CN118121633A - DNA hydrogel system for loading bone marrow mesenchymal stem cell-derived exosomes - Google Patents

Abstract

The invention relates to the technical field of biological medicines, and particularly provides a DNA hydrogel system for loading bone marrow mesenchymal stem cell-derived exosomes and a preparation method and application thereof. The DNA hydrogel is mainly formed by physically crosslinking linear ultra-long single-stranded DNA generated by a rolling circle amplification method through a chain entanglement effect, and a chemical crosslinking agent is not required to be added, so that the DNA hydrogel has good biocompatibility and injectability. According to the invention, the aptamer tgg2 of the targeted binding cartilage cell membrane protein FGFR1 is integrated into a DNA sequence for the first time so as to realize the functionalization of the hydrogel. Further studies have shown that the use of the hydrogel-entrapped exosomes of the present invention can significantly enhance the uptake capacity of chondrocytes, thereby inhibiting inflammation-induced apoptosis, promoting cell proliferation, migration and maintaining extracellular matrix (ECM) metabolic homeostasis. The present invention provides an alternative to preparing materials for novel OA treatment strategies for cartilage tissue repair.

Description

DNA hydrogel system for loading bone marrow mesenchymal stem cell-derived exosomes

Technical Field

The invention relates to the field of biomedical materials, in particular to a DNA hydrogel system for loading bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exos), a preparation method thereof and application thereof in osteoarthritis treatment.

Background

Osteoarthritis is a common chronic degenerative joint disease, which is clinically manifested mainly as joint pain, brief morning stiffness and joint cramps during exercise, often resulting in unstable joints and even disability, severely affecting the quality of life of the patient. Currently, osteoarthritis is considered to be a disease characterized mainly by degeneration of articular cartilage and involving the whole joint, including sub-cartilage remodeling, osteophyte formation, synovitis and lesions of structures such as ligaments, envelopes, etc., ultimately leading to joint failure. Popular research shows that over 5 hundred million people worldwide suffer from osteoarthritis, and the incidence rate is high due to the risk factors of modern society such as aging of the population, surge of obesity rate and the like. The economic loss of osteoarthritis in developed countries is up to $650 billion per year, and the direct medical costs are more than $1000 billion, which places a tremendous burden on the socioeconomic performance. However, there are a number of shortcomings with current clinical approaches for the treatment of osteoarthritis. For example, the first-line drug NSAIDs (non-steroidal anti-inflammatory drugs) commonly used in the clinic are relatively toxic and are prone to cause gastrointestinal and cardiovascular adverse effects. Joint replacement is mainly aimed at advanced patients with continuously worsened conditions, and osteoarthritis can be effectively cured, however, joint prosthesis is easy to wear or needs to be subjected to revision surgery and other risks. Recently emerging stem cell therapies have attracted attention, however, potential immune rejection and tumorigenicity have limited their wide clinical use. Therefore, the development of a novel therapeutic strategy for osteoarthritis is particularly urgent.

To date, exosomes have been widely used in osteoarthritis as an advanced cell-free treatment. Exosomes are extracellular vesicles secreted by the parent cell, having extracellular domains of surface lipids and transmembrane proteins similar to the parent cell, and an internal cytoplasmic composition, and thus are stably present in vivo. The exosomes not only inherit most of the functions of the parent cells, but also have lower immunogenicity and tumorigenicity, thus having less impact on intra-articular environmental homeostasis. Previous studies have shown that a variety of bioactive components carried by exosomes (proteins, nucleic acids, small molecule metabolites, etc.) play a key role in the treatment of osteoarthritis. The BMSCs-Exos contains long-chain non-coding RNA and microRNA, such as LNCRNA MEG-3, miR-136-5p and miR-6515-5p, can delay the aging and apoptosis of chondrocytes and promote the migration of the chondrocytes through various signal paths, so that the pathological microenvironment of osteoarthritis is improved. Although exosomes can be effective in alleviating osteoarthritis, they still have many limitations in clinical applications, such as insufficient targeting ability, short joint residence time, low bioavailability, etc., to accelerate clearance from the joint cavity.

Recently, the use of functional biomaterials such as hydrogels to entrap exosomes has become a promising strategy for regenerative medical treatment. Since DNA has good hydrophilicity and precise hydrogen bond-based base complementary pairing, this drives it to be one of the ideal sources of materials for constructing hydrogels. The DNA hydrogel not only has good biocompatibility, biodegradability and adjustable mechanical properties, but also has special intelligent programmability. Through flexible editing of DNA sequences, the responsiveness of the hydrogel to biological or non-biological stimuli such as temperature, pH value, metal ions, proteins, RNA, ATP and the like can be endowed, so that the specific identification of a target area and the controllable delivery of drugs are realized. Therefore, the use of DNA hydrogel in combination with exosomes can enhance the therapeutic effect of osteoarthritis by improving the problems of poor targeting ability, low bioavailability and the like.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a DNA hydrogel system for loading bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exos). The invention prepares an aptamer functionalized chondrocyte targeted DNA hydrogel based on RCA reaction for entrapment of BMSCs-Exos, and improves the treatment of osteoarthritis by enhancing the focus delivery efficiency and bioavailability of exosomes.

The aim of the invention can be achieved by the following technical scheme:

In a first aspect of the present invention, there is provided a BMSCs-Exos-loaded DNA hydrogel system woven from ultralong ssDNA strands based on RCA reactions, containing an Apt-tgg2 moiety capable of specifically targeting the cartilage cell membrane protein FGFR 1.

In a second aspect of the present invention, there is provided a method for preparing a BMSCs-Exos-loaded DNA hydrogel system, comprising the steps of:

Step 1, designing and synthesizing a DNA sequence: using a fluorescent group FAM to mark the 5 'end of the Apt-tgg2 to obtain FAM-Apt-tgg2 which emits green fluorescence under a specific excitation wavelength, inserting a DNA sequence reversely complementary with the Apt-tgg2 into ssDNA phosphorylated at the 5' end to obtain a template, and designing a primer reversely complementary with the 5 'end and the 3' end of the template according to a base complementary pairing principle;

Step 2, synthesizing a circ-DNA solution: mixing template dissolved in 10×T4DNAzyme buffer solution and primer in a molar ratio of 1:1, annealing by using a PCR thermal cycler, and setting parameters at 95 ℃ for 2min;65 ℃ for 2min; 60-20 ℃ and-1 ℃/min, then adding T4 ligase and incubating at 22 ℃ for 12 hours to obtain a circ-DNA solution;

Step 3, synthesizing DNA hydrogel based on RCA reaction: mixing a circ-DNA solution, dNTP, BSA, naCL and Phi29 DNA polymerase in a 10 Xphi 29 DNA polymerase buffer solution to prepare an RCA reaction system, incubating for 10 hours at 37 ℃ in a constant-temperature shaking table at 100rpm, and heating the product at 65 ℃ for 10 minutes to inactivate the Phi29 DNA polymerase to obtain DNA hydrogel;

step 4, extracting BMSCs-Exos: after BMSCs fusion degree reaches 60% -80%, changing a culture medium into an alpha-MEM culture medium containing 10% of exosome-free bovine serum, continuously culturing for 48 hours, collecting cell supernatant, extracting exosome from the cell supernatant by adopting an ultracentrifugation method, and sequentially obtaining the cell supernatant according to 300 Xg for 10 minutes; 2,000Xg, 10min; gradient centrifugation was performed at 10,000Xg for 30min, each time the supernatant was retained to remove cells and cell debris, the cell supernatant was filtered using a 0.22 μm microporous filter, followed by centrifugation at 120,000Xg in a ultra-high speed centrifuge at 4℃for 70min to obtain an exosome pellet and re-suspension using PBS;

Step 5, DNA hydrogel entrapping exosomes: and (3) mixing the DNA hydrogel prepared in the step (3) with the exosomes extracted in the step (4), and incubating at 37 ℃ for 30min to obtain a DNA hydrogel system loaded with BMSCs-Exos.

Further, the Apt-tgg2 described in step 1 was obtained based on cell-SELEX screening in previous studies.

Further, the Apt-tgg2, FAM-Apt-tgg2, primer and Template oligonucleotide sequences described in step 1 are as follows:

further, the concentration of the template solution and the primer solution in the step 2 is 1. Mu.M.

Further, the concentration of T4 ligase in step 2 was 2U/. Mu.l.

Further, the concentration of the circ-DNA solution in the RCA reaction system described in step 3 was 50nM, the concentration of dNTPs was 1mM, the concentration of BSA was 20mg/ml, the concentration of sodium chloride was 80mM, and the concentration of phi29DNA polymerase was 20U/. Mu.l.

Further, the ratio of DNA hydrogel volume to exosome mass described in step 5 was 0.5ml:0.2mg.

In a third aspect, the present invention provides an application of the BMSCs-Exos-loaded DNA hydrogel system in preparing a medicament for treating osteoarthritis, wherein the BMSCs-Exos-loaded DNA hydrogel system can play a role in treating osteoarthritis by inhibiting inflammation-induced chondrocyte apoptosis, promoting cell proliferation, migration and ECM synthesis.

Compared with the prior art, the invention has the following effects:

Firstly, the invention uses the aptamer functionalized chondrocyte targeting DNA hydrogel to encapsulate BMSCs-Exos, so as to remarkably enhance the efficiency of delivering the BMSCs to chondrocytes;

Secondly, the DNA hydrogel is formed by physical crosslinking through the chain entanglement of the ultralong ssDNA based on RCA reaction, and the DNA hydrogel has low cytotoxicity without introducing other toxic chemical crosslinking agents or photoinitiators, and has simple synthesis process and low cost, and is suitable for industrialized mass production;

Thirdly, the DNA hydrogel system loaded with BMSCs-Exos can promote cell proliferation, migration and ECM synthesis by inhibiting inflammation-induced chondrocyte apoptosis, thereby delaying the osteoarthritis process.

Drawings

FIG. 1 is a flow chart showing the preparation of a BMSCs-Exos-loaded DNA hydrogel system obtained in example 1 of the present invention.

FIG. 2 is a schematic diagram of the DNA hydrogel prepared in example 1 of the present invention.

FIG. 3 is a scanning electron microscope image of the DNA hydrogel prepared in example 1 of the present invention.

FIG. 4 is a transmission electron microscope image of BMSCs-Exos extracted in example 1 of the present invention.

FIG. 5 is a graph showing the particle size analysis of BMSCs-Exos extracted in example 1 of the present invention.

FIG. 6 shows a map of BMSCs-Exos marker proteins extracted in example 1 of the present invention.

FIG. 7 is a confocal microscopy image of the DNA hydrogel loaded BMSCs-Exos described in example 1 of the present invention.

FIG. 8 is a schematic diagram of the targeted delivery of BMSCs-Exos to chondrocytes by the DNA hydrogel described in example 2 of the present invention.

FIG. 9 shows an immunofluorescence co-localization map of Apt-tgg2 and the cartilage cell membrane protein FGFR1 described in example 2 of the present invention.

FIG. 10 is a confocal microscopy image of chondrocytes ingesting free or hydrogel-loaded BMSCs-Exos described in example 2 of the present invention.

FIG. 11 is a graph showing the effect of BMSCs-Exos-loaded DNA hydrogel system described in example 3 of the present invention on osteoarthritis chondrocyte degeneration.

FIG. 12 is an immunofluorescence staining chart of the effect of BMSCs-Exos-loaded chondrocyte-targeted DNA hydrogel system described in example 3 of the present invention on the metabolic homeostasis of osteoarthritis cartilage ECM.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

Example 1

(1) DNA sequence design and synthesis: apt-tgg2 was selected according to the cell-SELEX technique of the previous study. The 5' -end of the Apt-tgg2 is marked by a fluorescent group FAM to obtain the FAM-Apt-tgg2 which emits green fluorescence under a specific excitation wavelength. And inserting a DNA sequence reversely complementary to the Apt-tgg2 into the 5' -end phosphorylated ssDNA to obtain a template, and designing a primer reversely complementary to the 5' -end and the 3' -end of the template according to a base complementary pairing principle. All oligonucleotide sequences (see Table 1) were synthesized by Shanghai Biotechnology Inc.

(2) Synthesis of circ-DNA solution: for the synthesis of circ-DNA, a template (1. Mu.M) and a primer (1. Mu.M) dissolved in a 10 XT 4 DNA ligase buffer solution were mixed in a molar ratio of 1:1, and annealed by a PCR thermal cycler with parameters set at 95℃for 2min;65 ℃ for 2min; 60-20 ℃ and-1 ℃/min. Then T4 ligase (2U/. Mu.L) was added and incubated at 22℃for 12h to give a circle-DNA solution.

(3) Synthesis of DNA hydrogels based on RCA reactions: the circle-DNA (50 nM), dNTP (1 mM), BSA (20 mg/ml), naCL (80 mM) and Phi29 DNA polymerase (20U/. Mu.l) were mixed in a 10 Xphi 29 DNA polymerase buffer to prepare an RCA reaction system, which was incubated in a constant temperature shaker at 37℃and 100rpm for 10 hours, and then the product was heated at 65℃for 10 minutes to inactivate the Phi29 DNA polymerase to obtain a DNA hydrogel.

(4) Extraction of BMSCs-Exos: when the BMSCs fusion degree reaches 60% -80%, changing the culture medium into alpha-MEM culture medium containing 10% of exosome-free fetal bovine serum, and continuously culturing for 48 hours, and collecting cell supernatant. Exosomes were extracted from the cell supernatant using ultracentrifugation. Cell supernatants were sequenced at 300 Xg for 10min;2,000Xg, 10min; gradient centrifugation was performed at 10,000Xg for 30min, with each supernatant being retained to remove cells and cell debris. The cell supernatant was filtered using a 0.22 μm microporous filter, and then centrifuged twice at 120,000Xg in a super-high speed centrifuge at 4℃for 70min to obtain an exosome pellet and resuspended using PBS.

(5) Preparation of a DNA hydrogel System carrying BMSCs-Exos: combining the DNA hydrogel prepared in (3) with the exosomes extracted in (4) according to 0.5ml: after mixing at a ratio of 0.2mg, the mixture was incubated at 37℃for 30 minutes to obtain a BMSCs-Exos-loaded DNA hydrogel system.

(6) Fluorescent staining verifies DNA hydrogel-entrapped BMSCs-Exos: the DNA hydrogel prepared in (3) (SYBR Green II staining) was stained with BMSCs-Exos extracted in (4) (DiD staining) in an amount of 0.5ml: after mixing 0.2mg of the specific columns, the DNA hydrogel was incubated at 37℃for 30min and examined for entrapped BMSCs-Exos by confocal microscopy.

The preparation flow of the BMSCs-Exos-loaded chondrocyte-targeted DNA hydrogel system is shown in FIG. 1, and it can be seen from the figure that the template and the primer form circle-DNA under the catalysis of T4 ligase. After annealing the Primer to the circ-DNA, phi 29DNA polymerase performs template-dependent synthesis based on RCA reaction along the circ-DNA, replicating a large amount of ultralong ssDNA (P-RCA) with periodically repeated sequences, thereby physically crosslinking to form DNA hydrogel through chain entanglement and hydrogen bond interactions between bases. As shown in FIG. 2, the DNA hydrogel was changed from liquid to solid gel before and after RCA reaction. The scanning electron microscope image of fig. 3 shows the 3D porous microstructure of DNA hydrogels with pore sizes greater than 100nm, which facilitates the diffusion of exosomes in the hydrogels. Compared with other physical crosslinking methods based on base complementary pairing, the RCA reaction has high sensitivity and specificity, and can synthesize a large number of linear copies even with lower template concentration, so that the construction cost of the hydrogel is greatly reduced, and meanwhile, the precise programming of a DNA sequence can be realized in the replication process with high template dependence, so that the intelligent DNA hydrogel with controllable functions is prepared. In addition, the RCA reaction can be carried out under mild physiological conditions without adding other chemical crosslinking agents or photoinitiators, and the formed DNA hydrogel has good biocompatibility and biodegradability, so that the RCA reaction can be applied to in-situ gelling in vivo in the future so as to accelerate damaged tissue repair.

BMSCs-Exos was the earliest and most widely studied stem cell-derived exosomes, and the cargo contained therein, such as proteins, nucleic acids and small molecule metabolites, have remarkable anti-inflammatory, immunomodulatory and cartilage repair effects, so exosomes extracted from BMSCs supernatant are used as the main bioactive ingredient for the treatment of osteoarthritis. FIG. 4 shows a transmission electron microscope image showing BMSCs-Exos extracted from cell supernatants as cup-shaped spherical nanoparticles having a diameter of 100-150 nm and having a lipid bilayer membrane. The nanoparticle tracking analysis of FIG. 5 shows that the size distribution of BMSCs-Exos is 100-150 nm, consistent with the results of transmission electron microscopy analysis. The immunoblotting results of fig. 6 show that the exosome specific markers CD9, CD63 and TSG101 are highly expressed in the extracted nanoparticle lysate. These results indicate that the extracted BMSCs-Exos meet the exosome criteria described previously. To prepare a DNA hydrogel system carrying BMSCs-Exos, the two were mixed in a certain ratio, and confocal microscopy imaging of FIG. 7 showed that after mixing the DiD-labeled BMSCs-Exos was uniformly distributed in the SYBR Green II-labeled DNA hydrogel, indicating that it was successfully encapsulated in the DNA hydrogel.

Example 2

Steps (1) to (5) are the same as in example 1.

(6) And verifying chondrocyte targeting of Apt-tgg 2: chondrocytes were inoculated in 12-well plates, inoculated at a density of 2 to 3×10 ⁵ cells/well, cultured for 24h, the original medium was discarded, 1ml of complete medium containing 0.25 μ MFAM-Apt-gg2 was added, and incubated for 30min under light-shielding conditions. After the cells were gently rinsed 2-3 times with PBS after discarding the medium, 4% Paraformaldehyde (PFA) was added and the mixture was fixed at room temperature for 15min. The 4% PFA solution was discarded, immersed in PBS 2-3 times, blocked by adding 1% BSA at 37℃for 1 hour. The 1% BSA solution was discarded, washed 2-3 times with PBS, and the anti-FGFR 1 antibody diluted with 1% BSA was added and incubated with the cells at 37℃for 2h. The primary antibody was discarded, washed 2-3 times with PBS, the PE-labeled secondary antibody was diluted with PBS and incubated with the cells for 1h at 37℃and images were taken under an inverted fluorescence microscope.

(7) Chondrocyte uptake assay: BMSCs-Exos were labeled using the DiD staining kit according to the manufacturer's protocol. BMSCs-Exos were incubated with 10. Mu.M DiD at room temperature for 20min in the dark and the suspension was filtered using a 0.22 μm microporous filter. Unbound dye was then removed by centrifugation at 120,000Xg for 90 min. DiD-labeled free BMSCs-Exos or hydrogel-loaded BMSCs-Exos after 6h incubation with normal or IL-1β treated chondrocytes, cells were fixed at room temperature for 15min with 3.7% formaldehyde solution and then washed 2-3 times with PBS solution containing 0.1% Triton X-100. The microfilament green fluorescent probe was diluted with PBS containing 0.1% Triton X-100 and incubated with the cells at room temperature for 30min in the absence of light. The images were then observed and captured under a confocal microscope by washing 2-3 times with PBS containing 0.1% Triton X-100, staining with DAPI staining solution for 5 min.

This example essentially examined the ability of chondrocyte-targeted DNA hydrogels to significantly enhance the uptake of BMSCs-Exos by chondrocytes. To confer the ability of DNA hydrogels to target chondrocytes, the present invention integrates Apt-tgg2 capable of specifically binding to the chondrocyte membrane protein FGFR1 into a DNA sequence to achieve the functionalization of the hydrogels. Fig. 8 shows that Apt-tgg2 with a specific conformation contained in the DNA hydrogel sequence has a high affinity with chondrocyte surface membrane protein FGFR1, and that its loaded BMSCs-Exos is capable of targeted delivery to chondrocytes, thereby significantly enhancing the uptake capacity of BMSCs-Exos by chondrocytes. Fig. 9 shows that FAM-Apt-tgg2 and PE-labeled FGFR1 have good subcellular co-localization on the surface of cartilage cell membrane, which proves that Apt-tgg2 has specific recognition capability for FGFR 1. The confocal fluorescence microscopy imaging of fig. 10 shows that the intracellular fluorescence intensity of hydrogel-loaded BMSCs-Exos is significantly higher than that of free BMSCs-Exos, both in normal chondrocytes and inflammation-induced chondrocytes, further verifying the good chondrocyte targeting function of DNA hydrogels. Interestingly, we also found that DNA hydrogel-loaded BMSCs-Exos exhibited stronger fluorescence intensity in inflammation-induced chondrocytes than free BMSCs-Exos, which may be associated with increased expression levels of FGFR1 on the surface of osteoarthritis chondrocytes.

Example 3

Steps (1) to (5) are the same as in example 1.

(6) Dead-living staining detects cell proliferation: chondrocytes were seeded in 12-well plates at a density of 2-3×10 ⁵ cells/well, the normal group was not treated, the IL-1β group, exos group and Exos/Gel group were each continuously treated with 10ng/ml IL-1β for 3 days, and then whole culture medium of exosome serum containing equal volumes of PBS,10 μg/ml BMSCs-Exos and 10 μg/ml BMSCs-Exos/DNA hydrogel was added, respectively, and culture was continued for 3 days. The Calcein-AM, PI and 1 Xdetection buffer were then mixed according to 1:1:1000 were prepared as a staining working solution, and then cells were added and incubated at 37℃for 20min in the absence of light. The image was observed and captured with an inverted fluorescence microscope.

(7) Cell migration was detected by cross-well experiments: the cross-well chamber was used to perform a cross-well experiment on a 24-well plate. Normal group was added to the lower chamber with 700. Mu.L of complete medium containing 10% of exosome serum, IL-1. Beta. Group, exos group and Exos/Gel group were added to 700ul of complete medium containing 10% of exosome serum with equal volumes of PBS, 10. Mu.g/ml BMSCs-Exos and 10. Mu.g/ml BMSCs-Exos/DNA hydrogel, respectively, and then added to the lower chamber. Then, normal or IL-1β treated chondrocytes (8×10 ⁴ cells/well) were inoculated into an upper chamber containing 200. Mu.L of serum-free medium. After incubation for 24h, the cells were washed with PBS and then fixed with 4% PFA at room temperature for 15min. After washing 2-3 times with PBS, the cells were stained with 0.1% crystal violet for 15min. After washing 2-3 times with PBS, residual cells in the upper chamber are wiped off with a cotton swab. The images were observed and captured using an inverted fluorescence microscope.

(8) Real-time fluorescent quantitative PCR (qRT-PCR) detection of cartilage ECM metabolism related gene expression: chondrocytes were seeded in 12-well plates at a density of 2-3×10 ⁵ cells/well, untreated in normal groups, and treated continuously with 10ng/ml IL-1β for 3 days in both IL-1β, exos and Exos/Gel groups, followed by additional culture in whole serum medium containing equal volumes of PBS,10 μg/ml BMSCs-Exos and 10 μg/ml BMSCs-Exos/DNA hydrogel, respectively, for 48h. Total RNA was then extracted using TRIZOL reagent and reverse transcribed into cDNA using 5X PRIMESCRIPT RT MASTER Mix. The level of gene expression of ECM synthesis markers (type II collagen, aggrecan) and ECM degradation markers (MMP 13 (matrix metalloproteinase 13), ADAMTS-5 (thrombospondin metallopeptidase-5)) was detected using a fluorescent quantitative gene amplification instrument, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standard. mRNA expression levels of the target gene relative to the control group were quantitatively measured using 2 ^-ΔΔCt.

(9) Immunofluorescence detection of cartilage ECM metabolism-related protein expression: chondrocytes were seeded at a density of 2-3×10 ⁵ cells/well in 12-well plates, untreated in normal groups, and treated continuously with 10ng/ml IL-1β for 3 days in both IL-1β, exos and Exos/Gel groups, followed by additional culture in whole serum medium containing equal volumes of PBS,10 μg/ml BMSCs-Exos and 10 μg/ml BMSCs-Exos/DNA hydrogel, respectively, for 48h. Cells were gently rinsed with PBS and fixed with 4% PFA for 15min at room temperature. After washing 2-3 times with PBS, the cells were treated with 0.1% Triton-X100 cell permeabilizer for 10min. Chondrocytes were blocked with 1% BSA for 1h after 2-3 washes with PBS. Anti-type II collagen antibodies, anti-aggrecan antibodies, anti-MMP 13 antibodies and anti-ADAMTS-5 antibodies were diluted 1:100 with 1% BSA after 2-3 washes with PBS, and incubated with chondrocytes at 37℃for 2h, respectively. After washing 2-3 times with PBS, chondrocytes were incubated with secondary antibodies labeled with either the green fluorescent group FITC or the red fluorescent group Fluor594 for 1h. PBS washes 2-3 times were stained with DAPI for 5min at room temperature. The image was observed and captured using a confocal microscope.

This example examined mainly the effect of BMSCs-Exos loaded DNA hydrogel system on osteoarthritis chondrocyte degeneration, and in fig. 10, the result of dead-alive staining shows increased chondrocyte apoptosis and decreased cell proliferation activity in IL-1 β compared to control. Both Exos and Exos/Gel groups were able to inhibit inflammation-induced chondrocytes and promote cell proliferation, whereas Exos/Gel had significantly better therapeutic effects than Exos. Cell migration plays an important role in promoting cartilage ECM synthesis. The trans-well experiments showed that chondrocyte migration was significantly inhibited in the IL-1β group compared to the control group. Both group Exos and group Exos/Gel can rescue the inflammation-induced inhibition of cell migration, which can exert a stronger pro-migration effect. Cartilage ECM is composed mainly of type II collagen and aggrecan, and when chondrocytes are in inflammatory conditions, it can secrete a number of inflammatory factors such as MMP13 and ADAMTS-5 to accelerate ECM breakdown, resulting in imbalance in ECM metabolic homeostasis, thereby exacerbating cartilage degeneration. The qRT-PCR result shows that Exos/Gel group can obviously promote the expression of II-type collagen and aggrecan mRNA in the cartilage cells stimulated by IL-1 beta and simultaneously inhibit the expression of MMP-13 and ADAMTS-5, and the effect is obviously better than Exos group. FIG. 11 shows that the immunofluorescence staining results of group Exos/Gel also promote expression of cartilage ECM anabolism-related proteins (collagen type II, aggrecan) and inhibit expression of catabolism-related proteins (MMP 13, ADAMTS-5) at the protein level, and have a chondrogenic activity significantly superior to that of group Exos. These results indicate that the targeting DNA hydrogel can be used as an ideal carrier of the multifunctional bioactive component BMSCs-Exos for delaying osteoarthritis-induced chondrocyte degeneration.

Sequence listing

Claims (8)

1. A DNA hydrogel system loaded with BMSCs-Exos, characterized in that the DNA hydrogel system is woven from ultra-long ssDNA strands based on RCA reaction, comprising an Apt-tgg2 moiety capable of specifically targeting cartilage cell membrane protein FGFR 1.

2. A method for preparing the BMSCs-Exos-loaded DNA hydrogel system according to claim 1, comprising the specific steps of:

Step 1, designing and synthesizing a DNA sequence: the 5 'end of Apt-tgg2 is marked by using a fluorescent group FAM to obtain FAM-Apt-tgg2 which emits green fluorescence under a specific excitation wavelength, a DNA sequence reversely complementary with the Apt-tgg2 is inserted into ssDNA phosphorylated at the 5' end to obtain a template ssDNA (template), and primers reversely complementary with the 5 'end and the 3' end of the template are designed according to a base complementary pairing principle.

Step 2, synthesizing a circular DNA (circ-DNA) solution: mixing template dissolved in 10×T4DNAzyme buffer solution and primer at a molar ratio of 1:1, annealing with Polymerase Chain Reaction (PCR) thermal cycler, and setting parameters at 95deg.C for 2min;65 ℃ for 2min; 60-20 ℃ and-1 ℃/min, then adding T4 ligase and incubating at 22 ℃ for 12 hours to obtain a circ-DNA solution;

step 3, synthesizing DNA hydrogel based on RCA reaction: mixing a circ-DNA solution, dNTPs, bovine Serum Albumin (BSA), naCL and Phi29DNA polymerase in a 10 Xphi 29DNA polymerase buffer solution to prepare an RCA reaction system, incubating for 10 hours at 37 ℃ in a constant temperature shaking table at 100rpm, and heating the product at 65 ℃ for 10 minutes to inactivate the Phi29DNA polymerase to obtain DNA hydrogel;

Step 4, extracting BMSCs-Exos: after BMSCs fusion degree reaches 60% -80%, changing a culture medium into an alpha-MEM culture medium containing 10% of exosome-free bovine serum, continuously culturing for 48 hours, collecting cell supernatant, extracting exosome from the cell supernatant by adopting an ultracentrifugation method, and sequentially obtaining the cell supernatant according to 300 Xg for 10 minutes; 2000 Xg, 10min; gradient centrifugation was performed at 10000 Xg for 30min, each time the supernatant was retained to remove cells and cell debris, the cell supernatant was filtered using a 0.22 μm microporous filter, followed by centrifugation at 120000 Xg in a super-high speed centrifuge at 4℃for 70min to obtain an exosome pellet and re-suspension using PBS;

Step 5, DNA hydrogel entrapment BMSCs-Exos: and (3) mixing the DNA hydrogel prepared in the step (2) with the exosomes extracted in the step (4), and incubating at 37 ℃ for 30min to obtain a DNA hydrogel system loaded with BMSCs-Exos.

3. The method of claim 2, wherein the Apt-tgg2, FAM-Apt-tgg2, primer and Template oligonucleotide sequences described in step 1 are as follows:

4. the method of claim 2, wherein the concentration of the template solution and the primer solution in step 2 is 1 μm.

5. The method according to claim 2, wherein the concentration of the T4 ligase in the step 2 is 2U/. Mu.l.

6. The method according to claim 2, wherein the concentration of the circ-DNA solution in the RCA reaction system in the step 3 is 50nM, the concentration of dNTPs is 1mM, the concentration of BSA is 20mg/ml, the concentration of sodium chloride is 80mM, and the concentration of the phi29 DNA polymerase is 20U/. Mu.l.

7. The method of claim 2, wherein the ratio of volume of DNA hydrogel to exosome in step 5 is 0.5ml:0.2mg.

8. Use of a BMSCs-Exos loaded DNA hydrogel system according to claim 1 or prepared according to any one of claims 2 to 7 in the manufacture of a medicament for the treatment of osteoarthritis, wherein the BMSCs-Exos loaded DNA hydrogel system is capable of exerting an osteoarthritis treatment effect by inhibiting inflammation-induced chondrocyte apoptosis, promoting cell proliferation, migration and ECM synthesis.