GB2358637A

GB2358637A - Chitosan condensation products with a bisulphite addition compound

Info

Publication number: GB2358637A
Application number: GB0001734A
Authority: GB
Inventors: George Andrew Francis Roberts
Original assignee: BTG International Ltd
Current assignee: BTG International Ltd
Priority date: 2000-01-27
Filing date: 2000-01-27
Publication date: 2001-08-01
Also published as: WO2001055220A1; AU2001230333A1; EP1250359A1; GB0001734D0; JP2003523459A; US20030055211A1

Abstract

A condensation product of chitosan is formed by its reaction with a bisulphite addition compound HOCRR'SO<SB>3</SB>A wherein <SL> <LI> $ R and R' independently = H, an optionally substituted C<SB>1</SB>-C<SB>10</SB> hydrocarbon group or an optionally substituted alicyclic, aromatic carbocyclic or heterocyclic ring, or R and R' together = an optionally substituted alicyclic or heterocyclic ring, and <LI> $ A = an alkali metal or ammonium ion. </SL> The condensation product is a polymer comprising monomer units of formulae K, L, M and N: <EMI ID=1.1 HE=59 WI=144 LX=335 LY=1087 TI=CF> <PC>in the proportions <I>k, l, m</I> and <I>n</I>, respectively such that <EMI ID=1.2 HE=13 WI=40 LX=887 LY=1764 TI=CF> <PC>is at least 10% and is most preferably from 45 to 50%. The condensation product is an anionic polyelectrolyte and forms stable anionic solutions in water at neutral pH which may be mixed with other water soluble anionic polyelectrolytes, especially sodium alginate, to form admixtures or blends from which films, fibres, powders, sponges, gels and foams may be prepared. The chitosan may be regenerated from such products by acid or alkali treatment. The solubility of the condensation product also provides a method of extracting chitosan from chitin/calcium carbonate mixtures.

Description

2358637 TITLE Chitosan condensation products, their preparation and their

uses

DESCRIPTION

Technical Field

The invention relates to a condensation product of chitosan formed by its reaction with a bisulphite addition compound. The product is an anionic polyelectrolyte and forms stable anionic solutions in water at neutral pH. Such solutions may be mixed with other water soluble anionic polyelectrolytes, especially sodium alginate, to form very intimate admixtures or blends from which films, fibres, powders, sponges, gels and foams may be prepared. The chitosan may be regenerated from the condensation product in those films, fibres, powders, sponges, gels and foams by acid or alkali treatment during or subsequent to their production. The solubility of the condensation product also opens the way to a novel method for the extraction of chitosan from chitin/calcium carbonate mixtures.

Background of the Invention

Chitosan can be obtained on a commercial scale by deacetylation of the biopolymer chitin in concentrated alkaline solutions at elevated temperatures. Chitin is the main structural polymer in the exoskeletons of crustacea, molluscs and insects and is the principal fibrillar polymer in the cell wall of certain fungi and actinomycetes. The names "chitin" and "chitosan" do not refer to materials having discrete chemical structures but to a continuum of co-polymers of N-acetyl-D-glucosamine residues (K) and Dglucosamine residues (L):

CH 2 OH 0 0 -fHO:::2 J NH NHCOCH3 2 - (K) (L) The two materials may be distinguished by their solubility (chitosan) or insolubility (chitin) in dilute aqueous acid solutions.

Chitosan, being a base, forms a salt with acids so producing cationic polyelectrolyte structures whose solubility in water will depend on the nature of the acid anion involved. Chitosan is soluble in dilute HCI, HBr, HI, HN03 and HC104 but may be precipitated out of solution in HBr or HG by increasing the concentration of acid in the system. Chitosan is slightly soluble in dilute H3P04 but is insoluble in dilute H2S04 at room temperature, although chitosan sulphate is soluble in hot water. Chitosan is also soluble in aqueous solutions of a large number of organic acids. However, chitosan is readily hydrolysed on standing in acid and consequently such solutions have limited shelf lives.

The primary source material for chitin/chitosan extraction are crustacean exoskeletons (available as processing waste from the seafood industry). Current commercial extraction processes include a dernineralisation treatment with hydrochloric acid to remove the calcium carbonate, also present in substantial quantities in the source material. This demineralisation generates high volumes of calcium chloride effluent which must be disposed of in an environmentally safe manner.

Summary of the Invention

The invention provides a condensation product of chitosan and a bisulphite addition compound which has the formula HOCRR'SO3A wherein 0 each of R and R' independently represents a hydrogen atom, an optionally substituted linear or branched saturated or unsaturated hydrocarbon group having up to 10 carbon atoms or an optionally substituted alicyclic, aromatic carbocyclic or heterocyclic ring, or R and R' together represent an optionally substituted alicyclic or heterocyclic ring, and A represents an alkali metal or ammonium ion, which condensation product forms a stable anionic polyelectrolyte solution in water at neutral pH.

A may represent any alkali metal cation, especially lithium, sodium or potassium, or an ammonium ion. Preferably A represents a sodium ion. R and R' preferably represent hydrogen atoms. Where R and R' represent others from the moieties listed above, the preferred optional substituents are carboxy (COOH) and sulpho (S03H) groups as their salts.

It will be appreciated that the pH of distilled water may deviate from the standardized neutral value of 7. In this Specification the term 'neutral pH' is indicative of a value range of from 5 to 9.

Like chitosan itself, the condensation product of the invention does not have a discrete chemical structure. Rather, it is a continuum of monosubstituted D-glucosamine residues (M) and di-substituted D-glucosamine residues (N) C 20H CH OH_ 0 0 __O 0 2 HO:::

NHCRR'SO3A H05 N(CRR' S03A)2 J (M) (N) and includes a proportion of residual N-acetyl-D-glucosamine units (K) and Dglucosamine units (L). The relative proportions of these units (K) to (N) depend upon the degree of deacetylation of the chitosan used to prepare the condensation product, the amount of the bisulphite addition compound reacted with the chitosan and the reaction conditions. The condensation product is distinguished from chitosan by its ability to form a stable anionic polyelectrolyte solution in water at neutral pH.

The invention also provides a polymer comprising monomer units of formulae K, L, M and N (as hereinbefore defined) in proportions k, 1, m and n respectively such that:

m+2n x 100% t-! 10% k+l+m+n The above ratio implies a polymer produced by a method in which the number of HOCRR'SO3A molecules reacting with the chitosan is equal to or greater than 10% of the sugar residues. This ratio may be as high as 200% for a fully deacetylated chitosan in which all the glucosamine residues are saturated with bisulphite moieties. In this case k, I and m are all equal to zero. However, we prefer to have a polymer in which:

40%:! m+2n x I 00%:! 70% k+l+m+n Especially preferred is a polymer in which:

45%:! m+2n x 100%: 50% k+l+m+n The condensation product or polymer of the invention may be prepared by slurrying chitosan with either a solution of the bisulphite addition compound or with a solution containing precursors of the bisulphite addition compound. The amount of bisulphite addition compound required for a given weight of chitosan depends on the molecular weight of the chitosan and its physical structure. The lower the molecular weight of the chitosan, the lower the ratio required to achieve solubility. The more accessible the chitosan, e.g. if it has been dissolved up in dilute acid and then reprecipitated out by making the solution alkaline, the lower the ratio required. As a guide, however, it can be said that from 0. 5 to 3.5, more preferably from 1 to 2, moles of the bisulphite addition compound are used per equivalent of chitosan amine groups. These processes are within the scope of the invention.

The condensation product or polymer may be precipitated from the preparative reaction mixture by admixture with a water miscible organic solvent, e.g. acetone (the solvent being added to the reaction mixture, or the reaction mixture being added to the solvent). The precipitate can be collected and dried, suitably at WC under vacuum. The isolated condensation product (polymer) is stable and is convenient for transportation and storage. It may be re-dissolved in water at neutral pH, forming solutions having high concentration (up to 15% by weight). Such solutions remain stable for extended periods, certainly exceeding 12 months. The isolated condensation product (polymer) and its aqueous solutions also form a part of the invention.

The solutions according to the invention may be mixed with other water soluble anionic polyelectrolytes, both natural and man-made. Examples of these are sodium alginate, sodium carboxymethyl cellulose, carragheenan, sodium cellulose sulphate, chondroitin sulphates, dermatan sulphate, heparan sulphate, heparin, hyaluronic acid, keratan sulphate, pectin, poly(sodium acrylate) and poly(sodium. methacrylate). The solutions may also be mixed with water soluble proteins such as collagen and gelatine. The resultant mixed solutions, which also form part of the invention, are themselves stable.

The preferred ratio of condensation product or polymer to other water soluble anionic polyelectrolytes is from 5:1 to 1:5 by weight. The preferred other water soluble anionic polyelectrolyte is sodium alginate.

Mixtures of chitosan and alginate (a water soluble anionic polyelectrolyte) have been disclosed previously, e.g. US 5,836,970 (Pandit / The Kendall Company) and WO 99/01166 (Coloplast A/S). However both of these approaches use chitosan in the form of a cationic polyelectrolyte, in which form it interacts rapidly with the anionic alginate to give an insoluble polyelectrolyte complex. Thus the products obtained following these references consist of heterogeneous mixtures of the two components in which there is either no continuous phase or one of the components forms the continuous phase with the second component present in the form of particles suspended in this continuous phase. A layer of polyelectrolyte complex is formed at each of the interfaces between the two components. The particle size depends on the severity of the mixing treatment, while which component forms the continuous phase depends, inter alia, on the relative proportions of each and their molecular weight.

This contrasts with the blended solutions according to the invention since in these both components are present in the continuous phase in the form of a homogeneous blend, intimately mixed at the molecular level.

The mixed solutions can be used in conventional methods for the preparation of films, fibres, powders, sponges, foams and gels which themselves contain chitosan (as the condensation product or as itself, see below) and alginate (or other water soluble anionic polyelectrolytes) in the continuous phase in the form of a homogeneous blend, intimately mixed at the molecular level. The difference in structures may be clearly seen with the naked eye in films made by the different methods, and may also be objectively demonstrated by various scientific techniques. Owing to the homogeneous nature of the blends produced according to the invention, films and fibres produced from them have greater mechanical strength and flexibility, together with greater optical clarity.

It should be noted that films, fibres, powders, sponges, foams and gels can also be prepared from the solutions according to the invention unmixed with any other substances; such films, fibres, powders, sponges, foams and gels contain only chitosan (as the condensation product or as itself, see below).

Treatment of the condensation product of the invention with an acid or an alkali enables the chitosan to be regenerated. This is the case even when the condensation product has been formed into films, fibres, powders, sponges, foams and gels as described above. Indeed the regeneration treatment can be effected on the films, fibres, powders, sponges, foams and gels during their preparation or subsequent thereto.

In a preferred embodiment the invention also provides a method for preparing a polymer blend, the method comprising the steps of blending an aqueous solution as above defined with alginate in a weight ratio within the range of 5:1 to 1:5 treating the polymer blend so produced with an acid or alkali so as to regenerate chitosan or its salt.

The solubility of the condensation product of the invention in water at neutral pH also allows chitosan to be extracted from a chitin/calcium carbonate mixture, for example crustacean exoskeleton processing waste from the seafood industry. The extraction method comprises deacetylating the chitin in the said chitin/calcium carbonate mixture, slurrying the resultant chitosan/calcium carbonate mixture with a solution containing a sodium bisulphite addition compound as defined above or the precursors of such an addition compound and removing undissolved solids from the solution. The solution can be admixed with a water miscible organic solvent to precipitate the chitosan condensation product, which can be collected, washed and dried. Alternatively the pH of the solution can be raised to reconstitute the chitosan and precipitate it, after which it may be collected, washed with water and dried. These processes, which are within the scope of the invention, avoid the production of calcium chloride effluent.

The wide range of possible physical forms makes available a wide range of uses for the chitosan products of the invention, especially biomedical uses. These include wound and bum dressings, pharmaceutical excipients, transdermal controlled release drug patches, sutures and coatings therefor, adjuvants for calcium phosphate bone cements, scaffold materials for tissue engineering applications, medical device materials or coatings for conventional medical devices, surgical adhesion barriers, peridontal disease treatment, and the scaling of arterial puncture sites after catheterisation. Other potential nonmedical applications are found in the food and beverage industry, e.g. as preservatives, stabilisers and hydration control coatings, in the preparation of cosmetics and toiletries, in agriculture as a seed treatment and as a pesticide and in water treatment, particularly for heavy metal ion extraction, and as membranes for use in separation processes.

Detailed Description of the Invention The invention is illustrated by the following Examples.

Example 1 Preparation of a Chitosan Condensation Product 2 g of a high molecular weight (4.3 x 105) chitosan produced from prawn shell and having a level of deacetylation of approximately 72%, was slurried at room temperature with magnetic stirring in 100 mI of distilled water containing 2 g of HOCH2S03Na (sodium formaldehyde bisulphite) to give a 1.8: 1 ratio of moles of sodium formaldehyde bisulphite: equivalents of amine groups. The chitosan dissolved over a period of 3 to 4 hours to yield a clear smooth-flowing solution.

Expmple 2 Preparation of a Chitosan Condensation Product 2 g of chitosan, similar to that used in Example 1, was slurried at room temperature with magnetic stirring in 100 ml of distilled water containing 2 mI of a 30% solution of formaldehyde and 2.1 g of Na2S205 (sodium metabisulphite) as precursors of sodium formaldehyde bisulphite to give a 2.4: 1 ratio of moles of sodium formaldehyde bisulphite: equivalents of amine groups. The chitosan dissolved over a period of 3 to 4 hours to yield a clear smooth-flowing solution.

Expmple 3 Preparation of chitosan/sodium alginate and chitosan/carragheenan films 2 g of reprecipitated chitosan, prepared by addition of concentrated NH40H to a solution of the chitosan in 1% aqueous acid and having 82% deacetylation and a molecular weight of 8.2 x 104, was slurried at room temperature in 100 mi of distilled water containing 2 g of HOCH2SO3Na, giving a 1.5: I ratio of moles of sodium formaldehyde bisulphite: equivalents of amine groups solution. Separate aliquots (10 ml) of the resultant solution were then mixed with 10 ml of a 2% (w/v) aqueous solution of sodium alginate and 10 ml of a 2% (w/v) aqueous solution of carragheenan respectively. In the case of the chitosan condensation product/carragheenan blended solution, mixing was carried out at 40'C to ensure that the carragheenan solution was above its gelation temperature. Films were cast from the blended solutions and air-dried. The films were then steeped in 80% aqueous ethanol containing 0.5 % NaOH to regenerate the chitosan, washed in 80% aqueous ethanol and dried to give tough, flexible blend films having a transparency similar to that of films cast from the individual polymer solutions.

Example 4 Preparation of chitosan/sodium alginate and chitosan/carrajzheenan films Example 3 was repeated using 2 g of the reprecipitated chitosan and the solution process described in Example 2. After regenerating the chitosan the blend films had physical characteristics similar to those of the films prepared in Example 3.

Example 5 Isolation of the chitosan condensation product as a solid A solution of chitosan, prepared as described in Example 3, was poured into acetone to precipitate the chitosan condensation product. This was collected, washed with 70% aqueous ethanol and then dried at 60'C under vacuum. The isolated chitosan condensation product has been stored in sealed containers at room temperature for periods of up to 15 months. All stored samples remained readily soluble in water throughout the storage period. Solutions prepared from the isolated chitosan condensation product have also been stored at room temperature for periods for periods of up to 15 months and were not affected by microbial contamination.

Example 6 Isolation of the chitosan condensation product as a solid A solution of chitosan, prepared as described in Example 4, was poured into acetone to precipitate the chitosan condensation product. The precipitate was collected, dried and washed as described in Example 5 to obtain an isolated chitosan condensation product indistinguishable from that prepared in Example 5.

Example 7

Proaration of chitosan condensation product/sodium alginate and chitosan condensation product/carragheenan films 2 g of the reprecipitated chitosan sample prepared in Example 3 was slurried for 2 hours in 70% aqueous ethanol containing 2 g of HOCH2SO3Na, then rinsed with ftirther 70% aqueous ethanol and dried. Two portions (0. 3 g each) of the dried chitosan condensation product were dissolved in 10 ml aliquots of distilled water and mixed, separately, with 10 ml of a 2% (w/v) aqueous solution of sodium alginate and 10 ml of a 2% (w/v) aqueous solution of carragheenan. In the case of the chitosan condensation product/carragheenan blended solution, mixing was carried out at 40'C to ensure that the carragheenan solution was above its gelation temperature. Films were cast from the blended solutions and air-dried. The films produced were tough, flexible and transparent.

Example 8

Preparation of chitosan condensation lDroduct/sodium alginate and chitosan condensation product/carragheenan films Example 7 was repeated, but using 70% aqueous ethanol containing 2 ml of a 30% formaldehyde solution and 2.1 g of Na2S205 (sodium metabisulphite) as precursors of the HOCH2SO3Na. The blend films had physical characteristics similar to those of the films prepared in Example 7.

Example 9 Prevaration of chitosan/sodium alainate/sodium carboxmethyl cellulose films 4 g of a chitosan condensation product, prepared as described in Example 5, was dissolved in 100 ml of distilled water. A 10 ml aliquot of the solution was mixed with a 10 ml aliquot of a solution containing 2% (w/v) sodium carboxymethyl cellulose and 2% (w/v) sodium alginate, and films were cast from the mixed solution. The dried films were steeped in 80% aqueous ethanol containing 0.5% NaOH to regenerate the chitosan, washed in 80% aqueous ethanol to neutral and dried to give tough, flexible and transparent blend films.

Example 10 Preparation of chitosan/sodium alginate fibres A solution containing 10 g of a chitosan condensation product, prepared as described in Example 5, in 100 ml of distilled water was mixed with an equal volume of a solution containing 10 g of sodium alginate in 100 ml of distilled water. The concentrated solution was extruded through a fine nozzle into a bath of 10% aqueous CaC12 solution. The filaments formed were washed with distilled water, treated with 2% aqueous NaOH solution, rinsed in 50% aqueous ethanol and dried to give a strong, fibrous product.

Example I I Preparation of a spongy Mat of chitosan/sodium, alginate A solution containing 2% (w/v) of a chitosan condensation product, prepared as described in Example 5, and 2% (w/v) sodium alginate was subjected to freezing followed by removal of the water by sublimation ("freeze drying") to give an intimately mixed polymer blend in the form of a spongy mat. Treatment of the mat with 0.5% NaOH followed by rinsing in 50% aqueous ethanol converted the mat to one of chitosan/sodiurn alginate without loss of its physical structure.

Claims

1. A condensation product of chitosan and a bisulphite addition compound which has the formula HOCRTS03A wherein 0 each of R and R' independently represents a hydrogen atom, an optionally substituted linear or branched saturated or unsaturated hydrocarbon group having up to 10 carbon atoms or an optionally substituted alicyclic, aromatic =bocyclic or heterocyclic ring, or R and FV together represent an optionally substituted alicyclic or heterocyclic ring, and 4D A represents an alkali metal or ammonium. ion, which condensation product forms a stable anionic polyelectrolyte solution in water at neutral pH.

2. A condensation product according to claim I in which A represents a sodium ion.

3. A condensation product according to claim I or claim 2 in which R and W both represent hydrogen atoms.

4. A condensation product according to any preceding claim isolated as a solid.

5. A polymer comprising monomer units of formulae K, L, M and N:

CH 20H 0 0 HO!::

NHCOCH3 - K CH20H 0 HO NH 2 L CH 2-- f0-- 0 HO NHCRR'SO3A M CH20H 0 __-__o HO-- N(CRR'SO 3A)2 J N in the proportions k, 1, m and n, respectively, wherein 0 each of R and R' independently represents a hydrogen atom, an optionally substituted linear or branched saturated or unsaturated hydrocarbon group having up to 10 carbon atoms or an optionally substituted alicyclic, aromatic carbocyclic or heterocyclic ring, or R and R' together represent an optionally substituted alicyclic or heterocyclic ring, and A represents an alkali metal cation or ammonium ion, the proportions being such that the ratio:

rn + 2n - --- x 100%:?: 10% k+l+m+n

6. A polymer according to claim 5 in which A represents a sodium ion.

7. A polymer according to claim 5 or claim 6 in which:

40%:! m+2n x 100%:! 70% k+l+m+n

8. A polymer according to claim 7 in which:

45%: m+2n x 100%:5 50% k+l+m+n

9. A polymer according to any of claims 5 to 8 isolated as a solid.

10. An aqueous solution of a condensation product according to any of claims I to 3.

11. An aqueous solution of a polymer according to any of claims 5 to 8.

12. An aqueous solution prepared by dissolving a condensation product according to claim 4 in water.

13. An aqueous solution prepared by dissolving a polymer according to claim 9 in water.

14. An aqueous solution according to any of claims 10 to 13 flarther containing one or more other water soluble anionic polyelectrolytes.

15. An aqueous solution according to claim 14 in which the one or more other water soluble anionic polyelectrolytes are selected from sodium alginate, sodium carboxymethyl cellulose, carragheenan, sodium cellulose sulphate, chondroitin sulphates, dermatan sulphate, heparan sulphate, heparin, hyaluronic acid, keratan sulphate, pectin, poly(sodium acrylate) and poly(sodium. methacrylate).

16. An aqueous solution according to any of claims 10 to 15 further containing one or more water soluble proteins.

17. An aqueous solution according to claim 16 in which the one or more water soluble proteins are selected from collagen and gelatine.

18. A film, fibre, powder, sponge, foam or gel prepared from a solution according to any of claims 10 to 17.

19. Chitosan regenerated from a condensation product according to any of claims 1 to 4 by treating the condensation product with an acid or an alkali.

20. Chitosan regenerated from a polymer according to any of claims 5 to 9 by treating the polymer with an acid or an alkali.

21. A film, fibre, powder, sponge, foam or gel according to claim 18 which has been treated with an acid or alkali to regenerate chitosan from the condensation product therein.

22. A film, fibre, powder, sponge, foam or gel according to claim 18 which has been treated with an acid or alkali to regenerate chitosan from the polymer therein.

23. A film, fibre, powder, sponge, foam or gel according to claim 21 or claim 22 in which the treatment was effected during the preparation of the film, fibre, powder, sponge, foam or gel.

24. A film, fibre, powder, sponge, foam or gel according to claim 21 or claim 22 in which the treatment was effected subsequent to the preparation of the film, fibre, powder, sponge, foam or gel.

25. A method for the preparation of a condensation product according to any of claims I to 3, the method comprising slurrying chitosan with a solution of a bisulphite addition compound as defined in claim 1.

26. A method for the preparation of a condensation product according to any of claims I to 3, the method comprising slurrying chitosan with a solution containing precursors of a bisulphite addition compound as defined in claim 1.

27. A method according to claim 26 in which the precursors are sodium metabisulphite (Na2S205) and an aldehyde/ketone of the formula RR'CO wherein R and R'are as defined in claim 1.

28. A method according to any of claims 25 to Z7 in which from 0.5 to 3.5 moles of the bisulphite addition compound are used per equivalent of chitosan arnine groups.

29. A method according to any of claims 25 to 27 in which from I to 2 moles of the bisulphite addition compound are used per equivalent of chitosan amine groups.

30. A method for the preparation of a condensation product according to claim 4, the method comprising admixing an aqueous solution according to claim 10 with a water miscible organic solvent, collecting the resultant precipitate and drying it.

31. A method for the preparation of a polymer blend, the method comprising the steps of e blending an aqueous solution according to claim I I or claim 13 with alginate in a weight ratio within the range of 5:1 to 1:5, and treating the polymer blend so produced with an acid or alkali so as to regenerate chitosan or its salt.

32. A method for the extraction of chitosan from a chitin/calcium. carbonate mixture, the method comprising deacetylating the said chitin/calcium carbonate mixture, slurrying the resultant chitosan/calcium. carbonate mixture with a solution containing a bisulphite addition compound as defined in claim I or the precursors of such an addition compound, removing undissolved solids from the solution, treating the solution with acid or alkali to regenerate and precipitate chitosan, collecting the resultant precipitate and drying it.

33. A method for the preparation of a condensation product according to any of claims I to 3 from a chitin/calciurn carbonate mixture, the method comprising deacetylating the said chitin/calcium carbonate mixture, slurrying the resultant chitosan/calcium carbonate mixture with a solution containing a bisulphite addition compound as defined in claim I or the precursors of such an addition compound, removing undissolved solids from the solution, admixing the solution with a water miscible organic solvent, collecting the resultant precipitate and drying it.