Liposome-modified hemostatic sponge system

Abstract

The present invention discloses a hemostatic sponge system constructed by the combination of polypeptide-modified liposomes and alginate, comprising the following steps: (1) grafting a fatty chain to the amino terminus of a platelet-binding peptide; (2) inserting the modified peptide in step (1) into liposomes by means of hydrophobic interaction; (3) mixing the liposomes with an alginate solution and forming a hydrogel through double crosslinking; (4) the gel obtained in step (3) can be freeze-dried to obtain a hemostatic sponge. It can be inserted into the phospholipid bilayer of liposomes through peptide modification and embedded in the alginate sponge prepared by the double crosslinking method. Through the hemostatic performance of the modified liposomes and alginate acting in synergy, the coagulation components at the wound can be effectively enriched to accelerate the coagulation process, and the biocompatibility and non-toxicity of alginate can promote wound healing.

Description

Technical Field

[0001] The present invention relates to a hemostatic sponge system constructed by the combination of polypeptide-modified liposomes and alginate, which can be used for filling and hemostasis of grooves or irregular wounds. Background Art

[0002] When uncontrolled bleeding caused by trauma threatens life, effective hemostasis management is crucial for global military and civilian trauma care. Within minutes after uncontrollable bleeding occurs, the survival rate of the wounded can be improved through effective hemostasis interventions. As typical representatives of traditional hemostatic materials, gauze and bandages cannot achieve effective hemostasis in the face of extreme wound environments such as non-compressible penetrating arterial bleeding wounds and irregular deep arterial bleeding. In recent years, the research and development of new hemostatic materials have progressed more and more rapidly, and different forms of hemostatic materials have been developed to deal with different types of wound environments. For example, Patent CN202021887276 discloses a hemostatic dressing device carrying polypeptide liposomes, which uses a transmission component to enable medical staff to evenly apply the medicine on the bandage; Patent CN201510536381 discloses an amino acid liposome-containing hemostatic adhesive and its preparation method, which can actively provide nutrients or active substances for the wound surface; Patent CN202211268724 discloses a GelMA-DA / quaternized chitosan / glycerol composite hemostatic sponge material and its preparation method, which has good adhesion ability, suitable mechanical properties, good biocompatibility, significant antibacterial property and excellent coagulation and hemostasis performance. Compared with the above materials, firstly, we have selected different liposome modification methods and the hemostasis principles of the modifiers are completely different. Secondly, compared with chitosan, the excellent biocompatibility of the alginate we used is beyond doubt, and the high water absorption capacity of the sponge can effectively promote the aggregation of coagulation components in the blood and accelerate blood coagulation. Summary of the Invention

[0003] The purpose of the present invention is to construct a hemostatic sponge system, in which the polypeptide-modified liposomes are embedded in the double-crosslinked alginate. The present invention provides a hemostasis method that can be filled at irregular wounds such as grooves.

[0004] In order to achieve the above technical solution, a hemostatic sponge system constructed by the combination of polypeptide-modified liposomes and alginate according to the present invention specifically includes the following steps:

[0005] (1) Link a fatty chain to the amino terminus of the platelet-binding peptide;

[0006] (2) Insert the modified peptide in step (1) into the liposome by hydrophobic interaction;

[0007] (3) Mix the liposome with the alginate solution and form a hydrogel through double crosslinking;

[0008] (4) The gel obtained in step (3) can be freeze-dried to obtain a hemostatic sponge.

[0009] The fatty acid chain involved in the present invention can be C 16 or C 19 , and the platelet-binding peptide sequence is HHLGGAKQAGDV (Seq_1).

[0010] In step (3) involved in the present invention, the alginate concentration is 2%-10%, and the volume ratio of liposome to alginate is 1:5, 1:3 or 1:1.

[0011] In step (3) involved in the present invention, the double cross-linking is Ca 2+ cross-linking and photo-cross-linking, and AlgMA-LP is selected for photo-cross-linking.

[0012] In step (4) involved in the present invention, during the freeze-drying treatment, the pre-freezing temperature of the sample is -80°C, and the pre-freezing time is 48 h.

[0013] Compared with the prior art, the present invention has the following advantages: (1) low material cost, simple preparation method, easy to use and operate, and easy for large-scale production and storage; (2) the prepared hemostatic dressing has good biocompatibility and no cytotoxicity; (3) the alginate hemostatic sponge has excellent swelling performance, can absorb excessive body fluid at the wound, and maintain a suitable physiological environment at the wound; (4) the liposome-platelet binding peptide can cooperate with alginate to enrich coagulation components and improve the hemostasis efficiency. Description of the Drawings

[0014] Att Figure 1 is the modified peptide molecular structure of Example 1 involved in the present invention;

[0015] Att Figure 2 is the insertion of the modified peptide of Example 1 involved in the present invention into the liposome;

[0016] Att Figure 3 is the swelling property of the hemostatic sponge of Example 1 involved in the present invention;

[0017] Att Figure 4 is the coagulation time of the hemostatic sponge of Example 1 involved in the present invention;

[0018] Att Figure 5 is the cell experiment of the hemostatic sponge of Example 1 involved in the present invention.

[0019] Detailed implementation manners of the invention

[0020] The present invention will be further described below through specific examples and the accompanying drawings of the specification.

[0021] Example 1:

[0022] (1) Graft the fatty chain to the platelet-binding peptide

[0023] Using the Fmoc solid-phase synthesis method, start the synthesis from the C-terminus, and then graft the fatty chain C to the N-terminal part 16 , endowing it with hydrophobic properties. The molecular structure is as shown in the appendix Figure 1 .

[0024] (2) Insert the modified peptide into the liposome

[0025] Using hydrophobic interaction, dissolve the liposome into a film in chloroform, then add a surfactant to dissolve it and break the liposome membrane by ultrasonic treatment. Finally, add the modified peptide and use macroporous resin to absorb the surfactant on the inner surface of the liquid, so that the self-assembly of the modified peptide and the liposome is completed. The molecular structure is as shown in the appendix Figure 2 .

[0026] (3) Preparation of liposome-peptide and alginate hydrogel

[0027] Mix the liposome with a 2% (w / v) alginate solution at a volume ratio of 1:5, then add 2 mg of calcium carbonate powder, 10 mg of AlgMA, 2 mg of LP, and 3 mg of glucono-δ-lactone, stir at room temperature for 1 h, and then pour the solution into a 3-cm-diameter cell culture dish. After standing at room temperature for 3 h, irradiate it with a 365-nm ultraviolet lamp for 20 min

[0028] (4) Preparation of the hemostatic sponge

[0029] Place the ultraviolet-irradiated hydrogel in a -80 °C refrigerator for pre-freezing for 48 h, and then perform freeze-drying for 24 h

[0030] (5) Swelling performance of the hemostatic sponge

[0031] The swelling behavior of the hemostatic sponge was quantitatively studied by gravimetric analysis. Immerse the sponge (weight m0) in simulated body fluid at 37 °C, and at different time points, weigh the weight of the swollen sponge (m i ), and use the formula R = (m i – m0) / m0 (i = 1, 2, 3... in hours) to calculate the swelling rate. The results are as shown in the appendix Figure 3 .

[0032] (6) Coagulation experiment

[0033] In vitro coagulation: Place 5 mg of the sponge and 800 μL of antagonized blood in a 2-mL centrifuge tube, incubate at 37 °C, and measure the blood coagulation time

[0034] In vivo experiment: A cylindrical sponge with a diameter of 2 cm was placed on the cut wound of the femoral artery of a rat for filling, and the hemostasis index was the wound no longer oozing blood. The coagulation time was recorded. Sprague-Dawley (SD) rats were selected for the in vivo experiment. The results of the coagulation experiment are shown in the appendix Figure 4 as follows.

[0035] (7) Cell experiment

[0036] NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium (supplemented with 10% fetal bovine serum) in a CO2 incubator at 37°C with 5% humidity. The cell culture medium pre-incubated with the hemostatic sponge overnight was used to culture NIH-3T3 cells for 24 h. The cells were fluorescently photographed by the method of dual staining for live and dead cells, and the cell status was recorded. The results are shown in the appendix Figure 5 as follows.

[0037] Example 2:

[0038] This example is the same as Example 1 except that the alginate concentrations in step (3) are 5% and 10%.

[0039] Example 3:

[0040] This example is the same as Example 1 except that the volume ratios of liposome to alginate in step (3) are 1:3 and 1:1.

[0041] Tests showed that the hemostatic sponge prepared in Example 2 was close to that in Example 1 in other properties except for certain differences in swelling performance and coagulation time; the hemostatic sponge prepared in Example 3 was close to that in Example 1 in other properties except for certain differences in coagulation time.

Claims

1. A preparation method of a hemostatic sponge constructed by the composite of a polypeptide-modified liposome and alginate, characterized in that, Specifically, the following steps are included: (1) Graft a fatty chain C 16 to the amino terminus of a platelet-binding peptide with the amino acid sequence HHLGGAKQAGDV; (2) Utilize hydrophobic interaction to dissolve liposomes into a film in chloroform, then add a surfactant and dissolve it, break the liposome film by ultrasonic treatment, and finally add a modified peptide and absorb the surfactant in the liquid by using macroporous resin to complete the self-assembly of the modified peptide and liposomes; (3) Mix the liposome-peptide in step (2) with an alginate solution with a w / v of 2% at a volume ratio of 1:5, add 2 mg of calcium carbonate powder, 10 mg of AlgMA, 2 mg of LP, and 3 mg of glucono-δ-lactone, stir at room temperature for 1 h, then pour the solution into a cell culture dish with a diameter of 3 cm, let it stand at room temperature for 3 h, and irradiate it with a 365 nm ultraviolet lamp for 20 min; (4) Place the gel obtained in step (3) in a refrigerator at -80 °C for pre-freezing for 48 h, and then conduct freeze-drying for 24 h to obtain a hemostatic sponge.

2. The preparation method of the hemostatic sponge constructed by the composite of liposomes modified with polypeptides and alginate according to claim 1, characterized in that, In step (3), through Ca 2+ Double crosslinking with AlgMA-LP.

3. The preparation method of the hemostatic sponge constructed by the composite of liposomes modified with polypeptides and alginate according to claim 2, wherein, In step (4), the pre-freezing temperature is -80 °C and the pre-freezing time is 48 h.

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