Temperature-sensitive spray loaded with yak bone peptide as well as preparation method and application of temperature-sensitive spray

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a temperature-sensitive spray for promoting healing of a wound difficult to heal, in particular to a spray taking yak bone peptide as an active component and poloxamer as a temperature-sensitive matrix as well as a preparation method and application of the spray. The spray is prepared from the following components in percentage by weight / volume: 0.01 to 1.5 percent of yak bone peptide solution, 10 to 20 percent of poloxamer F127 and the balance of buffer solution. The spray is a solution capable of freely flowing in an environment below 25 DEG C, can be converted into semi-solid gel within 1 minute after being in contact with a wound surface at 32-37 DEG C, and has the advantages of convenience in use, in-situ gelation, good drug retention, capability of sustainably releasing drugs and capability of effectively promoting healing of the wound difficult to heal. The yak bone peptide has certain free radical scavenging and metal ion chelating capabilities, can relieve oxidative stress injury, optimizes a cell repair microenvironment, and can prevent chronic development of a wound healing process by regulating inflammatory factor expression and inhibiting early excessive inflammatory response.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a temperature-sensitive spray for promoting the healing of refractory wounds, in particular to a spray taking yak bone peptide as an active ingredient and poloxamer as a temperature-sensitive matrix, and a preparation method and application thereof. BACKGROUND

[0002] Refractory wounds, such as diabetic foot ulcers, venous leg ulcers and pressure sores, have become a serious clinical challenge due to their long healing period, susceptibility to infection and high treatment cost. The core pathological mechanism lies in the stagnation of the wound healing process in the persistent inflammatory stage, failing to normally enter the proliferation and remodeling period.

[0003] Currently, clinically commonly used wound dressings, such as traditional gauze, new functional dressings and even negative pressure treatment technology, all have different limitations. For example, traditional dressings are prone to adhesion to the wound surface, causing secondary damage, and lack the ability to actively promote healing; new dressings have limited ability to manage exudates and individualize drug delivery; negative pressure treatment is expensive and has poor portability, and lacks the "intelligent" biological function of regulating the microenvironment of the wound.

[0004] Yak bone peptide is a mixture of small peptides extracted from yak bone through biological enzymatic hydrolysis technology. Existing studies have shown that it has multiple biological activities such as promoting fibroblast proliferation, antioxidant and anti-inflammatory, but its application in the treatment of refractory wounds, especially in combination with modern intelligent dosage forms, is still in the blank.

[0005] Poloxamer F127 is a triblock copolymer with temperature-sensitive properties. Its solution is liquid at room temperature and can quickly change to gel near body temperature, making it very suitable as a matrix for spray to achieve convenient spraying and long-term retention of drugs in situ.

[0006] Therefore, the development of a new type of preparation that combines the multi-effect biological activity of yak bone peptide and the intelligent drug delivery advantage of temperature-sensitive gel has important significance and broad application prospects for solving the clinical problems of refractory wounds. SUMMARY

[0007] In view of the above problems, the present application provides a temperature-sensitive spray loaded with yak bone peptide and a preparation method and application thereof. The spray has the advantages of being convenient to use, in-situ gelation, good drug retention, sustained release of drugs and effective promotion of the healing of refractory wounds. The yak bone peptide solution used in the present application is YBP, and the prepared YBP / F127 spray is convenient, slow-release and has the effect of promoting the recovery of refractory wounds, and has good application prospects.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: The first aspect of the present application provides a temperature-sensitive spray loaded with yak bone peptide, namely YBP / F127 spray, wherein the yak bone peptide YBP is the main active ingredient, and the spray is composed of the active ingredient yak bone peptide YBP, the temperature-sensitive matrix poloxamer F127 and the solvent, and consists of the following ingredients by weight / volume percentage: 10%-20% of F127, 0.01-1.5% of YBP, and the balance of PBS.

[0009] Further, the content of F127 is 17% (w / v).

[0010] Further, the content of YBP is 0.5% (w / v).

[0011] The second aspect of the present application provides a preparation method of the above-mentioned YBP / F127 spray, which comprises the following steps: (1) dissolving poloxamer F127 in a buffer solution at 4°C, stirring until completely dissolved to obtain a poloxamer blank matrix solution; (2) dissolving yak bone peptide in the blank matrix solution obtained in step (1) and mixing uniformly at 4°C to obtain a temperature-sensitive solution loaded with yak bone peptide; (3) filtering the temperature-sensitive solution obtained in step to remove bacteria and filling in a spray device.

[0012] Further, the buffer solution in step a) is a phosphate buffer solution; and the stirring time is 12 hours.

[0013] Further, the filtering in step (c) is performed using a 0.22 μm microporous filter membrane.

[0014] The third aspect of the present application provides an antioxidant and anti-inflammatory pharmaceutical composition, wherein the effective ingredient comprises the temperature-sensitive solution prepared by the above-mentioned preparation method, namely YBP / F127 solution.

[0015] The fourth aspect of the present application provides the use of YBP / F127 spray preparation for promoting the recovery of difficult-to-heal wounds, preferably difficult-to-heal wounds, including but not limited to diabetic foot ulcers, venous leg ulcers, pressure sores and burn wounds.

[0016] The effect in wound repair is verified in some specific embodiments of the present application, and at a certain concentration, the YBP / F127 spray preparation can play a role in promoting the proliferation of fibroblasts, and can be used in the field of difficult-to-heal wound repair.

[0017] The present application has the following beneficial effects compared with the prior art: 1. The temperature-sensitive properties of poloxamer F127 are utilized to achieve the perfect combination of the convenience of liquid spraying and body temperature triggered gelation. After gelation, it can tightly adhere to irregular wounds, form a physical barrier, significantly improve the retention time of drugs on the wound surface, and avoid rapid loss due to flushing of exudate.

[0018] 2. The formed gel can act as a repository for yak bone peptide, achieving sustained and controlled release of the drug. Yak bone peptide itself has multiple functions of antioxidant, anti-inflammatory and promoting fibroblast / vascular endothelial cell proliferation and migration, which can break the vicious cycle of difficult-to-heal wounds from multiple targets and promote granulation tissue growth and re-epithelialization.

[0019] 3. Poloxamer is an FDA-approved pharmaceutical excipient with good biocompatibility. Yak bone peptide is derived from natural food sources and has high safety. The spray prepared by combining the two has no cytotoxicity and can provide a moist healing environment to reduce the pain of dressing change. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the solution-gel phase transition behavior of the prepared temperature-sensitive spray loaded with yak bone peptide.

[0021] Figure 2 is a temperature scanning diagram of 17% F127 loaded with yak bone peptide at 15-40℃.

[0022] Figure 3 is a viscosity change diagram of 17% F127 loaded with yak bone peptide at 4℃, 25℃ and 32℃.

[0023] Figure 4 is a scanning electron microscope (SEM) characterization diagram of yak bone peptide YBP / poloxamer F127 gel solution.

[0024] Figure 5 is a column chart of the effect of different concentrations of yak bone peptide YBP solution on the survival rate of 3T3 cells.

[0025] Figure 6 is a column chart of the effect of different concentrations of yak bone peptide YBP solution on the adhesion of 3T3 cells.

[0026] Figure 7 is a schematic diagram of the use of the temperature-sensitive spray prepared by the present application.

[0027] In the corresponding drawings, the yak bone peptide is abbreviated as YBP group, the poloxamer group is abbreviated as F127 group, and the yak bone peptide / F127 solution group is abbreviated as YBP@F127 group. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further explained below in combination with the embodiments. Based on the content disclosed in the present application, those skilled in the art can implement the present application by using other various specific embodiments, or any simple change or modification made by using the design structure and ideas of the present application also falls within the protection scope of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0029] In the following examples, the yak bone peptide solution was provided by Shanghai Fengyu Biological Technology Co., Ltd., the DMEM culture medium and the FBS were purchased from Shanghai Titan Technology Co., Ltd.

[0030] Example 1 Sample preparation: 17 g of poloxamer F127 was accurately weighed in a beaker. About 83 mL of pre-chilled phosphate buffered saline solution was measured, and the poloxamer F127 powder was slowly added to the cold water in small amounts under stirring until it was fully dissolved, and then placed in a 4℃ refrigerator overnight for swelling. A 17% (w / v) clear and transparent poloxamer blank gel was obtained. 5 mg / ml of yak bone peptide solution was added to the above blank gel, and shaken uniformly to obtain a uniform temperature-sensitive solution containing 0.5% yak bone peptide. The final solution was filtered through a 0.22 μm sterile microporous filter to remove bacteria, and then was divided into sterile spray bottles for storage at 4℃ for standby use. As shown in Figure 1 The solution-gel phase transition behavior diagram of the prepared yak bone peptide-loaded temperature-sensitive spray is shown in the figure. As can be seen from the figure, the yak bone peptide-loaded F127 is in a flowable solution state at 25℃, and is converted into a non-flowable gel state at 32℃.

[0031] Figure 7 The temperature-sensitive spray prepared by the present application is shown in the figure. The yak bone peptide-loaded F127 is in a flowable solution state at room temperature 25℃, and is sprayed out of the spray bottle at the wound site, and forms a gel at body temperature about 32℃, tightly adheres to the irregular wound surface, forms a physical barrier, significantly improves the residence time of the drug on the wound surface, and plays a role of anti-inflammatory, antioxidant and the like.

[0032] Example 2 The spray prepared in Example 1 was tested by using a rheometer to evaluate the viscosity change of the temperature-responsive hydrogel. The dynamic viscosity of the sample was measured at a constant shear rate (0.4 s -1 ), respectively, at fixed temperatures 4℃, 25℃ and 32℃. From the results of Figure 2 , it can be seen that the dynamic viscosity of the hydrogel at the physiological temperature of the human body (32℃) is always much greater than that at room temperature and low temperature.

[0033] Example 3 The aerosol prepared in Example 1 was tested using a rheometer to evaluate the rheological characteristics of the temperature-responsive hydrogel. The modulus of the hydrogel was measured at a constant frequency (6 Hz) as the temperature increased from 15 °C to 40 °C at a rate of 5 °C / min. Figure 3 Rheological analysis of the hydrogel showed that when the temperature was in the range of 30-40℃, the storage modulus G' was always greater than the loss modulus G", indicating that a relatively stable three-dimensional network structure was formed, which has elastic and solid-like rheological properties.

[0034] Example 4 The gel sample prepared in Example 1 of this invention was used to evaluate the microstructure of YBP@F127 using a scanning electron microscope (SEM) (TM3030Plus, Japan). First, the sample was frozen at -80°C for 2 hours, then dehydrated using a freeze dryer. The surface of the freeze-dried sample was then sputter-coated with gold, and the resulting images were analyzed using scanning electron microscopy. Figure 4 Scanning electron microscopy analysis showed that the addition of YBP did not significantly change the hydrogel network structure or pore size.

[0035] Example 5 3T3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and maintained at 37°C in an incubator at 95% aerosol / 5% CO2. Cells in logarithmic growth phase were used for experiments. Cell proliferation assay (MTT assay): The predicted number of cells were seeded in 96-well plates and cultured for 24 hours. YBP solution of various concentrations was administered, followed by further incubation with DMEM for 24 hours. 20 μL MTT reagent was added to each well, and incubation continued at 37°C for 4 hours. The medium was discarded, 150 μL LDMSO was added, and the plate was shaken for 10 minutes. The absorbance (OD value) was measured at 570 nm using a microplate reader. Figure 5 As shown, a solution with a concentration of 5 mg / ml significantly promoted cell proliferation, and therefore this concentration was determined to be the optimal working concentration for subsequent experiments.

[0036] Example 6 Different concentrations of YBP solution were plated in 96-well plates and incubated in an incubator at 37°C in an atmosphere of 95% air / 5% CO2for 2 h. Excess coating solution was removed from the wells and 1% BSA-PBS solution was added and incubated at 37°C in a 5% CO2cell incubator for 1 h. After removing excess liquid, the wells were washed three times with D-PBS and the washings discarded. The wells were sealed with sealing film and stored at 4°C until use. 3T3 cells, grown as described in Example 6, were seeded into the 96-well plates and incubated for 1 h. The cell solution was then removed and 20 μL of MTT reagent was added and the plates incubated at 37°C for 4 h. The medium was discarded and 150 μL of DMSO was added and the plates shaken for 10 min. The absorbance (OD value) was measured at 570 nm using a microplate reader as shown in Table 1. Figure 6 As shown in Table 1, the adhesion of the solution at a concentration of 5 mg / ml was significantly increased.

[0037] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. A thermosensitive spray loaded with yak bone peptides, characterized in that, The spray is composed of the active ingredient yak bone peptide, the thermosensitive matrix poloxamer F127, and a solvent; the mass-volume concentration of the yak bone peptide in the spray is 0.01%~1.5%, the mass-volume concentration of the poloxamer F127 in the spray is 10%~20%, and the remainder is solvent; the spray is in a solution state below 25°C and can be transformed into a semi-solid gel state at 32-37°C.

2. The temperature-sensitive spray according to claim 1, characterized in that, The mass-volume concentration of the yak bone peptide is 0.5%.

3. The temperature-sensitive spray according to claim 1, characterized in that, The mass-volume concentration of poloxamer F127 is 17%.

4. The temperature-sensitive spray according to claim 1, characterized in that, The solvent is a phosphate buffer solution.

5. A method for preparing a thermosensitive spray loaded with yak bone peptides as described in any one of claims 1-4, characterized in that, Includes the following steps: (a) Dissolve poloxamer F127 in buffer solution at 4–8°C and stir until completely dissolved to obtain a poloxamer blank matrix solution; (b) Dissolve yak bone peptide in the blank matrix solution obtained in step (a) and mix evenly at 4–8°C to obtain a thermosensitive solution loaded with yak bone peptide; (c) Filter the thermosensitive solution obtained in step (b) to remove bacteria and fill it into a spray device.

6. The method according to claim 5, wherein the buffer solution in step a) is a phosphate buffer solution and the stirring time is 12 hours; and the filtration in step (c) is performed using a 0.22 μm microporous membrane.

7. An antioxidant and anti-inflammatory pharmaceutical composition, characterized in that, Its active ingredient comprises the thermosensitive solution prepared according to claim 5 or 6.

8. Use of the thermosensitive spray loaded with yak bone peptides as described in any one of claims 1-3 in the preparation of a medicament for promoting wound healing.

9. The use according to claim 8, characterized in that, The wound is a difficult-to-heal wound.

10. The use according to claim 9, characterized in that, The difficult-to-heal wound is selected from at least one of diabetic wounds, venous leg ulcers, or pressure sores.

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