CN108837140B - Preparation method and application of earthworm protein microsphere nano wound compound - Google Patents
Preparation method and application of earthworm protein microsphere nano wound compound Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/501—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
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Abstract
The invention discloses a preparation method and application of a earthworm protein microsphere nano wound compound, which comprises the preparation of earthworm protein, the preparation of nano carbon spheres, the preparation of transition metal nano hollow microspheres and the preparation of the earthworm protein microsphere nano wound compound. Compared with the prior art, the invention embeds the protein into the transition metal nanometer hollow microsphere in a physical mode, thereby preparing the medical drug preparation with slow release, induction and multi-site effects to achieve the purpose of promoting wound repair.
Description
Technical Field
The invention relates to the technical field of protein medicine preparation, in particular to a preparation method and application of a earthworm protein microsphere nano wound compound.
Background
The number of patients with various wounds in modern society caused by traffic accidents, accidents (scalds, mechanical injuries and the like), medical operations and the like is increasing. Studies have shown that mortality due to trauma is the fourth in various diseases worldwide. According to statistics, the number of dead people caused by trauma in China is 10 million, and 100 million people are disabled, so that 100 hundred million RMB are directly and economically lost. This figure is still rising as society develops. Post-traumatic repair has therefore been a hot topic of research in the medical field. In recent years, researches show that the traditional Chinese medicine earthworm (earthworm) shows unique curative effect in this aspect. In recent years, a large number of research results show that the earthworm extract can achieve the effect of promoting wound healing by stimulating epithelial cell proliferation.
However, since the protein as a high molecular substance is very easily degraded in nature, the earthworm protein also has the problems of high consumption, high cost and relatively low drug effect in the using process. The nano-drug carrier generally has the structural characteristics of porosity, hollowness, multilayer and the like, and is easy to control the slow release of the drug. By selecting the types and the proportion of carrier materials, the release speed of the drug can be controlled, the drug-loaded nanoparticles with the slow release characteristic can be prepared, the half-life period of the drug is greatly prolonged, and the drug administration times and the drug dosage are reduced. In addition, the nano-drug carrier has large specific surface area and more connected or carried functional groups or active centers, and is easy to realize synchronization of therapeutic effect tracking and treatment. At present, products (Endorem, Feridex and Resovist) which are commercially available as superparamagnetic nanoparticles SPIO (superparamagnetic iron oxide nanoparticles) are all ferroferric oxide nanoparticles covered by polysaccharide, and the particle size of the whole particles is 50nm, but the materials do not have the condition of being used as drug slow release materials.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a preparation method and application of a earthworm protein microsphere nano wound compound.
In order to achieve the purpose, the invention is implemented according to the following technical scheme:
a preparation method of a earthworm protein microsphere nano wound compound comprises the following steps:
s1, preparation of earthworm protein: cleaning fresh Lumbricus with normal saline for three times, removing silt and dirt on body surface, shearing, crushing to obtain cell crushed slurry, adding physiological buffer solution into the cell crushed slurry, centrifuging at 4 deg.C for 5 min at low speed, and removing silt and digesta to obtain cell damaged mixture; preparing 10-70% gradient sucrose solution containing Tris-HCl buffer solution in a centrifugal tube, lightly placing the cell damage mixture on the upper end of the sucrose solution, centrifuging at 2500rpm, respectively collecting the upper layer component, the middle layer component and the lower layer component in the centrifugal tube, and preparing the earthworm protein mixture by a conventional ion exchange method and a gel chromatography method aiming at the middle layer component;
s2, preparing carbon nanospheres: weighing 40g of sucrose, dissolving in 100ml of deionized water, ultrasonically stirring overnight, heating to 200 ℃ to completely carbonize, repeatedly washing with deionized water and ethanol/acetone mixed solution to remove impurities in carbon, and ultrasonically dispersing for 72 hours to obtain nano carbon spheres for later use;
s3, preparing the transition metal nano hollow microsphere, which comprises the following steps:
s31, weighing 0.5mol of transition metal salt MXn, wherein M is transition metal, adding the transition metal salt MXn into the nano carbon spheres which are subjected to ultrasonic dispersion, continuing to perform ultrasonic dispersion in an ethanol/water mixed solution for 72 hours, removing the solution, adding a new ethanol/water mixed solution, and continuing to perform ultrasonic dispersion for 72 hours;
s32, adding 1g of ammonium chloride into S31, continuing to perform ultrasonic dispersion for 1 hour, heating at 150 ℃ for 12 hours, naturally cooling, filtering to obtain a precipitate, washing the precipitate for 3 times by using an ethanol/water mixed solution, and drying at a low temperature of 40 ℃ to obtain a dried product;
s33, heating the dried product prepared in the S32 to 700 ℃ at the heating rate of 0.5 ℃/min, maintaining for 5 hours, then cooling to 50 ℃ at the speed of 1 ℃/min, and cooling at room temperature to obtain the transition metal nano hollow microspheres;
s4, preparing a earthworm protein microsphere nano wound compound: weighing 1g of transition metal nanometer hollow microspheres, adding 2mg/ml earthworm protein mixed solution prepared by mixing earthworm protein mixture and solvent PBS, stirring for 24 hours at 4 ℃, centrifuging to remove supernatant, and depositing to obtain the prepared earthworm protein microsphere nanometer wound compound which is stored at 4 ℃.
Further, when the nano carbon spheres in the S31 are continuously subjected to ultrasonic dispersion in the ethanol/water mixed solution, the nano carbon spheres are mechanically stirred at 80 ℃ for 12 hours every 12 hours.
Further, the pH of the earthworm protein mixture in S4 is 7.5.
In addition, the invention also provides application of the earthworm protein microsphere nano wound compound, and the earthworm protein microsphere nano wound compound is used for healing animal wounds.
Compared with the prior art, the invention embeds the protein into the transition metal nanometer hollow microsphere in a physical mode, thereby preparing the medical drug preparation with slow release, induction and multi-site effects to achieve the purpose of promoting wound repair.
Drawings
FIG. 1(A) is a statistical chart of the 18-day healing rate of Blank group (Blank), positive control group (JWH) and earthworm nano-group (EW) in example 2 of the present invention; (B) a statistical chart of mortality at 3 days and 6 days after trauma of Blank group (Blank), positive control group (JWH) and earthworm nano-group (EW) in example 2 of the invention;
Detailed Description
The present invention will be further described with reference to specific examples, which are illustrative of the invention and are not to be construed as limiting the invention.
Example 1
A preparation method of a earthworm protein microsphere nano wound compound comprises the following steps:
s1, preparation of earthworm protein: cleaning fresh Lumbricus with normal saline for three times, removing silt and dirt on body surface, shearing, pressure crushing with Constant pressure crusher to obtain cell crushed slurry, adding physiological buffer solution (Tris-HCl, 20mM, pH 7.5) into the cell crushed slurry, centrifuging at 4 deg.C for 5 min at low speed, and removing silt and digestate in vivo to obtain cell crushed mixture; preparing 10-70% gradient sucrose solution containing Tris-HCl buffer solution in a centrifugal tube, lightly placing the cell damage mixture on the upper end of the sucrose solution, centrifuging at 2500rpm, respectively collecting the upper layer component, the middle layer component and the lower layer component in the centrifugal tube, and then preparing the earthworm protein mixture by conventional ion exchange method (NaCl 1M, Tris-HCl 20mM cation elution) and gel chromatography (20 mM Tris-HCl is used as eluent) aiming at the middle layer component;
s2, preparing carbon nanospheres: weighing 40g of sucrose, dissolving in 100ml of deionized water, ultrasonically stirring overnight, heating to 200 ℃ to completely carbonize, repeatedly washing with deionized water and ethanol/acetone mixed solution to remove impurities in carbon, and ultrasonically dispersing for 72 hours to obtain nano carbon spheres for later use;
s3, preparing the transition metal nano hollow microsphere, which comprises the following steps:
s31, weighing 0.5mol of transition metal salt MXn, wherein M is transition metal, adding the transition metal salt MXn into the nano carbon spheres which are subjected to ultrasonic dispersion, continuing to perform ultrasonic dispersion in an ethanol/water mixed solution for 72 hours, mechanically stirring at 80 ℃ for 12 hours every 12 hours, removing the solution, adding a new ethanol/water mixed solution, and continuing to perform ultrasonic dispersion for 72 hours;
s32, adding 1g of ammonium chloride into S31, continuing to perform ultrasonic dispersion for 1 hour, heating at 150 ℃ for 12 hours, naturally cooling, filtering to obtain a precipitate, washing the precipitate for 3 times by using an ethanol/water mixed solution, and drying at a low temperature of 40 ℃ to obtain a dried product;
s33, heating the dried product prepared in the S32 to 700 ℃ at the heating rate of 0.5 ℃/min, maintaining for 5 hours, then cooling to 50 ℃ at the speed of 1 ℃/min, and cooling at room temperature to obtain the transition metal nano hollow microspheres;
s4, preparing a earthworm protein microsphere nano wound compound: weighing 1g of transition metal nanometer hollow microspheres, adding 2mg/ml earthworm protein mixed solution with pH of 7.5, which is prepared by mixing earthworm protein mixture and solvent phosphate buffer saline solution PBS, stirring for 24 hours at 4 ℃, centrifuging to remove supernatant, and depositing to obtain the prepared earthworm protein microsphere nanometer wound compound, and storing at 4 ℃.
Example 2
The earthworm protein microsphere nano wound compound prepared in the example 1 can be used for healing animal wounds.
Verification example
Experiments were performed using 4-week-old Balb/C mice, which were adaptively bred for 1 week and then divided into 3 groups, respectively named: blank group (Blank), positive control group (JWH) and earthworm nano group (EW) were tested. 10% chloral hydrate is used for intraperitoneal injection and anesthesia, the experimental mouse is put in a prone position and is placed on a sterile sheet, and the skin on the back is disinfected by iodophor.
The mice were then mechanically depilated on their backs, and a circular incision (2 cm diameter) was made in the back using sterile ophthalmic scissors, creating an open wound model, which was placed in a cage for continued rearing.
The wound of the mouse is coated according to the wound area, and the dosage is 100mg/cm2(calculated by protein mass), the blank group is only coated with normal saline, the positive control group is coated with commercial drugs, and the earthworm nano group is coated with the earthworm protein microsphere nano wound compound prepared in the example 1. Feeding in a single cage, changing the medicine once every 3 days, continuously treating for 27 days, counting the wound healing condition every three days, taking pictures of the wounds of three groups of mice in equal proportion, and analyzing the wound area by ImageJ software; then, after the death of the over-anesthesia, a wound tissue sample is taken and subjected to H&E, staining and observing the recovery condition of the tissues.
As a result of the study, it was found that in the healing process, the earthworm nano-group (EW) coated with the compound healed faster 18 days after the wound than the other two groups, as shown in fig. 1 (a). The mortality rate of mice in the former 3 days is slightly reduced in the earthworm nano-group (EW) coated with the earthworm protein microsphere nano-wound complex compared with the Blank group (Blank) without the drug, while the mortality rate of mice coated with a positive control group (JWH) with a certain commercial drug is higher in the former period, and the mortality rate of mice coated with the positive control group (JWH) is statistically higher than that of the Blank group (Blank) and the earthworm nano-group (EW) coated with the earthworm protein microsphere nano-wound complex at 6 days, as shown in fig. 1 (B). In addition, H & E staining of the newly-born skin of the wound is observed to find that the earthworm nano-group (EW) coated with the earthworm protein microsphere nano-wound compound grows body hair earlier on the newly-born skin, H & E staining experiments are carried out on the newly-born tissue at 28 th day to find that the mouse tissue of the earthworm nano-group (EW) treated by the earthworm protein microsphere nano-wound compound is obviously better in order degree than that of the untreated blank group and the JWH treated positive control group (JWH), the mouse is completely killed by a respiratory anesthesia method at 30 th day, the tensile strength of the newly-born skin is counted empirically after the mice are killed, and the skin toughness of the experimental group treated by the earthworm protein microsphere nano-wound compound is higher than that of the other two groups.
The technical solution of the present invention is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention.
Claims (3)
1. A preparation method of a earthworm protein microsphere nano wound compound is characterized by comprising the following steps:
s1, preparation of earthworm protein: cleaning fresh Lumbricus with normal saline for three times, removing silt and dirt on body surface, shearing, crushing to obtain cell crushed slurry, adding physiological buffer solution into the cell crushed slurry, centrifuging at 4 deg.C for 5 min at low speed, and removing silt and digesta to obtain cell damaged mixture; preparing 10-70% gradient sucrose solution containing Tris-HCl buffer solution in a centrifugal tube, lightly placing the cell damage mixture on the upper end of the sucrose solution, centrifuging at 2500rpm, respectively collecting the upper layer component, the middle layer component and the lower layer component in the centrifugal tube, and preparing the earthworm protein mixture by a conventional ion exchange method and a gel chromatography method aiming at the middle layer component;
s2, preparing carbon nanospheres: weighing 40g of sucrose, dissolving in 100ml of deionized water, ultrasonically stirring overnight, heating to 200 ℃ to completely carbonize, repeatedly washing with deionized water and ethanol/acetone mixed solution to remove impurities in carbon, and ultrasonically dispersing for 72 hours to obtain nano carbon spheres for later use;
s3, preparing the transition metal nano hollow microsphere, which comprises the following steps:
s31, weighing 0.5mol of transition metal salt MXn, wherein M is transition metal, adding the transition metal salt MXn into the nano carbon spheres which are subjected to ultrasonic dispersion, continuing to perform ultrasonic dispersion in an ethanol/water mixed solution for 72 hours, removing the solution, adding a new ethanol/water mixed solution, and continuing to perform ultrasonic dispersion for 72 hours;
s32, adding 1g of ammonium chloride into S31, continuing to perform ultrasonic dispersion for 1 hour, heating at 150 ℃ for 12 hours, naturally cooling, filtering to obtain a precipitate, washing the precipitate for 3 times by using an ethanol/water mixed solution, and drying at a low temperature of 40 ℃ to obtain a dried product;
s33, heating the dried product prepared in the S32 to 700 ℃ at the heating rate of 0.5 ℃/min, maintaining for 5 hours, then cooling to 50 ℃ at the speed of 1 ℃/min, and cooling at room temperature to obtain the transition metal nano hollow microspheres;
s4, preparing a earthworm protein microsphere nano wound compound: weighing 1g of transition metal nanometer hollow microspheres, adding 2mg/ml earthworm protein mixed solution prepared by mixing earthworm protein mixture and solvent PBS, stirring for 24 hours at 4 ℃, centrifuging to remove supernatant, and depositing to obtain the prepared earthworm protein microsphere nanometer wound compound which is stored at 4 ℃.
2. The method for preparing the earthworm protein microsphere nano wound compound according to claim 1, which is characterized in that: and mechanically stirring the nano carbon spheres in the S31 at 80 ℃ for 12 hours every 12 hours when the nano carbon spheres continue to be dispersed in the ethanol/water mixed solution by ultrasound.
3. The method for preparing the earthworm protein microsphere nano wound compound according to claim 1, which is characterized in that: the pH of the earthworm protein mixed liquor in S4 is 7.5.
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| CN109529012A (en) * | 2019-01-10 | 2019-03-29 | 陕西中医药大学 | A kind of metal oxide nano bletilla striata pheretima albumen composition preparation method and application |
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