Naringin nano preparation for treating stem cell osteogenic differentiation disorder and preparation method of naringin nano preparation

Abstract

The invention relates to the field of biomedical nanotechnology, and discloses a naringin nano preparation for stem cell osteogenic differentiation disorder and a preparation method, the naringin nano preparation is composed of carboxymethyl chitosan, hydroformylated bletilla gum and naringin, and the mass ratio of carboxymethyl chitosan to hydroformylated bletilla gum to naringin is 10: 2.5: 1; the particle size of the nano preparation is 180-220 nm, and the Zeta potential is-23.5 + / -3.2 mV. According to the naringin nano-preparation for the stem cell osteogenic differentiation disorder, natural polysaccharide carboxymethyl chitosan and hydroformylated bletilla gum are adopted as a carrier and a cross-linking agent, the cytotoxicity of a traditional chemical cross-linking agent is completely avoided, the cell proliferation rate of a hydroformylated bletilla gum cross-linking system is larger than 120%, and the naringin nano-preparation has good biological safety; according to the prepared nano preparation, the solubility of the naringin is improved to 1.89 mg / mL from 0.25 mg / mL and is improved by about 7.56 times, and the dissolution rate is remarkably increased. The freeze-dried powder of the preparation can be stably stored for 9 months at 4 DEG C, and the particle size change rate after redissolution is less than 5%.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanotechnology, and in particular to a naringin nano-formulation for stem cell osteogenic differentiation disorder and its preparation method. Background Technology

[0002] Glucocorticoid-induced avascular necrosis of the femoral head is a common disabling bone disease caused by long-term or high-dose use of glucocorticoids. Its core pathological mechanism lies in the interference of hormones with the normal differentiation balance of bone marrow mesenchymal stem cells (BMSCs), namely, the overactivation of the peroxisome proliferator-activated receptor γ (PPARγ) pathway, which promotes the differentiation of BMSCs into adipocytes. At the same time, it inhibits the Runt-related transcription factor 2 (Runx2) / bone morphogenetic protein-2 (BMP-2) signaling pathway, resulting in osteogenic differentiation disorder, decreased bone repair capacity, and ultimately causing ischemic necrosis of bone tissue and collapse of the femoral head structure.

[0003] Naringin, a natural flavonoid glycoside, is widely found in plants such as Drynaria fortunei and Citrus aurantium. It possesses dual pharmacological activities, regulating bone metabolism, promoting osteogenic differentiation, and inhibiting adipogenic differentiation, making it potentially valuable for early intervention in SONFH. Naringin primarily activates osteogenic differentiation pathways such as BMP / Smad and Wnt / β-catenin, and acts as a natural weak agonist of the key adipogenic factor PPARγ. It competitively binds to PPARγ, avoiding excessive activation of the PPARγ signaling pathway by glucocorticoids, thus weakening the excessive lipogenesis and bone loss caused by potent activation, ultimately normalizing the expression and activity of PPARγ, thereby combating hormone-induced osteonecrosis. However, naringin itself has poor water and lipid solubility, low oral bioavailability (approximately 8.8%), rapid metabolism in vivo, short half-life, and lacks bone tissue targeting, making it difficult to achieve effective therapeutic concentrations at the lesion site.

[0004] Existing technologies have attempted to improve the solubility of naringin using nanoparticle drug delivery systems (such as liposomes and polymer nanoparticles), but the following problems still exist: they cannot penetrate the bone marrow sinusoidal barrier, resulting in low bone targeting efficiency; they also suffer from low encapsulation efficiency and drug loading, residual toxicity of the chemical cross-linking agents used, and inhibition of BMSCs activity, which limits their clinical application. Summary of the Invention

[0005] Given that the existing technologies mentioned above cannot penetrate the bone marrow sinusoidal barrier, have low bone targeting efficiency, low encapsulation rate and drug loading, residual toxicity of the chemical cross-linking agents used, and inhibition of BMSCs activity, and that their clinical application is somewhat limited, this invention is proposed.

[0006] Therefore, the purpose of this invention is to provide a naringin nano-formulation for osteogenic differentiation disorder of stem cells. The purpose is to use natural polysaccharide carboxymethyl chitosan and aldehyde-modified Bletilla striata gum as carriers and cross-linking agents, which completely avoids the cytotoxicity of traditional chemical cross-linking agents. The cell proliferation rate of the aldehyde-modified Bletilla striata gum cross-linking system is greater than 120%, and it has good biological safety.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a naringin nano-preparation for stem cell osteogenic differentiation disorder, composed of carboxymethyl chitosan, aldehyde-modified Bletilla striata gum and naringin, wherein the mass ratio of carboxymethyl chitosan, aldehyde-modified Bletilla striata gum and naringin is 10:2.5:1; the nano-preparation has a particle size of 180-220 nm and a zeta potential of -23.5±3.2 mV.

[0008] As a preferred embodiment of the naringin nano-formulation for stem cell osteogenic differentiation disorder described in this invention, wherein the degree of aldehyde substitution of the aldehyde-modified Bletilla striata gum is 35%-40%, and it is prepared by oxidizing Bletilla striata gum with sodium periodate.

[0009] A preferred embodiment of the method for preparing the naringin nano-formulation for stem cell osteogenic differentiation disorder described in this invention includes the following steps:

[0010] Step 1: Preparation of aldehyde-modified Bletilla striata gum: Mix 2wt% Bletilla striata gum solution with sodium periodate at a mass ratio of 100:2.0-2.5, react at 25℃ in the dark for 6-8 hours, add ethylene glycol to terminate the reaction, and obtain aldehyde-modified Bletilla striata gum powder after dialysis and freeze drying.

[0011] Step 2: Preparation of drug-loaded nanoparticles: Naringin solution and carboxymethyl chitosan solution are mixed, and then aldehyde-modified Bletilla striata gum solution is added. The mixture is stirred and reacted at 37°C for 30-60 minutes. After centrifugation, washing, and freeze-drying, the naringin nanoparticle formulation is obtained.

[0012] In a preferred embodiment of the method for preparing the naringin nano-formulation for stem cell osteogenic differentiation disorder described in this invention, the concentration of the naringin solution in step two is 1 mg / mL, the concentration of the carboxymethyl chitosan solution is 2 mg / mL and the pH is 5.5, and the concentration of the aldehyde-modified Bletilla striata gum solution is 1.5 mg / mL.

[0013] In a preferred embodiment of the method for preparing the naringin nano-formulation for stem cell osteogenic differentiation disorder described in this invention, in step two, the volume ratio of the naringin solution to the carboxymethyl chitosan solution is 1:5, and the volume ratio of the carboxymethyl chitosan solution to the aldehyde-modified Bletilla striata gum solution is 3:1.

[0014] In a preferred embodiment of the method for preparing naringin nanoparticles for osteogenic differentiation disorder of stem cells according to the present invention, mannitol and trehalose in a mass ratio of 2:1 are added as freeze-drying protectants during the freeze-drying process in step two.

[0015] As a preferred embodiment of the method for preparing naringin nanoparticles for stem cell osteogenic differentiation disorder according to the present invention, the naringin nanoparticles are used in the preparation of drugs for the prevention and / or treatment of hormone-induced femoral head necrosis.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] 1. This invention utilizes natural polysaccharide carboxymethyl chitosan and aldehyde-modified Bletilla striata gum as carriers and cross-linking agents, completely avoiding the cytotoxicity of traditional chemical cross-linking agents. The cell proliferation rate of the aldehyde-modified Bletilla striata gum cross-linking system is greater than 120%, exhibiting good biocompatibility. Furthermore, the prepared nano-formulation increases the solubility of naringin from 0.25 mg / mL to 1.89 mg / mL, an increase of approximately 7.56 times, and significantly accelerates the dissolution rate. The lyophilized powder of the formulation can be stably stored at 4°C for 9 months, with a particle size change rate of less than 5% after reconstitution.

[0018] 2. This invention achieves highly efficient encapsulation of naringin through the synergistic effect of iontophoresis and Schiff base cross-linking, with an encapsulation rate ≥87% and a drug loading >10.7%. In in vitro release experiments, the cumulative drug release rate reached 84% at pH 7.4 after 72 hours and 93% at pH 5.0 acidic conditions (simulating lysosomal environment), demonstrating ROS-responsive drug release characteristics.

[0019] 3. The nanoparticles of this invention have a particle size of 180-220 nm and a Zeta potential of -23.5 ± 3.2 mV, which meets the optimal range for intestinal absorption of orally administered nanoparticles. Through a triple mechanism of EPR effect, electrostatic adsorption of carboxymethyl chitosan to bone matrix Ca²⁺, and ROS-responsive release of aldehyde-modified Bletilla striata gum, precise enrichment of the drug in the osteonecrosis area is achieved, with a bone marrow / plasma drug concentration ratio of 0.11:1.

[0020] 4. The nano-formulation of the present invention can significantly enhance the ALP activity, calcium nodule formation and osteogenic-related gene (Runx2, BMP-2, OCN, OPN) and protein expression of BMSCs, while effectively inhibiting PPARγ2-mediated adipogenic differentiation. Its effect is significantly better than that of free naringin. At the same time, the preparation process does not require complex equipment, the raw materials used are widely available and inexpensive, the process is controllable, and it is suitable for large-scale production. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of Nar nanoformation of the stem cell osteogenic differentiation disordered naringin nanoformation of the present invention.

[0022] Figure 2 This is a TEM image of the Nar nanoformation of the stem cell osteogenic differentiation disorder of the present invention (showing a spherical structure).

[0023] Figure 3 The particle size distribution of the Nar nanoformation of the stem cell osteogenic differentiation disorder nanoformation of the present invention.

[0024] Figure 4 The in vitro drug release curve of the nano-formulation of naringin for osteogenic differentiation disorder of stem cells in this invention (84% release at pH 7.4 vs 93% release at pH 5.0 within 72 hours in PBS sustained-release solution).

[0025] Figure 5 HE staining image of the naringin nano-formulation for stem cell osteogenic differentiation disorder of the present invention (increased bone trabeculae and decreased adipocytes in the Nar nano-formulation group).

[0026] Figure 6 This is an ALP and alizarin red staining image of the naringin nano-formulation for osteogenic differentiation disorder of stem cells in this invention (proving that the Nar nano-formulation significantly enhances ALP activity and calcium nodule formation).

[0027] Figure 7 This invention relates to the osteogenic gene and protein expression (BMP-2 / Runx2 upregulation) of the naringin nano-formulation for osteogenic differentiation disorder of stem cells.

[0028] Figure 8 This is a schematic diagram illustrating the targeting of the bone marrow with the nano-formulation of naringin, a stem cell osteogenic differentiation disorder, according to the present invention. Detailed Implementation

[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0030] Example 1: Preparation and characterization of a Nar-loaded nanoformulation

[0031] Step 1: Preparation of Aldehyde-modified Bletilla Gum (BSG): Weigh 2g of purified Bletilla gum and dissolve it in 98mL of deoxygenated distilled water to prepare a 2wt% Bletilla gum solution. Add 2.2g of sodium periodate and stir at 25℃ in the dark (to avoid superoxidation) for 8h. Add 2mL of ethylene glycol to terminate the reaction for 2h. Transfer to a MWCO 3.5kDa dialysis bag and dialyze at 4℃ for 48h (with 6 water changes). Pre-freeze at -40℃ for 4h, then dry at -45℃ and 0.1mBar for 24h. The low temperature and low pressure environment helps water sublimate and improves drying efficiency. Then dry again at 25℃ for 6h to further remove residual moisture, finally obtaining crosslinking agent aldehyde-modified Bletilla gum powder. The degree of aldehyde substitution was determined to be 36.8% by the hydroxylamine method. Figure 1 );

[0032] Step 2: Preparation of drug-loaded nanoparticles: Dissolve 100 mg of carboxymethyl chitosan (CMC) (degree of deacetylation ≥90%, degree of substitution 0.8) in 50 mL of pH 5.5 acetate buffer, dissolve 20 mg of Nar in 20 mL of 10% ethanol aqueous solution (20 mg / 20 mL), add the solution dropwise to the carboxymethyl chitosan solution, then add aldehyde-modified Bletilla striata gum solution (50 mg / 33.3 mL), add dropwise at a rate of 1 mL / min, stir at 37℃ and 500 rpm for 30 min to obtain a nano-suspension;

[0033] Step 3: Purification and Lyophilization: After centrifuging the nano-suspension at 10,000 rpm for 15 minutes, wash twice with 5% mannitol solution by centrifugation. The washed precipitate is then added to a 5% mannitol-2% trehalose mixed solution (mass ratio 2:1) for a second lyophilization to obtain nanoparticles. No significant changes were observed in particle size, polydispersity index (PDI), and encapsulation efficiency when stored under refrigeration (2-8°C) and room temperature conditions. Figure 2 ).

[0034] Characterization of the Nar-loaded nanoparticles: 1. Particle size and potential: Dynamic light scattering (DLS) detected an average particle size of 205 ± 15 nm. Figure 3 PDI 0.28, Zeta potential -23.5 ± 3.2 mV Figure 2 ); 2. Drug loading and encapsulation efficiency: HPLC determined drug loading >10.7% and encapsulation efficiency ≥87%; 3. Cumulative release in PBS buffer over 72 hours: 84±3% (pH 7.4 simulating blood circulation) vs 93±2% (pH 5.0 simulating the acidic environment of lysosomes); Figure 4 ).

[0035] Example 2: Water solubility comparison test

[0036] 1. Experimental materials: Nar nanoparticles (Nar-NPs) were prepared according to the method in Example 1, and 10 mg was taken after lyophilization; free Nar prepared by our company with a purity >98%; Nar suspension (0.5% carboxymethyl chitosan-Na as suspending agent) or ordinary Nar nanoparticles.

[0037] 2. Solubility determination: The saturated solubility of naringin in different formulations was determined using the equilibrium solubility method. 10 mg of each of the above samples was added to 10 mL of deionized water and incubated in a 37℃ water bath with shaking at 100 rpm for 48 hours until dissolution equilibrium was reached. The solution was filtered through a 0.22 μm microporous membrane, and the filtrate was appropriately diluted before HPLC determination of the Nar concentration. The chromatographic conditions included: column: Diamonsil C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (25:75, v / v); flow rate: 1.0 mL / min; detection wavelength: 283 nm; column temperature: 30℃; injection volume: 20 μL.

[0038] 3. Experimental Results: The Nar nano-formulation prepared in this invention has a water solubility of 1.89 mg / mL, which is 7.56 times that of free Nar. The dissolution rate is increased by 17.75 times, and the time to reach dissolution equilibrium is shortened to 6-8 hours. It significantly improves the water solubility of naringin. The oral bioavailability is expected to be increased by 3-5 times, reducing the dosage and frequency of medication, and the onset of action is faster, which is beneficial for early intervention in the pathological process of SONFH (steroid-induced avascular necrosis of the femoral head).

[0039] Comparative study on water solubility

[0040] detection indicators ALP activity (U / mL) Calcium nodule (OD562) Runx2 protein PPARγ2 inhibition rate Hormone model group 7.82±1.43 0.35±0.09 0.18±0.01 - Free Nar group 9.22±1.65 0.47±0.02 0.41±0.02 Reduced by 57% Nanoparticles 16.59±2.12* 0.98±0.13* 0.59±0.05* Reduced by 71.8% Increase multiplier 1.8 times* 2.1 times* 1.4 times* Significant optimization

[0041] *P < 0.01, comparison of this nano-formulation with other groups

[0042] Example 3: In vivo experimental study of the effect of Nar nanoformulation on hormone-induced osteogenic differentiation disorder of BMSCs

[0043] 1. Experimental materials: Fifty male Sprague-Dawley (SD) rats aged 2 months and weighing 300-305g were randomly divided into 4 groups, including blank group (n=5), model group (n=15), free Nar group (n=15), and Nar nano-preparation group (n=15). Aldehyde-modified Bletilla striata gum, carboxymethyl chitosan and other solutions were prepared in accordance with the above method to prepare Nar nano-preparation.

[0044] 2. Experimental treatment: SD rats were used to establish a SONFH model by intraperitoneal injection of lipopolysaccharide (LPS) 20 μg / kg × 2 times and intramuscular injection of methylprednisolone (MPS) 40 mg / kg × 3 times. The free Nar group and the nano-formulation group were administered a solution of 30 mg / kg and 405 mg / kg (containing 30 mg Nar) daily by gavage for 6 weeks after the initial MPS injection. The blank group and the model group were given physiological saline. Body weight was recorded daily to adjust the dosage.

[0045] 3. Detection indicators: (1) Observe the survival, weight, food intake, excretion and whether there is anorexia, melena, or skin infection in rats. (2) After gavage administration, fast for 12 hours. Blood is collected from the tail vein in the morning of weeks 1, 2, 4 and 6 to detect partial thromboplastin time (APTT), tissue plasminogen activator (t-PA) / plasminogen activator inhibitor (PAI-1), low-density lipoprotein (LDL) / high-density lipoprotein (HDL), thrombomodulin (TM), glutathione (GSH) / lipid peroxidase (LPO) and alanine aminotransferase (GPT). (3) Rats are euthanized under anesthesia in week 6. Bilateral femurs are decalcified, cleared, embedded, sectioned and stained with HE. The incidence and severity of osteonecrosis (ON) in each group are calculated. The incidence of ON = number of ON+ rats in each group / number of rats in each group × 100%; the severity of ON = number of ON+ lesions / number of ON+ rats. (4) The ratio of trabecular bone area and the density of microvessels in ON were measured by ImageProPlus 6.0 software under a light microscope at 200x magnification. The ratio of trabecular bone area = trabecular bone area / total area of ​​ON × 100, and the density of microvessels = number of microvessels / total area of ​​ON. Eight fields of view were randomly selected for each specimen to calculate the adipocyte parameters: average diameter of adipocytes (µm) and density ( / mm2). The measurement was repeated 3 times.

[0046] 4. Experimental Results

[0047] (1) The incidence of ON in the model group (93.3%) > free Nar group (66.7%) > nano-preparation group (46.7%, P<0.01). The necrotic foci were concentrated in the femoral metaphysis. The severity of ON in each group was 2.4±0.7, 2.1±0.9, and 1.8±0.6, respectively. There was no statistically significant difference (P=0.147).

[0048] (2) Trabecular bone area ratio and microvessel density: blank group > nano-preparation group > free Nar group > model group ( Figure 5 (P < 0.01).

[0049] (3) Bone marrow adipocyte diameter and density: blank group < nano-preparation group < free Nar group < model group ( Figure 5 (P < 0.01).

[0050] (4) Serum indicators: The nano-prepared formulation was better able to restore hormone-induced abnormal serum APTT, TM, t-PA, PAI-1, LDL, HDL, GSH and LPO than free Nar, and was close to the level of the blank group.

[0051] Comparison of serum markers among different groups of rats (n=50, ±s)

[0052] index Time (week) Blank group Model group Free Nar group Nanoparticles p-value GPT (U / L) 1 36.3±0.8 120.9±0.5 83.6±0.7 67.6±0.6 <0.001 2 36.8±0.7 90.7±0.2 69.5±0.6 57.5±0.6 <0.001 4 36.9±0.5 69.1±0.4 58.6±0.8 45.2±0.5 <0.001 6 36.6±0.7 56.1±0.4 45.4±0.7 40.7±0.4 <0.001 p-value 0.476 <0.001 <0.001 <0.001 APTT (sec) 1 38.1±0.7 14.5±1.6 23.4±1.6 29.2±1.3 <0.001 2 38.2±0.8 27.7±1.7 31.7±1.7 33.6±1.5 <0.001 4 38.1±0.9 32.6±1.9 33.6±1.7 35.9±1.2 <0.001 6 38.3±1.1 35.1±2.0 36.2±1.9 37.0±1.5 0.009 p-value 0.374 <0.001 <0.001 <0.001 LDL / HDL 1 0.39±0.06 2.48±0.72 1.93±0.65 1.33±0.32 <0.001 2 0.40±0.07 1.58±0.75 1.18±0.57 0.97±0.41 <0.001 4 0.41±0.05 1.05±0.45 0.83±0.35 0.63±0.23 <0.001 6 0.40±0.06 0.96±0.52 0.76±0.42 0.57±0.27 <0.001 p-value 0.242 <0.001 <0.001 <0.001 tPA / PAI-1 1 0.78±0.09 0.27±0.09 0.47±0.09 0.64±0.07 <0.001 2 0.81±0.08 0.52±0.12 0.68±0.11 0.73±0.09 <0.001 4 0.80±0.07 0.63±0.07 0.73±0.12 0.79±0.08 <0.001 6 0.82±0.09 0.68±0.13 0.79±0.05 0.83±0.06 0.067 p-value 0.175 <0.001 <0.001 0.042 GSH / LPO 1 13.2±1.7 5.7±1.3 8.2±1.5 11.1±1.8 <0.001 2 13.4±1.6 9.1±1.3 10.3±1.5 11.6±1.3 0.007 4 13.3±1.5 9.9±1.2 10.7±1.6 11.9±1.5 0.032 6 13.5±1.3 11.5±1.1 11.6±1.4 12.1±1.2 0.133 p-value 0.226 0.009 0.032 0.052 TM (µg / L) 1 17.3±1.8 35.6±2.2 29.3±1.6 24.5±1.8 <0.001 2 17.5±2.1 28.4±2.3 24.3±1.8 20.9±1.7 <0.001 4 17.2±2.0 24.7±2.0 21.5±1.7 19.2±1.6 0.025 6 17.6±1.9 21.2±1.4 18.1±1.6 17.8±1.3 0.037 p-value 0.443 <0.001 0.008 0.015

[0053] Example 4: In vitro experiment on the effect of naringin on hormone-induced osteogenic differentiation disorder of BMSCs

[0054] 1. Experimental Materials

[0055] (1) Prepare Nar nano-formulations by self-made solutions of aldehyde-modified Bletilla striata gum, carboxymethyl chitosan, etc., according to the above method.

[0056] (2) The SONFH animal model was established according to the above method. The free Nar group and the nano-preparation group were administered the solution by gavage at 30 mg / kg and 405 mg / kg (containing 30 mg Nar) respectively for 6 weeks after the first injection of MPS. The blank group and the model group were given physiological saline. The body weight was recorded daily to adjust the dosage.

[0057] (3) Bone marrow was extracted from the anterior inferior iliac spine of rats in each group and subjected to gradient centrifugation to obtain mononuclear cell suspension. The cell concentration was adjusted with 10 mL of LG-DMEM, and the cells were seeded into culture dishes at 2.5 × 10⁵ nucleated cells / mL. The cells were cultured in a 37°C, 5% CO₂ incubator until they covered the bottom of the culture dish. Then, 7 × 10⁴ cells / mL were seeded into culture dishes and cultured for 2 weeks to prepare cell suspension. The cells were labeled according to the group number. The osteogenic differentiation of BMSCs was detected by ALP activity assay, alizarin red staining, qRT-PCR and Western blot.

[0058] 2. ALP activity assay: Blank group > Nanoparticle formulation group > Free Nar group > Model group ( Figure 6 (P<0.05).

[0059] 3. Alizarin Red staining: Compared with the model group and the free Nar group, the number of calcified nodules in the Nar nanoparticle group was significantly increased ( Figure 6 (P<0.05).

[0060] 4. qPCR detection: Compared with the model group and the free Nar group, the expression levels of Runx2, ALP, BMP-2, OCN and OPN genes in the nano-formulation group were significantly upregulated (P<0.05), while PPARγ2 expression was inhibited (P<0.05).

[0061] 5. Western blot detection ( Figure 7 The expression levels of Runx2, ALP, BMP-2, OCN, and OPN proteins in the nano-formulation group of BMSCs were higher than those in the model group and the free Nar group, while the expression of PPARγ2 protein decreased to 28.2% of that in the model group.

[0062] Summarize:

[0063] 1. The natural polysaccharide cross-linking system prepared by this invention completely avoids glutaraldehyde toxicity, and the cell proliferation rate of the aldehyde-modified Bletilla striata gum cross-linking system is >120%.

[0064] 2. The present invention provides Nar, a natural flavonoid glycoside found in common herbal medicines such as Citrus aurantium, Citrus reticulata peel, and Drynaria fortunei. It is a potent antioxidant with a purity of >99%. The nanoparticles prepared by the above technology have an encapsulation rate of ≥87%, which significantly improves the water solubility of Nar, increasing it from 0.25 mg / mL for free Nar to 1.89 mg / mL for nanoparticle formulations.

[0065] 3. The Nar nanoparticle formulation of this invention has a particle size of 180-220 nm, which conforms to the optimal range for intestinal absorption of orally administered nanoparticles. The zeta negative charge and high encapsulation efficiency can enhance colloidal stability, reduce the degradation of free drugs in the gastrointestinal tract, and promote intestinal adhesion; it also possesses bone-targeting properties. Figure 8 (1) Utilizing the vascular leakage in the osteonecrosis area, the drug is enriched in the osteonecrosis area through the EPR effect. (2) The negative charge of carboxymethyl chitosan is electrostatically adsorbed with Ca²⁺ in the bone matrix, and the ROS response release of aldehyde-modified Bletilla striata gel enhances the local concentration, thus making the bone marrow / plasma drug concentration ratio reach 0.11:1, directly targeting the target PPARγ of bone marrow BMSCs, and counteracting the hormone-induced osteogenic impairment and osteonecrosis mechanism of BMSCs.

[0066] 4. This formulation utilizes a triple mechanism of EPR effect, electrostatic targeting, and ROS-responsive release to achieve a bone marrow drug concentration of 11% of the plasma concentration after oral administration, which is higher than that of conventional oral nanoparticles (usually <10%), providing an efficient delivery strategy for the prevention and treatment of SONFH.

[0067] 5. The natural polysaccharide cross-linking system prepared by this invention exhibits good degradation performance. The pH of its degradation solution is slightly alkaline, between 7.5 and 7.7, which helps inhibit inflammatory responses at necrotic sites. When the nanoparticles are placed in a dialysis bag (MWCO 3.5-8 kDa) and immersed in PBS buffer at pH 7.4 to simulate blood circulation, 84% of Nar is cumulatively released after 72 hours. At pH 5.0, simulating an acidic lysosomal environment, 93% of Nar is cumulatively released. Figure 4 );

[0068] 6. This formulation utilizes iontophoresis, where the negatively charged carboxyl group (-COO⁻) of carboxymethyl chitosan forms hydrogen bonds with the phenolic hydroxyl group (-OH) of Nar, thus encapsulating the hydrophobic skeleton of flavonoid glycosides through hydrophobic interactions. Simultaneously, the Schiff base cross-linking (aldehyde-amino) of aldehyde-modified Bletilla striata gum forms a network structure, physically blocking Nar leakage and synergistically improving the encapsulation efficiency (≥87%) and drug loading (>10.7%).

[0069] 7. After adding mannitol-trehalose (mass ratio 2:1) as a protective agent, the freeze-dried powder of this preparation can be stored stably at 4°C for up to 9 months. When used, it can be reconstituted with physiological saline, and the particle size change rate is <5%S4.

[0070] 8. Compared with free Nar, nano-formulations are more effective at reversing osteogenic differentiation disorders and inhibiting adipogenic catabolism (as shown in the table below).

[0071] detection indicators ALP activity (U / mL) Calcium nodule (OD562) Runx2 protein PPARγ2 inhibition rate Hormone model group 7.82±1.43 0.35±0.09 0.18±0.01 - Free Nar group 9.22±1.65 0.47±0.02 0.41±0.02 Reduced by 57% Nanoparticles 16.59±2.12* 0.98±0.13* 0.59±0.05* Reduced by 71.8% Increase multiplier 1.8 times* 2.1 times* 1.4 times* Significant optimization

[0072] (*P<0.01, Nanoparticle formulation group vs. Free Nar group)

[0073] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A naringin nano-formulation for osteogenic differentiation disorder of stem cells, characterized in that, It is composed of carboxymethyl chitosan, aldehyde-modified leucovorin and naringin, wherein the mass ratio of carboxymethyl chitosan, aldehyde-modified leucovorin and naringin is 10:2.5:1; the particle size of the nano-formulation is 180-220 nm and the zeta potential is -23.5±3.2 mV.

2. The naringin nano-formulation for osteogenic differentiation disorder of stem cells according to claim 1, characterized in that: The aldehyde-substituted bleach gum has an aldehyde substitution degree of 35%-40% and is prepared by oxidizing bleach gum with sodium periodate.

3. A method for preparing a naringin nanoparticle formulation for osteogenic differentiation disorder of stem cells, wherein the formulation is applied to the naringin nanoparticle formulation for osteogenic differentiation disorder of stem cells as described in claim 2, characterized in that: Includes the following steps: Step 1: Preparation of aldehyde-modified Bletilla striata gum: Mix 2wt% Bletilla striata gum solution with sodium periodate at a mass ratio of 100:2.0-2.5, react at 25℃ in the dark for 6-8 hours, add ethylene glycol to terminate the reaction, and obtain aldehyde-modified Bletilla striata gum powder after dialysis and freeze drying. Step 2: Preparation of drug-loaded nanoparticles: Naringin solution and carboxymethyl chitosan solution are mixed, and then aldehyde-modified Bletilla striata gum solution is added. The mixture is stirred and reacted at 37°C for 30-60 minutes. After centrifugation, washing, and freeze-drying, the naringin nanoparticle formulation is obtained.

4. The method for preparing the naringin nano-formulation for osteogenic differentiation disorder of stem cells according to claim 3, characterized in that: In step two, the concentration of the naringin solution is 1 mg / mL, the concentration of the carboxymethyl chitosan solution is 2 mg / mL and the pH is 5.5, and the concentration of the aldehyde-modified bleach solution is 1.5 mg / mL.

5. The method for preparing the naringin nano-formulation for osteogenic differentiation disorder of stem cells according to claim 4, characterized in that: In step two, the volume ratio of the naringin solution to the carboxymethyl chitosan solution is 1:5, and the volume ratio of the carboxymethyl chitosan solution to the aldehyde-modified bleaching gum solution is 3:

1.

6. The method for preparing the naringin nano-formulation for osteogenic differentiation disorder of stem cells according to claim 5, characterized in that: In step two, mannitol and trehalose in a mass ratio of 2:1 are added as freeze-drying protectants during the freeze-drying process.

7. The use of the naringin nanoformulation as described in claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of hormone-induced avascular necrosis of the femoral head.

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