POLYCATIONIC POLYSACCHARIDE AND APPLICATION THEREOF

Abstract

A polycationic polysaccharide and an application thereof is disclosed. Specifically, the polycationic polysaccharide consists of a polysaccharide and a polyamine compound, and is a positively charged polycationic polysaccharide obtained by reacting a polysaccharide with an amine-containing or polyamine compound. The polycationic polysaccharide is applied in a biomedical functional material of an antibacterial biofilm, a biomedical device, and an antibacterial functional material.

Claims

1. A polycationic polysaccharide, the polycationic polysaccharide is a positively charged polycationic polysaccharide obtained by the reaction between a polysaccharide and a polyamine compound, wherein the polysaccharide has the following general formula: ##STR00152## wherein R1 to R5 are each independently selected from protected or unprotected hydroxyl group, protected or unprotected amino group, and sugar residue connected by glycosidic bond, and the sugar residue meets the requirements of Formula 1; the polyamine compound has the following general formula: ##STR00153## wherein R6 is selected from hydrogen atom or ##STR00154## R7 is selected from protected or unprotected amino group; R8 is selected from hydrogen atom or R6.

2. The polycationic polysaccharide according to claim 1, wherein the structural formula of the polycationic polysaccharide is one of the following structures: ##STR00155## formula 4 is a polycationic polysaccharide composed of a polysaccharide molecule with (1.fwdarw.6) glycosidic bond as the main chain and grafted by a polyamine compound; formula 5 is a polycationic polysaccharide composed of a polysaccharide molecule with (1.fwdarw.5) glycosidic bond as the main chain and grafted by a polyamine compound; formula 6 is a polycationic polysaccharide composed of a polysaccharide molecule with (1.fwdarw.4) glycosidic bond as the main chain and grafted by a polyamine compound; formula 7 is a polycationic polysaccharide composed of a polysaccharide molecule with (1.fwdarw.3) glycosidic bond as the main chain and grafted by a polyamine compound; wherein R6 is selected from hydrogen atom or ##STR00156## R7 is selected from protected or unprotected amino group; R8 is selected from hydrogen atom or R6.

3. The polycationic polysaccharide according to claim 1, wherein the molecular weight of the polyamine compound is less than 500 Daltons, and the polyamine compound is any one of the following compounds: TABLE-US-00003 Com- No pound Structure 1 com- pound 1 2 com- pound 2 3 com- pound 3 4 com- pound 4 5 com- pound 5 6 com- pound 6 7 com- pound 7 8 com- pound 8 9 com- pound 9 10 com- pound 10 11 com- pound 11 12 com- pound 12 13 com- pound 13 14 com- pound 14 15 com- pound 15 16 com- pound 16 17 com- pound 17 18 com- pound 18 19 com- pound 19 20 com- pound 20 21 com- pound 21 22 com- pound 22 23 com- pound 23 24 com- pound 24 25 com- pound 25 26 com- pound 26 27 com- pound 27 28 com- pound 28 29 com- pound 29 30 com- pound 30 31 com- pound 31 32 com- pound 32 33 com- pound 33 34 com- pound 34 35 com- pound 35 36 com- pound 36 37 com- pound 37 38 com- pound 38 39 com- pound 39 40 com- pound 40 41 com- pound 41 42 com- pound 42 43 com- pound 43 44 com- pound 44 45 com- pound 45 46 com- pound 46 47 com- pound 47 48 com- pound 48 49 com- pound 49 50 com- pound 50 51 com- pound 51 52 com- pound 52 53 com- pound 53 54 com- pound 54 55 com- pound 55 56 com- pound 56 57 com- pound 57 58 com- pound 58 59 com- pound 59 60 com- pound 60 61 com- pound 61 62 com- pound 62 63 com- pound 63 64 com- pound 64 65 com- pound 65 66 com- pound 66 67 com- pound 67 68 com- pound 68 69 com- pound 69 70 com- pound 70 71 com- pound 71 72 com- pound 72 73 com- pound 73 74 com- pound 74 75 com- pound 75 76 com- pound 76 77 com- pound 77 78 com- pound 78 79 com- pound 79 80 com- pound 80 81 com- pound 81 82 com- pound 82 83 com- pound 83 84 com- pound 84 85 com- pound 85 86 com- pound 86 87 com- pound 87 88 com- pound 88 89 com- pound 89 90 com- pound 90 91 com- pound 91 92 com- pound 92 93 com- pound 93 94 com- pound 94 95 com- pound 95 96 com- pound 96 97 com- pound 97 98 com- pound 98 99 com- pound 99 100 com- pound 100 101 com- pound 101 102 com- pound 102 103 com- pound 103 104 com- pound 104 105 com- pound 105 106 com- pound 106 107 com- pound 107 108 com- pound 108 109 com- pound 109 110 com- pound 110 111 com- pound 111 112 com- pound 112 113 com- pound 113 114 com- pound 114 115 com- pound 115 116 com- pound 116 117 com- pound 117 118 com- pound 118 119 com- pound 119 120 com- pound 120 121 com- pound 121 122 com- pound 122 123 com- pound 123 124 com- pound 124 125 com- pound 125 126 com- pound 126 127 com- pound 127 128 com- pound 128 129 com- pound 129 130 com- pound 130 131 com- pound 131 132 com- pound 132 133 com- pound 133 134 com- pound 134 135 com- pound 135 136 com- pound 136 137 com- pound 137 138 com- pound 138 139 com- pound 139 140 com- pound 140 141 com- pound 141 142 com- pound 142

4. The polycationic polysaccharide according to claim 3, wherein the number of sugar units in the structure of the polysaccharide is 2 to 2000.

5. A method of preparing an antibacterial material with the polycationic polysaccharide according to claim 1.

6. The method according to claim 5, wherein the antibacterial material achieves the effect of killing bacteria by destroying the biofilm structures of the bacteria.

7. The method according to claim 6, wherein the antibacterial material is applied for the preparation of a medicament or a medical device for the prevention or treatment of Gram-negative and/or Gram-positive bacterial infection.

8. The method according to claim 6, wherein the antibacterial material is applied as a biomedical functional material or a biomedical device.

9. The method according to claim 5, wherein the antibacterial material is an antibacterial functional material, including a daily chemical product, a packaging product, and a home improvement product with antibacterial functions.

10. An antibacterial agent, prepared from the polycationic polysaccharide according to claim 1 as an active ingredient and a pharmaceutically acceptable adjuvant.

11. (canceled)

12. (canceled)

13. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 is the infrared spectrogram of the polycationic polysaccharide in example 1 of the present disclosure.

[0037] FIG. 2 is the elemental analysis diagram of the polycationic polysaccharide in example 1 of the present disclosure.

[0038] FIG. 3 is the H NMR spectrum of the polycationic polysaccharide in example 1 of the present disclosure.

[0039] FIG. 4 is a graph showing the cytotoxicity results of the polycationic polysaccharide in example 1 of the present disclosure.

[0040] FIG. 5 is a graph showing the tissue toxicity results of the polycationic polysaccharide in example 1 of the present disclosure.

[0041] FIG. 6 is a diagram for the comparison of time in promoting wound healing for the polycationic polysaccharide of the present disclosure and the existing cationized polysaccharide.

[0042] FIG. 7 is a diagram for the comparison of cytotoxicity for the polycationic polysaccharides of the present disclosure constructed with saccharides from different sources.

[0043] FIG. 8 is a diagram for the comparison of time in promoting wound healing for the polycationic polysaccharides of the present disclosure constructed with saccharides from different sources.

DETAILED DESCRIPTION

[0044] The following examples are further descriptions of the present disclosure to be an illustration of the present technical content, but the essential content of the present disclosure is not limited to the following examples. Those of ordinary skill in the art can and shall know that any simple changes or substitutions based on the essential spirit of the disclosure shall be within the protection scope of the present disclosure claimed.

Example 1

[0045] A method for producing a polycationic polysaccharide, comprising the following steps: [0046] 1) Weighing 0.5 g of dextran with a molecular weight of 70,000 Daltons (purchased from Shanghai Macklin Biochemical Co., Ltd, cat #D806715), dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0047] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved dextran solution, and remaining reacting at room temperature for 2 hours; [0048] 3) Adding 2 g of diethylenetriamine dropwise to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0049] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitate three times with anhydrous ethanol, and drying it in vacuum for 48 hours to yield a product, which was stored under dry conditions for later use, and named as DETA-Dex.

[0050] The specific reaction process is shown in the following formula:

##STR00149##

[0051] The prepared polycationic polysaccharide was characterized by infrared spectroscopy:

[0052] 200 mg of potassium bromide and 2 mg of polycationic polysaccharide sample were weighted, and then ground in an agate mortar under baking with infrared lamp for the whole grinding process. The sample powder was placed into a mold and a pressure was applied up to 20 MPa. Maintained for 2 minutes, and then reduced the pressure to 0 slowly. The pressed sample tablet was took out and tested on a machine.

[0053] The results are shown in FIG. 1. Figure A is the infrared spectrum of the dextran, and Figure B is the infrared spectrum of the polycationic polysaccharide. In Figure B, the peak at 1711 cm.sup.?1 represents the stretching vibration peak of C?O in carbonyl, the peak at 1544 cm.sup.?1 represents the bending vibration peak of the nitrogen-hydrogen bond in primary amino group, and the peak at 1022 cm.sup.?1 represents the characteristic absorption peak of glucopyranose. The appearance of these peaks proved successful synthesis of the polycationic polysaccharide.

[0054] In addition, 5 mg of the prepared polycationic polysaccharide sample was weighted, then baked and ground fully under an infrared lamp. The sample powder was added to an elemental analyzer for testing. The results are shown in FIG. 2, where the synthesis of the polycationic polysaccharide can be considered successful if the nitrogen content is more than 7%.

[0055] The prepared polycationic polysaccharide was characterized by H NMR spectrum: [0056] 5 mg of dextran sample and 5 mg of polycationic polysaccharide sample were weighted, and fully dissolved in 500 ?l deuterated water respectively. Then the samples obtained were putted into a quartz NMR tube, and tested on a machine.

[0057] The results are shown in FIG. 3. Figure A is the H NMR spectrum of the dextran, in which the peak at 3.34-3.97 ppm is the peak generated by the hydrogen in the sugar ring of the dextran molecule. Figure B is the H NMR spectrum of the polycationic polysaccharide, in which the peak at 2.94-4.00 ppm is the peak generated by the hydrogen in the sugar ring of the polycationic polysaccharide, and the peak at 2.49-2.87 ppm is the peak generated by the hydrogen to which the carbon atom in ethylene amino group (?CH2CH2NH) in diethylene triamine grafted on the sugar ring of the polycationic polysaccharide is connected. The appearance of this peak indicates that the polycationic polysaccharide was successfully synthesized.

Example 2

[0058] Verification of cytotoxicity and tissue toxicity of the polycationic polysaccharide of the present disclosure.

Cytotoxicity

[0059] Human umbilical vein epithelial cell HUVEC was selected, and inoculated into 96-well plate of cell culture at 10.sup.4 cells/well, and then pre-cultured for 24 h. The cationized polysaccharide solution (cDex, derived from patent 201810714603.6) as prior art control group and the polycationic polysaccharide solution in this disclosure (named as DETA-Dex) were formulated with cell culture medium to a final concentration of 0.5 ?g/ml, 1 ?g/ml, 2.5 ?g/ml, 5 ?g/ml, 10 ?g/ml, 20 ?g/ml, 50 ?g/ml, 100 ?g/ml, respectively, and then added to the cell culture system for 30 min. After which, the cells were washed with cell culture medium for detection of cell activity.

[0060] Statistical results are shown in FIG. 4, showing that for human umbilical vein epithelial cell HUVEC and human skin fibroblast HFF-1, the polycationic polysaccharide of the present disclosure has lower cytotoxicity, and has better biocompatibility.

Tissue Toxicity

[0061] a. Establishment of a Mouse Back Trauma Model According to Literature Reports

[0062] Balb/c female mice were selected, weighed and recorded. The mice were randomized into groups with 10 mice per group. All the animals were intraperitoneally anesthetized with pentobarbital sodium. The back was dehaired and sterilized. At the thicker central part on the back of the mouse, a circular skin with a diameter of 0.5 cm was cut off to make a mouse back trauma model.

b. Medicament Treatment after Modeling

[0063] In order to detect the tissue toxicity of the polycationic polysaccharide of the present disclosure to wound tissue, an experiment was performed as follows: [0064] Blank control group: 100 ?l of physiological saline was smeared to the wound area during administration; [0065] Prior art control group: 100 ?l of 1 mg/ml cationized polysaccharide solution (c-Dextran, hereinafter referred to as c-Dex, derived from patent 201810714603.6) was smeared to the wound area during administration; [0066] Experimental group: 10 ?l of 1 mg/ml polycationic polysaccharide solution (named DETA-Dex) was smeared to the wound area during administration; [0067] The treated mice were placed in a warm, bright and comfortable environment to wait for them to wake up, and the wounds of the mice were examined 10 days later.

[0068] Statistical results are shown in FIG. 5, showing that the mice smeared with the polycationic polysaccharide solution of the present disclosure exhibited an accelerated wound healing, and there was no obvious swelling and ulceration around the wound, which indicates that smearing the polycationic polysaccharide of the present disclosure can promote healing of wound and has no significant tissue toxicity.

Example 3

[0069] Verification of therapeutic effect of the polycationic polysaccharide of the present disclosure on the model of wound infection by Pseudomonas aeruginosa.

a. Establishment of a Mouse Back Trauma Model According to Literature Reports

[0070] Balb/c female mice were selected, weighed and recorded. The mice were randomized into groups with 10 mice per group. All the animals were intraperitoneally anesthetized with pentobarbital sodium. The back was dehaired and sterilized. At the thicker central part on the back of the mouse, a circular skin with a diameter of 0.5 cm was cut off to make a mouse back trauma model.

b. Infection of Mouse by Pseudomonas aeruginosa after Modeling

[0071] Mice in each group were evenly smeared with Pseudomonas aeruginosa bacterial solution at the wound site at a dose of 10.sup.8 CFU/mouse, and the bacteria could form a complete biofilm within 72 hours.

c. Medicament Treatment

[0072] In order to detect the influence of the polycationic polysaccharide of the present disclosure on biofilm activity, an experiment was performed as follows: [0073] Blank control group: 100 ?l of physiological saline was smeared to the wound area during administration; [0074] Prior art control group: 100 ?l of 1 mg/ml cationized polysaccharide solution (cDex, derived from patent 201810714603.6) was smeared to the wound area during administration; [0075] Experimental group: 100 ?l of 1 mg/ml polycationic polysaccharide solution (named DETA-Dex) was smeared to the wound area during administration; [0076] The treated mice were placed in a warm, bright and comfortable environment to wait for them to wake up. The wounds of the mice were detected every day. The time for complete wound healing was recorded, and the mean and standard deviation SD of the time for wound healing were calculated.

[0077] Statistical results are shown in FIG. 6, showing that the mice smeared with the polycationic polysaccharide solution of the present disclosure exhibited an accelerated wound healing (the time for healing was the shortest), which indicates that smearing the polycationic polysaccharide of the present disclosure can quickly inhibit the proliferation and diffusion of bacteria and the formation of bacterial biofilm, effectively inhibit the production of endotoxin and exotoxin and the like by bacteria, and slow down development of disease.

Example 4

[0078] A method for constructing the polycationic polysaccharide of the present disclosure with mannan, comprising the following steps: [0079] 1) Weighing 0.5 g of mannan with a molecular weight of 70,000 Daltons (purchased from Shanghai Macklin Biochemical Co., Ltd, cat #M861453), dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0080] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved mannan solution, and remaining reacting at room temperature for 2 hours; [0081] 3) Adding 2 g of diethylenetriamine dropwise to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0082] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitate three times with anhydrous ethanol, and drying it in vacuum for 48 hours to yield a product, which was stored under dry conditions for later use, and named as DETA-Mannan.

##STR00150##

[0083] A method for constructing the polycationic polysaccharide of the present disclosure with chitosan, comprising the following steps: [0084] 1) Weighing 0.5 g of chitosan with a molecular weight of 70,000 Daltons (purchased from Shanghai Macklin Biochemical Co., Ltd, cat #C804726), dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0085] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved chitosan solution, and remaining reacting at room temperature for 2 hours; [0086] 3) Adding 2 g of diethylenetriamine dropwise to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0087] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitate three times with anhydrous ethanol, and drying it in vacuum for 48 hours to yield a product, which was stored under dry conditions for later use, and named as DETA-chitosan.

##STR00151##

[0088] A method for constructing the polycationic polysaccharide of the present disclosure with Bletilla striata polysaccharide, comprising the following steps: [0089] 1) Weighing 0.5 g of Bletilla striata polysaccharide (purchased from Lanzhou wotelaisi Biotechnology Co., Ltd.), dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0090] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved Bletilla striata polysaccharide solution, and remaining reacting at room temperature for 2 hours; [0091] 3) Adding 2 g of diethylenetriamine dropwise to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0092] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitate three times with anhydrous ethanol, and drying it in vacuum for 48 hours to yield a product, which was stored under dry conditions for later use, and named as DETA-B SP.

[0093] A method for constructing the polycationic polysaccharide of the present disclosure with konjac polysaccharide, comprising the following steps: [0094] 1) Weighing 0.5 g of konjac polysaccharide (purchased from Lanzhou wotelaisi Biotechnology Co., Ltd.), dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0095] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved konjac polysaccharide solution, and remaining reacting at room temperature for 2 hours; [0096] 3) Adding 2 ml of diethylenetriamine dropwise to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0097] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitate three times with anhydrous ethanol, and drying it in vacuum for 48 hours to yield a product, which was stored under dry conditions for later use, and named as DETA-KGM.

[0098] A method for constructing the polycationic polysaccharide of the present disclosure with amylose, comprising the following steps: [0099] 1) Weighing 0.5 g of amylose (Shanghai Macklin Biochemical Co., Ltd, cat #S817547), dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0100] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved amylose solution, and remaining reacting at room temperature for 2 hours; [0101] 3) Adding 2 g of diethylenetriamine dropwise to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0102] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitate three times with anhydrous ethanol, and drying it in vacuum for 48 hours to yield a product, which was stored under dry conditions for later use, and named as DETA-amylose.

[0103] A method for constructing the polycationic polysaccharide of the present disclosure with cellulose, comprising the following steps: [0104] 1) Weighing 0.5 g of cellulose (Shanghai Macklin Biochemical Co., Ltd, 25 ?m, cat #C804602), dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0105] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved cellulose solution, and remaining reacting at room temperature for 2 hours; [0106] 3) Adding 2 g of diethylenetriamine dropwise to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0107] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitate three times with anhydrous ethanol, and drying it in vacuum for 48 hours to yield a product, which was stored under dry conditions for later use, and named as DETA-cellulose.

[0108] A method for constructing the polycationic polysaccharide of the present disclosure with different polyamine compounds, comprising the following steps: [0109] 1) Weighing 0.5 g of dextran with a molecular weight of 70,000 Daltons, dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0110] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved dextran solution, and remaining reacting at room temperature for 2 hours; [0111] 3) Adding 2 g of compound 2 to compound 142 respectively to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0112] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as 2-Dex to 142-Dex respectively.

[0113] A method for constructing the polycationic polysaccharide of the present disclosure with dextran of different molecular weights, comprising the following steps: [0114] 1) Weighing 0.5 g of dextran with molecular weights of 360 Daltons, 50,000 Daltons, and 304,000 Daltons, respectively, dissolving each of them in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0115] 2) For each dissolved dextran solution, weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dextran solution, and remaining reacting at room temperature for 2 hours; [0116] 3) Adding 2 g of diethylenetriamine to the solution obtained above respectively, and remaining reacting at 25? C. for 24 hours; [0117] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as DETA-0.36, DETA-50, DETA-304 respectively.

[0118] A method for constructing the polycationic polysaccharide of the present disclosure with different polyamine compounds and mannan, comprising the following steps: [0119] 1) Weighing 0.5 g of dextran with a molecular weight of 70,000 Daltons, dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0120] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved dextran solution, and remaining reacting at room temperature for 2 hours; [0121] 3) Adding 2 g of compound 7, compound 15, compound 92 and compound 128 respectively to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0122] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as 7-Mannan, 15-Mannan, 92-Mannan, and 128-Mannan respectively.

[0123] A method for constructing the polycationic polysaccharide of the present disclosure with different polyamine compounds and chitosan, comprising the following steps: [0124] 1) Weighing 0.5 g of chitosan with a molecular weight of 70,000 Daltons, dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0125] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved chitosan solution, and remaining reacting at room temperature for 2 hours; [0126] 3) Adding 2 g of compound 2, compound 61 and compound 94 respectively to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0127] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as 2-chitosan, 61-chitosan, and 94-chitosan respectively.

[0128] A method for constructing the polycationic polysaccharide of the present disclosure with different polyamine compounds and Bletilla striata polysaccharide, comprising the following steps: [0129] 1) Weighing 0.5 g of Bletilla striata polysaccharide with a molecular weight of 70,000 Daltons, dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0130] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved Bletilla striata polysaccharide solution, and remaining reacting at room temperature for 2 hours; [0131] 3) Adding 2 g of compound 4, compound 14, compound 62 and compound 103 respectively to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0132] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as 4-BSP, 14-BSP, 62-BSP, and 103-BSP respectively.

[0133] A method for constructing the polycationic polysaccharide of the present disclosure with different polyamine compounds and konjac polysaccharide, comprising the following steps: [0134] 1) Weighing 0.5 g of konjac polysaccharide with a molecular weight of 70,000 Daltons, dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0135] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved konjac polysaccharide solution, and remaining reacting at room temperature for 2 hours; [0136] 3) Adding 2 g of compound 8, compound 44, compound 67 and compound 102 respectively to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0137] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as 8-KGM, 44-KGM, 67-KGM, and 102-KGM respectively.

[0138] A method for constructing the polycationic polysaccharide of the present disclosure with different polyamine compounds and amylose, comprising the following steps: [0139] 1) Weighing 0.5 g of amylose with a molecular weight of 70,000 Daltons, dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0140] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved amylose solution, and remaining reacting at room temperature for 2 hours; [0141] 3) Adding 2 g of compound 15, compound 37 and compound 112 respectively to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0142] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as 15-Amylose, 37-Amylose and 111-Amylose respectively.

[0143] A method for constructing the polycationic polysaccharide of the present disclosure with different polyamine compounds and cellulose, comprising the following steps: [0144] 1) Weighing 0.5 g of cellulose with a molecular weight of 70,000 Daltons, dissolving it in 25 ml dimethyl sulfoxide fully to obtain a mixed solution; [0145] 2) Weighing 1 g of NN-carbonyldiimidazole, directly adding it to the dissolved cellulose solution, and remaining reacting at room temperature for 2 hours; [0146] 3) Adding 2 g of compound 10, compound 49, compound 87, compound 102 and compound 140 respectively to the solution obtained above, and remaining reacting at 25? C. for 24 hours; [0147] 4) After the reaction completed, adding 5 times in volume of anhydrous ethanol to the solution obtained, stirring fully and then precipitating with centrifugation at 12,000 rpm for 10 minutes, then washing the obtained precipitates three times with anhydrous ethanol, and drying them in vacuum for 48 hours to yield products, which were stored under dry conditions for later use, and named as 10-Cellulose, 49-Cellulose, 87-Cellulose, 102-Cellulose, and 140-Cellulose respectively.

Example 5

[0148] Verification of cytotoxicity of the polycationic polysaccharide of the present disclosure.

[0149] Human umbilical vein epithelial cell HUVEC was selected, and inoculated into 96-well plate of cell culture at 10.sup.4 cells/well, and then pre-cultured for 24 h. The polycationic polysaccharide solution in this disclosure (named as DETA-Dex, DETA-Mannan, DETA-Chitosan, DETA-BSP, DETA-KGM, DETA-Amylose, DETA-Cellulose) were formulated with cell culture medium to a final concentration of 0.5 ?g/ml, 1 ?g/ml, 2.5 ?g/ml, 5 ?g/ml, 10 ?g/ml, 20 ?g/ml, 50 ?g/ml, 100 ?g/ml, respectively, and then added to the cell culture system for 30 min. After which, the cells were washed with cell culture medium for detection of cell activity.

[0150] Statistical results are shown in FIG. 7, showing that for human umbilical vein epithelial cell HUVEC, the polycationic polysaccharide of the present disclosure has lower cytotoxicity, and has better biocompatibility. For actual application, higher concentrations and larger doses can achieve better therapeutic effects.

Example 6

[0151] Verification of therapeutic effect of the polycationic polysaccharides of the present disclosure constructed with different polysaccharides on the model of wound infection by Pseudomonas aeruginosa.

a. Establishment of a Mouse Back Trauma Model According to Literature Reports

[0152] Balb/c female mice were selected, weighed and recorded. The mice were randomized into groups with 10 mice per group. All the animals were intraperitoneally anesthetized with pentobarbital sodium. The back was dehaired and sterilized. At the thicker central part on the back of the mouse, a circular skin with a diameter of 0.5 cm was cut off to make a mouse back trauma model.

b. Infection of Mouse by Pseudomonas aeruginosa after Modeling

[0153] Mice in each group were evenly smeared with Pseudomonas aeruginosa bacterial solution at the wound site at a dose of 10.sup.8 CFU/mouse, and the bacteria could form a complete biofilm within 72 hours.

c. Medicament Treatment

[0154] In order to detect the influence of the polycationic polysaccharides of the present disclosure on biofilm activity, an experiment was performed as follows: [0155] Blank control group: physiological saline was smeared to the wound area during administration; [0156] Experimental group: 100 ?l of 1 mg/ml polycationic polysaccharide solution (named as 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, 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, 140-Cellulose) was smeared to the wound area during administration; [0157] The treated mice were placed in a warm, bright and comfortable environment to wait for them to wake up. The wounds of the mice were detected every day. The time for complete wound healing was recorded, and the mean and standard deviation SD of the time for wound healing were calculated.

[0158] Statistical results are shown in FIG. 8 and Table 1, showing that all the mice smeared with the polycationic polysaccharide solution of the present disclosure constructed with polysaccharides from different sources exhibited an accelerated wound healing, which indicates that smearing the polycationic polysaccharide of the present disclosure can quickly inhibit the proliferation and diffusion of bacteria and the formation of bacterial biofilm, effectively inhibit the production of endotoxin and exotoxin and the like by bacteria, and slow down development of disease.

TABLE-US-00002 TABLE 1 Time for wound healing of mice treated with polycationic polysaccharide Name of polycationic Time for wound Standard polysaccharide healing (day) deviation 2-Dex 15.64 1.42 3-Dex 15.21 1.18 4-Dex 13.97 0.95 5-Dex 15.80 0.92 6-Dex 13.84 0.54 7-Dex 15.58 0.51 8-Dex 14.96 0.87 9-Dex 15.24 1.08 10-Dex 13.84 0.87 11-Dex 14.35 0.58 12-Dex 15.22 1.05 13-Dex 14.50 0.98 14-Dex 14.88 1.26 15-Dex 15.22 1.15 16-Dex 14.83 0.78 17-Dex 15.99 1.47 18-Dex 15.91 1.25 19-Dex 14.78 1.31 20-Dex 13.66 0.91 21-Dex 14.66 1.04 22-Dex 14.21 0.78 23-Dex 14.23 0.96 24-Dex 14.24 1.20 25-Dex 14.44 1.39 26-Dex 16.01 1.30 27-Dex 14.75 1.19 28-Dex 15.87 1.14 29-Dex 13.58 0.88 30-Dex 14.11 1.47 31-Dex 15.71 0.82 32-Dex 15.78 1.11 33-Dex 14,81 1.03 34-Dex 15.67 1.31 35-Dex 14.70 0.66 36-Dex 14.81 0.53 37-Dex 13.73 0,83 38-Dex 13.58 0.78 30-Dex 15.55 0.78 40-Dex 14.09 1.39 41-Dex 13.60 0.59 42-Dex 14.88 0.68 43-Dex 14.47 0.87 44-Dex 14.14 0.97 45-Dex 14.89 0.68 46-Dex 14.19 1.32 47-Dex 14.12 0.93 48-Dex 15.95 1.42 49-Dex 13.73 1.37 50-Dex 15.58 1.13 51-Dex 14.00 0.51 52-Dex 15.78 1.39 53-Dex 14.57 0.95 54-Dex 15.48 0.93 55-Dex 15.53 0.99 56-Dex 14.84 1.14 57-Dex 14.29 0.64 58-Dex 13.61 0.92 59-Dex 15.62 1.48 60-Dex 13.87 1.28 61-Dex 15.71 0.84 62-Dex 14.69 0.57 63-Dex 15.68 1.48 64-Dex 15.65 0.99 65-Dex 14.52 0.74 66-Dex 15.98 0.85 67-Dex 15.65 1.18 68-Dex 15.90 1.33 69-Dex 15.41 0.85 70-Dex 13.81 0.66 71-Dex 14.48 1.49 72-Dex 15.34 1.13 73-Dex 13.95 1.10 74-Dex 15.66 1.47 75-Dex 13.61 0.54 76-Dex 13.76 1.31 77-Dex 14.12 1.32 78-Dex 15.39 0.76 79-Dex 15.61 0.62 80-Dex 15.96 0.98 81-Dex 15.10 0.73 82-Dex 15.06 1.21 83-Dex 14.12 1.32 84-Dex 15.86 1.04 85-Dex 13.66 0.89 86-Dex 15.92 1.45 87-Dex 15.85 1.13 88-Dex 14.13 0.81 89-Dex 15.07 1.25 90-Dex 14.21 1.23 91-Dex 15.18 0.77 92-Dex 14.61 0.60 93-Dex 15.27 1.14 94-Dex 14.16 1.43 95-Dex 15.38 1.26 96-Dex 14.39 0.82 97-Dex 16.01 1.25 98-Dex 14.28 0.59 99-Dex 14.87 1.22 100-Dex 13.83 0.71 101-Dex 15.10 0.65 102-Dex 15.08 1.10 103-Dex 15.22 1.27 104-Dex 14.22 1.02 105-Dex 14.49 1.36 106-Dex 14.63 0.74 107-Dex 15.36 1.28 108-Dex 14.17 0.79 109-Dex 15.30 1.04 110-Dex 15.20 0.86 111-Dex 15.13 1.25 112-Dex 14.65 0.76 113-Dex 14.77 0.86 114-Dex 15.66 0.57 115-Dex 14.51 1.40 116-Dex 15.81 1.45 117-Dex 13.91 1.19 118-Dex 15.34 1.43 119-Dex 14.34 1.39 120-Dex 14.63 0.68 121-Dex 15.66 1.42 122-Dex 14.21 0.90 123-Dex 13.69 1.16 124-Dex 15.14 1.35 125-Dex 15.25 1.23 126-Dex 13.58 0.67 127-Dex 14.10 0.54 128-Dex 15.09 1.30 129-Dex 14.08 0.58 130-Dex 15.90 0.91 131-Dex 14.77 1.17 132-Dex 14.97 1.47 133-Dex 15.58 1.48 134-Dex 14.39 1.28 135-Dex 15.96 0.95 136-Dex 14.01 1.31 137-Dex 15.26 0.73 138-Dex 15.35 1.19 139-Dex 14.99 0.71 140-Dex 14.16 1.20 141-Dex 13.93 0.91 142-Dex 15.69 1.38 DETA-0.36 17.38 2.41 DETA-50 13.2 2.67 DETA-304 16.31 1.38 7-Mannan 15.77 2.11 15-Mannan 15.54 1.71 92-Mannan 14.84 1.00 128-Mannan 14.99 1.33 2-chitosan 15.14 0.55 61-chitosan 14.95 1.51 94-chitosan 15.52 1.14 4-BSP 15.92 0.33 14-BSP 14.75 1.27 62-BSP 14.09 1.46 103-BSP 14.45 2.35 8-KGM 13.98 1.31 44-KGM 15.16 1.28 67-KGM 14.86 2.34 102-KGM 14.01 1.76 15-Amylose 15.12 0.27 37-Amylose 14.04 1.71 111-Amylose 14.99 1.86 10-Cellulose 14.41 1.99 49-Cellulose 14.49 1.25 87-Cellulose 15.36 0.53 102 -Cellulose 15.45 1.77 140-Cellulose 15.19 0.76

[0159] The above descriptions are only preferred examples of the present disclosure and are not intended to limit the present disclosure. Any modification, equivalent replacement or improvement made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.