Pyramid-type thermal assembly efficacy skin care nanoparticle based on DNA nucleic acid and application of pyramid-type thermal assembly efficacy skin care nanoparticle

Abstract

The invention discloses a pyramid type thermal assembly efficacy skin care nanoparticle based on DNA nucleic acid and application thereof. According to the invention, skin-care active components are loaded into a nucleic acid framework in a thermal assembly innovation mode to obtain a'pyramid-shaped 'functional skin-care nano system, and the embedding rate, stability, infiltration capacity and bioavailability of the system are improved. According to the invention, four sections of nucleotide single chains with the length of 59 basic groups are designed as a tetrahedral framework sequence, so that the synthesis cost is reduced. According to the invention, a blending autonomous thermal assembly mode is developed to load skin-care active substances into a tetrahedral frame to prepare a'pyramid-shaped 'functional skin-care nano system, so that the embedding efficiency of the skin-care active substances is improved. Whitening active substances are wrapped, zebra fish experiments prove that the pyramid-shaped functional skin care (whitening) nanoparticles can more remarkably inhibit melanin synthesis and relieve and improve skin melanin pigmentation, and meanwhile, the system is proved to have a functional skin care effect.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic production, and in particular to a DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticle and application thereof. Background Art

[0002] As consumers' demands for skin health and beauty continue to increase, the market for functional skin care cosmetics is growing rapidly. These products not only focus on basic moisturizing and cleansing functions, but also place special emphasis on the treatment and improvement of specific skin problems (such as whitening, wrinkles, acne, etc.). Although functional skin care cosmetics have made significant progress in ingredient selection and formula design, they face the key problem of insufficient permeability.

[0003] Take melanin as an example. It exists in the basal layer of the epidermis of human skin and is mainly produced by melanocytes. Under ultraviolet radiation, melanin will change and form a protective substance that can reduce skin damage. In addition, the content and distribution of melanin in the basal layer directly affect the color of the skin. With the metabolic process, melanin will gradually migrate and accumulate in the epidermis. However, common whitening products are often not ideal for removing deep melanin.

[0004] These problems mainly stem from factors such as the limitation of the skin barrier, the stability of active ingredients, the limitations of the carrier system, and individual differences. In order to overcome these challenges, researchers are developing new delivery systems and technologies, such as nanocarriers, liposomes, microneedle patches, etc., to improve the penetration efficiency and bioavailability of active ingredients, thereby achieving better skin care results.

[0005] Nucleic acid vectors are carriers with specific spatial configurations formed by one or more nucleotide chains through complementary base pairing. Since nucleic acid molecules have the characteristics of high biosafety and good water solubility, many studies have used them in related fields to reduce drug toxicity and improve water solubility. Nucleic acid frameworks of different configurations can provide different characteristics for drug delivery, such as achieving slow release of drugs and improving drug penetration. In short, using nucleic acid frameworks as active substance carrier skeletons can significantly improve the efficiency and biosafety of substances, providing new possibilities for future drug development and treatment methods.

[0006] In the prior art, in order to enhance the efficacy of skin care products to solve a series of skin problems such as preventing abnormal accumulation of melanin in the skin, the conventional carriers commonly used often have some toxic side effects and are difficult to penetrate into deep tissues. This may lead to poor use experience and safety issues, such as "accidental injury" to normal tissues, aggravation of skin problems, single effect, and repeated and unsatisfactory effects. These problems will undoubtedly greatly increase the cost of use. In contrast, new carriers such as nucleic acid frameworks have shown great potential in improving therapeutic effects and safety. For example, the national invention patent application CN117202937A discloses a nucleic acid cross skeleton that carries drugs through sequence terminal groups, the national invention patent application CN116459270A discloses that the use of nucleic acid tetrahedrons (tetrahedral framework nucleic acids, tFNA) as transport carriers can achieve efficient entry of composite drugs into cells through biological membranes to play a role, and the national invention patent application CN114569628A discloses the use of nucleic acid tetrahedrons as transport carriers to carry drugs to improve skin problems. Although the above two-dimensional nucleic acid cross skeleton has a high affinity for nucleic acids, the number of drugs it can carry is very limited and it cannot achieve beneficial penetration performance; while the tetrahedron as a three-dimensional spatial structure can significantly improve the penetration efficiency, but its long sequence leads to high synthesis costs, and its room temperature incubation method will also limit the encapsulation efficiency to a certain extent. In order to solve the above problems, it is urgent to develop a thermal assembly nanosystem based on a DNA carrier framework with higher encapsulation efficiency, better penetration effect, better stability, lower cost, and better activity effect. Summary of the invention

[0007] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a DNA nucleic acid pyramid-shaped thermally assembled functional skin care nanoparticle.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticles.

[0009] Another object of the present invention is to provide the application of the above-mentioned DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticles.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A DNA nucleic acid pyramid-shaped thermally assembled functional skin care nanoparticle, in which skin care active ingredients are loaded on a nucleic acid framework with a tetrahedral structure;

[0012] Among them, the nucleic acid framework is composed of four single-stranded nucleic acids.

[0013] The tetrahedral structure is a pyramid-shaped spatial configuration.

[0014] The length of the single-stranded nucleic acid is preferably 40 to 59 nt.

[0015] The sequences of the four single-stranded nucleic acids are preferably as shown in SEQ ID NO.1 to SEQ ID NO.4.

[0016] The skin care active ingredient is preferably an ingredient with whitening effect; more preferably α-arbutin.

[0017] The preparation method of the above-mentioned DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticles comprises the following steps:

[0018] (1) mixing four single-stranded nucleic acids in a molar ratio of 1:1:1:1 to obtain a nucleic acid mixed solution;

[0019] (2) heating the nucleic acid mixed solution to melt the nucleic acid to obtain a hot mixed solution of nucleic acid containing free single strands;

[0020] (3) uniformly mixing the solution containing the skin care active ingredient and the hot mixed solution of the nucleic acid obtained in step (2), and rapidly cooling the mixture to facilitate annealing of the single-stranded nucleic acid to form a DNA vector framework;

[0021] (4) The system obtained in step (3) is then oscillated to facilitate the loading of skin care active ingredients on the DNA carrier framework, thereby obtaining skin care nanoparticles based on the pyramid-shaped thermal assembly of DNA nucleic acids.

[0022] The solvent in the nucleic acid mixed solution described in step (1) is preferably a binding buffer.

[0023] The composition of the binding buffer is preferably as follows: 20-100 mM Tris-HCl, 5-20 mM MgSO4, pH 7.4-7.6; more preferably as follows: 50 mM Tris-HCl, 8 mM MgSO4, pH 7.4-7.6.

[0024] The nucleic acid concentration in the nucleic acid mixed solution described in step (1) is preferably 1 to 5 μM, and more preferably 2.5 μM.

[0025] The melting conditions in step (2) are preferably: melting temperature 80-100°C, melting time 5-20 min; more preferably: melting temperature 95°C, melting time 10 min.

[0026] The solution containing skin care active ingredients in step (3) is an aqueous solution; its concentration is preferably 1-5 mM; more preferably 2.5 mM.

[0027] The skin care active ingredient is a heat-resistant skin care active ingredient; preferably a heat-resistant whitening ingredient; more preferably α-arbutin.

[0028] The skin care active ingredient and the nucleic acid in step (3) are preferably mixed in a ratio of 1:250 to 1000; more preferably in a ratio of 1:500 to 650.

[0029] The specific operation of the rapid cooling described in step (3) is as follows: cooling the uniformly mixed solution at 0-8°C for 10-60 minutes; more preferably, cooling the uniformly mixed solution at 0°C for 30 minutes.

[0030] The oscillation conditions in step (4) are preferably: a rotation speed of 150 to 250 rpm, a temperature of 0 to 30° C., and a time of 1 to 6 h; more preferably: a rotation speed of 150 to 250 rpm, a temperature of 20 to 30° C., and a time of 2 to 4 h.

[0031] The above-mentioned DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticles are used in the preparation of skin care products.

[0032] The principle of the present invention is:

[0033] The content and distribution of melanin in the basal layer directly affect the color of the skin, leading to common skin problems such as uneven skin tone and spots. Arbutin is a natural whitening ingredient, and α-arbutin is widely used because of its excellent thermal stability and safety. It can inhibit the production of melanin through various pathways, including inhibiting the activity of tyrosinase, thereby reducing the accumulation of melanin. However, once melanin is further metabolized and gradually migrates and accumulates in the epidermis of the skin, the effect of using arbutin through ordinary transdermal penetration will be greatly reduced.

[0034] The nucleic acid nanoframe is a "pyramid-shaped" three-dimensional space shape formed by folding nucleic acid chains. It has strong rigidity and in addition to having good biological affinity, the specific structure can freely pass through the phospholipid cell membrane and various biological barriers, and has great potential for application in biological organisms. The present invention adopts 4 independently designed single-stranded nucleic acid sequences of fixed length, and forms a stable "pyramid-shaped" functional whitening nanosystem by blending with α-arbutin for autonomous thermal assembly. The present invention develops an autonomous thermal assembly method for blending active substances with 4 nucleic acid sequences, which improves the drug loading of the substance and the stability of the system. The present invention uses α-arbutin as a whitening active substance to prepare a nucleic acid functional whitening system, making full use of the membrane permeability of the "pyramid-shaped" nucleic acid, improving the permeability and whitening activity of the whitening system, and having the characteristics of low toxicity, and greatly reducing the preparation cost.

[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0036] (1) The present invention designs four tetrahedral nucleic acid single strands with fixed chain lengths, which significantly reduces the synthesis cost while ensuring the loading space inside the framework, and successfully forms a framework structure through assembly.

[0037] (2) The present invention proposes a thermal assembly method of nucleic acid tetrahedrons and active substances. When the four nucleic acid single strands are completely unzipped, the active substances are added to form a mixed system, which participates in the complementary pairing process of nucleic acid bases to achieve thermal assembly, thereby improving the encapsulation capacity of nucleic acid tetrahedrons for active substances.

[0038] (3) The present invention is the first to assemble α-arbutin and nucleic acid tetrahedron to prepare a nucleic acid efficacy whitening system, which has better whitening efficacy activity. Nucleic acid tetrahedron as a whitening system provides a better biomembrane penetration effect, and the whitening activity is significantly improved compared with free α-arbutin, and it effectively inhibits melanin production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the preparation process of Example 2.

[0040] Figure 2 It is a PAGE gel electrophoresis verification result diagram of nucleic acid self-assembly products; wherein, lane M is DNA Marker, lanes 1 to 4 are four single-stranded nucleic acids, lane 5 is the product of Example 1, and lane 6 is the product of Comparative Example 1.

[0041] Figure 3 This is a graph showing the results of Zetasizer detection of the particle sizes of the nucleic acid tetrahedral framework and nanoparticle B sample group obtained in Example 1.

[0042] Figure 4 This is a transmission electron microscope (TEM) observation result of nanoparticle B.

[0043] Figure 5 This is the result of melanin formation in zebrafish fry after treatment with different group systems. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

[0045] Example 1

[0046] In this embodiment, self-assembly to form a nucleic acid tetrahedral framework structure with a fixed chain length comprises the following steps:

[0047] (1) Sequence design: The designed sequence is a 59 nt nucleotide single strand (the sequence is shown in Table 1), which effectively reduces the sequence synthesis cost while ensuring the framework size and loading space;

[0048] (2) Nucleic acid system: All four single-stranded nucleotides were diluted to 2.5 μM with binding buffer and mixed in a sterilized, enzyme-inactivated 0.2 mL PCR tube at a molar ratio of 1:1:1:1;

[0049] (3) Framework assembly: The nucleic acid reaction system was heated to 95°C using a PCR reactor and maintained for 10 min, and then quickly placed in ice for 20 min to obtain nucleic acid tetrahedrons;

[0050] (4) Electrophoresis verification: 8% PAGE gel electrophoresis was performed to preliminarily verify the self-assembly of the nucleic acid tetrahedral framework;

[0051] (5) Particle size verification: Use a particle size analyzer to verify the size of the nucleic acid tetrahedral framework and further verify the framework assembly.

[0052] The binding buffer was 1×TM Buffer with the following composition: 50 mM Tris-HCl, 8 mM MgSO4, pH 7.4-7.6.

[0053] Table 1. Sequences of the four nucleotide single strands of Example 1

[0054]

[0055]

[0056] Example 2

[0057] The present invention uses α-arbutin with better heat resistance and stability as a whitening raw material, and obtains nanoparticles with whitening effect by self-heat assembly through blending. The process is as follows Figure 1 As shown, the specific preparation steps are as follows:

[0058] (1) Preparing an α-arbutin free system: dissolving α-arbutin in ultrapure water to obtain an α-arbutin working solution with a concentration of 2.5 mM for later use;

[0059] (2) Nucleic acid chain melting: All four single-stranded nucleotides were diluted to 2.5 μM with binding buffer, mixed in a sterilized enzyme-inactivated EP tube at a molar ratio of 1:1:1:1, and maintained at 95°C for 10 min to completely melt and form free single strands;

[0060] (3) preparing a nucleic acid hot mixed solution: further diluting the α-arbutin working solution with pure water, and mixing the obtained α-arbutin diluted solution with the four completely melted nucleotide single-stranded mixtures obtained in step (2) at a molar ratio of 1:250, 1:500, and 1:1000, respectively;

[0061] (4) Forming a nucleic acid framework: After the liquids in step (3) are mixed, the hot mixed liquids of different concentrations are immediately placed at 0° C. for 30 minutes, and the nucleic acids form a special tetrahedral structure based on the principle of base complementary pairing;

[0062] (5) Preparing a whitening particle sample group: The nucleic acid framework blend system obtained in step (4) was transferred to a room temperature shaker and stabilized at a rotation speed of 200 rpm for 3 hours to obtain nanoparticles A (the ratio of α-arbutin to single-chain nucleotides was 1:250), nanoparticles B (the ratio of α-arbutin to single-chain nucleotides was 1:500) and nanoparticles C (the ratio of α-arbutin to single-chain nucleotides was 1:1000) samples with whitening efficacy;

[0063] (6) Detection of embedding rate: The sample obtained in step (5) was centrifuged at 14,000×g for 10 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, and the liquid outside the ultrafiltration tube was taken for detection of α-arbutin embedding rate.

[0064] The background of the mixed solution in step (4) is 10 mM Tris-HCl, 1.6 mM MgSO4, pH 7.4-7.6.

[0065] Comparative Example 1

[0066] The same preparation method as in Example 1 is used, except that:

[0067] The composition and length (63 nt) of the nucleotide sequences are different, and the sequences are shown in Table 2.

[0068] Table 2 Comparative Example 1 Sequences of four nucleotide single strands

[0069]

[0070] Comparative Example 1 is obtained by assembling the tetrahedral framework in the same manner as in Example 1.

[0071] Comparative Example 2

[0072] The nucleic acid tetrahedral framework was prepared according to the same sequence as in Example 1, except that a conventional incubation method different from that of the sample group in Example 2 was used, and the steps were as follows:

[0073] (1) Dissolve α-arbutin in ultrapure water to obtain an α-arbutin working solution with a concentration of 2.5 mM for later use;

[0074] (2) maintaining the four nucleotide single strands at 95° C. for 10 min, and then rapidly cooling them in ice for 20 min to obtain the nucleic acid tetrahedron;

[0075] (3) further diluting the α-arbutin working solution with pure water, and mixing the obtained α-arbutin dilution solution with the nucleic acid tetrahedral framework obtained in step (2) at a molar ratio of 1:250, 1:500, and 1:1000, respectively;

[0076] (4) Finally, the mixture was incubated in a shaker at 200 rpm for 3 h to obtain nanoparticles D (the ratio of α-arbutin to single-chain nucleotides was 1:250), nanoparticles E (the ratio of α-arbutin to single-chain nucleotides was 1:500), and nanoparticles F (the ratio of α-arbutin to single-chain nucleotides was 1:1000) with whitening effects.

[0077] Effect Example 1

[0078] The tetrahedral framework difference of Example 1 was verified, and the gel electrophoresis results of Example 1 and Comparative Example 1 were compared, and the sizes of Example 1 and Comparative Example 1 were compared. The specific comparison results are as follows:

[0079] (1) Gel electrophoresis comparison results: 8% PAGE gel electrophoresis results are as follows Figure 2 As shown, there is not much difference between the electrophoresis results of Example 1 (left) and Comparative Example 1 (right); the two nucleic acid tetrahedral frameworks have formed a rigid structure, which significantly reduces the swimming speed of the framework in the gel, so the band position is obviously higher.

[0080] (2) Size comparison results: The particle size analyzer test results are shown in Table 3. The sizes of the nucleic acid tetrahedral frameworks assembled in Example 1 and Comparative Example 1 are both about 11 nm, with no significant difference.

[0081] Table 3 Particle size comparison between comparative example 1 and example 1

[0082]

[0083] The above results show that the fixed chain length sequence (59nt) designed by the present invention has a significantly lower synthesis cost while ensuring that it has a similar framework size, framework structure and material loading space as that in Comparative Example 1 compared with the nucleic acid sequence in Comparative Example 1.

[0084] Effect Example 2

[0085] (1) Detection of embedding rate:

[0086] After the samples prepared in Example 2 and Comparative Example 2 were centrifuged using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, the embedding rate was calculated by testing the total α-arbutin concentration standard curve and the α-arbutin (Arbutin) concentration remaining in the ultrafiltration tube extracellular solution.

[0087] The residual α-arbutin concentration in the ultrafiltration tube extracellular fluid was detected using a BioTek multifunctional microplate reader. 2 When the fluorescence intensity is greater than 5000, the standard curve equation (1) is:

[0088] IF 外液Arbutin =1441.17+30.09c Arbutin -0.0079c 2 Arbutin (1)

[0089] The residual α-arbutin concentration in the ultrafiltration tube extracellular fluid was detected using a BioTek multifunctional microplate reader. When the fluorescence intensity was less than 5000, the standard curve equation (2) was:

[0090] IF 外液Arbutin =-151.97+41.88c Arbutin (2)

[0091] IF external fluid Arb is the fluorescence intensity of α-arbutin in the external fluid;

[0092] c Arbutin is the concentration of α-arbutin, in μmol / L.

[0093] Since the fluorescence intensity of K blank is high, the error of directly calculating the encapsulation efficiency by fluorescence intensity is too large. Therefore, the fluorescence intensity of α-arbutin in the external solution (IF external solution Arb) is converted into α-arbutin concentration (c Arbutin ) and then calculate the encapsulation efficiency.

[0094] Comparison results of differences in embedding efficiency of α-arbutin:

[0095] The α-arbutin embedding verification method is as follows:

[0096] E Arb =[1-(c r-Arbutin / c t-Arbutin )]×100% (3)

[0097] E Arbutin is the encapsulation efficiency of α-arbutin, in %;

[0098] c r-Arbutin is the residual α-arbutin concentration, in μmol / L;

[0099] c t-Arbutin is the total α-arbutin concentration, in μmol / L.

[0100] The results are shown in Table 4, which shows that: (1) the nucleic acid systems of Example 2 and Comparative Example 2 both successfully encapsulated α-arbutin; (2) at the same encapsulation ratio, the encapsulation rate of the system prepared by the mixed self-heat assembly incubation method of Example 2 was higher than the encapsulation rate of the system prepared by the conventional incubation method of Comparative Example 2.

[0101] Table 4 Comparison of α-arbutin embedding efficiency between comparative example 2 and example 2

[0102]

[0103] (2) Size detection of nanoparticles before and after encapsulation of α-arbutin

[0104] The sizes of the nucleic acid tetrahedral framework obtained in Example 1 and the nanoparticles B obtained from the sample group in Example 2 were measured using a Zetasizer particle size analyzer. The particle size comparison results are shown in FIG. Figure 3 The particle size comparison results are shown in Table 5. It can be seen that the size of the nucleic acid whitening system successfully loaded with arbutin is about 21.04 nm, which is significantly larger than that of Example 1, indicating that the sample group successfully encapsulated arbutin.

[0105] Table 5 Particle size comparison between Example 1 and nanoparticle B sample group

[0106]

[0107]

[0108] (3) Electron microscope morphology observation:

[0109] The size and morphology of nanoparticles B were observed using a JEM-1400FLASH transmission electron microscope. The results of transmission electron microscopy are as follows: Figure 4 As shown, polygonal particles such as triangles and rhombuses can be observed, with a size of about 20nm, which is consistent with the size of the nucleic acid whitening system. The polygonal shape is formed due to the special configuration of the nucleic acid tetrahedron. The above results can verify the successful assembly of the nucleic acid whitening system with arbutin wrapped in tetrahedrons, and the transmission electron microscope size and particle size analyzer test results confirm each other.

[0110] The results of the effect sample group show that the self-heating assembly method of the blend proposed in the present invention effectively improves the loading efficiency of α-arbutin compared with the conventional incubation method used in Comparative Example 2; the nanoparticles B have a significantly larger size than the nucleic acid tetrahedron framework of Example 1, indicating that arbutin is successfully embedded; the shape structure of the nanoparticles B in the sample group conforms to the characteristics of nucleic acid tetrahedrons.

[0111] Effect Example 3

[0112] According to the embedding efficiency comparison results, the hot mixed liquid ratio of the sample group was optimized, and the mixing ratio of 1:625 was selected to prepare the corresponding nanoparticles G for zebrafish related experimental results verification and comparison, including the detection of zebrafish embryo lethality, melanin deposition and melanin inhibition rate. The specific test comparison method is as follows:

[0113] The breeding of zebrafish was carried out in accordance with the zebrafish whitening experimental standard (T / HPCIA005-2022), and sexually mature, healthy and deformed wild-type zebra fish were used for spawning. Sexually mature wild-type zebrafish were selected and placed in a spawning box with a baffle at a male-female ratio of 1:2, and cultured overnight in a constant temperature incubator (28°C); the baffle was removed the next day, mating for 1 hour under light to lay eggs, and then foreign matter in the culture dish was removed and placed in a constant temperature incubator (28°C) for culture. Embryos (gastrula stage) with normal development at 6-8hpf (hours post-fertilization, hpf) were selected for subsequent melanin removal experiments. Normally developed gastrula embryos were sucked with a plastic pipette and added to a 24-well cell culture plate at a ratio of 10 embryos per well. 2 mL of nanoparticle G sample solution (containing 500 μM arbutin) was added to each well and two parallel groups were set up. The cover was then placed in a constant temperature incubator (28°C) for culture. A blank control group and a positive control group of the same concentration were set up at the same time. The fry can be hatched after the embryo culture is about 66 hpf. The fry in this state are observed and photographed, and the embryonic mortality rate is calculated based on the observation results; after the embryo culture is 72 hpf, methylcellulose with a concentration of 4.5% is dripped on a slide to fix the fry. Two fry in each group are selected to take pictures of melanin in specific parts of the head, and the gray value is measured using the software ImageJ. All experiments were repeated three times.

[0114] (1) Comparison results of embryo mortality after sample treatment:

[0115] The survival rate of zebrafish fry was observed after zebrafish embryos were treated with different system groups. After the blank control group (water), the positive control group (500μM arbutin) and the nanoparticle G sample group (500μM arbutin, i.e. the experimental group) were treated with embryos at 66hpf, the survival rate of the three groups of fry was 95% (Table 6). After excluding experimental errors, the samples did not show toxicity at this concentration; in addition, it can be observed from the results that the melanin deposition of the fry in the sample group was significantly lower than that of the other two groups of fry, indicating that the sample initially showed excellent whitening activity.

[0116] Table 6 Calculation of mortality rate after sample treatment and culture

[0117]

[0118] (2) Comparison results of melanin deposition:

[0119] The zebrafish larvae were photographed under a microscope and stitched together. Figure 5 shown. Figure 5 The results showed that the fry hatched from embryos treated in the blank control group had normal body shape and normal melanin pigmentation on the head; Figure 5 The results showed that the larvae hatched from embryos treated with 500 μM arbutin in the positive control group had normal body shape and the melanin deposition on the head was comparable to that in the blank group, with no significant difference. Figure 5 The results showed that the zebrafish fry hatched after embryos were treated with the nanoparticle G sample group (500 μM arbutin) had normal body shape, and the melanin deposition on the head was significantly different from that of the first two groups, and the whitening activity was significantly improved.

[0120] (3) Comparison results of melanin inhibition rate:

[0121] ImageJ was used to measure the gray value, and the measurement results were converted into numerical values ​​and the melanin inhibition rate was calculated. The melanin inhibition rate was analyzed based on the gray value. The melanin inhibition rate calculation formula is:

[0122] Melanin inhibition rate = [1-(G 样品处理 / G 阴性对照 )]×100% (4)

[0123] The unit of melanin inhibition rate is %;

[0124] G 阴性对照 is the gray value of the head of zebrafish larvae in the negative control group;

[0125] G 样品处理 This is the grayscale value of the zebrafish larvae head after sample processing.

[0126] The calculation results are shown in Table 7. The melanin inhibition rate of the positive control group (500 μM arbutin) is 16.65%, and the melanin inhibition rate of the nanoparticle sample group (500 μM arbutin) at the same arbutin concentration is 89.5%.

[0127] Table 7 Comparison of melanin inhibition rate after sample treatment and culture

[0128]

[0129] The results of Example 3 show that the nanoparticles G provided by the present invention have low toxicity characteristics, and the nanoparticles G have more significant whitening activity than free α-arbutin.

[0130] In summary, the thermally assembled skin care nanoparticles based on the DNA vector framework provided by the present invention, wherein the nucleic acid framework not only provides better penetration efficiency and stability for the whitening system, but also effectively reduces the synthesis cost by designing a tetrahedral sequence of 59 nucleotides (nt) while ensuring the framework size, structure and material loading space. The present invention has developed a blended autonomous thermal assembly method for α-arbutin with high thermal stability, which significantly improves the loading efficiency and achieves a higher α-arbutin embedding rate while ensuring the effect of the active substance. The present invention uses the whitening effect verified by the zebrafish experiment to verify that the "pyramid-type" efficacy whitening nanoparticles have a more significant whitening effect than the free α-arbutin of the same concentration, and significantly reduces the deposition of melanin.

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticle, characterized in that: Skin care active ingredients are loaded on a nucleic acid framework with a tetrahedral structure; Among them, the nucleic acid framework is composed of four single-stranded nucleic acids; The length of the single-stranded nucleic acid is 40 to 59 nt.

2. The DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticles according to claim 1, characterized in that: The sequences of the four single-stranded nucleic acids are shown in SEQ ID NO.1 to SEQ ID NO.4; The skin care active ingredient is an ingredient with whitening effect.

3. The DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticles according to claim 1, characterized in that: The skin care active ingredient is alpha-arbutin.

4. The method for preparing the DNA nucleic acid pyramid-shaped thermal assembly functional skin care nanoparticles according to any one of claims 1 to 3, characterized in that The steps include: (1) mixing four single-stranded nucleic acids in a molar ratio of 1:1:1:1 to obtain a nucleic acid mixed solution; (2) heating the nucleic acid mixed solution to melt the nucleic acid to obtain a hot mixed solution of nucleic acid containing free single strands; (3) uniformly mixing the solution containing the skin care active ingredient and the nucleic acid hot mixed solution obtained in step (2), and rapidly cooling the mixture to obtain a DNA vector framework; (4) The system obtained in step (3) is then oscillated to facilitate the loading of skin care active ingredients on the DNA carrier framework, thereby obtaining skin care nanoparticles based on the pyramid-shaped thermal assembly of DNA nucleic acids.

5. The method for preparing skin care nanoparticles based on DNA nucleic acid pyramid-shaped thermal assembly according to claim 4, characterized in that: The solvent in the nucleic acid mixed solution described in step (1) is a binding buffer; The binding buffer is composed of: 20-100 mM Tris-HCl, 5-20 mM MgSO4, pH 7.4-7.6; The nucleic acid concentration in the nucleic acid mixed solution described in step (1) is 1 to 5 μM; The solution containing skin care active ingredients in step (3) is an aqueous solution.

6. The method for preparing skin care nanoparticles based on DNA nucleic acid pyramid-shaped thermal assembly according to claim 4, characterized in that: The melting conditions in step (2) are: melting temperature 80-100°C, melting time 5-20 min; The specific operation of the rapid cooling described in step (3) is as follows: cooling the uniformly mixed solution at 0 to 8° C. for 10 to 60 minutes; The oscillation conditions in step (4) are: rotation speed 150-250 rpm, temperature 0-30° C., and time 1-6 h.

7. The method for preparing skin care nanoparticles based on DNA nucleic acid pyramid-shaped thermal assembly according to claim 6, characterized in that: The melting conditions in step (2) are: melting temperature 95°C, melting time 10 min; The specific operation of the rapid cooling described in step (3) is as follows: cooling the uniformly mixed solution at 0° C. for 30 min; The oscillation conditions in step (4) are: rotation speed 150-250 rpm, temperature 20-30° C., and time 2-4 h.

8. The method for preparing skin care nanoparticles based on DNA nucleic acid pyramid-shaped thermal assembly according to claim 4, characterized in that: The skin care active ingredient described in step (3) and the nucleic acid described are mixed in a ratio of 1:250 to 1000.

9. Use of the DNA nucleic acid pyramid-shaped thermally assembled functional skin care nanoparticles according to any one of claims 1 to 3 in the preparation of skin care products.

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