cellu selected from the group consisting of alanine (ALA), histi lose, polyethylene oxide, polypropylene oxide, poly(ethylene dine (HIS), arginine (ARG), lysine (LYS), aspartic acid oxide-co-propylene oxide) copolymers, poly(ethylene oxide (ASP), glutamine (GLN), glycine (GLY), hydroxyproline co-propylene oxide-co-ethylene oxide) copolymers, polyvi (HYP), isoleucine (ILE), leucine (LEU), norleucine (NLE), nyl alcohol, and polycaprolactone diols, or derivatives phenylalanine (PHE), proline (PRO), serine (SER), threonine thereof. (THR), tyrosine (TYR), and valine (VAL). 72. The method of claim 61, wherein said aqueous polymer 77. The composition of claim 43, wherein said composi composition turns into a gel at a temperature above 10°C. tion is loaded with cells selected from the group consisting of 73. The method of claim 61, wherein said aqueous polymer chondrocytes (articular cartilage), fibrochondrocytes (menis composition turns into a gel at a temperature above 37°C. cus), ligament fibroblasts (ligament), skin fibroblasts (skin), 74. The method of claim 61, wherein said polymer solution tenocytes (tendons), myofibroblasts (muscle), mesenchymal further comprises a Solid particulate additive, said solid par stem cells and keratinocytes (skin). ticulate additive being added to the polymer solution of step a) or b). c c c c c