Complexes of polypeptide-nucleic acid tetrahedra and methods of making and using the same

Abstract

This invention discloses a polypeptide-nucleic acid tetrahedron complex, its preparation method, and its applications. The complex comprises a nucleic acid tetrahedron and a polypeptide, wherein the polypeptide is nonapeptide-1 with the amino acid sequence shown in SEQ ID NO:13; the nucleic acid tetrahedron is self-assembled from four single-stranded DNA molecules of the same length. This invention combines the transdermal function of the nucleic acid tetrahedron, which inhibits melanin production, with the melanocyte-targeting function of nonapeptide-1. This allows the TDN-NA-1 complex to possess both autonomous transdermal capability and melanocyte-targeting ability, further combining the melanin-inhibiting effects of both to achieve a stronger ability to inhibit melanin production, thereby achieving precise whitening and possessing broad application prospects in the cosmetics field.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to polypeptide-nucleic acid tetrahedral complexes, their preparation methods, and applications. Background Technology

[0002] Nucleic acid tetrahedra (also called tetrahedral DNA nanostructures, TDNs, or tetrahedral framework nucleic acids, tFNA) are composed of four equal-length single-stranded DNA strands mixed in equal amounts. Each face of the tetrahedron consists of three short sequences of single-stranded DNA (ssDNA) paired with the sequence of the same ssDNA strand. Each side consists of 20 bases, forming a three-dimensional DNA structure with controllable size and rigidity.

[0003] Due to their good controllability in size and shape, simple preparation, high mechanical strength, and good biocompatibility and membrane permeability [1], TDNs have broad application prospects in drug delivery, disease treatment and biomedical imaging. At present, TDNs have been shown to deliver small molecule drugs, peptides and oligonucleotides. Relying on their biological and structural characteristics in cell permeation, TDNs can enter cells through porous protein internalization with low electrostatic repulsion, but this way of entering cells is nonspecific and random.

[0004] The first step in melanin formation is the binding of alpha-MSH (melanocyte-stimulating hormone) to melanocortin receptor 1 (MC1R) on melanocytes, which then activates tyrosinase, further leading to melanin production. Nonapeptide-1 (also known as a skin-whitening peptide) is a small peptide containing nine amino acids. It is a biomimetic peptide of natural alpha-MSH, which competitively binds to MC1R, inhibiting tyrosinase activation and thus suppressing melanin production.

[0005] Nonapeptide-1 has a high affinity for the MC1R receptor, which makes it highly effective at targeting melanocytes in the skin. However, nonapeptide-1 itself does not have the ability to directly penetrate the skin. Summary of the Invention

[0006] To address the technical problems of existing technologies, such as the lack of targeting capabilities of nucleic acid tetrahedra and the lack of autonomous transdermal properties of nonapeptide-1, this invention provides a polypeptide-nucleic acid tetrahedron complex, its preparation method, and its applications.

[0007] Specifically, the present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] A first aspect of the present invention provides a complex comprising a nucleic acid tetrahedron and a polypeptide; wherein the polypeptide is 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 of the same length.

[0009] In some embodiments, the sequences of the four single-stranded DNAs are shown in SEQ ID NO:1-4, respectively;

[0010] Alternatively, the sequences of the four single-stranded DNAs are shown in SEQ ID NO:5-8, respectively;

[0011] Alternatively, the sequences of the four single-stranded DNAs are shown in SEQ ID NO:9-12, respectively.

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

[0013] In some embodiments, the nucleic acid nanoparticles also include a carrier and / or a cross-linking agent.

[0014] In this invention, the carrier is conventional in the art, such as pharmaceutically acceptable carriers, including but not limited to buffers, antioxidants, preservatives, proteins, peptides, polymers, amino acids, and sugars. In some embodiments, the polymer can form a scaffold or particulate structure under specific conditions.

[0015] In this invention, the crosslinking agent can be conventional in the art, used to crosslink the nucleic acid nanoparticles or nucleic acid tetrahedra of this invention internally, crosslink intermolecularly, or crosslink with the scaffold or particle structure, or to enable the carrier to form a stable spatial structure.

[0016] A third aspect of the present invention provides a pharmaceutical composition comprising a complex as described in the first aspect of the present invention or nucleic acid nanoparticles as described in the second aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipients.

[0017] A fourth aspect of the present invention provides a method for preparing a complex as described in the first aspect of the present invention, the method 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; its pH is 7.5-8.5.

[0019] In some implementations, the molar ratio of the nucleic acid tetrahedron to the polypeptide is 1:1 to 1:1000.

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

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

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

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

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

[0025] In some specific implementations, the buffer system consists of 10 mM Tris-HCl, 1 mM EDTA, 50 mM MgCl2, and pH 8.0.

[0026] In some implementations, the reaction is carried out at 95°C for at least 10 minutes.

[0027] In some implementations, the termination condition is 4°C.

[0028] In some implementations, the termination reaction is carried out at 4°C for 20 minutes.

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

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

[0031] In some embodiments, the target cells are cells expressing melanocortin 1 receptors.

[0032] In some specific implementations, the target cells are melanocytes.

[0033] A sixth aspect of the invention provides the use of the complexes described in the first aspect, the nucleic acid nanoparticles described in the second aspect, or the pharmaceutical compositions described in the third aspect in the preparation of products for transdermal administration.

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

[0035] In some implementations, the product is a product that inhibits melanin production.

[0036] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0037] The reagents and raw materials used in this invention are all commercially available.

[0038] The positive and progressive effects of this invention are as follows:

[0039] This invention involves a simple combination of self-assembled nucleic acid tetrahedrons and nonapeptide-1, followed by ultrafiltration to obtain the TDN-NA-1 complex. Physicochemical characterization demonstrates that the two can form a tight complex. The targeting ability of the TDN-NA-1 complex was demonstrated at the cellular level, its transdermal properties at the animal level, and its stronger ability to inhibit melanin production at the molecular level. This invention combines the transdermal function of the nucleic acid tetrahedron (which inhibits melanin production) with the melanocyte-targeting function of nonapeptide-1, giving the TDN-NA-1 complex both autonomous transdermal capability and melanocyte-targeting ability. Furthermore, the combined melanin-inhibiting effects of both enhance its ability to suppress melanin production, thus achieving precise whitening and possessing broad application prospects in the cosmetics field. Attached Figure Description

[0040] Figure 1 The results of TDN self-assembly agarose gel electrophoresis are shown.

[0041] Figure 2 The results of agarose gel electrophoresis of TDN-NA-1 complexes with different molar ratios are shown.

[0042] Figure 3 The image shows the UV absorption peaks of the TDN-NA-1 complex.

[0043] Figure 4 The results of agarose gel electrophoresis of the TDN-NA-1 complex are shown.

[0044] Figure 5 The results of agarose gel electrophoresis of Cy5 fluorescently modified TDN and TDN-NA-1.

[0045] Figure 6 shows the membrane penetration test results of TDN-NA-1; scale bar: 100μm.

[0046] Figure 7 shows the transdermal efficacy test results of TDN-NA-1; scale bar: 200 μm.

[0047] Figure 8 shows the detection results of the TDN-NA-1 complex targeting different cells.

[0048] Figure 9 The results show the mRNA expression levels of genes related to melanin expression.

[0049] Figure 10 This is the result of melanin expression level detection. Detailed Implementation

[0050] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

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

[0052] 1. Main reagents

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

[0054] 2. Main instruments and equipment

[0055] Benchtop refrigerated centrifuge, metal bath, electrophoresis apparatus, gel cutter, nanoparticle size analyzer

[0056] 3. Experimental Procedure

[0057] 3.1 TDNs ssDNA Synthesis

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

[0059] Table 1. TDN Sequences

[0060]

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

[0062] 3.2 ssDNA lysis and quantification

[0063] The synthesized ssDNA was in dry powder form. After centrifugation at 12000 rpm for 5 min, 152 μL of purified water was added and the mixture was left at room temperature for 1 min. After thorough vortexing and centrifugation, 2 μL of each ssDNA was taken and quantified using a UV spectrophotometer. The concentration of each ssDNA was calculated based on the A260 value and extinction coefficient, and the concentration was normalized to 30 μM.

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

[0065] Take 0.2 mL centrifuge tubes and add S1, S2, S3, S4, S1+S2, S1+S2+S3, and S1+S2+S3+S4 to a final concentration of 2 μM, respectively. Add 10× reaction buffer (composed of 100 mM Tris-HCl, 10 mM EDTA, 500 mM MgCl2, pH 8.0) and purified water to a final volume of 30 μL. The specific self-assembly systems are shown in Table 2 below.

[0066] Table 2. TDN Self-Assembly System

[0067]

[0068] After thoroughly vortexing and centrifuging the prepared self-assembled system, incubate it at 95°C for 10 min on a standard PCR instrument, and immediately place it on ice for 1 h to obtain the prepared TDNs. Store at 4°C for later use.

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

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

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

[0072] All TDN-NA-1 complex was transferred to a 10 kDa ultrafiltration tube. 300 μL of 1×Reaction buffer was added, and the mixture was mixed by pipetting. The tube was centrifuged at 10,000 rpm for 5 min, and the waste liquid in the collection tube was discarded. Then, 400 μL of 1×Reaction buffer was added to the ultrafiltration tube, and the mixture was mixed by pipetting. The tube was centrifuged at 10,000 rpm for 5 min, and the waste liquid in the collection tube was discarded. The ultrafiltration tube was then inverted into a new collection tube, and centrifuged at 10,000 rpm for 5 min. The liquid at the bottom of the collection tube was the TDN-NA-1 complex. The volume was adjusted to 100 μL using 1×Reaction buffer to achieve a final TDN concentration of 1 μM.

[0073] 3.5 Identification of the TDN-NA-1 complex

[0074] 3.5.1 Agarose gel electrophoresis

[0075] Take 4 μL of TDN-NA-1 complex, add 1 μL of 5× non-denaturing loading buffer, mix well, centrifuge, and then perform 2% agarose gel electrophoresis at 120V for 30 min. The electrophoresis results are shown in the figure. Figure 2 .

[0076] Analysis of the agarose gel electrophoresis results showed that, compared with the TDNs control, the electrophoresis rate of the TDN-NA-1 complex was significantly slower and the electrophoretic bands were larger, indicating that nonapeptide-1 was incorporated into the TDNs.

[0077] 3.5.2 Detection of Absorption Peaks by Ultraviolet Spectrophotometer

[0078] 2 μL of TDNs (1 μM), nonapeptide-1 (300 μM), TDN-NA-1 complex (1 μM TDN), and filtrate waste liquid were respectively analyzed by ultraviolet spectrophotometer. Simultaneously, wavelengths of 260 nm and 280 nm were scanned. The results are shown in [Figure number missing]. Figure 3 Analysis of the absorption peaks showed that, compared with the TDNs control, the TDN-NA-1 complex exhibited a significant absorption peak at 280 nm, indicating that nonapeptide-1 was incorporated into the TDNs.

[0079] 3.5.3 Particle size detection

[0080] Take 20 μL of TDN-NA-1 complex, add it to 180 μL of 1×Reaction buffer, mix and centrifuge, then add it to a detection dish. The particle size of TDNs and TDN-NA-1 is measured on a nanoparticle size analyzer (brand Malvern PANalytical, model Zetasizer Ultra). The particle size of TDN-NA-1 is larger than that of TDNs, which is consistent with the results of agarose gel electrophoresis, indicating that nonapeptide-1 is incorporated into TDNs.

[0081] Example 2: Nonapeptide-1 composed of different nucleic acid tetrahedrons

[0082] 1. Main reagents

[0083] Purified water, 1×Reaction buffer, TDN 1, TDN 7, TDN 8, Nonapeptide-1

[0084] 2. Main instruments and equipment

[0085] Benchtop refrigerated centrifuge, metal bath, electrophoresis apparatus, gel cutting apparatus

[0086] 3. Experimental Procedure

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

[0088] Nonapeptide-1 (NA-1) was dissolved in purified water and then mixed with different TDNs, including TDN 1, TDN 7 and TDN 8. The final concentration of TDNs after mixing was 500 nM. The molar ratio of TDNs to NA-1 was selected as 1:300 (TDN 500 nM, nonapeptide-1 150 μM). The total volume was made up to 200 μL with 1×reaction buffer. The mixture was then mixed at 4 °C for 16 hours using a rotary mixer to form the TDN-NA-1 complex.

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

[0090] All TDN-NA-1 complex was transferred to a 10 kDa ultrafiltration tube. 300 μL of 1×Reaction buffer was added, and the mixture was mixed by pipetting. The tube was centrifuged at 10,000 rpm for 5 min, and the waste liquid in the collection tube was discarded. Then, 400 μL of 1×Reaction buffer was added to the ultrafiltration tube, and the mixture was mixed by pipetting. The tube was centrifuged at 10,000 rpm for 5 min, and the waste liquid in the collection tube was discarded. The ultrafiltration tube was then inverted into a new collection tube, and centrifuged at 10,000 rpm for 5 min. The liquid at the bottom of the collection tube was the TDN-NA-1 complex. The volume was adjusted to 100 μL using 1×Reaction buffer to achieve a final TDN concentration of 1 μM.

[0091] 3.3 TDN-NA-1 complex agarose gel electrophoresis

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

[0093] 4. Analysis of experimental results: Analysis of agarose gel electrophoresis results showed that different TDNs could be combined with nonapeptide-1.

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

[0095] 1. Main reagents

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

[0097] 2. Main instruments and equipment

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

[0099] 3. Experimental Procedure

[0100] 3.1 Self-assembly of Cy5 fluorescently modified TDNs

[0101] S1 in the nucleic acid tetrahedron was labeled with Cy5 fluorescence, and according to the self-assembly system in Example 1, the corresponding S2 / S3 / S4 were denatured and renatured to self-assemble into a nucleic acid tetrahedron.

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

[0103] Nonapeptide-1 (NA-1) was dissolved in purified water and then mixed with TDNs. The final concentration of TDNs after mixing was 500 nM and the final concentration of nonapeptide-1 was 60 μM. The total volume was made up to 500 μL using 1×Reaction buffer. The mixture was then mixed at 4 °C for 16 hours to form the TDN-NA-1 complex.

[0104] All TDN-NA-1 complex was transferred to a 10 kDa ultrafiltration tube and centrifuged at 10,000 rpm for 5 min. The remaining volume was approximately 100 μL; this was discarded. 400 μL of 1×Reaction buffer was added to the ultrafiltration tube and mixed thoroughly by pipetting. The tube was centrifuged at 10,000 rpm for 5 min, and the remaining liquid was discarded. The ultrafiltration tube was then inverted into the collection tube and centrifuged at 10,000 rpm for 5 min. The liquid at the bottom of the collection tube was the TDN-NA-1 complex. The volume was adjusted to 250 μL using 1×Reaction buffer to achieve a final TDN concentration of 1 μM. Electrophoresis was performed on a 2% agarose gel at 120 V for 30 min. The electrophoresis results are shown below. Figure 5 .

[0105] 3.3 Cell Culture

[0106] 3.3.1 The cultured B16F10 cells were seeded into 48-well cell culture plates, with 5 × 10^4 cells per well, in 10% FBSDMEM complete medium, and cultured overnight at 37°C with 5% CO2 to stabilize the cells.

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

[0108] 3.3.3 After the culture is completed, remove the culture medium, wash with 1×PBS to remove residual reagents;

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

[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 3 times with PBS;

[0111] 3.3.6 Add 250 μL of PBS to each membrane and observe the membrane penetration effect under a fluorescence microscope. The results are shown in Figure 6.

[0112] 4. Experimental results analysis: Within the same time period, compared with the Cy5 fluorescently modified TDN group, Cy5 fluorescently modified TDN-NA-1 accumulated more on the cell surface and more TDN entered the cell interior; there was no significant difference in the transmutation effect after different nucleic acid tetrahedra were combined with nonapeptide-1.

[0113] Example 4: Transdermal efficacy test of TDN-NA-1 complex

[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] Cryostat, fluorescence microscope

[0118] 3. Experimental Procedure

[0119] 3.1 Mouse treatment

[0120] Purchase four 6-8 week old BALB / c mice and allow them to acclimatize to the environment for two days. After rubbing the backs of the mice, remove the fur. Take 10 μL of the corresponding nucleic acid tetrahedron and 10 mg of Aquaphor lotion, mix them thoroughly, and apply the mixture to the skin of the mice in an area of ​​2 cm × 2 cm. Keep each mouse in a separate cage in the dark for 24 hours. The experimental groups are set up as shown in Table 3 below:

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

[0122] Grouping mouse quantity NC 1 Cy5 modifies TDN-S1 1 Cy5 modifies TDN 8 1 Cy5 modified TDN 1-NA-1 complex 1 Cy5 modified TDN 7-NA-1 complex 1 Cy5-modified TDN 8-NA-1 complex 1

[0123] 3.2 Preparation of skin cryopreservation tissue

[0124] Mice were euthanized by cervical dislocation, and the skin on their backs was removed and fixed in 4% paraformaldehyde for 30 minutes. The skin was then cut into 2 mm wide pieces and placed in a disposable embedding cassette containing OCT embedding medium. The cassette was then flash-frozen in liquid nitrogen for 10 seconds to fix the skin in the embedding medium with the cross-section facing upwards. The cassette was then placed at -80°C overnight to allow the embedding medium to freeze completely.

[0125] 3.3 Frozen sections of skin tissue

[0126] Turn on the cryostat (Leica, model CM1950) one hour in advance to allow the instrument temperature to drop to around -25°C before cryosectioning. The sections are 20 μm thick and laid flat on the adhesive slides (Shitai).

[0127] 3.4 Staining of sections

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

[0129] 3.5-inch cover

[0130] Add 10 μL of fluorescent anti-aging agent to a glass slide, carefully cover the slide with a coverslip, and then observe the sample tissue under a fluorescence microscope after mounting.

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

[0132] 4. Experimental Results Analysis: The TDN 8-NA-1 complex can penetrate the stratum corneum and enter the skin well, with no difference in transdermal effect compared to TDN 8 itself. This indicates that the nonapeptide-1 complexed with TDN does not affect the transdermal effect of TDN. Comparing different TDN-NA-1 complexes, the TDN 8-NA-1 complex has a stronger transdermal effect, accumulating more fluorescence deep in the skin, while the TDN 1-NA-1 and TDN 7-NA-1 complexes have slightly weaker transdermal effects.

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

[0134] 1. Main reagents

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

[0136] 2. Main instruments and equipment

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

[0138] 3. Experimental Procedure

[0139] 3.1 Human skin melanoma cells (SK-MEL-1 cells), human immortalized keratinocytes (HaCaT cells), human monocytes (THP-1 cells), and mouse melanoma cells (B16F10 cells) were seeded into 48-well cell culture plates at 5 × 10^4 cells per well and cultured overnight at 37°C with 5% CO2 to stabilize the cells.

[0140] 3.2 Replace with complete culture medium containing Cy5 fluorescently modified TDN-NA-1 (final concentration 250 nM) and continue culturing for 24 hours, with an equal volume of 1×TM buffer added as a blank control.

[0141] 3.3 After the culture was completed, the culture medium was removed directly from HaCaT cells and B16F10 cells, and they were washed once with 1×PBS to remove residual reagents.

[0142] All SK-MEL-1 and THP-1 cells were aspirated into 1.5 mL centrifuge tubes, centrifuged at 800 g for 5 min, and the supernatant was removed. The cells were washed once with 1×PBS and centrifuged again to remove residual reagents. The cells were resuspended in 50 μL of 1×PBS and added to a glass slide. The cells were placed in a 50 °C oven for 10 min to allow the liquid to evaporate completely.

[0143] 3.4 Add 250 μL of 4% paraformaldehyde directly to the culture wells of HaCaT cells and B16F10 cells for 15 min to fix them. Discard the paraformaldehyde and wash with PBS 3 times for 5 min each time.

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

[0145] 3.5 Take an appropriate amount of 1 μg / mL DAPI solution and add it to the culture well or drop it onto the glass slide. Stain the nuclei for 10 min, then discard the DAPI solution and wash with PBS 3 times for 2 min each time.

[0146] 3.6 Add 250 μL of 1×PBS to the culture wells of HaCaT cells and B16F10 cells and keep them moist; add an appropriate amount of fluorescent anti-aging agent to the slides of SK-MEL-1 cells and THP-1 cells, and carefully cover with a coverslip; take pictures under a fluorescence microscope and analyze the fluorescence intensity. The results are shown in Figure 8.

[0147] 4. Analysis of experimental results: Fluorescence intensity analysis showed that after the combination of nucleic acid tetrahedron and nonapeptide-1, more fluorescence accumulated in the cell membrane and intracellular space of SK-MEL-1 cells, HaCat cells, and B16F10 cells expressing MC1R, while the fluorescence intensity of THP-1 cells did not change much, proving that the TDN 8-NA-1 combination has clear targeting.

[0148] Example 6: Detection of mRNA expression levels of genes related to melanin expression inhibition by the TDN-NA-1 complex.

[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 instruments and equipment

[0152] Mini centrifuge, benchtop refrigerated centrifuge, real-time PCR instrument

[0153] 3. Experimental Procedure

[0154] 3.1 Cell Culture

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

[0156] 3.2 Total RNA Extraction

[0157] Collect all cell suspensions, centrifuge at 2000 rpm for 5 min, discard the culture medium, wash once with 1×PBS, add 300 μL of lysis buffer, lyse the cells thoroughly, and transfer them to 1.5 mL centrifuge tubes for total RNA extraction according to the cell / tissue total RNA extraction kit (YEASEN, catalog number 19221ES60).

[0158] 3.3 Total RNA detection

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

[0160] The primer and probe sequences for all genes 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 Jierui Biotechnology Co., Ltd. The synthesized primers and probes were in dry powder form. After centrifugation at 12,000 rpm for 5 min, the corresponding volume of enzyme-free water was added according to the COA. After incubation at room temperature for 1 min, the mixture was thoroughly vortexed and centrifuged to obtain a 100 μM stock solution.

[0164] Take 10 μL of primer and probe stock solution and add it to 90 μL of enzyme-free water. Vortex thoroughly to mix and centrifuge to obtain a 10 μM working solution. Store at -20℃ 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 thoroughly vortexing and centrifuging the prepared reaction system, aliquot it into 96-well plates and perform the reaction on a real-time PCR instrument (Shanghai Hongshi, model SLAN-96S). The reaction program is shown in Table 6 below:

[0169] Table 6. PCR reaction procedure

[0170]

[0171] 3.3.5 After the reaction, using the GAPDH gene Ct value as an internal reference, the Ct values ​​of the TYR gene, TRP-1 gene, and TRP-2 gene in all detection wells were calculated using 2^-(ΔΔCt). The bar chart results are shown below. Figure 9 .

[0172] 4. Experimental Results Analysis: Analysis of the mRNA expression levels of melanin-related genes showed that the TDN 8-NA-1 complex was able to better inhibit the expression of melanin-related gene mRNA in melanoma cells, with inhibition rates stronger than those of the nonapeptide-1 group and the TDN 8 group alone. However, the melanin expression inhibition rates of the TDN 1-NA-1 complex and the TDN 7-NA-1 complex were not stronger. The reason for this is that TDN 1 and TDN 7 themselves do not have the ability to inhibit melanin synthesis, while TDN 8 and nonapeptide-1 both have the ability to inhibit melanin synthesis. When the two are combined, they exhibit better whitening ability.

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

[0174] 1. Main reagents

[0175] DMEM 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, benchtop refrigerated centrifuge, real-time PCR instrument

[0178] 3. Experimental Procedure

[0179] 3.1 Cell Culture

[0180] Cultured SK-MEL-1 cells were seeded in 6-well cell culture plates at a density of 2 × 10^3 cells per well. After 12 hours of incubation in 2 mL DMEM complete medium at 37°C and 5% CO2, the medium was replaced with fresh DMEM complete medium. TDN 1-NA-1 complex, TDN 7-NA-1 complex, and TDN 8-NA-1 complex were added to the medium at a final concentration of 250 nM, respectively. A negative control group was set up without any other components, and a positive control group was set up with a final concentration of 20 μM nonapeptide-1. Each reaction was performed in triplicate. After 48 hours of incubation at 37°C and 5% CO2, all cell suspensions were collected, centrifuged at 2000 rpm for 5 min, and the old medium was discarded. 3 mL of fresh complete medium containing the corresponding sample was added, and the cells were incubated for 72 hours. The old medium was discarded again, and 3 mL of fresh complete medium containing the corresponding sample was added, and the cells were incubated for another 48 hours.

[0181] Collect all cell suspensions, centrifuge at 2000 rpm for 5 min, discard the culture medium, wash once with 1×PBS, add 200 μL of melanin extraction solution (1M NaOH solution containing 10% DMSO) to each well, vortex vigorously to mix, place in an 80℃ metal bath and incubate for 60 min, cool, vortex thoroughly to mix, centrifuge, and aspirate 150 μL of the solution into each 96-well microplate. Measure the absorbance at 405 nm for each well using a microplate reader, and determine the total protein content in the lysate of each sample.

[0182] 3.2 Data Processing

[0183] 3.2.1 Calculation of relative melanin content

[0184] According to the formula: Relative melanin content = Melanin measurement A 405 The relative melanin content of each well was obtained by dividing the total protein content by the total protein content value, and the average of the relative melanin content of the three replicates was calculated. A bar chart of melanin expression levels is shown below. Figure 10 .

[0185] According to the formula: Inhibition rate = (Negative control A) 405 -Test Group A 405 ) / (Negative control group A) 405 )×100%, calculate the melanin expression inhibition rate of all wells, and the specific inhibition rates are shown in Table 7 below.

[0186] Table 7. Results of melanin determination and analysis

[0187]

[0188] 4. Analysis of experimental results: Analysis of melanin expression levels ( Figure 10The TDN 8-NA-1 complex can better inhibit melanin expression in melanoma cells, while the TDN 1-NA-1 complex and the TDN 7-NA-1 complex do not show a stronger inhibition rate of melanin expression. The reason for this is that TDN 1 and TDN 7 themselves do not have the ability to inhibit melanin synthesis, while TDN 8 and nonapeptide-1 both have the ability to inhibit melanin synthesis. When the two are combined, they show better whitening ability.

[0189] References:

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

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

Claims

1. A complex, characterized in that, The complex comprises a nucleic acid tetrahedron and a polypeptide; wherein the polypeptide is nonapeptide-1 with an amino acid sequence as shown in SEQ ID NO: 13; the nucleic acid tetrahedron is self-assembled from four single-stranded DNAs, the sequences of which are shown in SEQ ID NO: 1-4 respectively; Alternatively, the sequences of the four single-stranded DNAs are shown in SEQ ID NO: 5-8, respectively; Alternatively, the sequences of the four single-stranded DNAs are shown in SEQ ID NO: 9-12, respectively.

2. A nucleic acid nanoparticle, characterized in that, The nucleic acid nanoparticles comprise the complex as described in claim 1.

3. The nucleic acid nanoparticles as described in claim 2, characterized in that, The nucleic acid nanoparticles also contain a carrier and / or a cross-linking agent.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the complex as described in claim 1 or the nucleic acid nanoparticles as described in claim 2 or 3, and excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The excipients are pharmaceutically acceptable carriers.

6. A method for preparing the complex as described in claim 1, characterized in that, The method includes the step of mixing the nucleic acid tetrahedron and the polypeptide in a solution.

7. The method as described in claim 6, characterized in that, The solution comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA and 20-100 mM MgCl2; its pH is 7.5-8.

5.

8. The method as described in claim 6 or 7, characterized in that, The molar ratio of the nucleic acid tetrahedron to the polypeptide is 1:50-1:

400.

9. The method as described in claim 8, characterized in that, The molar ratio of the nucleic acid tetrahedron to the polypeptide is 1:50, 1:100, 1:200, 1:300, or 1:

400.

10. The method as described in claim 6, characterized in that, The method further includes the step of preparing the nucleic acid tetrahedron, which involves mixing and reacting four types of single-stranded DNA in a buffer system; and terminating the reaction after the single-stranded DNA forms double-stranded DNA.

11. The method as described in claim 10, characterized in that, The four types of single-stranded DNA were mixed at equimolar concentrations.

12. The method as described in claim 10 or 11, characterized in that, The buffer system comprises 5-20 mM Tris-HCl, 0.5-10 mM EDTA, and 20-100 mM MgCl2; its pH is 7.5-8.5; and / or, The reaction is carried out at 95°C for at least 10 min; and / or the reaction is terminated at 4°C for 20 min.

13. The method as described in claim 12, characterized in that, The buffer system consists of 10 mM Tris-HCl, 1 mM EDTA, 50 mM MgCl2, and pH 8.

0.

14. A method for delivering polypeptides to cells, characterized in that, The method comprises contacting the complex of claim 1, the nucleic acid nanoparticles of claim 2 or 3, or the pharmaceutical composition of claim 4 or 5 with target cells; the method is not for diagnostic or therapeutic purposes.

15. The method as described in claim 14, characterized in that, The target cells are cells that express melanocortin 1 receptors.

16. The method as described in claim 14 or 15, characterized in that, The target cells are melanocytes.

17. The use of the complex of claim 1, the nucleic acid nanoparticles of claim 2 or 3, or the pharmaceutical composition of claim 4 or 5 in the preparation of a product for transdermal application, wherein the product is a product that inhibits melanin production.

18. The application as described in claim 17, characterized in that, The product in question is either a cosmetic or a pharmaceutical.

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