Photo-thermal response intelligent drug release microsphere as well as preparation method and application thereof

Abstract

The invention discloses photo-thermal response intelligent drug release microspheres as well as a preparation method and application thereof. The microspheres comprise cationic starch, MXene nanosheets, hyaluronic acid methacrylate and Kartogenin (KGN), wherein the cationic starch encapsulates KGN through a spiral cavity of the cationic starch to form an inclusion compound. The preparation method of the microspheres comprises the following steps: dissolving cationic starch in a PBS (Phosphate Buffer Solution), adding KGN, and carrying out ultrasonic treatment to form an inclusion compound; mixing the obtained inclusion compound with hyaluronic acid methacrylate and MXene nanosheets, and preparing monodisperse liquid drops by adopting a micro-fluidic technology; and carrying out ultraviolet crosslinking to obtain the microspheres. The microspheres disclosed by the invention show good photo-thermal response performance under irradiation of near-infrared light, can realize controllable release of drugs, and have excellent cartilage tissue adhesion and biocompatibility. Through the synergistic effect of the medicine and the stem cells, the microspheres can effectively delay the progress of osteoarthritis and improve the joint function, and a new strategy is provided for treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a photothermal responsive intelligent drug-releasing microsphere and a preparation method and application thereof. Background Art

[0002] Osteoarthritis is a chronic degenerative joint disease that affects millions of people worldwide. The disease is characterized by progressive degeneration of articular cartilage, which can cause joint pain, stiffness, and loss of function in patients. Currently, intra-articular injection is one of the effective means of treating osteoarthritis, but this method of administration has significant limitations. Since the drug in the joint cavity is quickly cleared, the drug utilization rate is low and frequent injections are required, which not only increases the patient's pain, but also makes it difficult to achieve the desired therapeutic effect. Therefore, it is of great significance to develop a delivery system that can achieve long-term drug delivery in the joint cavity.

[0003] Studies in recent years have shown that the residence time of drugs in the joint cavity plays a key role in the therapeutic effect of osteoarthritis. Traditional drug delivery systems are often unable to maintain therapeutic concentrations, and most drugs are cleared through the synovial circulation within hours after injection. To address this problem, researchers have tried to develop a variety of materials with tissue adhesion to prolong the retention time of drugs. These methods include modifying the drug delivery system with adhesive groups and developing biomimetic adhesion materials. However, achieving controlled drug release and effective tissue adhesion in a dynamic joint environment still faces major challenges.

[0004] In the treatment strategy of osteoarthritis, both drug intervention and cell therapy show good application prospects. Among them, Kartogenin (KGN), as a small molecule drug, has been shown to promote chondrocyte differentiation and cartilage regeneration by regulating the CBFβ-RUNX1 pathway. However, KGN has a narrow therapeutic window and has problems such as poor solubility and rapid clearance, which limits its clinical application. Researchers have adopted a variety of delivery strategies, such as nanoparticles, hydrogels, and microspheres, but it is still challenging to achieve efficient drug loading and controlled release. Similarly, bone marrow mesenchymal stem cells show important value in cartilage repair due to their multidirectional differentiation potential, immunomodulatory effects, and ability to secrete therapeutic factors. However, current delivery methods make it difficult to maintain sufficient cell retention and survival, especially in the joint environment where inflammation and mechanical stress coexist.

[0005] Although photothermal therapy has opened up new possibilities for controlled drug delivery, the existing technology still lacks an intelligent delivery system that can simultaneously achieve precise delivery of drugs and stem cells, has good tissue adhesion, and can be photothermally controlled. Therefore, it is urgent to develop a new multifunctional delivery system to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a photothermal responsive smart drug-releasing microsphere and its preparation method and application. In order to solve the technical problems of short drug retention time, low utilization rate and poor cell survival rate in the prior art, the present invention provides a smart microsphere system that can simultaneously realize the coordinated delivery of drugs and stem cells and has photothermal controlled drug release function, which is a photothermal responsive smart drug-releasing microsphere for preparing the treatment of osteoarthritis.

[0007] The basic structure of the photothermal responsive smart drug-releasing microspheres includes cationic starch, MXene nanosheets, hyaluronic acid methacrylate and KGN. The cationic starch is obtained by modifying starch with GTAC. The modified cationic starch can encapsulate KGN through its spiral cavity and provide the positive charge required for binding with cartilage tissue. The MXene nanosheets are selectively etched by Ti 3 AlC 2 The hyaluronic acid methacrylate is prepared and used to provide photothermal response performance. The hyaluronic acid methacrylate is used as a matrix material to provide biocompatibility and lubricity.

[0008] Furthermore, the preparation method of the photothermal responsive intelligent drug-releasing microspheres comprises the following steps: dissolving the cationic starch in PBS buffer, adding KGN thereto and forming an inclusion complex by ultrasonic treatment; mixing the inclusion complex with the hyaluronic acid methacrylate and the MXene nanosheets, and preparing monodisperse droplets by microfluidic technology; and finally obtaining the microspheres by ultraviolet crosslinking.

[0009] Furthermore, the microspheres exhibit excellent photothermal conversion performance under near-infrared light irradiation. When the microspheres are irradiated with 808nm near-infrared light, the MXene nanosheets can convert light energy into heat energy, causing the local temperature to rise to about 42°C. The temperature increase can trigger the controlled release of the drug, and the release behavior can be precisely regulated by adjusting the illumination parameters.

[0010] Furthermore, the mild thermal stimulation can induce the expression of heat shock protein HSP70, which protects chondrocytes. At the same time, the positive charge on the surface of the microspheres can electrostatically adsorb with cartilage tissue, significantly prolonging their retention time in the joint cavity.

[0011] Furthermore, the microspheres have a porous structure and can be used for loading and delivery of bone marrow mesenchymal stem cells. The average particle size of the microspheres is 85 μm, and the surface pore size is about 2 μm. The porous structure can provide a microenvironment for the attachment and growth of bone marrow mesenchymal stem cells, thereby achieving efficient delivery of cells. Experimental verification shows that the microspheres have excellent biocompatibility and controllable degradation performance.

[0012] Furthermore, the microspheres can be administered by intra-articular injection, with a dose of 1 mg / kg, once every two weeks, and near-infrared light irradiation is performed once a week for 10 minutes, and the drug is released on demand under the control of near-infrared light. Through the synergistic effect of drugs and stem cells, the microsphere system can effectively delay the progression of osteoarthritis and improve joint function.

[0013] Furthermore, the microspheres can increase the local temperature to 42±0.5°C under near-infrared light irradiation, inducing the expression of heat shock protein HSP70.

[0014] Furthermore, the surface of the microspheres carries a positive charge, which can electrostatically adsorb to cartilage tissue and prolong the retention time.

[0015] Furthermore, the drug loading efficiency of the microspheres is 7.1 wt %, and the cumulative release within 14 days under the control of near-infrared light can reach 90%.

[0016] Compared with the prior art, the photothermal responsive smart drug-releasing microspheres of the present invention solve many problems existing in traditional drug delivery systems through structural innovation and functional integration. The microspheres can not only achieve efficient loading and controlled release of drugs, but can also be used for stem cell delivery, providing a new strategy and solution for the treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the technical solution of the present invention and constitute a part of the specification. Together with the specific implementation methods of the present application, they are used to explain the technical solution of the present invention and do not constitute a limitation on the technical solution of the present invention.

[0018] Figure 1 The figure is a schematic diagram of the preparation process and mechanism of action of the photothermal responsive smart drug-releasing microspheres of the present invention. Figure 1 A shows the preparation of cationic starch and its inclusion process with KGN, Figure 1 B shows the transmission electron microscopy image of the final cationic starch-KGN inclusion complex (NCA@KGN). Figure 1 C shows the Zeta potential distribution diagram of NCA@KGN. Figure 1 D shows the process flow of preparing microspheres by microfluidics. Figure 1 E shows an optical microscope photo of the final microspheres. Figure 1 F is the scanning electron microscope image of the microspheres. Figure 1 G shows the distribution of each element in the microsphere, Figure 1 H shows the particle size distribution of microspheres, Figure 1 I shows the pore size distribution of microspheres, Figure 1 J is the swelling curve of the microspheres in PBS, Figure 1K shows the swelling rate of freeze-dried H / M-NCA@KGN microspheres in PBS buffer over time.

[0019] Figure 2 The photothermal performance and drug release behavior of the microspheres of the present invention are characterized in FIG. Figure 2 A shows the temperature distribution thermal imaging diagram under near-infrared light of different power densities. Figure 2 B is a comparison of the heating curves of microspheres containing MXene and those without MXene. Figure 2 C is the temperature rise curve under different lighting conditions. Figure 2 D shows the photothermal imaging results in animals. Figure 2 E is the quantitative analysis result of the photothermal effect in vivo, Figure 2 F is the temperature change curve under multiple light cycles, Figure 2 G shows the UV-Vis analysis results of drug loading and release behavior, Figure 2 H is the quantitative analysis result of drug loading efficiency, Figure 2 I is the drug release curve under near-infrared light control, Figure 2 J is the fluorescence image of Nile Red released by near-infrared triggering. Figure 2 K is the in vivo fluorescence imaging showing the retention and degradation of DIR-labeled microspheres over a 4-week period.

[0020] Figure 3 The figure is an evaluation diagram of the biocompatibility and cell delivery performance of the microspheres of the present invention. Figure 3 A shows the results of CCK-8 cell activity detection, Figure 3 B is the live and dead cell staining result. Figure 3 C is the quantitative analysis of cell counting results. Figure 3 D shows a fluorescence microscope photo of bone marrow mesenchymal stem cells on the surface of microspheres. Figure 3 E is a schematic diagram of cell-material interaction. Figure 3 F shows the migration of cells into osteoarthritic cartilage tissue.

[0021] Figure 4 The figure is a graph showing the evaluation results of the microspheres of the present invention promoting cartilage differentiation. Figure 4 AB show the results of RT-PCR analysis of cartilage markers COL-2a and SOX9. Figure 4 C is the immunofluorescence staining of COL-2a protein. Figure 4 DE is the result of Western blot analysis, Figure 4 F is the quantitative analysis result of fluorescence intensity, Figure 4 G is a schematic diagram of the mechanism of near-infrared light-induced chondrogenic differentiation.

[0022] Figure 5This is a diagram for evaluating the protective effect of the microspheres of the present invention in an in vitro osteoarthritis model. Figure 5 A shows the immunofluorescence staining results of cartilage matrix-related proteins. Figure 5 BC is the result of RT-PCR analysis. Figure 5 DE is the result of protein quantitative analysis. Figure 5 F is the result of flow cytometry analysis. Figure 5 GH is the analysis result of HSP70 expression, Figure 5 I is the quantitative analysis result of chondrocyte apoptosis rate.

[0023] Figure 6 This is a graph showing the evaluation results of the microspheres of the present invention in the treatment of osteoarthritis in vivo. Figure 6 A shows the animal experiment scheme, Figure 6 B is the quantitative analysis result of osteophyte volume. Figure 6 C is the X-ray evaluation result, Figure 6 D is the micro CT reconstructed image, Figure 6 E is the three-dimensional reconstruction result of cartilage structure.

[0024] Figure 7 The figure is a histological and molecular level evaluation result of the protective effect of the microspheres of the present invention on cartilage tissue. Figure 7 A shows the results of H&E staining, safranin O staining and toluidine blue staining, Figure 7 B is the immunofluorescence analysis result of cartilage markers. Figure 7 C is the OARSI score result. Figure 7 D is the analysis result of GAG content, Figure 7 EF is the quantitative analysis result of COL-2a and SOX9 expression levels.

[0025] Figure 8 Schematic diagram of the preparation process of the photothermal responsive smart drug-releasing microspheres of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is only used to illustrate the present invention and is not intended to limit the scope of the present invention. It should be understood that the features described in the present invention can be combined with each other without conflict. Example

[0027] refer to Figure 1-8 As shown, preparation of photothermal responsive smart drug release microspheres 1.1 Preparation of cationic starch Dissolve 2.5 g of amylose in 10 mL of deionized water. Prepare 1 mL of 1.0 M NaOH solution, add 2.8 g of GTAC, and stir the resulting mixture at 60 ° C at 500 rpm for 3 hours. Then neutralize the reaction solution with 1% acetic acid, and collect the precipitate by centrifugation at 1000 rpm for 5 minutes. Wash the precipitate repeatedly with 95% ethanol until no chloride ions are detected in the supernatant. The obtained cationic starch is vacuum dried at 60 ° C, dissolved in deionized water, ultrasonically treated at 20 kHz for 2 hours, filtered through a 0.22 μm membrane, and freeze-dried to obtain cationic starch (NCA).

[0028] 1.2 Preparation of MXene nanosheets Weigh 1.5 g LiF and dissolve it in 25 mL hydrochloric acid solution, stir at room temperature for 10 minutes. Gradually add 1.25 g Ti 3 AlC 2 , react at 40 ° C for 24 hours. The reaction mixture was cooled to room temperature, centrifuged at 3500 rpm for 5 minutes, and the supernatant was discarded. The precipitate was repeatedly washed with deionized water until the pH of the supernatant reached 6. 75 mL of deionized water was added to the precipitate, and after ultrasonication for 1 hour, it was centrifuged at 3500 rpm for 30 minutes, and the supernatant containing MXene nanosheets was collected for use.

[0029] 1.3 Preparation of NCA@KGN inclusion complex 100 mg NCA was dissolved in 10 mL PBS, and an appropriate amount of KGN in DMSO was added. The mixture was treated with a 20 kHz probe sonicator for 10 minutes, and the resulting solution was placed in a 3.5 kDa dialysis bag and dialyzed in PBS (pH 7.4) for 24 hours to remove unencapsulated drugs. The NCA@KGN inclusion complex was obtained by freeze drying.

[0030] 1.4 Preparation of photothermal responsive smart drug release microspheres Microspheres were prepared by microfluidic technology. The aqueous phase consisted of 5wt% hyaluronic acid methacrylate, 0.1wt% NCA@KGN, 0.3wt% MXene nanosheets and 0.5wt% photoinitiator LAP. The oil phase was paraffin oil containing 5wt% Span80. The flow rates of the aqueous and oil phases were controlled by a dual-channel syringe pump, and monodisperse droplets were collected. The crosslinking was carried out by 405nm ultraviolet light, and the oil phase was removed by washing with acetone and deionized water to obtain photothermal responsive smart drug release microspheres.

[0031] Physicochemical characterization of microspheres 2.1 Morphology and structure characterization The morphology of NCA@KGN was observed by transmission electron microscopy, and the results showed that it was irregularly spherical with an average particle size of about 55nm. Dynamic light scattering test showed that the hydrated particle size of NCA@KGN was 90nm and the Zeta potential was +45mV. Optical microscopy and scanning electron microscopy observations showed that the prepared microspheres were regular spherical, with a porous structure, uniform particle size distribution, an average particle size of 85μm, and a surface pore size of about 2μm. Element distribution analysis confirmed that C, N, O and Ti elements were evenly distributed in the microspheres.

[0032] 2.2 Photothermal performance evaluation The microspheres (200 μg / mL) were dispersed in PBS and subjected to a temperature increase experiment under 808 nm near-infrared light irradiation (0.5-1.5 W / cm²). Microspheres containing 0.3 wt% MXene can raise the solution temperature to 48 ± 0.5 °C under 1.0 W / cm² light, while the control group without MXene showed no obvious temperature change. In vivo illumination experiments showed that the microspheres can maintain the local temperature at 42 ± 0.5 °C and maintain stable photothermal conversion efficiency after multiple illumination cycles.

[0033] 2.3 Evaluation of drug loading and release performance NCA@KGN was characterized by UV-Vis spectroscopy. The results showed that NCA itself had no obvious absorption peak, while NCA@KGN showed the characteristic absorption peak of KGN at 276nm, confirming the successful encapsulation of the drug. The drug loading determination showed that the drug loading efficiency of NCA was about 8.2wt%, and the loading efficiency of the microspheres was 7.1wt% (based on the same amount of NCA). The drug release experiment showed that a single near-infrared light irradiation (0.5W / cm², 10 minutes) could trigger a rapid release of about 20±5% of the drug. After four cycles of illumination during the 14-day release period, the cumulative release reached 90%, while the control group without illumination released only 38%.

[0034] 2.4 Biocompatibility evaluation The CCK-8 method was used to evaluate the cytotoxicity of microspheres to bone marrow mesenchymal stem cells. The cells were co-cultured with microspheres for 1, 3, and 5 days before testing. The results showed that there was no significant difference in the cell proliferation activity of each experimental group, indicating that the microspheres had good biocompatibility. The live and dead cell staining results further confirmed the safety of the microspheres, and fluorescence microscopy showed that the cells adhered and grew well on the surface of the microspheres.

[0035] In vitro evaluation of therapeutic effects 3.1 Chondrogenic differentiation induction experiment Bone marrow mesenchymal stem cells were co-cultured with microspheres for 14 days, during which they were irradiated with near-infrared light (0.5 W / cm²) four times. RT-PCR analysis showed that the expression levels of cartilage markers COL-2a and SOX9 were significantly upregulated in the H / M-NCA@KGN+NIR group. Immunofluorescence staining and Western blot analysis further confirmed the above results, indicating that the system can effectively promote the differentiation of stem cells into chondrocytes.

[0036] 3.2 Evaluation of anti-inflammatory protective effects An IL-1β-induced in vitro osteoarthritis model was established, and the protective effect of the microspheres was evaluated using a Transwell co-culture system. The results showed that H / M-NCA@KGN+NIR treatment significantly inhibited cartilage matrix degradation, maintained a high Aggrecan expression level, and reduced the expression of MMP-13. Flow cytometry analysis showed that the treatment could effectively reduce the apoptosis rate of chondrocytes. Mechanistic studies found that mild photothermal effect can induce the expression of HSP70, thereby exerting a protective effect.

[0037] In vivo evaluation of therapeutic efficacy 4.1 Animal model establishment and drug administration regimen Eight-week-old male SD rats were selected and osteoarthritis models were established by medial meniscus resection combined with anterior cruciate ligament transection. The model rats were randomly divided into five groups: sham operation group, saline group, H / M-NCA group, H / M-NCA@KGN group and H / M-NCA@KGN+NIR group. The drug was administered on the second day after surgery at a dose of 1 mg / kg, injected into the joint cavity once every two weeks, and NIR treatment (808nm, 0.5W / cm², 10 minutes) was performed once a week. The animals were killed for evaluation after 8 weeks of treatment.

[0038] 4.2 Radiological evaluation X-ray examination results showed that the joint space width of the H / M-NCA@KGN+NIR group was well maintained and osteophyte formation was less. Micro-CT analysis further confirmed that this group had the best joint structure protection. Three-dimensional reconstruction results showed that its subchondral bone structure integrity was better than that of other treatment groups.

[0039] 4.3 Histological evaluation HE staining, safranin O staining, and toluidine blue staining results showed that the cartilage matrix protection in the H / M-NCA@KGN+NIR group was the most obvious. OARSI score and GAG content analysis quantitatively confirmed the results. Immunofluorescence analysis showed that the expression of COL-2a and SOX9 in this group was close to that of the sham operation group, indicating that it has a good cartilage protection effect. No obvious pathological changes in major organs were found in the biosafety evaluation.

[0040] The above examples fully demonstrate the application value of the present invention in the treatment of osteoarthritis, but the protection scope of the present invention is not limited thereto. Any non-substantial improvements and equivalent substitutions made by those skilled in the art on the basis of the present invention shall fall within the protection scope of the present invention.

Claims

1. A photothermal responsive smart drug-releasing microsphere, characterized in that: The microspheres include cationic starch, MXene nanosheets, hyaluronic acid methacrylate and KGN; wherein the cationic starch encapsulates the KGN through its spiral cavity to form an inclusion complex.

2. The photothermal responsive smart drug-releasing microspheres according to claim 1, characterized in that: The cationic starch is prepared by modifying amylose with GTAC.

3. The photothermal responsive smart drug-releasing microspheres according to claim 1, characterized in that: The MXene nanosheets are prepared by selectively etching Ti3AlC2.

4. The photothermal responsive smart drug-releasing microspheres according to claim 1, characterized in that: The mass fraction of the hyaluronic acid methacrylate is 5wt%, the mass fraction of the cationic starch and KGN inclusion complex is 0.1wt%, the mass fraction of the MXene nanosheet is 0.3wt%, and the rest is water.

5. The photothermal responsive smart drug-releasing microspheres according to claim 1, characterized in that: The microspheres have a porous structure, an average particle size of the microspheres is 85 μm, and a surface pore size of about 2 μm, and can be used for loading and delivering bone marrow mesenchymal stem cells.

6. The method for preparing photothermal responsive smart drug-releasing microspheres according to claim 1, characterized in that: The following steps are involved: (1) Dissolve cationic starch in PBS buffer, add KGN and form inclusion complex by ultrasonic treatment; (2) mixing the inclusion complex obtained in step (1) with hyaluronic acid methacrylate and MXene nanosheets, and preparing monodisperse droplets using microfluidics technology; (3) The microspheres are obtained by ultraviolet cross-linking.

7. The preparation method according to claim 6, characterized in that The oil phase of the microfluidic technology in step (2) is paraffin oil containing 5wt% Span80.

8. The preparation method according to claim 6, characterized in that: The wavelength of the ultraviolet light in step (3) is 405 nm.

9. The use of the photothermal responsive smart drug-releasing microspheres according to claim 1, characterized in that: Used to prepare microspheres for the treatment of osteoarthritis, and control drug release through near-infrared light irradiation.

10. The use according to claim 9, characterized in that: The wavelength of the near infrared light is 808nm, and the power density is 0.5-1.5W / cm².

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