Immunoregulation stent with anti-tumor and bone repair functions as well as preparation method and application of immunoregulation stent

Abstract

The invention provides an anti-tumor and bone repair immunoregulation stent as well as a preparation method and application thereof, and belongs to the technical field of tissue engineering implant materials. According to the invention, an HBC network containing MCSF / CSF-1R and CD47 / SIPR alpha pathway blocking drug co-delivery system is assembled on an aldehyde-rich 3D printing calcium phosphate skeleton, and a therapeutic implant capable of activating macrophages swallowing tumor cells in stages and promoting bone regeneration is developed. The implant shows effective combination with the surface of a monocyte / macrophage, meanwhile, M2 polarization of bone marrow-derived monocyte is remarkably inhibited, and the phagocytic ability of the macrophage to tumor cells is enhanced. The two drugs loaded by the co-delivery system can play a synergistic role to efficiently resist tumors; the decomposed delivery carrier can promote bone regeneration. In an in-vivo tumor treatment model, the treatment stent significantly reduces local M2 macrophages and inhibits tumor growth. In an in vivo tissue regeneration model, the treatment scaffold promotes uniform bone growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering implant materials, and specifically relates to an immunomodulatory scaffold with both anti-tumor and bone repair functions, and a preparation method and application thereof. Background Art

[0002] After surgical resection of malignant bone tumors, complex, large bone defects require repair, but this is accompanied by the challenge of low survival rates after recurrence. Developing therapeutic implants for repairing individualized bone defects and inhibiting tumor recurrence is crucial to improving patient survival and quality of life.

[0003] 3D-printed regenerative implants have highly designable shapes and internal structures, are easy to carry with therapeutic systems, and have excellent application potential in the postoperative treatment of bone tumors. Self-setting calcium phosphate cement (CPC) can be rapidly recrystallized into degradable nanohydroxyapatite through ion exchange after mixing with water at room temperature, showing excellent formability and osteoconductivity in personalized bone defect repair applications. Regenerative implants customized with bioinks formulated with CPC as the main ingredient have also shown excellent effects in promoting bone tissue regeneration; however, such implants currently lack the ability to inhibit tumor recurrence. Studies have shown that tumor-associated macrophages (TAMs) with impaired anti-tumor function play an important role in promoting tumor immune escape and recurrence. Therefore, how to introduce macrophage regulatory functions into 3D-printed calcium phosphate scaffolds has become a focus of research on the postoperative treatment of bone tumors.

[0004] Tumor cells secrete macrophage colony stimulating factor (MCSF), which induces M2 polarization of tumor-associated macrophages (TAMs), a key mechanism promoting tumor recurrence. MCSF exerts its biological effects by activating cell surface receptors, leading to autophosphorylation of the receptor cFMS kinase, which in turn initiates intracellular signaling pathways. Therefore, colony stimulating factor 1 receptor (CSF-1R) inhibitors competitively inhibit the binding of adenosine triphosphate to cFMS kinase, showing the potential to mitigate immune evasion by reducing the number of TAMs.

[0005] While effective doses of CSF-1R inhibitors delivered via scaffolds can modulate the polarized phenotype of local macrophages, the "don't eat me" signal CD47 expressed by tumor cells binds to the receptor signaling regulator protein-α (SIRPα) on the macrophage surface, inhibiting their phagocytic function and potentially hindering the regulatory effects of CSF-1R inhibitors. Recent studies have demonstrated that SIRPα antibodies can specifically bind to the macrophage surface and simultaneously block the CD47 / SIRPα signaling interaction. The construction of a nanodelivery system that simultaneously enriches CSF-1R inhibitors and SIRPα antibodies would be an effective platform for reducing M2 tumor-associated macrophages at the tumor site and enhancing their tumor phagocytic capacity. Therefore, integrating a dual-pathway blocking delivery system targeting MCSF / CSF-1R and CD47 / SIRPα signaling into 3D-printed CPC scaffolds offers the potential to enhance local macrophage tumor phagocytosis, thereby inhibiting tumor recurrence.

[0006] Mesoporous silica-based nanoparticles (MSNs) have good biocompatibility and a high specific surface area. They are carriers with immune adjuvant functions, not only suitable for the enrichment of immunomodulatory drugs, but also can promote the development of naive T cells by acting on dendritic cells. However, MSNs have a limited number of binding sites for antibody molecular chains with both positive and negative charges, which may cause the delivery system to easily disintegrate, thereby reducing the therapeutic effect. Although chemical modification of antibodies to MSNs is stable, this may lead to antibody inactivation due to conformational changes.

[0007] Recent studies have found that appropriately modifying the surface of aminated nanocarriers with phenylboronic acid (PBA) can stabilize protein binding and maintain its activity through electrostatic interactions, boron-nitrogen coordination bonds, and cation-π interactions. Therefore, aminated MSNs (MSN-NH2-PBA) modified with PBA exhibit the potential to efficiently enrich CSF-1R inhibitors and SIRPα antibodies, promising the construction of a structurally stable dual-drug co-delivery system. However, a short in vivo residence time is a major drawback of nanoparticle-based immunotherapy drug delivery systems. In the absence of significant tumor recurrence, most nanoparticles used for conventional intravenous administration may be metabolized and excreted by the body within 24 hours, and simply dispersing the nanoparticles on a stent does not alleviate this problem.

[0008] To prolong drug release, a common strategy involves incorporating delivery systems into bone repair materials to formulate bioinks and create therapeutic scaffolds. However, drug release from calcium phosphate-based scaffolds occurs gradually as the material degrades, requiring a long time and being difficult to control. Notably, macrophages, which possess anti-tumor capabilities, secrete proinflammatory cytokines such as interleukin-6, interleukin-1, and tumor necrosis factor-α (TNF-α), which play a crucial role in the inflammatory response. Excessive local inflammatory cytokines can damage surrounding soft and hard tissues. Furthermore, bone repair materials exposed to the tumor microenvironment early during implantation may adsorb a significant amount of immunosuppressive signaling factors, thereby impacting the therapeutic process. Therefore, constructing a drug delivery system loaded with CSF-1R inhibitors and SIRPα antibodies, achieving controlled and regulated drug release from the scaffold material, and effectively leveraging the scaffold's bone repair and anti-tumor effects remains a major challenge in this approach.

[0009] Therefore, how to develop an immunomodulatory scaffold based on calcium phosphate scaffold that has both anti-tumor and bone repair functions, achieve controlled release of drugs on the scaffold, efficiently realize the bone repair function of the calcium phosphate scaffold, and provide the scaffold with excellent anti-tumor function has become a technical problem that needs to be solved urgently. Summary of the Invention

[0010] The present invention aims to address the above-mentioned technical problems and thus provides an immunomodulatory scaffold with both anti-tumor and bone repair properties, as well as its preparation method and application. The technical purpose of the present invention is to provide a calcium phosphate scaffold based on a calcium phosphate scaffold that combines anti-tumor immunomodulatory and bone repair functions. As a dual-drug delivery system, it can effectively exert the functions and effects of both drugs, synergistically enhancing their effectiveness and achieving efficient repair of tissue defects after bone tumor surgery.

[0011] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0012] The present invention first provides a method for preparing an immunomodulatory scaffold having both anti-tumor and bone repair properties, comprising the following steps:

[0013] (1) dispersing the amino-modified mesoporous silica in anhydrous methanol and reacting with 4-(bromomethyl)phenylboronic acid to prepare phenylboronic acid-modified amino-modified mesoporous silica nanoparticles;

[0014] (2) dispersing the nanoparticles obtained in step (1) in dimethyl sulfoxide containing a CSF-1R inhibitor, stirring the reaction to obtain inhibitor-loaded nanoparticles, which were then dispersed in PBS buffer, incubated with SIPRɑ antibodies, and centrifuged to obtain dual-drug nanoparticles loaded with the inhibitor and the antibody;

[0015] (3) An aqueous solution containing methacryloylated hyaluronic acid, methacryloylated oxidized hyaluronic acid, and a photoinitiator is used as a curing liquid, and is uniformly mixed with calcium phosphate powder in a certain liquid-solid ratio to prepare a bio-ink, and a calcium phosphate scaffold is prepared by 3D printing. The dual-drug nanoparticles obtained in step (2) are dispersed in a hydroxybutyl chitosan solution, and the calcium phosphate scaffold is immersed in the above solution for in situ cross-linking to prepare an immunomodulatory scaffold with anti-tumor and bone repair properties.

[0016] Currently, 3D-printed implants face the challenge of reducing recurrence rates in the postoperative treatment of bone tumors. This is closely related to the implant's inability to reverse the M2 polarization of macrophages and the loss of tumor phagocytosis caused by residual tumor cells. The method provided by the present invention enriches a CSF-1R pathway inhibitor within the porous structure of a co-delivery system, immobilizes a SIRPɑ antibody on its surface via boron-nitrogen coordination bonds, and assembles the co-delivery system onto an aldehyde-rich 3D-printed calcium phosphate scaffold using dynamic covalent bonds. This results in the development of a multifunctional therapeutic implant that phase-activates local tumor-phagocytic macrophages and promotes bone tissue regeneration.

[0017] Experiments in this invention demonstrate that phenylboronic acid-modified mesoporous silica nanoparticles, as drug carriers, can efficiently enrich CSF-1R inhibitors, stably bind antibodies, and retain their biological activity. The co-delivery system, assembled onto a calcium phosphate scaffold via a hydroxybutyl chitosan (HBC) network, enables controlled sustained release of the drug in the target treatment area. This effectively blocks the interaction between tumor cells and macrophages via the MCSF / CSF-1R and CD47 / SIPRα signaling pathways, thereby inhibiting M2 polarization of macrophages, preserving their phagocytic activity, and suppressing tumor recurrence. The gradual disintegration of the scaffold assembly exposes the calcium-phosphorus-silicon-based core, effectively promoting bone tissue repair. Therefore, the present invention's therapeutic scaffold, stably assembled with a co-delivery system for targeted regulation of local tumor phagocytosis by macrophages, provides a new engineering strategy for the treatment of bone tumors after surgery, inhibiting tumor immune escape and promoting bone tissue repair.

[0018] As shown in the experimental results of the present invention, the dual-drug loaded therapeutic stent significantly improved the anti-tumor effect compared with a single drug, and its bone repair effect was also better; the stent only needed to load a smaller amount of drug to achieve a highly effective anti-tumor effect. It can be seen that the two drugs exerted a synergistic effect, jointly improving the stent of the present invention's ability to inhibit tumor immune escape and recurrence, as well as bone repair.

[0019] Furthermore, the preparation method of the amino-modified mesoporous silica in step (1) is based on a sol-gel method using ethyl orthosilicate as a silicon source, hexadecyltrimethylammonium bromide as a template, and ammonium fluoride as an ammonia source.

[0020] Furthermore, the amino-modified mesoporous silica in step (1) reacts with 4-(bromomethyl)phenylboronic acid in a mass ratio of 1:0.4-4.

[0021] Furthermore, the CSF-1R inhibitor in step (2) is GW2580.

[0022] Furthermore, the weight ratio of the nanoparticles to the CSF-1R inhibitor in step (2) is 10:1.

[0023] Furthermore, the stirring reaction in step (2) is carried out at room temperature for 12 hours, and the co-incubation is carried out at 37° C. for 0.5 hours.

[0024] Furthermore, in step (3), the proportions of the methacrylated hyaluronic acid, methacrylic acid oxidized hyaluronic acid and photoinitiator in the curing solution are 4-10%, 1-10% and 0.5-2% respectively.

[0025] Furthermore, the concentration of the dual-drug nanoparticles in step (3) is 2-40 mg / mL, and the concentration of the hydroxybutyl chitosan solution is 1-2 wt%.

[0026] The second purpose of the present invention is to provide an immunomodulatory scaffold having both anti-tumor and bone repair properties prepared by the above method.

[0027] A third object of the present invention is to provide the use of the above-mentioned immunomodulatory scaffold with both anti-tumor and bone repair properties in the preparation of implants for treating bone defects after bone tumor surgery.

[0028] The beneficial effects of the present invention are as follows:

[0029] (1) The present invention assembles an HBC network containing MCSF / CSF-1R and CD47 / SIPRɑ pathway blocking drug co-delivery system on an aldehyde-rich 3D printed calcium phosphate skeleton based on dynamic covalent bonds, and develops a therapeutic implant that can activate macrophages that phagocytize tumor cells and promote bone regeneration in stages. By enriching CSF-1R inhibitors in the porous core of MSN-NH2-PBA and firmly fixing SIRPɑ antibodies on its surface, a targeted drug co-delivery system for macrophages is constructed. After assembling SIPRα antibodies, the drug delivery system shows effective binding to the surface of monocytes / macrophages. In combination with the release of CSF-1R inhibitors, the system significantly inhibits the M2 polarization of bone marrow-derived mononuclear cells (BMMs) and enhances the phagocytic ability of macrophages to tumor cells. After optimizing the assembly process, the HBC solution containing the co-delivery system is uniformly cross-linked to the calcium phosphate scaffold to form a slow-release hydrogel layer, and the therapeutic components can be gradually released from the scaffold while maintaining its regulatory function on macrophages. In an in vivo tumor therapy model, the therapeutic scaffold, which effectively released the co-delivery system, significantly reduced local M2 macrophages and inhibited tumor growth. After 8 weeks of subcutaneous implantation in normal mice, the therapeutic scaffold demonstrated good biocompatibility and promoted uniform bone growth.

[0030] (2) The dual drug delivery system of the present invention has a synergistic effect between the two drugs, which significantly improves the bone repair effect and anti-tumor effect of the scaffold compared with the loading of a single drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Characterization of the co-delivery system; (a) Schematic diagram of the preparation process of the co-delivery system; (b) TEM images of MSN, MSN-NH2 and MSN-NH2-PBA; (c) XPS spectrum of MSN-NH2; (d) Thermogravimetric curves of MSN-NH2 and MSN-NH2-PBA; (e) Zeta potential of MSN, MSN-NH2 and MSN-NH2-PBA; (f) EDS surface scanning image of MSN-NH2-PBA; (g) Digital photo showing the color of MSN-NH2-PBA / GW2580 / anti-SIPRɑ collected by centrifugation; (h) The ratio of GW2580 released from MSN-NH2-PBA / GW2580 / anti-SIPRɑ (n=3); (i) Confocal microscopy image showing that after 24 hours of culture, MSN-NH2-PBA / GW2580 / anti-SIPRɑ adhered to the surface of RAW264.7 cells.

[0032] Figure 2Screening of biosafety concentrations of nanoparticle delivery systems and their regulatory effects on macrophage proliferation, polarization, and phagocytosis; (a) Schematic diagram describing the dose screening and functional validation experimental process of the co-delivery system; (b) Effects of each group of modifications and particle concentrations on the viability of RAW264.7, BMMs, and BMSCs after 4 and 24 h of culture; (c) Effects of the co-delivery system on BMMs proliferation after 4 and 24 h of culture; (d) Confocal microscopy images showing that after 24 h of co-culture, MSN-NH2-PBA / GW2580 / a nti-SIPRɑ adhered to the surface of BMMs; (e) PCR results showed the effect of the delivery system on the expression of BMMs polarization-related genes (iNOs and CD206) compared with the MCSF+IL4+IL10 group (n=3, **P<0.01****P<0.0001); ##P<0.01, ##P<0.001 (compared with the MSN-NH2-PBA group); (f) Microscopic images showed the phagocytic effect of RAW264.7 cells on tumor cells after 24 hours of culture with the delivery system.

[0033] Figure 3 Characterization of the assembled co-delivery system scaffold; (a) Schematic diagram describing the preparation process of the therapeutic scaffold; (b) Microscope and scanning electron microscope images showing the porous structure and surface morphology of CPC and assembled co-delivery carrier CPC scaffold; (c) Scanning electron microscope images showing the changes in the scaffold surface morphology and co-delivery carrier release kinetics after immersion in PBS solution for 1 day and 7 days; (d) EDS image showing the element distribution on the scaffold surface after immersion in PBS solution for 7 days; (e) XRD spectrum showing the main phases on the scaffold surface after immersion for 7 days; (f) Fluorescence microscope image showing that MSN-NH2-PBA / GW2580 / anti-SIPRɑ is uniformly distributed on the scaffold surface; (g) The proportion of GW2580 released from the scaffold within 7 days (n=3).

[0034] Figure 4Figure 3 Effects of scaffold-released components on BMMs proliferation, polarization, and osteoclastogenesis; (a) Schematic diagram showing the process of scaffold-released substances regulating macrophage behavior; (b) Effects of released substance components on BMMs proliferation after 24 h of culture (n=3, ****P<0.0001 compared with the control group; ##P<0.01, #### compared with the CPC group P<0.0001; 00 compared with the MSN-NH2-PBA / GW2580 group P<0.01); (c) CD206 immunofluorescence staining and related (d) Statistical analysis results showing the effects of released substance components on BMMs polarization. (e) PCR results showed that the co-delivery system released by the scaffolds affected the gene expression of polarized macrophages (n=3, ****P<0.0001 compared with the MCSF+IL4+IL10 group; #### compared with the CPC group, P<0.0001); (f) TRAP staining, correlation statistical analysis and confocal microscopy images showed that the co-delivery system released by the scaffolds affected the osteoclastogenesis of BMMs after 3 days of culture (n=3, ****P<0.0001; #### compared with the CPC group, P<0.0001).

[0035] Figure 5 Figure 3 Effects of the co-delivery system scaffold on the function of BMSCs; (a) Schematic diagram describing the study; (b) CCK-8 assay results showing the effects of scaffold-released components on BMSC proliferation; (c) Confocal microscopy images showing the adhesion morphology of BMSCs on the scaffold after 3 days of culture; (d) ALP staining and (e) OPN immunofluorescence staining results and related statistical analysis showed the effects of released components on the osteogenic differentiation of BMSCs after 7 days of culture (n=3, **P<0.01, compared with the CPC group).

[0036] Figure 6To assemble a co-delivery system scaffold to effectively activate tumor-eating macrophages in a 4T1 breast tumor model in vitro and in vivo; (a) Confocal fluorescence microscopy shows the effect of components released from the therapeutic scaffold on the phagocytic activity of macrophages against tumor cells; (b) The percentage of RAW264.7 cells phagocytosing 4T1 cells after co-culture (n=3, ****P<0.0001 compared with the control group; #P<0.05, #### compared with the CPC group P<0.0001; 000 compared with the MSN-NH2-PBA / GW2580 group P<0.001); (c) Tumor size curve shows the effect of the scaffold on the phagocytic activity of macrophages against tumor cells. (a) Effects of scaffold material components on tumor growth, and the weight change curve shows the biocompatibility of the material components (n=3); (b) Tumor size curve shows the effect of the assembled co-delivery system scaffold on tumor growth, and the weight change curve shows the biocompatibility of the scaffold (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group); (c) Immunofluorescence staining images and related quantitative results show the distribution of M1 and M2 macrophages in tumors (n=3, *P<0.05, ****P<0.0001 compared with the CPC group).

[0037] Figure 7 To assemble the co-delivery system scaffold for biosafety and tissue regeneration performance; (a) Digital photograph of the scaffold site 8 weeks after implantation; (b) HE staining and Masson staining results showing the tissue regeneration performance of the implanted scaffold; Green arrows indicate the location of new bone; Immunofluorescence staining images of the implanted scaffold (c, d) and related quantitative results (e, f); (*P < 0.05, compared with the CPC group).

[0038] Figure 8 Schematic diagram of the fabrication process for assembling a co-delivery system scaffold and its role in postoperative treatment of bone tumors; (a) preparation of the targeted co-delivery system and (b) its assembly on a 3D-printed calcium phosphate framework; after implantation of the therapeutic scaffold, (c) the first stage regulates the phenotype and phagocytic function of macrophages; and (d) the second stage provides a microenvironment for bone repair. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the scope of protection of the present invention.

[0040] Example 1

[0041] 1. Experimental Materials and Methods

[0042] 1. Synthesis of PBA-modified nanoparticles

[0043] MSN-NH2 nanoparticles (MSN-NH2) with a size of approximately 100 nm were synthesized using the following method: 1.82 g of hexadecyltrimethylammonium bromide and 3.0 g of ammonium fluoride were dissolved in 500 mL of deionized water. 9 mL of tetraethyl orthosilicate was added dropwise to the solution with stirring at 80°C and the reaction continued for 2 hours. After the reaction was completed and cooled overnight, the product was collected by centrifugation and washed multiple times with ethanol and deionized water. The washed product was dispersed in a mixture of 4 mL of hydrochloric acid and 200 mL of ethanol and reacted at 90°C for 24 hours. After the reaction, the product was washed several times with water and ethanol and then freeze-dried for later use. The morphology of the nanoparticles was evaluated using transmission electron microscopy (TEM; TALOS F200X, Thermo Fisher Scientific, USA). The elemental distribution and content of MSN-NH2 were determined by X-ray photoelectron spectroscopy (XPS; AXIS UltraDLD, Shimadzu, Japan) and energy-dispersive spectroscopy (EDS, TALOS F200X). Subsequently, the MSN-NH2 particles were uniformly dispersed in anhydrous methanol and 4-(bromomethyl)phenylboronic acid (1mM / 100mg MSN-NH2) was added for 24h. The product was dialyzed with methanol and distilled water and then freeze-dried and collected (MSN-NH2-PBA). Morphological evaluation was performed using TEM and the degree of modification was quantified by thermogravimetric analysis (TGA5500, TA, USA).

[0044] 2. Assembly of the Nanodelivery System

[0045] GW2580 (TargetMol, USA), a small molecule inhibitor with highly selective pathway-blocking properties, was used as a model for the development of drugs that block the MCSF / CSF-1R pathway. Due to the hydrophobicity of GW2580 and the hydrophilicity of the SIPRɑ antibody, we first enriched GW2580 within the hydrophobic core of MSN-NH2-PBA. Then, through electrostatic interactions, boron-nitrogen coordination bonds, and cation-π interactions, the anti-SIPRɑ was stably assembled onto the surface of the MSN-NH2-PBA to create a co-delivery system. This assembly design allows the anti-SIPRɑ on the surface of the co-delivery system to readily bind to target cells and block the pathway. Furthermore, the binding of the surface antibody can delay the release of GW2580 to a certain extent due to steric hindrance, thereby prolonging the duration of the blocking effect.

[0046] The specific steps are as follows: 10 mg of MSN-NH2-PBA particles were dispersed in 1 mL of dimethyl sulfoxide (Solaibao, China) containing 1 mg of GW2580 and stirred at 25°C for 12 h. The inhibitor-loaded MSN-NH2-PBA (denoted as MSN-NH2-PBA / GW2580) was then collected by centrifugation and freeze-dried. The inhibitor-loaded nanoparticles were evenly dispersed in 1 mL of phosphate-buffered saline (PBS, Hyclone, USA), incubated with anti-SIPRɑ at 37°C for 0.5 h, and then collected by centrifugation to obtain the dual-drug co-delivery system (denoted as MSN-NH2-PBA / GW2580 / anti-SIPRɑ).

[0047] To facilitate antibody labeling, nanoparticles were prepared using APC-anti-SIPRɑ (Biolgend, USA) for in vitro experiments. The particles were immersed in PBS on a shaker at 37°C, and the amount of GW2580 released from the carrier was calculated. The released solution was collected at specific time points and fresh buffer was added. The absorbance at 280 nm was measured by UV-visible spectroscopy (Eppendorf, Germany) to calculate the amount of inhibitor released.

[0048] 3. Cell culture and targeted delivery

[0049] Mouse bone marrow monocytes and rat bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured. RAW264.7 (National Certified Cell Line Collection, China) cells expressing SIPRɑ on their cell membranes and BMMs, two classic mouse monocyte / macrophage cell lines used for immunomodulatory drug performance studies, were used as experimental models to explore the efficacy of the anti-SIPRɑ assembly delivery system. Cells were seeded in 96-well plates (1×10 4 Cells were cultured in normal culture medium for 2 hours before being switched to medium containing MSN-NH2-PBA / GW2580 / anti-SIPRɑ. After 12 hours of incubation, cells were fixed with 4% paraformaldehyde (Wuhan Saiwei Biotechnology Co., Ltd., China). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Saiwei Biotechnology Co., Ltd.), and the binding of nanoparticles to cells was observed using a confocal fluorescence microscope (Zeiss, Germany).

[0050] 4. Cell Viability Assay

[0051] The effects of different modification groups and particle dosages on the activity of RAW264.7 cells, bone marrow-derived mononuclear cells, and bone marrow mesenchymal stem cells were observed. 4 cells / well) and were cultured in the presence of different concentrations (0, 12.5, 25, 50, and 100 μg mL-1 ) in the culture medium containing dispersed particles. After incubation for 4 h and 24 h, cell viability was detected using the Cell Counting Kit-8 (CCK-8, Shanghai Yuli Biotechnology Co., Ltd., China). -1 MSN-NH2-PBA, MSN-NH2-PBA / GW2580 and MSN-NH2-PBA / GW2580 / anti-SIPRɑ were cultured with RAW264.7 cells and BMMs, respectively, and their effects on cell viability were observed (n=3).

[0052] 5. Regulation of BMMs polarization

[0053] BMMs were cultured in M2 polarization induction medium (M2 PICM) containing different particle groups to investigate their regulatory effects on polarization-related immunosuppression. M2 PICM was prepared according to existing literature methods, and BMMs were seeded in 24-well plates (5×10 4 The cells were cultured in the induction medium containing different particle groups for 24 h, and then reverse transcription polymerase chain reaction (RT-PCR) was performed to analyze the expression of inducible nitric oxide synthase (iNOs) and CD206 markers in macrophages.

[0054] 6. In vitro tumor phagocytosis assay

[0055] RAW264.7 cells were labeled with APC-conjugated CD-11b antibody (BioLegend) and then seeded in 12-well plates (5 × 10 4 cells / well). The cells were cultured in M2 induction medium containing 20 ng / mL IL-4 (Peprotech, USA) for 24 h. Subsequently, different groups of particles were incubated with the cells for 24 h. 4T1 breast cancer cells (National Certified Cell Line Collection Center) were labeled with green CMFDA live cell tracer (China Yisheng Biotechnology (Shanghai) Co., Ltd.) and incubated with macrophages (5×10 4 After co-culture with 400 cells / well for 12 h, the phagocytosis of tumor cells by RAW264.7 cells was observed using a confocal microscope.

[0056] 7. 3D printing of CPC brackets

[0057] Hydroxybutyl chitosan (HBC), methacrylated hyaluronic acid (HAMA) and methacrylated hyaluronic acid (OHAMA) were synthesized according to existing literature methods. A curing solution containing 5wt% HAMA, 2wt% OHAMA and 0.5% w / v photoinitiator was prepared and mixed with CPC powder (Shanghai Ruibang Biomaterial Co., Ltd., China) at a specific solid-liquid ratio to prepare a bio-ink. The bio-ink was transferred to a bio-printer (Cellink, Sweden) and the CPC scaffold was printed using an established method. After re-curing, it was dried and stored. A classic cube model (10×10×2mm) was designed and printed. 3 ) and circular models (d = 8 mm, h = 2 mm) for in vitro and in vivo experiments. In the bioprinter system, the model packing density was set to 26%, which was used to produce scaffolds with a pore size of approximately 350 μm, a pore size found to be beneficial for nutrient transport and bone regeneration.

[0058] 8. Preparation and Characterization of Immunomodulatory Scaffolds

[0059] Based on the above-mentioned safe and effective dose experimental results, the nanoparticles were dispersed in a 1 wt% HBC solution at specific concentrations and assembled with OHAMA contained within the scaffolds (50 μg / scaffold for in vitro cell experiments and 1 mg / scaffold for in vitro materials science experiments and in vivo experiments). To evaluate the effects of in situ cross-linking of the hydrogel mask layer on the scaffold structure and the dispersion of the nanoparticles, the scaffold morphology was examined using optical microscopy (Olympus Corporation, Japan) and scanning electron microscopy (SEM, TESCAN, Czech Republic).

[0060] Subsequently, the loading stability of the nanoparticles on the scaffolds was investigated. After immersion in PBS for 1 and 7 days, the surface morphology and elemental distribution of the assembled nanoparticle scaffolds were analyzed using scanning electron microscopy and energy dispersive spectroscopy, respectively. After immersion in PBS for 7 days, the scaffolds were further analyzed by X-ray diffraction (D8 ADVANCE DaVinci, Bruker, Germany).

[0061] 9. Inhibitor release kinetics

[0062] Each nanoparticle-loaded scaffold in a 24-well plate was soaked in 2 mL of PBS and incubated at 37°C. At specific time points, the supernatant from each well was collected and stored, and an equal amount of fresh buffer was added. Subsequently, the absorbance of the supernatant was measured using a UV spectrophotometer (Eppendorf) to analyze the concentration and release kinetics of GW2580.

[0063] 10. Extract Preparation

[0064] Extracts from different groups of scaffolds were prepared, and the concentrations of calcium, phosphorus, and silicon ions were measured. Subsequently, various conditioned mediums were prepared for experiments on BMMs proliferation, polarization, osteoclast differentiation, and BMSCs osteogenic differentiation.

[0065] 11. Regulation of BMMs polarization by the scaffold release system

[0066] First, the effect of scaffold-released components on BMMs viability was detected by co-culturing cells with the extract. BMMs were seeded in 96-well plates (1×10 4 Cells were co-cultured with extracts from different scaffold groups. After 1 and 3 days of culture, cell viability was assessed using a CCK-8 assay. Cells cultured in medium containing 30 ng / mL macrophage colony-stimulating factor (Peprotech) served as a control group.

[0067] Subsequently, the effects of scaffold-released components on BMM polarization were investigated. 4 After culturing BMMs at 400 nm (1 / 4 cell / well) for 24 h, the cells were subjected to immunofluorescence staining to detect CD206, a specific marker of M2 macrophages, and the results were observed under a confocal microscope.

[0068] Next, the effect of the released components of the scaffold on reversing polarization-related immunosuppression was analyzed. 4 BMMs were cultured at 400 cells / well. After 24 h of culture, the expressions of cell markers TNF-α and CD206 were detected by RT-PCR.

[0069] 12. Inhibit osteoclast formation in BMMs

[0070] The effects of components released from the scaffold on osteoclast formation were determined. BMMs were treated with scaffold extract (96-well plate, 1×10 4 Cells were cultured for 1 and 3 days (10 cells / well). Tartrate-resistant acid phosphatase (TRAP) staining was followed by observation under a light microscope. The percentage of TRAP-positive area was quantified using ImageJ software. Cell morphology was observed under a confocal microscope after staining with rhodamine-phalloidin (Wuhan Saiwei Biotechnology Co., Ltd.) and DAPI.

[0071] 13. Effects on BMSCs Function

[0072] (1) Cell viability

[0073] Different scaffold groups (24-well plates, 1×10 4 BMSCs were cultured in the extract of 100 μg / well (μg / well). After 1 and 3 days of culture, the cell proliferation was detected using the CCK-8 kit.

[0074] (2) Cell adhesion on the scaffold

[0075] 1×10 4 Each BMSC was cultured on the scaffold for 3 days and stained with rhodamine-phalloidin and DAPI after fixation. The cell morphology was observed under a confocal microscope.

[0076] (3) Cell osteogenic differentiation function

[0077] BMSCs were treated with extracts from different scaffold groups (24-well plate, 5 × 10 4 A portion of the cells were stained with alkaline phosphatase (ALP) (Biyuntian Biotechnology Co., Ltd.), and another portion was immunostained with osteopontin (OPN, Abcam). The cell nuclei were stained with DAPI and observed under a confocal microscope.

[0078] 14. Effect of stent material composition on tumor growth

[0079] Twelve 4-week-old female BALB / c mice (Beijing Biotechnology Co., Ltd.) were divided into three groups: (1) untreated group; (2) CPC group; and (3) CPC / MSN-NH2-PBA group. Each mouse was injected with 4T1 breast cancer cells (1×10 6 When the average tumor volume is about 100 mm 3 At 4 p.m., a stent was implanted near the tumor. The body weight and tumor volume of each mouse were measured at regular intervals after surgery. The animals were sacrificed 10 days after implantation.

[0080] 15. Anti-tumor effect of stents

[0081] In vitro studies of the stent-delivery system regulating macrophage phagocytosis of tumor cells were conducted as described above and analyzed by confocal microscopy. Based on the confocal microscopy results, the phagocytic percentage was calculated by dividing the number of phagocytic cells by the total number of macrophages.

[0082] Tumor models were established and scaffolds were implanted as described above. Nine four-week-old female BALB / c mice were divided into three groups: 1) untreated; 2) CPC / MSN-NH2-PBA / GW2580; and 3) CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ. Body weight and tumor volume were measured at regular intervals after surgery. Animals were sacrificed 10 days after implantation. Animal experiments were performed in accordance with the guidelines and regulations of the Institutional Animal Care and Use Committee of Shanghai Junbo Biotechnology Co., Ltd.

[0083] To evaluate the scaffold's regulatory effect on resident macrophages, tumor samples were collected and processed into sections. CD206 and iNOs expression was detected by immunofluorescence staining and observed by confocal microscopy. Relative mean fluorescence intensity was quantified using ImageJ software.

[0084] 16. In vivo biosafety and osteogenic performance

[0085] Nine four-week-old female BALB / c mice were divided into three groups: 1) CPC group; 2) CPC / MSN-NH2-PBA / GW2580 group; and 3) CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ group. Stents were implanted subcutaneously in the dorsal pockets of anesthetized mice. Eight weeks after implantation, the animals were euthanized, and the stents were harvested for subsequent evaluation.

[0086] The scaffolds were prepared into histological sections. The sections were stained with hematoxylin and eosin (HE) and Masson's trichrome and observed under a microscope. Immunofluorescence staining was performed to detect osteopontin (OPN) and osteocalcin (OCN, Abcam) antibodies.

[0087] 17. Statistical Analysis

[0088] The data were expressed as mean ± standard deviation. GraphPad Prism 9.5 software was used for statistical analysis.

[0089] 2. Experimental Results and Discussion

[0090] 1. Synthesis and characterization of mesoporous silica nanoparticles

[0091] Branched MSNs were synthesized and amino modified. Figure 1 b) Detection of the morphology of nanoparticles. TEM image ( Figure 1 Figure b) shows that both types of particles exhibit highly uniform particle size and the expected dendritic pore structure. Amino modification does not affect the shape and internal pore structure of the particles. In addition, absorption peaks of silicon, oxygen, and nitrogen were observed in the XPS graph of the particles, indicating the successful modification of NH2 groups ( Figure 1 (c) Based on this, 4-(bromomethyl)phenylboronic acid was reacted with MSN-NH2 to prepare PBA-modified MSN-NH2 (MSN-NH2-PBA). TEM results showed that the nanoparticles remained morphologically unchanged after PBA modification, with particle sizes remaining around ~100 nm at each modification stage. Further experiments were conducted to analyze the PBA modification effect.

[0092] Thermogravimetric analysis results showed that ( Figure 1In (d), when the temperature is increased to 600℃, the weight loss of MSN-NH2 and MSN-NH2-PBA is 17% and 21%, respectively. The Zeta potentials of MSN, MSN-NH2 and MSN-NH2-PBA are (-9.61±0.03), (27.07±1.11) and (5.32±1.29) mV, respectively. Figure 1 (e) The reduction in mass added by the PBA group on MSN-NH2 and the decrease in the zeta potential indicate successful modification. In the EDS surface scanning image of MSN-NH2-PBA, silicon, oxygen, nitrogen, and boron elements are evenly distributed, and their morphology is consistent with the shape of the nanoparticles ( Figure 1 f), indicating that the modification of the PBA group is uniform.

[0093] Because branched MSNs have numerous pores within them, they are suitable for accumulating small molecule drugs. Therefore, CSF-1R inhibitors were added to them. CD47 is a transmembrane protein present at high levels on the surface of tumor cells and plays a crucial role in regulating the phagocytic activity of macrophages. When it interacts with the receptor SIRPα on macrophages, it sends a "don't eat me" signal, weakening the phagocytic function of macrophages.

[0094] This study incorporated SIRPα antibodies onto the surface of delivery nanoparticles to inhibit the CD47 / SIRPα axis interaction. The loading performance of MSN-NH2-PBA for both the inhibitor and the antibody was first investigated. For ease of tracing, APC-anti-SIRPα was initially used as an antibody research model. After the nanoparticles were assembled at room temperature and collected by centrifugation, the antibody was visible as a blue color in the precipitate collected at the bottom of the centrifuge tube, demonstrating their stable and efficient binding ( Figure 1 f). Subsequently, we studied the release of GW2580 in a dual-pathway inhibitory drug delivery system. Figure 1 As shown in Figure g, the inhibitor was gradually released within 1 week without a burst release. We speculate that this phenomenon is due to the gating effect of the surface-assembled antibody through volume blocking. After culturing the assembled delivery system with RAW264.7 cells for 1 day, the binding of nanoparticles to cells under the action of anti-SIPRɑ was preliminarily observed. According to the results of confocal microscopy observation ( Figure 1 (h) APC fluorescence shows cell morphology. Based on this result, we speculate that although the antibody is assembled on the nanoparticles, it can still function normally when binding to SIPRɑ on the surface of RAW264.7 cells.

[0095] 2. Dose optimization of drug delivery system

[0096] The effects of group modification and particle concentration on the viability of RAW264.7, BMMs and BMSCs were investigated. Figure 2In a), the dosage of the drug delivery system was optimized. First, different concentrations of MSN, MSN-NH2, and MSN-NH2-PBA were co-cultured with cell culture; the CCK-8 test results were as follows: Figure 2 As shown in Figure (b), after 4 hours of incubation with different particle concentrations, the survival rate of all cell types was high. After 24 hours of co-culture, the cytotoxicity of the particles showed a clear concentration-dependent effect. Cell viability decreased with increasing concentration. The sensitivity of different cell types to particle concentration was relatively similar. When the particle concentration exceeded 25 μg / mL, cell viability decreased to below 70%. In addition, amino and PBA modification of MSNs had no significant effect on cell viability. Based on these results, nanoparticles with a concentration of 20 μg / mL were prepared for subsequent experiments.

[0097] 3. Regulation of cell polarization and tumor phagocytosis.

[0098] Next, we preliminarily evaluated the effect of the drug delivery system on the activity of BMMs. Figure 2 As shown in Figure c, further loading of inhibitors and antibodies at this particle dose did not affect cell viability. After BMMs were co-cultured with MSN-NH2-PBA / GW2580 / anti-SIPRɑ for 24 h, significant fluorescent antibodies were observed on the cell surface ( Figure 2 d), thus confirming the potential of MSN-NH2-PBA / GW2580 / anti-SIPRɑ to specifically bind to BMMs and regulate related pathways. Subsequently, BMMs were co-cultured with M2 polarization induction culture medium containing MSN-NH2-PBA, MSN-NH2-PBA / GW2580, and MSN-NH2-PBA / GW2580 / anti-SIPRɑ for 24 hours to analyze the effect of the nanodelivery system on cell polarization. Figure 2 PCR analysis in Figure 5 shows that CD206 expression on M2 macrophages was significantly reduced in the GW2580-loaded nanoparticle group compared with the control group (P < 0.001), effectively inhibiting M2 polarization of BMMs. This indicates that the inhibitor maintains its original activity within the delivery system. Compared with the MSN-NH2-PBA / GW2580 group, CD206 expression in the MSN-NH2-PBA / GW2580 / anti-SIPRɑ group was reduced, and the inhibitory effect on M2 polarization was enhanced. This may be related to the assembly of the anti-SIPRɑ, which facilitates the rapid accumulation of the delivery system on cells. Compared with the control group, iNOS expression was increased in both the MSN-NH2-PBA / GW2580 and MSN-NH2-PBA / GW2580 / anti-SIPRɑ groups, promoting M1 polarization of cells, with the latter exhibiting a stronger effect than the former.

[0099] The main function of the introduced anti-SIPRɑ is to block the CD47 / SIPRɑ signal. Therefore, we selected 4T1 tumor cells expressing CD47 as a research model to study the regulatory effect of the drug delivery system on macrophage phagocytosis. Figure 2 As shown in center (f), RAW264.7 cells and tumor cells were randomly distributed in the control and MSN-NH2-PBA groups. However, macrophages were observed recruiting and phagocytosing tumor cells in the MSN-NH2-PBA / GW2580 and MSN-NH2-PBA / GW2580 / anti-SIPRɑ culture media. Compared with the MSN-NH2-PBA / GW2580 group, more phagocytic cell clusters were observed in the MSN-NH2-PBA / GW2580 / anti-SIPRɑ group. This finding is consistent with previous studies reporting inhibition of intercellular CD47 / SIPRɑ signaling and demonstrates the effectiveness of our anti-SIPRɑ assembly. These findings demonstrate that the synergistic blockade of MCSF / CSF-1R and CD47 / SIPRɑ by the delivery system modulates macrophage phenotype and enhances phagocytic capacity, leading to anti-tumor activity.

[0100] 4. Characteristics of assembled drug delivery system stents

[0101] Tumor resection results in complex bone defects, which can be clinically filled with ready-to-use polymethyl methacrylate (PMMA) bone cement. However, it is difficult to degrade in vivo and does not promote bone remodeling. During postoperative treatment of malignant tumors, such as osteosarcoma, a common disease in adolescents, this residual bioinert material may impair normal development and growth. Therefore, bioactive implants that precisely fill defects, promote autologous bone regeneration, and gradually degrade have been a focus of research over the past three decades. Self-setting calcium phosphate bone cement is a new clinically active bone cement composed primarily of dicalcium phosphate and tetracalcium phosphate. Upon contact with water at room temperature, it rapidly hydrates and solidifies to form osteoconductive, bone-like hydroxyapatite. When mixed with a curing fluid, calcium phosphate bone cement exhibits excellent formability and can be injected and prepared into porous scaffolds to fill defects and promote bone regeneration. During bone repair, the solidified product is degraded by osteoclast secretions and subsequently metabolized by the body. Our previous research has shown that CPC-based bioinks can be easily combined with 3D printing technology to create structures with adjustable shapes, internal porous structures, and mechanical properties, allowing for precise filling of complex defects and promoting bone tissue repair. Therefore, we used previously established methods to prepare a bone repair core skeleton using CPC-based bioink.

[0102] HAMA-based photocrosslinking curing solution was introduced into the bio-ink to improve its formability, and OHAMA was further introduced to provide crosslinkable aldehyde groups ( Figure 3 (a) The composite bio-ink showed excellent printability, e.g. Figure 3 As shown in (b), the printed scaffold has a transparent porous structure with a pore size of approximately 400 μm to promote fluid exchange, nutrient transport, and bone ingrowth. Due to the hydration reaction of CPC, characteristic needle-shaped nanohydroxyapatite is visible within the scaffold.

[0103] To address the need for phased, localized, and stable delivery of postoperative tumor immune escape suppression therapy, this delivery system was incorporated into a HBC solution and assembled onto an aldehyde-rich CPC scaffold via a Schiff base reaction. Due to its excellent biocompatibility and tunable cross-linking and degradation properties, the hydrogel effectively preserves the bioactivity of the delivered therapeutic agent over a specified time period and protects it from rapid hydrolytic degradation or enzymatic breakdown. The Schiff base cross-linking reaction occurs rapidly at room temperature, making it suitable for the stable assembly of the delivery system in three dimensions.

[0104] The optimized HBC solution was loaded with different doses of MSN-NH2-PBA and then assembled on the stent to observe the assembly effect. It can be seen from the naked eye that the drug-loaded nanoparticles of each dose were well assembled in the stent, maintaining pore connectivity and no obvious drug-loaded particle leakage. Then, optical microscopy and scanning electron microscopy were used to observe the assembly of the mask sustained-release layer on the stent (1 mg of particles per stent). According to optical microscopy observation ( Figure 3 In (b), the scaffold has a uniform pore size of approximately 400 μm. Scanning electron microscopy results show that the HBC solution loaded with drug delivery particles assembles on the CPC scaffold to form a uniform cross-linked mask layer, with the nanoparticles evenly distributed within the mask layer.

[0105] 5. Regulation of BMMs proliferation, polarization, and osteoclastogenesis

[0106] Recent studies have shown that there is a strong correlation between MCSF in the microenvironment and the proliferation, phenotypic changes and osteoclast differentiation of BMMs. Therefore, we analyzed the effects of scaffold-released drug delivery systems on the proliferation, polarization and osteoclast differentiation of BMMs ( Figure 4 First, cells were incubated with extracts from different scaffolds to examine their activity. Figure 4 Figure (b) shows that culture medium from the CPC scaffold did not affect the activity of BMMs, while the extract containing the drug delivery system effectively inhibited their proliferation (P < 0.001). The released MSN-NH2-PBA / GW2580 exhibited a stronger inhibitory effect on BMM proliferation than the MSN-NH2-PBA / GW2580 / anti-SIPRɑ combination (P < 0.01). GW2580 inhibited cell proliferation in a concentration-dependent manner, likely due to the assembly of anti-SIPRɑ on the nanoparticles, which further sustained the release of GW2580.

[0107] According to the immunofluorescence staining results ( Figure 4 In Figures c and d), CPC extract induced BMMs to polarize toward CD206+M2. In contrast, the number of M2 phenotype BMMs in the extract culture medium containing the sustained-release drug delivery system was significantly lower than that in the CPC extract culture medium (P<0.001), indicating that the sustained-release drug delivery system exhibited a stronger effect than the CPC scaffold in regulating cell polarization. Subsequently, BMMs were co-cultured with M2 polarization-inducing medium containing scaffold-released components to evaluate the ability of the drug delivery system to reverse immunosuppression. Figure 4 As shown in Figure (e), compared with the control group, the gene expression of CD206 (M2 macrophages) was significantly decreased in the CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ groups (P < 0.001). Compared with the CPC / MSN-NH2-PBA / GW2580 group, CD206 expression was decreased in the CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ group. CSF-1R inhibitors released from the scaffolds significantly inhibited the M2 polarization of BMMs, and this inhibitory effect was further enhanced after the addition of anti-SIPRɑ. This phenomenon may be due to the targeted delivery induced by anti-SIPRɑ, which led to the enrichment of the inhibitor around the cells. In addition, the delivered inhibitors promoted the M1 polarization of BMMs. The expression of M1 macrophage genes (TNF-ɑ) induced by CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ scaffolds was higher than that of the control group.

[0108] The MCSF / CSF-1R pathway is closely related to the differentiation of monocytes into osteoclasts. Excessive secretion of MCSF will lead to an increase in the number of local osteoclasts, disrupting the balance of bone metabolism in the defect site, thereby promoting tumor growth and affecting tissue repair. After 3 days of co-culture, the effects of scaffold-released components on the differentiation of BMMs into osteoclasts were investigated. Figure 4 The TRAP staining results in (f) show that CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ scaffold-released drug delivery systems have a significant inhibitory effect on osteoclastogenesis in BMMs. The number of TRAP-positive osteoclasts in the CPC / MSN-NH2-PBA / GW2580 group and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ group was significantly lower than that in the control group and CPC group ( Figure 4 In addition, observation of multinucleated osteoclasts under confocal microscopy further confirmed the TRAP staining results ( Figure 4 (h) Multinuclear osteoclasts were observed in both the control and CPC groups, but the number of multinuclear osteoclasts was significantly decreased in the CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ groups.

[0109] 6. Effects on BMSCs Function

[0110] In addition to anti-tumor properties, scaffolds designed for postoperative treatment of bone tumors must also be able to promote bone tissue regeneration. Although the immunomodulatory function of scaffold drug delivery systems has been demonstrated, their impact on bone regeneration remains unclear. Therefore, we investigated the effects of components released from the scaffold at intermediate stages on BMSC activity and osteogenic differentiation ( Figure 5 (a). Figure 5 As shown in Figure b, during the 3-day co-culture with the extracts of each scaffold, BMSCs maintained a high level of viability. There was no statistically significant difference between the experimental groups. Consistent with previous findings, during the co-culture, cells adhered and spread better on the scaffold containing the HBC mask layer ( Figure 5 (c) Then, the effect of the scaffold-released components on the alkaline phosphatase activity of BMSCs was investigated. Figure 5 As shown in Figure d, there was no statistically significant difference in the ALP-positive area between the CPC group, CPC / MSN-NH2-PBA / GW2580 group, and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ group. The CPC / MSN-NH2-PBA / GW2580 group and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ group showed stronger positive staining. Therefore, we further examined the expression of OPN in BMSCs after 7 days of co-culture with the scaffold-released components by immunofluorescence staining ( Figure 5 (e) OPN expression levels were higher in the CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ groups than in the CPC group (P < 0.01). These results suggest that the release of components from the scaffold during the intermediate phase does not adversely affect the activity and osteogenic differentiation of BMSCs. Appropriate silicon ion concentrations can enhance the osteogenic differentiation of BMSCs. Silicon ions dissociated from the delivery vehicle in the culture medium may be within an appropriate concentration range, enabling the scaffold sustained-release system to promote the osteogenic differentiation of BMSCs.

[0111] 7. Anti-tumor effects in vivo and in vitro

[0112] M2 polarization-induced RAW264.7 cells were first cultured with the scaffold extract alone, and then co-cultured with tumor cells to observe the regulatory effect of the scaffold release system on the performance of macrophages in phagocytosis of tumor cells. Figure 6 As shown in Figures (a) and (b), the phagocytic percentages of RAW264.7 cells in the control, CPC, CPC / MSN-NH2-PBA / GW2580, and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ groups were 4.82±1.09, 4.53±1.42, 8.04±1.64, and 15.40±0.71, respectively. Macrophages cultured with the CPC scaffold-released component did not exhibit phagocytic activity against tumor cells and could not reverse immunosuppression. In contrast, the CPC / MSN-NH2-PBA / GW2580 scaffold-released component enhanced the phagocytic function of RAW264.7 cells (P<0.05), which may be related to a decrease in the number of M2 phenotype RAW264.7 cells. Cells treated with the CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ scaffold-released component exhibited the most significant tumor phagocytic characteristics (P<0.0001). This phenomenon can be attributed to two factors: first, the presence of anti-SIPRɑ promoted the targeted delivery of GW2580 to macrophages; second, the binding of anti-SIPRɑ to macrophages effectively inhibited the CD47 / SIPRɑ immune escape interaction between tumor cells and macrophages, thereby enhancing phagocytic activity.

[0113] The mouse subcutaneous 4T1 breast tumor model is a widely used model for studying the efficacy of immunotherapy and was therefore used to evaluate the in vivo immunomodulatory effects of the scaffold. Excessive use of nanoparticles may lead to adverse reactions, including renal impairment and potentially life-threatening consequences. Therefore, we first investigated the biosafety of the scaffold material components and their effects on tumor growth. CPC and CPC / MSN-NH2-PBA scaffolds were implanted subcutaneously in mice with a volume of nearly 100 mm. 3 The changes of tumor and mouse body weight were carefully detected and recorded at designated time points. Figure 6 The results in Figure c show that neither the scaffold nor the carrier material significantly interfered with tumor growth or affected the weight maintenance of the mice. Subsequently, we established an identical tumor model to study the effects of the scaffold assembled into the co-delivery system on macrophages and tumor growth in vivo. The CPC / MSN-NH2-PBA / GW2580 scaffold group showed the expected anti-tumor effect ( Figure 6(d) Starting from the 7th day, the tumor volume in the CPC / MSN-NH2-PBA / GW2580 scaffold group was significantly smaller than that in the untreated group (P<0.01). The CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ scaffold targets macrophages through antibodies, regulating the phagocytic function of macrophages and showing more effective inhibition of tumor growth than the CPC / MSN-NH2-PBA / GW2580 scaffold and the untreated group (P<0.0001). On the 5th day after inoculation, the tumor volume in the CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ group was significantly smaller than that in the untreated group (P<0.05). There was no significant difference in the body weight of mice among the groups, further demonstrating the good biocompatibility of the scaffold. Figure 6 d). Mice were killed 10 days after surgery and tumors were removed for histological analysis. Figure 6 Figure (e) shows that, consistent with the observed trend of tumor growth inhibition, the number of iNOs+ M1 macrophages in tumors treated with CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ scaffolds was higher than that in the control group, while the number of CD206+ M2 macrophages was lower than that in the control group (P<0.0001). In addition, the number of M2 macrophages in tumor tissues in the CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ group was significantly reduced compared with the CPC / MSN-NH2-PBA / GW2580 group (P<0.05). These results suggest that scaffolds delivering a dual-pathway blocking system have the potential to modulate local macrophage phenotype and inhibit tumor growth.

[0114] 8. Histocompatibility and osteogenic properties

[0115] CPC, CPC / MSN-NH2-PBA / GW2580, and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ scaffolds were implanted subcutaneously in mice to investigate their effects on tissue regeneration. The survival rate of mice following scaffold implantation was 100%, and physiological functions remained normal. Eight weeks after implantation, the scaffolds were harvested for further analysis. Figure 7 As shown in Figure a, no abnormalities such as redness, swelling, or tissue necrosis were observed at the implantation sites of the scaffolds. The shape and structure of the scaffolds remained intact, and blood vessels were visible. HE and Masson staining showed that new bone tissue infiltrated the scaffolds in all experimental groups. The bone tissue formed in the CPC group was randomly distributed, while the new bone tissue in the CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ groups was mainly distributed around each printed filament ( Figure 7 (middle b). Fluorescence staining results showed ( Figure 7 In the CPC / MSN-NH2-PBA / GW2580 and CPC / MSN-NH2-PBA / GW2580 / anti-SIPRɑ groups, the OPN and OCN-positive areas within the scaffolds were significantly higher than those in the CPC group (P < 0.05). These results suggest that the scaffolds assembled into the co-delivery system have good tissue compatibility, and the gradual release of dual immunomodulatory drugs does not affect normal tissue function in the long term. Furthermore, this may be due to the gradual dissociation of silicon ions from MSN-NH2-PBA after local administration, which allows the scaffolds to exhibit osteoinductive activity and promote uniform regeneration of surrounding bone tissue.

[0116] 3. Conclusion

[0117] The present invention is based on the dynamic covalent bonding of an HBC network containing MCSF / CSF-1R and CD47 / SIPRɑ pathway blocking drug co-delivery systems assembled on an aldehyde-rich 3D printed calcium phosphate skeleton. A therapeutic implant that can activate macrophages that phagocytize tumor cells and promote bone regeneration has been developed. Figure 8 ). As a functional carrier, MSN-NH2-PBA can efficiently enrich CSF-1R inhibitors and stably combine with anti-SIPRɑ to form a co-delivery system. After the introduction of the HBC network, the co-delivery system was uniformly assembled in the masked sustained-release layer on the surface of the calcium phosphate skeleton. The co-delivery system can be gradually released from the scaffold while maintaining the biological activity of the therapeutic drug, thereby blocking related cell interactions, significantly inhibiting the M2 polarization of BMMs, and promoting its phagocytosis of tumor cells. The two drugs in the co-delivery system of the present invention exert a synergistic effect, significantly improving the bone repair effect and anti-tumor effect of the scaffold compared to the loading of a single drug. In the in vivo tumor model, the therapeutic scaffold assembled with the co-delivery system effectively reduced the number of local M2 macrophages and inhibited tumor growth. After 8 weeks of subcutaneous implantation in normal mice, the therapeutic scaffold showed good biocompatibility and promoted uniform new bone formation.

Claims

1. A method for preparing an immunomodulatory scaffold with both anti-tumor and bone repair properties, characterized in that: The following steps are involved: (1) dispersing the amino-modified mesoporous silica in anhydrous methanol and reacting with 4-(bromomethyl)phenylboronic acid to prepare phenylboronic acid-modified amino-modified mesoporous silica nanoparticles; (2) dispersing the nanoparticles obtained in step (1) in dimethyl sulfoxide containing a CSF-1R inhibitor, stirring the reaction to obtain inhibitor-loaded nanoparticles, which were then dispersed in PBS buffer, incubated with SIPRɑ antibodies, and centrifuged to obtain dual-drug nanoparticles loaded with the inhibitor and the antibody; (3) An aqueous solution containing methacryloylated hyaluronic acid, methacryloylated oxidized hyaluronic acid, and a photoinitiator is used as a curing liquid, and is uniformly mixed with calcium phosphate powder in a certain liquid-solid ratio to prepare a bio-ink, and a calcium phosphate scaffold is prepared by 3D printing. The dual-drug nanoparticles obtained in step (2) are dispersed in a hydroxybutyl chitosan solution, and the calcium phosphate scaffold is immersed in the above solution for in situ cross-linking to prepare an immunomodulatory scaffold with anti-tumor and bone repair properties.

2. The preparation method according to claim 1, characterized in that The amino-modified mesoporous silica in step (1) is prepared by using ethyl orthosilicate as a silicon source, hexadecyltrimethylammonium bromide as a template, and ammonium fluoride as an ammonia source.

3. The preparation method according to claim 1, characterized in that The amino-modified mesoporous silica in step (1) reacts with 4-(bromomethyl)phenylboronic acid in a mass ratio of 1:0.4-4.

4. The preparation method according to claim 1, characterized in that The CSF-1R inhibitor in step (2) is GW2580.

5. The preparation method according to claim 1, characterized in that The weight ratio of the nanoparticles to the CSF-1R inhibitor in step (2) is 10:

1.

6. The preparation method according to claim 1, characterized in that The stirring reaction in step (2) is carried out at room temperature for 12 hours, and the co-incubation is carried out at 37° C. for 0.5 hours.

7. The preparation method according to claim 1, characterized in that In step (3), the weight ratios of the methacrylated hyaluronic acid, the methacrylic acid oxidized hyaluronic acid and the photoinitiator in the curing solution are 4-10%, 1-10% and 0.5-2% respectively.

8. The preparation method according to claim 1, characterized in that The concentration of the dual-drug nanoparticles in step (3) is 2-40 mg / mL, and the concentration of the hydroxybutyl chitosan solution is 1-2 wt%.

9. An immunomodulatory scaffold with both anti-tumor and bone repair properties prepared by the method according to any one of claims 1 to 8.

10. Use of the immunomodulatory scaffold with both anti-tumor and bone repair properties according to claim 9 in the preparation of an implant for treating bone defects after bone tumor surgery.

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