Hemostatic and antibacterial hydrogel powder with adhesion and self-propelling properties and preparation method of hemostatic and antibacterial hydrogel powder

Abstract

The invention discloses hemostatic antibacterial hydrogel powder with adhesion and self-propelling properties and a preparation method thereof. The hemostatic antibacterial hydrogel powder is prepared by blending polymer hydrogel powder and CaCO3 powder. The hydrogel powder can be used for stopping bleeding and promoting wound healing, has body fluid or blood-induced adhesion performance, and can be used as a physical barrier to block a bleeding opening, promote blood coagulation on the surface of the wound and accelerate surface hemostasis, meanwhile, bubbles released by dissolving CaCO3 can drive surrounding materials to enter the interior of the wound, blood coagulation factors are further released, and the healing of the wound is promoted. And blood coagulation cascade in the wound is promoted, so that internal and external double-effect hemostasis is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a hemostatic and antibacterial hydrogel powder with adhesion and self-propelling properties and a preparation method thereof. Background Art

[0002] When encountering severe bleeding caused by sudden situations, massive blood loss often leads to serious complications in the body, including hypotension and multiple organ dysfunction, and in severe cases, loss of life. Common hemostatic materials (such as hemostatic sponges, hemostatic gauze, hemostatic powder, etc.) are not suitable for emergency situations of severe bleeding: sponges and gauze are not suitable for irregular wounds with heavy or incompressible bleeding, because they cannot enter deep bleeding sites to promote hemostasis. At the same time, sponges and gauze do not have adhesion properties. When applied to the bleeding site, additional pressure needs to be applied to promote hemostasis. When applied, hemostatic powder cannot form a stable scab and even dissolves in the blood. In addition, it is easy to be washed away when facing the high-pressure blood flow gushing out of the bleeding wound, resulting in unsatisfactory hemostasis effect, thus missing the platinum rescue time. Therefore, there is an urgent need for faster and more effective hemostatic repair strategies.

[0003] Adhesive hemostatic materials (adhesives) can adhere to the wound site when encountering body fluids or blood, forming a physical barrier to block the bleeding and accelerate hemostasis. However, most commercial bioadhesives have limitations, such as low biocompatibility, slow adhesion formation, poor mechanical matching with tissues, and weak adhesive properties. In addition, some hydrogel adhesives require light curing, additional coupling agents, or thrombin-mediated coagulation, and only stop bleeding on the surface, but may not be effective for deep tissue bleeding. Summary of the invention

[0004] In order to solve the two difficulties that hemostatic materials only stop bleeding on the wound surface and have low biosafety in emergency situations, the present invention provides a hemostatic antibacterial hydrogel powder with adhesion and self-propelling properties and a preparation method thereof, the purpose of which is to enable the obtained powder to quickly absorb blood after contacting the bleeding site to form an adhesive hydrogel, which can effectively adhere to the wound site and promote blood coagulation on the wound surface. At the same time, the acidic environment created by the hydrogel powder when it encounters blood promotes the dissolution of calcium-based inorganic compounds, and the convection generated by the released bubbles and the surrounding materials drives part of the materials into the wound, while releasing coagulation factors to promote the coagulation cascade inside the wound, thereby achieving internal and external dual-effect hemostasis.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] The invention discloses a method for preparing a hemostatic and antibacterial hydrogel powder with adhesion and self-propelling properties, comprising the following steps:

[0007] Step 1: Preparation of modified citric acid

[0008] Add citric acid to DMF and stir until dissolved, then add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir at room temperature for 24 to 48 hours, then remove DMF by rotary evaporation to obtain modified citric acid;

[0009] Step 2: Preparation of polymer hydrogel powder

[0010] Weigh chitosan and dissolve it in 0.1-0.3M acetic acid solution, add dopamine, stir at 50-70°C for 4-6 hours, then add modified citric acid, continue stirring until hydrogel is formed, freeze-dry and grind with a ball mill to obtain polymer hydrogel powder;

[0011] Step 3: Preparation of hemostatic and antibacterial hydrogel powder

[0012] The polymer hydrogel powder obtained in step 2 was mixed with CaCO 3 After the powders are mixed, they are ball-milled and passed through a 300-mesh sieve to obtain hemostatic and antibacterial hydrogel powder.

[0013] Further, in step 1, the molar ratio of citric acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 2-3:7-9:4-5;

[0014] Furthermore, in step 2, the mass ratio of chitosan, dopamine and modified citric acid is 2:1 to 2:1 to 2. In the process of synthesizing the polymer hydrogel powder, adjusting the mass ratio of chitosan to dopamine and / or modified citric acid can adjust the adhesion properties of the obtained polymer hydrogel powder.

[0015] Further, in step 3, CaCO 3 The powder accounts for 5%-20% of the mass of the polymer hydrogel powder.

[0016] The hemostatic antibacterial hydrogel powder of the present invention has the function of hemostasis and / or promoting wound healing. The mechanism is as follows: N-hydroxysuccinimide (NHS) is grafted onto citric acid and then combined with chitosan and dopamine. The introduction of NHS and dopamine provides adhesion properties for the polymer hydrogel system, so that when applied to the bleeding site, it can quickly absorb blood, self-crosslink in situ to form an adhesive hydrogel, adhere to the wound site, and promote hemostasis on the wound surface. When the hemostatic antibacterial hydrogel powder contacts the blood at the wound site, citric acid and acetic acid provide an acidic environment for calcium carbonate, promote the dissolution of calcium carbonate, and the released carbon dioxide bubbles drive the surrounding materials into the deep wound tissue, while further releasing the coagulation factor Ca 2+, promoting the coagulation cascade inside the wound, thereby achieving the purpose of internal and external dual-effect hemostasis. The present invention starts from these two aspects to obtain a hydrogel powder with adhesion and self-propelling properties. The powder can quickly gel under the triggering of liquid, quickly form high shear strength and high toughness adhesion with the wound tissue, seal the wound, complete hemostasis, and prevent bacterial infection and oxidative stress in the wound, which is beneficial to wound healing in the later stage of hemostasis.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. The polymer hydrogel powder of the present invention improves the adhesion performance of the hydrogel system through amide reaction and hydrogen bonding force, and has high shear strength and interface toughness. Through tissue adhesive shear lap test and peel test, it can form good adhesion with wet tissue in a short time (shear strength>9kPa, interface toughness can be up to more than 30J / m 2 ), and can adhere to irregular surgical wounds with a large amount of tissue and blood, quickly promoting hemostasis on the wound surface.

[0019] 2. The addition of calcium carbonate to the hemostatic antibacterial hydrogel powder of the present invention provides the possibility of promoting deep hemostasis of wounds. The inherent acidity of the hydrogel itself provides dissolution conditions for calcium carbonate, and the generation and separation of bubbles drive the material into the wound, while releasing Ca 2+ , promoting internal hemostasis of the wound.

[0020] 3. The hemostatic and antibacterial hydrogel powder of the present invention requires liquid contact to trigger an adhesion effect and will not adhere to any object in a dry environment.

[0021] 4. The hemostatic and antibacterial hydrogel powder of the present invention has good emergency hemostatic performance. In the liver scratch model of SD rats, the time for the hemostatic and antibacterial hydrogel powder to achieve effective hemostasis by covering the wound surface (about 38 seconds) is much shorter than the hemostasis time of the commercially available product Ppg (about 153 seconds).

[0022] 5. The hemostatic and antibacterial hydrogel powder of the present invention has simple preparation conditions and is extremely convenient to use, store and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 CPCN@CaCO in the embodiment 3 Schematic diagram of the structure and formation of hemostatic antibacterial hydrogel powder.

[0024] Figure 2 The CaCO obtained in the example 3 SEM images of powders before and after grinding.

[0025] Figure 3is the interfacial toughness of CPCN hydrogel powder prepared at different mass ratios of chitosan and dopamine in the example ( Figure 3 (a)) and shear strength ( Figure 3 (b))Data graph.

[0026] Figure 4 is the interfacial toughness of CPCN hydrogel powder prepared at different mass ratios of chitosan and modified citric acid in the example ( Figure 4 (a)) and shear strength ( Figure 4 (b))Data graph.

[0027] Figure 5 CPCN@CaCO obtained with different calcium carbonate addition amounts in the examples 3 Interfacial toughness of hydrogel powders ( Figure 5 (a)) and shear strength ( Figure 5 (b))Data graph.

[0028] Figure 6 CPCN@CaCO obtained with different calcium carbonate addition amounts in the examples 3 Comparison of bubble generation of hydrogel powders.

[0029] Figure 7 CPCN hydrogel powder prepared in the example and CPCN@CaCO 3 SEM images of hydrogel powder.

[0030] Figure 8 CPCN@CaCO prepared in the example 3 The morphological photos of hydrogel powder before and after adding hydrogel, Figure 8 (a) before adding water, Figure 8 (b) After adding water.

[0031] Fig. 9 CPCN@CaCO prepared in the example at different tilt angles 3 Data graph of the migration distance of hydrogel powder in blood and PBS.

[0032] Fig.10 The blood coagulation photos of different experimental groups in the in vitro hemostasis test of the embodiment ( Fig.10 (a)) and coagulation time ( Fig.10 (b)).

[0033] Fig.11 is the coagulation time of different experimental groups in the in vivo hemostasis test of the embodiment ( Fig.11 (a)) and bleeding volume statistics ( Fig.11 (b)).

[0034] Fig.12These are photographs of blood loss on sterile filter paper of different experimental groups in the in vivo hemostasis test of the example.

[0035] Fig.13 The bacterial growth conditions of different groups in the antibacterial performance test of the embodiment ( Fig.13 (a)) and bacterial survival rate ( Fig.13 (b)).

[0036] Fig.14 The wound healing conditions of different groups in the wound healing test of MRSA infection in the embodiment ( Fig.14 (a)) and the changes in mouse body weight ( Fig.14 (b)). DETAILED DESCRIPTION

[0037] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0038] Example 1

[0039] like Figure 1 As shown, this embodiment prepares the hemostatic antibacterial hydrogel powder with adhesion and self-propelling properties according to the following steps:

[0040] Step 1: Preparation of modified citric acid

[0041] 2.6 mmol of citric acid was added to 15 mL of DMF and stirred until dissolved; 8.3 mmol of N-hydroxysuccinimide (NHS) and 4.6 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) powder were added to the system, stirred at room temperature for 24 h, and then DMF was removed by rotary evaporation to obtain modified citric acid (CA-NHS).

[0042] Step 2: Preparation of polymer hydrogel powder

[0043] 0.5 g chitosan (CS) was weighed and dissolved in 20 mL 0.1 M acetic acid solution, 0.25 g dopamine (DA) was added, and the mixture was stirred at 60° C. for 5 hours. Then 0.25 g CA-NHS was added and the mixture was stirred continuously to form a hydrogel. The polymer hydrogel powder (CPCN) was obtained by freeze drying and ball mill grinding.

[0044] Step 3: Preparation of calcium carbonate powder

[0045] Weigh 345mgNa 2 CO 3 and 485 mg CaCl 2 ·2H 2O were dissolved in 10 mL of deionized water, and then Na 2 CO 3 The solution was added to the rapidly stirred CaCl 2 The solution was stirred for 10 minutes and then centrifuged for washing (centrifugation at 3000 rpm for 5 minutes each time, the supernatant was discarded, the lower precipitate was obtained, and the washing was repeated 3 to 5 times), and then freeze-dried to obtain the precipitated CaCO 3 Precursor; then ground by ball mill (60W power operation 240s, interruption 60s, repeated 4 times), such as Figure 2 As shown, CaCO 3 The CaCO particles change from larger to smaller particles. 3 powder.

[0046] Step 4: Preparation of hemostatic and antibacterial hydrogel powder

[0047] 100 mg of the polymer hydrogel powder obtained in step 2 was mixed with 10 mg of CaCO 3 After the powders were mixed, they were ball-milled and sieved through a 300-mesh sieve to obtain the hemostatic and antibacterial hydrogel powder (CPCN@CaCO 3 ).

[0048] The obtained hemostatic antibacterial hydrogel powder was subjected to the following performance tests:

[0049] 1. Adhesion performance

[0050] The tensile load of the hydrogel powder obtained under the lap shear state was determined using the standard test method ASTM F2255-2005 (2015) for the strength characteristics of tissue adhesives: the fresh pig skin graft preparation procedure specified in the pharmaceutical industry standard YY / T 0729.1-2009 was used, the pig skin was 20 mm long, 10 mm wide, and 2 mm thick, and the hydrogel powder was fixed in two pieces of pig skin. The test was carried out using an Instron model 5943 universal testing machine, the measurement temperature was 25°C, the shear tensile speed was 5 mm / min, and each group of samples was measured at least three times, and the corresponding F tensile load was recorded, and the data results of shear strength and interface toughness were statistically analyzed.

[0051] (1) To compare the effects of different mass ratios of chitosan to dopamine and modified citric acid on the adhesion properties of the obtained polymer hydrogel powders, the amount of chitosan in step 2 was kept unchanged, and the mass of dopamine or CA-NHS was adjusted to obtain polymer hydrogel powders with different chitosan / dopamine and chitosan / modified citric acid mass ratios. The data results of the shear strength and interface toughness are shown in Figure 2. Figure 3 and Figure 4As shown, it can be seen that the optimal mass ratios of chitosan / dopamine and chitosan / modified citric acid when synthesizing polymer hydrogel powder are both 2:1.

[0052] (2) To compare polymer hydrogel powder and CaCO 3 Effect of different mass ratios of powder on the adhesion properties of hemostatic antibacterial hydrogel powder. Keep the amount of polymer hydrogel powder in step 4 unchanged and adjust the CaCO 3 The mass of powder obtained CaCO 3 The shear strength and interface toughness data of samples with different mass percentages of powder in polymer hydrogel powder are as follows: Figure 5 As shown in the figure, it can be seen that CPCN and CaCO 3 Different mass ratios have a great influence on the adhesion properties of the samples. 3 As the content of CaCO increases, the shear strength and interface toughness of the hydrogel powder will gradually decrease. 3 When the addition amount is 10%, the shear strength and interface toughness of the mixed hydrogel powder are the largest. Then, by adding the hydrogel powder into water and observing the bubble generation, the optimal proportion of calcium carbonate is further evaluated based on the amount of bubbles generated by different calcium carbonate addition amounts. Figure 6 As shown in the figure, when the calcium carbonate content is 20%, the most bubbles are generated, and then as the calcium carbonate content increases, the amount of bubbles generated decreases. When the calcium carbonate content is 10%, the difference between the bubbles generated and the content of 20% is small. Combining the shear strength, interface toughness and bubble generation data, when CaCO 3 When the incorporation amount is 10%, the overall performance of the mixed hydrogel powder is the best.

[0053] 2. Appearance

[0054] Figure 7 The polymer hydrogel powder CPCN and the hemostatic antibacterial hydrogel powder CPCN@CaCO obtained in step 2 and step 4 3 The SEM image shows the successful introduction of calcium carbonate.

[0055] The hemostatic antibacterial hydrogel powder CPCN@CaCO obtained in step 4 3 (The photos are as follows Figure 8 (a)) by adding water, the powder can quickly trigger the formation of gel (the photo of which is shown in Figure 8 (as shown in (b)).

[0056] 3. Self-propulsion performance

[0057] 30 mg CPCN@CaCO 3The powder material was placed in a plastic tube with a diameter of 1 to 2 mm, one end of which was blocked. The tube was then placed horizontally and 500 μL of PBS buffer or rabbit blood was injected into the tube to make the material contact with the liquid. The plastic tube was then tilted at different angles (0° for horizontal and 90° for vertical). The CPCN@CaCO 3 The distance the powder moves in the liquid is used to evaluate its self-propulsion performance. Fig. 9 As shown, it can be seen that CPCN@CaCO 3 The powder has excellent self-propelling properties.

[0058] 4. In vitro hemostatic performance

[0059] (1) Extracorporeal hemostasis

[0060] The coagulation time of whole blood (rabbit blood) in 96-well plates was monitored to evaluate the effects of blank group, gauze group, Ppg group, CaCO 3 Powder group, CPCN powder group, and CPCN@CaCO 3 The in vitro coagulation ability of the powder group was tested in 6 experimental groups. The specific method was as follows: 10 mg of different materials were added to a 96-well plate, and then 100 μL of whole blood was added, and coagulation photos were taken (e.g. Fig.10 (a)) and recorded the coagulation time of each group (as shown in Fig.10 (b)). It can be seen that CPCN@CaCO 3 The powder can stop bleeding quickly.

[0061] (2) Intracorporeal hemostasis

[0062] The liver perforation hemostasis model of SD rats was established: blank group, gauze group, Ppg group, CaCO 3 Powder group, CPCN powder group, CPCN@CaCO 3 There were 6 experimental groups in total in the powder group. All experimental groups maintained the same dosage of hemostatic materials, and 3 SD rats in each group were used as parallel controls. After the rats were anesthetized and fixed, the abdominal cavity was opened layer by layer to expose the liver, and the tissue fluid and blood on the surface of the liver were absorbed with a sterile cotton swab. Use a perforator to perforate close to the hepatic aorta. After 3 seconds of free bleeding, immediately cover the hemostatic material of the corresponding component (gauze group) or apply it to the surface of the rat liver wound, and lay sterile filter paper underneath. After starting the timing, it is necessary to confirm whether the wound is coagulated every 3 seconds, take photos and videos of the wound, and record the coagulation time (such as Fig.11 After the experiment, the sterile filter paper was photographed (as shown in Fig.12 The difference in mass before and after the sterile filter paper was used to stop bleeding was measured and the amount of bleeding in the rat liver was calculated (as shown in the figure Fig.11The experimental operation should be repeated three times or more.

[0063] It can be seen that compared with the gauze and Panax notoginseng (Ppg) hemostatic powder hemostatic materials on the market, the prepared hemostatic antibacterial hydrogel powder with adhesion and self-propelling properties has the shortest hemostatic time (about 38s) and blood loss (about 150mg), thanks to its good adhesion and self-propelling properties; at the same time, compared with CaCO 3 Powder group and CPCN powder group, CPCN@CaCO 3 The results showed that the combination of the two groups could maximize their respective functions, thereby increasing adhesion and self-propulsion properties and shortening hemostasis time.

[0064] 5. Antibacterial properties

[0065] The coating plate method was used to evaluate the blank group, gauze group, Ppg group, CaCO 3 Powder group, CPCN powder group, CPCN@CaCO 3 The antibacterial properties of the powder group against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) were tested in 6 experimental groups. The specific method was as follows: first, different hemostatic materials with the same concentration (250 μg / mL) were prepared, and the plates were coated after incubation with MRSA / E. coli at 37°C for 4 h, and then cultured in a 37°C incubator for 24 h to observe the bacterial growth conditions of different groups (e.g. Fig.13 (a)), and calculated the bacterial survival rate of each group (as shown in Fig.13 (b)). The results show that CPCN@CaCO 3 There was almost no bacterial growth on the powder group plates, and the antibacterial rates against MRSA / E.coli were both over 95%.

[0066] 6. MRSA-infected wound healing

[0067] Establishment of MRSA bacterial infection wound model: blank group, Panax notoginseng (Ppg) group, CaCO 3 Powder group, CPCN powder group, CPCN@CaCO 3 There were 5 experimental groups in total. All experimental groups maintained the same concentration of hemostatic materials. Three balb / c mice in each group were used as parallel controls. A circular wound of about 1 cm was cut on the back of the balb / c mice, and then 50 μL of MRSA bacterial solution was dripped to infect the wound. One day later, different hemostatic materials (50 μL) of the same concentration (250 μg / mL) were dripped into the wound site. The wound healing status of each group at different times was recorded by taking pictures (such as Fig.14 (a)) and the changes in mouse body weight (as shown in Fig.14(b)). The results show that CPCN@CaCO 3 The powder group had a good ability to promote the healing of MRSA-infected wounds, and the normal growth changes of the mouse weight curve indicated that CPCN@CaCO 3 The powder has good biosafety.

[0068] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a hemostatic and antibacterial hydrogel powder having adhesion and self-propelling properties, characterized in that: The steps include: Step 1: Preparation of modified citric acid Add citric acid to DMF and stir until dissolved, then add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir at room temperature for 24 to 48 hours, then remove DMF by rotary evaporation to obtain modified citric acid; Step 2: Preparation of polymer hydrogel powder Weigh chitosan and dissolve it in 0.1-0.3M acetic acid solution, add dopamine, stir at 50-70°C for 4-6 hours, then add modified citric acid, continue stirring until hydrogel is formed, freeze-dry and grind with a ball mill to obtain polymer hydrogel powder; Step 3: Preparation of hemostatic and antibacterial hydrogel powder The polymer hydrogel powder obtained in step 2 is mixed with CaCO3 powder, ball-milled and sieved to obtain hemostatic and antibacterial hydrogel powder.

2. The preparation method according to claim 1, characterized in that: The hemostatic and antibacterial hydrogel powder can be rapidly triggered to form a hydrogel when encountering liquid.

3. The preparation method according to claim 1, characterized in that: In step 1, the molar ratio of citric acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 2-3:7-9:4-5.

4. The preparation method according to claim 1, characterized in that: In step 2, the mass ratio of chitosan, dopamine and modified citric acid is 2:1-2:1-2.

5. The preparation method according to claim 1, characterized in that: In step 3, the CaCO3 powder accounts for 5%-20% of the mass of the polymer hydrogel powder.

6. A hemostatic and antibacterial hydrogel powder having adhesion and self-propelling properties obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the hemostatic and antibacterial hydrogel powder according to claim 6 in the preparation of hemostatic and / or wound healing promoting drugs.

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