A nanocomposite, injectable self-enhanced hydrogel gene delivery system and preparation method and use thereof

Abstract

The present invention provides a nanocomposite, an injectable self-enhancing hydrogel gene delivery system, and a preparation method and use thereof, which belong to the field of medicine. The present invention uses mitochondrial targeted antioxidant peptides and hyaluronic acid to coat miR-21 inhibitors and branched polyethyleneimine complexes to obtain a nanocomposite. The present invention further combines the nanocomposite with a hydrogel containing MMP-2-substrate peptides and tannic acid to obtain an injectable self-enhancing hydrogel gene delivery system. The nanocomposite and the injectable self-enhancing hydrogel gene delivery system can inhibit inflammation, scavenge reactive oxygen species, inhibit apoptosis of nasopharyngeal carcinoma cells, and regulate the metabolic homeostasis of the extracellular matrix in the targeted IVD, and have good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a nanocomposite, an injectable self-enhanced hydrogel gene delivery system, and a preparation method and use thereof. Background Art

[0002] Degeneration (IDD) of intervertebral discs (IVDs) is closely associated with changes in the mechanical and biochemical characteristics of the degenerating nucleus pulposus (NP) microenvironment. The occurrence and progression of NP degeneration is largely believed to be due to inflammatory responses caused by aging, trauma, and mechanical loading. Exposure of NP cells (NPCs) to proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), can trigger these cells to undergo apoptosis or enter a senescent state, leading to the disruption of normal IVD homeostasis, impaired collagen synthesis, and degradation of the extracellular matrix (ECM), resulting in increased concentrations of reactive oxygen species (ROS), local acidification, and overexpression of matrix metalloproteinases (mMPs), a disintegrin-like metalloproteinase with type I platelet-binding protein motifs (ADAMTs), and other enzymes with degradation functions in the IV microenvironment. Therefore, efforts to restore normal NPC function, eliminate local inflammation, and promote ECM regeneration are expected to effectively reverse IDD. Researchers have been hoping to develop an ideal drug delivery carrier in the nucleus pulposus, but a large number of research results show that due to the complex tissue structure, poor blood supply and complex mechanical environment in the intervertebral disc, existing materials such as natural materials: decellularized matrix, alginate, peptide hydrogel, and synthetic materials: hyaluronic acid, PLGA, etc., cannot meet the requirements of constructing functionalized nucleus pulposus in vitro. Internationally, a large number of scientists have conducted preclinical studies on some nucleus pulposus replacement scaffold materials that can carry cell growth, including fibrin gel (GelfoamTM), hyaluronic acid sponge (HyStem®, HYAFF) and peptide hydrogel (Puramatrix®), but they all have problems such as inappropriate mechanical strength, poor biocompatibility, inability to achieve injectable requirements, immune response with the host, high cost, and complex manufacturing process. At present, there is still no mature functionalized drug delivery product for clinical use in China.

[0003] MicroRNA (miRNA) is a class of small non-coding RNAs consisting of 18-24 nucleotide sequences. It is widely present in eukaryotes and is involved in gene regulation. Mature miRNA guides the RISC complex (miRISC) in the cytoplasm to target mRNAs with partially complementary sequences in the 3'-UTR region, resulting in transcriptional silencing or mRNA degradation, thereby regulating various physiological activities of cells. Imbalance in miRNA regulation plays an important role in the occurrence of intervertebral disc degeneration. A large number of studies have shown that abnormal expression of multiple miRNAs will lead to inflammation of cells inside the nucleus pulposus, ECM degradation, apoptosis of nucleus pulposus cells, etc., and accelerate the degeneration of the nucleus pulposus of the intervertebral disc through different mechanisms of action. Therefore, regulating the function of nucleus pulposus cells through miRNAs gene therapy has great application prospects in the repair of degenerated nucleus pulposus of intervertebral discs.

[0004] MiRNA gene therapy can be divided into miRNA replacement therapy when miRNAs are deficient and miRNA inhibition therapy when they are overexpressed, depending on the expression of miRNAs themselves. However, the clinical utility of miRNA continues to be hampered by its poor stability, limited transfection efficiency, tendency to distribute in a nonspecific manner, and its potential for off-target effects. In the application of miRNA nanodelivery vectors, although cationic polymers have shown excellent transfection efficiency in delivering miRNAs, their cytotoxicity limits its clinical application.

[0005] In recent years, people have been trying to use drug delivery systems such as microspheres, nano-micelles, exosomes, and acellular matrices as nucleus pulposus replacement scaffold materials. However, due to the lack of mechanical properties, complex preparation processes, and expensive raw materials, they have not been able to meet all of the above requirements. Hydrogel materials have continued to receive attention in recent years and have been widely used in various tissue repair fields. The inherent permeability, water absorption, degradability, good biocompatibility, and huge drug-loading controlled release capacity and mechanical support potential of injectable hydrogels have great application potential in the selection of nucleus pulposus replacement scaffold materials.

[0006] However, there is a potential risk of extravasation of hydrogel materials after injection when injectable hydrogels are used as drug delivery carriers for intervertebral disc regeneration. In order to solve this problem, on the one hand, it is necessary to increase the elastic modulus of the hydrogel itself. However, hydrogels with increased elastic modulus often cannot be injected through thinner puncture needles, and increasing the diameter of the puncture needle will increase the damage to the annulus fibrosus of the intervertebral disc, resulting in this contradiction that cannot be resolved. Therefore, it is urgent to develop a high elastic modulus hydrogel with "in situ gelation after injection". On the other hand, in order to reduce the potential risk of extravasation of hydrogel materials after injection, it is urgent to optimize the mechanical properties of the hydrogel, improve the biocompatibility of the hydrogel, shorten the gelation time of the hydrogel, and develop a more efficient hydrogel delivery system. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a nanocomposite, an injectable self-enhanced hydrogel gene delivery system and a preparation method and use thereof.

[0008] The invention provides a nanocomplex, which is prepared by using oxidized hyaluronic acid, a miRNA inhibitor, polyethyleneimine and a mitochondrial targeted antioxidant peptide as raw materials, wherein the ratio of the number of amino groups in the polyethyleneimine to the number of phosphate groups in the miRNA inhibitor is 20-40:1; the ratio of the number of H-derived carboxylic acid groups in the oxidized hyaluronic acid to the number of phosphate groups in the miRNA inhibitor is 5-15:1; and the molar ratio of the mitochondrial targeted antioxidant peptide to the miRNA inhibitor is 1:1-3.

[0009] Furthermore, the ratio of the number of amino groups in the polyethyleneimine to the number of phosphate groups in the miRNA inhibitor is 30:1; the ratio of the number of H-derived carboxylic acid groups in the oxidized hyaluronic acid to the number of phosphate groups in the miRNA inhibitor is 10:1; and the molar ratio of the mitochondrial targeted antioxidant peptide to the miRNA inhibitor is 1:2.

[0010] Further, the miRNA inhibitor is a miR-21 inhibitor;

[0011] The polyethyleneimine is branched polyethyleneimine;

[0012] The mitochondria-targeted antioxidant peptide is Szeto-Schiller 31;

[0013] The preparation method of oxidized hyaluronic acid comprises the following steps: hyaluronic acid, water and an oxidant react under light-proof conditions, dialyze, and freeze-dry to obtain oxidized hyaluronic acid.

[0014] Furthermore, the mass ratio of the hyaluronic acid to the oxidant is 1000-3000:1; the oxidant is periodate; the reaction temperature is 10-40°C, the reaction time is 0.5-1.5 hours; the dialysis time is 2-4 days;

[0015] The mass ratio of the hyaluronic acid to the oxidant is 2000:1; the oxidant is sodium periodate; the reaction temperature is 15-35°C, the reaction time is 1 hour; and the dialysis time is 3 days.

[0016] The present invention also provides a method for preparing the above-mentioned nanocomposite, the method comprising the following steps:

[0017] (1) reacting the miRNA inhibitor with polyethyleneimine to prepare a complex;

[0018] (2) The oxidized hyaluronic acid, the complex and the mitochondria-targeted antioxidant peptide are reacted to obtain a nanocomplex.

[0019] Furthermore, in step (1), the reaction temperature is 10-40°C and the reaction time is 0.5-1.5 hours;

[0020] In step (2), the reaction conditions are: incubation for 3 to 5 hours under constant stirring nitrogen.

[0021] Further,

[0022] In step (1), the reaction temperature is 15-35°C and the reaction time is 1 hour;

[0023] In step (2), the reaction conditions are: incubation for 4 hours under constant stirring nitrogen.

[0024] The present invention also provides an injectable self-reinforced hydrogel, which is prepared using the above-mentioned nanocomposite, acrylate monomers, matrix metalloproteinases, and tannic acid as raw materials; the mass ratio of the matrix metalloproteinase to tannic acid is 1-3:1; the mass volume ratio of the matrix metalloproteinase to the acrylate monomers is 1:4-8 mg / mL; and the mass ratio of the nanocomposite to the acrylate monomers is 1:30-50.

[0025] Furthermore, the mass ratio of the matrix metalloproteinase to tannic acid is 2:1; the mass ratio of the matrix metalloproteinase to the acrylate monomer is 10:5.32; and the mass ratio of the nanocomposite to the acrylate monomer is 1:35.5.

[0026] Furthermore, the acrylic acid ester monomer is methyl methacrylate.

[0027] The present invention also provides a method for preparing the injectable self-reinforced hydrogel, which comprises the following steps: mixing the nanocomposite, acrylate monomers, matrix metalloproteinases and tannic acid, adjusting pH, and incubating to obtain the injectable self-reinforced hydrogel.

[0028] Furthermore, the pH is adjusted to a range of 7 to 8, the incubation temperature is 35 to 45° C., and the incubation time is 20 to 30 hours.

[0029] Furthermore, the pH range of the adjustment is 7.5, the incubation temperature is 37° C., and the incubation time is 24 hours.

[0030] The present invention also provides the use of the nanocomposite and the injectable self-reinforcing hydrogel in preparing a drug for preventing and / or treating intervertebral disc degeneration.

[0031] The present invention has achieved the following beneficial effects:

[0032] The present invention uses the natural polysaccharide hyaluronic acid (HA) as the carrier shell for the first time, and combines the miR-21 inhibitor / bPEI nanocomplex with the hydrogel delivery system. The present invention utilizes the slow characteristics of the thiol-ene click reaction to design a hydrogel with the characteristic of "in situ gelation after injection". The hydrogel is injected into the intervertebral disc in the form of a liquid, so that it spontaneously gels again. The gelation time and mechanical strength of this hydrogel construction mode are adjustable, which reduces the potential risk of extravasation of the hydrogel material after injection, and can be well used for intervertebral disc regeneration.

[0033] The present invention uses MMP-2-substrate peptide (MMP-2 sp) as a crosslinker and synthesizes a hydrogel containing tannic acid (TA) by thiol-ene reaction. The obtained hydrogel has good injection performance, MMP-2 reaction performance and self-enhancement performance. Then, the nanocomposite of miR-21 inhibitor (Antagomir-21) / 25Ka branched polyethyleneimine (bPEI) is produced by electrostatic adsorption, and then natural polysaccharide hyaluronic acid (HA) and novel mitochondrial targeted antioxidant peptide Szeto-Schiller 31 (ss-31) are selected as the outer layer coating of the miR-21 inhibitor / bPEI (PH) nanocomposite to produce miR-21 inhibitor / bPEI / oHA / ss-31 (PHs) nanocomposite. These PHs target degenerated NPCs and are internalized through receptor-mediated endocytosis that responds to HA, play the role of inhibiting inflammation, scavenging ROS, inhibiting NPC apoptosis, and regulating the metabolic homeostasis of degenerated NPCs, thereby promoting IVD regeneration.

[0034] The level of MMP-2 is highly correlated with the degree of denaturation, and the injectable self-reinforcing hydrogel of the present invention can be triggered by MMP-2 to release PHs on demand and in an environmentally responsive manner. Subsequently, the oHA-coated complex with CD44 targeting ability can further promote the absorption of PH by degenerated NPC. During delivery, the miR-21 inhibitor effectively inhibits apoptosis and promotes ECM regeneration by targeting Spry1. At the same time, the ss-31 peptide dissociates from PHs and rapidly targets the inner mitochondrial membrane, restoring mitochondrial homeostasis, enhancing intracellular ATP production, inhibiting mROS production, and inhibiting the activity of the mROS-nLRP3-IL-1β / Caspase1 pathway. Therefore, the platform is effective in inhibiting inflammation, scavenging reactive oxygen species, inhibiting apoptosis of nasopharyngeal carcinoma cells, and regulating the metabolic homeostasis of the extracellular matrix in targeted IVDs.

[0035] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0036] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Physicochemical properties of PHs: (a) Schematic overview of the PHs synthesis process; (b) HA and oHA 1 H NMR spectroscopy; (c) size distribution of the indicated nanocarrier formulations in PBS; (d) imaging of miR / bPEI and PHs formulations by TEM (scale bar: 50 nm); (e, f) nanoparticle size and zeta potential values; (g) change in nanoparticle size over time when incubated in PBS; (h) analysis of the interaction between bPEI and miR-21 inhibitor; (i) evaluation of RNase resistance of naked miR-21 inhibitor and PHs (* P < 0.05, n = 3).

[0038] Figure 2 Physicochemical properties of hydrogels: (a) Schematic overview of the hydrogel synthesis process; (b) HA and MeHA 1 (d) Overview of the hydrogel production process and the MMP-2 responsiveness, injectability, and plasticity properties of the final hydrogels; (e) Imaging of the hydrogels by SEM (scale bar: 200 μm), inset: PHs (scale bar: 400 nm); (f, g) Rheological characterization of the hydrogels to assess the storage (G') and loss (G") moduli and gelation process, (f) 37 °C, frequency 1 Hz, strain 1%; (g) oscillatory strain sweep; (h) Schematic overview of the injection of precursor solutions and MMP-2-mediated release of PHs after gelation in the case of IDD.

[0039] Figure 3Figure 4: CD44-mediated cellular uptake of PHs: (a) Analysis of the distribution of Cy3-miR-21 inhibitor in NPCs (scale bar: 20 μm) after transfection with various vectors; (b) Semi-quantitative analysis with corresponding markers; (c) Measurement of miR-21 expression using qPCR; (d) Analysis of CD44 levels in NPCs stimulated with TBHP by (e) immunofluorescence (scale bar: 20 μm) and (f) Western immunoblotting; (g) Intracellular distribution of Cy3-miR-21 inhibitor (red) and FAM-miR-21 inhibitor (green) in NPCs cultured with equal amounts of FAM-miR / bPEI and Cy3-PHs (scale bar: 20 μm); (h) Schematic diagram of how CD44 promotes the internalization of PHs; (i) Ratio of red and green fluorescence intensity; (j) TEM for detecting PHs internalized by NPCs (scale bar: 1 μm) (* P < 0.05, ** P < 0.01, ns: not significant).

[0040] Figure 4 Evaluation of PHs lysosomal escape, ECM regeneration, and anti-apoptotic activity: (a) evaluation of colocalization between FAM-labeled nanocarriers and LysoTracker Red to determine the ability of PHs to escape the lysosomal compartment (scale bar: 20 μm); (b) representative flow cytometry plots and (c) corresponding quantification of indicated treatment groups; (d) representative TUNEL staining and immunofluorescence detection of Col II and MMP-13 in indicated groups (scale bar: 50 μm); (e) Western immunoblotting and (f) corresponding semiquantitative analysis of protein expression levels in NPCs transfected with miR-21 mimics and / or si-Spry1 constructs; (g) Western immunoblotting and (h) corresponding semiquantitative analysis of protein levels in TBHP-treated NPCs in indicated treatment groups (* P < 0.05, ** P < 0.01, ns: not significant).

[0041] Figure 5(a) Fluorescence confocal microscopy-based analysis of ss-19 and PH / ss-19 uptake by healthy and TBHP-stimulated NPCs, and the colocalization of ss-19 was assessed using MitoTracker Green (scale bar: 20 μm); (b) Representative confocal laser scanning microscopy images of JC-1 staining in NPCs of the treatment groups (scale bar: 50 μm); (c) Confocal images of mROS and cROS levels in TBHP-stimulated NPCs of the designated treatment groups (scale bar: 50 μm), and transmission electron microscopy (TEM) was used to observe the mitochondrial structure of each group (scale bar: 1 μm), with white triangles and white arrows indicating mitochondria and internalized PHs, respectively; (d) Representative immunofluorescence staining results of NLRP3 and IL-1β in each group (scale bar: 50 μm).

[0042] Figure 6 Figure 3: Biocompatibility, degradability and gene release of hydrogels: (a) Schematic overview of the ROS / RNS scavenging properties of the prepared hydrogels; (b, c) The antioxidant activity of TA-loaded hydrogels was confirmed by (b) DPPH and (c) OH scavenging assays; (d) Release kinetics of TA and miR-21 inhibitors under a series of microenvironmental conditions; (e) NPCs were stimulated with TBHP and cell viability was determined; (f) Live / Dead and DCFH-DA staining (scale bar: 50 μm) analysis; (g) In vitro degradation results of hydrogels under different conditions; (h) Real-time fluorescence imaging of the Cy3-miR-21 inhibitor construct after injection into the IVD; (i) Corresponding semi-quantitative in vivo image analysis results (* P < 0.05, ** P < 0.01, ns: not significant. n = 3).

[0043] Figure 7 Figure 3: In vivo therapeutic effects of PHs in the IDD rat model: (a) Schematic overview of the experimental methods using model rats; (b) X-ray images and (c) MRI images of the caudal intervertebral disc of rats after 4 and 8 weeks of treatment; (d) changes in disc height index (DHI) at 4 and 8 weeks of treatment; (e) changes in MRI grade at 4 and 8 weeks of treatment; (f) representative H&E, Safranin-O / Fast Green (scale bar: 1 mm), and TUNEL staining (scale bar: 100 μm) images at 4 and 8 weeks in the indicated treatment groups; (g) histological grade analysis of the indicated groups at 4 and 8 weeks of treatment; (h) semi-quantitative analysis of TUNEL-positive cells; (i) representative immunofluorescence staining results of Col II, MMP-13, and TNF-α in different treatments after 8 weeks; (jl) representative quantitative results of Col II, MMP-13, and TNF-α (scale bar: 500 μm) (* P < 0.05, ** P<0.01, n = 3). DETAILED DESCRIPTION

[0044] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0045] The "room temperature" referred to in the present invention is 25±10°C.

[0046] Example 1. Preparation of the Nanocomposite of the Present Invention

[0047] 1.0 g of HA was dissolved in 100 mL of deionized water to obtain a 10 mg / mL HA solution. Sodium periodate (0.5 mg, 5 mL) was then dropped into the HA solution and stirred at room temperature for 6 hours while avoiding light, and then the unreacted periodate was inactivated by adding 1 mL of ethylene glycol. After stirring at room temperature for 1 hour, the reaction was purified by dialysis water for 3 days and then freeze-dried to obtain oxidized HA (oHA). Then 11 μg of miR-21 inhibitor (Antagomir-21) was mixed with 25Ka branched polyethyleneimine (bPEI) in 0.5 mL of deionized water at a N / P ratio of 30:1, and the solution was incubated at room temperature for 1 hour to obtain a complex of miR-21 inhibitor and bPEI (miR / bPEI). The N / P ratio is defined by the ratio between the number of amino groups in bPEI and the number of phosphate groups in the miR-21 inhibitor. The prepared miR / bPEI was combined with 0.5 mL of oHA at a concentration of 0.78 mg / mL at a C / P ratio of 10:1 to obtain a mixture of miR / bPEI and oHA (PH). The C / P ratio is defined by the ratio between the number of H-derived carboxylic acid groups and the number of phosphate groups present in the miR-21 inhibitor. The mixture PH was then incubated for 4 hours under constant stirring nitrogen, and 0.2 mL of ss-31 peptide solution at a concentration of 1 mg / mL was added and incubated for 4 hours to mix thoroughly to obtain a miR / bPEI / oHA / ss-31 (PHs) nanocomplex solution.

[0048] Example 2: Preparation of the injectable self-reinforcing hydrogel of the present invention

[0049] Methyl methacrylate (MeHA) was prepared from methacrylic anhydride using a conventional route. The specific steps were as follows: HA (3 g, 7.61 mmol, 1.0 eq) was dissolved in deionized water (300 mL) and cooled on ice. The pH was adjusted to 8.5 and methacrylic anhydride (2.25 mL, 15.1 mmol, 2 eq) was added. Under vigorous stirring, NaOH was added to maintain the pH at 7.5-8.5 for 8 hours. On the next day, 1.125 mL of methacrylic anhydride was added and the pH was maintained at 7.5-8.5 for an additional 4 hours to obtain a MeHA (13.3 mg / mL) solution.

[0050] Then, a MMP-2 sp / TA mixture containing 100 mg MMP-2 sp and 50 mg tannic acid (TA) was prepared, and 100 μL of this MMP-2 sp / TA mixture was added to 400 μL MeHA (13.3 mg / mL) in a dropwise manner, and the mixture was continuously stirred, and then 10 μL PHs solution (5% w / v) was added dropwise. The pH of the mixture was adjusted to about 7.5 using NaOH (0.1 M) to obtain a precursor solution, which was incubated at 37°C for 24 hours to form an injectable self-reinforced hydrogel, i.e., a hydrogel containing TA with a ratio of MMP-2 sp (100 mg / ml): MeHA (13.3 mg / ml) (v:v) = 2:8, and the final PHs concentration of the resulting hydrogel was 1.09 mg / ml.

[0051] Example 3: Preparation of the injectable self-reinforcing hydrogel of the present invention

[0052] The method of reference example 2 was followed, except that 100 μL of MMP-2 sp (100 mg / ml) was replaced with 50 μL to prepare a hydrogel containing TA with a ratio of MMP-2 sp (100 mg / ml): MeHA (13.3 mg / ml) (v:v) = 1:8 (see Table 1).

[0053] The following is a control sample preparation.

[0054] Comparative Example 1: Preparation of the injectable self-reinforcing hydrogel of the present invention

[0055] Referring to the method of Example 2, the only difference is that tannic acid is not added, and a TA-free MMP-2 sp (100 mg / ml): MeHA (13.3 mg / ml) (v:v) = 2:8 hydrogel is prepared (see Table 1).

[0056] Comparative Example 2: Preparation of the injectable self-reinforcing hydrogel of the present invention

[0057] The method of Reference Example 4 was used, except that tannic acid was not added, and a TA-free hydrogel with a ratio of MMP-2 sp (100 mg / ml): MeHA (13.3 mg / ml) (v:v) = 1:8 was prepared (see Table 1).

[0058] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0059] Experimental Example 1: Screening of hydrogel composition ratio

[0060] 1. Experimental methods

[0061] The gelation time of the hydrogels prepared in Examples 2 and 3 was compared with that in Control Examples 1 and 2. The specific experimental operation was as follows: a modular compact rheometer was used to measure the storage and loss moduli (G′ and G′′) of the hydrogels. The intersection time of the two curves was the gel time, and the time when the two curves remained unchanged was the complete gel time.

[0062] 2. Experimental results

[0063] According to the results in Table 1, it can be seen that adding TA and increasing the proportion of MMP-2 sp can shorten the initial gelation time and complete gelation time of the hydrogel. When MMP-2 sp (100 mg / ml): MeHA (13.3 mg / ml) (v:v) = 2:8 is selected and TA is added to prepare the hydrogel, the initial gelation time and complete gelation time of the hydrogel can be significantly shortened.

[0064] Table 1. Gelation time to study hydrogel formation

[0065]

[0066] Experimental Example 2: Physicochemical properties of nanocomposites and injectable self-reinforced hydrogels

[0067] 1. Experimental methods

[0068] The results were performed using a Bruker Avance II 400 MHz instrument. 1 H NMR spectroscopy. Fourier transform infrared spectroscopy (FTIR) at 400–4000 cm -1 The chemical structure of the polymers was analyzed within a range of 100 nm. The zeta potential and size distribution of the nanoparticles were determined using a Malvern Zetasizer Nano-ZS90 instrument. The structure of the freeze-dried hydrogels was evaluated by scanning electron microscopy. The rheology of the hydrogels was evaluated using an MCR302 modular compact rheometer (Anton Paar GmbH, Graz, Austria).

[0069] 2. Experimental results

[0070] (1) Physicochemical properties of PHs

[0071] like Figure 1 As shown, the oHA samples 1 The presence of C=O in the H NMR spectrum confirmed the successful synthesis of oHA ( Figure 1 b). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results showed that the size distribution of PH and PHs complexes was larger than that of miR / bPEI complexes ( Figure 1 c, d). Figure 1 As shown in e, both PH and PHs complexes exhibited an average hydrodynamic diameter of approximately 400 nm. Relative to the positively charged miR / bPEI nanocomplex (53.27 ± 1.82), the zeta potentials of PH and PHs complexes were -25.47 ± 1.06 and -18.47 ± 0.64, respectively ( Figure 1 f). The particle size of the PHs complex and miR / bPEI can remain stable for a long time ( Figure 1 g). In the gel retardation assay, the N / P ratio of miR / bPEI nanocomplex was greater than 30, showing excellent loading effect ( Figure 1 h). To confirm that the PHs complex is sufficient to protect the encapsulated miR-21 inhibitor from degradation, PHs and naked miR-21 inhibitor samples were incubated in the presence of ribonuclease (RNase). The results showed that the naked miR-21 inhibitor was rapidly degraded, while the miR-21 inhibitor present in PHs could maintain its integrity for up to 300 minutes during RNase treatment ( Figure 1 i).

[0072] These results confirmed that miR / bPEI and PHs modified with oHA, ss-31, and miR / bPEI were successfully synthesized, the PHs had uniform morphology, were highly stable, and exhibited excellent miRNA loading efficiency.

[0073] (2) Physicochemical properties of hydrogels

[0074] like Figure 2 As shown in b, 1 The H NMR spectrum showed clear characteristic resonances corresponding to the vinyl group of methacrylate (5.5-6.5 ppm) and the methyl group of methacrylic anhydride (1.8 ppm), confirming the successful grafting of methacrylic anhydride onto HA. The FTIR spectrum showed that the C=O of HA was at 1670 cm -1 The stretching vibration at 1550cm -1 This is consistent with the results of successfully prepared MeHA ( Figure 2c). The injectable self-reinforcing hydrogel was prepared according to Example 2. This hydrogel can be easily injected and exhibits good plasticity in liquid form ( Figure 2 d). Scanning electron microscopy (SEM) analysis of the microstructural characteristics of the obtained hydrogel revealed that the structure was uniform, with a pore size between 100 and 300 μm, and that the encapsulation of PHs did not adversely affect the structural properties of the hydrogel ( Figure 2 e). Figure 2 f shows that the sol-gel transition begins after about 6 min when the storage (G′) and loss (G′′) moduli are equal, eventually reaching a peak level of mechanical strength after about 6.7 h and showing a final G′ of about 700 Pa when verified by oscillatory strain sweeps ( Figure 2 g). The G′ of the hydrogel prepared without TA is slightly lower than that of the hydrogel containing TA, which confirms that the addition of TA can enhance the mechanical properties of the final hydrogel ( Figure 2 g).

[0075] Experimental Example 3: Performance Test of Nanocomposite

[0076] 1. Experimental methods

[0077] (1) Analysis of PHs cell uptake and in vivo escape

[0078] After extraction and culture of rat NP cells (NPC), 200 μL of miR / bPEI, PH, and PHs at 5 μg / mL were added to the cell culture medium in 6-well plates. The cells were then imaged by confocal laser scanning microscopy (CLSM) after 6 hours to detect the uptake of miR-21 inhibitors, and the miRNA delivery efficiency was evaluated by qPCR to analyze the miR-21 expression level. As a control, cells were transfected with miR-21 inhibitors and Lipofectamine RNAiMAX transfection reagent (Invitrogen). After incubation with PHs for 2 hours, LysoTracker red was used to stain the endosomal compartment, and DAPI (Invitrogen) was used for cell staining, and then imaged using an inverted fluorescence microscope.

[0079] (2) Analysis of intracellular ss-31 peptide release and mitochondrial protective effect

[0080] PHs containing the fluorescent ss-13 analog ss-19 (PH / ss-19) were prepared. Then, healthy or TBHP-treated NPCs were treated with PH / ss-19 or ss-19 (20 μg / mL) for 6 h, after which mitochondria were stained with MitoTracker Green (Beyotime, China) for 30 min, and cells were imaged by CLSM. The changes in mitochondrial membrane potential after different nanoparticle treatments were detected using the Mitochondrial Membrane Potential Detection Kit JC-1 (Beyotime, China). NPCs were seeded in 24-well plates and cultured for 24 h. After 4 h of TBHP treatment, the culture medium was replaced with fresh medium containing PBS, PH, or PHs, followed by incubation for 4 h. Cells were stained using JC-1 according to the manufacturer's instructions. Cells were washed twice with JC-1 wash buffer before CLSM capture. The above co-incubation operation was repeated in the same way. The intracellular ATP level was detected using an ATP content detection kit (Beijing Solarbio Technology Co., Ltd., China). After staining with MitoSOX Red (MKBio, China) and DCFH-DA (Beyotime, China) for 30 min, the intracellular ROS (cROS) and mitochondrial ROS (mROS) levels were observed under CLSM.

[0081] 2. Experimental results

[0082] like Figure 3 As shown in a, compared with miR / bPEI complexes, PH and PHs complexes coated with HA showed high uptake levels comparable to commercial liposome transfection reagents. TEM also confirmed that PHs had been successfully internalized by NPCs ( Figure 3 j). The expression level of intracellular miR-21 was analyzed by qPCR, showing the reduction of miR-21 expression in cells transfected with Lipofectamine or PHs ( Figure 3 b, c). When CD44 expression in NPCs treated with TBHP was analyzed by immunofluorescence staining and western blotting, a significant upregulation of this glycoprotein was observed after TBHP treatment relative to untreated NPCs ( Figure 3 df), consistent with its upregulation in other cells under inflammatory conditions. To further confirm the CD44 targeting ability of PH, NPCs were incubated in the presence of miR / bPEI and PH, and the consequences of this transfection were evaluated. The results showed that higher levels of Cy3-PH were found to accumulate in TBHP-stimulated NPCs in the presence of miR / bPEI and PH relative to control cells, and the addition of anti-CD44 antibodies was sufficient to disrupt this trend ( Figure 3Together, these data confirm that CD44 is expressed at high levels in the setting of inflammation-induced NPC degeneration and that CD44 upregulation may allow NPCs to better uptake HA-coated nanoparticles ( Figure 3 h).

[0083] like Figure 4 As shown in a, NPCs were incubated with PHs and miR / bPEI complexes containing FAM-miR-21 inhibitor, and lysosomal compartments were stained with LysoTracker Red. Clear colocalization between miR / bPEI complexes and lysosomes was observed, and most PHs were able to escape from these lysosomes, demonstrating the superiority of PHs over miR / bPEI in lysosomal escape. Figure 4 Flow cytometry and TUNEL staining showed that the control group showed a small number of apoptotic NPCs, while the untreated group had significantly more apoptotic dead NPCs ( Figure 4 eh).

[0084] Figure 5 a shows that ss-19 transport efficiency is higher in normal NPCs after combination with PHs compared to ss-19 alone. NPCs subjected to TBPH-induced degeneration exhibited higher levels of cytoplasmic ss-19. Loss of mitochondrial membrane potential (ΔΨm) is a hallmark of mitochondrial dysfunction, so the effect of PHs nanoparticle treatment on ΔΨm was assessed in this NPC degeneration model using JC-1 staining. The ratio of red to green fluorescence was relatively high in healthy NPCs, consistent with normal ΔΨm, whereas this ratio was reduced after TBPH treatment ( Figure 5 b). PHs treatment significantly restored the ΔΨm of these cells, whereas PH alone had little effect on this potential. Given that ROS contribute to mitochondrial stress, the levels of mitochondrial and cellular ROS (mROS and cROS) were analyzed. In response to TBHP stimulation, the levels of mROS and cROS increased significantly ( Figure 5 c) Due to the action of ss-31 peptide, PHs can effectively inhibit the production of ROS. TEM showed that TBHP treatment caused severe mitochondrial damage, including inner membrane rupture, cristae loss, and outer membrane rupture. Mitochondrial swelling and damage were still obvious in PH-treated cells, and cells treated with PHs significantly improved mitochondrial structural damage and ensured the integrity of cristae and outer membrane ( Figure 5 c). Figure 5 d shows that even after PH treatment, the levels of inflammatory markers IL-1β and Caspase-1 in TBHP-stimulated NPCs were still elevated, and the levels of IL-1β and Caspase-1 were significantly reduced after PHs treatment with ss-31.

[0085] The above results indicate that the PHs complex can be taken up at high levels in NPC cells and has significant advantages in lysosomal escape. The PHs complex can also restore the loss of mitochondrial membrane potential, effectively inhibit ROS production, and significantly reduce IL-1β and Caspase-1 levels.

[0086] Experimental Example 4: Performance test of hydrogel

[0087] 1. Experimental methods

[0088] DPPH and OH·Scavenging experiment:

[0089] Equal amounts of hydrogels with different components (hydrogel without TA, hydrogel without TA + PHs, complete hydrogel + PHs) were added into 2 mL EP tubes and incubated at 37 °C for 4 h. Then, equal amounts of supernatant were taken, added into DPPH and OH· test kits, and the absorbance was quantitatively measured to evaluate the DPPH and OH· scavenging ability.

[0090] TA and miRNA-21 in vitro release experiment:

[0091] 0.2 g of the hydrogel prepared in Example 2 was loaded into a microfiltration membrane (0.45 μm; n=3) or a dialysis bag (MWCO=8000DA; n=3), and then fully immersed in PBS (pH 7.4, pH 6.5, or pH 6.5+1 μg / mL MMP-2) at 37°C, and the supernatant samples were collected over time. The TA release rate was determined by UV-visible spectrophotometry, and the Cy3 fluorescence (Ex: 550 nm, Em: 570 nm) was determined by a Bio Synergy H1 microplate reader.

[0092] In vitro degradation experiment of hydrogel:

[0093] An equal amount of hydrogel was placed in a 2 mL EP tube and PBS (with or without MMP-2) was added. The tube was incubated at 37°C with shaking. The hydrogel supernatant was removed at different time points. The surface was wiped dry and loaded. The residual hydrogel ratio = hydrogel mass / hydrogel initial mass*100%.

[0094] Hydrogel biocompatibility experiment:

[0095] Cell viability was detected by CCK-8 method and live / dead immunofluorescence staining. The specific operation was as follows: after sterilization radiation, the hydrogel (0.2 g / ml) was immersed in complete DMEM cell culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Strep) and extracted for 24 hours. Then it was diluted to 25% with fresh complete DMEM (pH 7.4). 8000 mouse L929 fibroblasts were seeded in 96-well plates and incubated at 37°C (5% CO2) for 24 hours. Then the diluted hydrogel extract was added to the cells and then incubated for another 24 hours and 48 hours. At each time point, cell proliferation and morphology were evaluated by CCK-8 and calcein-AM / PI staining according to the kit instructions. Finally, the cell morphology was observed under a fluorescence microscope and photographed.

[0096] miRNA-21 in vivo retention experiment:

[0097] The present invention uses Cy3 to label the miR-21 inhibitor, and the hydrogel encapsulating the miR-21 inhibitor and the PH complex is used as a control group. The hydrogel / PHs and Cy3-miR-21 inhibitor preparations are implanted into the caudate nucleus intervertebral disc (Co 2 / 3, 4 / 5, 6 / 7) of the model rats, and then the Cy3-miR-21 inhibitor is detected using an IVIS spectral system (PerkinElmer, USA).

[0098] 2. Experimental results

[0099] like Figure 6 As shown in the figure, the hydrogel without TA had the lowest scavenging efficiency of DPPH and OH·. After adding PHs, the scavenging efficiency increased slightly, but it was still less than 40%. The complete hydrogel with TA (hydrogel + PHs group) showed excellent scavenging efficiency of DPPH and OH·, with a scavenging rate of more than 80% ( Figure 6 b, c), it can be seen that the addition of TA greatly improved the ROS / RNS scavenging ability of the hydrogel ( Figure 6 a).

[0100] The results of the in vitro release curves of TA and miRNA-21 showed that TA could be rapidly released from the hydrogel regardless of whether it was in an acidic environment or not, indicating that TA, as a small water-soluble molecule, could quickly move and release in the hydrogel network, and that an acidic environment (pH 6.5) could accelerate the release of TA. As the miRNA-21 inhibitor was encapsulated in PHs, PHs could be released from the hydrogel network only in the MMP-2 environment. The latter was cleaved under acidic stimulation (simulating the environment inside the cell) and further released from the dialysis bag, thereby achieving a slow and intelligent release ( Figure 6d). Therefore, the hydrogel system can achieve the effect of rapid release of TA and sustained release of miRNA-21 inhibitors on demand.

[0101] The results of the hydrogel in vitro degradation experiment showed that compared with the control group, the degradation rate of the hydrogel increased significantly after the addition of MMP-2, and the degradation was basically complete in 14 days, indicating that the hydrogel has a sensitive MMP-2 response property ( Figure 6 g).

[0102] The results of hydrogel biocompatibility showed that the complete hydrogel (hydrogel + PHs group) could largely protect L929 cells from the stimulation of TBHP and significantly improve cell activity ( Figure 6 e), reduce the number of dead cells ( Figure 6 f). Although the hydrogel without TA (the hydrogel without TA + PHs group) has a slight L929 cell protection function, the effect is obviously not as good as that of the complete hydrogel. The above results suggest that the addition of TA enables the hydrogel delivery system of the present invention to have a significant ability to remove ROS and protect cell survival activity.

[0103] The results of the miRNA-21 in vivo residual experiment showed that ( Figure 6 h, i) In the hydrogel + PHs group, the Cy3 fluorescently labeled miRNA-21 inhibitor can be retained in the body for up to 14 days thanks to the dual protection of hydrogel and PHs. In the treatment group with only hydrogel or PHs protection, the miRNA-21 inhibitor can be retained for about 7 days, while the naked miRNA-21 inhibitor is cleared in the body within 3 days.

[0104] The above results show that the hydrogel gene delivery system of the present invention has super strong protection for miRNA-21 inhibitors and can achieve a long-term sustained release effect.

[0105] Experimental Example 5: Analysis of the therapeutic effect of injectable self-reinforcing hydrogel in vivo

[0106] 1. Experimental methods

[0107] Establishment of intervertebral disc degeneration model and material implantation:

[0108] Male SD rats (300-350 g) were anesthetized by continuous inhalation of isoflurane using an animal anesthesia device. The Co 7 / 8 and 8 / 9 intervertebral discs were punctured in the center of the intervertebral disc with a 23G needle, rotated for 5 s, and maintained for 30 s. Subsequently, 20 μL of PBS, hydrogel precursor solution, PH suspension, and PH-loaded hydrogel precursor solution (NC group, hydrogel group, PH group, and hydrogel+PHs group, respectively) were injected into the intervertebral disc through a 25G needle.

[0109] Clinical efficacy observation:

[0110] X-ray and MRI scans of the coccyx were performed in vivo at different time points to observe the changes in intervertebral height, and quantitative scores were obtained based on the T2-weighted images in MRI and the degeneration grade. After the rats were sacrificed, H&E and safranin O-fast green tissue staining, as well as immunofluorescence staining and quantitative analysis of Tunel, Col II, MMP-13, and TNF-α were performed.

[0111] 2. Experimental results

[0112] The changes of degenerative intervertebral disc were observed at 4 and 8 weeks after modeling ( Figure 7 a), and the results are as follows.

[0113] X-ray results showed that compared with the negative control group (NC), the hydrogel + PHs group significantly restored the intervertebral disc height, and the level of intervertebral disc height recovery was similar to that of the normal group (control). Figure 7 b, d) Although the simple hydrogel group or PHs group increased the intervertebral disc height to a certain extent, the repair ability was limited.

[0114] MRI results showed that the hydrogel + PHs group could significantly increase the water content of the nucleus pulposus tissue after modeling (signal intensity increased significantly), and the MRI score was close to that of the normal group ( Figure 7 c, e), indicating that the hydrogel and PHs play a full role in the repair process of nucleus pulposus tissue.

[0115] The results of H&E and Safranin O-Fast Green tissue staining showed that after treatment in the hydrogel + PHs group, the nucleus pulposus tissue and the annulus fibrosus tissue were well demarcated, and the structure inside the nucleus pulposus tissue was clear. In the NC group, the hydrogel group, or the PHs treatment group, the boundaries between the nucleus pulposus tissue and the annulus fibrosus were blurred and difficult to identify. The tissue score of the hydrogel + PHs group was the highest, close to that of the normal group ( Figure 7 f, g).

[0116] The results of immunofluorescence staining of Tunel, Col II, MMP-13 and TNF-α showed that after treatment in the hydrogel + PHs group, the proportion of Tunel-positive cells was the lowest (i.e., the lowest apoptotic activity of nucleus pulposus cells), the expression of Col II, a representative protein of ECM synthesis, was the highest, and the expression of MMP-13, a representative protein of ECM degradation, and TNF-α, a representative protein of inflammatory factors, was the lowest ( Figure 7 f, hl).

[0117] The above results show that under the dual synergistic effect of hydrogel and PHs, the degenerated nucleus pulposus tissue can effectively reproduce the ECM metabolic balance, inhibit the inflammatory response and the apoptotic activity of nucleus pulposus cells, and jointly achieve the regeneration and repair effect of the degenerated nucleus pulposus from multiple aspects.

[0118] In summary, the present invention provides a nanocomposite, an injectable self-enhancing hydrogel gene delivery system, and a preparation method and use thereof. The present invention uses mitochondrial targeted antioxidant peptides and hyaluronic acid to coat miR-21 inhibitors and branched polyethyleneimine complexes to obtain a nanocomposite. The present invention further combines the nanocomposite with a hydrogel containing MMP-2-substrate peptides and tannic acid to obtain an injectable self-enhancing hydrogel gene delivery system. The nanocomposite and the injectable self-enhancing hydrogel gene delivery system can inhibit inflammation, scavenge reactive oxygen species, inhibit apoptosis of nasopharyngeal carcinoma cells, and regulate the metabolic homeostasis of the extracellular matrix in the targeted IVD, and have good application prospects.

Claims

1. An injectable self-reinforcing hydrogel, characterized in that: The invention is prepared in water with nanocomplex, methacrylated hyaluronic acid, matrix metalloproteinase and tannic acid as raw materials; the matrix metalloproteinase is MMP-2-substrate peptide; the mass ratio of MMP-2-substrate peptide to tannic acid is 1-3:1; the mass ratio of MMP-2-substrate peptide to methacrylated hyaluronic acid is 10:3-7; the mass ratio of nanocomplex to methacrylated hyaluronic acid is 1:30-50; the nanocomplex is prepared with oxidized hyaluronic acid, miRNA inhibitor, polyethyleneimine and mitochondrial targeted antioxidant peptide as raw materials, wherein the ratio of the number of amino groups in polyethyleneimine to the number of phosphate groups in miRNA inhibitor is 20-40:1; the ratio of the number of H-derived carboxylic acid groups in oxidized hyaluronic acid to the number of phosphate groups in miRNA inhibitor is 5-15:1; the molar ratio of mitochondrial targeted antioxidant peptide to miRNA inhibitor is 1:1-3.

2. The injectable self-reinforcing hydrogel according to claim 1, characterized in that: The ratio of the number of amino groups in the polyethyleneimine to the number of phosphate groups in the miRNA inhibitor is 30:1; the ratio of the number of H-derived carboxylic acid groups in the oxidized hyaluronic acid to the number of phosphate groups in the miRNA inhibitor is 10:1; and the molar ratio of the mitochondrial targeted antioxidant peptide to the miRNA inhibitor is 1:

2.

3. The injectable self-reinforcing hydrogel according to claim 1, characterized in that: The miRNA inhibitor is a miR-21 inhibitor; The polyethyleneimine is branched polyethyleneimine; The mitochondria-targeted antioxidant peptide is Szeto-Schiller 31; The preparation method of oxidized hyaluronic acid comprises the following steps: hyaluronic acid, water and an oxidant react under light-proof conditions, dialyze, and freeze-dry to obtain oxidized hyaluronic acid.

4. The injectable self-reinforcing hydrogel according to claim 1, characterized in that: The preparation method of the nanocomposite comprises the following steps: (1) reacting the miRNA inhibitor with polyethyleneimine to prepare a complex; (2) The oxidized hyaluronic acid, the complex and the mitochondria-targeted antioxidant peptide are reacted to obtain a nanocomplex.

5. The injectable self-reinforcing hydrogel according to claim 4, characterized in that: In step (1), the reaction temperature is 10 to 40° C. and the reaction time is 0.5 to 1.5 hours; In step (2), the reaction conditions are: incubation for 3 to 5 hours under nitrogen with constant stirring.

6. The injectable self-reinforcing hydrogel according to claim 5, characterized in that: In step (1), the reaction temperature is 15-35° C. and the reaction time is 1 hour; In step (2), the reaction conditions are: incubation for 4 hours under nitrogen with constant stirring.

7. A method for preparing the injectable self-reinforcing hydrogel according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: mixing the nanocomplex, methacrylated hyaluronic acid, MMP-2-substrate peptide and tannic acid, adjusting pH, and incubating to obtain the product.

8. The method according to claim 7, characterized in that The pH value is adjusted to a range of 7 to 8, the incubation temperature is 35 to 45° C., and the incubation time is 20 to 30 hours.

9. Use of the injectable self-reinforcing hydrogel according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating intervertebral disc degeneration.

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