A temperature-mediated self-contracting hemostatic microneedle hydrogel and its preparation and application

Abstract

The invention discloses a temperature-mediated self-contracting hemostatic microneedle hydrogel and its preparation and application, and belongs to the technical field of biomaterials. The invention prepares double-bond hydroxybutyl chitosan by modifying chitosan polymer with hydroxybutyl and double bonds, and injects it into a microneedle mold after mixing with a photoinitiator LAP and performing photocrosslinking. The obtained microneedle hydrogel can be fixed on the surface of a lubricated organ and has temperature sensitivity. When under body temperature conditions, the microneedle hemostatic hydrogel will shrink and deform, causing the tissue in contact (rivet) to tighten to a certain extent, thereby achieving the purpose of physically blocking the bleeding wound and reducing the amount of bleeding in the first place.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a temperature-mediated self-contracting hemostatic microneedle hydrogel and a preparation method and application thereof. Background Art

[0002] Bleeding is the main complication after trauma. Massive bleeding can quickly cause a decrease in effective blood circulation, insufficient tissue perfusion, and dysfunction of important organs. It can be seen that bleeding is a difficult problem that threatens life safety during trauma treatment and surgery. How to effectively stop bleeding is the focus of emergency treatment and clinical research.

[0003] Traditional hemostatic materials are mainly gauze, but gauze has poor hemostatic effect and may cause tissue damage or necrosis. Another disadvantage is that this type of hemostatic material is mostly used for surface hemostasis, cannot be used for hemostasis of internal organs, and cannot stay in the body for a long time.

[0004] In recent years, research on natural polymer hemostatic hydrogel materials has made rapid progress. Due to its good biosafety and degradability, it can be used for hemostatic treatment of surface wounds or internal bleeding. Surface bleeding wounds are relatively rough and easy to dry, and it is relatively easy to achieve rapid hemostasis after the application of hemostatic materials; however, the surface of internal organs is rich in oil and fat, and most of them are covered with physiological lubricants. After the application of hemostatic materials, it is not easy to fix on their surface, and the peristalsis of internal organs further increases the probability of hemostatic materials slipping off. If effective fixation cannot be achieved, no matter how good the hemostatic effect is, it cannot be exerted, which is also an important reason for the poor hemostatic effect of hemostatic hydrogels. Summary of the invention

[0005] To solve the related problems, the primary purpose of the present invention is to provide a method for preparing a temperature-mediated self-contracting hemostatic microneedle hydrogel.

[0006] Another object of the present invention is to provide a temperature-mediated self-contracting hemostatic microneedle hydrogel obtained by the above preparation method.

[0007] Another object of the present invention is to provide an application of the temperature-mediated self-contracting hemostatic microneedle hydrogel.

[0008] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0009] A method for preparing a temperature-mediated self-contracting hemostatic microneedle hydrogel, which is based on chitosan polymer and is modified with hydroxybutyl and double bonds to prepare double-bond hydroxybutyl chitosan (HBC_m), which is mixed with a photoinitiator and injected into a microneedle mold and photocrosslinked. The obtained microneedle hydrogel can be fixed on the surface of a lubricated organ and has temperature sensitivity. When under body temperature conditions, the microneedle hemostatic hydrogel will shrink and deform, causing the contact (rivet) tissue to tighten to a certain extent, achieving the purpose of physically blocking the bleeding wound and reducing the amount of bleeding in the first place. Specifically comprising the following steps:

[0010] (1) fully dissolving hydroxybutyl chitosan in water, adding methacrylic anhydride dropwise, and obtaining a double-bond hydroxybutyl chitosan solution after the reaction is complete;

[0011] (2) adjusting the pH of the double-bond hydroxybutyl chitosan solution obtained in step (1) to neutral, dialyzing, and freeze-drying to obtain double-bond hydroxybutyl chitosan;

[0012] (3) fully dissolving the double-bond hydroxybutyl chitosan obtained in step (2) in water to obtain a double-bond hydroxybutyl chitosan solution;

[0013] (4) adding a photoinitiator to the double-bond hydroxybutyl chitosan solution obtained in step (3) to obtain a hydrogel prepolymer solution;

[0014] (5) Injecting the hydrogel prepolymer obtained in step (4) into the microneedle mold, photocrosslinking, and demolding to obtain the temperature-mediated self-contracting hemostatic microneedle hydrogel.

[0015] Furthermore, the hydroxybutyl chitosan described in step (1) is prepared by the following method: alkalizing chitosan with a NaOH solution to obtain alkalized chitosan; dispersing the alkalized chitosan in an isopropanol aqueous solution, adding 1,2-butylene oxide dropwise, and reacting; adjusting the pH of the reaction solution to neutral, dialyzing, filtering, and freeze-drying to obtain hydroxybutyl chitosan.

[0016] The weight average molecular weight MW of the chitosan is 50,000-1,000,000; further, the weight average molecular weight MW is 300,000.

[0017] The concentration of the NaOH solution is 20% to 70%; further, the concentration of the NaOH solution is 50%.

[0018] The alkalization treatment time is 6 to 48 hours; further, the time is 24 hours.

[0019] The volume ratio of isopropanol to water in the isopropanol aqueous solution is 1-2:1-2; further, the volume ratio is 1:1.

[0020] The ratio of chitosan to 1,2-butylene oxide is 2-6 g: 40-120 mL; further, the ratio is 4 g: 80 mL.

[0021] The reaction conditions are: temperature 40°C to 70°C, time 6 to 24 hours; further, the conditions are: temperature 60°C, time 12 hours.

[0022] The reagent used for pH adjustment is 1% HCl solution.

[0023] Furthermore, the ratio of hydroxybutyl chitosan and methacrylic anhydride in step (1) is 1-4:3-9 by mass; further, the ratio is 2:6.4 by mass.

[0024] Furthermore, the dissolution in step (1) is carried out at room temperature. The room temperature in the present invention refers to 20-30°C, and further, the room temperature refers to 25°C.

[0025] Furthermore, the reaction conditions in step (1) are: temperature 40°C to 80°C, time 12 to 24 hours, and rotation speed 500 to 1500 rpm; further, the conditions are: temperature 60°C, time 24 hours, and rotation speed 1000 rpm.

[0026] Furthermore, the reagent used for pH adjustment in step (2) is sodium bicarbonate.

[0027] Furthermore, the dialysis time in step (2) is 3 to 4 days.

[0028] Furthermore, the freeze-drying in step (2) is carried out at a temperature of -20°C to -80°C; further, at a temperature of -60°C.

[0029] Furthermore, the mass concentration of the double-bond hydroxybutyl chitosan solution in step (3) is 2% to 10%; further, the mass concentration is 5%.

[0030] Furthermore, the photoinitiator described in step (4) is any one of a water-soluble photoinitiator or a fat-soluble photoinitiator; further, the photoinitiator is a water-soluble photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphite) or a fat-soluble photoinitiator Irgacure2959.

[0031] Furthermore, the amount of the photoinitiator added in step (4) is calculated based on a concentration of 1 to 2 wt % in the system; further, the amount of the photoinitiator added is calculated based on a concentration of 2 wt % in the system.

[0032] Furthermore, the microneedles corresponding to the microneedle mold described in step (5) are single-sided needles or double-sided needles, and the specific shape is: needle tip diameter 2-10 μm, needle height 100-1000 μm, base diameter 200-1000 μm, center spacing 100 μm-1000 μm; further, the needle tip diameter is 5 μm, the needle height is 600 μm, the base diameter is 500 μm, and the center spacing is 500 μm.

[0033] Furthermore, the conditions for the photocrosslinking in step (5) are: wavelength 365 nm, time 30±2 seconds.

[0034] Neutral in the present invention means pH=7.

[0035] The water in the present invention refers to ultrapure water.

[0036] A temperature-mediated self-contracting hemostatic microneedle hydrogel is obtained by the above preparation method.

[0037] The application of the above temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products.

[0038] Furthermore, the hemostatic product is a visceral hemostatic product.

[0039] Existing hemostatic hydrogels are prone to loose adhesion, slippage, and ectopicity when dealing with bleeding in smooth interface tissues, and cannot be effectively fixed at the target location, making it even more difficult to achieve subsequent hemostatic effects. Advantages of the hemostatic microneedle hydrogel technology prepared by the method of the present invention:

[0040] (1) The microneedle structure enables the present invention to have a better riveting effect on the application interface, especially for visceral tissues with smooth surfaces. After attachment, it is not easy to slip out of place, which is more advantageous for hemostasis of organ bleeding.

[0041] (2) Body temperature conditions trigger the microneedle hydrogel to shrink and deform, which further mediates the tightening of the adhesion interface. When applied to the soft organ surface, it will cause the bleeding site to tighten and close, reduce the amount of bleeding in the first place, and improve the hemostatic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of temperature-mediated hemostatic microneedle hydrogel actively tightening the bleeding wound surface to quickly reduce bleeding;

[0043] Figure 2 It is an intuitive morphology and microscopic morphology of the planar structure hydrogel (HBC_mGel) and microneedle hydrogel (HBC_mMicroneedleGel) constructed based on double-bond hydroxybutyl chitosan (HBC_m);

[0044] Figure 3The temperature-mediated hemostatic microneedle hydrogel (HBC_mmicroneedleGel) contraction experiment analysis diagram; wherein, (A) is the hydrogel morphology at different temperatures; (B) is the hydrogel area contraction data bar chart at different temperatures;

[0045] Figure 4 Schematic diagram of the tensile test after the pig liver is bonded together by the microneedle hydrogel (HBC_md-microneedleGel); a is the morphology of the double-sided structure temperature-mediated hemostatic microneedle hydrogel; b is the schematic diagram of the tensile test structure; c is the tensile test morphology; d is the morphology of the pig liver after the test;

[0046] Figure 5 Comparison of tensile strength of microneedle hydrogel (HBC_md-microneedleGel), double bond modified gelatin (GelMA), and planar structure hydrogel (HBC_mGel) after bonding to pig liver; A is the stress test graph of microneedle hydrogel; B is the stress test graph of double bond modified gelatin and planar structure hydrogel;

[0047] Figure 6 Comparison of liver hemostasis time of double bond modified gelatin (GelMA), planar structure hydrogel (HBC_mGel), and microneedle hydrogel (HBC_mmicroneedleGel). DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0049] The experimental methods without specific experimental conditions in the examples are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials, reagents, equipment, etc. used are those obtained from commercial sources unless otherwise specified.

[0050] The microneedle morphology corresponding to the microneedle mold used in the following examples is: a needle tip diameter of 5 μm, a needle height of 600 μm, a base diameter of 500 μm, and a center spacing of 500 μm.

[0051] The molecular weight of chitosan used in the following examples is MW=300,000.

[0052] Example 1: Preparation of temperature-mediated self-contracting hemostatic microneedle hydrogel (HBC_mMicroneedleGel)

[0053] Step 1: Synthesis of hydroxybutyl chitosan (HBC)

[0054] (1) How long does the purified chitosan undergo alkalinization treatment with 50% NaOH solution? 24 h;

[0055] (2) 4 g of alkalized chitosan was dispersed in 80 mL of isopropanol aqueous solution (volume ratio of 1:1), 80 mL of 1,2-butylene oxide was added dropwise, and the mixture was reacted at 60 °C for 12 h;

[0056] (3) Using 1% HCl to adjust the pH to neutral, dialyze, filter, and freeze-dry to obtain hydroxybutyl chitosan, which is referred to as HBC.

[0057] Step 2: Synthesis of double bond hydroxybutyl chitosan (HBC_m)

[0058] (1) Dissolve 2 g HBC in ultrapure water until fully dissolved at room temperature, and add 6.4 g methacrylic anhydride dropwise;

[0059] (2) Control the reaction temperature to 60°C, stir vigorously at 1000 rpm / min, and continue the reaction for 24 h;

[0060] (3) adding solid sodium bicarbonate to adjust the pH to neutral;

[0061] (4) Dialysis for 3-4 days;

[0062] (5) Freeze-drying was performed at -60°C to obtain double-bond hydroxybutyl chitosan, which was recorded as HBC_m.

[0063] Step 3: Preparation of temperature-mediated self-contracting hemostatic microneedle hydrogel

[0064] (1) Weigh HBC_m and prepare it into 5% concentration with ultrapure water, and stir continuously at 4°C until dissolved;

[0065] (2) adding a water-soluble photoinitiator LAP to prepare a solution with a concentration of 2%;

[0066] (3) Inject into the microneedle mold and transfer to 365nm light cross-linking for 30 seconds to form gel;

[0067] (4) Demolding, the obtained thermosensitive hydrogel patch with microneedle structure is the temperature-mediated self-contracting hemostatic microneedle hydrogel, denoted as HBC_mMicroneedleGel.

[0068] Comparative Example 1: Preparation of planar temperature-sensitive hydrogel (HBC_mGel)

[0069] (1) Weigh double-bond hydroxybutyl chitosan (HBC_m), prepare a 5 wt% solution with ultrapure water, and stir continuously at 4°C until dissolved;

[0070] (2) adding a water-soluble photoinitiator LAP to prepare a solution with a concentration of 2 wt %;

[0071] (3) Transfer to 365 nm light cross-linking for 30 seconds to form a gel, which is recorded as HBC_mGel.

[0072] Comparative Example 2: Preparation of double bond modified gelatin (GelMA) hydrogel

[0073] (1) dissolving double bond modified gelatin (GelMA) in deionized water to prepare a solution with a concentration of 20 wt%;

[0074] (2) adding a water-soluble photoinitiator LAP to prepare a solution with a concentration of 2 wt %;

[0075] (3) Transfer to 365 nm light cross-linking for 30 seconds to form a gel, which is recorded as GelMA.

[0076] Effect Example 1: Temperature-mediated microneedle hydrogel contraction experiment

[0077] (1) Take HBC_mMicroneedleGel prepared in Example 1;

[0078] (2) Place on a glass slide at 25°C, outline the edge of the hydrogel, and calculate the initial area of ​​the hydrogel;

[0079] (3) Transfer to 37°C and incubate for 3 min. Calculate the area based on the edge of the shrunken hydrogel.

[0080] (4) Compare the changes in hydrogel area under 25℃ and 37℃ conditions.

[0081] like Figure 3 As shown in the figure, the initial area of ​​the hydrogel at 25°C is 138.9 mm 2 After incubation at 37°C, the area contraction was 116.7 mm 2 It can be seen that the hydrogel of the present invention is temperature sensitive, and the microneedle hemostatic hydrogel will shrink and deform when placed at body temperature.

[0082] Effect Example 2: Microneedle hydrogel adhesion (fixation) experiment on internal organs

[0083] 1. Tensile strength test of thermosensitive hemostatic microneedle hydrogel with double-sided microneedle structure

[0084] (1) According to the method of Example 1, a double-sided microneedle mold was used to prepare a thermosensitive hemostatic microneedle hydrogel (HBC_md-microneedleGel) with a double-sided microneedle structure;

[0085] (2) Using pig liver as the experimental object, two pieces of pig liver were bonded together using HBC_md-microneedleGel;

[0086] (3) Place in a tensile testing machine to test the tensile strength;

[0087] (4) At the same time, the tensile strength of double bond modified gelatin (GelMA) and planar structure thermosensitive hydrogel (HBC_mGel) was compared.

[0088] like Figure 4 , 5 As shown in the figure, the tensile strength of the HBC_md-microneedleGel group with microneedle structure was 8.495 kPa, while the tensile strengths of the HBC_mGel group and GelMA group without microneedle structure were 0.555 kPa and 0.343 kPa, respectively, and the bonding strength was significantly lower than that of the HBC_md-microneedleGel group. HBC_md-microneedleGel showed good rivet adhesion effect on the liver surface.

[0089] Effect Example 3: Experiment on the application of hemostatic microneedle hydrogel in hemostasis of mouse liver

[0090] (1) Double bond modified gelatin (GelMA) and planar structure thermosensitive hydrogel (HBC_mGel) were prepared according to the methods of Comparative Examples 1 and 2, and thermosensitive hemostatic microneedle hydrogel (HBC_mmicroneedleGel) was prepared according to the method of Example 1;

[0091] (2) After the mouse was anesthetized, the liver was exposed and a small incision was made with a surgical blade to cause local bleeding in the liver;

[0092] (3) The prepared hydrogel patch was applied to the bleeding site of the liver, the bleeding condition was observed, and the time when the bleeding stopped was recorded.

[0093] like Figure 6 As shown in the figure, the results showed that in the HBC_mmicroneedleGel group, the liver wound stopped bleeding after 10 seconds, while in the HBC_mGel group and the GelMA group, the bleeding stopped after 12.8 seconds and 16.3 seconds, respectively. The liver hemostasis effect of HBC_md-microneedleGel was improved, and HBC_md-microneedleGel with microneedle structure and thermosensitive properties had a positive effect on the hemostasis of organ bleeding.

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Application of a temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products, characterized in that: The temperature-mediated self-contracting hemostatic microneedle hydrogel is obtained by a preparation method comprising the following steps: (1) fully dissolving hydroxybutyl chitosan in water, adding methacrylic anhydride dropwise, and obtaining a double-bond hydroxybutyl chitosan solution after the reaction is complete; (2) adjusting the pH of the double-bond hydroxybutyl chitosan solution obtained in step (1) to neutral, dialyzing, and freeze-drying to obtain double-bond hydroxybutyl chitosan; (3) fully dissolving the double-bond hydroxybutyl chitosan obtained in step (2) in water to obtain a double-bond hydroxybutyl chitosan solution; (4) adding a photoinitiator to the double-bond hydroxybutyl chitosan solution obtained in step (3) to obtain a hydrogel prepolymer solution; (5) injecting the hydrogel prepolymer obtained in step (4) into the microneedle mold, photocrosslinking, and demolding to obtain the temperature-mediated self-contracting hemostatic microneedle hydrogel; The ratio of hydroxybutyl chitosan and methacrylic anhydride in step (1) is 1-4:3-9 by mass; The mass concentration of the double-bond hydroxybutyl chitosan solution described in step (3) is 2% to 10%; The microneedles corresponding to the microneedle mold described in step (5) are single-sided needles or double-sided needles, and the specific shape is: needle tip diameter 2-10 μm, needle height 100-1000 μm, base diameter 200-1000 μm, and center spacing 100-1000 μm.

2. The use of the temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products according to claim 1, characterized in that: The photoinitiator in step (4) is a water-soluble photoinitiator LAP or a fat-soluble photoinitiator Irgacure 2959; The amount of the photoinitiator added in step (4) is calculated based on its concentration in the system being 1 to 2 wt %.

3. Use of the temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products according to claim 1 or 2, characterized in that: The reaction conditions in step (1) are: temperature 40°C to 80°C, time 12 to 24 h, and rotation speed 500 rpm to 1500 rpm; The freeze-drying described in step (2) is carried out at a temperature of -20°C to -80°C.

4. Use of the temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products according to claim 1 or 2, characterized in that: The mass ratio of hydroxybutyl chitosan and methacrylic anhydride in step (1) is 2:6.4; The reaction conditions in step (1) are: temperature 60°C, time 24 h, and rotation speed 1000 rpm; The reagent used for pH adjustment in step (2) is sodium bicarbonate; The dialysis time in step (2) is 3 to 4 days; The mass concentration of the double-bond hydroxybutyl chitosan solution described in step (3) is 5%; The amount of the photoinitiator added in step (4) is calculated based on its concentration in the system being 2 wt %; The microneedle corresponding to the microneedle mold described in step (5) is a single-sided needle or a double-sided needle, and the specific shape is: needle tip diameter 5 μm, needle height 600 μm, base diameter 500 μm, center spacing 500 μm; The conditions for the photocrosslinking described in step (5) are: wavelength 365 nm, time 30±2 seconds.

5. Use of the temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products according to claim 1 or 2, characterized in that: The hydroxybutyl chitosan described in step (1) is prepared by the following method: alkalizing chitosan with a NaOH solution to obtain alkalized chitosan; dispersing the alkalized chitosan in an isopropanol aqueous solution, adding 1,2-butylene oxide dropwise, and reacting; adjusting the pH of the reaction solution to neutral, dialyzing, filtering, and freeze-drying to obtain hydroxybutyl chitosan.

6. The use of the temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products according to claim 5, characterized in that: The molecular weight of the chitosan is MW=50000-1000000; The concentration of the NaOH solution is 20% to 70%; The alkalization treatment time is 6 to 48 hours; The volume ratio of isopropanol to water in the isopropanol aqueous solution is 1-2:1-2; The ratio of chitosan to 1,2-butylene oxide is 2-6 g: 40-120 mL; The reaction conditions are: temperature 40°C to 70°C, time 6 to 24 hours.

7. The use of the temperature-mediated self-contracting hemostatic microneedle hydrogel in the preparation of hemostatic products according to claim 5, characterized in that: The chitosan has a weight average molecular weight MW=300000; The concentration of the NaOH solution is 50%; The alkalization treatment time is 24h; The volume ratio of isopropanol to water in the isopropanol aqueous solution is 1:1; The ratio of chitosan to 1,2-butylene oxide is 4 g:80 mL; The reaction conditions are: temperature 60°C, time 12 h; The reagent used for pH adjustment is 1% HCl solution.

8. Use of the temperature-mediated self-contracting hemostatic microneedle hydrogel according to claim 1 or 2 in the preparation of hemostatic products, characterized in that: The hemostatic product is a visceral hemostatic product.

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