Gel for preventing recurrence and promoting bone repair after osteosarcoma surgery and preparation method and application thereof

Abstract

The application discloses a gel for preventing recurrence and promoting bone repair after osteosarcoma surgery and a preparation method thereof, and comprises the following steps: preparing a mixed solution A of doxorubicin and zinc nitrate; preparing a mixed solution B of 2-methylimidazole and siRNA; adding the mixed solution B into the mixed solution A under stirring, and reacting to obtain a red precipitate; washing and centrifuging the red precipitate to remove the un-encapsulated reactants; vacuum drying the solid precipitate obtained through centrifugal separation to obtain RDZ; modifying catechol on chitosan to obtain catechol chitosan; and adding the RDZ suspension into the catechol chitosan solution, mixing, homogenizing to obtain the catechol chitosan gel loaded with RDZ.

Description

Technical Field

[0001] This invention belongs to the field of gel technology for preventing recurrence and promoting bone repair after osteosarcoma surgery, specifically relating to a gel for preventing recurrence and promoting bone repair after osteosarcoma surgery, its preparation method and application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Osteosarcoma (OS) is a common bone cancer in children and young adults, typically a highly malignant tumor. The most common site of occurrence is the distal femur, followed by the proximal tibia and proximal humerus. Amputation was once the primary treatment for osteosarcoma. However, with advancements in diagnostic and therapeutic techniques, limb-sparing surgery combined with chemotherapy has become the main approach. Nevertheless, poor tumor cell responsiveness often hinders treatment efficacy, and postoperative local tumor recurrence and metastasis remain challenging. Furthermore, the removal of tumor segments leads to extensive bone defects, posing significant repair problems. Due to these clinical difficulties, a qualified osteosarcoma treatment requires a prominent anti-tumor approach and advanced osteogenic techniques.

[0004] Methotrexate (MTX), doxorubicin (ADM), cisplatin (DDP), and ifosfamide (IFO) have become the four cornerstone drugs in chemotherapy for osteosarcoma. However, for patients with high-grade malignancy who are not sensitive to conventional chemotherapy, chemotherapy drugs are of little effect, and considering the bone repair issues after osteosarcoma surgery, local administration of high doses of chemotherapy drugs is clearly not advisable. Compared with systemic administration, local administration can effectively increase the local concentration of chemotherapy drugs and reduce side effects on other organs and tissues.

[0005] Immunotherapy has garnered significant attention due to its efficacy in treating various cancers. Numerous preclinical trials support its application in overall survival (OS). Notably, PD-L1 expression is positively correlated with drug resistance, TIL count, and osteosarcoma cell proliferation. The PD-1 / PD-L1 interaction has demonstrated clinical benefits for various malignancies. Combining it with immune checkpoint inhibitors can not only enhance anti-tumor efficacy but also reduce the impact of high-dose chemotherapy on bone repair.

[0006] Hydrogels, as excellent carriers for sustained drug release, hold great promise for postoperative drug delivery. Clinically, 3D-printed polycaprolactone (PCL) scaffolds with relatively high mechanical strength are widely used in tissue engineering. However, their limited biological function and strong hydrophobicity restrict the application of PCL scaffolds. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a gel for preventing recurrence and promoting bone repair after osteosarcoma surgery, and a method for preparing the gel therein.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing a gel for preventing recurrence and promoting bone repair after osteosarcoma surgery, comprising the following steps:

[0010] Prepare a mixed solution A of doxorubicin and zinc nitrate;

[0011] Prepare a mixed solution B of 2-methylimidazole and RNA;

[0012] Mixed solution B was added to mixed solution A with stirring, and a red precipitate was obtained after the reaction.

[0013] Wash and centrifuge the red precipitate to remove unloaded reactants;

[0014] The solid precipitate obtained by centrifugation was dried under vacuum to obtain RDZ;

[0015] Catechol was used to modify chitosan to obtain catechin chitosan;

[0016] Add the RDZ suspension to the catechin chitosan solution, mix well, homogenize, and obtain the RDZ-loaded catechin chitosan gel.

[0017] To address the clinical challenge of high recurrence rates and large-segment bone defects after osteosarcoma surgery, this invention provides a viscous hydrogel that can be coated onto a scaffold for local delivery of chemotherapeutic drugs and siPD-L1 (siRNA that silences PD-L1). Because RNA therapy is inherently unstable and cannot cross cell membranes due to its negative charge, various chemical modifications or the use of suitable carriers are required to improve its pharmacokinetics and pharmacodynamics. The metal-organic framework ZIF-8 is a biocompatible nanomaterial that can respond to acidic environments, overcoming enzymatic and acidic digestion through endosome escape, successfully maintaining the physical and chemical integrity of the embedded RNA, and offering significant advantages in improving RNA delivery efficiency. Furthermore, studies have shown that ZIF-8 NPs released from the hydrogel can upregulate the production and secretion of alkaline phosphatase, collagen 1, and osteocalcin, promoting osteogenic differentiation of rBMSCs. Therefore, ZIF-loaded chemotherapy drugs doxorubicin and siPD-L1 were used to synthesize nanoparticles RDZ. RDZ was then mixed into a gel to exert a dual effect of preventing recurrence and promoting bone repair after osteosarcoma surgery.

[0018] Catechol-modified chitosan gel has the advantages of strong adhesion and good biocompatibility, and has great potential as a scaffold coating. When coated on PCL scaffolds, it can effectively improve the hydrophilicity, bioactivity and osteogenic differentiation capacity of the scaffolds.

[0019] To address the issue of ZIF's stable and effective encapsulation of siRNA and DOX, the proportions of DOX, siRNA, 2-methylimidazole, and zinc ions in the synthesized ZIF were adjusted to obtain a ZIF with stable crystal form, uniform particle size, and the ability to simultaneously encapsulate RNA and doxorubicin (DOX). This ZIF is more stable in solution and more sensitive to pH, allowing RDZ to better achieve lysosomal escape, enabling precise and efficient delivery of RNA, and allowing DOX and RNA to exert a better synergistic drug delivery effect.

[0020] To address the clinical challenges of high recurrence rates and large-segment bone defects after osteosarcoma surgery, this study leverages the advantages of ZIF nanoparticles, which can both load drugs and promote bone repair, by incorporating ZIF into a viscous hydrogel. To achieve sufficient drug loading and increase the ZIF concentration in the gel solution, the concentration and mixing ratio of the ZIF solution and CA-CS mixture were adjusted. A homogenizer was used to thoroughly mix the ZIF and CA-CS solutions, ensuring better dispersion of the ZIF solution within the gel and resulting in a uniformly dispersed ZIF gel without compromising gel adhesion.

[0021] In some embodiments, the RNA is siRNA, circRNA, or shRNA with a nucleotide count of less than 100 kDa.

[0022] In some embodiments, in mixed solution A, the concentration of doxorubicin is 3-7 mg / mL; and the concentration of zinc nitrate is 50-100 mg / mL.

[0023] Preferably, in mixed solution A, the concentration of doxorubicin is 5.3-5.5 mg / mL; and the concentration of zinc nitrate is 60-70 mg / mL.

[0024] In some embodiments, the concentration of 2-methylimidazole in mixed solution B is 200-250 mg / mL; the concentration of siRNA is 30-50 μM.

[0025] Preferably, in mixed solution B, the concentration of 2-methylimidazole is 220-230 mg / mL; and the concentration of siRNA is 30-40 μM.

[0026] In some embodiments, the volume ratio of mixed solution A to mixed solution B is 0.5-1:3-4.

[0027] Preferably, the reaction time of mixed solution A and mixed solution B is 20-40 min, and the reaction temperature is 20-40℃.

[0028] More preferably, the reaction time of mixed solution A and mixed solution B is 25-35 min, and the reaction temperature is 25-35℃.

[0029] In some embodiments, the method for preparing catechin-modified chitosan includes the following steps:

[0030] Add EDC solution and HCA solution to chitosan solution, adjust the pH of the solution to 4.8, protect from light, stir and react at 20-30℃ for 10-14 hours to obtain reaction solution;

[0031] The reaction solution was placed in a dialysis bag and dialyzed first with hydrochloric acid solution containing NaCl, then with pure water. After dialysis, the reaction solution was freeze-dried to obtain catechol-modified chitosan.

[0032] Preferably, the concentration of chitosan in the reaction solution is 1.5-2 g / L; the concentration of EDC is 0.8-1.0 g / L; and the concentration of HCA is 0.5-1.0 g / L.

[0033] Secondly, the present invention provides a gel for preventing recurrence and promoting bone repair after osteosarcoma surgery, which is prepared by the aforementioned preparation method.

[0034] Thirdly, the present invention provides the application of the gel for preventing recurrence and promoting bone repair after osteosarcoma surgery in the postoperative period of osteosarcoma.

[0035] Fourthly, the present invention provides a scaffold comprising a PCL scaffold substrate and the gel coated on the surface of the PCL scaffold substrate.

[0036] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0037] RZ particles loaded with sufficient RNA using the one-pot method readily aggregated, producing noticeable precipitation after 2 hours. However, by adding a certain amount of DOX (doxorubicin) to the one-pot method, the positively charged DOX could neutralize the strong negative charge of the RNA to some extent, resulting in more stable RDZ particles. Compared to RZ, RDZ particles are less prone to aggregation and precipitation, have more uniform particle size, and exhibit more stable PDI changes.

[0038] RDZ has a high absolute value of Zata potential, resulting in better dispersibility and stability in solution. Under an electron microscope, RDZ exhibits distinct crystal characteristics, with a rhomboid morphology, good dispersibility, and a particle size of 400 nm.

[0039] After adding DOX, the diffraction peaks of DZ matched those of ZIF, and the crystallinity and diffraction peak intensities did not change significantly compared to ZIF. However, after adding RNA, the diffraction peak intensities decreased, indicating that the addition of RNA affects the crystal structure of ZIF, which corresponds to the TEM results.

[0040] The maximum amount of RNA that ZIF can carry shows that when the mass ratio of ZIF to RNA is 30:1, the RNA can be completely encapsulated. When the mass ratio of DOX-loaded ZIF to RNA is 40:1, the RNA can be completely encapsulated. Furthermore, the RDZ formed by encapsulation can protect the RNA from the influence of RNase, thus playing a role in effectively encapsulating and delivering RNA.

[0041] The encapsulation efficiency and drug loading of RDZ for DOX were determined using fluorescence spectrophotometry. The method was validated for specificity, intra-day and inter-day precision, and recovery. The encapsulation efficiency of RDZ for DOX was found to be 86 ± 1.06%, and the drug loading was 6.5%. Using the same measurement method, the cumulative release behavior of DOX by RDZ and DZ was investigated at pH 5.0 and 7.0. Both DZ and RDZ showed faster release under acidic conditions at pH 5.0. RDZ exhibits pH responsiveness, facilitating lysosomal escape of RNA and enabling precise RNA delivery. Due to the interaction between DOX and Zn... 2+ A relatively strong coordination bond will form between them. Under acidic conditions, only 38% of the DOX in DZ is released after 9 hours. However, the addition of RNA weakens the interaction between DOX and Zn. 2+ The coordination bonds between them allow up to 80% of the DOX in RDZ to be released, which is more conducive to the drug entering the cell and exerting its effects quickly, and to better synergize with siRNA on tumor cells.

[0042] After mixing a certain amount of RDZ into the gel, a gel with good injectability can be prepared. Using SEM to observe RDZ@Gel at a microscopic level, the gel exhibits a typical honeycomb structure with pores ranging from 100 to 500 μm, meeting the requirements for cell adhesion and growth, which is conducive to stem cell adhesion and promotes bone repair. RDZ is evenly distributed on the surface of the CA-CS gel, indicating that a gel with uniform RDZ dispersion has been successfully prepared.

[0043] After the prepared hydrogel was damaged, the polymer chains in the composite gel could pass through the water medium in the hydrogel matrix and reform chemical bonds to bond together. After 12 hours, the two gels could be completely fused together, showing a good self-healing effect.

[0044] Rheological properties were characterized by a high-temperature and high-pressure rheometer. The storage modulus (G') of both RDZ@Gel and Gel was significantly greater than the loss modulus (G”), indicating that they have solid elastic properties. Moreover, after adding RDZ to Gel, the G' of RDZ@Gel gel was much higher than G”, indicating that the addition of RDZ significantly enhanced the mechanical strength of the gel.

[0045] The gel has good biocompatibility and good adhesion to PCL scaffolds, which can help improve the hydrophobicity of the scaffolds and enhance their biocompatibility.

[0046] RDZ loaded with chemotherapy drugs has a good effect on killing tumor cells.

[0047] ZIF (PRZ) loaded with siPD-L1 can effectively silence the PD-L1 protein and is a good vector for delivering siRNA.

[0048] Gel+RDZ has better bone repair effects and good in vivo anti-tumor effects. Attached Figure Description

[0049] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0050] Figure 1 The results of the stability test of RZ and RDZ are shown in the figure; (a) the red solution on the left is RDZ and the white solution on the right is RZ; (b) a comparison of the changes in PDI of RZ and RDZ determined by DLS method.

[0051] Figure 2 This is a comparison diagram of the particle size and potential of nanoparticles; (a) particle size of nanoparticles; (b) potential of nanoparticles;

[0052] Figure 3 These are the particle size distribution and TEM images of RDZ.

[0053] Figure 4 This is the XRD pattern of the nanoparticles;

[0054] Figure 5 These are agarose gel retardation experiments verifying the encapsulation of RNA by nanoparticles; (a) is an agarose gel displacement diagram with different weight ratios of RZ to RNA; (b) is an agarose gel displacement diagram with different weight ratios of RDZ to RNA.

[0055] Figure 6 This is a graph demonstrating the protective effect of RDZ on RNA;

[0056] Figure 7These are comparison charts of DOX release from DZ and RDZ at different pH levels; (a) Comparison chart of DOX release from DZ in PBS at different pH levels. (b) Comparison chart of DOX release from RDZ in PBS at different pH levels.

[0057] Figure 8 This is a SEM image of Gel+RDZ;

[0058] Figure 9 This is a characterization diagram of the self-healing properties of Gel+RDZ;

[0059] Figure 10 These are rheological property characterization diagrams of Gel and Gel+RDZ;

[0060] Figure 11 These are SEM images of the scaffold with adhered Gel+RDZ. (A) is before adhesion, and (B) is after adhesion.

[0061] Figure 12 This is a comparison diagram of the in vitro biocompatibility of the gel;

[0062] Figure 13 This is a comparison chart of the in vitro antitumor effects of nanoparticles;

[0063] Figure 14 This is a graph showing the protein silencing efficiency of RDZ;

[0064] Figure 15 These are representative microCT 3D reconstruction images (top row) and sectional views (bottom row) of femoral defects in rats from different treatment groups. A is at 4 weeks post-surgery; B is at 8 weeks post-surgery.

[0065] Figure 16 Yes (Quantitative analysis of bone tissue at the defect site: BV / TV bone volume fraction (A), Tb.sp trabecular separation (B);

[0066] Figure 17 This is a comparison chart of tumor recurrence volume measurements in different treatment groups. Detailed Implementation

[0067] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0068] The present invention will be further described below with reference to the embodiments.

[0069] Example 1

[0070] Preparation of RDZ

[0071] 1) RNA-DOX@ZIF-8 (RDZ) containing both RNA and DOX was prepared using a one-pot method. To avoid degradation of siRNA by RNases, all solutions were prepared using DEPC water.

[0072] 2) First, prepare a 10 mg / mL DOX drug solution and mix it with a 148 mg / mL zinc nitrate hexahydrate solution at a volume ratio of 15:13 to prepare solution A.

[0073] 3) Mix 328 mg / ml of 2-methylimidazole solution with 125 μM of siRNA (siPD-L1, sequence 5′-GAGGUAAUCUGGACAAACATT-3′) solution at a volume ratio of 2.3:1 to prepare solution B.

[0074] 4) Take 3.3 mL of solution B and add 0.7 mL of solution A at room temperature and stirring speed of 7000 rpm. The reaction solution changes from purple to red. After reacting for 30 min, a pink suspension is obtained. Centrifuge at 10000 rpm for 5 min to obtain a red precipitate.

[0075] 5) Wash the precipitate twice with deionized water at the same speed to remove unloaded reactants, and then vacuum dry for 6 hours to obtain RDZ.

[0076] Preparation of RZ and DZ

[0077] RZ is prepared in the same way as RDZ, except that the DOX solution in solution A is replaced with DEPC water, while the other steps remain the same. DZ can be prepared by replacing the RNA solution in solution B with DEPC water.

[0078] Synthesis of Catechol Chitosan (CA-CS)

[0079] 1) Weigh 0.1g of chitosan (MW=150000) into 50ml of hydrochloric acid solution (0.05M) and stir for 30min to dissolve it completely.

[0080] 2) Weigh 0.1g of 3,4-dihydroxyphenylpropionic acid (HCA) and 0.1g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and dissolve them in 2ml of ethanol solution (1:1, V / V).

[0081] 3) Add 0.8 mL of EDC solution and 0.6 mL of HCA solution to the chitosan solution. The solution turns pale yellow. Adjust the pH of the reaction solution to 4.8 with NaOH solution to obtain a yellow solution. React at room temperature, in a sealed and dark environment, with a stirring rate of 7000 rpm for 12 h.

[0082] 4) The reaction solution after 12 hours of reaction was placed into a dialysis bag (molecular weight cutoff 5000), and dialyzed with hydrochloric acid solution (5mM) containing 0.6g NaCl for 24 hours, and then dialyzed with pure water for 6 hours. During the dialysis process, magnetic stirring was used to accelerate the dialysis. After freeze-drying, catechol-modified chitosan (CA-CS) was obtained.

[0083] Preparation of RDZ-loaded catechol-chitosan (CA-CS) gel

[0084] 56 mg of CA-CS was dissolved in 1 mL of PBS to obtain a 5.6 wt% CA-CS solution. RDZ was then dissolved in 1 mL of PBS at a concentration of 0.08–0.32 wt%. The RDZ was dispersed using an ultrasonic cell disruptor at 20% power for 10 seconds to obtain a well-dispersed RDZ suspension in PBS. The RDZ solution was added to the CA-CS solution, and the two were mixed in equal volumes. The mixture was homogenized for 10 seconds to obtain a CA-CS gel loaded with RDZ.

[0085] 4. Prepare Gel+RDZ with a concentration of 0.08-0.32wt% and a CA-CS concentration of 5-6wt%. Mix CA-CS and RDZ using a homogenizer.

[0086] 5. The gel-coated scaffold was immersed for 1 hour, and the scaffold material was PCL (polycaprolactone).

[0087] ZIF (RZ) loaded with sufficient RNA using the one-pot method readily aggregates. Adding a certain amount of DOX (doxorubicin) to the one-pot method, due to the positive charge of DOX, can neutralize the strong negative charge of RNA to some extent, resulting in a more stable RDZ. The prepared RZ and RDZ were dispersed in PBS solution, and their stability was observed at room temperature. Samples were taken at 0, 3, 6, 12, and 24 hours to determine the polydispersity index (PDI). It was found that the RZ solution aggregated after 2 hours, producing a significant precipitation. Figure 1 (a) White solution. RDZ does not aggregate or precipitate in PBS solution after 24 hours, as shown. Figure 1 The red solution in (a) is more stable. This can also be seen by measuring the PDI of the two nanoparticles in PBS, such as... Figure 1 (b) The PDI of RZ had already become 0.3 after 3h, indicating that the particle size distribution was uneven and that agglomeration occurred, making it unable to remain stable in PBS. In contrast, the PDI of RDZ remained at around 0.2 for 24h, indicating a uniform particle size distribution.

[0088] like Figure 2As shown, the prepared ZIF particle size was 258.66 ± 20.03 nm. The particle size did not change significantly after adding DOX, with the DZ particle size being 223.33 ± 10.21 nm. The ZIF particle size increased after adding RNA, with the RZ particle size being 402.33 ± 28.15 nm and the RDZ particle size being 412.66 ± 12.65 nm, indicating that the addition of RNA had a greater impact on the ZIF structure. The prepared ZIF potential was 5.39 ± 0.52 mV, and the DZ potential was 14 ± 0.2 mV. The addition of negatively charged RNA caused a zeta potential flip, indicating successful RNA loading. The RZ potential was 19.7 ± 0.83 mV, and the RDZ potential was -24.3 ± 0.52 mV. The RDZ exhibited a higher absolute zeta potential, indicating better dispersibility and stability in solution. Figure 3 As shown, RDZ exhibits obvious crystal characteristics under an electron microscope, with a rhomboid morphology, good dispersibility, and a particle size of about 400 nm, which is consistent with the particle size measured by DLS.

[0089] To further investigate the effects of DOX and RNA loading on the ZIF structure, X-ray diffraction spectra of the prepared ZIF, DZ, and RDZ were examined. Figure 4 The results showed that after the addition of DOX, the diffraction peaks of DZ and RNA matched those of ZIF, and the crystallinity and diffraction peak intensity did not change significantly compared with ZIF, indicating that the addition of DOX and RNA had little effect on the crystal structure of ZIF.

[0090] To investigate the affinity of ZIF for siRNA, RZ and RDZ with different mass ratios to siRNA were prepared and electrophoresed on agarose gels. After 20 minutes of electrophoresis, the gels were placed in a gel imaging system for photographic observation. Figure 5 The results showed that, compared with free siRNA, RNA loading was significantly delayed at an RZ:RNA mass ratio of 30:1, indicating complete RNA encapsulation. An RDZ:RNA mass ratio of 68:1 could completely encapsulate the RNA. To evaluate the protective effect of RDZ against RNase A digestion of siRNA, RZ or RDZ was incubated with RNase A (35 mU) at 37°C for 1 hour, followed by denaturation of RNase A by adding 50 mM sodium dodecyl sulfate (SDS) at 60°C for 5 minutes. 50 mg / mL heparin solution was then added to the reaction mixture, followed by incubation for another 10 minutes to displace the siRNA from the RZ or RDZ. Figure 6As shown, no RNA was detected after incubation with RNase for 1 hour, indicating that the RNA had been degraded. In contrast, RDZ, after incubation with RNase for 1 hour, protected the RNA from the effects of RNase. Even after heparin was used to remove the RNA from the RDZ, it was still detectable, demonstrating that RDZ has a good protective effect on RNA.

[0091] The encapsulation efficiency and drug loading of RDZ for DOX were determined using fluorescence spectrophotometry. The method was validated for specificity, intra-day and inter-day precision, and recovery. The encapsulation efficiency of RDZ for DOX was found to be 86%. + The drug loading was 1.06%, and the total drug content was 6.5%. Using the same measurement method, the cumulative DOX release behavior of RDZ and DZ was investigated at pH 5.0 and 7.0, respectively. Figure 7 As shown, both DZ and RDZ release more rapidly under acidic conditions (pH 5.0). RDZ exhibits pH responsiveness, facilitating lysosomal escape of RNA and enabling precise RNA delivery. Due to the differences between DOX and Zn... 2+ A relatively strong coordination bond will form between them. Under acidic conditions, only 38% of the DOX in DZ can be released after 9 hours. However, after the addition of RNA, the interaction between DOX and Zn is weakened. 2+ The coordination bonds between them allow up to 80% of the DOX in RDZ to be released, which is more conducive to the drug entering the cell and exerting its effects quickly, and to better synergize with siRNA on tumor cells.

[0092] like Figure 8 As shown, a gel with good injectability can be prepared by mixing a certain amount of RDZ into the gel. Using SEM to observe RDZ@Gel at a microscopic level, the gel exhibits a typical honeycomb structure with pores ranging from 100 to 500 μm, meeting the requirements for cell adhesion and growth, which is conducive to stem cell adhesion and promotes bone repair. RDZ is evenly distributed on the surface of the CA-CS gel, indicating that a gel with uniform RDZ dispersion has been successfully prepared.

[0093] The self-healing properties of the gel were investigated by staining it with methylene blue. Figure 9 As shown, 6 hours after the hydrogel is damaged, the polymer chains in the composite gel can pass through the water medium in the hydrogel matrix and reform chemical bonds to bond together. After 12 hours, the two gels can be completely fused together, showing a good self-healing effect.

[0094] To further investigate whether the addition of RDZ affects gelation and the changes in gel properties after the addition of RDZ, rheological characterization was performed using a high-temperature, high-pressure rheometer, such as... Figure 10As shown, the storage modulus (G') of both RDZ@Gel and Gel is significantly greater than the loss modulus (G”), exhibiting solid elastic properties. Furthermore, after adding RDZ to Gel, the G' of RDZ@Gel gel is much higher than G”, indicating that the addition of RDZ significantly enhances the mechanical strength of the gel.

[0095] To improve the hydrophobicity and biocompatibility of PCL scaffolds, the gel coating needs to have good adhesion to the scaffold and be uniform. For example... Figure 11 As shown, after fully immersing the PCL scaffold in the gel solution for 1 hour, it can be seen that the gel has completely adhered to the scaffold. The gel scaffold was freeze-dried for microscopic observation, which also shows that the gel can fill the gaps in the scaffold and a layer of gel has been uniformly adhered to the scaffold. The gel has good adhesion to the scaffold, which can help improve the hydrophobicity of the scaffold and enhance its biocompatibility.

[0096] The biocompatibility of the gels was investigated. CA-CS gels loaded with 0.08%, 0.16%, and 0.32% ZIF (w / v) were prepared. 1 g of gel was placed in 10 ml of DMEM complete medium and extracted at 37°C for 24 h. The extract was obtained under aseptic conditions. The extract was then co-cultured with L929 cells for 48 hours, and cell viability was assessed using CCK8 reagent. Figure 12 The results showed that Gel+0.08% ZIF, Gel+0.16% ZIF and Gel+0.32% ZIF did not affect cell survival and had good biocompatibility.

[0097] The in vitro tumor-killing effect of nanoparticles was evaluated by comparing mouse osteosarcoma cells (K7M2 / wt) with human osteosarcoma cells (HOS). K7M2 cells were cultured at 8 × 10⁻⁶ cells / wt. 3 Cells were seeded at a density of 1:1 in each well of a 96-well plate. After 24 hours of incubation, the prepared ZIF, RZ, DZ, and RDZ were aseptically diluted to a specific concentration with complete culture medium and added to the wells. After 48 hours of incubation, cell viability was measured using the CCK8 assay to evaluate the in vitro antitumor effect of RDZ. Figure 13 As shown, the results indicate that DZ and RDZ loaded with chemotherapy drugs have good tumor cell killing effects.

[0098] K7M2 cells were used at a rate of 1×10 5 The protein was seeded at a density of 100% in each well of a 6-well plate. After 24 hours of incubation, the prepared ZIF (NRZ) containing negative control RNA and the ZIF (PRZ) containing PD-L1 siRNA were aseptically diluted to a certain concentration with complete culture medium and added to the wells. After 48 hours of incubation, the protein was extracted and detected by Western blotting. Figure 14The results showed that, compared to free siRNA and NRZ loaded with negative control RNA, ZIF(PRZ) loaded with siPD-L1 can effectively silence the PD-L1 protein and is a good vector for delivering siRNA.

[0099] A rat tibial bone defect model was established, and the rats were divided into three groups. PCL scaffolds, scaffolds coated with Gel+ZIF, and scaffolds coated with Gel+RDZ were implanted at the defect site, respectively. Rats were sacrificed at 4 or 8 weeks, and the tibias were harvested for micro-CT scanning. Three-dimensional reconstruction analysis of the defect site was performed. Figure 15 As shown, at 4 weeks post-surgery, the defect was not fully healed. In the control group (without gel) and the Gel+DOX group (scaffold coated with DOX gel), the defect remained large. The RDZ gel group, due to the addition of ZIF, showed better bone repair due to the ZIF released from the gel promoting osteogenic differentiation of stem cells, resulting in significantly increased bone mass. At 8 weeks post-surgery, the cortical bone of the defect had largely healed, with the Gel+RDZ group showing the best healing and the highest bone formation at the defect site. Quantitative analysis yielded bone volume fraction (BV / TV) and trabecular bone separation (Tb.sp). The DOX in the Gel+DOX group, being less abundant, did not significantly affect bone repair and showed little difference from the control group using only the scaffold. The Gel+RDZ group, due to the addition of ZIF, could release Zn... 2+ It promotes bone repair. The BV / TV increased significantly in the 8-week group compared to the control group, and Tb.sp decreased significantly, indicating that Gel+RDZ has a better bone repair effect.

[0100] The in vivo antitumor effect of gels was evaluated by establishing a mouse subcutaneous tumor recurrence model. After resection of the subcutaneous tumor in tumor-bearing mice, GeL+ZIF, Gel+RZ, GeL+DZ, and GeL+RDZ were injected, respectively. Postoperative tumor recurrence was observed, and the volume of the recurring tumor in the mice was measured. Figure 17 The Gel+RDZ group shown had the fewest tumor recurrences and the best recurrence prevention effect.

[0101] Example 2

[0102] Preparation of RDZ

[0103] RNA-DOX@ZIF-8 (RDZ) containing both RNA and DOX was prepared using a one-pot method. To avoid RNase degradation of the siRNA, all solutions were prepared using DEPC water.

[0104] First, a 10 mg / mL DOX drug solution is prepared and mixed with a 130 mg / mL zinc nitrate hexahydrate solution at a volume ratio of 15:10 to form solution A.

[0105] Solution B was prepared by mixing 300 mg / ml of 2-methylimidazole solution with 150 μM of siRNA solution at a volume ratio of 2.3:1.

[0106] Take 3.3 mL of solution B and add 0.7 mL of solution A while stirring at 7000 rpm at room temperature. The reaction solution changes from purple to red. After reacting for 35 min, a pink suspension is obtained. Centrifuge at 10000 rpm for 5 min to obtain a red precipitate.

[0107] The precipitate was washed twice by centrifugation with deionized water at the same speed to remove unencapsulated reactants, and then vacuum dried for 6 hours to obtain RDZ.

[0108] Preparation of RZ and DZ

[0109] RZ is prepared in the same way as RDZ, except that the DOX solution in solution A is replaced with ultrapure water (DEPC water), while the other steps remain the same. DZ can be prepared by replacing the RNA solution in solution B with DEPC water.

[0110] Synthesis of Catechol Chitosan (CA-CS)

[0111] Weigh 0.1 g of chitosan (MW = 150000) into 50 ml of hydrochloric acid solution (0.05 M) and stir for 30 min to dissolve it completely.

[0112] Weigh 0.1 g of 3,4-dihydroxyphenylpropionic acid (HCA) and 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and dissolve them in 2 ml of ethanol solution (1:1, V / V).

[0113] Add 0.8 mL of EDC solution and 0.6 mL of HCA solution to the chitosan solution. The solution turns pale yellow. Adjust the pH of the reaction solution to 4.8 with NaOH solution to obtain a yellow solution. React at room temperature, in a sealed and dark environment, with stirring at 7000 rpm for 12 h.

[0114] The reaction solution after 12 hours was placed in a dialysis bag (molecular weight cutoff 5000), dialyzed for 24 hours with hydrochloric acid solution (5mM) containing 0.6 g NaCl, and then dialyzed for 6 hours with pure water. During the dialysis process, magnetic stirring was used to accelerate the dialysis. After freeze-drying, catechol-modified chitosan (CA-CS) was obtained.

[0115] Preparation of RDZ-loaded catechol-chitosan (CA-CS) gel

[0116] 56 mg of CA-CS was dissolved in 1 mL of PBS to obtain a 5.6 wt% CA-CS solution. RDZ was then dissolved in 1 mL of PBS at a concentration of 0.32 wt%. The solution was ultrasonically dispersed for 10 seconds at 20% power using an ultrasonic cell disruptor to obtain a well-dispersed RDZ suspension in PBS. The RDZ solution was added to the CA-CS solution, and the two were mixed in equal volumes. The mixture was homogenized for 10 seconds to obtain a CA-CS gel loaded with RDZ.

[0117] Example 3

[0118] Preparation of RDZ

[0119] RNA-DOX@ZIF-8 (RDZ) containing both RNA and DOX was prepared using a one-pot method. To avoid RNase degradation of the siRNA, all solutions were prepared using DEPC water.

[0120] First, a 10 mg / mL DOX drug solution is prepared and mixed with a 130 mg / mL zinc nitrate hexahydrate solution at a volume ratio of 15:15 to form solution A.

[0121] Solution B was prepared by mixing 350 mg / ml of 2-methylimidazole solution with 200 μM of siRNA solution at a volume ratio of 2.3:1.

[0122] Take 3 mL of solution B and add 1 mL of solution A while stirring at 7000 rpm at room temperature. The reaction solution changes from purple to red. After reacting for 20 min, a pink suspension is obtained. Centrifuge at 10000 rpm for 5 min to obtain a red precipitate.

[0123] The precipitate was washed twice by centrifugation with deionized water at the same speed to remove unencapsulated reactants, and then vacuum dried for 6 hours to obtain RDZ.

[0124] Preparation of RZ and DZ

[0125] RZ is prepared in the same way as RDZ, except that the DOX solution in solution A is replaced with ultrapure water (DEPC water), while the other steps remain the same. DZ can be prepared by replacing the RNA solution in solution B with DEPC water.

[0126] Synthesis of Catechol Chitosan (CA-CS)

[0127] Weigh 0.1 g of chitosan (MW = 150000) into 50 ml of hydrochloric acid solution (0.05 M) and stir for 30 min to dissolve it completely.

[0128] Weigh 0.1 g of 3,4-dihydroxyphenylpropionic acid (HCA) and 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and dissolve them in 2 ml of ethanol solution (1:1, V / V).

[0129] Add 0.8 mL of EDC solution and 0.6 mL of HCA solution to the chitosan solution. The solution turns pale yellow. Adjust the pH of the reaction solution to 4.8 with NaOH solution to obtain a yellow solution. React at room temperature, in a sealed and dark environment, with stirring at 7000 rpm for 12 h.

[0130] The reaction solution after 12 hours was placed in a dialysis bag (molecular weight cutoff 5000), dialyzed for 24 hours with hydrochloric acid solution (5mM) containing 0.6 g NaCl, and then dialyzed for 6 hours with pure water. During the dialysis process, magnetic stirring was used to accelerate the dialysis. After freeze-drying, catechol-modified chitosan (CA-CS) was obtained.

[0131] Preparation of RDZ-loaded catechol-chitosan (CA-CS) gel

[0132] 56 mg of CA-CS was dissolved in 1 mL of PBS to obtain a 5.6 wt% CA-CS solution. RDZ was then dissolved in 1 mL of PBS at 0.1 wt%. The solution was ultrasonically dispersed for 10 seconds at 20% power using an ultrasonic cell disruptor to obtain a well-dispersed RDZ suspension in PBS. The RDZ solution was added to the CA-CS solution, and the two were mixed in equal volumes. The mixture was homogenized for 10 seconds to obtain a CA-CS gel loaded with RDZ.

[0133] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a gel for osteosarcoma postoperative recurrence prevention and bone repair promotion, characterized by: It comprises the following steps: Preparation of mixed solution A of doxorubicin and zinc nitrate; Preparation of mixed solution B of 2-methylimidazole and siRNA; Add mixed solution B to mixed solution A under stirring, react, and obtain red precipitate; Wash and centrifuge the red precipitate to remove un-encapsulated reactants; Vacuum dry the solid precipitate obtained by centrifugation to obtain RDZ; Modification of chitosan with catechol to obtain catechol chitosan; Add RDZ suspension to catechol chitosan solution, mix, homogenize, and obtain catechol chitosan gel loaded with RDZ; The volume ratio of mixed solution A to mixed solution B is 0.5-1:3-4, the reaction time of mixed solution A and mixed solution B is 20-40 min, and the reaction temperature is 20-40℃. In mixed solution A, the concentration of doxorubicin is 3-7 mg / mL, and the concentration of zinc nitrate is 50-100 mg / mL. The RNA is siRNA, circRNA or shRNA with a molecular weight of less than 100 kd. In mixed solution B, the concentration of 2-methylimidazole is 200-250 mg / mL, and the concentration of siRNA is 30-50 μM. The preparation method of catechol-modified chitosan comprises the following steps: Add EDC solution and HCA solution to chitosan solution, adjust the pH value of the solution to 4.8, avoid light, and stir at 20-30℃ for 10-14 h to obtain reaction liquid. Pack the reaction liquid into a dialysis bag, dialyze with hydrochloric acid solution containing NaCl first, then dialyze with pure water, freeze-dry the reaction liquid after dialysis, and catechol-modified chitosan is obtained.

2. The process for the preparation of gel for osteosarcoma post-operative prevention of recurrence and promotion of bone repair according to claim 1, characterized in that: In mixed solution A, the concentration of doxorubicin is 5.3-5.5 mg / mL, and the concentration of zinc nitrate is 60-70 mg / mL.

3. The process for the preparation of gel for osteosarcoma post-operative prevention of recurrence and promotion of bone repair as claimed in claim 1, wherein: In mixed solution B, the concentration of 2-methylimidazole is 220-230 mg / mL, and the concentration of siRNA is 30-40 μM.

4. The process for the preparation of gel for osteosarcoma post-operative prevention of recurrence and promotion of bone repair as claimed in claim 1 wherein: The reaction time of mixed solution A and mixed solution B is 25-35 min, and the reaction temperature is 25-35℃.

5. The process for the preparation of gel for osteosarcoma post-operative prevention of recurrence and promotion of bone repair as claimed in claim 1, wherein: In the reaction liquid, the concentration of chitosan is 1.5-2 g / L, the concentration of EDC is 0.8-1.0 g / L, and the concentration of HCA is 0.5-1.0 g / L.

6. A gel for osteosarcoma post-surgical recurrence prevention and bone repair promotion, characterized by: Prepared by the preparation method of any one of claims 1-5.

7. A stent, characterized by: It comprises a PCL scaffold matrix and the gel of claim 6 coated on the surface of the PCL scaffold matrix.

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