DNA nucleic acid pyramid type heat assembly efficacy skin care nanoparticles and application thereof

Abstract

The application discloses a DNA nucleic acid pyramid type heat assembly efficacy skincare nanoparticle and application thereof. The application carries skin care active ingredients into a nucleic acid framework through a heat assembly innovative mode to obtain a "pyramid type" efficacy skincare nano system, and improves embedding rate, stability, penetration capacity and bioavailability of the system. The application designs four nucleotide single strands with a length of 59 bases as tetrahedral framework sequences, and reduces synthesis cost. The application develops a blending self-heat assembly mode to load skin care active substances in the tetrahedral framework to prepare the "pyramid type" efficacy skincare nano system, and improves embedding efficiency of the skin care active substances. The application wraps whitening active substances, and verifies that the pyramid type efficacy skincare (whitening) nanoparticle can more significantly inhibit melanin synthesis, relieve and improve skin melanin deposition through a zebra fish experiment, and proves that the system has the efficacy skincare effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic production, and particularly relates to a DNA nucleic acid pyramid type heat assembly efficacy skin care nanoparticle and application thereof. BACKGROUND

[0002] With the increasing demand for skin health and beauty from consumers, the market for efficacy skin care cosmetics is rapidly growing. These products not only focus on basic moisturizing and cleaning functions, but also emphasize the treatment and improvement effects on specific skin problems such as whitening, wrinkles, acne, etc. Although significant progress has been made in the selection of ingredients and the design of formulations for efficacy skin care cosmetics, they still face the key problem of insufficient penetration.

[0003] For example, melanin exists in the basal layer of the epidermis of human skin and is mainly produced by melanocytes. Under the irradiation of ultraviolet light, melanin changes and forms 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 gradually migrates and accumulates to the epidermis. However, the common whitening products often do not have ideal effects on the removal of deep melanin.

[0004] These problems are mainly caused by the limitations of the skin barrier, the stability of active ingredients, the limitations of carrier systems, 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, so as to achieve better skin care effects.

[0005] Nucleic acid carriers are carriers with specific spatial configurations formed by one or several nucleotide chains through base complementary pairing. Due to the characteristics of high biological safety and good water solubility of nucleic acid molecules, many studies have used them in related fields to reduce drug toxicity and improve water solubility. Different configurations of nucleic acid frameworks can provide different characteristics for drug delivery, such as achieving slow release of drugs, improving penetration ability of drugs, etc. In summary, using nucleic acid framework as the carrier skeleton of active substances can significantly improve the action efficiency and biological safety of the substances, and provides new possibilities for future drug research 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 caused by preventing abnormal accumulation of skin melanin, the conventional carriers commonly used usually have some toxic side effects and are difficult to penetrate into deep tissues. This can cause poor use experience and safety problems, such as "injury" to normal tissues, aggravation of skin problems, single effect, repeated and unsatisfactory effect, etc. These problems will undoubtedly greatly increase the use cost, compared with the new type of carrier such as nucleic acid framework which shows great potential in improving treatment effect and safety, such as the nucleic acid cross skeleton carrying drugs through the sequence end group disclosed in the national invention patent application CN117202937A, the nucleic acid tetrahedral framework (tFNA) as a transport carrier can realize the high-efficiency of the composite drug into cells to play a role through biological membranes, and the nucleic acid tetrahedral framework as a transport carrier to carry drugs to improve skin problems disclosed in the national invention patent application CN114569628A. Although the above two-dimensional nucleic acid cross skeleton has high affinity of nucleic acid, the number of drugs carried is very limited and the beneficial penetration performance cannot be achieved; and the tetrahedron as a three-dimensional spatial structure can significantly improve the penetration efficiency, but the long sequence leads to high synthesis cost, and the room temperature incubation method will also limit the wrapping efficiency to some extent. In order to solve the above problems, it is urgent to develop a DNA carrier framework-based hot assembly nanosystem with higher wrapping efficiency, better penetration effect, better stability, lower cost and better activity effect. SUMMARY

[0007] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a DNA nucleic acid pyramid type hot assembly efficacy skin care nanoparticle.

[0008] Another purpose of the present application is to provide a preparation method of the above-mentioned DNA nucleic acid pyramid type hot assembly efficacy skin care nanoparticle.

[0009] Still another purpose of the present application is to provide the application of the above-mentioned DNA nucleic acid pyramid type hot assembly efficacy skin care nanoparticle.

[0010] The purpose of the present application is achieved by the following technical solutions:

[0011] A DNA nucleic acid pyramid type hot assembly efficacy skin care nanoparticle is loaded with skin care active ingredients on a nucleic acid framework in a tetrahedral structure.

[0012] The nucleic acid framework is composed of four single-stranded nucleic acids.

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

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

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

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

[0017] The preparation method of the skin care nanoparticles based on DNA nucleic acid pyramid thermal assembly efficacy described above comprises the following steps:

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

[0019] (2) The nucleic acid mixed solution is heated for melting to obtain a nucleic acid thermal mixture containing free single-stranded nucleic acids;

[0020] (3) The solution containing the skin care active ingredient is mixed uniformly with the nucleic acid thermal mixture obtained in step (2) and rapidly cooled to facilitate the annealing of single-stranded nucleic acids to form a DNA carrier framework;

[0021] (4) The system obtained in step (3) is further oscillated to facilitate the loading of the skin care active ingredient on the DNA carrier framework to obtain skin care nanoparticles based on DNA nucleic acid pyramid thermal assembly efficacy.

[0022] The solvent in the nucleic acid mixed solution 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, it is as follows: 50 mM Tris-HCl, 8 mM MgSO4, pH 7.4-7.6.

[0024] The concentration of nucleic acids in the nucleic acid mixed solution in step (1) is preferably 1-5 μM; more preferably, it is 2.5 μM.

[0025] The conditions for melting in step (2) are preferably as follows: melting temperature 80-100℃, melting time 5-20 min; more preferably, the melting temperature is 95℃ and the melting time is 10 min.

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

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

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

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

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

[0031] The application of the above-mentioned skin care nanoparticles based on the DNA nucleic acid pyramid type thermal assembly efficacy in the preparation of skin care products.

[0032] The principle of the present application 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, color spots, etc. Arbutin, as a natural whitening ingredient, alpha-arbutin in it is widely used due to its excellent thermal stability, safety, etc. It can inhibit the generation of melanin through multiple pathways, including inhibiting the activity of tyrosinase, thereby reducing the accumulation of melanin. However, once the melanin is further metabolized and gradually migrates and accumulates to the epidermis of the skin, the effect of using arbutin through ordinary transdermal penetration will be greatly reduced.

[0034] The nucleic acid nano framework is a "pyramid type" three-dimensional spatial shape formed by folding nucleic acid chains, which has strong rigidity and, in addition to good biological affinity, the specific structure can freely pass through phospholipid cell membranes and various biological barriers, and has great potential in biological applications. The present application uses a self-designed 4-segment fixed-length single-stranded nucleic acid sequence, which is self-assembled with alpha-arbutin to form a stable "pyramid type" efficacy whitening nano system. The present application develops a blending self-assembly method of active substances and 4 nucleic acid sequences, which improves the drug loading capacity and system stability. The present application uses alpha-arbutin as a whitening active substance to prepare a nucleic acid efficacy whitening system, which fully utilizes the membrane penetration of the "pyramid type" nucleic acid, improves the penetration, whitening activity of the whitening system, and has the characteristics of low toxicity, and greatly reduces the preparation cost.

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

[0036] (1) The application designs four segments of fixed length of tetrahedral nucleic acid single strands, which significantly reduces the synthesis cost while ensuring the loading space inside the framework, and successfully forms a framework structure by assembling.

[0037] (2) The application proposes a thermal assembly method of nucleic acid tetrahedron and active substances, which adds active substances to form a blending system when the four segments of nucleic acid single strands are completely dissociated, participates in the complementary pairing process of nucleic acid bases to realize thermal assembly, and improves the loading capacity of nucleic acid tetrahedron to active substances.

[0038] (3) The application first assembles alpha-arbutin and nucleic acid tetrahedron to prepare a nucleic acid whitening system with better whitening efficacy activity. The nucleic acid tetrahedron provides better bio-membrane penetration effect as a whitening system, and the whitening activity is significantly improved compared with free alpha-arbutin, and the melanin production is effectively inhibited. BRIEF DESCRIPTION OF DRAWINGS

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

[0040] Figure 2 is a PAGE gel electrophoresis verification result diagram of the self-assembly product of nucleic acid; wherein, lane M is DNA Marker, lanes 1-4 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 is a result diagram of Zetasizer detection of the particle size of the nucleic acid tetrahedron framework and nanoparticle B sample obtained in Example 1.

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

[0043] Figure 5 is a result diagram of melanin formation of zebra fish larvae after treatment of different groups of systems. DETAILED DESCRIPTION

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

[0045] Example 1

[0046] The self-assembly of the nucleic acid tetrahedron framework structure with fixed chain length in this embodiment includes the following steps:

[0047] (1) Sequence design: design 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: four single-stranded nucleotides were each diluted to 2.5 mM with binding buffer, mixed in a sterile and enzyme-free 0.2 mL PCR tube at a molar ratio of 1:1:1:1;

[0049] (3) Frame assembly: the nucleic acid reaction system was heated to 95°C and maintained for 10 min using a PCR reaction instrument, and then quickly cooled on ice for 20 min to obtain a nucleic acid tetrahedron;

[0050] (4) Electrophoresis verification: an 8% PAGE gel was prepared for electrophoresis to preliminarily verify the self-assembly of the nucleic acid tetrahedron frame;

[0051] (5) Particle size verification: the size of the nucleic acid tetrahedron frame was verified using a particle size analyzer to further verify the frame assembly.

[0052] The binding buffer is 1xTM Buffer, which is composed of 50mM Tris-HCl, 8mM MgSO4, pH 7.4-7.6.

[0053] Table 1. Sequence of four nucleotide single strands in Example 1

[0054]

[0055]

[0056] Example 2

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

[0058] (1) Configuration of α-arbutin free system: α-arbutin was dissolved in ultrapure water to obtain an α-arbutin working solution with a concentration of 2.5 mM, which was prepared for use;

[0059] (2) Nucleic acid strand dissociation: four single-stranded nucleotides were each diluted to 2.5 mM with binding buffer, mixed in a sterile and enzyme-free EP tube at a molar ratio of 1:1:1:1, and completely dissociated at 95°C for 10 min to form free single strands;

[0060] (3) Preparation of nucleic acid hot mixture: the α-arbutin working solution was further diluted with pure water, and the obtained α-arbutin diluent was mixed with the four completely dissociated nucleotide single strand mixture obtained in step (2) at a molar ratio of 1:250, 1:500, and 1:1000, respectively.

[0061] (4) Forming nucleic acid framework: after mixing in step (3), the hot mixed solution of different concentration ratios was immediately placed at 0°C for 30 min, and the nucleic acid formed a tetrahedral special structure by base complementary pairing principle;

[0062] (5) Preparation of whitening particle sample group: the nucleic acid framework blend system obtained in step (4) was transferred to a constant temperature shaker, and was stably shaken at a speed of 200 rpm for 3 h to obtain whitening nanoparticles A (the ratio of α-arbutin to nucleotide single strand was 1:250), nanoparticles B (the ratio of α-arbutin to nucleotide single strand was 1:500) and nanoparticles C (the ratio of α-arbutin to nucleotide single strand was 1:1000);

[0063] (6) Detection of embedding rate: the sample obtained in step (5) was centrifuged at 14000 x g for 10 min with an ultrafiltration centrifuge tube with a molecular weight cut-off of 30 kDa, and the outer liquid of the ultrafiltration tube was used for α-arbutin embedding rate detection.

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

[0065] Comparative Example 1

[0066] According to the same preparation method of Example 1, the difference is that:

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

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

[0069]

[0070] By the same tetrahedral framework assembly method as Example 1, Comparative Example 1 is obtained.

[0071] Comparative Example 2

[0072] According to the same sequence of Example 1 to prepare the nucleic acid tetrahedral framework, the difference is that a different conventional incubation method is used instead of the sample group of Example 2, and the steps are as follows:

[0073] (1) Dissolve α-arbutin in ultrapure water to obtain a 2.5 mM α-arbutin working solution for standby;

[0074] (2) The four nucleotide single strands were maintained at 95°C for 10 min, and then quickly placed in ice for 20 min to obtain the nucleic acid tetrahedron;

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

[0076] (4) finally, incubate in a shaker at a speed of 200 rpm for 3 h to obtain nanoparticles D (the ratio of α-arbutin to nucleotide single strand is 1:250), nanoparticles E (the ratio of α-arbutin to nucleotide single strand is 1:500), and nanoparticles F (the ratio of α-arbutin to nucleotide single strand is 1:1000) with whitening effect.

[0077] Effect Example 1

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

[0079] (1) Gel electrophoresis comparison results: 8% PAGE gel electrophoresis results are shown in Figure 2 It can be seen that the electrophoresis results of Example 1 (left) and Comparative Example 1 (right) have no great difference; the two nucleic acid tetrahedral frameworks significantly reduce the migration speed of the framework in the gel due to the formation of a rigid structure, so the band position is obviously high.

[0080] (2) Size comparison results: The particle size instrument detection results are shown in Table 3, and the size of the nucleic acid tetrahedral framework assembled by Example 1 and Comparative Example 1 is about 11 nm, and there is no obvious difference.

[0081] Table 3 Particle size comparison of Comparative Example 1 and Example 1

[0082]

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

[0084] Effect Example 2

[0085] (1) Detection of embedding rate:

[0086] After centrifuging the samples prepared by Example 2 and Comparative Example 2 with an ultrafiltration centrifuge tube with a molecular weight cut-off of 30 kDa, the embedding rate is calculated by testing the total α-arbutin concentration standard curve and the α-arbutin (Arbutin) concentration remaining in the liquid outside the ultrafiltration tube.

[0087] The BioTek multifunctional enzyme marker was used to detect the residual a- arbutin concentration in the ultrafiltration tube external liquid. In order to make the fitting equation R 2 The standard curve equation (1) is as follows when the fluorescence intensity is greater than 5000:

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

[0089] The BioTek multifunctional enzyme marker was used to detect the residual a- arbutin concentration in the ultrafiltration tube external liquid. In order to make the fitting equation R

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

[0091] IF external liquid Arb is the external liquid a- arbutin fluorescence intensity;

[0092] c Arbutin is the a- arbutin concentration, with units of μmol / L.

[0093] Since the K blank fluorescence intensity is high, directly using the fluorescence intensity to calculate the encapsulation rate error is too large, therefore, the external liquid a- arbutin fluorescence intensity (IF external liquid Arb) is converted into a- arbutin concentration (c Arbutin ) and then the encapsulation rate is calculated.

[0094] Comparison results of a- arbutin embedding efficiency difference:

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

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

[0097] E Arbutin is the a- arbutin encapsulation rate, with units of %;

[0098] c r-Arbutin is the residual a- arbutin concentration, with units of μmol / L;

[0099] c t-Arbutin is the total a- arbutin concentration, with units of μmol / L.

[0100] The results are shown in Table 4, and it can be seen that: (1) the nucleic acid systems of Example 2 and Comparative Example 2 successfully encapsulate α-arbutin; (2) at the same embedding ratio, the embedding rate of the system prepared by the blending self-thermal assembly incubation method of Example 2 is higher than that of the system prepared by the conventional incubation method of Comparative Example 2.

[0101] Table 4 Comparison of α-arbutin embedding efficiency of Comparative Example 2 and Example 2

[0102]

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

[0104] The size of the nucleic acid tetrahedral framework obtained in Example 1 and the nanoparticles B obtained in the sample group of Example 2 was detected by a Zetasizer particle size instrument, and the particle size comparison results are shown in Table 5. Figure 3 As can be seen from the particle size comparison results shown in Table 5, 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 embedded arbutin.

[0105] Table 5 Particle size comparison of Example 1 and the sample group of nanoparticles B

[0106]

[0107]

[0108] (3) Electron microscope morphology observation:

[0109] The size and morphology of nanoparticles B were observed by a JEM-1400FLASH transmission electron microscope, and the transmission electron microscope observation results are shown in Table 6. Figure 4 As can be seen from the transmission electron microscope observation results shown in Table 6, triangular and rhombic polygon-shaped particles can be observed, with a size of about 20 nm, 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 that the nucleic acid whitening system successfully encapsulates arbutin, and the transmission electron microscope size is consistent with the particle size instrument detection results.

[0110] The results of the sample group show that the blending self-thermal assembly method proposed in the present application effectively improves the loading efficiency of α-arbutin compared with the conventional incubation method of Comparative Example 2. Nanoparticles B have a significantly larger size than the nucleic acid tetrahedral framework of Example 1, indicating that arbutin is successfully embedded. The shape structure of the sample group of nanoparticles B is consistent with the characteristics of the nucleic acid tetrahedron.

[0111] Effect Example 3

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

[0113] The breeding of zebrafish refers to the zebrafish whitening experiment standard (T / HPCIA005-2022), and sexually mature, healthy and non-deformed wild type zebrafish are used for spawning. Sexually mature wild type zebrafish are selected and placed in a spawning box with a baffle at a male to female ratio of 1:2, and cultured overnight in a constant temperature incubator (28℃). The next day, the baffle was removed and the fish were mated for 1h under light to spawn, then the culture dish was cleaned of foreign matter and placed in a constant temperature incubator (28℃) for culture. Select 6-8hpf (hours post-fertilization, hpf) normally developed embryos (gastrula stage) for subsequent melanin removal experiments. The normally developed gastrula stage embryos were aspirated with a plastic pipette, and 10 embryos per well were added to a 24-well cell culture plate, 2mL of nanoparticle G sample solution (containing 500μM arbutin) was added to each well, and two parallel groups were set up, and then covered with a cover plate and placed in a constant temperature incubator (28℃) for culture. At the same time, a blank control group and a positive control group of the same concentration were set up. After about 66hpf of embryo culture, the larvae were hatched, and the larvae in this state were observed and photographed, and the mortality rate of the embryos was calculated according to the observation results; after 72hpf of embryo culture, 4.5% methyl cellulose was added to the glass slide for fixation of the larvae, and 2 larvae per group were selected for melanin photography of the head of the larvae, and the gray value was determined by software ImageJ. All experiments were repeated three times.

[0114] (1) Embryo mortality rate comparison results after sample treatment:

[0115] After observing the survival rate of zebrafish larvae treated with different system groups, the survival rates of the larvae in 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 all 95% (Table 6) after 66hpf of embryo treatment, excluding experimental errors, the sample did not show toxicity at this concentration; in addition, it can be observed from the results that the melanin deposition of the larvae in the sample group was significantly lower than that in the other two groups, indicating that the sample showed excellent whitening activity.

[0116] Table 6 Mortality rate calculation after sample treatment and culture

[0117]

[0118] (2) Melanin deposition comparison results:

[0119] The zebrafish larvae were photographed by using a microscope and spliced, and the results are shown in Figure 5 . Figure 5 The results show that the zebrafish larvae hatched after the embryos in the blank control group were treated have normal body shape and normal melanin deposition in the head. Figure 5 The results show that the zebrafish larvae hatched after the embryos in the 500 μM arbutin positive control group were treated have normal body shape, and the melanin deposition in the head is equivalent to that in the blank group, without significant difference. Figure 5 The results show that the zebrafish larvae hatched after the embryos in the nanoparticle G sample group (500 μM arbutin) were treated have normal body shape, and the melanin deposition in the head is significantly different from that in the previous two groups, and the whitening activity is significantly improved.

[0120] (3) Comparison of melanin inhibition rate

[0121] The gray value was determined by using ImageJ, the determination results were converted into numerical values, and the melanin inhibition rate was calculated. The melanin inhibition rate was analyzed based on the gray value, and the melanin inhibition rate calculation formula was:

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

[0123] The unit of the melanin inhibition rate is %.

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

[0125] G 样品处理 is the gray value of the head of the zebrafish larvae after sample treatment.

[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) under 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 Effect Implementation Example 3 show that the nanoparticle G provided by the application has low toxicity characteristics, and the nanoparticle G has more significant whitening activity compared with free α-arbutin.

[0130] In summary, the present application provides a hot assembly skin care nanoparticle based on a DNA carrier framework, 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 59 nucleotide (nt) tetrahedral sequence while ensuring the framework size, structure and material loading space. The present application develops a blending self-assembly method for high thermal stability alpha-arbutin, which significantly improves the loading efficiency and achieves higher alpha-arbutin embedding rate under the premise of ensuring the effect of active substances. The present application uses zebrafish experiments to verify the whitening effect, which verifies that the "pyramid type" functional whitening nanoparticles have more significant whitening effect compared with free alpha-arbutin of the same concentration, and significantly reduce the deposition of melanin.

[0131] The above embodiment is a preferred embodiment of the present application, but the embodiments of the present application are not limited by the above embodiment, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be included in the protection scope of the present application.

Claims

1. A DNA nucleic acid pyramid type thermal assembly efficacy skin care nanoparticle based on, characterized by: Is loaded with skin care active ingredients on the nucleic acid framework in the form of tetrahedral structure; Wherein, the nucleic acid framework is composed of four single-stranded nucleic acids; The sequence of the four single-stranded nucleic acids is shown in SEQ ID NO. 1-4; The skin care active ingredient is alpha-arbutin; The DNA nucleic acid pyramid type thermal assembly efficacy skin care nanoparticle is prepared by the following preparation method: (1) Four single-stranded nucleic acids are mixed in a molar ratio of 1:1:1:1 to obtain a nucleic acid mixed solution; (2) The nucleic acid mixed solution is heated to denature to obtain a nucleic acid thermal mixture containing free single strands; (3) The solution containing the skin care active ingredient is mixed uniformly with the nucleic acid thermal mixture obtained in step (2) and rapidly cooled to obtain a DNA carrier framework; (4) The system obtained in step (3) is oscillated to facilitate the loading of the skin care active ingredient on the DNA carrier framework to obtain the DNA nucleic acid pyramid type thermal assembly efficacy skin care nanoparticle.

2. The method for preparing DNA nucleic acid-based gold pyramid type heat assembly efficacy skin care nanoparticles according to claim 1, characterized in that Including the following steps: (1) Four single-stranded nucleic acids are mixed in a molar ratio of 1:1:1:1 to obtain a nucleic acid mixed solution; (2) The nucleic acid mixed solution is heated to denature to obtain a nucleic acid thermal mixture containing free single strands; (3) The solution containing the skin care active ingredient is mixed uniformly with the nucleic acid thermal mixture obtained in step (2) and rapidly cooled to obtain a DNA carrier framework; (4) The system obtained in step (3) is oscillated to facilitate the loading of the skin care active ingredient on the DNA carrier framework to obtain the DNA nucleic acid pyramid type thermal assembly efficacy skin care nanoparticle.

3. The preparation method of the DNA nucleic acid pyramid type thermal assembly efficacy skin care nanoparticle according to claim 2, characterized in that: The solvent in the nucleic acid mixed solution in step (1) is a binding buffer; The composition of the binding buffer is as follows: 20-100 mM Tris-HCl, 5-20 mM MgSO4, pH 7.4-7.6; The nucleic acid concentration in the nucleic acid mixed solution in step (1) is 1-5 μM; The solution containing the skin care active ingredient in step (3) is an aqueous solution.

4. The preparation method of the DNA nucleic acid pyramid type thermal assembly efficacy skin care nanoparticle according to claim 2, characterized in that: The denaturation conditions in step (2) are as follows: denaturation temperature 80-100 ℃, denaturation time 5-20 min; The rapid cooling in step (3) is specifically as follows: the uniformly mixed solution is placed in 0-8 ℃ for 10-60 min; The oscillation conditions in step (4) are as follows: rotation speed 150-250 rpm, temperature 0-30 ℃, time 1-6 h.

5. The preparation method of the DNA nucleic acid pyramid type thermal assembly efficacy skin care nanoparticle according to claim 4, characterized in that: The denaturation conditions in step (2) are as follows: denaturation temperature 95 ℃, denaturation time 10 min; The rapid cooling in step (3) is specifically as follows: the uniformly mixed solution is placed in 0 ℃ for 30 min; The oscillation conditions in step (4) are: rotation speed 150-250 rpm, temperature 20-30℃, time 2-4h. 6.The preparation method of the DNA nucleic acid pyramid type heat assembly efficacy skin care nanoparticles according to claim 2, characterized in that: The skin care active ingredient and the nucleic acid in step (3) are in a ratio of 1:250-1000. 7.The application of the DNA nucleic acid pyramid type heat assembly efficacy skin care nanoparticles in claim 1 in the preparation of skin care products.

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