Injectable Composite Hydrogel for Integrated Diagnosis and Treatment of Osteosarcoma and Its Preparation Method

Abstract

The present invention discloses an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma and a preparation method thereof. The hydrogel is an ionic and covalent double-crosslinked sodium alginate / polyacrylamide / carboxymethyl chitosan / liposome nanoparticles / magnesium particles double network hydrogel, which not only has excellent mechanical and injectable properties, but also can be implanted into the bone defect site by injection. The hydrogel will first release nanomedicines at body temperature. The released ferroferric oxide nanoparticles can be used as a contrast agent to locate the lesion, and then produce hydroxyl radicals through the Fenton reaction, thereby eliminating tumor cells; then, through in vitro ultrasonic heating, the magnesium particles react with water to produce hydrogen and magnesium ions, thereby achieving anti-inflammatory and anti-tumor effects, and finally regulating the differentiation of mesenchymal stem cells to promote bone repair. Therefore, the present invention expands the application of hydrogels in the field of tumor treatment while realizing the integration of staged bone tumor diagnosis, treatment, and repair.

Description

Technical Field

[0001] The present invention belongs to the field of material technology, and specifically relates to an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma and a preparation method thereof. Background Art

[0002] Osteosarcoma is the most common primary bone malignancy, clinically arising in the distal femur and proximal tibia. Current treatment strategies for osteosarcoma primarily rely on surgical resection of the primary tumor combined with adjuvant chemoradiotherapy. However, the risk of metastasis and the severe side effects of chemotherapeutic drugs hinder its continued application. Notably, surgical resection of osteosarcoma, in addition to the inevitable risk of tumor recurrence and metastasis, often results in large bone defects and articular cartilage loss, making postoperative healing difficult. During surgery, injectable fillers can better adapt to irregular defects, simplify procedures, save time, and improve surgical success rates. Furthermore, monitoring for osteosarcoma recurrence is crucial. In this context, diagnosing tumor recurrence while simultaneously reconstructing the bone defect and eliminating residual tumor cells is crucial for effective osteosarcoma treatment. Therefore, developing biomedical materials that integrate injectable, diagnostic, therapeutic, and repair functions to address the underlying pathology is a promising strategy for clinical bone tumor treatment.

[0003] The tumor microenvironment is a unique microenvironment found in tumor tissue, characterized by mild acidity, hypoxia, and overexpression of hydrogen peroxide. The Fenton reaction, which converts hydrogen peroxide into hydroxyl radicals under acidic conditions through the catalysis of ferrous ions, has been extensively explored in cancer treatment in recent years. Compared to traditional treatments, the Fenton reaction's mechanism of tumor treatment is closely integrated with the tumor microenvironment, offering advantages unmatched by traditional approaches, such as high selectivity and endogenous activation. The 3D-printed MnPSe-3 nanosheet composite scaffold described in Patent CN 114939187A utilizes the Fenton effect to generate hydroxyl radicals for tumor treatment. Another example is the microspheres co-loaded with doxorubicin (DOX) and ferric oxide (Fe₃O₄) nanoparticles, described in Patent CN 111450267A, which utilize DOX and the Fenton effect to synergistically treat tumors. Furthermore, Fe₃O₄ also exhibits magnetic resonance imaging capabilities, enabling rapid lesion localization. However, bone reconstruction after eliminating residual tumors is equally important. Not only is bone tissue difficult to reconstruct, but the Fenton effect can also cause secondary damage to normal tissues.

[0004] Using magnesium as a reactant to produce hydrogen for tumor treatment is an emerging therapeutic approach that holds promise for resolving these issues. Magnesium reacts with water at elevated temperatures (45°C) to produce hydrogen and magnesium ions. Hydrogen can eliminate inflammation within tissues and inhibit tumors, while magnesium ions stimulate cellular pathways, promoting stem cell bone differentiation and ultimately achieving bone regeneration. Combining these mechanisms could potentially enable the diagnosis, treatment, and repair of bone tumors, a significant advancement in bone tumor treatment and repair. Summary of the Invention

[0005] Based on this, the present invention provides an injectable composite hydrogel for integrated osteosarcoma diagnosis and treatment, and its preparation method. This composite hydrogel exhibits excellent mechanical, anti-inflammatory, imaging, and osteogenic properties, enabling early tumor localization and treatment, as well as later bone repair, expanding the application of hydrogels in tumor treatment.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] An injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma, specifically an ionically covalently double-crosslinked sodium alginate / polyacrylamide / carboxymethyl chitosan / liposome / magnesium particle double-network hydrogel. The liposomes contained therein can produce a Fenton effect, exerting an anti-tumor effect; while the magnesium particles can produce hydrogen through chemical reactions, thereby exerting anti-inflammatory and bone repair effects.

[0008] The preparation method of the injectable composite hydrogel comprises the following steps:

[0009] (1) Dipalmitoylphosphatidylcholine (DPPC), ferrosoferric oxide (Fe3O4), diphenylphosphoryl azide (DPPA), 1,2-bis(diphenylphosphino)ethane (DPPE), and DSPE-PEG-2000 were mixed in proportion, dissolved in chloroform (CHCl3), and ultrasonically treated for 1-30 min to obtain solution S1;

[0010] (2) Solution S1 was subjected to rotary evaporation to remove the organic solution, and deionized water was added to disperse the solution S2;

[0011] (3) Add doxorubicin hydrochloride (DOX), propylene glycol block polyether (F-68) and perfluorohexane (PFH) to solution S2 and mix well to obtain solution S3;

[0012] (4) Ultrasonicate solution S3 using a cell disruptor for 5-30 min to obtain solution S4;

[0013] (5) Sodium alginate (Alg), acrylamide (AAm), carboxymethyl chitosan (CMC) and hydroxyapatite (HA) were added to distilled water and stirred to obtain solution S5;

[0014] (6) Add the following to solution S5: N,N' -methylenebisacrylamide (MBAA) and tetramethylethylenediamine (TEMED), stirred evenly to obtain solution S6;

[0015] (7) Add ammonium persulfate (APS), solution S4, and magnesium powder (Mg) to solution S6 in sequence while stirring, and stir evenly to obtain a prepolymer solution S7;

[0016] (8) The prepolymerized solution S7 is polymerized at room temperature to obtain the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0017] Furthermore, the mass ratio of DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 used in step (1) is (5-15):(3-8):(2-10):(2-10):(2-10), and the amount of CHCl3 added is 0.1-0.2 mL / mg.

[0018] Furthermore, the amount of deionized water used in step (2) is 0.01-0.1 mL per mg of evaporated product.

[0019] Furthermore, in the solution S3 of step (3), the concentration of DOX is 0.002-0.080 g / mL, the concentration of F-68 is 0.002-0.070 mg / mL, and the concentration of PFH is 0.002-0.070 mg / mL.

[0020] Furthermore, in the solution S5 of step (5), the concentration of Alg is 0.015-0.050 g / mL, the concentration of AAm is 0.030-0.500 g / mL, the concentration of CMC is 0.025-0.090 g / mL, and the concentration of HA is 0.015-0.200 g / mL; and the stirring time is 3-24 h.

[0021] Furthermore, in the solution S6 of step (6), MBAA The concentration of dapoxetine is 0.05-0.20 mg / mL, and the concentration of TEMED is 0.10-0.60 mg / mL.

[0022] Furthermore, in step (7), the amount of ammonium persulfate added to solution S6 is 1.50-2.00 mg / mL, the amount of solution S4 added is 10-100 μL / mL; the amount of magnesium powder added is 1-10 mg / mL; and the stirring time is 1-12 h.

[0023] Furthermore, the polymerization time in step (8) is 1-10 min.

[0024] The injectable composite hydrogel prepared above has excellent mechanical, anti-inflammatory, imaging and osteogenic properties, and also exhibits excellent anti-tumor properties. Therefore, it can be used for the integrated diagnosis and treatment of osteosarcoma. Specifically:

[0025] (1) The ferroferric oxide nanoparticles contained in the composite hydrogel synthesized by the present invention can serve as a contrast agent to enhance the nuclear magnetic resonance imaging signal, thereby being used for lesion localization;

[0026] (2) The present invention utilizes hydrogel as a carrier to load liposomes containing DOX and ferroferric oxide, so that the synthesized composite hydrogel has an excellent tumor elimination effect at body temperature and can be used as a drug to eliminate tumor cells;

[0027] (3) The composite hydrogel synthesized by the present invention has excellent mechanical properties and can be used as a filler to fill bone defects and provide mechanical support;

[0028] (4) The composite hydrogel synthesized in the present invention has good anti-inflammatory and osteogenesis effects at 45°C, and can promote bone regeneration in the later stage of repair.

[0029] The perfluorohexane in the liposomes of the composite hydrogel system of the present invention undergoes a phase transition under the influence of ultrasound, enabling the precise and controllable release of the encapsulated drug. The introduced doxorubicin and ferroferric oxide undergo a Fenton effect at body temperature, generating hydroxyl radicals for efficient tumor clearance. Furthermore, at higher temperatures (45°C), the magnesium particles in the system react with water to produce hydrogen and magnesium ions, thereby achieving anti-inflammatory and bone repair capabilities. Furthermore, it can also inhibit tumor migration and prevent tumor recurrence. Therefore, the composite hydrogel of the present invention not only eliminates tumor cells but also achieves anti-inflammatory and efficient bone repair effects, achieving an integrated approach to diagnosis, treatment, and repair of bone tumors, and has broad application prospects in the field of bone tumor treatment.

[0030] Compared with the prior art, the present invention has the following excellent effects:

[0031] (1) The composite hydrogel synthesized in the present invention overcomes existing clinical defects and organically combines the three functions of diagnosis, treatment, and repair into one system, thereby reducing the risk of secondary surgery. It also has the property of being injectable, which improves the convenience of operation and the efficiency of treatment.

[0032] (2) The composite hydrogel synthesized by the present invention can regulate the concentration of hydroxyl radicals in the system by regulating temperature, thereby playing a role in treating inflammation in tissues. It is an artificially controllable and intelligent integrated material that can adapt to different repair environments;

[0033] (3) The composite hydrogel synthesized by the present invention has excellent mechanical properties and fatigue resistance and can adapt to different defect conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The preparation flow chart (A) and the action mechanism diagram (B) of the composite hydrogel of the present invention are shown.

[0035] Figure 2 The projection electron micrograph (A), projection electron micrograph (B), infrared spectrum (C), optical microscope (D), zeta potential (E), particle size distribution (F, G), hysteresis curve (H), relaxation curve (I), and XPS (J) of the liposomes prepared in Example 1.

[0036] Figure 3 Scanning electron microscope image (A), average pore size and pore analysis image (B), elemental analysis image (C), element scanning image (D), infrared spectrum image (E), contact angle analysis image (F) and swelling rate analysis image (G) of the composite hydrogel prepared in Example 1.

[0037] Figure 4 Figure 1 shows the cross-linking mechanism and adhesion mechanism of the composite hydrogel prepared in Example 1 (A), adhesion diagram (B), adhesion quantitative test diagram (C), compression quantitative test diagram (D), tensile quantitative test diagram (E), compression displacement-stress curve diagram (F), tensile displacement-stress curve diagram (G), reciprocating displacement-stress curve diagram after 1000 compression cycles (H), reciprocating displacement-stress curve diagram after 1000 stretching cycles (I), compression and stretching schematic diagram (J), quantitative diagrams of dissipated energy during 1000 reciprocating cycles (K, L), and amplitude test diagram (M).

[0038] Figure 5 The graphs of gelation time and temperature changes at different APS concentrations (A, B), gelation time at different temperatures (C), injectability proof graph (D), gelation rheological properties analysis graph at different temperatures (EG), self-healing performance analysis graph (IJ), shape adaptability graph (K) and self-healing mechanism analysis graph (L) of the composite hydrogel prepared in Example 1.

[0039] Figure 6Diagram showing the mechanism of the composite hydrogel prepared in Example 1 affecting the generation of hydroxyl radicals (A, D), the effects of liposomes, magnesium particles, and both on the level of hydroxyl radicals at different temperatures (BH), biocompatibility characterization (K), live-death staining, and macrophage differentiation (L). DETAILED DESCRIPTION

[0040] The following is combined with Figure 1 , the technical solution of the present invention is described in detail.

[0041] The present invention discloses an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma, the preparation method of which comprises the following steps:

[0042] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of (5-15):(3-8):(2-10):(2-10):(2-10), dissolved in CHCl3 at a rate of 0.1-0.2 mL / mg, and ultrasonically treated for 1-30 min to obtain solution S1;

[0043] (2) Solution S1 was subjected to rotary evaporation to remove the organic solvent, and the resulting residue was dispersed in deionized water at a rate of 0.01-0.1 mL / mg to obtain solution S2;

[0044] (3) Add DOX, F-68, and PFH to solution S2 and mix to obtain solution S3; wherein the concentration of DOX is 0.002-0.080 g / mL, the concentration of F-68 is 0.002-0.070 mg / mL, and the concentration of PFH is 0.002-0.070 mg / mL;

[0045] (4) Ultrasonicate solution S3 using a cell disruptor for 5-30 min to obtain solution S4;

[0046] (5) Add Alg, AAm, CMC, and HA to distilled water and stir for 3-24 h to make them uniform, to obtain solution S5; wherein the concentration of Alg is 0.015-0.050 g / mL, the concentration of AAm is 0.030-0.500 g / mL, the concentration of CMC is 0.025-0.090 g / mL, and the concentration of HA is 0.015-0.200 g / mL;

[0047] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6; wherein, MBAA The concentration of is 0.05-0.20 mg / mL, and the concentration of TEMED is 0.10-0.60 mg / mL;

[0048] (7) APS, solution S4, and magnesium powder were added to solution S6 in the following order: 1.50-2.00 mg / mL, 10-100 μL / mL, and 1-10 mg / mL, respectively, while stirring. The mixture was stirred for 1-12 h to obtain a prepolymer solution S7.

[0049] (8) The prepolymerized solution S7 was polymerized at room temperature for 1-10 min to obtain an injectable composite hydrogel Alg / PAAm / CMC / Lip / Mg.

[0050] By optimizing the ratios of Alg, AAm, HA, liposome nanoparticles, and magnesium powder, the present invention achieves a composite hydrogel with not only strong mechanical properties but also the ability to perform magnetic resonance imaging and control the system's repair state at different temperatures. This allows for anti-inflammatory and bone repair-promoting effects, thereby enabling a phased recovery of tissues, first diagnosing cancer cells, then treating cancer cells, and finally bone repair. Overall, the composite hydrogel synthesized in this invention possesses the mechanical, anti-inflammatory, and osteogenic properties of liposomes encapsulating Fe3O4 nanoparticles and DOX, which can treat tumors at body temperature (37°C). Furthermore, at higher temperatures (45°C), this allows for lesion detection and tumor elimination in the early stages of the treatment process, and anti-inflammatory and bone regeneration in the later stages.

[0051] To further understand the present invention, an injectable integrated diagnostic and therapeutic composite hydrogel for treating osteosarcoma and a preparation method thereof provided by the present invention are described below in conjunction with examples. The scope of protection of the present invention is not limited by the following examples.

[0052] Example 1

[0053] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 10:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0054] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0055] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.003 g / mL DOX, 0.003 mg / mL F-68, and 0.004 mg / mL PFH;

[0056] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0057] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0058] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.12 mg / mL MBAA and 0.40 mg / mL TEMED;

[0059] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0060] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0061] Figure 2 This is a characterization spectrum of the liposomes prepared in Example 1. From the characterization contents, it can be concluded that liposomes with uniform particle size, good dispersibility, nuclear magnetic resonance imaging and paramagnetism were successfully prepared.

[0062] Figure 3 This is a basic characterization graph of the composite hydrogel prepared in Example 1. From the various characterizations, it can be concluded that a double-network composite hydrogel was successfully prepared, and the composite hydrogel has high porosity and hydrophilicity, which is conducive to cell adhesion and proliferation.

[0063] Figure 4 This is a characterization diagram of the mechanical properties of the composite hydrogel prepared in Example 1. From the various characterizations, it can be concluded that the prepared composite hydrogel has good mechanical properties and fatigue resistance, and can be used as a bone defect filler.

[0064] Figure 5 This is a functional performance characterization diagram of the composite hydrogel prepared in Example 1. From the various characterizations, it can be concluded that the composite hydrogel prepared has the properties of injectability, self-healing, and shape adaptability, and can be used as an injectable bone defect filler.

[0065] Figure 6 This is a chemical property characterization diagram of the composite hydrogel prepared in Example 1. From these characterizations, it can be concluded that the composite hydrogel prepared has the function of increasing hydroxyl radical levels at body temperature and reducing hydroxyl radical levels at 45°C. It also has good biocompatibility and can be used as an injectable filler material for tumor treatment, anti-inflammatory, and repair promotion.

[0066] Example 2

[0067] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 8:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0068] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0069] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.003 g / mL DOX, 0.003 mg / mL F-68, and 0.004 mg / mL PFH;

[0070] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0071] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0072] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.12 mg / mL MBAA and 0.40 mg / mL TEMED;

[0073] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0074] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0075] Example 3

[0076] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 8:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0077] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0078] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.005 g / mL DOX, 0.003 mg / mL F-68, and 0.004 mg / mL PFH;

[0079] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0080] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0081] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.12 mg / mL MBAA and 0.40 mg / mL TEMED;

[0082] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0083] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0084] Example 4

[0085] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 8:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0086] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0087] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.005 g / mL DOX, 0.004 mg / mL F-68, and 0.004 mg / mL PFH;

[0088] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0089] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0090] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.12 mg / mL MBAA and 0.40 mg / mL TEMED;

[0091] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0092] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0093] Example 5

[0094] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 8:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0095] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0096] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.005 g / mL DOX, 0.004 mg / mL F-68, and 0.004 mg / mL PFH;

[0097] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0098] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0099] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.15 mg / mL MBAA and 0.40 mg / mL TEMED;

[0100] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0101] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0102] Example 6

[0103] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 8:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0104] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0105] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.005 g / mL DOX, 0.004 mg / mL F-68, and 0.004 mg / mL PFH;

[0106] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0107] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0108] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.15 mg / mL MBAA and 0.50 mg / mL TEMED;

[0109] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0110] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0111] Example 7

[0112] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 8:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0113] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0114] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.005 g / mL DOX, 0.004 mg / mL F-68, and 0.004 mg / mL PFH;

[0115] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0116] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0117] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.15 mg / mL MBAA and 0.50 mg / mL TEMED;

[0118] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0119] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0120] Comparative Example 1

[0121] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 10:10:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0122] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0123] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.003 g / mL DOX, 0.003 mg / mL F-68, and 0.004 mg / mL PFH;

[0124] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0125] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0126] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.12 mg / mL MBAA and 0.40 mg / mL TEMED;

[0127] (7) 1.60 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0128] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0129] Comparative Example 2

[0130] (1) DPPC, Fe3O4, DPPA, DPPE, and DSPE-PEG-2000 were mixed in a mass ratio of 8:4:3:3:3, dissolved in CHCl3 at a volume of 0.1 mL / mg, and ultrasonically treated for 20 min to obtain solution S1;

[0131] (2) Solution S1 was evaporated to remove the organic solvent, and the resulting product was dispersed in deionized water at a rate of 0.02 mL / mg to obtain solution S2;

[0132] (3) DOX, F-68, and PFH were added to solution S2 and mixed to obtain solution S3 containing 0.003 g / mL DOX, 0.003 mg / mL F-68, and 0.004 mg / mL PFH;

[0133] (4) Solution S3 was sonicated for 20 min using a cell disruptor to obtain solution S4;

[0134] (5) Alg, AAm, CMC, and HA were added to distilled water and stirred for 12 h to make them uniform, obtaining solution S5 containing 0.0317 g / mL Alg, 0.333 g / mL AAm, 0.00583 g / mL CMC, and 0.0867 g / mL HA;

[0135] (6) MBAA and TEMED were added to solution S5 in sequence and stirred evenly to obtain solution S6 containing 0.12 mg / mL MBAA and 0.40 mg / mL TEMED;

[0136] (7) 3.0 mg / mL APS, 50 μL / mL solution S4, and 2.5 mg / mL magnesium powder were added to solution S6 in sequence while stirring, and stirred for 2 h to make it uniform, to obtain prepolymer solution S7;

[0137] (8) The prepolymerized solution S7 was polymerized at room temperature for 3 min to obtain an injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma.

[0138] The hydroxyl radical generation performance of the composite hydrogels prepared in Example 2 and Comparative Example 1 was tested. The results showed that, when the concentrations of other components remained unchanged, the group with a higher concentration of ferrosoferric oxide had a stronger hydroxyl radical generation ability and better tumor clearance effect. However, excessive hydroxyl radicals can lead to excessive tissue inflammation, and the subsequent hydrogen generation cannot offset the generated hydroxyl radicals, which is not able to effectively eliminate inflammation and promote bone repair.

[0139] The gelation properties of the composite hydrogels prepared in Example 2 and Comparative Example 2 were tested. The results showed that, while the concentrations of other components remained constant, increasing the APS concentration accelerated crosslinking and enhanced mechanical properties. However, this resulted in excessively high gelation temperatures and lower porosity, hindering tissue growth.

[0140] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma, characterized in that: The composite hydrogel is specifically an ion-covalent double-crosslinked sodium alginate / polyacrylamide / carboxymethyl chitosan / liposome / magnesium particle double network hydrogel. The liposome contained in the hydrogel can produce a Fenton effect and exert an anti-tumor effect; while the magnesium particles can produce hydrogen through a chemical reaction, thereby exerting anti-inflammatory and bone repair effects. The preparation of the composite hydrogel comprises the following steps: (1) Dipalmitoylphosphatidylcholine, ferrosoferric oxide, diphenylphosphoazide, 1,2-bis(diphenylphosphino)ethane, and phosphatidylethanolamine were mixed in proportion, dissolved in chloroform, and ultrasonically treated for 1-30 min to obtain solution S1; (2) Solution S1 was subjected to rotary evaporation to remove the organic solution, and deionized water was added to disperse the solution S2; (3) Add doxorubicin hydrochloride, propylene glycol block polyether and perfluorohexane to solution S2 and mix well to obtain solution S3; (4) Ultrasonicate solution S3 using a cell disruptor for 5-30 min to obtain solution S4; (5) Sodium alginate, acrylamide, carboxymethyl chitosan, and hydroxyapatite were added to distilled water and stirred to obtain solution S5; (6) Add the following to solution S5: N,N' -methylenebisacrylamide and tetramethylethylenediamine, stirring evenly to obtain solution S6; (7) Add ammonium persulfate, solution S4, and magnesium powder to solution S6 in sequence while stirring, and stir evenly to obtain a prepolymer solution S7; (8) polymerizing the prepolymerized solution S7 at room temperature to obtain the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma; The mass ratio of dipalmitoylphosphatidylcholine, ferrosoferric oxide, diphenylphosphoazide, 1,2-bis(diphenylphosphino)ethane, and phosphatidylethanolamine used in step (1) is (5-15):(3-8):(2-10):(2-10):(2-10); In the solution S3 of step (3), the concentration of doxorubicin hydrochloride is 0.002-0.080 g / mL, the concentration of propylene glycol block polyether is 0.002-0.070 mg / mL, and the concentration of perfluorohexane is 0.002-0.070 mg / mL; In the solution S5 of step (5), the concentration of sodium alginate is 0.015-0.050 g / mL, the concentration of acrylamide is 0.030-0.500 g / mL, the concentration of carboxymethyl chitosan is 0.025-0.090 g / mL, and the concentration of hydroxyapatite is 0.015-0.200 g / mL; In the solution S6 of step (6), N,N' -The concentration of methylenebisacrylamide is 0.05-0.20 mg / mL, and the concentration of tetramethylethylenediamine is 0.10-0.60 mg / mL.

2. A method for preparing the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma according to claim 1, characterized in that: The following steps are involved: (1) Dipalmitoylphosphatidylcholine, ferrosoferric oxide, diphenylphosphoazide, 1,2-bis(diphenylphosphino)ethane, and phosphatidylethanolamine were mixed in proportion, dissolved in chloroform, and ultrasonically treated for 1-30 min to obtain solution S1; (2) The organic solution of solution S1 was removed by rotary evaporation, and deionized water was added to disperse the solution S2; (3) Add doxorubicin hydrochloride, propylene glycol block polyether and perfluorohexane to solution S2 and mix well to obtain solution S3; (4) Ultrasonicate solution S3 using a cell disruptor for 5-30 min to obtain solution S4; (5) Sodium alginate, acrylamide, carboxymethyl chitosan, and hydroxyapatite were added to distilled water and stirred to obtain solution S5; (6) Add the following to solution S5: N,N' -methylenebisacrylamide and tetramethylethylenediamine, stirring evenly to obtain solution S6; (7) Add ammonium persulfate, solution S4, and magnesium powder to solution S6 in sequence while stirring, and stir evenly to obtain a prepolymer solution S7; (8) polymerizing the prepolymerized solution S7 at room temperature to obtain the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma; The mass ratio of dipalmitoylphosphatidylcholine, ferrosoferric oxide, diphenylphosphoazide, 1,2-bis(diphenylphosphino)ethane, and phosphatidylethanolamine used in step (1) is (5-15):(3-8):(2-10):(2-10):(2-10), and the amount of chloroform added is 0.1-0.2 mL / mg; In the solution S3 of step (3), the concentration of doxorubicin hydrochloride is 0.002-0.080 g / mL, the concentration of propylene glycol block polyether is 0.002-0.070 mg / mL, and the concentration of perfluorohexane is 0.002-0.070 mg / mL; In the solution S5 of step (5), the concentration of sodium alginate is 0.015-0.050 g / mL, the concentration of acrylamide is 0.030-0.500 g / mL, the concentration of carboxymethyl chitosan is 0.025-0.090 g / mL, and the concentration of hydroxyapatite is 0.015-0.200 g / mL; the stirring time is 3-24 h; In the solution S6 of step (6), N,N' -The concentration of methylenebisacrylamide is 0.05-0.20 mg / mL, and the concentration of tetramethylethylenediamine is 0.10-0.60 mg / mL.

3. The method for preparing the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma according to claim 2, characterized in that: The amount of deionized water used in step (2) is 0.01-0.1 mL per mg of evaporated product.

4. The method for preparing the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma according to claim 2, characterized in that: In step (7), the amount of ammonium persulfate added to solution S6 is 1.50-2.00 mg / mL, the amount of solution S4 added is 10-100 μL / mL; the amount of magnesium powder added is 1-10 mg / mL; and the stirring time is 1-12 h.

5. The method for preparing the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma according to claim 2, characterized in that The polymerization time in step (8) is 1-10 min.

6. Use of the injectable composite hydrogel for integrated diagnosis and treatment of osteosarcoma according to claim 1 in the preparation of an osteosarcoma diagnostic and therapeutic agent, characterized in that: The composite hydrogel can serve as a contrast agent and simultaneously exert the effects of anti-tumor and anti-inflammatory at the implantation site and promoting bone repair.

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