CD63 aptamer-driven DNA hydrogel and its preparation method and application

Abstract

CD63 aptamer-driven DNA hydrogels and their preparation methods and applications belong to the technical field of hydrogel materials. To address the technical problems of low delivery efficiency and targeting, short drug retention, and poor permeability in existing hydrogels, the present invention provides a CD63 aptamer-driven DNA hydrogel. The DNA hydrogel is composed of three Y-shaped DNA chains with nucleotide sequences as shown in SEQ ID NOs. 1-3, L1+L2 (SEQ ID NO. 4), a CD63 aptamer (SEQ ID NO. 5), and L2 (SEQ ID NO. 6). The DNA hydrogel provided by the present invention can efficiently load exosomes, has good biocompatibility and mechanical properties, and reduces side effects caused by excessive local drug concentration. It can be used to load exosomes to promote skin wound healing and treat osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel materials, and in particular relates to a DNA hydrogel and a preparation method and application thereof. Background Art

[0002] Hydrogels are three-dimensional hydrophilic polymer networks that can serve as drug delivery systems, providing a moist therapeutic environment, promoting tissue growth, and controlling the release of exosomes. Commonly used hydrogels in the prior art include photocurable hydrogels, such as gelatin methacryloyl (GelMA), hyaluronic acid methacryloyl (HAMA), and thermosensitive hydrogels, also known as thermoresponsive hydrogels. Among these, gelatin methacryloyl hydrogels suffer from low mechanical properties and poor controllable degradation, resulting in poor retention and activity of loaded exosomes. Furthermore, gelatin methacryloyl hydrogels are also known to be highly viscous. Thermosensitive hydrogels also suffer from poor mechanical properties, inadequate biocompatibility, slow response, difficulty in controlled drug release, and uncontrollable stability and degradation. Thermoresponsive hydrogels undergo a property change above the critical solution temperature (LSTC), exhibiting a pronounced sol-gel transition and auto-shrinkage.

[0003] Therefore, those skilled in the art are eager to develop a hydrogel with high delivery efficiency and targeting, which can stably release loaded exosomes for a long time. Summary of the Invention

[0004] In order to solve the technical problems of low delivery efficiency and targeting, short drug retention and poor permeability of hydrogels in the prior art, the present invention provides a CD63 aptamer-driven DNA hydrogel and its preparation method and application.

[0005] One of the objectives of the present invention is to provide a method for preparing a CD63 aptamer-driven DNA hydrogel, the preparation method comprising the following steps:

[0006] S1: The three DNA chains Y1, Y2, and Y3 were placed in 0.2 M PB buffer at a molar ratio of 1:1:1 and mixed to obtain a Y-shaped scaffold; the PB buffer was pH 7.4 and 300 mM sodium chloride;

[0007] S2: The L1+L2 DNA strand, CD63 aptamer, and L2 DNA strand were placed in 0.2 M PB buffer at a molar ratio of 1:1:1 and mixed to obtain a linker; the PB buffer had a pH of 7.4 and contained 300 mM sodium chloride.

[0008] S3: The Y-shaped scaffold obtained in S1 and the linker obtained in S2 were incubated at 90°C for 10 minutes respectively. The Y-shaped scaffold and the linker cooled to room temperature were mixed evenly in a volume ratio of 2:3 and allowed to stand to obtain a CD63 aptamer-driven DNA hydrogel.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the Y1 DNA chain in S1 is shown as SEQ ID NO.1.

[0010] In a preferred embodiment of the present invention, the nucleotide sequence of the Y2 DNA chain in S1 is shown as SEQ ID NO.2.

[0011] In a preferred embodiment of the present invention, the nucleotide sequence of the Y3 DNA chain in S1 is shown as SEQ ID NO.3.

[0012] In a preferred embodiment of the present invention, the nucleotide sequence of the L1+L2 DNA chain in S2 is shown as SEQ ID NO.4.

[0013] In a preferred embodiment of the present invention, the nucleotide sequence of the CD63 aptamer in S2 is shown as SEQ ID NO.5.

[0014] In a preferred embodiment of the present invention, the nucleotide sequence of the L2 DNA chain in S2 is shown as SEQ ID NO.6.

[0015] A second object of the present invention is to provide a CD63 aptamer-driven DNA hydrogel, wherein the DNA hydrogel is obtained by the above-mentioned preparation method.

[0016] The third object of the present invention is to provide the use of the above-mentioned CD63 aptamer-driven DNA hydrogel in loading exosomes to promote skin wound healing.

[0017] A fourth object of the present invention is to provide the use of the above-mentioned CD63 aptamer-driven DNA hydrogel in the treatment of osteoarthritis, wherein the use is carried out by injecting the CD63 aptamer-driven DNA hydrogel carrying exosomes.

[0018] Beneficial effects of the present invention: The present invention provides a CD63 aptamer-driven DNA hydrogel, which is composed of three Y-shaped DNA chains with nucleotide sequences shown in SEQ ID NO.1-3, as well as L1+L2 (SEQ ID NO.4), a CD63 aptamer (SEQ ID NO.5) and L2 (SEQ ID NO.6).

[0019] The DNA hydrogel prepared by the present invention is different from traditional linear DNA cross-linking. The designed Y-shaped branches provide multiple terminal complementary sequences, which can combine with each other through base complementary pairing to form a stable structure. They can also perform multi-point binding with the connecting chain (L-shaped single chain) through base complementary pairing to form a three-dimensional network cross-linking structure, reducing the random entanglement of molecular chains, improving the mechanical strength of the hydrogel, and making it suitable for long-term drug delivery.

[0020] L1 and L2 are linear single-stranded DNAs that bind to the ends of the Y-shaped scaffold through complementary pairing to form "YL" cross-linking nodes and construct the main framework of the hydrogel; 1) The L1+L2 combination can extend the network length, so that the porosity and drug release rate of the hydrogel meet the requirements; the CD63 aptamer (AptCD63) embedded in the CD63-L1 chain can specifically recognize the exosome surface marker CD63 protein, ensuring that the exosomes can be efficiently anchored in the hydrogel; 2) The L2 chain acts as a universal connecting unit, participating in the L1+ L2 pairs with and binds to CD63-L1, increasing the density of cross-linking points and improving the mechanical properties of the gel; by mixing L2 and CD63-L1, the aptamer is evenly distributed to avoid local uneven drug loading; 3) The CD63 aptamer is a single-stranded DNA that folds to form a specific three-dimensional conformation and binds to the specific epitope of the CD63 protein on the surface of exosomes, giving the DNA hydrogel the ability to specifically target exosomes, accurately capturing and fixing exosomes, improving delivery efficiency and targeting, and avoiding the leakage problem of traditional physical encapsulation.

[0021] Since the base pairing and double helix structure of DNA are most stable at neutral pH (about 7.4), the present invention chooses to use 0.2 M PB (pH 7.4, 300 mM sodium chloride) buffer to enable DNA chains to better perform base complementary pairing and prevent acidic or alkaline conditions from causing hydrolysis or denaturation of DNA chains; in addition, the ionic strength in 0.2 M PB buffer can accelerate the dynamic hybridization process of DNA chains, allowing the Y-shaped scaffold and the connecting chain to quickly form a three-dimensional network. The sustained-release properties conferred by the three-dimensional network structure and uniform pores of the hydrogel can stably release the loaded exosomes for a long time.

[0022] Mixing the Y-shaped scaffold and the linker in a volume ratio of 2:3 can ensure that each Y-shaped scaffold has a sufficient number of linkers bound to it, which can form a stable three-dimensional network while avoiding excessive cross-linking that leads to gel brittleness or too small pore size.

[0023] It can be seen that the CD63 aptamer-driven DNA hydrogel provided by the present invention can efficiently load exosomes, has good biocompatibility and mechanical properties, and its sustained release effect can reduce side effects caused by excessive local drug concentration. It can not only load exosomes to promote wound healing, providing a new strategy for the clinical treatment of chronic wounds, but also can be used to treat osteoarthritis by injecting CD63 aptamer-driven DNA hydrogel carrying exosomes.

[0024] The CD63 aptamer-driven DNA hydrogel provided by the present invention solves the technical problems of traditional drug delivery systems that are easily and quickly cleared from wound sites, insufficient local drug concentration, and poor exosome permeability caused by the skin barrier; compared with single therapy, the CD63 aptamer-driven DNA hydrogel provided by the present invention not only overcomes the limitations of the difficulty of exosome preservation in the body and the insufficient mechanical properties of traditional carriers, but also achieves precise drug loading and controllable release through sequence programmability, forming a uniform drug distribution while maintaining the stability of the active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In the following figures, Apt CD63 represents CD63 aptamer, Y scaffold represents Y-shaped scaffold, GS / exo represents ginsenoside-loaded exosomes, Hyd and Hydrogel both represent hydrogels, GS represents ginsenoside, and exo represents exosomes.

[0026] Figure 1 Schematic diagram of the construction of the CD63 aptamer-driven DNA hydrogel in Example 1;

[0027] Figure 2 This is a state diagram of the CD63 aptamer-driven DNA hydrogel in Example 1; wherein Gel is gel and Solution is solution;

[0028] Figure 3 This is the SEM image of the CD63 aptamer-driven DNA hydrogel in Example 1;

[0029] Figure 4 This is a rheological analysis graph of the CD63 aptamer-driven DNA hydrogel in Example 1; the abscissa "Wavelength" is the wavelength, and the ordinate "Circular dichroisn" is the circular dichroism spectrum;

[0030] Figure 5This is the non-denaturing polyacrylamide gel electrophoresis image of the CD63 aptamer-driven DNA hydrogel in Example 1; lane L is a 100 bp gradient standard reference, lane 1 is Y1, lane 2 is Y1+Y2, lane 3 is Y1+Y2+Y3, lane 4 is (L1+L2), lane 5 is CD63-L1, lane 6 is L2, lane 7 is (L1+L2)+CD63-L1, lane 8 is (L1+L2)+CD63-L1+L2, and lane 9 is the annealed sample in lane 3 plus the annealed sample in lane 8;

[0031] Figure 6 This is a circular dichroism characterization result diagram of the exo-sequence connected to the CD63 aptamer in Example 1; the abscissa Angularfrequeney is the angular frequency;

[0032] Figure 7 This is a schematic diagram of the principle of CD63 aptamer-driven DNA hydrogel loaded with GS / exo exosomes promoting skin wound healing in Example 2;

[0033] Figure 8 This is a statistical chart of skin transmittance in different treatment groups within 48 hours in Example 2; the vertical axis "skintransmittance" is the skin transmittance;

[0034] Figure 9 This is a statistical chart of skin retention rates within 48 hours for different treatment groups in Example 2; the vertical axis is skin retention rate;

[0035] Figure 10 This is a statistical chart of transdermal rates of different treatment groups in Example 2 within 48 hours; the vertical axis "transdermalrate" is the transdermal rate;

[0036] Figure 11 This is a diagram showing the skin wound healing status of mice in different treatment groups in Example 2;

[0037] Figure 12 This is a statistical chart of the degree of skin wound healing in mice in different treatment groups in Example 2; the vertical axis Wound area represents the wound area;

[0038] Figure 13 Figure 2 shows the results of VEGFA expression analysis in skin wound tissues of mice in different treatment groups; A is a Western Blot analysis gel; B is a statistical graph of VEGFA expression by Western Blot analysis; the vertical axis is the relative expression of VEGFA; C is a statistical graph of VEGFA expression by RT-qPCR analysis; the vertical axis is the relative mRNA expression;

[0039] Figure 14 Figures 2A and 2B are the results of tissue staining of skin wounds of mice in different treatment groups in Example 2; A is the H&E staining image, and B is the Masson staining image;

[0040] Figure 15 Schematic diagram of the CD63 aptamer-driven DNA hydrogel loaded with exosomes for the treatment of osteoarthritis in Example 3; wherein Col2 is type II collagen and Sox9 is a transcription factor;

[0041] Figure 16 This is a Western blot analysis of the protein levels of anabolic, catabolistic and inflammatory factors in chondrocytes induced by IL-1β in Example 3;

[0042] Figure 17 Figures 2 and 3 are the quantitative analysis results of the Western blotting results in Example 3; Figure A is the quantitative analysis result of MMP13 protein, Figure B is the quantitative analysis result of MMP3 protein, Figure C is the quantitative analysis result of iNOS protein, Figure D is the quantitative analysis result of COX2 protein, Figure E is the quantitative analysis result of COL2 protein, and Figure F is the quantitative analysis result of SOX9 protein;

[0043] Figure 18 This is a graph showing the results of evaluating cell viability using the CCK 8 assay in Example 3;

[0044] Figure 19 Figures are the results of immunohistochemical analysis in Example 3; A is a representative image of immunohistochemical analysis of the expression levels of aggrecan and MMP13 in explants maintained by IL-1β after 72 hours of treatment, and B is a quantitative immunohistochemical analysis. DETAILED DESCRIPTION

[0045] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0046] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0047] The experimental steps involved in the following examples are:

[0048] 1) Masson staining:

[0049] The samples were fixed with 4% paraformaldehyde and dehydrated in a gradient of ethanol (75%, 85%, 90%, 95%, and 100%) for 5 minutes. After dehydration, the samples were cleared with xylene and embedded in paraffin. 4-μm-thick sections were prepared, dried in a 60°C oven for 3 hours, and stored at room temperature until use. The sections were attached to slides and baked at 60°C for 2-3 hours to ensure firm adhesion. The slides were then dewaxed by placing them in xylene I and xylene II for 10 minutes each. Subsequently, the sections were placed in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each to remove the xylene. The sections were then hydrated in a gradient of ethanol (95%, 80%, and 70%) for 3 minutes each and finally rinsed twice with distilled water. The sections were then immersed in hematoxylin solution for 3 minutes to stain the cell nuclei blue. Afterwards, the sections were rinsed with tap water for 10 minutes for bluing. Sections were then placed in a Ponceau acid fuchsin stain for 10 minutes, staining collagen and muscle fibers red. Sections were immersed in a phosphomolybdic acid solution for 3 minutes to fade the acid fuchsin on the collagen fibers, while retaining the red color on muscle fibers. Sections were then placed in an aniline blue stain for 5 minutes, staining the collagen fibers blue. Sections were treated with a 1% glacial acetic acid solution for 1 minute to enhance the staining and brighten the color. Sections were then dehydrated in 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II, each for 5 minutes, and then transparentized in xylene I and xylene II. Finally, the sections were mounted with neutral gum, dried, and observed under a microscope.

[0050] 2) H&E staining:

[0051] The samples were fixed with 4% paraformaldehyde and dehydrated stepwise through ethanol gradient (75%, 85%, 90%, 95%, 100%) for 5 min. After dehydration, the samples were cleared with xylene and embedded in paraffin. 4 μm thick sections were prepared, dried in a 60°C oven for 3 h, and stored at room temperature for later use. When dewaxing the sections, the samples were first immersed in xylene I and II solutions for 5 min each, then hydrated with reverse ethanol gradient (100%, 95%, 80%, 70%) for 5 min. The tissue sections were stained with hematoxylin for 5 min, rinsed with distilled water until there was no chromatin residue in the background, differentiated with 1% hydrochloric acid ethanol solution for 3 s, and quickly rinsed with running water to terminate the reaction. The sections were stained with eosin solution for 3 min, showing a characteristic pink color. After staining, the sections were again stained with ethanol gradient (70%, 80%, 95%, 100%). %), dehydrated for 3 min, and finally transparentized with xylene, mounted with optical resin and covered with a coverslip. After the mounting agent is completely cured, the sample can be prepared for microscopic morphological observation.

[0052] Western blot experiment:

[0053] Lyse the cells or tissues to be tested using RIPA lysis buffer (containing 1% protease inhibitor cocktail), incubate on ice for 30 minutes for complete lysis, separate the total protein by centrifugation, and collect the supernatant; quantify the total protein concentration using a BCA protein assay kit, and plot a standard curve using bovine serum albumin (BSA) as the standard; mix the protein sample with loading buffer (containing β-mercaptoethanol or DTT) and denature in a boiling water bath for 5-10 minutes; perform electrophoresis on a 10% SDS-PAGE gel at a voltage of typically 80-120 V until the bromophenol blue indicator reaches the bottom of the gel; transfer the proteins on the gel to a PVDF membrane (polyvinylidene fluoride membrane) using the wet transfer method; transfer conditions: constant current 200-300 mA for 1 hour, using TBS-T (Tris-buffered saline solution containing 5% skim milk powder, 0.1% The membrane was blocked with Tween-20 at room temperature for 1 hour to reduce nonspecific binding; the membrane was incubated overnight on a shaker at 4°C using the primary antibody diluent (the primary antibody must specifically bind to the target protein); the membrane was washed three times with TBS-T for 5-10 minutes each time to remove unbound primary antibody; the membrane was incubated with an HRP-labeled secondary antibody that matched the species of the primary antibody at room temperature for 45 minutes (the secondary antibody dilution ratio is usually 1:5000-1:20000); the membrane was incubated with a chemiluminescent substrate (such as ECL reagent) and the signal intensity was detected using a chemiluminescent imaging system; an internal control (such as GAPDH) was used to normalize the expression level of the target protein. The grayscale value of the bands was analyzed using ImageJ software to calculate the relative expression of the target protein and the internal control.

[0054] Example 1: Preparation of a CD63 aptamer-driven DNA hydrogel

[0055] S1: Three DNA strands, Y1 (nucleotide sequence shown in SEQ ID NO. 1), Y2 (nucleotide sequence shown in SEQ ID NO. 2), and Y3 (nucleotide sequence shown in SEQ ID NO. 3), were placed in 0.2 M PB buffer at a molar ratio of 1:1:1 and mixed uniformly. The PB buffer had a pH of 7.4 and contained 300 mM sodium chloride to obtain a Y-shaped scaffold.

[0056] S2: The L1+L2 DNA strand (nucleotide sequence shown in SEQ ID NO. 4), the CD63 aptamer (nucleotide sequence shown in SEQ ID NO. 5), and the L2 DNA strand (nucleotide sequence shown in SEQ ID NO. 6) were placed in 0.2 M PB buffer at a molar ratio of 1:1:1 (pH = 7.4, 300 mM sodium chloride) to obtain a linker;

[0057] S3: The Y-shaped scaffold obtained in S1 and the linker obtained in S2 were incubated at 90°C for 10 minutes, and the Y-shaped scaffold and the linker cooled to room temperature were mixed evenly in a volume ratio of 2:3 and allowed to stand to obtain a CD63 aptamer-driven DNA hydrogel. The construction process is as follows: Figure 1 shown.

[0058] Effect experiment:

[0059] In this example, gel electrophoresis was used to characterize the hybridization pattern between DNAs. The CD63 aptamer-driven DNA hydrogel prepared in this example was dissolved in a buffer containing 20 mM Tris-HCl (pH = 7.4), 5 mM MgCl2, and 300 mM NaCl. The final concentration of the DNA hydrogel was 3 M. The gel was incubated at 90°C for 10 minutes and annealed to 25°C at a cooling rate of -1°C / min. The gel was mixed with 2× loading buffer at a ratio of 1:1 and loaded onto a 10% polyacrylamide gel containing 1× TBE buffer and 10 mM sodium chloride. Electrophoresis was performed at 90 V for 55 minutes, stained with SYBR Gold, and the image was scanned on an iBrightFL1000 instrument. Scanning electron microscopy and circular dichroism spectroscopy were performed.

[0060] In this example, DNA hydrogels were synthesized and characterized to effectively load exosomes. Figure 2 As shown, after the L scaffold was introduced, the hydrogel formed a cross-linked structure, and when the container containing the CD63 aptamer-driven DNA hydrogel prepared in this example was inverted at the bottom of the syringe bottle, the hydrogel remained stable.

[0061] like Figure 3 As shown, scanning electron microscopy images at 100 nm and 50 nm magnifications show that the DNA hydrogel prepared in this example exhibits a well-defined three-dimensional network structure with uniform pores.

[0062] like Figure 4 As shown, circular dichroism characterization showed that the exosomes encapsulated by the CD63 aptamer-driven DNA hydrogel (Atp CD63) prepared in this example exhibited a significant shift in the absorption peak compared to that of the exosomes alone, indicating that the aptamer successfully captured the exosomes within the connection.

[0063] like Figure 5 As shown, the results of 10% polyacrylamide gel electrophoresis (PAGE) showed that the migration speed of the 9-mer was slower than that of the linear template, confirming that the template was successfully synthesized.

[0064] like Figure 6 As shown, the rheological characterization of the hydrogel showed that its shear storage modulus (G') was higher than its shear loss modulus (G''), indicating that the hydrogel exhibited typical properties of hydrogels.

[0065] Example 2: Application of CD63 aptamer-driven DNA hydrogel in promoting skin wound healing by loading ginsenoside exosomes

[0066] GS was loaded into the double-layer membrane structure of MSC-exo to form MSC-exo loaded with GS (GS / exo). The CD63 aptamer-driven DNA hydrogel prepared in Example 1 was used to effectively load the above-mentioned ginsenoside-loaded exosomes (GS / exo) to obtain the DNA hydrogel loaded with ginsenoside exosomes. The specific application process is as follows: Figure 7 shown.

[0067] Effect experiment:

[0068] The ex vivo permeation experiment was performed using the Franz diffusion cell method. Using saline as the receiving medium, the skin of the back of mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) was removed and the subcutaneous fat was removed. The skin was then mounted on the system with the stratum corneum facing up and the dermis facing down. The cells were divided into three groups, each consisting of six mouse skin tissues. Free ginsenoside (GS), ginsenoside-loaded exosomes (GS / exo), and CD63 aptamer-driven DNA hydrogel-loaded ginsenoside exosomes (GS / exo+Hyd) prepared in Example 1 were administered at a dose of 200 mg / kg to the top of the skin defined by the upper cells. The cells were sealed and kept in a 37°C water bath with a stirring speed of 300 rpm. At 0.5, 2, 4, 8, 12, 24, 36, and 48 h, 1 mL of culture medium was removed for detection and supplemented with the same volume of fresh culture medium. The collected culture medium was mixed with 0.5 mL of acetonitrile to break the emulsion, centrifuged, and the supernatant was collected. The supernatant was supplemented with methanol to 2 mL and passed through a 0.22 μm filter membrane; the GS content in the filtrate was determined by HPLC, and the skin permeability, skin retention and total permeability (skin permeability + skin retention) were calculated.

[0069] The ex vivo transdermal penetration of free ginsenoside (GS), ginsenoside-loaded exosomes (GS / exo), and CD63 aptamer-driven DNA hydrogel-loaded ginsenoside exosomes (GS / exo+Hyd) was evaluated over a 48-h time span. Figure 8-10 As shown in the data, although the strategy of loading GS into exosomes effectively promoted the skin absorption of GS, the experimental group of the CD63 aptamer-driven DNA hydrogel loaded with ginsenoside exosomes (GS / exo+Hyd) provided by the present invention had the highest skin permeability, skin retention rate and transdermal rate within 48 hours, which were much higher than those of the GS group and the GS / exo group. It can be seen that the CD63 aptamer-driven DNA hydrogel provided by the present invention further enhanced the skin penetration of GS.

[0070] 3. Application of CD63 aptamer-driven DNA hydrogel loaded with ginsenoside exosomes to promote skin wound healing in diabetic patients

[0071] In this example, in order to verify the in vivo healing properties of the CD63 aptamer-driven DNA hydrogel prepared in Example 1, a full-thickness skin defect mouse model was constructed. Six-week-old ICR mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were acclimated for 7 days, fasted for 12 hours, and then intraperitoneally injected with streptozotocin (STZ) at a dose of 150 mg / kg. On the 7th day, blood glucose levels were randomly measured, and successful induction of diabetes was defined as a random blood glucose level of 16.7 mmol / L. Thirty-six successfully induced diabetic mice were randomly divided into six groups: Con, GS, MSC-exo, GS / exo, CD63 aptamer-driven DNA hydrogel prepared in Example 1 (Hydrogel), and CD63 aptamer-driven DNA hydrogel loaded with ginsenoside exosomes (GS / exo+Hyd) prepared in Example 1. The backs of the mice were shaved and disinfected, and circular skin excision wounds (diameter 6 mm) were created. To minimize the effects of skin contraction on wound healing, an inner diameter rubber ring and skin adhesive were used to secure the wound site. Each group was given a different treatment, and wound images were taken on days 0, 7, and 14 after treatment. Wound area (mm²) and healing rate were quantified and analyzed using ImageJ software for statistical analysis. On days 7 and 14, wound tissues were collected for H&E and Masson staining. Skin tissues were lysed, and protein extracts were collected for Western blot analysis. Experiments were performed in triplicate to ensure reliability and reproducibility, and the results were statistically analyzed using GraphPad Prism.

[0072] like Figure 11 As shown, on day 14, the wound area of ​​the skin wounds in the Hydrogel group was significantly reduced, with epithelial tissue covering the tumor skin and no obvious scar formation. However, the wound area of ​​the skin wounds in the Con, GS, MSC-exo, and GS / exo groups was not completely healed, and the red, exposed wound in the center was still not covered by the tumor epithelium. The skin wounds of the mice in the GS / exo+Hyd group were almost completely healed, with new skin covering the original open wound, no scar tissue on the surface, and surrounding hair regeneration.

[0073] like Figure 12 As shown in the data, the wound contraction rates of the skin wounds of mice in the GS / exo+Hyd group were 75% and 90% after 7 and 14 days of treatment, respectively. It can be seen that the area of ​​the skin wounds was significantly reduced, indicating that the CD63 aptamer-driven DNA hydrogel provided by the present invention can effectively promote wound healing and has potential application prospects in wound dressings.

[0074] like Figure 13As shown, Western blot analysis results revealed a significant upregulation of VEGF expression in the drug-loaded hydrogel group of the GS / exo+Hyd group, which was further confirmed by RT-qPCR detection results.

[0075] like Figure 14 As shown, the results of H&E and Masson staining showed that on the 7th day, the skin tissues all showed varying degrees of inflammatory response. Compared with the control group, the infiltration of inflammatory cells in the skin tissues of the GS, MSC-exo, GS / exo and GS / exo+Hyd groups was reduced, among which the skin tissues of the GS / exo+Hyd group showed the most significant healing. On the 14th day, granulation tissue formation and neovascularization were observed in all experimental groups, indicating that wound repair had entered the remodeling stage; however, it is worth noting that the GS / exo+Hyd group showed a higher density of fibroblasts, well-arranged tumor tissue and a thinner epidermal layer. It can be seen that the CD63 aptamer-driven DNA hydrogel provided by the present invention has good biocompatibility, antimicrobial and antioxidant properties, provides a favorable microenvironment for wound repair, and thus effectively promotes wound healing.

[0076] In summary, the CD63 aptamer-driven DNA hydrogel provided by the present invention not only improves the poor skin permeability of GS, but also achieves a synergistic effect between GS and MSC-exo in wound repair in diabetic patients (DM). It also prolongs the residence time of GS / exo in the wound and increases the bioavailability of GS / exo. The CD63 aptamer specifically binds to the CD63 protein on the surface of exosomes, achieving efficient anchoring and loading, providing a favorable microenvironment for wound repair, and thus effectively promoting wound healing.

[0077] Example 3: Application of CD63 aptamer-driven DNA hydrogel in the treatment of osteoarthritis

[0078] Bone marrow mesenchymal stem cell exosomes (BMSC) have the potential to treat bone defects. The DNA hydrogel prepared in Example 1 was used to effectively load BMSC (BMSC@Hyd experimental group), prolonging the residence time of BMSC in the bone defect and increasing the bioavailability of BMSC. Specifically, the CD63 aptamer-driven DNA hydrogel prepared in Example 1 carrying BMSC exosomes was injected. The process is as follows. Figure 15 shown.

[0079] Effect experiment:

[0080] 1. Western Blot Analysis

[0081] Cells were scraped off with a cell wiper and mixed with radioimmunoprecipitation assay buffer (RIPA) containing phosphatase inhibitors and protease inhibitors in a 100:1:1 mass ratio, and cell lysates were collected. The cell lysate was treated with an ultrasonic disruptor and centrifuged at 12,000 rpm for 30 min at 4°C. After centrifugation, the supernatant was collected and loaded with loading buffer at a ratio of 4:1, refrigerated for 5 min, and denatured at 95°C for 5 min. The total protein was then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (8.0-12.5%) and transferred to a polyvinylidene difluoride membrane. The unbound sites on the membrane were blocked with 5% bovine serum albumin for 1 hour, the target band was cut from the membrane and incubated with a specific primary antibody at 4°C overnight, and the membrane was washed three times with tris-buffered saline containing 0.1% Tween 20 for 15 minutes, incubated with a specific secondary antibody at room temperature for 1 hour, and washed again three times with tris-buffered saline containing 0.1% Tween 20 for 15 minutes. Western blot was used. Western blots were developed using an ECL substrate kit (purchased from Thermo Fisher Scientific, USA) and a Bio-Rad scanner, and the intensity of the bands was quantified using digital image analysis software.

[0082] like Figure 16-17 As shown in the figure, the results of Western blot analysis of the protein levels of anabolic, catabolic and inflammatory factors in IL-1β-induced chondrocytes showed that after 48 hours of treatment with BMSC@Hyd (DNA hydrogel-loaded BMSC exosomes prepared in Example 1) loaded with 0.5, 1.0, 2.5 and 5.0 μM LRRK 2-IN-1, BMSC@Hyd promoted the expression of COL 2 and SOX 9 in a dose-dependent manner and inhibited the protein levels of iNOS, COX 2, MMP 3 and MMP 13. The upregulation of catabolic and inflammatory factors and the downregulation of anabolic factors induced by IL-1β in chondrocytes were reversed in an LRRK 2-IN-1-dependent manner.

[0083] 2. In vitro cytotoxicity assay

[0084] The in vitro cytotoxicity of LRRK 2-IN-1 was measured using a cell counting kit-8 (CCK 8, purchased from Solebro Reagents). Briefly, chondrocytes were cultured in 96-well plates (5000-10,000 cells / well) for 24 hours and then treated with BMSC@Hyd containing LRRK 2-IN-1 for 24 hours. CCK 8 reagent was added to the plate, and the absorbance value at a wavelength of 450 nm was detected using a microplate reader.

[0085] like Figure 18As shown, no toxicity was observed in the cells after 24 h of treatment with BMSC@Hyd, indicating that the chondrocytes remained in good condition throughout the 48 h culture period.

[0086] It can be seen that the CD63 aptamer-driven DNA hydrogel-loaded BMSC exosomes (BMSC@Hyd) provided by the present invention improves the chondrocyte targeting effect, has a significant effect in promoting chondrocyte growth, inhibiting chondrocyte proliferation and inflammation, and does not inhibit the vitality of chondrocytes.

[0087] 3. Quantitative Immunohistochemical Analysis

[0088] Paraffin-embedded sections of decalcified mouse cartilage tissue (BMSC@Hyd-treated for 72 hours and mouse cartilage tissue without BMSC@Hyd) were dewaxed and blocked with 5% bovine serum albumin for 1 hour. They were then incubated with primary antibodies against the anabolic factor aggrecan and the catabolic factor MMP13 at 4°C overnight; the sections were incubated with secondary antibodies, developed, and observed under a microscope. A similar immunohistochemical analysis protocol was performed on human OA cartilage samples to analyze the expression of aggrecan and MMP13.

[0089] like Figure 19 As shown in Figure 3, immunohistochemical analysis showed that BMSC@Hyd treatment effectively increased the expression of aggrecan and inhibited the expression of MMP13 in human bone defect cartilage after 72 h, indicating that BMSC@Hyd has certain potential in the treatment of human bone defects.

[0090] In summary, the CD63 aptamer-driven DNA hydrogel provided by the present invention can load BMSC exosomes. The CD63 aptamer specifically binds to the CD63 protein on the surface of exosomes to achieve efficient anchoring and loading. The DNA hydrogel can be applied to a bone defect mouse model by injection to promote bone tissue regeneration.

[0091] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for preparing a CD63 aptamer-driven DNA hydrogel, characterized in that: The preparation method comprises the following steps: S1: The three DNA chains Y1, Y2, and Y3 were placed in 0.2 M PB buffer at a molar ratio of 1:1:1 and mixed to obtain a Y-shaped scaffold; the PB buffer was pH 7.4 and 300 mM sodium chloride; S2: The L1+L2 DNA strand, CD63 aptamer, and L2 DNA strand were placed in 0.2 M PB buffer at a molar ratio of 1:1:1 and mixed to obtain a linker; the PB buffer had a pH of 7.4 and contained 300 mM sodium chloride. S3: The Y-shaped scaffold obtained in S1 and the linker obtained in S2 were incubated at 90°C for 10 minutes, respectively. The Y-shaped scaffold and the linker cooled to room temperature were mixed evenly in a volume ratio of 2:3 and allowed to stand to obtain a CD63 aptamer-driven DNA hydrogel. The nucleotide sequence of the Y1 DNA strand in S1 is shown in SEQ ID NO.1; The nucleotide sequence of the Y2 DNA strand in S1 is shown in SEQ ID NO. 2; The nucleotide sequence of the Y3 DNA strand in S1 is shown in SEQ ID NO. 3; The nucleotide sequence of the L1+L2 DNA strand in S2 is shown in SEQ ID NO.4; The nucleotide sequence of the CD63 aptamer in S2 is shown in SEQ ID NO.5; The nucleotide sequence of the L2 DNA chain in S2 is shown in SEQ ID NO.

6.

2. A CD63 aptamer-driven DNA hydrogel, characterized in that: The DNA hydrogel is obtained by the preparation method according to claim 1.

3. Use of the CD63 aptamer-driven DNA hydrogel according to claim 2 in the preparation of a drug for promoting skin wound healing by loading exosomes.

4. Use of the CD63 aptamer-driven DNA hydrogel according to claim 2 in loading exosomes to prepare a drug for treating osteoarthritis.

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