Fluid gelatin particles with combined size as well as preparation method and application of fluid gelatin particles

Abstract

The invention relates to fluid gelatin particles with combined sizes as well as a preparation method and application of the fluid gelatin particles. The specific preparation method of the fluid gelatin particles with the combined size comprises the following steps: carrying out physical or chemical crosslinking to obtain gelatin particles or gelatin sponge, then carrying out mechanical grinding, then sieving by using screens with different pore diameters, collecting gelatin particles with different sizes, and finally combining the particles with different sizes according to a proper and determinable volume ratio to obtain the fluid gelatin particles with the combined size. The combined size fluid gelatin particles provided by the invention are pre-swelled to obtain the hemostatic fluid gelatin. And the pre-swelled gelatin particles have good flowability and extremely low swelling property. The fluid gelatin particles with the combined size have good biocompatibility, biological activity and lacuna plugging effect, and can be used for development of fluid hemostatic materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical engineering and biomaterials, and particularly relates to a fluid gelatin particle of combined size, a preparation method and application thereof. BACKGROUND

[0002] At present, intraoperative bleeding is still a great challenge to surgeons, especially compared with open surgery, with the wide use of minimally invasive, endoscopic and surgical robots, the visual field change caused by the surgical process limits the hemostasis operation. In addition, there are complex and irregular wounds, deep interstitial bleeding, extensive bleeding wounds, especially bleeding sites involving important nerve tissue, etc. in surgical operations, making intraoperative rapid and effective hemostasis more challenging. Due to the above factors, common hemostatic materials such as gauze, hemostatic sponge, hemostatic dressing, hemostatic microspheres and hemostatic powder often cannot meet the clinical practice. For example, gauze, gelatin sponge, collagen sponge and dressing mainly stop bleeding through physical compression of the wound site, but they are not suitable for irregularly shaped wounds, deep wounds or narrow wounds, and the swelling of the liquid-absorbing materials may cause compression and occupation of important nerve tissue if not used properly. Powder-type hemostatic materials applied to diffuse wounds lack fluidity and cannot penetrate into the bleeding surface of minimally invasive surgery and deep interstitial spaces. It is worth noting that effective control of bleeding during surgery is very important in various surgical operations, and timely and effective control of intraoperative bleeding can shorten the operation time, reduce the probability of blood transfusion, and reduce the incidence of postoperative complications, so the development of rapid, effective and safe sealing hemostatic materials has extremely important significance and clinical value.

[0003] Fluid-type hemostatic materials, especially fluid gelatin materials, have certain fluidity and can be applied to target sites through a syringe, have good shape compliance for bleeding wounds, have the advantages of clinical ease of operation, small injury, fast hemostasis, etc., and are suitable for scenes where traditional hemostatic techniques are ineffective or cannot be applied. Fluid gelatin hemostatic materials not only can block and compress the bleeding wound through physical compression, but also can play a biological hemostatic function by absorbing various hemostatic factors in the body, and its swelling rate is only 20% or less of its own volume, which will not cause compression of important tissues such as nerves caused by swelling of traditional materials. At the same time, it can be uniformly mixed with thrombin in a short time to improve the hemostatic effect of fluid gelatin and reduce the time required for hemostasis. Fluid gelatin hemostatic materials have been widely used in neurosurgery, spinal surgery and minimally invasive surgery, and have achieved satisfactory clinical hemostatic effect.

[0004] Generally, fluid gelatin is produced by cross-linking gelatin sponges, then mechanically pulverizing the cross-linked sponges into particles, and finally mixing the pulverized gelatin powder with a solvent to create a fluid form. Although some fluid gelatin formulations have limited particle size, they often suffer from a wide particle size range, are frequently mixtures of multiple sizes, and have uncertain and inconsistent particle composition ratios across different size ranges. This not only causes batch-to-batch variations but also affects clinical hemostatic efficacy. Therefore, controlling the size composition of gelatin particles is crucial for achieving better clinical hemostatic effects with fluid gelatin hemostatic materials. Summary of the Invention

[0005] The purpose of this invention is to provide fluid gelatin particles of various sizes, their preparation method, and applications. Specifically, the filling of cavitary bleeding surfaces with fluid gelatin particles can be approximated as a particle filling problem in millimeter-scale curved spaces. It is well known that particles of different sizes and particle combinations have different filling effects on curved surfaces. Based on this, this application, after examining gelatin particles and particle combinations of various size ranges, provides fluid gelatin particles of various sizes with good hemostatic effects, their preparation method, and applications. Specifically, cross-linked gelatin particles or sponges obtained by physical or chemical cross-linking methods are mechanically ground, then sieved using sieves of different pore sizes to obtain gelatin particles of different size ranges. These particles are then mixed in a suitable proportion to obtain fluid gelatin particles of various sizes. The fluid gelatin particles of various sizes provided by this invention have good biocompatibility and the ability to seal and stop bleeding in curved spaces. They can be used in the development of fluid hemostatic materials and for the embolization treatment of diseases.

[0006] This invention provides fluid gelatin particles of various sizes, characterized in that these particles are composed of physically or chemically cross-linked gelatin particles. The gelatin particles can be prepared from type A or type B gelatin, and the gel strength of the gelatin is 200-300 Bloom g. The physical cross-linking is primarily thermal cross-linking at a temperature of 100-180°C, preferably under vacuum conditions. The chemical cross-linking includes, but is not limited to, cross-linking using aldehydes, genipin, and carbodiimides.

[0007] This invention provides a fluid gelatin particle with a combination of sizes, characterized in that the fluid gelatin particle with a combination of sizes is prepared by mixing gelatin particles of different size ranges. The gelatin particles of different sizes are obtained by mechanically grinding cross-linked gelatin particles or gelatin sponges and sieving them through sieves of different mesh sizes, preferably 20 mesh, 30 mesh, 40 mesh, and 60 mesh sieves. The gelatin particles of different sizes include: large particles (approximately 830-550 micrometers in diameter) collected after passing through a 20-mesh sieve and then a 30-mesh sieve; medium particles (approximately 550-380 micrometers in diameter) collected after passing through a 30-mesh sieve and then a 40-mesh sieve; and small particles (approximately 380-250 micrometers in diameter) collected after passing through a 40-mesh sieve and then a 60-mesh sieve.

[0008] This invention provides a fluid gelatin particle with a combination of sizes, characterized in that the fluid gelatin particle with a combination of sizes is prepared by mixing gelatin particles of different size ranges as described above. Preferably, the fluid gelatin particle with a combination of sizes is prepared by mixing particles of two sizes in a certain volume ratio, and preferably, the volume ratio of the larger particle to the smaller particle is 2-4:1.

[0009] The present invention provides a method for preparing fluid gelatin particles of combined sizes, characterized by comprising the following preparation process: S1. Place an appropriate amount of gelatin granule raw material in a stainless steel container and place it in a vacuum drying oven. After vacuuming, perform high-temperature crosslinking at 100-180℃ for 4-10 hours. Alternatively, the gelatin granule raw material can be suspended in a crosslinking solution and crosslinked for 8-48 hours under stirring at 60-500 rpm. The high-temperature crosslinked gelatin granules can also be further suspended in a crosslinking solution for re-crosslinking. Alternatively, gelatin granules can be fully dissolved in deionized water at a concentration of 10-20%, and then freeze-dried to obtain gelatin sponge. The obtained gelatin sponge can then be physically or chemically crosslinked as described above.

[0010] S2, the cross-linked gelatin particles or gelatin sponges obtained in S1 are mechanically ground or pulverized to obtain gelatin particles. The harvested gelatin particles are sequentially passed through 20, 30, 40 and 60 mesh sieves. The particles that do not pass through the 20 mesh sieve and then the 30 mesh sieve are collected as large-sized particles; the particles that do not pass through the 30 mesh sieve and then the 40 mesh sieve are collected as medium-sized particles; and the particles that do not pass through the 40 mesh sieve and then the 60 mesh sieve are collected as small-sized particles.

[0011] S3, the gelatin particles of different sizes obtained in S2 are physically mixed in different volume ratios to obtain fluid gelatin particles of combined sizes. The volume ratio of larger particles to smaller particles is 2-4:1.

[0012] The steps described above in the method for preparing fluid gelatin particles of combined sizes provided by the present invention can also be adjusted as follows: the obtained gelatin particle raw material is first mechanically ground, then sieved to collect particles of different sizes, then crosslinked according to the above physical or chemical methods to obtain fluid gelatin particles of different sizes, and finally physically mixed according to the above method to obtain fluid gelatin particles of combined sizes.

[0013] This invention provides a combination of sizes of fluid gelatin particles, which are cross-linked gelatin particles with a certain degree of swelling. By suspending and pre-swelling these combined-size fluid gelatin particles in a stable suspension, including water for injection, physiological saline, and phosphate buffer, a hemostatic fluid gelatin can be obtained. The pre-swelled gelatin particles retain their particle shape while ensuring the flowability of subsequent products. The swelling ratio of the particle size is less than 20% within 12 hours, which can meet the requirements for hemostasis of cavitary bleeding surfaces and avoidance of nerve compression. Specifically, this invention provides combined-size fluid gelatin particles, which can be obtained by controlling the volume ratio of gelatin particles of different sizes. This stabilizes the particle composition ratio across different size ranges, avoids batch-to-batch variations, and enhances its clinical hemostatic effect. Furthermore, by controlling the volume ratio of different size gelatin particles, an embolic agent suitable for tumor embolization therapy can be obtained. Attached Figure Description Figure 1 This figure shows the leakage results of gelatin particles of different sizes and their combinations in phenol red aqueous solution in Example 2. In the figure, 1 represents only large-sized particles; 2 represents only medium-sized particles; 3 represents only small-sized particles; 4 represents a 1:2 combination of large and medium-sized particles; 5 represents a 1:2 combination of medium and small-sized particles; 6 represents a 1:2 combination of large and medium-sized particles; 7 represents a 2:1 combination of large and medium-sized particles; 8 represents a 1:2 combination of large and small-sized particles; and 9 represents a 2:1 combination of medium and small-sized particles.

[0014] Figure 2 The image shows typical morphology (A) of microscopic photographs of particles of different sizes after 1 hour of pre-swelling and 12 hours of swelling in Example 3, as well as the volume swelling rate calculated from particles of different sizes after 12 hours of swelling. L represents large particles; M represents medium particles; and S represents small particles.

[0015] Figure 3The results of the coagulation test in Example 4 are shown below. From left to right: large particles alone (No. 1), medium particles alone (No. 2), small particles alone (No. 3), a combination of medium and small particles (2:1) (No. 4), a combination of large and small particles (1:2) (No. 5), a combination of large and medium particles (2:1) (No. 6), a combination of large and medium particles (1:2) (No. 7), a combination of large and small particles (2:1) (No. 8), and a combination of medium and small particles (1:2) (No. 9). Detailed Implementation

[0016] The present invention will now be described in further detail with reference to embodiments, so that those skilled in the art can better understand and implement the present invention.

[0017] Example 1: (1) Weigh a certain amount of type B gelatin granule raw material with a freeze strength of 250 Bloom g, dry it at 55°C, and then transfer it to a vacuum drying oven. After vacuuming, set the temperature to 150°C and crosslink it at high temperature for 5 hours. Physically crosslinked gelatin granules are obtained. (2) Transfer the gelatin granules to a ball mill cup, add an appropriate amount of grinding beads, grind at 600 rpm for 1 hour, and collect the ground gelatin granules. (3) Pass the collected gelatin granules through 20, 30, 40 and 60 mesh sieves. Collect the un-sieved particles after passing through a 20 mesh sieve and then through a 30 mesh sieve. The un-sieved particles after passing through a 30 mesh sieve and then through a 40 mesh sieve are the medium-sized particles. The un-sieved particles after passing through a 40 mesh sieve and then through a 60 mesh sieve are the small-sized particles. (4) Mix large-sized particles with medium-sized particles, large-sized particles with small-sized particles, and medium-sized particles with small-sized particles in ratios of 2:1 and 1:2 respectively to obtain fluid gelatin particles with different size combinations.

[0018] Example 2: (1) A 5 mL pipette tip was vertically fixed on a test tube rack, and a centrifuge tube was fixed below it. (2) Approximately 1.5 mL of each of the various sizes of gelatin particles and combined sizes obtained in Example 1 was taken into the 5 mL pipette tip, and 4 mL of phenol red aqueous solution was added to the tip. The volume of phenol red water collected in the centrifuge tube below was observed after adding phenol red water to different particles. As a result, within 1 minute, different sizes of particles or combined sizes of particles had different leakage effects on the phenol red solution. Approximately 2 mL of phenol red aqueous solution from large particles alone leaked into centrifuge tube (1); approximately 1 mL of phenol red aqueous solution from medium particles alone leaked into centrifuge tube (2); and approximately 0.5 mL of phenol red aqueous solution from small particles alone leaked into centrifuge tube (2). 1 mL of phenol red aqueous solution leaked into the centrifuge tube (3), but the leakage of phenol red aqueous solution gradually increased over time, reaching about 3 mL after 12 hours; about 1.5 mL of phenol red aqueous solution leaked into the centrifuge tube for the 1:2 combination of large and medium-sized particles (4); about 1 mL of phenol red aqueous solution leaked into the centrifuge tube for the 1:2 combination of medium and small-sized particles (5); about 1.5 mL of phenol red aqueous solution leaked into the centrifuge tube for the 1:2 combination of large and medium-sized particles (6); about 0.5 mL of phenol red aqueous solution leaked into the centrifuge tube for the 2:1 combination of large and medium-sized particles (7); about 1 mL of phenol red aqueous solution leaked into the centrifuge tube for the 1:2 combination of large and small-sized particles (8); and almost no phenol red aqueous solution leaked into the centrifuge tube for the 2:1 combination of medium and small-sized particles, with only some particles dripping into the centrifuge tube (9). These results indicate that particle size affects the filling and closure of curved space, with the 2:1 combination of medium and small-sized particles showing the best closure effect.

[0019] Example 3: (1) Take appropriate amounts of large, medium, and small-sized particles obtained in Example 1 and suspend them in phosphate buffer. (2) At 1, 2, 4, 6, and 12 hours after suspension, take a small amount and transfer it to a glass slide for observation and photography under a microscope. (3) Measure the size of the photographed particles using ImageJ software. Based on the average particle size after 1 hour of pre-suspension (S0), the average particle size obtained at 12 hours of suspension is S12. Calculate the particle size according to the formula... Volume swelling ratio = (S12-S0) / S0 * 100%.

[0020] Example 4: (1) Weigh a certain amount of type B gelatin granule raw material, dry it at 55°C, and then transfer it to a 2% glutaraldehyde crosslinking solution for crosslinking for 12 hours. Obtain chemically crosslinked gelatin granules. (2) Transfer the crosslinked gelatin granules to a centrifuge tube, add deionized water, and centrifuge and wash repeatedly at least 3 times. Then transfer it to a stainless steel pan and dry it at 50°C. (3) Disperse the dried gelatin granules and transfer them to a ball mill cup. Add an appropriate amount of grinding beads and grind at 600 rpm for 1 hour. Collect the ground gelatin granules. (4) Pass the collected gelatin granules through 20, 30, 40 and 60 mesh sieves. Collect the un-sieved particles after passing through a 20 mesh sieve and then through a 30 mesh sieve as large-sized particles; collect the un-sieved particles after passing through a 30 mesh sieve and then through a 40 mesh sieve as medium-sized particles; and collect the un-sieved particles after passing through a 40 mesh sieve and then through a 60 mesh sieve as small-sized particles. (5) Mix large-sized particles with medium-sized particles, large-sized particles with small-sized particles, and medium-sized particles with small-sized particles in ratios of 2:1 and 1:2 respectively to obtain fluid gelatin particles with different size combinations.

[0021] Example 5: (1) At room temperature, 5 mL of anticoagulated pig blood was added to a centrifuge tube. (2) The large, medium, and small-sized particles and combinations of different sizes obtained in Example 4 were added to pig blood at a volume ratio of 1:3. The coagulation was observed, and a photo was taken after ten minutes. It can be seen that the coagulation effect of different sizes and combinations of different sizes of particles is different. From left to right, they are: simple large-sized particles (1), simple medium-sized particles (2), simple small-sized particles (3), medium and small-sized (2:1) combination particles (4), large and small-sized (1:2) combination particles (5), large and medium-sized (2:1) combination particles (6), large and medium-sized (1:2) combination particles (7), large and small-sized (2:1) combination particles (8), and medium and small-sized (1:2) combination particles (9). It is evident that small-sized particles (No. 3), large and medium-sized (2;1) combined particles (No. 6), and medium and small-sized (2;1) combined particles (No. 4) all produced significant coagulation, especially the medium and small-sized (2;1) combined particles (No. 4) which formed dense coagulation clots.

Claims

1. A combination size fluid gelatin particle, process for its preparation and use, characterized in that It is prepared by cross-linking, mechanical grinding, and sieving to collect gelatin particles of different sizes, and combining the particles of different sizes in a suitable and determinable volume ratio.

2. The combination size fluid gel particles of claim 1 wherein The raw material of the gelatin particles can be type A gelatin or type B gelatin, and the gelatin has a bloom strength of 200-300 (Bloom).

3. The combination size fluid gel particles of claim 1 wherein The gelatin particles are obtained by mechanical grinding of gelatin particles cross-linked by physical or chemical methods or gelatin sponge.

4. The combination size fluid gel particles of claim 1 wherein The mechanically ground gelatin particles are sieved through screens of different mesh sizes to collect them.

5. The combination size fluid gel particles of claim 1 wherein The combined size gelatin particles are obtained by combining two or more sizes of particles.

6. The combination sized fluid gelatin particles of claim 1, wherein The method comprises the following steps: (1) a suitable amount of gelatin particle raw material is placed in a stainless steel container and placed in a vacuum drying box, vacuumized, and then cross-linked at a high temperature of 100-180°C for 4-10 hours; alternatively, the gelatin particle raw material is proportionally suspended in a cross-linking liquid solution, and cross-linked under stirring at 60-500 rpm for 8-48 hours; alternatively, the high-temperature cross-linked gelatin particles are further suspended in a cross-linking liquid for re-cross-linking; alternatively, the gelatin particles are fully dissolved in deionized water at a concentration of 20-40%, and further freeze-dried to obtain gelatin sponge, which is further cross-linked by physical or chemical methods in the above manner; (2) the cross-linked gelatin particles or gelatin sponge obtained in step (1) are mechanically ground or crushed to obtain gelatin particles. The harvested gelatin particles are sequentially sieved through screens of 20, 30, 40, and 60 mesh, and the particles that do not pass through the 20-mesh screen after sieving through the 20-mesh screen and then the 30-mesh screen are collected as large-size particles; the particles that do not pass through the 30-mesh screen after sieving through the 30-mesh screen and then the 40-mesh screen are collected as medium-size particles; and the particles that do not pass through the 40-mesh screen after sieving through the 40-mesh screen and then the 60-mesh screen are collected as small-size particles; (3) the gelatin particles of different sizes obtained in step (2) are physically mixed in different volume ratios to obtain combined size fluid gelatin particles. The volume ratio of the large-size particles to the small-size particles is 2-4:

1. Preferably, the combination is as follows: large size: medium size is 2-4:1, and medium size: small size is 2-4:

1.

7. The combination sized fluid gelatin particles of claim 1, wherein Alternatively, the gelatin particle raw material is mechanically ground, then sieved to collect particles of different sizes, then cross-linked by the above physical or chemical methods to obtain fluid gelatin particles of different sizes, and finally physically mixed to obtain combined size fluid gelatin particles.

8. A combination sized fluid gelatin particle according to claim 1, process for its preparation and use, characterized in that The prepared combined size fluid gelatin particles can be suspended in physiological saline, water for injection, and physiological saline or water for injection containing thrombin and antibacterial agents to prepare a hemostatic material with fluid properties.

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