Grafted sacran polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the field of functional polymer materials, and particularly relates to grafted polysaccharide, a preparation method thereof and application of the grafted polysaccharide in the field of biomedical materials. According to the preparation method, polysaccharide carboxyl is activated through the condensing agent firstly, then the polysaccharide carboxyl reacts with the terminal reaction type PEG for grafting, the grafted polysaccharide SA is successfully prepared, the preparation method is simple and convenient, aftertreatment is easy, the reaction is conducted in a water system environment, and the preparation method is environmentally friendly and free of toxicity and harm to the human body. According to the grafted polysaccharide SA, the dissolving property of the SA is improved, the dissolving temperature is reduced to 60 DEG C, the solubility is improved by 60%, and the dissolving time is greatly shortened. The grafted polysaccharide SA can be blended with a cationic material to form a uniform / stable solution, polyelectrolyte is not generated, and the grafted polysaccharide SA has high application value in the field of medical dressings.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of functional polymer materials, and particularly relates to a grafted polysaccharide, a preparation method thereof and application thereof in the field of biomedical materials. BACKGROUND

[0002] Skin is the largest organ of the human body, mainly composed of epidermis and dermis. In recent years, skin damage has become a major public health problem. Traditional medical bandages and gauze ignore the management of wound inflammation and skin regeneration, and are difficult to meet the requirements of clinical application. Polysaccharide dressings with good biocompatibility, three-dimensional porous structure and water retention and absorption capacity have become a hot research object.

[0003] Existing biologically derived polysaccharide dressings mainly include alginate and hyaluronic acid, but they only play a role in absorbing exudates in the process of wound healing, and have no antibacterial effect, which easily leads to wound infection. However, the addition of cationic antibacterial materials such as ε-polylysine (ε-PLL) and the like easily produces polyelectrolyte complexes (PECs), and then it is difficult to form a uniform and stable film auxiliary, which limits the application of biologically derived polysaccharides in the field of medical materials.

[0004] Sacran (hereinafter referred to as SA), a sulfated polysaccharide, is extracted from Aphanothece sacrum, and its molecular weight exceeds 100 million g / mol, which is a supermolecule. From the chemical structure, it is an amphoteric polyanionic polysaccharide composed of multiple sugar units, and contains functional groups such as carboxylic acid groups and sulfate groups on the molecular chain. Sacran has shown important application value in the fields of cosmetics, biomedicine and advanced materials due to its super strong moisturizing property, film forming property, anti-inflammatory activity and material forming ability. Its water retention capacity is 4 times that of hyaluronic acid, and its safety has been recognized. It has been put into practical application as a cosmetic raw material; SA has a wound healing effect compared with other polysaccharides. However, natural SA also has some deficiencies that limit its application; first, its low solubility caused by excessive self-aggregation requires long time high temperature treatment to obtain a uniform solution. Second, its huge molecular weight causes significant steric hindrance, and the electrostatic interaction of the negative charges on the molecular chain, resulting in low chemical reactivity of Sacran, making it difficult to be chemically modified; in addition, like other anionic polysaccharides, it easily forms polyelectrolyte complexes with cationic antibacterial materials (as shown in Comparative Example 3), which seriously limits its application.

[0005] Therefore, it is necessary to develop a new material based on Sacran to solve this problem. SUMMARY

[0006] In order to solve the problems in the prior art, the graft sacran polysaccharide is developed based on Sacran, the dissolution speed and solubility are greatly improved, the SA has the ability of blending with the cationic antibacterial polymer at a low concentration and fast dissolution, and the problem that the polysaccharide and the cationic antibacterial polymer are difficult to blend in the prior art is solved.

[0007] In order to achieve the above-mentioned purpose, the technical method adopted by the present application is as follows: The carboxyl group of SA is grafted with a terminal reactive PEG oligomer; specifically, the carboxyl group of the polysaccharide is activated by using a condensing agent, and then grafted with a terminal reactive PEG to achieve the purpose of being able to blend with an antibacterial cation without appearing a polyelectrolyte complex. Further, a microneedle patch is prepared, the preparation method is simple, post-processing is easy, and the environment is friendly.

[0008] The first aspect of the present application provides a graft sacran polysaccharide, which is a dendritic branched structure, and is prepared by grafting a terminal reactive polyethylene glycol to a sacran polysaccharide main skeleton through a chemical bond; The grafting degree of the graft sacran polysaccharide is 0.7%-37.5%.

[0009] In some embodiments, the graft sacran polysaccharide has a structure as shown in formula (I): (I); In formula (I), ; Further, in formula (I), i is independently an integer of 21-452, and n and m are independently integers of 8000-28000.

[0010] In some embodiments, the weight average molecular weight of the graft sacran polysaccharide is 1.000*10 7 to 3.002*10 7 ; in some preferred embodiments, the weight average molecular weight of the graft sacran polysaccharide is 1.002*10 7 to 3.002*10 7 ; The molecular weight of the natural sacran polysaccharide is between 1.00*10 7 and 3.00*10 7 ; in some preferred embodiments of the present application, the natural sacran with a molecular weight of 1.00*10 7 is used as a raw material, and after grafting to different degrees, the molecular weight is between 1.000*10 7 and 1.002*10 7When using other molecular weight natural sacran as raw material, the molecular weight changes after grafting.

[0011] In some embodiments, the branched degree of the grafted sacran polysaccharide is 0.7%-37.5%.

[0012] Further, the solubility of the grafted sacran polysaccharide aqueous solution changes with temperature. In some embodiments, the solubility temperature of the grafted sacran polysaccharide is 60-80℃. Further, the concentration of the grafted SA aqueous solution ranges from 0-0.8%; further, the concentration of the grafted sacran polysaccharide aqueous solution ranges from 0.01%-0.8%.

[0013] Further, the dissolution time (60℃) of the grafted SA is no more than 8 hours; in some embodiments, the dissolution time is no more than 4 hours.

[0014] In some embodiments, the grafted SA is configured into aqueous solution in different mass fraction ratios. The prepared grafted SA aqueous solution has a concentration ranging from 0.01% to 0.8%, which is significantly improved compared with the original SA.

[0015] The second aspect of the present application provides a preparation method of the grafted sacran polysaccharide, which is grafted by chemical bond between the carboxyl on the sacran polysaccharide molecular chain and the terminal reactive polyethylene glycol; specifically comprising the following steps: S11. Dissolving SA in a solvent, heating and stirring to completely dissolve to obtain an SA solution; S12. Adding a condensing agent to the SA solution obtained in step S11 to form a reaction intermediate at low temperature; S13. Adding terminal reactive polyethylene glycol to the intermediate obtained in step S12, and dialyzing after reaction for a period of time to obtain grafted SA.

[0016] The preparation method of the grafted sacran polysaccharide of the present application utilizes the condensing agent to form an intermediate to improve the reaction activity and then participate in the reaction to obtain grafted SA, and the carboxyl reacts with the amino to generate amide.

[0017] In some embodiments, the terminal reactive polyethylene glycol is terminal amino polyethylene glycol.

[0018] In some embodiments, in step S11, the reaction conditions are 60-90℃; and the stirring time is 24-72h; In some embodiments, in step S12, the condensing agent is selected from a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) or N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCL) and N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide (Sulfo-NHS).

[0019] In some preferred embodiments, the condensing agent is a combination of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0020] In some embodiments, in step S12, the condensing agent N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride is added first, followed by the condensing agent N-hydroxysuccinimide.

[0021] In some implementations, the low temperature condition in step S12 is 0°C-8°C.

[0022] Further, in step S13, the terminal reactive polyethylene glycol is selected from one or a combination of amino-polyethylene glycol-amino (NH2-PEG-NH2), amino-polyethylene glycol monomethyl ether (Mpeg-NH2), and dopamine-polyethylene glycol-amino (Dopamine-PEG-NH2).

[0023] In some embodiments, the M of the NH2-PEG-NH2 W (Weight-average molecular weight) is 400-20000; In some embodiments, the Mpeg-NH2 of the Mpeg-NH2 W The range is 1000-20000; In some embodiments, the M of the Dopamine-PEG-NH2 W The value is 1000-4000, preferably 2000.

[0024] In some preferred embodiments, the preparation method of grafted SA includes the following steps: (1) dissolving SA in water, heating and stirring for 72 h to completely dissolve it to obtain an SA solution; (2) pouring the SA solution into a three-necked flask, adding EDCL and NHS, and forming a reaction intermediate at 0℃-8℃; (3) adding Mpeg-NH2 (Mw=1000) to the intermediate obtained in step (2), reacting for 24 h, and then dialysis to obtain grafted SA.

[0025] In some embodiments, when the amount of SA is 5 parts by mass, the amount of the condensing agent EDC (or EDCI) in step S12 is 0.038-0.382 parts by mass; and the amount of the condensing agent NHS (or Sulfo-NHS) is 0.023-0.23 parts by mass. In some embodiments, when the amount of SA is 5 parts by mass, the amount of the terminal reaction type polyethylene glycol in step S13 is 0.2-8 parts by mass.

[0026] A third aspect of the present application provides the use of the grafted sacran polysaccharide in biomedical materials.

[0027] In some embodiments, the grafted sacran polysaccharide can be blended with a cationic antibacterial material to form a uniform and stable solution; further, the grafted sacran polysaccharide is used for preparing a medical dressing.

[0028] The cationic antibacterial material is selected from polylysine, carboxymethyl chitosan, polyquaternary ammonium salt, polyhexamethylene guanidine, and some antibacterial metal cations, such as zinc ions, copper ions, and the like.

[0029] In some embodiments, the grafted sacran polysaccharide is used for preparing a microneedle patch; and the preparation method specifically comprises steps S21 and S22. S21: polylysine (PLL) is added to the grafted SA, and the tip is shaped by pouring into a microneedle mold; S22: polyvinyl alcohol (PVA) is heated and dissolved in a solvent, and then is laid on the surface of the tip shaped in S21 to prepare a microneedle patch.

[0030] In some embodiments, the heating condition in step S22 is 80-100℃.

[0031] Further, the present application can control the arrangement density and depth of the microneedles by controlling the concentration of the grafted SA and PLL, so as to adapt to complex application scenarios.

[0032] Advantages: The present application has at least the following advantages: (1) The present application first provides a grafted polysaccharide SA, which enriches the variety and application range of SA. The grafted polysaccharide has a molecular weight of 1.002×10 7 to 3.002×10 7 , and a branching degree of 0.7%-37.5%, which improves the solubility of SA, reduces the solubility temperature, and increases the solubility by 60%, and reduces the solubility time to 3% of the unmodified SA.

[0033] (2) The application also provides a preparation method of the grafted polysaccharide SA, which comprises the following steps: firstly, activating carboxyl groups of the polysaccharide by using a condensing agent, and then reacting with a terminal reactive PEG to graft, so that the grafted polysaccharide SA is successfully prepared, the preparation method is simple, and post-treatment is easy; the modification of the SA is carried out in a water-based environment, and there is no safety hazard of toxic reagents and environmental pollution.

[0034] (3) The application further provides application of the grafted polysaccharide SA, the grafted polysaccharide SA of the application maintains good biocompatibility and degradability of SA, good water retention performance and greatly improved solubility; and after modification, the grafted polysaccharide SA can be blended with cationic antibacterial materials to form a uniform / stable solution, and has a wide application prospect in biomedical materials.

[0035] The grafted polysaccharide SA of the application can be blended with cationic polysaccharides to prepare a microneedle patch, so that the dual effects of antibiosis and healing promotion are realized, and the application prospect is wide. BRIEF DESCRIPTION OF DRAWINGS

[0036] The accompanying drawings are included to provide a further understanding of the application, and constitute a part of the specification, which together with the embodiments of the application, are used to explain the application, and do not constitute a limitation to the application. In the drawings: Figure 1 A structure diagram of the grafted polysaccharide prepared in the application; Figure 2 A step schematic diagram of the grafted polysaccharide prepared in examples 1-6; Figure 3 An infrared spectrum diagram of the grafted polysaccharide prepared in example 1; Figure 4 An X-ray electron energy spectrum diagram of the grafted polysaccharide prepared in example 1: Figure 5 A viscosity curve diagram of the grafted polysaccharide prepared in example 1: Figure 6 A nuclear magnetic resonance hydrogen spectrum diagram of the grafted polysaccharide in the preparation process of example 1, wherein a: SA + N- (3-dimethylaminopropyl) -N'-ethyl carbodiimide hydrochloride, b: SA + N- (3-dimethylaminopropyl) -N'-ethyl carbodiimide hydrochloride + N-hydroxy succinimide; c: the grafted SA prepared in example 1; Figure 7 A blending effect diagram of example 14 and comparative example 1 with polylysine; Figure 8 A blending effect diagram of example 15 and comparative example 2 with carboxymethyl chitosan; Figure 9Effect diagram of blending hyaluronic acid and polylysine for Comparative Example 3; Figure 10 Effect diagram of preparing microneedle patch for the embodiment of the present application. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be described clearly and completely in combination with the drawings and specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. The experimental methods in the following embodiments are all conventional methods in the art, unless otherwise specified. However, it is easy for those skilled in the art to understand that the specific material ratio, process conditions and their results described in the embodiments are only used to illustrate the present application, and should not and will not limit the present application described in detail in the claims.

[0038] The experimental methods described in the following embodiments are all conventional methods, unless otherwise specified. The reagents and instruments can be obtained from commercial channels, unless otherwise specified.

[0039] Example 1 Preparation of grafted SA: SA 5 g, amino polyethylene glycol monomethyl ether (M W =1000) 0.4 g (0.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000). The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water and stir at 80°C for 72 h to obtain a 0.5% SA solution.

[0040] (2) Take 36 ml of 0.5% SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and then add 0.4 g of amino polyethylene glycol monomethyl ether (M W =1000) after 1 h of reaction, and react for 24 h.

[0041] (3) Put the final solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and dialyze for 7 days to obtain grafted SA.

[0042] Example 2 Preparation of grafted SA: SA 5 g, amino polyethylene glycol monomethyl ether (M W= 2000) 0.8 g (0.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0043] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 0.8 g of aminopolyethylene glycol monomethyl ether (M W = 2000), and react for 24 h.

[0044] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and after dialysis for 7 days, finally obtain grafted SA.

[0045] Example 3 Preparation of grafted SA: SA 5 g, aminopolyethylene glycol monomethyl ether (M W = 5000) 2 g (0.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0046] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 2 g of aminopolyethylene glycol monomethyl ether (M W = 5000), and react for 24 h.

[0047] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and after dialysis for 7 days, finally obtain grafted SA.

[0048] Example 4: Preparation of grafted SA: SA 5 g, aminopolyethylene glycol monomethyl ether (M W=10000) 4 g (0.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0049] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and then add 4 g of amino polyethylene glycol monomethyl ether (M W =10000) after 1 h of reaction, and react for 24 h.

[0050] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 14000), dialyze for 7 days to obtain grafted SA.

[0051] Example 5 Preparation of grafted SA: SA 5 g, amino polyethylene glycol monomethyl ether (M W =13000) 5.2 g (0.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0052] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and then add 5.2 g of amino polyethylene glycol monomethyl ether (M W =13000) after 1 h of reaction, and react for 24 h.

[0053] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 14000), dialyze for 7 days to obtain grafted SA.

[0054] Example 6 Preparation of grafted SA: SA 5 g, amino polyethylene glycol monomethyl ether (M W= 20000) 8 g (0.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 25000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0055] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 8 g of amino-polyethylene glycol-amino (M W = 20000), and react for 24 h.

[0056] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 25000), and after dialysis for 7 days, finally obtain grafted SA.

[0057] Example 7 Preparation of grafted SA: SA 5 g, amino-polyethylene glycol-amino (M W = 400) 0.08 g (0.2 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0058] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 0.08 g of amino-polyethylene glycol-amino (M W = 400), and react for 24 h.

[0059] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and after dialysis for 7 days, finally obtain grafted SA.

[0060] Example 8 Preparation of grafted SA: SA 5 g, amino-polyethylene glycol-amino (M W=1000) 0.2 g (0.2 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0061] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 0.2 g of amino-polyethylene glycol-amino (M W =400), and react for 24 h.

[0062] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and after dialysis for 7 days, finally obtain grafted SA.

[0063] Example 9 Preparation of grafted SA: SA 5 g, amino-polyethylene glycol-amino (M W =2000) 0.4 g (0.2 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0064] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 0.4 g of amino-polyethylene glycol-amino (M W =2000), and react for 24 h.

[0065] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and after dialysis for 7 days, finally obtain grafted SA.

[0066] Example 10 Preparation of grafted SA: SA 5 g, amino-polyethylene glycol-amino (M W=5000) 1 g (0.2 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% SA solution.

[0067] (2) Take 36 ml of the 0.5% SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 1 g of amino-polyethylene glycol-amino (M W =5000), and react for 24 h.

[0068] (3) Place the solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and after dialysis for 7 days, finally obtain grafted SA.

[0069] Example 11 Preparation of grafted SA: SA 5 g, amino-polyethylene glycol-amino (M W =10000) 2 g (0.2 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% SA solution.

[0070] (2) Take 36 ml of the 0.5% SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 2 g of amino-polyethylene glycol-amino (M W =10000), and react for 24 h.

[0071] (3) Place the solution obtained in (2) into a dialysis bag (molecular weight 14000), and after dialysis for 7 days, finally obtain grafted SA.

[0072] Example 12 Preparation of grafted SA: SA 5 g, amino-polyethylene glycol-amino (M W=20000) 4 g (0.2 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 25000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0073] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 4 g of amino-polyethylene glycol-amino (M W =20000), and react for 24 h.

[0074] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 25000), and after dialysis for 7 days, finally obtain grafted SA.

[0075] Example 13 Preparation of grafted SA: SA 5 g, dopamine-polyethylene glycol-amino (M W =2000) 0.8 g (0.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride 0.382 g (2 mmol), N-hydroxysuccinimide 0.46 g (2 mmol), deionized water 1 L, dialysis bag (molecular weight 8000-14000) The method comprises the following steps: (1) Dissolve 5 g of SA in 1 L of deionized water, stir at 80°C for 72 h to obtain a 0.5% concentration of SA solution.

[0076] (2) Take 36 ml of 0.5% concentration of SA solution, add 0.382 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.46 g of N-hydroxysuccinimide, and after 1 h of reaction, add 0.8 g of dopamine-polyethylene glycol-amino (M W =2000), and react for 24 h.

[0077] (3) Put the last solution obtained in (2) into a dialysis bag (molecular weight 8000-14000), and after dialysis for 7 days, finally obtain grafted SA.

[0078] Example 14 (modified SA prepared in Example 1 is blended with polylysine) Preparation of grafted SA and PLL blend: 1 g of grafted SA (prepared in Example 1), polylysine 0.02 g (0.0057 mmol), deionized water 1 L, comprising the following steps: (1) Dissolve 1g of grafted SA in 1L of deionized water and stir at 80°C for 1h to obtain a 0.1% SA solution.

[0079] (2) Take 20 ml of 0.1% grafted SA solution, add 0.02 g of polylysine, stir thoroughly for 10 min and let stand.

[0080] Comparative Example 1: (Unmodified SA blended with polylysine) Preparation of SA and polylysine blend: 1g SA, 0.02g (0.0057mmol) polylysine, 1L deionized water, including the following steps: (1) Dissolve 1g of SA in 1L of deionized water and stir at 80°C for 72h to obtain a 0.1% SA solution.

[0081] (2) Take 20 ml of 0.1% SA solution, add 0.02 g of polylysine, stir thoroughly for 30 min and let stand.

[0082] like Figure 7 As shown, the left figure is a mixed solution of grafted SA and PLL (Example 14), and the right figure is a mixed solution of ungrafted SA and PLL (Comparative Example 1). It can be seen that after the SA carboxyl group is modified, the generation of polyelectrolyte complex can be significantly reduced, forming a homogeneous and stable solution.

[0083] Example 15 (The modified SA obtained in Example 8 was blended with carboxymethyl chitosan) Preparation of a blend of grafted SA and carboxymethyl chitosan: 1 g grafted SA (obtained in Example 8), 0.02 g carboxymethyl chitosan, 1 L deionized water, including the following steps: (1) Dissolve 1g of grafted SA in 1L of deionized water and stir at 80°C for 1h to obtain a 0.1% SA solution.

[0084] (2) Take 20 ml of 0.1% grafted SA solution, add 0.02 g of carboxymethyl chitosan, stir thoroughly for 10 min and let stand.

[0085] Comparative Example 2: (Unmodified SA blended with carboxymethyl chitosan) Preparation of SA and carboxymethyl chitosan blend: 1g SA, 0.02g carboxymethyl chitosan, 1L deionized water, including the following steps: (1) Dissolve 1g of SA in 1L of deionized water and stir at 80°C for 72h to obtain a 0.1% SA solution.

[0086] (2) Take 20 ml of 0.1% SA solution, add 0.02 g of carboxymethyl chitosan, stir thoroughly for 30 min and let stand.

[0087] like Figure 8 As shown, the left figure is a blend solution of grafted SA and carboxymethyl chitosan (Example 15), and the right figure is a blend solution of ungrafted SA and carboxymethyl chitosan (Comparative Example 2). It can be seen that after the SA carboxyl group is modified, the generation of polyelectrolyte complex can be significantly reduced, forming a homogeneous and stable solution.

[0088] Comparative Example 3: (Hyaluronic acid and polylysine blend) The experimental method is the same as that of Comparative Example 1 / 2; like Figure 9 As shown, a large amount of polyelectrolyte complex is generated in a mixed solution of hyaluronic acid and polylysine, which severely limits its application.

[0089] like Figures 7-9 As shown, the original SA, like existing anionic polysaccharides, produces polyelectrolyte complexes when blended with cationic antibacterial materials, which greatly limits its application scenarios; while the grafted SA of the present invention can form a homogeneous and stable solution when blended with cationic antibacterial materials, overcoming the defects of existing materials.

[0090] Test example: Figure 1 A schematic diagram of the grafted SA structure of the present invention is shown.

[0091] In some embodiments, the method for preparing grafted SA according to the present invention is as follows: Figure 2 As shown in Examples 1-6.

[0092] The synthesis of grafted SA was demonstrated by Fourier transform infrared spectroscopy and organic elemental analysis of grafted and ungrafted SA.

[0093] like Figure 3 As shown, Fourier transform infrared spectroscopy was used to analyze and compare the infrared characteristic absorption peaks of SA before and after grafting after amidation reaction. Due to the partial amidation grafting of SA, the stretching vibration peak of the carboxyl carbonyl group at 1700 cm-1 decreased, while a new stretching vibration peak of the amide bond carbon-oxygen double bond appeared at 1600 cm-1, which is consistent with the results after grafting.

[0094] The test results of Examples 1-13 and the original SA are shown in Table 1. After the grafting reaction, the proportion of carbon and hydrogen elements increased significantly. In contrast, the proportion of nitrogen element in the ungrafted SA increased from 0.46% to 1.97% in Example 1, 1.91% in Example 2, 2.08% in Example 8, and 1.94% in Example 13, which is consistent with the changes after the grafting reaction.

[0095] The maximum concentration of ungrafted SA can reach 0.5% when stirred at 80°C for 3 days. The maximum concentration of the grafted SA prepared in Examples 1-13 can reach 0.8% at 60°C, and the dissolution time only needs 2-8 hours. The dissolution performance is significantly improved.

[0096] Table 1 The X-ray electron spectrogram of the grafted polysaccharide prepared in Example 1 is shown in Figure 4 After PEG grafting, a clear N 1s peak (~400eV) appears. Since SA itself contains almost no detectable N, the appearance of the N 1s peak directly proves the successful grafting of SA.

[0097] As shown in Figure 5 After PEG grafting, the viscosity of SA decreases, because the PEG chains shield the electrostatic repulsion between the charges on the polysaccharide backbone through steric hindrance effect. The charge repulsion is the main driving force for chain stretching, and once it is shielded, the polysaccharide chain will shrink from the stretched state to a more compact structure. The hydrodynamic volume of the whole molecule decreases. In addition, the grafted molecules after shrinking are more compact and are not easy to entangle with each other, so the flow resistance is greatly reduced, and the viscosity decreases.

[0098] As shown in Figure 6 After PEG modification, the c graph has a typical amide bond characteristic peak at 8ppm compared with the a and b graphs, which also indicates the successful grafting of SA.

[0099] The present application prepares a new type of grafted polysaccharide SA, with a molecular weight of 1.002×10 7 to 3.002×10 7 , a branching degree of 0.7%-37.5%, which improves the dissolution performance of SA, reduces the dissolution temperature to 60°C, increases the solubility by 60%, and greatly reduces the dissolution time. The preparation method of the present application first activates the carboxyl group of polysaccharide with a condensing agent, and then reacts with a terminal reactive PEG to successfully prepare the grafted polysaccharide SA. The preparation method is simple, and the reaction is carried out in an aqueous environment, which is environmentally friendly and harmless to human body. The grafted polysaccharide SA of the present application maintains the good biocompatibility and degradability of SA, good water retention performance, and significantly improves the dissolution performance; and after modification, it can be blended with cationic materials to form a uniform / stable solution without polyelectrolyte, providing a more extensive application scenario, and having high application value in the field of medical dressings.

[0100] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A grafted sacran polysaccharide, characterized by, The sacran polysaccharide is grafted with a terminal reactive polyethylene glycol through a chemical bond to form a dendritic branched structure. The degree of grafting of the grafted sacran polysaccharide branch is 0.7%-37.5%.

2. The grafted sacran polysaccharide according to claim 1, characterized in that, weight average molecular weight of 1.002 x 10 7 to 3.002 x 10 7 .

3. The grafted sacran polysaccharide of claim 1, wherein The concentration of the aqueous solution of the grafted sacran polysaccharide is 0.01%-0.8%.

4. The method for preparing the grafted sacran polysaccharide according to any one of claims 1 to 3, characterized by, The sacran polysaccharide is grafted with a terminal reactive polyethylene glycol through a chemical bond to form a dendritic branched structure. S11. Dissolve SA in a solvent, heat and stir to completely dissolve to obtain an SA solution; S12. Add a condensing agent to the SA solution obtained in step S11 to form a reaction intermediate at low temperature; S13. Add a terminal reactive polyethylene glycol to the intermediate obtained in step S12, and dialyze after reaction for a period of time to obtain a grafted SA.

5. The preparation method of the grafted sacran polysaccharide according to claim 4, characterized in that, In step S11, the reaction conditions are 60-90°C, and the stirring time is 24-72h.

6. The preparation method of the grafted sacran polysaccharide according to claim 4, characterized in that, In step S12, the condensing agent is selected from the combination of EDC or EDCL and NHS or Sulfo-NHS; And / or, in step S12, the low temperature condition is 0°C-8°C.

7. The method of preparing a grafted sacran polysaccharide according to claim 4, wherein In step S13, The terminal reactive polyethylene glycol is selected from one or a combination of amino-polyethylene glycol-amino (NH2-PEG-NH2), amino polyethylene glycol monomethyl ether (Mpeg-NH2), dopamine-polyethylene glycol-amino (Dopamine-PEG-NH2).

8. The grafted sacran polysaccharide of any one of claims 1-3, or the grafted sacran polysaccharide prepared by the method of any one of claims 4-7, is applied in a biomedical material.

9. Use according to claim 8, characterized in that, It can be blended with a cationic antibacterial material to form a uniform and stable solution; And / or, the cationic antibacterial material is selected from polylysine, carboxymethyl chitosan, polyquaternary ammonium salt, polyhexamethylene guanidine, and an antibacterial metal cation.

10. Use according to claim 9, characterized in that, It is applied to prepare a microneedle patch, and the preparation method comprises the following steps: S21: Add polylysine to the grafted SA, pour into a microneedle mold to shape the tips; S22: Dissolve polyvinyl alcohol in a solvent by heating at 80°C-100°C, and then spread on the surface of the shaped tips in S21 to prepare a microneedle patch.

Images