Polycationic polysaccharides and their applications

Abstract

The present invention relates to a polycationic polysaccharide and its uses, more specifically, the polycationic polysaccharide is composed of two parts, a polysaccharide and a polyamine compound, and employs a positively charged polycationic polysaccharide obtained by reaction between a polysaccharide and an amine or a compound containing a polyamine, and the application of the polycationic polysaccharide as a biomedical functional material for antibacterial biofilms, a biomedical device, and a material with antibacterial function.

Description

[Technical field]

[0001] The present invention relates to the field of biomedical technology, in particular to polycationic polysaccharides and their application as antibacterial materials and as drugs for treating chronic inflammatory diseases. [Background technology]

[0002] Natural polymeric polysaccharides have good biocompatibility and bioactivity, and are widely used in clinical and biopharmaceutical fields. At present, semi-synthetic cationized modified natural polysaccharides have been widely studied, and the amino groups modified on the surface have a high density of positive charges after protonation in aqueous solution, so they can be used as carriers to transport nucleic acid drugs. At the same time, many literatures have reported that polysaccharide structures containing a large number of positive charges have excellent antibacterial performance. Cationized polysaccharides bind to biological macromolecules through charge interactions, affecting the relevant functions of biological molecules and changing the activity of biological molecules.

[0003] Biofilm is a bacterial group that adheres to the surface of an object by secreting extracellular large molecules from various kinds of gram-negative or gram-positive bacteria to encase the bacteria, and is prone to causing various infections. The biofilm coating can protect the cells with extracellular large molecules to resist the body's immune defense, and at the same time, can improve resistance to antibiotics by 10 to 1000 times. The existence of biofilm greatly increases the difficulty of killing bacteria in traditional medicine, and promotes the occurrence of chronic or secondary infections. Therefore, it is currently necessary to find a method that can prevent the formation of biofilm, treat diseases associated with biofilm, sterilize and disinfect biomedical devices, and provide new biofunctional materials with better sterilization effect.

[0004] At present, in the course of clinical treatment, the formation and growth of biofilms are still a problem to be solved. Prevention and treatment of bacterial biofilms are still limited to killing bacteria with antibiotics to reduce the formation of biofilms, but the presence of biofilms protects bacteria from antibiotics, creating a vicious cycle. Researchers have found that polycationic polysaccharides are more effective than conventional antibacterial biofilm drugs due to their excellent antibacterial activity and unique biological activity, such as the ability to promote wound repair, due to their unique structure and carried positive charge. Therefore, polycationic polysaccharides are applied as drugs to treat infections caused by bacterial biofilms and related chronic inflammatory diseases. They are also developed and applied as bactericidal coating agents and new antibacterial functional materials for biomedical devices. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the problems existing in the prior art as mentioned above, the present invention provides polycationic polysaccharides, and the application of the prepared polycationic polysaccharides in biomedical functional materials, biomedical devices and materials with antibacterial functions. [Means for solving the problem]

[0006] The present invention adopts the following technical solutions:

[0007] A positively charged polycationic polysaccharide obtained by a reaction between a polysaccharide and a polyamine compound, the polysaccharide being represented by the following formula:

[0008] [ka]

[0009] among them,

[0010] R1 to R5 are each independently selected from a protected or unprotected hydroxyl group, a protected or unprotected amino group, and a sugar residue linked by a glycosidic bond, and the sugar residue satisfies the requirements of Formula 1.

[0011] The polyamine compound is represented by the following formula.

[0012] [ka]

[0013] among them,

[0014] R6 is a hydrogen atom or [ka] Selected from;

[0015] R7 is selected from a protected or unprotected amino group;

[0016] R8 is selected from a hydrogen atom or R6.

[0017] Preferably, the structural formula of the polycationic polysaccharide is:

[0018] [ka]

[0019] [ka] and

[0020] Formula 4 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of (1→6) glycosidic bonds;

[0021] Formula 5 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of (1→5) glycosidic bonds;

[0022] Formula 6 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of (1→4) glycosidic bonds;

[0023] Formula 7 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of (1→3) glycosidic bonds;

[0024] among them,

[0025] R6 is a hydrogen atom or [ka] Selected from;

[0026] R7 is selected from a protected or unprotected amino group;

[0027] R8 is selected from a hydrogen atom or R6.

[0028] Preferably, the polyamine compound has a molecular weight of less than 500 Daltons and is one of the following compounds:

[0029] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0030] Preferably, the polysaccharide structure contains between 2 and 2000 sugar units.

[0031] The present invention also discloses the application of the above polycationic polysaccharide as an antibacterial material.

[0032] Preferably, the antimicrobial material acts to kill bacteria by disrupting the structure of the bacterial biofilm.

[0033] Preferably, the antimicrobial material is used in the manufacture of a medicament or medical device for preventing and treating gram-negative and / or gram-positive bacterial infections.

[0034] Preferably, the antibacterial material is used as a biomedical functional material or a biomedical device.

[0035] Preferably, the antibacterial material is an antibacterial functional material, including daily chemical products, packaging products, and home improvement products having antibacterial functions.

[0036] The present invention also discloses an antibacterial agent produced by using the above-mentioned polycationic polysaccharide as an active ingredient together with medically acceptable auxiliary materials.

[0037] The present invention also discloses an antibacterial medical device produced by using the above-mentioned polycationic polysaccharide as an active ingredient and adding medically acceptable auxiliary materials.

[0038] The present invention also discloses the application of the above polycationic polysaccharides as drugs for treating chronic inflammatory diseases.

[0039] Preferably, the medication for treating chronic inflammatory disease includes medication for preventing surgical wound infection, medication for preventing burn wound infection. Effect of the Invention

[0040] Compared with the prior art (e.g., the solution with Chinese application number 201810714603.6), the polycationic polysaccharide of the present invention has better antibacterial and anti-inflammatory properties, promotes wound healing, and has lower cytotoxicity, and may be used in biomedical devices and biopharmaceutical functional materials. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 is an infrared spectrum of the polycationic polysaccharide in Example 1 of the present invention. [Diagram 2] FIG. 2 is an elemental analysis diagram of the polycationic polysaccharide in Example 1 of the present invention. [Diagram 3] FIG. 3 is a 1H NMR spectrum of the polycationic polysaccharide in Example 1 of the present invention. [Figure 4] FIG. 4 is a graph showing the results of cytotoxicity of the polycationic polysaccharide in Example 1 of the present invention. [Diagram 5] FIG. 5 is a graph showing the results of tissue toxicity of the polycationic polysaccharide in Example 1 of the present invention. [Figure 6] FIG. 6 is a comparison diagram showing the time taken for the polycationic polysaccharide of the present invention to promote wound healing compared to a conventional cationic polysaccharide. [Figure 7] FIG. 7 is a comparative graph of the cytotoxicity of the polycationic polysaccharides of the present invention constructed with saccharides of various origins. [Figure 8] FIG. 8 is a time-series graph showing the effect of the polycationic polysaccharides of the present invention, constructed from saccharides of various origins, on promoting wound healing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The following examples are provided as further explanations of the technical content of the present invention, but the substantial content of the present invention is not limited to the following examples. Those skilled in the art should be aware that any simple modifications and substitutions based on the substantial spirit of the present invention are within the scope of protection claimed by the present invention.

[0043] Example 1

[0044] A method for producing a polycationic polysaccharide, comprising the steps of:

[0045] 1) 0.5 g of dextran having a molecular weight of 70,000 daltons (purchased from Shanghai McKinley Biochemical Technology Co., Ltd., product number D806715) was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0046] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved dextran solution, and allow to react at room temperature for 2 hours.

[0047] 3) Further, 2 g of diethylenetriamine is added dropwise to the above solution, followed by reaction at 25°C for 24 hours.

[0048] 4) After the reaction is completed, add 5 times the volume of anhydrous ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with anhydrous ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named DETA-Dex.

[0049] The specific reaction process is shown in the following formula:

[0050] [ka]

[0051] The produced polycationic polysaccharides are characterized by infrared spectroscopy.

[0052] Take 200mg of potassium bromide and 2mg of polycationic polysaccharide sample, and finely grind it in an agate mortar. The whole grinding process is completed by baking under an infrared light lamp. The sample powder is put into a mold and pressurized to 20MPa, with a residence time of 2 minutes. The pressure is slowly reduced to 0, and the pressed sample tablet is taken out and measured by the instrument.

[0053] The results are shown in FIG. 1, where A is the infrared spectrum of dextran and B is the infrared spectrum of polycationic polysaccharide. -1 The peak at 1544 cm represents the stretching vibration peak of the carbonyl carbon-oxygen double bond. -1 The peak at 1022 cm represents the bending vibration peak of the nitrogen-hydrogen bond of the primary amino group. -1 The peaks in the graph indicate the characteristic absorption peaks of glucopyranose, and the appearance of these peaks proves that the synthesis of polycationic polysaccharide was successful.

[0054] In addition, take a 5 mg sample of the prepared polycationic polysaccharide, heat the sample with an infrared lamp, and polish it thoroughly. The powder of the sample is added to an elemental analyzer for measurement. The results are shown in Figure 2. When the nitrogen element content is greater than 7%, it can be confirmed that the synthesis of polycationic polysaccharide is successful.

[0055] The produced polycationic polysaccharides are characterized by 1H NMR spectrum.

[0056] 5 mg of a dextran sample and 5 mg of a polycationic polysaccharide sample are taken and thoroughly dissolved in 500 μl of deuterated water, placed in a quartz nuclear magnetic tube, and measured by the device.

[0057] The results are shown in Figure 3, where Figure A is the 1H NMR spectrum of dextran, and the peaks at 3.34-3.97 ppm are peaks obtained by hydrogen in the sugar ring of the dextran molecule. Figure B is the 1H NMR spectrum of polycationic polysaccharide, and the peaks at 2.94-4.00 ppm are peaks obtained by hydrogen in the sugar ring of the polycationic polysaccharide, and the peaks at 2.49-2.87 ppm are peaks obtained by hydrogen linked to the carbon atom of the ethyleneamino group (-CH2CH2NH) in the diethylenetriamine grafted to the sugar ring in the polycationic polysaccharide. The appearance of these peaks indicates that the synthesis of polycationic polysaccharide has been successful.

[0058] Example 2

[0059] The cytotoxicity and tissue toxicity of the polycationic polysaccharide of the present invention are confirmed.

[0060] cytotoxicity

[0061] Select and harvest human umbilical vein endothelial cells (HUVECs) from 10 4Inoculate the cells into a 96-well cell culture plate at 1 / hole and pre-culture for 24h. The cationized polysaccharide solution of the control group of the prior art (cDex, derived from China Patent 201810714603.6) and the polycationic polysaccharide solution of the present invention (named DETA-Dex) are mixed with cell culture medium at final concentrations of 0.5μg / ml, 1μg / ml, 2.5μg / ml, 5μg / ml, 10μg / ml, 20μg / ml, 50μg / ml, and 100μg / ml, respectively, and then put into the cell culture system. After 30min, wash the cells with cell culture medium and measure the activity of the cells.

[0062] The statistical results, as shown in FIG. 4, indicate that the polycationic polysaccharide of the present invention has less cytotoxicity to human umbilical vein endothelial cells HUVEC and human dermal fibroblast cells HFF-1, and has better biocompatibility.

[0063] tissue toxicity

[0064] A mouse back wound model based on literature reports

[0065] Balb / c female mice were taken, weighed and recorded, and randomly divided into groups, 10 mice per group. After intraperitoneal anesthesia with sodium pentobarbital, the hair on the back was removed and disinfected, and a round skin with a diameter of 0.5 cm was incised in the thick part of the central skin of the mouse back as a mouse back wound model.

[0066] b. After the model is created, it is administered.

[0067] To determine the histotoxicity of the polycationic polysaccharides of the invention on wound tissue, the following procedure is followed:

[0068] Blank control group: 100 μl of saline was applied to the wound area at the time of dosing.

[0069] Prior art control group: When administering, 100 μl of 1 mg / ml cationic polysaccharide solution (c-Dextran, hereinafter referred to as c-Dex for short, derived from Chinese Patent 201810714603.6) was applied to the wound area.

[0070] Experimental group: At the time of administration, 100 μl of 1 mg / ml polycationic polysaccharide solution (named DETA-Dex) was applied to the wound area.

[0071] The treated mice were placed in a warm, bright and comfortable environment and allowed to wake up. After 10 days, the wounds of the mice were examined.

[0072] The statistical results are shown in FIG. 5. The mice to which the polycationic polysaccharide solution of the present invention was applied were able to hasten the wound healing process. There was no obvious swelling or ulceration near the wound, which demonstrated that the application of the polycationic polysaccharide of the present invention can rapidly promote wound healing and has no obvious tissue toxicity to the tissue.

[0073] Example 3

[0074] The therapeutic effect of the polycationic polysaccharide of the present invention on a wound model infected with Pseudomonas aeruginosa is confirmed.

[0075] A mouse back wound model based on literature reports

[0076] Balb / c female mice were taken, weighed and recorded, and randomly divided into groups, 10 mice per group. After intraperitoneal anesthesia with sodium pentobarbital, the hair on the back was removed and disinfected, and a round skin with a diameter of 0.5 cm was incised in the thick part of the central skin of the mouse back as a mouse back wound model.

[0077] b. After creating the model, infect the mice with Pseudomonas aeruginosa.

[0078] Each group of mice was injected with 108 Pseudomonas aeruginosa and bacterial solution were evenly applied at a dose of CFU / animal, and after 72 hours the bacteria were able to form a complete biofilm.

[0079] c. Administration treatment

[0080] To determine the effect of the polycationic polysaccharides of the invention on biofilm activity, proceed as follows:

[0081] Blank control group: 100 μl of saline was applied to the wound area at the time of dosing.

[0082] Prior art control group: When administering, 100 μl of 1 mg / ml cationic polysaccharide solution (cDex, derived from China Patent 201810714603.6) was applied to the wound area.

[0083] Experimental group: At the time of administration, 100 μl of 1 mg / ml polycationic polysaccharide solution (named DETA-Dex) was applied to the wound area.

[0084] The treated mice were placed in a warm, bright and comfortable environment and allowed to wake up. The wound healing status of the mice was checked daily, and the time until the wound was completely healed was measured, and the mean and standard deviation (SD) of the wound healing time were calculated.

[0085] The statistical results, as shown in FIG. 6, indicate that the mice to which the polycationic polysaccharide solution of the present invention was applied were able to accelerate the wound healing process (shortest healing time), which explains that application of the polycationic polysaccharide of the present invention can quickly inhibit bacterial proliferation and spread and the formation of bacterial biofilm, effectively inhibit the production of endotoxins, exotoxins, etc. by bacteria, and slow the progression of diseases.

[0086] Example 4

[0087] The method for producing the polycationic polysaccharide of the present invention constructed from mannan comprises the following steps:

[0088] 1) 0.5 g of mannan having a molecular weight of 70,000 daltons (purchased from Shanghai McKinley Biochemical Technology Co., Ltd., product number M861453) was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0089] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved mannan solution and allow to react at room temperature for 2 hours.

[0090] 3) Further, 2 g of diethylenetriamine is added dropwise to the above solution, followed by reaction at 25°C for 24 hours.

[0091] 4) After the reaction is completed, add 5 times the volume of absolute ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with absolute ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named DETA-Mannan.

[0092] [ka]

[0093] The method for preparing the polycationic polysaccharide of the present invention constructed with chitosan comprises the following steps:

[0094] 1) 0.5 g of chitosan having a molecular weight of 70,000 daltons (purchased from Shanghai McKinley Biochemical Technology Co., Ltd., product number C804726) was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0095] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved chitosan solution and allow to react at room temperature for 2 hours.

[0096] 3) Further, 2 g of diethylenetriamine is added dropwise to the above solution, followed by reaction at 25°C for 24 hours.

[0097] 4) After the reaction is completed, add 5 times the volume of absolute ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with absolute ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named DETA-Chitosan.

[0098] [ka]

[0099] The method for preparing the polycationic polysaccharide of the present invention made of Bletilla striata polysaccharide comprises the following steps:

[0100] 1) 0.5 g of white polysaccharide (purchased from Lanzhou Wotl Biotechnology Co., Ltd.) was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0101] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved white sugar solution, and allow to react at room temperature for 2 hours.

[0102] 3) Further, 2 g of diethylenetriamine is added dropwise to the above solution, followed by reaction at 25°C for 24 hours.

[0103] 4) After the reaction is completed, add 5 times the volume of anhydrous ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with anhydrous ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named DETA-BSP.

[0104] The method for producing the polycationic polysaccharide of the present invention constructed from konjac polysaccharide comprises the following steps:

[0105] 1) 0.5 g of konjac polysaccharide (purchased from Lanzhou Ivory Biotechnology Co., Ltd.) was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0106] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved konjac polysaccharide solution, and allow to react at room temperature for 2 hours.

[0107] 3) Further, 2.5 mL of diethylenetriamine is added dropwise to the above solution, followed by reaction at 25°C for 24 hours.

[0108] 4) After the reaction is completed, add 5 times the volume of anhydrous ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with anhydrous ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named DETA-KGM.

[0109] The method for producing the polycationic polysaccharide of the present invention constructed from amylose comprises the following steps:

[0110] 1) 0.5 g of amylose (purchased from Shanghai McKinley Biochemical Technology Co., Ltd., product number S817547) was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0111] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved amylose solution and allow to react at room temperature for 2 hours.

[0112] 3) Further, 2 g of diethylenetriamine is added dropwise to the above solution, followed by reaction at 25°C for 24 hours.

[0113] 4) After the reaction is completed, add 5 times the volume of absolute ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the precipitate three times with absolute ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named DETA-Amylose.

[0114] The method for producing the polycationic polysaccharides of the present invention constructed from cellulose comprises the following steps:

[0115] 1) 0.5 g of cellulose (purchased from Shanghai McKinley Biochemical Technology Co., Ltd., 25 μm, product number C804602) was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0116] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved cellulose solution, and allow to react at room temperature for 2 hours.

[0117] 3) Further, 2 g of diethylenetriamine is added dropwise to the above solution, followed by reaction at 25°C for 24 hours.

[0118] 4) After the reaction is completed, add 5 times the volume of absolute ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with absolute ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named DETA-Cellulose.

[0119] The method for preparing the polycationic polysaccharides of the present invention constructed with different polyamine compounds comprises the following steps:

[0120] 1) 0.5 g of dextran having a molecular weight of 70,000 daltons was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0121] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved dextran solution, and allow to react at room temperature for 2 hours.

[0122] 3) Further, 2 g of each of Compound 2 to Compound 142 is added to the above solution, followed by reaction at 25° C. for 24 hours.

[0123] 4) After the reaction is completed, add 5 volumes of absolute ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the precipitate three times with absolute ethanol and then vacuum dry for 48 hours to obtain the product, which is dried and stored for future use and named 2-Dex to 142-Dex.

[0124] The method for preparing the polycationic polysaccharides of the present invention constructed with dextrans of different molecular weights comprises the following steps:

[0125] 1) 0.5 g of dextrans with molecular weights of 360 daltons, 50,000 daltons, and 304,000 daltons were weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0126] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved dextran solution, and allow to react at room temperature for 2 hours.

[0127] 3) Further, 2 g of diethylenetriamine is added to each of the above solutions, followed by reaction at 25°C for 24 hours.

[0128] 4) After the reaction is completed, add 5 times the volume of anhydrous ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with anhydrous ethanol and then vacuum dry for 48 hours to obtain the products, which are dried and stored for future use and named DETA-0.36, DETA-50, and DETA-304, respectively.

[0129] The method for preparing the polycationic polysaccharides of the present invention constructed with different polyamine compounds and mannan comprises the following steps:

[0130] 1) 0.5 g of dextran having a molecular weight of 70,000 daltons was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0131] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved dextran solution, and allow to react at room temperature for 2 hours.

[0132] 3) Further, 2 g each of compound 7, compound 15, compound 92, and compound 128 are added to the above solution, followed by reaction at 25° C. for 24 hours.

[0133] 4) After the reaction is completed, add 5 times the volume of anhydrous ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with anhydrous ethanol and then vacuum dry for 48 hours to obtain the products, which are dried and stored for future use and named 7-Mannan, 15-Mannan, 92-Mannan, and 128-Mannan.

[0134] The method for preparing the polycationic polysaccharides of the present invention constructed with different polyamine compounds and chitosan includes the following steps:

[0135] 1) 0.5 g of chitosan having a molecular weight of 70,000 daltons was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0136] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved chitosan solution and allow to react at room temperature for 2 hours.

[0137] 3) Further, 2 g each of Compound 2, Compound 61, and Compound 94 are added to the above solution, followed by reaction at 25° C. for 24 hours.

[0138] 4) After the reaction is completed, add 5 times the volume of absolute ethanol to the obtained solution and stir thoroughly. Then, centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the precipitate three times with absolute ethanol and vacuum dry for 48 hours to obtain the products, which are dried and stored for future use and named 2-chitosan, 61-chitosan, and 94-chitosan.

[0139] The method for preparing the polycationic polysaccharides of the present invention constructed with different polyamine compounds and polysaccharides includes the following steps:

[0140] 1) 0.5 g of each of white and polysaccharides having a molecular weight of 70,000 daltons was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0141] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved white sugar solution, and allow to react at room temperature for 2 hours.

[0142] 3) Further, 2 g each of compound 4, compound 14, compound 62, and compound 103 are added to the above solution, followed by reaction at 25° C. for 24 hours.

[0143] 4) After the reaction is completed, add 5 times the volume of anhydrous ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with anhydrous ethanol and then vacuum dry for 48 hours to obtain the products, which are dried and stored for future use and named 4-BSP, 14-BSP, 62-BSP, and 103-BSP.

[0144] The method for preparing the polycationic polysaccharide of the present invention constructed with different polyamine compounds and konnyaku polysaccharide comprises the following steps:

[0145] 1) 0.5 g of konjac polysaccharide having a molecular weight of 70,000 daltons was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0146] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved konjac polysaccharide solution, and allow to react at room temperature for 2 hours.

[0147] 3) Further, 2 g each of compound 8, compound 44, compound 67, and compound 102 are added to the above solution, followed by reaction at 25° C. for 24 hours.

[0148] 4) After the reaction is completed, add 5 times the volume of anhydrous ethanol to the obtained solution, stir thoroughly, and then centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the obtained precipitate three times with anhydrous ethanol and then vacuum dry for 48 hours to obtain the products, which are dried and stored for future use and named 8-KGM, 44-KGM, 67-KGM, and 102-KGM.

[0149] The method for preparing the polycationic polysaccharides of the present invention constructed with different polyamine compounds and amylose comprises the following steps:

[0150] 1) 0.5 g of amylose having a molecular weight of 70,000 daltons was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0151] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved amylose solution and allow to react at room temperature for 2 hours.

[0152] 3) Further, 2 g each of compound 15, compound 37, and compound 111 are added to the above solution, followed by reaction at 25° C. for 24 hours.

[0153] 4) After the reaction is completed, add 5 times the volume of absolute ethanol to the obtained solution and stir thoroughly. Then, centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the precipitate three times with absolute ethanol and vacuum dry for 48 hours to obtain the products, which are dried and stored for future use and named 15-Amylose, 37-Amylose, and 111-Amylose.

[0154] The method for preparing the polycationic polysaccharides of the present invention constructed with different polyamine compounds and cellulose comprises the following steps:

[0155] 1) 0.5 g of cellulose having a molecular weight of 70,000 daltons was weighed out and thoroughly dissolved in 25 mL of dimethyl sulfoxide to obtain a mixed solution.

[0156] 2) Weigh out 1 g of N'N-carbonyldiimidazole and add it directly to the dissolved cellulose solution, and allow to react at room temperature for 2 hours.

[0157] 3) Further, 2 g each of compound 10, compound 49, compound 87, compound 102, and compound 140 are added to the above solution, followed by reaction at 25° C. for 24 hours.

[0158] 4) After the reaction is completed, add 5 times the volume of absolute ethanol to the obtained solution and stir thoroughly. Then, centrifuge the precipitate at 12,000 rpm for 10 minutes. Wash the precipitate three times with absolute ethanol and vacuum dry for 48 hours to obtain the product. Dry the product and store it for future use. Name the product as 10-Cellulose, 49-Cellulose, 87-Cellulose, 102-Cellulose, and 140-Cellulose.

[0159] Example 5

[0160] The cytotoxicity of the polycationic polysaccharide of the present invention is confirmed.

[0161] Select and harvest human umbilical vein endothelial cells (HUVECs) from 10 4 The cells are inoculated into a 96-well cell culture plate in a single well and pre-cultured for 24 hours. The polycationic polysaccharide solutions of the present invention (named DETA-Dex, DETA-Mannan, DETA-Chitosan, DETA-BSP, DETA-KGM, DETA-Amylose, and DETA-Cellulose) are mixed with cell culture medium to give final concentrations of 0.5 μg / ml, 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml, and 100 μg / ml, respectively, and then placed into a cell culture system. After 30 minutes, the cells are washed with cell culture medium, and the activity of the cells is measured.

[0162] The statistical results are shown in Figure 7, which indicates that the polycationic polysaccharide of the present invention has less cytotoxicity and better biocompatibility to human umbilical vein endothelial cells (HUVECs). In practical use, higher concentrations and larger dosages can be adopted to achieve better therapeutic effects.

[0163] Example 6

[0164] The therapeutic effect of the polycationic polysaccharides of the present invention constructed with different polysaccharides on a wound model infected with Pseudomonas aeruginosa is confirmed.

[0165] A mouse back wound model based on literature reports

[0166] Balb / c female mice were taken, weighed and recorded, and randomly divided into groups, 10 mice per group. After intraperitoneal anesthesia with sodium pentobarbital, the hair on the back was removed and disinfected, and a round skin with a diameter of 0.5 cm was incised in the thick part of the central skin of the mouse back as a mouse back wound model.

[0167] b. After creating the model, infect the mice with Pseudomonas aeruginosa.

[0168] Each group of mice was injected with 10 8 Pseudomonas aeruginosa and bacterial solution were evenly applied at a dose of CFU / animal, and after 72 hours the bacteria were able to form a complete biofilm.

[0169] c. Administration treatment

[0170] To determine the effect of the polycationic polysaccharides of the invention on biofilm activity, proceed as follows:

[0171] Blank control group: saline was applied to the wound area at the time of administration.

[0172] Experimental group: When administered, 100 μl of 1 mg / ml polycationic polysaccharide solution (DETA-Dex, DETA-Mannan, DETA-Chitosan, DETA-BSP, DETA-KGM, DETA-Amylose, DETA-Cellulose, 2-Dex to 142-Dex, DETA-0.36, DETA-50, DETA-304, 7-Mannan, 15-Mannan, 92-Mannan, 128-Mannan) was applied to the wound area. The following coatings (named nan, 2-chitosan, 61-chitosan, 94-chitosan, 4-BSP, 14-BSP, 62-BSP, 103-BSP, 8-KGM, 44-KGM, 67-KGM, 102-KGM, 15-Amylose, 37-Amylose, 111-Amylose, 10-Cellulose, 49-Cellulose, 87-Cellulose, 102-Cellulose, and 140-Cellulose) were applied to the tissue.

[0173] The treated mice were placed in a warm, bright and comfortable environment and allowed to wake up. The wound healing status of the mice was checked daily, and the time until the wound was completely healed was measured, and the mean and standard deviation (SD) of the wound healing time were calculated.

[0174] The statistical results are shown in FIG. 8 and Table 1. The results show that the wounds of the mice to which the polycationic polysaccharide solution of the present invention, which is composed of polysaccharides of different origins, was applied could hasten the healing process. This demonstrates that the application of the polycationic polysaccharide of the present invention can quickly inhibit the proliferation and spread of bacteria and the formation of bacterial biofilms, effectively inhibit the production of endotoxins, exotoxins, etc. by bacteria, and slow the progression of diseases.

[0175] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0176] The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A positively charged polycationic polysaccharide obtained by a reaction between a polysaccharide and a polyamine compound, the number of sugar units contained in the structure of the polysaccharide being 2 to 2000, and the structural formula of the polycationic polysaccharide being any one of the following structures: 【Chemistry 1】 【Chemistry 2】 Formula 4 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of (1→6) glycosidic bonds; Formula 5 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of a (1→5) glycosidic bond; Formula 6 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of (1→4) glycosidic bonds; Formula 7 is a polycationic polysaccharide in which a polyamine compound is grafted onto a polysaccharide molecule having a main chain of (1→3) glycosidic bonds; The molecular weight of the polyamine compound is less than 500 daltons; and The polyamine compound is any one of the following compounds: 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 1-9】 【Table 1-10】 【Table 1-11】 【Table 1-12】 【Table 1-13】 【Table 1-14】 【Table 1-15】

2. Use of the polycationic polysaccharide according to claim 1 as an antibacterial material, The antibacterial material is used for producing medicines or medical devices for preventing and treating infections caused by gram-negative and / or gram-positive bacteria.

3. The use according to claim 2, characterized in that the antibacterial material is a biomedical functional material or a biomedical device.

4. 13. An antibacterial agent produced by adding medically acceptable auxiliary materials to the polycationic polysaccharide according to claim 1 as an active ingredient.

5. 13. An antibacterial medical device produced using the polycationic polysaccharide according to claim 1 as an active ingredient and adding medically acceptable auxiliary materials.

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