A tetrahedral framework nucleic acid-lipoic acid complex and its uses

Abstract

The present invention discloses a tetrahedral framework nucleic acid-lipoic acid complex, which is formed by the complexation of tetrahedral framework nucleic acid and lipoic acid; the molar ratio of the tetrahedral framework nucleic acid to lipoic acid is 1:40 to 1:480. The present invention uses tFNA as a carrier for the co-delivery of lipoic acid, transports lipoic acid into cells, inhibits the production of reactive oxygen species, regulates the apoptotic protein pathway Bcl-2 / Bax / Caspase-3, inhibits the expression of the inflammatory pathway NF-κB, effectively treats the oxidative stress and inflammation caused by photo-damage, achieves the purpose of treating skin photo-damage, has a clear mechanism, obvious effects, and has good application prospects.

Description

Technical Field

[0001] The present invention specifically relates to a tetrahedral framework nucleic acid-lipoic acid complex and its uses. Background Art

[0002] Skin photo-damage is a common skin disease, manifested as skin erythema, dryness, edema, desquamation, and its incidence increases year by year with the destruction of the environment and the increase in ultraviolet radiation. Skin photo-damage is an acute inflammatory disease induced by oxidative stress damage after ultraviolet (UV) radiation. The state of photo-damage that is not controlled for a long time will further damage the skin, cause skin aging, and even lead to malignant lesions. Therefore, it is crucial to control and treat skin photo-damage caused by ultraviolet exposure.

[0003] At present, there is no standard treatment method for skin photo-damage. Relatively speaking, local antioxidant administration has the least trauma, but it is necessary to overcome the deficiency of skin bioavailability. Therefore, developing a new antioxidant drug delivery system for the histological damage and collagen fiber degradation of skin photo-damage and locally administering drugs through transdermal effect is an important research direction for treating skin photo-damage.

[0004] Lipoic acid (LA) is a physiological antioxidant, which plays a key role in energy metabolism and has unique antioxidant properties. It is used in dermatology and cosmetics to repair skin damage and prevent premature skin aging. When applied locally, LA can reduce erythema caused by exposure to ultraviolet radiation and reduce the damage caused by ultraviolet rays. However, LA has low water solubility, poor photo-stability and chemical stability, and low bioavailability and tissue permeability, which affect its effect in the treatment of skin diseases. Developing an effective carrier can enable LA to play a better role, which is of great significance for improving the application value of LA in the treatment of skin diseases.

[0005] Tetrahedral framework nucleic acid (tFNA) is a novel bio-nanomaterial self-assembled by four DNA single strands with specific sequences through base pairing. Due to its structural stability, cell permeability, tissue permeability and biocompatibility, it has broad application prospects in drug delivery, disease treatment and biomedical imaging. However, due to the special structure of tetrahedral framework nucleic acid and its certain activity, whether tetrahedral framework nucleic acid can successfully load small molecule drugs, how the two interact after loading, whether it is an additive effect, an antagonistic effect, or a synergistic effect, these are all unknown, and the effects of different small molecule drugs may vary greatly. At present, there is no research on the loading of lipoic acid by tetrahedral framework nucleic acid, and there is no relevant report on whether tetrahedral framework nucleic acid can successfully load lipoic acid and play a synergistic effect. Summary of the Invention

[0006] To solve the above problems, the present invention provides a tetrahedral framework nucleic acid - lipoic acid complex, which is composed of tetrahedral framework nucleic acid and lipoic acid; the molar ratio of the tetrahedral framework nucleic acid to lipoic acid is 1:40 to 1:480.

[0007] Further, the molar ratio of the tetrahedral framework nucleic acid to lipoic acid is 1:80 to 1:320.

[0008] Even further, the molar ratio of the tetrahedral framework nucleic acid to lipoic acid is 1:160.

[0009] Further, the four DNA single - strand sequences of the tetrahedral framework nucleic acid are as shown in SEQ ID NO.1 - 4.

[0010] The present invention also provides a preparation method of the aforementioned tetrahedral framework nucleic acid - lipoic acid complex, which includes the following steps:

[0011] Take the tetrahedral framework nucleic acid and lipoic acid and add them to a solvent according to the said molar ratio for mixing, and then perform ultrafiltration to obtain the product.

[0012] Further, the solvent is PBS and / or DMSO.

[0013] Further, the synthesis method of the tetrahedral framework nucleic acid is: Add four DNA single - strands with equimolar ratio to TM buffer, maintain at 95°C for 10 min, and quickly cool down to 4°C and maintain for more than 20 min to obtain the product.

[0014] Further, the temperature of the mixing is 4°C and the time is 4 - 6 h.

[0015] The present invention also provides the use of the aforementioned tetrahedral framework nucleic acid - lipoic acid complex in the preparation of a drug for treating skin photo - damage.

[0016] Further, the drug is a transdermal administration preparation for reducing skin photo - damage, restoring normal skin appearance and / or restoring skin elasticity.

[0017] Even further, the drug is a transdermal administration preparation for reducing skin photo - damage induced by ultraviolet radiation.

[0018] The tetrahedral framework nucleic acid-lipoic acid complex of the present invention uses tFNA as a carrier for the co-delivery of lipoic acid, transports lipoic acid into cells, inhibits the production of reactive oxygen species, regulates the apoptotic protein pathway Bcl-2 / Bax / Caspase-3, inhibits the expression of the inflammatory pathway NF-κB, effectively treats the oxidative stress and inflammation caused by photo-damage, and achieves the purpose of treating skin photo-damage. The mechanism is clear and the effect is obvious. The complex of the present invention has good tissue permeability and biocompatibility, can be administered locally non-invasively through the transdermal administration route, and achieves excellent effects, having good application prospects.

[0019] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0020] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0021] Figure 1 The synthesis method and characterization results of the TLA complex: A is a schematic diagram of the synthesis process of the tFNA and TLA complex; B is the PAGE verification of the successful synthesis of tFNA and TLA; C is the atomic force microscope image of the tFNA and TLA complex; D is the particle size result of the tFNA and TLA complex; E is the potential result of tFNA, LA and the TLA complex; F is the absorption spectrum of the TLA complex synthesized with different molar ratios of tFNA and LA between 230 nm and 380 nm wavelengths; G is the ratio of the fluorescence excited by the binding of the TLA complex synthesized with different molar ratios of tFNA and LA to the Gel-Red dye to the fluorescence excited by the binding of pure tFNA to the Hochest dye; H is the ratio of the fluorescence excited by the binding of the TLA complex synthesized with different molar ratios of tFNA and LA to the Gel-Red dye to the fluorescence excited by the binding of pure tFNA to the Gel-Red dye.

[0022] Figure 2Establishment of the cell photo-damage model and the treatment results of TLA on cell viability before and after photo-damage: A shows the results of photo-damage modeling of HDF cells; B shows the proliferation activity results of normal HDF cells treated with tFNA and TLA complexes (tFNA:LA molar ratio = 1:80, 1:160, 1:320); C shows the proliferation activity results of HDF cells after photo-damage modeling treated with tFNA and TLA complexes (tFNA:LA molar ratio = 1:160); D shows the migration activity results and statistics of HDF cells after photo-damage modeling treated with tFNA and TLA complexes (tFNA:LA molar ratio = 1:160).

[0023] Figure 3 Detection of the endocytosis performance of the TLA complex in photo-damaged cells: A shows the confocal microscopy detection results of the entry of tFNA and TLA into HDF cells after photo-damage modeling at different time points (Cy5: red; cytoskeleton: green; nucleus: blue); B shows the flow cytometry detection results and statistics of the entry of tFNA and TLA into HDF cells after photo-damage modeling at different time points.

[0024] Figure 4 Regulation of oxidative stress and apoptosis of photo-damaged cells by the TLA complex: A shows the results and statistics of oxidative stress-induced apoptosis of HDF cells after photo-damage modeling treated with tFNA and TLA; B shows the fluorescence microscopy results of reactive oxygen species production in HDF cells after photo-damage modeling treated with tFNA and TLA; C shows the microplate reader detection results of reactive oxygen species production in HDF cells after photo-damage modeling treated with tFNA and TLA; D shows the confocal microscopy detection results of Bcl-2 protein expression in HDF cells after photo-damage modeling treated with tFNA and TLA (Bcl-2: red; cytoskeleton: green; nucleus: blue); E shows the confocal microscopy detection results of Bax protein expression in HDF cells after photo-damage modeling treated with tFNA and TLA (Bax: red; cytoskeleton: green; nucleus: blue); F shows the confocal microscopy detection results of Caspase-3 protein expression in HDF cells after photo-damage modeling treated with tFNA and TLA (Caspase-3: red; cytoskeleton: green; nucleus: blue); G shows the results of detecting the expression of apoptosis-related proteins in photo-damaged cells by Western blotting; H shows the quantitative analysis of the Western blotting results.

[0025] Figure 5Regulation of the TLA complex on the inflammatory pathway and the expression of inflammatory proteins and collagen-related proteins in photo-damaged cells: A shows the confocal microscopy detection results of the expression of NF-κB protein in HDF cells after photo-damage modeling with tFNA and TLA treatment (NF-κB: red; cytoskeleton: green; nucleus: blue); B shows the confocal microscopy detection results of the expression of p-NF-κB protein in HDF cells after photo-damage modeling with tFNA and TLA treatment (p-NF-κB: red; cytoskeleton: green; nucleus: blue); C shows the results of detecting the expression of the inflammatory pathway in photo-damaged cells by Western blotting; D shows the quantitative analysis of the Western blotting results; E shows the results of detecting the expression of inflammatory proteins and collagen-related proteins in photo-damaged cells by Western blotting; F shows the quantitative analysis of the Western blotting results.

[0026] Figure 6 Establishment of a mouse model of skin photo-damage and treatment results: A shows the macroscopic images of the results of skin photo-damage mouse modeling and after treatment; B shows the fluorescence section detection results of tissue permeability after topical administration of tFNA and TLA (Cy5: red; nucleus: blue).

[0027] Figure 7 Results of sections of mice after skin photo-damage treatment: A shows the H&E staining results after topical administration of tFNA and TLA; B shows the Masson staining results after topical administration of tFNA and TLA; C shows the statistical results of the epidermal thickness of each group in the H&E staining results; D shows the statistical results of the collagen volume fraction of each group in the Masson staining results; E shows the statistical results of the overall skin condition considering the epidermal thickness and collagen volume fraction of each group. Detailed implementation methods

[0028] The raw materials and equipment used in the specific implementation methods of the present invention are all known products and are obtained by purchasing commercially available products.

[0029] Example 1. Preparation of tetrahedral framework nucleic acid-lipoic acid (TLA) complex

[0030] 1. Synthesis of tetrahedral framework nucleic acid

[0031] Add equimolar amounts of four special-sequence DNA single strands (so that the final concentration of each of the four DNA single strands after dissolution is 1000 nM) to TM buffer (50 mM MgCl2 and 10 mM Tris-HCl, pH 8.0). After a series of heating procedures (95 °C for 10 minutes; 4 °C for 20 minutes), the assembly of tetrahedral framework nucleic acid (tFNA) is completed. The specific sequences of the DNA single strands are shown in Table 1 below.

[0032] Table 1. Specific sequences of DNA single strands required for tFNA synthesis

[0033]

[0034] 2. Synthesis of TLA Complex

[0035] Dissolve lipoic acid (LA) in DMSO, and then mix it with tFNA (the concentration of tFNA is 1000 nM, and the molar ratio of tFNA to LA is 1:40, 1:80, 1:160, 1:320 or 1:480). Shake the mixed solution at 4 °C for 6 hours, and then ultrafilter it with a 30 kd ultrafiltration tube to obtain the product.

[0036] LA will be loaded onto tFNA through groove binding and intercalation binding to form a TLA complex drug system. The schematic diagram of the specific synthesis process is as shown in Figure 1 Figure A. According to the usage, generally dilute the TLA complex in PBS so that the TLA complex is 250 nM in terms of nucleic acid concentration.

[0037] The beneficial effects of the TLA complex prepared in Example 1 are demonstrated by the following specific test examples.

[0038] Test Example 1. Related Characterization of Tetrahedral Framework Nucleic Acid-Lipoic Acid (TLA) Complex

[0039] 1. Characterization Method

[0040] Verify whether the tFNA and TLA complexes are successfully synthesized by polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM), and observe their size and shape. Dynamic light scattering DLS (Nano ZS, Malvern, England) is used to measure the particle size and Zeta potential of the tFNA and TLA complexes. The absorbance curves of tFNA, LA and TLA complexes are depicted with a super differential spectrophotometer. The binding fluorescence of Hochest and GelRed is detected by a Varioskan LUX microplate reader (Thermo Scientific, USA).

[0041] 2. Characterization Results

[0042] As shown in the schematic diagram in Figure 1 Figure A, the synthesis of the TLA complex is divided into two steps: (1) tFNA is self-assembled from four equimolar amounts of ssDNA, and the successful synthesis of tFNA is detected by PAGE ( Figure 1 Figure B); (2) tFNA is complexed with LA by shaking for 6 h to obtain the TLA complex, and the successful synthesis of the TLA complex is also verified by PAGE. In addition, it can be observed that there are no obvious by-products in both tFNA and TLA ( Figure 1 Figure B).

[0043] Through AFM detection, the tFNA molecule was observed to have a characteristic triangular pyramid structure, while the TLA complex had an oval structure. The sizes of tFNAs and TLA were both around 15 nm( Figure 1 C).

[0044] The particle size and zeta potential of the tFNA and TLA complexes were detected by DLS, which also confirmed the successful synthesis of the TLA complex. In Figure 1 D, the size of tFNA was 12.2 ± 1.5 nm, while the size of the TLA complex was 18.3 ± 3.7 nm. In Figure 1 E, the zeta potential of tFNA was -8.1 ± 1.9 mV, while the zeta potential of the TLA complex was -18.0 ± 2.5 mV. These differences indicated that the new nanoparticle TLA complex had been synthesized.

[0045] Meanwhile, by changing the amount of LA in the solution, the molar ratios of tFNA and LA in the solution for preparing the TLA complex were 1:40, 1:80, 1:160, 1:320, and 1:480 respectively. The TLA complexes with different LA contents were prepared according to the method of the example. Figure 1 F showed that the peak position of the absorption spectrum of the TLA complex had a red shift and a decrease compared with that of tFNA, indicating the successful synthesis of the TLA complex; as the proportion of LA increased, the absorption peak of TLA showed a trend of decreasing first and then increasing, and the decrease was most obvious when the molar ratio of tFNA and LA was 1:160.

[0046] The binding methods between LA and tFNA were competitively determined by using Hochest dye and Gel-Red dye respectively. When tFNA was incubated with 1x Hochest or 1x GelRed for 20 minutes, the fluorescence of the corresponding dye was excited by the groove binding (Hochest) or intercalative binding (Gel-Red) of the dye to the nucleic acid structure of tFNA. The TLA complexes prepared from different molar ratios of tFNA and LA were incubated with Hochest or Gel-Red dye together. The fluorescence intensity of Hochest( Figure 1 G) and Gel-Red( Figure 1 H) showed a trend of decreasing first and then increasing with the increase of the LA concentration, and the decrease was most obvious when the molar ratio of tFNA and LA was 1:160. This meant that LA could interact with tFNA through groove binding and intercalative binding to form the TLA complex.

[0047] Generally speaking, the above results indicated that the present invention had constructed a novel nano-drug system, the TLA complex, for the first time, in which LA was bound to tFNA through groove binding and intercalative binding modes. The prepared TLA complex had better stability and improved dissolution rate.

[0048] Test Example 2: Improvement of Cell Viability by TLA Treatment

[0049] 1. Experimental Method

[0050] (1) Cultivation of Human Dermal Fibroblasts and Establishment of Photoaging Model

[0051] The human dermal fibroblast cell line HDF cells were cultured in complete medium containing high-glucose Dulbecco's modified Eagle's medium (H-DMEM, HyClone, Logan, USA), 10% fetal bovine serum (FBS, HyClone, Logan, USA), and 1% penicillin-streptomycin solution (HyClone, Logan, USA), and cultured in a 37 °C cell incubator containing 5% CO2.

[0052] Under the PBS solution environment, the cells were irradiated with a narrow-spectrum UVB ultraviolet lamp (311 nm, TL20W / 01, Philips, USA) at a fixed distance above the cells to induce photoaging of HDF cells. Drug treatment was carried out 24 hours after the end of ultraviolet lamp irradiation. Generally, tFNA, LA, and TLA were used to treat the cells for 24 hours.

[0053] (2) Cell Proliferation Activity of HDF Cells in Normal or Photoaging State after TLA Treatment

[0054] HDF cells were seeded in 96-well plates. For the determination of the proliferation activity of normal state HDF cells, the cells were treated with serum-free H-DMEM medium containing 250 nM tFNA or 250 nM TLA complexes prepared from different molar ratios of tFNA and LA (the molar ratios of tFNA and LA were 1:80, 1:160, 1:320 respectively, as in Example 1) for 24 hours, and the blank group was treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 24 hours. Then the cells were cultured in 10% CCK-8 solution (KeyGEN Biotech) at 37 °C for 1 hour. The cell viability was measured by the OD value at 450 nm.

[0055] HDF cells were seeded in 96-well plates. For the determination of the proliferation activity of HDF cells in a photodamaged state, the cell photodamage model was established according to the method described in Experimental Example 2 (1). 24 hours after the completion of the modeling, the cells were treated with serum-free H-DMEM medium containing 250nM tFNA, 40μM LA, or 250nM TLA complex (tFNA and LA molar ratio of 1:160, as in Example 1) for 24 hours. The blank group (normal cells) and the control group (photodamaged cells) were treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 24 hours. The cells were then cultured in 10% CCK-8 solution (KeyGEN Biotech) at 37°C for 1 hour. Cell viability was measured by the OD value at 450nm.

[0056] (3) Cell migration activity of photodamaged HDF cells after TLA treatment

[0057] HDF cells were seeded in 6-well plates and treated with cell photodamage modeling according to the method described in Experimental Example 2(1). 24 hours after the modeling, a "cross" scratch was made on the bottom of the well plate using a sterile pipette. After washing with PBS, the cells were continuously treated with serum-free H-DMEM medium containing 250nM tFNA, 40μM LA, or 250nM TLA complex (tFNA and LA molar ratio of 1:160, as in Example 1) for 48 hours. The blank group (normal cells) and the control group (photodamaged cells) were treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 48 hours. Cell migration changes at 0, 12, 24, and 48 hours after administration were observed and recorded using a microscope (Olympus, Japan).

[0058] 2. Experimental results

[0059] Figure 2 A. Visible ultraviolet radiation-induced photodamage of HDF cells affects the cell morphology, leading to weakened cell adhesion, cell deformation and shrinkage, and increased cytoplasmic content ( Figure 2 A) Cell proliferation and migration abilities both affect damage repair and are important research factors in photoremediation. Figure 2 B shows that tFNA and TLA at different synthesis ratios can promote the proliferation of normal HDF cells, among which TLA-160 and TLA-320 show significant pro-proliferation effects (combined with the test results of tFNA loading LA efficiency in Experimental Example 1, TLA-160 was selected for subsequent experiments). Light damage can reduce HDF cell proliferation by about 50%, but after the cells are treated with 250nM TLA, the cell proliferation activity is restored, and the difference is statistically significant compared with the control group ( Figure 2C), indicating that TLA can promote the proliferation of photo-damaged cells. The viability state of cells also includes cell migration ability. Figure 2 The results of the scratch test in D showed that the migration speed of HDF cells was significantly reduced after photo-damage. After treating photo-damaged cells with TLA, the migration speed of the cells at the 12, 24, and 48 h time points increased significantly and was able to exceed the migration speed of normal state HDF cells.

[0060] Test Example 3, Cellular uptake experiment of tFNA and TLA

[0061] 1. Experimental method

[0062] Using the single-stranded S1 labeled with Cy5 (S1-Cy5), the Cy5-labeled tFNA and TLA complex was prepared according to the method described in Example 1 of the present invention (in the solution for preparing the TLA complex, tFNA: 250 nM, LA: 40 μM).

[0063] To examine the cellular uptake performance of the tFNA and TLA complex, HDF cells were seeded in 6-well plates, and the cells were subjected to photo-damage modeling treatment according to the method described in Test Example 2(1). After 24 hours of the end of the modeling, the photo-damaged cells were treated with the Cy5-labeled tFNA and TLA complex for 6 or 12 hours. Then the cells were collected and rinsed 3 times with PBS. Finally, the proportion of cells with fluorescent Cy5 among all cells was obtained by flow cytometry (CytoFLEX, Beckman Coulter Inc., Brea, USA). Images of the Cy5-labeled tFNA and TLA complex dispersed in the cells were obtained by confocal microscopy (Olympus, Tokyo, Japan).

[0064] 2. Experimental results

[0065] The entry of the Cy5-labeled tFNA and TLA complex into photo-damaged HDF cells was examined using flow cytometry and confocal microscopy. From the images of the laser confocal microscope ( Figure 3 A), it was observed that the Cy5-labeled tFNAs and TLA complex were widely distributed in the cytoplasm of the cells, and the intracellular material distribution increased with the prolongation of the time of treating the cells. According to the results of flow cytometry ( Figure 3 B), it was detected that after treating the cells for 12 hours, the proportion of cells containing the TLA complex and tFNA fluorescence reached about 70%. This phenomenon indicates that although the cellular uptake ability of TLA is slightly weaker than that of tFNA after complexing tFNA with LA, the TLA complex still has excellent cellular uptake ability.

[0066] Test Example 4, TLA reduces cell apoptosis by inhibiting ROS production and regulating the anti-apoptotic signaling pathway

[0067] 1. Experimental methods

[0068] (1) Apoptosis of HDF cells after TLA treatment in the photo-damaged state

[0069] HDF cells were seeded in 6-well plates. After culturing the HDF cells and inducing photo-damage according to the method described in Test Example 2(1), the cells were continuously treated with serum-free H-DMEM medium containing 250 nM tFNA, 40 μM LA, or 250 nM TLA complex (the molar ratio of tFNA to LA was 1:160, as in Example 1) for 24 hours. The blank group (normal cells) and the control group (photo-damaged cells) were treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 24 hours. The cells were digested with 0.25% trypsin without EDTA, and the cells were collected and washed by centrifugation at 1000 rpm for 5 minutes. The cells were stained with FITC-Annexin V and PI dye under light avoidance conditions, and the apoptosis of the cells was detected by flow cytometry (CytoFLEX, Beckman Coulter Inc., Brea, USA).

[0070] (2) Production of reactive oxygen species in HDF cells after TLA treatment in the photo-damaged state

[0071] After culturing the HDF cells and inducing photo-damage according to the method described in Test Example 2(1), the cells were continuously treated with serum-free H-DMEM medium containing 250 nM tFNA, 40 μM LA, or 250 nM TLA complex (the molar ratio of tFNA to LA was 1:160, as in Example 1) for 24 hours. The blank group (normal cells) and the control group (photo-damaged cells) were treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 24 hours. After washing the cells with PBS, the cells were stained with DCFH-DA reactive oxygen species dye under light avoidance conditions. Finally, images of the generation of intracellular reactive oxygen species in the cells were obtained by fluorescence microscopy (Olympus, Tokyo, Japan). The fluorescence intensity of the cells after staining was detected by a Varioskan LUX microplate reader (Thermo Scientific, USA), and the production of reactive oxygen species in different groups of cells was calculated.

[0072] (3) Regulation of apoptosis-related signaling pathways in photo-damaged HDF cells by TLA

[0073] Bcl-2, Bax, and Caspase-3 are key proteins in the cell apoptosis-related signaling pathway. MAPK is also one of the key proteins involved in cell apoptosis. These proteins were used as target proteins, and GAPDH was used as the internal reference protein for the experiment. After culturing HDF cells and inducing photo-damage according to the method described in Experimental Example 2(1), the cells were continuously treated with serum-free H-DMEM medium containing 250 nM tFNA, 40 μM LA, or 250 nM TLA complex (the molar ratio of tFNA to LA was 1:160, as in Example 1) for 24 hours. The blank group (normal cells) and the control group (photo-damaged cells) were treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 24 hours. After the drug treatment was completed, total proteins in the samples were extracted, and the protein expressions of MAPK, Bcl-2, Bax, and Caspase-3 were detected by Western blotting. After the drug treatment was completed, the cells were subjected to immunofluorescence staining, and confocal microscopy (Olympus, Tokyo, Japan) was used to obtain the expression images of Bcl-2, Bax, and Caspase-3 proteins in the cells.

[0074] 2. Experimental Results

[0075] Figure 4 A It can be seen that after UVB-induced photo-damage of HDF cells, apoptosis caused by oxidative stress increased significantly. However, TLA treatment effectively inhibited the activation of cell apoptosis. Figure 4 B, C It can be seen that HDF cells produce only a small amount of reactive oxygen species under normal conditions. After photo-damage, HDF cells produced a large amount of reactive oxygen species, and the intracellular reactive oxygen species content increased by about 2.5 times, and the cells entered the state of oxidative stress. After treatment with TLA, the oxidative stress state of photo-damaged HDF cells was alleviated, and the intracellular reactive oxygen species level basically returned to normal. Figure 4 D It can be seen that the anti-apoptotic protein Bcl-2 in normal HDF cells was mainly expressed in the cytoplasm, while its expression in photo-damaged cells was significantly reduced. After treatment with TLA, the expression of Bcl-2 was restored and mainly localized in the nucleus. In addition, the pro-apoptotic protein Bax ( Figure 4 E) and the apoptosis execution protein Caspase-3 ( Figure 4 F) were highly expressed in the cytoplasm of photo-damaged HDF cells, while after treatment with TLA, the expressions of Bax and Caspase-3 in the cells were significantly decreased. Detection by Western blotting ( Figure 4Similar results were also shown in (G, H). The expression of MAPK protein was activated in photo-damaged cells and inhibited after TLA treatment. This protein plays a crucial role in the MAPK signaling pathway, and its activation can also induce apoptosis and the production of ROS, as well as promote the activation of the NF-κB signaling pathway. Comprehensive analysis suggests that TLA can effectively reduce the apoptosis of photo-damaged HDF cells by regulating the MAPK and Bcl-2 / Bax / Caspase-3 signaling pathways and inhibiting the production of intracellular reactive oxygen species.

[0076] Experimental Example 5: TLA inhibits the inflammatory response by inhibiting the NF-κB signaling pathway and promotes cell secretion

[0077] 1. Experimental method

[0078] (1) Regulation of the inflammatory-related signaling pathways of photo-damaged HDF cells by TLA

[0079] The NF-κB signaling pathway is a key signaling pathway for regulating cellular inflammatory responses. Among them, the proteins that play key roles are mainly NF-κB, p-NF-κB, and IκB. These proteins were used as target proteins, and GAPDH was used as the internal reference protein for the experiment. After culturing HDF cells and inducing photo-damage according to the method described in Experimental Example 2(1), the cells were continuously treated with serum-free H-DMEM medium containing 250 nM tFNA, 40 μM LA, or 250 nM TLA complex (the molar ratio of tFNA to LA was 1:160, as in Example 1) for 24 hours. The blank group (normal cells) and the control group (photo-damaged cells) were treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 24 hours. After the drug treatment was completed, total proteins in the samples were extracted, and the protein expressions of NF-κB, p-NF-κB, and IκB were detected by Western blotting. After the drug treatment was completed, the cells were subjected to immunofluorescence staining, and confocal microscopy (Olympus, Tokyo, Japan) was used to obtain the expression images of NF-κB and p-NF-κB proteins in the cells.

[0080] (2) Regulation of the inflammatory response and collagen-related proteins of photo-damaged HDF cells by TLA

[0081] Since the NF-κB signaling pathway is a key signaling pathway that regulates cellular inflammatory responses, changes in its expression can affect the production of downstream inflammatory proteins and collagen-related proteins. The inflammatory protein IL-1β, the collagen-related proteins TGF-β and Collagen-1 were selected as target proteins, and GAPDH was used as an internal reference protein for the experiment. After culturing HDF cells and establishing a photo-damage model according to the method described in Experimental Example 2(1), the cells were continuously treated with serum-free H-DMEM medium containing 250 nM tFNA, 40 μM LA or 250 nM TLA complex (the molar ratio of tFNA and LA was 1:160, as in Example 1) for 24 hours. The blank group (normal cells) and the control group (photo-damaged cells) were treated with serum-free H-DMEM medium containing 1 / 4 volume of PBS for 24 hours. After the drug treatment was completed, total proteins in the samples were extracted, and the protein expressions of IL-1β, TGF-β and Collagen-1 were detected by Western blotting.

[0082] 2. Experimental Results

[0083] UVB radiation-induced photo-damage can activate the NF-κB signaling pathway in HDF cells. Confocal microscopy ( Figure 5 A, B) and Western blotting ( Figure 5 C, D) both found that TLA treatment could down-regulate the expressions of NF-κB and p-NF-κB, inhibit the degradation of IκB, and thus inhibit the activation of the NF-κB signaling pathway in photo-damaged cells. At the same time, the NF-κB signaling pathway affects the expressions of many downstream proteins, especially inflammatory factors and collagen. The results of Western blotting ( Figure 5 E, F) showed that after cell photo-damage, the protein expression of IL-1β was significantly increased, but after TLA treatment, its expression was inhibited. In addition, TLA treatment enhanced the expression of TGF-β, which is an important cell growth factor in fibroblasts, can regulate procollagen transcription, and plays an important role in collagen synthesis. Collagen-1 is produced by fibroblasts and is one of the most abundant proteins in the skin. After cell photo-damage, the protein expression of Collagen-1 decreased significantly, which was closely related to the increase in inflammatory factors caused by the NF-κB and MAPK signaling pathways. After TLA treatment, the expression of Collagen-1 increased significantly, which was beneficial to the recovery of photo-damage.

[0084] Experimental Example 6. Animal Experiment

[0085] 1. Experimental Method

[0086] (1) Establishment and treatment of an animal model of skin photo-damage

[0087] In vivo experiments used BALB / C mice (female, 20 - 25 g, 6 - 8 weeks old). The animal experiments were approved by the Ethics Committee of West China School of Stomatology, Sichuan University. The mice were raised in an SPF - level environment with regular day - night cycles and free access to food. After shaving and depilation of the mice, the mice were randomly divided into 5 groups (3 mice in each group): blank group (normal mice), control group (mice with photo - damage model), tFNA group (mice with photo - damage model), LA group (mice with photo - damage model), and TLA group (mice with photo - damage model). The mice were irradiated with UVB (540 mJ / cm 2 / d, once a day for 3 days) to establish a photo - damaged skin model on the back of the mice, and the apparent condition of the back skin of the mice was photographed and recorded.

[0088] For each group of mice, normal saline, tFNA, LA, or TLA complex (prepared according to the method described in Example 1, with a tFNA concentration of 1 μM and an LA concentration of 160 μM in the solution during preparation) was mixed with Aquaphor ointment (Eucerin, Germany) at a mass ratio of 1:1, and then evenly applied to the back skin of the mice at 0.5 g / mouse. The drug was administered once every other day for 4 times. After the drug administration was completed, the apparent condition of the back skin of the mice was photographed and recorded. 24 hours after the last drug administration, the mice were sacrificed, and the back skin was paraffin - embedded and sectioned, followed by fluorescence staining, H&E staining, and Masson staining.

[0089] (2) Tissue penetration of tFNA and TLA

[0090] To investigate the tissue penetration of tFNAs material and TLA complex in the photo - damaged skin of mice, tFNAs - Cy5 and TLA - Cy5 were used for transdermal drug delivery (the steps of mouse skin modeling and drug delivery were carried out according to the method described in Test Example 6.(1)). After collecting the back skin tissue samples of the mice, they were frozen - embedded and sectioned, stained with DAPI dye, and the distribution of Cy5 fluorescence in the skin tissue was observed through a confocal microscope (Olympus, Tokyo, Japan) to verify the tissue penetration performance of tFNAs and TLA complex in the photo - damaged skin.

[0091] (3) Detection of the therapeutic effect of TLA on skin photo - damage

[0092] To investigate the therapeutic effect of TLA complex on the photo - damaged skin of mice, after collecting the back skin tissue samples of the mice, they were paraffin - embedded and sectioned, and then H&E staining and Masson staining were performed respectively. The change in the epidermal thickness and the change in the collagen volume fraction in the skin tissue were observed through a slide scanner (Leica, Germany) to verify the therapeutic effect of TLA complex on the photo - damaged skin tissue.

[0093] 2. Experimental results

[0094] Figure 6 A It can be seen that after the establishment of the skin photo-damage model in the back of the mice, the skin became red, with poor elasticity, and erythema and scabs were visible. After drug treatment, large-scale scabs appeared in the modeling area of the control group, and the surrounding skin was red and congested. The scab area in the LA group was smaller, and there was no obvious congestion in the surrounding skin; the skin in the tFNA group was slightly red, and the scabs had basically fallen off; in contrast, in the TLA group, the skin erythema was significantly improved, the scabs had completely fallen off, there was no obvious skin redness, and the healing speed after photo-damage was faster.

[0095] The tissue penetration of tFNAs and TLA into the skin of mice as shown by fluorescence staining. After administration 4 times, a large amount of Cy5 fluorescence was detected in both the epidermis and dermis of the skin of mice in the tFNAs treatment group; the Cy5 fluorescence in the TLA group was mainly concentrated in the epidermis, but it could still enter the dermis ( Figure 6 B).

[0096] After photo-damage, the epidermis of the skin thickened, inflammatory infiltration occurred in the dermis, and the boundary between the epidermis and the dermis was disordered. After TLA treatment, the thickness of the epidermis decreased relatively, and there was a small amount of inflammatory cell infiltration ( Figure 7 A). In addition, the arrangement of collagen fibers in the dermis of the skin was relatively disordered, and it was difficult to observe the normal woven bundle structure. The collagen deposition and fiber arrangement pattern in the TLA group were more orderly compared with the control group ( Figure 7 B). The thickness of the epidermis epithelium after treatment was measured and statistically analyzed. The thickness of the epidermis in the control group increased by about 5 times. Compared with the control group, the average thickness of the epidermis in the TLA group decreased significantly, and it was statistically significant compared with the control group, the tFNAs group, and the LA group ( Figure 7 C). After calculating and statistically analyzing the collagen volume fraction (CVF) of the skin after treatment, it was found that the collagen volume fraction in the control group decreased by about 50%, and there were also obvious decreases in the tFNA group and the LA group, while the TLA group restored the collagen volume fraction to the normal level, showing statistical differences compared with the control group, the tFNA group, and the LA group ( Figure 7 D). Considering that both CVF and epidermal thickness are important histological changes in skin photo-damage, we analyzed the correlation between these two factors in different groups ( Figure 7 E). It can be found that the TLA group had a lower epidermal thickness and a higher collagen fiber content, and the data distribution was similar to that of the blank group, indicating that the TLA treatment had the best recovery effect on photo-damaged skin tissue and played a synergistic effect.

[0097] In summary, the present invention provides a tetrahedral framework nucleic acid-lipoic acid complex, which uses tFNA as a carrier for the co-delivery of lipoic acid, transports lipoic acid into cells, enhances the cell viability of photo-damaged HDF cells, inhibits cell apoptosis and the production of reactive oxygen species, regulates the apoptotic protein pathway Bcl-2 / Bax / Caspase-3, inhibits the expression of the inflammatory pathway NF-κB, effectively treats the oxidative stress and inflammation caused by photo-damage, achieves the purpose of treating skin photo-damage, with a clear mechanism and obvious effects. The complex of the present invention has good tissue permeability and biocompatibility, can be administered non-invasively locally through the transdermal drug delivery route, has excellent therapeutic effects on skin photo-damage, and has good application prospects.

Claims

1. A tetrahedral framework nucleic acid-lipoic acid complex, characterized in that: It is composed of the complex of tetrahedral framework nucleic acid and lipoic acid; the molar ratio of the tetrahedral framework nucleic acid to lipoic acid is 1:160; The four DNA single-strand sequences of the tetrahedral framework nucleic acid are shown as SEQ ID NO. 1-4.

2. A method for preparing the tetrahedral framework nucleic acid-lipoic acid complex according to claim 1, characterized in that: It includes the following steps: Take the tetrahedral framework nucleic acid and lipoic acid and add them to a solvent according to the said molar ratio for mixing, and then perform ultrafiltration to obtain the product.

3. The preparation method according to claim 2, wherein: The solvent is PBS and / or DMSO.

4. The preparation method according to claim 2, wherein: The synthesis method of the tetrahedral framework nucleic acid is: add four DNA single strands with equimolar ratio to TM buffer solution, maintain at 95 °C for 10 min, and quickly cool down to 4 °C and maintain for more than 20 min to obtain the product.

5. The preparation method according to claim 2, characterized in that: The temperature of the mixing is 4 °C and the time is 4-6 h.

6. Use of the tetrahedral framework nucleic acid-lipoic acid complex according to claim 1 in the preparation of a drug for treating skin photo-damage.

7. The use according to claim 6, characterized in that: The drug is a transdermal administration preparation for reducing skin photo-damage, restoring the normal appearance of the skin and / or restoring skin elasticity.

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