CN115177547A - MiRNA freeze-drying protective agent with repairing function and preparation method of freeze-drying balls of miRNA freeze-drying protective agent - Google Patents
MiRNA freeze-drying protective agent with repairing function and preparation method of freeze-drying balls of miRNA freeze-drying protective agent Download PDFInfo
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- CN115177547A CN115177547A CN202210875526.9A CN202210875526A CN115177547A CN 115177547 A CN115177547 A CN 115177547A CN 202210875526 A CN202210875526 A CN 202210875526A CN 115177547 A CN115177547 A CN 115177547A
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Abstract
The invention provides a mi RNA freeze-drying protective agent with a repairing function and a preparation method of a freeze-drying ball thereof, and relates to the technical field of skin care products. The mi RNA freeze-drying protective agent with the repairing function comprises 0.1-0.8 part of oligopeptide, 1-5 parts of amino acid, 0.05-0.1 part of hyaluronate, 5-20 parts of polysaccharide and 1-5 parts of polyalcohol by mass fraction, and the pH value of a freeze-drying protective agent mixed solution is 5-8. The freeze-dried ball finished product prepared from various repair components and the protective agent mixed solution has the advantages that after the freeze-drying process optimization and the freeze-drying protective agent optimization are carried out, the surface of the freeze-dried ball is free of cracks and smoother, and the appearance of the freeze-dried ball is smooth and blue, so that the freeze-dried ball is more attractive and beautiful.
Description
Technical Field
The invention relates to the technical field of skin care products, in particular to a miRNA freeze-drying protective agent with a repairing function and a preparation method of a freeze-drying ball thereof.
Background
MicroRNAs (miRNAs) are an endogenous, regulatory-functional, non-coding RNA species found in eukaryotes, the size of which is about 20-25 nucleotides in length. miRNA is the most important part of siRNA in exosomes, where the site where mature miRNA binds to its complementary mRNA regulates gene expression through base pairing. miRNAs are well conserved in species evolution, and miRNAs found in plants, animals and fungi are only expressed at specific tissues and developmental stages, and miRNA tissue specificity and timing determine tissue and cell functional specificity, suggesting that miRNAs play multiple roles in the regulation of cell growth and development processes. At present, the physiological functions of part of miRNAs are deeply researched, for example, miRNAs regulate cell growth and tissue differentiation, so that the miRNAs are related to development and diseases in the life process. A series of studies have shown that: miRNAs play an important role in cell growth and apoptosis, blood cell differentiation, homeobox gene regulation, neuronal polarity, insulin secretion, brain morphogenesis, cardiogenesis, late embryonic development and other processes.
Mesenchymal Stem Cells (MSCs) are undifferentiated Cells with multidirectional differentiation potential and self-renewal capacity, play an important role in skin wound repair and tissue regeneration, and are the medical means with the most clinical application value at present. It has been found that the role of stem cells in wound repair and tissue regeneration is primarily related to their paracrine capacity, not their differentiation capacity. Paracrine of stem cells is achieved primarily by exosomes. Exosomes are microvesicles secreted by cells, and the most important role is to transmit intercellular communication signals. The exosome regulation is mainly realized by miRNA to regulate immune response, regulate physiological reactions such as proliferation, migration, differentiation, apoptosis and the like of target cells, promote angiogenesis and maintain the steady state of the internal environment of an organism.
At present, the formula and the process of the cosmetic freeze-dried powder or freeze-dried ball products on the market mainly take the solution components to be formed by freeze-drying, and the protection of the structure and the performance of the active components in the processes of pre-freezing and dehydration in the freeze-drying process is rarely considered. The preparation of the freeze-dried balls is relatively extensive in the industry, the manual preparation and split charging method is mainly carried out by using a mould, the mould basically has the steps of hemispherical freezing forming and manual ball synthesis, the operation is complex, and the complex steps can cause the problems of high large-scale production cost, poor repeatability, high manpower requirement, more pollution sources brought by human beings and the like.
Based on the problems, the invention aims to provide a freeze-drying protective agent which can effectively protect the activity of miRNA in the freeze-drying process and has the functions of moisturizing skin care products, whitening, repairing and the like, and a preparation method of freeze-drying balls of the freeze-drying protective agent, so as to obtain miRNA freeze-drying ball products which are high in activity, easy to dissolve, smooth and attractive in surface, high in hardness and capable of being stored and transported at normal temperature.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a miRNA freeze-drying protective agent with a repairing function and a preparation method of a freeze-drying ball thereof, so that the problems that an active substance is unstable and easy to inactivate in a liquid system and the like are solved, and a freeze-drying ball product which has a good protective effect on a bioactive component in a freeze-drying process, is simple in shape, smooth and attractive in appearance, high in hardness, free of powder falling and easy to dissolve is obtained.
(II) technical scheme
In order to realize the purpose, the invention is realized by the following technical scheme: the miRNA freeze-drying protective agent with the repairing function comprises 0.1-0.8 part of oligopeptides, 1-5 parts of amino acids, 0.05-0.1 part of hyaluronate, 5-20 parts of polysaccharides and 1-5 parts of polyhydric alcohols by mass, and the pH value of a freeze-drying protective agent mixed solution is 5-8.
Preferably, the oligopeptides are one or more of tripeptide-1-copper, acetyl hexapeptide, acetyl octapeptide-1, pentapeptide-3, dipeptide snake toxin and pentapeptide-3, palmitoyl tripeptide-1, palmitoyl hexapeptide-6, palmitoyl tripeptide-5 and hexapeptide-9.
Preferably, the amino acid is one or more of glycine, histidine, glutamic acid and arginine.
Preferably, the hyaluronate is enzyme-cleaved hydrolyzed sodium hyaluronate.
Preferably, the polysaccharide is one or more of maltodextrin, trehalose, sucrose, resistant dextrin, maltose, lactose, maltotriose, pullulan, starch and cellulose.
Preferably, the polyalcohol is one or more of mannitol, xylitol, inositol, sorbitol and polyethylene glycol.
Preferably, the preparation method of the miRNA freeze-dried spheres with the repair function comprises the following steps:
s1, degerming
Sterilizing and cooling a high-temperature material at high temperature, adding oligopeptide, hyaluronic acid and miRNA, dissolving, fully dissolving the solution, mixing uniformly to ensure the fluidity of the solution, and filtering and sterilizing the solution by 0.25um to prepare a miRNA freeze-dried mixed solution;
s2, freezing and forming
Quantitatively injecting the mixed miRNA mixed solution into a freeze-dried ball mould which is provided with a hole at the top and has the diameter of 7-20mm and can be vertically separated by using an aseptic automatic liquid separator with a 0.7mm needle, and placing the mould in a liquid nitrogen deep freezing environment in the liquid separation process to ensure that the part in contact with the mould is quickly frozen and prevent overflowing to influence the surface smoothness;
s3, subpackaging
Freezing for 1h, taking out the mold in a sterile environment, opening the mold after the temperature rises, and subpackaging into penicillin bottles by using automatic subpackaging and covering equipment;
s4. Prefreezing
Placing the subpackaged formed freeze-dried balls on a pre-freezing plate layer of a freeze dryer, and pre-freezing for 1-4h at the temperature of-50 to-70 ℃;
s5, sublimation drying
Carrying out sublimation drying on the freeze-dried balls subjected to pre-freezing treatment, wherein the temperature rise rate of sublimation drying is 30min, the temperature rises to-35 ℃ to-20 ℃, the temperature is kept for 20-36h, and the vacuum degree is kept at 0-10pa;
s6, resolving and drying
Carrying out resolution drying on the freeze-dried ball subjected to sublimation drying treatment, wherein the temperature rising rate of the resolution drying is 1 ℃/h, the temperature rises to 10-25 ℃, the temperature is kept for 5-15h, and the vacuum degree is 10-30pa;
s7. Gland packing
And after the cold freezing is finished, filling inert gas into the vacuum environment, sealing the vacuum environment by using the in-place press plug, and taking out the press cover for packaging.
Preferably, the inert gas filled in the finished freeze-dried ball is one of nitrogen and carbon dioxide.
Preferably, the water content of the finished freeze-dried ball prepared from the mixed solution of the various repair components and the protective agent is 1-4%.
Preferably, the core components such as miRNA and the like are more stable after the freeze-drying protective agent is optimized in the freeze-drying ball finished product prepared from the mixed solution of the various repair components and the protective agent.
(III) advantageous effects
The invention provides a miRNA freeze-drying protective agent with a repairing function and a preparation method of a freeze-drying ball thereof. The method has the following beneficial effects:
1. according to the miRNA freeze-drying protective agent with the repairing function and the preparation method of the freeze-drying ball, the freeze-drying ball finished product prepared from various repairing components and protective agent mixed liquor has no cracks on the surface and is smoother after the freeze-drying process optimization and the freeze-drying protective agent optimization, and the appearance of the freeze-drying ball is smooth and blue, and is more attractive and beautiful due to the blue addition of oligopeptide copper ions.
2. According to the miRNA freeze-drying protective agent with the repairing function and the preparation method of the freeze-drying balls, freeze-drying ball finished products prepared from various repairing components and protective agent mixed liquor have more excellent hardness after being subjected to freeze-drying procedures and freeze-drying protective agent optimization, are not prone to powder falling, chip falling and splitting into blocks, can be stored at normal temperature for a long time, can reduce transportation cost and prolong the use quality guarantee period of users, and are more convenient to use and simpler to operate.
3. According to the miRNA freeze-drying protective agent with the repairing function and the preparation method of the freeze-drying ball thereof, various cosmetic liquid products prepared by the method can be freeze-dried into solid preparations through the freeze-drying process of the method and the protective agent, the operation method is simple, large-scale production can be expanded, the used components of various cosmetics can be freeze-dried, and the miRNA freeze-drying protective agent can be used after being dissolved with a solvent after being dried and formed, so that the operation is very simple and convenient.
4. The freeze-dried pellet prepared by the method solves the stability problem of the miRNA product, and simultaneously solves the problems of poor water solubility, hardness, appearance smoothness and the like of the freeze-dried pellet under the condition of ensuring high activity of functional substances of the product.
Drawings
FIG. 1 is a graph showing the Ct value variation of miRNA of each formula before and after lyophilization according to the present invention;
FIG. 2 is a graph of Ct values for qPCR of each formulation before and after lyophilization according to the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the embodiment of the invention provides a miRNA freeze-drying protective agent with a repairing function, which comprises 0.1-0.8 part of oligopeptide, 1-5 parts of amino acid, 0.05-0.1 part of hyaluronate, 5-20 parts of polysaccharide and 1-5 parts of polyol by mass, wherein the pH value of a freeze-drying protective agent mixed solution is 5-8.
The oligopeptides are one or more of tripeptide-1-copper, acetyl hexapeptide, acetyl octapeptide-1, pentapeptide-3, dipeptide snake toxin and pentapeptide-3, palmitoyl tripeptide-1, palmitoyl hexapeptide-6, palmitoyl tripeptide-5 and hexapeptide-9, the amino acid is one or more of glycine, histidine, glutamic acid and arginine, the hyaluronate is enzyme-cleaved hydrolyzed sodium hyaluronate, the polysaccharides are one or more of maltodextrin, trehalose, sucrose, resistant dextrin, maltose, lactose, maltotriose, pullulan, starch and cellulose, and the polyalcohol is one or more of mannitol, xylitol, inositol, sorbitol and polyethylene glycol.
The preparation method of the miRNA freeze-dried spheres with the repairing function comprises the following steps:
s1, degerming
Sterilizing and cooling a high-temperature material at high temperature, adding oligopeptide, hyaluronic acid and miRNA, dissolving, fully dissolving the solution, mixing uniformly to ensure the fluidity of the solution, and filtering and sterilizing the solution by 0.25um to prepare a miRNA freeze-dried mixed solution;
s2, freezing and forming
Quantitatively injecting the mixed miRNA liquid mixture into a freeze-dried ball mould which is provided with an opening at the top and has the diameter of 7-20mm and can be separated up and down by using an aseptic automatic liquid distributor with a 0.7mm needle, wherein in the liquid separation process, the mould is placed in a liquid nitrogen deep freezing environment to ensure that the part in contact with the mould is quickly frozen and prevent overflow from influencing the surface smoothness;
s3, subpackaging
Freezing for 1h, taking out the mold in a sterile environment, opening the mold after the temperature rises, and subpackaging into penicillin bottles by using automatic subpackaging and capping equipment;
s4. Prefreezing
Placing the subpackaged formed freeze-dried balls on a pre-freezing plate layer of a freeze dryer, and pre-freezing for 1-4h at the temperature of-50 to-70 ℃;
s5, sublimation drying
Carrying out sublimation drying on the freeze-dried balls subjected to pre-freezing treatment, wherein the temperature rise rate of sublimation drying is 30min, the temperature rises to-35 ℃ to-20 ℃, the temperature is kept for 20-36h, and the vacuum degree is kept at 0-10pa;
s6, resolving and drying
Resolving and drying the freeze-dried ball subjected to sublimation drying treatment at a heating rate of 1 ℃/h to 10-25 ℃, keeping the temperature for 5-15h and a vacuum degree of 10-30pa;
s7. Gland packing
And after the cold freezing is finished, filling inert gas into the vacuum environment, sealing the vacuum environment by using the in-place press plug, and taking out the press cover for packaging.
The inert gas filled in the finished freeze-dried ball is one of nitrogen and carbon dioxide, the water content of the finished freeze-dried ball prepared from the mixed liquid of various repairing components and the protective agent is 1-4%, and the core components of miRNA and the like are more stable after the freeze-dried protective agent is optimized.
Example 2:
freeze-drying protection mixed liquid preparation
The following lyophilized protection mixtures of mirnas were prepared, respectively:
and (3) mixing liquid A: 10 parts of maltodextrin, 5 parts of trehalose, 2 parts of sorbitol, 3 parts of mannitol, 3 parts of glycine, 0.1 part of enzymolysis sodium hyaluronate, 0.2 part of tripeptide-1-copper and 0.2 part of acetyl hexapeptide;
and (3) mixed liquid B: 5 parts of maltodextrin, 10 parts of trehalose, 2 parts of sorbitol, 3 parts of mannitol, 3 parts of glycine, 0.1 part of enzymolysis sodium hyaluronate, 0.2 part of tripeptide-1-copper and 0.2 part of acetyl hexapeptide;
and (3) mixed liquid C: 5 parts of maltodextrin, 5 parts of trehalose, 5 parts of sorbitol, 5 parts of mannitol, 3 parts of glycine, 0.2 part of enzymolysis sodium hyaluronate, 0.1 part of tripeptide-1-copper and 0.2 part of acetyl hexapeptide;
and (3) mixing liquid D: 10 parts of resistant dextrin, 5 parts of trehalose, 4 parts of sorbitol, 1 part of mannitol, 3 parts of glycine, 0.1 part of enzymolysis sodium hyaluronate, 0.2 part of tripeptide-1-copper and 0.2 part of acetyl hexapeptide;
and (3) mixing liquid E: 10 parts of maltodextrin, 5 parts of trehalose, 3 parts of glycine, 2 parts of sorbitol, 2 parts of mannitol, 0.1 part of high-molecular sodium hyaluronate and 0.2 part of acetyl hexapeptide;
and (3) mixing liquid F: 10 parts of maltodextrin, 4 parts of trehalose, 3 parts of glycine, 2 parts of sorbitol, 2 parts of mannitol, 0.1 part of tripeptide-1-copper and 0.2 part of acetyl hexapeptide.
Example 3:
preparing freeze-dried balls and freeze-drying:
1) Preparing a protective agent mixed solution: respectively dissolving and sterilizing protective agent substances prepared from the formulas A, B, C, D, E and F step by step, sterilizing polysaccharide and polyalcohol at high temperature to prevent pollution, cooling, adding oligopeptide, hyaluronic acid and core components to dissolve, fully dissolving the solution, mixing uniformly to ensure the fluidity of the solution, filtering and sterilizing through a 0.25-0.45 um microporous filter membrane to prepare miRNA freeze-drying protective agent mixed solution A, B, C, D, E and F;
2) Forming and subpackaging freeze-dried balls: preserving the temperature of the sterilized freeze-drying protective agent mixed liquor at 4 ℃ for 1.5h in advance, then quantitatively injecting the mixed miRNA freeze-drying protective agent mixed liquor into a pre-precooled freeze-drying ball mould placed in a liquid nitrogen deep freezing environment by using an aseptic automatic dispenser with a 0.7mm needle, taking out the mould in the aseptic environment after freezing for 3h, demoulding the quick freeze-drying ball, and subpackaging the quick freeze-drying ball into a penicillin bottle by using automatic subpackaging and capping equipment;
3) Pre-freezing: placing the subpackaged formed freeze-dried balls in a pre-freezing plate layer of a freeze dryer, setting the temperature to be raised to the eutectic point temperature, keeping the eutectic point temperature for 2 hours, and then cooling to-65 ℃ for pre-freezing for 3 hours;
4) Sublimation drying: carrying out sublimation drying on the freeze-dried balls subjected to pre-freezing treatment, wherein the temperature rising rate of sublimation drying is increased to-30 ℃ within 30min, the temperature is maintained for 25h, and the vacuum degree is maintained at 10pa;
5) And (3) resolving and drying: carrying out resolution drying on the freeze-dried balls subjected to sublimation drying treatment, wherein the temperature rise rate of the resolution drying is 1 ℃/h, the temperature is raised to 20 ℃, the temperature is maintained for 10h, and the vacuum degree is 30pa;
6) Filling: and after vacuum freeze-drying is finished, filling inert gas into the vacuum environment, sealing by a position pressure plug, taking out the gland and packaging.
Comparative example:
1) Preparing a protective agent mixed solution: in the current freeze-dried ball product of cosmetics, the mixed liquid of the product is mainly excipient, the protection of core components is less important, or the protection of bioactive substances is less important, and more matters are concerned about the weight and the color of the product;
2) Forming and subpackaging freeze-dried balls: at present, freeze-dried balls with small diameters and low liquid adding amount generally use a liquid separation system, liquid nitrogen is used for forming, and automatic liquid separation forming and subpackaging can be realized, however, freeze-dried ball products with diameters larger than 8mm are mainly manufactured into spheres or other shapes by means of die forming, particularly the spheres, after the spheres are formed by means of half-sphere forming, two hemispheres are combined and frozen to form the spheres, more forming and subpackaging needs depend on manual operation, the operation is complex, the efficiency is low, and the large-scale batch production needs are difficult to meet;
3) Pre-freezing: placing the subpackaged formed freeze-dried balls in a pre-freezing plate layer of a freeze dryer, and pre-freezing for 3 hours at the temperature of-65 ℃;
4) Sublimation drying: carrying out sublimation drying on the freeze-dried ball subjected to pre-freezing treatment, wherein the temperature rise rate of the sublimation drying is 30min, the temperature is increased to-40 ℃, the temperature is maintained for 40h, and the vacuum degree is maintained at 10pa;
5) And (3) resolving and drying: carrying out resolution drying on the freeze-dried ball subjected to sublimation drying treatment, wherein the temperature rising rate of the resolution drying is 1 ℃/h, the temperature is raised to 10 ℃, the temperature is maintained for 20h, and the vacuum degree is 30pa;
6) Filling: and after vacuum freeze-drying is finished, filling inert gas into the vacuum environment, sealing by using a position press plug, taking out the press cover and packaging.
In the above process, there are the following problems:
firstly, the freeze-dried ball is synthesized mainly by hand in the forming process, the operation time is long, the freeze-dried ball is easy to melt in the process, or the temperature is raised too high to exceed the eutectic point temperature, so that the change of the glass state enables the inactivation of active miRNA or other active substances, meanwhile, the melting causes the problems of product shape deformation, microorganism pollution caused by manual introduction and the like, and the method has low efficiency and cannot meet the requirement of large-scale production;
secondly, the pre-freezing process is simple, and the pre-freezing process of pre-cooling and raising the temperature of the eutectic point and reducing the temperature is not carried out, tests show that the pre-freezing is more uniform in the process of reducing the temperature and pre-freezing after pre-cooling at 4 ℃ and forming at low temperature and raising the temperature to the eutectic point before pre-freezing for 2 hours, and the surface of a freeze-dried product is smooth, and the freeze-dried product is easy to shrink, crack and peel and wrinkle on the surface after direct pre-freezing;
and thirdly, the temperature of the freeze-drying program is set on the surface, and the eutectic point temperature, the glass transition temperature tg and the disintegration temperature Tc are tested, so that the freeze-drying program can be set, the freeze-drying time is shortened, and the freeze-drying efficiency is improved.
Example 4:
appearance test of lyophilized pellets
And (3) performing apparent detection on the freeze-dried balls prepared by the freeze-dried formulas A, B, C, D, E and F, wherein detection indexes comprise the integrity, the surface gloss, the crack and the color expression of the freeze-dried balls and the color and the uniformity of the freeze-dried balls, the test is mainly recorded by visual observation, and the test results are as shown in the following table 1:
table 1 appearance test results of freeze-dried balls
The results in the table show that the freeze-dried balls prepared by the three formulas A, D and E have beautiful appearance, bright color and higher integrity.
Friability testing of freeze-dried balls
And (4) preparing freeze-dried balls of the formulas A, B, C, D, E and F after freeze-drying. Performing friability detection, and in order to detect whether the formula of the freeze-dried balls can keep excellent appearance in the transportation process of the freeze-dried finished products, adopting a roller method to perform a test method, wherein the test method specifically comprises the following steps: 10 freeze-dried balls are accurately weighed and then placed into a test roller of a friability tester, wherein the test roller is 289mm in diameter and 34mm in thickness, and the rotation speed is 25 rpm, and the test is carried out for 4 minutes. After the roller test was completed, the shape and integrity of the freeze-dried balls were observed and the weight of the freeze-dried balls was measured, with the test results as shown in table 2 below:
TABLE 2 friability test results for freeze-dried balls
The above table shows that most of the above formulas have high mechanical strength, and maintain good integrity under the action of machinery, wherein a, D, E and F are excellent, which indicates that the freeze-dried balls of the above formulas can sufficiently resist the external force during transportation and use, and reduce the possibility of powder falling due to damage caused by external force, and better selection of freeze-drying protective agent and excipient is used.
Solubility testing of lyophilized pellets
And (3) carrying out dissolution speed detection on the freeze-dried balls prepared by the freeze-dried formulas A, B, C, D, E and F after freeze-drying is finished, and selecting an optimal formula in order to compare the influences of the freeze-dried balls with different formulas on the water solubility and the dissolution speed of a freeze-dried finished product, wherein the test method comprises the following steps: 5ml of sterile water is added into a freeze-dried penicillin bottle, timing is started, the complete disintegration time and the complete dissolution time of the freeze-dried penicillin in the water solution are tested under the condition of not shaking, and the test results are shown in the following table 3:
table 3 solubility test results of the lyophilized pellet
The results in the table show that most of the above formulations have high water-soluble disintegration performance and dissolution effect, and the freeze-dried balls can be rapidly disintegrated within 10S after being dissolved in water, because the complete dissolution is slightly longer than the disintegration time due to the presence of hyaluronic acid, wherein A, B, D, E and F all have excellent performances. The formula F has the highest dissolving speed because no hyaluronic acid exists, and the dissolving speed of the enzymolysis hyaluronic acid is higher than that of the common high-molecular hyaluronic acid and low-molecular hyaluronic acid.
Moisture testing of lyophilized pellets
The freeze-dried balls prepared by the freeze-dried formulas A, B, C, D, E and F are subjected to water and water activity detection, the water content of the freeze-dried balls can influence the stability of miRNA and bioactive substances in the freeze-dried balls, the shelf life of freeze-dried ball products and the like, and the test method comprises the following steps: the freeze-dried ball product is subjected to water content measurement by a constant weight method, the water activity is measured by a Rotanick water activity meter, and the test results are shown in the following table 4:
table 1 moisture test results of freeze-dried spheres
The results in the table show that the optimized freeze-drying process has good performance in the aspects of water content, water activity and the like due to the common freeze-drying process no matter from the aspects of freeze-drying time and freeze-drying effect, and the two indexes have great influence on the stability of the storage device of the freeze-drying ball.
Example 5:
the miRNA testing method comprises the following steps: and (3) determining miRNA by adopting a qPCR method of a stem-loop method.
The specific method comprises the following steps: designing a Stem-loop reverse transcription Primer (Stem-loop RT Primer) to carry out reverse transcription on miRNA to obtain cDNA, selecting snRNA U6 as an internal reference, taking a certain amount of cDNA to carry out real-time fluorescence quantitative PCR (real-time qPCR), and comparing Ct values of the qPCR to obtain the amount of the miRNA.
And (3) testing the activity of the freeze-dried miRNA spheres after freeze-drying: dissolving and uniformly mixing the freeze-dried mixed solution A, B, C, D, E and F formula substances, polysaccharide and polyhydric alcohol, sterilizing, cooling, adding amino acid, hyaluronic acid and oligopeptide, uniformly mixing, adding miRNA, fully mixing at a low temperature, performing microfiltration, performing freeze-drying ball forming, performing forming and subpackaging, performing pre-freezing by adopting a low-temperature pre-freezing temperature-rising eutectic point in a temperature-reducing mode, performing vacuum freeze-drying, performing primary sublimation drying and desorption drying, filling inert gas, and sealing and packaging. And (3) carrying out Ct value test on the miRNA in each formula before freeze-drying, carrying out Ct value detection in the same method after drying, and comparing the change of the Ct value test to determine the freeze-drying damage degree of the miRNA, wherein the higher the Ct value is, the less the miRNA amount is.
The test result is shown in figure 1, and the result shows that the protective effect of the protective agent formulas A, E and F on miRNA is obvious, and the Ct value change before and after freeze-drying is small.
And (3) testing the stability of the miRNA freeze-dried pellet product: and (3) placing the dried freeze-dried ball product in a condition of 45 ℃ and 75% of relative humidity for carrying out an accelerated test on the storage period, storing for 6 months for carrying out a stability test, and measuring the Ct value by adopting a stem loop method-qPCR (quantitative polymerase chain reaction) method of miRNA after the storage period is up to evaluate the stability.
The test result is shown in figure 2, and the result shows that the freeze-dried miRNA is relatively stable at normal temperature, has better storage stability in a dry environment, and has better stability under the condition that the water content and water activity of the product are lower.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. The miRNA freeze-drying protective agent with the repairing function is characterized by comprising 0.1-0.8 part of oligopeptide, 1-5 parts of amino acid, 0.05-0.1 part of hyaluronate, 5-20 parts of polysaccharide and 1-5 parts of polyol by mass, and the pH value of a freeze-drying protective agent mixed solution is 5-8.
2. The miRNA freeze-drying protective agent with repair function and the preparation method of the freeze-drying balls thereof as claimed in claim 1, wherein the oligopeptides are one or more of tripeptide-1-copper, acetyl hexapeptide, acetyl octapeptide-1, pentapeptide-3, dipeptide snake toxin and pentapeptide-3, palmitoyl tripeptide-1, palmitoyl hexapeptide-6, palmitoyl tripeptide-5 and hexapeptide-9.
3. The miRNA freeze-drying protective agent with repair function and the preparation method of the freeze-drying balls thereof according to claim 1, wherein the amino acid is one or more of glycine, histidine, glutamic acid and arginine.
4. The miRNA freeze-drying protective agent with repairing function and the preparation method of the freeze-drying ball thereof according to claim 1, wherein the hyaluronate is enzyme-cleaved and hydrolyzed sodium hyaluronate.
5. The miRNA freeze-drying protective agent with repair function and the preparation method of the freeze-drying balls thereof according to claim 1, wherein the polysaccharides are one or more of maltodextrin, trehalose, sucrose, resistant dextrin, maltose, lactose, maltotriose, pullulan, starch and cellulose.
6. The miRNA freeze-drying protective agent with repairing function and the preparation method of the freeze-drying balls thereof according to claim 1, wherein the polyalcohol is one or more of mannitol, xylitol, inositol, sorbitol and polyethylene glycol.
7. A preparation method of miRNA freeze-dried spheres with repair functions is characterized by comprising the following steps:
s1, degerming
Sterilizing and cooling a high-temperature material at high temperature, adding oligopeptide, hyaluronic acid and miRNA, dissolving, fully dissolving the solution, mixing uniformly to ensure the fluidity of the solution, and filtering and sterilizing the solution by 0.25um to prepare a miRNA freeze-dried mixed solution;
s2, freezing and forming
Quantitatively injecting the mixed miRNA liquid mixture into a freeze-dried ball mould which is provided with an opening at the top and has the diameter of 7-20mm and can be separated up and down by using an aseptic automatic liquid distributor with a 0.7mm needle, wherein in the liquid separation process, the mould is placed in a liquid nitrogen deep freezing environment to ensure that the part in contact with the mould is quickly frozen and prevent overflow from influencing the surface smoothness;
s3, subpackaging
Freezing for 1h, taking out the mold in a sterile environment, opening the mold after the temperature rises, and subpackaging into penicillin bottles by using automatic subpackaging and capping equipment;
s4. Prefreezing
Placing the packaged formed freeze-dried balls on a pre-freezing plate layer of a freeze dryer, and pre-freezing for 1-4h at the temperature of-50 ℃ to-70 ℃;
s5, sublimation drying
Carrying out sublimation drying on the freeze-dried balls subjected to pre-freezing treatment, wherein the temperature rise rate of sublimation drying is 30min, the temperature rises to-35 ℃ to-20 ℃, the temperature is kept for 20-36h, and the vacuum degree is kept at 0-10pa;
s6, resolving and drying
Resolving and drying the freeze-dried ball subjected to sublimation drying treatment at a heating rate of 1 ℃/h to 10-25 ℃, keeping the temperature for 5-15h and a vacuum degree of 10-30pa;
s7. Gland packing
And after the freezing is finished, filling inert gas into the vacuum environment, sealing by a position press plug, and taking out the gland for packaging.
8. The method for preparing a miRNA freeze-dried pellet with a repairing function according to claim 7, wherein the inert gas filled in the finished freeze-dried pellet is one of nitrogen and carbon dioxide.
9. The method for preparing the miRNA freeze-dried microspheres with repairing functions of claim 7, wherein the water content of the freeze-dried microspheres prepared from the mixed solution of various repairing components and the protective agent is between 1 and 4 percent.
10. The method for preparing the miRNA freeze-dried spheres with the repairing function according to claim 7, wherein the core components miRNA and the like are more stable after the freeze-drying protective agent is optimized in the freeze-dried sphere finished product prepared from the mixed solution of the various repairing components and the protective agent.
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