Compositions for targeted delivery of chemotherapeutic drugs to bone marrow and methods of making and using the same

Abstract

This invention belongs to the field of bone marrow targeted technology, specifically relating to a composition for bone marrow-targeted delivery of chemotherapeutic drugs, its preparation method, and its application. The composition for bone marrow-targeted delivery of chemotherapeutic drugs includes anionic phospholipids, cholesterol, drugs, an organic solvent phase, and an aqueous phase. The preparation method includes: oil phase preparation: dissolving anionic phospholipids, cholesterol, and drugs in an organic solvent to achieve a lipid concentration of 10-30 mg / mL; aqueous phase preparation: obtaining a clear aqueous solution; nanoparticle preparation by injection method: adding the oil phase dropwise to the aqueous phase, homogenizing and emulsifying, and then homogenizing again to obtain a nanoparticle suspension; nanoparticle preparation by thin-film dispersion method: removing the organic solvent using a rotary evaporator to form a uniform thin film, adding the aqueous phase dropwise to the oil phase, homogenizing and emulsifying, and then homogenizing again to obtain a nanoparticle suspension; purification: ultrafiltration, centrifugation, washing, and resuspending to obtain a purified anionic lipid nanoparticle composition. This invention, through ligand-free preparation and utilizing the charge properties of the anionic lipids themselves, enables efficient and specific delivery of chemotherapeutic drugs to the bone marrow, while maintaining good safety and stability.

Description

Technical Field

[0001] This invention belongs to the field of bone marrow targeting technology, specifically relating to compositions for bone marrow-targeted delivery of chemotherapeutic drugs, their preparation methods, and applications. Background Technology

[0002] Chemotherapy is a primary treatment for hematologic malignancies, such as acute myeloid leukemia (AML) and multiple myeloma (MM). However, conventionally administered intravenously distributed chemotherapy drugs lack tissue specificity, leading to severe systemic toxicity (such as cardiotoxicity, neurotoxicity, and bone marrow suppression). Furthermore, the drug concentration reaching the bone marrow lesion is limited, affecting treatment efficacy. Bone marrow is a vital hematopoietic and immune organ, but bone marrow-related diseases such as AML, myelodysplastic syndromes (MDS), and MM are difficult to treat, mainly due to the presence of the bone marrow-blood barrier, which makes it difficult for conventional drugs to effectively target bone marrow tissue. While existing chemotherapy drugs (such as anthracyclines and anthraquinones) have some efficacy, systemic administration easily causes side effects such as bone marrow suppression and cardiotoxicity, and the low drug concentration in the bone marrow easily leads to drug resistance and relapse.

[0003] Liposomes, as a nanomedicine delivery system, can enhance drug accumulation in tumor tissues to some extent by improving penetration and retention effects. However, traditional long-circulating liposomes (such as liposomes encapsulating doxorubicin) still have insufficient targeting ability for bone marrow. Nanocarriers such as liposomes have been explored for bone marrow targeting, but most rely on targeting peptides or antibody modifications (such as the DT7 peptide in patent CN114344276A and the bispecific antibody in patent CN115887491A), which are costly and complex in process. Anionic liposomes (such as patent CN117159402A) are used in cosmetics, but their repair efficacy does not involve bone marrow targeting and does not utilize negative charge properties. The bone marrow microenvironment is rich in positively charged components such as calcium ions and alkaline phosphatase. Anionic lipids can achieve targeting through electrostatic interactions, but current technologies have not systematically optimized this strategy. In addition, bone marrow-related diseases are diverse, including AML, MM, MDS, osteomyelitis, osteoporosis, etc., but current technologies lack broad-spectrum targeting platforms. Summary of the Invention

[0004] To address the problems in the prior art, this invention provides a composition for bone marrow-targeted delivery of chemotherapeutic drugs, its preparation method, and its application. By using a lipid nanosystem with the charge properties of anionic lipids without ligand modification, the invention achieves efficient and specific drug delivery to the bone marrow while maintaining good safety and stability.

[0005] The technical problem solved by this invention is achieved by the following technical solution:

[0006] The present invention aims to provide a composition for bone marrow-targeted delivery of chemotherapeutic drugs, comprising anionic phospholipids, cholesterol, drugs, an organic solvent phase and an aqueous phase.

[0007] Furthermore, by weight percentage, the composition comprises 3%-12% anionic phospholipids, 1%-5% cholesterol, 1%-20% pharmaceuticals, 5%-30% organic solvents, and the balance water, and the composition has a zeta potential of -10mV to -40mV.

[0008] Furthermore, the anionic phospholipid is at least one of the following: phosphatidylserine series, phosphatidylglycerol derivatives, cardiolipin variants, phosphate ester derivatives, and sulfonic acid modified phospholipids; the cholesterol is at least one of the following: cholesterol sulfate derivatives, carboxylic acid modified cholesterol, and carboxylic acid modified cholesterol.

[0009] The drug is selected from at least one of small molecule chemotherapy drugs, biological agents and macromolecule drugs, kinase inhibitors and anti-infective drugs;

[0010] The organic solvent phase can be chloroform, anhydrous ethanol, dichloromethane, methanol, a chloroform-methanol mixture, tert-butanol, or ethanol, with a concentration of 50%-100%. The aqueous phase can be 50-300 mM ammonium sulfate solution, 3-5 mM sucrose octaphosphate solution, phosphate buffer (PBS, pH 7.4), HEPES buffer, physiological saline (0.9% NaCl), or deionized water.

[0011] A method for preparing a composition for bone marrow-targeted delivery of chemotherapy drugs includes the following steps:

[0012] S1. Oil phase preparation: Anionic phospholipids, cholesterol, and drugs are dissolved in an organic solvent in proportion to form a homogeneous lipid mixture solution.

[0013] S2. Preparation of aqueous phase: Stir the aqueous phase until it is completely dissolved to obtain a clear aqueous solution;

[0014] S3. Preparation of nanoparticles: Nanoparticles are prepared by injecting or dispersing the aqueous and oil phases using the injection method or thin film dispersion method.

[0015] S4. Purification: Ultrafiltration centrifugation, washing and resuspending to obtain purified anionic lipid nanocomposite.

[0016] Furthermore, in the oil phase preparation, the molar ratio of anionic phospholipid:cholesterol:drug is 70-85:15-25:5-10.

[0017] Furthermore, in the oil phase preparation, the concentration of the lipid mixture solution formed is 10-30 mg / mL.

[0018] Furthermore, in the preparation of the aqueous phase: prepare 150-300 mM ammonium sulfate solution, 3-5 mM sucrose octaphosphate solution, phosphate buffer (PBS, pH 7.4), HEPES buffer, physiological saline (0.9% NaCl) or deionized water.

[0019] Furthermore, the method for preparing nanoparticles using the injection method includes: adding the aqueous phase dropwise to the oil phase under stirring at 300-500 rpm, with the dropwise addition rate controlled in the range of 1-6 mL / min; then homogenizing at 5000-10000 rpm for 3-5 minutes to preliminarily emulsify the mixture; and further circulating the mixture 2-3 times under a high-pressure microjets at a pressure of 20000-25000 PSI to obtain a nanoparticle suspension with uniform particle size.

[0020] Furthermore, the method for preparing nanoparticles using the thin-film dispersion method includes: removing the organic solvent from the oil phase using a rotary evaporator, allowing the lipids to form a uniform, thin, and transparent lipid film on the inner wall of the flask. While stirring at 300-500 rpm, the aqueous phase is added dropwise to the oil phase at a rate controlled within the range of 1-6 mL / min. The mixture is then homogenized at 5000-10000 rpm for 3-5 minutes to initially emulsify the mixture. Further, the mixture is circulated 2-3 times using a high-pressure microfluidic homogenizer at a pressure of 20000-25000 PSI to obtain a suspension of nanoparticles with uniform particle size.

[0021] Further purification involves centrifuging at 4000-6000 ×g for 10-20 minutes using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10-100 kDa to remove unencapsulated drugs and free components. The mixture is then washed 2-3 times with PBS and finally resuspended in 8-20% sucrose solution to obtain the purified anionic lipid nanocomposite.

[0022] The use of compositions for bone marrow-targeted delivery of chemotherapeutic drugs or methods for preparing such compositions in the preparation of drugs for treating acute myeloid leukemia, multiple myeloma, or osteoporosis.

[0023] Composition of the composition for bone marrow-targeted delivery of chemotherapy drugs:

[0024] By weight percentage, it includes anionic phospholipids (3%-12%), cholesterol (1%-5%), drugs (1%-20%), organic solvent concentration (5%-30%), and the balance water. The zeta potential ranges from -10mV to -40mV, and this range is also an important factor in achieving bone marrow targeting.

[0025] Types of anionic phospholipids:

[0026] Naturally derived anionic phospholipids include: phosphatidylserine (PS) series (such as soybean-derived phosphatidylserine, brain-derived phosphatidylserine, synthetic dipalmitoylphosphatidylserine, and dioleoylphosphatidylserine), phosphatidylglycerol (PG) derivatives (such as distearylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, and palmitoyloleoylphosphatidylglycerol), cardiolipin variants (such as bovine heart-derived cardiolipin, synthetic cardiolipin, and hydrogenated cardiolipin), phosphatidic acid (PA), phosphate ester derivatives, and sulfonic acid-modified phospholipids.

[0027] Synthetic anionic phospholipids: phosphate ester derivatives (dipalmitoyl phosphatidic acid, dioleoyl phosphatidic acid, phosphatidylinositol and their phosphorylated derivatives), sulfonic acid modified phospholipids (cerebrothiolipin, synthetic sulfonic acid phospholipids), etc.

[0028] Types of anionic cholesterol:

[0029] Cholesterol sulfate derivatives: cholesterol sulfate, cholesterol monosuccinate (CHEMS), cholesterol monosuccinate, cholesterol phosphate, etc.

[0030] Cholesterol modified with carboxylic acids: cholesterol carboxylic acid derivatives, polyethylene glycol-modified anionic cholesterol, etc.

[0031] Types of drugs:

[0032] Small molecule chemotherapy drugs include: anthracyclines (daunorubicin, doxorubicin, vincristine), anthraquinones (such as doxorubicin, epirubicin, daunorubicin, mitoxantrone, loxoantrone), topoisomerase inhibitors (irinotecan, topotecan, etoposide), alkylating agents (such as cyclophosphamide, melphalan, bendamustine), antimetabolites (such as gemcitabine, cytarabine, 5-fluorouracil), anti-inflammatory and immunomodulatory drugs (such as dexamethasone, prednisolone, indomethacin, celecoxib, methotrexate, leflunomide), and metabolic regulators (teriparatide, zoledronic acid, alendronate sodium), etc.

[0033] Biologics and macromolecular drugs: proteasome inhibitors (such as bortezomib, carfilzomib, ixazomib), immunomodulators (such as lenalidomide, pomalidomide, thalidomide), kinase inhibitors (such as imatinib, dasatinib, ruxolitinib), antibody drugs (such as trastuzumab, rituximab, daratumumab, denosumab), etc.

[0034] Anti-infective drugs: antibiotics (such as vancomycin, cefazolin, levofloxacin, clindamycin), antifungal drugs (such as amphotericin B, voriconazole, caspofungin).

[0035] Preparation method:

[0036] Oil phase preparation: Lipids and drugs are dissolved in an organic solvent to form a film. Anionic phospholipids, cholesterol, and drugs are dissolved in an organic solvent in a ratio (usually a molar ratio of anionic phospholipids:cholesterol:drug = 70-85:15-25:5-10) to achieve a lipid mixture solution concentration of 10-30 mg / mL.

[0037] Aqueous phase preparation: Prepare 150-300 mM ammonium sulfate solution, 3-5 mM sucrose octaphosphate solution, phosphate buffer (PBS, pH 7.4), HEPES buffer, physiological saline (0.9% NaCl) or deionized water, and stir until completely dissolved to obtain a clear aqueous phase solution.

[0038] Nanoparticle formation by injection: The aqueous phase is added dropwise to the oil phase under stirring at 300-500 rpm, with the drop rate controlled in the range of 1-6 mL / min. Then, the mixture is homogenized at 5000-10000 rpm for 3-5 minutes to initially emulsify the mixture. The mixture is then further circulated 2-3 times under a high-pressure microjets at a pressure of 20000-25000 PSI to obtain a suspension of nanoparticles with uniform particle size.

[0039] Nanoparticle formation via thin-film dispersion: The organic solvent in the oil phase is removed by rotary evaporation, allowing the lipids to form a uniform, thin, and transparent lipid film on the inner wall of the flask. The aqueous phase is then added dropwise to the oil phase while stirring at 300-500 rpm, with the addition rate controlled in the range of 1-6 mL / min. The mixture is then homogenized at 5000-10000 rpm for 3-5 minutes to initially emulsify the mixture. Further homogenization is achieved by circulating the mixture 2-3 times using a high-pressure microfluidic homogenizer at 20000-25000 PSI pressure to obtain a suspension of nanoparticles with uniform particle size.

[0040] Purification: Ultrafiltration centrifugation. Centrifuge at 4000-6000 ×g for 10-20 minutes using ultrafiltration centrifuge tubes (molecular weight cutoff 10-100 kDa) to remove unencapsulated drug and free components. Wash 2-3 times with PBS (pH 7.4), and finally resuspend in 8-20% sucrose solution to obtain the purified anionic lipid nanocomposite.

[0041] Expanded indications:

[0042] The composition is suitable for the following bone marrow-related diseases, all of which benefit from a dual-targeting mechanism:

[0043] Malignant hematologic disorders: Acute myeloid leukemia (AML), multiple myeloma (MM), myelodysplastic syndromes (MDS), myelofibrosis, and lymphoma with bone marrow involvement.

[0044] Benign bone marrow diseases: osteoporosis, osteomyelitis, bone marrow edema syndrome, bone marrow necrosis.

[0045] Other: Post-bone marrow transplant complications (such as GVHD), bone marrow involvement in autoimmune diseases, and hereditary bone marrow diseases (such as thalassemia).

[0046] The expanded indications are based on the characteristics of the bone marrow microenvironment and the homing mechanism of inflammatory cells, and experiments have shown improved targeting efficiency.

[0047] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0048] 1. The composition of the present invention for bone marrow-targeted delivery of chemotherapy drugs does not rely on exogenous ligand modification. It achieves preferential distribution to the bone marrow through the surface and interface characteristics of the anionic lipid system itself, and has a highly efficient bone marrow (bone marrow microenvironment, bone marrow cavity) targeting ability. It can be applied to the preparation of drugs for treating bone marrow-related diseases and expand its indications.

[0049] 2. The preparation method of the composition for bone marrow-targeted delivery of chemotherapy drugs of the present invention is applicable to various drug loading processes. By adjusting the component ratio and process parameters (such as temperature, pressure, etc.), the encapsulation efficiency (typically > 90%) and particle size (50-150 nm) can be optimized. It exhibits high stability, with a particle size change of < 10% after 3 months of storage at 4°C.

[0050] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0051] Figure 1 This is a TEM image of the anionic lipid nanoparticles with cholesterol succinate monoester (CHEMS) as a component in this invention.

[0052] Figure 2 The particle size (a) and potential diagram (b) of the anionic lipid nanoparticles with CHEMS as a component in this invention are shown.

[0053] Figure 3This is a TEM image of the anionic lipid nanoparticles of the present invention, which are composed of sodium 1,2-distearate-sn-glycerol-3-phosphate glycerol (DSPG).

[0054] Figure 4 This is a particle size potential diagram of anionic lipid nanoparticles with DSPG as a component in this invention.

[0055] Figure 5 This is an image showing the uptake of anionic lipid nanoparticles in C1498 cells in Example 5 of the present invention.

[0056] Figure 6 This is a diagram showing the cytotoxicity of anionic lipid nanoparticles on C1498 cells in Example 6 of the present invention.

[0057] Figure 7 For in vivo targeting verification in Example 7 of the present invention: in vivo imaging (a); tissue fluorescence image (b).

[0058] Figure 8 Figure (a) shows the weight changes of each group of AML mice in Example 8 of this invention, and figure (b) shows the spleen weight.

[0059] Figure 9 The figures show the blood biochemical tests (a) and blood routine tests (b) of each group of AML mice in Example 8 of this invention.

[0060] Figure 10 The present invention includes flow cytometry measurements of cell invasion in spleen (Sp), bone marrow (BM), and peripheral blood (PB) (a); quantitative analysis of invasive leukemia cells (bd); and white blood cell count in peripheral blood of AML mice (e). Detailed Implementation

[0061] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0062] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0063] Example 1: Preparation of daunorubicin lipid nanoparticles with CHEMS as a component

[0064] Step (1) Dissolve DSPC, CHEMS, and MPEG-DSPE2000 in anhydrous ethanol at a molar ratio of 79:20:1 to make the lipid mixture solution concentration reach approximately 20 mg / mL.

[0065] Step (2) At 55°C, the lipid solution was rapidly injected into a solution containing 250 mM ammonium sulfate and continuously stirred in a water bath for 30 min. The prepared empty liposomes were cooled and then filtered through a 0.22 μm filter membrane.

[0066] Step (3) Place the above liposomes in a dialysis bag with a molecular weight cutoff of 10 kDa, use 10% sucrose solution as the dialysis medium, dialyze overnight, change the solution every 5 hours to replace the external aqueous phase, and establish a pH gradient inside and outside the lipid membrane.

[0067] In step (4), the phospholipid to daunorubicin (DNR) mass ratio is 10:1. The DNR solution is added to the liposomes in step (3). This process is carried out by stirring continuously in a 60°C water bath for 30 min to obtain anionic lipid nanoparticles (CHEMS-LNP) loaded with daunorubicin.

[0068] Example 2: Characterization of anionic lipid nanoparticles with CHEMS as a component

[0069] I. Observation of lipid nanoparticle morphology under transmission electron microscopy

[0070] A small amount of lipid nanoparticles was dropped onto a copper grid covered with a carbon film. After drying at room temperature, 2.0% phosphotungstic acid was added for staining for 5 min. After allowing it to dry naturally, it was observed under a transmission electron microscope and photographed. The electron microscope images are shown below. Figure 1 As shown, liposomes contain a single lipid membrane and have a spherical appearance.

[0071] II. Determination of Particle Size and Potential

[0072] Take an appropriate amount of the lipid nanoparticle solution to be tested, dilute it 10 times with pure water, and then use a particle size potentiometer to investigate and measure its particle size, polydispersity index (PDI), and zeta potential. Figure 2 This indicates that the daunorubicin lipid nanoparticles have a particle size of approximately 100 nm, a PDI of less than 0.2, and a Zeta potential of -20 mV.

[0073] Example 3: Preparation of carfilzomib lipid nanoparticles with DSPG as a component

[0074] Step (1) Dissolve DSPC, DSPG, and MPEG-DSPE2000 in anhydrous ethanol at a molar ratio of 75:20:5 to make the lipid mixture solution concentration reach approximately 20 mg / mL.

[0075] Step (2) At 55°C, the lipid solution was rapidly injected into a solution containing 250 mM ammonium sulfate and continuously stirred in a water bath for 30 min. The prepared empty liposomes were cooled and then filtered through a 0.22 μm filter membrane.

[0076] Step (3) Place the above liposomes in a dialysis bag with a molecular weight cutoff of 10 kDa, use 10% sucrose solution as the dialysis medium, dialyze overnight, change the solution every 5 h to replace the external aqueous phase, and establish a pH gradient inside and outside the lipid membrane.

[0077] In step (4), the carfilzomib solution was added to the liposomes from step (3) with a mass ratio of 10:1 of phospholipid to carfilzomib. This process was carried out by stirring continuously in a 60°C water bath for 30 min to obtain anionic lipid nanoparticles (Carfilzomib-LNP) loaded with carfilzomib.

[0078] Example 4: Characterization of anionic lipid nanoparticles with DSPG as a component

[0079] I. Observation of lipid nanoparticle morphology under transmission electron microscopy

[0080] A small amount of lipid nanoparticles was dropped onto a copper grid covered with a carbon film. After drying at room temperature, 2.0% phosphotungstic acid was added for staining for 5 min. After allowing it to dry naturally, it was observed under a transmission electron microscope and photographed. The electron microscope images are shown below. Figure 3 As shown, liposomes contain a single lipid membrane and have a spherical appearance.

[0081] II. Determination of Particle Size and Potential

[0082] Take an appropriate amount of the lipid nanoparticle solution to be tested, dilute it 10 times with pure water, and then use a particle size potentiometer to investigate and measure its particle size, polydispersity index (PDI), and zeta potential. Figure 4 This indicates that the carfilzomili lipid nanoparticles have a particle size of approximately 100 nm, a PDI of less than 0.2, and a zeta potential of -20 mV.

[0083] Example 5: Uptake efficiency of anionic lipid nanoparticles in inflammatory cells

[0084] In T25 flasks containing 80%-90% RAW264.7 cells, cells were mixed by blowing off 2 mL of serum-free DMEM medium. 5 μL of cell suspension was added to six wells of a 96-well plate. After overnight incubation, the cells adhered. The supernatant was removed, and the cells were washed three times with PBS. 100 μL of free DNR, Chol-LNP, and CHEMS-LNP were added to two wells of each group (final DNR concentration 5 μg / mL). After incubation for 4 h, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of 4% paraformaldehyde was added to each well for fixation for 15 min. The supernatant was discarded, and the cells were washed three times with PBS. 100 μL of prepared DAPI staining solution was added, and staining was performed for 10 min. The supernatant was discarded, and the cells were washed three times with PBS. Finally, 100 μL of PBS was added to each well, and fluorescence images were captured using an inverted microscope.

[0085] Figure 5 This indicates that, compared with free DNR and Chol-LNP, RAW264.7 cells showed better uptake of anionic lipid nanoparticles, demonstrating the specific uptake of anionic lipid nanoparticles by macrophages.

[0086] Example 6: Cytotoxicity experiment of anionic lipid nanoparticles on C1498 cells

[0087] Take 5 × 10⁸ C1498 cells in logarithmic growth phase. 4 Cells were seeded per well in 96-well plates. DNR, Chol-LNP, and CHEMS-LNP were diluted with serum-free DMEM medium and added to the 96-well plates to make the final concentrations of DNR 0.5, 1, 2, 4, 8, and 16 μg / mL, respectively. After incubating the cells in a 37°C, 5% CO2 incubator for 24 h, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for another 2 h. The absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated.

[0088] Figure 6 The results showed that anionic lipid nanoparticles were more toxic to C1498 cells than free DNR and Chol-LNP.

[0089] Example 7: In vivo targeting validation of anionic lipid nanoparticles

[0090] By injecting 10 via the tail vein of C57BL / 6 mice 6An AML mouse model was established using C1498 cells, which were randomly divided into three groups. Chol-LNP and CHEMS-LNP, loaded with DID fluorescent dye, were prepared, respectively. Free DID, DID-Chol-LNP, and DID-CHEMS-LNP were injected intravenously into C57 mice (approximately 2 μg DID per mouse). In vivo fluorescence imaging was performed on anesthetized mice at 1, 3, 6, and 8 hours post-injection. After imaging, the mice were sacrificed, and major organs and hind limbs were dissected for in vitro organ imaging.

[0091] Figure 7 The results showed that the CHEMS-LNP group had a stronger enrichment in the femur and tibia of AML mice than other groups, demonstrating the excellent bone-targeting ability of anionic lipid nanoparticles.

[0092] Example 8: Biosafety of anionic lipid nanocompositions loaded with daunorubicin

[0093] By injecting 10 via the tail vein of C57BL / 6 mice 6 An AML mouse model was established using C1498 cells. The mice were divided into four groups of six mice each: (1) saline group; (2) free DNR group; (3) Chol-LNP group; and (4) CHEMS-LNP group. One week after modeling, the mice were administered the drug every two days for a total of six times (the single dose of DNR was 5 mg / kg). Body weight was recorded every two days during the drug administration period. One week after the drug administration was completed, the mice were dissected, and the heart, liver, spleen, lungs, kidneys, bones, and peripheral blood were collected. The heart, liver, spleen, lungs, kidneys, and femur were stained with H&E.

[0094] The experimental results showed that the body weight of mice in each group did not change significantly. The spleen weight statistics showed that the spleen enlargement of mice treated with CHEMS-LNP was reduced, indicating that the lipid nanoparticles had good biocompatibility. Figure 8 ).

[0095] Blood biochemical analysis showed that liver function markers (ALT, GGT) and kidney function markers (BUN, CREA, GLU) in all groups of mice were within the normal range; and routine blood tests indicated no toxicity to platelets (PLT) and red blood cells (RBC), indicating that the lipid nanoparticles have excellent biocompatibility. Figure 9 ).

[0096] Example 9: Anionic lipid nanocompositions loaded with daunorubicin for acute myeloid leukemia

[0097] By injecting 10 via the tail vein of C57BL / 6 mice 6An AML mouse model was established using C1498 cells. The mice were divided into four groups of six each: (1) saline group; (2) free DNR group; (3) Chol-LNP group; and (4) CHEMS-LNP group. The mice were administered the drug every two days for a total of six times (DNR dose was 5 mg / kg per administration) one week after modeling. Leukemia cells extracted from the spleen, bone marrow, and peripheral blood were labeled with PE-anti-mouse-CD45 to indicate infiltration and treatment efficacy. The spleens of the mice were weighed.

[0098] Flow cytometry and its quantitative analysis showed that AML cells infiltrated the spleen, bone marrow, and peripheral blood in the CHEMS-LNP group the least; WBC counts in peripheral blood also indicated that the CHEMS-LNP group had the best treatment effect. Figure 10 ).

[0099] This invention relates to a composition for bone marrow-targeted delivery of chemotherapeutic drugs, with anionic lipids as the core components, including phospholipids and cholesterol. It achieves highly efficient bone marrow targeting through a dual mechanism: binding to the bone marrow microenvironment via negative charge electrostatic interaction, and active transport mediated by inflammatory cells. It can encapsulate various chemical drugs, including small-molecule chemotherapeutic agents, biologics, and anti-infectives, for a wide range of bone marrow-related diseases, such as hematological malignancies and benign bone marrow disorders. Experiments show that this invention can significantly increase drug concentration in the bone marrow by 3-5 times, enhance efficacy, and reduce systemic toxicity. This invention has advantages such as simple preparation, strong targeting, and broad indications, and possesses high clinical translational value.

[0100] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0101] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A composition for targeted delivery of chemotherapy drugs to bone marrow, characterized in that, It includes anionic phospholipids, cholesterol, drugs, organic solvent phases, and aqueous phases.

2. The composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in claim 1, characterized in that: The composition comprises, by weight percentage, 3%-12% anionic phospholipids, 1%-5% cholesterol, 1%-20% pharmaceuticals, 5%-30% organic solvent phase, and the balance being an aqueous phase, and has a zeta potential of -10mV to -40mV.

3. The composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in claim 1, characterized in that: The anionic phospholipids are at least one of the following: phosphatidylserine series, phosphatidylglycerol derivatives, cardiolipin variants, phosphate ester derivatives, and sulfonic acid-modified phospholipids; the cholesterol is at least one of the following: cholesterol sulfate derivatives and carboxylic acid-modified cholesterol. The organic solvent phase can be chloroform, anhydrous ethanol, dichloromethane, methanol, a chloroform-methanol mixture, tert-butanol, or ethanol, with a concentration of 50%-100%. The aqueous phase can be 50-300 mM ammonium sulfate solution, 3-5 mM sucrose octaphosphate solution, phosphate buffer, HEPES buffer, physiological saline, or deionized water.

4. The method for preparing the composition for bone marrow-targeted delivery of chemotherapeutic drugs according to any one of claims 1-3, characterized in that: Includes the following steps: S1. Oil phase preparation: Anionic phospholipids, cholesterol, and drugs are dissolved in an organic solvent to form a homogeneous lipid mixture solution; S2. Preparation of aqueous phase: Stir the aqueous phase until it is completely dissolved to obtain a clear aqueous solution; S3. Preparation of nanoparticles: Nanoparticles are prepared by injecting or dispersing the aqueous and oil phases using the injection method or thin film dispersion method. S4. Purification: Ultrafiltration centrifugation, washing and resuspending to obtain purified anionic lipid nanocomposite.

5. The method for preparing the composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in claim 4, characterized in that: In the oil phase preparation, the molar ratio of anionic phospholipid:cholesterol:drug is 70-85:15-25:5-10.

6. The method for preparing the composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in claim 5, characterized in that: In the oil phase preparation, the concentration of the lipid mixture solution formed is 10-30 mg / mL.

7. The method for preparing the composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in claim 4, characterized in that: The method for preparing nanoparticles by injection includes: adding the aqueous phase dropwise to the oil phase under stirring at 300-500 rpm at a rate of 1-6 mL / min; then homogenizing at 5000-10000 rpm for 3-5 minutes to preliminarily emulsify the mixture; and then circulating the mixture 2-3 times under a high-pressure microjets at a pressure of 20000-25000 PSI to obtain a suspension of nanoparticles with uniform particle size.

8. The method for preparing the composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in claim 4, characterized in that: The method for preparing nanoparticles using thin-film dispersion includes: removing organic solvents from the oil phase using a rotary evaporator, allowing the lipids to form a uniform, thin, and transparent lipid film on the inner wall of the flask; adding the aqueous phase dropwise to the oil phase while stirring at 300-500 rpm at a rate of 1-6 mL / min; then homogenizing the mixture at 5000-10000 rpm for 3-5 minutes to preliminarily emulsify the mixture; and further circulating the mixture 2-3 times using a high-pressure microfluidic homogenizer at a pressure of 20000-25000 PSI to obtain a suspension of nanoparticles with uniform particle size.

9. The method for preparing the composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in claim 4, characterized in that: During purification, ultrafiltration centrifuge tubes with a molecular weight cutoff of 10-100 kDa were centrifuged at 4000-6000 ×g for 10-20 minutes to remove unencapsulated drugs and free components. The mixture was washed 2-3 times with PBS and finally resuspended in 8-20% sucrose solution to obtain purified anionic lipid nanocomposite.

10. The use of the composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in any one of claims 1-3 or the method for preparing the composition for bone marrow-targeted delivery of chemotherapeutic drugs as described in any one of claims 4-9 in the preparation of drugs for treating acute myeloid leukemia, multiple myeloma or osteoporosis.

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