LU503425A1 - Low-cost temperature-sensitive hydrogel for wound treatment and preparation method thereof - Google Patents

Abstract

The invention relates to the field of hydrogel dressings, and in particular to a low-cost temperature-sensitive hydrogel for wound treatment and a preparation method thereof. The invention aims to solve a problem of poor solubility and pH limitation of the existing hydrogel wound dressing made of chitosan. The method includes: step 1, preparing N-2-HACC; and step 2, adding a sodium hyaluronate solution dropwise to an acetic acid solution of N-2-HACC in an ice bath with stirring, and then quickly adding a sodium β-glycerophosphate solution to have a reaction, thus obtaining a N-2-HACC-HA hydrogel. The invention adopts a chitosan derivative N- 2-HACC which has better solubility under neutral and slightly acidic conditions and also can reduce skin irritation. The hydrogel of the invention has low cost, excellent effect of promoting wound healing, can form gel in a short time when the temperature rises to 37 ℃, and has good clinical operability.

Description

DESCRIPTION 0503425

Low-cost temperature-sensitive hydrogel for wound treatment and preparation method thereof

Background of the Present Invention

[0001] The present invention relates to the field of hydrogel dressings, and in particular to a low- cost temperature-sensitive hydrogel for wound treatment and a preparation method thereof.

Description of Related Arts

[0002] In daily life, trauma may be caused everywhere, and the problem of wound healing is becoming more and more important clinically. Due to the different conditions, the degree of injury of wounds is different. Moreover, the wounds are also susceptible to infection and easily disrupt the stability of the body's internal environment. The complex environment in which the skin is located makes it difficult for traditional wound dressings to meet people's needs for high- quality wound repair and management needs. Therefore, there is an urgent need for a novel biomedical dressing that can accelerate wound healing.

[0003] As a wound dressing, hydrogel can not only function as a barrier to prevent the invasion of exogenous foreign bodies, but also provide a moist and acidic environment for wounds, accelerate wound healing, and reduce the change frequency and consumption of the dressing.

Hydrogel also has good water absorption, biocompatibility, and air permeability, promotes the dissolution of necrotic tissue and dead skin, and effectively exerts its autolytic debriding effect.

Hydrogel can be closely attached to uneven wounds without adhesion, and can be easily removed from the wounds without causing secondary damage. In addition, various drugs and growth factors can be introduced into a three-dimensional network structure of hydrogel to further accelerate wound healing. Hydrogel dressing is easy to use, can be directly applied to wounds, and has the advantages of easy cleaning and no residue. Therefore, the application prospect of hydrogel wound dressing or as drug carrier in promoting wound healing is very broad.

[0004] Chitosan that has biocompatibility, biodegradability, non-toxicity, broad-spectrum 0508425 antibacterial properties and can stop bleeding, reduce inflammation and promote wound healing is an ideal material for hydrogel wound dressings, but because of its poor solubility, limited by pH, it can only be dissolved under the condition of pH lower than 6, which greatly limits its wide application.

Summary of the Present Invention

[0005] The present invention aims to solve a problem of poor solubility and pH limitation of the existing hydrogel wound dressing made of chitosan, and provides a low-cost temperature- sensitive hydrogel for wound treatment and a preparation method thereof.

[0006] The low-cost temperature-sensitive hydrogel for wound treatment according to the present invention 1s made of N-2-hydroxypropyltrimethylammonium chloride chitosan, acetic acid, sodium hyaluronate and sodium B-glycerophosphate.

[0007] The present invention further provides a preparation method of a low-cost temperature- sensitive hydrogel for wound treatment, including the following steps:

[0008] step 1, dissolving chitosan in an acetic acid solution, stirring the obtained solution thoroughly, adding a NaOH solution dropwise to adjust pH to 9, soaking for 0.5-1 h, filtering the solution with suction, washing the obtained precipitate with deionized water until neutral, and freeze-drying the precipitate in vacuum to obtain dried chitosan;

[0009] dispersing the dried chitosan in isopropanol, stirring the obtained solution thoroughly, and heating the solution up to 80-85 °C; adding an isopropanol solution of 2,3- epoxypropyltrimethylammonium chloride dropwise within 30 min, stirring the obtained solution at a constant temperature of 80-85°C for 9-10 h at a stirring speed of 500-550 r/min, then cooling the solution to room temperature, resting the solution still for 1-2 h, adding 4 °C absolute ethanol, soaking for 0.5-1 h, filtering the solution with suction, freeze-drying the precipitate in vacuum to constant weight, thus obtaining N-2-hydroxypropyltrimethylammonium chloride chitosan (N-2-HACC); and

. . . . . . . LU503425

[0010] step 2, adding a sodium hyaluronate solution dropwise to an acetic acid solution of N-2-

HACC in an ice bath with stirring for 30-40 min, then quickly adding sodiumf- glycerophosphat solution, and continuing to stir the solution to have reaction for 30-40 min to obtain N-2-HACC-

HA hydrosol. In clinical application, the hydrosol is heated to 37 °C by wound temperature and can be cross-linked with ions to form hydrogel in 40 s.

[0011] Further, in step 1, a volume ratio of acetic acid to deionized water in the acetic acid solution is (1-15):(20-40).

[0012] Further, in step 1, a ratio of the mass of chitosan to the volume of acetic acid in the acetic acid solution is 1 g:(1-15) mL.

[0013] Further, in step 1, a mass ratio of chitosan to the dried chitosan is 1:(4-10).

[0014] Further, in step 1, a ratio of the mass of the dried chitosan to the volume of isopropanol is 1 g:(S-15) mL.

[0015] Further, in step 1, a ratio of the mass of the dried chitosan to the volume of the isopropanol solution of epoxypropyltrimethylammonium chloride is 1 g: (5-15) mL; the isopropanol solution of epoxypropyltrimethylammonium chloride has a concentration of 0.18-0.2 g/mL.

[0016] Further, in step 1, a ratio of the mass of the dried chitosan to the volume of absolute ethanol is 1 g:(10-30) mL.

[0017] Further, in step 2, a volume ratio of the acetic acid solution of N-2-HACC to the sodium hyaluronate solution is (1-5):1.

[0018] Further, in step 2, a volume ratio of the acetic acid solution of N-2-HACC to the sodium

B-glycerophosphate solution is (1-10):1.

[0019] Further, in step 2, a ratio of the mass of N-2-HACC in the acetic acid solution of N-2-

HACC to the volume of the acetic acid solution is 1 g: (5-25) mL; the acetic acid solution has a concentration of 0.1-0.12 mol/L.

[0020] Further, in step 2, a ratio of the mass of sodium hyaluronate to the volume of deionized 0508425 water in the sodium hyaluronate solution 1s 1 g: (1000-2000) mL.

[0021] Further, in step 2, a ratio of the mass of sodium B-glycerophosphate to the volume of deionized water in the sodium B-glycerophosphate solution is 1g: (1-5) mL.

[0022] Beneficial effects of the present invention:

[0023] The method of the present invention adopts a chitosan derivative N-2- hydroxypropyltrimethylammonium chloride chitosan. Compared with chitosan, the derivative has better solubility under neutral and slightly acidic conditions, is more suitable for preparing gel, and can reduce irritation to the skin.

[0024] The structure of the hydrogel of the present invention is a porous three-dimensional network structure, and this structure is conducive to the exchange of water, gas and some small molecular substances, which provides a guarantee for the hydrogel to be used as a wound dressing or a wound repair drug. Moreover, this three-dimensional network structure is suitable for use as a carrier for drug delivery. Because the method introduces positively charged quaternary ammonium salts, the antibacterial, anti-inflammation effects of chitosan and the effect of promoting wound healing are further improved. On the basis of ion cross-linking of chitosan to form a gel, a three-dimensional self-assembly effect formed by the attraction of positive and negative charges is further introduced, so that the formed gel has better porosity.

This porous three-dimensional network structure has a large specific surface area and strong adsorption force, thereby being more suitable for drug carrying. In the hydrogel, antibacterial agents such as nano silver and nano gold can be added to reduce inflammation or vascular endothelial growth factors to promote wound healing according to special wound healing needs.

[0025] The N-2-HACC-HA hydrogel prepared according to the present invention can store a large amount of water, and has good water storage performance, and this indicates that the N-2-

HACC-HA hydrogel can keep the wound moist and provide a good environmental basis for cell proliferation and tissue proliferation in the wound, which is conducive to wound healing.

Moreover, this feature also provides feasibility support for carrying soluble drugs or vaccine 0508425 antigens on the N-2-HACC-HA hydrogel.

[0026] If sodium B-glycerophosphate is only added to N-2-HACC during the preparation process of the hydrogel according to the present invention, the performance of the gel will be unstable.

Although the addition of sodium a-glycerophosphate can make the performance of hydrogels stable, the high price of sodium a-glycerophosphate greatly increases the cost of hydrogels. After a large number of experiments for screening and research, temperature-sensitive gels made from

N-2-HACC, sodium hyaluronate (HA) and sodium B-glycerophosphate have better wound healing effect. The sodium hyaluronate used in the present invention has good hygroscopicity and can enhance the permeability of the hydrogels. In addition, the sodium hyaluronate can form a self-assembled structure with N-2-HACC and reduce the crystallization of N-2-HACC, thereby improving the hydrophilicity of the hydrogels. Moreover, the sodium hyaluronate can increase the cross-linking degree of the hydrogels and improve the stability of the hydrogels.

[0027] In addition, since the sodium hyaluronate has low cost and its price is comparable to that of sodium B-glycerophosphate, the cost of the hydrogel is ultimately reduced.

[0028] The hydrogel of the present invention has excellent wound healing promoting effect and good temperature sensitivity. Before use, it is a hydrosol that is easy to apply. After application, due to the body temperature of the human body or animals (pets), the sol forms a hydrogel in a short time (about 40 s).

[0029] The present invention uses N-2-HACC, sodium hyaluronate and sodium ß- glycerophosphate as raw materials, all of which are non-toxic and have no side effects. The cytotoxicity test showed that the freeze-dried hydrogel powder did not present obvious toxicity to PK-15 cells, and when its concentration reached 12.5%, the cell viability could still reach 90% or above, indicating that N-2-HACC-HA hydrogel has good security.

Brief Description of the Drawings

[0030] Fig. 1 shows the morphology of a N-2-HACC-HA hydrogel under a scanning electron microscope in Example 1;

[0031] Fig. 2 is a further enlarged view of a part defined by a box in Fig. 1; 0508425

[0032] Fig. 3 1s a further enlarged view of the part defined by the box in Fig. 2;

[0033] Fig. 4 shows cytotoxicity results of N-2-HACC-HA of different concentrations to PK-15;

[0034] Fig. 5 shows in vitro hemolysis test results of N-2-HACC-HA of different concentrations;

[0035] Fig. 6 shows the effect of N-2-HACC-HA solutions of different concentrations on the proliferation of DF-1;

[0036] Fig. 7 shows the wound healing situation of treated SD rats;

[0037] Fig. 8 shows pathological observation images of skin tissue wounds of rats in examples; and

[0038] Fig. 9 shows the pH dependence of water solubility of chitosan and N-2-HACC.

Detailed Description of the Preferred Embodiment

[0039] The technical solution of the present invention is not limited to the specific embodiments set forth below, but also includes any combination of the specific embodiments.

[0040] Specific embodiment 1: A low-cost temperature-sensitive hydrogel for wound treatment in this example was made of N-2-hydroxypropyltrimethylammonium chloride chitosan (N-2-

HACC), acetic acid, sodium hyaluronate and sodium B-glycerophosphate.

[0041] The hydrogel of the present invention has excellent wound healing promoting effect and good temperature sensitivity. Before use, it is a sol that is easy to apply. After application, due to the body temperature of the human body or animals (pets), the sol forms a gel in a short time (about 40 s).

[0042] According to the present invention, N-2-HACC, sodium hyaluronate and sodium ß- glycerophosphate were used as raw materials, and all of them were non-toxic and free of side effects. The cytotoxicity test showed that the freeze-dried hydrogel powder did not present obvious toxicity to PK-15 cells, and when its concentration reached 12.5%, the cell viability could still reach 90% or above, indicating that N-2-HACC-HA hydrogel has good security.

[0043] Specific embodiment 2: A preparation method of a low-cost temperature-sensitive 0508425 hydrogel for wound treatment included the following steps:

[0044] step 1, dissolving chitosan in an acetic acid solution, stirring the obtained solution thoroughly, adding a NaOH solution dropwise to adjust pH to 9, soaking for 0.5-1 h, filtering the solution with suction, washing the obtained precipitate with deionized water until neutral, and freeze-drying the precipitate in vacuum to obtain dried chitosan;

[0045] dispersing the dried chitosan in isopropanol, stirring the obtained solution thoroughly, and heating the solution up to 80-85 °C; adding an isopropanol solution of 2,3- epoxypropyltrimethylammonium chloride dropwise within 30 min, stirring the obtained solution at a constant temperature of 80-85°C for 9-10 h at a stirring speed of 500-550 r/min, then cooling the solution to room temperature, resting the solution still for 1-2 h, adding 4 °C absolute ethanol, soaking for 0.5-1 h, filtering the solution with suction, freeze-drying the precipitate in vacuum to constant weight, thus obtaining N-2-hydroxypropyltrimethylammonium chloride chitosan (N-2-HACC); and

[0046] step 2, adding a sodium hyaluronate solution dropwise to an acetic acid solution of N-2-

HACC in an ice bath with stirring for 30-40 min, , then quickly adding sodiumf- glycerophosphat solution, continuing to stir the solution to have reaction for 30-40 min to obtain

N-2-HACC-HA hydrosol. In clinical application, the hydrosol is heated to 37 °C by wound temperature and can be cross-linked with ions to form hydrogel in 40 s..

[0047] The N-2-HACC-HA hydrogel can store a large amount of water, and has better water storage performance because N-2-HACC has better hydration compatibility compared with chitosan. Moreover, sodium hyaluronate has good water absorption performance. More importantly, the sodium hyaluronate solution is added dropwise before ionic crosslinking, so that the three-dimensional self-assembly formed by the positive and negative charge attraction of N- 2-HACC and sodium hyaluronate destroys the spatial regularity of the two polymers, inhibits polymer crystallization, and increases the affinity between water molecules and polymer chains.

In addition, the porous three-dimensional structure jointly formed by self-assembly and ion

Co. . . . . . . LU503425 cross-linking has a high specific surface area, so the gel obtained in the present invention can store a large amount of water.

[0048] Specific embodiment 3: According to this embodiment, in step 1, the volume ratio of acetic acid to deionized water in the acetic acid solution was (1-15):(20-40). Other steps and parameters in this embodiment were the same as those in the specific embodiment 2.

[0049] Specific embodiment 4: According to this embodiment, in step 1, the ratio of the mass of chitosan to the volume of acetic acid in the acetic acid solution was 1 g:(1-15) mL. Other steps and parameters in this embodiment were the same as those in the specific embodiment 2 or 3.

[0050] Specific embodiment 5: According to this embodiment, in step 1, the mass ratio of chitosan to the dried chitosan was 1:(4-10). Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 4.

[0051] Specific embodiment 6: According to this embodiment, in step 1, the ratio of the mass of the dried chitosan to the volume of isopropanol was 1 g:(5-15) mL. Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 5.

[0052] Specific embodiment 7: According to this embodiment, in step 1, the ratio of the mass of the dried chitosan to the volume of the isopropanol solution of epoxypropyltrimethylammonium chloride was 1 g: (5-15) mL. Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 6.

[0053] Specific embodiment 8: According to this embodiment, the concentration of the isopropanol solution of epoxypropyltrimethylammonium chloride was 0.18-0.2 g/mL. Other steps and parameters in this embodiment were the same as those in the specific embodiment 7.

[0054] Specific embodiment 9: According to this embodiment, in step 1, the ratio of the mass of the dried chitosan to the volume of absolute ethanol was 1 g:(10-30) mL. Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 8.

[0055] Specific embodiment 10: According to this embodiment, in step 2, the volume ratio of the acetic acid solution of N-2-HACC to the sodium hyaluronate solution was (1-5):1. Other steps and parameters in this embodiment were the same as those in one of the specific embodiments + 903425 to 9.

[0056] Specific embodiment 11: According to this embodiment, in step 2, the volume ratio of the acetic acid solution of N-2-HACC to the sodium B-glycerophosphate solution was (1-10):1.

Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 10.

[0057] Specific embodiment 12: According to this embodiment, in step 2, the ratio of the mass of N-2-HACC in the acetic acid solution of N-2-HACC to the volume of the acetic acid solution was 1 g: (5-25) mL. Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 11.

[0058] Specific embodiment 13: According to this embodiment, the concentration of the acetic acid solution was 0.1-0.12 mol/L. Other steps and parameters in this embodiment were the same as those in the specific embodiment 12.

[0059] Specific embodiment 14: According to this embodiment, in step 2, the ratio of the mass of sodium hyaluronate to the volume of deionized water in the sodium hyaluronate solution was 1 g: (1000-2000) mL. Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 13.

[0060] Specific embodiment 15: According to this embodiment, in step 2, the ratio of the mass of sodium B-glycerophosphate to the volume of deionized water in the sodium ß- glycerophosphate solution was 1 g: (1-5) mL. Other steps and parameters in this embodiment were the same as those in one of the specific embodiments 2 to 14.

[0061] The following examples of the present invention are described in detail, and the following examples are implemented on the premise of the technical solutions of the present invention, and detailed implementations and specific operating procedures are provided, but the scope of the present invention is not limited to the following examples.

[0062] Example 1

. .. LU503425

[0063] The preparation method of a low-cost temperature-sensitive hydrogel for wound treatment in this example included the following steps:

[0064] 1. Preparation of N-2-HACC

[0065] (1) Soaking treatment of chitosan

[0066] 6 g of chitosan was dissolved in an acetic acid solution and the obtained solution was stirred for 1 h by a constant-speed stirrer. À 15 mol/L NaOH solution was then added dropwise to adjust pH to 9. The chitosan was soaked for 0.5 h, the solution was then filtered with suction, the obtained precipitate was washed with deionized water until neutral, and then freeze-dried in vacuum to obtain 25 g of dried chitosan. The acetic acid solution herein was made from 66.6 mL of acetic acid and 173.4 mL of deionized water.

[0067] (2) Ring-opening reaction of chitosan and glycidyltrimethylammonium chloride

[0068] The dried chitosan obtained in step (1) was dispersed in 50 mL of an isopropanol solution, the obtained solution was stirred thoroughly and then placed in an 80 °C water bath. An isopropanol solution of 2,3-epoxypropyltrimethylammonium chloride was then added dropwise within 30 min, the obtained solution was stirred at a constant temperature of 80 °C for 9 h at a stirring speed of 500 r/min to have a reaction, then cooled to room temperature and rested still for 1 h. 150 mL of 4 °C absolute ethanol was then added for soaking for 0.5 h, the solution was filtered with suction using absolute ethanol, the precipitate was freeze-dried in vacuum to constant weight, thus obtaining N-2-hydroxypropyltrimethylammonium chloride chitosan (N-2-

HACC). The isopropanol solution of 2,3-epoxypropyltrimethylammonium chloride was prepared from 9 g of 2,3-epoxypropyltrimethylammonium chloride and 50 mL of isopropanol.

[0069] 2. Preparation of an acetic acid solution of N-2-HACC

[0070] 0.8 g of N-2-HACC was weighed and completely dissolved in 10 mL of 0.1 mol/L acetic acid and the obtained solution was then placed in a refrigerator with a temperature of 4 °C until the temperature of the solution was constant.

[0071] 3. Preparation of a sodium hyaluronate solution

[0072] 0.005 g of sodium hyaluronate was weighed and completely dissolved in 8 mL of 0508425 deionized water and the obtained solution was then placed in a refrigerator with a temperature of 4 °C until the temperature of the solution was constant.

[0073] 4. Preparation of a sodium B-glycerophosphate solution

[0074] 2 g of sodium B-glycerophosphate was weighed and completely dissolved in 2 mL of deionized water and the obtained solution was then placed in a refrigerator with a temperature of 4 °C until the temperature of the solution was constant.

[0075] 5. Preparation of a N-2-HACC-HA hydrogel

[0076] The sodium hyaluronate solution was added dropwise to the acetic acid solution of N-2-

HACC in an ice bath with stirring for 30 min, and the sodium B-glycerophosphate solution was then quickly added to have an ion cross-linking reaction for 30 min, thus obtaining the N-2-

HACC-HA hydrogel.

[0077] Figs. 1-3 show the morphology of the N-2-HACC-HA hydrogel under a scanning electron microscope of different magnifications in this example. From Fig. 1, it can be seen that the structure of the N-2-HACC-HA hydrogel is a porous three-dimensional network structure, and this structure is conducive to the exchange of water, gas and some small molecular substances, which provides a guarantee for the N-2-HACC-HA hydrogel to be used as a wound dressing or a wound repair drug. From Fig. 3, it can be seen that there are still a large number of nanoscale pores in the N-2-HACC-HA hydrogel, which further improves the ability of the hydrogel to retain water and exchange gases and some small molecules.

[0078] Table 1 shows the test results of the water content of the N-2-HACC-HA hydrogel in this example. From the data in Table 1, it can be seen that the water content of the hydrogel is (89.62+0.09)%, which means that the N-2-HACC-HA hydrogel can store a large amount of water and has good water storage performance. This indicates that the N-2-HACC-HA hydrogel can keep the wound moist and provide a good environmental basis for cell proliferation and tissue proliferation in the wound, which is conducive to wound healing. Moreover, this feature

. a . . . LU503425 also provides feasibility support for carrying soluble drugs, antibacterial agents, growth promoting factors or gene of protein on the N-2-HACC-HA hydrogel.

[0079] Table 1

Sample 1 2 3 Mean

Weight before drying (g) 5.685 5.532 5.466 5.561+0.112

Weight after drying (g) 0.595 0.576 0.560 0.577+0.018

Water content (%) 89.53 89.59 89.75 89.62+0.09

[0080] Fig. 4 shows cytotoxicity results of N-2-HACC-HA of different concentrations to PK-15.

When the concentration of N-2-HACC-HA is 0.39%, 3.125%, 6.25% and 12.5%, the cell viability reaches 90% or above, and there is no significant difference from the control group (p>0.05). When the concentration of N-2-HACC-HA is 0.78% and 1.56%, the cell viability of

PK-15 is higher than that of the control group, and there is significant difference from the control group (p<0.05). It shows that N-2-HACC-HA can promote the growth of PK-15 in an appropriate concentration range. However, when the concentration reaches 25% and 50%, there is significant difference from the control group in terms of cell viability (p<0.05), and it may be due to the high osmotic pressure caused by the high concentration of sodium B-glycerophosphate solution that has some influence on the cell viability, but in this case, the cell viability is still high, respectively (91.04+3.07)% and (88.21+4.72)%. This indicates that when the concentration of N-2-HACC-HA is high, its cytotoxicity is still low. The above results show that N-2-HACC-

HA has little effect on the viability of PK-15 and thus has good biological safety.

[0081] Example 2

[0082] The preparation method of a temperature-sensitive hydrogel for wound treatment in this example included the following steps:

[0083] 1. Preparation of N-2-HACC

[0084] (1) Soaking treatment of chitosan

. . . . . . . . LU503425

[0085] 6 g of chitosan was dissolved in an acetic acid solution and the obtained solution was stirred for 1 h by a constant-speed stirrer. A 15 mol/L NaOH solution was then added dropwise to adjust pH to 9. The chitosan was soaked for 0.5 h, the solution was then filtered with suction, the obtained precipitate was washed with deionized water until neutral, and then freeze-dried in vacuum to obtain 25 g of dried chitosan. The acetic acid solution herein was made from 66.6 mL of acetic acid and 173.4 mL of deionized water.

[0086] (2) Ring-opening reaction of chitosan and glycidyltrimethylammonium chloride

[0087] The dried chitosan obtained in step (1) was dispersed in 50 mL of an isopropanol solution, the obtained solution was stirred thoroughly and then placed in an 80 °C water bath. An isopropanol solution of 2,3-epoxypropyltrimethylammonium chloride was then added dropwise within 30 min, the obtained solution was stirred at a constant temperature of 80 °C for 9 h at a stirring speed of 500 r/min to have a reaction, then cooled to room temperature and rested still for 1 h. 150 mL of 4 °C absolute ethanol was then added for soaking for 0.5 h, the solution was filtered with suction using absolute ethanol, the precipitate was freeze-dried in vacuum to constant weight, thus obtaining N-2-hydroxypropyltrimethylammonium chloride chitosan (N-2-

HACC). The isopropanol solution of 2,3-epoxypropyltrimethylammonium chloride was prepared from 9 g of 2,3-epoxypropyltrimethylammonium chloride and 50 mL of isopropanol.

[0088] 2. Preparation of a lactic acid solution of N-2-HACC

[0089] 0.4g of N-2-HACC was weighed and completely dissolved in 6 mL of 0.1 mol/L lactic acid and the obtained solution was then placed in a refrigerator until the temperature of the solution was constant at 4 °C.

[0090] 3. Preparation of a sodium B-glycerophosphate solution

[0091] 0.5 g of sodium a-glycerophosphate and 1 g of sodium B-glycerophosphate was weighed and completely dissolved in 4 mL of ultrapure water and the obtained solution was then placed in a refrigerator until the temperature of the solution was constant at 4 °C.

[0092] 4. Preparation of a N-2-HACC hydrogel

[0093] The sodium glycerophosphate solution was added dropwise to the lactic acid solution of 903425

N-2-HACC in an ice bath with stirring to have an ion cross-linking reaction for 30 min, thus obtaining the N-2-HACC hydrogel.

[0094] The N-2-HACC-HA hydrogel prepared in Example 1 and the N-2-HACC hydrogel prepared in Example 2 were subjected to a local skin sensitization test, and the results of the local skin sensitization test are shown in Table 2. Neither the N-2-HACC hydrogel group nor the

N-2-HACC-HA hydrogel group had skin erythema or edema on Day 1, Day 7 and Day 14 after induction administration, and on Day 14 after the last induction, no skin erythema or edema occurred 24 h and 48 h after the challenge administration, with a skin reaction score of 0 and a sensitization rate of 0. The negative control group had no skin erythema or edema 24 h and 48 h after the challenge administration, with a skin reaction score of 0 and a sensitization rate of 0.

According to the evaluation regulations, the sensitization intensity of the N-2-HACC-HA hydrogel was determined to be no sensitization.

[0095] Table 2

Time/h Score of N-2-HACC hydrogel group Score of N-2-HACC-HA Score of negative control

Core of skin reaction 24 0 0 0

Core of skin reaction 48 0 0 0

Sensitization rate (%) 0 0 0

Sensitization intensity No sensitization No sensitization No sensitization

[0096] FIG. 5 shows the in vitro hemolysis test results of the N-2-HACC-HA hydrogel of different concentrations. It can be seen that during the observation period, the positive control group (test tube No. 7) is clear red, and hemolysis occurs, while the different doses of N-2-

HACC-HA hydrogel group (test tube Nos. 1-5) and negative control group (test tube No. 6) do not appear hemolysis, different erythrocyte sedimentation velocities in test tubes Nos. 1-5 are caused by the increase of solution viscosity with the increase of hydrogel concentration. The

. ._ LU503425 results show that the N-2-HACC-HA hydrogel does not cause hemolysis of red blood cells, is safe to use at the current dose, can be in direct contact with blood, and can be applied to wound dressings. The components in test tubes Nos. 1-7 are shown in Table 3.

[0097] Table 3

Group 1 2 3 4 5 6 7

Mass of gel (mg) 01 02 03 04 05 Negative control Positive control

Administration volume (mL)0.1 02 03 04 05

Normal saline (mL) 24 23 22 21 20 25 0

Deionized water (mL) 0 0 0 0 0 0 2.5 2% red blood cell suspension (mL) 25 25 25 25 25 25 25

[0098] FIG. 6 shows the effect of N-2-HACC-HA gel of different concentrations on the proliferation of DF-1. The N-2-HACC-HA hydrogel has a great effect on the cell viability of

DF-1 as the concentration of the N-2-HACC-HA hydrogel increases. When the concentration of the N-2-HACC-HA hydrogel is 0.78%, the cell viability is 108.44% (p<0.01), indicating that the

N-2-HACC-HA hydrogel significantly promotes the proliferation of DF-1. When the concentration of the N-2-HACC-HA hydrogel is between 0.98% and 6.25%, the cell viability is always higher than or equal to that of the control group. When the concentration of the N-2-

HACC-HA hydrogel is 1.56%, the cell viability reaches the maximum, which is 111.27% (p<0.05). All this indicates that within a certain concentration range, the N-2-HACC-HA hydrogel can promote the proliferation of DF-1. The low cell viability of the N-2-HACC-HA hydrogel at a high concentration may be caused by the difference in osmotic pressure between the inside and outside of the cell. A high concentration causes a high osmotic pressure, which in turn leads to cell rupture and death and reduces the cell viability of DF-1 cells. It can be seen from the figure that when the concentration is 50%, the cell viability of DF-1 is above 80%, indicating that the N-2-HACC-HA hydrogel has no obvious cytotoxicity to DF-1.

[0099] SD rats after skin punching were randomly divided into four groups: negative control 0508425 group (Control), liquid carboxyning wound dressing group (SN group), N-2-HACC-HA hydrogel group (N-2-HACC-HA group) and N-2-HACC hydrogel group (N-2-HACC group), 14 rats in each group. Wounds of the Negative control group were not treated, wounds of the SN group were treated with a liquid carboxyning wound dressing, wounds of the N-2-HACC group were treated with the N-2-HACC hydrogel prepared in Example 2, and wounds of the N-2-

HACC-HA group were treated with the N-2-HACC-HA hydrogel prepared in Example 2.

[00100] FIG. 7 shows the wound healing situation of treated SD rats. It can be seen from the figure that the wounds of rats in each treatment group have improved in varying degrees after administration. On Day 3 after administration, the wound surfaces of rats in each treatment group are dark red, and the wounds begin to scab. On Day 5 after administration, the wounds of rats in the N-2-HACC-HA group and the N-2-HACC group change from red to dark red, and the scabs begin to dry and harden. On Day 7, the wound scabs of rats in the N-2-HACC-HA group fall off, and the wound healing accelerates. On Day 13, the skin wounds of the rats in the N-2-

HACC-HA group are basically completely healed, while the skin wounds of rats in the N-2-

HACC group and the SN group are not completely healed. The scabs on the wound surfaces of rats in the negative control group begin to darken and harden on Day 7 and then fell off on Day 10, and the wounds shrink on Day 15. After administration, the scabs on the wound surfaces of the rats in the SN group begin to harden on Day 5, then turn dark red and become dry on Day 7; on Day 10, the scabs begin to shrink and the wound surfaces are healed further, and on Day 15, the scabs disappear, scars are formed, and the wounds are basically healed. In summary, at the same time after administration, the wound healing rate of the N-2-HACC-HA group was higher than that of other groups. On Day 13, there were rats with completely healed wounds in the N-2-

HACC-HA group. However, rats in the other groups were not completely healed. On Day 14, there were rats with completely healed wounds in the N-2-HACC group. On Day 15, the rats in the N-2-HACC-HA group and the N-2-HACC group were all healed completely, while there were no rats with completely healed wounds in the control group and the SN group on Day 15.

[00101] The wound healing rates of SD rats are shown in Table 4. On Day 3 after 0508425 administration, the mean wound healing rates of the rats in the N-2-HACC-HA group, the negative control group, the N-2-HACC group and the SN group are 35.00%, 13.32%, 22.58% and 19.96%, respectively. The wound healing rate of the rats in the N-2-HACC-HA group 1s significantly higher than that of rates in the negative control group (p<0.001). The wound healing rate of rats in the N-2-HACC-HA group 1s significantly higher than that of rats in the SN group (p<0.01). The wound healing rate of rats in the N-2-HACC-HA group 1s significantly higher than that of rats in the N-2-HACC group (p<0.05), while the wound healing rate of rats in the N-2-HACC group is not significantly different from that of rats in the carboxyning group (p>0.05). All this indicates that after 3 days of continuous administration, both the N-2-HACC-

HA hydrogel and the N-2-HACC hydrogel can promote wound healing, but the N-2-HACC-HA hydrogel is significantly better than the N-2-HACC hydrogel in the effect of promoting wound healing,

[00102] Table 4

Group Wound healing rate on Day 3 after administration (%) Wound healing rate on Day 7 after administration (%) Wound healing rate on Day 11 after administration (%) Wound healing rate on Day 15 after administration (%)

Control 13.32+0.80 37.63+2.59 6749987 89.03+2.84

SN group 19.96+1.62 44.59+4.16 71.2849.54 92.19+1.86

N-2-HACC group 22.58+1.80 48.34+£590 83.13+4.66 100

N-2-HACC-HA group 35.00+2.72 70.916,29 92.75+3.73 100

[00103] On Day 7 after administration, the mean wound healing rates of the rats in the N- 2-HACC-HA group, the negative control group, the N-2-HACC group and the SN group are 70.91%, 37.63%, 48.34% and 44.59%, respectively. The wound healing rate of the rats in the N- 2-HACC-HA group is significantly higher than that of rates in the negative control group (p<0.001). The wound healing rate of rats in the N-2-HACC-HA group 1s significantly higher than that of rats in the other two groups (p<0.001). The wound healing rate of rats in the N-2-

HACC-HA group is significantly higher than that of rats in the N-2-HACC group (p<0.05). All 0508425 this indicates that after 7 days of continuous administration, both the N-2-HACC-HA hydrogel and the N-2-HACC hydrogel can effectively promote wound healing, but the N-2-HACC-HA hydrogel 1s significantly better than the N-2-HACC hydrogel and the SN group in the effect of promoting wound healing.

[00104] On Day 11 after administration, the mean wound healing rates of the rats in the N- 2-HACC-HA group, the negative control group, the N-2-HACC group and the SN group are 92.75%, 67.49%, 83.13% and 71.28%, respectively. The wound healing rate of the rats in the N- 2-HACC-HA group 1s significantly higher than that of rates in the negative control group (p<0.001). The wound healing rate of rats in the N-2-HACC-HA group 1s significantly higher than that of rats in the other two groups (p<0.001). The wound healing rate of rats in the N-2-

HACC group 1s significantly higher than that of rats in the negative control group (p<0.001). The wound healing rate of rats in the N-2-HACC group is significantly higher than that of rats in the

SN group (p<0.005). The wound healing rate of rats in the N-2-HACC-HA group is significantly higher than that of rats in the N-2-HACC group (p<0.05). All this indicates that after 11 days of continuous administration, both the N-2-HACC-HA hydrogel and the N-2-HACC hydrogel can effectively promote wound healing, but the N-2-HACC-HA hydrogel 1s significantly better than the N-2-HACC hydrogel and the SN group in the effect of promoting wound healing.

[00105] On Day 15 after administration, the mean wound healing rates of the rats in the N- 2-HACC-HA group, the negative control group, and the SN group are 100%, 89.03%, 48.34% and 92.19%, respectively. The three groups are not significantly different from the negative control group in terms of wound healing rate of rats (p>0.05), indicating that the three groups of drugs have the same healing effect on wounds in the skin of rats after 15 days of continuous administration.

[00106] FIG. 8 shows pathological observation images of skin tissue wounds of rats. No lesions were found in the skin tissue of rats in each group, indicating that the N-2-HACC-HA hydrogel and the N-2-HACC hydrogel are safe as wound dressings and do not cause lesions in the skin tissue. As can be seen from the figure, inflammatory cell infiltration appears earlier in the skin tissue around the wounds of the rats in the N-2-HACC-HA group and the N-2-HACC 0508425 group than in the negative control group, with new fibroblasts and new blood vessels beginning to appear on Day 5 after administration and epidermal formation occurring earlier. By comparing the skin tissues around the wounds of the rats in the N-2-HACC-HA group, the N-2-HACC group, and the SN group, inflammatory cell infiltration in the skin tissues around the wounds of the rats in these three groups all occur on Day 3 after administration. On Day 5 after administration, inflammatory cell infiltration increases, and new blood vessels and fibroblasts appear in the dermal tissue. All this indicates that the three have similar effects in the inflammatory stage and in promoting formation of new blood vessels. However, from Day 7 after administration, the N-2-HACC-HA group is significantly superior in promoting epidermal formation, granulation tissue hyperplasia, and formation of new blood vessels, compared to the

N-2-HACC hydrogel group and the SN group in terms of changes in the skin tissue around the wounds, and this indicates that the N-2-HACC-HA hydrogel has a stronger effect on fibroblast chemotaxis and in promoting epidermal formation, granulation tissue hyperplasia, and formation of new blood vessels. These results are consistent with trends from external observations of wound healing and wound healing rates.

[00107] The present invention adopts a chitosan derivative N-2- hydroxypropyltrimethylammonium chloride chitosan as the raw material for preparing a hydrogel, and compared with chitosan, the derivative has better solubility under neutral and slightly acidic conditions.

[00108] It is found in this present invention that if sodium B-glycerophosphate is only added to the acetic acid solution of N-2-HACC during the preparation process of the hydrogel, the performance of the hydrogel will be unstable, and a-glycerophosphate must be also added.

The price of a-glycerophosphate is dozens of times that of sodium B-glycerophosphate, which increases the preparation cost of the hydrogel. After a large number of experiments for screening and research, the present invention adopts N-2-HACC, sodium hyaluronate and sodium P- glycerophosphate to prepare the temperature-sensitive hydrogel. The prices of sodium hyaluronate and sodium B-glycerophosphate are comparable, which reduces the cost and 0508425 achieves better wound healing effect.

[00109] The pH dependence of water solubility of chitosan and N-2-HACC 1s shown in

FIG. 9, where Curve a represents pH dependence of N-2-HACC, curve b represents the pH dependence of chitosan. It can be seen from FIG. 9 that the light transmittance of the chitosan solution drops suddenly when the pH>6, and gradually decreases and tends to be gentle when the pH is 8 or above; however, the light transmittance of the N-2-HACC solution is relatively stable all the time, and it decreased slightly when the pH reaches 8.5, and the light transmittance remains almost unchanged when the pH continues to increase. The results show that the water solubility of chitosan is greatly affected by pH, while N-2-HACC can exist in almost all pH conditions, which greatly expands its application.

Claims (14)

CLAIMS LU503425

1. A low-cost temperature-sensitive hydrogel for wound treatment, made of N-2- hydroxypropyltrimethylammonium chloride chitosan, acetic acid, sodium hyaluronate and sodium P-glycerophosphate.

2. A preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 1, the method comprising the following steps: step 1, dissolving chitosan in an acetic acid solution, stirring the obtained solution thoroughly, adding a NaOH solution dropwise to adjust pH to 9, soaking for 0.5-1 h, filtering the solution with suction, washing the obtained precipitate with deionized water until neutral, and freeze- drying the precipitate in vacuum to obtain dried chitosan; dispersing the dried chitosan in isopropanol, stirring the obtained solution thoroughly, and heating the solution up to 80-85 °C; adding an isopropanol solution of 2,3- epoxypropyltrimethylammonium chloride dropwise within 30 min, stirring the obtained solution at a constant temperature of 80-85°C for 9-10 h at a stirring speed of 500-550 r/min, then cooling the solution to room temperature, resting the solution still for 1-2 h, adding 4 °C absolute ethanol, soaking for 0.5-1 h, filtering the solution with suction, freeze-drying the precipitate in vacuum to constant weight, thus obtaining N-2-hydroxypropyltrimethylammonium chloride chitosan; and step 2, adding a sodium hyaluronate solution dropwise to an acetic acid solution of N-2-HACC in an ice bath with stirring for 30-40 min, then quickly adding sodiumf- glycerophosphatsolution, continuing to stir the solution to have reaction for 30-40 min to obtain N-2-HACC-HA hydrosol. In clinical application, the hydrosol is heated to 37 °C by wound temperature and can be cross-linked with ions to form hydrogel in 40 s.

3. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 2, wherein in step 1, a volume ratio of acetic acid to deionized water in the acetic acid solution is (1-15):(20-40).

4. The preparation method of the low-cost temperature-sensitive hydrogel for wound 0508425 treatment according to claim 2 or 3, wherein in step 1, a ratio of the mass of chitosan to the volume of acetic acid in the acetic acid solution is 1 g:(1-15) mL.

5. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 4, wherein in step 1, a mass ratio of chitosan to the dried chitosan is 1:(4-10).

6. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 5, wherein in step 1, a ratio of the mass of the dried chitosan to the volume of isopropanol is 1 g:(5-15) mL.

7. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 6, wherein in step 1, a ratio of the mass of the dried chitosan to the volume of the isopropanol solution of epoxypropyltrimethylammonium chloride is 1 g:(5-15)

mL.

8. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 7, wherein the isopropanol solution of epoxypropyltrimethylammonium chloride has a concentration of 0.18-0.2 g/mL.

9. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 8, wherein in step 2, a volume ratio of the acetic acid solution of N- 2-HACC to the sodium hyaluronate solution is (1-5):1.

10. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 9, wherein in step 2, a volume ratio of the acetic acid solution of N- 2-HACC to the sodium B-glycerophosphate solution is (1-10):1.

11. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 10, wherein in step 2, a ratio of the mass of N-2-HACC to the volume of the acetic acid solution is 1 g: (5-25) mL.

. _ L 42

12. The preparation method of the low-cost temperature-sensitive hydrogel for wound 903425 treatment according to claim 11, wherein the acetic acid solution has a concentration of 0.1-0.12 mol/L.

13. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 12, wherein in step 2, a ratio of the mass of sodium hyaluronate to the volume of deionized water in the sodium hyaluronate solution is 1 g: (1000-2000) mL.

14. The preparation method of the low-cost temperature-sensitive hydrogel for wound treatment according to claim 13, wherein in step 2, a ratio of the mass of sodium P- glycerophosphate to the volume of deionized water in the sodium B-glycerophosphate solution is Ig: (1-5) mL.