Polypeptide-nucleic acid tetrahedron compound as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide-nucleic acid tetrahedron compound as well as a preparation method and application thereof. The compound comprises a nucleic acid tetrahedron and polypeptide, wherein the polypeptide is nonapeptide-1 with an amino acid sequence as shown in SEQ ID NO: 13; the nucleic acid tetrahedron is formed by self-assembly of four single-stranded DNAs with the same length. According to the compound, the transdermal function of nucleic acid tetrahedron capable of inhibiting melanogenesis and the melanocyte targeting function of nonapeptide-1 are combined, so that the TDN-NA-1 compound has the melanocyte targeting property while having the autonomous transdermal ability, and the blackness inhibiting effects of the nucleic acid tetrahedron and the nonapeptide-1 are combined, so that the effect of inhibiting melanogenesis is achieved. Therefore, the whitening composition has a stronger capability of inhibiting melanin generation, so that the purpose of precise whitening is achieved, and the whitening composition has a wide application prospect in the field of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polypeptide-nucleic acid tetrahedron complex and a preparation method and application thereof. BACKGROUND

[0002] A nucleic acid tetrahedron (also called a tetrahedral DNA nanostructure, TDN, or a tetrahedral framework nucleic acid, tFNA) is composed of 4 equal-length single-stranded DNAs. Each face of the tetrahedron is composed of 3 short sequences of single-stranded DNA (ssDNA) paired with the same sequence of ssDNA, and each edge is composed of 20 bases, forming a size-controllable and rigid three-dimensional DNA structure.

[0003] TDNs have a good controllability in size and shape, are simple to prepare, have high mechanical strength, and have good biocompatibility and membrane permeability, etc., and thus have a broad application prospect in drug delivery, disease treatment, and biomedical imaging. At present, TDNs have been proved to be capable of delivering small molecule drugs, polypeptides, and oligonucleotide drugs. TDNs can enter cells through porous endocytosis by virtue of their biological and structural characteristics in cell penetration, but this way of entering cells is non-specific and random.

[0004] The first step of melanin formation is the combination of melanocyte-stimulating hormone (alpha-MSH) and melanocortin receptor 1 (MC1R) on melanocytes, and then the activation of tyrosinase to further form melanin. Nonapeptide-1 (also called whitening peptide) is a small molecule peptide containing 9 amino acids, which is a biomimetic peptide of natural melanocyte-stimulating hormone (alpha-MSH). It combines with melanocortin receptor 1 (MC1R) in a competitive manner, hinders the activation of tyrosinase, and then inhibits the formation of melanin.

[0005] The high affinity of nonapeptide-1 to MC1R receptor makes it have good targeting to melanocytes in the skin, but nonapeptide-1 itself does not have the ability to directly penetrate the skin. SUMMARY

[0006] To solve the technical problems that the nucleic acid tetrahedron does not have targeting and nonapeptide-1 itself does not have the ability to autonomously penetrate the skin in the prior art, the present application provides a polypeptide-nucleic acid tetrahedron complex and a preparation method and application thereof.

[0007] Specifically, the present application solves the above technical problems through the following technical solutions.

[0008] The first aspect of the present application provides a complex comprising a nucleic acid tetrahedron and a polypeptide; wherein the polypeptide is a nonapeptide-1 with an amino acid sequence as shown in SEQ ID NO: 13; and the nucleic acid tetrahedron is self-assembled from four single-stranded DNAs with the same length.

[0009] In some embodiments, the sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 1-4, respectively;

[0010] Alternatively, the sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 5-8, respectively;

[0011] Alternatively, the sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 9-12, respectively.

[0012] The second aspect of the present application provides a nucleic acid nanoparticle comprising the complex as described in the first aspect of the present application.

[0013] In some embodiments, the nucleic acid nanoparticle further comprises a carrier and / or a crosslinking agent.

[0014] In the present application, the carrier is conventional in the art, for example, a pharmaceutically acceptable carrier, including but not limited to a buffer, an antioxidant, a preservative, a protein, a polypeptide, a high molecular polymer, an amino acid, and a sugar. In some embodiments, the high molecular polymer can form a scaffold or a particle structure under certain conditions.

[0015] In the present application, the crosslinking agent can be conventional in the art, for use in the internal crosslinking, intermolecular crosslinking of the nucleic acid nanoparticle or the nucleic acid tetrahedron of the present application, or the crosslinking with the scaffold or the particle structure, or for use in the carrier to form a stable spatial structure.

[0016] The third aspect of the present application provides a pharmaceutical composition comprising the complex as described in the first aspect of the present application or the nucleic acid nanoparticle as described in the second aspect of the present application, and a pharmaceutically acceptable carrier and / or adjuvant.

[0017] The fourth aspect of the present application provides a method for preparing the complex as described in the first aspect of the present application, comprising the step of mixing the nucleic acid tetrahedron and the polypeptide in a solution.

[0018] In some embodiments, the solution comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA, and 20-100 mM MgCl2; and has a pH of 7.5-8.5.

[0019] In some embodiments, the molar ratio of the nucleic acid tetrahedron and the polypeptide is 1:1-1:1000.

[0020] In some embodiments, the molar ratio of the nucleic acid tetrahedron and the polypeptide is 1:50-1:400.

[0021] In some embodiments, the molar ratio of the nucleic acid tetrahedron and the polypeptide is 1:50, 1:100, 1:200, 1:300 or 1:400.

[0022] In some embodiments, the method further comprises the step of preparing the nucleic acid tetrahedron, comprising mixing and reacting four single-stranded DNAs in a buffer system; terminating the reaction after the single-stranded DNAs form double-stranded DNAs.

[0023] In some embodiments, the four single-stranded DNAs are mixed at equimolar concentration.

[0024] In some embodiments, the buffer system comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA and 20-100 mM MgCl2; and has a pH of 7.5-8.5.

[0025] In some specific embodiments, the buffer system has a composition of 10 mM Tris-HCl, 1 mM EDTA, 50 mM MgCl2, pH 8.0.

[0026] In some embodiments, the reaction is at 95°C for at least 10 min.

[0027] In some embodiments, the reaction is terminated at 4°C.

[0028] In some embodiments, the reaction is terminated at 4°C for 20 min.

[0029] The fifth aspect of the present application provides a method of delivering a polypeptide to a cell, the method comprising contacting a complex as described in the first aspect of the present application, a nucleic acid nanoparticle as described in the second aspect of the present application or a pharmaceutical composition as described in the third aspect of the present application with a target cell.

[0030] In some embodiments, the method is for non-diagnostic or therapeutic purposes.

[0031] In some embodiments, the target cell is a cell expressing melanocortin 1 receptor.

[0032] In some specific embodiments, the target cell is a melanocyte.

[0033] The sixth aspect of the present application provides use of the complex of the first aspect, the nucleic acid nanoparticle of the second aspect or the pharmaceutical composition of the third aspect in the preparation of a product for transdermal administration.

[0034] In some embodiments, the product is a cosmetic or a pharmaceutical product.

[0035] In some embodiments, the product is a product for inhibiting melanin production.

[0036] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining preferred embodiments of the present application.

[0037] The reagents and raw materials used in the present application are commercially available.

[0038] The positive progress effect of the present application is that:

[0039] The nucleic acid tetrahedron and nonapeptide-1 after self-assembly are simply compounded and subjected to ultrafiltration to obtain the TDN-NA-1 complex. It is proved by physicochemical characterization that the two can form a close complex, and it is proved at the cellular level that the TDN-NA-1 complex has targeting property, at the animal level that the TDN-NA-1 complex has transdermal property, and at the molecular level that the TDN-NA-1 complex has stronger ability to inhibit melanin production. Thus, the transdermal function of the nucleic acid tetrahedron with the function of inhibiting melanin production and the melanocyte targeting function of nonapeptide-1 are combined, so that the TDN-NA-1 complex has the functions of autonomous transdermal ability and melanocyte targeting, and the inhibiting effects of the two are combined, so that it has stronger ability to inhibit melanin production, thereby achieving the purpose of precise whitening, and having wide application prospects in the field of cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is the agarose gel electrophoresis result of TDN self-assembly.

[0041] Figure 2 It is the agarose gel electrophoresis result of TDN-NA-1 complex with different molar ratios.

[0042] Figure 3 It is the ultraviolet absorption peak graph of TDN-NA-1 complex.

[0043] Figure 4 It is the agarose gel electrophoresis result of TDN-NA-1 complex.

[0044] Figure 5 It is the agarose gel electrophoresis result of Cy5 fluorescently modified TDN and TDN-NA-1.

[0045] Figure 6 is the result of the transdermal penetration effect detection of TDN-NA-1; scale: 100 μm.

[0046] Figure 7 is the result of the transdermal penetration effect detection of TDN-NA-1; scale: 200 μm.

[0047] Figure 8 is the result of the different cell targeting effect detection of TDN-NA-1 complex.

[0048] Figure 9 Figure 9 is the result of the melanin expression related gene mRNA expression detection.

[0049] Figure 10 Figure 10 is the result of the melanin expression detection. DETAILED DESCRIPTION

[0050] The present application will be further described in the following examples, but the present application is not limited in the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to the conventional methods and conditions, or according to the product instructions.

[0051] Example 1: Preparation of nucleic acid tetrahedron-nonapeptide-1 (TDN-NA-1) complex

[0052] 1. Main reagents

[0053] Purified water, 10x Reaction buffer, synthetic TDN ssDNA single strand, nonapeptide-1

[0054] 2. Main instruments and equipment

[0055] Tabletop refrigerated centrifuge, metal bath, electrophoresis instrument, gel cutting instrument, nanoparticle size analyzer

[0056] 3. Experimental steps

[0057] 3.1 TDNs ssDNA synthesis

[0058] The ssDNA sequence is synthesized by Shanghai Jeery Bioengineering Co., Ltd., and the specific sequence is shown in Table 1 below:

[0059] Table 1. TDN sequence

[0060]

[0061] The sequence of nonapeptide-1 is MPFRWFKPV (SEQ ID NO: 13)

[0062] 3.2 ssDNA dissolution and quantification

[0063] The ssDNA synthesized back was in dry powder state, after centrifugation at 12000 rpm for 5 min, 152 μL purified water was added, and after standing at room temperature for 1 min, it was fully vortexed, mixed and centrifuged; 2 μL was taken for quantitative determination by ultraviolet spectrophotometer, and the concentration of each ssDNA was calculated according to the A260 value and the extinction coefficient, and the concentration was normalized to 30 μM;

[0064] 3.3 Detection of self-assembly effect of nucleic acid tetrahedron (TDNs)

[0065] Take 0.2 mL centrifuge tube, respectively add S1, S2, S3, S4, S1+S2, S1+S2+S3, S1+S2+S3+S4 with final concentration of 2 μM, 10×Reaction buffer (composition: 100 mM Tris-HCl, 10 mM EDTA, 500 mM MgCl2, pH 8.0), purified water to make up the volume to 30 μL, and the specific self-assembly system is as follows in Table 2:

[0066] Table 2. TDN self-assembly system

[0067]

[0068] After the prepared self-assembly system was fully vortexed, mixed and centrifuged, it was incubated at 95℃ for 10 min on a general PCR instrument, immediately placed on ice for 1 h, which was the prepared TDNs, and stored at 4℃ for standby.

[0069] Take 4 μL of the self-assembled TDNs, add 1 μL of 5×non-denaturing loading buffer, mix and centrifuge, and then perform 2% agarose gel electrophoresis at 120 V for 30 min, and the electrophoresis result is shown in Figure 1 .

[0070] 3.4 Preparation of TDN-NA-1 complex

[0071] The nonapeptide-1 (NA-1, whose amino acid sequence is MPFRWFKPV (SEQ ID NO: 13)) was dissolved in purified water, and then mixed with TDNs (after mixing, the concentration of TDNs was 500 nM, and the molar ratio of TDNs to NA-1 was 1:50, 1:100, 1:200, 1:300, 1:400, and 1×Reaction buffer was used to make up the total volume of 200 μL), and then rotated and mixed on a rotating mixer at 4℃ for 16 hours to form TDN-NA-1 complex.

[0072] The TDN-NA-1 complex was all sucked into a 10 kd ultrafiltration tube, 300 μL of 1 x Reaction buffer was added, mixed by blowing, centrifuged at 10000 rpm for 5 min, and the waste liquid in the collection tube was discarded; 400 μL of 1 x Reaction buffer was added to the ultrafiltration tube, mixed by blowing, centrifuged at 10000 rpm for 5 min, and the waste liquid in the collection tube was discarded; the ultrafiltration tube was inverted into a new collection tube, centrifuged at 10000 rpm for 5 min, and the liquid at the bottom of the collection tube was the TDN-NA-1 complex, which was adjusted to 100 μL in volume using 1 x Reaction buffer, so that the final concentration of TDN was 1 μM.

[0073] 3.5 TDN-NA-1 complex identification

[0074] 3.5.1 Agarose gel electrophoresis

[0075] 4 μL of TDN-NA-1 complex was taken respectively, 1 μL of 5 x non-denaturing loading buffer was added, mixed and centrifuged, and then 2% agarose gel electrophoresis was performed, electrophoresis was performed at 120 V for 30 min, and the electrophoresis result was seen Figure 2 .

[0076] From the analysis of the agarose gel electrophoresis result, compared with the TDNs control, the electrophoretic rate of the TDN-NA-1 complex was obviously slower, and the electrophoretic band was larger, which indicated that the nonapeptide-1 was complexed into the TDNs.

[0077] 3.5.2 Ultraviolet spectrophotometer absorption peak detection

[0078] 2 μL of TDNs (concentration 1 μM), nonapeptide-1 (concentration 300 μM), TDN-NA-1 complex (TDN concentration 1 μM) and filtration waste liquid were taken respectively for ultraviolet spectrophotometer detection, and scanning was performed at 260 nm and 280 nm, and the result was seen Figure 3 . From the absorption peak analysis, compared with the TDNs control, the TDN-NA-1 complex had an obvious absorption peak at 280 nm, which indicated that the nonapeptide-1 was complexed into the TDNs.

[0079] 3.5.3 Particle size detection

[0080] 20 μL of TDN-NA-1 complex was taken, mixed with 180 μL of 1 x Reaction buffer, and then added to a detection dish, and the particle size of TDNs and TDN-NA-1 was detected on a nanoparticle size instrument (brand Malvern PANalytical, model Zetasizer Ultra), the particle size of TDN-NA-1 was larger than that of TDNs, which was consistent with the result of agarose gel electrophoresis, indicating that the nonapeptide-1 was complexed into the TDNs.

[0081] Example 2: Different nucleic acid tetrahedron complexed nonapeptide-1

[0082] 1. Main reagents

[0083] Purified water, 1 x Reaction buffer, TDN 1, TDN 7, TDN 8, nonapeptide-1

[0084] 2. Main instruments and equipment

[0085] Tabletop refrigerated centrifuge, metal bath, electrophoresis instrument, gel cutting instrument

[0086] 3. Experimental steps

[0087] 3.1 Preparation of TDN-NA-1 complex

[0088] Dissolve nonapeptide-1 (NA-1) in purified water, then mix with different TDNs, including TDN 1, TDN 7 and TDN 8. After mixing, the final concentration of TDNs is 500 nM, and the molar ratio of TDNs to NA-1 is selected to be 1:300 (TDN 500 nM, nonapeptide-1 150 μM). Use 1 x Reaction buffer to make up the total volume of 200 μL. Rotate and mix on a rotating mixer at 4°C for 16 hours to form a TDN-NA-1 complex.

[0089] 3.2 Purification of TDN-NA-1 complex

[0090] Collect all the TDN-NA-1 complex into a 10 kd ultrafiltration tube, add 300 μL of 1 x Reaction buffer, mix by blowing, centrifuge at 10,000 rpm for 5 min, and discard the waste liquid in the collection tube. Add 400 μL of 1 x Reaction buffer to the ultrafiltration tube, mix by blowing, centrifuge at 10,000 rpm for 5 min, and discard the waste liquid in the collection tube. Invert the ultrafiltration tube into a new collection tube and centrifuge at 10,000 rpm for 5 min. The liquid at the bottom of the collection tube is the TDN-NA-1 complex. Adjust the volume to 100 μL using 1 x Reaction buffer to make the final concentration of TDN 1 μM.

[0091] 3.3 Agarose gel electrophoresis of TDN-NA-1 complex

[0092] Take 4 μL of TDN-NA-1 complex, add 1 μL of 5 x non-denaturing loading buffer, mix and centrifuge, then perform 2% agarose gel electrophoresis at 120 V for 30 min. The electrophoresis results are shown in Figure 4 .

[0093] 4. Analysis of experimental results: from the results of agarose gel electrophoresis, different TDNs can be compounded with nonapeptide-1.

[0094] Example 3: Detection of the membrane penetration effect of TDN-NA-1 complex

[0095] 1. Main reagents

[0096] Cy5 fluorescent modified TDN-S1 ssDNA single strand, Cy5 fluorescent modified TDN, Cy5 fluorescent modified TDN 1-NA-1 complex, Cy5 fluorescent modified TDN 7-NA-1 complex, Cy5 fluorescent modified TDN 8-NA-1 complex.

[0097] 2. Main instruments and equipment

[0098] Tabletop refrigerated centrifuge, metal bath, cell incubator, fluorescence microscope.

[0099] 3. Experimental steps

[0100] 3.1 Cy5 fluorescent modified TDN self-assembly

[0101] Cy5 fluorescent labeling of S1 in nucleic acid tetrahedron, and according to the self-assembly system in Example One, S2 / S3 / S4 are self-assembled into nucleic acid tetrahedron through denaturation and renaturation.

[0102] 3.2 Preparation of Cy5 fluorescent modified TDN-NA-1 complex

[0103] Dissolve nonapeptide-1 (NA-1) in pure water, then mix with TDNs. The final concentration of TDNs is 500 nM, the final concentration of nonapeptide-1 is 60 μM, and 1×Reaction buffer is used to make up the total volume of 500 μL. Rotate and mix on a rotary mixer at 4°C for 16 hours to form TDN-NA-1 complex.

[0104] Collect all TDN-NA-1 complex into a 10 kd ultrafiltration tube, centrifuge at 10000 rpm for 5 min, the remaining volume is about 100 μL, discard the waste liquid in the collection tube; add 400 μL of 1×Reaction buffer to the ultrafiltration tube, mix by blowing, centrifuge at 10000 rpm for 5 min, discard the waste liquid in the collection tube; invert the ultrafiltration tube into the collection tube, centrifuge at 10000 rpm for 5 min, the liquid at the bottom of the collection tube is TDN-NA-1 complex, adjust the volume to 250 μL using 1×Reaction buffer to make the final concentration of TDNs 1 μM. 2% agarose gel electrophoresis, 120V electrophoresis for 30 min, the electrophoresis result is shown in Figure 5 .

[0105] 3.3 Cell culture

[0106] 3.3.1 Incubate B16F10 cells in 48-well cell culture plates at 5 x 10^4 cells per well, 10% FBS DMEM complete medium, 37°C, 5% CO2 overnight to stabilize the cells;

[0107] 3.3.2 Replace with Cy5 fluorescently modified TDN 8 (final concentration 250 nM), Cy5 fluorescently modified TDN 1-NA-1 (final concentration 250 nM), Cy5 fluorescently modified TDN 7-NA-1 (final concentration 250 nM), Cy5 fluorescently modified TDN 8-NA-1 (final concentration 250 nM) complete medium, continue to culture for 24 hours, with the addition of Cy5 modified TDN-S1 at a final concentration of 250 nM as a negative control, and the addition of an equal volume of 1 x TM buffer as a blank control.

[0108] 3.3.3 After the end of the incubation, remove the medium, wash with 1 x PBS, and remove residual reagents;

[0109] 3.3.4 Add 250 μL of 4% paraformaldehyde for fixation for 15 min, discard the paraformaldehyde, and wash with PBS 3 times;

[0110] 3.3.5 Add 200 μL of 1 μg / mL DAPI solution to stain the nuclei for 10 min, discard the DAPI solution, and wash with PBS 3 times;

[0111] 3.3.6 Add 250 μL of PBS, and observe the penetration effect under a fluorescence microscope, with the results shown in Figure 6.

[0112] 4. Analysis of experimental results: Compared to the Cy5 fluorescently modified TDN group, more Cy5 fluorescently modified TDN-NA-1 accumulated on the cell surface, and more TDN entered the interior of the cells within the same time; there was no significant difference in the penetration effect after compounding the different nucleic acid tetrahedra with nonapeptide-1.

[0113] Example 4: Detection of the Transdermal Penetration Effect of TDN-NA-1 Complexes

[0114] 1. Main reagents

[0115] Cy5 fluorescently modified TDN-S1 ssDNA single strand, Cy5 fluorescently modified TDN 8, Cy5 fluorescently modified TDN 1-NA-1 complex, Cy5 fluorescently modified TDN 7-NA-1 complex, Cy5 fluorescently modified TDN 8-NA-1 complex.

[0116] 2. Main instruments and equipment

[0117] Freezing microtome, fluorescence microscope

[0118] 3. Experimental procedure

[0119] 3.1 Mouse treatment

[0120] Four 6-8 week old BALB / C mice were purchased and acclimated for 2 days. After the mice were acclimated, the back hair of the mice was removed, 10 μL of the corresponding nucleic acid tetrahedron + 10 mg of Aquaphor were mixed and applied to the skin of the mice, with an area of 2 cm x 2 cm. The mice were housed in single cages in the dark for 24 hours. The experimental groups were set up as shown in Table 3.

[0121] Table 3. TDN-NA-1 complex transdermal test grouping

[0122] Grouping Mouse amount NC 1 only Cy5 modified TDN-S1 1 only Cy5 modified TDN 8 1 only Cy5 modified TDN 1-NA-1 complex 1 only Cy5 modified TDN 7-NA-1 complex 1 only Cy5 modified TDN 8-NA-1 complex 1 only

[0123] 3.2 Skin freezing tissue preparation

[0124] The mice were sacrificed by cervical dislocation, and the back skin was removed and fixed in 4% paraformaldehyde for 30 min. The skin was cut into 2 mm wide pieces and placed in a disposable embedding box with OCT embedding agent. The skin was quickly frozen in liquid nitrogen for 10 s to fix it in the embedding agent, with the cross-section facing up. The embedding agent was then completely frozen at -80°C overnight.

[0125] 3.3 Skin tissue frozen section

[0126] The frozen section machine (brand Leica, model CM1950) was turned on one hour in advance to reduce the temperature of the instrument to about -25°C. The frozen sections were then cut to a thickness of 20 μM and placed on adhesive glass slides (brand Shetai).

[0127] 3.4 Section staining

[0128] The cut sections were fixed in 4% paraformaldehyde for 15 min and then washed with PBS three times for 5 min each time. The cell nuclei were stained with 1 μg / mL DAPI for 10 min, and then washed with PBS three times for 5 min each time.

[0129] 3.5 Mounting

[0130] Fluorescent anti-aging agent 10 μL was added to the glass slide, and a coverslip was carefully placed on the glass slide. The sample tissue was then observed under a fluorescence microscope after mounting.

[0131] The experimental results are shown in Figure 7.

[0132] 4. Experimental result analysis: TDN 8-NA-1 complex can pass through the stratum corneum into the skin very well, and there is no difference in transdermal effect with TDN 8 itself, which shows that the nine-peptide-1 complex will not affect the transdermal of TDN. Comparing different TDN-NA-1 complexes, TDN 8-NA-1 complex has stronger transdermal effect and more fluorescence is accumulated in the deep skin, while TDN 1-NA-1 complex and TDN 7-NA-1 complex have slightly worse transdermal effect.

[0133] Example 5: Detection of different cell targeting effects of TDN 8-NA-1 complex

[0134] 1. Main reagents

[0135] Cy5 fluorescent modified TDN 8-NA-1 complex.

[0136] 2. Main instruments and equipment

[0137] Tabletop refrigerated centrifuge, metal bath, cell incubator, fluorescence microscope.

[0138] 3. Experimental steps

[0139] 3.1 Human skin melanoma cells (SK-MEL-1 cells), human immortal keratinocytes (HaCaT cells), human mononuclear cells (THP-1 cells), and mouse melanoma cells (B16F10 cells) were inoculated into 48-well cell culture plates at 5x10^4 cells per well, and incubated at 37°C with 5% CO2 overnight to stabilize the cells.

[0140] 3.2 Replace the complete medium containing Cy5 fluorescent modified TDN-NA-1 (final concentration 250nM) and continue to culture for 24 hours. Add the same volume of 1xTM buffer as a blank control.

[0141] 3.3 After the incubation, HaCaT cells and B16F10 cells were directly removed from the culture medium, washed once with 1xPBS, and the residual reagents were removed.

[0142] SK-MEL-1 cells and THP-1 cells were all aspirated into 1.5mL centrifuge tubes, centrifuged at 800g for 5min, washed once with 1xPBS and centrifuged, and the residual reagents were removed. 50μL of 1xPBS was used to resuspend the cells, which were then added dropwise to the glass slides and placed in a 50°C oven for 10min to completely volatilize the liquid.

[0143] 3.4 250μL of 4% paraformaldehyde was directly added to the culture wells of HaCaT cells and B16F10 cells and fixed for 15min. The paraformaldehyde was discarded and washed with PBS for 3 times, 5min each time.

[0144] Add an appropriate amount of 4% paraformaldehyde to the cell area on the slide, cover all cells, fix for 15 min, rinse with PBS 3 times, 5 min each time;

[0145] 3.5 Add an appropriate amount of 1 μg / mL DAPI solution to the culture hole or drop on the slide, respectively, and stain the nucleus for 10 min. Discard the DAPI solution and rinse with PBS 3 times, 2 min each time.

[0146] 3.6 Add 250 μL of 1×PBS to the HaCaT cell and B16F10 cell culture hole to keep it moist. Add an appropriate amount of fluorescent anti-aging agent to the SK-MEL-1 cell and THP-1 cell slide, and carefully cover it with a cover glass. Take a photo under the fluorescence microscope and analyze the fluorescence intensity. The results are shown in Figure 8.

[0147] 4. Analysis of experimental results: From the fluorescence intensity analysis, after the nucleic acid tetrahedron and nonapeptide-1 complex, the cell membrane and intracellular aggregation of SK-MEL-1 cells, HaCat cells and B16F10 cells expressing MC1R were more fluorescent, while the fluorescence intensity of THP-1 cells changed little, proving that TDN 8-NA-1 complex has clear targeting.

[0148] Example 6: Detection of TDN-NA-1 complex inhibiting the expression of mRNA of melanin expression related genes

[0149] 1. Main reagents

[0150] DMEM medium, fetal bovine serum, SK-MEL-1 cells, nonapeptide-1, TDN, TDN-NA-1 complex, total RNA extraction kit, C203P1 Multiplex One Step RT-qPCR Probe Kit (UDG Plus).

[0151] 2. Main equipment

[0152] Mini centrifuge, benchtop refrigerated centrifuge, fluorescence quantitative PCR instrument

[0153] 3. Experimental steps

[0154] 3.1 Cell culture

[0155] SK-MEL-1 cells were inoculated in 48-well cell culture plates at 10^3 cells per well in 250 μL of DMEM complete medium, and incubated at 37°C in 5% CO2 for 12 hours. Then, the medium was replaced with fresh DMEM complete medium containing TDN 8, TDN 1-NA-1 complex, TDN 7-NA-1 complex, and TDN 8-NA-1 complex at a final concentration of 250 nM, respectively, and a negative control group without other components was set up. A positive control group was set up by adding a final concentration of 20 μM nonapeptide-1. Each reaction was set up in duplicate, and incubated at 37°C in 5% CO2 for 72 hours.

[0156] 3.2 Total RNA extraction

[0157] All cell suspensions were collected and centrifuged at 2000 rpm for 5 min. After the medium was discarded, the cells were washed once with 1×PBS, and 300 μL of lysis buffer was added to lyse the cells. After the lysis, the cells were transferred to 1.5 mL centrifuge tubes, and total RNA was extracted according to the total RNA extraction kit (YEASEN, item number 19221ES60).

[0158] 3.3 Total RNA detection

[0159] 3.3.1 After the total RNA of the extracted cells was quantified, it was diluted to 5 ng / μL, and the mRNA expression of the tyrosinase (TYR) gene, DHICA oxidase (TRP-1) gene, and dopachrome isomerase (TRP-2) gene was detected.

[0160] The sequences of all gene primers and probes are shown in Table 4 below:

[0161] Table 4. Primer and probe sequences

[0162]

[0163] 3.3.2 All primer and probe sequences were synthesized by Shanghai Jeery Biological Engineering Co., Ltd. The synthesized primers and probes were in dry powder form, which were centrifuged at 12000 rpm for 5 min, and the appropriate volume of enzyme-free water was added according to the COA. After standing at room temperature for 1 min, the mixture was thoroughly vortexed and centrifuged to obtain a 100 μM stock solution.

[0164] 10 μL of the primer and probe stock solution was added to 90 μL of enzyme-free water, and the mixture was thoroughly vortexed and centrifuged to obtain a 10 μM working solution, which was stored at -20°C for later use.

[0165] 3.3.3 The reaction system is shown in Table 5 below:

[0166] Table 5. PCR reaction system

[0167]

[0168] 3.3.4 After the prepared reaction system was fully vortexed and mixed, it was centrifuged and then was aliquoted into a 96-well plate, and was reacted on a fluorescence quantitative PCR instrument (brand: Shanghai Hongshi, model: SLAN-96S), and the reaction procedure was shown in Table 6:

[0169] Table 6. PCR reaction procedure

[0170]

[0171] 3.3.5 After the reaction was completed, the Ct value of the GAPDH gene was used as an internal reference to calculate the 2^-(ΔΔCt) value of the Ct value of the TYR gene, the TRP-1 gene and the TRP-2 gene in all detection wells, and the column chart results were shown in Figure 9 .

[0172] 4. Experimental result analysis: from the analysis of the mRNA expression amount of the melanin expression related genes, the TDN 8-NA-1 complex could better inhibit the expression of the melanin related gene mRNA in the melanoma cells, and the inhibition rate was stronger than that of the single nonapeptide-1 group and the TDN 8 group; while the TDN 1-NA-1 complex and the TDN 7-NA-1 complex did not have stronger melanin expression inhibition rate, and the reason was that TDN 1 and TDN 7 did not have the ability to inhibit melanin synthesis, while TDN 8 and nonapeptide-1 had the ability to inhibit melanin synthesis, and after the combination of the two, better whitening ability was shown.

[0173] Example 7: TDN-NA-1 complex inhibits melanin expression

[0174] 1. Main reagents

[0175] DMEM culture medium, fetal bovine serum, SK-MEL-1 cells, PBS, TDN-NA-1 complex, total RNA extraction kit, One-Step RT-qPCR Probe Kit (UDG Plus)

[0176] 2. Main instruments and equipment

[0177] Mini centrifuge, table-top refrigerated centrifuge, fluorescence quantitative PCR instrument

[0178] 3. Experimental steps

[0179] 3.1 Cell culture

[0180] SK-MEL-1 cells were inoculated in 6-well plate cell culture plates at 2x10^3 cells per well in 2 mL of DMEM complete medium, and incubated at 37°C, 5% CO2 for 12 hours. New DMEM complete medium was then added, and a final concentration of 250 nM of TDN 1-NA-1 complex, TDN 7-NA-1 complex, and TDN 8-NA-1 complex was added to the medium, respectively. A negative control group without other components was set up, and a final concentration of 20 μM nonapeptide-1 was added to the positive control group. Each reaction was set up in triplicate, and incubated at 37°C, 5% CO2 for 48 hours. All cell suspensions were then collected, centrifuged at 2000 rpm for 5 min, and the old medium was discarded. New complete medium containing the corresponding samples was added at 3 mL, and incubated for 72 h. The old medium was then discarded again, and new complete medium containing the corresponding samples was added at 3 mL, and incubated for another 48 h.

[0181] All cell suspensions were collected, centrifuged at 2000 rpm for 5 min, and washed once with 1xPBS. Melanin extraction solution (1 M NaOH solution containing 10% DMSO) was added at 200 μL per well, and mixed vigorously. The mixture was incubated in a 80°C metal bath for 60 min. After cooling, the mixture was vortexed thoroughly and centrifuged. 150 μL of the solution was transferred to a 96-well enzyme plate, and the absorbance value at 405 nm was detected on an enzyme marker. The total protein content in each sample lysate was also determined.

[0182] 3.2 Data processing

[0183] 3.2.1 Calculation of relative melanin content

[0184] According to the formula: relative melanin content = melanin determination A 405 value / total protein determination value, the relative melanin content of each well was obtained, and the average value of the relative melanin content of three replicates was calculated. The melanin expression column chart is shown in Figure 10 .

[0185] According to the formula: inhibition rate = (negative control A 405 - test group A 405 ) / (negative control group A 405 ) x 100%, the melanin expression inhibition rate of all wells was calculated, and the specific inhibition rate is shown in Table 7 below.

[0186] Table 7. Melanin determination analysis results

[0187]

[0188] 4. Experimental result analysis: from the melanin expression analysis Figure 10), TDN 8-NA-1 complex can better inhibit the expression of melanin in melanoma cells, while TDN 1-NA-1 complex and TDN 7-NA-1 complex do not have stronger melanin expression inhibition rate, analyze the reason TDN 1 and TDN 7 itself does not have the ability to inhibit melanin synthesis, and TDN 8 and nonapeptide-1 both have the ability to inhibit melanin synthesis, after combination, better whitening ability is shown.

[0189] Reference:

[0190] [1] Christian Wiraja, et al. Framework nucleic acids as programmable carrier for transdermal drug delivery [J]. NATURE COMMUNICATIONS, 2019, 10 (1147).

[0191] Although the specific embodiments of the present application are described above, those skilled in the art should understand that these are only illustrative, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present application. Therefore, the protection scope of the present application is defined by the appended claims.

Claims

1. A composite, characterized in that, The complex comprises a nucleic acid tetrahedron and a polypeptide; wherein the polypeptide is a nonapeptide-1 with an amino acid sequence as shown in SEQ ID NO: 13; and the nucleic acid tetrahedron is self-assembled from four single-stranded DNAs with the same length.

2. The composite of claim 1, wherein, The sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 1-4, respectively; Alternatively, the sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 5-8, respectively; Alternatively, the sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 9-12, respectively.

3. A nucleic acid nanoparticle, characterized in that, The nucleic acid nanoparticle comprises the complex of claim 1 or 2; and optionally further comprises a carrier and / or a cross-linking agent.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the complex of claim 1 or 2 or the nucleic acid nanoparticle of claim 3, and a pharmaceutically acceptable carrier and / or excipient.

5. A method of preparing the complex according to claim 1 or 2, characterized in that, The method comprises the step of mixing the nucleic acid tetrahedron and the polypeptide in a solution. Preferably, the solution comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA and 20-100 mM MgCl2, and has a pH of 7.5-8.

5.

6. The method of claim 5, wherein, The molar ratio of the nucleic acid tetrahedron and the polypeptide is 1:1-1:1000; preferably 1:50-1:400, such as 1:50, 1:100, 1:200, 1:300 or 1:

400.

7. The method of claim 5 or 6, wherein, The method further comprises the step of preparing the nucleic acid tetrahedron, comprising mixing and reacting four single-stranded DNAs in a buffer system; and terminating the reaction after the single-stranded DNAs form double-stranded DNAs; and the four single-stranded DNAs are preferably mixed at equimolar concentrations. Preferably, the buffer system comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA and 20-100 mM MgCl2, and has a pH of 7.5-8.5; and the buffer system preferably comprises 10 mM Tris-HCl, 1 mM EDTA, 50 mM MgCl2, pH 8.0; and / or, The reaction is performed at 95℃ for at least 10 min; and / or, the reaction is terminated at 4℃ for 20 min.

8. A method of delivering a polypeptide to a cell, comprising, The method comprises contacting the complex of claim 1 or 2, the nucleic acid nanoparticle of claim 3 or the pharmaceutical composition of claim 4 with a target cell; and the method is preferably for non-diagnostic or therapeutic purposes. Preferably, the target cell is a cell expressing melanocortin 1 receptor; such as a melanocyte.

9. Use of the complex of claim 1 or 2, the nucleic acid nanoparticle of claim 3 or the pharmaceutical composition of claim 4 in the preparation of a product for transdermal administration.

10. Use according to claim 9, wherein the compound is ###0002### The product is a cosmetic or a pharmaceutical product; and / or, the product is a product for inhibiting melanin production. The complex comprises a nucleic acid tetrahedron and a polypeptide; wherein the polypeptide is a nonapeptide-1 with an amino acid sequence as shown in SEQ ID NO: 13; and the nucleic acid tetrahedron is self-assembled from four single-stranded DNAs with the same length. The sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 1-4, respectively; Alternatively, the sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 5-8, respectively; Alternatively, the sequences of the four single-stranded DNAs are as shown in SEQ ID NOs: 9-12, respectively. The nucleic acid nanoparticle comprises the complex of claim 1 or 2; and optionally further comprises a carrier and / or a cross-linking agent. The pharmaceutical composition comprises the complex of claim 1 or 2 or the nucleic acid nanoparticle of claim 3, and a pharmaceutically acceptable carrier and / or excipient. The method comprises the step of mixing the nucleic acid tetrahedron and the polypeptide in a solution. Preferably, the solution comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA and 20-100 mM MgCl2, and has a pH of 7.5-8.

5. The molar ratio of the nucleic acid tetrahedron and the polypeptide is 1:1-1:1000; preferably 1:50-1:400, such as 1:50, 1:100, 1:200, 1:300 or 1:

400. The method further comprises the step of preparing the nucleic acid tetrahedron, comprising mixing and reacting four single-stranded DNAs in a buffer system; and terminating the reaction after the single-stranded DNAs form double-stranded DNAs; and the four single-stranded DNAs are preferably mixed at equimolar concentrations. Preferably, the buffer system comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA and 20-100 mM MgCl2, and has a pH of 7.5-8.5; and the buffer system preferably comprises 10 mM Tris-HCl, 1 mM EDTA, 50 mM MgCl2, pH 8.0; and / or, The reaction is performed at 95℃ for at least 10 min; and / or, the reaction is terminated at 4℃ for 20 min. The method comprises contacting the complex of claim 1 or 2, the nucleic acid nanoparticle of claim 3 or the pharmaceutical composition of claim 4 with a target cell; and the method is preferably for non-diagnostic or therapeutic purposes. Preferably, the target cell is a cell expressing melanocortin 1 receptor; such as a melanocyte.

9. Use of the complex of claim 1 or 2, the nucleic acid nanoparticle of claim 3 or the pharmaceutical composition of claim 4 in the preparation of a product for transdermal administration. The product is a cosmetic or a pharmaceutical product; and / or, the product is a product for inhibiting melanin production.

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