Title:  Temperature-controlled pH-dependent formation of ionic polysaccharide gels

United States Patent:  6,344,488

Assignee:  Bio Syntech (Laval, CA)

Filed:  February 12, 1999

Foreign Application Priority Data:  Aug 04, 1997[CA] (2212300)

The present invention relates a temperature-controlled pH-dependant formation of ionic polysaccharide gels, such as chitosan/organo-phosphate aqueous systems, and methods of preparation thereof. While chitosan aqueous solutions are pH-dependant gelating systems, the addition of a mono-phosphate dibasic salt of polyol or sugar to a chitosan aqueous solutions leads to further temperature-controlled pH-dependant gelation. Solid organo-phosphate salts (1-20% w/v) are added and dissolved at low temperature (10^oC.) within 0.5 to 4.0% w/v chitosan in aqueous acidic solutions. Aqueous chitosan/organo-phosphate solutions are initially stored at low temperatures (4^oC.), then endothermally gelated within the temperature range of 30 to 60^oC. Chitosan/organo-phosphate solutions rapidly turn into gels at the desired gelation temperature. Gelation can be ex vivo within any receivers or molds, or in situ in animals or humans (in vivo) so as to fill a tissue defect or cavity.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a temperature-controlled pH-dependant formed polysaccharide gel which could be used to encapsulate cells and cellular material while retaining their biological activity.

Another aim of the present invention is to provide a polysaccharide gel which would retain its solid or gel state at the physiological temperature or 37^oC.

Another aim of the present invention is to provide a method for the preparation of such a polysaccharide gel.

In accordance with the present invention there is provided a polysaccharide based gel which comprises:

a) 0.1 to 5.0% by weight of chitosan or a chitosan derivative; and

b) 1.0 to 20% by weight of a salt of polyol or sugar selected from the group consisting of mono-phosphate dibasic salt, mono-sulfate salt and a mono-carboxylic acid salt of polyol or sugar;

wherein said gel is induced and stable within a temperature range from 20 to 70^oC. and is adapted to be formed and/or gelated in situ within a tissue, organ or cavities of an animal or a human.

The salt may be any of the following or in any of the following combination:

a) a mono-phosphate dibasic salt selected from the group consisting of glycerol, comprising glycerol-2-phosphate, sn-glycerol 3-phosphate and L-glycerol-3-phosphate salts;

b) a mono-phosphate dibasic salt and said polyol is selected from the group consisting of histidinol, acetol, diethylstilbestrol, indole-glycerol, sorbitol, ribitol, xylitol, arabinitol, erythritol, inositol, mannitol, glucitol and a mixture thereof;

c) a mono-phosphate dibasic salt and said sugar is selected from the group consisting of fructose, galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fuculose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose, threose, xylulose, hexose, methylthio-ribose, methylthio-deoxy-ribulose, and a mixture thereof;

d) a mono-phosphate dibasic salt and said polyol is selected from the group consisting of palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, arachidonoyl-glycerol, and a mixture thereof; and

e) glycerophosphate salt is a selected from the group consisting of glycerophosphate disodium, glycerophosphate dipotassium, glycerophosphate calcium, glycerophosphate barium and glycerophosphate strontium.

A preferred gel in accordance with one embodiment of the present invention is selected from the group consisting of chitosan-.beta.-glycerophosphate, chitosan-.alpha.-glycerophosphate, chitosan-glucose-1-glycerophosphate, and chitosan-fructose-6-glycerophosphate.

The solid particulate or water-soluble additives may be incorporated within said polysaccharide gel prior to the gelation.

The drugs, polypeptides or non-living pharmaceutical agents may be incorporated within said polysaccharide gel prior to the gelation.

The living microorganisms, plant cells, animal cells or human cells may be encapsulated within said polysaccharide gel prior to the gelation.

The gel may be formed in situ sub-cutaneously, intra-peritoneally, intra-muscularly or within biological connective tissues, bone defects, fractures, articular cavities, body conduits or cavities, eye cul-de-sac, or solid tumors.

The gel of the present invention may be used as a carrier for delivering pharmaceutical agents in situ.

In accordance with the present invention there is also provided a method for producing a polysaccharide gel solution of the present invention, which comprises the steps of:

a) dissolving a chitosan or a chitosan derivative within an aqueous acidic solution of a pH from about 2.0 to about 5.0 to obtain an aqueous polysaccharide composition having a concentration of 0.1 to 5.0% by weight of a chitosan or of a chitosan derivative;

b) dissolving 1.0 to 20% by weight of a salt of polyol or sugar, wherein said salt is selected from the group consisting of mono-phosphate dibasic salt, mono-sulfate salt and a mono-carboxylic acid salt, in said aqueous polysaccharide composition of step a) to obtain a polysaccharide gel solution, wherein said polysaccharide gel has a concentration of 0.1 to 5.0% by weight of a chitosan or a chitosan derivative, and a concentration of 1.0 to 20% by weight of a salt of a polyol or sugar, and has a pH from about 6.4 to about 7.4.

This method may further comprises a step c) after step b),

c) heating said polysaccharide gel solution at a solidifying temperature ranging from about 20^oC. to about 80^oC. until formation of a polysaccharide gel.

A pharmaceutical agent may be added to the polysaccharide gel solution of step b).

The method may further comprises a step i) after step b),

i) dispensing for gelation the polysaccharide gel solution into a desired receiver, either in a mold or within a tissue, organ or body cavity.

The aqueous acidic solution may be prepared from organic or inorganic acids selected from the group consisting of acetic acid, ascorbic acid, salicylic acid, phosphoric acid, hydrochloric acid, propionic acid, formic acid, and a mixture thereof.

The polysaccharide gel solution may be kept in a stable ungelled liquid form at a temperature ranging from about 0^oC. to about 20^oC.

The solidifying temperature is ranging from about 37^oC. to about 60^oC., preferably about 37^oC.

The molecular weight of chitosan is ranging from about 10,000 to 2,000,000.

The polysaccharide gel is thermoirreversible or thermoreversible by adjusting the polysaccharide gel pH, when the pH of said polysaccharide gel solution is >6.9, or when the pH of said polysaccharide gel solution is <6.9.

The solid particulate additives may be added to the polysaccharide gel solution of step b).

The polysaccharide gel solution may be introduced within an animal or human body by injection or endoscopic administration, and gelled in situ at a temperature of about 37^oC.

In accordance with the present invention there is also provided the use of the polysaccharide gel for producing biocompatible degradable materials used in cosmetics, pharmacology, medicine and/or surgery.

The gel may be incorporated as a whole, or as a component, into implantable devices or implants for repair, reconstruction and/or replacement of tissues and/or organs, either in animals or humans.

The gel may be used as a whole, or as a component of, implantable, transdermal or dermatological drug delivery systems.

The gel may be used as a whole, or as a component of, opthalmological implants or drug delivery systems.

The gel may be used for producing cells-loaded artificial matrices that are applied to the engineering and culture of bioengineered hybrid materials and tissue equivalents.

The loaded cells may be selected from the group consisting of chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligament), skin fibroblasts (skin), tenocytes (tendons), myofibroblasts (muscle), mesenchymal stem cells and keratinocytes (skin).

The cells-loaded gel and derived products are devoted to the culture and engineering of artificial articular cartilage and cartilaginous tissues and organs, either for surgical or laboratory testing applications.

The cells-loaded gel and derived products are devoted to the processing and engineering of living artificial substitutes for ligaments, tendons, skin, bone muscles and any metabolic organs, either for surgical or laboratory testing applications.

The cells-loaded gel and derived products are applied as living substitutes for the replacement of articular cartilages, fibrocartilages, cartilaginous organs, ligaments, tendons, bone tissues or skin.

The cells-loaded hydrogel is gelated in situ to induce an ectopic formation of fibrocartilage-like or cartilage-like tissues.

In accordance with the present invention there is also provided the use of loaded polysaccharide gel as injectable or implantable gel biomaterials which act as supports, carriers, reconstructive devices or substitutes for the formation in situ of bone-like, fibrocartilage-like or cartilage-like tissues at a physiological location of an animal or a human.

The polysaccharide gel solution may be used for producing a derived gel or hydrogel by 1) incorporating and dissolving at least one complementary polymer within said polysaccharide gel solution, and 2) by allowing said polysaccharide and complementary polymer to interact for a sufficient period of time to turn into a clear three-dimensional gel within a temperature range between 20^oC. to 60^oC.

The complementary polymer is a non-ionic water-soluble polysaccharide or a hydroxyalkyl cellulose.

For the purpose of the present invention the following terms and expressions are defined below.

The term "polysaccharide gel solution" is intended to mean a polysaccharide solution in a stable ungelled liquid form at a temperature ranging from about 0^oC. to about 15^oC. which can be gelated or changed to a gel state when heated at the gelating temperature.

The term "gelating temperature" is intended to mean any temperature ranging from about 20^oC. to about 80^oC., preferably between 37^oC. to about 60^oC., and more preferably at about the physiological temperature or 37^oC.

The expression "salts of polyols or sugars" is intended to mean mono-phosphate di-basic salts, mono-sulfate salts-and mono-carboxylic acid salts of polyols or sugars.

The present invention include method of forming different gelated materials, those materials being either molded (customized shapes, tubes, membranes, films . . . ) or formed in situ within biological environments (filling of tissue defects).

In a preferred embodiment, the chitosan/organo-phosphate aqueous solution has a pH above the pKa of chitosan and turn into solid gel upon thermal stimulation. This polysaccharide gel can be used as a carrier for drugs or as a non-living therapeutics delivery system, as substituting materials for tissues and organs and as encapsulants for living cells or microorganisms. Chitosan/organo-phosphate gel matrices are rapidly formed at temperatures between 30 to 60^oC. Chitosan/organo-phosphate aqueous systems are used as injectable filling materials, injected and gelated in situ for filling and repairing tissue defects.

Glycerol-2-phosphate, glycerol-3-phosphate and glucose-1-phosphate based salts are the preferred disclosed salts in accordance with the present invention.

Chitosan/polyol- or sugar-phosphate and chitosan/polyol- or sugar-sulfate gels can be applied to surgical reconstructive and regeneration uses and drug delivery purposes. They provide thermally reversible or irreversible bioerodible polymeric gels with biologically well-known and compatible components for a broad range of medical/biotechnological applications.

Claim 1 of 28 Claims

What is claimed is:

1. A polysaccharide based gel solution which comprises:

a) 0.1 to 5.0% by weight of chitosan; and

b) 1.0 to 20% by weight of a salt of polyol or sugar selected from the group consisting of monophosphate dibasic salt of polyol or sugar;

wherein said gel solution is a solution at pH between 6.5 and 7.4 at a temperature below 20^oC., and forms a gel within a temperature range from 20 to 70^oC.

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