Drug delivery system for treating osteoporosis caused by cell aging as well as preparation method and application of drug delivery system

Abstract

The invention relates to a drug delivery system for treating osteoporosis caused by cell aging as well as a preparation method and application of the drug delivery system. The drug delivery system comprises a bone targeting ligand modified drug carrier and an anti-bone cell aging drug entrapped in the drug carrier, the bone targeting ligand is selected from any one of alendronate sodium, bisphosphate and analogues thereof, tetracycline, tartrate-resistant acid phosphatase TRAP binding peptide, pregnancy region protein or e-selectin; the anti-osteocyte senescence medicine comprises any one or a combination of at least two of dasatinib, quercetin, metformin, L-ascorbic acid, aspirin or fisetin. According to the prepared drug delivery system, the bioavailability of the drug can be effectively improved, targeted release of the drug in bone tissue is achieved, senescent cell enrichment is reduced, and the bone microenvironment is improved, so that a new strategy is provided for treating osteoporosis caused by cell senescence.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a drug delivery system for treating osteoporosis caused by cell aging, and a preparation method and application thereof. Background Art

[0002] Musculoskeletal disorders include a wide range of conditions that affect an individual's motor system, ranging from sudden, transient problems (such as fractures, sprains and strains) to lifelong conditions such as persistent functional limitations and disabilities. Typical symptoms are pain (usually constant) and limitations in mobility, dexterity and overall function, and reduced ability to work. Musculoskeletal disorders include: Joint problems: such as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, ankylosing spondylitis; Bone problems: such as osteoporosis, osteopenia, fragility fractures, traumatic fractures; Muscle problems, such as sarcopenia; Spinal problems: such as back and neck pain; Problems in multiple parts or systems of the body: such as musculoskeletal localized and widespread pain and inflammatory diseases (connective tissue diseases and vasculitis, such as systemic lupus erythematosus).

[0003] Osteoporosis (OP) is a common skeletal disease characterized by decreased bone density, which leads to an increased risk of fractures, seriously affects the patient's quality of life, and may bring about the risk of disability or death. The risk of bone loss in cancer patients increases significantly after receiving radiotherapy and chemotherapy, which not only increases the risk of bone metastasis, but also aggravates the severity of OP. At present, the clinical treatment of osteoporosis mainly uses anti-resorptive therapy, including bisphosphonates and bone morphogenetic protein drugs. These drugs slow bone loss by inhibiting bone resorption, but they need to be used for a long time, and some patients may experience side effects such as gastrointestinal discomfort, muscle pain, and osteonecrosis after stopping the drug.

[0004] Nano drug delivery system is one of the classic drug delivery systems, with good biocompatibility and biodegradability, which can effectively improve the solubility and bioavailability of the loaded drugs. Compared with traditional treatment methods, it can achieve targeted release of drugs in bone tissue and has a more lasting therapeutic effect. Therefore, it is particularly important to develop an intervention measure that can effectively improve the solubility and bioavailability of drugs. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a drug delivery system for treating osteoporosis caused by cellular senescence, a preparation method and applications thereof, and specifically to provide a drug delivery system for treating osteoporosis caused by cellular senescence, a preparation method thereof and applications thereof in the preparation of drugs for treating osteoporosis caused by cellular senescence.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a drug delivery system for treating osteoporosis caused by cell aging, the drug delivery system comprising: a drug carrier modified with a bone-targeting ligand and an anti-osteocellular aging drug encapsulated in the drug carrier;

[0008] The bone targeting ligand is selected from any one of alendronate, bisphosphonates and their analogs, tetracycline, tartrate-resistant acid phosphatase TRAP binding peptide, pregnancy zone protein or e-selectin;

[0009] The anti-osteoblast aging drug includes any one of dasatinib, quercetin, metformin, L-ascorbic acid, aspirin or fisetin, or a combination of at least two of them.

[0010] The specific drug delivery system for treating osteoporosis caused by cell senescence prepared by the present invention can actively target bone tissue while delivering anti-aging drugs, thereby achieving the targeted release of bone tissue drugs. Bone targeting can be achieved by modifying drug carriers with bone targeting ligands. The drug delivery system can effectively improve the bioavailability of anti-osteocellular senescence drugs, can be used for targeted synergistic drug delivery, reduce the local enrichment of senescent cells in bone tissue, and thus reduce bone loss and slow down the progression of osteoporosis.

[0011] Preferably, the drug carrier comprises any one of liposomes, polylactic acid, polylactic acid-coglycolic acid, polycaprolactone, hydroxyapatite, calcium carbonate, calcium hydroxide or exosomes, preferably liposomes;

[0012] Preferably, the bone targeting ligand is alendronate sodium;

[0013] Preferably, the bone-targeting ligand-modified drug carrier is an alendronate-targeted modified liposome carrier;

[0014] Preferably, the anti-osteoblast aging drug comprises a combination of Dasatinib and Quercetin.

[0015] In the present invention, the binding force between liposomes and bone minerals is further enhanced by the introduction of alendronate, so that the drug delivery system has bone targeting, and the targeted modification of alendronate to liposomes is achieved by first modifying alendronate sodium to DSPE-PEG-NHS, and then mixing and reacting with other raw materials for preparing the drug delivery system. This modification method has a higher preparation efficiency than other modification methods. In addition, the drug delivery system can achieve specific accumulation of drugs in bone tissue, thereby improving the local concentration and therapeutic effect of the drugs, and reducing the distribution of drugs in non-target tissues, thereby achieving targeted release of drugs in bone tissue, thereby effectively improving the bioavailability of drugs, reducing potential damage to healthy tissues, and providing an effective technical solution for treating osteoporosis caused by cell aging.

[0016] Preferably, the raw materials for preparing the sodium alendronate targeted modified liposome carrier include: hydrogenated soybean lecithin, sodium alendronate modified DSPE-PEG-NHS, DSPE-PEG2000, and cholesterol.

[0017] Preferably, the mass ratio of the hydrogenated soybean lecithin, DSPE-PEG-NHS modified with sodium alendronate, DSPE-PEG2000, cholesterol, and anti-osteocytic aging drug is (20-40):(2-6):(1-5):(0.2-2):(0.21-2.1);

[0018] The above “20-40” may be, for example, 20, 22, 25, 27, 30, 32, 35, 38, 40, etc.;

[0019] The above-mentioned “2-6” may be, for example, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.;

[0020] The above-mentioned “1-5” may be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0021] The above-mentioned “0.2-2” may be, for example, 0.2, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc.;

[0022] The above-mentioned "0.21-2.1" can be, for example, 0.21, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.8, 2, 2.1, etc. Other specific point values ​​within this numerical range can be selected, and they will not be elaborated here.

[0023] The drug delivery system prepared by the present invention using the raw materials within the above-mentioned specific mass ratio range has a more stable structure, ensuring the stable delivery of the loaded drug, thereby further improving the therapeutic efficiency of the drug.

[0024] Preferably, the mass ratio of dasatinib to quercetin is (0.001-0.1):(0.2-2);

[0025] Among them, the above-mentioned "0.001-0.1" can be, for example, 0.001, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.09, 0.1, etc. Other specific point values ​​within the numerical range can be selected, and they will not be repeated here one by one;

[0026] The above-mentioned "0.2-0.5" can be, for example, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc. Other specific point values ​​within this numerical range can be selected, and they will not be elaborated here one by one.

[0027] The combination of dasatinib and quercetin within a specific mass ratio range has a better therapeutic effect on osteoporosis caused by cell aging.

[0028] Preferably, the particle size of the drug delivery system is 50-200nm, for example, it can be 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0029] In a second aspect, the present invention provides a method for preparing the drug delivery system according to the first aspect, the preparation method comprising the following steps:

[0030] The hydrogenated soybean lecithin, DSPE-PEG-NHS modified with sodium alendronate, DSPE-PEG2000, cholesterol, anti-osteocytic aging drugs and organic solvent are mixed to obtain a mixed solution, and after evaporating and removing the organic solvent to form a film, the film is hydrated and extruded to obtain the drug delivery system;

[0031] The organic solvent includes chloroform and / or methanol.

[0032] Preferably, the preparation method of the alendronate-modified DSPE-PEG-NHS comprises: reacting DSPE-PEG-NHS active lipid with alendronate under alkaline conditions to synthesize alendronate-modified DSPE-PEG-NHS, namely DSPE-PEG-Aln.

[0033] Preferably, the pH of the reaction is 7.5-9, for example, it can be 7.5, 7.6, 7.8, 8, 8.2, 8.5, 8.8, 9, etc. Other specific points within this numerical range can be selected and will not be described here one by one.

[0034] Preferably, the reaction temperature is 22-27°C, for example, it can be 22°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 26°C, 27°C, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0035] Preferably, the reaction time is 20-30h, for example, it can be 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0036] Preferably, the solvent used for the hydration includes PBS solution.

[0037] Preferably, the hydration process is performed with ultrasound.

[0038] Preferably, the hydration time is 8-15 min, for example, it can be 8 min, 8.5 min, 9 min, 9.5 min, 10 min, 10.5 min, 11 min, 11.5 min, 12 min, 12.5 min, 13 min, 13.5 min, 14 min, 14.5 min, 15 min, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0039] Preferably, the extrusion granulation is performed by first passing through a 400 nm polycarbonate membrane and then passing through a 200 nm polycarbonate membrane.

[0040] Preferably, dialysis is performed after the extrusion granulation.

[0041] Preferably, the solvent used for the dialysis includes PBS solution.

[0042] In a third aspect, the present invention provides a use of the drug delivery system according to the first aspect or the preparation method according to the second aspect in preparing a drug for treating osteoporosis caused by cell aging.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The specific drug delivery system for treating osteoporosis caused by cell senescence prepared by the present invention can actively target bone tissue while delivering anti-aging drugs, thereby achieving targeted release of bone tissue drugs. Bone targeting can be achieved by modifying drug carriers with bone-targeting ligands. The drug delivery system can effectively improve the bioavailability of anti-aging drugs, can deliver drugs in a targeted and coordinated manner, reduce the local accumulation of senescent cells in bone tissue, and thus reduce bone loss and slow down the progression of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1-1 is a particle size distribution diagram of the drug-loaded liposomes prepared in Examples 1-3 and Comparative Example 1;

[0046] Figure 1-2 is a scanning electron micrograph of the drug-loaded liposomes prepared in Examples 1-3 and Comparative Example 1;

[0047] Figure 2-1 1 is a graph showing the in vitro binding test results of the drug-loaded liposomes prepared in Example 1 and Comparative Example 1 and hydroxyapatite;

[0048] Figure 2-2 is a graph showing the test results of the binding degree between the drug-loaded liposomes prepared in Example 1 and Comparative Example 1 and hydroxyapatite in mice;

[0049] Figure 3 are the 3D images of the femurs of mice in each group by micro-CT;

[0050] Figure 4 This is a statistical graph of the survival time of each group of mice after drug treatment. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0052] The DSPE-PEG-NHS active lipid involved in the following examples is the product purchased from Aladdin with the product number D163643-500mg; alendronate sodium is the product purchased from Xi'ans with the product number A-21244; DSPE-PEG2000 is the product purchased from HARVEYBIO with the product number 147867-65-0; hydrogenated soybean lecithin is the product purchased from Nippon Seika Co., Ltd. with the product number NO1003; cholesterol is the product purchased from J&K / B&K with the product number 156754;

[0053] Dasatinib was purchased from McLean with the product number D828602-1g; quercetin was purchased from McLean with the product number Q817162-50g; metformin was purchased from McLean with the product model number 1115-70-4; L-ascorbic acid (VC) was purchased from McLean with the product model number 50-81-7; aspirin was purchased from McLean with the product model number 50-78-2; and fisetin was purchased from McLean with the product model number F809597-5g.

[0054] The experimental results were statistically analyzed using Graphpad. **** represents p < 0.0001, ** represents p < 0.01, and * represents p < 0.05.

[0055] Example 1

[0056] This embodiment provides a drug-loaded liposome, and the preparation method thereof is as follows:

[0057] (1) 34 mg of DSPE-PEG-NHS active lipid and 19.5 mg of sodium alendronate were added to a phosphate buffer solution at pH 8 for reaction. After stirring at 25° C. for 24 h, the reaction solution was transferred to a dialysis bag (1000 Da) and dialyzed with deionized water for 3 days. Subsequently, the reaction solution was collected and freeze-dried to obtain a white solid powder of DSPE-PEG-NHS modified with sodium alendronate, namely DSPE-PEG-Aln.

[0058] (2) Hydrogenated soybean lecithin, alendronate-modified DSPE-PEG-NHS, DSPE-PEG2000, cholesterol, dasatinib (D) and quercetin (Q) in a mass ratio of 30:4:2:1:0.008:1.2 were mixed and dissolved in 8 mL of a mixed organic solvent solution of chloroform and methanol (volume ratio of 4:1), and then the organic solvent was rotary evaporated under vacuum conditions. After a yellow film appeared at the bottom of the beaker, it was hydrated with PBS solution (pH 7.4) and ultrasonicated for 10 minutes to obtain crude liposomes. The crude liposomes were repeatedly extruded with 400 nm and 200 nm polycarbonate membranes, and dialyzed in PBS for 10 hours (molecular weight cutoff of 3500 Da) to remove unencapsulated free drugs, thereby obtaining the drug-loaded liposomes, namely Aln-Lipo-DQ.

[0059] Example 2

[0060] This embodiment provides a drug-loaded liposome, and the preparation method thereof is as follows:

[0061] (1) 34 mg of DSPE-PEG-NHS active lipid and 19.5 mg of sodium alendronate were added to a phosphate buffer solution at pH 8.5 for reaction. After stirring at 24°C for 26 h, the reaction solution was transferred to a dialysis bag (1000 Da) and dialyzed with deionized water for 3 days. Subsequently, the reaction solution was collected and freeze-dried to obtain a white solid powder of DSPE-PEG-NHS modified with sodium alendronate, namely DSPE-PEG-Aln.

[0062] (2) Hydrogenated soybean lecithin, DSPE-PEG-Aln, DSPE-PEG2000, cholesterol, dasatinib (D) and quercetin (Q) in a mass ratio of 25:5:3:0.8:0.006:1.5 were mixed and dissolved in 8 mL of a mixed organic solvent solution of chloroform and methanol (volume ratio of 3:1), and then the organic solvent was rotary evaporated under vacuum conditions. After a yellow film appeared at the bottom of the beaker, it was hydrated with PBS solution (pH 7.4) and ultrasonicated for 8 minutes to obtain crude liposomes. The crude liposomes were repeatedly extruded with 400 nm and 200 nm polycarbonate membranes, and dialyzed in PBS for 10 hours (molecular weight cutoff of 3500 Da) to remove unencapsulated free drugs, thereby obtaining the drug-loaded liposomes, namely Aln-Lipo-DQ.

[0063] Example 3

[0064] This embodiment provides a drug-loaded liposome, and the preparation method thereof is as follows:

[0065] (1) 34 mg of DSPE-PEG-NHS active lipid and 19.5 mg of sodium alendronate were added to a phosphate buffer solution at pH 8.5 for reaction. After stirring at 28°C for 20 h, the reaction solution was transferred to a dialysis bag (1000 Da) and dialyzed with deionized water for 3 days. Subsequently, the reaction solution was collected and freeze-dried to obtain a white solid powder of DSPE-PEG-NHS modified with sodium alendronate, namely DSPE-PEG-Aln.

[0066] (2) Hydrogenated soybean lecithin, DSPE-PEG-Aln, DSPE-PEG2000, cholesterol, dasatinib (D) and quercetin (Q) in a mass ratio of 35:5:1:1.5:0.003:1 were mixed and dissolved in 8 mL of a mixed organic solvent solution of chloroform and methanol (volume ratio of 5:1), and then the organic solvent was rotary evaporated under vacuum conditions. After a yellow film appeared at the bottom of the beaker, it was hydrated with PBS solution (pH 7.4) and ultrasonicated for 12 minutes to obtain crude liposomes. The crude liposomes were repeatedly extruded with 400 nm and 200 nm polycarbonate membranes, and dialyzed in PBS for 10 hours (molecular weight cutoff of 3500 Da) to remove unencapsulated free drugs, thereby obtaining the drug-loaded liposomes, namely Aln-Lipo-DQ.

[0067] Comparative Example 1

[0068] This comparative example provides a drug-loaded liposome, and its preparation method is different from that of Example 1 only in that step (1) is not performed, and the alendronate-modified DSPE-PEG-NHS in step (2) is replaced by DSPE-PEG2000 of equal molar mass that is not modified with alendronate and mixed with other preparation raw materials to react, and the drug-loaded liposome prepared is Lipo-DQ.

[0069] Test Example 1

[0070] In this test example, the particle size of the drug-loaded liposomes prepared in Examples 1-3 and Comparative Example 1 was tested by dynamic light scattering, and the surface morphology of the drug-loaded liposomes was analyzed by SEM electron microscopy. Figure 1-1 (Particle size distribution) and Figure 1-2 (Electron microscope image).

[0071] The results showed that the drug-loaded liposomes prepared in each group showed a good polydispersity index, and the spherical morphology of the drug-loaded liposomes was verified by transmission electron microscopy images, and the particle size was close to 200nm.

[0072] Test Example 2

[0073] This test example tests the in vitro binding of liposomes to hydroxyapatite (HAp)

[0074] (1) Test sample:

[0075] Aln-Lipo-Cy5.5: The preparation method thereof is different from that of Example 1 only in that dasatinib and quercetin are replaced with Cy5.5 fluorescent dye;

[0076] Lipo-Cy5.5: The preparation method thereof is different from that of Comparative Example 1 only in that dasatinib and quercetin are replaced by Cy5.5 fluorescent dye.

[0077] (2) Test method: Aln-Lipo-Cy5.5 and Lipo-Cy5.5 (0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL) of different concentrations were incubated with HAp at 25°C for 4 h. After the incubation, the mixture was centrifuged and separated into two parts: supernatant and HAp precipitate. The fluorescence intensity of Aln-Lipo-Cy5.5 and Lipo-Cy5.5 in the HAp supernatant was measured using a fluorescence spectrophotometer to test the binding rate of drug-loaded liposomes with HAp. The results are as follows: Figure 2-1 shown.

[0078] As can be seen from the figure, the binding rate of the drug-loaded liposomes prepared in the present invention with HAp is as high as 60%, while the binding rate of the drug-loaded liposomes prepared in Comparative Example 1 with HAp is only 10%. Hydroxyapatite is the main component of bone matrix. Through the degree of binding between the drug-loaded liposomes and hydroxyapatite, it can be known that the drug-loaded liposomes prepared in the present invention have excellent bone targeting, so as to increase the concentration of the drug in the target site and enhance the therapeutic effect.

[0079] (2) In vivo binding degree between the drug-loaded liposomes prepared in Example 1 and Comparative Example 1 and hydroxyapatite (HAp).

[0080] Six 8-week-old mice (C57bl / 6, male) were selected and divided into Example 1 group and Comparative Example 1 group, with 3 mice in each group. 200 μL of 5 mg / mL concentration of Aln-Lipo-Cy5.5 and Lipo-Cy5.5 liposome suspensions were injected intravenously into the tail of the mice, respectively. The distribution of Cy5.5 fluorescence signals in the femur and tibia of the mice was detected by in vivo imaging system (IVIS) on the 1st day and the 7th day after injection. The results are shown in FIG. Figure 2-2 shown.

[0081] Depend on Figure 2-2 It can be seen that on the 1st day and the 7th day after injection, the fluorescence intensity of the liposomes in the femur and tibia of the mice in the Example 1 group was significantly higher than that in the Comparative Example 1. It can be seen that the liposomes prepared by the present invention have excellent bone targeting, and can deliver drugs to bone tissue in a targeted manner, thereby achieving specific accumulation of drugs in bone tissue, thereby improving the local concentration of drugs and therapeutic effects.

[0082] Test Example 3

[0083] This test example tests the therapeutic effects of the drug-loaded liposomes prepared in Example 1 and Comparative Example 1 on osteoporotic mice.

[0084] (a) Animal modeling and grouping

[0085] Dox

[0086] Doxorubicin was dissolved in physiological saline and then sterilized by filtration through a 0.22 μm filter membrane. Twenty 8-week-old mice (C57bl / 6, male) were selected for modeling, and 5 mg / kg of doxorubicin solution was intraperitoneally injected once a week for 4 consecutive weeks to obtain an osteoporosis mouse model.

[0087] The mice were randomly divided into 4 groups: (1) model group: mice were injected with 200 μL of normal saline via tail vein; (2) conventional treatment group: mice were orally administered with 5 mg / kg (body weight) of dasatinib and 50 mg / kg (body weight) of quercetin; (3) comparative example 1 group: mice were injected with 200 μL of 12 mg / mL concentration of the drug-loaded liposome suspension prepared in comparative example 1 via tail vein; (4) example 1 group: mice were injected with 200 μL of 12 mg / mL concentration of the drug-loaded liposome suspension prepared in example 1 via tail vein; and a control group was set up consisting of 8-week-old mice (C57bl / 6, male) without modeling injected with 200 μL of normal saline.

[0088] Induction radiation (IR)

[0089] Radiation (IR) therapy-induced bone aging model: 8-week-old mice (C57bl / 6, male) were anesthetized and placed under an irradiator with only their legs exposed. The rest of the mouse body parts were covered with lead plates and irradiated at a dose of 15 Gy. Drug treatment was performed three days later.

[0090] The mice were randomly divided into 4 groups: (1) model group: mice were injected with 200 μL normal saline via tail vein; (2) conventional treatment group: mice were orally administered with 5 mg / kg (body weight) dasatinib and 50 mg / kg (body weight) quercetin; (3) comparative example 1 group: mice were injected with 200 μL of the drug-loaded liposome suspension prepared in comparative example 1 via tail vein; (4) example 1 group: mice were injected with 200 μL of the drug-loaded liposome suspension prepared in example 1 via tail vein; and a control group was set up consisting of 8-week-old mice (C57bl / 6, male) without modeling and injected with 200 μL normal saline.

[0091] Bilateral ovariectomy (OVX) model

[0092] Eight-week-old bilaterally ovariectomized model mice were randomly divided into four groups: (1) model group: mice were injected with 200 μL of normal saline via tail vein; (2) conventional treatment group: mice were orally administered with 5 mg / kg (body weight) of dasatinib and 50 mg / kg (body weight) of quercetin; (3) comparative example 1 group: mice were injected with 200 μL of the drug-loaded liposome suspension prepared in comparative example 1 via tail vein; (4) example 1 group: mice were injected with 200 μL of the drug-loaded liposome suspension prepared in example 1 via tail vein; and a control group was set up consisting of 8-week-old wild-type mice (C57bl / 6, male) injected with 200 μL of normal saline.

[0093] Aged mice model

[0094] Wild-type (C57bl / 6, male) mice aged 22 months were randomly divided into 4 groups: (1) wild-type group: mice were injected with 200 μL of normal saline via tail vein; (2) conventional treatment group: mice were orally administered with 5 mg / kg (body weight) of dasatinib and 50 mg / kg (body weight) of quercetin; (3) comparative example 1 group: mice were injected with 200 μL of the drug-loaded liposome suspension prepared in comparative example 1 via tail vein; (4) example 1 group: mice were injected with 200 μL of the drug-loaded liposome suspension prepared in example 1 via tail vein.

[0095] (b) Experimental data collection and analysis

[0096] After the treatment, the mice in each group of experiments were dissected, the femurs were collected, fixed in 4% paraformaldehyde for 24 hours, and then placed vertically in a 12mmde scanning tube. The distal femoral epiphysis was scanned using Skyscan 1176 (Skyscan). All scanning parameters are as follows: the scanner voltage is 50kV, the current is 200μA, and the image resolution is set to 6μm per pixel. After the scan, 200 slice thicknesses out of 150 slices below the growth plate were selected for analysis and 3D reconstruction. Data analysis software (CTAn) was used to determine the trabecular volume fraction (BV / TV; %), trabecular number (Tb.N; 1 / mm), trabecular separation (Tb.Th; mm), trabecular thickness (Tb.Th; mm), and structural model index (SMI). Subsequently, further three-dimensional reconstruction was performed using image reconstruction software, and the results are shown in the figure. Figure 3 As shown. Figure 3 The results showed that significant osteoporosis and bone loss occurred in the bone tissue of mice treated with doxorubicin or radiotherapy, as well as bilaterally ovariectomized model mice and elderly mice, while the bone loss of each group of mice treated with drugs was improved. More complete trabecular bone results were observed in the treated mice in the Example 1 group, and the micro-CT (μCT) 3D image of its femur was closest to the femur tissue of normal mice in the control group. It can be seen that the drug delivery system of the drug-loaded liposomes prepared in Example 1 has excellent effects in promoting bone formation, inhibiting bone resorption, and restoring the balance of the bone remodeling process.

[0097] Test Example 4

[0098] This test case tests the survival of irradiated mice after drug treatment.

[0099] Forty-eight 8-week-old mice (C57bl / 6, male) were anesthetized and placed in an irradiator and irradiated at a dose of 8 Gy.

[0100] The mice were randomly divided into 6 groups: S1: model group, mice were injected with 200 μL normal saline via tail vein; S2: D+Q group, mice were orally administered with 5 mg / kg (body weight) dasatinib and 50 mg / kg (body weight) quercetin; S3: fisetin group, mice were orally administered with 100 mg / kg fisetin; S4: aspirin group, mice were orally administered with 50 mg / kg aspirin; S5: L-ascorbic acid group, mice were orally administered with 100 mg / kg L-ascorbic acid; S6: In the metformin group, mice were orally administered 250 mg / kg of metformin; at the same time, a control group was set up, which consisted of 8-week-old mice (C57bl / 6, male) that were not irradiated and orally administered 200 μL of normal saline. The mice in each group were cultured in an environment with a temperature of 22-25°C for 25 days, during which they were free to eat and drink. The survival of the mice in each group was observed, and the survival rate of the mice was calculated. Among them, the day of irradiation was set as day 0, and the mice were given drug treatment on days -2, 1, and 4, respectively. The statistical results are as follows Figure 4 shown.

[0101] Depend on Figure 4 The results showed that compared with the S1 group (model group), each drug treatment group significantly improved the survival rate of irradiated mice. Among them, the S5 group (L-ascorbic acid group) showed the most significant survival advantage, with the survival rate increased to 50%, much higher than the 0% of the S1 group (model group). The survival rates of the S2 group (D+Q group) and the S4 group (aspirin group) were both 37.5%, which were also significantly higher than those of the model group. The survival rates of the S3 group (fisetin group) and the S6 group (metformin group) increased slightly, both at 25%, but were still better than the model group. Overall, each treatment group improved the survival rate of irradiated mice, especially the L-ascorbic acid group, which performed most significantly, and the D+Q group also demonstrated good intervention potential for radiotherapy-induced aging and related complications.

[0102] In summary, the specific drug delivery system for treating osteoporosis caused by cell senescence prepared by the present invention can actively target bone tissue while delivering anti-aging drugs, thereby achieving targeted release of bone tissue drugs. Bone targeting can be achieved by modifying drug carriers with bone-targeting ligands. The drug delivery system can effectively improve the bioavailability of anti-aging drugs, can deliver drugs in a targeted and coordinated manner, reduce the local enrichment of senescent cells in bone tissue, and thus reduce bone loss and slow down the progression of osteoporosis.

[0103] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0104] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0105] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A drug delivery system for treating osteoporosis caused by cell aging, characterized in that: The drug delivery system comprises: a drug carrier modified with a bone-targeting ligand and an anti-osteocytic aging drug contained in the drug carrier; The bone targeting ligand is selected from any one of alendronate, bisphosphonates and their analogs, tetracycline, tartrate-resistant acid phosphatase TRAP binding peptide, pregnancy zone protein or e-selectin; The anti-osteoblast aging drug includes any one of dasatinib, quercetin, metformin, L-ascorbic acid, aspirin or fisetin, or a combination of at least two of them.

2. The drug delivery system according to claim 1, characterized in that The drug carrier comprises any one of liposomes, polylactic acid, polylactic acid-coglycolic acid, polycaprolactone, hydroxyapatite, calcium carbonate, calcium hydroxide or exosomes, preferably liposomes; Preferably, the bone targeting ligand is alendronate sodium; Preferably, the bone-targeting ligand-modified drug carrier is an alendronate-targeted modified liposome carrier; Preferably, the anti-osteoblast aging drug comprises a combination of dasatinib and quercetin.

3. The drug delivery system according to claim 2, characterized in that The raw materials for preparing the sodium alendronate targeted modified liposome carrier include: hydrogenated soybean lecithin, sodium alendronate modified DSPE-PEG-NHS, DSPE-PEG2000, and cholesterol.

4. The drug delivery system according to claim 2 or 3, characterized in that The mass ratio of the hydrogenated soybean lecithin, DSPE-PEG-NHS modified with sodium alendronate, DSPE-PEG2000, cholesterol, and the anti-osteocytic aging drug is (20-40):(2-6):(1-5):(0.2-2):(0.21-2.1); Preferably, the mass ratio of dasatinib to quercetin is (0.001-0.1):(0.2-2).

5. The drug delivery system according to any one of claims 1 to 4, characterized in that The particle size of the drug delivery system is 50-200 nm.

6. A method for preparing a drug delivery system according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: The hydrogenated soybean lecithin, DSPE-PEG-NHS modified with sodium alendronate, DSPE-PEG2000, cholesterol, anti-osteocytic aging drugs and organic solvent are mixed to obtain a mixed solution, and after evaporating and removing the organic solvent to form a film, the film is hydrated and extruded to obtain the drug delivery system; The organic solvent includes chloroform and / or methanol.

7. The preparation method according to claim 6, characterized in that The preparation method of the alendronate sodium modified DSPE-PEG-NHS comprises: reacting the DSPE-PEG-NHS active lipid with alendronate sodium under alkaline conditions to synthesize the alendronate sodium modified DSPE-PEG-NHS, i.e., DSPE-PEG-Aln; Preferably, the pH of the reaction is 7.5-9; Preferably, the reaction temperature is 22-27°C; Preferably, the reaction time is 20-30 hours.

8. The preparation method according to claim 6, characterized in that: The solvent used for the hydration includes PBS solution; Preferably, the hydration process is performed by ultrasound; Preferably, the hydration time is 8-15 min; Preferably, the extrusion granulation is firstly passed through a 400nm polycarbonate membrane and then passed through a 200nm polycarbonate membrane.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The extrusion granulation is followed by dialysis; Preferably, the solvent used for the dialysis includes PBS solution.

10. Use of the drug delivery system according to any one of claims 1 to 5 or the preparation method according to any one of claims 6 to 9 in preparing a drug for treating osteoporosis caused by cell senescence.

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