US20050283004A1

US20050283004A1 - Alkylsulfonated polyaminosaccharides

Info

Publication number: US20050283004A1
Application number: US10/871,890
Authority: US
Inventors: I-Chien Wei; Kung-Hsing Lee; Hsien-Ming Hung
Original assignee: Hopax Chemicals Manufacturing Co Ltd
Current assignee: Hopax Chemicals Manufacturing Co Ltd
Priority date: 2004-06-18
Filing date: 2004-06-18
Publication date: 2005-12-22
Also published as: AU2005202652B2; US8263763B2; AU2005202652A1; TWI305535B; KR101155884B1; CN1880343A; CA2510020C; KR20060048401A; JP2006002158A; JP5415663B2; CN100509859C; EP1607406A1; US20060025583A1; TW200602355A; CA2510020A1; JP2013100523A; JP5797673B2

Abstract

Alkylsulfonated polyaminosaccharides, such as alkylsulfonated chitosan, having a sultone substituent covalently bonded to the amino group and a known size and degree of substitution may be produced by methods of the invention. Such methods may include heating of the polyaminosaccharide in a polar solvent, such as alcohol, ether, or alcoholether to reflux, then adding an alkyl sultone. Alkylsulfonated polyaminosaccharides of the present invention may be used in a wide variety of applications. Alkylsulfonated chitosan is particularly useful in wound healing.

Description

FIELD OF THE INVENTION

The present invention relates to alkylsulfonated polyaminosaccharides, methods of making such molecules, and their uses, particularly in wound healing. Specific aspects of the invention relate to alkylsulfonated chitosan.

BACKGROUND

Polyaminosaccharides are of considerable interest in a number of fields ranging from medicine and agriculture to water treatment and cleaning products. However, the use of such molecules is somewhat limited by difficulties in dissolving them in water. Thus, various approaches have been used to increase solubility of polyaminosaccharides in water.
Early methods often involved adding an organic or inorganic acid to the polyaminosaccharides to render them water soluble. However, the pH of the resulting solution was then very acidic, decreasing its usefulness in many biological applications. Increasing the pH of the solution tended to cause precipitation of the polyaminosaccharides.
Other methods have involved addition of a sulfonic acid group to the polyaminosaccharides. However, these methods have often resulted in serious degradation of the starting materials. Additionally, such methods have tended to produce a pool of modified polyaminosaccharides with high variability in important properties, such as polymer length and degree of substitution. While such modified polyaminosaccharides may exhibit increased water solubility, their use in applications benefiting from precise knowledge of polyaminosaccharide properties is either severely curtailed or requires additional processing.
Chitosan is a polyaminosaccharide of particular interest in a number of applications. Like many polyaminosaccharides, chitosan may be readily harvested from naturally occurring materials. The primary source of chitosan is presently discarded shells of lobsters and crayfish or shrimp, although it may also be obtained from the shells of crabs and other crustaceans as well as from insect shells and fungi. Chitosan is normally non-toxic and is compatible with a variety of living systems, including human tissues. However, like many other polyaminosaccharides, chitosan exhibits only limited solubility in water.
Acids have been added to chitosan to increase its water solubility. It has also been sulfonated, which results in a molecule with a chemical structure similar to that of heparin, a powerful anticoagulant.
One previous method for sulfonating chitosan involves the use of chlorosulfonic acid as a sulfonating agent in a low-polar solvent such as pyrimidine. An organic base is then added as an acid receptor for the sulfonation reaction. However, chlorosulfonic acid is not easy to use and it sulfonates not only at the amino group of the polyaminosaccharide, but also at other sites, such as the hydroxy groups. Additionally, the need to supply an acid receptor complicates the reaction. Overall, the reaction is difficult to carry out and often results in low yield of poorly characterized and unpredictable product.
Other methods have utilized alkyl sultones, which are considerably easier to use than chlorosulfonic acid. However, these methods add the alkyl sultones to chitosan in an aqueous acetic acid solution. H₂O reacts readily with alkyl sultones in the aqueous solution and hydrolyzes them, resulting in substantial loss of this reactant.
Additionally, the hydrolyzed alkyl sultones form highly acidic alkylsulfonic acid, which then degrades the chitosan. As a result, large amounts of alkyl sultone and chitosan are required for this method and yield is very poor, making the reaction economically unviable for many applications. Additionally, because of degradation, the length of the resulting alkylsulfonated chitosan varies widely, even when a uniform starting reagent is used. The degree of substitution of the amino group with an alkyl sultone also varies widely and cannot be reliably predicted from the outset. Finally, a significant amount of bonding between the chitosan and sultone resulting from the above method is actually ionic bonding, which is disrupted fairly easily by changes in the chemical environment. Although some covalent bonding, which is much more stable, does occur, it is infrequent and does not represent a significant portion of the bonding between the chitosan and the alkyl sultone.
Accordingly, additional methods for sulfonation of polyaminosaccharides, particularly chitosan are needed.

SUMMARY

The present invention relates to methods of producing sulfonated polyaminosaccharides by reaction of the polyaminosaccharides with an alkyl sultone in an organic solvent. The reaction occurs at reflux temperature. After reaction, the alkylsulfonated polyaminosaccharides may become less soluble or insoluble in the solution and may thus be readily recovered.
The resulting alkylsulfonated polyaminosaccharides may be of substantially the same length as prior to reaction. In addition, they may be sulfonated almost exclusively on the amino group and the degree of substitution may substantially correspond to that predicted at the outset of the reaction.
Alkylsulfonated polyaminosaccharides of the present invention may be non-toxic and biocompatible. They may be formed into films or non-woven structures, supplied in solution, or supplied and used in any other manner suitable for a given application. Specifically, some alkylsulfonated polyaminosaccharides of the present invention may be used for medical devices, personal care products, cosmetics, oral care products, odor control products, agricultural agents, water treatment, cleaning products, biochemistry products, contact lens solutions, and other medical products.
In selected embodiments of the present invention the alkylsulfonated polyaminosaccharides may include an alkylsulfonated chitosan. This alkylsulfonated chitosan may be structurally similar to heparin. Various forms may be used in a variety of manners, such as medical applications, including wound healing, disinfectants, water treatment, enzyme immobilization, and cosmetics.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood though the following drawings, taken in connection with the detailed description below, which provide further information regarding certain embodiments of the invention.
FIG. 1 illustrates the reaction of chitosan and an alkyl sultone to produce alkylsulfonated chitosan, according to an embodiment of the present invention.
FIG. 2 is a graph showing the effect of various quantities of alkylsulfonated chitosan powder, according to an embodiment of the present invention, on the weight of rats. (N=6)
FIG. 3 is a graph showing irritation of rat skin by alkylsulfonated chitosan powder, according to an embodiment of the present invention. Symbols designate the weight of alkylsulfonated chitosan powder per 2.5×2.5 cm area. The control includes no chitosan. (N=6)
FIG. 4 is a graph showing the inflammatory effects of alkylsulfonated chitosan according to an embodiment of the present invention as measured by PGE₂production by fibroblasts. The concentration of PGE₂was measured 24 hours after fibroblasts were treated with alkylsulfonated chitosan. The number shown represents the mean±standard deviation. (N=6)
FIG. 5 illustrates the effects of (a) alginate (Kaltostat®), (b) unmodified chitosan sponge, (c) unmodified chitosan fibers, and (d) 80% substituted alkylsulfonated chitosan, according to an embodiment of the present invention, on wound healing in rats. The FIGURE shows conditions of panniculus carnosis on the 21st day after operation.
FIG. 6 illustrates the cellular morphology of a clone L929 mouse fibroblasts negative control. Cells were grown on a Millipore AP250 1000 filter.
FIG. 7 illustrates the cellular morphology of clone L929 mouse fibroblasts grown around Kaltostat®.
FIG. 8 illustrates the cellular morphology of clone L929 mouse fibroblast grown around chitosan sponge.
FIG. 9 illustrates the cellular morphology of clone L929 mouse fibroblasts grown around chitosan fiber.
FIG. 10 illustrates the cellular morphology of clone L929 mouse fibroblasts grown around alkylsulfonated chitosan dressing according to an embodiment of the present invention.
FIG. 11 illustrates a histological analysis to analyze the cytotoxicity to cells grown around Kaltostat® (crystal violet stain).
FIG. 12 illustrates a histological analysis to analyze the cytotoxicity to cells grown around chitosan sponge (crystal violet stain).
FIG. 13 illustrates a histological analysis to analyze the cytotoxicity to cells grown around chitosan fiber (crystal violet stain).
FIG. 14 illustrates a histological analysis to analyze the cytotoxicity to cells grown around alkylsulfonated chitosan dressing according to an embodiment of the present invention (crystal violet stain).

DETAILED DESCRIPTION

The present invention includes alkylsulfonated polyaminosaccharides and methods for their creation and use. In selected embodiments, it relates to alkylsulfonated chitosan.
In specific embodiments of the present invention, the polyaminosaccharide is placed in an organic solvent. The polyaminosaccharide may be pre-processed to influence results of the reaction. For example, it may be deacetylated to allow access to the amino groups. It may also have protective groups to limit the degree of substitution, although in many embodiments this is not necessary. Reactive groups may be provided to facilitate other desirable reactions with the alkyl sultones. The polyaminosaccharide may be of any size, but in specific embodiments it may have a molecular weight of between 300 and 200,000, more specifically between 3,000 and 140,000.
In certain embodiments, the organic solvent may be a highly polar solvent. For example, alcohol, ether, etheralcohol and mixtures thereof may be used as a solvent. In specific embodiments the solvent may have less than 10% water. In certain embodiments, the alcohol may be methanol, ethanol, isopropanol or butanol or the ether may be methoxypropanol.
The mixture of polyaminosaccharide and solvent is next heated to reflux temperature (this may correspond with the boiling temperature of the mixture). For example, the reflux temperature may be between 50-150° C. Alkyl sultones are added to the solution. They may be added gradually or all at once. The alkyl sultones normally react almost exclusively with the amino groups of the polyaminosaccharide, with little to no reaction with the hydroxy groups. Further the alkyl sultones normally primarily form covalent bonds with the amino groups of the completed alkyl sulfonated polyaminosuccharide, which are far more resistant to changes in the chemical environment than ionic bonds. Ionic bonds are the most common type of bond formed in many previous methods. The present invention may result in at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of sultone-amino group bonds being covalent bonds.
In certain embodiments of the present invention, the alkyl sultones have a general formula of:

where R₁may be CH₂, CH or C₂H₄; R₂may be CH₂or CH; and R₃may be H or CH₃. In more specific embodiments, R₁is CH₂, R₂is CH₂, and R₃is H; R₁is CH, R₂is CH, and R₃is H; R₁is CH₂, R₂is CH₂, and R₃is CH₃; R₁is C₂H₄, R₂is CH₂, and R₃is H. More specifically, the alkyl sultone may be 1,3-propane sultone, 1,3-propene sultone, 1,4-butane sultone, or 2,4-butane sultone.
Only one type of alkyl sultone may be added, or a combination of two or more alkyl sultones may be used. Selection of the alkyl sultone and any combinations may be based on the desired properties and uses of the alkylsulfonated polyaminosaccharides to be produced. For example, selection of the alkyl sultone may be based on the need to perform further chemistry on the polyaminosaccharides, which may be facilitated by the presence of a specific alkyl group or influenced by steric effects of large alkyl groups.
The relative amounts of polyaminosaccharide and alkyl sultone used in the reaction may be selected to give a desired degree of substitution of the polyaminosaccharides. The degree of substitution of alkylsulfonated polyaminosaccharides in selected embodiments of the present invention may be between 10% and 80%, between 5% and 60%, or between 30% and 40%. The degree of substitution of alkylsulfonated polyaminosaccharides in selected embodiments of the present invention may also be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The degree of substitution obtained using methods of the present invention tends to be consistent in many embodiments and is thus predictable through routine experimentation with various polyaminosaccharides and alkyl sultones. The degree of substitution may be influenced by the amount and nature of the alkyl sultone added, although in many embodiments substantially all of the sultone reacts with the polyaminosaccharide. The general length of the polyaminosaccharides also influences the degree of substitution, as longer molecules will have a larger number of sultones for a given degree of substitution when compared to a shorter molecule. Length of polyaminosaccharides may be readily determined based on various measurements known to the art, such as molecular weight. Reflux temperature, gradual versus immediate addition of the alkyl sultone, and reaction time may additionally influence the degree of substitution of the polyaminosaccharide.
In selected embodiments, the mole ratio of polyaminosaccharides to alkyl sultone may be between approximately 1:4 and 4:1. In more specific embodiments it may be between 1:2 and 2:1.
After reaction, the alkylsulfonated polyaminosaccharide may be removed by filtration and dissolved in water or another solvent or crystallized. In many embodiments of the present invention, the yield of this reaction is quite high, such as at least 50%, at least 80%, at least 90%, at least 95% or even at least 99%.
After reaction, the size of the alkylsulfonated polyaminosaccharide may be known to a specificity normally accepted in the relevant field of use. In specific embodiments, the known size will generally correspond with the size of the original polyaminosaccharide because the reaction does not cause substantial degradation of the polyaminosaccharide.
Alkylsulfonated polyaminosaccharides of the present invention may be used for a variety of purposes such as those described elsewhere in the present application. In order to facilitate these uses, they may be solubilized in water or an aqueous solvent to form solutions of different viscosities. They may also be used to form films or three-dimensional structures. Although a variety of forms may be sufficient for long-term storage, powdered or lyophilized alkylsulfonated polyaminosaccharides may be particularly well suited to avoid degradation and other problems associated with storage.
While any suitable polyaminosaccharide may be used in the present invention, a number of specific embodiments relate to the use of chitosan. Chitosan is a long polymer of the general formula:
Chitosan of any type, including the α, β, linear or branched types may be used in the present invention. It may be sulfonated on the amino groups. This renders the molecule more hydrophilic, produces cationic groups, and may stabilize the molecule.
More specifically, chitosan may be reacted with an alkyl sultone as depicted in FIG. 1. Using this reaction, if 100 g of chitosan having a molecular weight of approximately 161 is used, then amounts of sultone indicated in Table 1 may be used to obtain the indicated degree of substitution. Table 1 shows that the degree of substitution of 1,3-propane sultone on chitosan is an approximately linear relationship to the amount of 1,3 propane sultone reactant.

TABLE 1

Amount of 1,3-Propane Sultone Reactant

Used to Obtain A Selected Degree of Substitution

of Chitosan (M.W. 161) in a Methanol Solvent

Degree of Substitution* Amount of Sultone Reactant

0 0

10 7.6 g

30 22.8 g

70 53.1 g

80 60.7 g

*Expressed as percentage of available amino groups occupied by a sultone after reaction.
As expected for a reaction in which nearly all sultone added reacts, the relationship between degree of substitution of the chitosan and the amount of sultone added may be approximately linear, particularly for degrees of substitution between 10% and 80%.
Alkylsulfonated chitosan produced by the above methodologies may be dissolved in water. This process is aided by the application of heat and stirring. The resulting solution is a weak acid with a transparent, light yellow color. The viscosity of the solution may be influenced by the amount of alkylsulfonated chitosan added. Alkylsulfonated chitosan may also be formed into a transparent, elastic film. In certain exemplary embodiments of the present invention, the elastic film may be produced by the dissolving the alkylsulfonated chitosan in a solvent, such as water, then evaporating the solvent. Other exemplary embodiments may use an oven to evaporate the solvent. In one embodiment the solution of alkylsulfonated chitosan is placed in an oven for one day. The thickness of this film may be varied, for example by controlling the concentration of the solution. Alkylsulfonated chitosan films may be used, inter alia, in medicine, medical devices, cosmetics and food. Alkylsulfonated chitosan may also be formed into weaves of nano-fibers similar to those formed from chitosan in U.S. Pat. No. 6,638,918. It may then be put to similar uses. Additionally, microcapsules of alkylsulfonated chitosan may be formed and used in a manner similar to that described for chitin in U.S. Pat. No. 6,242,099. Hydrogels may be formed as described using chitosan as the cationic polysaccharide in U.S. Pat. No. 5,858,392. Finally, the alkylsulfonated chitosan of the present invention may be used to coat natural fibers as described in U.S. 2003/0134120.
Alkylsulfonated chitosan has been shown to inhibit streptomycin-resistant Staphylococcus aureus, E. coli CCRC 10675, Pseudomonas aeruginosa CCRC 12450 and Candida albicans CCRC 20511 and may be able to inhibit or kill various other harmful microorganisms. The minimum inhibiting concentration of alkylsulfonated chitosan streptomycin-resistant Staphylococcus aureus is 0.38 mg/ml; for E. coli CCR 10675 it is 0.094 mg/ml; for Pseudomonas aeruginosa CCRC 12450 it is 0.38 mg/ml; and for Candida albicans CCRC 20511 it is 0.19 mg/ml. These minimum inhibiting concentrations are generally lower than those of unmodified chitosan. Therefore, alkylsulfonated chitosan may be used in place of chitosan in applications where anti-microbial effects are desirable to obtain a better product. Antimicrobial effects are largely independent of the size of the alkylsulfonated chitosan, the degree of substitution, or the sultone substituents. Specifically, because of its anti-microbial properties, alkylsulfonated chitosan may be useful in personal care products, food products, cleaning agents, agricultural agents, cosmetics, medicines and medical devices. For example, alkylsulfonated chitosan of the present invention may be used in place of chitosan in combination with elecampane as an anti-bacterial and anti-inflammatory agent as described in U.S. Pat. No. 6,521,628. Fibers similar to anti-bacterial rayon fibers such as described in U.S. Pat. No. 6,497,927 may be made using alkylsulfonated chitosan of the present invention.
Cleaning products, such as the mold and mildew remover of U.S. 2003/0176306 may be made with the alkylsulfonated chitosan of the present invention.
Alkylsulfonated chitosan of the present invention may also be used to enhance resistance of plants to disease, as described in U.S. Pat. No. 6,413,910. It may also be used to control plant diseases as described in U.S. Pat. No. 6,060,429 and U.S. Pat. No. 5,374,627.
While the alkylsulfonated chitosan of the present invention is normally an anti-microbial agent, some organisms, such as those described in U.S. Pat. No. 5,208,159, may actually grow well in it. It may also be used to encapsulate cells not harmed by the anti-microbial effects as is described for chitosan in U.S. Pat. No. 4,647,536.
Additionally, alkylsulfonated chitosan exhibits little to no toxicity to larger organisms, such as mammals. The acute oral toxicity (LD₅₀) amount for alkylsulfonated chitosan in rats is at least 5 g/kg, which is sufficient for it to be considered non-toxic. Additionally, alkylsulfonated chitosan does not appear to have significant effects on the weight of rats when ingested regularly. Therefore, alkylsulfonated chitosan may be used as an additive in foods or medicines. For example, alkylsulfonated chitosan of the present invention may be used in drug carriers or vaccines as described for unmodified chitosan in U.S. Pat. No. 6,497,904, cationic chitosan in U.S. Pat. No. 6,391,318 and various chitosans in U.S. Pat. No. 5,629,011. It may also be labeled with a radionuclide and used in radiation therapy as is described for chitosan in U.S. Pat. No. 5,762,903. Use in place of the cationic derivatives of native chitosan in the anti-inflammatory, anti-viral and anti-fungal agent of U.S. Pat. No. 6,562,802 may also be possible. Cationic derivatives of native chitosan used for receptor-mediated delivery in U.S. Pat. No. 5,129,887 may also be replaced with alkylsulfonated chitosan of the present invention.
Water treatment applications of chitosan have been described in U.S. Pat. No. 5,336,415 and U.S. Pat. No. 5,543,056, in which the alkylsulfonated chitosan of the present invention may also be used.
In yet another example, alkylsulfonated chitosan of the present invention may be used in the place of chitosan in oral care and odor control compositions such as those described in U.S. 2003/0104020. It may also be used in place of native chitosan in food coatings, preservatives, or gelled emulsions as described in U.S. 2003/0203084, U.S. Pat. No. 6,200,619, U.S. Pat. No. 4,223,023 and U.S. Pat. No. 6,238,720, or it may be used as a drug, weight loss agent, or food additive as described in U.S. Pat. No. 6,495,142, U.S. Pat. No. 5,098,733 and U.S. Pat. No. 5,976,550.
Further, alkylsulfonated chitosan does not irritate dermal tissue in rats or induce irregular or unusual growth. Thus it is appropriate to use in personal care products, cleaning products, cosmetics and medical devices. For example, alkylsulfonated chitosan of the present invention may be used with collagen to form beauty packs, as is described for chitosan in JP 6,048,917 and JP 5,345,712. It may be used in place of chitosan in an anti-aging cream as described in U.S. 2003/0104020 or in acne creams as described in U.S. Pat. No. 6,451,773. Use in place of chitosan in agents to aid in removal of adhesives from the epidermis as described in U.S. Pat. No. 6,461,635 is also possible.
In other applications, the alkylsulfonated chitosan of the present invention may be used in place of the cation residue of chitosan in a leukoreduction filter as described in U.S. Pat. No. 6,497,927. Other filtration media may also be made from chitosan as described in U.S. Pat. No. 5,618,622.
Alkylsulfonated chitosan of the present invention may also be used in place of the water-soluble salt of chitosan employed in the hair care products and methods of U.S. Pat. No. 4,202,881 and U.S. Pat. No. 4,134,412. It may also be used in place of differently modified chitosan to form nail polish as described in U.S. Pat. No. 4,954,619. Use of chitosans, which may be replaced with the alkylsulfonated chitosan of the present invention, in contact lens solutions is described in U.S. 2002/0177577.
Chitosan has been previously explored as a potential wound dressing or anticoagulant. Alkylsulfonated chitosan produced using a method of the present invention (FIG. 5D) has been shown to be more effective in promoting wound healing than presently marketed alginate wound dressings (Kaltostat®, ConvaTec, Ltd., UK) (FIG. 5A), chitosan sponges (FIG. 5B) and chitosan fibers (Chinatex, Co. Ltd., Taiwan)(FIG. 5C). As FIG. 5 clearly shows, the 80% sultone-substituted chitosan of the present invention produced markedly superior wound healing in rats.
Thus alkylsulfonated chitosan may be used in place of chitosan or other modified chitosan in a number of wound healing applications. For example, U.S. Pat. No. 3,911,116, U.S. Pat. No. 4,532,134 and U.S. Pat. No. 3,914,413 all discuss the use of chitin or modified chitin in the promotion of wound healing. The alkylsulfonated chitosan of the present application, if used in the same manner as chitin in any of these three patents, would be expected to provide superior results. Similarly, the artificial skin described in U.S. Pat. No. 5,166,187 may be made with the alkylsulfonated chitosan of the present invention with expected improvements in the final product.
An example method of the present invention includes a method of wound healing in a wounded mammal comprising applying an alkylsufonated chitosan to the wound. In certain exemplary embodiments, the wound may include open wounds, bleeding wounds, open ulcers, vascular grafts, vascular patches, bleeding saturated areas, bleeding cardiac valve areas, and any combination thereof. The method may further include inhibiting fibroplasia and/or promoting tissue regeneration in vascular grafts. Other embodiments of the present method have the alkylsulfonated chitosan in the form of a film, a fiber, a sponge, a bubble, a non-woven, a liquid, a powder and any combination thereof. One embodiment of the present method applies the alkylsulfonated chitosan to the wound for at least 14 days. Another embodiment applies the alkylsulfonated chitosan to the wound for at least 21 days.
Finally, the alkylsulfonated chitosan of the present invention may be used in place of the chitosan of U.S. 2002/0189022 to treat textiles after washing.
In all of the above examples, alkylsulfonated chitosan may be used in place of chitosan to achieve superior results. Thus none of the above discussion should be construed as indicating that the patents discussed encompass, teach or suggest any aspect of the present invention.
The following examples are provided to further explain specific examples of the invention. They are not intended to represent all aspect of the invention in its entirety. Variations will be apparent to one skilled in the art.

EXAMPLES

Example 1

Reagents Used

In the following examples, unless otherwise indicated, chitosan with a molecular weight of between 10,000 and 200,000 was used. Chitosan in the following examples generally includes poly(D-glucosamine) with about 75% to about 85% deacetylation. 1,3-propane sultone or 1,4-butane sultone was used as the alkyl sultone. The solvent for reaction was methanol, n-butanol or methoxypropanol.

Example 2

Test of Previous Methods—Comparative Example

The following reaction was described in a study of the “Semi-IPN of Sulfonated Polyurethane and Chitosan”, NTU, 2001. Six grams (g) of chitosan powder was dissolved in 594 g of 2 Wt % acetic acid solution and filtered. The filtrate was placed in a four-neck flask and blanketed with nitrogen. The filtrate was brought to 30 degrees Celsius (° C.) and stirred at 200 revolutions per minute (rpm). Ninety milliliters (ml) of 1,3-propane sultone was added to the flask slowly and stirred for 6 hours. The reaction product was precipitated by adding 1000 ml of acetone and separated by filtration. The product was then washed with a large amount of methanol and acetone several times. It was dried in a vacuum oven. After drying, 9.3 g of water-soluble sulfonated chitosan was obtained, representing a yield of 96%. However, the reaction process was in acidic conditions and the pH of the solution was low enough to cause chitosan degrade.
To prove the water-soluble sulfonated chitosan produced according to methods of NTU publication were sulfonated primarily with ionic bonds, the process below was performed. First, the sulfur content of product was determined to equal 9.79% by weight. Then 2 g product was added to 50 ml pure water and the solution was stirred to ensure that sulfonated chitosan was dissolved in water. The solution was maintained overnight at room temperature (pH was 3.1), then ammonium solution (20%) was added until pH was 11˜12 to form a gel-like precipitation that was separated out. After that, the product was filtered and then washed with water and 50% methanol and finally with pure methanol. After drying the product in a vacuum oven (75° C.), the weight was 1.05 g. The sulfur content of final product was measured to be 0.48%. Therefore, the reduction of sulfur content shows that the sultone was attached to the chitosan primarily through ionic bonds.

Example 3

Reaction of High Molecular Weight Chitosan with 1,3-Propane Sultone

161 g of high molecular weight chitosan (MW around 140,000) was placed in a flask and 700 ml of methanol was added and stirred. The solution was heated to reflux temperature (65-67° C.), then 122 g of 1,3-propane sultone was added slowly in drops. After all of the 1,3-propane sultone was added, reflux was continued for 4 hours. The flask was then cooled to room temperature and the product was filtered and rinsed with methanol several times. The product was then dried overnight in a vacuum oven. After drying, 310 g of alkylsulfonated chitosan was obtained, representing a yield of 99.7%. Since the reaction process of alkylsulfonated chitosan was in an almost neutral condition (pH 5˜6), no degradation of chitosan was observed.
To prove the water-soluble sulfonated chitosan produced according to methods of Example 3 was sulfonated primarily with covalent bonds, the same process as described above in Example 2 was performed. After adding ammonia solution, the alkylfonated chitosan was precipitated, filtered, washed, and dried. Then the sulfur content was analyzed. Because the sulfur content was only reduced from 8% to 5%, the alkylsulfonated chitosan produced according to the method of Example 3 was sulfonated primarily with covalent bonds.

Example 4

Reaction of Low Molecular Weight Chitosan with 1,3-Propane Sultone

161 g of low molecular weight chitosan (MW around 30,000) was placed in a flask and 700 ml of methanol was added and stirred. The solution was heated to reflux temperature (65-67° C.), then 122 g of 1,3-propane sultone was added slowly in drops. After all of the 1,3-propane sultone was added, reflux was continued for 4 hours. The flask was then cooled to room temperature and the product was filtered and rinsed with methanol several times. The product was then dried overnight in a vacuum oven. After drying, 280.2 g of alkylsulfonated chitosan was obtained, representing a yield of 99.0%.

Example 5

Reaction of High Molecular Weight Chitosan with 1,4-Butane Sultone

161 g of high molecular weight chitosan (MW around 140,000) was placed in a flask and 700 ml of n-butanol was added and stirred. The solution was heated to reflux temperature (110° C.), then 136 g of 1,4-butane sultone was added slowly in drops. After all of the 1,4-butane sultone was added, reflux was continued for 8 hours. The flask was then cooled to room temperature and the product was filtered and rinsed with methanol several times. The product was then dried overnight in a vacuum oven. 252.5 g of alkylsulfonated chitosan was obtained, representing a yield of 85%.

Example 6

Reaction of Low Molecular Weight Chitosan with 1,4-Butane Sultone

161 g of low molecular weight chitosan (MW around 30,000) was placed in a flask and 700 ml of n-butanol was added and stirred. The solution was heated to reflux temperature (110° C.), then 136 g of 1,4-butane sultone was added slowly in drops. After all of the 1,4-butane sultone was added, reflux was continued for 8 hours. The flask was then cooled to room temperature and the product was filtered and rinsed with methanol several times. The product was then dried overnight in a vacuum oven. 246.5 g of alkylsulfonated chitosan was obtained, representing a yield of 83%.

Example 7

Reaction of Very Low Molecular Weight Chitosan with 1,3-Propane Sultone

16.1 g of very low molecular weight chitosan (MW around 10,000) was placed in a flask and 200 ml of 1-methoxy-2-propanol was added and stirred. The solution was heated to reflux temperature (110-115° C.), then 12.3 g of 1,3-propane sultone was added slowly in drops. After all of the 1,3-propane sultone was added, reflux was continued for 4 hours. The flask was then cooled to room temperature and the product was filtered and rinsed with methanol several times. The product was then dried overnight in a vacuum oven. 26.3 g of alkylsulfonated chitosan was obtained, representing a yield of 93.1%.

Example 8

Anti-Microbial Effects

A 48-well plate was used for the present experiment although only the first 24 wells were used. All wells had a total volume of 1 ml. The first well received only 0.5 ml of sterilized water and 0.5 ml of luria broth (LB). The second well received 0.5 ml of alkylsulfonated chitosan sulfonated with 1,3-propane sultone was dissolved to 0.3% wt in 2 wt % acetic acid solution as well as 0.5 ml of bacterial or yeast culture (1 ml overnight bacterial or yeast solution+100 ml LB medium). The third well received 0.5 ml of alkylsulfonated chitosan dissolved to 0.3% wt in 2 wt % acetic acid solution and 0.5 ml of sterilized water. After mixing 0.5 ml was transferred from the third well to the fourth well. 0.5 ml of sterilized water was then added to the fourth well and after mixing, 0.5 ml was transferred to the fifth well. Such serial dilutions were repeated through the twenty-fourth well. 0.5 ml of bacterial or yeast culture was added to the third to twenty-fourth wells.

A separate 48-well plate was used for each microorganism to be tested. Streptomycin-resistant Staphylococcus aureus, E. coli CCRC 10675, Pseudomonas aeruginosa CCRC 12450 and Candida albicans CCRC 20511 were all tested. Because microorganism growth renders the solution more opaque, spectroscopy may be used to detect microorganism growth. Scanning of the plate to detect the highest dilution/lowest concentration of alkylsulfonated chitosan that prevents further growth of the microorganism revealed the minimum inhibitory concentration (MIC) for each microorganism. The results, as well as the known MIC for unmodified chitosan, are presented below in Table 2.

TABLE 2


Minimum Inhibitory Concentration of
Alkylsulfonated Chitosan for Selected
Microorganisms

	Alkylsulfonated	Unmodified
Microorganism	Chitosan MIC	Chitosan MIC

Streptomyacin-resistant	0.38 mg/ml	0.63 mg/ml
Staphylococcus aureus
E. coli CCRC 10675	0.094 mg/ml	0.625 mg/ml
Pseudomonas aeruginosa CCRC	0.38 mg/ml	1.25 mg/ml
12450
Candida albicans CCRC 20511	0.19 mg/ml	0.31 mg/ml

Example 9

Toxicity and Weight Effects of Alkylsulfonated Chitosan

Twenty-four 7-week-old rats were divided into four groups. All rats were deprived of food overnight prior to commencement of the experiment. Rats were then fed a specified amount of powdered alkylsulfonated chitosan sulfonated with 1,3-propane sultone. All members of each group received 2 g/kg, 3 g/kg, 4 g/kg or 5 g/kg of the powder. Rats were then observed for 7 days after the experiment to determine survival and weight. The experiment was designed to determine the LD₅₀value for the alkylsulfonated chitosan (the amount that produced death in 50% of the rats). However, none of the rats in the experiment died, indicating that the LD₅₀value is well above 5 g/kg. Because any substance with an LD₅₀above 5 g/kg is deemed to be non-toxic, further studies were not necessary. Additionally, as shown in FIG. 2, the weight of the rats was also not substantially affected, providing further indication of the lack of serious harm in the animals.

Example 10

Skin Irritation Effects of Alkylsulfonated Chitosan

The backs of 6-8-week-old Wistar rats were shaved and allowed the recover for 24 hours. Either 0.25 g or 0.5 g of alkylsulfonated chitosan powder sulfonated by 1,3-propane sultone was added to a small amount of petroleum jelly and applied to gauze which was then fixed to a 2.5×2.5 cm²area of the back with ventilated tape. A control rat receiving only petroleum jelly was also prepared. Color of the skin under the test area and outside of the test area was measured and visually observed at 24, 48, 72, 96 and 120 hours after application began. Change in chromatism (Δa*) measured by a Chrommeter was recorded. The level of irritation was also observed visually and assigned a value of 0-3. Photographs were taken at each measurement time.

Table 3 and FIG. 3 present the results of this test. The level of irritation as determined chromatographically was highest after the first day for both test samples and the control rat, likely indicating the residual effects of shaving or irritation produced by petroleum jelly. However, statistical analysis indicated no obvious change over any of the days (p>0.05). Additionally, no skin irritation was observed in any of the rats on any day, so the assigned irritation value for visible observation was consistently 0.

TABLE 3


Chromatism and Visual Observations of Rat
Skin Treated with Alkylsulfonated Chitosan

Control

0.25 g powder

0.5 g powder

Day	Δa*	Visual	Δa*	Visual	Δa*	Visual

1	1.516	0	1.323	0	1.603	0
	(0.534)		(0.411)		(0.403)
2	0.251	0	0.132	0	0.112	0
	(0.456)		(0.314)		(0.201)
3	0.360	0	−0.310	0	−0.013	0
	(1.361)		(0.410)		(1.330)
4	−0.454	0	−0.307	0	−0.747	0
	(0.883)		(0.256)		(0.717)
5	−0.504	0	−0.447	0	−0.644	0
	(0.730)		(0.717)		(0.519)

*Standard deviation is indicated in parentheses.
N = 6.

Example 11

Fibroblast Inflammation Effects of Alkylsulfonated Chitosan

Dermal cells were cultured in vitro and the concentration of prostaglandin (PGE₂), an indicator of inflammation, was measured through optical density at 405 nm after addition of alkylsulfonated chitosan 1,3-propane sultone. Ethanol, which does not inflame fibroblasts and cause release of PGE₂was used as a negative control. Phorbol myristate acetate (PMA), a powerful inflammatory agent, was used as a positive control. 0.001, 0.002, 0.003 or 0.004 w/w % alkylsulfonated chitosan was added to the culture in the experiment. FIG. 4 shows the results, which indicate that the alkylsulfonated chitosan induced no more PGE₂release than ethanol and significantly less than PMA, further confirming that the alkylsulfonated chitosan is not an inflammatory agent.

Example 12

In Vitro Cytotoxicity of Alkylsulfonated Chitosan

L929 mouse fibroblast cells were cultured to confluence in 6 cm plates. A sterile sample of one of four dressings was added to each plate. The dressings included a control millipore filler (FIG. 6) and four experimental dressings including Kaltostat® (FIG. 7), chitosan sponge (FIG. 8), chitosan fiber (FIG. 9), and 4 milligrams (mg) film of alkylsulfonated chitosan sulfonated with 1,3-propane sultone (FIG. 10). Samples were added to the center of the dish, which was then incubated for one day. Cellular morphology was inspected microscopically. The morphology of cells around the four experimental dressings were the same with the negative control, which shows the dressings are all biocompatible.
Histological analysis using 2% crystal violet stain was also performed the analyze cytotoxicity. The cells grown around all four dressings were stained with crystal violet which stains only cells. (FIGS. 11 thru 14). Growth around the dressings was normal, which shows the materials are not toxic.

Example 13

In Vivo Wound Healing Experiments

Chitosan sponges used in these experiments were prepared by Chinatex by dissolving 5 w/w % chitosan in 2 Wt % acetic acid_(aq)then placing the mixture in a 20 cm×20 cm×0.3 cm container. The mixture was freeze dried, then neutralized with 5% NaOH_(aq). The sponge was then washed in deionized water until neutral then freeze dried again.
Chitosan fibers were prepared by Chinatex by preparing the same initial mixture, then filtering it with a metal filter. After degassing, 5% NaOH_(aq)was added and the mixture was wet-spun. The resulting fibers were washed with deionized water until neutral then dried.
Sprague-Dawley rats weighting around 250-300 g were anesthetized and their backs were shaved and disinfected. A wound of approximately 3 cm×3 cm was made using a surgical knife, removing the skin down to the panniculus carnosus. A dressing made of Kaltostat®, a chitosan sponge, chitosan fiber, or alkylsulfonated chitosan film was then applied to the wound and covered with gauze. A 6 cm×7 cm piece of Tegaderm (3M, Minnesota), was then applied and secured with an elastic bandage wound around the rat. The rats were kept in isolation and provided with food and water ad libidum. Healing of the wound was assessed after 3, 7, 14 and 21 days. The bandages were not replaced during this period, although they were temporarily removed for inspection.
All dressings appeared to not be cytotoxic. Repair rate of the panniculus carnosis was calculated by (area of wound immediately after operation—area of wound on day n)/(area of wound immediately after operation). After 14 days, all dressings exhibited approximately 70% repair rate. However, by day 21 the repair rates were all almost 100%, except when chitosan fiber was used. However, as FIG. 5 clearly shows, only the wound treated with alkylsulfonated chitosan was fully closed and exhibits far more advanced healing. Thus alkylsulfonated chitosan exhibits superior wound healing ability when compared with alginate or unmodified chitosan.
The above description relates to only selected exemplary embodiments of the present invention, variations will be apparent to one skilled in the art.

Claims

1. A method of making an alkylsulfonated polyaminosaccharide comprising:

adding a polyaminosaccharide to an organic solvent to form a mixture;

heating the mixture to a reflux temperature;

reacting an alkyl sultone with the polyaminosaccharide in the mixture to form a covalent bond between the alkyl sultone and an amino group of the polyaminosaccharide resulting in an alkylsulfonated polyaminosaccharide.

2. The method of claim 1, wherein the polyaminosaccharide further comprises a deacetylated polyaminosaccharide.

3. The method of claim 1, wherein the polyaminosaccharide has a molecular weight of between 300 and 200,000.

4. The method of claim 3, wherein the polyaminosaccharide has a molecular weight of between 3,000 and 140,000.

5. The method of claim 1, wherein the polyaminosaccharide comprises chitosan.

6. The method of claim 1, wherein the organic solvent comprises an alcohol, ether, or etheralcohol.

7. The method of claim 6, further comprising the alcohol, ether, or etheralcohol selected from the group consisting of: methanol, ethanol, isopropanol, butanol, methoxypropanol and any combination thereof.

8. The method of claim 1, further comprising the reflux temperature between 50 and 150° C.

9. The method of claim 1, further comprising the alkyl sultone having a formula of:

wherein R₁is CH₂, CH or C₂H₄; R₂is CH or CH₂; and R₃is H or CH₃.

10. The method of claim 9, wherein R₁is CH₂, R₂is CH₂, and R₃is H.

11. The method of claim 9, wherein R₁is CH, R₂is CH, and R₃is H.

12. The method of claim 9, wherein R₁is CH₂, R₂is CH₂, and R₃is CH₃.

13. The method of claim 9, wherein R₁is C₂H₄, R₂is CH₂, and R₃is H.

14. The method of claim 1, further comprising the alkyl sultone selected from the group consisting of: 1,3-propane sultone, 1,3-propene sultone, 1,4-butane sultone, 2,4-butane sultone and any combination thereof.

15. The method of claim 1, further comprising the alkylsulfonated polyaminosaccharide having a known degree of substitution with the alkyl sultone.

16. The method of claim 15, further comprising a degree of substitution between 10% and 80%.

17. The method of claim 15, further comprising a degree of substitution between 5% and 60%.

18. The method of claim 15, further comprising a degree of substitution between 30% and 40%.

19. The method of claim 1, further comprising the mixture having a mole ratio of polyaminosaccharide to alkyl sultone of between 1:4 and 4:1.

20. The method of claim 1, further comprising the mixture having a mole ratio of polyaminosaccharide to alkyl sultone of between 1:2 and 2:1.

21. The method of claim 1, further comprising precipitating the alkylsulfonated polyaminosaccharide.

22. The method of claim 21, further comprising collecting the precipitated alkylsulfonated polyaminosaccharide by filtration.

23. The method of claim 21, further comprising crystallizing the alkylsulfonated polyaminosaccharide.

24. The method of claim 1, further comprising obtaining a reaction yield of at least 50%.

25. The method of claim 1, further comprising obtaining a reaction yield of at least 80%.

26. The method of claim 1, further comprising obtaining a reaction yield of at least 99%.

27. A method of making an alkylsulfonated chitosan comprising:

adding a chitosan to an organic solvent to form a mixture;

heating the mixture to a reflux temperature;

reacting an alkyl sultone with the chitosan in the mixture to form a covalent bond between an alkyl sultone and an amino group of the chitosan resulting in an alkylsulfonated chitosan.

28. The method of claim 27, wherein the chitosan is selected from the group consisting of: α chitosan, β chitosan, linear chitosan, branched chitosan and any combination thereof.

29. The method of claim 27, further comprising the organic solvent selected from the group consisting of: methanol, ethanol, isopropanol, butanol, methoxypropanol and any combination thereof.

30. The method of claim 27, further comprising the reflux temperature between 60 and 140° C.

31. The method of claim 27, wherein the alkyl sultone comprises 1,3-propane sultone or 1,4-butane sultone.

32. The method of claim 27, further comprising the alkylsulfonated chitosan having a known degree of substitution with the alkyl sultone.

33. A alkylsulfonated chitosan made by the process of:

adding a chitosan to an organic solvent to form a mixture;

heating the mixture to a reflux temperature;

34. A formulation comprising an alkylsulfonated chitosan having a known size and degree of substitution and at least 50% of alkyl sultones attached to amino groups of the chitosan attached through a covalent bond.

35. The formulation of claim 34, further comprising the alkylsulfonated chitosan in an aqueous solution.

36. The formulation of claim 34, further comprising the alkylsulfonated chitosan in a film.

37. The formulation of claim 34, further comprising the alkylsulfonated chitosan in a powder form.

38. The formulation of claim 34, further comprising the formulation operable to promote wound healing in a wounded mammal.

39. The formulation of claim 34, further comprising the formulation operable to inhibit the growth of a micro-organism.

40. The formulation of claim 39, further comprising the micro-organism selected from the group consisting of:

streptomycin-resistant Staphylococcus aureus, E. coli CCRC 10675, Pseudomonas aeruginosa CCRC 12450 and Candida albicans CCRC 20511, and any combination thereof.

41. The formulation of claim 34, further comprising the formulation having no toxic effects in mammals.

42. The formulation of claim 34, further comprising the formulation having no inflammatory effects in mammals.

43. The formulation of claim 34, wherein the formulation is suitable for use in an application that uses chitosan.

44. The formulation of claim 34, wherein the formulation is suitable for use in a personal care product.

45. The formulation of claim 44, wherein the personal care product is selected from the group consisting of: a hair care product, a nail polish, an oral care product, an odor control composition and a contact lens solution, and any combination thereof.

46. The formulation of claim 34, wherein the formulation is suitable for use in a food product.

47. The formulation of claim 46, wherein the food product is selected from the group consisting of: a preserving agent, a film-like product, a health food, a dietary food, a food coating, a gelled emulsion, a weight-loss agent, a food additive and any combination thereof.

48. The formulation of claim 34, wherein the formulation is suitable for use in cleaning products.

49. The formulation of claim 34, wherein the formulation is suitable for use in an agricultural product.

50. The formulation of claim 49, wherein the agricultural product is selected from the group consisting of: a feed additive, an animal food additive, a plant care product, a fertilizer, a grass cultivator, a disease inhibitor, an anti-fungal agent and any combination thereof.

51. The formulation of claim 34, wherein the formulation is suitable for use in a cosmetic.

52. The formulation of claim 51, wherein the cosmetic is selected from the group consisting of: a beauty pack, an anti-aging cream, an acne cream, a make-up, a facial cleansing product and a maintenance product and any combination thereof.

53. The formulation of claim 51, wherein the cosmetic is selected from the group consisting of: a gel, a cream, a grease, a spray, a bubble, a non-woven, a liquid, a powder and any combination thereof.

54. The formulation of claim 34, wherein the formulation is suitable for use as a medicine.

55. The formulation of claim 54, wherein the medicine is selected from the group consisting of: an antibacterial agent, an anti-fungal agent, an anti-inflammatory agent, an anti-tumor agent, an anti-cholesterol agent, an anti-viral agent, a drug carrier, a vaccine, a microcapsule, a hydrogel, a fibrin adhesive, a fibrin sealant and any combination thereof.

56. The formulation of claim 34, wherein the formulation is suitable for use in medical devices.

57. The formulation of claim 56, wherein the medical device is a radiation therapy device or a leukocyte removal device.

58. The formulation of claim 34, wherein the formulation is suitable for use in weaves of nano-fibers.

59. The formulation of claim 34, wherein the formulation is suitable for use in a textile.

60. The formulation of claim 59, wherein the textile is selected from the group consisting of: a natural fiber, a synthetic fiber and any combination thereof.

61. The formulation of claim 34, wherein the formulation is suitable for use in a water-treatment application.

62. The formulation of claim 61, wherein the water treatment application is selected from the group consisting of: a heavy metal absorber, a lipid absorber, a protein absorber and any combination thereof.

63. The formulation of claim 34, wherein the formulation is suitable for use in a biochemistry product.

64. The formulation of claim 63, wherein the biochemistry product is selected from the groups consisting of: an enzyme purifier, a enzyme holder, an exchangeable resin, a TLC material and any combination thereof.

65. An alkylsulfonated chitosan having a known size and degree of substitution and at least 50% of alkyl sultone attached to amino groups of the chitosan attached through a covalent bond.

66. A method of promoting wound healing in a wounded mammal comprising applying an alkylsulfonated chitosan to the wound.

67. The method of claim 66, wherein the wound is selected from the group consisting of: an open wound, a bleeding wound, an open ulcer, a vascular graft, a vascular patch, a bleeding sutured area, a bleeding cardiac valve area and any combination thereof.

68. The method of claim 66, further comprising inhibiting fibroplasia.

69. The method of claim 66, further comprising promoting tissue regeneration in vascular grafts.

70. The method of claim 66, further comprising the alkylsulfonated chitosan in the form of a film.

71. The method of claim 66, further comprising the alkylsulfonated chitosan in a form selected from the group consisting of: a fiber, a sponge, a gel, a cream, a grease, a spray, a bubble, a non-woven, a liquid, a powder and any combination thereof.

72. The method of claim 66, wherein the alkylsulfonated chitosan is applied for at least 14 days.

73. The method of claim 66, wherein the alkylsulfonated chitosan is applied for at least 21 days.