Preparation method of coix lacryma-jobi vinasse protein peptide-chitosan composite hemostatic gel

Abstract

The invention discloses a preparation method of coix lacryma-jobi grain stillage protein peptide-chitosan composite hemostatic gel. The preparation method comprises the following steps: modifying and extracting coix lacryma-jobi grain stillage protein peptide, preparing a composite base material dispersion liquid, constructing a cross-linking system, performing CBD-thrombin non-covalent anchoring, and performing gel forming to finally obtain the composite hemostatic gel. Rapid hemostasis is achieved through double mechanisms of physical capture and chemical activation, the method is suitable for high-pressure and irregular wounds, and the defect that pure chitosan is prone to collapsing is overcome; the biocompatibility is good, the anti-inflammatory function and the repair promoting function are achieved, and hemostasis and repair integration is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hemostatic gel preparation, and particularly relates to a preparation method of a coix rice lees protein peptide-chitosan composite hemostatic gel. BACKGROUND

[0002] Chitosan, as a natural cationic polysaccharide, has become one of the core substrates in the field of hemostatic materials due to its excellent biocompatibility, biodegradability and broad-spectrum antibacterial properties. Its hemostatic mechanism is clear. Through the electrostatic interaction between the positive charge in the molecular structure and the negative charge on the surface of the red blood cell membrane, red blood cells are quickly induced to aggregate, and platelet adhesion and coagulation factor cascade reactions are activated to accelerate blood clot formation. Currently, chitosan-based hemostatic products have achieved large-scale commercial application and are widely used in surgical operations, emergency trauma and other scenarios.

[0003] However, the inherent defects of pure chitosan materials still restrict their application in complex scenarios. The mechanical strength is insufficient, and in the case of high pressure bleeding in arteries or irregular wounds in deep parts, the material is prone to structural collapse and rupture after absorbing liquid, making it difficult to maintain hemostatic effect for a long time. The functional dimension is single, and only relies on physical and chemical effects to achieve coagulation, lacking the biological activity of actively regulating the microenvironment of the wound and promoting tissue repair, and the adaptability to infected wounds and chronic wounds is poor. The degradation controllability is poor, and the natural degradation period is usually 1-2 weeks, which does not match the 2-4 week period of wound tissue repair. Early degradation may lead to secondary bleeding, and slow degradation may cause foreign body reactions.

[0004] In recent years, the application potential of bioactive peptides in the field of hemostasis has gradually emerged. For example, fibrin polypeptides can enhance the adhesion of materials to the wound through cross-linking reaction, and improve the stability of hemostasis. However, the specific hemostatic function of coix rice lees protein peptide has not been systematically studied. The commonly used hemostatic materials in current clinical practice include gauze, sponge, hydrogel, powder and other dosage forms, but all have significant technical bottlenecks. Traditional materials such as gelatin sponge have a sharp decrease in mechanical strength after absorbing liquid, which easily leads to breakage and shedding, resulting in hemostatic failure. Oxidized cellulose materials have a faster hemostatic speed, but lack antibacterial activity, increasing the risk of wound infection. Synthetic materials such as cyanoacrylate have strong adhesion, but have poor biocompatibility, which may cause local inflammatory reactions after surgery and affect wound healing. More importantly, single-component hemostatic materials generally have an imbalance in function. Single chitosan material cannot balance fast coagulation and tissue adhesion strength, and single protein material lacks long-term structural stability and antibacterial ability, which cannot meet the clinical needs and urgently needs to develop a multi-component collaborative hemostatic system. Therefore, a preparation method of a coix rice lees protein peptide-chitosan composite hemostatic gel is needed. SUMMARY

[0005] The application aims to solve the problems in the prior art and provides a preparation method of a coix seed vinasse protein peptide-chitosan composite hemostatic gel.

[0006] To achieve the above-mentioned object, the application adopts the following technical scheme: The application comprises the following steps: A. Modification extraction of coix seed vinasse protein peptide: using the alkaline proteolysis-supercritical fluid extraction combined technology, taking coix seed vinasse as raw material, under the conditions of substrate concentration 20%, enzyme addition amount 1300 u / g, 45 DEG C, pH 9.0, enzymolysis for 6 h, and then purified by supercritical CO2 fluid extraction, the coix seed vinasse protein peptide with uniform molecular weight is obtained; B. Preparation of composite substrate dispersion liquid: the modified extracted coix seed vinasse protein peptide is dissolved in a 5%-10% hydrophobic group modified chitosan derivative solution, 0.5 g / L-5 g / L two-dimensional titanium dioxide nanosheet dispersion liquid is added, and the mixture is dispersed for 30 min by ultrasonic with a power of 60 W to obtain a mixed dispersion liquid, wherein the mass ratio of coix seed vinasse protein peptide to hydrophobic group modified chitosan derivative is (1:5)-(1:15), and the two-dimensional titanium dioxide nanosheet dispersion liquid accounts for 5%-15% of the volume of the mixed dispersion liquid; C. Construction of crosslinking system: L-arginine-based degradable polyurethane and o-phthaldehyde grafted pre-crosslinked hyaluronic acid are added to the mixed dispersion liquid, and then 0.5% photoinitiator is added after uniform stirring, wherein the addition amount of L-arginine-based degradable polyurethane is 5%-10% of the mass of the mixed dispersion liquid, and the addition amount of o-phthaldehyde grafted pre-crosslinked hyaluronic acid is 1%-3%; D. CBD-thrombin non-covalent anchoring: CBD-thrombin complex solution with a concentration of 1 mg / mL-5 mg / mL is added to the crosslinking system, and incubated at 4 DEG C and 150 r / min for 30-60 min, so as to realize non-covalent anchoring of CBD-thrombin in the gel precursor network through hydrophobic interaction and hydrogen bond, and the addition amount of CBD-thrombin is 0.1%-0.5% of the total mass of the mixed system; E. Gel forming: the mixed system is irradiated by a 365 nm ultraviolet lamp for 3-5 min for solidification, and then subjected to 3-5 times of freeze-thaw cycles to finally obtain the composite hemostatic gel.

[0007] Further, 0.1%-0.3% metal ion chelating agent is added in the alkaline proteolysis process in step A, the pressure of supercritical CO2 extraction is 30-50 MPa, the temperature is 35-45 DEG C, and the extraction time is 2-3 h.

[0008] Further, in step B, the hydrophobic group modified chitosan derivative is palmitoyl chitosan, and chitosan and palmitoyl chloride are reacted according to a modified raw material molar ratio of 1:(0.1-0.3), and the molar substitution degree of the palmitoyl chitosan is 5%-20%.

[0009] Further, in step C, the L-arginine-based degradable polyurethane DLPU is obtained by polymerization reaction of PEG400, isophorone diisocyanate and L-arginine according to a molar ratio of 1:(2.0-2.5):(0.8-1.2), the polymerization reaction is carried out in anhydrous N,N-dimethylformamide (DMF) solvent, and the reaction is carried out at 80℃ under reflux for 4-6h, and then the product is purified by dialysis and freeze-dried.

[0010] Further, in step E, the freeze-thaw cycle reaction condition is: freezing solidification at-20℃ for 18h to form a gel, and standing at room temperature for 4h to thaw, and repeating 3 times, and the gel porosity is 60%-80%.

[0011] In another aspect, the yiyi distiller's grains protein peptide-chitosan composite hemostatic gel is prepared by the preparation method, and comprises the following components and contents: a hydrophobic group modified chitosan derivative 5%-15wt%, a modified yiyi distiller's grains protein peptide 0.5%-3wt%, two-dimensional titanium oxide nanosheet 0.05%-0.5wt%, L-arginine-based degradable polyurethane 5%-10wt%, o-phthaldehyde grafted pre-crosslinked hyaluronic acid 1%-3wt%, CBD-thrombin 0.1%-0.5wt%, and the balance is medical grade deionized water.

[0012] Further, the CBD-thrombin is anchored in the gel network through non-covalent binding.

[0013] 8. The application of the yiyi distiller's grains protein peptide-chitosan composite hemostatic gel in claim 6, and the application comprises: (1) being used for preparing a hemostatic dressing for acute trauma, surgical wound surface and low coagulation state; (2) being used for the hemostasis and repair integrated treatment of infectious wound surface and chronic refractory wound surface.

[0014] Compared with the prior art, the yiyi distiller's grains protein peptide-chitosan composite hemostatic gel has the following beneficial effects: The yiyi distiller's grains protein peptide-chitosan composite hemostatic gel realizes rapid hemostasis through the dual mechanisms of physical capture and chemical activation, adapts to high pressure and irregular wound surface, solves the defect that pure chitosan is easy to collapse, has good biocompatibility, has anti-inflammatory and repair functions, and realizes hemostasis and repair integration. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is a schematic diagram of the preparation method of the yiyi distiller's grains protein peptide-chitosan composite hemostatic gel. Figure 2 Figure 1 is a schematic diagram of an optical microscope image of Example 1 of the present application; DETAILED DESCRIPTION The present application is further described below and in the specific examples, which illustrate the present application by way of example but not by way of limitation.

[0016] As Figure 1 shown in the present embodiment, the following steps are included: Example 1 The mass ratio of the yiyi vinasse protein peptide and palmitoyl chitosan was 1:10; the concentration of the two-dimensional titanium oxide nanosheet dispersion liquid was 1g / L, and the volume ratio of the two-dimensional titanium oxide nanosheet dispersion liquid in the mixed dispersion liquid was 10%; the added amount of the L-arginine-based degradable polyurethane (DLPU) was 8% of the mass of the mixed dispersion liquid; the added amount of the o-phthaldehyde grafted pre-crosslinked hyaluronic acid was 2%; the added amount of the CBD-thrombin was 0.3wt% of the total mass of the mixed system; and the added amount of the photoinitiator I2959 was 0.5% of the mixed system.

[0017] The palmitoyl chitosan was prepared according to a chitosan and palmitoyl chloride molar ratio of 1:0.2, and the molar substitution degree was 12%; the DLPU was prepared according to a molar ratio of PEG400:isophorone diisocyanate (IPDI):L-arginine of 1:2.2:1.0; and the freeze-thaw cycle condition was -20℃ freezing for 18h / room temperature thawing for 4h, repeated for 3 times.

[0018] After detection, as Figure 2As shown, the micrometer-scale uniform porous structure (50-100 pm) can quickly capture blood components such as red blood cells and platelets, accelerating blood clot formation; the two-dimensional titanium oxide nanosheet uniformly dispersed in the DLPU-chitosan interpenetrating network strengthens the adsorption and activation ability of the gel to blood components, and cooperates with the non-covalent anchoring of CBD-thrombin to construct a dual hemostatic mechanism of physical capture and chemical activation, making the in vitro coagulation time ≤60 s and the in vivo hemostatic time ≤84 s, which is significantly better than traditional chitosan materials. The core performance of the composite hemostatic gel prepared in Example 1 is as follows: the in vitro coagulation time is 52.3±3.8 s, which is significantly shorter than that of pure chitosan gel (138.5±9.6 s) and commercially available chitosan hemostatic gel (96.4±7.2 s); the shear strength is 78.6±4.1 kPa, the tensile strength is 115.4±5.7 kPa, and the elongation at break is 203.2±9.5%, which is far superior to the mechanical properties of pure chitosan gel (shear strength 43.2±3.5 kPa, tensile strength 67.4±4.8 kPa); the hemolysis rate is 2.1±0.3%, which meets the standard of medical material hemolysis rate <5% and is lower than that of commercially available products (4.1±0.6%); the 5-day live cell rate of rat adipose-derived stem cells in a 37℃, 5% CO2 environment is 97.5±1.2%, without obvious cytotoxicity; the gel porosity is 72.4±3.6%, which is beneficial to nutrient exchange and metabolic product discharge; the in vitro degradation rate of DLPU is 71.3±4.2% in 90 days, which is suitable for the wound repair period.

[0019] In the in vivo rat liver hemorrhage model test, the hemostatic time of the gel of the present embodiment is 78.5±6.2 s, and the bleeding volume is 0.42±0.08 mL, which shows a very significant advantage in hemostatic efficiency and bleeding volume control compared with pure chitosan gel (hemostatic time 156.8±10.3 s, bleeding volume 1.23±0.15 mL) and commercially available products (hemostatic time 112.5±8.7 s, bleeding volume 0.87±0.12 mL) (P<0.01).

[0020] Example 2 The mass ratio of coix seed vinasse protein peptide to palmitoyl chitosan is 1:8; the concentration of the two-dimensional titanium oxide nanosheet dispersion is 3 g / L, which accounts for 12% of the volume proportion of the mixed dispersion; the addition amount of DLPU is 10% of the mass of the mixed dispersion; the addition amount of o-phthaldehyde grafted pre-crosslinked hyaluronic acid is 3%; the addition amount of CBD-thrombin is 0.5wt% of the total mass of the mixed system; and the addition amount of photoinitiator I2959 is 0.5% of the mixed system.

[0021] The palmitoyl chitosan is prepared by reacting chitosan with palmitoyl chloride at a molar ratio of 1:0.25, and the molar substitution degree is 16%; the DLPU is prepared by polymerization at a molar ratio of PEG400:IPDI:L-arginine = 1:2.4:1.1; the freeze-thaw cycle conditions are the same as in Example 1.

[0022] The mechanical properties of the gel of this example 2 are the best: the shear strength is 85.3±3.9kPa, the tensile strength is 127.8±6.3kPa, and the elongation at break is 191.5±8.8%, the shear strength is increased by 8.5% compared with Example 1, which can effectively resist external impact and friction on the wound surface in high-pressure bleeding scenarios; the in vitro clotting time is 48.7±4.2s, which is the fastest among the three examples, due to the synergistic effect of the higher CBD-thrombin addition and the cation of DLPU; the hemolysis rate is 2.5±0.4%, and the 5-day live cell rate is 96.8±1.5%, which still meets the biocompatibility requirements; the gel porosity is 68.7±3.2%, which is slightly lower than that of Example 1, but still can guarantee the air permeability of the wound microenvironment; the 90-day in vitro degradation rate of DLPU is 68.5±3.9%, which meets the controllable degradation requirements.

[0023] In the in vivo test, the hemostatic time of the gel of this example is 72.3±5.8s, and the bleeding volume is 0.38±0.07mL, which further shortens the clotting time and bleeding volume compared with Example 1, and is especially suitable for high-pressure bleeding scenarios such as arterial bleeding, and the blood clot stability is significantly better than that of the control group, and there is no postoperative secondary bleeding phenomenon.

[0024] Example 3 The mass ratio of coix seed vinasse protein peptide to palmitoyl chitosan is 1:12; the concentration of two-dimensional titanium dioxide nanosheet dispersion liquid is 0.8g / L, and the volume proportion of which in the mixed dispersion liquid is 8%; the addition amount of DLPU is 6% of the mass of the mixed dispersion liquid; the addition amount of o-phthaldehyde grafted pre-crosslinked hyaluronic acid is 1%; the addition amount of CBD-thrombin is 0.2wt% of the total mass of the mixed system; and the addition amount of photoinitiator I2959 is 0.5% of the mixed system.

[0025] The palmitoyl chitosan is prepared by reacting chitosan with palmitoyl chloride at a molar ratio of 1:0.15, and the molar substitution degree is 8%; the DLPU is prepared by polymerization at a molar ratio of PEG400:IPDI:L-arginine = 1:2.1:0.9; and the freeze-thaw cycle conditions are the same as in Example 1.

[0026] The biocompatibility of the gel of the present example performs best: the hemolysis rate is only 1.8±0.2%, which is the lowest value among the three groups of examples, and the damage to red blood cells is minimal; the 5-day live cell rate reaches 98.2±1.1%, which can significantly promote the proliferation of rat adipose stem cells and provide cell-level support for wound repair; the in vitro coagulation time is 55.6±4.5s, which still meets the requirement of rapid coagulation ≤60s; the shear strength is 73.8±3.7kPa, the tensile strength is 108.6±5.2kPa, and the elongation at break is 215.7±10.2%, the higher elongation at break enables it to better fit irregular wounds and adapt to the deformation of active parts such as joints; the gel porosity is 76.5±3.8%, which is the highest value among the three groups of examples, and is beneficial to the absorption of wound exudate and drug release; the 90-day in vitro degradation rate of DLPU is 74.6±4.5%, which is slightly faster than the other two groups, and is more suitable for the long-term repair needs of chronic wounds.

[0027] In the in vivo test, the hemostasis time of the gel of the present example is 83.7±6.5s, and the bleeding volume is 0.45±0.09mL, although the hemostasis speed is slightly slower than that of Example 2, the postoperative inflammatory reaction of the wound is extremely slight, and the pathological section shows that the new granulation tissue grows more vigorously at 7 days after operation, and the number of neovascularization is significantly more than that of the control group.

[0028] The composite gel solves the core defect of insufficient mechanical strength of pure chitosan material, and the linear viscoelastic region ensures the structural stability of the gel in the physiological environment; the high storage modulus of 80kPa and the yield strain of 3% enable it to withstand the external impact of high-pressure bleeding scenes and adapt to the activity and irregular shape of the wound, avoiding the collapse of the structure after liquid absorption; the characteristics of rapid gelation within 3 minutes take into account the timeliness and convenience of clinical emergency hemostasis, and are suitable for complex scenes such as acute trauma and surgical wounds.

[0029] As Figure 2 The continuous interconnected porous structure and uniform dispersion of each component in the present example reduce the stimulation of the material to the wound, and cooperate with the anti-inflammatory activity of the coix wine lees protein peptide and the degradable characteristics of DLPU, so that the gel has a hemolysis rate <5% and a 5-day live cell rate of rat adipose stem cells >95%, which meets the medical biocompatibility standard; the porous structure also provides channels for nutrient exchange and cell migration, the DLPU degradation product L-arginine can promote neovascularization, and the coix wine lees protein peptide provides repair nutrients.

[0030] The coix wine lees protein peptide-chitosan composite hemostatic gel as a delivery carrier Hydrophilic small molecules (thioflavin T, rhodamine B and doxorubicin) and / or hydrophobic small molecules (nile red and paclitaxel) are dissolved in the above solution according to the designed content (Mg) and composition. After the solvent is volatilized, a self-assembled gel loaded with guest molecules is obtained.

[0031] The content of the guest molecules in the self-assembled gel was determined by gravimetric method and UV-Vis spectroscopy. The mass of the self-assembled gel was obtained by drying 200 μL of the oligomer solution, and the average value (MFF-GA) was obtained from three samples. For the samples loaded with guest molecules, 200 μL of water was added to the dried samples and shaken. After 1 day, the supernatant was obtained by centrifugation at 8000 rpm for 5 min, and the UV-Vis spectroscopy test was performed.

[0032] When the initial concentration of the guest molecules was 1 mg / mL, the adsorption efficiency of the hydrophilic thioflavin T was about 94.5%, and the loading efficiency was about 17.4%. The adsorption efficiency of the hydrophobic Nile red was 100%, and the loading efficiency was 18.3%. When the hydrophilic thioflavin T and the hydrophobic Nile red were added at the same time, the adsorption efficiency was about 100%, and the loading efficiency was about 30.9%, which was significantly better than the traditional nanomedicine carrier (it is difficult to simultaneously load hydrophilic and hydrophobic guest molecules, and the loading efficiency is difficult to exceed 20%). When the initial concentration was increased to 2 mg / mL, the loading efficiency of Nile red was increased to 30.9%, indicating that the loading amount of the guest molecules can be changed by changing the initial concentration of the self-assembled gel.

[0033] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and the inventive concept of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for preparing a coix seed lees protein peptide-chitosan composite hemostatic gel, characterized in that, Includes the following steps: A. Modification and extraction of Coix lacryma-jobi fermented grain protein peptides: Using alkaline enzymatic hydrolysis-supercritical fluid extraction technology, Coix lacryma-jobi fermented grains were used as raw material. The enzyme was hydrolyzed for 6 hours under the conditions of substrate concentration of 20%, enzyme addition of 1300u / g, 45℃ and pH 9.

0. Then, the mixture was purified by supercritical CO2 fluid extraction to obtain Coix lacryma-jobi fermented grain protein peptides with uniform molecular weight. B. Preparation of composite substrate dispersion: The modified and extracted coix seed lees protein peptide was dissolved in a 5%-10% (w / w) solution of hydrophobic group modified chitosan derivative, and 0.5 g / L-5 g / L of two-dimensional titanium dioxide nanosheet dispersion was added. The mixture was ultrasonically dispersed at 60 W for 30 min to obtain a mixed dispersion. The mass ratio of coix seed lees protein peptide to hydrophobic group modified chitosan derivative was (1:5)-(1:15), and the two-dimensional titanium dioxide nanosheet dispersion accounted for 5%-15% of the volume of the mixed dispersion. C. Constructing the crosslinking system: Add L-arginine-based biodegradable polyurethane and phthalaldehyde-grafted pre-crosslinked hyaluronic acid to the mixed dispersion, stir evenly, and then add 0.5% photoinitiator, wherein the amount of L-arginine-based biodegradable polyurethane added is 5%-10% of the mass of the mixed dispersion, and the amount of phthalaldehyde-grafted pre-crosslinked hyaluronic acid added is 1%-3%; D. Non-covalent anchoring of CBD-thrombin: A CBD-thrombin composite solution with a concentration of 1 mg / mL-5 mg / mL was added to the cross-linking system and gently incubated at 4℃ and 150 r / min for 30-60 min. Non-covalent anchoring of CBD-thrombin in the gel precursor network was achieved through hydrophobic interactions and hydrogen bonds. The amount of CBD-thrombin added was 0.1%-0.5% of the total mass of the mixed system. E. Gel molding: The mixed system is cured by irradiation with a 365nm ultraviolet lamp for 3-5 minutes, and then subjected to 3-5 freeze-thaw cycles to finally obtain a composite hemostatic gel.

2. The preparation method of the coix seed lees protein peptide-chitosan composite hemostatic gel according to claim 1, characterized in that... In step A, 0.1%-0.3% of a metal ion chelating agent is added to the alkaline proteolytic process. The supercritical CO2 extraction pressure is 30-50 MPa, the temperature is 35-45℃, and the extraction time is 2-3 h.

3. The method for preparing a coix seed lees protein peptide-chitosan composite hemostatic gel according to claim 1, characterized in that, The hydrophobic group modified chitosan derivative mentioned in step B is palmitoyl chitosan. Chitosan and palmitoyl chloride react according to the molar ratio of modified raw materials 1:(0.1-0.3), and the molar substitution degree of the palmitoyl chitosan is 5%-20%.

4. The preparation method of the coix seed lees protein peptide-chitosan composite hemostatic gel according to claim 1, characterized in that... In step C, the L-arginine-based biodegradable polyurethane is produced by polymerizing PEG400, isophorone diisocyanate, and L-arginine in a molar ratio of 1:(2.0-2.5):(0.8-1.2). The polymerization reaction is carried out in anhydrous N,N-dimethylformamide (DMF) solvent and refluxed at 80°C for 4-6 hours. The product is purified by dialysis and then freeze-dried.

5. The preparation method of the coix seed lees protein peptide-chitosan composite hemostatic gel according to claim 1, characterized in that, The freeze-thaw cycle reaction conditions described in step E are as follows: freeze-cure at -20℃ for 18 hours to form a gel, thaw at room temperature for 4 hours, repeat 3 times, and the gel porosity is 60%-80%.

6. A coix seed lees protein peptide-chitosan composite hemostatic gel, characterized in that, Prepared by the method described in claim 1, the product comprises the following components and contents: 5%-15wt% hydrophobic modified chitosan derivative, 0.5%-3wt% modified coix seed lees protein peptide, 0.05%-0.5wt% two-dimensional titanium dioxide nanosheets, 5%-10wt% L-arginine-based biodegradable polyurethane, 1%-3wt% phthalaldehyde-grafted pre-crosslinked hyaluronic acid, 0.1%-0.5wt% CBD-thrombin, with the balance being medical-grade deionized water.

7. The composite hemostatic gel according to claim 6, characterized in that, The CBD-thrombin is anchored in the gel network via non-covalent binding.

8. The application of the Coix Seed Wine Residue Protein Peptide-Chitosan Composite Hemostatic Gel as described in claim 6, characterized in that, The applications include: (1) Used to prepare hemostatic dressings for acute trauma, surgical wounds and hypocoagulable conditions; (2) Used for integrated hemostasis and repair treatment of infected wounds and chronic non-healing wounds.

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