Improved hyaluronic acid injection formulations for inflammation targeting and stem cell capture, and methods of making and uses thereof

Abstract

The application provides an improved hyaluronic acid injection preparation for inflammation targeting and stem cell capture, a preparation method and use thereof, and belongs to the field of medicine. The injection preparation is prepared from unsaturated bond modified hyaluronic acid, stem cell targeting DNA tetrahedron and thiol modified inflammation targeting aptamer as raw materials. The injection preparation can be targeted to accumulate at an osteoarthritis site, capture mesenchymal stem cells in situ, recruit mesenchymal stem cells to the osteoarthritis site, promote differentiation of stem cells into cartilage, promote cartilage repair, play a role in treating osteoarthritis, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to an improved hyaluronic acid injection preparation for inflammation targeting and stem cell capture, and a preparation method and use thereof. BACKGROUND

[0002] Osteoarthritis (OA) is the most common cause of disability in the elderly, and its core pathological manifestations are chronic mild inflammation and structural damage of various tissues in the joint, including hyaline articular cartilage, subchondral bone, ligament, meniscus, joint capsule, synovial membrane, usually accompanied by pain and dysfunction. The pathogenesis of OA is complex and heterogeneous, and existing medical technology cannot reverse the destructive process of OA or restore damaged cartilage. The first-line drugs for OA patients in clinical practice include non-steroidal anti-inflammatory drugs, analgesics (paracetamol, tramadol, duloxetine) and intra-articular injection drugs (natural cartilage extracellular matrix hyaluronic acid (HA), glucocorticoids, and platelet-rich plasma, etc.). However, these drugs only aim to relieve symptoms and do not substantially interrupt the destructive process of OA or repair damaged cartilage, and may also cause uncontrollable systemic side effects. End-stage OA patients generally use joint replacement, but this treatment is expensive and the improvement in function is not satisfactory.

[0003] Hyaluronic acid (HA) is a polysaccharide composed of D-glucuronic acid and N-acetylglucosamine, and is the main component of the natural cartilage extracellular matrix. Numerous studies have shown that the role of HA in the joint includes enhancing the lubricity of the joint surface, reducing the concentration of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), and reducing joint pain. In addition, HA can alleviate cartilage damage caused by inflammatory mediators by binding to the CD44 receptor on the cell surface, thereby protecting the articular cartilage. Although HA shows certain application potential in the treatment of OA, like other non-steroidal anti-inflammatory drugs and analgesics, its therapeutic effect is mainly limited to relieving symptoms, and cannot fundamentally block the destructive process of OA or repair damaged cartilage. In addition, these treatment methods can cause uncontrollable systemic side effects. Therefore, there is an urgent need to develop new OA treatment strategies.

[0004] Stem cell therapy shows great advantages in the treatment of OA. Mesenchymal stromal cells / Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic stem cells with self-renewal ability. Many clinical trials use MSCs for treatment, and common indications include autoimmune diseases such as graft-versus-host disease, multiple sclerosis, amyotrophic lateral sclerosis, myocardial infarction, osteoarthritis, neurodegenerative diseases, cardiovascular diseases and orthopedic diseases. However, many clinical trial reports have failed to develop, and the reasons include: allogeneic stem cell programs have ethical issues, donor heterogeneity and risk of immune rejection; artificial induction programs have low induction efficiency, limited differentiation potential and tumorigenic risk. In contrast, autologous stem cells can avoid the above problems, and because they will not circulate as commodities, they can avoid problems caused by cryopreservation and cryogenic transport, and reduce the potential risk of pathogen transmission. However, the application of autologous stem cells still has some unresolved problems, including: 1) Isolation of autologous stem cells from the patient's body can cause secondary damage; 2) In vitro culture causes treatment delay, reduced differentiation potential, and potential contamination risk; 3) Mechanical shear force during injection can cause membrane rupture; 4) Lack of 3D structure to support cell viability after infusion can result in low cell retention. Therefore, it is urgent to explore and develop a strategy to solve the damage and contamination problems of autologous stem cells during in vitro isolation, culture, and injection, as well as the retention problem at the target site after in vivo infusion. SUMMARY

[0005] The purpose of the present application is to provide an improved hyaluronic acid injection preparation for inflammation targeting and stem cell capture, and a preparation method and use thereof.

[0006] The present application provides an improved hyaluronic acid injection preparation, which is prepared from unsaturated bond modified hyaluronic acid, thiol modified stem cell targeting DNA tetrahedron and thiol modified inflammation targeting aptamer.

[0007] Further, the unsaturated bond modified hyaluronic acid is hyaluronic acid modified by methacrylic anhydride;

[0008] The stem cell targeting DNA tetrahedron is a DNA tetrahedron modified by a stem cell targeting aptamer;

[0009] The inflammation targeting aptamer is DTA64.

[0010] Further, the preparation method of the hyaluronic acid modified by methacrylic anhydride comprises the following steps: reacting hyaluronic acid with methacrylic anhydride, and the pH value of the reaction is 7-9;

[0011] The stem cell targeting aptamer is Apt19S.

[0012] The nucleotide sequence of the thiol-modified inflammation targeting aptamer is shown as SEQ ID NO. 15.

[0013] Further, the thiol-modified stem cell targeting DNA tetrahedron is a tetrahedral structure formed by four DNA single strands with nucleotide sequences shown as SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10 respectively and an Apt19S single strand with nucleotide sequence shown as SEQ ID NO. 12.

[0014] Further, the molar ratio of the four DNA single strands is 1:1:1:1, and the molar ratio of the DNA single strand with nucleotide sequence shown as SEQ ID NO. 1 to the Apt19S single strand with nucleotide sequence shown as SEQ ID NO. 12 is 1:(1-3).

[0015] Further, the ratio of the unsaturated bond-modified hyaluronic acid, the thiol-modified stem cell targeting DNA tetrahedron and the thiol-modified inflammation targeting aptamer is (10-30) mg:(0.1-5) nmol:(0.1-5) nmol.

[0016] Further, the ratio of the unsaturated bond-modified hyaluronic acid, the thiol-modified stem cell targeting DNA tetrahedron and the thiol-modified inflammation targeting aptamer is 20 mg:1 nmol:1 nmol.

[0017] The application also provides a method for preparing the improved hyaluronic acid injection preparation, which comprises the following steps: mixing the unsaturated bond-modified hyaluronic acid, the thiol-modified stem cell targeting DNA tetrahedron and the thiol-modified inflammation targeting aptamer and then reacting to obtain the improved hyaluronic acid injection preparation.

[0018] The application also provides the use of the improved hyaluronic acid injection preparation in the preparation of a drug for treating and / or preventing osteoarthritis.

[0019] Further, the drug is a drug for targeting enrichment to an osteoarthritis site, in-situ capture of mesenchymal stem cells, recruitment of mesenchymal stem cells to the osteoarthritis site, promotion of differentiation of stem cells into cartilage, and / or promotion of cartilage repair.

[0020] In the application, the DNA tetrahedron nanostructure (Tetrahedral DNA Nanostructure) is abbreviated as TDN.

[0021] In the application, the injection preparation can be an injection solution or a powder injection.

[0022] The present application has the following beneficial effects:

[0023] The therapeutic effect of the commonly used clinical first-line drug hyaluronic acid injection is mainly limited to relieving symptoms, and cannot fundamentally block the destructive process of OA or repair the damaged cartilage. The TDN cell capture structure functionalized hyaluronic acid injection system provided by the present application can target enrichment to the osteoarthritis site, capture mesenchymal stem cells in situ, recruit mesenchymal stem cells to the osteoarthritis site, promote stem cell differentiation into cartilage, rebuild damaged cartilage, promote cartilage repair, and play a role in treating osteoarthritis, and has good application prospect.

[0024] The TDN cell capture structure functionalized hyaluronic acid injection system provided by the present application avoids the ethical and pollution risks of exogenous stem cell implantation, reduces the loss of vitality and treatment delay caused by in vitro isolation and expansion of stem cells, and avoids the tumorigenic risk of in vitro induction of stem cells.

[0025] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above-mentioned basic technical ideas of the present application.

[0026] The above content of the present application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above-mentioned subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Synthesis and characterization of TDN-based membrane binding structures. (A) Schematic diagram of membrane binding structure with programmable ligand number; (B) 4-valent membrane binding device, i.e. MD simulation calculation, zeta potential, dynamic light scattering images of T0, T1, T2 and T3 structures, respectively; (C) Agarose gel electrophoresis characterization of band migration of different membrane binding structures; (D) AFM images of T0 and T3 membrane binding structures (scale, 100 nm).

[0028] Figure 2. Different membrane-bound structures interact with the bone marrow mesenchymal stem cell membrane. (A) Schematic diagram of the interaction of different membrane-bound structures with bone marrow mesenchymal stem cells; (B) Confocal images of BMSC cells treated with different membrane-bound structures for 30 min (green: cell membrane stained with Dio green dye; red: membrane-bound structure labeled with cy-5 fluorescein; yellow: co-localization of membrane-bound structure and cell membrane); (C) Intracellular fluorescence density and cell membrane fluorescence density statistics in B; (D) Confocal images of L929 cells treated with T3 membrane-bound structures for 30 min, replacing the Apt19S sequence in T3 with a nonsense sequence, and confocal images of L929 cells treated for 30 min; (E) Representative trajectories of dissipative particle dynamics simulation of the interaction process of different membrane-bound devices with cell membranes; (F) Interaction energy distribution between different membrane-bound devices and cell membranes (*P < 0.05, **P < 0.005, *P < 0.0005).

[0029] Figure 3 . Structure characterization of HADT injection system. (A) Schematic diagram of the design of HADT injection system: thiol-modified T3 structure and thiol-modified DTA64 structure are connected to methyl methacrylate hyaluronic acid by click chemistry; (B-C) Fluorescent gel electrophoresis and fluorescent microscopy results preliminarily verify the preparation of HADT; (D-E) Nuclear magnetic hydrogen spectrum and XPS energy spectrum analysis confirm the conjugation of thiolated T3 structure and thiolated DTA64 structure with HAMA; (F) Rheological analysis of the modified HADT injection system; (G) Dispersion tribology test (I) of the modified HADT injection system (scale: 200 μm. *P < 0.05, **P < 0.005, *P < 0.005).

[0030] Figure 4 . In vitro evaluation of the injection solution. (A) Loading efficiency of HA and T3 using physical mixing or chemical mixing; (B) Capture efficiency of 250 nM HAT and 500 nM HAT capturing complementary aptamer; (C) Statistics of the results of Figure A; (D) Statistics of the results of Figure B; (E) HAT capturing Cy 5-labeled Apt 19S complementary sequence (Apt19S #) the results of HAT alone capturing BMSC cells and L929 cells; (G) the statistical results of Figure E; (H) the statistical results of Figure F; (I) the results of HAT capturing BMSC in mixed BMSC and L929 cell conditions; (J) the results of Transwell cell migration experiment demonstrating the effect of HAT recruiting BMSC cells, transwell experiment schematic; (K) the statistical results of Figure I; (L) the statistical results of Figure J; (M) the results of captured BMSC cells growing on T25 culture flask and HAT; (N) the schematic of HAT capturing DiO labeled BMSC cells and DiD labeled L929 cells; (O) BMSC cells cultured in 500 nM T3, 500 nM DTA64, 1% wt concentration HA consisting of HA, HAD, HAT, HADT for 24 hours and 48 hours showed no significant difference in activity compared to control medium (*P<0.05, **P<0.005, ***P<0.0005).

[0031] Figure 5 In vivo therapeutic effect evaluation of HADT injection system. (A) Schematic diagram of rat osteoarthritis model modeling; (B) in vivo fluorescence colocalization results of fam labeled T3 structure, cy5 labeled DTA64 structure, and cy3 labeled inflammatory protein DEK, which proved that HADT injection solution injected into the joint cavity was enriched at the osteoarthritis inflammation site under the targeting effect of DTA64; (C) behavioral experiment of gait analysis proved that HADT significantly improved the rat's stride length and foot contact area; (D) statistical results of Figure C; (E) micro-CT analysis results showed that HADT injection solution improved the number of osteophytes and the smoothness of the bone surface; (F) trabecular analysis results showed that HADT injection solution improved the osteophyte formation and cartilage surface discontinuity of the joint surface; (G) scanning electron microscopy showed the roughness of the joint surface (*P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005).

[0032] Figure 6Histological evaluation of HADT injection system after in vivo treatment. (A-B) HE and masson staining results at 2 months showed that the cartilage thickness of the HADT injection system group increased, and the subchondral bone matrix hardness increased; (C) CD29 and CD44 staining results at 2 months showed that the HADT injection group retained a large number of CD29 and CD44 positive multipotent stem cells locally; (D) Tunel staining results showed that the HADT injection improved the apoptosis of local tissue cells of osteoarthritis; (E) MMP13 staining results showed that the HADT injection reduced the expression of destructive enzymes in the local tissues of osteoarthritis; (F) COL I staining results showed that the HADT injection reduced the proliferation of reactive type I collagen in the local tissues of osteoarthritis; (G) COL II staining results showed that the HADT injection increased the expression of type II collagen in the joint components; (H-I) Statistical results of figures A, B: HE and masson staining results at 2 months showed that the OARSI score and Mankin score of the HADT injection system group decreased significantly; (J) Statistical results of figure C; (K) Statistical results of figure D; (L) Statistical results of figure E; (M) Statistical results of figure F; (N) Statistical results of figure G. DETAILED DESCRIPTION

[0033] The raw materials and equipment used in the present application are known products, which can be obtained by purchasing commercially available products.

[0034] Unless otherwise specified, the operation of the present application is carried out at room temperature (25±5℃).

[0035] Example 1, Preparation of TDN cell capture structure functionalized hyaluronic acid injection system

[0036] Table 1. Nucleotide sequence

[0037]

[0038]

[0039] 1. Synthesis of TDN cell capture structure

[0040] Four designed special single-stranded SH-S1, S2b, S3b, S4b with sticky ends (the final concentration after addition is 1 μM, the sequence is shown in Table 1) and Apt19S-b* single-stranded (the final concentration after addition is 3 μM, the sequence is shown in Table 1) are added to TM buffer solution (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) to form a 100 μL synthesis system, and the temperature of the PCR thermal cycler is set to maintain at 95℃ for 10 minutes, then quickly reduced to 4℃, and cooled for 20 minutes, to complete the folding, and obtain thiolated TDN-Apt19S*3 (SH-T3).

[0041] 2. Preparation of inflammation targeting and cell capture structure functionalized hyaluronic acid injection

[0042] 5g HA was mixed with 10.4g methacrylic anhydride (MA) liquid, 10g sodium hydroxide was used to adjust the pH of the reaction solution to 8, to prepare HA-MA injection. The thiolated DTA64 (i.e. SH-DTA64, 500nM) aptamer, thiolated TDN cell capture structure SH-T3 (500nM) was added to 1ml of HA-MA injection (containing 10mg HA-MA) and reacted for 4 hours, to obtain TDN cell capture structure functionalized hyaluronic acid (abbreviated as DTA64-HA-T3 or HADT) injection system.

[0043] The following is the preparation method of the control sample.

[0044] Example 1, synthesis of TDN basic structure

[0045] Four single-stranded S1, S2, S3, S4 (the final concentration after addition was 1 μM, and the sequence is shown in Table 1) were added to TM buffer buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) to form a 100 μL synthesis system, and the temperature of the PCR thermal cycler was set to maintain at 95°C for 10 minutes, then quickly reduced to 4°C, cooled for 20 minutes, folded to obtain TDN (T0).

[0046] Example 2, synthesis of control TDN cell capture structure T1

[0047] Four designed special single-stranded S1, S2b, S3b, S4b with sticky ends (the final concentration after addition was 1 μM, and the sequence is shown in Table 1) and the corresponding Apt19S-b* single-stranded (the final concentration after addition was 1 μM, and the sequence is shown in Table 1) were added to TM buffer buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) to form a 100 μL synthesis system, and the temperature of the PCR thermal cycler was set to maintain at 95°C for 10 minutes, then quickly reduced to 4°C, cooled for 20 minutes, folded to obtain TDN-Apt19S*1 (T1).

[0048] Example 3, synthesis of control TDN cell capture structure T2

[0049] Four specially designed single strands S1, S2b, S3b, S4b with sticky ends (the final concentration after adding is 1 μM, and the sequence is shown in Table 1) and Apt19S-b* single strand (the final concentration after adding is 2 μM, and the sequence is shown in Table 1) are added into 100 μL of a synthesis system in TM buffer solution (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0), and the temperature of the PCR thermal cycler is set to be stably maintained at 95°C for 10 minutes, then quickly reduced to 4°C, and cooled for 20 minutes to complete the folding, thereby obtaining TDN-Apt19S*2 (T2).

[0050] Synthesis of control TDN cell capture structure T3

[0051] Four specially designed single strands S1, S2b, S3b, S4b with sticky ends (the final concentration after adding is 1 μM, and the sequence is shown in Table 1) and Apt19S-b* single strand (the final concentration after adding is 3 μM, and the sequence is shown in Table 1) are added into 100 μL of a synthesis system in TM buffer solution (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0), and the temperature of the PCR thermal cycler is set to be stably maintained at 95°C for 10 minutes, then quickly reduced to 4°C, and cooled for 20 minutes to complete the folding, thereby obtaining TDN-Apt19S*3 (T3).

[0052] 5g of HA is mixed with 10.4g of methacrylic anhydride (MA) liquid, 10g of sodium hydroxide is used to adjust the pH of the reaction solution to 8, and HA-MA is prepared. 10mg of HA-MA is added with thiolated DTA64 (i.e., SH-DTA64, the sequence is shown in Table 1, 500nM) aptamer, and the reaction is carried out for 4 hours, thereby obtaining 1ml of inflammation-targeted HA injection (referred to as HAD).

[0053] Preparation of TDN cell capture structure functionalized hyaluronic acid injection: thiolated TDN cell capture structure SH-T3 (500nM) is added into 1ml of HA-MA injection (containing 10mg of HA-MA) and reacted for 4 hours, thereby obtaining TDN cell capture structure functionalized hyaluronic acid (referred to as TDN-HA or HAT) injection system.

[0054] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0055] Experimental Example 1, Structure characterization of TDN basic structure and TDN capture structure

[0056] 1. Experimental method

[0057] (1) Polyacrylamide gel electrophoresis (PAGE) and agarose gel electrophoresis: 8% non-denaturing polyacrylamide gel or 1.5% agarose gel was configured, and TDN-Apt19S*3 (T3), TDN-Apt19S*2 (T2), TDN-Apt19S*1 (T1), TDN (TO), S1, S2b, S3b, S4b samples mixed with loading buffer were added to the PAGE gel lane respectively, and 20 bp DNA Maker was added. Adjust the appropriate electrophoresis instrument parameters (~ 80V, 80min), stop running the gel when the Marker band runs to the bottom edge of the gel. The whole gel was placed in the pre-configured Gel Red nucleic acid staining solution for 10 minutes in the dark. Expose and take pictures using the gel imaging system to determine the synthesis and yield of different designed TDN capture structures.

[0058] (2) Atomic force microscope (AFM): After synthesizing different TDNs and TDN capture structure units, dilute 10-100 times with TM buffer or 1xTE buffer (10mM Tris, 1mM EDTA, pH 8.0). Take 10-20 μL of the diluted solution and drop it on a mica sheet. After setting the appropriate parameters, scan the sample and take pictures.

[0059] (3) Transmission electron microscope: After synthesizing different TDNs and TDN capture structure units, dilute 10-100 times with TM buffer or 1xTE buffer (10mM Tris, 1mM EDTA, pH 8.0). Add the synthesized TDNs and TDN capture structures to the copper mesh, let it stand for a few minutes, then add phosphotungstic acid staining solution to the copper mesh and stain for 2 minutes, then remove the phosphotungstic acid solution. Carefully add pure water to the copper mesh and wash twice to remove excess phosphotungstic acid solution, then remove the remaining water, let it dry, and then observe and take pictures.

[0060] (4) Capillary electrophoresis: Fill the capillary with running buffer and set the appropriate voltage (18-20V). Dilute the stock solution of different designed TDN capture structures with running buffer to obtain good peak shape.

[0061] 2. Experimental results

[0062] The experimental results are shown in Figure 1 TDN capture structures (T3, T2, T1) and TDN basic structure (TO) were successfully synthesized.

[0063] Experimental Example 2, Performance verification and optimization of TDN capture structure

[0064] 1. Experimental method

[0065] 1.1 Biocompatibility verification

[0066] The femur of SD rat was taken and the bone marrow mesenchymal stem cells were obtained by enzymatic digestion. The cells were identified by morphological observation and flow cytometry. L929 (fibroblast cells) were purchased from cell bank and cultured in standard culture environment (10% fetal bovine serum, 100 U / mL penicillin / streptomycin double antibody, 5% CO2, 37°C). The above-mentioned multipotent stem cells and terminal differentiation negative control L929 cells were inoculated in 96-well plates or 6-well plates at a concentration of 5x10 4-8 4 ×10 4 The cells were cultured for 24h, then different designed TDN capture structures were added at concentrations of 250nM and 500nM respectively. CCK8 kit, apoptosis detection kit, live and dead cell staining, cell morphology observation and other techniques were used to determine the biocompatibility of TDN capture structures at different concentrations.

[0067] 1.2 Optimization of TDN capture structure

[0068] The TDN structure was optimized using all-atom molecular dynamics tools. The all-atom model of TDN capture structure was constructed by molecular dynamics tool Visual Molecular Dynamics 1.93. The binding strength and capture efficiency of the target binding site were analyzed and quantitatively characterized. The TDN capture structure was optimized by coarse-grained molecular dynamics model (dissipative particle dynamics analysis).

[0069] 1.3 Performance verification and screening of TDN capture structure

[0070] (1) Gel electrophoresis: bone marrow mesenchymal stem cells were used as positive cells to screen the binding strength and capture efficiency of TDN capture structure. Specifically, after synthesis of TDN capture structure, it was mixed with positive cell suspension for 30 min. After centrifugation to precipitate the cells, high performance liquid chromatography, gel electrophoresis and other methods were used to quantitatively analyze the TDN capture structure remaining in the supernatant, so as to determine the binding strength and efficiency (TDN capture efficiency = (synthesis yield - supernatant residual amount) / synthesis yield).

[0071] (2) Fluorescent staining and confocal microscopy: Dio and Did dyes were used to label the membranes of positive and negative cells respectively. Cy5-labeled TDN capture structure carrying 0-3 Apt19S, Cy5-labeled TDN capture structure carrying 0-3 meaningless DNA sequences, and equal amount of Apt19S single strand and positive and negative control cells were mixed. The binding label of different designed materials and different cell membranes was observed by confocal laser scanning microscope.

[0072] 2. Experimental results

[0073] Experimental results are as follows Figure 2 As shown in BC, it can be seen that TDN and T1 structures are mainly distributed inside the cell, while T2 and T3 are mainly distributed in the cell membrane. Moreover, the total cell membrane fluorescence and the proportion of cell membrane fluorescence of T3 are higher than those of T2. The optimal TDN cell capture structure T3 was selected.

[0074] Experiment Example 3: Performance Verification and Optimization of the Injection System

[0075] The yield, mechanical properties, and stability of the structure were determined using gel electrophoresis, high-performance liquid chromatography, immunofluorescence, Fourier transform infrared spectroscopy, and nuclear magnetic resonance hydrogen spectroscopy.

[0076] (1) Characterization by 1H NMR and XPS: The results confirmed the conjugation of thiolized T3 and thiolized DTA64 structures with HAMA.

[0077] (2) Validation of the capture and enrichment structure performance: After synthesizing the capture and enrichment structure, the corresponding concentration of simple culture medium and the test sample were placed in the lower chamber of the Transwell. Positive cells and negative cells were placed in the upper chamber of the Transwell and incubated for 24h and 48h, respectively. Cells were fixed with paraformaldehyde, cell nuclei were stained with DAPI, and cells that did not migrate to the lower chamber were wiped off with cotton swabs. The number of cells passing through the bottom wall of the Transwell chamber in multiple fields of view was observed and counted using a fluorescence microscope, and the cell enrichment efficiency and enrichment specificity of different materials were compared.

[0078] (3) Biocompatibility verification and optimization: Bone marrow mesenchymal stem cells were cultured in a standard culture environment (10% fetal bovine serum, 100 U / mL penicillin / streptomycin antibiotics, 5% CO2, 37℃). At a concentration of 5 × 10⁻⁶ cells / mL... 4- 8 × 10⁴ cells / mL were seeded into 96-well or 6-well plates. After culturing for 24 h, different designed capture structures were added at concentrations of 250 nM and 500 nM, respectively. The effect of the capture and enrichment process on cell viability was evaluated by combining the CCK8 assay.

[0079] Experimental results are as follows Figure 3 , 4 As shown, it can be seen that HA, HAD, HAT, and HADT were successfully chemically synthesized, and the modified HA injection solution has similar rheological characteristics and lubricating properties to the original HA injection solution. Figure 3 FG). Furthermore, chemical bonding has a higher bonding efficiency than physical mixing. Figure 4 A) and target molecule capture efficiency ( Figure 4 B). HAT also exhibits good target molecule and target cell capture and recruitment capabilities, specifically good biocompatibility. The optimal concentration of TDN-HA was determined to be 500 nM. Figure 4 ).

[0080] Experimental Example 4: Verification of the performance of the injection system in an osteoarthritis model

[0081] (1) Construction of an osteoarthritis model: After the SD rats were anesthetized, the skin was prepared, a local antiseptic was applied to the skin over the knee joint, and the skin was incised transversely on the lateral side of the right knee joint. After the joint capsule was opened, the cartilage was kept moist with a saline solution, the patella was dislocated medially, and the anterior cruciate ligament of the rat was incised with a microsurgical knife to establish an anterior cruciate ligament transection (ACLT) osteoarthritis model in the right knee joint.

[0082] (2) Observation of the in vivo metabolism of the sample under test using small-animal live imaging: Using the constructed rat osteoarthritis model, the Cy5 fluorescent group-modified injection solution under test was injected into the joint cavity of the rat, and the fluorescence intensity in the joint cavity of the live rat and the fluorescence intensity of the ex vivo joint tissue were recorded at 1 day, 3 days, 5 days, 1 week, and 2 weeks to determine the in vivo metabolism of the sample under test.

[0083] (3) Evaluation of the therapeutic effect of the sample under test in osteoarthritis (OA): Normal saline, HA injection, HAD injection, HAT injection, and HADT injection were injected into the joint cavity of the OA rat. Samples were harvested at 1 month and 2 months, and the repair effect of OA in the OA rat in different groups was analyzed by micro-CT, HE staining, Masson staining, OARSI scoring, and mechanical evaluation.

[0084] The experimental results are shown in Figure 5 and Figure 6 It can be seen that the HADT injection solution has good inflammation targeting ability Figure 5 B), promotes the recovery of stride length and foot contact area in the gait analysis behavioral experiment Figure 5 C-D), and improves the discontinuity and roughness of the joint surface of the rat after modeling Figure 5 E-G). At the same time, the in vivo histological staining results Figure 6 show that the Mankin’s score and OARSI score decrease after treatment; the expression of CD29 and Cd44 stem cell markers increases significantly; the number of apoptotic cells in tunel staining decreases; the expression of MMP13, a key enzyme for the degradation of type II collagen in articular cartilage, decreases; the reactive type I collagen proliferation decreases, and the expression of type II collagen, the main component of articular cartilage, increases; indicating that the improved HADT injection solution improves the symptoms of osteoarthritis and promotes cartilage repair in vivo.

[0085] In conclusion, the application provides a hyaluronic acid injection for inflammation targeting and stem cell capturing. The hyaluronic acid injection can be targeted to the osteoarthritis site, in-situ capture mesenchymal stem cells, recruit mesenchymal stem cells to the osteoarthritis site, promote differentiation of stem cells into cartilage, promote cartilage repair, play a role in treating osteoarthritis, and has good application prospect.

Claims

1. A modified hyaluronic acid injection formulation, characterized in that, The modified hyaluronic acid injection formulation is prepared from unsaturated bond-modified hyaluronic acid, thiol-modified stem cell-targeting DNA tetrahedron, and thiol-modified inflammation-targeting aptamer. The thiol-modified stem cell-targeting DNA tetrahedron is a tetrahedral structure formed by four DNA single strands with nucleotide sequences as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.10, and an Apt19S single strand with nucleotide sequence as shown in SEQ ID NO.

12. The nucleotide sequence of the thiol-modified inflammation-targeting aptamer is shown in SEQ ID NO.15; The unsaturated bond-modified hyaluronic acid is hyaluronic acid modified with methacrylic anhydride; The molar ratio of the four DNA single strands is 1:1:1:1, and the molar ratio of the DNA single strand with the nucleotide sequence shown in SEQ ID NO.1 to the Apt19S single strand with the nucleotide sequence shown in SEQ ID NO.12 is 1:(1-3). The ratio of the unsaturated bond-modified hyaluronic acid, the thiol-modified stem cell-targeting DNA tetrahedron, and the thiol-modified inflammation-targeting aptamer is (10-30) mg: (0.1-5) nmol: (0.1-5) nmol.

2. The modified hyaluronic acid injection formulation according to claim 1, characterized in that, The stem cell-targeted DNA tetrahedron is a DNA tetrahedron modified with a stem cell-targeted aptamer.

3. The modified hyaluronic acid injection formulation according to claim 2, characterized in that, The method for preparing the hyaluronic acid modified with methacrylic anhydride includes the following steps: reacting hyaluronic acid with methacrylic anhydride to obtain the hyaluronic acid; the pH value of the reaction is 7-9.

4. The modified hyaluronic acid injection formulation according to claim 1, characterized in that, The ratio of the unsaturated bond-modified hyaluronic acid, the thiol-modified stem cell-targeting DNA tetrahedron, and the thiol-modified inflammation-targeting aptamer is 20 mg: 1 nmol: 1 nmol.

5. A method for preparing the modified hyaluronic acid injection formulation according to any one of claims 1-4, characterized in that, The method includes the following steps: mixing unsaturated bond-modified hyaluronic acid, thiol-modified stem cell-targeting DNA tetrahedron, and thiol-modified inflammation-targeting aptamer and reacting them to obtain a modified hyaluronic acid injection formulation.

6. Use of the modified hyaluronic acid injection formulation of any one of claims 1-4 in the preparation of a medicament for the treatment and / or prevention of osteoarthritis.

7. The use according to claim 6, characterized in that, The drug is a drug that targets and enriches itself at osteoarthritis sites, captures mesenchymal stem cells in situ, recruits mesenchymal stem cells to osteoarthritis sites, promotes stem cell differentiation into cartilage, and / or promotes cartilage repair.

Images