CN112043818A - Medicine for skin wound and preparation method and application thereof - Google Patents

Abstract

The invention relates to a medicament for treating wound, a preparation method and application thereof, wherein the medicament contains polypeptide and benzalkonium chloride, and the polypeptide has an amino acid sequence shown as SEQ ID NO.1 or an amino acid sequence which is deleted, substituted or added with one amino acid compared with the sequence of the SEQ ID NO. 1. The polypeptide and benzalkonium chloride contained in the medicine for treating skin wounds can form stable gel, and the polypeptide and the benzalkonium chloride have synergistic effect and are beneficial to repairing skin wounds, particularly repairing more than two-stage burn.

Description

A kind of medicine for skin wound and its preparation method and application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a medicine for skin wounds and a preparation method and application thereof.

Background technique

Skin wound repair has always been a difficult problem in the field of clinical medicine. The skin is the largest organ of the human body, a physical barrier between the human body and the external environment, and undertakes many important physiological functions, including perspiration, heat sensation and pain sensation. Once the skin is damaged, it will cause the internal and external environment of the body to become unbalanced, resulting in secondary malnutrition, multiple organ failure and even death. Traditional pro-wound repair drugs have certain limitations, such as high cost, relatively low activity and easy to cause hypertrophic scar healing. Therefore, it is very important and necessary to search for novel pro-wound repair drugs. Compared with traditional wound repairing drugs, polypeptides have the characteristics of high activity, high specificity and high stability. Chinese patent application CN110624097A discloses a small molecule peptide from pearl oyster that can repair skin wounds.

In recent years, more and more nano-drugs (preparations) have entered the sequence of innovative pharmaceutical research and development. Innovative drugs with nano-materials as carriers and the transformation of traditional drugs into new nano-formulations have rapidly become one of the important research contents in the field of nano-biomedicine. Compared with traditional drugs, nano-drugs have better solubility, targeting and slow-controlling properties. There are now many varieties of nano-drugs on the market, such as liposomes, nanoemulsions, nanoparticles, and polymer hydrogels.

Supramolecular hydrogels are aggregates formed by the self-assembly of carbohydrate derivatives, base pairs or small amino acid molecules through non-covalent bonds, and the water content is usually as high as 95%. Hydrogels. Because of its high biocompatibility, low biotoxicity, easy degradation, and easy removal in vivo, it can be called a "traceless" framework for medical engineering. At the same time, the supramolecular structure's plasticity, length tunability, aspect ratio controllability, and high-sequence-level self-assembly can all be "tailored" according to the requirements of the in vivo membrane environment and biological expression signals. Moreover, the three-dimensional nanostructures formed by the self-assembly of gelling factors are in the same order of magnitude as the extracellular matrix, which can provide an environment close to the human body for the migration, growth and differentiation of cells. These advantages make supramolecular hydrogels attractive in the fields of drug delivery, cell culture, and tissue engineering.

In the past two decades, the application of small-molecule hydrogels in the field of medicine has aroused extensive research interest and achieved fruitful research results. The applicant of the present invention attempts to combine small molecule hydrogels and small molecule peptides to propose a drug for skin wound repair using small molecule peptides as carriers.

SUMMARY OF THE INVENTION

In order to provide a drug for skin wounds with good biocompatibility, the present invention provides a polypeptide which can form a gel with active pharmaceutical ingredients and has the effect of repairing wounds. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

One aspect of the present invention relates to a medicament for wounding, which is characterized by containing a polypeptide and benzalkonium chloride, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO. 1 or is similar to the sequence of SEQ ID NO. 1 An amino acid sequence in which any one amino acid is deleted, replaced, or one amino acid added.

In a preferred embodiment of the present invention, the medicament includes a pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, the drug is a gel.

In a preferred embodiment of the present invention, the gelling agent is a hydrogel, and the hydrogelling agent contains CaCl ₂ . The present invention is more favorable for the formation of hydrogel by containing CaCl ₂ .

In a preferred embodiment of the present invention, the mass ratio of the polypeptide and the benzalkonium chloride in the drug is between 10:1 and 1:10, preferably between 5:1 and 1:2. , more preferably between 2:1 and 1:1. The present invention helps to form stable hydrogel by controlling the mass ratio of the polypeptide to benzalkonium chloride.

In a preferred embodiment of the present invention, the pH of the hydrogel is between 7-9. By controlling the pH value of the hydrogel, the present invention can avoid irritation to the wound without affecting the formation of the hydrogel.

Another aspect of the present invention also relates to the preparation method of the above-mentioned medicine, and the preparation method comprises the following steps:

The polypeptide and benzalkonium chloride are dissolved in buffer solution, and hydrogel is formed by shearing.

In a preferred embodiment of the present invention, the pH of the buffer solution is 7-9 and contains CaCl ₂ .

Another aspect of the present invention also relates to the application of the above-mentioned medicament in the preparation of a medicament for repairing skin wounds.

In a preferred embodiment of the present invention, the skin wound is a burn; preferably, the burn is a second degree or higher burn.

beneficial effect

The polypeptide Ser-His-Ser-Gly-Phe-Pho and benzalkonium chloride contained in the medicament for skin wounds of the present invention can form a stable gel, and the synergistic effect of the two can help repair skin wounds, especially Repair burns of level 2 and above.

Description of drawings

FIG. 1 is a graph showing the results of shear rheology experiments of examples of the present invention.

Detailed ways

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. . Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels.

The polypeptide involved in the present invention is Ser-His-Ser-Gly-Phe-Pho, and the peptide sequence is also derived from the small molecule peptide of pearl oyster. The polypeptides used in the examples were prepared by Shanghai Sangong by solid-phase synthesis.

Example 1: Preparation of hydrogels

According to the formula in Table 1 below, the prepared polypeptide was mixed with benzalkonium chloride, and then mixed with a PBS solution containing 100 mM CaCl ₂ and pH 8.0 to prepare a mixed solution, and the obtained mixed solution became viscous after stirring. Hydrogels.

Table 1: Hydrogel ratio

Numbering Polypeptide (wt%) Benzalkonium chloride (wt%%) Mass ratio of peptide to benzalkonium chloride 1 3% 0 - 2 2.5% 0.5% 5:1 3 2.0% 1% 2:1 4 1.5% 1.5% 1:1 5 1.0% 2.0% 1:2

The prepared gel was subjected to a frequency sweep rheology experiment under the condition of 25° C. and 1% strain, and the frequency sweep range was 1 Hz-100 Hz. The experimental results are shown in FIG. 1 . G represents the shear storage modulus and G' represents the shear loss modulus, when G is significantly higher than G' in the entire scanning range, indicating the formation of a hydrogel. It can be seen from the results in Fig. 1 that the polypeptide of the present invention is mixed with benzalkonium chloride in a certain proportion to form a hydrogel with high mechanical strength. Although the specific mechanism of hydrogel formation is not known, it is speculated that the hydrogel is formed due to the interaction between the amino group and carboxyl group in the polypeptide and the ammonium in benzalkonium chloride through hydrogen bonding and van der Waals force.

Example 2: Pharmacodynamic Experiment

1. Animal model preparation:

Hair removal: 5% sodium pentobarbital 4mg/20g, intraperitoneal injection anesthesia, trim back long hair, apply barium sulfide depilatory evenly on the back, remove the hair after a while, and wash the hair removal area with water. After 24 hours, the patients were similarly anesthetized, and the scalded wound was prepared. A water bottle with a temperature of 90°C was placed lightly on the back skin of the mice, and the scalding time was 15s, and then the rats were kept in separate cages at a room temperature of 25°C, with free access to water. At 48 hours post-injury, the appearance of the wounds of the rats was observed, and the skin samples of the wounds were taken, routinely fixed, stained with HE, and observed by light microscope histology to check whether the degree of epidermal burn conformed to the deep second-degree burn wound at this time.

2. Wound repair experiment:

The secretions and necrotic tissue from the wounds of the mouse model were removed, and the wounds were cleaned and disinfected with iodophor solution. According to different groups, 2% benzalkonium chloride saline solution (control group) and hydrogel were sprayed respectively. Apply once every 12 hours in the early burn, and once a day after 3 days. In addition, when the dressing is changed on the 3rd day after the operation, the wound should be cleaned with a saline cotton ball containing gentamicin to remove the liquefied material, and then continue to be applied, once a day, until the skin pieces gradually expand and merge with each other and heal. Calculate the healing rate=(original wound area-unhealed wound area)/original wound area, and the experimental results are shown in Table 2 below.

Table 2: Statistics of healing rate of each experimental example

The preferred embodiments of the present invention have been described above, but are not intended to limit the present invention. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (10)

1. A medicament for wounding, characterized by comprising a polypeptide and benzalkonium chloride, the polypeptide having the amino acid sequence shown in SEQ ID NO. 1 or a deletion or replacement compared to the sequence of SEQ ID NO. 1 Any one of the amino acids or the amino acid sequence with one amino acid added. 2. The medicament of claim 1 comprising a pharmaceutically acceptable carrier. 3. The medicine according to claim 1, which is a gel. 4. The medicine according to claim 3, wherein the gel is a hydrogel, and the hydrogel contains CaCl ₂ . 5. The medicine according to claim 4, wherein the mass ratio of the polypeptide and the benzalkonium chloride in the medicine is between 10:1 and 1:10, preferably between 5:1 and 1:2 between, more preferably between 2:1 and 1:1. 6. The medicament of claim 4, wherein the pH of the hydrogel is between 7-9. 7. A method for preparing a medicament for wounding, the medicament comprising a polypeptide and benzalkonium chloride, the polypeptide having the amino acid sequence shown in SEQ ID NO. 1 or a sequence similar to that of SEQ ID NO. 1 Compared with the amino acid sequence of deletion, replacement of any one amino acid or addition of one amino acid; the preparation method comprises the following steps: The polypeptide and benzalkonium chloride are dissolved in buffer solution, and hydrogel is formed by shearing. 8. The preparation method according to claim 7, wherein the pH value of the buffer solution is 7-9, and contains CaCl ₂ . 9. The application of the medicament according to any one of claims 1-6 in the preparation of a medicament for repairing skin wounds. 10. The application according to claim 9, wherein the skin wound is a burn; preferably, the burn is a second degree or higher burn.

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