A jatrophine-chitosan / calcium carbonate hemostatic sponge and a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and discloses a acutine-chitosan / calcium carbonate hemostatic sponge as well as a preparation method and application thereof. The acutine and chitosan are condensed to obtain acutine-chitosan, and then the acutine-chitosan is reacted with sodium carbonate, calcium hydroxide and urea to form the acutine-chitosan / calcium carbonate hemostatic sponge. In-vivo and in-vitro experiments verify that the sponge can be used for hemostasis and antibiosis, and due to the pH sensitivity of the acutine-chitosan, the acutine can be intelligently controlled to be released under acidic conditions (cancer acidic environment), so that the sponge can be used for rapid hemostasis during cancer surgery, and also helps to prevent recurrence of liver cancer after surgery. Therefore, the multifunctional acutine-chitosan / calcium carbonate hemostatic sponge of the application provides a 'one-stop' synergistic strategy for intraoperative hemostasis and postoperative prevention of tumor recurrence of liver cancer, and has great clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an abrin-chitosan / calcium carbonate hemostatic sponge, its preparation method, and its application. Background Technology

[0002] Liver cancer is the fifth most common malignant tumor worldwide, accounting for 9.1% of all cancer deaths globally. The incidence of primary liver cancer continues to rise, a trend that may persist for decades. To date, surgical resection remains the preferred and most effective treatment for liver cancer. However, massive bleeding is inevitable during the removal of highly vascularized liver tumors, making hemostasis crucial. While many reported or commercially available hemostatic agents, such as tissue adhesives, glutaraldehyde-crosslinked albumin, zeolite-based QuickClot, fibrin bandages, or gelatin hemostatic agents, are highly effective at stopping bleeding from superficial wounds, current clinical practice for hemostasis during malignant liver tumor resection still primarily relies on simple gauze packing. This significantly increases the risk of intraoperative mortality, places a greater burden on the blood supply of the healthcare system, and studies have shown that unavoidable tissue damage and bleeding during tumor resection can lead to cancer cell metastasis via the bloodstream, greatly increasing the risk of tumor recurrence. Therefore, providing a comprehensive product that can both stop bleeding during tumor resection and help prevent postoperative tumor recurrence is of great significance.

[0003] *Abrus precatorius* possesses various pharmacological effects, including antitumor, antioxidant, antibacterial, antiviral, anti-inflammatory, analgesic, and immunomodulatory properties. Currently, it is used clinically as a single herb or in combination with traditional Chinese medicine (TCM) formulas, primarily in combination with other herbs to enhance its efficacy. Abrine, extracted from *Abrus precatorius*, has important hepatoprotective functions. The article "Abrine, an IDO1 inhibitor, suppresses the immune escape and enhances the immunotherapy of anti-PD-1 antibody in hepatocellular carcinoma" points out that the specific IDO1 inhibitor absrine can inhibit IFN-γ, PBMCs, or the IDO1-induced IDO1-jak1-stat1 signaling pathway. By inhibiting CD47 expression, it enhances macrophage phagocytosis, effectively suppressing tumor immune escape and reducing PD-L1 expression in HCC (hepatocellular carcinoma) cells. Combined with anti-PD-1 antibodies, it has a synergistic therapeutic effect on HCC. However, there are currently no reports on the combined use of abrin with chitosan / calcium carbonate to control the release of abrin in a cancerous environment, and its use for intraoperative hemostasis and prevention of postoperative tumor recurrence. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for preparing abrin-chitosan / calcium carbonate hemostatic sponge.

[0005] The second objective of this invention is to provide a basidiosin-chitosan / calcium carbonate hemostatic sponge prepared according to the above preparation method.

[0006] The third objective of this invention is to provide the application of the above-mentioned abrin-chitosan / calcium carbonate hemostatic sponge in the preparation of antibacterial, hemostatic, and liver cancer recurrence prevention products.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for preparing abrin-chitosan / calcium carbonate hemostatic sponge, the method comprising the following steps: S1. Abrusatine and chitosan are condensed together to obtain absinine-chitosan;

[0008] S2. The abrin-chitosan from step S1 is combined with sodium carbonate, calcium hydroxide, and urea in a one-pot process to synthesize abrin-chitosan / calcium carbonate hemostatic sponge.

[0009] Chitosan is rich in amino groups, which dissolve under acidic conditions. Meanwhile, the structure of abrin is similar to that of amino acids, possessing both amino and carboxyl groups. Under pH 4-6, the amino groups of chitosan and the carboxyl groups of abrin undergo a condensation reaction, binding chitosan and abrin via non-covalent bonds to obtain abrin-chitosan. This abrin-chitosan approach allows for intelligent controlled release of abrin under acidic conditions, avoiding the problem of uncontrolled release caused by directly adding abrin to chitosan / calcium carbonate, which can result in an initial burst of release followed by insufficient effective drug concentration and reduced drug utilization.

[0010] Calcium carbonate ions are known to be key clotting factors in blood, participating in the coagulation process. A sponge prepared by combining abrin-chitosan with sodium carbonate can not only be used for hemostasis and antibacterial purposes, but also, due to the pH sensitivity of abrin-chitosan, can intelligently control the release of abrin under acidic conditions (the acidic environment of cancer). Using this sponge during cancer surgery can also help prevent recurrence after liver cancer surgery.

[0011] Further, in step S1, the condensation reaction is carried out by reacting abrin alkaloid with chitosan under alkaline conditions at a temperature of 65-75°C for 20-40 minutes; the pH is then adjusted to 4.5-6.3 to obtain the final product.

[0012] Furthermore, in step S1, the condensation reaction is carried out by reacting abrin with chitosan under acidic conditions at 70°C for 30 minutes; the pH is then adjusted to 4.5–6.3 to obtain the final product.

[0013] Specifically, the acidic conditions are those containing 1% glacial acetic acid. Abrus precatorius alkaloid is added to a 1% glacial acetic acid-chitosan solution (1 mL of glacial acetic acid and 1 g of chitosan are added to 100 mL of water, pH approximately 3.3–3.5) and heated in a water bath at 70°C.

[0014] Furthermore, in step S2, the mass ratio of sodium carbonate, calcium hydroxide, and urea is 2.5–2.8:1.6–2:1.

[0015] Furthermore, in step S2, the mass ratio of sodium carbonate, calcium hydroxide, and urea is 2.65:1.85:1.

[0016] The present invention also provides abrin-chitosan / calcium carbonate hemostatic sponge prepared by the above preparation method.

[0017] This invention demonstrates the excellent antibacterial properties of the abrin-chitosan / calcium carbonate hemostatic sponge through in vitro contact testing. Furthermore, animal models of in vivo hemostasis and postoperative recurrence during ectopic hepatocellular tumor resection further validate the sponge's excellent performance in rapid hemostasis and prevention of tumor recurrence after tumor resection. This multifunctional abrin-chitosan / calcium carbonate hemostatic sponge provides a "one-stop" synergistic strategy for intraoperative hemostasis and postoperative prevention of tumor recurrence, possessing significant clinical application potential.

[0018] Therefore, the present invention also provides the application of the above-mentioned abrin-chitosan / calcium carbonate hemostatic sponge in the preparation of antibacterial products.

[0019] Furthermore, the antibacterial activity is against Staphylococcus aureus and / or Escherichia coli.

[0020] The present invention also provides the application of the above-mentioned abrin-chitosan / calcium carbonate hemostatic sponge in the preparation of products for hemostasis.

[0021] The present invention also provides the application of the above-mentioned abrin-chitosan / calcium carbonate hemostatic sponge in the preparation of products for preventing liver cancer recurrence.

[0022] The present invention also provides a product comprising the above-mentioned abrin-chitosan / calcium carbonate hemostatic sponge.

[0023] Furthermore, the product also includes other medically acceptable excipients.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an abrin-chitosan / calcium carbonate hemostatic sponge. The process involves first synthesizing abrin and chitosan through a condensation reaction to obtain abrin-chitosan, which is then reacted with sodium carbonate, calcium hydroxide, and urea to form the abrin-chitosan / calcium carbonate hemostatic sponge. This avoids the problem of not being able to intelligently control the release of abrin by directly adding abrin to chitosan / calcium carbonate. In vitro and in vivo experiments have verified that this sponge can not only be used for hemostasis and antibacterial purposes, but also, due to the pH sensitivity of abrin-chitosan, can intelligently control the release of abrin under acidic conditions (the acidic environment of cancer). Using this sponge during cancer surgery can quickly stop bleeding and also helps prevent postoperative recurrence of liver cancer. Therefore, the multifunctional abrin-chitosan / calcium carbonate hemostatic sponge of this invention provides a "one-stop" synergistic strategy for intraoperative hemostasis and postoperative prevention of tumor recurrence in liver cancer, and has great clinical application potential. Attached Figure Description

[0025] Figure 1 This is the dissolved state of abrin-chitosan. Figure 1 In the diagram, A represents chitosan; B represents abrin-chitosan; C represents chitosan dissolved in acetic acid solution; and D represents abrin-chitosan dissolved in acetic acid solution.

[0026] Figure 2 The results show the detection of the amount of abrin released by QPCA-1 under different pH conditions.

[0027] Figure 3 These are the results of the QPCA antibacterial performance test. Among them, Figure 3 In the figure, A represents the antibacterial test results of different sponges against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli; B represents a representative image of a bacterial culture plate. ***p<0.001.

[0028] Figure 4 The results are from a mouse tail amputation model hemostasis test using QPCA. Among them, Figure 4 In the figure, A represents a representative image; B represents the quantitative statistics of blood loss. ***p<0.001.

[0029] Figure 5 This is a QPCA trial for the prevention of in vivo recurrence after tumor resection. Among them, Figure 5 In the figures, A represents representative photographs of tumor resection and hemostasis surgery (green arrow: tumor exposure, yellow arrow: hemostasis); B represents quantitative statistics of blood loss in tumor resection bleeding models under different treatment methods; C represents survival curves of different groups of mice in the 14-day experiment; D represents photographs of tumor recurrence in each group of mice 14 days after surgery; E represents tumor weight. *p<0.05, ***p<0.001. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] Chitosan molecular structure: ; Abrus alkaline molecular structure formula: .

[0033] Example 1: Preparation of Abrus precatorius alkaloid-chitosan / calcium carbonate hemostatic sponge (QPCA) I. Experimental Methods 1. Add 500 mg of abrin to 150 mL of a 1% glacial acetic acid-chitosan solution (1 mL of glacial acetic acid and 1 g of chitosan are added to 100 mL of water). Heat in a 70°C water bath. The abrin reacts with the chitosan for approximately half an hour. Adjust the pH of the solution to 4.5–6.3 with potassium hydroxide to obtain a precipitate. Filter under vacuum, remove the filtrate, wash (soak) the precipitate with water until neutral, and then filter again. Finally, freeze-dry to obtain a brownish-yellow solid powder, as shown below. Figure 1 As shown. Dissolving it in acetic acid solution yielded the following results. Figure 1 As shown, the obtained abrin-chitosan structure is stable and uniform, indicating successful synthesis.

[0034]

[0035] 2. Subsequently, 2g of the successfully synthesized abrin-chitosan, 10.6g of sodium carbonate, 7.4g of calcium hydroxide, and 4g of urea were dissolved in 100mL of water to synthesize in situ abrin-chitosan / calcium carbonate hydrogel in a one-pot method.

[0036] As above, hydrogels with abacus alkaloid addition of 0 (using only 0.5 g of chitosan), abacus alkaloid-chitosan addition of 0.5, 1, 1.5, and 2 g were prepared respectively. The hydrogels were soaked in deionized water to remove excess unreacted substances, and the soaking and washing were repeated 5 times until the pH of the soaking solution was neutral. Then, the sponges were obtained by freeze drying and named QPCA-0, QPCA-1, QPCA-2, QPCA-3, and QPCA-4 respectively.

[0037] 1g of QPCA-1 was used to verify the amount of abrin alkaloid released by the drug-eluting sponge under different pH conditions, and the result was detected by high performance liquid chromatography. Figure 2 As shown, abrin can rapidly release abrin under acidic conditions.

[0038] Chitosan is rich in amino groups, which dissolve under acidic conditions. Meanwhile, the structure of abrin is similar to that of amino acids, possessing both amino and carboxyl groups. Under pH conditions of 4-6, the amino groups on chitosan and the carboxyl groups of abrin undergo a condensation reaction, combining chitosan and abrin via non-covalent bonds to obtain abrin-chitosan. Using this in the preparation of hydrogels / sponges can not only be used for hemostasis, but also, due to the pH sensitivity of abrin-chitosan, it can intelligently control the release of abrin under acidic conditions (the acidic environment of cancer). Using this hydrogel / sponge during cancer surgery can also help prevent postoperative recurrence of liver cancer.

[0039] Example 2: Antibacterial performance test of QPCA I. Experimental Methods Postoperative infection is a common risk in clinical surgeries such as tumor resection. This invention employs a contact method to simulate the antibacterial effect of the sponge (QPCA) prepared in Example 1 upon contact with bacteria. Sponges with different drug loading concentrations from Example 1 were placed in 24-well culture plates, and 10 μL of bacteria (Escherichia coli and Staphylococcus aureus) at a concentration of 10 μL were added to each plate. 6 A bacterial suspension of CFU / mL was dropped onto a sponge. As a control, 10 μL of bacterial suspension was added to wells without sponges. After incubating the 24-well plate at 37°C for 2 hours, 1 mL of PBS was added to each well to resuspend the bacteria. 200 μL of the bacterial suspension was spread onto nutrient agar plates and incubated at 37°C for 24 hours. The number of colonies on the nutrient agar plates was then counted. Six replicates were set up for each group. The inhibition rate R was calculated as R = (Qc - Qh) / Qc × 100%. Where: Qh represents the number of bacteria in the different concentrations of abrin; Qc represents the number of bacteria in the control group.

[0040] II. Experimental Results The results are as follows Figure 3 As shown, this indicates that the sponge has good antibacterial ability against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Further analysis showed that with increasing absinin content in the sponge, the inhibition rate against Staphylococcus aureus remained relatively stable (p > 0.05), while the inhibition rate against Escherichia coli significantly increased (p < 0.05). Figure 3 A in the figure indicates that the addition of abrin-chitosan gives the sponge good antibacterial properties, and the addition of abrin to the sponge system is more helpful in inhibiting Escherichia coli. Figure 3 Figure B shows a representative bacterial culture plate. Similarly, it can be seen that the sponge containing abrin-chitosan significantly reduced or even eliminated bacterial colonies on the culture plate, while the control group was full of bacteria.

[0041] Example 3: Hemostasis test of a mouse tail amputation model using QPCA I. Experimental Methods The hemostatic effect of QPCA was evaluated using a mouse tail amputation model. Forty-eight female DBA / 1 (Kunming) mice (5-6 weeks old) were randomly divided into eight groups. Animals were anesthetized with isoflurane and fixed to a surgical board. The tails were severed at 50% of their length using a scalpel, and a 15-second window was allowed to allow for normal blood loss. The tail incision was then covered with gauze, gelatin sponge, and sponge (QPCA from Example 1 of this invention) of equal volume under slight pressure, and the amount of bleeding was recorded. Mice with untreated wounds served as the control group. All animal experiments were approved by the Animal Research Committee of Guangxi University of Chinese Medicine, and all procedures were strictly performed in accordance with animal experimental ethics requirements.

[0042] II. Experimental Results From representative images ( Figure 4 As shown in A), the blood loss in all QPCA groups was less than that in the control group. Quantitative results further indicated that, compared to the blank control group, gauze group, and gelatin sponge group, the blood loss in all QPCA groups was significantly reduced. p <0.001. There was no significant difference in hemostatic effect among all doses of QPCA ( p >0.05), indicating that the sponge prepared in this invention has a hemostatic promoting effect ( Figure 4 (B in the text). Furthermore, all mice were still alive at the end of the test.

[0043] Example 4: QPCA trial for the prevention of in vivo recurrence after tumor resection I. Experimental Methods To verify the effects of abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4) in Example 1 on in vivo hemostasis and prevention of recurrence after surgical resection of ectopic hepatocellular carcinoma, an in vivo mouse model of ectopic hepatocellular carcinoma was established. Forty 6-week-old female BALB / c mice were used to establish a subcutaneous HCC recurrence model. H22 hepatocellular carcinoma cells (1×10⁻⁶) were introduced from the mice. 6 0.2 mL PBS was subcutaneously injected into the right axilla of mice. When the tumor volume reached 130 mm, 3Afterwards, all visible tumors were surgically removed. Blood loss was recorded during the surgery using gauze, commercial gelatin hemostatic sponges, and abrin-chitosan / calcium carbonate hemostatic sponges (QPCA-4). Mice with removed tumors were randomly divided into four groups (n=12): (i) no treatment after removal; (ii) daily intraperitoneal injection of fluorouracil (5-Fu) (0.02 g / kg); (iii) implantation of abrin-chitosan / calcium carbonate hemostatic sponges (QPCA-4) at the removal site; (iv) implantation of abrin-chitosan / calcium carbonate hemostatic sponges (QPCA-4) at the removal site and daily intraperitoneal injection of 5-Fu (0.02 g / kg). After 14 days, all mice were euthanized, and regenerated tumor tissue from the axilla was isolated for further observation.

[0044] II. Experimental Results About one week after subcutaneous injection of H22 cells, approximately 130 mm of cells grew in the axilla of mice. 3 Tumors of various sizes Figure 5 In the figures, A represents the tumor 0 days before resection, the tumor exposure during surgery, the bleeding after resection, the hemostasis status after adding cryotherapy fluid, and the final wound closure. During the surgical resection, gauze and gelatin hemostatic sponge groups were used as controls to test the hemostatic effect of abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4) during tumor resection. Figure 5 As shown in B, the blood loss of the abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4) was significantly different from that of the blank group, gauze group, and commercial gelatin hemostatic sponge (all p < 0.001), which means that the abrin-chitosan / calcium carbonate hemostatic sponge provided in Example 1 of this invention not only reduced the risk of death from intraoperative bleeding in mice, but also greatly reduced the risk of cancer cell metastasis and blood metastasis.

[0045] Postoperative treatment included injection of 5-FU and / or implantation of abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4). The survival curves of mice after 14 days were as follows: Figure 5 As shown in C, due to the impact of tumor recurrence, only 6 mice in the control group survived to the end of the experiment, 9 mice in the 5-Fu group survived, 8 mice in the abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4) group survived, and all mice in the abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4) + 5-Fu group survived. Mice were euthanized on day 14, and axillary regenerated tumor tissue was isolated for further evaluation. Figure 5 As shown in D, there were significant differences in tumor morphology among the different treatment groups. The tumors in the QPCA-4+5-Fu group were the smallest, and further weight analysis was performed. Figure 5In the E group, the mean weight of recurrent tumors in the abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4) + 5-Fu group was significantly lower than that in the blank control group (p<0.001), abrin-chitosan / calcium carbonate hemostatic sponge (QPCA-4) (p<0.001) and 5-Fu group (p<0.01).

[0046] In summary, it can be demonstrated that by adjusting the pH to 4-6 to induce a condensation reaction between the amino groups on chitosan and the carboxyl groups of abrin, chitosan and abrin are combined via non-covalent bonds to obtain abrin-chitosan. Using abrin-chitosan to prepare abrin-chitosan / calcium carbonate hemostatic sponge avoids the problem of uncontrolled release of abrin caused by directly adding abrin to chitosan / calcium carbonate, and does not affect the original antibacterial and hemostatic effects of the chitosan / calcium carbonate sponge. Furthermore, due to the pH sensitivity of abrin-chitosan, it can intelligently control the release of abrin under acidic conditions (the acidic environment of cancer). Using this sponge in intraoperative hemostasis and postoperative treatment of cancer can also help inhibit postoperative recurrence of liver cancer, improve mouse survival rates, and exhibit a good synergistic effect when used in combination with 5-FU.

Claims

1. A method for preparing a bromelain-chitosan / calcium carbonate hemostatic sponge, characterized in that, The preparation method includes the following steps: S1. Abrusatine and chitosan are condensed together to obtain absinine-chitosan; S2. The abrin-chitosan from step S1 is combined with sodium carbonate, calcium hydroxide, and urea in a one-pot process to synthesize abrin-chitosan / calcium carbonate hemostatic sponge.

2. The preparation method according to claim 1, characterized in that, In step S1, the condensation reaction involves reacting abrin with chitosan under acidic conditions at a temperature of 65–75°C for 20–40 minutes, followed by adjusting the pH to 4.5–6.3 to obtain the final product.

3. The preparation method according to claim 2, characterized in that, In step S1, the condensation reaction is carried out by reacting abrin with chitosan under acidic conditions at 70°C for 30 minutes; then the pH is adjusted to 4.5-6.3 to obtain the final product.

4. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of sodium carbonate, calcium hydroxide, and urea is 2.5–2.8:1.6–2:

1.

5. The preparation method according to claim 4, characterized in that, In step S2, the mass ratio of sodium carbonate, calcium hydroxide, and urea is 2.65:1.85:

1.

6. The abrin-chitosan / calcium carbonate hemostatic sponge prepared by any of the preparation methods described in claims 1 to 5.

7. The use of the abrin-chitosan / calcium carbonate hemostatic sponge according to claim 6 in the preparation of antibacterial products.

8. The application according to claim 7, characterized in that, The antibacterial activity is against Staphylococcus aureus and / or Escherichia coli.

9. The use of the abrin-chitosan / calcium carbonate hemostatic sponge according to claim 6 in the preparation of products for hemostasis.

10. The use of the abrin-chitosan / calcium carbonate hemostatic sponge of claim 6 in the preparation of a product for preventing recurrence of liver cancer.

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