The present invention relates to biocompatible polysaccharide gel compositions, and in particular chitosan compositions in vaccines, for drug delivery, tissue augmentation, cell culture, encapsulation of viable cells, cosmetic use, orthopaedic use, use as biomaterials, wound healing devices, thickener and additive in the food industry, use as glues, lubricants, drilling and servicing fluids. The gels are prepared by covalently cross-linked chitosan gels having a low degree of deacetylation. By choosing chitosans of specific degrees of deacetylation and using efficient cross-linking conditions gels with interesting and unexpected biological and physical properties could be obtained. This is in contrast to other cross-linked chitosan hydrogels, made from standard chitosan and using typical cross-linking protocols. The gels according to the invention can be made to have very low toxicity and they can be made to degrade rapidly. Another striking feature of said gels is that they do not precipitate when subjected to neutral and alkaline conditions. They also possess a rigidity which allows for further mechanical processing into e.g. injectable so called “crushed gels”, useful in a vast number of applications.
A hydrogel could be defined as a colloidal gel in which water is the dispersion medium. Hydrogels are widely used in many fields and have become billion dollar industries in several areas. Typically hydrogels are made from water soluble polymers, which have been either isolated from natural sources or obtained by synthesis or by chemical modifications of natural polymers. These polymers are selected for their physical and biological properties and are used alone or in combinations depending on the desired product properties. Some polymers have physical properties that make them suitable for medical use whereas others are used in the food industry, mechanical processing and manufacturing industry, as lubricants, drilling and servicing fluids, in cosmetics, biomaterial applications, in biotechnology as cell scaffolds and more. The different applications require different qualities of the polymers and many technical applications are based on crude bulk qualities available at low cost, whereas highly purified qualities, often at high cost, are required for medical applications. Sometimes the physical properties of the polymeric solution, such as viscosity, are the main parameter of interest, whereas in other applications the biological and toxicological properties become more prominent for its function in the intended application.
Some polymers are used for filling purposes in tissue augmentation compositions in which gels are used either alone or together with solid beads. In other uses, e.g. wound healing, drug delivery, vaccine vehicles other polymers with other properties are desired to meet the medical demand. In general, properties like viscosity, anti-microbial activity, adhesive or water absorbing/retaining capacity are all properties that have to be considered. Water retention capacity and swelling are typically of great importance in food applications in which the polymer is used either as a thickener or as a solubility enhancer and stabiliser of other agents. There is a wide variety of polymers found in medical products, both synthetic and polymers of natural origin. In many applications it is important that the polymers degrade and are eliminated without causing unwanted side-effects. Even though biodegradability is not always necessary, a good biocompatibility is crucial in order to avoid side-reactions like inflammation, immunological reactions, or rejection of the material. Therefore it is not surprising that naturally occurring, non-toxic polysaccharides are used in medical products, as they have excellent physical properties in combination with interesting biological and medical properties and are usually available at high purity and low cost. Commonly used polysaccharides are e.g. cellulose, alginates, chitosan, hyaluronic acid, starch or derivatives thereof.
In medicine, gels and ointments are used e.g. for delivery of drugs, cosmetic purposes, or to give anti-bacterial barriers to avoid infection. Hydrogels have often relevant solubility and biological properties and are consequently found in a vast set of products. Gel forming polysaccharides like hyaluronic acid, derivatives of cellulose, and alike have become profitable industrial areas. Hyaluronic acid is an example of a polymer which could be used as such since it spontaneously forms hydrogels when used in low concentration water solutions at physiological pH. Other polymers like cellulose cannot be used, as such, and have to be chemically modified to get the desired properties. When preparing hydrogels from polysaccharides, a typical protocol involves dissolution of the polymer in an aqueous solution in low concentrations, often between 0.5 and 3% (w/w). When higher viscosities are desired this can be achieved by either adding more polymer to the solution, if solubility permits, or by cross-linking the polymers. Cross-linking gives polymers of higher molecular weight and consequently of higher viscosity. Cross-linking can be performed in different ways, using covalent, ionic or hydrophobic strategies and a huge number of approaches are available. In general when the product of such a cross-linking reaction is intended for medical use it is desirable to keep the cross-linking level as low as possible, since there is a risk for introducing immunological reactions toward the linker and it may also compromise the biodegradability.
Immunology and allergy. The immune system can be divided into innate and adaptive immunity. The innate or non-specific immunity is an inherent resistance manifested by a species that has not been immunised by infection or vaccination. Adaptive or acquired immunity is a type of immunity in which there is an altered reactivity against the antigen that stimulated it and which generates antigen-specific immunological memory. The immunity may be active i.e. a result of an acquired infection or a vaccination or it can be passive i.e. acquired from a transfer of antibodies. Passive vaccination with antibodies has several drawbacks: Injection of foreign substances may give rise to an immune response against the injected antibodies. Monoclonal antibodies must be injected in a large amount which makes this therapy very expensive. The treatment has to be sustained to maintain its function. Active vaccination to induce antibody formation and immunological memory is most often preferred. Most natural immunogens are proteins with a molecular weight above 5 kDa. Even immunogenic molecules may not generate the level of immunity desired. To increase the intensity of the immune response immunogens are combined with adjuvants. Adjuvants are agents that enhance the immune response without generating unwanted antibodies against the adjuvant. If the immunogen is still unable to generate an acceptable immune response, it may be conjugated to a carrier that is more immunogenic. Small molecules with molecular weight ranging from 0.1 to 2 kDa are often too small to be recognised by the immune system and thereby difficult to use as such in immunisations. One way of circumventing this is to bind them covalently to larger carrier molecules. Vaccination can be oral, nasal, subcutaneous, submucosal, sublingual, or intramuscular.
The recognition and destruction of foreign cells by T- and NK cells is termed cell-mediated immunity (TH1 immune response). Humoral immunity is associated with B-cells (TH2 immune response). Aluminium hydroxide has been reported to selectively activate TH2 cells whereas Freund's complete adjuvant activates TH1 cells. Chitosan has been shown to enhance both humoral and cell-mediated immune response (Vaccine 3, 379-384, 1985).
The innate immune system recognises a wide spectrum of pathogens without a need for prior exposure. The main cells responsible for innate immunity, monocytes/macrophages and neutrophils, phagocytose microbial pathogens and trigger the innate, inflammatory, and adaptive immune responses. Toll-like receptors (TLRs) are a family of type I transmembrane proteins involved in the recognition of a wide range of microbes. They play a key role in the innate immune system. TLRs are a type of pattern recognition receptors (PRRs) and recognise molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). Macrophage receptors are also considered to be pattern-recognition receptors. The macrophage mannose receptor recognises hexoses with equatorially placed hydroxyl groups at carbons C3 and C4, positions enabling the recognition of mannose, fructose, N-acetylglucosamine and glucose (Curr. Opin. Immunol. 10, 50-55, 1998).
Allergy is a very common disorder affecting approximately one fourth to one third of the population in industrial countries, e.g. more than 50 million Americans suffer from allergic diseases. The treatment strategy by far most commonly used today is to target the effector mechanisms of allergy, e.g. by oral ingestion of antihistamines or by topical corticosteroids. Antihistamine and corticosteroid treatment can be effective in relieving allergy symptoms, but their use leads to exposing the entire body to the pharmaceutical product, and they may produce unpleasant or even harmful side effects. Allergen-specific immunotherapy is the only treatment in use that targets the underlying causes of allergy and that gives long-lasting symptom relief. It may thus be considered as the only curative treatment for allergic disease. This treatment may be given as subcutaneous injections or sublingually. Carried out by injecting allergen extracts subcutaneously it has a well-documented effect, while the efficacy of sublingual allergen-specific immunotherapy is less documented.
Allergic diseases such as asthma and rhinitis are caused by an inappropriate immune response to otherwise harmless environmental antigens, i.e. allergens. The most common form is Immunoglobulin (Ig) E-mediated allergy, characterised by the presence of allergen-specific IgE. There are currently two general strategies to treat IgE-mediated allergies, pharmacological therapy and allergen-specific immunotherapy. Pharmacological treatment includes treatment with topical corticosteroids, especially in the case of allergic asthma and eczema. However, 10-20% of the patients with allergic asthma do not respond to steroid treatment. Other common anti-allergy drugs target effector mechanisms of IgE-mediated allergy, e.g. antihistamines, antileukotrienes and chromones. The only curative therapy of IgE-mediated allergy, i.e. the only treatment that gives long-lasting relief of symptoms, is allergen specific immunotherapy (ASIT). In contrast to pharmaceutical treatment, ASIT has also been shown to reduce airway inflammation and protect against development into chronic asthma (J Allergy Clin Immunol. 1998 102(4 Pt 1), 558-62). The treatment is based on the repeated administration of allergen in order to induce allergen-specific unresponsiveness. At present, allergen extracts prepared from natural sources and adsorbed to aluminium hydroxide (alum) are commonly used in ASIT. Alum delays the release of allergen and acts as an adjuvant. However, there are some drawbacks linked to the use of allergen extracts and alum. Many injections with low allergen doses are required during a time period of 3-5 years. To solve problems like induction of new sensitisations and adverse side-effects to extracts, recombinant allergens have been proposed for use in ASIT (Adv Immunol. 2004; 82:105-53, Nat Rev Immunol. 2006 October; 6(10):761-71). Recombinant allergens can be modified in different ways with the aim to achieve safer and more efficient protocols for ASIT. Examples of such novel strategies are to create so called hypoallergens, i.e. allergens with reduced IgE binding capacity but retained T-cell activity, vaccination with allergen-derived peptides or coupling of allergens to immunomodulating agents such as immunostimulatory oligonucleotides containing CpG motifs (Nat Rev Immunol. 2006 October; 6(10):761-71, Curr Opin Immunol. 2002 December; 14(6):718-27). Alum is known to cause granuloma at the injection site and to mainly stimulate Th2 responses. Consequently alternative adjuvants are needed for ASIT.
Adjuvants are substances that enhance the ability of an antigen to elicit an immune response. Even though extensive efforts to develop new adjuvants for human vaccines are made, the only widely used adjuvant is still aluminium hydroxide. It has been shown that aluminium adjuvants can cause neuron death. The development of novel adjuvants is desirable in order to maximise the efficiency of new vaccines. An ideal adjuvant should give long lasting expression of functionally active antibodies, elicit cell-mediated immunity and enhance the production of memory T and B lymphocytes with highly specific immuno-reactivity against an antigen. It should provide both an immediate defense and a protection against future challenges with an antigen. It should also be biodegradable, non-toxic and not give rise to an immune response directed towards the adjuvant itself.
Vaccinations should give a long-lasting effect, fast antibody production and high antibody titres.
The use of chitin and chitosan as adjuvant has been mentioned in U.S. Pat. No. 4,372,883 and U.S. Pat. No. 4,814,169. The use of chitosan in vaccines in the form of solutions, dispersions, powders or microspheres has been described in U.S. Pat. No. 5,554,388, U.S. Pat. No. 5,744,166, and WO 98/42374. Cross-linking of chitosan switches the immune response from a TH2 towards a mixed TH1/TH2 response. The use of chitosan solutions mixed with antigens for immunisations show that chitosan is equipotent to Freund's incomplete adjuvant and superior to aluminium hydroxide (Vaccine 11, 2085-2094, 2007).
Drug delivery. Drug delivery is a very intense research area and a lot of money is today spent on finding new and improved formulations that deliver pharmaceutical active ingredients like low molecular drugs, genes, and vaccines more specifically and at the same time minimises unwanted side effects. Old drugs become new in new and improved formulations.
The properties of chitosan, physical and biological, have made it very suitable for delivery of pharmaceutically active components and as a delivery vehicle for e.g. vaccines, gene fragments and micro-RNA. Useful and important features of chitosan is that it to bind to all living tissue, has muco-adhesive properties, is degradable and opens tight junctions between cells. By taking advantage of these properties, drug delivery over the mucous membrane can be dramatically improved. Drug formulations based on chitosan technology are today under development for different purposes e.g. as vaccine carriers, drug releasing hydrogels, membranes, gauze and more. Chitosan has shown to be useful in e.g. colon delivery (H. Tozaki, et. al, J. Pharm, Sci., 86, 1016-1021, 1997) and intranasal delivery of insulin (U.S. Pat. No. 5,744,166). Chitosan has also been used as a carrier in gene delivery (MacLaughlin, et. al, J. Controlled Release, 56, 259-272, 1998).
Some formulations are designed to give a sustained release over time whereas release from others are more instant. When a hydrogels of chitosan are used it has been found that cross-linking is preferred since gels without cross-linkers have a tendency to dissolve. Another advantage of using cross-linking is that the release rate from the gel can be altered by using different degrees of cross linking. Chitosan can be used for development of new formulations for e.g. oral, dermal, subcutaneous, buccal, sublingual, nasal, rectal, vaginal and intra muscular administration.
Many drugs that are administered in an unmodified faun by conventional systemic routes fail to reach the target organs in an effective concentration, or are not effective over a length of time due to a facile metabolism. By use of a Drug Delivery Systems (DDS), it is possible to overcome these problems.
Cancer drugs are often characterised by a short plasma half-life and/or by remarkable side effects. An approach to reduce these problems may be via focal administration, i.e. local drug delivery at the site of the cancer via implantation/injection of a DDS containing the chemotherapeutic agent. In comparison with systemic administration, the extent of side effects will decrease and the total effect of the drug will increase.
When developing a DDS for focal therapy of cancer, several technical factors have to be taken into consideration, namely biocompatibility, biodegradable (importance depends on disease, site of application and number of administrations), sterility/sterilisation, compatibility with drugs and pharmaceutical excipients, ease of administration (via syringe is preferred), flexibility regarding the dose, drug load, dose positioning, ability to control release rate of drug and patient acceptability, as well as consideration of regulatory hurdles, CoG (cost of goods), and IPRs.
By injection of the DDS with drug results in the localisation of a greater amount of the loaded drug at the tumour site, thus improving cancer therapy and reducing the harmful non-specific side effects of chemotherapeutics.
Tissue augmentation. Tissue augmentation can be used for both medical and cosmetic purposes. A medical application is, for example, augmentation of tissues in order to obtain improved function of the tissue. Examples of tissues that can be strengthened by injection of bulking agents are the vocal cords, the oesophagus, urethra or rectum. In the area of cosmetic surgery, soft tissue augmentation may be used to correct defects as scars and wrinkles and to enlarge for example lips or breasts. A variety of different materials, both non-biodegradable and biodegradable, has been used to repair or augment soft tissue. Examples of materials used for permanent soft tissue augmentation are silicone, Gore-Tex, and ePTFE. Examples of biodegradable materials are collagen, autologous fat, cross-linked hyaluronic acid, and synthetic polymers.
Silicone is one of the most frequently used materials for permanent soft tissue augmentation. Adverse reactions to liquid injectable silicone include granulomatous reactions, inflammatory reactions, and drifting. These reactions can occur years after initial treatment. Furthermore, since injectable silicone is a permanent filler, the above complications can become a serious problem since the substance will not be metabolised and the reaction can persist despite treatment.
Collagen is one of the most frequently used injectable materials, both for cosmetic applications and as a bulking agent for e.g. urinary incontinence. Collagen, however, has several drawbacks. It degrades rapidly and approximately 3% of the population show delayed hypersensitivity reactions, which makes it necessary to perform allergy tests over a period of time prior to injection. Furthermore, collagen of bovine origin may transmit viral diseases.
Autologous fat injections are well known. These materials also have disadvantages. Fat injected into facial lines and wrinkles have caused loss of vision and embolism in some patients. Furthermore autologous fat is readily absorbed by the body.
Cross-linked hyaluronic acid products are used both for cosmetic treatments and as bulking agents for the treatment of e.g. urinary incontinence (UI) and vesicoureteral reflux (VUR).
A common approach in the design of bulking agents is to use spheres of a non-biodegradable material dispersed in a biologically degradable carrier. Examples include carbon-coated beads in a beta-glucan gel, hydroxyapatite spheres in carboxymethyl cellulose, polytetrafluoroethylene particles and poly(lactic-co-glycolic acid) (PLGA) microspheres. One risk with particle injections is the potential particle migration to distant organs such as brain and lungs.
The existing materials are not optimal and there is a continuing search for new materials for tissue augmentation applications, materials that are injectable through thin needles, biocompatible, non-toxic and with suitable residence time in the tissue.
Chitosan gels for soft tissue augmentation have been described (WO 97/04012, EP 1 333 869).
Chitosan gels have also been used in the cultivation of cells and for incorporation of viable cells to be used in e.g. cartilage tissue engineering as described in for example Biomaterials. 2000; 21(21):2165-61, J Biomed Mater Res A. 2007; 83(2):521-9, and Biochimie. 2006; 88(5): 551-64.
In cosmetics chitosan has been used in for example skin creams (US 20060210513, US 20040043963) and to decrease skin irritation caused by shaving (U.S. Pat. No. 6,719,961).
Chitosan may also be used as a lubricant (Nature. 2003, 425:163-165). The use of chitosan as a thickener has been described in e.g. Environ Sci Technol. (2002) 36(16):3446-54 and Nanotechnology (2006) 17 3718-3723. It has also been used as a glue (Biomacromolecules. 1(2):252-8 (2000) and Fertil Steril, 84, 75-81 (2005)) and as a dietary supplement (U.S. Pat. No. 5,098,733, U.S. Pat. No. 5,976,550, U.S. Pat. No. 6,238,720 and U.S. Pat. No. 6,428,806).
In addition to the medical applications, viscoelastic chitosan hydrogels may be used as pseudoplastic, shear thinning chitosan-containing fluids, and a method of enhancing the thermal stability of such fluids is described in for example U.S. Pat. No. 6,258,755,
Chitin is next to cellulose the most abundant polysaccharide on earth. It is found in hard structures and strong materials in which it has a function of a reinforcement bar. Together with calcium salts, some proteins and lipids it builds up the exoskeletons of marine organisms like crustaceans and arthropods. It is also found in the cell walls of some bacteria and sponges and build up the hard shells and wings of insects. Commercially, chitin is isolated from crustacean shells, which is a waste product from the fish industry. Chitosan is a linear polysaccharide composed of 1,4-beta-linked D-glucosamine and N-acetyl-D-glucosamine residues. Chitin in itself is not water soluble, which strongly limits its use. However, treatment of chitin with strong alkali gives the partly deacetylated and water-soluble derivative chitosan which can be processed in a number of different physical forms, e.g. films, sponges, beads, hydrogels, membranes. Chitosans in their base form, and in particular those of high molecular weight, and/or high degrees of N-deacetylation, are practically insoluble in water, however its salt with monobasic acids tend to be water-soluble. The average pKa of the glucosamine residues is about 6.8 and the polymer forms water-soluble salts with e.g. HCl, acetic acid, and glycolic acid. The solubility of chitosan depends on several factors, both intrinsic as e.g. chain length, degree of deacetylation, acetyl group distribution within the chains, but also external conditions such as ionic strength, pH, temperature, and solvent. From literature it is known that a degree of acetylation of about 50% is optimal for solubility. When making gels and water solutions in an acidic environment there is a practical limit set by the solubility of the specific chitosan, which is dependent on its molecular weight and its degree of N-deacetylation. However, the amount of chitosan in an aqueous medium is typically in a range from 1-10%, or 1-5%, by weight based on the weight of the liquid medium, with the amount tending towards the higher end of the range if low molecular weight chitosans are used (Carbohydr. Polym. 25, 65-70, 1994).
The inherent properties of chitosan, being biodegradable, non-toxic and anti-microbial in combination with its cationic and hydrophilic nature makes it attractive in pharmaceutical formulations. However, its poor solubility at physiological conditions has limited its practical use. Scientists have circumvented this shortcoming of solubility by making chemically modified chitosan derivatives with superior solubility properties at physiological pH e.g. sulphated chitosan, N-carboxymethyl chitosan, O-carboxymethyl chitosan and N,O-carboxymethyl chitosan (Int J Biol Macromol. (4), 177-80, 1994, Carbohydr Res. 302(1-2):7-12, 1997).
A consequence of introducing chemical substituents on chitosan will be changed biological properties e.g. altered degradation rate and the risk for introducing groups that will have a negative impact on biocompatibility and toxicity. This problem has been addressed in U.S. Pat. No. 6,344,488 in which glycerophosphate is used as a solubility enhancer and thus allows preparation of chitosan hydrogels at physiological pH, without the modification of the chitosan structure.
Chitosan solutions can be cross-linked under acidic conditions, typically at suitable for Shiff base formation (pH 4-5), to form hydrogels. A huge number of different cross-linkers with different structures and reactivities have been used. Several cross-linking agents have been used in order to form gels from liquid chitosan, for example glycosaminoglycans such as hyaluronic acid and chondroitin sulfate (Ann. Pharm. Fr. 58 47-53, 2000), glutaraldehyde (Ind. Eng. Chem. Res. 36: 3631-3638, 1997), glyoxal (U.S. Pat. No. 5,489,401), diethyl squarate (Macromolecules 31:1695-1601, 1998), diepoxides such as diglycidyl ether (U.S. Pat. No. 5,770,712), tripolyphosphate (J Appl Polym Sci 74: 1093-1107, 1999), genipin (J Polym Sci A: Polym Chem 38: 2804-2814, 2000, Biomaterials. 23:181-191, 2002), formaldehyde (J. Polym. Sci. Part A: Polym. Chem. 38, 474, 2000, Bull. Mater. Sci., 29, 233-238, 2006). When a hydrogel is the desired product it is mandatory that chitosan and its derivative remain in solution and that precipitation thereof is avoided. Attempts to adjust the pH of cross-linked chitosan hydrogels, to physiologically acceptable levels, result in precipitation and insoluble materials of limited use. It is desirable to keep the degree of cross-linking as low as possible, both for toxicology reasons and also because a high degree of cross-linking may alter the behaviour of chitosan completely (Eur J Pharm Biopharm. 2004, 57(1):19-34. Review).
A specific group of hydrogels are the viscoelastic gels, gels that are viscous and at the same time show elastic properties. A viscoelastic gel will deform and flow under the influence of an applied shear stress, but when the stress is removed the liquid will slowly recover from some of the deformation. This is used in e.g. ophthalmology, tissue augmentation, and cosmetic surgery. The viscoelasticity of the gels allow for mechanical processing which includes the preparation of crushed gels. Viscoelastic gels of hyaluronic acid are e.g. used in eye surgery, wrinkle filling or in the treatment of urinary incontinence.
Chitosan, a natural polyelectrolyte. The three dimensional orientation of a polyelectrolyte in an aqueous environment will be dependent on e.g. its nature/chemical composition, size, concentration and charge density, i.e. the number of charges and the distance between its charged groups. The spatial interactions of any polyelectrolyte in a solution will be controlled by enthalpy and the molecule will strive to adapt a low energy state in which it is most stable. This energy minimisation process involves different types of interactions, either intra-molecular (within the same molecule) or inter-molecular (between molecules). Examples of intra-molecular interactions are hydrogen bonds, hydrophobic interactions and interactions between charged groups on the polymer. Typical inter-molecular interactions are solvent interactions and interactions with other molecules. Irrespectively of the type of interaction involved, the driving force for these interactions is to find energetically favourable conformations of the polyelectrolyte.
When a polyelectrolyte contains charged groups having the same type of charge, e.g. positive, the groups will repel each other. In order to reduce its internal energy the polyelectrolyte molecule will strive to separate its internal charges as much as possible, which will lead to a stretched polymer chain. These stretched polymers will not only be more “space demanding”, they will also have a relatively high state of energy harboured in the constrained linkages between atoms.
On the other hand, if the polyelectrolyte contains charges of opposite signs, they will attract each other and form internal salt bridges which will result in a different three dimensional orientation of the polymer, i.e., different parts of the polymer are brought closer to each other. In a polymer without any charges, there are no ionic interactions and consequently its three dimensional orientation will depend on its ability to foam stabilising hydrogen bonds and hydrophobic interactions within the molecule and with the surrounding molecules and the media. In contrast to the polyelectrolytes, the uncharged polymers that do not contain any high energetic repulsive forces, from some kind of “random coil” structure in which their internal energy has been minimised and their relative energy content is lower than that of the polyelectrolytes.
Physically, ionic interactions (charges) are much stronger and involve more energy than other interactions like, hydrogen bonds, van der Waals forces and hydrophobic interactions. The relative impact of the former on the molecular orientation is thus large and will in many cases overshadow the impact of the other types of forces involved.
The chitosan polymer with its mix of N-acetyl-glucosamine and glucosamine residues could theoretically be a neutral polymer but in most practically and biologically relevant situations it will be protonated, since the pKa value for the glucosamines in chitosan is approximately 6.8. However, in contrast to polyelectrolytes bearing permanently charged groups, the charge density of a chitosan polymer can be varied and will be directly dependent on the pH of a water solution. Practically, most commercially available and unmodified chitosans are insoluble in water solutions when the pH is above approximately 6 and above this pH they will precipitate from an aqueous solution. The precipitation is energetically driven as the chitosan molecule requires a large number of charges on its molecular backbone to form an energetically favourable state of solvatisation. If this can not be accomplished; the molecules will precipitate from solution and faun more stable precipitates. In the precipitate, the chitosan chains have been brought together which allows for energy optimisations by molecular interactions between and within the chitosan molecules.
In order to increase the viscosity of a chitosan solution chemical cross-linking can be used. In such a reaction the chitosan chains are linked together to form larger network like aggregates. During such a reaction the viscosity successively increases and the solution becomes more gel-like in its structure. There is a large number of cross-linking procedures described for chitosan in solution and they have in common that the chitosan is dissolved in an acidic water phase and the cross-linking reaction takes place in at low pH, typically from 4-5. The low pH used implies that the chitosan chains are in their protonated form and they are consequently in a “stretched” form when cross-linked. The resulting cross-linked gel is then technically a macro structure of protonated and stretched chitosan chains. When such a macro network, is brought to neutral or alkaline conditions, it will gradually lose its charges, collapse and eventually precipitate. This is to some extent expected, since when standard chitosans, (degree of deacetylation between approximately 80-95%) are brought to a pH above 6 they precipitate. The cross-linking in itself has generated even larger electrolyte structures which will be even more demanding to stabilise in a water solution from an energy point of view. This is because positive charges have been brought closer together in the junction points between chains and thus will be even more difficult to stabilise with solvating water molecules. Consequently they are even more prone to precipitate than the individual chains when solution conditions are turned in a less energetically optimal way, e.g. pH is raised. Through the cross-links the macro gel structure has been locked in a stretched and energetically unfavourable state, which physically does not allow rearrangement to coils and other conformations that can contribute to more energetically favoured conformations resulting in higher stability of the system.
The precipitation of chitosan gels, formed in acidic conditions, is easily experimentally confirmed by subjecting a lump of such a cross-linked chitosan gel to a pH above seven or even higher values, i.e. pH 7-14. Immediately when such a lump is placed in a buffer of higher pH the surface of the lump becomes whitish by a thin layer of precipitate and as diffusion goes on, the lump becomes more and more whitish until it is fully precipitated.
However, and surprisingly we have found that this collapse of cross-linked chitosan gel macro structures can be circumvented by using low deacetylated chitosan of specific degrees of deacetylation and cross-linking the chitosan chains in an energetically less constrained conformation. Gels produced according to this procedure can be treated with 1 M sodium hydroxide without forming precipitates. A corresponding non-cross-linked gel will precipitate when treated with 1M NaOH.
By using the higher solubility of these specific chitosans, pH can be brought much higher during the cross-linking reactions. The advantages of doing this are numerous. Firstly the protonation of the chitosan chain becomes low and the chitosan polymer is almost neutral at pH above eight, allowing the formation of less constrained and more random coil like network in the solution. When the chitosan is subjected to cross-linking in this state the resulting gel structure will be built up by individual chitosan chains of higher flexibility and which will make them more easily reorganised to more energetically favoured macro structures when conditions are changed. Secondly, the possibility to use a higher pH is beneficial in terms of substantially increased reactivity of the amino groups on the glucosamine residues. This makes the couplings more efficient and enables the use of much lower concentrations of cross-linking reagents to reach a defined degree of cross-linking. Another benefit is that the side reactions are kept low. These cross-linked gels have several advantages compared to chitosan gels prepared at low pH and from standard grade chitosans (degree of deacetylation 80-95%). The fact that they do not precipitate at physiological conditions implies that they are more accessible to degrading enzymes, which leads to fast degradation of the gels, but also other properties as described in the present specification.