Preparation method of ultrasonic-guided injectable degradable hemostatic hydrogel

Abstract

The invention discloses a preparation method of injectable hemostatic glue, which comprises the following steps: step 1, synthesizing oxidized hyaluronic acid: dissolving hyaluronic acid in distilled water, adding NaIO4, stirring, dialyzing a reaction product after the reaction is finished, and then freeze-drying to obtain the oxidized hyaluronic acid; the epsilon-polylysine and the oxidized hyaluronic acid obtained in the step 1 are dissolved in a PBS buffer solution, an oxidized hyaluronic acid solution and an epsilon-polylysine solution are mixed, the epsilon-polylysine / oxidized hyaluronic acid hydrogel can be obtained, and the pH value of the mixed solution is preferably 5.0-13.0. According to the method, the hemostatic gel solution can be injected to visceral hemorrhage and external hemorrhage, is gelled in situ at a wound, can be automatically degraded in vivo, and can be taken out without an operation. Taking a liver hemorrhagic point as an example, the hydrogel effectively solves the problem of closed bleeding of the liver. The preparation method provided by the invention is mild in condition and convenient to operate, and gel forming is easy to control.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and in particular relates to a method for preparing an injectable and degradable hemostatic hydrogel. Background Art

[0002] For liver laceration caused by accident or explosion, the skin surface is intact, but there is severe bleeding inside the liver. The bleeding wound is irregular in shape, and internal bleeding is usually incompressible. Traditional hemostatic materials such as gauze and sponges are usually not injectable and need to be pressed to stop bleeding; α-cyanoacrylate rapid medical glue is currently mainly used for liver hemostasis. Although this medical glue can be injected, it is non-degradable and needs to be removed by subsequent surgery. It will release heat when it forms gel at the bleeding site, which will cause more serious damage to the patient. Fibrin-based glue is one of the most widely used adhesives in clinical applications, but it has the risk of spreading diseases and allergic reactions. The method of administering drugs by cutting the wound on the skin surface is easy to cause secondary damage to the patient. Therefore, it is urgent to develop a new type of liver hemostatic material that is both injectable and degradable. During the period from the closure of the liver wound to the healing, the closed wound is still at risk of cracking. Therefore, it is necessary to design a tissue adhesive that can both effectively close the wound and assist in post-closure care of the wound. This tissue adhesive needs to have the functions of traditional tissue adhesives, such as sufficient bonding strength and low toxicity, and at the same time, it needs to be able to degrade when necessary to prevent new injuries caused by removal. We designed a multifunctional tissue adhesive that is injectable, biocompatible and degradable, which can achieve efficient wound closure and assist in wound care after closure.

[0003] ε-Polylysine is a natural biological metabolic product. It has good bactericidal ability and thermal stability. Hyaluronic acid is a high molecular weight anionic polysaccharide derived from the glycosaminoglycan family. As one of the main components of human connective tissue, hyaluronic acid is also involved in tissue repair. There are many modification sites for hyaluronic acid, which can be modified by grafting and connecting with reactive groups. Summary of the invention

[0004] The object of the present invention is to provide an in-situ formed adhesive hydrogel cross-linked by dynamic bonds, and to adjust the injection resistance, dispersibility, curing time, strength modulus, and tissue adhesion in a wide range by multiple dynamic bond control mechanisms, so as to achieve the excellent performance of low resistance in vitro, high dispersibility in vivo, tissue modulus matching, and high flow rate bleeding anti-scourability. The present invention selects hyaluronic acid and ε-polylysine as raw materials, introduces aldehyde groups into hyaluronic acid, and selects the Schiff base bond (imine bond) between the aldehyde group and the amino group as the dynamic bond of the cross-linked hydrogel. The present invention uses ε-polylysine (PL) and oxidized hyaluronic acid (OHA) as model molecules to construct a hydrogel system cross-linked by dynamic bonds. Both ε-polylysine (PL) and oxidized hyaluronic acid (OHA) have excellent biocompatibility and degradability. We designed this dynamic cross-linked structure to give the hydrogel adhesive better mechanical strength and bonding strength while maintaining its injectable and degradable capabilities, while also having good antibacterial capabilities. Therefore, the hydrogel adhesive can exhibit the expected multifunctionality, can efficiently close closed liver wounds and assist in promoting healing after wound closure.

[0005] The present invention creates a closed liver bleeding model for a 50kg experimental pig to simulate the closed bleeding of the human liver. First, the rotary cutting gun is penetrated from the surface of the experimental pig to the liver through ultrasonic positioning, and a suitable liver modeling position is found from the ultrasonic image, and then rotary cutting is performed. The same position is rotary cut twice in different directions, and the rotary cutting diameter is 5.7mm and the rotary cutting length is 2cm. Then the microblood flow is switched to observe obvious active bleeding of the liver. After 30s, the 20cm long 18G puncture needle is moved to the bleeding site of the liver through ultrasonic positioning, and the bleeding is observed through the microblood flow after the hemostatic glue is injected. The results show that the adhesive hydrogel formed in situ by dynamic bond cross-linking of the present invention can efficiently close the closed liver wound and assist in promoting healing after wound closure, achieving the excellent performance of low resistance in vitro, high diffusivity in vivo, tissue modulus matching and high flow rate bleeding anti-scouring.

[0006] In order to achieve the above object, the present invention provides a preparation method based on hemostatic glue, comprising the following steps:

[0007] Step 1: Synthesis of oxidized hyaluronic acid (OHA): dissolve hyaluronic acid (HA) in distilled water, add NaIO4 and stir, dialyze the reaction product after the reaction is completed, and then freeze-dry to obtain OHA;

[0008] Step 2: Synthesis of PL / OHA hydrogel: ε-polylysine (PL) and OHA obtained in step 1 are dissolved in PBS buffer respectively, and the OHA solution is mixed with the PL solution to obtain ε-polylysine / oxidized hyaluronic acid (PL / OHA) cross-linked hydrogel adhesive, i.e., ε-polylysine / oxidized hyaluronic acid hydrogel (PL / OHA hydrogel). Furthermore, before the OHA solution and the PL solution are mixed, the pH of the ε-polylysine solution and / or the oxidized hyaluronic acid solution is adjusted so that the pH of the solution after the ε-polylysine solution and the oxidized hyaluronic acid solution are mixed is 5 to 13, preferably 5 to 9, preferably 9 to 13, preferably pH 7.0 to 8.5, and the preferred pH adjustment range is 5.0, 5.5, 6.0, 6.5, 7.0, 7.3, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.3, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0 and ranges therebetween.

[0009] Preferably, before the OHA solution and the PL solution are mixed, the pH of the PL solution is adjusted to 10-14, preferably 10-12, 12-13, 13-14, and more preferably 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 and ranges therebetween.

[0010] Preferably, in any of the above items, before the OHA solution is mixed with the PL solution, the pH of the OHA solution is adjusted to less than 9, more preferably the pH of the OHA solution is adjusted to 7.5-8, and even more preferably the pH is 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 and ranges therebetween.

[0011] Any of the above items is preferably that before the OHA solution and the PL solution are mixed, the pH of the PL solution is adjusted to 12-13, and the pH of the OHA solution is adjusted to 7.5-8. Preferably, NaON is used to adjust the pH of the OHA solution and the PL solution. After the pH is adjusted, the OHA solution and the PL solution are mixed to obtain a mixed solution with a pH of 5.0-13.0 (further preferably 5-9, 9-13, 7.0-8.5); further, the pH of the mixed solution is preferably 7.5-8. In the technical scheme of the present invention, considering the characteristics of the aldehyde group of OHA being easily denatured under high alkalinity, in order to ensure the success of gelation, the present invention preferably controls the pH of the OHA solution to be below 9, and the more preferred range is pH 7.5-8. Under this premise, since the amino group of the PL solution will not be denatured under high alkalinity, in order to ensure that the pH of the mixed solution is 5.0-13.0 (further preferably 5-9, 9-13, 7.0-8.5), therefore, the preferred pH range of the PL solution is 12-13.

[0012] Any of the above is preferably that in step 1, the mass ratio of HA:NaIO4 is 8:4 to 8:9. More preferably, it is 8:4, 8:5, 8:6, 8:7, 8:8, 8:9 and the range therebetween.

[0013] Any of the above is preferably that in step 1, the reaction time of hyaluronic acid (HA) and NaIO4 is 0 to 72 hours, more preferably 0 to 24 hours, and preferably 0, 5, 10, 15, 20, 24, 36, 48, 72 hours and ranges therebetween.

[0014] Any of the above is preferably that in step 1, after the reaction is completed, the reaction product is placed in a dialysis bag, and the molecular weight cutoff of the dialysis bag is 1000 to 100000, preferably 1000 to 14000. Preferably, the molecular weight cutoff of the dialysis bag is 1000, 5000, 9000, 14000, 15000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 and the range therebetween.

[0015] Preferably, in any of the above items, in step 2, the mass ratio of PL:OHA is 1:1 to 1:2.5, and more preferably 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5 and ranges therebetween.

[0016] The HA or PL selected in the present invention is a conventional product in the prior art, and any molecular weight of HA or PL is suitable for the present invention. Preferably, the molecular weight of HA is preferably 50,000 to 500,000, and the molecular weight of PL is preferably 3,500 to 4,000. Further preferably, the molecular weight of OHA is 50,000, 100,000, 200,000, 300,000, 400,000, 500,000 and the range therebetween; the molecular weight of PL is preferably 3,500, 3,600, 3,700, 3,800, 3,900, 4,000 and the range therebetween.

[0017] The present invention does not need to add a crosslinking agent, but promotes gelation by adjusting the pH of the solution. The preferred method of adjusting the pH is to add a NaOH solution.

[0018] Any of the above is preferably that in step 2, the pH is adjusted using 1-20 mol / L NaOH, preferably 2-5 mol / L, preferably 1, 2, 3, 4, 5, 8, 10, 12, 15, 18, 20 mol / L and ranges therebetween.

[0019] The present invention achieves control of the gelling time by adjusting the amount of NaOH added, that is, by controlling the pH of the final solution.

[0020] Preferably, the gelling time is controlled by adjusting the pH of the mixed solution to meet the needs of different treatment scenarios. Preferably, the method of adjusting the pH of the mixed solution includes: directly adjusting the pH of the mixed solution, or adjusting the pH of the OHA solution or PL solution before mixing.

[0021] Preferably, in any of the above items, in step 2, the gelation time (i.e., gelation time) of the obtained PL / OHA hydrogel is 0 to 1 h. Preferably, the pH of the solution after the PL solution and the OHA solution are mixed is adjusted to 5 to 13, so that the gelation time of the obtained PL / OHA hydrogel is 0 to 1 h. Preferably, in any of the above items, the gelation time is 10 to 180 s. Further preferably, the final pH of the solution after the PL solution and the OHA solution are mixed is adjusted to 7 to 8.5.

[0022] Preferably, any of the above items has a gelling time of 10s to 40s. Further preferably, the pH of the solution after mixing is adjusted to 7 to 8.

[0023] Preferably, in any of the above items, the gelling time is 10s to 20s. Preferably, the pH of the solution after mixing is 7.5 to 8.

[0024] Any of the above items is preferably adjusted to adjust the pH of the solution before mixing so that the pH of the solution after mixing is 7-8.5 and the gelation time is 10-180s. The preferred scheme is that the pH of the PL solution is adjusted to 12-13, the pH of the OHA solution is preferably 6-7, and the gelation time is 3-10min; the pH of the OHA solution is preferably 7-8, and the gelation time is 1-3min; the pH of the OHA solution is preferably 8-9 and the gelation time is 1-60s.

[0025] In a preferred embodiment of the present invention, oxidized hyaluronic acid OHA is synthesized according to the following method:

[0026] Weigh 8.0 g of HA and dissolve it in 400 mL of distilled water, and stir for 4 hours to completely dissolve it. Then, weigh 5 to 8 g of NaIO4 (more preferably 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g or 8 g of NaIO4) and dissolve it in the reaction system, stir continuously, and react for 24 hours. After the reaction is completed, the reaction product is placed in a dialysis bag and dialyzed against distilled water for 72 hours, and then OHA is obtained by freeze drying.

[0027] In a preferred embodiment of the present invention, PL / OHA hydrogel is synthesized according to the following method:

[0028] 180-200 mg of PL was weighed and dissolved in 200 μL of 20 mM PBS buffer (pH = 7.2-7.4), and then 60-85 μL of sodium hydroxide solution was added thereto to obtain a PL solution (pH = 12-13). 200-400 mg of OHA was dissolved in 1 mL of 20 mM PBS buffer (pH = 7.2-7.4). The pH of the OHA solution was then adjusted to 7.5-8 by sodium hydroxide solution. Finally, the OHA solution (pH = 7.5-8) was mixed with the PL solution (pH = 12-13) to obtain a PL / OHA cross-linked hydrogel adhesive.

[0029] In another preferred embodiment of the present invention, PL / OHA hydrogel is synthesized according to the following method:

[0030] 180-200 mg of PL was weighed and dissolved in 200 μL of 20 mM PBS buffer (pH = 7.2-7.4), and then 70-85 μL of sodium hydroxide was added thereto to obtain a PL solution (pH = 12-13). 200-400 mg of OHA was dissolved in 1 mL of 20 mM PBS buffer (pH = 7.2-7.4). The pH of the OHA solution was then adjusted to 7.5-8 by NaOH solution. Finally, the OHA solution (pH = 7.5-8) was mixed with the PL solution (pH = 12-13) to obtain a PL / OHA cross-linked hydrogel adhesive.

[0031] The present invention also provides an ultrasound-guided injectable hemostatic hydrogel prepared by any of the preparation methods described above, namely, a PL / OHA hydrogel.

[0032] Preferably, the present invention provides an ultrasound-guided injectable wound-sealing hemostatic hydrogel prepared by any of the preparation methods described above, namely, a PL / OHA hydrogel.

[0033] The present invention also provides the use of any of the above-mentioned preparation methods in the preparation of ultrasound-guided injectable hemostatic drugs.

[0034] Preferably, the present invention provides the use of any of the above-mentioned preparation methods in the preparation of ultrasound-guided injectable wound-sealing hemostatic drugs.

[0035] The present invention also provides a double-channel syringe for use with any of the above-mentioned PL / OHA hydrogels.

[0036] The present invention also provides a syringe for use with the hemostatic hydrogel, wherein the syringe is a double-channel syringe, comprising a first sleeve, a second sleeve, a first push rod, a second push rod and a syringe connector; the first push rod and the second push rod are connected; the syringe connector is a three-way structure, comprising a first interface, a second interface and a third interface; the first interface and the second interface of the syringe connector are respectively connected to the first sleeve and the second sleeve, and the third interface of the syringe connector is connected to an injection needle.

[0037] Preferably, the first sleeve is connected to the second sleeve.

[0038] Further preferably, a diameter ratio of the first sleeve to the second sleeve is 1-2:1.

[0039] Further preferably, a diameter ratio of the first sleeve to the second sleeve is 1.5:1.

[0040] More preferably, the diameter of the first sleeve is 12-17 mm, and the diameter of the second sleeve is 7-13 mm.

[0041] Further preferably, the length of the first sleeve or the second sleeve is 50-55 mm.

[0042] Further preferably, the inner diameter of the gel outlet of the first sleeve or the second sleeve is 2 mm.

[0043] Further preferably, the inner diameters of the first interface and the second interface are the same as the outer diameter of the gel outlet of the first sleeve or the second sleeve, both of which are 4 mm.

[0044] Further preferably, the inner diameter of the third interface is 2 mm.

[0045] The invention provides a method for preparing an injectable hemostatic glue, which can effectively solve the problem of organ bleeding and external bleeding, and is degradable, especially solving the problem of closed bleeding in the liver, and provides a new method for liver hemostasis. The preparation method of the invention obtains a degradable hydrogel that can be autonomously degraded in the body without surgical removal.

[0046] The hemostatic hydrogel of the invention has mild preparation conditions, is easy to operate, and has easy control of gelation. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The figure is a schematic diagram of the synthesis route of PL / OHA self-healing hydrogel in preferred embodiment 1 of the present invention.

[0048] Figure 2Fourier transform infrared spectra of PL, OHA and PL / OHA hydrogels in preferred embodiment 1 of the present invention.

[0049] Figure 3 The NMR spectra of PL, OHA and PL / OHA hydrogel in the preferred embodiment 1 of the present invention are shown.

[0050] Figure 4 This is the rheological frequency spectrum of the PL / OHA hydrogel in the preferred embodiment 3 of the present invention.

[0051] Figure 5 This is the rheological time spectrum of the PL / OHA hydrogel in the preferred embodiment 3 of the present invention.

[0052] Figure 6 It is a schematic diagram of the assembly of the PL solution and the OHA solution in the double-channel syringe shown in the preferred embodiment 1 of the present invention.

[0053] Figure 7 This is an assembly diagram of a double-channel syringe in preferred embodiment 2 of the present invention.

[0054] Figure 8 This is a diagram of PL / OHA hydrogel injection in preferred embodiment 2 of the present invention.

[0055] Fig. 9 This is the gelation diagram of PL / OHA hydrogel in preferred embodiment 2 of the present invention.

[0056] Fig.10 This is a portable ultrasound comparison chart in preferred embodiment 4 of the present invention.

[0057] Fig.11 This is a comparison diagram of microblood flow in preferred embodiment 4 of the present invention.

[0058] Fig.12 This is the bleeding surface of the liver after dissection in the preferred embodiment 4 of the present invention.

[0059] Fig.13 The gelation conditions of different sodium hydroxide addition amounts in preferred embodiment 5 of the present invention are shown.

[0060] Fig.14 It is a side view of the first sleeve and the second sleeve in preferred embodiment 6 of the present invention.

[0061] Fig.15 This is a side view of the first push rod and the second push rod in preferred embodiment 6 of the present invention.

[0062] Fig.16 It is a side view of the syringe connector described in the preferred embodiment 6 of the present invention. DETAILED DESCRIPTION

[0063] The present invention is further described in detail below through the accompanying drawings and specific embodiments.

[0064] Implementation Case 1

[0065] like Figure 1 The following is a schematic diagram of the synthesis route of the PL / OHA self-healing hydrogel of the present invention. The specific method is:

[0066] Step 1: Synthesis of OHA:

[0067] Weigh 8.0g HA and dissolve it in 400mL distilled water, stir for 4h to completely dissolve it. Then, weigh 5-8g NaIO4 (preferably, weigh 6.5g NaIO4) and dissolve it in the reaction system, stir continuously, and react for 24h. After the reaction is completed, place the reaction product in a dialysis bag and dialyze it against distilled water for 72h, and then obtain OHA by freeze drying. The dialysis bag is preferably MWCO: 8-14kDa.

[0068] Step 2: Assemble the two-channel gel:

[0069] 2 g of PL was weighed and dissolved in 2 mL of 20 mM PBS (pH = 7.2-7.4) solution, and then an appropriate amount of 4 M sodium hydroxide was added thereto to adjust the pH to 12-13.

[0070] 1.775 g of OHA was dissolved in 5 mL of 20 mM PBS buffer (pH=7.2-7.4), and the pH of the OHA solution was then adjusted to 7.5-8 by NaOH solution (4 M).

[0071] The PL solution and OHA solution were assembled in Figure 6 In the double-channel syringe shown, 1.5 mL of the formed PL solution is transferred to the small channel of the double-channel syringe, and 4.1 mL of the OHA solution is transferred to the large channel of the double-channel syringe.

[0072] Step 3: Synthesis of PL / OHA hydrogel:

[0073] The push rod of the double-channel syringe is pushed to extrude the PL solution and the OHA solution through the syringe needle in proportion (preferably, the mass ratio of PL:OHA in the mixed solution injected into the needle is 1:1 to 1:2, preferably, the mass ratio of PL:OHA is 1:1, 1:1.2, 1:1.5, 1:1.8) to form a PL / OHA hydrogel.

[0074] The obtained hydrogel is subjected to property testing, wherein:

[0075] Figure 2 Shown are Fourier transform infrared spectra of PL, OHA, and PL / OHA hydrogels; Figure 3 NMR spectra of PL, OHA and PL / OHA hydrogels.

[0076] Example 2

[0077] The dual-channel gel was prepared and assembled according to the method provided in Example 1, and the injectability and dispersibility were tested.

[0078] Assemble the double-channel syringe with an 18G puncture needle (such as Figure 7 As shown), the injectability of the PL / OHA hydrogel adhesive is tested by pushing the piston at a speed of 3-4 mL / min (preferably, the injectability of the PL / OHA hydrogel adhesive is tested by pushing the piston at a speed of 3.2 mL / min, 3.4 mL / min, 3.6 mL / min or 3.8 mL / min). Figure 8 It can be seen that the PL / OHA mixed solution after the double-channel components converged can be injected through a 20 cm long 18G medical puncture needle, indicating that the PL / OHA hydrogel has excellent injectability and can gel in an irregular mold within 1 minute after the PL / OHA mixed solution is injected (such as Fig. 9 As shown), it shows that PL / OHA hydrogel has a good solidification speed, can provide a suitable injection time and good gelation and dispersibility, and the gel strength after gelation meets the hemostatic strength requirements and meets the requirements of non-traumatic liver hemostasis.

[0079] Example 3

[0080] Rheological properties test of PL / OHA hydrogel.

[0081] To test the frequency spectrum of PL / OHA hydrogel, 0.1928g polylysine was weighed and added to 193μL 20mM PBS buffer, and then an appropriate amount of 4M sodium hydroxide solution was added to adjust the pH to 12-13, and vortexed to a transparent solution; 0.355g oxidized hyaluronic acid was dissolved in 1mL 20mM PBS, and then the pH was titrated to 7.5 with 4M NaOH solution, and transferred to the cavity of the syringe, the needle model of the syringe was 18G, and the hydrogel was pushed onto the rheometer platform. The frequency spectrum frequency range was set to 0.1-10Hz, and the time spectrum time was set to 0-6min, and the injection-molded hydrogel modulus was tested. The results are as follows: Figure 4 , 5 shown.

[0082] Example 4

[0083] 1) Synthesis of OHA:

[0084] 8.0 g HA was weighed and dissolved in 400 mL distilled water, and stirred for 4 h to completely dissolve. Subsequently, 5-8 g NaIO4 (preferably, 7 g NaIO4) was weighed and dissolved in the reaction system, and the mixture was stirred continuously for 24 h. After the reaction, the reaction product was placed in a dialysis bag and dialyzed against distilled water for 72 h, and then OHA was obtained by freeze drying.

[0085] 2) Assemble the dual-channel gel:

[0086] 2 g of PL was weighed and dissolved in 2 mL of 20 mM PBS (pH = 7.2-7.4) solution, and then an appropriate amount of 4 M sodium hydroxide was added thereto to adjust the pH to 12-13. 1.775 g of OHA was dissolved in 5 mL of 20 mM PBS buffer (20 mM, pH = 7.2-7.4). The pH of the OHA solution was then adjusted to 7.5-8 by NaOH solution (4 M). 2 mL of the formed PL solution was transferred to the small channel of the double-channel syringe, and 5.47 mL of the OHA solution was transferred to the large channel of the double-channel syringe.

[0087] 3) Hemostatic performance test:

[0088] In order to verify that PL / OHA hydrogel has good injectability and liver hemostasis performance, pigs weighing about 50kg were used as experimental subjects to create a liver closed penetrating injury bleeding model. First, the pigs were anesthetized, and the internal liver position of the pigs was found using portable ultrasound. Then, under the guidance of portable ultrasound, a rotary cutter (diameter 5.7mm) was used to cut the pig liver, and a wound with a diameter of 5.7mm and a length of 2cm was cut at the liver position. Rotary cuts were performed twice in different directions at the same position. Pay attention to avoid the liver artery as much as possible. The bleeding points of the experimental pigs' livers were monitored in real time using portable ultrasound and micro-blood flow instruments throughout the process. The rotary cutter was pulled out in time after rotary cutting. After the experimental pigs' livers were bleeding for about 10 to 20 seconds, a 20cm long 18G medical puncture needle was positioned to the bleeding site of the liver under ultrasound guidance, and then PL / OHA hydrogel was injected. The portable ultrasound ( Fig.10 ) and micro-blood flow ( Fig.11 ) and anatomical diagrams ( Fig.12 ) analysis showed that PL / OHA hydrogel has excellent hemostatic properties.

[0089] Implementation Case 5

[0090] 1) Synthesis of OHA:

[0091] 8.0 g HA was weighed and dissolved in 400 mL distilled water, and stirred for 4 h to completely dissolve. Subsequently, 5-8 g NaIO4 (preferably, 6 g NaIO4) was weighed and dissolved in the reaction system, and the mixture was stirred continuously for 24 h. After the reaction, the reaction product was placed in a dialysis bag and dialyzed against distilled water for 72 h, and then OHA was obtained by freeze drying.

[0092] 2) Determination of gelation time with different amounts of NaOH added:

[0093] Weigh 2g PL and dissolve it in 2mL 20mM PBS (pH=7.2-7.4) solution. Dissolve 1.775g OHA in 5mL 20mM PBS buffer (pH=7.2-7.4). Then adjust the pH of the OHA solution to 5-6 with NaOH solution (4M). Transfer 300μL of the formed PL solution to a centrifuge tube containing 1mL OHA solution, add different amounts of NaOH and vortex for 20s to observe the gel formation. From Table 1 and Fig.13 It can be seen that with the increase of NaOH addition, the solution pH gradually increases and the gelation time gradually shortens. The gelation time is the shortest when the pH of the mixed solution is between 7.5 and 8.5, which is only 10 to 20 seconds. As the amount of NaOH added increases, the gelation time gradually increases, proving that the gelation time can be controlled by adjusting the amount of NaOH added.

[0094] Table 1:

[0095]

[0096] Note: The pH range endpoint values ​​described in the table are inclusive at the left endpoint and exclusive at the right endpoint.

[0097] According to Example 5, when the final pH of the solution after the PL solution and the OHA solution are mixed is preferably 7-8.5, the gelation time is controlled within 1 min; more preferably, the gelation time is 10s-40s, and the preferred pH of the mixed solution is 7-8; more preferably, the gelation time is 10s-20s, and the preferred pH of the mixed solution is 7.5-8.

[0098] Example 6

[0099] Example 6 provides a syringe applied to the present invention, which is a double-channel syringe, including a first sleeve, a second sleeve, a first push rod, a second push rod and a syringe connector; the first push rod is connected to the second push rod; the syringe connector is a three-way structure, including a first interface, a second interface and a third interface; the first interface and the second interface of the syringe connector are respectively connected to the first sleeve and the second sleeve, and the third interface of the syringe connector is connected to an injection needle.

[0100] Fig.14 It is a side view of the first sleeve and the second sleeve.

[0101] Preferably, the first sleeve is connected to the second sleeve.

[0102] Further preferably, a diameter ratio of the first sleeve to the second sleeve is 1-2:1.

[0103] Further preferably, a diameter ratio of the first sleeve to the second sleeve is 1.5:1.

[0104] Further preferably, the diameter of the first sleeve is 14.94 mm, and the diameter of the second sleeve is 9.36 mm.

[0105] Further preferably, the length of the first sleeve or the second sleeve is 52.5 mm.

[0106] Further preferably, the inner diameter of the gel outlet of the first sleeve or the second sleeve is 2 mm.

[0107] Fig.15 It is a side view of the first push rod and the second push rod.

[0108] Fig.16 A side view of the syringe connector.

[0109] Preferably, the inner diameters of the first interface and the second interface are the same as the outer diameter of the gel outlet of the first sleeve or the second sleeve, both of which are 4 mm.

[0110] Further preferably, the inner diameter of the third interface is 2 mm.

[0111] Example 7

[0112] Example 7 provides a preferred embodiment of synthesizing PL / OHA hydrogel:

[0113] 180-200 mg of PL was weighed and dissolved in 200 μL of 20 mM PBS buffer (pH = 7.2-7.4), and then 60-85 μL of sodium hydroxide solution was added thereto to obtain a PL solution (pH = 12-13). 200-400 mg of OHA was dissolved in 1 mL of 20 mM PBS buffer (pH = 7.2-7.4). The pH of the OHA solution was then adjusted to 7.5-8 by sodium hydroxide solution. Finally, the OHA solution (pH = 7.5-8) was mixed with the PL solution (pH = 12-13) to obtain a PL / OHA cross-linked hydrogel adhesive.

[0114] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing an injectable hemostatic glue, comprising the following steps: Step 1: Synthesizing oxidized hyaluronic acid: dissolving hyaluronic acid in distilled water, adding NaIO4 and stirring, dialyzing the reaction product after the reaction is completed, and then freeze-drying to obtain the oxidized hyaluronic acid; Step 2: Synthesis of ε-polylysine / oxidized hyaluronic acid hydrogel: ε-polylysine and the oxidized hyaluronic acid obtained in step 1 are dissolved in PBS buffer respectively, and the oxidized hyaluronic acid solution is mixed with the ε-polylysine solution to obtain the ε-polylysine / oxidized hyaluronic acid cross-linked hydrogel adhesive; before mixing, the pH of the ε-polylysine solution and / or the oxidized hyaluronic acid solution is adjusted so that the pH of the solution after the ε-polylysine solution and the oxidized hyaluronic acid solution are mixed to be 5.0 to 13.

0.

2. The method according to claim 1, characterized in that In step 1, the mass ratio of hyaluronic acid to NaIO4 is 8:4 to 8:

9.

3. The method according to claim 1, characterized in that In step 1, the reaction time of hyaluronic acid and NaIO4 is 0 to 72 hours.

4. The method according to claim 1, characterized in that In step 1, after the reaction is completed, the reaction product is placed in a dialysis bag, and the molecular weight cutoff of the dialysis bag is 1000 to 100000.

5. The method according to claim 2, characterized in that In step 2, after the solutions are mixed, the mass ratio of ε-polylysine to oxidized hyaluronic acid is 1:1 to 1:2.

5.

6. The method according to claim 2, characterized in that In step 2, the pH is adjusted using 1 to 20 mol / L NaOH.

7. The ultrasound-guided injectable hydrogel prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the preparation method according to any one of claims 1 to 6 in the preparation of ultrasound-guided injectable hemostatic drugs.

9. A syringe used in conjunction with the hemostatic hydrogel according to claim 7, characterized in that: It includes a first sleeve, a second sleeve, a first push rod, a second push rod and a syringe connector; the first push rod and the second push rod are connected; the syringe connector is a three-way structure, including a first interface, a second interface and a third interface; the first interface and the second interface of the syringe connector are respectively connected to the first sleeve and the second sleeve, and the third interface of the syringe connector is connected to an injection needle.

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