TWI496791B - Crosslinked chitosan and its preparation method - Google Patents

Description

Cross-linked chitosan and preparation method thereof

The present invention relates to a cross-linked chitosan and a preparation method thereof, and more particularly to a chitosan which produces a crosslinked structure by formation of a guanamine bond and a preparation method thereof.

Chitosan has been widely used in biomedical applications due to its antibacterial activity, hemostasis, tissue regeneration, and high biocompatibility, for example, as a skeleton of biomedical materials.

In order to enhance the efficacy of chitosan, many studies in the prior art have focused on the modification of chitosan, and the biological materials currently known for modification include collagen, alginic acid, gelatin and the like.

Further, if chitosan is physically or chemically cross-linked with other components, a product having better mechanical strength and having special biomedical use can be obtained. For example, if glutaraldehyde is used as a cross-linking agent to covalently crosslink chitosan, a special microparticle structure can be produced, which can be used not only in drug delivery systems but also in tissue engineering. Scaffolds, see Gupta, KC, et al., Glutaraldehyde cross-linked chitosan microspheres for controlled release of centchroman. Carbohydr Res, 2007. 342(15): p. 2244-52.

In addition to the aforementioned applications, it is currently known to apply chitosan to wound dressings under appropriate crosslinking conditions. For example, the inventors of the present invention have discovered that chitosan can be formed into a polyelectrolyte complex with polyglutamic acid, and after lyophilization, an opaque porous scaffold material that can be applied to a wound dressing is obtained. The chitosan/poly glutamic acid composite scaffold described above has a significant contribution to wound healing compared to wounds without dressings. From the tissue section of the wound, chitosan has an inhibitory effect on inflammatory cells and has a stimulating effect on the proliferation of fibroblasts, while polyglutamic acid can enhance the growth of epithelial cells. This can be seen in Lin Yuyong, the application of chitosan/polyglutamic acid composite scaffold in wound dressing, Master's thesis of Institute of Polymer Science and Engineering, National Taiwan University, July 2009.

In addition, researchers have also cross-linked chitosan with collagen, polyphosphoric acid, hyaluronic acid, etc. to obtain wound dressings with different properties and different biomedical applications.

However, due to the aforementioned techniques for modifying chitosan, there are still some unsatisfactory ones, such as the cross-linking agent glutaraldehyde is toxic, most of the wound dressings produced have no transparency, and the preparation process is more efficient. Low...etc. How to propose a novel modified chitosan and its preparation method and apply it to wound dressing or water gel is a positive breakthrough for researchers in this field.

Inspired by the shortcomings of the prior art, the inventor exhausted his mind and researched it, and based on his years of experience in the industry, he developed a cross-linked chitosan and its preparation method as a new A generation of water gel or wound dressing that has satisfactory transparency, water absorption, mechanical strength, chemical stability, and the like.

One of the objects of the present invention is to provide a crosslinked polysaccharide comprising glucosamine and N-acetylglucosamine, and the monomers are linked to each other by β(1-4) glycosidic bonds, at least in part. The glucosamine monomer forms a guanamine bond with the crosslinking agent.

In the above-mentioned crosslinked polysaccharide, the crosslinking agent preferably includes a dibasic acid or an anhydride thereof, and is preferably substituted with an amine group.

In the above crosslinked polysaccharide, the crosslinking agent is preferably selected from the group consisting of malonic acid, succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, aspartic acid, glutamic acid, and at least two A group consisting of a polylactic acid of a carboxyl group, and a polylactic acid type includes a lactic acid oligomer.

In addition, in a preferred embodiment, the molecular weight of the polysaccharide before crosslinking is between 60 kDa and 800 kDa, and the degree of deacetylation (DD) is greater than 80%, and the -NH ₃ ⁺ pair -COO ^- The repeating unit ratio is between 1:1 and 9:1. In another preferred embodiment, the crosslinking agent can be a lactic acid oligomer having at least two carboxyl groups and preferably having a molecular weight of between 300 and 3000, more preferably about 500, 1000 or 2000.

The crosslinked polysaccharide described above can be applied to a hygroscopic material such as a diaper, a feminine article, a mask, etc., and is preferably used as a wound dressing.

A further object of the present invention is to provide a water gel comprising chitosan, wherein at least a part of the glucosamine monomer forms a guanamine bond with a crosslinking agent, and the crosslinking agent is preferably a natural small molecule acid or an anhydride thereof. And may be selected from the group consisting of malonic acid, succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, aspartic acid, glutamic acid, and polylactic acid having at least two carboxyl groups.

It is still another object of the present invention to provide a crosslinked chitosan having a partial structure as shown in Formula I:

In a preferred embodiment, X is a substituted or unsubstituted C ₁ -C ₅ alkylene group, and is preferably substituted with an amine group.

Further, it is an object of the present invention to provide a process for preparing a crosslinked chitosan comprising using a dibasic acid or a dibasic acid anhydride to form a crosslinked structure by formation of a guanamine bond.

In the above method, the crosslinking structure is preferably produced in the presence of carbodiimide, and more preferably in N,N-(3-dimethylaminopropyl)-N'-ethyl-carbon two. It is carried out in the presence of an imine and N-hydroxy-succinimide. Further, N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide and N-hydroxy-succinimide are preferably dissolved in an alcohol such as ethanol.

To fully clarify the objects, features, and advantages of the present invention, those of ordinary skill in the art of the present invention can understand the invention and practice the invention. A detailed description of the present invention will be given.

Noun definition

In the present invention, "chitosan" refers to a product obtained by deacetylation of chitin, which belongs to a linear polysaccharide, and the monomers are randomly distributed glucosamine and N-acetylglucosamine. They are joined to each other by a β(1-4) glycosidic bond.

In the present invention, "de-acetylation degree" means the molar ratio occupied by glucosamine in the chitosan molecule.

In the present invention, "crosslinking" means that two or more specific functional groups between or within a polymer molecular chain form a bridging structure by a crosslinking agent. For example, an amine group on a glucosamine monomer in a chitosan molecular chain can be used as a crosslinking agent by a dibasic acid, and a glucosamine monomer in another molecular chain of chitosan The amine group is crosslinked.

In the present invention, "water gel" means a water-insoluble polymer material which is swellable in water to retain water in its structure. Moreover, as is known to those of ordinary skill in the art to which the present invention pertains, the water gel system is not the same as xerogel, sponge, or aerogel.

Water gel preparation

0.8 g of chitosan powder was dissolved in 20 mL of a 2.0% (v/v) aqueous acetic acid solution at room temperature, followed by stirring until the powder was completely dissolved to obtain 4.0% (w/w) chitosan. The solution was then allowed to stand overnight for removal of bubbles in the solution. In one embodiment, the chitosan used has an average molecular weight of 300 kDa and a deacetylation degree of 97%, but the average molecular weight may also range from 60 kDa to 800 kDa, and the degree of deacetylation may be greater than 80%.

The glutamic acid, succinic acid, and succinic anhydride are separately dissolved in deionized water, and then the dibasic acid or its anhydride solution is dispersed in the chitosan solution and uniformly stirred. Next, N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide (EDC) and N-hydroxy-succinimide (NHS) as catalysts are added to promote Formation of a guanamine bond. The solution was then stirred at 60 ° C to obtain a clear water gel.

In the present invention, the formation of a guanamine bond may not be carried out in the presence of a catalyst. For example, the formation of a guanamine bond may be carried out in an environment of high temperature and reduced pressure without using a catalyst. Further, in order to enhance the progress of the crosslinking reaction, N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide and N-hydroxy-succinimide are preferably dissolved in ethanol. in.

In the present invention, the ratio of the repeating unit of the chitosan/dibasic acid or its anhydride may be from 90/10 to 50/50, but not limited thereto, such as 80/20, 75/25, 70. /30, 67/33, 60/40, etc. For convenience of explanation, the following is a list of the weight of chitosan and dibasic acid used in the preparation of different repeating unit ratios and the gel fraction of the water gel, wherein C50S50 represents chitosan (chitosan) The ratio of repeating units for succinic acid (represented by S) is 50:50, C80S20 represents the ratio of repeating units of chitosan to succinic acid is 80:20, and C50G50 represents chitosan. The ratio of sugar to glutamic acid (represented by G) is 50:50, and so on; and the gel fraction is calculated by the following formula.

Gel rate = (the original weight of the water gel - the weight of the water gel after three days of soaking in water) the original weight of the water gel.

Property analysis

In order to analyze the physicochemical properties of chitosan/succinic acid water gel, chitosan/succinic anhydride water gel and chitosan/glutamic acid water gel with different repeating unit ratios, The water gel is analyzed as follows:

A. FTIR spectroscopy

The prepared water gel was analyzed using an FTIR spectrometer (Perkin-Elmer Spectrum RX1 System) to examine the signal changes of the amine group and the carboxyl group in the composition.

The test results showed that the crosslinked water gel showed absorption peaks at 1631 cm ^-1 and 1587 cm ^-1 , which confirmed that the indole bond was formed during the crosslinking.

B. Expansion ratio

The prepared water gel is first immersed in 0.05M phosphate buffered physiological saline solution (PBS). After reaching the steady state, the surface liquid is sucked by the filter paper, and then the wet weight of the water gel after expansion is measured, and below Calculate the expansion rate of water gel:

Expansion ratio = (wet weight - dry weight) dry weight

The dry weight and the wet weight respectively represent the weight measured before and after the water gel is immersed in the PBS.

The results showed that all of the water gel reached a steady state three hours after immersion in PBS, and the expansion rate increased as the proportion of repeating units increased. In other words, the chitosan/crosslinking agent is an 80/20 water gel having a swelling ratio greater than that of a chitosan/crosslinking agent of 50/50. When the repeating unit ratio is 50/50, since the number of crosslinks formed is the largest, it contains less pores and absorbs less water, and the swelling ratio is the lowest. In addition, the measurement results showed a water gel containing chitosan/glutamic acid (pKa of 2.10 and 4.07) compared to chitosan/succinic acid (pKa of 4.20 and 5.60) and chitosan/ Succinic anhydride has a lower expansion ratio. In addition, the test results also found that the water gel obtained by using a dibasic acid and a dibasic acid anhydride as a crosslinking agent has substantially the same expansion ratio.

C. Compression modulus

A universal testing machine was used to measure the compression modulus of the water gel. During the test, the water gel was compressed at a rate of 0.5 mm/min and analyzed by the slope of 5%-35% deformation in a compressive stress-strain diagram.

The analysis results show that the compressive strength of chitosan is increased by the formation of crosslinked structure, and the 50/50 water gel of chitosan/crosslinking agent shows the highest compression modulus, and the result is the same as the expansion ratio. The analysis results are consistent, that is, the 50/50 water gel of chitosan/crosslinking agent has the smallest pore size because of the highest degree of crosslinking. In addition, chitosan/glutamic acid exhibits a higher compression modulus due to higher reactivity and higher degree of crosslinking.

D. Thermal properties analysis

D1. Thermogravimetric Analysis (TGA)

The cracking temperature of the water gel was analyzed using a Perkin Elmer 7 series thermogravimetric analyzer. The temperature was increased from 100 ° C to 800 ° C at a rate of 10 ° C / min, and the temperature and sample quality were continuously recorded during the process.

The analysis results show that the weight loss of the water gel changes from two stages to one stage with the formation of the bond. In addition, since the thermal stability of the dibasic acid is inferior to that of chitosan, the 5% cleavage temperature decreases as the amount of the dibasic acid increases, and the residual weight is also inversely proportional to the amount of the dibasic acid.

D2. Differential Scanning Thermal Analysis (DSC)

The temperature record of the water gel was drawn by Perkin Elmer's Pyris 6 differential scanning calorimeter. The measurement was carried out at a heating rate of 10 ° C / min and with nitrogen purge, and the temperature was raised from 50 ° C to 200 ° C.

It can be observed from the DSC temperature record that the weight loss of water vapor increases as the ratio of dibasic acid increases, which is due to the fact that the dibasic acid is more hydrophilic than chitosan. Further, from the phenomenon of peak shift, it is known that the water content of the material increases. It can also be seen from the temperature record that as the crosslink density increases, the glass transition temperature Tg of the material also increases or disappears because the crosslinked structure destroys the arrangement of chitosan.

E. Biodegradation analysis

The simulated sample weight was measured over time under simulated physiological conditions to thereby assess the biodegradation rate of the sample. After measuring the initial weight of the sample, the sample was immersed in 20 ml of 0.1 M PBS containing 58,100 units/ml lysozyme at 37 ° C for 28 days, after which the weight loss percentage was calculated.

The results showed that the degradation of chitosan was the most serious when the cross-linking was not carried out, the weight loss was about 33%, and the degradation rate was quite stable. In contrast, the chitosan/crosslinking agent is 50/50 water gel, there is no obvious degradation phenomenon, there may be two reasons: one, chitosan and cross-linking agent form a tight cross-linking structure; The material has a small pore size, so its surface area in contact with lysozyme is also small.

Animal experiment

A. Cutting experiment

ICR male rats (30-35g) were first anesthetized with ether, and after shaving, a 1 cm wide wound was formed on the back of the mouse with scissors, and the above chitosan/crosslinking agent was 50/50 water gel and A cast material made of uncrosslinked pure chitosan was placed on the wound to observe changes in the wound.

Figure 1 is a photograph of the third day of wound staining by H&E staining, in which Figure 1a is the control group, ie no applicator is used; Figure 1b is the use of un-crosslinked chitosan coating. The 1c~1e plan uses 50/50 chitosan/glutamic acid dressing, 50/50 chitosan/succinic anhydride dressing and 50/50 chitosan / Succinic acid applicator. In the figure, Epi denotes the epidermal layer; K denotes keratin; N denotes a new blood vessel; F denotes a hair follicle; In denotes an inflammatory cell; and the arrow points to keratin.

It can be found from Fig. 1 that on the third day after wound formation, the epidermis and stratum corneum of the 50/50 chitosan/glutamic acid dressing are thicker than other cast materials, and the blood vessels are The appearance of angiogenesis has begun, and the aforementioned phenomenon is not observed in the wounds of the control group. For chitosan/glutamic acid dressings, since glutamic acid has a positively charged amine group, cells can be more easily attached to the dressing, thereby accelerating the repair and regeneration of the tissue.

B. Burning experiment

A moderately scalded wound of 2 cm x 1.5 cm was formed on the back of the ICR male (30-35 g), and chitosan/glutamic acid dressings of different repeating unit ratios were placed on the wound to observe changes in the wound.

Figure 2 is a photograph of the wound on the sixth day stained by H&E staining, in which Fig. 2a is the control group, that is, no applicator is used; and Fig. 2b is the chitosan coating without cross-linking. The 2c~2f plans are those using 80/20, 75/25, 67/33, 50/50 chitosan/glutamic acid perfusate.

It can be found from Fig. 2 that on the sixth day after the wound is formed, all the wounds coated with chitosan/glutamic acid dressing are more complete (thick) than the control group. The repair of the wound stratum corneum of the pure chitosan-coated material is still in progress (thin). In addition, there was a phenomenon of wound infiltration in the control group (inflamed cells), while the other five groups had subsided. If it is put on a few In the wound of glycan/glutamic acid dressing, it can be found that the dermis layers of 75/25, 67/33 and 50/50 are in the process of repair, and the dermis layer of CS50/G50 is the most complete.

The present invention has been disclosed in its preferred embodiments, and it should be understood by those of ordinary skill in the art that the present invention is not intended to limit the scope of the invention. It should be noted that variations and permutations equivalent to those of the embodiments are considered to be within the scope of the invention. Therefore, the scope of the invention is defined by the scope of the following claims.

Fig. 1a to Fig. 1e are photographs obtained by dyeing the wound on the third day by H&E staining; Fig. 2a to Fig. 2f are photographs obtained by dyeing the wound on the sixth day by H&E staining.

Claims (13)

A crosslinked polysaccharide comprising glucosamine and N-acetylglucosamine, and the monomers are linked to each other by a β(1-4) glycosidic bond, wherein at least a portion of the glucosamine monomer and the crosslinking agent are formed. Amidoxime bond. The crosslinked polysaccharide of claim 1, wherein the crosslinking agent comprises a dibasic acid or an anhydride thereof. The crosslinked polysaccharide according to claim 1, wherein the crosslinking agent is selected from the group consisting of malonic acid, succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, aspartic acid, glutamine A group consisting of an acid and a polylactic acid having at least two carboxyl groups. A water gel comprising chitosan wherein at least a portion of the glucosamine monomer forms a guanamine linkage with a crosslinking agent. The water gel of claim 4, wherein the crosslinking agent is selected from the group consisting of malonic acid, succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, aspartic acid, glutamic acid, and a group consisting of at least two carboxylated lactic acid oligomers. A cross-linked chitosan having a partial structure as shown in Formula I: Wherein X is a substituted or unsubstituted C ₁ -C ₅ alkylene group. The cross-linked chitosan as described in claim 6 wherein X is substituted with an amine group. A method for preparing a cross-linked chitosan, comprising: using a dibasic acid or a dibasic acid anhydride to form a crosslinked structure of chitosan by the formation of a guanamine bond. The method of claim 8, wherein the crosslinked structure is produced by N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide and N-hydroxy-amber. It is carried out in the presence of quinone. The method of claim 9, wherein N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide and N-hydroxy-succinimide are dissolved in ethanol in. The method of claim 8, wherein the dibasic acid or dibasic acid anhydride is selected from the group consisting of malonic acid, succinic acid, succinic anhydride, glutaric acid, glutaric anhydride, aspartic acid, glutamine. A group consisting of an acid and a polylactic acid having at least two carboxyl groups. The method of claim 8, wherein the dibasic acid or the dibasic acid anhydride is added to the chitosan solution to form a crosslinked structure. A cross-linked chitosan prepared according to the method of any one of claims 8-12.