CN111714451B

CN111714451B - Anti-angiogenesis micelle for preventing scar generation and preparation method thereof

Info

Publication number: CN111714451B
Application number: CN202010457550.1A
Authority: CN
Inventors: 冯文学; 李杨洪
Original assignee: Ruixi Chongqing Biotechnology Co ltd
Current assignee: Ruixi Chongqing Biotechnology Co ltd
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2022-11-29
Anticipated expiration: 2040-05-26
Also published as: CN111714451A

Abstract

The invention relates to an anti-angiogenesis micelle for preventing scar generation and a preparation method thereof, belonging to the technical field of pharmaceutical preparations. The micelle comprises the following components in percentage by weight: 8-15% of poly beta-arbutin, 4-22% of acceptable auxiliary materials in the skin external preparation and the balance of water. The poly beta-arbutin is added into the micelle, and the substance has high antioxidant activity, can inactivate ROS, and can ensure that mRNA expression and protein secretion of VEGF can not be normally carried out, thereby inhibiting the growth of redundant blood vessels in the wound healing process and further playing a role in preventing scars. In addition, the capacity of the micelle for preventing scars is further improved by adding the active ingredients. The micelle has simple components and better biocompatibility to tissues, and the preparation method is simple, easy to operate and suitable for expanded production.

Description

Anti-angiogenesis micelle for preventing scar generation and preparation method thereof

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an anti-angiogenesis micelle for preventing scar generation and a preparation method thereof.

Background

Scar (scar) is a general term for the morphological and histopathological changes of the appearance of normal skin tissue caused by various traumas, and is an inevitable product in the process of repairing human traumas. When the growth of the scar exceeds a certain limit, various complications can occur, such as disfigurement, dysfunction of functional activities and the like, which bring great physical and mental pains to patients, in particular to the scar left after burns, scalds and serious trauma.

The normal wound healing process is a network regulation process which is jointly participated by a series of physiological and biochemical changes, cells, cell factors, extracellular matrix and the like and mutually regulated, the normal physiological healing process can be influenced and changed by various pathological and physiological conditions, and the excessive proliferation of scars is the result of pathological healing.

A great deal of research indicates that excessive proliferation of fiber cells in scar tissues is accompanied by a great deal of collagen deposition in the tissues, and due to the large form and polarity of the collagen, the activity of key enzymes in the collagen biosynthesis is obviously enhanced, but the matrix degradation is reduced, so the scar formation may be related to the synthesis and reduction imbalance of the collagen, namely, the keloid formation is mainly caused by imbalance between the proliferation and apoptosis of the fiber cells and is influenced by various factors.

At present, the scar treatment mainly adopts operation, radiotherapy and drug treatment, and no specific treatment method exists. Among them, reduction of collagen formation and promotion of apoptosis are the main approaches of drug therapy. Blood circulation is the basis for tissues, organs and cells to perform normal physiological functions and recover from pathological conditions, and also affects the healing of skin wounds and the remodeling of scars. Previous studies have shown that keloid invasive growth and its collagen expression are closely related to changes in blood supply and microcirculation, and anti-angiogenic therapy may be a promising therapeutic approach.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide an anti-angiogenic micelle that prevents scar formation; the second purpose is to provide a preparation method of the anti-angiogenesis micelle for preventing scar generation.

In order to achieve the purpose, the invention provides the following technical scheme:

1. an anti-angiogenesis micelle for preventing scar generation, which comprises the following components in percentage by weight: 8-15% of poly beta-arbutin, 4-22% of acceptable auxiliary materials in the skin external preparation and the balance of water.

Preferably, the micelle further comprises 0.1-3% by weight of an active ingredient.

Preferably, the active ingredient is at least one of ethacridine, etodolac, ketoprofen, pranoprofen, flurbiprofen, meloxicam, acltalide, sulindac, celecoxib, tiaprofenic acid, tenoxicam, naproxen, piroxicam, felbinac, acemetacin, ampiroxicam, amfenac, ibuprofen, zaltoprofen, diclofenac, indomethacin, moxifloxacin, loxoprofen, clobenzaprine, or lornoxicam.

Preferably, the preparation method of the poly beta-arbutin comprises the following steps:

adding beta-arbutin and cuprous chloride-tetramethylethylenediamine into Tris-HCl buffer solution in oxygen atmosphere, reacting at 60-80 deg.C for 1-2d, dialyzing and purifying for 12-24h, and freeze drying.

Preferably, the concentration of the Tris-HCl buffer solution is 0.05mol/L, and the pH value is 7-9; the mass ratio of the beta-arbutin to the cuprous chloride-tetramethylethylenediamine is 1:3-7.

Preferably, the adjuvants include a humectant, a thickener, and a preservative; the humectant accounts for 3-10% of the total weight of the micelle, the thickener accounts for 0.1-2% of the total weight of the micelle, and the preservative accounts for 1-10% of the total weight of the micelle.

Preferably, the humectant is at least one of 1,3-butanediol, 1,3-propanediol, sodium lactate, glycerol, hydroxyethyl urea, sodium hyaluronate, chitin derivative or plant polysaccharide extract; the thickening agent is at least one of polyacrylate cross-linked polymer-6, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, sodium polyacrylate, polyacrylic acid polymer, xanthan gum or hydroxyethyl cellulose; the preservative is at least one of phenoxyethanol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate or sorbitol.

2. The preparation method of the anti-angiogenesis micelle for preventing scar generation comprises the following steps:

dissolving poly beta-arbutin, active ingredients and acceptable auxiliary materials in the skin external preparation in an organic phase, then dripping a water phase at the speed of 10-30 drops/min under the conditions of ice bath and stirring, continuously stirring for 12-24h, dialyzing for 6-12h, and dissolving in water after freeze drying.

Preferably, the volume ratio of the organic phase to the aqueous phase is 1.

Preferably, the organic phase is dimethyl sulfoxide; the aqueous phase is water.

Preferably, the water-soluble substance after freeze-drying is: and testing the content of each component in the freeze-dried product, setting the dosage of the product and water according to the actual requirement on the content of each component in the micelle, and dissolving the product in water.

The invention has the beneficial effects that: the invention provides an anti-angiogenesis micelle for preventing scar generation and a preparation method thereof, wherein poly beta-arbutin is added into the micelle, due to an adjacent effect and a conjugated system, the hydroxyl bond dissociation bond energy of phenolic hydroxyl on the beta-arbutin is reduced, H atoms on hydroxyl (-OH) on a benzene ring are easy to fall off from molecules, the anti-oxidation activity is increased, the poly beta-arbutin has high anti-oxidation activity, and the poly beta-arbutin is combined with peroxy Radicals (ROO), alkoxy Radicals (RO), hydroxyl radicals (& OH) and the like to lose the activity. ROS activates STAT3 to transcriptionally regulate VEGF, mRNA expression and protein secretion of VEGF are promoted, angiogenesis is promoted, poly beta-arbutin with high antioxidant activity can inactivate ROS through the action, mRNA expression and protein secretion of VEGF cannot be normally carried out, accordingly, growth of redundant blood vessels in a wound healing process is inhibited, and the effect of preventing scars is achieved. In addition, the capacity of the micelle for preventing scars is further improved by adding the active ingredients. The micelle has simple components and better biocompatibility to tissues, and the preparation method is simple, easy to operate and suitable for expanded production.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Example 1

An anti-angiogenesis micelle for preventing scar generation comprises the following components in percentage by weight: 10% of poly beta-arbutin, 1% of active ingredient (clobenzaprine), 6.1% of acceptable auxiliary materials in the skin external preparation (5% of humectant [1,3-butanediol ], 0.1% of thickening agent [ xanthan gum ], 1% of preservative [ propyl p-hydroxybenzoate ], and the balance of purified water. The micelle is prepared by the following method:

(1) Adding beta-arbutin and cuprous chloride-tetramethylethylenediamine into a Tris-HCl buffer solution with the concentration of 0.05mol/L and the pH value of 8 in an oxygen atmosphere, reacting at 60 ℃ for 2d, dialyzing and purifying for 18h, and finally freeze-drying to prepare poly beta-arbutin, wherein the mass ratio of the beta-arbutin to the cuprous chloride-tetramethylethylenediamine is 1:5;

(2) Dissolving the poly beta-arbutin prepared in the step (1), the active ingredient (clobenzaprine) and acceptable auxiliary materials (humectant [1,3-butanediol ], thickener [ xanthan gum ] and preservative [ propyl p-hydroxybenzoate ]) in dimethyl sulfoxide, then dropwise adding purified water at a speed of 10 drops/min under the conditions of ice bath and stirring, wherein the volume ratio of dimethyl sulfoxide to dropwise added purified water is 1.

Example 2

An anti-angiogenesis micelle for preventing scar generation comprises the following components in percentage by weight: 15% of poly beta-arbutin, 3% of active ingredient (pranoprofen), 6% of acceptable auxiliary materials (humectant [ sodium hyaluronate ], thickener [ sodium polyacrylate ]1%, preservative [ phenoxyethanol ] 2%) in the skin external preparation, and the balance of purified water. The micelle is prepared by the following method:

(1) Adding beta-arbutin and cuprous chloride-tetramethylethylenediamine into a Tris-HCl buffer solution with the concentration of 0.05mol/L and the pH value of 7 in an oxygen atmosphere, reacting for 1d at 80 ℃, dialyzing and purifying for 24h, and finally freeze-drying to prepare poly beta-arbutin, wherein the mass ratio of the beta-arbutin to the cuprous chloride-tetramethylethylenediamine is 1:7;

(2) Dissolving the poly beta-arbutin prepared in the step (1), the active ingredient (pranoprofen) and acceptable auxiliary materials (humectant [ sodium hyaluronate ], thickener [ sodium polyacrylate ] and preservative [ phenoxyethanol ]) in the skin external preparation in dimethyl sulfoxide, then dropwise adding purified water at the speed of 30 drops/min under the ice bath and stirring conditions, wherein the volume ratio of dimethyl sulfoxide to dropwise added purified water is 1.

Example 3

An anti-angiogenesis micelle for preventing scar generation comprises the following components in percentage by weight: 8% of poly beta-arbutin, 0.1% of an active ingredient (ibuprofen), 13% of acceptable auxiliary materials (a humectant [ glycerin ], a thickener [ hydroxyethyl cellulose ]2%, a preservative [ sorbitol ]) in the skin external preparation, and the balance of purified water. The micelle is prepared by the following method:

(1) Adding beta-arbutin and cuprous chloride-tetramethylethylenediamine into a Tris-HCl buffer solution with the concentration of 0.05mol/L and the pH value of 9 in an oxygen atmosphere, reacting at 70 ℃ for 1.5 days, dialyzing and purifying for 12 hours, and finally freeze-drying to prepare poly beta-arbutin, wherein the mass ratio of the beta-arbutin to the cuprous chloride-tetramethylethylenediamine is 1:3;

(2) Dissolving the poly beta-arbutin prepared in the step (1), the active ingredient (ibuprofen) and acceptable auxiliary materials (humectant [ glycerin ], thickener [ hydroxyethyl cellulose ] and preservative [ sorbitol ]) in the skin external preparation in dimethyl sulfoxide, then dropwise adding purified water at a speed of 20 drops/min under ice bath and stirring conditions, wherein the volume ratio of the dimethyl sulfoxide to the dropwise added purified water is 1.

Comparative examples

According to the weight percentage, 10 percent of beta-arbutin, 1 percent of active ingredient (clobenzaprine) and 6.1 percent of acceptable auxiliary materials (humectant [1,3-butanediol ]5 percent, thickener [ xanthan gum ]0.1 percent and preservative [ propyl p-hydroxybenzoate ]1 percent) in the skin external preparation are added into purified water and evenly mixed to prepare the beta-arbutin solution.

Example 4

Encapsulation efficiency and drug loading measurements of micelles

The products obtained in examples 1 to 3 after freeze-drying were redissolved in dimethyl sulfoxide, the absorbance of the active substance in each micelle was measured by uv-vis spectrophotometry, the content of the corresponding active substance was calculated using the regression equation of the standard curve of the active substance, and the Encapsulation Efficiency (EE) and the drug Loading Capacity (LC) were calculated according to the following formulas, and the results are shown in table 1.

TABLE 1 encapsulation efficiency and drug loading measurements

As can be seen from Table 1, the encapsulation efficiency of the active substances in the micelles in examples 1 to 3 is 73.7 +/-1.9%, 69.87 +/-2.1% and 71.9 +/-1.2% in sequence, and the drug loading is 25.7 +/-0.8%, 23.7 +/-0.6% and 27.1 +/-0.4% in sequence, which indicates that the encapsulation efficiency and the drug loading of the micelles in the invention are better, and the higher drug loading is the key for ensuring the curative effect.

Example 5

Micelle particle diameter and potential measurement

The micelle solutions prepared in examples 1 to 3 were placed in specific cuvettes, respectively, and the particle size, particle size dispersion coefficient (PDI) and Zeta potential of the micelles were measured using a nano-particle size and potential analyzer, the temperature was set at 25 ℃ at a test angle of 90 °, and the test results are shown in table 2.

TABLE 2 particle diameter, particle diameter Dispersion coefficient (PDI) and Zeta potential measurement results

As can be seen from table 2, the micelles in examples 1 to 3 have uniform particle size distribution, good dispersibility, and negative charges, indicating better stability.

Example 6

Determination of micellar antioxidant Properties

The DPPH free radical has a maximum absorption peak near the wavelength of 517nm, when the DPPH free radical reacts with the antioxidant, the absorption value at the wavelength of 517nm is reduced, the reduction degree and the received electrons (the activity of the antioxidant for removing the free radicals) are in a quantitative relation, and the reaction progress is easily monitored by a spectrophotometer.

Taking 3mL of the micellar solution of example 1 and the β -arbutin solution of the comparative example, respectively, the DPPH elimination rate was calculated by first measuring the wavelength at 517nm when DPPH was not added to the two solutions, then measuring the wavelength at 517nm after each addition of 0.5 μ g DPPH, and the measurement results are shown in Table 3, and the DPPH radical scavenging calculation formula is:

in the formula: a. The _{Control group} The absorbance of the free radical was determined for the solution without DPPH

A _{Sample (I)} For the addition of DPPH solution the radical absorbance.

TABLE 3 DPPH Elimination Rate measurement results

As can be seen from Table 3, the elimination rate of DPPH free radical in the micellar solution of example 1 is nearly 50%, and the elimination rate of DPPH free radical in the beta-arbutin solution of the comparative example is nearly 17%, which indicates that poly-beta-arbutin has better antioxidant property than beta-arbutin.

Example 7

Micelle to scar microangiosum and fibroblast proliferation and apoptosis assay

The back of 30 nude mice is disinfected by 75% ethanol, a 0.5cm incision is made at the scapula on the right side, subcutaneous lacuna is formed by latent separation, scar tissues are cut into the size of 5mm multiplied by 8mm after the epidermis is removed, the incision is externally applied with eye ointment and then exposed, the nude mice after operation are transferred into respective cages for breeding, after 20 days, 6 transplant bodies are observed to have white surface color, the transplant bodies and the skin adhesion are not reliably removed, the skin on the surfaces of the other 24 keloid blocks is ruddy, the positions of the scar tissues are fixed and closely adhered with the surrounding skin, blood vessels deeply grow from the periphery, the keloid blocks are shown to be completely alive, namely the nude mouse models are successfully made, the 24 keloid nude mouse models are divided into 3 groups, and each group is 8 and are respectively marked as an example 1 group, a comparative example group and a blank control group. Three groups were injected subcutaneously around keloid scar at postoperative 21, 23, 25, 27d with 0.4mg/0.2mL of the micellar solution of example 1, respectively; in the comparative example, the micelle solution was 0.4mg/0.2mL, and the sodium chloride solution having a concentration of 0.9% was 0.2mL. The number of microvasculature around scar (MVC), fibroblast Proliferation Index (PI), fibroblast Apoptosis Index (AI) were tested daily, and the results showed that the number of microvasculature around scar (MVC) was reduced in the example 1 group after day 10, and the difference was statistically significant compared to the blank control group; the fibroblast Proliferation Index (PI) is obviously reduced compared with a blank control group (P < 0.05), and the fibroblast Apoptosis Index (AI) is obviously increased compared with the blank control group (P < 0.05); compared with the comparative example group, the group of example 1 obviously inhibits the growth of microvessels, reduces the proliferation index of fibroblasts, and promotes the apoptosis of fibroblasts, and the combination of example 6 shows that compared with beta-arbutin, the poly beta-arbutin of the invention has stronger inoxidizability, the capabilities of inhibiting the proliferation of blood vessels and fibroblasts, increases the apoptosis of fibroblasts, and effectively prevents the formation of scars. Data for the number of Microvessels (MVC), fibroblast Proliferation Index (PI), and fibroblast Apoptosis Index (AI) measurements at day 10 around each scar group are shown in table 4.

TABLE 4 microangiocount of scar and fibroblast proliferation and apoptosis assay results for each group

Finally, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An anti-angiogenesis micelle for preventing scar generation is characterized by comprising the following components in percentage by weight: 8 percent of poly beta-arbutin, 0.1 percent of ibuprofen as an active ingredient, 13 percent of acceptable auxiliary material humectant in the skin external preparation and the balance of purified water; wherein, the acceptable auxiliary material humectant consists of 10 percent of glycerin, 2 percent of thickening agent hydroxyethyl cellulose and 1 percent of preservative sorbitol;

the micelle is prepared by the following method:

(2) Dissolving poly beta-arbutin prepared in the step (1), active ingredient ibuprofen and the acceptable auxiliary material humectant in the skin external preparation consisting of glycerol, thickener hydroxyethyl cellulose and preservative sorbitol in dimethyl sulfoxide, then dripping purified water at the speed of 20 drops/min under ice bath and stirring conditions, wherein the volume ratio of the dimethyl sulfoxide to the dripped purified water is 1;

the encapsulation rate of active substances in the micelle is 71.9 +/-1.2 percent, and the drug loading rate is 27.1 +/-0.4 percent.