Preparation method and application of a gene delivery system for synergistic treatment of bone metastases

Abstract

This invention discloses a method for preparing a gene delivery system for synergistic treatment of bone metastases and its application. The invention uses polyethyleneimine (PEI) as the backbone material. First, mannose is linked by disulfide bonds, then alendronate (ALN) is linked by Schiff base bonds to synthesize a bone-targeting synergistic gene vector, PSMCA. Finally, the therapeutic gene miR-34a is loaded onto the vector, resulting in a gene delivery system for synergistic treatment of bone metastases. The prepared gene delivery system can target and deliver the therapeutic gene to the bone metastases site. It exerts a synergistic therapeutic effect on tumors by releasing the drug through a hierarchical response to the bone metastases microenvironment and intracellular redox environment, showing promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to a method for preparing a gene delivery system for the synergistic treatment of bone metastases and its application. This delivery system targets and delivers therapeutic genes to the bone tumor site while synergistically inhibiting osteoclast resorption and tumor cell proliferation, thus breaking the vicious cycle between bone resorption and tumor proliferation to synergistically treat bone metastases. This type of nanomedicine delivery system can be used for gene therapy of bone tumors, especially bone metastases. Background Technology

[0002] Bone metastases are highly malignant tumors. Malignant tumor cells originating in extra-skeletal organs and tissues breach the basement membrane and invade surrounding tissues. They then colonize bone tissue via the bloodstream or lymphatic system, growing into metastatic lesions. Bone is one of the most common sites of metastasis for malignant tumors. Once cancer cells spread to bone tissue, treatment usually relies on relatively conservative methods such as systemic chemotherapy, targeted therapy, or immunotherapy to control the progression of metastatic cancer, improve the patient's quality of life, and prolong survival. However, due to the unique physiological structure of bone tissue and the development of treatment resistance, bone metastases remain a major bottleneck in the treatment of malignant tumors. Tumor cells that metastasize and colonize bone tissue activate precursor cells in the bone matrix by secreting various activating factors. This leads to the excessive release of receptor activator for nuclear factor-κB ligand (RANKL), promoting osteoclast activation and maturation, affecting the normal morphology of bone tissue, and causing osteolysis. Following osteolysis, growth factors within the bone tissue are released, which in turn promote tumor cell growth, resulting in a vicious cycle of mutually reinforcing growth between tumor cells and osteoclasts, thus increasing the difficulty of treating bone metastases. Furthermore, due to the unique structure of bone tissue and the presence of the bone marrow-blood barrier, conventional vectors struggle to effectively deliver therapeutic genes to the lesion site in bone tissue. Therefore, developing an effective drug delivery system to address the challenges of gene therapy and the characteristics of bone metastases is of significant theoretical and practical importance.

[0003] Gene therapy is a novel disease treatment method that delivers specific therapeutic genes to target cells to exert their effects, potentially offering a cure for diseases. Among these, miRNAs (miRNAs) are a class of post-transcriptional regulatory factors that can specifically bind to target mRNAs, inhibiting the expression of target proteins and thus regulating gene expression, thereby affecting tumor cell proliferation, apoptosis, and migration. As a therapeutic entity, miRNAs, with their mechanism of action and high specificity, can regulate many aspects of tumor cell development and progression, representing a novel approach to cancer treatment. However, miRNA therapy faces challenges such as short half-life, susceptibility to degradation by nucleases in the blood, non-specific distribution, and off-target effects, limiting the drug-like potential of miRNAs and hindering the development of suitable delivery vectors.

[0004] Gene delivery vectors are generally classified into viral and non-viral vectors. Viral vectors, which have high transfection efficiency, have limited loading capacity and targeting capabilities, and are accompanied by side effects such as carcinogenicity, immunogenicity, and susceptibility, making the development of non-viral vectors extremely important. Polyethyleneimine (PEI), due to its high content of primary and secondary amines, high cationicity, and protonation properties, has become the gold standard for gene transfection. Although PEI shows great promise for delivering therapeutic genes, its targeting and intracellular release capabilities still need to be improved to achieve both precise gene release and high transfection efficiency, while avoiding off-target effects. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a gene delivery system for the synergistic treatment of bone metastases and its application. The constructed nano-delivery system for the synergistic treatment of bone metastases exhibits good storage stability and bone targeting. This nano-delivery system can target and deliver the therapeutic gene miR-34a to the site of bone metastases, releasing alendronate to synergistically inhibit osteoclast resorption and tumor cell proliferation, significantly improving the therapeutic effect of bone metastases and providing new ideas for the research of anti-bone metastases drugs.

[0006] One objective of this invention is to provide a method for preparing a gene delivery system for synergistic treatment of bone metastases. The method is characterized by first synthesizing thiol-containing Man-cys via reductive amination; then, linking Man-cys to the surface of branched polyethyleneimine (PEI) rich in amino groups via amidation to obtain a disulfide-containing product PSM; subsequently, linking p-carboxybenzaldehyde (CBA) to the surface of alendronate PSM via amidation to obtain an aldehyde-containing product PSMC; finally, linking alendronate (ALN) to the surface of PSMC via reductive amination to obtain a bone-targeting synergistic gene carrier PSCMA; and finally, electrostatically adsorbing the obtained PSCMA with the therapeutic gene miR-34a to obtain the gene delivery system PSMCA / miR-34a for synergistic treatment of bone metastases.

[0007] The present invention discloses a method for preparing a gene delivery system for synergistic treatment of bone metastases, which specifically includes the following steps:

[0008] Step (1) Dissolve cysteine ​​and mannose in an aqueous solution of sodium hydroxide in a certain proportion. After stirring the reaction at a specific temperature for a certain time, place the reaction system in an ice bath and add a certain amount of NaBH4. After the reaction stabilizes, place it at a specific temperature for a certain time. After the reaction is completed, adjust the pH of the reaction system with 6M HCl solution, evaporate under reduced pressure, add a certain amount of methanol to dissolve, let stand overnight, filter, evaporate the filtrate under reduced pressure, add a certain amount of grade III water to dissolve and transfer to a dialysis bag (MWCO 200), dialyze with 2000mL grade III water for 6h, change the water once every 2h, evaporate by rotary evaporation and freeze dry to obtain a pale yellow solid Man-cys.

[0009] Step (2): A certain amount of PEI and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) were dissolved in dimethyl sulfoxide (DMSO). The SPDP solution was slowly added to the PEI solution, and the mixture was stirred at room temperature for a certain period of time. A certain amount of Man-cys was dissolved in DMSO and slowly added to the above reaction solution. After stirring at room temperature for a certain period of time, the reaction solution was transferred to a dialysis bag (MWCO 7000) and dialyzed against 2000 mL of Grade III water for 48 h, with the water changed every 6 h. The solution was then rotary evaporated and freeze-dried to obtain a white solid PSM.

[0010] In step (3), CBA, 1-ethyl-(3-dimethylaminopropyl)-3-carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) were dissolved in DMSO in a certain proportion. After stirring at room temperature for a certain period of time, a certain amount of PSM was dissolved in grade III water and slowly added to the above reaction solution. The reaction was continued to be stirred at room temperature for a certain period of time. The reaction solution was then transferred to a dialysis bag (MWCO7000) and dialyzed with 2000 mL of grade III water for 48 h. The water was changed every 6 h. The solution was then rotary evaporated and freeze-dried to obtain white solid PSMC.

[0011] Step (4) Dissolve a certain amount of ALN and PSMC in Grade III water in a certain proportion. Slowly add the PSMC solution to the ALN solution and stir the reaction at a specific temperature for a certain time. Transfer the reaction solution into a dialysis bag (MWCO 7000) and dialyze it in 2000 mL of Grade III water for 24 h. Change the water every 6 h. Rotary evaporation and freeze drying are used to obtain white solid PSMCA.

[0012] Step (5) Prepare a solution of miR-34a and PSMCA with DEPC water to a certain concentration. Mix the two solutions according to the experimental dosage, vortex rapidly for 30 seconds, and let stand at room temperature for 20 minutes to obtain a PSMCA / miR-34a nanoparticle solution.

[0013] In the specific preparation, in step (1), the molar ratio of cysteine ​​to mannose is 5:1 to 1:5, the specific reaction temperature is 10 to 100℃, and the reaction time is 1 to 10 h; the molar ratio of NaBH4 to cysteine ​​is 5:1 to 1:5, the specific reaction temperature after adding NaBH4 is 0 to 50℃, and the reaction time is 2 to 150 h; after the reaction is completed, the pH of the reaction system is adjusted to 1 to 5 with 6M HCl solution; after rotary evaporation under reduced pressure, the volume of methanol solution added is 40 to 100 mL, it is left to stand overnight, the methanol is removed under reduced pressure, and the volume of grade III water added is 2 to 20 mL.

[0014] In step (2), the mass ratio of PEI to SPDP is 50:1 to 1:1, the PEI used is linear polyethyleneimine (LPEI) or branched polyethyleneimine (BPEI) with a molecular weight range of 600 to 50 kDa, and the reaction time at room temperature is 2 to 24 h; the mass ratio of Man-cys to PEI used is 1:1 to 1:60, and the reaction time at room temperature is 2 to 24 h.

[0015] In step (3), the molar ratio of CBA, EDAC and NHS is 3:1:1 to 1:3:3, the reaction time at room temperature is 0.5 to 4 h, the mass ratio of CBA to PSM is 10:1 to 1:100, and the reaction time at room temperature is 2 to 24 h.

[0016] In step (4), the mass ratio of ALN to PSMC is 1:1 to 1:80, the specific reaction temperature is 20 to 100°C, and the reaction time is 2 to 24 hours.

[0017] In step (5), the concentration of miR-34a is 0.01 to 1 mg / mL, the concentration of PSMCA is 0.1 to 2 mg / mL solution, and the mass ratio of PSMCA and miR-34a when mixed is 1:1 to 20:1.

[0018] The second objective of this invention is to provide a gene delivery system for the synergistic treatment of bone metastases and its application in drugs for treating bone tumors.

[0019] This invention has the following outstanding effects:

[0020] (1) The gene delivery vector PSMCA was synthesized for the first time. Alendronic acid has both bone-targeting and anti-bone resorption functions. The gene delivery system PSMCA / miR-34a for synergistic treatment of bone metastases was prepared by loading the therapeutic gene miR-34a. The particle size and anti-bone metastasis activity of the PSMCA / miR-34a delivery system were evaluated.

[0021] (2) The gene delivery system prepared in this invention has a particle size of approximately 180 nm, and its particle size and PDI remain relatively stable within 14 days, demonstrating good stability. In vivo studies on its anti-bone metastasis activity have shown that the gene delivery system can inhibit tumor proliferation, reduce bone destruction, and has a significant synergistic therapeutic effect on bone metastases.

[0022] (3) In summary, this gene delivery system can deliver genes to bone tumor sites in a targeted manner, and through the synergistic therapeutic effect of alendronate and miR-34a, inhibit the activity of osteoclasts and tumor cells, break the vicious cycle between bone tissue and tumor cells, and synergistically treat bone metastases, which has good clinical application prospects. Attached Figure Description

[0023] Figure 1A This is the proton NMR spectrum of the delivery carrier for the synergistic treatment of bone metastases;

[0024] Figure 1B This is the phosphomagnetic resonance spectrum of the delivery carrier for synergistic treatment of bone metastases;

[0025] Figure 1C This is the infrared spectrum of the delivery carrier for the synergistic treatment of bone metastases;

[0026] Figure 2A This is a particle size distribution map of a gene delivery system for the synergistic treatment of bone metastases;

[0027] Figure 2B This is a stability diagram of a gene delivery system for synergistic treatment of bone metastases;

[0028] Figure 3A This is an in vivo imaging result of bone-targeting affinity of a gene delivery system for synergistic treatment of bone metastases, n=3.

[0029] Figure 3B This is a semi-quantitative fluorescence intensity map of the bone-targeting affinity results of a gene delivery system for synergistic treatment of bone metastases. ** p<0.01 (compared to the PEI / FAM-miR-34a group);

[0030] Figure 4A These are photographs of ex vivo tumors showing the anti-tumor effects of a gene delivery system for synergistic treatment of bone metastases, n=8.

[0031] Figure 4B The tumor weight statistics are the anti-tumor results of the gene delivery system for synergistic treatment of bone metastases. * p<0.05, ** p<0.01, compared with the saline group; &p<0.05, compared with the PEI / miR-34a group; # p<0.05 (compared to the PSM / miR-34a group);

[0032] Figure 4C This is a statistical result of the tumor suppression rate of the gene delivery system for synergistic treatment of bone metastases. ** p<0.01, compared with the PEI / miR-34a group; && p<0.01 (compared to the PSM / miR-34a group);

[0033] Figure 5A The results of Micro-CT 3D bone reconstruction of bone tissue in tumor-bearing mice that inhibited bone destruction using a gene delivery system that synergistically treats bone metastases, n=8;

[0034] Figure 5B The results are Micro-CT images of the tibia of tumor-bearing mice that inhibited bone destruction using a gene delivery system for synergistic treatment of bone metastases, n=3. Detailed Implementation

[0035] The specific embodiments of the present invention will be further described below. The specific embodiments described below provide a more detailed explanation of the technical problems and solutions solved by the present invention. It should be understood that the following descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0036] Example 1

[0037] Preparation of bone-targeted synergistic delivery carrier PSMCA

[0038] 3.63 g (30 mmol) of cysteine ​​and 5.40 g (30 mmol) of mannose were dissolved in 10 mL of sodium hydroxide solution and reacted at 65 °C for 5 h with stirring. Under ice-water bath conditions, 3.00 g (79 mmol) of sodium borohydride was added, and stirring continued at 25 °C for 120 h. Under ice-water bath conditions, the pH of the reaction solution was adjusted to 1 with 6 M hydrochloric acid solution. Water was removed by vacuum distillation, and 80 mL of methanol was added to dissolve the resulting white solid. The solution was allowed to stand overnight. After filtration, methanol was removed from the filtrate under vacuum, yielding a white solid. This solid was dissolved in 10 mL of purified water and dialyzed in a dialysis bag (MWCO 200) for 6 h, with water changed every 2 h. The solution was then freeze-dried to obtain 4.71 g of a pale yellow solid, Man-cys, with a yield of 55%.

[0039] 448 mg of branched polyethyleneimine and 37 mg of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester were dissolved in 10 mL and 2 mL of dimethyl sulfoxide, respectively. The 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester solution was added to the polyethyleneimine solution, and the mixture was stirred at room temperature for 12 h. 33.6 mg of Man-cys was dissolved in 2 mL of dimethyl sulfoxide and added to the above reaction mixture, and the mixture was stirred at room temperature for 12 h. The mixture was filtered, and the supernatant was transferred to a dialysis bag (MWCO 7000) and dialyzed for 48 h, with water changed every 6 h. After freeze-drying, 394 mg of PEI-SPDP-Man-cys (PSM) solid was obtained, with a yield of 80%.

[0040] 105 mg (0.86 mmol) of p-carboxybenzaldehyde was dissolved in 10 mL of dimethyl sulfoxide. 161 mg (0.84 mmol) of 1-ethyl-(3-dimethylaminopropyl)-3-carbodiimide salt and 97 mg (0.84 mmol) of N-hydroxysuccinimide were then added to the p-carboxybenzaldehyde solution. The mixture was stirred at room temperature for 2 h. 379 mg of PSM was dissolved in 10 mL of purified water and added to the p-carboxybenzaldehyde reaction solution. The mixture was reacted at room temperature for 12 h. The reaction solution was then dialyzed in a dialysis bag (MWCO 7000) for 24 h, with the water changed every 6 h. The solution was freeze-dried to obtain 407 mg of solid PSM-CBA (PSMC), with a yield of 84%.

[0041] 189 mg of alendronate was dissolved in 8 mL of purified water, and 371 mg of PSMC was dissolved in 16 mL of purified water. The alendronate solution was added, and the mixture was reacted at 65 °C for 12 h. After the reaction solution cooled to room temperature, it was placed in a dialysis bag (MWCO 7000) and dialyzed for 24 h, with water changed every 6 h. The solution was then freeze-dried to obtain 524 mg of solid PSMC-ALN (PSMCA), with a yield of 93%. The structure of PSMCA was identified using 1H and 1N NMR spectroscopy and infrared spectroscopy. The results are as follows: Figure 1A , Figure 1B , Figure 1C As shown, the synthesized product and the designed support have the same structure.

[0042] Example 2

[0043] Preparation and characterization of PSMCA / miR-34a gene delivery system for bone metastases

[0044] Solutions of miR-34a and PSMCA were prepared using DEPC-treated water at concentrations of 0.5 mg / mL and 1 mg / mL, respectively. The two solutions were mixed according to the experimental concentrations (PSMCA to miR-34a mass ratio of 5), vortexed, and allowed to stand at room temperature for 20 min to obtain a PSMCA / miR-34a nanoparticle solution. The particle size, multidiffusion coefficient, and storage stability of the PSMCA / miR-34a nanoparticle solution were determined using a laser particle size analyzer.

[0045] Experimental results: Morphology measurements of PSMCA / miR-34a nanoparticles showed a particle size of 180 nm and a multiple diffusion coefficient of 0.206. Figure 2A On days 1, 7, and 14, the particle size and PDI of the nanoparticles were below 200 nm and 0.250, respectively. Figure 2B This indicates that the nanoparticle solution has good storage stability.

[0046] Example 3

[0047] In vitro bone targeting evaluation of the PSMCA / miR-34a gene delivery system for bone metastases

[0048] PEI / FAM-miR-34a and PSMCA / FAM-miR-34a nanoparticles were prepared, with FAM-miR-34a at a concentration of 10 μg / mL. Tibial bones from healthy mice were placed in 1 mL of the nanoparticle solution and incubated on a shaker at 37°C, 100 rpm for 3 h. After incubation, 2 mL of PBS was added for washing, and this process was repeated three times. The Lumina X5 in vivo imaging system was used to detect the fluorescence intensity on the tibial surface and to investigate the adsorption of different nanoparticles on the tibial bone.

[0049] Experimental Results: The adsorption of fluorescently labeled nanoparticles on isolated tibiae was observed using an in vivo imaging system. Figure 3A It can be seen that the fluorescence intensity of the PSMCA / FAM-miR-34a group is higher than that of the PEI / FAM-miR-34a group. Quantitative analysis of the fluorescence intensity showed that ( Figure 3B The fluorescence intensity of the PSMCA / FAM-miR-34a group was about 3.1 times that of the PEI / FAM-miR-34a group, indicating that PSMCA / FAM-miR-34a adsorbed more on the tibia, further demonstrating that the ALN-modified carrier PSMCA has good bone affinity.

[0050] Example 4

[0051] In vivo antitumor experiment of PSMCA / miR-34a gene delivery system for bone metastases

[0052] 50 μL (2 × 10⁻⁶) was injected intramedullary into the right tibia of 4-6 week old female BALB / c mice. 8 A bone metastasis orthotopic animal model was established using 4 T1-Luc cells / mL. Treatment began on the fourth day after modeling, with tumor-bearing mice receiving intravenous injections of Saline, PEI / miR-34a, PSM / miR-34a, and PSMCA / miR-34a nanoparticle solutions, respectively. Mice were sacrificed at the planned time points, and all tumor-bearing tibiae were collected, photographed, and weighed. The tumor inhibition rate for each treatment group was calculated. Tumor inhibition rate (%) = (mean tumor weight in the Saline group - tumor weight in each experimental group) / mean tumor weight in the Saline group × 100%

[0053] Experimental results: In vivo anti-tumor results showed that, compared with the Saline group, the final volume of bone metastases in mice treated with PEI / miR-34a and PSM / miR-34a was reduced, and the bone metastases in mice treated with PSMCA / miR-34a were the smallest. Figure 4A Compared to the Saline group, the weight of bone metastases was significantly reduced after treatment with each nanoparticle group, and tumor growth was inhibited. The PSMCA / miR-34a group had the lowest tumor weight after treatment compared to PEI / miR-34a and PSM / miR-34a. Figure 4B This indicates that the PSMCA / miR-34a delivery system exerts a synergistic antitumor effect. Similarly, the tumor inhibition rate results ( Figure 4C The results showed that, compared with PEI / miR-34a and PSM / miR-34a, the PSMCA / miR-34a group had a significant inhibitory effect on tumor proliferation after treatment.

[0054] Example 5

[0055] In vivo inhibition of bone destruction by the gene delivery system PSMCA / miR-34a for bone metastases

[0056] After establishing an animal model of bone metastasis, mice were divided into groups (Saline, PEI / miR-34a, PSM / miR-34a, and PSMCA / miR-34a nanoparticle solutions) and treated. Mice were sacrificed at the planned time, and the right hind limbs of all tumor-bearing mice were collected. After removing the epidermal tissue, all samples were scanned using Micro-CT to observe bone destruction in each group.

[0057] Experimental results: Micro-CT scan results show that ( Figure 5AAfter treatment, the Saline group mice showed complete tibial and femoral ends in the right hind limb, while the PEI / miR-34a and PSM / miR-34a groups showed intact femoral end structures. The PSM / miR-34a group mice exhibited relatively better bone integrity and reduced bone destruction, demonstrating the synergistic therapeutic effect of PSM / miR-34a on bone metastases. Further 3D reconstruction of the tibial cross-section was performed. Figure 5B The results showed that in the Saline group, the periosteum of tumor-bearing mice was spiky and rough, the cortical bone was deformed, and the trabeculae were completely dissolved. In the PEI / miR-34a and PSM / miR-34a groups, the cortical bone surface of tumor-bearing mice was rough, with some lacking complete morphology. The number of trabeculae was greater than in the Saline group, but the spacing between trabeculae was larger, the thickness was uneven, and the trabeculae were severely dissolved, thinned, and broken, with almost no complete or continuous trabeculae. In contrast, the tibial cortex of tumor-bearing mice in the PSM / miR-34a group showed depressions and visible bone defects, and the number of trabeculae was greater. The results indicate that treatment with PEI / miR-34a, PSM / miR-34a, and PSM / miR-34a can inhibit the destruction of bone tissue by tumor cells to a certain extent. Moreover, PSM / miR-34a showed the most significant effect in treating bone metastases, significantly reducing the destruction of the cortical bone in tumor-bearing mice.

Claims

1. A method for preparing a gene delivery system for the synergistic treatment of bone metastases, characterized in that, First, Man-cys containing thiol groups was synthesized by reductive amination. Then, Man-cys was linked to the surface of polyethyleneimine (PEI) containing abundant amino groups by amidation to obtain PSM containing disulfide bonds. Next, p-carboxybenzaldehyde (CBA) was linked to the surface of PSM by amidation to obtain PSMC containing aldehyde groups. Finally, alendronate (ALN) was linked to the surface of PSMC by reductive amination to obtain the bone-targeting synergistic gene carrier PSCMA. The obtained PSCMA was electrostatically adsorbed with the therapeutic gene miR-34a to obtain the gene delivery system PSMCA / miR-34a for synergistic treatment of bone metastases. Specifically, the following steps are included: Step (1): Cysteine ​​and mannose were dissolved in an aqueous solution of sodium hydroxide in a certain proportion. After stirring at a specific reaction temperature for a certain time, the reaction system was placed in an ice bath. A certain amount of NaBH4 was added. After the reaction stabilized, the reaction was carried out at a specific reaction temperature for a certain time. After the reaction was completed, the pH of the reaction system was adjusted with 6 M HCl solution. The system was then evaporated under reduced pressure. A certain amount of methanol was added to the reaction system to dissolve the solid. The system was left to stand overnight. After filtration, the filtrate was evaporated under reduced pressure to obtain a white solid. A certain amount of Grade III water was added to the white solid mixture to dissolve the solid. The mixture was then transferred to a MWCO 200 dialysis bag and dialyzed with 2000 mL of Grade III water for 6 h. The water was changed every 2 h. The solid was then evaporated and freeze-dried to obtain a pale yellow solid, Man-cys. Step (2): Mix a certain amount of PEI and 3-(2-pyridyldithio)propionic acid N 1-Hydroxysuccinimide ester (SPDP) was dissolved in dimethyl sulfoxide (DMSO). The SPDP solution was slowly added to PEI solution, and the mixture was stirred at room temperature for a certain period of time. A certain amount of Man-cys was dissolved in DMSO and slowly added to the above reaction solution. After stirring at room temperature for a certain period of time, the reaction solution was transferred to an MWCO7000 dialysis bag and dialyzed with 2000 mL of Grade III water for 48 h, with the water changed every 6 h. The solution was then rotary evaporated and freeze-dried to obtain a white solid PSM. Step (3): Sequentially add CBA, 1-ethyl-(3-dimethylaminopropyl)-3-carbodiimide salt (EDAC) and N 1-Hydroxysuccinimide (NHS) was dissolved in DMSO in a certain proportion and stirred at room temperature for a certain time. Then, a certain amount of PSM was dissolved in Grade III water and slowly added to the above reaction solution. The reaction solution was stirred at room temperature for a certain time. The reaction solution was then transferred to an MWCO 7000 dialysis bag and dialyzed with 2000 mL of Grade III water for 48 h. The water was changed every 6 h. The solution was then obtained by rotary evaporation and freeze drying to obtain white solid PSMC. Step (4): Dissolve a certain amount of ALN and PSMC in Grade III water in a certain ratio, slowly add the PSMC solution to the ALN solution, stir the reaction at a specific reaction temperature for a certain time, transfer the reaction solution into a MWCO 7000 dialysis bag, dialyze with 2000 mL of Grade III water for 24 h, change the water every 6 h, and obtain white solid PSMCA by rotary evaporation and freeze drying. Step (5): Prepare a specific concentration solution of miR-34a and PSMCA with DEPC water. Mix the two solutions in proportion according to the experimental dosage, mix them by pipetting, and let stand at room temperature for 20 min to obtain the gene delivery system PSMCA / miR-34a for synergistic treatment of bone metastases.

2. The method for preparing the gene delivery system for synergistic treatment of bone metastases according to claim 1, characterized in that, In step (1), the molar ratio of cysteine ​​to mannose is 5:1 to 1:5, the specific reaction temperature is 10 to 100℃, and the reaction time is 1 to 10 h; the molar ratio of NaBH4 to cysteine ​​is 5:1 to 1:5, the specific reaction temperature after adding NaBH4 is 0 to 50℃, and the reaction time is 2 to 150 h; after the reaction is completed, the pH of the reaction system is adjusted to 1 to 5 with 6 M HCl solution; after rotary evaporation under reduced pressure, the volume of methanol solution added is 40 to 100 mL, the mixture is allowed to stand overnight, the methanol is removed under reduced pressure, and the volume of grade III water added is 2 to 20 mL.

3. The method for preparing the gene delivery system for synergistic treatment of bone metastases according to claim 1, characterized in that, In step (2), the mass ratio of PEI to SPDP is 50:1 to 1:1, the PEI used is linear polyethyleneimine (LPEI) or branched polyethyleneimine (BPEI) with a molecular weight range of 600 to 50 kDa, and the reaction time at room temperature is 2 to 24 h; the mass ratio of Man-cys to PEI used is 1:1 to 1:60, and the reaction time at room temperature is 2 to 24 h.

4. The method for preparing the gene delivery system for synergistic treatment of bone metastases according to claim 1, characterized in that, In step (3), the molar ratio of CBA, EDAC and NHS is 3:1:1 ~ 1:3:3, the reaction time at room temperature is 0.5 ~ 4 h, the mass ratio of CBA to PSM is 10:1 ~ 1:100, and the reaction time at room temperature is 2 ~ 24 h.

5. The method for preparing the gene delivery system for synergistic treatment of bone metastases according to claim 1, characterized in that, In step (4), the mass ratio of ALN to PSMC is 1:1 to 1:80, the specific reaction temperature is 20 to 100℃, and the reaction time is 2 to 24 h.

6. The method for preparing the gene delivery system for synergistic treatment of bone metastases according to claim 1, characterized in that, In step (5), the concentration of miR-34a is 0.01 ~ 1 mg / mL, the concentration of PSMCA is 0.1 ~ 2 mg / mL solution, and the mass ratio of PSMCA and miR-34a when mixed is 1:1 ~ 20:

1.

7. The use of the gene delivery system for synergistic treatment of bone metastases prepared by any one of claims 1 to 6 in the preparation of drugs for treating bone metastases.

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