Biomimetic photosynthetic nanoparticles, methods of making and using the same

Abstract

The application discloses a kind of bionic photosynthetic nanoparticles and its preparation method and application.The bionic photosynthetic nanoparticles are prepared by thylakoid nanoparticle suspension and bone marrow mesenchymal stem cell membrane suspension mixing, and are prepared after oscillation incubation.The bionic photosynthetic nanoparticles and the biological energy hydrogel prepared by the bionic photosynthetic nanoparticles are applied to prepare the biological medical material or drug for repairing osteoporotic bone defect, can simultaneously, continuously and targetedly provide energy supply for osteoporotic bone defect site, relieve oxidative stress and improve hypoxic microenvironment, thereby synergistically promote mitochondrial function recovery and bone marrow mesenchymal stem cell osteogenic differentiation, effectively break the pathological vicious cycle of osteoporotic bone defect, and finally realize efficient bone regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and bone tissue regeneration materials technology, specifically relating to a biomimetic photosynthetic nanoparticle, its preparation method and application. Background Technology

[0002] Repairing osteoporotic bone defects is a major clinical challenge in orthopedics. Its high incidence and disability rate severely impact patients' quality of life and impose a heavy medical burden. The fundamental reason why bone defects are difficult to repair in osteoporosis lies in the imbalance of bone homeostasis and the decline in bone regeneration potential. Its pathological process is complex, involving multiple factors such as energy metabolism disorders, increased oxidative stress, and a hypoxic microenvironment. Bone tissue regeneration is a highly energy-dependent process, including osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), bone matrix synthesis, and mineralization, all of which require a continuous and stable supply of adenosine triphosphate (ATP) as an energy basis. Under osteoporotic conditions, the energy metabolism of osteocytes and BMSCs is severely impaired, manifested as insufficient ATP production. This is accompanied by mitochondrial dysfunction, chronic oxidative stress, and a hypoxic microenvironment in the bone defect area, collectively forming a vicious cycle of "oxidative stress-mitochondrial damage-energy supply inhibition," severely hindering bone repair.

[0003] To address intracellular energy deficiency in pathological states, current technologies attempt to directly supplement exogenous adenosine triphosphate (ATP), but its efficacy is minimal due to poor cell membrane permeability and extremely short in vivo half-life. In recent years, biomaterials based on cellular energy homeostasis regulation have gradually emerged. For example, some bioactive materials providing tricarboxylic acid cycle intermediates or phosphate sources can directly improve mitochondrial energy supply; while biomaterials containing antioxidants such as selenium and manganese dioxide indirectly regulate energy metabolism to promote bone repair by scavenging reactive oxygen species (ROS). However, the pathological characteristics of osteoporotic bone defects simultaneously encompass energy deficiency, mitochondrial damage, and hypoxia, and currently lack a single biomaterial system that can simultaneously meet all three therapeutic needs. Therefore, developing novel therapeutic systems with combined energy supply, mitochondrial repair, and oxygen delivery functions is crucial to overcoming the challenges in repairing osteoporotic bone defects. In addition, to improve local delivery, natural polymer materials with excellent biocompatibility, controllable degradation and good mechanical properties, such as silk fibroin (SF) and its modified products, such as methacrylamide silk fibroin (SilMA), have been extensively studied for constructing hydrogel scaffolds that can fill bone defects and support cell growth.

[0004] Thylakoids are the site of photosynthesis in plants, efficiently converting light energy into chemical energy under illumination, simultaneously producing adenosine triphosphate (ATP), reduced coenzyme II (NADPH), and oxygen. This characteristic offers a revolutionary approach to simultaneously addressing the three major challenges of energy deficiency, oxidative stress, and hypoxia. However, the main technological bottleneck currently faced is how to safely and specifically deliver heterologous thylakoids to bone defect sites and maintain their functional activity in the in vivo environment. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the primary objective of this invention is to provide a method for preparing biomimetic photosynthetic nanoparticles. This method is simple, easy to operate, and suitable for industrial production.

[0006] The second objective of this invention is to provide a biomimetic photosynthetic nanoparticle.

[0007] A third objective of this invention is to provide a bioenergy hydrogel prepared using biomimetic photosynthetic nanoparticles. This bioenergy hydrogel comprises biomimetic photosynthetic nanoparticles (BNs or BTUPs) loaded therein and a substrate hydrogel, wherein the substrate hydrogel is at least one of methacrylamide silk fibroin (SilMA) or protocatechuic aldehyde-grafted silk fibroin-phenylboronic acid-grafted chondroitin sulfate (SDPC) hydrogel. This bioenergy hydrogel integrates energy supply, antioxidant properties, oxygen supply, and biocompatibility, effectively breaking the vicious cycle of osteoporosis and bone defects, and significantly promoting bone regeneration.

[0008] A fourth objective of this invention is to provide the application of at least one of biomimetic photosynthetic nanoparticles and bioenergy hydrogels prepared using biomimetic photosynthetic nanoparticles in the preparation of biomedical materials or drugs for repairing osteoporotic bone defects. When applied to medical devices or drugs for repairing osteoporotic bone defects, these biomimetic photosynthetic nanoparticles and bioenergy hydrogels can simultaneously, continuously, and targetedly provide energy to the osteoporotic bone defect site, alleviate oxidative stress, and improve the hypoxic microenvironment, thereby synergistically promoting mitochondrial function recovery and osteogenic differentiation of bone mesenchymal stem cells (BMSCs), ultimately achieving efficient bone regeneration.

[0009] The primary objective of this invention can be achieved by the following technical solution:

[0010] A method for preparing biomimetic photosynthetic nanoparticles includes the following steps: mixing a suspension of thylakoid nanoparticles (TNs or TUPs) with a mass concentration of 0.5–5 mg / mL with a suspension of bone marrow mesenchymal stem cell membranes (BM) with a mass concentration of 0.5–5 mg / mL at a volume ratio of 1:0.3–3; incubating with shaking; and then extruding sequentially through polycarbonate membranes at 1000 nm, 400 nm, and 200 nm; followed by centrifugation and purification to obtain biomimetic photosynthetic nanoparticles (BNs or BTUPs); wherein the thylakoid nanoparticle suspension is prepared from thylakoids extracted from plant leaves containing chlorophyll.

[0011] Preferably, the preparation method of the thylakoid nanoparticle suspension is as follows:

[0012] Take cleaned and destemmed plant leaves, crush them in pre-cooled extraction buffer, filter them, centrifuge the filtrate, precipitate them, resuspend them in washing buffer, spread the resuspended solution on a pre-prepared Percoll density gradient solution, centrifuge them, collect the green thylakoid bands located at the gradient interface, wash them with washing buffer, and obtain purified thylakoids.

[0013] The purified thylakoids were resuspended in osmotic shock buffer, sonicated in an ice bath, and extruded sequentially through 1000 nm, 400 nm, 200 nm and 100 nm polycarbonate membranes to prepare a thylakoid nanoparticle suspension.

[0014] Alternatively, the purified thylakoids are resuspended in osmotic shock buffer, sonicated in an ice bath, and thoroughly mixed with upconversion nanoparticle (UP) suspension at a mass ratio of 1:0.3–3. After shaking and incubation, the mixture is extruded sequentially through 1000 nm, 400 nm, and 200 nm polycarbonate membranes, centrifuged, and resuspended to prepare a thylakoid nanoparticle suspension.

[0015] Preferably, the plant leaves are spinach leaves.

[0016] Preferably, the upconversion nanoparticle (UPs) suspension is prepared by resuspending UPs in distilled water, the mass concentration of the upconversion nanoparticle (UPs) suspension is 2~8 mg / mL, and the upconversion nanoparticle (UPs) is water-soluble core-shell upconversion nanoparticle (X-SHB-650), purchased from Hangzhou Xinqiao Biotechnology Co., Ltd.

[0017] Preferably, the pre-cooled extraction buffer consists of 330 mmol / L sorbitol, 50 mmol / L N-(2-hydroxyethyl)piperazine-N'-2-sulfonic acid (HEPES-KOH) pH 7.6, 5 mmol / L magnesium chloride (MgCl2), and 0.1% w / v bovine serum albumin (BSA); the washing buffer consists of 300 mmol / L sorbitol, 50 mmol / L N-(2-hydroxyethyl)piperazine-N'-2-sulfonic acid (HEPES-KOH) pH 7.6, 5 mmol / L magnesium chloride (MgCl2), 2 mmol / L ethylenediaminetetraacetic acid (EDTA), and 10 mmol / L L-ascorbic acid sodium salt; the Percoll density gradient solution has an upper layer of 40% Percoll and a lower layer of 80% Percoll; the osmotic shock buffer consists of 10 mmol / L... Composed of N-(2-hydroxyethyl)piperazine-N'-2-sulfonic acid (HEPES-KOH) pH 7.6, 10 mmol / L magnesium chloride (MgCl2), and 10 mmol / L sodium L-ascorbate.

[0018] Preferably, in the preparation process of the biomimetic photosynthetic nanoparticles, the temperature of the oscillation incubation is 4-8°C, the oscillation incubation time is 10-30 minutes; the temperature of the centrifugation is 4-6°C, the centrifugation speed is 3000-15000g, and the centrifugation time is 10-30 minutes.

[0019] Preferably, in the preparation process of the thylakoid nanoparticle suspension, the centrifugation temperature is 4-8°C, the centrifugation speed is 1000-10000g, and the centrifugation time is 5-10 minutes; the ice bath ultrasonication time is 3-5 minutes.

[0020] Preferably, the bone marrow mesenchymal stem cell membrane (BM) suspension is prepared as follows:

[0021] Human bone marrow mesenchymal stem cells (hBMSCs) cultured to the logarithmic growth phase are collected, resuspended in pre-cooled hypotonic lysis buffer, incubated on ice, and then the cells are lysed to prepare cell lysate. The cell lysate is centrifuged to remove cell nuclei and unbroken cell debris, and the supernatant is centrifuged again; the resulting precipitate is the hBMSC cell membrane suspension. Preferably, the hBMSCs are obtained by collecting donor bone marrow tissue samples, mixing with phosphate-buffered saline (PBS) buffer, adding dropwise to Percoll cell separation medium, centrifuging at low temperature to remove impurities, adding more PBS buffer, centrifuging again, washing, discarding the supernatant, and obtaining the cell suspension. The cell suspension is then seeded into sterile culture flasks for proliferation culture to obtain human bone marrow mesenchymal stem cells (hBMSCs).

[0022] Preferably, the hypotonic lysis buffer is composed of 1 mmol / L sodium bicarbonate (NaHCO3), 0.2 mmol / L ethylenediaminetetraacetic acid (EDTA), and 1 mmol / L benzyl sulfonyl fluoride (PMSF), and the pH of the hypotonic lysis buffer is 7.4.

[0023] Preferably, in the method for preparing the bone marrow mesenchymal stem cell membrane suspension, the incubation time on ice is 20-30 minutes; the shaking incubation temperature is 4-8°C, and the shaking incubation time is 10-30 minutes; the centrifugation temperature is 4-6°C, the centrifugation speed is 3000-15000g, and the centrifugation time is 10-30 minutes.

[0024] The second objective of this invention can be achieved by the following technical solution:

[0025] A biomimetic photosynthetic nanoparticle was prepared according to the above preparation method.

[0026] The third objective of this invention can be achieved by the following technical solution:

[0027] A bioenergy hydrogel prepared using biomimetic photosynthetic nanoparticles comprises a substrate hydrogel and biomimetic photosynthetic nanoparticles (BNs or BTUPs), wherein the substrate hydrogel is at least one of methacrylamide silk fibroin (SilMA) or protocatechuic aldehyde grafted silk fibroin-phenylboronic acid grafted chondroitin sulfate (SDPC) hydrogel.

[0028] Preferably, the mass concentration of the biomimetic photosynthetic nanoparticles, calculated as chlorophyll, is 2.5-20 μg / mL.

[0029] Preferably, when the substrate hydrogel is a methacrylamide silk fibroin (SilMA) hydrogel, the methacrylamide silk fibroin hydrogel is formed by crosslinking a 10%-30% methacrylamide silk fibroin solution by irradiation with 405nm ultraviolet light for 10-60s in the presence of a photoinitiator.

[0030] When the substrate hydrogel is a protocatechuic aldehyde grafted fibroin-phenylboronic acid grafted chondroitin sulfate (SDPC) hydrogel, the protocatechuic aldehyde grafted fibroin-phenylboronic acid grafted chondroitin sulfate (SDPC) hydrogel is formed by cross-linking protocatechuic aldehyde grafted fibroin (SF-DB) with a mass concentration of 20% and phenylboronic acid grafted chondroitin sulfate (PBA-CS) with a mass concentration of 20% in a volume ratio of 1:0.2 to 5 by vortex mixing.

[0031] Preferably, the method for preparing the methacrylamide silk fibroin (SilMA) is as follows:

[0032] Silk fibroin was dissolved in LiBr solution at a constant temperature of 60-70℃ by stirring. Then, glycidyl methacrylate was added and stirred in the dark to form a mixed solution. The solution was filtered through a mesh screen, dialyzed, and freeze-dried to finally obtain SilMA.

[0033] Preferably, the mass concentration of the silk fibroin fiber in the mixed solution is 0.1–0.5 g / mL, the mass concentration of the LiBr solution in the mixed system is 0.5–1.5 g / mL, and the mass concentration of the glycidyl methacrylate in the mixed system is 0.5–2 g / mL.

[0034] Preferably, the silk fibroin fiber is obtained by repeatedly softening silkworm cocoons in a boiling alkaline solution, washing with deionized water, and air-drying.

[0035] Preferably, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0036] The fourth objective of this invention can be achieved by the following technical solution:

[0037] The application of at least one of biomimetic photosynthetic nanoparticles and bioenergy hydrogels prepared using biomimetic photosynthetic nanoparticles in the preparation of biomedical materials or drugs for repairing osteoporotic bone defects.

[0038] Preferably, the application includes the bioenergy hydrogel generating adenosine triphosphate (ATP), reduced coenzyme II (NADPH), and oxygen through photosynthesis under visible light (such as 630nm red light) or near-infrared light (such as 808nm near-infrared light), thereby increasing the energy level of cells in the lesion site, clearing reactive oxygen species, improving hypoxia, and thus promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone tissue regeneration.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] (1) Functional integration innovation: This invention combines plant thylakoids with complete photosynthetic function with tissue-engineered hydrogels for the first time, creating a "bioenergy factory" that can simultaneously and actively provide ATP (energy), NADPH (antioxidant) and oxygen (metabolic substrate), namely bioenergy hydrogel; this design addresses the three core obstacles of osteoporosis bone repair in principle, realizing multiple synergistic treatments of a single material system;

[0041] (2) High-quality targeted delivery and biocompatibility: This invention utilizes the cell membrane of bone marrow mesenchymal stem cells (BMSCs) to biomimeticly coat thylakoids. The resulting biomimetic photosynthetic nanoparticles are composed of thylakoid nanoparticles wrapped by cell membranes derived from bone marrow mesenchymal stem cells. The biomimetic structure of these photosynthetic nanoparticles endows them with excellent homologous targeting and immune evasion capabilities, enabling them to effectively evade the recognition and clearance of the immune system. Furthermore, through homologous targeting, they can be preferentially taken up by bone marrow mesenchymal stem cells (BMSCs) at the bone defect site, significantly improving the precision and biocompatibility of the treatment and reducing the risk of immune rejection that may be caused by heterologous materials.

[0042] (3) Reversing mitochondrial dysfunction: In vitro experiments conducted by this invention have confirmed that, under visible light irradiation, the bioenergy hydrogels of this invention (SilMA@BNs, SDPC@BTUPs and SilMA / SDPC@BTUPs) can significantly increase the levels of ATP and NADPH in damaged BMSCs, effectively remove excess reactive oxygen species, restore mitochondrial membrane potential and respiratory function, and fundamentally restart the cell's "energy engine".

[0043] (4) Significantly promotes bone regeneration: In vitro osteogenic induction experiments of bioenergy hydrogel showed that the material can strongly promote osteogenic differentiation of BMSCs, manifested by increased alkaline phosphatase activity, large-scale formation of calcium nodules and upregulation of expression of key osteogenic genes (RUNX2, OPN, OCN); In the osteoporotic rat skull defect model, after implantation of bioenergy hydrogel and local light irradiation, the amount of new bone formation, bone density and bone volume fraction were significantly improved, which proved its excellent in vivo osteogenic ability.

[0044] (5) It can serve as an ideal tissue engineering scaffold: Methacrylamide silk fibroin has good biocompatibility, adjustable degradation rate and mechanical properties. Its three-dimensional porous structure can realize the continuous and controllable release of biomimetic photosynthetic nanoparticles, and can also provide a good microenvironment for cell migration, adhesion and growth, thus achieving a perfect unity of "active drug delivery" and "passive scaffold support".

[0045] (6) Solving the problem of limited penetration depth of visible light in tissues in vivo: By integrating thylakoids with upconversion nanoparticles (UPs) to prepare upconversion thylakoid nanoparticles (TUPs), and then using bone marrow mesenchymal stem cell membranes for biomimetic coating, biomimetic photosynthetic nanoparticles (BTUPs) are prepared. UPs can be used to convert near-infrared light (NIR) in situ into visible light, endowing them with photosynthetic capabilities under NIR irradiation. NIR has stronger tissue penetration than visible light, thus solving the problem of limited penetration depth of visible light in tissues in vivo. In addition, the photosynthetic function of thylakoids depends on visible light excitation, but the tissue penetration depth of visible light is limited. Deep defects in human bone tissue (especially weight-bearing bones such as the femur) are difficult for visible light to penetrate, which poses a great challenge to the photosynthetic function of thylakoids driven by visible light. This invention breaks through the light transmission limitations when applying thylakoids to deep bone tissues such as the femur, and constructs a multifunctional bone repair system. This system can convert highly penetrating near-infrared (NIR) light in situ into visible light, thereby enabling thylakoids to autonomously activate and function in deep bone tissues in vivo. It also has efficient targeted delivery and immune escape capabilities, and can synergistically provide energy, antioxidant and oxygen supply, ultimately achieving systematic reshaping and efficient repair of the pathological microenvironment of osteoporotic bone defects. Attached Figure Description

[0046] Figure 1 The following are the characterization results of the cell membrane camouflaged thylakoid nanoparticles (BNs) in Example 1. A shows the microstructure of simple thylakoid nanoparticles (TNs) and BNs under transmission electron microscopy; B shows the particle size distribution of TNs, BMs, and BNs as determined by NTA; C shows the Zeta potential measurement; D shows the fluorescence colocalization: DiO fluorescent dye labels BM (green), and PKH26 fluorescent dye labels TNs (red); E shows the quantitative analysis of fluorescence colocalization; F shows the retention of protein components verified by Coomassie brilliant blue staining; G shows the Western blot verification of photosynthetic protein PsbA and cell membrane characteristic protein Na. + / K + Graph showing the retention of ATPase;

[0047] Figure 2The figures shown are the cellular uptake and functional verification results of cell membrane camouflaged thylakoid nanoparticles (BNs) in Example 2. A shows the CCK-8 assay results under no light; B shows the CCK-8 assay results under light; C shows the uptake of TNs and BNs in BMSCs, THP-1, and RAW264.7 cells; D shows the cellular uptake fluorescence quantitative analysis; E shows the intracellular ATP content detection; F shows the intracellular NADPH content detection; G shows the H2O2 scavenging assay; H shows the intracellular oxygen content level detected by the oxygen-sensitive fluorescent probe; and I shows the oxygen content detection fluorescence quantitative analysis. Figure 3 This image shows the effect of cell membrane-masked thylakoid nanoparticles (BNs) on the repair of mitochondria in bone marrow mesenchymal stem cells (BMSCs) damaged by H2O2 under light irradiation, as verified in in vitro cell experiments in Example 3. In the image, A shows the intracellular ROS content; B shows the quantitative analysis of intracellular ROS fluorescence intensity; C shows the fluorescence imaging of mitochondrial membrane potential; D shows the red / green fluorescence intensity ratio corresponding to the JC-1 polymer / monomer form for membrane potential detection; E shows the mitochondrial ROS content; and F shows the quantitative analysis of mitochondrial ROS fluorescence intensity.

[0048] Figure 4 The figures shown are the results of the in vitro osteogenic differentiation experiment in Example 4. A represents ALP staining after osteogenic induction of BMSCs under different concentrations of BNs treatment; B represents the quantitative analysis of ALP-positive area; C represents the Alizarin Red S staining of mineralized nodules after osteogenic induction of BMSCs under different concentrations of BNs treatment; and D represents the quantitative analysis of calcium deposition based on absorbance at 562 nm after the mineralized nodules dissolve.

[0049] Figure 5 The diagram shows the genome-wide effects of BNs revealed by transcriptome sequencing in Example 5. In this diagram, A is the KEGG pathway enrichment analysis of upregulated genes in the BNs group compared to the H2O2 group; and B is the GO biological process enrichment analysis of upregulated genes in the BNs group compared to the H2O2 group.

[0050] Figure 6 This is a schematic diagram illustrating the construction of the bioenergy hydrogel in Example 6, where A represents the FTIR spectra of SF and SilMA; B represents the FTIR spectra of SF and SilMA. 1HNMR spectrum; C shows the swelling properties of SilMA hydrogels at concentrations of 10%, 20%, and 30%; D shows the in vitro degradation of SilMA hydrogels at concentrations of 10%, 20%, and 30%; E shows the in vitro cumulative release kinetics of BNs in SilMA@BNs hydrogels at different concentrations; F shows the compressive elastic modulus of SilMA hydrogels measured at different strain levels; G shows representative compressive stress-strain curves.

[0051] Figure 7 The images shown in Example 6 illustrate the porous structure of the bioenergy hydrogel and its cell compatibility verification. A is a scanning electron microscope image of the microstructure of SilMA and the bioenergy hydrogel (SilMA@BNs); B is a CCK-8 cell proliferation assay after co-culturing BMSCs with SilMA or SilMA@BNs hydrogel for 1, 3, and 5 days; C is the live / dead staining results (green: live cells; red: dead cells) after co-culturing BMSCs with SilMA or SilMA@BNs hydrogel for 1, 3, and 5 days.

[0052] Figure 8 Figure 1 shows the evaluation results of skull defect regeneration in animal experiments in Example 7; Figure 2 shows representative Micro-CT three-dimensional and two-dimensional coronal reconstruction images of the skull defect site at 6 and 12 weeks post-operation, with red circles marking the new bone area; Figure 3 shows Micro-CT quantitative analysis of bone regeneration at 6 and 12 weeks post-operation, including bone density and bone volume fraction; Figure 4 shows H&E staining, where NB represents new bone tissue, OB represents original bone tissue, and Gel represents the remaining hydrogel component; Figure 5 shows Masson trichrome staining.

[0053] Figure 9 The image shows H&E stained sections of the major organs (heart, liver, spleen, lung, and kidney) of female SD rats 12 weeks post-surgery in Example 7.

[0054] Figure 10 Figure 8 shows the characterization results of the upconversion photosynthetic nanoparticles (BTUPs) described in Example 8. Figure A shows the microstructure of upconversion nanoparticles (UPs), upconversion thylakoid nanoparticles (TUPs), and BTUPs under transmission electron microscopy. Figure B shows the particle size distribution of UPs, TUPs, and BTUPs determined by NTA. Figure C shows the fluorescence colocalization: DiO fluorescent dye labeled BM (green), and PKH26 fluorescent dye labeled TUPs (red). Figure D shows the quantitative analysis of fluorescence colocalization. Figure E shows the retention of protein components verified by Coomassie brilliant blue staining. Figure F shows the Western blot verification of photosynthetic protein PsbA and cell membrane characteristic protein Na. + / K +Graph showing the retention of ATPase;

[0055] Figure 11 The diagram shows the in vitro NIR activation effect of BTUPs in Example 8; where A is the intracellular ATP content detection diagram; B is the intracellular NADPH content detection diagram; C is the intracellular oxygen content level detection diagram using oxygen-sensitive fluorescent probes; and D is the fluorescence quantitative analysis diagram of oxygen content detection.

[0056] Figure 12 The figure shows the characterization results of protocatechuic aldehyde grafted silk fibroin (SF-DB) and phenylboronic acid grafted chondroitin sulfate (PBA-CS) in Example 9. In this figure, A shows the FT-IR spectra of silk fibroin (SF), protocatechuic aldehyde (DB), and SF-DB; B shows the FT-IR spectra of SF and SF-DB. 1 HNMR spectrum; C is the FTIR spectrum of chondroitin sulfate (CS), aminophenylboronic acid (PBA), and PBA-CS; D is the FTIR spectrum of CS and PBA-CS. 1 HNMR spectrum;

[0057] Figure 13 The image shows the adhesion of the BTUPs-loaded protocatechuic aldehyde-grafted silk fibroin-phenylboronic acid-grafted chondroitin sulfate (SDPC@BTUPs) hydrogel to the bone tissue surface in Example 9.

[0058] Figure 14 Figure 9 shows the results of the in vitro osteogenic differentiation experiment of SDPC@BTUPs hydrogel. A shows ALP staining after osteogenic induction, with hBMSCs co-cultured with SDPC@BTUPs hydrogels loaded with different BTUPs concentrations; B shows the quantitative analysis of ALP-positive area; C shows Alizarin Red S staining of mineralized nodules after osteogenic induction, with hBMSCs co-cultured with SDPC@BTUPs hydrogels loaded with different BTUPs concentrations; D shows the quantitative analysis of calcium deposition based on absorbance at 562 nm after the mineralized nodules dissolve.

[0059] Figure 15 This is a cell compatibility verification diagram of the bioenergy hydrogel (SilMA / SDPC@BTUPs) in Example 10. In this diagram, A shows the proliferation detection of CCK-8 cells after co-culturing hBMSCs with methacrylamide silk fibroin-protocatechuic aldehyde grafted silk fibroin-phenylboronic acid grafted chondroitin sulfate (SilMA / SDPC) or SilMA / SDPC@BTUPs hydrogel for 1, 3, and 5 days; B shows the live / dead staining results (green: live cells; red: dead cells) after co-culturing hBMSCs with SilMA / SDPC or bioenergy hydrogel (SilMA / SDPC@BTUPs) for 1, 3, and 5 days.

[0060] Figure 16 Figure A shows the evaluation results of femoral defect regeneration in animal experiments in Example 11; A is a representative Micro-CT three-dimensional reconstruction image of the femoral defect site at 4 and 8 weeks postoperatively; B is a Micro-CT quantitative analysis of bone regeneration at 4 and 8 weeks postoperatively, including bone density, bone volume fraction, and number of trabeculae.

[0061] Figure 17 Figure A shows the evaluation results of femoral defect regeneration in animal experiments in Example 11; A is an H&E staining image; B is a Masson trichrome staining image.

[0062] Figure 18 The image shows H&E stained sections of the major organs (heart, liver, spleen, lung, and kidney) of the female SD rats in Example 11, 8 weeks after surgery. Detailed Implementation

[0063] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0064] In this embodiment of the invention, the thylakoid nanoparticles are simple thylakoid nanoparticles (TNs) or upconversion thylakoid nanoparticles (TUPs); the biomimetic photosynthetic nanoparticles are cell membrane camouflaged thylakoid nanoparticles (BNs) or upconversion photosynthetic nanoparticles (BTUPs); the bioenergy hydrogel is SilMA@BNs hydrogel (BNs-loaded methacrylamide silk fibroin) or SDPC@BTUPs hydrogel (BTUPs-loaded protocatechuic aldehyde-grafted silk fibroin / phenylboronic acid-grafted chondroitin sulfate hydrogel) or SilMA / SDPC@BTUPs hydrogel (BTUPs-loaded methacrylamide silk fibroin-protocatechuic aldehyde-grafted silk fibroin-phenylboronic acid-grafted chondroitin sulfate hydrogel); the upconversion nanoparticles (UPs) are model X-SHB-650, purchased from Hangzhou Xinqiao Biotechnology Co., Ltd.

[0065] Example 1

[0066] The preparation method of the biomimetic photosynthetic nanoparticles (cell membrane camouflaged thylakoid nanoparticles, BNs) described in this embodiment includes the following steps:

[0067] (1) Preparation of thylakoid nanoparticle (TN) suspension: Fresh plant leaves (spinach leaves) were taken, washed and destemmed, and placed overnight in the dark at 4℃ to reduce starch grain damage; 50g of spinach leaves were taken and 50 mL of pre-cooled extraction buffer (330 mmol / L sorbitol, 50 mmol / L HEPES-KOH pH 7.6, 5 mmol / L MgCl2, 0.1% w / v BSA) were added and homogenized with a high-speed homogenizer. The homogenate was filtered through a fine cotton cloth and the filtrate was centrifuged at 3000g for 10 minutes at 4℃; the precipitate was resuspended in washing buffer (300 mmol / L sorbitol, 50 mmol / L HEPES-KOH pH 7.6, 5 mmol / L MgCl2, 2 mmol / L EDTA, 10 mmol / L L-ascorbic acid sodium); the resuspended solution was spread on a pre-prepared Percoll density gradient solution, with the upper layer being 40%... Percoll (v / v), with the lower layer being 80% Percoll (v / v); centrifuged at 3000g for 10 minutes at 4°C; the green thylakoid bands at the gradient interface were carefully collected and washed with washing buffer; the purified thylakoid precipitate was resuspended in osmotic shock buffer (10 mmol / L HEPES-KOH pH 7.6, 10 mmol / L MgCl2, 10 mmol / L L-ascorbic acid sodium) and sonicated on ice for 3 minutes; subsequently, the suspension was extruded sequentially through 400 nm, 200 nm, and 100 nm polycarbonate membranes to obtain a thylakoid particle (TN) suspension with uniform particle size; the absorbance was measured at 645 nm and 665 nm using a UV-Vis spectrophotometer, and the chlorophyll content was calculated according to the formula to determine the concentration;

[0068] (2) Preparation of bone marrow mesenchymal stem cell membrane (BM) suspension: Human bone marrow mesenchymal stem cells (hBMSCs) cultured to the logarithmic growth phase were collected using the hypotonic lysis method and resuspended in pre-cooled hypotonic lysis buffer (1 mmol / L NaHCO3, 0.2 mmol / L EDTA, 1 mmol / L PMSF, pH 7.4) and incubated on ice for 20 minutes. Subsequently, the cells were repeatedly aspirated with an insulin syringe to break them up and prepare cell lysate. The cell lysate was centrifuged at 3000g for 5 minutes at 4°C to remove cell nuclei and unbroken cell debris. The supernatant was then centrifuged at 15000g for 30 minutes at 4°C. The resulting precipitate was the BM membrane, which was resuspended in sterile PBS and stored at -80°C for later use to prepare the BM suspension (1 mg / mL). Western blot analysis confirmed that the membrane was rich in the plasma membrane marker protein Na+. + / K + -ATPase, while the cytoplasmic protein β-tubulin signal was weak, indicating high extraction purity;

[0069] (3) Preparation of biomimetic photosynthetic nanoparticles (BNs): The thylakoid nanoparticle (TNs) suspension prepared above was mixed with the BM membrane suspension at a volume ratio of 2:1 and gently shaken and incubated at 4°C for 30 minutes to allow the membrane material to initially combine with the TNs; then, the mixed suspension was extruded sequentially through polycarbonate membranes with pore sizes of 1000 nm, 400 nm and 200 nm. The extruded suspension was centrifuged at 10000g for 10 minutes, the precipitate was collected and resuspended in sterile deionized water to obtain biomimetic photosynthetic nanoparticles (BNs).

[0070] like Figure 1 The above are the characterization results of the biomimetic photosynthetic nanoparticles (BNs) described in this embodiment. Figure 1 As shown in Figure A, transmission electron microscopy (TEM) observation revealed that TNs are spherical or ellipsoidal in shape, with visible lamellar structures inside; while BNs are spherical, encapsulated by a distinct membrane structure, with a slightly increased diameter, confirming the successful encapsulation of BM; as shown in Figure A. Figure 1 As shown in Figure B, nanoparticle tracking analysis (NTA) results indicate that the average particle sizes of TNs, BMs, and BNs are 129.8 nm, 198.9 nm, and 212.7 nm, respectively, with uniform particle size distribution; Figure 1 As shown in Figure C, Zeta potential measurements revealed that the potentials of TNs were approximately -6.7 mV, BM was approximately -10.6 mV, and BNs was approximately -10.0 mV. The potential values ​​of TNs and BNs are closer to those of BM, further supporting the encapsulation. Figure 1 As shown in Figures D and E, TNs were labeled with PKH26 (red fluorescence), and BMs were labeled with DiO (green fluorescence). Observation using a laser confocal microscope (CLSM) showed that the red and green fluorescence in the BNs were highly colocalized; as shown... Figure 1 As shown in Figures F and G, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), Coomassie brilliant blue staining, and Western blotting analysis confirmed that BNs retained both the thylakoid photosynthetic core protein PsbA and the membrane protein Na derived from BM. + / K + -ATPase, while cytoplasmic protein contamination is minimal.

[0071] Example 2: In vitro cell compatibility, targeted uptake and biological function verification of BNs

[0072] Cell compatibility was further tested based on Example 1 using the CCK-8 assay. First, donor bone marrow tissue samples were collected and rapidly transferred to a sterile 50 mL centrifuge tube. An equal volume of PBS buffer was added, and the tube was gently inverted to thoroughly mix the bone marrow sample with the buffer, breaking up any cell clumps. Then, the mixture was slowly added dropwise to the top layer of twice the volume of Percoll cell separation medium. The centrifuge tube was placed at 4°C and centrifuged at 400 g for 20 minutes. After centrifugation, the intermediate cloud-like mononuclear cell layer formed after centrifugation was carefully aspirated using a sterile pipette and transferred to a new sterile centrifuge tube. Ten volumes of PBS buffer were added to the tube, and the cells were gently resuspended by pipetting. The tube was then centrifuged at 250 g for 10 minutes for washing. The supernatant was discarded, and the cell suspension was seeded into sterile culture flasks for proliferation culture to obtain human bone marrow mesenchymal stem cells (hBMSCs). Human bone marrow mesenchymal stem cells (hBMSCs) in the logarithmic growth phase were seeded into 96-well plates and treated with different concentrations of BNs (0.25, 0.5, 1.0, 2.0, 4.0 μg / mL, based on chlorophyll content). A light group (630 nm red LED illumination for 30 minutes daily) and a dark group were established. Results were analyzed after 1, 2, and 3 days of culture. Figure 2 Results A and B showed that BNs at all concentrations did not inhibit cell proliferation within 72 hours; under light conditions, medium and high concentrations of BNs showed a trend of promoting proliferation on day 3, indicating their good cell compatibility and light-dependent biological activity.

[0073] Cellular targeted uptake: PKH26-labeled TNs (1 μg / mL) and BNs (1 μg / mL) were respectively combined with hBMSCs (2 × 10⁻⁶). 4 (cells / well), human mononuclear macrophages (THP-1, 2 × 10⁶ cells / well), 5 (cells / well) and mouse macrophages (RAW 264.7, 2 × 10⁻⁶ cells / well) 5 (each well) was incubated for 6 hours in a 24-well plate. Figure 2 As shown in C and D, CLSM observation and quantitative fluorescence analysis revealed that the uptake efficiency of BNs in hBMSCs was significantly higher than that in unencapsulated TNs; conversely, the uptake of BNs in both macrophage types was significantly lower than that in TNs. This demonstrates that BM encapsulation endows BNs with homologous targeting ability and immune escape properties.

[0074] Energy metabolism and antioxidant function:

[0075] ATP and NADPH supplementation: A model of oxidative stress in hBMSCs induced by H2O2 (200 μmol / L, 2 hours) was established. Figure 2The E and F tests revealed that the intracellular ATP and NADPH levels in the H2O2 model group were significantly reduced. However, after treatment with BNs and red light irradiation, the intracellular ATP level recovered by about 45%, and the NADPH level was also effectively increased, demonstrating that BNs can directly replenish energy and reducing power exogenously through photosynthesis.

[0076] ROS removal and oxygen production: such as Figure 2 In vitro H2O2 scavenging experiments by G showed that BNs can directly decompose H2O2, with a scavenging rate of up to 63%. The degree of intracellular hypoxia was detected using the oxygen-sensitive probe Ru(dpp)3Cl2. Figure 2 The H and I results showed that BNs treatment significantly improved the hypoxic state of cells. This confirms that BNs exert their antioxidant and microenvironment-improving effects through their catalase-like activity and photosynthetic oxygen production capacity.

[0077] Example 3: The protective effect and mechanism of BNs on mitochondrial function

[0078] Further ROS removal and mitochondrial structure protection based on Example 1: such as Figure 3 As shown in Figures A and B, the DCFH-DA fluorescent probe detection showed that BNs can effectively scavenge H2O2-induced total intracellular ROS.

[0079] Restoring mitochondrial function: such as Figure 3 As shown in C and D, the use of the MitoSOX Red probe revealed that BNs significantly reduced the level of mitochondrial-derived superoxide (mtROS).

[0080] Example 4: BNs promote osteogenic differentiation of hBMSCs in vitro

[0081] Further evaluation of osteogenic differentiation phenotype was conducted based on Example 1: In osteogenic induction medium, BNs treatment significantly restored the osteogenic capacity of hBMSCs inhibited by H2O2. Figure 4 As shown in Figures A and B, alkaline phosphatase (ALP) staining revealed that bone nuclei (BNs) enhanced early osteogenic activity in a concentration-dependent manner, with 1 μg / mL showing the best effect. Figure 4 As shown in C and D, Alizarin Red S (ARS) staining revealed a significant increase in the number and area of ​​calcium nodules in the BNs-treated group.

[0082] Example 5: Transcriptome sequencing reveals the genome-wide effects of BNs.

[0083] Based on Example 1, transcriptome sequencing analysis was further performed on hBMSCs from the control group, H2O2 model group, and H2O2+BNs treatment group. Figure 5As shown in Figure A, KEGG pathway enrichment analysis revealed that BN treatment significantly upregulated pathways related to oxidative phosphorylation and mitophagy. Figure 5 As shown in Figure B, GO analysis also revealed a significant enrichment of genes related to processes such as mitochondrial organization and respiratory chain complex assembly after BN treatment. These data confirm, at the genome-wide level, that BNs may exert a protective effect by enhancing mitochondrial energy metabolism and activating mitochondrial quality control.

[0084] Example 6: Preparation, characterization, and biocompatibility of bioenergy hydrogel (SilMA@BNs hydrogel)

[0085] (1) Synthesis and characterization of methacrylamide silk fibroin (SilMA): The specific preparation method of SilMA is as follows: Prepare 2L of 0.02 mol / L Na2CO3 solution, and after boiling, add 40g of silkworm cocoons and boil in boiling water for 30 minutes, stirring once every 5 minutes to obtain softened silk; take out the silk and put it into deionized water to rub and wash 3 times, and then put it into Na2CO3 solution again to boil, and the operation is the same as before; after the silk is fully degummed, rub and wash it again with deionized water, wring it dry and air dry for 1-2 days to obtain purified silk fibroin fiber; weigh 10g of dried silk fibroin fiber and slowly add 50mL of LiBr solution (0 The solution was stirred at 60°C until completely dissolved. Then, 2.9 mL of glycidyl methacrylate (1.04 g / mL) was added to the solution, and the mixture was stirred at 60°C in the dark for 3 hours to form a mixed solution. The resulting mixed solution was filtered through a 70 μm mesh sieve, placed in a dialysis bag (molecular weight cutoff 12000-14000 Da), and dialyzed in deionized water for 4 days. After dialysis, the solution was freeze-dried to obtain SilMA. Figure 6 As shown in Figures A and B, the ¹H NMR and FT-IR spectra confirm the successful grafting of the methacryloyl group;

[0086] (2) Preparation and performance optimization of SilMA@BNs hydrogels: BNs (10 μg / mL) were mixed with SilMA solutions (containing 0.25% LAP photoinitiator) of different concentrations (10%, 20%, 30% w / v), and crosslinked into a gel after irradiation with 405nm ultraviolet light for 30 seconds; Figure 6 As shown in C to G, the system evaluation shows that 20% SilMA achieves the best balance in terms of swelling ratio, in vitro degradation rate, BNs sustained-release performance (approximately 28% released after 48 hours), and compressive modulus, and is therefore selected as the optimal concentration.

[0087] (3) Hydrogel morphology and biocompatibility: such as Figure 7Scanning electron microscopy (SEM) showed that the 20% SilMA@BNs hydrogel possessed a uniformly interconnected porous structure, and BNs were successfully loaded into the network; Figure 7 CCK-8 experiment in China B and Figure 7 Live / dead cell staining of C confirmed that SilMA@BNs, like pure SilMA, are non-cytotoxic to hBMSCs and support good cell adhesion and proliferation.

[0088] Example 7: In vivo bone repair efficacy of SilMA@BNs in an osteoporotic rat model of skull defects

[0089] Further research was conducted based on Example 6:

[0090] (1) Animal model establishment and grouping: Female SD rats underwent bilateral ovariectomy (OVX) and osteoporosis model was naturally formed after 8 weeks. Then, a critical bone defect with a diameter of 5 mm was created on the top of the skull. They were randomly divided into four groups: non-osteoporosis sham operation group (Control), osteoporosis blank group (OVX), simple hydrogel group (OVX+SilMA), and combined treatment group (OVX+SilMA@BNs). SilMA was used at a concentration of 20% w / v, and the BNs loading concentration in SilMA@BNs was 10 μg / mL. The SilMA or SilMA@BNs mixed solution was filled into the defect site using a syringe and then photocured for 30 seconds. The defect sites of each group were irradiated with 630 nm red light for 30 minutes every day for one week after surgery.

[0091] (2) Micro-CT evaluation of bone regeneration effect: such as Figure 8 As shown in Figures A and B, Micro-CT three-dimensional reconstruction and quantitative analysis at 6 and 12 weeks post-operation showed that the amount of new bone (bone volume fraction, BV / TV) and bone mineral density (BMD) in the OVX+SilMA@BNs group were significantly higher than those in the OVX group and the OVX+SilMA group, and continued to increase over time, indicating that it has a continuous bone-promoting capacity.

[0092] (3) Histological analysis: such as Figure 8 C and D H&E and Masson trichrome staining showed that continuous new bone formation was observed in the OVX+SilMA@BNs group at 6 weeks, and the new bone was more mature and collagen deposition was abundant at 12 weeks, with good matching between material degradation and bone ingrowth.

[0093] (4) In vivo biosafety: such as Figure 9 As shown, 12 weeks post-surgery, no pathological damage or inflammation was found in the H&E stained sections of the major organs (heart, liver, spleen, lungs, and kidneys), demonstrating that SilMA@BNs hydrogel has good in vivo biocompatibility.

[0094] Example 8: Preparation of biomimetic photosynthetic nanoparticles (upconversion photosynthetic nanoparticles, BTUPs) and their near-infrared (NIR) activation effect

[0095] (1) Preparation of thylakoid nanoparticle (TUP) suspension: Fresh plant leaves (spinach leaves) were taken, washed and destemmed, and placed overnight in the dark at 4℃ to reduce starch grain damage; 50g of spinach leaves were taken and 50 mL of pre-cooled extraction buffer (330 mmol / L sorbitol, 50 mmol / L HEPES-KOH pH 7.6, 5 mmol / L MgCl2, 0.1% w / v BSA) were added and homogenized with a high-speed homogenizer. The homogenate was filtered through a fine cotton cloth and the filtrate was centrifuged at 3000g for 10 minutes at 4℃; the precipitate was resuspended in washing buffer (300 mmol / L sorbitol, 50 mmol / L HEPES-KOH pH 7.6, 5 mmol / L MgCl2, 2 mmol / L LEDTA, 10 mmol / L L-ascorbic acid sodium); the resuspended solution was spread on a pre-prepared Percoll density gradient solution, with the upper layer being 40%. Percoll (v / v), lower layer 80% Percoll (v / v); centrifuged at 3000g for 10 minutes at 4°C; carefully collected the green thylakoid bands at the gradient interface and washed with washing buffer; finally, the purified thylakoid precipitate was rinsed with osmotic shock buffer (10 mmol / L HEPES-KOH pH 7.6, 10 mmol / L MgCl2, 10 mmol / L... The thylakoid nanoparticles (TUPs) were resuspended in sodium L-ascorbate and sonicated in an ice bath for 3 minutes. Subsequently, the upconversion nanoparticles (UPs, Hangzhou Xinqiao Biotechnology Co., Ltd., X-SHB-650) were resuspended in distilled water to prepare a UPs suspension (4 mg / mL). The UPs suspension was thoroughly mixed with the thylakoid resuspending at a mass ratio of -2:1 and gently shaken and incubated at 4°C for 30 minutes. The mixed suspension was extruded sequentially through polycarbonate membranes with pore sizes of 1000 nm, 400 nm, and 200 nm. The extruded suspensions were centrifuged at 10000 g for 10 minutes, and the precipitate was resuspended to obtain a thylakoid nanoparticle (TUPs) suspension. The absorbance was measured at wavelengths of 645 nm and 665 nm using a UV-Vis spectrophotometer, and the chlorophyll content was calculated according to the formula to determine the concentration.

[0096] (2) Preparation of bone marrow mesenchymal stem cell membrane (BM) suspension: Human bone marrow mesenchymal stem cells (hBMSCs) cultured to the logarithmic growth phase were collected using the hypotonic lysis method and resuspended in pre-cooled hypotonic lysis buffer (1 mmol / L NaHCO3, 0.2 mmol / L EDTA, 1 mmol / L PMSF, pH 7.4) and incubated on ice for 20 minutes. Subsequently, the cells were repeatedly aspirated with an insulin syringe to break them up and prepare cell lysate. The cell lysate was centrifuged at 3000g for 5 minutes at 4°C to remove cell nuclei and unbroken cell debris. The supernatant was then centrifuged at 15000g for 30 minutes at 4°C. The resulting precipitate was the BM membrane, which was resuspended in sterile PBS and stored at -80°C for later use to prepare the BM suspension (1 mg / mL). Western blot analysis confirmed that the membrane was rich in the plasma membrane marker protein Na+. + / K + -ATPase, while the cytoplasmic protein β-tubulin signal was weak, indicating high extraction purity;

[0097] (3) Preparation of biomimetic photosynthetic nanoparticles (BTUPs): The thylakoid nanoparticle (TUPs) suspension prepared above was mixed with the BM membrane suspension at a volume ratio of 2:1 and gently shaken and incubated at 4°C for 30 minutes to allow the membrane material to initially combine with TNs; then, the mixed suspension was extruded sequentially through polycarbonate membranes with pore sizes of 1000 nm, 400 nm and 200 nm. The extruded suspension was centrifuged at 10000 g for 10 minutes, the precipitate was collected and resuspended in sterile deionized water to obtain biomimetic photosynthetic nanoparticles (BTUPs).

[0098] like Figure 10 The above are the characterization results of the biomimetic photosynthetic nanoparticles (BTUPs) described in this embodiment. Figure 10 As shown in Figure A, TEM observation reveals that UPs exhibit a hexagonal structure; TUPs nanoparticles also exhibit a hexagonal structure, but their outer periphery is covered by a membrane structure, which proves the effective encapsulation of UPs by TK; BTUPs exhibit a typical "core-shell" structure, with TUPs acting as the core and tightly encapsulated by BM, maintaining an overall spherical shape, and a double-layer membrane structure visible on the outermost layer; as shown in Figure A. Figure 10 As shown in Figure B, the NTA measurement results indicate that the average particle sizes of UPs, TUPs, and BTUPs are 129.8 nm, 183.9 nm, and 220.2 nm, respectively, showing a gradual increase in particle size and uniform distribution. Figure 10 As shown in C and D, PKH26-labeled TUPs (red) and DiO-labeled BMs (green) exhibit fluorescent co-localization in BTUPs; as Figure 10As shown in Figures E and F, SDS-PAGE Coomassie Brilliant Blue staining and Western Blot analysis confirmed that BTUPs retain both the thylakoid photosynthetic core protein PsbA and the BM-derived membrane protein Na. + / K + -ATPase, while cytoplasmic protein contamination is minimal.

[0099] (4) In vitro NIR activation effect of BTUPs

[0100] In vitro NIR activation effect assessment: such as Figure 11 Detection tests A and B revealed that after BTUP treatment followed by NIR irradiation (5 minutes of continuous irradiation followed by a 1-minute interval, repeated for 6 cycles), intracellular ATP levels significantly increased, approximately 1.5 times that of the control group; simultaneously, NADPH levels also significantly increased, approximately 1.9 times that of the control group; in addition, as... Figure 11 Results C and D showed that BTUP treatment also significantly improved intracellular hypoxia. This demonstrates that BTUPs, under NIR excitation, can directly regulate the levels of corresponding intracellular parameters through the production of ATP, NADPH, and oxygen via photosynthesis.

[0101] Example 9: Preparation, characterization, osteogenic efficacy and biocompatibility of bioenergy hydrogel (SDPC@BTUPs hydrogel)

[0102] (1) Synthesis and characterization of SF-DB: Prepare 2L of 0.02 mol / L Na2CO3 solution, boil it, add 40g of silkworm cocoons, and boil for 30 minutes, stirring once every 5 minutes to obtain softened silk; take out the silk and put it into deionized water to rub and wash 3 times, then put it into Na2CO3 solution again to boil, and the operation is the same as before; after the silk is fully degummed, rub and wash it again with deionized water, wring it dry and air dry for 1-2 days to obtain purified silk fibroin fiber; weigh 10g of dried silk fibroin fiber and slowly add it to 50mL of LiBr solution (0.81g / mL), and boil at 6 Stir at 0℃ until completely dissolved to form a homogeneous silk fibroin (SF) solution; then weigh 250 mg of protocatechuic aldehyde (DB) and add it to 10 mL of deionized water, heat to 60℃ and stir thoroughly to dissolve; slowly add the DB solution to the SF solution, then stir at 60℃ in the dark for 3 hours to form a mixed solution; after the reaction is complete, put the mixed solution into a dialysis bag (molecular weight cutoff 3500 Da) and dialyze in deionized water at 4℃ for 4 days; after dialysis, freeze-dry the solution to finally obtain SF-DB lyophilized powder; Figure 12 As shown in Figures A and B, the ¹H NMR and FT-IR spectra confirm the successful preparation of SF-DB;

[0103] (2) Synthesis and characterization of PBA-CS: 7.0 g of chondroitin sulfate (CS) was weighed and placed in 700 mL of deionized water and stirred thoroughly until CS was completely dissolved; 3.276 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.925 g of N-hydroxysuccinimide (NHS) were slowly added to the CS aqueous solution, the pH was adjusted to 4.5, and the reaction was stirred in the dark for 1 hour; 0.46 g of aminophenylboronic acid (PBA) was added to 5 mL In dimethyl sulfoxide (DMSO), stir until completely dissolved to obtain a PBA solution; slowly add the PBA solution dropwise to the above CS mixture, adjust the pH to 4.5, continue to wrap the beaker with aluminum foil to protect it from light, and stir the reaction overnight at room temperature; allow PBA and activated CS to fully undergo an amidation reaction to form a PBA-CS solution; after the reaction is complete, put the solution into a dialysis bag (molecular weight cutoff 3500 Da), and dialyze it in deionized water at 4°C for 3 days; after dialysis, freeze-dry the solution to finally obtain PBA-CS lyophilized powder; Figure 12 As shown in C and D, the ¹H NMR and FT-IR spectra confirm the successful preparation of SF-DB;

[0104] (3) Preparation of SDPC@BTUPs hydrogel and its in vitro promotion of osteogenic differentiation of hBMSCs: SF-DB (200 mg / mL) and PBA-CS (200 mg / mL) were dissolved separately in PBS solution. The solutions were then vortexed with BTUPs (5–40 μg / mL) at a ratio of SF-DB:PBA-CS = 2:1 to obtain the SDPC@BTUPs hydrogel, which appeared as shown in the image. Figure 13 As shown, co-culturing with SDPC@BTUPs hydrogel in osteogenic induction medium significantly promoted the osteogenic capacity of hBMSCs. Figure 14 As shown in Figures A and B, alkaline phosphatase (ALP) staining revealed that BTUPs enhanced early osteogenic activity in a concentration-dependent manner, with 20 μg / mL showing the best effect. Figure 14 As shown in C and D, Alizarin Red S (ARS) staining revealed that SDPC@BTUPs hydrogel significantly increased the number and area of ​​calcium nodules.

[0105] Example 10: Preparation, characterization, osteogenic efficacy and biocompatibility of bioenergy hydrogel (SilMA / SDPC@BTUPs hydrogel)

[0106] Preparation and biocompatibility of SilMA / SDPC@BTUPs hydrogel: The SDPC@BTUPs hydrogel prepared in Example 9 was injected into a mold; subsequently, a 20% SilMA solution containing 0.25% LAP was prepared and injected into the upper layer of the mold (the lower layer was the SDPC@BTUPs hydrogel, with a volume ratio of 1:1). The mixture was then photocured using a 405nm UV lamp for 30 seconds. After demolding, the SilMA / SDPC@BTUPs hydrogel was obtained. Biocompatibility of the SilMA / SDPC@BTUPs hydrogel: as shown... Figure 15 CCK-8 experiment in China and Figure 15 Live / dead cell staining of hBMSCs confirmed that the SilMA / SDPC@BTUPs hydrogel was non-cytotoxic to hBMSCs and supported good cell adhesion and proliferation.

[0107] Example 11: Bone repair efficacy of bioenergy hydrogel (SilMA / SDPC@BTUPs hydrogel) in a rat model of femoral defect with osteoporosis.

[0108] (1) Animal model establishment and grouping: Female SD rats underwent bilateral ovariectomy (OVX) and osteoporosis model was naturally formed 8 weeks later. Then, a bone defect with a diameter of 3 mm and a depth of 3 mm was constructed in the distal femur. They were randomly divided into three groups: osteoporosis blank group (OVX), simple hydrogel group (OVX+SilMA / SDPC), and compound treatment group (OVX+SilMA / SDPC@BTUPs). SilMA was selected at a concentration of 20% w / v, and the BTUPs loading concentration in SDPC@BTUPs was 20 μg / mL. SilMA / SDPC and SilMA / SDPC@BTUPs hydrogels were prepared using a cylindrical mold (diameter 3 mm, depth 3 mm) (the volume ratio of the upper SilMA to the lower SDPC or SDPC@BTUPs was 1:1). The corresponding materials were then filled into the defect area. The defect sites of each group were irradiated with NIR for 30 minutes every day for one week after surgery.

[0109] (2) Micro-CT evaluation of bone regeneration effect: such as Figure 16 As shown in Figures A and B, Micro-CT three-dimensional reconstruction and quantitative analysis at 4 and 8 weeks post-operation showed that the bone mineral density and bone volume fraction of the OVX+SilMA / SDPC@BTUPs group were significantly higher than those of the OVX group and the OVX+SilMA / SDPC group, indicating that it has a sustained bone-promoting ability.

[0110] (3) Histological analysis: such as Figure 17H&E and Masson trichrome staining of A and B showed that continuous new bone formation was observed in the OVX+ SilMA / SDPC@BTUPs group at 4 weeks, and the formation of new bone was more significant and collagen deposition was abundant at 8 weeks, with good matching between material degradation and bone ingrowth.

[0111] (4) In vivo biosafety: such as Figure 18 As shown, 8 weeks post-surgery, no pathological damage or inflammation was found in the H&E stained sections of the major organs (heart, liver, spleen, lungs, and kidneys), demonstrating that the SilMA / SDPC@BTUPs system has good in vivo biocompatibility.

[0112] (5) Conclusion

[0113] The specific embodiments of this invention fully demonstrate that bone nuclei (BNs) constructed by coating thylakoids with hBMSC membranes can effectively achieve targeted delivery and immune evasion, and simultaneously exert a triple function of energy supply (ATP), antioxidation (NADPH, ROS scavenging), and oxygen supply (O2) under visible light activation. BNs protect mitochondrial structure by scavenging ROS, fundamentally restoring mitochondrial function from a quality control perspective, thereby restarting the cell's "energy engine" and ultimately efficiently promoting osteogenic differentiation of BMSCs. The bioenergy hydrogel (SilMA@BNs hydrogel) formed by loading BNs onto photocrosslinkable SilMA hydrogel possesses excellent physicochemical properties, biocompatibility, and sustained-release characteristics, demonstrating outstanding ability to promote bone regeneration and angiogenesis in an osteoporotic rat skull defect model. To address the challenge of photodynamic activation of deep bone tissue in clinical applications, upconversion nanoparticles (UPs) are further introduced into thylakoid and cell membrane components to obtain biomimetic photosynthetic nanoparticles (BTUPs) with NIR activation effects. SDPC@BTUPs hydrogels or SilMA / SDPC@BTUPs hydrogels, formed by loading BTUPs onto SDPC hydrogels or SilMA / SDPC hydrogels, can effectively achieve photodynamic activation of deep bone tissues such as the rat femur. This hydrogel provides a novel bioenergy therapy with multi-target synergistic effects for treating osteoporotic bone defects.

[0114] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic photosynthetic nanoparticle, characterized in that, The process includes the following steps: mixing a suspension of thylakoid nanoparticles with a mass concentration of 0.5–5 mg / mL with a suspension of bone marrow mesenchymal stem cell membranes with a mass concentration of 0.5–5 mg / mL at a volume ratio of 1:0.3–3; incubating with shaking; and then extruding the mixture sequentially through polycarbonate membranes at 1000 nm, 400 nm, and 200 nm. After centrifugation and purification, biomimetic photosynthetic nanoparticles are obtained. The thylakoid nanoparticle suspension is prepared from thylakoids extracted from plant leaves containing chlorophyll.

2. The method of claim 1, wherein the method further comprises the step of: The preparation method of the thylakoid nanoparticle suspension is as follows: Take cleaned and destemmed plant leaves, crush them in pre-cooled extraction buffer, filter them, centrifuge the filtrate, precipitate them, resuspend them in washing buffer, spread the resuspended solution on a pre-prepared Percoll density gradient solution, centrifuge them, collect the green thylakoid bands located at the gradient interface, wash them with washing buffer, and obtain purified thylakoids. The purified thylakoids were resuspended in osmotic shock buffer, sonicated in an ice bath, and extruded sequentially through 1000 nm, 400 nm, 200 nm and 100 nm polycarbonate membranes to prepare a thylakoid nanoparticle suspension. Alternatively, the purified thylakoids are resuspended in osmotic shock buffer, sonicated in an ice bath, and thoroughly mixed with upconversion nanoparticle suspension at a mass ratio of 1:0.3–3. The mixture is then incubated with shaking and extruded sequentially through 1000 nm, 400 nm, and 200 nm polycarbonate membranes. After centrifugation and resuspension, a thylakoid nanoparticle suspension is prepared.

3. The method for preparing biomimetic photosynthetic nanoparticles according to claim 2, characterized in that, The pre-cooled extraction buffer consists of 330 mmol / L sorbitol, 50 mmol / L N-(2-hydroxyethyl)piperazine-N'-2-sulfonic acid (pH 7.6), 5 mmol / L magnesium chloride, and 0.1% bovine serum albumin (w / v). The washing buffer consists of 300 mmol / L sorbitol, 50 mmol / L N-(2-hydroxyethyl)piperazine-N'-2-sulfonic acid (pH 7.6), 5 mmol / L magnesium chloride, 2 mmol / L ethylenediaminetetraacetic acid (EDTA), and 10 mmol / L sodium L-ascorbate. The Percoll density gradient solution has a 40% (v / v) Percoll upper layer and an 80% (v / v) Percoll lower layer. The osmotic shock buffer consists of 10 mmol / L N-(2-hydroxyethyl)piperazine-N'-2-sulfonic acid (pH 7.6), 10 mmol / L magnesium chloride, and 10 mmol / L sodium L-ascorbate.

4. The method for preparing biomimetic photosynthetic nanoparticles according to claim 1, characterized in that, The method for preparing the bone marrow mesenchymal stem cell membrane suspension is as follows: Human bone marrow mesenchymal stem cells cultured to the logarithmic growth phase were collected, resuspended in pre-cooled hypotonic lysis buffer, incubated on ice, and then the cells were lysed to prepare cell lysate. The cell lysate was centrifuged to remove cell nuclei and unbroken cell debris, and the supernatant was centrifuged. The resulting precipitate was the bone marrow mesenchymal stem cell membrane suspension.

5. The method for preparing biomimetic photosynthetic nanoparticles according to claim 4, characterized in that, The human bone marrow mesenchymal stem cells were obtained by collecting bone marrow tissue samples from donors, mixing them with phosphate buffered saline solution, adding them dropwise to Percoll cell separation medium, centrifuging at low temperature to remove impurities, adding more phosphate buffered saline solution, centrifuging again, washing, discarding the supernatant to obtain a cell suspension, and inoculating the cell suspension into sterile culture flasks for proliferation culture to obtain human bone marrow mesenchymal stem cells.

6. The method for preparing biomimetic photosynthetic nanoparticles according to claim 4, characterized in that, The hypotonic lysis buffer consists of 1 mmol / L sodium bicarbonate, 0.2 mmol / L ethylenediaminetetraacetic acid, and 1 mmol / L benzyl sulfonyl fluoride, and the pH of the hypotonic lysis buffer is 7.

4.

7. A biomimetic photosynthetic nanoparticle, characterized in that, It is prepared according to any one of claims 1 to 6.

8. A bioenergy hydrogel prepared using biomimetic photosynthetic nanoparticles, characterized in that, The invention comprises a substrate hydrogel and the biomimetic photosynthetic nanoparticles of claim 7, wherein the substrate hydrogel is at least one of methacrylamide silk fibroin hydrogel or protocatechuic aldehyde-grafted silk fibroin-phenylboronic acid-grafted chondroitin sulfate hydrogel.

9. The bioenergy hydrogel prepared using biomimetic photosynthetic nanoparticles according to claim 8, characterized in that, When the substrate hydrogel is a methacrylamide silk fibroin hydrogel, the methacrylamide silk fibroin hydrogel is formed by cross-linking a methacrylamide silk fibroin solution with a mass concentration of 10%-30% under 405nm ultraviolet light in the presence of a photoinitiator. When the substrate hydrogel is a protocatechuic aldehyde grafted fibroin-phenylboronic acid grafted chondroitin sulfate hydrogel, the protocatechuic aldehyde grafted fibroin-phenylboronic acid grafted chondroitin sulfate hydrogel is formed by cross-linking protocatechuic aldehyde grafted fibroin with a mass concentration of 20% and phenylboronic acid grafted chondroitin sulfate with a mass concentration of 20% in a volume ratio of 1:0.2-5 by vortex mixing.

10. The use of at least one of the biomimetic photosynthetic nanoparticles according to claim 7 and the bioenergy hydrogels prepared using biomimetic photosynthetic nanoparticles according to claims 8-9 in the preparation of biomedical materials or drugs for repairing osteoporotic bone defects.

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