CN115624497B - Liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, and preparation method and application thereof - Google Patents
Liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, and a preparation method and application thereof, and relates to the technical field of liposomes. The invention provides a liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, which is of a core-shell structure, wherein the core of the core-shell structure is deoxyribonucleic acid or ribonucleic acid, and the shell of the core-shell structure comprises the following components: phosphate species, cholesterol, solubilizing agents, stabilizers and targeting ligands. The invention adopts phospholipid composite technology to carry out bionic encapsulation on deoxyribonucleic acid or ribonucleic acid, and prepares a novel liposome preparation for encapsulating the deoxyribonucleic acid or ribonucleic acid, thereby greatly improving the stability and whitening activity of the liposome preparation. The method provided by the invention is simple to operate and high in feasibility, and is expected to become a bright spot in the field of cosmetics in the 21 st century.
Description
Technical Field
The invention relates to the technical field of liposome, in particular to liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, and a preparation method and application thereof.
Background
Deoxyribonucleic acid (DNA) is an organic compound with a complex molecular structure. Deoxyribonucleic acid exists in the nucleus as a component of the chromosome and is the main genetic material of organisms. As early as 40 years ago, students were extracting DNA from gland tissues of male sturgeons for research of treating radiation injury, and later applied it in cosmetics to stimulate cell repair activity. The DNA salt with lower concentration can increase the quantity of fibroblasts and keratinocytes, reduce skin wrinkles and improve skin elasticity. Ribonucleic acids (abbreviated as RNA), genetic information vectors present in biological cells and in part viruses and viroids. RNA is a long-chain molecule formed by condensing ribonucleotides through phosphodiester bonds, one ribonucleotide molecule is composed of phosphoric acid, ribose and bases, the RNA is generally a single-chain long molecule and does not form a double-helix structure, but many RNAs also need to form a certain secondary structure or even a tertiary structure through a base pairing principle to perform biological functions.
Liposomes (lipomes) refer to the microvesicles formed by encapsulating a drug within a lipid bilayer. By utilizing the characteristic that liposome can be fused with cell membrane, the medicine is delivered into the cell. Liposomes are useful as carriers for anticancer agents, which interact with cells in a large number, and are often used in cosmetics as carriers for moisturizers, whiteners, and nutrients. Thus, the preparation of the active substance as liposomes encapsulating deoxyribonucleic acid or ribonucleic acid can produce the following effects on the skin: (1) The active substances are left between epidermis and dermis, so that adverse reactions of the whole body can be effectively avoided. (2) The chemical stability of the active substance is improved, and as some active substances are easily oxidized, unstable to light and heat and easily affected by pH, the active substances are isolated from the external environment by encapsulating the active substances in liposomes, thereby improving the stability of the active substances. (3) The moisturizing effect, research shows that the liposome can well keep skin moisture, and even the liposome without the encapsulated active substances can activate cells and inhibit moisture loss in the deep layer of the skin. (4) Skin care effect, because the structure of human skin is relatively dense, macromolecular substances with biological activity cannot be absorbed. Liposomes, due to their structural characteristics, can fuse with cells in the skin, making them more accessible through the skin to cells deep in the skin and accumulate in the cells. In addition, compared with the skin care product without the liposome, the skin care product with the liposome can remarkably reduce the roughness of the skin.
At present, no research on the coating and combination of deoxyribonucleic acid or ribonucleic acid and liposome exists, and whether the combined product has good efficacy or not is examined.
Disclosure of Invention
Based on this, the present invention aims to overcome the above-mentioned shortcomings of the prior art and provide a liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, and a preparation method and application thereof. The invention provides a liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, which is prepared by biomimetically encapsulating the deoxyribonucleic acid or ribonucleic acid by adopting a phospholipid composite technology, and has the advantages of greatly improving the stability and whitening activity. The method is simple to operate and high in feasibility, and is expected to become a bright spot in the field of cosmetics in the 21 st century. The invention is to hide deoxyribonucleic acid or ribonucleic acid in liposome vesicle, and the liposome vesicle has high stability, so that the chance of contacting the active ingredient with external unstable factors is reduced.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a liposome encapsulating deoxyribonucleic acid or ribonucleic acid, the liposome being of a core-shell structure, the core of the core-shell structure being deoxyribonucleic acid or ribonucleic acid, the shell of the core-shell structure comprising the following components: phosphate species, cholesterol, solubilizing agents, stabilizers and targeting ligands.
The liposome for encapsulating deoxyribonucleic acid or ribonucleic acid can greatly improve the stability of the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid, the deoxyribonucleic acid or ribonucleic acid is occluded in a liposome vesicle, the encapsulation effect of the liposome vesicle is utilized, the contact opportunity of active components with external unstable factors is reduced, the stability of the product is improved, and the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid is expected to become a bright point in the field of 21 st century cosmetics. The liposome of the invention can greatly improve the whitening activity, and the active ingredients can be continuously and slowly released between the epidermis and the dermis after the active ingredients encapsulated by the liposome are absorbed through the epidermis, so that the bioavailability is improved, and the purposes of long acting and irritation reduction are achieved. The liposome for encapsulating deoxyribonucleic acid or ribonucleic acid provided by the invention can generate whitening targeting, adopts targeting ligand and the like to carry out self-assembly and encapsulation with the liposome on the surface of the liposome, and targets and identifies skin melanin and melanoma cells to generate a specific whitening effect.
As a preferred embodiment of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the shell of the core-shell structure comprises the following components in parts by weight: 140-180 parts of phosphate substances, 10-30 parts of cholesterol, 140-200 parts of solubilizer, 140-200 parts of stabilizer and 2.5-40 parts of targeting ligand. As a preferred embodiment of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the shell of the core-shell structure comprises the following components in parts by weight: 160 parts of phosphate substances, 20 parts of cholesterol, 160 parts of solubilizer, 160 parts of stabilizer and 10 parts of targeting ligand.
After a lot of experimental researches, the inventor finds that in the preparation process of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, the selection of the weight parts of each component is the key for influencing the success of the preparation of the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid, and the particle size and PDI (particle size distribution index) of the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid prepared by the selection of the specific weight parts are smaller, so that the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid prepared by the preparation method is more stable.
As a preferred embodiment of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the phosphate ester substance is soybean lecithin.
As a preferred embodiment of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the solubilizing agent is polyethylene glycol 400. As a preferred embodiment of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the stabilizer is glycerol.
After a lot of experimental researches, the inventor finds that cholesterol has the capacity of adjusting the fluidity of a double-layer membrane and can well improve the stability of the liposome. The solubilizer exists on the surface of the liposome, so that the circulation time of the liposome in the systemic circulation is increased, the medicine is protected from metabolic inactivation and degradation, and the intracellular uptake of liposome vesicles is increased. The stabilizer can prevent precipitation and coagulation, and increase stability of liposome vesicles. In the process of preparing the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, under the combined action of the phosphate substance, cholesterol, a solubilizer, a stabilizer and a targeting ligand, the particle size and PDI (particle size distribution index) of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid are smaller, and the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid is more stable. Meanwhile, the prepared liposome for encapsulating deoxyribonucleic acid or ribonucleic acid has high encapsulation rate, good whitening effect, good B16 cell activity inhibition effect and good melanin secretion inhibition effect.
As a preferred embodiment of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the targeting ligand is at least one of hemoglobin, sodium hyaluronate, and folic acid. As a preferred embodiment of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the targeting ligand is sodium hyaluronate.
After a lot of experimental studies, the inventors found that the selection of the targeting ligand affects the particle size and PDI (particle size distribution index) of the finally prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid, and affects the whitening effect of the finally prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid. Under the selection of the specific targeting ligand, the particle size and PDI (particle size distribution index) of the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid are smaller, and the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid is more stable, has better effect of inhibiting melanin secretion and has better whitening effect.
In another aspect, the present invention provides a method for preparing the liposome encapsulating deoxyribonucleic acid or ribonucleic acid, comprising the steps of:
(1) Dissolving the phosphate substance and cholesterol, uniformly mixing, and performing rotary steaming to obtain a film;
(2) Adding the solubilizer, the stabilizer, the targeting ligand, the deoxyribonucleic acid or the ribonucleic acid into the film obtained in the step (1), adding a buffer solution, uniformly mixing, and performing ultrasonic treatment to obtain a suspension;
(3) And (3) passing the suspension obtained in the step (2) through a microporous filter membrane, and obtaining the liposome encapsulating the deoxyribonucleic acid or ribonucleic acid after a liposome extrusion system.
The invention adopts a film hydration method to prepare liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, and under the preparation method of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, the particle size and PDI (particle size distribution index) of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid are smaller, and the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid is more stable.
As a preferred embodiment of the method for preparing a liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the invention, in the step (1), the solvent used in the dissolution is absolute ethanol, and the spin evaporation temperature is 40-60 ℃; the film is a yellowish film.
In the preferred embodiment of the method for preparing a liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, in the step (2), the buffer is one of a phosphate buffer, a citrate buffer, a carbonate buffer, and an acetate buffer. As a preferred embodiment of the method for preparing a liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, in the step (2), the buffer is a phosphate buffer.
After a great deal of experimental study, the inventor finds that the buffer solution plays a good role in buffering and further maintains the stability of the liposome structure.
As a preferred embodiment of the preparation method of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid, in the step (2), the mixing temperature is 25-35 ℃, the mixing time is 15-25min, and the ultrasonic time is 2-4min; the suspension is a milky white suspension.
As a preferred embodiment of the method for preparing a liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, in the step (3), the diameter of the microporous filter membrane is 0.2-0.6 μm.
As a preferred embodiment of the method for preparing a liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to the present invention, the diameter of the microporous filter membrane is 0.45 μm.
In yet another aspect, the invention provides the use of said deoxyribonucleic acid or ribonucleic acid encapsulated liposomes in cosmetics.
As a preferred embodiment of the application of the present invention, the cosmetic includes face cream, eye cream, toner, essence, or the like.
After a large number of experimental researches, the inventor finds that the specific liposome for encapsulating deoxyribonucleic acid or ribonucleic acid provided by the invention has high stability and good whitening effect, and has wide application in cosmetics.
Compared with the prior art, the invention has the beneficial effects that: (1) The invention adopts phospholipid composite technology to carry out bionic encapsulation on deoxyribonucleic acid or ribonucleic acid, and prepares a novel liposome preparation for encapsulating the deoxyribonucleic acid or ribonucleic acid, thereby greatly improving the stability and whitening activity of the liposome preparation. The method is simple to operate and high in feasibility, and is expected to become a bright spot in the field of cosmetics in the 21 st century. (2) The liposome for encapsulating deoxyribonucleic acid or ribonucleic acid designed by the research can greatly improve the stability, and the probability of contacting an active ingredient with external unstable factors is reduced due to the encapsulation effect of the liposome vesicle because the deoxyribonucleic acid or ribonucleic acid is occluded in the liposome vesicle, so that the stability of the product is improved. (3) The liposome of the encapsulated deoxyribonucleic acid or ribonucleic acid can greatly improve the whitening activity, and the active ingredient reservoir is formed between the epidermis and the dermis after the active substance encapsulated by the liposome is absorbed through the epidermis, so that the active ingredient can be continuously and slowly released, the bioavailability is improved, and the purposes of long acting and irritation reduction are achieved. (4) The liposome for encapsulating deoxyribonucleic acid or ribonucleic acid designed by the research can generate whitening targeting, and the targeting ligand albumin and the like are adopted to carry out self-assembly and encapsulation on the surface of the liposome, so that skin melanin and melanoma cells are targeted and identified, and a specific whitening effect is generated.
Detailed Description
The objects, technical solutions and advantages of the present invention will be further described with reference to specific examples, which are intended to be illustrative of the contents of the present invention in detail, not limiting the present invention. All other embodiments, which can be made by those skilled in the art without the inventive effort, are intended to be within the scope of the present invention. The experimental methods used in the examples and comparative examples of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used, if otherwise specified, are commercially available.
Examples and comparative examples
The components and parts by weight of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid in the embodiment of the invention are shown in the following tables 1 to 6, and the preparation method of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid comprises the following steps:
(1) Dissolving the phosphate substance and cholesterol in a eggplant-shaped bottle, uniformly mixing, and performing rotary evaporation, wherein the solvent used in the dissolving process is absolute ethyl alcohol, the rotary evaporation temperature is 50 ℃, and a layer of light yellow uniform film is obtained on the inner wall of a container;
(2) Adding the solubilizer, the stabilizer, the targeting ligand, the deoxyribonucleic acid or the ribonucleic acid into the container with the film in the step (1), simultaneously adding a buffer solution, mixing and reacting at the temperature of 30 ℃, mixing and reacting for 20min, and carrying out ultrasonic treatment for 3min to obtain milky suspension;
(3) And (3) passing the suspension obtained in the step (2) through a microporous filter membrane with the diameter of 0.45 mu m, and obtaining the liposome encapsulating the deoxyribonucleic acid or ribonucleic acid after passing through a liposome extrusion system.
TABLE 1
TABLE 2
TABLE 3 Table 3
TABLE 4 Table 4
TABLE 5
TABLE 6
Comparative example 1
The composition and weight parts of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid in the comparative example are completely the same as those of the embodiment 1, the preparation method of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid is different, the embodiment 1 adopts an ethanol injection method, and the preparation method of the liposome encapsulating deoxyribonucleic acid or ribonucleic acid in the embodiment 1 comprises the following steps:
(1) Dissolving the phosphate substance and cholesterol in an eggplant-shaped bottle, wherein the solvent used in dissolving is absolute ethyl alcohol, so as to obtain a lipid solution 1;
(2) Adding the solubilizer, the stabilizer, the targeting ligand, the buffer solution, the deoxyribonucleic acid or the ribonucleic acid into a 100mL beaker, placing the beaker in a magnetic stirrer, wherein the water bath temperature is 60 ℃, the rotating speed of the magnetic stirrer is 300r/min, slowly adding the lipid solution 1 in the step (1) into the beaker by using a syringe, continuously stirring for 1 hour, and fixing the volume by using a 25mL volumetric flask to obtain a lipid solution 2;
(3) And (3) passing the lipid solution 2 in the step (2) through an ice-water bath and ultrasonic treatment for 60s, passing through a microporous filter membrane with the diameter of 0.45 mu m, and passing through a liposome extrusion system to obtain the liposome encapsulating the deoxyribonucleic acid or ribonucleic acid.
Performance test 1 particle size and PDI (particle size distribution index) test of liposomes encapsulating deoxyribonucleic acid or ribonucleic acid.
Test standard: the particle size is 100-1000nm, and the PDI is less than 0.8.
The testing process comprises the following steps: taking a proper amount of deoxyribonucleic acid or ribonucleic acid liposome, diluting the liposome with water for injection by a proper multiple, and measuring the particle size and PDI of the liposome by using a Zetasizer Nano-ZS90 Nano-particle sizer. Test results: as shown in tables 7-13.
TABLE 7
TABLE 8
From Table 8, it is clear that examples 1 and examples 6 to 10 investigate the effect of the kind of targeting ligand on the particle size and PDI (particle size distribution index) of the prepared DNA or ribonucleic acid encapsulated liposome when the DNA or ribonucleic acid is encapsulated. The results show that when the targeting ligand is folic acid, the particle size and PDI (particle size distribution index) of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid are larger, and the stability of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid is slightly lower. When the targeting ligand is sodium hyaluronate or albumin, the particle size and PDI (particle size distribution index) of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid are smaller, and the stability of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid is better.
TABLE 9
Table 10
TABLE 11
Table 12
TABLE 13
| Performance of | Example 1 | Comparative example 1 |
| Particle size (nm) | 456.4±40.7 | 1333±87.6 |
| PDI (particle size distribution index) | 0.518±0.13 | 0.454±0.15 |
As is clear from the comprehensive comparison of tables 9 to 12, it is clear from Table 9 that examples 1 and 1-1 to 1-4 are examined for the effect of the amount of sodium hyaluronate used when sodium hyaluronate is selected as a targeting ligand in the case of encapsulating deoxyribonucleic acid on the particle size and PDI (particle size distribution index) of the finally produced liposome encapsulating deoxyribonucleic acid or ribonucleic acid. As is clear from Table 9, when sodium hyaluronate is selected as the targeting ligand, example 1, and when sodium hyaluronate is 10mg, the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid has a relatively uniform PDI (particle size distribution index) distribution with a relatively small particle size, and the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid has relatively high stability.
As is clear from Table 10, examples 8, 8-1 to 8-4 are examined for the effect of the amount of sodium hyaluronate used in the case of selecting sodium hyaluronate as a targeting ligand in the case of encapsulating ribonucleic acid on the particle size and PDI (particle size distribution index) of finally produced liposomes encapsulating deoxyribonucleic acid or ribonucleic acid. As is clear from Table 10, in example 8, when sodium hyaluronate is selected as the targeting ligand, and when sodium hyaluronate is 10mg, the particle size and PDI (particle size distribution index) of the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid are small, and the stability of the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid is high.
As is clear from Table 11, examples 6 and 6-1 to 6-4 are examined for the effect of the amount of sodium hyaluronate used in the preparation of the target ligand for the encapsulation of deoxyribonucleic acid or ribonucleic acid-encapsulated liposomes and PDI (particle size distribution index). As is clear from Table 11, when sodium hyaluronate is selected as the targeting ligand, example 6, and when sodium hyaluronate is 10mg, the particle size and PDI (particle size distribution index) of the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid are small, and the stability of the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid is high.
As is clear from Table 12, examples 9 and 9-1 to 9-4 are examined for the effect of the amount of sodium hyaluronate used in the preparation of the final liposome encapsulating deoxyribonucleic acid or ribonucleic acid on the particle size and PDI (particle size distribution index) when the targeting ligand is albumin. As is clear from Table 12, when sodium hyaluronate is selected as the targeting ligand, example 9, and when sodium hyaluronate is 10mg, the particle size and PDI (particle size distribution index) of the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid are small, and the stability of the prepared liposome encapsulating deoxyribonucleic acid or ribonucleic acid is high.
As is clear from Table 13, the particle size and PDI (particle size distribution index) of the liposomes encapsulating deoxyribonucleic acid or ribonucleic acid prepared by the different preparation methods were different, and the thin film hydration method was used in example 1 of the present invention, and the specific preparation process was as described above, and the ethanol injection method was used in comparative example 1, and the specific preparation process was as described above. As is clear from Table 13, the particle size and PDI (particle size distribution index) of the DNA or ribonucleic acid encapsulated liposome prepared by the thin film hydration method used in the examples are smaller, and the DNA or ribonucleic acid encapsulated liposome prepared by the thin film hydration method used in the examples is more stable.
Performance test 2 encapsulation efficiency test of liposomes encapsulating deoxyribonucleic acid or ribonucleic acid.
The testing process comprises the following steps: precise measurement of appropriate amounts of examples 1, 6 and 68. EXAMPLE 9 liposomes encapsulating deoxyribonucleic acid or ribonucleic acid, and diluting with PBS in a centrifuge tube, centrifuging for 3min, collecting the appropriate amount of supernatant in an EP tube, adding a small amount of PBS solution, and determining the free drug concentration C 1 . Adding the rest liposome solution encapsulating deoxyribonucleic acid or ribonucleic acid into a centrifuge tube, adding appropriate amount of methanol, performing ultrasonic demulsification, adding small amount of the solution into EP tube, adding PBS solution, and determining total concentration C of the medicine 0 . Encapsulation efficiency%1-C as per formula 1 /C 0 The encapsulation efficiency was calculated by x 100%.
Test results: as shown in table 14.
TABLE 14
| Performance of | Example 1 | Example 6 | Example 8 | Example 9 |
| Encapsulation efficiency (%) | 53.49±2.20 | 19.54±1.50 | 54.50±1.72 | 36.05±1.43 |
As is clear from the above table, the liposome prepared in example 1 and example 8, which encapsulates deoxyribonucleic acid or ribonucleic acid, has higher encapsulation efficiency and better stability. In the process of preparing the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid, when the targeting ligand selects sodium hyaluronate, the liposome for encapsulating the deoxyribonucleic acid or ribonucleic acid is prepared to be more stable.
Performance test 3 targeting test.
The testing process comprises the following steps: after culturing melanoma B16 cells to logarithmic phase, counting cells under microscope, and adjusting cell concentration to 1×10 5 Each mL, followed by seeding the cells into 96-well plates, adding 100. Mu.L of cell fluid per well, 1X 10 per well 4 About each cell, DMEM medium containing 10% FBS was added at 37℃and 5% CO 2 Culturing for 24 hours in an incubator; after cell wall-attached growth, discarding the supernatant, respectively adding 100 mu L of whole culture medium for preparing liposome by different targeting ligands into a 96-well plate, wherein the concentration of the drug is 0.1mg/mL,0.2mg/mL and 0.4mg/mL, simultaneously, respectively culturing 1min,5min,10min,20min and 40min post-treatment cells by a blank control group without adding drugs, detecting the concentration value of the liposome, and determining the optimal detection time of the liposome. After the optimal reaction time and concentration are determined, the medicine-containing culture medium is removed, PBS is used for washing 3 times, 0.5mL of sterile water is added for repeated freeze-thawing 3 times in an ultralow temperature refrigerator at-80 ℃, cell lysate is collected, centrifugation is carried out for 3min (11000 r/min), supernatant is taken out, and the concentration of liposome is detected. Kinetic parameters were calculated according to the mie equation:
V＝Vmax[S]/(Km+[S])＝C/t
where V is the uptake rate, vmax is the maximum uptake rate, km is the Mitsubishi constant, and S is the substrate concentration. C is the concentration of liposomes in the supernatant and t is the cell treatment time.
The biological targeting performance of the targeting ligand is examined through the cell uptake rate of B16 cells on liposome, namely, the faster the cell uptake rate is, the better the targeting performance is, so that the targeting liposome preparation with the best activity is screened.
Test results: as shown in table 15.
TABLE 15
| Performance of | Example 1 | Example 6 | Example 8 | Example 9 | Blank space |
| Cell uptake Rate (μg/ml/min) | 0.10615 | 0.05305 | 0.08725 | 0.0625 | 0.007078 |
As can be seen from the above table, the liposome cells prepared in example 1 and example 8, which encapsulate deoxyribonucleic acid or ribonucleic acid, have a faster uptake rate and a better targeting property. In the process of preparing the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid, when the targeting ligand is sodium hyaluronate, the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid is prepared and has better targeting performance.
Performance test 4 liposome stability test of encapsulated deoxyribonucleic acid or ribonucleic acid.
The testing process comprises the following steps: the long-term stability of the liposomes encapsulating deoxyribonucleic acid or ribonucleic acid prepared in example 1 and example 8 was examined using a stability test box, and the stability of the liposomes encapsulating deoxyribonucleic acid or ribonucleic acid prepared in example 1 and example 8 was examined for 10 days at 25℃and 60% relative humidity by examining the encapsulation efficiency, particle size, and PDI (particle size distribution index) of the liposomes encapsulating deoxyribonucleic acid or ribonucleic acid prepared in example 1 and example 8 within 10 days, and the relevant indexes were examined on day 0, day 5, and day 10, respectively.
Test results: as shown in table 16.
Table 16
| Long term (25 ℃,60% RH) | Example 1 | Example 8 |
| Encapsulation (%) 0 days | 53.49±2.18 | 54.5±2.54 |
| For 5 days | 51.23±3.98 | 51.21±4.12 |
| For 10 days | 50.17±4.31 | 50.39±7.15 |
| Particle size (nm) 0 day | 456.4±51.3 | 331.5±47.56 |
| For 5 days | 498.3±23.64 | 387.4±36.57 |
| For 10 days | 478.6±41.32 | 367.9±40.21 |
| PDI (particle size distribution index) (0 days) | 0.518±0.13 | 0.353±0.09 |
| For 5 days | 0.464±0.18 | 0.397±0.21 |
| For 10 days | 0.543±0.28 | 0.419±0.18 |
As can be seen from the above table, long-term stability examination of the encapsulated deoxyribonucleic acid or ribonucleic acid liposomes prepared in examples 1 and 8 of the present invention for 10 days revealed that the encapsulated deoxyribonucleic acid or ribonucleic acid liposomes prepared in examples 1 and 8 are stable in encapsulation efficiency, particle size and PDI (particle size distribution index) for 10 days, and the encapsulated deoxyribonucleic acid or ribonucleic acid liposomes prepared in examples 1 and 8 of the present invention have excellent long-term stability.
Performance test 5 liposome whitening test of encapsulated deoxyribonucleic acid or ribonucleic acid.
The testing process comprises the following steps: the inhibition activity of tyrosinase in vitro was studied. Briefly, tyrosinase inhibition was detected using a solution of L-DOPA as substrate. Glabridin was used as a positive control. Liposomes (40. Mu.L) at different concentrations were added to 96-well plates with a substrate solution containing 40. Mu.L (0.453 g/L) and 80. Mu.L potassium phosphate buffer (PBS, pH 6.8), incubated in a 37℃water bath for 10min, and then 200U/mL tyrosinase 40. Mu.L was added to each well and mixed by shaking at room temperature in the absence of light for 15min. The absorbance values were then measured and recorded rapidly with a Multiskan Go microplate reader at 475nm wavelength and measured 3 times in parallel to calculate the inhibition rate.
Tyrosinase inhibition rate (%) = ((A-B) - (C-D))/(A-B) ×100%
Wherein A is the absorbance value of 120. Mu.L of PBS buffer+40. Mu. L L-DOPA, B is 80. Mu.L of PBS buffer+40. Mu. L L-DOPA solution+40. Mu.L of tyrosinase solution, C is 40. Mu.L of sample solution+80. Mu.L of PBS buffer+40. Mu. L L-DOPA solution, and D is 40. Mu.L of sample solution+40. Mu.L of PBS buffer+40. Mu. L L-DOPA solution+40. Mu.L of tyrosinase solution.
Test results: as shown in table 17.
TABLE 17
| Performance of | Example 1 | Example 6 | Example 8 | Example 9 | Blank group |
| Inhibition rate | 7.59±1.15 | 4.16±1.10 | 17.69±2.30 | 5.72±0.53 | -11.87±1.26 |
Tyrosinase is a key enzyme in the skin melanin synthesis process, so it can directly affect the rate of melanin synthesis. The present invention measures the inhibition of tyrosinase to examine the whitening activity of liposomes encapsulating deoxyribonucleic acid or ribonucleic acid. As can be seen from the above table, the tyrosinase inhibitor activities of examples 1, 6, 8 and 9 were all significantly improved compared with the blank group, suggesting that the compositions had a certain whitening activity. Among them, tyrosinase inhibitors of example 1 and example 8 were more active, thus demonstrating that the liposomes encapsulating deoxyribonucleic acid or ribonucleic acid prepared in example 1 and example 8 were more effective in whitening skin.
Performance test 6 anti-melanoma cell activity performance test.
The testing process comprises the following steps: b16 cells were grown to log phase, digested with pancreatin, centrifuged, washed with PBS and seeded in 96-well plates at 1 x 10 per well 4 About each cell, DMEM medium containing 10% FBS was added at 37℃and 5% CO 2 Culturing for 24 hours in an incubator; liposome solutions (example 1, example 6, example 8, example 9) encapsulating deoxyribonucleic acid or ribonucleic acid with different concentrations of DNA and RNA: 6 compound wells per dose, 100 μl per well was added to 96 well plates and incubated for 24h; the blank group (with the same amount of culture medium) was treated without drug; after the completion of the culture, 10. Mu.L of MTT solution and 90. Mu.L of DMEM medium were added to each well, and after 4 hours of culture, the supernatant was discarded, 100. Mu.L of LDMSO was added to each well, and the mixture was homogenized to be sufficiently dissolved, and the OD value was measured at a wavelength of 490nm using an ELISA. Cell activity was expressed as the ratio of the mean OD values of the dosing group to the blank group.
Test results: as shown in table 18.
TABLE 18
| Performance of | Example 1 | Example 6 | Example 8 | Example 9 | Blank space |
| Cell Activity | 62±5.87 | 90±4.31 | 96±2.19 | 159±7.11 | 89±8.88 |
The B16 cells are one of main sources of skin melanin, so that the secretion of the melanin can be effectively inhibited by inhibiting the activity of the B16 cells, and the purpose of whitening is achieved. From the table, it can be found that the liposome prepared in example 1 and encapsulating deoxyribonucleic acid or ribonucleic acid has a better B16 cell activity inhibition effect.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and 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 the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (9)
1. The liposome for encapsulating deoxyribonucleic acid or ribonucleic acid is characterized by being of a core-shell structure, wherein a core of the core-shell structure is deoxyribonucleic acid or ribonucleic acid, and a shell of the core-shell structure is prepared from the following components in parts by weight: 140-180 parts of phosphate substances, 10-30 parts of cholesterol, 140-200 parts of solubilizer, 140-200 parts of stabilizer and 2.5-40 parts of targeting ligand;
the solubilizer is polyethylene glycol 400;
the stabilizer is glycerol;
the targeting ligand is sodium hyaluronate;
the preparation method of the liposome for encapsulating deoxyribonucleic acid or ribonucleic acid is characterized by comprising the following steps:
(1) Dissolving the phosphate substance and cholesterol, uniformly mixing, and performing rotary steaming to obtain a film;
(2) Adding the solubilizer, the stabilizer, the targeting ligand, the deoxyribonucleic acid or the ribonucleic acid into the film obtained in the step (1), adding a buffer solution, uniformly mixing, and performing ultrasonic treatment to obtain a suspension;
(3) And (3) passing the suspension obtained in the step (2) through a microporous filter membrane, and obtaining the liposome encapsulating the deoxyribonucleic acid or ribonucleic acid after a liposome extrusion system.
2. The liposome encapsulating deoxyribonucleic acid or ribonucleic acid as claimed in claim 1, wherein the shell of the core-shell structure is prepared from the following components in parts by weight: 160 parts of phosphate substances, 20 parts of cholesterol, 160 parts of solubilizer, 160 parts of stabilizer and 10 parts of targeting ligand.
3. The liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to claim 1, wherein in the step (1), the solvent used in the dissolution is absolute ethanol, and the spin evaporation temperature is 40-60 ℃; the film is a yellowish film.
4. The liposome encapsulating deoxyribonucleic acid or ribonucleic acid of claim 1, wherein in step (2), the buffer is one of phosphate buffer, citrate buffer, carbonate buffer, acetate buffer.
5. The liposome encapsulating deoxyribonucleic acid or ribonucleic acid of claim 1, wherein in step (2), the buffer is a phosphate buffer.
6. The liposome encapsulating deoxyribonucleic acid or ribonucleic acid as claimed in claim 1, wherein in the step (2), the mixing temperature is 25-35 ℃, the mixing time is 15-25min, and the ultrasonic time is 2-4min; the suspension is a milky white suspension.
7. The liposome encapsulating deoxyribonucleic acid or ribonucleic acid as claimed in claim 1, wherein in the step (3), the diameter of the microporous filter membrane is 0.2-0.6 μm.
8. The liposome encapsulating deoxyribonucleic acid or ribonucleic acid as claimed in claim 7, wherein the diameter of the microporous filter membrane is 0.45 μm.
9. Use of a liposome encapsulating deoxyribonucleic acid or ribonucleic acid according to any one of claims 1 to 2 for the preparation of a cosmetic.
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