Dual-regulation nano drug delivery system based on near-infrared light response and application thereof

Abstract

The invention provides a near-infrared light NIR response-based dual-regulation nano drug delivery system and application thereof, the nano drug delivery system takes mesoporous silica as a carrier, and alendronate sodium is coupled on the surface of the carrier to increase bone targeting; mesopores in the carrier are blocked by rhodamine RH, nitric oxide NO can be consumed to generate rhodamine B, and bioactive drugs in the carrier are released. The invention constructs a systemic drug carrier with bone targeting and diabetic bone microenvironment response release, and solves the problems that the bone healing ability of diabetic patients is significantly reduced, and traditional local treatment is difficult to effectively improve systemic bone microenvironment disorder; the problems that an existing local drug system cannot be released according to needs, local NO and NO in bone marrow are difficult to efficiently remove, and macrophages are difficult to transform to repair-promoting M2 type are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the field of diabetic bone microenvironment responsive drugs, and more specifically to an AgSr-MSNs based on near-infrared light (NIR) response with NO / pH dual regulation and its application in diabetic bone repair. Background Technology

[0002] Diabetes mellitus (DM) is a metabolic disease characterized primarily by chronic hyperglycemia. The International Diabetes Federation predicts that by 2030, more than 10% of the global population will be affected by diabetes. Diabetic patients often experience a significant decline in bone repair capacity, and their bone healing impairment contrasts sharply with that of the healthy population, highlighting the urgent need to develop bone regeneration and repair strategies specifically for metabolic disorders.

[0003] The primary cause of impaired bone regeneration in diabetes is immune dysregulation during tissue repair, particularly the abnormal shift in macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2). This dysregulation disrupts the spatiotemporal coordination of downstream repair processes such as angiogenesis and osteoblast differentiation, thereby hindering bone healing. Therefore, regulating macrophage polarization may be an effective strategy for bone regeneration in diabetes. Studies have found that M1 macrophages in diabetic bone marrow and bone defect areas exhibit significantly elevated expression of inducible nitric oxide synthase (iNOS), producing 30 times more nitric oxide (NO) than M2 macrophages. This suggests that clearing NO or inhibiting iNOS may have significant anti-inflammatory and macrophage reprogramming effects. However, current diabetic bone repair strategies are mostly limited to local interventions at fracture or bone defect sites, neglecting the sustained impact of the systemic diabetic bone microenvironment on the repair process, resulting in limited efficacy. Therefore, developing strategies to systematically regulate the diabetic bone microenvironment is crucial for achieving efficient and sustainable bone regeneration.

[0004] Mesoporous silica nanoparticles (MSNs) have become an important delivery platform in the field of bone regeneration due to their high specific surface area, tunable mesoporous structure, and excellent drug loading capacity. Their surface chemistry facilitates the modification of stimulus-responsive capping agents (such as pH, temperature, or phototriggered mechanisms) to achieve spatiotemporally controlled drug release. Furthermore, the modular design of MSNs can simultaneously load multiple bioactive ions (such as Sr²⁺), overcoming the limitations of traditional ion delivery systems. Strontium ions (Sr²⁺) exhibit significant osteogenic efficacy in bone regeneration and repair. Sr²⁺ upregulates osteogenic markers of mesenchymal stem cells (such as ALP and OCN) by activating the MAPK / ERK1 / 2 signaling pathway, while simultaneously inhibiting adipogenesis and promoting mineralized nodule formation. In addition, Sr²⁺ also has immunomodulatory effects, driving macrophage polarization towards a pro-regenerative phenotype and stimulating angiogenesis. Ag₂S quantum dots possess a mild and controllable photothermal effect, enabling multifunctional synergistic therapy in bone repair. Therefore, this inspired us to construct a whole-body nanocarrier that responds to the M1 polarization state of macrophages and the acidic microenvironment of diabetes, restoring the whole-body state coupled with "immunity-osteogenesis-angiogenesis".

[0005] Patent application CN202310359956.X discloses a GMP / GP smart hydrogel periosteum with dual NIR and enzyme responses and its preparation method. This smart hydrogel periosteum can achieve intelligent controlled release of angiogenic peptides and osteogenic factors through dual NIR and enzyme responses to meet the repair needs at different time stages of fracture repair, showing great application potential in fracture repair and allogeneic bone integration for large bone defects. However, this type of hydrogel periosteum is mainly used for fracture repair. Because diabetic patients have significantly reduced bone healing capacity, local treatment is difficult to effectively improve systemic bone microenvironment dysregulation. Existing local drug systems cannot release on demand, efficiently clear local NO, and promote the conversion of macrophages to the pro-repair M2 type. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the shortcomings of existing technologies, this invention provides a NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response and its application. It constructs a systemic drug carrier with bone targeting and release response to the diabetic bone microenvironment, solving the problems of significantly reduced bone healing capacity in diabetic patients, the inability of traditional local treatments to effectively improve systemic bone microenvironment dysregulation, and the inability of existing local drug systems to release on demand, efficiently remove local NO, and promote the transformation of macrophages into pro-repair M2 types.

[0008] (II) Technical Solution

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] A NO / pH dual-regulated nanoparticle drug delivery system based on near-infrared (NIR) light response is described. The system uses strontium-doped mesoporous silica Ag₂S@SrMSNs as a carrier, with rhodamine RH loaded within the mesoporous channels. The nanoparticles are then modified with monoamino-substituted β-cyclodextrin (β-CD) and alendronate sodium (Ald) via a Schiff base reaction, resulting in the RH-CD@Ag₂S@SrMSNs-COOH / Ald nanoparticle drug delivery system, abbreviated as AgSr-MSNs. The system uses mesoporous silica as a carrier, with alendronate sodium coupled to the carrier surface to enhance bone targeting. The mesopores on the carrier are sealed with rhodamine RH, which consumes nitric oxide (NO) to generate rhodamine B. The mesoporous silica nanoparticles degrade more rapidly under acidic conditions, releasing the bioactive drug within.

[0011] This invention also provides a method for preparing a NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response, comprising the following steps:

[0012] Synthesis of S1,Ag2S quantum dots (Ag2S QDs)

[0013] The synthesis of Ag₂S QDs employed a modified thermal decomposition method: An empty 100 mL three-necked flask was prepared, washed, dried, and ensured to be free of water. First, a vacuum was drawn for 5 min, followed by purging with nitrogen for approximately 10 min to expel all air from the flask. Under nitrogen atmosphere, 76.8 mg of arsenic reagent (C₅H₁₀NS₂Ag), 30 g of 1-octadecene (ODE), and 6 g of 1-dodecyl mercaptan (1-DT) were added sequentially. The mixture was vigorously stirred with a magnetic stirrer, then heated to 100 °C and held for 10 min using a heating mantle. The temperature was then continuously increased to 170 °C and held for 10 min to allow the formation of Ag₂S QDs under nitrogen conditions, resulting in a dark black solution. Finally, 30 mL of n-hexane was added to terminate the reaction, and the mixture was cooled to room temperature. Ag2S QDs were collected by centrifugation at 11,000 rpm for 15 min, followed by washing three times with acetone (11,000 rpm × 15 min), and finally dispersed in chloroform. The QDs were then temporarily stored in a refrigerator at 4°C or dried in a vacuum oven at 70°C and stored away from light for further research.

[0014] ② Synthesis of Ag2S@SrMSNs

[0015] 10 mg of dried Ag₂S QDs were redispersed in 5 mL of chloroform and sonicated to obtain solution I. 0.205 g of CTAB and solution I were dispersed in a beaker containing 20 mL of triple-distilled water and stirred until a milky white homogenate was formed, yielding solution II. Solution II was heated to 70 °C in an oil bath under rapid stirring to evaporate the chloroform, yielding a clear, transparent brown solution III (Ag₂S / CTAB solution). Solution III was transferred to a 250 mL flask, and triple-distilled water was added to a final volume of 100 mL, yielding solution IV. 0.056 g of NaOH was added to solution IV and stirred at room temperature to obtain solution V. Solution V was then heated to 80 °C, and 500 mL of tetraethyl orthosilicate (TEOS) was added, followed immediately by 0.5 mL of SrCl₂·6H₂O. Rapid stirring (1000 rpm) was maintained for 1 min, then the stirring speed was reduced, and the mixture was gently stirred at 80 °C (100 rpm) for 8 h. The sample was collected by centrifugation at 10,000 rpm for 10 min and washed repeatedly with water and anhydrous ethanol (10,000 rpm × 10 min) to obtain sample Ag2S@SrSiO2 (non-mesoporous). Template removal using CTAB was then performed. The product was redispersed in 50 mL of anhydrous ethanol containing 0.5 g ammonium nitrate, and then refluxed overnight at 75°C. The product was collected by centrifugation and washed repeatedly with water and anhydrous ethanol to obtain product Ag2S@SrMSNs. It can be freeze-dried for later use.

[0016] ③ Modification of Ag2S@SrMSNs

[0017] 10 mg of Ag2S@SrMSNs were dispersed in 100 mL of a methanol solution containing 5 mg of carboxyl-polyethylene glycol-silane (COOH-PEG-Silane). The mixture was gently stirred at 70 °C using a heated magnetic stirrer, refluxed overnight, and then collected by centrifugation (10,000 rpm × 15 min). The product was then washed repeatedly with water and anhydrous ethanol to obtain carboxyl-modified Ag2S@SrMSNs (Ag2S@SrMSNs-COOH). Ag2S@MMSNs-COOH was dispersed in 20 mL of RH PBS solution (concentration 0.5 mg / mL) and magnetically stirred for 24 hours at room temperature in the dark. The RH-loaded Ag2S@MMSNs-COOH was collected by centrifugation (10,000 rpm, 15 min). The product prepared above was dissolved in 10 mL of water to obtain solution I. 0.1 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.1 mmol of N-hydroxysuccinimide (NHS) were added to solution I, and the mixture was shaken to mix thoroughly. The mixture was then stirred with a magnetic stirrer at room temperature for 15 min (to activate the carboxyl groups of the nanoparticles) to obtain solution II. Subsequently, 0.1 mmol of sodium alendronate and 0.1 mmol of monoamino-substituted β-CD were added to solution II, and the mixture was stirred at room temperature in the dark for 24 h. Finally, the final product (RH-CD@Ag2S@SrMSNs-COOH / Ald) was collected by centrifugation and washing.

[0018] Note: RH-CD@Ag2S@SrMSNs-COOH / Ald is abbreviated as AgSr-MSNs.

[0019] In the preparation process of the above-mentioned nano-drug delivery system, the amount of Ag2S@SrMSNs used was 10 mg, the amount of alendronate sodium Ald and cyclodextrin β-CD used was 0.1 mM, the amount of rhodamine RH used was 10 mg, and the loading rate was 6.3%.

[0020] This invention also provides the application of a dual-regulated nano-drug delivery system based on near-infrared (NIR) light response in the preparation of drugs for bone repair in diabetes.

[0021] (III) Beneficial Effects

[0022] The beneficial effects of this invention are:

[0023] The systemic drug carrier AgSr-MSNs constructed in this invention has bone-targeting and diabetic bone microenvironment-responsive release properties. In vitro and in vivo experiments have confirmed its effectiveness in bone targeting. It is proposed to target the bone via tail vein system administration, which can not only achieve immune regulation at diabetic bone defect sites, but also achieve immune dysregulation at sites far from bone defect sites.

[0024] This invention first discovered that the level of iNOS (a key rate-limiting enzyme in NO production) in M1 macrophages at the site of diabetic bone defects is significantly increased, with fluorescence intensity approximately 15 times that of wild-type control mice. Based on the acidic microenvironment of diabetes, the mesoporous silica nanoparticles constructed in this invention exhibit accelerated degradation under acidic conditions. Furthermore, the mesopores are sealed with rhodamine RH (which consumes NO to generate Rh B), thereby achieving on-demand release, clearing local NO, and promoting macrophage polarization towards the M2 type, demonstrating promising clinical application prospects.

[0025] This invention addresses the problem of significantly reduced bone healing capacity in diabetic patients, where traditional local treatments are ineffective in improving systemic bone microenvironment dysregulation. By designing a near-infrared (NIR) responsive NO / pH dual-regulation nano-drug delivery system, it can effectively target and remove local NO from bone injuries and promote the transformation of macrophages into the pro-repair M2 type, demonstrating a special efficacy in the recovery treatment of diabetic bone defects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 A schematic diagram of the synthesis steps of AgSr-MSNs;

[0028] Figure 2 Figure 1 shows the experimental results of AgSr-MSNs releasing their bioactive drugs in response to an acidic environment. In figure 2, a represents transmission electron microscopy (TEM) images of AgSr-MSNs after incubation at pH 7.4 and pH 6.5 for 1, 4, and 7 days; b represents the percentage of free RhB in the solution; and c represents the percentage of free Sr in the solution. 2+ Concentration percentage;

[0029] Figure 3 Figure 1 shows the effect of AgSr-MSNs on NO in macrophages. Figure 2 shows the uptake of AgSr-MSNs by macrophages under lipopolysaccharide (LPS) and high glucose conditions; Figure 3 shows the change in NO concentration in macrophages after incubation of AgSr-MSNs with macrophages for different time periods.

[0030] Figure 4Experimental results show the bone targeting ability of AgSr-MSNs. Figure a shows the in vitro bone targeting ability of AgSr-MSNs; figure b shows the organ enrichment of AgSr-MSNs at different time points.

[0031] Figure 5 Figure 1 shows the in vitro and in vivo biocompatibility assessment of AgSr-MSNs. Figure 2 shows the effect of different concentrations of AgSr-MSNs nanoparticles on the cell viability of Raw 264.7 macrophages, bone marrow stromal stem cells (BMSCs), and endothelial cells (human umbilical vein endothelial cells, HUVECs); Figure 3 shows cell viability staining after incubation of MSNs nanoparticles with Raw 264.7, BMSCs, and HUVECs; Figure 4 shows the hemolysis assay of different concentrations of AgSr-MSNs nanoparticles; Figure 5 shows the detection of blood biochemical indicators; and Figure 6 shows the in vivo biosafety assessment of MSNs nanoparticles.

[0032] Figure 6 This is an experimental diagram showing the effect of AgSr-MSNs combined with NIR irradiation on promoting M2 macrophage polarization. In the diagram, ab represents the immunofluorescence staining of CD86 (a marker of M1 macrophages) after MSNs were incubated with macrophages; cd represents the immunofluorescence staining of CD206 (a marker of M2 macrophages) after MSNs were incubated with macrophages.

[0033] Figure 7 ALP and ARS staining patterns show how AgSr-MSNs enhance in vitro bone formation by promoting macrophage M2 polarization.

[0034] Figure 8 This is an in vitro angiogenesis experiment showing how AgSr-MSNs enhance macrophage M2 polarization.

[0035] Figure 9 This image shows experimental results of AgSr-MSNs promoting bone regeneration in diabetic patients. Figure a shows a schematic diagram of the animal drug administration; figure b shows micro-CT images at 7 and 21 days post-administration; and figures cd show bone parameter analysis from the micro-CT scan. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0037] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0038] Example 1, Synthesis of AgSr-MSNs

[0039] Synthesis of S1, Ag2S QDs

[0040] The synthesis of Ag₂S QDs employed a modified thermal decomposition method: An empty 100 mL three-necked flask was prepared, washed, dried, and ensured to be free of water. First, a vacuum was drawn for 5 min, followed by purging with nitrogen for approximately 10 min to expel all air from the flask. Under nitrogen atmosphere, 76.8 mg of arsenic reagent (C₅H₁₀NS₂Ag), 30 g of 1-octadecene (ODE), and 6 g of 1-dodecyl mercaptan (1-DT) were added sequentially. The mixture was vigorously stirred with a magnetic stirrer, then heated to 100 °C and held for 10 min using a heating mantle. The temperature was then continuously increased to 170 °C and held for 10 min to allow the formation of Ag₂S QDs under nitrogen conditions, resulting in a dark black solution. Finally, 30 mL of n-hexane was added to terminate the reaction, and the mixture was cooled to room temperature. Ag2S QDs were collected by centrifugation at 11,000 rpm for 15 min, followed by washing three times with acetone (11,000 rpm × 15 min), and finally dispersed in chloroform. The QDs were then temporarily stored in a refrigerator at 4°C or dried in a vacuum oven at 70°C and stored away from light for further research.

[0041] ② Synthesis of Ag2S@SrMSNs

[0042] 10 mg of dried Ag₂S QDs were redispersed in 5 mL of chloroform and sonicated to obtain solution I. 0.205 g of CTAB and solution I were dispersed in a beaker containing 20 mL of triple-distilled water and stirred until a milky white homogenate was formed, yielding solution II. Solution II was heated to 70 °C in an oil bath under rapid stirring to evaporate the chloroform, yielding a clear, transparent brown solution III (Ag₂S / CTAB solution). Solution III was transferred to a 250 mL flask, and triple-distilled water was added to a final volume of 100 mL, yielding solution IV. 0.056 g of NaOH was added to solution IV and stirred at room temperature to obtain solution V. Solution V was then heated to 80 °C, and 500 mL of tetraethyl orthosilicate (TEOS) was added, followed immediately by 0.5 mL of SrCl₂·6H₂O. Rapid stirring (1000 rpm) was maintained for 1 min, then the stirring speed was reduced, and the mixture was gently stirred at 80 °C (100 rpm) for 8 h. The sample was collected by centrifugation at 10,000 rpm for 10 min and washed repeatedly with water and anhydrous ethanol (10,000 rpm × 10 min) to obtain sample Ag2S@SrSiO2 (non-mesoporous). Template removal using CTAB was then performed. The product was redispersed in 50 mL of anhydrous ethanol containing 0.5 g ammonium nitrate, and then refluxed overnight at 75°C. The product was collected by centrifugation and washed repeatedly with water and anhydrous ethanol to obtain product Ag2S@SrMSNs. It can be freeze-dried for later use.

[0043] ③ Modification of Ag2S@SrMSNs

[0044] 10 mg of Ag2S@SrMSNs were dispersed in 100 mL of a methanol solution containing 5 mg of carboxyl-polyethylene glycol-silane (COOH-PEG-Silane). The mixture was gently stirred at 70 °C using a heated magnetic stirrer, refluxed overnight, and then collected by centrifugation (10,000 rpm × 15 min). The product was then washed repeatedly with water and anhydrous ethanol to obtain carboxyl-modified Ag2S@SrMSNs (Ag2S@SrMSNs-COOH). Ag2S@MMSNs-COOH was dispersed in 20 mL of RH PBS solution (concentration 0.5 mg / mL) and magnetically stirred for 24 hours at room temperature in the dark. The RH-loaded Ag2S@MMSNs-COOH was collected by centrifugation (10,000 rpm, 15 min). The product prepared above was dissolved in 10 mL of water to obtain solution I. 0.1 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.1 mmol of N-hydroxysuccinimide (NHS) were added to solution I, and the mixture was shaken to mix thoroughly. The mixture was then stirred with a magnetic stirrer at room temperature for 15 min (to activate the carboxyl groups of the nanoparticles) to obtain solution II. Subsequently, 0.1 mmol of sodium alendronate and 0.1 mmol of monoamino-substituted β-CD were added to solution II, and the mixture was stirred at room temperature in the dark for 24 h. Finally, the final product (RH-CD@Ag2S@SrMSNs-COOH / Ald) was collected by centrifugation and washing.

[0045] A schematic diagram of the above experimental process is shown below. Figure 1 .

[0046] Example 2: AgSr-MSNs release their bioactive drugs in response to acidic environments.

[0047] like Figure 2 As shown, the synthesized AgSr-MSNs were placed in PBS buffer solutions at pH 6.5 and pH 7.4, respectively, to simulate the physiological environment and the acidic environment of diabetic bone marrow. AgSr-MSNs were dispersed in the two pH buffer systems to prepare suspensions. The suspensions were then completely dispersed using an ultrasonic cleaner and placed in 6-well plates, shaken at a constant temperature of 37°C to simulate the in vivo environment. Samples were taken on days 1, 4, and 7, and the degradation of the material was observed using TEM. Additionally, the two suspensions were collected and analyzed using inductively coupled plasma atomic emission spectrometry (ICP-AES). ICP detection primarily studies the composition of substances by detecting radiation energy; it has extremely high sensitivity and minimal interference, with a detection limit typically in the range of ppb (ng / mL).

[0048] The detection procedure is as follows: First, the AgSr-MSNs degradation solutions treated at different pH and for different times are filtered to obtain a clear solution, which is then diluted to 15 mL with deionized water. Then, the sample is detected using an inductively coupled plasma optical emission spectrometer (ICP-OES (ICAP6300 DUO); ThermoFisher Scientific; UK) to obtain the corresponding signal intensity and calculate the Sr concentration in the solution. 2+ Content. The amount of Rh B generated was determined by ultraviolet-visible spectrophotometry (UV-Vis) to characterize the degradation of the material.

[0049] from Figure 2 It can be seen that, compared to the neutral environment of pH 7.4, the degradation of AgSr-MSNs is accelerated under acidic conditions (pH 6.5). Figure 2 a), while the Rh B generated by the reaction of RH and NO in the mesopores increases ( Figure 2 b), leading to the Sr inside it 2+ Release acceleration ( Figure 2 c). Ultimately, this achieves a response to the acidic microenvironment of diabetes and high NO release Sr. 2+ The effect.

[0050] Example 3, AgSr-MSNs respond to NO in macrophages

[0051] 500 μg / mL AgSr-MSNs were incubated with macrophages for 4 h and 8 h under LPS (1 μg / mL) and a high glucose environment (35 mmol / L), respectively. Cells were fixed with 4% paraformaldehyde at the corresponding time points, and intracellular fluorescence intensity was observed under a confocal microscope (excitation wavelength 550 nm; emission wavelength 575 nm). The content of NO metabolites in the cell culture supernatant was then detected using the Griess method after 1 and 3 days to calculate the remaining intracellular NO content. Experimental results are shown below. Figure 3 It can be seen that as time goes on, the amount of AgSr-MSNs phagocytosed by macrophages increases ( Figure 3 a) Increased NO consumption ( Figure 3 b).

[0052] Example 4, AgSr-MSNs bone targeting

[0053] In vitro bone targeting: Since the main component of bone is hydroxyapatite (HAP), HAP was used as a model component to simulate bone tissue in the in vitro binding experiment to evaluate the in vitro targeting ability of AgSr-MSNs. Different nanomaterials, modified with or without the targeting molecule sodium alendronate (Ald), were incubated with HAP tablets at 37°C in a shaker for 3 hours. The tablets were then washed three times with PBS. Finally, images were taken using a NIR-II imaging system. In vivo targeting: AgSr-MSNs were injected intravenously into 10-week-old male C57 / BL6J mice (25 mg / kg). Mice were sacrificed at 24, 48, and 72 hours, and their hearts were perfused with warm PBS. The hearts, livers, spleens, lungs, and kidneys of the mice were collected for NIR-II imaging to observe their dynamic distribution in vivo. The experimental results are shown below. Figure 4 It can be seen that AgSr-MSNs coupled with Ald can clearly cluster on HAP ( Figure 4 a). In vivo tracer experiments ( Figure 4 b) also shows that AgSr-MSNs can significantly target bone, especially at 48 hours.

[0054] Example 5: AgSr-MSNs combined with NIR irradiation exhibit good biocompatibility in vivo and in vitro.

[0055] 1) In vitro biocompatibility assessment

[0056] Different concentrations (0, 50, 100, 200, 300, 400, 500, 600, and 700 μg / mL) of AgSr-MSNs were cultured in Raw 264.7 macrophages, bone marrow stromal stem cells (BMSCs), and endothelial cells (HUVECs) under NIR irradiation for 1, 4, and 7 days. The cell culture medium was then replaced with serum-free cell culture medium containing CCK-8 reagent (Beyotime, China) at a ratio of 1:10, and incubated at 37°C in the dark for 30 minutes. The absorbance was then measured at a wavelength of 450 nm.

[0057] Raw 264.7, BMSCs, and HUVECs cells were incubated for 7 days using the method described above. Cells were then fixed with 4% paraformaldehyde and washed once with PBS. 100 μl of Calcein AM / PI staining working solution (Beyotime, China; ratio: Calcein AM 1 μl : PI 1 μl : buffer 100 μl) was added to each well of a 96-well plate and incubated at 37°C for 45 minutes in the dark (live cells were stained green, dead cells were stained red). The staining working solution was discarded, and the cells were washed once with PBS and observed under a fluorescence microscope.

[0058] Fresh anticoagulated whole blood was collected from mouse hearts, washed three times with warm PBS, and the supernatant was discarded. The erythrocyte pellet was resuspended in PBS, and then the erythrocyte suspension was incubated with PBS, ddH2O, and 500 μg / ml AgSr-MSNs at 37°C for 1 hour. Hemolysis was observed. The absorbance of the supernatant at 560 nm was measured using a microplate reader, and the hemolysis rate was calculated using the following formula:

[0059] Hemolysis rate (%) = (OD positive control - OD negative control) / (OD sample - OD negative control) × 100%

[0060] 2) In vivo biocompatibility assessment

[0061] AgSr-MSNs were injected intravenously into 10-week-old male C57 / BL6J mice (25 mg / kg) every 3 days for 3 consecutive weeks. Mice were euthanized, and cardiac blood was collected to detect aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA2), and blood urea nitrogen (UREAL) in the blood (using Nanjing Jiancheng Biological Kits). Simultaneously, the hearts, livers, spleens, lungs, and kidneys of the mice were collected and fixed in 4% paraformaldehyde for 7 days. The sections were then embedded in paraffin, sectioned, and baked in a 55°C oven for 2 hours. They were then subjected to two cycles of ethanol (20 minutes each), followed by 95% ethanol (5 minutes each), 85% ethanol (5 minutes each), and 75% ethanol (5 minutes each). After high-temperature retardation, hematoxylin and eosin (H&E) staining was performed, and the pathological structures of each organ were observed under a microscope.

[0062] Different concentrations of AgSr-MSNs were incubated with key cells for bone defect repair (Raw 264.7, BMSCs, and HUVECs). The results showed that concentrations of AgSr-MSNs below 500 μg / ml had no significant effect on cell viability, but cell viability was inhibited at concentrations above 500 μg / ml. Figure 5 a). Subsequently, 500 μg / ml of different forms of MSNs were co-incubated with Raw 264.7, BMSCs, and HUVECs, respectively, and then the cells were stained with calcein (live cells were stained green, and dead cells were stained red). Results are shown below. Figure 5 Experimental results showed that no dead cells were observed after treatment with these nanoparticles. Figure 5 b). Subsequent hemolysis experiments also showed that no significant hemolysis was observed with 500 μg / ml AgSr-MSNs (b). Figure 5c), which ensures the safety of in vivo circulating administration. Then, AgSr-MSNs were injected via tail vein into diabetic mice with femoral bone defects for 3 weeks, followed by NIR irradiation. Blood was collected to detect blood biochemical liver and kidney function indicators (aspartate aminotransferase AST, alanine aminotransferase ALT, creatinine CREA2, and blood urea nitrogen UREAL). The results showed that AST, ALT, CREA2, and UREAL were all within the normal reference range. Figure 5 d). H&E staining of the heart, liver, spleen, lungs, and kidneys all indicated that the nanoparticles had no significant toxicity to liver and kidney function or other vital organs. Figure 5 e). All the above results indicate that AgSr-MSNs have good biocompatibility in vitro and in vivo.

[0063] Example 6: AgSr-MSNs combined with NIR irradiation can regulate the immune phenotype (promote macrophage M2 polarization).

[0064] like Figure 6 MSNs (500 μg / mL) at different stages were incubated with bone marrow macrophages under LPS and high glucose conditions for 48 hours. Cells were then fixed with 4% paraformaldehyde for 20 minutes and washed once with PBS. Cell membranes were permeated with 0.1% Triton X-100 (BioFroX) for 10 minutes, incubated with 10% donkey serum (Solarbio) for 30 minutes, and then incubated overnight at 4°C with primary antibodies CD86 and CD206 (ABclonal, 1:100 dilution). Cells were washed twice with PBS, and then the secondary antibody conjugated with 488 and Cy3 (ABclonal, 1:150 dilution) was diluted with universal antibody diluent (NCM, Biotech) to prepare the working solution. Cells were incubated in the dark for 1 hour. Cells were washed twice, incubated with DAPI solution (Solarbio) in the dark for 10 minutes, washed once with PBS, blocked with an anti-fluorescence quencher, and the cellular immunophenotypic transformation was observed under a microscope.

[0065] MSNs were incubated with macrophages, and the expression of markers for M1 macrophages (CD86) and M2 macrophages (CD206) was detected. Results are shown below. Figure 6 Compared with the control group, AgSr-MSNs combined with NIR irradiation significantly reduced the fluorescence intensity of CD86. Figure 6 a, b), while increasing the fluorescence intensity of CD206 ( Figure 6 (c, d). This demonstrates that AgSr-MSNs combined with NIR irradiation significantly inhibited macrophage M1 polarization and promoted macrophage M2 polarization.

[0066] Example 7: AgSr-MSNs combined with NIR irradiation promote in vitro bone formation by regulating macrophage M2 polarization.

[0067] First, we extracted bone marrow stromal cells (BMSCs) from mouse bone marrow. The specific steps were as follows: Four-week-old male C57 / BL6J mice were purchased from the Experimental Animal Center of Tongji Medical College and euthanized. The mouse skin was cut open, and the intact femur and tibia with muscle were separated and soaked in 75% alcohol for 10-15 minutes. The femur and tibia were removed, and the soft tissue around the bones was separated, taking care to maintain the integrity of the bones. The separated bones were soaked in DMEM / F12 complete medium (Gbico), and the bone at both ends was slightly cut to expose the red bone marrow. The bone marrow cavity was repeatedly flushed with a 1 ml syringe (Shanghai Jinta) until the bone turned completely white. The bone marrow cell suspension was filtered through a 70 μm filter. Then, it was centrifuged at 1200 rpm for 5 minutes. The cell pellet was resuspended in red blood cell lysis buffer (Solarbio), incubated for 5-10 minutes, and centrifuged at 1200 rpm for 5 minutes. The cells were resuspended in PBS and washed once. The cells were then resuspended in DMEM / F12 complete medium and seeded in T25 cell culture flasks (Nest), designated P0. After confluence, the cells were digested, and P3-P5 were used for subsequent experiments.

[0068] First, different MSNs were incubated with macrophages under LPS and high glucose conditions for 48 hours, and the macrophage conditioned medium was collected. Then, the collected conditioned medium was mixed with osteoblast differentiation medium (Cyagen) to treat BMSCs. The medium was changed fresh every 3 days. After 14 days, the cells were stained with alkaline phosphatase (ALP). The specific steps were as follows: the cells were washed once with warm PBS, then fixed with 4% paraformaldehyde for 20 minutes, and washed once with PBS. The staining working solution was prepared according to the BCIP / NBT alkaline phosphatase staining kit (Beyotime) instructions, and incubated on a shaker at room temperature for 30 minutes. The cells were washed once with PBS and observed under a microscope.

[0069] First, different MSNs were incubated with macrophages under LPS and high glucose conditions for 48 hours, and the macrophage conditioned medium was collected. Then, the collected conditioned medium was mixed with osteoblast differentiation medium (Cyagen) to treat BMSCs. The medium was changed fresh every 3 days. After 21 days, the cells were stained with Alizarin Red ARS, following these steps: Cells were washed once with warm PBS, then fixed in 4% paraformaldehyde for 20 minutes, and washed once with PBS. ARS staining working solution (Cyagen) was incubated with the cells for 10 minutes. After washing once with PBS, the cells were observed under a microscope.

[0070] Subsequently, the cell culture supernatant after MSN incubation with macrophages was used to treat BMSCs, and changes in their osteoblast differentiation and mineralization potential were observed. Figure 7 Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining patterns showed that macrophage culture supernatant treated with AgSr-MSNs combined with NIR significantly promoted the differentiation of BMSCs into osteoblasts (increased ALP-positive cells) and mineralization (increased ARS-positive cells). This indicates that AgSr-MSNs combined with NIR irradiation promotes in vitro bone formation by regulating macrophage M2 polarization.

[0071] Example 8: AgSr-MSNs combined with NIR irradiation promote in vitro angiogenesis by regulating macrophage M2 polarization.

[0072] First, different MSNs were incubated with macrophages under LPS and high glucose conditions for 48 hours, and the macrophage conditioned medium was collected. Then, 50 μl of Corning gel was added to each well of a pre-chilled 96-well plate. The plates were incubated at 37°C for 30 minutes to allow solidification. The macrophage conditioned medium was then mixed with ECM (Gbico) to resuspend the HUVECs at a density of 1 × 10⁻⁶. 4 Cells were seeded at 100 μl per well in 96-well plates coated with matrix gel. The cells were incubated at 37°C for 6 hours, after which the culture medium was discarded. Cells were washed once with PBS. Then, the cells were incubated for 30 minutes in Calcein AM staining working solution (Beyotime) at 37°C in the dark, followed by one wash with PBS. Microscopic images were taken for observation. Results are shown below. Figure 8 It is evident that AgSr-MSNs combined with NIR irradiation can significantly improve the impaired in vitro tube-forming ability of endothelial cells under LPS and high glucose conditions.

[0073] Example 9: AgSr-MSNs combined with NIR irradiation promotes the regeneration of bone defects in diabetic patients.

[0074] All animal experiments were conducted in strict accordance with the experimental protocol approved by the Animal Experiment Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IACUC No. 4539). Ten-week-old male C57BL / 6J mice were used to induce a diabetic model via intraperitoneal injection of streptozotocin (STZ, Solarbio, 60 mg / kg, dissolved in 0.1 M pH 4.5 citrate-phosphate buffer). Blood glucose levels were monitored by tail vein sampling; a blood glucose level >16.7 mmol / L for three consecutive days was considered a successful diabetes model. Diabetic mice underwent distal femoral bone defect surgery under sterile conditions: after anesthesia with 1% sodium pentobarbital, the surgical area was shaved and disinfected with povidone-iodine. A 1 cm skin incision was made along the longitudinal axis of the femur, the intermuscular space was bluntly dissected to expose the distal femur, a 1.5 mm bone defect was prepared using a surgical drill, the incision was sutured in layers, and disinfected again. Treatment with tail vein administration began on postoperative day 3. The experimental groups were: wild-type control group (PBS), diabetic group (PBS), and diabetic AgSr-MSNs treatment group (100 μl, 25 mg / kg combined with NIR irradiation). Administration was twice weekly for two weeks. Animals were humanely euthanized three weeks postoperatively, and specimens were collected for analysis.

[0075] To observe the effect of AgSr-MSNs combined with NIR irradiation on in vivo regeneration of diabetic bone defects. A mouse model of distal femoral shaft bone defect was established. After surgery, AgSr-MSNs were administered via the tail vein in combination with NIR irradiation, and the in vivo efficacy was observed. Figure 9 a). Results are shown in Figure 9 It can be seen that AgSr-MSNs combined with NIR irradiation can significantly accelerate the bone regeneration process at the femoral defect site at 7 and 21 days. Figure 9 This demonstrates that AgSr-MSNs combined with NIR irradiation promote in vivo regeneration of bone defects in diabetic patients.

[0076] In summary, this invention provides a NO / pH dual-regulated nanodrug delivery system based on NIR response and its application, constructing a systemic drug carrier with bone targeting and release response to the diabetic bone microenvironment. This addresses the problems of significantly reduced bone healing capacity in diabetic patients, the difficulty of effectively improving systemic bone microenvironment dysregulation with traditional local treatments, and the inability of existing local drug systems to release on demand, efficiently clear local NO, and promote the transformation of macrophages into pro-repair M2 types.

[0077] Finally, it should be noted that the above embodiments are only for illustrating the present invention and not for limiting the scope of protection of the present invention. Furthermore, after reading the technical content of this invention, those skilled in the art can make various modifications, alterations, or variations to the present invention, and all such equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response, characterized in that, The nano-drug delivery system uses strontium ion-doped mesoporous silica Ag2S@SrMSNs as a carrier, with rhodamine RH loaded into the mesoporous channels. The nanoparticles are then modified with monoamino-substituted β-cyclodextrin β-CD and alendronate sodium Ald via a Schiff base reaction to obtain the nano-drug delivery system RH-CD@Ag2S@SrMSNs-COOH / Ald, abbreviated as AgSr-MSNs.

2. The preparation method of the NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response as described in claim 1, characterized in that, Includes the following steps: Synthesis of S1, Ag2S QDs The synthesis of Ag₂S QDs employed a modified thermal decomposition method. A dry three-necked flask was purged with nitrogen. Under nitrogen atmosphere, arsenic reagent C₅H₁₀NS₂Ag, 1-octadecene ODE, and 1-dodecyl mercaptan 1-DT were added sequentially. After stirring, the mixture was heated to 100°C and held for 10 min, then continuously heated to 170°C and held for 10 min to generate Ag₂S QDs under nitrogen conditions. The final solution turned dark black. The reaction was then terminated by adding n-hexane and cooled to room temperature. The Ag₂S QDs were collected by centrifugation, washed with acetone, and finally dispersed in chloroform. The solution was then dried under vacuum at 4°C or 70°C and stored protected from light. Synthesis of S2, Ag2S@SrMSNs The dried Ag2S QDs obtained in step S1 were redispersed in chloroform and dispersed evenly using an ultrasonic machine to obtain solution I. Hexadecyltrimethylammonium bromide (CTAB) and solution I were dispersed in a beaker containing triple-distilled water and stirred to form a milky white slurry to obtain solution II. Solution II was heated to 70°C in an oil bath under stirring at 1000 rpm to evaporate the chloroform and obtain a clear and transparent brown solution III, namely the Ag2S / CTAB solution. Transfer solution III to a flask, then add triple-distilled water to obtain solution IV; add NaOH to solution IV and stir at room temperature to obtain solution V; Solution V was then heated to 80°C, tetraethyl orthosilicate (TEOS) was added, followed by SrCl2·6H2O, and stirred at 1000 rpm for 1 min. The stirring speed was then reduced, and the mixture was stirred at 80°C and 100 rpm for 8 h. The sample was collected by centrifugation and washed with water and anhydrous ethanol to obtain Ag2S@SrSiO2. The template CTAB was then removed, and the product was redispersed in anhydrous ethanol containing ammonium nitrate. The mixture was then refluxed at 75°C, and the product was collected by centrifugation and washed with water and anhydrous ethanol to obtain Ag2S@SrMSNs. S3, Ag2S@SrMSNs modification The Ag2S@SrMSNs obtained in step S2 were dispersed in a methanol solution containing carboxyl-polyethylene glycol-silane COOH-PEG-Silane, stirred at 70°C, refluxed, and finally centrifuged at 10000 rpm for 15 min to collect the product. Then, washing with water and anhydrous ethanol yields Ag2S@SrMSNs containing carboxyl groups, i.e., Ag2S@SrMSNs-COOH; Ag2S@MMSNs-COOH was dispersed in 20 mL of 0.5 mg / mL RH PBS solution and magnetically stirred for 24 hours at room temperature in the dark. The RH-loaded Ag2S@MMSNs-COOH was collected by centrifugation at 10,000 rpm for 15 min. The product prepared above was dissolved in water to obtain solution I. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to solution I, and the mixture was shaken to mix thoroughly. The mixture was stirred at room temperature for 15 min to activate the carboxyl groups of the nanoparticles, yielding solution II. Subsequently, alendronate sodium (Ald) and monoamino-substituted β-CD were added to solution II, and the mixture was stirred at room temperature in the dark for 24 hours. h, and finally centrifugation and washing were performed to collect the final product RH-CD@Ag2S@SrMSNs-COOH / Ald, to obtain the nano-drug delivery system RH-CD@Ag2S@SrMSNs-COOH / Ald, abbreviated as AgSr-MSNs.

3. The preparation method of the NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response according to claim 2, characterized in that, In the preparation process of the nano-drug delivery system, the amount of Ag2S@SrMSNs used is 10 mg, the amount of alendronate sodium Ald and cyclodextrin β-CD used is 0.1 mM each, the amount of rhodamine RH used is 10 mg, and the loading rate is 6.3%.

4. The preparation method of the NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response according to claim 2, characterized in that, In step S1, when collecting Ag2S QDs, the centrifugation speed is 11000 rpm and the time is 15 min; the centrifugation washing speed is 11000 rpm and the time is 15 min, and the washing is performed three times.

5. The preparation method of the NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response according to claim 2, characterized in that, In step S2, when collecting the sample Ag2S@SrSiO2, the centrifugation speed is 10000 rpm and the time is 10 min. When washing with water and anhydrous ethanol, the centrifugation speed is 10000 rpm and the time is 10 min.

6. The application of the NO / pH dual-regulated nano-drug delivery system based on near-infrared (NIR) light response according to any one of claims 1-5 in the preparation of drugs for bone repair in diabetes.

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