A composite biomaterial loaded with multi-modal reprogramming therapeutic agents, methods of making and use thereof in the treatment of osteoarthritis

Abstract

The application provides a composite biomaterial loaded with multi-mode reprogramming therapeutic agents, a preparation method thereof and application thereof in treating osteoarthritis, and belongs to the technical field of biological medical materials, drug delivery, regenerative medicine and epigenetics. The composite biomaterial comprises sodium isoamyl barbital, an epigenetic reprogramming small molecule VC6TF and a lipid nanoparticle encapsulating a reprogramming factor mRNA. The composite material has a precise construction idea and a systematic in-vivo and in-vitro performance and safety evaluation system, so that it can treat osteoarthritis by precisely reversing the epigenetic age of joint cells.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials, drug delivery, regenerative medicine and epigenetics, and particularly to a composite biomaterial loaded with a multimodal reprogramming therapeutic agent, its preparation method and its application in the treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a chronic, disabling disease characterized by degenerative changes in articular cartilage, inflammation of the synovium, and osteophyte formation. Current mainstream treatments (such as NSAIDs and glucocorticoid injections) can only provide palliative relief of symptoms and cannot stop or reverse the disease progression.

[0003] Recent studies have revealed two key pathological mechanisms: first, early chondrocyte apoptosis caused by mitochondrial dysfunction and excessive reactive oxygen species (ROS); and second, senescence of joint cells (chondrocytes and synovial cells) and the related loss of epigenetic information, which are among the fundamental factors driving the progression of osteoarthritis (OA). For the former, amobarbital sodium, as a reversible inhibitor of mitochondrial complex I, can effectively reduce ROS bursts and protect cell viability. However, its high lipid solubility and rapid systemic distribution make it difficult to maintain a long-term effective therapeutic concentration within the joint cavity. For the latter, some epigenetic reprogramming techniques (such as using Yamanaka factor OSK or combinations of small chemical molecules) have been shown to reverse the epigenetic age of cells and restore their function without altering their cell identity, providing a revolutionary approach to the disease modification therapy of OA.

[0004] However, existing technologies have significant bottlenecks: Limitations of monotherapy: Sodium amobarbital alone cannot reverse established cellular senescence phenotypes; and reprogramming intervention alone may not adequately address the acute damage caused by ROS bursts in the early stages of OA.

[0005] Delivery and stability challenges: Whether it's small molecule drugs (such as sodium amobarbital), small molecule reprogrammed compounds, or large molecule reprogrammed factors (such as mRNA), they all face problems such as rapid clearance, degradation, or instability within the joint cavity. Physically mixed drug delivery methods struggle to achieve uniform drug distribution, stable protection, and synergistic controlled release.

[0006] The safety window is unknown: reprogramming intervention carries the risk of "over-reprogramming," leading to loss of cell identity or tumorigenesis. For OA treatment, the extent of epigenetic reversal (i.e., the "treatment window") that enables effective tissue repair and is absolutely safe has not yet been clearly defined in vivo.

[0007] Lack of integrated platform: There is currently a lack of a standardized delivery and evaluation platform that can combine "acute phase cell protection" (through drugs) with "long-term aging reversal" (through reprogramming) and systematically compare and optimize different reprogramming strategies (small molecules vs. genes, different factor combinations).

[0008] Therefore, there is an urgent need in this field for an innovative biomaterial and delivery system that can synergistically load active ingredients with multiple mechanisms of action, solve the problems of stability and targeted delivery, and establish safe and effective synergistic treatment plans and precise epigenetic reversal targets through rigorous in vivo studies. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a precise method for constructing composite hyaluronic acid hydrogel materials that can synergistically load and stably deliver small molecule drugs, reprogrammed small molecules, and reprogramming factor nucleic acids.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a composite biomaterial loaded with a multimodal reprogramming therapeutic agent, the composite biomaterial comprising sodium amobarbital, the epigenetic reprogramming small molecule VC6TF, and lipid nanoparticles encapsulating reprogramming factor mRNA.

[0011] Preferably, the epigenetic reprogramming small molecule VC6TF includes four or five of the following: valproic acid, CHIR99021, E-616452, transphenylcyclopropane, and trichomoniasis.

[0012] Preferably, the reprogramming factor mRNA includes at least three of OCT4, SOX2, KLF4, GLIS1, and LIN28, and does not include c-MYC.

[0013] This invention also provides a method for preparing the composite biomaterial loaded with the multimodal reprogramming therapeutic agent, wherein sodium amobarbital solution, epigenetic reprogramming small molecule VC6TF solution and lipid nanoparticle solution encapsulating reprogramming factor mRNA are added sequentially to a gel matrix, the mixed complex is filtered to remove bacteria, and the pH is adjusted to obtain the composite biomaterial loaded with the multimodal reprogramming therapeutic agent.

[0014] Preferably, the hyaluronic acid used to prepare the gel matrix has a molecular weight of 500~3200kDa and a swelling time of ≥7h.

[0015] Preferably, the final concentration of sodium amobarbital in the gel matrix is ​​0.5~1.5 mg / mL; The final concentrations of the epigenetic reprogramming small molecule VC6TF in the gel matrix were 150-350 μM valproic acid, 5-20 μM CHIR99021, 5-20 μM E-616452, 2-10 μM transphenylcyclopropane, and 25-100 μM trichomoniasis. The total final concentration of reprogramming factor mRNA in the lipid nanoparticles encapsulating reprogramming factor mRNA in the gel matrix is ​​20~100 μg / mL; the molar ratio of the reprogramming factor mRNA is OCT4:SOX2:KLF4:GLIS1:LIN28=(1.5-6):1:1:1:1.

[0016] Preferably, the method for preparing the lipid nanoparticles encapsulated with reprogramming factor mRNA is as follows: a lipid ethanol solution and an mRNA aqueous solution are mixed in a microfluidic manner to obtain a primary LNP suspension; the primary LNP suspension is then concentrated by dialysis and ultrafiltration to obtain lipid nanoparticles encapsulated with reprogramming factor mRNA.

[0017] Preferably, the lipid ethanol solution comprises lipid components in the following molar ratio: 45%~55% DLin-MC3-DMA, 8%~15% DSPC, 35%~45% cholesterol, and 1.0%~2.5% DMG-PEG2000; the lipid components are dissolved in anhydrous ethanol to obtain a lipid ethanol solution with a total lipid concentration of 8~15 mM; The total concentration of reprogramming factor mRNA in the lipid nanoparticles is 0.3~1.0 mg / mL.

[0018] The present invention also provides the application of the composite biomaterial loaded with the multimodal reprogramming therapeutic agent or the composite biomaterial loaded with the multimodal reprogramming therapeutic agent prepared according to the preparation method in the preparation of a medicament for treating osteoarthritis.

[0019] Preferably, the extent to which the composite biomaterial reverses the epigenetic age of joint cells is controlled between 57% and 77%, and it does not induce abnormal proliferation or loss of cell identity in joint tissues.

[0020] This invention provides a composite biomaterial loaded with a multimodal reprogramming therapeutic agent. Using hyaluronic acid (HA) hydrogel as a carrier, it can simultaneously load and stably deliver a small molecule therapeutic drug (sodium amobarbital), the epigenetic reprogramming small molecule VC6TF, and reprogramming factor mRNA. Its precise construction approach and systematic in vitro and in vivo performance and safety evaluation system enable it to treat osteoarthritis (OA) by precisely reversing the epigenetic age of joint cells.

[0021] This invention validates the material's performance through a comprehensive evaluation system encompassing physicochemical properties, in vitro drug release, in vivo pharmacodynamics / pharmacokinetics, epigenetic effects, and systemic safety. This composite material achieves sustained drug release and protection, significantly prolonging the intra-articular retention of the active ingredient. Validated in an osteoarthritis (OA) animal model, the material exhibits synergistic therapeutic effects in "cell protection" and "aging reversal," and for the first time, clearly defines a window of epigenetic age reversal (57%–77%) that combines highly efficient repair with high safety for intra-articular treatment. Based on this window of epigenetic age reversal, precise and safe treatment options for osteoarthritis can be provided. Attached Figure Description

[0022] Figure 1 A simplified flowchart of the invention. Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] Preparation of composite biomaterials

[0026] a) Matrix preparation: 1.0 g of hyaluronic acid (Zimmer Biomet, Warsaw, IN) with a weight average molecular weight of 1.5 MDa was dissolved in 100 mL of pre-cooled sterile phosphate buffer (PBS, pH 7.4). The mixture was stirred and swollen for 16 h under light-protected, inert gas (nitrogen) protection and at a speed of 1000 rpm (which can be selected from 800 to 1500 rpm; 1000 rpm was used in this example) to form a homogeneous, transparent HA-based gel with suitable viscoelasticity.

[0027] b) Formulation or preparation of active ingredients

[0028] i. Sodium amobarbital (Bausch Health, Bridgewater, NJ) solution, prepared with PBS to a final gel concentration of 0.775 mg / mL.

[0029] ii. Epigenetic reprogramming small molecule VC6TF (or C1) solution, with the following components in the gel: 250 μM valproic acid (VPA), 10 μM CHIR99021, 10 μM E-616452 (RepSox), 5 μM tranylcypromine, and 50 μM forskolin, in PBS containing 5% DMSO, all purchased from Sigma-Aldrich.

[0030] iii. Preparation of lipid nanoparticles encapsulating reprogramming factor mRNA (OSKGL mRNA-LNP): 1. Lipid Ethanol Solution: Accurately weigh the following molar ratios: DLin-MC3-DMA (cationic lipid) 50%, DSPC (auxiliary lipid) 10%, cholesterol 38.5%, and DMG-PEG2000 (PEG-modified lipid) 1.5%. Dissolve all four lipids together in anhydrous ethanol, vortex and gently heat (e.g., 37°C water bath) to aid dissolution, and prepare a clear lipid ethanol solution with a total lipid concentration of 10 mM. Aliquot and store at -80°C protected from light.

[0031] 2. Aqueous mRNA phase: Chemically modified OSKGL mixed mRNA (OCT4, SOX2, KLF4, GLIS1, and LIN28 mRNA mixed in an equimolar ratio of 3:1:1:1:1) was dissolved in 10 mM sodium citrate buffer (pH 4.0) to prepare an aqueous mRNA solution with a total concentration of 0.2 mg / mL. The solution was then placed on ice for later use.

[0032] 3. Microfluidic Mixing: A commercially available staggered herringbone microfluidic chip was used. The total flow rate was set to 3 mL / min, and the flow rate ratio of the lipid ethanol phase to the mRNA aqueous phase was 1:3 (i.e., ethanol phase 0.75 mL / min, aqueous phase 2.25 mL / min, final mixing volume ratio 1:3). The two phase solutions were injected into the two inlets of the chip via a syringe pump, and instantaneously mixed within the chip channels. The milky white dispersion collected at the outlet is the primary LNP suspension.

[0033] 4. Dialysis and Concentration: Immediately transfer the primary LNP suspension to a dialysis bag with a molecular weight cutoff of 100 kDa. Dialyze with 4 L of 1× PBS buffer (pH 7.4) at 4°C. Change the dialysate every 30 minutes for the first 2 hours; then continue dialysis for 2 hours. After dialysis, concentrate the mRNA by centrifugation at 2000 g at 4°C using a 100 kDa MWCO ultrafiltration centrifuge tube until the mRNA concentration reaches 0.5 mg / mL.

[0034] 5. Sterilization and Characterization: The concentrated LNP suspension was aseptically filtered through a 0.22 μm PES membrane. Sampling and Measurement: Dynamic light scattering (DLS) showed an average particle size of (102 ± 5) nm and a polydispersity index (PDI) of 0.12; the encapsulation efficiency was determined to be (98.5 ± 1.2)% using the RiboGreen kit.

[0035] c) Active ingredient compounding: Under low temperature conditions (2~8°C, 4°C in this example) on a magnetic stirrer, stir at 300 rpm for 2 hours. Slowly add the solutions or dispersions of the three active ingredients sequentially to the HA-based gel obtained in step a) (containing 0.775 mg / mL sodium amobarbital, 250 μM valproic acid, 10 μM CHIR99021, 10 μM E-616452, 5 μM transphenylcyclopropane, 50 μM salvia miltiorrhizin, and 50 μg / mL OSKGL-mRNA), ensuring thorough mixing without bubbles. Filter the mixed complex through a 0.22 μm PES membrane and adjust its pH to 7.2 with sterile NaHCO3 solution (the pH can be selected within the range of 6.5~7.8). Aseptically dispense the final product into pre-filled syringes and store at 2-8°C.

[0036] Example 2

[0037] Stability verification of sodium amobarbital

[0038] The composite biomaterial prepared in Example 1 and a free sodium amobarbital solution (prepared according to step b of Example 1) were placed in a 60°C accelerated stability test chamber. Samples were taken at 0, 12, 24, and 48 hours, and three parallel samples (n=3) were tested at each time point to determine the sodium amobarbital content. The results are shown in Tables 2 and 3. The content of the free drug decreased to about 75% of the initial value after 48 hours, while the drug content in the composite gel remained above 95%, demonstrating that the HA hydrogel matrix has a significant stabilizing effect on sodium amobarbital.

[0039] Methods: HPLC-UV method was used for the quantitative analysis of sodium amobarbital. Chromatographic conditions: A C18 column was used with methanol-water (containing 0.1% trifluoroacetic acid) as the mobile phase, isocratic elution, a flow rate of 0.3 mL / min, and a detection wavelength of 220 nm. This method was validated, and its specificity, linearity, precision, and accuracy all met the requirements of ICH Q2(R1) guidelines.

[0040] Table 1: Key Indicators for Validation of the HPLC-UV Method (used for content determination in this example)

[0041] Table 2. Accelerated stability test results of sodium amobarbital (60℃, ambient humidity, protected from light)

[0042] Table 3. Results of stability studies on multiple batches of final products

[0043] Notes: 1. Temperature, humidity, and light exposure: According to standard practice in drug stability studies, long-term and intermediate-term condition studies are typically conducted under light-protected and humidity-controlled conditions. The light exposure conditions (4500±500 lx) in the influencing factors study are typical requirements of the ICH guidelines. The conclusion that "the product is light-stable" means that it needs to be protected from light during routine storage and use, but is stable under short-term exposure to indoor light. 2. The "Preset Standards / Compliance Ranges" in the table are set based on the quality requirements for sterile preparations in the pharmacopoeia and ICH guidelines.

[0044] Example 3

[0045] Animal Model and Grouping: An osteoarthritis (OA) model was induced in SD rats via medial meniscectomy (MNX). Four weeks post-surgery, rats were randomly divided into six groups (n=10): sham-operated group (HA gel), model control group (HA gel), sodium amobarbital monotherapy group (HA gel loaded), C1 small molecule group (HA gel loaded), OSKGL mRNA-LNP monotherapy group (HA gel loaded), and the composite biomaterial group from Example 1. All groups were prepared according to the method described in Example 1, with the same final concentration of the active ingredient. A single intra-articular injection of 50 μL of the corresponding formulation was administered. Animals were sacrificed on day 28 post-administration.

[0046] a) Pain was assessed by weight-bearing tests, OARSI scores were obtained by histological sections, and the proportion of p16+ senescent cells was detected by immunohistochemistry.

[0047] I. Weight-bearing test to assess pain

[0048] 1. Experimental Design

[0049] Instrument: Dynamic load testing system (IITC Life Science Incapacitance Tester).

[0050] Principle: This method utilizes a high-sensitivity sensor platform to measure the weight distribution of rats in their hind limbs in real-time and non-invasively during free movement. Pain caused by osteoarthritis leads animals to instinctively reduce weight-bearing on the affected limb.

[0051] Testing environment: a quiet behavioral laboratory with soundproofing and dim lighting. Animals were allowed to acclimatize to the environment for 30 minutes before the test.

[0052] Test process: 1) Place the rats individually in the test chamber (the bottom is the sensor platform).

[0053] 2) Allow the animal to explore freely and stand upright for 5 minutes.

[0054] 3) The system software automatically records the cumulative load value of the left and right hind limbs within this 5-minute period (unit: grams × seconds).

[0055] 4) Each animal was tested three times, with an interval of at least one hour between each test, and the average value was used for analysis.

[0056] Evaluation indicators: Weight-bearing difference of the affected limb: (weight-bearing value of the healthy limb) - (weight-bearing value of the affected limb). The larger this value is, the more severe the asymmetry in weight-bearing caused by pain.

[0057] Weight-bearing ratio of the affected limb: (weight-bearing value of the affected limb) / (total weight-bearing value of both hind limbs) × 100%. This ratio is close to 50% in normal healthy rats, and significantly decreased in OA model rats.

[0058] 2. Evaluation Results

[0059] Table 4. Difference in weight-bearing capacity of affected limbs in rats from different treatment groups (unit: grams × seconds)

[0060] Table 5. Weight-bearing ratio and pain improvement in the affected limb of rats in different treatment groups.

[0061] The improvement rate in Table 5 is calculated as follows: Improvement rate = [(weight-bearing ratio of treatment group - weight-bearing ratio of model group) / (weight-bearing ratio of sham surgery group - weight-bearing ratio of model group)] × 100%; Taking the composite group as an example: [(46.3 - 30.2) / (49.5 - 30.2)]×100% ≈ 83.4%.

[0062] Conclusion: The combined therapy of this invention can significantly restore the weight-bearing capacity of the affected limb, with an improvement rate of 83.4%, which is close to the normal level of sham surgery. Moreover, the effect is significantly better than any single therapy group (p < 0.01), which directly proves its excellent synergistic effect of pain relief and functional repair.

[0063] II. OARSI Scoring Criteria and Methods

[0064] 1. Scoring System

[0065] This study used the internationally recognized OARSI (Osteoarthritis Research Society International) histological scoring system to quantitatively assess cartilage lesions in the rat knee joint.

[0066] 2. Section preparation and staining

[0067] After fixation, decalcification, and paraffin embedding, knee joint specimens were sectioned into 5 μm thick sections along the sagittal plane. The sections were stained with hematoxylin and eosin (H&E) and safranin O-fast green to assess cell morphology and proteoglycan content, respectively.

[0068] 3. OARSI Histological Scoring System (for rat models)

[0069] 3.1 Scoring Principles

[0070] For each knee joint, two regions, the medial tibial plateau and the medial femoral condyle, are scored separately. The sum of the scores for the two regions is the OARSI total score for that knee joint, with a total score range of 0-12.

[0071] 3.2 Detailed scoring rules for individual body parts

[0072] Table 6 below describes the lesion severity levels and corresponding scores for individual scoring sites (such as the tibial plateau or femoral condyle): Table 6 Scoring Details

[0073] Total score calculation: The score range for each site (tibial plateau / femoral condyle) is 0-6 points, and the total score for both sites ranges from 0-12 points. The higher the score, the more severe the osteoarthritis pathology.

[0074] 4. OARSI scoring results

[0075] Table 7. Joint OARSI scores and improvement rates in rats of different treatment groups

[0076] OARSI score improvement rate calculation formula: Improvement rate = [(Model group score - Treatment group score) ÷ (Model group score - Sham surgery group score)] × 100%; Taking the compound group as an example: Improvement rate = [(9.8-1.8) ÷ (9.8-0.5)] × 100%, = (8.0 ÷ 9.3) × 100% ≈ 86.0%; Conclusion: The combination therapy of the present invention can significantly improve cartilage pathology, with an OARSI score improvement rate of 86.0%, which is significantly better than any single therapy group (p < 0.01).

[0077] 5. Calculation and analysis of synergistic effect coefficient

[0078] To quantitatively assess whether the combination of the three drugs produces a synergistic effect, rather than just an additive effect, the Bliss independence model was used to calculate the Coefficient of Drug Interaction (CDI).

[0079] 1. Calculation Principle

[0080] Assuming the effects of each drug are independent, the expected effect of combined drug use (E) exp ) for: E exp =1-(1-E A )×(1-E B )×(1-Ec) Among them, EA, EB, and EC represent the improvement rates (improvement rate / 100) of each monotherapy group.

[0081] Real effect (E) obs () represents the improvement rate measured in the combined treatment group.

[0082] The Coefficient of Synergy (CDI) is defined as: CDI = E obs / E exp ; CDI > 1.15: indicates a strong synergistic effect; 1 < CDI ≤ 1.15: indicates a weak synergistic or additive effect; CDI < 1: indicates an antagonistic effect.

[0083] 2. Calculation process

[0084] Based on the data in Table 7, the improvement rate is converted into an improvement percentage: EA (sodium amobarbital) = 0.280 EB (C1 small molecule) = 0.215, EC(OSKGL mRNA-LNP) = 0.314, Eobs (Example 1 Composite Biomaterial Group) = 0.860.

[0085] Calculate the expected effects: Eexp=1-(1-0.280)×(1-0.215)×(1-0.314)=1-(0.720×0.785×0.686)=1-0.387=0.613, that is, the expected improvement rate is 61.3%.

[0086] Calculate the synergistic effect coefficient: CDI = 0.8600.613 ≈ 1.40.

[0087] Conclusion: The synergistic effect coefficient (CDI) was 1.40, significantly greater than 1.15, demonstrating that the combined use of sodium amobarbital, C1 small molecules, and OSKGL mRNA-LNP produced a strong synergistic therapeutic effect in improving OA cartilage pathology, rather than a simple additive effect. This provides crucial quantitative evidence for the core advantage of this invention—"synergistic treatment, root cause intervention."

[0088] III. Immunohistochemical detection of the proportion of p16+ senescent cells

[0089] 1. Experimental Design

[0090] Sample preparation: On day 28 after drug administration, rat knee joints were taken, fixed, decalcified, and embedded in paraffin. 5μm thick serial sections were prepared along the sagittal plane.

[0091] Immunohistochemical staining: 1. Antigen retrieval: The sections were subjected to heat-mediated antigen retrieval in citrate buffer (pH 6.0).

[0092] 2. Sealing: Use 3% BSA to seal at room temperature for 30 minutes.

[0093] 3. Primary antibody incubation: Add mouse anti-rat p16INK4a monoclonal antibody (Abcam, 1:200 dilution) and incubate overnight at 4°C.

[0094] 4. Secondary antibody incubation: Add HRP-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 1 hour.

[0095] 5. Staining and Counterstaining: The cell nuclei were counterstained with hematoxylin using a DAB staining kit.

[0096] 6. Mounting observation: Mount with dehydrated, clear, neutral resin.

[0097] Image Analysis and Quantitative Analysis: 1. Image acquisition: Under a 200x optical microscope, five non-overlapping fields of view were randomly selected from the weight-bearing area of ​​the articular cartilage for each slice and photographed.

[0098] 2. Cell counting: Using ImageJ software, count cells manually or semi-automatically in each field of view. p16 positive cell count: chondrocytes with nuclei stained brownish-yellow or brownish-red; Total chondrocyte count: All chondrocytes in the field of view (nuclei stained blue with hematoxylin).

[0099] 3. Proportion Calculation: Percentage of p16+ cells in each field of view = (Number of p16-positive cells / Total number of chondrocytes) × 100% The proportion of p16+ cells in each animal is the average of the proportions in the animal's five fields of view.

[0100] Evaluation metrics: percentage of p16+ senescent cells (%) and clearance rate.

[0101] 2. Evaluation Results

[0102] Table 8. Proportion and clearance rate of p16+ senescent cells in articular cartilage of different treatment groups

[0103] The clearance rate is calculated as follows: [(Model group proportion - Treatment group proportion) / (Model group proportion - Sham surgery group proportion)] × 100%; Taking the composite group as an example: [(65.8 - 9.8) / (65.8 - 3.2)] × 100% ≈ 89.5% (consistent with the conclusion of >85% in Table 8).

[0104] Conclusion: The composite material of this invention can efficiently remove senescent cells in articular cartilage, with a clearance rate of over 85%, significantly superior to each single-drug group. This provides direct cellular evidence for "aging reversal" and is highly consistent with the results of epigenetic age reversal (66%) and pathological score improvement (>80%), together forming a complete chain of evidence for the invention's "active reversal of aging and achievement of disease modification".

[0105] The above experimental results show that the compound group of the present invention is significantly superior to any single-drug group or dual-drug group in terms of weight balance, improvement of OARSI score (improvement rate >80%) and clearance of senescent cells (clearance rate >85%) (p < 0.01), and proves that there is a synergistic effect among the active ingredients.

[0106] b) Epigenetic analysis: Articular cartilage was obtained by microdissection, DNA was extracted and whole-genome methylation sequencing was performed, epigenetic age was calculated using the established chondrocyte-specific DNA methylation clock, and the reversal magnitude relative to the model control group was calculated.

[0107] Table 9. Epigenetic window period data in the rat MNX-OA model.

[0108] in conclusion: 1. Window period effectiveness: The epigenetic age reversal rate in the G6 group was concentrated in 66%±5%, which fell entirely within the preset window of 57%-77%. At this time, the best cartilage repair effect and the highest senescent cell clearance rate were achieved, and there were no safety issues.

[0109] 2. Window period boundary verification: Lower limit verification: The reversal rate (<57%) in groups G3 and G4 was accompanied by insufficient tissue repair.

[0110] Upper limit verification: Although the reversal rate of G7 group (>77%) was acceptable in some indicators, it triggered a clear security event (abnormal tissue proliferation).

[0111] Verification of the necessity of the carrier: Due to the lack of sustained-release carrier, the reversal amplitude in group G8 was discrete and the mean did not reach the window, the efficacy was unstable and accompanied by inflammation, which proves that the HA hydrogel carrier is indispensable for achieving "precise window treatment".

[0112] 3. Safety: Under the preferred embodiment (G6) of the present invention, no treatment-related local or systemic adverse reactions were observed.

[0113] Key findings: Effective treatment is accompanied by a significant reversal of the epigenetic age of chondrocytes. When the reversal rate is below approximately 57%, tissue repair is insufficient; when the reversal rate is above approximately 77%, signs of safety risks, such as abnormal tissue structure or ectopic cell clusters, are observed. Therefore, precisely controlling the epigenetic age reversal rate within the range of 57% to 77% (preferably 60%-75%) is crucial for achieving safe and efficient OA treatment. The preferred combination of this invention can stably control the reversal rate within the median range of this "golden window" (e.g., 66% ± 5%).

[0114] Conclusion: The combined treatment regimen of this invention can stably guide treatment to the safe and efficient "golden window," achieving the best treatment benefit-risk ratio.

[0115] Example 4

[0116] Determination of key physicochemical properties and regulatory effects on chondrocyte epigenetic age and OA pathology of HA hydrogel platforms loaded with different combinations of reprogramming factors.

[0117] HA hydrogel platforms loaded with different combinations of reprogramming factors were prepared according to Example 1. The determination and calculation of the epigenetic age reversal magnitude, OARSI score improvement rate (vs. G2), and p16+ senescent cell clearance rate were performed according to Example 3.

[0118] The results of the physicochemical property determination are shown in Table 10, and the regulatory effects on the epigenetic age of chondrocytes and OA pathology are shown in Table 11.

[0119] Table 10: Key physicochemical properties of HA hydrogel platforms with different combinations of reprogramming factors

[0120] Conclusion: This preparation process ensures that hydrogels loaded with different combinations of factors have a highly consistent physicochemical basis, eliminating the interference caused by differences in formulations to subsequent biological comparisons.

[0121] Table 11. Regulatory effects of different combinations of reprogramming factors on epigenetic age of chondrocytes and OA pathology.

[0122] Example 5: In vivo pharmacokinetic study

[0123] 1. Experimental Objective

[0124] The pharmacokinetic characteristics of the composite biomaterial of the present invention in animal models were evaluated, including the drug release delay effect and tissue distribution characteristics, and its local retention and system safety were verified.

[0125] 2. Laboratory animals and grouping

[0126] (1) Beagle dog model (for plasma pharmacokinetics study): Healthy adult beagle dogs, weighing 8-12 kg, half male and half female, were divided into the composite gel group of this invention (loaded with sodium amobarbital, VC6TF small molecule and OSKGL-mRNA-LNP) and the free mixed control group (containing the same three active ingredients but without gel matrix, i.e. the G8 group formulation in Example 3), with n=6 in each group.

[0127] (2) SD rat model (for tissue distribution study): Healthy adult SD rats, weighing 200~250 g, were divided into the composite gel group of this invention and the free sodium amobarbital solution group, with n=8 in each group.

[0128] 3. Dosing regimen

[0129] Intra-articular injection in one knee. The administration volume was 0.5 mL / joint for beagle dogs and 50 µL / joint for SD rats. The final concentration of sodium amobarbital was 0.775 mg / mL (consistent with Example 1).

[0130] 4. Sample Collection and Processing

[0131] (1) Beagle plasma samples: Blood was collected from the forelimb vein at 0.083 (5 min), 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 48 h after administration, and the plasma was separated and stored at -80℃.

[0132] (2) Tissue samples from SD rats: Local tissues (synovium, ligaments, meniscus) of the treated joints and major organs such as heart, liver, spleen, lung, kidney, and brain were collected at 4 h and 24 h after drug administration. After homogenization, the supernatant was collected and stored at -80℃.

[0133] 5. Detection Methods

[0134] The concentration of sodium amobarbital in the sample was determined using a validated HPLC-MS / MS method. A C18 column was used with gradient elution of 0.1% formic acid-water-acetonitrile mobile phase. Electrospray ionization (ESI+) was employed in positive ion mode, with the monitored ion pair being m / z 219.1 → 162.1. This method complies with relevant guidelines.

[0135] 6. Data Analysis

[0136] Pharmacokinetic parameters were calculated using a non-compartmental model. Tissue distribution results are expressed as drug concentrations in each tissue.

[0137] 7. Experimental Results

[0138] The experimental data are summarized in the table below (Table 12).

[0139] Table 12 In vivo pharmacokinetic data

[0140] Example 6: Toxicology / Safety Evaluation Study

[0141] To ensure the preclinical safety of the composite biomaterial of this invention for intra-articular injection, we systematically conducted the following safety evaluation studies in accordance with Good Laboratory Practice (GLP) and related guidelines (such as ICH S4, S6, and S8).

[0142] 1. Repeated-dose toxicity studies (rodents and non-rodents)

[0143] 1.1 Repeated-dose toxicity test in SD rats

[0144] Objective: To assess the potential toxicity of the material under short-term repeated intra-articular administration and to determine the no-observed-adverse-effect level (NOAEL).

[0145] Animals and grouping: Healthy SD rats, half male and half female, weighing 180-220g, were randomly divided into a solvent control group (blank HA gel) and a test sample group (composite gel loaded with active ingredient, concentration as in Example 1). The number of animals in each group was sufficient to meet statistical requirements (usually n=10 / sex / group).

[0146] Dosage regimen: Once weekly for three consecutive weeks, intra-articular injection in one knee. The injection volume was determined based on species conversion to ensure that the drug exposure (based on body surface area) reached a sufficient multiple of the expected clinical dose. The injection concentration in the test group was calculated based on the saturation concentration of sodium amobarbital (0.775 mg / mL).

[0147] Observation and testing: Clinical observation: Observe the animal's general condition, behavior, feeding, and feces twice a day.

[0148] Weight and food intake: Recorded twice a week.

[0149] Hematology and serum biochemistry: Blood was collected 24 hours after the last administration and after the recovery period for complete blood cell count and biochemical indicators such as coagulation function, liver and kidney function, and electrolytes.

[0150] Gross anatomy and histopathology: Animals were euthanized 24 hours after the last administration and at the end of the recovery period for a full autopsy to observe for any abnormalities in the organs. Special attention was paid to collecting samples from the injection joints (including synovium, cartilage, and ligaments), local lymph nodes, and major organs such as the heart, liver, spleen, lungs, kidneys, and brain. These samples were then paraffin-embedded, sectioned, and stained with H&E. Pathologists reviewed the slides blinded.

[0151] Key Results and Conclusions: In all animals, the test substance was well tolerated at the maximum saturation concentration (0.775 mg / mL), and no toxic reactions related to the test substance were observed. Based on this, the NOAEL for SD rats was determined to be 0.775 mg / mL.

[0152] 1.2 Repeated-dose toxicity test in beagle dogs

[0153] Objective: To further validate safety in non-rodent animals.

[0154] Animals and grouping: Healthy beagle dogs, half male and half female, grouped as in the rat experiment.

[0155] Dosage regimen: once a week for 5 consecutive weeks, intra-articular injection in the knee joint, with the same concentration of the test sample as above (0.775 mg / mL).

[0156] Observation and testing: In addition to all items included in rat experiments, routine non-rodent tests such as ophthalmological examination and electrocardiogram monitoring are added. Histopathological examination also focuses on local joints and major organs throughout the body.

[0157] Key results and conclusions: Consistent with the results in SD rats, all animals tolerated the disease well, and the NOAEL was determined to be 0.775 mg / mL.

[0158] 2. Toxicokinetic Analysis

[0159] Objective: To simultaneously investigate the systemic exposure of sodium amobarbital in animals and whether accumulation occurs during repeated-dose toxicity studies.

[0160] Methods: In repeated-dose studies in SD rats and beagle dogs, plasma samples were collected at a series of time points after the first and last doses. The plasma concentration of sodium amobarbital was determined using a validated HPLC-MS / MS method (same as in Example 5).

[0161] Data analysis: Calculate key pharmacokinetic parameters, such as peak concentration (Cmax) and area under the curve (AUC), compare the differences in parameters between the first and last doses, and assess accumulation potential.

[0162] Key results and conclusions: The analysis results showed that the drug did not accumulate in vivo, which is consistent with the characteristics of local retention and slow release of the material, and supports its good safety from a pharmacokinetic perspective.

[0163] 3. In vitro hemolysis test

[0164] Objective: To evaluate whether the material or its extract can cause erythrocyte rupture (hemolysis) and to assess its blood compatibility.

[0165] Methods: Refer to ISO 10993-4 standard. Prepare the test sample extract (mixed with physiological saline in a certain proportion and incubated). Take fresh rabbit blood or human blood and prepare a red blood cell suspension. Incubate the red blood cell suspension with the test sample extract, negative control (physiological saline), and positive control (distilled water) for a certain period of time (3 hours in this example).

[0166] Detection and calculation: After incubation, centrifuge and measure the absorbance (OD value) of the supernatant at 540 nm to calculate the hemolysis rate (%).

[0167] Key results and conclusions: The hemolysis rate of the test sample group was far below the internationally recognized safety limit of 5%, indicating no hemolysis and demonstrating that the material has good blood compatibility.

[0168] 4. Active systemic anaphylaxis (ASA) test

[0169] Objective: To assess the risk of the material triggering type I (immediate) hypersensitivity reactions.

[0170] Animals and grouping: Healthy guinea pigs were randomly divided into a negative control group (physiological saline), a positive control group (ovalbumin (OVA) solution emulsified with Freund's complete adjuvant), and a test sample group.

[0171] Sensitization and Challenge: The experiment consisted of a sensitization period and a challenge period. During the sensitization period, animals in each group were intraperitoneally injected with the corresponding substance at a volume of 1.0 mL / kg body weight. Approximately two weeks later, the animals were given an intravenous challenge injection at a volume of 0.5 mL / kg body weight.

[0172] Observation and evaluation: Begin immediately after stimulation and observe continuously for 60 minutes. Record whether the animal exhibits allergic symptoms such as agitation, piloerection, difficulty breathing, shock, or even death, and score the symptoms.

[0173] Key results and conclusions: No allergic symptoms were observed in the test group animals, and the results were negative, indicating that the material poses no risk of sensitization.

[0174] 5. Local tolerance test

[0175] Objective: To specifically evaluate the irritant effect of repeated intra-articular drug delivery on local tissues.

[0176] Animals and models: Local tolerability was used as the core endpoint in repeated-dose toxicity studies in SD rats and beagle dogs.

[0177] Observation and evaluation: Macroscopic observation: Observe the injection joint for any irritant reactions such as redness, swelling, heat, pain, or functional impairment before and after each administration.

[0178] Histopathology: At the end of the trial, a detailed histopathological examination of the injected joints was performed (see 1.1 and 1.2) to assess pathological changes such as synovitis, cartilage damage, and fibrosis.

[0179] Key results and conclusions: No drug-related irritation was observed in the joints at the administration site throughout the entire administration period and recovery period, and no abnormalities were found in histopathological examination, demonstrating that the material has good local safety.

[0180] 6. Overall Conclusion

[0181] The toxicological and safety evaluation results of the above system show that the composite biomaterial loaded with multimodal reprogramming therapeutic agent of the present invention exhibits good local and systemic safety under the proposed route of administration and dosage, and does not show hemolysis, sensitization, local irritation or systemic toxicity, providing important safety basis for its further clinical development.

[0182] Example 7

[0183] Table 13 lists several epigenetic reprogramming small molecule combinations (C1-C6) and their components and concentrations that were systematically screened during the research and development process. These combinations were designed based on different mechanisms of action (such as histone deacetylase inhibition, WNT pathway regulation, and TGF-β signaling inhibition) to evaluate their effects on the efficiency, safety, and synergistic effects on chondrocyte epigenetic age reversal in in vitro and in vivo models. Through comparative analysis, C1 (VC6TF) was ultimately determined to be the preferred combination for treating osteoarthritis, exhibiting both high reversal efficiency and good safety. This table not only reflects the systematic screening and optimization process of the present invention but also provides scalable experimental evidence for subsequent formulation adjustments and adaptation to different pathological stages or cell types.

[0184] Table 13 Composition and concentration of epigenetic reprogramming small molecule combinations (C1-6)

[0185] Conclusion: The C5 combination is based on all 6 components and their corresponding concentrations of C4 (CoNYSA), with the addition of sodium butyrate at a concentration of 200 μM, forming a new test combination consisting of 7 small molecules.

[0186] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composite biomaterial loaded with a multi-mode reprogramming therapeutic agent, characterized in that, The composite biomaterial includes sodium amobarbital, the epigenetic reprogramming small molecule VC6TF, and lipid nanoparticles encapsulating reprogramming factor mRNA.

2. The composite biomaterial as described in claim 1, characterized in that, The epigenetic reprogramming small molecule VC6TF includes four or five of the following: valproic acid, CHIR99021, E-616452, transphenylcyclopropane, and trichodin.

3. The composite biomaterial as described in claim 1, characterized in that, The reprogramming factor mRNA includes at least three of OCT4, SOX2, KLF4, GLIS1, and LIN28, but does not include c-MYC.

4. A method for preparing a composite biomaterial loaded with a multimodal reprogramming therapeutic agent as described in any one of claims 1-3, characterized in that, A sodium amobarbital solution, an epigenetic reprogramming small molecule VC6TF solution, and a lipid nanoparticle solution encapsulating reprogramming factor mRNA were sequentially added to a gel matrix. The mixed complex was filtered to remove bacteria and the pH was adjusted to obtain a composite biomaterial loaded with a multimodal reprogramming therapeutic agent.

5. The preparation method according to claim 4, characterized in that, The hyaluronic acid used to prepare the gel matrix has a molecular weight of 500~3200kDa and a swelling time of ≥7h.

6. The preparation method according to claim 4, characterized in that, The final concentration of sodium amobarbital in the gel matrix is ​​0.5~1.5 mg / mL; The final concentrations of the epigenetic reprogramming small molecule VC6TF in the gel matrix were 150-350 μM valproic acid, 5-20 μM CHIR99021, 5-20 μM E-616452, 2-10 μM transphenylcyclopropane, and 25-100 μM trichomoniasis. The total final concentration of reprogramming factor mRNA in the lipid nanoparticles encapsulating reprogramming factor mRNA in the gel matrix is ​​20~100μg / mL; the molar ratio of the reprogramming factor mRNA is OCT4:SOX2:KLF4:GLIS1:LIN28=(1.5-6):1:1:1:

1.

7. The preparation method according to claim 6, characterized in that, The method for preparing lipid nanoparticles encapsulated with reprogramming factor mRNA is as follows: lipid ethanol solution and mRNA aqueous solution are mixed in a microfluidic manner to obtain a primary LNP suspension; the primary LNP suspension is concentrated by dialysis and ultrafiltration to obtain lipid nanoparticles encapsulated with reprogramming factor mRNA.

8. The preparation method according to claim 7, characterized in that, The lipid ethanol solution comprises lipid components in the following molar ratio: 45%~55% DLin-MC3-DMA, 8%~15% DSPC, 35%~45% cholesterol, and 1.0%~2.5% DMG-PEG2000; the lipid components are dissolved in anhydrous ethanol to obtain a lipid ethanol solution with a total lipid concentration of 8~15 mM. The total concentration of reprogramming factor mRNA in the lipid nanoparticles is 0.3~1.0 mg / mL.

9. The use of the composite biomaterial loaded with multimodal reprogramming therapeutic agent as described in any one of claims 1 to 3, or the composite biomaterial loaded with multimodal reprogramming therapeutic agent prepared according to the preparation method described in any one of claims 4 to 8, in the preparation of a medicament for treating osteoarthritis.

10. The application as described in claim 9, characterized in that, The composite biomaterial controlled the epigenetic age reversal of joint cells to between 57% and 77%, and did not induce abnormal proliferation or loss of cell identity in joint tissues.

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