CD63 aptamer driven DNA hydrogel as well as preparation method and application thereof

Abstract

The invention discloses CD63 aptamer driven DNA hydrogel as well as a preparation method and application thereof, and belongs to the technical field of hydrogel materials. In order to solve the technical problems of relatively low delivery efficiency and targeting property, short drug retention and relatively poor permeability of hydrogel in the prior art, the invention provides a CD63 aptamer driven type DNA hydrogel, the DNA hydrogel is composed of three Y-type DNA chains, L1 + L2 (SEQ ID NO.4), a CD63 aptamer (SEQ ID NO.5) and L2 (SEQ ID NO.6), and the nucleotide sequences of the three Y-type DNA chains are shown as SEQ ID NO.1-3. The DNA hydrogel provided by the invention can efficiently load the exosome, has good biocompatibility and mechanical properties, and reduces side effects caused by overlarge local drug concentration; the exosome-loaded hydrogel can be applied to exosome-loaded promotion of skin wound healing and treatment of osteoarthritis.

Description

Technical Field

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

[0002] A hydrogel is a three-dimensional hydrophilic polymer network that can be used as a drug delivery system to provide a moist treatment environment, promote tissue growth, and control 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, or heat-responsive hydrogels. Among them, gelatin methacryloyl has problems such as low mechanical properties of the hydrogel, poor controllable degradability, resulting in poor retention ability and activity of the loaded exosomes, and large viscosity of the gelatin methacryloyl hydrogel; thermosensitive hydrogels have problems such as poor mechanical properties, insufficient biocompatibility, slow response speed, difficulty in drug controlled release, and uncontrollable stability and degradation; heat-responsive hydrogels change in performance when the temperature is greater than the lower critical solution temperature (LCST), resulting in obvious sol-gel transitions and self-shrinking properties.

[0003] Therefore, those skilled in the art are eager to develop a hydrogel with high delivery efficiency and targeting, and capable of stably releasing 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 residence time, and poor permeability of hydrogels in the prior art, the present invention provides a CD63 aptamer-driven DNA hydrogel, a preparation method thereof, and an application thereof.

[0005] One of the purposes of the present invention is to provide a preparation method of a CD63 aptamer-driven DNA hydrogel, and the preparation method includes the following steps: S1: Mix three DNA strands Y1, Y2, and Y3 evenly in a 0.2 M PB buffer solution according to a molar ratio of 1:1:1 to obtain a Y-shaped scaffold; the PB buffer solution is pH = 7.4 and 300 mM sodium chloride; S2: Mix the L1 + L2 DNA strand, the CD63 aptamer, and the L2 DNA strand evenly in a 0.2 M PB buffer solution according to a molar ratio of 1:1:1 to obtain a linker; the PB buffer solution is pH = 7.4 and 300 mM sodium chloride; S3: Incubate the Y-shaped scaffold obtained in S1 and the linker obtained in S2 at 90 °C for 10 minutes respectively. Mix the cooled-to-room-temperature Y-shaped scaffold and linker evenly at a volume ratio of 2:3, and let it stand to obtain the CD63 aptamer-driven DNA hydrogel.

[0006] In a preferred embodiment of the present invention, the nucleotide sequence of the Y1 DNA strand in S1 is shown as SEQ ID NO.1.

[0007] In a preferred embodiment of the present invention, the nucleotide sequence of the Y2 DNA strand in S1 is shown as SEQ ID NO.2.

[0008] In a preferred embodiment of the present invention, the nucleotide sequence of the Y3 DNA strand in S1 is shown as SEQ ID NO.3.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the L1+L2 DNA strand in S2 is shown as SEQ ID NO.4.

[0010] In a preferred embodiment of the present invention, the nucleotide sequence of the CD63 aptamer in S2 is shown as SEQ ID NO.5.

[0011] In a preferred embodiment of the present invention, the nucleotide sequence of the L2 DNA strand in S2 is shown as SEQ ID NO.6.

[0012] The second object of the present invention is to provide a CD63 aptamer-driven DNA hydrogel, which is obtained by the above preparation method.

[0013] The third object of the present invention is to provide the application of the above CD63 aptamer-driven DNA hydrogel in loading exosomes to promote skin wound healing.

[0014] The fourth object of the present invention is to provide the application of the above CD63 aptamer-driven DNA hydrogel in the treatment of osteoarthritis, and the application is carried out by injecting the CD63 aptamer-driven DNA hydrogel carrying exosomes.

[0015] The beneficial effects of the present invention: The present invention provides a CD63 aptamer-driven DNA hydrogel, which is composed of three Y-shaped DNA strands with nucleotide sequences shown as SEQ ID NOs.1-3, and L1+L2 (SEQ ID NO.4), CD63 aptamer (SEQ ID NO.5) and L2 (SEQ ID NO.6).

[0016] The DNA hydrogel prepared by the present invention is different from traditional linear DNA crosslinking. The designed Y-shaped branches provide multiple terminal complementary sequences, which can bind to each other through base complementary pairing to form a stable structure. It can also perform multi-point binding with the linker chain (L-shaped single-stranded DNA) through base complementary pairing, forming a three-dimensional network crosslinked structure to reduce the random entanglement of molecular chains, improve the mechanical strength of the hydrogel, and is suitable for long-term drug loading.

[0017] L1 and L2 are linear single-stranded DNAs, which bind to the ends of the Y-shaped scaffold through complementary pairing to form a "Y-L" crosslinking node and construct the main framework of the hydrogel; 1) The L1+L2 combination can extend the network length, making the porosity and drug release rate of the hydrogel meet the requirements; The CD63 aptamer (AptCD63) is embedded in the CD63-L1 chain, which can specifically recognize the CD63 protein on the surface of exosomes, ensuring that exosomes can be efficiently anchored in the hydrogel; 2) The L2 chain serves as a general linker unit, participating in both the L1+L2 pairing and binding to CD63-L1, increasing the crosslinking point density and improving the mechanical properties of the gel; By mixing L2 with CD63-L1, it ensures uniform distribution of aptamers and avoids uneven local drug loading; 3) The CD63 aptamer is a single-stranded DNA, which forms a specific three-dimensional conformation through folding and binds to a specific epitope of the CD63 protein on the surface of exosomes, endowing the DNA hydrogel with the ability to specifically target exosomes, precisely capturing and fixing exosomes, improving the delivery efficiency and targeting, and avoiding the leakage problem of traditional physical encapsulation.

[0018] Since the base pairing and double helix structure of DNA are most stable at neutral pH (about 7.4), the present invention selects to use 0.2 M PB (pH 7.4, 300 mM sodium chloride) buffer solution, enabling the DNA strands to perform better base complementary pairing and preventing hydrolysis or denaturation of the DNA strands caused by acidic or alkaline conditions; In addition, the ionic strength in the 0.2 M PB buffer solution can accelerate the dynamic hybridization process of the DNA strands, enabling the Y-shaped scaffold and the linker chain to quickly form a three-dimensional network. The slow-release characteristics endowed by the three-dimensional network structure and uniform pores of the hydrogel can stably release the loaded exosomes for a long time.

[0019] 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 linker bindings, which can not only form a stable three-dimensional network but also avoid excessive crosslinking resulting in gel brittleness or too small pore size.

[0020] 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 slow-release effect can reduce the side effects caused by excessive local drug concentration. It can not only load exosomes to promote wound healing and provide a new strategy for the clinical treatment of chronic wounds, but also treat osteoarthritis by injecting the CD63 aptamer-driven DNA hydrogel carrying exosomes.

[0021] The CD63 aptamer-driven DNA hydrogel provided by the present invention solves the technical problems of traditional drug delivery systems being easily and rapidly cleared at the wound site, insufficient local drug concentration, and poor exosome permeability caused by the skin barrier. Compared with single therapies, the CD63 aptamer-driven DNA hydrogel provided by the present invention not only overcomes the limitations of difficult preservation of exosomes in vivo and insufficient mechanical properties of traditional carriers, but also realizes precise drug loading and controllable release through sequence programmability, forming a uniform drug distribution while maintaining the stability of active ingredients. Description of the Drawings

[0022] In the following drawings, Apt CD63 represents the CD63 aptamer, Y scaffold represents the Y-shaped scaffold, GS / exo represents exosomes loaded with ginsenosides, Hyd and Hydrogel both represent hydrogels, GS represents ginsenosides, and exo represents exosomes.

[0023] Figure 1 It is a schematic diagram for constructing the CD63 aptamer-driven DNA hydrogel in Example 1; Figure 2 It is a state diagram of the CD63 aptamer-driven DNA hydrogel in Example 1; where Gel is the gel and Solution is the solution; Figure 3 It is an SEM image of the CD63 aptamer-driven DNA hydrogel in Example 1; Figure 4 It is a rheological analysis diagram 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; Figure 5 It is a non-denaturing polyacrylamide gel electrophoresis diagram of the CD63 aptamer-driven DNA hydrogel in Example 1; the L lane is the 100 bp ladder standard reference, the 1st lane is Y1, the 2nd lane is Y1+Y2, the 3rd lane is Y1+Y2+Y3, the 4th lane is (L1+L2), the 5th lane is CD63-L1, the 6th lane is L2, the 7th lane is (L1+L2)+CD63-L1, the 8th lane is (L1+L2)+CD63-L1+L2, and the 9th lane is the annealed sample of the 3rd lane + the annealed sample of the 8th lane; Figure 6 CD63 aptamer-linked exonuclease circular dichroism characterization results in Example 1; the abscissa Angularfrequeney is the angular frequency; Figure 7 Schematic diagram of CD63 aptamer-driven DNA hydrogel loaded with GS / exo exosomes promoting skin wound healing in Example 2; Figure 8 Statistical chart of skin transmittance of different treatment groups within 48 hours in Example 2; the ordinate skintransmittance is the skin transmittance; Figure 9 Statistical chart of skin retention rate of different treatment groups within 48 hours in Example 2; the ordinate skinretention is the skin retention rate; Figure 10 Statistical chart of transdermal rate of different treatment groups within 48 hours in Example 2; the ordinate transdermalrate is the transdermal rate; Figure 11 Diagram of the skin wound healing of mice in different treatment groups in Example 2; Figure 12 Statistical chart of the skin wound healing degree of mice in different treatment groups in Example 2; the ordinate Wound area is the wound area; Figure 13 Diagram of the VEGFA expression results of skin wound tissue analysis of mice in different treatment groups in Example 2; A is the gel diagram of Western Blot analysis; B is the statistical chart of VEGFA expression in Western Blot analysis; the ordinate Therelative expression of VEGFA is the relative expression level of VEGFA; C is the statistical chart of VEGFA expression detected by RT-qPCR analysis; the ordinate Relative mRNA expression is the relative mRNA expression level; Figure 14 Diagram of the staining results of skin wound tissue of mice in different treatment groups in Example 2; A is the H&E staining diagram, and B is the Masson staining diagram; Figure 15 Schematic diagram of CD63 aptamer-driven DNA hydrogel loaded with exosomes for the treatment of osteoarthritis in Example 3; where Col2 is type II collagen and Sox9 is a transcription factor; Figure 16 Western blot analysis diagram of the protein levels of anabolism, catabolism, and inflammatory factors in IL-1β-induced chondrocytes in Example 3; Figure 17Quantitative analysis result graph of the Western blot results in Example 3; A is the quantitative analysis result graph of MMP13 protein, B is the quantitative analysis result graph of MMP3 protein, C is the quantitative analysis result graph of iNOS protein, D is the quantitative analysis result graph of COX2 protein, E is the quantitative analysis result graph of COL2 protein, and F is the quantitative analysis result graph of SOX9 protein; Figure 18 Result graph of evaluating cell viability by CCK-8 assay in Example 3; Figure 19 Result graph 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 graph of immunohistochemical quantitative analysis. Detailed implementation manners

[0024] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Those related can obviously make changes or appropriate alterations and combinations to 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.

[0025] In order to make the purpose, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific implementation manners. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0026] The experimental steps involved in the following examples: 1) Masson staining: Fix the sample to be tested with 4% paraformaldehyde, and dehydrate it step by step through gradient ethanol (75%, 85%, 90%, 95%, 100%) for 5 min; after the dehydration process is completed, the sample is treated with xylene for transparency and then embedded in paraffin. Prepare a 4-μm-thick section, dry it in an oven at 60 °C for 3 h, and store it at room temperature for later use; attach the section to a glass slide, bake the slide at 60 °C for 2 - 3 h to firmly attach the section. Then, place the glass slide in xylene I and xylene II for 10 min each for dewaxing treatment; subsequently, place the section in absolute ethanol I and absolute ethanol II for 5 min each to remove xylene; then place the section in 95%, 80%, 70% gradient ethanol for 3 min each for hydration, and finally rinse it with distilled water twice; immerse the section in hematoxylin stain for 3 minutes to stain the cell nuclei blue. Then rinse it with tap water for 10 min for bluing; place the section in ponceau acid fuchsin stain for 10 min to stain collagen fibers, muscle fibers, etc. red; immerse the section in phosphomolybdic acid solution for 3 min to fade the acid fuchsin on the collagen fibers, while the muscle fibers, etc. still retain red; transfer the section to aniline blue stain for 5 min to stain the collagen fibers blue; treat the section with 1% acetic acid aqueous solution for 1 min to enhance the staining effect and make the color more vivid. The section is dehydrated through 95% ethanol I, 95% ethanol II, absolute ethanol I, and absolute ethanol II in sequence, 5 min for each level, and then placed in xylene I and xylene II for transparency in sequence; finally, seal the section with neutral balsam, and observe it under a microscope after drying.

[0027] 2) H&E staining: Fix the sample to be tested with 4% paraformaldehyde, and dehydrate it step by step through gradient ethanol (75%, 85%, 90%, 95%, 100%) for 5 min; after the dehydration process is completed, the sample is treated with xylene for transparency and then embedded in paraffin. Prepare a 4-μm-thick section, dry it in an oven at 60 °C for 3 h, and store it at room temperature for later use; when dewaxing the section, first immerse the sample in xylene I and II solutions for 5 min each, and then hydrate it with reverse gradient ethanol (100%, 95%, 80%, 70%) for 5 min. Stain the tissue section with hematoxylin for 5 min, rinse it with distilled water until there is no chromatin residue in the background, differentiate it with 1% hydrochloric acid ethanol solution for 3 s, quickly rinse it with running water to terminate the reaction, and stain it with eosin stain for 3 min to present a characteristic pink color; after the staining is completed, the section is dehydrated again through gradient ethanol (70%, 80%, 95%, 100%) for 3 min, and finally treated with xylene for transparency, sealed with optical resin and covered with a coverslip. After the sealant is completely cured, the prepared sample can be used for microscopic morphological observation.

[0028] Western blot experiment: Lyse the cells or tissues to be tested with RIPA lysis buffer (containing 1% protease inhibitor mixture), incubate on ice for 30 minutes for sufficient lysis, separate the total protein by centrifugation, and collect the supernatant; use a BCA protein assay kit to quantify the total protein concentration, and draw a standard curve with bovine serum albumin (BSA) as the standard; mix the protein sample with the loading buffer (containing β-mercaptoethanol or DTT), and denature it in a boiling water bath for 5-10 minutes; perform electrophoresis using a 10% SDS-PAGE gel, usually at a voltage of 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 membrane conditions: constant current of 200-300 mA for 1 hour, block at room temperature for 1 hour with TBS-T (Tris-buffered saline solution containing 0.1% Tween-20) containing 5% skim milk powder to reduce non-specific binding; incubate overnight on a shaker at 4°C with the primary antibody dilution solution, and the primary antibody needs to specifically bind to the target protein; wash the membrane 3 times with TBS-T, 5-10 minutes each time, to remove the unbound primary antibody; incubate at room temperature for 45 minutes with an HRP-labeled secondary antibody that is species-matched to the primary antibody, and the dilution ratio of the secondary antibody is usually 1:5000-1:20000, incubate the membrane with a chemiluminescent substrate (such as ECL reagent), and detect the signal intensity through a chemiluminescent imaging system; an internal reference (such as GAPDH) is used to normalize the expression level of the target protein, and the gray value of the band is analyzed using ImageJ software to calculate the relative expression of the target protein to the internal reference.

[0029] Example 1: Preparation of a CD63 aptamer-driven DNA hydrogel S1: Place 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), in 0.2 M PB buffer and mix evenly according to a molar ratio of 1:1:1. The PB buffer has a pH of 7.4 and 300 mM sodium chloride to obtain a Y-shaped scaffold; S2: Place 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) in 0.2 M PB buffer and mix evenly according to a molar ratio of 1:1:1. The PB buffer has a pH of 7.4 and 300 mM sodium chloride to obtain a linker; S3: Incubate the Y-shaped scaffold obtained in S1 and the linker obtained in S2 at 90 °C for 10 minutes respectively. Mix the above-mentioned Y-shaped scaffold and linker cooled to room temperature evenly at a volume ratio of 2:3, and let it stand to obtain the CD63 aptamer-driven DNA hydrogel. The construction process is as Figure 1 shown.

[0030] Effect experiment: In this example, gel electrophoresis technology was used to characterize the hybridization form 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, incubated at 90 °C for 10 minutes, and annealed to 25 °C at a cooling rate of -1 °C / min; 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, and electrophoresed at 90 V for 55 minutes, stained with SYBR Gold, and scanned the image on an iBright FL1000 instrument. Scanning electron microscopy and circular dichroism spectroscopy analysis were performed.

[0031] In this example, in order to effectively load exosomes, a DNA hydrogel was synthesized and characterized; as Figure 2 shown, after introducing the L scaffold, the hydrogel formed a cross-linked structure, and when the container filled with the CD63 aptamer-driven DNA hydrogel prepared in this example was inverted at the bottom of the syringe bottle, the hydrogel still remained stable.

[0032] As Figure 3 shown, scanning electron microscope images at magnifications of 100 nm and 50 nm show that the DNA hydrogel prepared in this example exhibits a well-defined three-dimensional network structure with uniform pores.

[0033] As Figure 4 shown, circular dichroism spectroscopy characterization shows that compared with exosomes alone (exo), the exosomes encapsulated by the CD63 aptamer-driven DNA hydrogel (Atp CD63) prepared in this example show a significant shift in the absorption peak, indicating that the aptamer successfully captured the exosomes inside the linker.

[0034] As Figure 5 shown, the results of 10% polyacrylamide gel electrophoresis (PAGE) show that the migration speed of 9-mer is slower than that of the linear template, confirming the successful synthesis of the template.

[0035] As Figure 6As shown, the rheological characterization of the hydrogel shows that its shear storage modulus (G') is higher than its shear loss modulus (G''), indicating that the hydrogel exhibits typical properties of a hydrogel.

[0036] Example 2: Application of CD63 aptamer-driven DNA hydrogel in promoting skin wound healing by loading ginsenoside exosomes Ginsenoside (GS) was loaded into the bilayer 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 a DNA hydrogel loaded with ginsenoside exosomes. The specific application process is as Figure 7 shown.

[0037] Effect experiment: An in vitro permeation experiment was carried out using the Franz diffusion cell method. Saline was used as the receiving medium. The skin on the back of mice (purchased from: Liaoning Changsheng Biotechnology Co., Ltd.) was removed, and the subcutaneous fat was removed; then the skin was installed on the system with the stratum corneum facing up and the dermis facing down; it was divided into 3 groups, with 6 pieces of mouse skin tissue in each group. Free ginsenoside (GS), ginsenoside-loaded exosomes (GS / exo), and CD63 aptamer-driven DNA hydrogel loaded with ginsenoside exosomes (GS / exo+Hyd) prepared in Example 1 were applied to the top of the skin defined by the upper cells at a dose of 200 mg / kg; sealed and maintained 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 the culture medium was taken out for detection, and the same volume of fresh culture medium was added. The collected culture medium was mixed with 0.5 mL of acetonitrile to break the emulsion, centrifuged, and the supernatant was taken; the supernatant was supplemented to 2 mL with methanol and passed through a 0.22 μm filter membrane; the content of GS in the filtrate was determined by HPLC, and the skin permeability, skin retention, and total permeability (skin permeability + skin retention) were calculated.

[0038] The in vitro percutaneous permeation of free ginsenoside (GS), ginsenoside-loaded exosomes (GS / exo), and CD63 aptamer-driven DNA hydrogel loaded with ginsenoside exosomes (GS / exo+Hyd) was evaluated over a 48-hour time span, as Figures 8 - 10As shown, although the strategy of loading GS into exosomes effectively promoted the skin absorption of GS, the experimental group of 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 the 48-hour period, far 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.

[0039] 3. Application of CD63 Aptamer-Driven DNA Hydrogel in Promoting Skin Wound Healing of Diabetic Patients by Loading Ginsenoside Exosomes In this example, in order to verify the wound-healing promoting characteristics in vivo of the CD63 aptamer-driven DNA hydrogel prepared in Example 1, a mouse model with full-thickness skin defects was constructed; 6-week-old ICR mice (purchased from: Liaoning Changsheng Biotechnology Co., Ltd.) were acclimated for 7 days, fasted for 12 hours, and then streptozotocin (STZ) was intraperitoneally injected at a dose of 150 mg / kg; on the 7th day, the blood glucose level was randomly measured, and successful induction of diabetes was defined as a random blood glucose level of 16.7 mmol / L; 36 successfully induced diabetic mice were randomly divided into 6 groups: Con, GS, MSC-exo, GS / exo, the CD63 aptamer-driven DNA hydrogel prepared in Example 1 (Hydrogel), and the CD63 aptamer-driven DNA hydrogel loaded with ginsenoside exosomes prepared in Example 1 (GS / exo+Hyd); the backs of the mice were shaved and disinfected, and circular skin excision wounds (6 mm in diameter) were created. To minimize the effect of skin contraction on wound healing, an inner diameter rubber ring and skin adhesive were used to fix the wound site. Different treatments were given to each group, and wound images were taken on days 0, 7, and 14 after treatment; the 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; the skin tissues were lysed, and protein extracts were collected for Western blot analysis. The experiments were performed in triplicate to ensure reliability and reproducibility, and the results were statistically analyzed using GraphPad Prism.

[0040] As Figure 11As shown, on the 14th day, the wound area of the skin wound in the Hydrogel group mice was significantly reduced, the epithelial tissue covered the tumor skin, and no obvious scar formation was observed; while in the Con, GS, MSC-exo, and GS / exo group mice, the wound area of the skin wound did not completely heal, and the red exposed wound in the center was still not covered by the tumor epithelium. In the mice of the GS / exo+Hyd group, the skin wound was almost completely healed, the new skin covered the original open wound, there was no scar tissue on the surface, and hair regeneration occurred around.

[0041] As Figure 12 shown, the wound contraction rates of the skin wounds in the GS / exo+Hyd group mice were 75% and 90% respectively after 7 days and 14 days of treatment. It can be seen that the area of the skin wound 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.

[0042] As Figure 13 shown, the results of Western blot analysis revealed a significant up-regulation of VEGF expression in the drug-loaded hydrogel group of the GS / exo+Hyd group, and it was further proved by the results of RT-qPCR detection.

[0043] As Figure 14 shown, the results of H&E and Masson staining showed that on the 7th day, the skin tissues all showed varying degrees of inflammatory reactions. 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 decreased. Among them, the skin tissue of the GS / exo+Hyd group showed the most significant healing. On the 14th day, granulation tissue formation and new blood vessel formation were visible in each experimental group, indicating that the wound repair had entered the remodeling stage; but it should be noted that the GS / exo+Hyd group showed a higher density of fibroblasts, well-arranged tumor tissues, and a thinner epidermal layer. It can be seen that the CD63 aptamer-driven DNA hydrogel provided by the present invention has good biocompatibility, anti-microbial and anti-oxidant properties, provides a favorable microenvironment for wound repair, and thus effectively promotes wound healing.

[0044] In summary, the CD63 aptamer-driven DNA hydrogel provided by the present invention not only improves the poor skin permeability of GS, but also realizes the synergistic effect of GS and MSC-exo in wound repair under the diabetic (DM) state; prolongs the residence time of GS / exo at the wound site and increases the bioavailability of GS / exo; the CD63 aptamer specifically binds to the CD63 protein on the surface of exosomes to achieve efficient anchoring and loading, provides a favorable microenvironment for wound repair, and thus effectively promotes wound healing.

[0045] Example 3: Application of CD63 Aptamer-Driven DNA Hydrogel in the Treatment of Osteoarthritis 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), prolong the residence time of BMSC at the bone defect site, and increase the bioavailability of BMSC. Specifically, by injecting the CD63 aptamer-driven DNA hydrogel prepared in Example 1 carrying BMSC exosomes, the process is as Figure 15 shown.

[0046] Effect experiment: 1. Western blot analysis Cells were scraped off using a cell scraper, and the cells were mixed with radioimmunoprecipitation assay buffer (RIPA) containing phosphatase inhibitors and protease inhibitors supplied at a mass ratio of 100:1:1, and cell lysates were collected; the cell lysates were treated with an ultrasonic crusher and centrifuged at 12,000 rpm for 30 min at 4 °C. After centrifugation, the supernatant was collected, and loading buffer was added at a ratio of 4:1, refrigerated for 5 min, and denatured at 95 °C for 5 min; then, the total protein was 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 h. The targeted bands were cut from the membrane and incubated with specific primary antibodies overnight at 4 °C, and the membrane was rinsed three times for 15 min with tris-buffered saline containing 0.1% Tween 20, incubated with specific secondary antibodies at room temperature for 1 h, and washed three times again with tris-buffered saline containing 0.1% Tween 20 for 15 min; The Western blot was developed using a Western 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.

[0047] As Figures 16 - 17 shown, the results of Western blot analysis of the protein levels of anabolic, catabolic, and inflammatory factors in IL-1β-induced chondrocytes showed that after treatment with BMSC@Hyd (DNA hydrogel prepared in Example 1 loaded with BMSC exosomes) loaded with 0.5, 1.0, 2.5, and 5.0 μM LRRK 2-IN-1 for 48 h, 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 a LRRK 2-IN-1-dependent manner.

[0048] 2. In vitro cytotoxicity assay The in vitro cytotoxicity of LRRK 2-IN-1 was measured using a Cell Counting Kit-8 (CCK 8, purchased from: Solarbio Science & Technology Co., Ltd.). Briefly, chondrocytes were cultured in a 96-well plate (5000 - 10,000 cells / well) for 24 hours, then treated with BMSC@Hyd of LRRK 2-IN-1 for 24 hours. The CCK 8 reagent was added to the plate, and the absorbance value at a wavelength of 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0049] As Figure 18 shown, no toxicity was observed in the cells after treatment with BMSC@Hyd for 24 hours, indicating that chondrocytes remained in good condition throughout the 48-hour culture period.

[0050] It can be seen that the CD63 aptamer-driven DNA hydrogel loaded with BMSC exosomes (BMSC@Hyd) provided by the present invention improves the targeting effect on chondrocytes, has a significant effect in promoting chondrocyte growth, inhibiting chondrocyte proliferation and inflammation, and does not inhibit chondrocyte viability.

[0051] 3. Immunohistochemical quantitative analysis After dewaxing and decalcifying the paraffin-embedded mouse cartilage tissue (mouse cartilage tissue treated with BMSC@Hyd for 72 hours and mouse cartilage tissue without BMSC@Hyd), it was blocked with 5% bovine serum albumin for 1 h, and then incubated overnight at 4°C with primary antibodies against the anabolic factor aggrecan and the catabolic factor MMP13. 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.

[0052] As Figure 19 shown, immunohistochemical analysis showed that treatment with BMSC@Hyd for 72 hours effectively increased the expression of aggrecan and inhibited the expression of MMP13 in human bone defect cartilage, indicating that BMSC@Hyd has certain potential in the treatment of human bone defects.

[0053] 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.

[0054] The content not described in detail in the specification of the present invention is well-known technology to those skilled in the art. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A preparation method of a CD63 aptamer-driven DNA hydrogel, characterized in that The preparation method includes the following steps: S1: Mix three DNA strands, Y1, Y2, and Y3, evenly in a 0.2 M PB buffer solution according to a molar ratio of 1:1:1 to obtain a Y-shaped scaffold; the PB buffer solution has a pH of 7.4 and 300 mM sodium chloride. S2: Mix the L1+L2 DNA strand, the CD63 aptamer, and the L2 DNA strand evenly in a 0.2 M PB buffer solution according to a molar ratio of 1:1:1 to obtain a linker; the PB buffer solution has a pH of 7.4 and 300 mM sodium chloride. S3: Incubate the Y-shaped scaffold obtained in S1 and the linker obtained in S2 at 90 °C for 10 minutes respectively. Mix the cooled Y-shaped scaffold and the linker evenly according to a volume ratio of 2:3, and let it stand to obtain a CD63 aptamer-driven DNA hydrogel.

2. The preparation method according to claim 1, wherein, The nucleotide sequence of the Y1 DNA strand described in S1 is shown as SEQ ID NO.

1.

3. The preparation method according to claim 1, wherein The nucleotide sequence of the Y2 DNA strand described in S1 is shown as SEQ ID NO.

2.

4. The preparation method according to claim 1, wherein, The nucleotide sequence of the Y3 DNA strand described in S1 is shown as SEQ ID NO.

3.

5. The preparation method according to claim 1, wherein The nucleotide sequence of the L1+L2 DNA strand described in S2 is shown as SEQ ID NO.

4.

6. The preparation method according to claim 1, wherein The nucleotide sequence of the CD63 aptamer described in S2 is shown as SEQ ID NO.

5.

7. The preparation method according to claim 1, wherein, The nucleotide sequence of the L2 DNA strand described in S2 is shown as SEQ ID NO.

6.

8. A CD63 aptamer-driven DNA hydrogel, characterized in that, The DNA hydrogel is obtained by using the preparation method described in any one of claims 1 to 7.

9. Use of the CD63 aptamer-driven DNA hydrogel described in claim 8 in promoting skin wound healing by loading exosomes.

10. Use of the CD63 aptamer-driven DNA hydrogel according to claim 8 in the treatment of osteoarthritis, characterized in that, The use is carried out by injecting the CD63 aptamer-driven DNA hydrogel carrying exosomes.

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