CN114949181B - A repairing lotion containing epidermal growth factor microcapsules and preparation method thereof - Google Patents
A repairing lotion containing epidermal growth factor microcapsules and preparation method thereof Download PDFInfo
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Abstract
本发明提供一种含有表皮生长因子微胶囊的修复乳及其制备方法，通过微胶囊技术将表皮生长因子包裹在具有囊壁结构的微胶囊中，再将微胶囊加入基液中制备修复乳，一方面，本发明的微胶囊囊壁结构具有很好的机械性能，提供的制备方法能够很好地将水性的表皮生长因子成功包裹到囊壁结构中，另一方面，本发明的表皮生长因子在使用过程中能够避免被外界物质所氧化，保持较长时间的药效。
The present invention provides a repairing lotion containing epidermal growth factor microcapsules and a preparation method thereof. The epidermal growth factor is encapsulated in a microcapsule with a capsule wall structure by microencapsulation technology, and the microcapsule is added into a base liquid to prepare the repairing lotion. On the one hand, the capsule wall structure of the microcapsule of the present invention has good mechanical properties, and the preparation method provided can successfully encapsulate the aqueous epidermal growth factor into the capsule wall structure. On the other hand, the epidermal growth factor of the present invention can avoid being oxidized by external substances during use and maintain the efficacy for a long time.
Description
Technical Field
The invention relates to the technical field of medicine manufacturing, in particular to an epidermal growth factor microcapsule and a preparation method of repair emulsion containing the same.
Background
Basic research and clinical practice prove that the wound healing process of human body is mainly regulated and controlled by growth factors, and in the wound healing process, certain growth factors are exogenously supplemented or endogenous growth factor activity is stimulated by the exogenously growth factors, so that the effect of promoting wound repair can be achieved. For example, some chronic wound surfaces which are difficult to heal are not healed for a long time, and the main reason is that the wound surfaces lack inflammatory reaction and the release and growth stimulation effect of endogenous growth factors, and meanwhile, tissue repair cells (epithelial cells and fibroblasts) are in a dormant state, and when the platelet-derived growth factors, the fibroblast growth factors, the epidermal growth factors and the like are exogenously applied, the tissue repair cells can be directly acted, and the repair process is started. Among them, epidermal growth factor plays an important role in wound repair. The existing researches show that the epidermal growth factor is a stimulus for cell division and proliferation, can promote the migration of wound cells, accelerate the growth of granulation tissues and increase the components such as DNA, RNA, collagen fibers, hyaluronic acid and the like in the granulation. Epidermal growth factor is capable of stimulating proliferation and migration of keratin cells required for wound healing. Animal and clinical studies show that the epidermal growth factor can obviously promote wound healing, especially has better effect on some difficult-to-heal wounds, and the prior studies find that after severe skin injury caused by burns and the like occurs, it is very important to cover the wounds with proper dressing, so that the loss of body fluid and protein of the wounds can be reduced, bacterial infection and secondary tissue injury can be prevented, and in some cases, a bracket can be provided for proliferation cells to promote wound healing. This has been a research hotspot in the field of wound repair of severe burns and the like in the last two decades. The wound dressing loaded with the growth factors has good application prospect, wherein the wound dressing is represented by a novel wound dressing containing the epidermal growth factors.
The epidermal growth factor is stable to heat, can be kept stable for a long period of time at-20 ℃ and is still stable after being boiled for 30min at 100 ℃ in a neutral solution, but is inactivated in 0.1NNaOH or 0.2NHCI at 100 ℃. Resistant to digestion by trypsin, chymotrypsin and pepsin. The epidermal growth factor molecule does not contain alanine, phenylalanine and lysine. Arginine at position 53 and leucine at position 52 are often removed by proteases, so that the EGF amino-terminal 40 amino-acids have the full activity of the factor. The spatial structure of EGF molecule can be divided into N section and C section which are relatively independent, and these two sections are relatively rigid, and are respectively connected by two pairs of disulfide bonds and one pair of disulfide bonds which are relatively flexible, so that the spatial positions of two sections are relatively variable, and the whole molecule is relatively flexible.
The biological effect of the epidermal growth factor ① is that epithelial cell division and proliferation are promoted, and the epidermal growth factor can be applied to skin wound healing. ② Epidermal growth factor is associated with certain cancerous lesions. ③ EGF regulates endocrine glands. ④ Has regulation and control effects on respiratory system. ⑤ Plays an important role in the occurrence and development of diabetes. ⑥ Has effects on reproductive system, on male, and epidermal growth factor affecting testis development and spermatogenesis, and on female, epidermal growth factor regulating ovary development and reproductive function.
However, the biggest problem of the epidermal growth factor used as the exogenous growth factor in the use process of wound healing treatment is that the epidermal growth factor is easily oxidized by external substances such as air and the like, and then loses activity, so that the acting time is short and the treatment effect is poor.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides repair emulsion containing an epidermal growth factor microcapsule and a preparation method thereof.
In order to achieve the aim, the technical scheme is that the repair emulsion containing the epidermal growth factor microcapsule comprises a repair emulsion matrix, and is characterized in that the matrix also comprises the epidermal growth factor microcapsule, the microcapsule comprises a wall material and a capsule core material, the capsule core material is an epidermal growth factor stock solution, the wall material is a polymer formed by crosslinking genipin citrate and gelatin chitosan, and the chemical structural formula of the polymer is shown as formula (I):
In the formula (I), R1 to R9 are selected from eighteen different amino acids of glycine, alanine, serine, aspartic acid, glutamic acid amino, proline, arginine, histidine, tyrosine, cystine, leucine, threonine, methionine, valine, phenylalanine, tryptophan, glutamic acid and lysine.
Preferably, it is characterized in that in the formula (I):
R5 and R6 are selected from eighteen residues of different amino acids glycine, alanine, serine, aspartic acid, glutamic acid amino, proline, arginine, histidine, tyrosine, cystine, leucine, threonine, methionine, valine, phenylalanine, tryptophan, glutamic acid and lysine;
R3 and R7 are residues of lysine or arginine;
r2, R4 and R8 are residues of aspartic acid or glutamic acid amino;
r1 and R9 are serine, threonine or tyrosine residues.
The invention also provides a preparation method of the repair emulsion containing the epidermal growth factor microcapsule, which specifically comprises the following steps:
(1) Preparing a repair milk matrix;
(2) Preparing an epidermal growth factor microcapsule, which comprises the following specific steps of;
a. dissolving gelatin in acetic acid aqueous solution to obtain gelatin acetic acid solution;
b. Adding chitosan into gelatin acetic acid solution, stirring to dissolve chitosan to obtain gelatin/chitosan mixed solution, adding epidermal growth factor stock solution, and adjusting pH to 5.8-6.2;
c. adding soybean lecithin as a surfactant into vegetable oil, heating and uniformly stirring;
d. adding the gelatin chitosan mixed solution prepared in the step b into the vegetable oil after the step c for heating and emulsifying, closing heating after the emulsification is completed, and naturally cooling to room temperature;
e. adding genipin solution into the solution system after the step d for crosslinking reaction, and obtaining genipin Ping Mingjiao chitosan microcapsules after full reaction;
f. Adding a citric acid aqueous solution into the reaction system after the step e, adding glacial acetic acid, regulating the pH to 2-3, and fully reacting under the protection of N 2 to obtain an epidermal growth factor microcapsule;
(3) And (3) adding microcapsules with the capsule core material being the epidermal growth factor into the repairing emulsion matrix in the step (1), and uniformly mixing to obtain the repairing emulsion containing the epidermal growth factor microcapsules.
Preferably, the step (1) specifically includes the following steps:
a1. mixing fructus Rosae Davuricae oil, oleum Olivarum, vaseline, isopropyl myristate and glyceryl monostearate, and stirring at 200-300rpm at 85deg.C to obtain a first mixture;
b1. mixing glycerol, polyethylene glycol 400 and triethanolamine, and stirring at 200-300rpm at 85deg.C to obtain a second mixture;
c1. Adding the second mixture into the first mixture, adding essence, antiseptic and deionized water, stirring at 2000-3000rpm for 30-60min, cooling to room temperature at 400-500rpm, and obtaining the repairing emulsion matrix.
Preferably, the rose hip oil is characterized in that the rose hip oil is prepared from, by weight, 10-20 parts of rose hip oil, 10-20 parts of olive oil, 6-10 parts of Vaseline, 1-3 parts of isopropyl myristate and 1-3 parts of glyceryl monostearate, 3-7 parts of glycerin, 3-7 parts of polyethylene glycol 400, 3-6 parts of triethanolamine, 0.2-0.8 part of essence, 0.2-0.8 part of preservative and the balance deionized water. It is further preferred that 15 parts of rose hip oil, 15 parts of olive oil, 8 parts of vaseline, 2 parts of isopropyl myristate and 2 parts of glyceryl monostearate are mixed according to weight parts, 5 parts of glycerin, 400 parts of polyethylene glycol, 4 parts of triethanolamine, 0.5 part of essence, 0.5 part of preservative and 43 parts of deionized water.
Preferably, the preservative is methyl parahydroxybenzoate or a complex of methyl parahydroxybenzoate and ethyl parahydroxybenzoate.
Preferably, the step f further includes:
g. And d, standing the reaction system after the step f, pouring out the upper oil phase, centrifuging, and separating out the oil phase to obtain the purified epidermal growth factor microcapsule.
Preferably, in the step b, the pH is preferably adjusted to 6, the vegetable oil in the step c is one or more of corn oil, olive oil, soybean oil and peanut oil, and the specific step of the step e is that 0.5% of genipin aqueous solution is added into the solution system after the step d, and the mixture is reacted at room temperature for 15 h, so that the genipin crosslinked gelatin/chitosan microcapsule is obtained after full reaction.
Preferably, the specific step of the step f is that citric acid is added into the reaction system of the step e, glacial acetic acid is added, the pH is regulated to 2-3, the reaction temperature is increased to 40 ℃ under the protection of N 2, and the reaction is carried out for 8: 8 h and then cooled to room temperature, so that the epidermal growth factor microcapsule is obtained.
Preferably, the components in the step (2) are 45-55 parts of gelatin, 3-7 parts of chitosan, 250-350 parts of 1.0% acetic acid solution, 40-60 parts of soybean phospholipid, 30-40 parts of 0.5% genipin, 5-15 parts of 1.0% citric acid and 2500-3000 parts of vegetable oil. Further preferably, the composition comprises, by weight, 50 parts of gelatin, 5 parts of chitosan, 300 parts of 1.0% acetic acid solution, 50 parts of soybean phospholipid, 35 parts of 0.5% genipin, 10 parts of 1.0% citric acid and 2500-3000 parts of vegetable oil.
Preferably, the mass ratio of the gelatin to the chitosan is 8:1 to 12:1, the volume ratio of the water phase to the oil phase is 1:3 to 1:5, and more preferably, the mass ratio of the gelatin to the chitosan is 10:1, and the volume ratio of the water phase to the oil phase is 1:4.
Preferably, the mass ratio of the matrix to the epidermal growth factor microcapsule is 80-90:20-10.
Compared with the prior art, the preparation method has the beneficial effects that the epidermal growth factor is wrapped in the microcapsule with the capsule wall structure by the microcapsule technology, on one hand, the microcapsule capsule wall structure has good mechanical property, and the preparation method can successfully wrap the water-based epidermal growth factor in the capsule wall structure, and on the other hand, the epidermal growth factor can be prevented from being oxidized by external substances in the use process, and the drug effect is maintained for a long time.
Drawings
FIG. 1 is a graph of isoelectric points of gelatin, chitosan, and chitosan/gelatin of example 1 at different mass ratios;
FIG. 2 is a thermogravimetric plot of gelatin, chitosan, gelatin/chitosan complex, genipin cross-linked gelatin chitosan and citric acid secondary cross-linked gelatin/chitosan.
FIG. 3 shows the morphology of the microcapsules under a microscope during the preparation of the microcapsules by using genipin as a cross-linking agent and gelatin and chitosan as capsule walls, wherein (a) is emulsified for 30min, (b) is emulsified for 60min, (c) is cross-linked for 3h, and (d) is cross-linked for 15 h.
FIG. 4 is a graph showing the absorbance of cells at different concentrations of EGF in example 5.
FIG. 5 is a photograph taken by a fluorescence microscope of example 5, wherein (a) is a control group, (b) is an EGF microcapsule, (c) is an EGF stock solution, and (d) is an EGF stock solution.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
EXAMPLE 1 isoelectric point measurement of gelatin, chitosan, gelatin-chitosan composite
1.1 Isoelectric point of gelatin
0.5 G gelatin was weighed, added with 100ml deionized water, and stirred at 50 ℃ water bath temperature until completely dissolved, yielding a 0.5% gelatin solution. The pH was adjusted using 0.001 mol/L HCl solution and 0.001 mol/L NaOH solution, and the conductivity of the gelatin solution at different pH values was recorded using a pH meter and conductivity meter.
1.2 Isoelectric point of chitosan
0.5 G chitosan is weighed and added into 100ml of 0.01 mol/L HCl solution, and the mixture is stirred at room temperature until the chitosan is completely dissolved, thus obtaining 0.5 percent chitosan solution. The pH of the solution was adjusted using a 0.001 mol/L NaOH solution and the conductivity of the gelatin solution at different pH values was recorded using a pH meter and conductivity meter.
1.3 Isoelectric point of chitosan/gelatin composite material
100 ML of a 1.0% gelatin solution was placed in a beaker and magnetically stirred with a water bath at 50 ℃. Adding a certain volume of 1.0% chitosan solution into the gelatin solution, and stirring for 1h to obtain gelatin/chitosan uniform mixed solution. The volume ratio of chitosan to gelatin is 4:100, 10:100, 20:100, 50:100, 75:100, 100:100 respectively. The pH of the mixed solution was changed using a 1.0% HCl solution and a 0.1 mol/L NaOH solution, and the conductivity of the gelatin/chitosan solution was measured at different pH values using a pH meter and a conductivity meter.
The isoelectric points of the gelatin, the chitosan and the chitosan/gelatin with different mass ratios are measured according to the method shown in the table 1, and the isoelectric point curves of the gelatin, the chitosan and the chitosan/gelatin with different mass ratios are shown in the table 1.
TABLE 1 isoelectric points of gelatin, chitosan and different mass ratios of isoelectric points of chitosan/gelatin
| CS/GE mass ratio | 1:0Chitosan | 1:25 | 1:10 | 1:2 | 1:1 | 0:1Gelation |
| Isoelectric point/EEP value | 5.5 | 6.0 | 3.5 | 4.5 | 4.0 | 5.0 |
Example 2 preparation method of epidermal growth factor microcapsule repair emulsion
(1) Preparing a repair milk matrix;
a. Mixing 15g of rose-hip oil, 15g of olive oil, 8g of vaseline, 2g of isopropyl myristate and 2g of glycerol monostearate, and uniformly stirring at 200-300rpm at 85 ℃ to obtain a first mixture;
b. Mixing 5g of glycerol, 5g of polyethylene glycol 400 and 4g of triethanolamine, and uniformly stirring at 200-300rpm at 85 ℃ to obtain a second mixture;
c. Adding the second mixture into the first mixture, adding 0.5g essence, 0.5g preservative and 43g deionized water, stirring at 2000-3000rpm for 30-60min, cooling to 400-500rpm, and cooling to room temperature to obtain a repairing milk matrix;
(2) Preparing an epidermal growth factor microcapsule, which comprises the following specific steps of;
a. 5.0g of gelatin was weighed into a beaker and dissolved in 30mL of 1.0% aqueous acetic acid at 37℃in a water bath;
b. After gelatin is completely dissolved, adding 0.5g of chitosan into gelatin solution, stirring and dissolving to obtain uniform gelatin/chitosan mixed solution, adding an epidermal growth factor stock solution, and adopting 5.0% ammonia water solution to adjust pH to 6;
In this step, the pH is adjusted to 6 because gelatin is an amphoteric polymer, its isoelectric point is 5.0, pH is greater than its isoelectric point, gelatin molecule is negatively charged, i.e., -NH 3 + has a portion which is combined with-OH - to convert to-NH 2, so that the content of-COO - (negative charge) in gelatin molecule is greater than the content of-NH 3 + (positive charge), and molecule is negatively charged. When gelatin is in a medium less than the isoelectric point, the gelatin molecule becomes positively charged, i.e., a portion of-COO - is bound to-H + and converted to-COOH, so that the-NH 3 + (positive charge) content of the gelatin molecule is greater than the-COO - (negative charge) content, and the molecule becomes positively charged. Therefore, the pH value of the system is regulated to 6, gelatin is negatively charged, chitosan is positively charged due to protonation of free ammonia genes on molecules of the chitosan in an acidic medium, so that the negatively charged gelatin and the positively charged chitosan undergo complex coacervation reaction due to electrostatic interaction, and the ionization reaction of the gelatin at different pH values is shown as the formula:
The protonation reaction process of chitosan in an acidic medium is shown in a formula III:
the complex coacervation reaction of gelatin and chitosan is shown in formula IV:
c. Taking 272m corn oil in a three-neck flask, adding 5.0g of soybean lecithin, heating to 36 ℃, and uniformly stirring, wherein the soybean lecithin is an ampholytic surfactant which can be extracted from soybeans and is natural and nontoxic;
d. adding gelatin/chitosan mixed solution into corn oil for emulsification, wherein the emulsification temperature is 37 ℃ and the emulsification time is 60 min;
e. the gelatin can undergo sol-gel transition, swelling when the temperature is higher than 35 ℃, and gelling when the temperature is lower than 35 ℃. Thereby reducing the temperature to room temperature, being beneficial to forming a relatively fixed shell film of the particles due to gelatin gel, improving the stability of the particles and being beneficial to the next cross-linking reaction;
f. And e, adding 3.5mL of 0.5% genipin solution into the reaction system cooled in the step e, and reacting at normal temperature for 15 h.
In the step, genipin can be subjected to crosslinking reaction with a polymer containing free amino groups, free amino groups on chitosan and gelatin can be subjected to nucleophilic attack on an allyl carbon atom at the C-3 position of genipin under an acidic condition, and a dihydropyran ring is opened to form heterocyclic amine;
The cross-linking reaction process of genipin and chitosan is shown as formula V:
;
g. Adding 1mL of 1% citric acid aqueous solution into the reaction system after the step f, adding glacial acetic acid, adjusting the pH to 2-3, and under the protection of N 2, increasing the reaction temperature to 40 ℃, reacting 8-h, and cooling to room temperature to obtain the epidermal growth factor microcapsule;
in the step, genipin reacts with free amino groups on gelatin and chitosan to generate crosslinking, free hydroxyl groups are also present on chitosan and gelatin molecules, citric acid is added into a reaction system, under certain conditions, carboxyl groups on the citric acid and free hydroxyl groups on macromolecules generate esterification reaction, and the microcapsule taking gelatin and chitosan as capsule walls is subjected to secondary crosslinking, wherein the structural formula is shown in formula I:
h. And standing the microcapsule obtained by the reaction for 2 h, depositing gelatin/chitosan microcapsule on the lower layer, pouring corn oil on the upper layer, pouring out the upper oil phase, taking the lower microcapsule, centrifuging, and separating out the oil phase to obtain the secondary crosslinking microcapsule. And finally transferring the microcapsule into a wide-mouth bottle, and sealing and preserving.
(3) And (3) adding the microcapsule with the capsule core material being the epidermal growth factor into the matrix in the step (1), and uniformly mixing to obtain the repair emulsion containing the epidermal growth factor microcapsule.
Example 3 thermogravimetric analysis (TG)
3.1 Analytical method
2-6 Mg samples are weighed, tested by adopting a TG/DSC synchronous thermal analyzer, and the temperature is raised from room temperature to 600 ℃ at the rate of 10 ℃ per minute, and the gas atmosphere is nitrogen.
3.2 Analysis results
The thermal decomposition temperatures of the different samples are shown in table 2,
TABLE 2 thermal decomposition temperatures of different samples
| Sample of | Thermal decomposition temperature/°c |
| Gelatin | 284 |
| Chitosan | 226 |
| Gelatin/chitosan | 275 |
| Genipin cross-linked gelatin/chitosan | 290 |
| Citric acid secondary cross-linked microcapsule | 293 |
Fig. 2 is a thermal weight graph of gelatin, chitosan, gelatin/chitosan complex, genipin cross-linked gelatin/chitosan and citric acid secondary cross-linked microcapsule, and as can be seen from table 2 and fig. 2, the thermal decomposition temperature of the microcapsule obtained by citric acid secondary cross-linking is maximum, reaching 293 ℃, and the thermal stability is higher than that of genipin primary cross-linked gelatin/chitosan. Therefore, the microcapsule wall after citric acid secondary crosslinking has stronger strength and better thermal stability.
The reason for the above results is that the example 2 of this patent adopts a double cross-linking agent, the first cross-linking adopts genipin, which is the product of hydrolysis of geniposide by beta-glucosidase, and is an excellent natural biological cross-linking agent. The second cross-linking adopts citric acid which naturally exists in fruits such as lemon and orange, one citric acid molecule contains three carboxyl groups and one hydroxyl group, and under certain reaction conditions, the citric acid can perform esterification reaction with the hydroxyl groups on gelatin and chitosan, and the gelatin/chitosan on the microcapsule wall can be further cross-linked and solidified, so that the strength of the microcapsule wall is improved, and the thermal stability of the microcapsule is improved.
EXAMPLE 4 optical microscopy of microcapsules during preparation
And sucking a proper amount of cured microcapsule solution on a glass slide by using a suction tube, observing under a model WV-CP240/G optical microscope, and photographing and recording.
Fig. 3 is a time comparison chart of a microcapsule microscope in the process of preparing microcapsules by taking genipin as a cross-linking agent and gelatin and chitosan as capsule walls, and the chart shows that in the emulsification stage, the particle size of particles gradually becomes smaller along with the increase of the emulsification time, the particle size distribution is narrowed, and when the emulsification time reaches 60 min, the particle size is smaller, and the stability is better. Therefore, the emulsification time is preferably 60 min. After emulsification is completed, genipin is added for crosslinking, the wall of the capsule is gradually formed after crosslinking for 3 hours, and the crosslinking time reaches 15 h, so that the stable gelatin/chitosan microcapsule is obtained.
Example 5 cell experiments of epidermal growth factor microcapsules
The experimental method comprises the following steps:
After the single-layer fibroblast is digested, the cell density is diluted to 10000/mL by adding a high-sugar DMEM culture medium, and the culture medium is inoculated into a 96-well plate at 100 mu L/well, 6 compound wells are arranged on each culture medium, and the culture medium is placed into a 37 ℃ and 5% CO 2 culture box for culture for 24 hours.
EGF stock solution was diluted by corresponding factors with medium to obtain 100. Mu.L/L, 50. Mu.L/L, 10. Mu.L/L, 5. Mu.L/L, 1. Mu.L/L EGF and medium mixed solution, which was added to the cell plate, 100. Mu.L was added to each well, 6 duplicate wells were set for each concentration, and 100. Mu.L medium was added to the blank group. After incubation for 24h in a 37℃5% volume fraction CO 2 incubator, 20. Mu.L of MTT solution was added to each well and incubated for 4h in a 37℃5% volume fraction CO 2 incubator.
The liquid in the cell plate was aspirated by a liquid suction pump, 150. Mu.L of DMSO was added to each well, and the mixture was homogenized on a shaker for 10min, and absorbance at 490nm was measured with an enzyme-labeled instrument.
The treatment method of the epidermal growth factor microcapsule comprises taking excessive epidermal growth factor microcapsule, centrifuging, and washing to remove oil on the surface of the microcapsule. According to the feeding ratio of the synthetic microcapsule, the mass of the microcapsule which is 10 mu L/L growth factor to be weighed is calculated, the microcapsule which is 10 times of the mass is weighed according to the calculation result, a certain amount of DMEM high sugar culture medium is added, the microcapsule is broken through beating, the embedded growth factor is dissolved in the culture medium, the supernatant is centrifugated, the supernatant is taken and diluted to a theoretical value of 10 mu L/L (the microcapsule is not broken completely and is the theoretical value), the microcapsule is added into a cell plate, 6 compound holes are formed, and the rest operation is the same as EGF stock solution experiment.
The fluorescent microscope is used for shooting pictures of L929 cells under three treatment modes of a blank control group, an epidermal growth factor microcapsule and an epidermal growth factor stock solution.
Experimental results:
And measuring absorbance at 490nm by using an enzyme-labeled instrument, removing points with larger deviation, and averaging residual absorbance values to represent the absorbance at the concentration of the epidermal growth factor, wherein the obtained cell absorbance diagrams of the epidermal growth factor with different concentrations are shown in figure 4. As can be seen from the graph, the absorbance gradually increases with the increase of the concentration of the EGF without microencapsulation, which means that the cell proliferation is faster, and the absorbance of the microencapsulated EGF is smaller than that of the stock solution with the same concentration, because the microencapsulation is not totally broken by beating, and the EGF concentration in the culture solution is lower than the theoretical value of 10 mu L/L, so the absorbance is lower than that of the stock solution.
The comparison of the photographs taken by the fluorescence microscope is shown in FIG. 5, in which (a) is a control group, (b) is an EGF microcapsule, (c) is an EGF stock solution, and (d) is an EGF stock solution, and it is clear from the comparison that the proliferation of cells cultured in the culture solution added with EGF microcapsule is significantly faster than that in the control group, but slower than that in the culture solution added with EGF stock solution. This shows that the growth factor is successfully embedded and is not inactivated, and the problem that the growth factor is easy to inactivate when meeting an oxidant can be solved.
In summary, compared with the prior art, the invention has the beneficial effects that the microcapsule wall structure of the microcapsule has good mechanical property on one hand, and the preparation method can successfully wrap the water-based epidermal growth factor into the microcapsule wall structure on the other hand, and can avoid being oxidized by external substances in the use process, so that the drug effect is maintained for a long time.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (9)
1. The repair emulsion containing the epidermal growth factor microcapsule comprises a repair emulsion matrix, and is characterized in that the matrix also comprises the epidermal growth factor microcapsule, the microcapsule comprises a wall material and a capsule core material, the capsule core material is an epidermal growth factor stock solution, the wall material is a polymer formed by crosslinking citric acid/genipin and gelatin/chitosan, and the chemical structural formula of the polymer is shown as formula (I):
In the formula (I), R1 to R9 are selected from eighteen different amino acids of residues of glycine, alanine, serine, aspartic acid, glutamic acid amino, proline, arginine, histidine, tyrosine, cystine, leucine, threonine, methionine, valine, phenylalanine, tryptophan, glutamic acid and lysine;
The preparation method of the repair emulsion containing the epidermal growth factor microcapsule specifically comprises the following steps:
(1) Preparing a repair milk matrix;
(2) The preparation method of the epidermal growth factor microcapsule comprises the following specific steps:
a. Dissolving gelatin in acetic acid aqueous solution to obtain gelatin acetic acid solution;
b. Adding chitosan into gelatin acetic acid solution, stirring to dissolve chitosan to obtain gelatin/chitosan mixed solution, adding epidermal growth factor stock solution, and adjusting pH to 5.8-6.2;
c. Adding soybean lecithin as a surfactant into vegetable oil, heating and uniformly stirring;
d. Adding the gelatin chitosan mixed solution prepared in the step b into the vegetable oil after the step c for heating and emulsifying, closing heating after the emulsification is completed, and naturally cooling to room temperature;
e. adding genipin solution into the solution system after the step d for crosslinking reaction, and obtaining genipin Ping Mingjiao chitosan microcapsules after full reaction;
f. Adding a citric acid aqueous solution into the reaction system after the step e, adding glacial acetic acid, regulating the pH to 2-3, and fully reacting under the protection of N 2 to obtain an epidermal growth factor microcapsule;
(3) And (3) adding microcapsules with the capsule core material being the epidermal growth factor into the repairing emulsion matrix in the step (1), and uniformly mixing to obtain the repairing emulsion containing the epidermal growth factor microcapsules.
2. The repair emulsion containing epidermal growth factor microcapsule according to claim 1, wherein in the formula (I):
R5 and R6 are selected from eighteen residues of different amino acids glycine, alanine, serine, aspartic acid, glutamic acid amino, proline, arginine, histidine, tyrosine, cystine, leucine, threonine, methionine, valine, phenylalanine, tryptophan, glutamic acid and lysine;
R3 and R7 are residues of lysine or arginine;
r2, R4 and R8 are residues of aspartic acid or glutamic acid amino;
r1 and R9 are serine, threonine or tyrosine residues.
3. The repair emulsion containing the epidermal growth factor microcapsule according to claim 1, wherein the step (1) specifically comprises the following steps:
a1. mixing fructus Rosae Davuricae oil, oleum Olivarum, vaseline, isopropyl myristate and glyceryl monostearate, and stirring at 200-300rpm at 85deg.C to obtain a first mixture;
b1. Mixing glycerol, polyethylene glycol 400 and triethanolamine, and stirring at 200-300rpm at 85deg.C to obtain a second mixture;
c1. adding the second mixture into the first mixture, adding essence, antiseptic and deionized water, stirring at 2000-3000rpm for 30-60min, cooling to room temperature at 400-500rpm, and obtaining the repairing emulsion matrix.
4. The repair emulsion containing the epidermal growth factor microcapsule according to claim 3, wherein the repair emulsion comprises, by weight, 10-20 parts of rose hip oil, 10-20 parts of olive oil, 6-10 parts of vaseline, 1-3 parts of isopropyl myristate and 1-3 parts of glyceryl monostearate, 3-7 parts of glycerin, 3-7 parts of polyethylene glycol 400, 3-6 parts of triethanolamine, 0.2-0.8 part of essence, 0.2-0.8 part of preservative and the balance deionized water.
5. A repair emulsion containing epidermal growth factor microcapsules according to claim 3, wherein said preservative is methylparaben or a complex of methylparaben and ethylparaben.
6. The repair emulsion containing epidermal growth factor microcapsules of claim 1, further comprising, after step f:
g. And d, standing the reaction system after the step f, pouring out the upper oil phase, centrifuging, and separating out the oil phase to obtain the purified epidermal growth factor microcapsule.
7. The repair emulsion containing the epidermal growth factor microcapsule according to claim 1, wherein in the step b, the pH is regulated to 6, the vegetable oil in the step c is one or more of corn oil, olive oil, soybean oil and peanut oil, and the specific step of the step e is that 0.5% of genipin aqueous solution is added into the solution system after the step d, and the solution system is reacted for 15 hours at room temperature, and the genipin cross-linked gelatin/chitosan microcapsule is obtained after the full reaction.
8. The repair emulsion containing the epidermal growth factor microcapsule according to claim 1, wherein the specific step of the step f is that citric acid is added into the reaction system after the step e, glacial acetic acid is added, the pH is regulated to 2-3, the reaction temperature is increased to 40 ℃ under the protection of N 2, and the reaction is carried out for 8 hours and then cooled to room temperature, so that the epidermal growth factor microcapsule is obtained.
9. The repair emulsion containing the epidermal growth factor microcapsule according to claim 1, wherein the components in the step (2) are 45-55 parts by weight of gelatin, 3-7 parts by weight of chitosan, 250-350 parts by weight of 1.0% acetic acid solution, 40-60 parts by weight of soybean phospholipid, 30-40 parts by weight of 0.5% genipin, 5-15 parts by weight of 1.0% citric acid and 2500-3000 parts by weight of vegetable oil.
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