A method of eliminating a protein corona from a nanoparticle

Abstract

The present application relates to a method for eliminating the protein corona on the surface of nanoparticles. Specifically, the present application provides a method for eliminating the protein corona on the protein corona modified nanoparticles, which comprises the step of: subjecting the protein corona modified nanoparticles to ultrasonic irradiation treatment, thereby eliminating the protein corona on the protein corona modified nanoparticles, wherein the nanoparticles encapsulate perfluoropentane. The ultrasonic irradiation can eliminate the protein corona on the surface of the nanoparticles, overcome the masking effect of the protein corona on the ligand modified on the surface of the nanoparticles, and avoid the protein corona from hindering the binding of the ligand modified on the surface of the nanoparticles to the cell receptors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically to a method for eliminating the protein crown on the surface of nanoparticles. Background Technology

[0002] Ligand / receptor-mediated drug-loaded nanoparticles, such as nanoparticles and liposomes, represent a strategy to enhance the antitumor efficacy of drugs, particularly for low-permeability solid tumors like liver or pancreatic cancer. In these tumors, the vascular endothelial cells are well-organized and densely packed, with small intercellular spaces. Even when drugs are delivered to the blood vessels of these tumors, the well-organized and densely packed tumor vascular endothelial cells hinder their effective penetration into the tumor microenvironment. Therefore, when treating low-permeability tumors, traditional enhanced permeability and retention (EPR) effects are insufficient to effectively penetrate the tumor from the gaps in tumor vessels, thus failing to exert an effective antitumor effect. For low-permeability solid tumors, ligands modified on the surface of drug-loaded nanoparticles specifically bind to receptors on the surface of tumor vascular endothelial cells. This ligand / receptor binding mediates endocytosis and exocytosis of the drug-loaded nanoparticles by the tumor vascular endothelial cells, promoting the penetration of the nanoparticles from the bloodstream into the tumor site and enhancing the antitumor effect.

[0003] However, in protein-containing environments such as serum-containing culture media or blood, nanoparticles adsorb proteins on their surface, forming a protein corona. This protein corona acts as a fluid biological barrier, masking the ligands on the nanoparticle surface and hindering their binding to ligands on cells, such as tumor vascular endothelial cells or tumor cells. This prevents ligand / receptor-mediated endocytosis and exocytosis of nanoparticles. For example, in blood, because the protein corona masks the ligands on the drug-loaded nanoparticle surface, it hinders the binding of ligands on the surface of drug-loaded nanoparticles to receptors on the surface of tumor vascular endothelial cells, thus preventing ligand-modified nanoparticles from penetrating from the blood to the tumor site and reducing the anticancer effect of the drug. This is the main reason why existing ligand-modified nanoparticles cannot effectively penetrate from tumor blood vessels to the tumor site to exert targeted therapy. Furthermore, in experiments that screen or identify potential cell-targeting ligands by modifying the surface of nanoparticles with test ligands, the modified nanoparticles often need to be incubated with cells in protein-containing conditions (such as serum-containing medium) to screen and identify cell-targeting ligands. However, because the protein crown formed by the adsorption of proteins by nanoparticles in protein-containing conditions (such as serum-containing medium) masks the test ligands on the surface of the nanoparticles, it is often impossible to effectively and accurately screen and identify whether the test ligands can target cells in vitro in protein-containing conditions (such as serum-containing medium), especially when the amount of test ligand is low, which easily leads to false negative results, thus limiting the development of ligands.

[0004] In addition, existing drug-loaded nanoparticles have many drawbacks, such as low biocompatibility, easy retention in lysosomes, and degradation of nanoparticles and their loaded drugs by various enzymes in lysosomes, thereby reducing therapeutic efficacy, etc.

[0005] Therefore, there is a need in the art to develop a method to eliminate the protein crown on the surface of nanoparticles to overcome the masking effect of the protein crown on the surface-modified ligands of nanoparticles, thereby effectively enabling targeted therapy of ligand-modified nanoparticles or screening, identification and development of potential ligands in protein-containing conditions (such as serum-containing culture media). Summary of the Invention

[0006] The purpose of this invention is to provide a nanoparticle in which ultrasonic irradiation can effectively eliminate the protein crown on the surface of the nanoparticle, thereby overcoming the masking effect of the protein crown on the ligands modified on the surface of the nanoparticle.

[0007] In a first aspect, the present invention provides nanoparticles loaded with perfluoropentane.

[0008] Preferably, the nanoparticles are nanoparticles or liposomes.

[0009] Preferably, the nanoparticles are coated with perfluoropentane.

[0010] Preferably, the nanoparticles include nanomaterials.

[0011] Preferably, the nanomaterials include amphiphilic materials.

[0012] Preferably, the nanomaterials include nanoparticles and / or lipid materials of liposomes.

[0013] Preferably, the amphiphilic material includes amphiphilic nanoparticles and / or lipid materials of liposomes.

[0014] Preferably, the liposomes comprise lipid materials.

[0015] Preferably, the lipid material comprises one or more of the following: 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPE), soybean lecithin, phosphatidylcholine (PC, lecithin), cholesterol, phosphatidylethanolamine (PE, cephalin), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), dicetylphosphatidylphosphate (DCP), dimyristic phosphatidylcholine (DMPC), distearylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), and dioleoylphosphatidylcholine (DOPC).

[0016] Preferably, the lipid material comprises 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0017] Preferably, the distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) is selected from the group consisting of: DSPE-PEG600, DSPE-PEG800, DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG4000, DSPE-PEG6000, or combinations thereof.

[0018] Preferably, the DPPC is 1-10 parts by weight, more preferably 2-8 parts by weight, even more preferably 4-6 parts by weight, and most preferably 3 parts by weight.

[0019] Preferably, the DSPE-PEG is 0.5-8 parts by weight, more preferably 1-5 parts by weight, even more preferably 1-3 parts by weight, and most preferably 2 parts by weight.

[0020] Preferably, the perfluoropentane is 0.01-0.5 parts by weight, more preferably 0.02-0.2 parts by weight, even more preferably 0.05-0.15 parts by weight, even more preferably 0.08-0.12 parts by weight, and most preferably 0.1 parts by weight.

[0021] Preferably, the weight ratio of DPPC to DSPE-PEG is 0.2-8:1, more preferably 0.5-5:1, even more preferably 1-2:1, even more preferably 1.3-1.7:1, and most preferably 1.5:1.

[0022] Preferably, the volume weight ratio (ml:mg) of the perfluoropentane to the DPPC is 1:20-40, more preferably 1:25-35, even more preferably 1:27-32, and most preferably 1:30.

[0023] Preferably, the nanoparticles include drug-loaded nanoparticles.

[0024] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[0025] Preferably, the drug is in the form of 0.5-8 parts by weight, more preferably 1-5 parts by weight, even more preferably 1-3 parts by weight, and most preferably 2 parts by weight.

[0026] Preferably, the weight ratio of DPPC to the drug is 0.2-8:1, more preferably 0.5-5:1, even more preferably 1-2:1, even more preferably 1.3-1.7:1, and most preferably 1.5:1.

[0027] Preferably, the drug includes a drug that is unstable in lysosomes.

[0028] Preferably, the drug includes drugs that are retained and / or degraded by lysosomes.

[0029] Preferably, the degradation includes degradation by lysosomal enzymes.

[0030] Preferably, the drug comprises a drug that is degraded by lysosomal enzymes.

[0031] Preferably, the drug targets the cytoplasm or the nucleus.

[0032] Preferably, the drug comprises a gene or a protein.

[0033] Preferably, the gene is selected from the group consisting of DNA, RNA, or a combination thereof.

[0034] Preferably, the drug includes an anticancer drug.

[0035] Preferably, the anticancer drug includes a chemical drug.

[0036] Preferably, the anticancer drug is selected from the group consisting of gemcitabine, cytarabine, doxorubicin, fluorouracil, or combinations thereof.

[0037] Preferably, the drug comprises a free drug form or a prodrug form.

[0038] Preferably, the drug includes a prodrug.

[0039] Preferably, the prodrug comprises a prodrug formed by modifying a prodrug carrier with free drug.

[0040] Preferably, the prodrug comprises a free drug and a prodrug carrier connected by chemical bonds.

[0041] Preferably, the drug includes a hydrophobic drug or a hydrophilic drug.

[0042] Preferably, the free drug includes a hydrophobic drug or a hydrophilic drug.

[0043] Preferably, the free drug includes an anticancer drug.

[0044] Preferably, the prodrug carrier includes a hydrophobic carrier or a hydrophilic carrier.

[0045] Preferably, the prodrug carrier includes a higher fatty acid carrier or a higher fatty alcohol carrier.

[0046] Preferably, the prodrug carrier comprises a higher fatty acid containing 12-26 (preferably 14-22, more preferably 16-20) carbon atoms.

[0047] Preferably, the prodrug carrier comprises a higher fatty alcohol containing 12-26 (preferably 14-22, more preferably 16-20) carbon atoms.

[0048] Preferably, the higher fatty acid carrier is selected from the group consisting of: palmitic acid (hexadecanoic acid), pearlitic acid (heptadecanoic acid), stearic acid (octadecanoic acid), oleic acid (octadecenoic acid), linoleic acid (octadecadienoic acid), linolenic acid (octadecanetrienoic acid), arachidic acid (eicosanoic acid), eicosapentaenoic acid, benzanoic acid (docosahexaenoic acid), DHA (docosahexaenoic acid), ligninic acid (tetracosanoic acid), or combinations thereof.

[0049] Preferably, the oleic acid includes trans-oleic acid.

[0050] Preferably, the higher fatty alcohol carrier is selected from the group consisting of: palmitol, stearyl alcohol, oleyl alcohol, linoleyl alcohol, linolenic acid alcohol, arachidonic acid alcohol, eicosapentaenoic acid, betaine alcohol, docosahexaenoic acid, or combinations thereof.

[0051] Preferably, the prodrug comprises an amphiphilic prodrug.

[0052] Preferably, the amphiphilic prodrug is used as a nanomaterial of nanoparticles.

[0053] Preferably, the amphiphilic prodrug is used as a nanomaterial of nanoparticles.

[0054] Preferably, the amphiphilic prodrug is used as the lipid material of the liposome.

[0055] Preferably, the amphiphilic prodrug is a lipid bilayer.

[0056] Preferably, the amphiphilic prodrug comprises a pharmaceutical active ingredient as a hydrophilic portion and a prodrug carrier as a hydrophobic portion.

[0057] Preferably, the amphiphilic prodrug comprises a pharmaceutical active ingredient as a hydrophobic portion and a prodrug carrier as a hydrophilic portion;

[0058] Preferably, the prodrug comprises:

[0059] DC

[0060] Wherein, "D" represents the active pharmaceutical ingredient, "C" represents the prodrug carrier, and "-" is the connecting bond.

[0061] Preferably, the active pharmaceutical ingredient includes a drug that is unstable in lysosomes.

[0062] Preferably, the active pharmaceutical ingredient includes a hydrophobic active pharmaceutical ingredient or a hydrophilic active pharmaceutical ingredient.

[0063] Preferably, the active pharmaceutical ingredient includes active pharmaceutical ingredients that are retained and / or degraded by lysosomes.

[0064] Preferably, the degradation includes degradation by lysosomal enzymes.

[0065] Preferably, the active pharmaceutical ingredient includes an active pharmaceutical ingredient that has been degraded by lysosomal enzymes.

[0066] Preferably, the target site of the active pharmaceutical ingredient is in the cytoplasm or the cell nucleus.

[0067] Preferably, the active pharmaceutical ingredient includes a gene or a protein.

[0068] Preferably, the gene is selected from the group consisting of DNA, RNA, or a combination thereof.

[0069] Preferably, the active pharmaceutical ingredient includes an anticancer drug.

[0070] Preferably, the prodrug comprises:

[0071]

[0072] Wherein, R represents an anticancer drug, including gemcitabine, cytarabine, doxorubicin, fluorouracil, or combinations thereof.

[0073] Preferably, the drug comprises gemcitabine transoleate.

[0074] Preferably, the prodrug comprises gemcitabine trans oleate.

[0075] Preferably, the gemcitabine trans oleate has the following structure:

[0076]

[0077] Preferably, the nanoparticles further include water, a buffer solution, and / or perfluoropentane.

[0078] Preferably, the nanoparticles encapsulate water, a buffer solution, and / or perfluoropentane.

[0079] Preferably, the lipid bilayer of the liposome encapsulates water, a buffer solution, and / or perfluoropentane.

[0080] Preferably, the buffer solution comprises a phosphate buffer containing glycerol.

[0081] Preferably, the glycerol-containing phosphate buffer contains 5-15% glycerol by volume, more preferably 8-12%, and even more preferably 10%.

[0082] Preferably, the concentration of the phosphate buffer is 5-15 mM, more preferably 8-12 mM, and even more preferably 10 mM, based on the concentration of phosphate.

[0083] Preferably, the pH of the glycerol-containing phosphate buffer solution is 7.2-7.6, more preferably 7.4.

[0084] Preferably, the lipid bilayer is coated with perfluoropentane and / or a phosphate buffer containing glycerol.

[0085] Preferably, the encapsulation efficiency of the drug-loaded nanoparticles is ≥90%, more preferably ≥95%, more preferably ≥99%, and most preferably 100%.

[0086] Preferably, the drug loading of the drug-loaded nanoparticles is 8-15 wt%, more preferably 9-11 wt%.

[0087] A second aspect of the present invention provides a method for preparing nanoparticles as described in the first aspect of the present invention, the method comprising the steps of:

[0088] (1) Dissolve the nanomaterials in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0089] (2) After immersing the nanoparticle membrane in perfluoropentane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain nanoparticles.

[0090] Preferably, the nanoparticles are drug-loaded nanoparticles, and the method for preparing the drug-loaded nanoparticles includes the following steps:

[0091] (1) Dissolve nanomaterials and drugs in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0092] (2) After immersing the nanoparticle membrane in perfluoropentane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain nanoparticles.

[0093] Preferably, the nanoparticles are liposomes, and the method for preparing the liposomes includes the following steps:

[0094] (1) Dissolve the lipid material in an organic solvent, remove the organic solvent, and obtain a lipid membrane;

[0095] (2) After immersing the lipid membrane in perfluoropentane, add a buffer solution to hydrate the lipid membrane and stir to obtain liposomes.

[0096] Preferably, the nanoparticles are drug-loaded liposomes, and the method for preparing the drug-loaded liposomes includes the following steps:

[0097] (1) The lipid material and the drug are dissolved in an organic solvent, and the organic solvent is removed to obtain a lipid membrane;

[0098] (2) After immersing the lipid membrane in perfluoropentane, add a buffer solution to hydrate the lipid membrane and stir to obtain liposomes.

[0099] Preferably, in step (1), the organic solvent is selected from the group consisting of chloroform, dichloromethane, or combinations thereof.

[0100] Preferably, in step (1), the weight-to-volume ratio (mg:ml) of DPPC to the organic solvent is 1:0.2-5, more preferably 1:0.5-2, even more preferably 1:0.5-1.5, even more preferably 1:0.8-1.2, and most preferably 1:1.

[0101] Preferably, the volume ratio of the perfluoropentane to the buffer solution is 1:30-70, more preferably 1:40-60, even more preferably 1:45-55, and even more preferably 1:48-52.

[0102] Preferably, in step (1), the weight-to-volume ratio (mg:ml) of the drug to the organic solvent is 1:2-5, more preferably 1:1-2, more preferably 1:1.3-1.7, and more preferably 1:1.5.

[0103] Preferably, in step (1), the organic solvent is removed by rotary vacuum evaporation.

[0104] Preferably, in step (1), the organic solvent is removed by rotary vacuum evaporation at 35-40°C.

[0105] Preferably, in step (2), perfluoropentane is immersed in the lipid membrane at low temperature.

[0106] Preferably, in step (2), the hydration is carried out at a low temperature.

[0107] Preferably, in step (2), the stirring includes the following steps:

[0108] First stir at a low temperature, then stir again after raising the temperature.

[0109] Preferably, the low temperature is 2-10°C, more preferably 2-6°C, and most preferably 4°C.

[0110] Preferably, the stirring time at low temperature is 0.2-0.8h, more preferably 0.4-0.6h, and even more preferably 0.5h.

[0111] Preferably, the temperature rise is 20-40°C, more preferably 25-35°C, and even more preferably 28-32°C.

[0112] Preferably, the stirring time after the temperature is increased is 0.5-1.5h, more preferably 0.8-1.2h, and even more preferably 1h.

[0113] Preferably, the stirring includes stirring while the container is open.

[0114] Preferably, the stirring removes unencapsulated perfluoropentane.

[0115] Preferably, the stirring includes stirring with a magnetic stir bar.

[0116] Preferably, during the stirring process after the temperature is raised, the container holding the stirring liquid is in an open state.

[0117] Preferably, the stirring after raising the temperature can remove unencapsulated perfluoropentane.

[0118] Preferably, the liposomes are in the form of liposome nanodroplets.

[0119] Preferably, the method for preparing the liposomes includes the following steps:

[0120] (1) DPPC and DSPE-PEG are dissolved in the organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0121] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain liposomes.

[0122] Preferably, the method for preparing the liposomes includes the following steps:

[0123] (1) 2.8-3.2 mg DPPC and 1.8-2.2 mg DSPE-PEG were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0124] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃ for 0.3-0.7h, and then stir in a round-bottom flask in a water bath at 28-32℃ for 0.8-1.2h under open conditions to obtain liposomes.

[0125] Preferably, the method for preparing the drug-loaded liposomes includes the following steps:

[0126] (1) DPPC, DSPE-PEG and the drug are dissolved in an organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0127] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain drug-loaded liposomes.

[0128] Preferably, the method for preparing the drug-loaded liposomes includes the following steps:

[0129] (1) 2.8-3.2 mg DPPC, 1.8-2.2 mg DSPE-PEG and 1.8-2.2 mg of drug were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation, forming a lipid film in the round-bottom flask;

[0130] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃ for 0.3-0.7h, and then stir in a round-bottom flask in a water bath at 28-32℃ for 0.8-1.2h under open conditions to obtain drug-loaded liposomes.

[0131] In a third aspect, the present invention provides ligand-modified nanoparticles, the ligand-modified nanoparticles comprising the nanoparticles as described in the first aspect of the present invention; and ligands.

[0132] Preferably, the ligand-modified nanoparticles include ligand-modified nanoparticles or ligand-modified liposomes.

[0133] Preferably, the ligand includes a targeting ligand.

[0134] Preferably, the surface of the nanoparticles contains ligands.

[0135] Preferably, the surface includes an outer surface.

[0136] Preferably, the surface of the nanoparticles includes the outer surface of the nanoparticles.

[0137] Preferably, the outer surface of the nanoparticles contains ligands.

[0138] Preferably, the ligand modification is performed on nanomaterials.

[0139] Preferably, the ligand is modified on the nanomaterial of the nanoparticles.

[0140] Preferably, the ligand is modified on the surface of the nanoparticles.

[0141] Preferably, the modification includes physical modification and / or chemical modification.

[0142] Preferably, the modification includes physical adsorption, chemisorption, and / or coupling.

[0143] Preferably, the ligand is adsorbed on the surface of the nanoparticles.

[0144] Preferably, the adsorption includes physical adsorption and / or chemical adsorption.

[0145] Preferably, the ligand is coupled to the nanomaterial on the surface of the nanoparticles.

[0146] Preferably, the ligand comprises a receptor that targets a cell or the cell surface.

[0147] Preferably, the ligand includes a ligand that targets tumor vascular cells and / or tumor cells.

[0148] Preferably, the ligand comprises a polypeptide or protein ligand.

[0149] Preferably, the ligand comprises RGD peptide and / or NGR peptide.

[0150] Preferably, the lipid material includes ligand-modified lipid materials.

[0151] Preferably, the ligand is coupled to the nanomaterial of the nanoparticles.

[0152] Preferably, the ligand is coupled to a lipid material.

[0153] Preferably, the coupling includes chemical coupling.

[0154] Preferably, the ligand is coupled to distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) to form DSPE-PEG-ligand.

[0155] Preferably, the ligand is coupled to a portion of the nanomaterial (such as a lipid material).

[0156] Preferably, the DSPE-PEG-ligand includes DSPE-PEG-RGD and / or DSPE-PEG-NGR.

[0157] Preferably, the ligand coupling on the lipid material includes DSPE-PEG-RGD and / or DSPE-PEG-NGR.

[0158] Preferably, the DSPE-PEG-RGD is selected from the group consisting of: DSPE-PEG600-RGD, DSPE-PEG800-RGD, DSPE-PEG1000-RGD, DSPE-PEG2000-RGD, DSPE-PEG4000-RGD, DSPE-PEG6000-RGD, or a combination thereof.

[0159] Preferably, the DSPE-PEG-NGR is selected from the group consisting of: DSPE-PEG600-NGR, DSPE-PEG800-NGR, DSPE-PEG1000-NGR, DSPE-PEG2000-NGR, DSPE-PEG4000NGR, DSPE-PEG6000-NGR, or a combination thereof.

[0160] Preferably, the DSPE-PEG-ligand is 1-10 parts by weight, more preferably 2-8 parts by weight, even more preferably 4-6 parts by weight, and most preferably 3 parts by weight.

[0161] Preferably, the weight ratio of the DSPE-PEG-ligand to the DPPC is 1:0.2-5, more preferably 1:0.5-2, even more preferably 1:0.5-1.5, even more preferably 1:0.8-1.2, and most preferably 1:1.

[0162] Preferably, the ligand-modified nanoparticles have a particle size of 120-260 nm, more preferably 160-210 nm, even more preferably 170-200 nm, and even more preferably 180-200 nm.

[0163] Preferably, the potential of the ligand-modified nanoparticles is -2 to -18 mV, more preferably -2 to -15 mV, and even more preferably -5 to -12 mV.

[0164] Preferably, the ligand comprises a ligand that targets a cell surface receptor.

[0165] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor vascular cells.

[0166] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor cells.

[0167] Preferably, the surface includes an outer surface.

[0168] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[0169] Preferably, the cell surface receptor includes extracellular receptors.

[0170] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[0171] Preferably, the ligand includes a ligand that mediates the uptake of ligand-modified nanoparticles by cells.

[0172] Preferably, the ligand includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[0173] Preferably, the ligand includes a ligand that mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[0174] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site.

[0175] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site via endocytosis and exocytosis.

[0176] Preferably, the ligand targets vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of ligand-modified nanoparticles by vascular endothelial cells.

[0177] Preferably, the ligand can mediate the endocytosis of ligand-modified nanoparticles in the blood by tumor vascular cells, and after endocytosis, it can be exocytodated outside the tumor blood vessels (such as the tumor tissue microenvironment).

[0178] A fourth aspect of the present invention provides a method for preparing ligand-modified nanoparticles as described in the third aspect of the present invention, the method comprising:

[0179] The ligand is modified onto the nanoparticles to obtain ligand-modified nanoparticles.

[0180] Preferably, the method for preparing the ligand-modified nanoparticles includes:

[0181] (1) Dissolve the ligand-modified nanomaterial in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0182] (2) After immersing perfluoropentane into the nanoparticle membrane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain ligand-modified nanoparticles.

[0183] Preferably, the method for preparing the ligand-modified nanoparticles includes:

[0184] (1) Dissolve the ligand-modified nanomaterial and drug in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0185] (2) After immersing perfluoropentane into the nanoparticle membrane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain ligand-modified nanoparticles.

[0186] Preferably, the ligand-modified nanomaterial includes one or more of DPPC, DSPE-PEG, and DSPE-PEG-ligand.

[0187] Preferably, the ligand-modified nanomaterial includes one or more of DPPC, DSPE-PEG, DSPE-PEG-RGD, and DSPE-PEG-NGR.

[0188] Preferably, the method for preparing the ligand-modified liposomes includes the following steps:

[0189] (1) DPPC, DSPE-PEG-ligand and DSPE-PEG are dissolved in the organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0190] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain ligand-modified liposomes.

[0191] Preferably, the method for preparing the ligand-modified liposomes includes the following steps:

[0192] (1) 2.8-3.2 mg DPPC, 2.8-3.2 mg DSPE-PEG-ligand and 1.8-2.2 mg DSPE-PEG were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0193] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃, and then stir in a round-bottom flask in a water bath at 28-32℃ under open conditions to obtain ligand-modified liposomes.

[0194] Preferably, the method for preparing the ligand-modified liposomes includes the following steps:

[0195] (1) DPPC, DSPE-PEG-ligand, DSPE-PEG and the drug are dissolved in an organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0196] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain ligand-modified liposomes.

[0197] Preferably, the method for preparing the ligand-modified liposomes includes the following steps:

[0198] (1) 2.8-3.2 mg DPPC, 2.8-3.2 mg DSPE-PEG-ligand, 1.8-2.2 mg DSPE-PEG and 1.8-2.2 mg drug were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation, forming a lipid film in the round-bottom flask;

[0199] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃, and then stir in a round-bottom flask in a water bath at 28-32℃ under open conditions to obtain ligand-modified liposomes.

[0200] Preferably, step (1) is as described in the second aspect of the present invention above.

[0201] Preferably, step (2) is as described in the second aspect of the present invention above.

[0202] In a fifth aspect, the present invention provides a composition comprising nanoparticles as described in the first aspect of the present invention and / or ligand-modified nanoparticles as described in the third aspect of the present invention.

[0203] Preferably, the composition is a pharmaceutical composition.

[0204] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0205] Preferably, the composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0206] Preferably, the composition is an injectable formulation, an oral formulation, or a topical formulation.

[0207] Preferably, the injectable formulation is an intravascular injection formulation.

[0208] Preferably, the injectable formulation is an intravenous injection formulation, an arterial injection formulation, an intratumoral injection formulation, a tumor intravascular injection formulation, or a tumor microenvironment injection formulation.

[0209] Preferably, the intravenous injection preparation is an upper limb intravenous preparation or a lower limb intravenous preparation.

[0210] In a sixth aspect, the present invention provides the use of nanoparticles as described in the first aspect of the present invention and / or ligand-modified nanoparticles as described in the third aspect of the present invention for preparing compositions for the prevention and / or treatment of diseases.

[0211] Preferably, the nanoparticles include drug-loaded nanoparticles.

[0212] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[0213] Preferably, the disease is an indication for the drug.

[0214] Preferably, the drug includes an anticancer drug.

[0215] Preferably, the drug is as described in the first aspect of the present invention.

[0216] Preferably, the disease includes a tumor.

[0217] Preferably, the tumor includes human tumors (such as human tumors) or non-human mammalian tumors.

[0218] Preferably, the tumor includes a low-permeability tumor.

[0219] Preferably, the tumor includes tumors with low vascular permeability.

[0220] Preferably, the tumor includes a solid tumor.

[0221] Preferably, the tumor includes a solid tumor with low vascular permeability.

[0222] Preferably, the tumor includes liver cancer.

[0223] Preferably, the tumor includes human liver cancer.

[0224] Preferably, the liver cancer cells include Huh7 cells and / or HepG2 cells.

[0225] Preferably, the tumor includes pancreatic cancer.

[0226] Preferably, the pancreatic cancer includes pancreatic adenocarcinoma.

[0227] Preferably, the pancreatic cancer includes pancreatic cancer in situ.

[0228] Preferably, the pancreatic cancer includes pancreatic adenocarcinoma in situ.

[0229] Preferably, the pancreatic cancer includes pancreatic ductal adenocarcinoma.

[0230] Preferably, the pancreatic cancer includes human pancreatic ductal adenocarcinoma.

[0231] Preferably, the pancreatic cancer cells include BxPC-3 cells.

[0232] Preferably, the tumor includes tumors with poor enhanced permeability and retention (EPR effect).

[0233] Preferably, the low permeability of the tumor vessels includes low drug penetration from the tumor vessels to the tumor site.

[0234] Preferably, the low permeability of the tumor vessels includes low drug penetration from the intercellular spaces of the tumor vessels to the tumor site.

[0235] Preferably, the tumor vessels with low permeability include one or more characteristics selected from the group consisting of:

[0236] (a) The tumor vascular cells are well-formed and densely packed; and / or

[0237] (b) The tumor vascular cells have small intercellular spaces.

[0238] Preferably, the vascular cells include vascular endothelial cells.

[0239] Preferably, the composition is a pharmaceutical composition.

[0240] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0241] Preferably, the dosage form of the composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0242] Preferably, the dosage form of the composition is an injectable formulation, an oral formulation, or a topical formulation.

[0243] Preferably, the injectable formulation is an intravascular injection formulation.

[0244] Preferably, the injectable formulation is an intravenous injection formulation, an arterial injection formulation, an intratumoral injection formulation, a tumor intravascular injection formulation, or a tumor microenvironment injection formulation.

[0245] Preferably, the intravenous injection preparation is an upper limb intravenous preparation or a lower limb intravenous preparation.

[0246] Preferably, the treatment includes inhibition, reduction, relief, reversal, or eradication.

[0247] In a seventh aspect, the present invention provides a system or apparatus for treating a disease, said system or apparatus comprising nanoparticles as described in the first aspect of the present invention and / or ligand-modified nanoparticles as described in the third aspect of the present invention; and an ultrasound device.

[0248] Preferably, the system or device further includes a manual or label, which states:

[0249] In the process of treating a disease by applying nanoparticles as described in the first aspect of the present invention and / or ligand-modified nanoparticles as described in the third aspect of the present invention to a desired object, ultrasound irradiation is performed on the lesion site (such as a tumor site).

[0250] Preferably, the nanoparticles include drug-loaded nanoparticles.

[0251] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[0252] Preferably, the ultrasonic device includes an ultrasonic transducer.

[0253] Preferably, the object includes a human or a non-human mammal.

[0254] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0255] Preferably, the disease is an indication for the drug.

[0256] Preferably, the drug includes an anticancer drug.

[0257] Preferably, the drug is as described in the first aspect of the present invention.

[0258] Preferably, the disease includes a tumor.

[0259] Preferably, the tumor is as described in the sixth aspect of the present invention.

[0260] Preferably, the application is by injection, oral administration, or topical application.

[0261] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[0262] Preferably, the injection is administered via intravascular injection.

[0263] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[0264] An eighth aspect of the present invention provides a method for preventing and / or treating a disease by applying nanoparticles as described in the first aspect of the present invention and / or ligand-modified nanoparticles as described in the third aspect of the present invention to a desired object, thereby preventing and / or treating the disease.

[0265] Preferably, the nanoparticles include drug-loaded nanoparticles.

[0266] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[0267] Preferably, the object includes a human or a non-human mammal.

[0268] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0269] Preferably, the disease is an indication for the drug.

[0270] Preferably, the disease includes a tumor.

[0271] Preferably, the drug includes an anticancer drug.

[0272] Preferably, the drug is as described in the first aspect of the present invention.

[0273] Preferably, the tumor is as described in the sixth aspect of the present invention.

[0274] Preferably, after applying nanoparticles as described in the first aspect of the invention and / or ligand-modified nanoparticles as described in the third aspect of the invention to the desired object, the lesion site (such as a tumor site) is subjected to ultrasonic irradiation.

[0275] Preferably, the application is by injection, oral administration, or topical application.

[0276] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[0277] Preferably, the injection is administered intravascularly.

[0278] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[0279] In a ninth aspect, the present invention provides a protein crown-modified nanoparticle, said protein crown-modified nanoparticle comprising the nanoparticles as described in the first aspect of the present invention and / or the ligand-modified nanoparticles as described in the third aspect of the present invention; and a protein crown.

[0280] Preferably, the nanoparticles are loaded with perfluoropentane.

[0281] Preferably, the proteins in the protein crown include serum proteins, plasma proteins, and / or tissue proteins.

[0282] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[0283] Preferably, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0284] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0285] Preferably, the cattle include fetal cattle.

[0286] Preferably, the serum and / or plasma includes fetal bovine serum and / or fetal bovine plasma.

[0287] Preferably, the protein crown-modified nanoparticles include isolated or ex vivo protein crown-modified nanoparticles.

[0288] Preferably, the protein crown modification is performed on nanoparticles as described in the first aspect of the invention and / or on ligand-modified nanoparticles as described in the third aspect of the invention.

[0289] Preferably, the protein crown modification is applied to the surface of nanoparticles as described in the first aspect of the invention and / or ligand-modified nanoparticles as described in the third aspect of the invention.

[0290] Preferably, the surface includes an outer surface.

[0291] Preferably, the modification includes physical modification and / or chemical modification.

[0292] Preferably, the modification includes physical adsorption, chemisorption, and / or coupling.

[0293] Preferably, the modification includes adsorption.

[0294] In a tenth aspect, the present invention provides a method for preparing protein crown-modified nanoparticles as described in the ninth aspect of the present invention, the method comprising the steps of:

[0295] The nanoparticles as described in the first aspect of the present invention and / or the ligand-modified nanoparticles as described in the third aspect of the present invention are incubated with proteins to separate the protein crown-modified nanoparticles.

[0296] Preferably, the method described is an in vitro method or an in vivo method.

[0297] Preferably, the method is a non-diagnostic and non-therapeutic method.

[0298] Preferably, the incubation is in vitro or in vivo.

[0299] Preferably, the incubation includes incubation in conditions containing protein.

[0300] Preferably, the protein-containing conditions include blood, serum, plasma, and / or culture medium.

[0301] Preferably, the incubation includes incubation in a culture medium.

[0302] Preferably, the culture medium comprises a liquid culture medium.

[0303] Preferably, the culture medium includes a cell culture medium.

[0304] Preferably, the culture medium contains protein.

[0305] Preferably, the proteins include serum proteins, plasma proteins, and / or tissue proteins.

[0306] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[0307] Preferably, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0308] Preferably, the culture medium includes a culture medium containing serum, plasma, and / or tissue protein.

[0309] Preferably, the culture medium includes a serum-containing culture medium.

[0310] Preferably, the serum comprises fetal bovine serum.

[0311] Preferably, the plasma includes fetal bovine plasma.

[0312] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[0313] Preferably, the culture includes in vitro culture.

[0314] Preferably, the serum, plasma, and / or tissue proteins include serum, plasma, and / or tissue proteins from humans or non-human mammals.

[0315] Preferably, the incubation includes incubation in blood, serum, or plasma.

[0316] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[0317] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[0318] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0319] Preferably, the cattle include fetal cattle.

[0320] Preferably, the incubation time is 0.25-6h, more preferably 0.25-4h, even more preferably 0.25-2h, more preferably 0.25-1h, more preferably 0.25-0.5h, for example 0.5-1h.

[0321] Preferably, the separation includes gel chromatography separation.

[0322] Preferably, the gel comprises a dextran gel.

[0323] Preferably, the separation includes size exclusion chromatography.

[0324] In an eleventh aspect, the present invention provides a method for eliminating the protein crown of protein crown-modified nanoparticles, the method comprising the steps of:

[0325] The protein crown-modified nanoparticles are subjected to ultrasonic irradiation to eliminate the protein crown of the nanoparticles.

[0326] Preferably, the nanoparticles are loaded with perfluoropentane.

[0327] Preferably, the method includes an in vitro method or an in vivo method.

[0328] Preferably, the method includes non-therapeutic and / or non-diagnostic methods.

[0329] Preferably, the protein crown-modified nanoparticles are as described in the ninth aspect of the present invention.

[0330] Preferably, the elimination includes reduction or removal.

[0331] Preferably, the reduction includes a reduction in protein content.

[0332] Preferably, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in protein-free conditions.

[0333] Preferably, the protein-free conditions include physiological saline, PBS buffer, or serum-free culture medium.

[0334] Preferably, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in conditions containing protein.

[0335] Preferably, the protein-containing conditions include blood, serum, plasma, and / or culture medium.

[0336] Preferably, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in blood, serum, or plasma.

[0337] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[0338] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[0339] Preferably, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in a culture medium.

[0340] Preferably, the proteins in the protein crown include serum proteins, plasma proteins, and / or tissue proteins.

[0341] Preferably, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0342] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[0343] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0344] Preferably, the cattle include fetal cattle.

[0345] Preferably, the serum comprises fetal bovine serum.

[0346] Preferably, the plasma includes fetal bovine plasma.

[0347] Preferably, the culture medium comprises a liquid culture medium.

[0348] Preferably, the culture medium includes a cell culture medium.

[0349] Preferably, the culture medium includes a protein-containing culture medium.

[0350] Preferably, the culture medium contains protein.

[0351] Preferably, the proteins include serum proteins, plasma proteins, and / or tissue proteins.

[0352] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[0353] Preferably, the culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

[0354] Preferably, the culture medium includes a serum-containing culture medium.

[0355] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[0356] Preferably, the cells include tumor cells and / or tumor vascular cells.

[0357] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[0358] Preferably, the tumor vascular cells include ECDHCC cells.

[0359] Preferably, the cells include cells that need to be cultured or grown in conditions containing proteins.

[0360] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[0361] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[0362] Preferably, the culture includes in vitro culture.

[0363] Preferably, the acoustic intensity of the ultrasonic irradiation is 0.1-40 W / cm². 2 The optimal concentration is 0.1-20 W / cm². 2 More preferably 0.2-15W / cm 2 More preferably 0.5-10W / cm 2 Better 1-5W / cm 2 Better 1-3W / cm 2 The optimal concentration is 1.5-2.5 W / cm². 2 Optimal 1.8-2.2W / cm 2 Better 2.0W / cm 2 .

[0364] Preferably, the frequency of the ultrasonic irradiation is 0.02-30MHz, more preferably 0.1-20MHz, even more preferably 0.2-15MHz, even more preferably 0.5-10MHz, even more preferably 1-8MHz, even more preferably 1-5MHz, most preferably 2-4MHz, even more preferably 2.5-3.5MHz, even more preferably 2.8-3.2MHz, and even more preferably 3MHz.

[0365] Preferably, the duty cycle of the ultrasonic irradiation is 10-80%, more preferably 20-80%, more preferably 30-70%, more preferably 35-65%, more preferably 40-60%, most preferably 45-55%, more preferably 48-52%, and more preferably 50%.

[0366] Preferably, the ultrasonic irradiation time is greater than 2 minutes, more preferably greater than 5 minutes, more preferably greater than 10 minutes, more preferably greater than 15 minutes, for example 15-20 minutes.

[0367] A twelfth aspect of the present invention provides a method for screening or identifying potential ligands targeting cell or cell surface receptors, the method comprising the steps of:

[0368] (I) Ligands are modified onto nanoparticles to obtain ligand-modified nanoparticles;

[0369] (II) Incubate the cell or cell surface receptor with the ligand-modified nanoparticles of step (I), subject them to ultrasonic irradiation, and determine the binding of the ligand-modified nanoparticles of step (I) or the ligand of the ligand-modified nanoparticles to the cell or cell surface receptor, thereby screening or identifying whether the ligand of step (I) is a potential ligand for targeting the cell or cell surface receptor.

[0370] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[0371] Preferably, the cell surface receptor includes extracellular receptors.

[0372] Preferably, the nanoparticles are loaded with perfluoropentane.

[0373] Preferably, the nanoparticles are as described in the first aspect of the present invention.

[0374] Preferably, the ligand-modified nanoparticles in step (I) are the ligand-modified nanoparticles described in the third aspect of the present invention.

[0375] Preferably, the ligand in step (I) includes the ligand to be tested.

[0376] Preferably, step (II) includes:

[0377] (II) Incubate the cell or cell surface receptor with the ligand-modified nanoparticles of step (I), subject them to ultrasonic irradiation, and determine whether the ligand-modified nanoparticles of step (I) or the ligand of the ligand-modified nanoparticles bind to the cell or cell surface receptor, thereby screening or identifying whether the ligand of step (I) is a potential ligand for targeting the cell or cell surface receptor.

[0378] Preferably, in step (II), if the ligand-modified nanoparticle or the ligand of the ligand-modified nanoparticle binds to a cell or a cell surface receptor, then the ligand in step (I) is a potential ligand targeting the cell or cell surface receptor.

[0379] Preferably, in step (II), if the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles do not bind to the cell or cell surface receptor, then the ligand in step (I) is not a potential ligand for targeting the cell or cell surface receptor.

[0380] Preferably, the method further includes setting up a control group, which comprises nanoparticles without ligand modification, and measuring the binding of the nanoparticles without ligand modification to cells or cell surface receptors.

[0381] Preferably, the method further includes setting up a control group, which comprises unmodified nanoparticles and other conditions are the same as those for ligand-modified nanoparticles, and measuring the binding of unmodified nanoparticles to cells or cell surface receptors.

[0382] Preferably, if the binding force B1 between the ligand-modified nanoparticle or the ligand of the ligand-modified nanoparticle and the cell or cell surface receptor is greater than the binding force B0 between the unmodified nanoparticle and the cell or cell surface receptor, then the ligand in step (I) is a potential ligand for targeting the cell or cell surface receptor.

[0383] Preferably, step (II) includes:

[0384] (II-1) In the test group, cells or cell surface receptors were incubated with ligand-modified nanoparticles from step (I) and subjected to ultrasonic irradiation. The binding force B1 between the ligand-modified nanoparticles from step (I) or the ligands of the ligand-modified nanoparticles and the cells or cell surface receptors was measured. A control group was set up, which included nanoparticles without ligand modification and other measurement conditions were the same as those in the test group. The binding force B0 between the nanoparticles without ligand modification and the cells or cell surface receptors was measured.

[0385] (II-2) If the binding force B1 between the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles in step (I) and the cell or cell surface receptor is greater than the binding force B0 between the unmodified nanoparticles and the cell or cell surface receptor, then the ligand in step (I) is a potential ligand for targeting the cell or cell surface receptor.

[0386] Preferably, if the binding force B1 between the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles in step (I) and the cell or cell surface receptor is similar to the binding force B0 between the unmodified nanoparticles and the cell or cell surface receptor, then the ligand in step (I) is not a potential ligand for targeting the cell or cell surface receptor.

[0387] Preferably, the targeting includes specific targeting or non-specific targeting.

[0388] Preferably, "greater than" includes significantly greater than.

[0389] Preferably, "greater than" includes significantly greater than and statistically significant.

[0390] Preferably, "greater than" means that the ratio (B1 / B0) of the binding force B1 between the ligand of the ligand-modified nanoparticle or the ligand of the ligand-modified nanoparticle and the cell or cell surface receptor to the binding force B0 between the unmodified nanoparticle and the cell or cell surface receptor is >1.0, more preferably ≥1.2, more preferably ≥1.5, more preferably ≥2, more preferably ≥3, more preferably ≥5, more preferably ≥10, more preferably ≥15, more preferably ≥20, more preferably ≥30, more preferably ≥50, more preferably ≥80, more preferably ≥100, more preferably ≥80, more preferably ≥150, more preferably ≥200, more preferably ≥500, more preferably ≥1000, more preferably ≥5000, more preferably ≥10000.

[0391] Preferably, B1 / B0 is 1.5-10000, more preferably 2-500, even more preferably 2-200, even more preferably 2-100, even more preferably 2-50, and even more preferably 5-30.

[0392] Preferably, "greater than" means that the binding force B1 of the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles to the cell or cell surface receptor in the test group with biological reproducibility is greater than the binding force B0 of the unmodified nanoparticles to the cell or cell surface receptor in the control group with biological reproducibility, and the P value is less than 0.05 after t-test.

[0393] Preferably, the ligand comprises a polypeptide or protein ligand.

[0394] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[0395] Preferably, the binding includes affinity.

[0396] Preferably, the binding force includes affinity.

[0397] Preferably, the ligand includes a potential ligand.

[0398] Preferably, the ligand-modified nanoparticles include in vitro or isolated ligand-modified nanoparticles.

[0399] Preferably, the cell or cell surface receptor includes in vitro or isolated cells or cell surface receptors.

[0400] Preferably, the method includes an in vitro method or an in vivo method.

[0401] Preferably, the method includes non-therapeutic and / or non-diagnostic methods.

[0402] Preferably, the incubation is in vitro incubation or in vivo incubation.

[0403] Preferably, the body refers to the body of a human or non-human mammal.

[0404] Preferably, the incubation includes incubation in conditions containing protein.

[0405] Preferably, the incubation involves incubating the cell or cell surface receptor with the ligand-modified nanoparticles of step (I) in a protein-containing environment.

[0406] Preferably, the conditions include in vivo conditions or in vitro conditions.

[0407] Preferably, the cells include cells that need to be cultured or grown in conditions containing proteins.

[0408] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[0409] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[0410] Preferably, the culture includes in vitro culture.

[0411] Preferably, the conditions containing the protein include blood, serum, plasma, cell tissue microenvironment, or culture medium.

[0412] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[0413] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[0414] Preferably, the proteins include serum proteins, plasma proteins, and / or tissue proteins.

[0415] Preferably, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0416] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[0417] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0418] Preferably, the cattle are fetal cattle.

[0419] Preferably, the serum comprises fetal bovine serum.

[0420] Preferably, the plasma includes fetal bovine plasma.

[0421] Preferably, the culture medium comprises a liquid culture medium.

[0422] Preferably, the culture medium includes a cell culture medium.

[0423] Preferably, the culture medium includes a protein-containing culture medium.

[0424] Preferably, the culture medium contains protein.

[0425] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[0426] Preferably, the culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

[0427] Preferably, the culture medium includes a serum-containing culture medium.

[0428] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[0429] Preferably, the incubation involves incubating the nanoparticles modified with the ligands of step (I) with cell or cell surface receptors in a culture medium containing serum, plasma and / or tissue proteins.

[0430] Preferably, the incubation involves incubating the cell or cell surface receptor with the ligand-modified nanoparticles of step (I) in a serum-containing culture medium.

[0431] Preferably, the ligand includes a ligand that targets a cell or a cell surface receptor.

[0432] Preferably, the cells include tumor cells and / or tumor vascular cells.

[0433] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[0434] Preferably, the tumor includes human tumors (such as human tumors) or non-human mammalian tumors.

[0435] Preferably, the tumor includes a low-permeability tumor.

[0436] Preferably, the tumor includes tumors with low vascular permeability.

[0437] Preferably, the tumor includes a solid tumor.

[0438] Preferably, the tumor includes a solid tumor with low vascular permeability.

[0439] Preferably, the tumor includes liver cancer.

[0440] Preferably, the tumor includes human liver cancer.

[0441] Preferably, the liver cancer cells include Huh7 cells and / or HepG2 cells.

[0442] Preferably, the tumor includes pancreatic cancer.

[0443] Preferably, the pancreatic cancer includes pancreatic adenocarcinoma.

[0444] Preferably, the pancreatic cancer includes pancreatic cancer in situ.

[0445] Preferably, the pancreatic cancer includes pancreatic adenocarcinoma in situ.

[0446] Preferably, the pancreatic cancer includes pancreatic ductal adenocarcinoma.

[0447] Preferably, the pancreatic cancer includes human pancreatic ductal adenocarcinoma.

[0448] Preferably, the pancreatic cancer cells include BxPC-3 cells.

[0449] Preferably, the tumor vascular cells include ECDHCC cells.

[0450] Preferably, the tumor includes tumors with poor enhanced permeability and retention (EPR effect).

[0451] Preferably, the low permeability of the tumor vessels includes low drug penetration from the tumor vessels to the tumor site.

[0452] Preferably, the low permeability of the tumor vessels includes low drug penetration from the intercellular spaces of the tumor vessels to the tumor site.

[0453] Preferably, the tumor vessels with low permeability include one or more characteristics selected from the group consisting of:

[0454] (a) The tumor vascular cells are well-formed and densely packed; and / or

[0455] (b) The tumor vascular cells have small intercellular spaces.

[0456] Preferably, the vascular cells include vascular endothelial cells.

[0457] Preferably, the ligand is capable of binding to cells or cell surface receptors.

[0458] Preferably, the binding includes specific binding or non-specific binding.

[0459] Preferably, the receptor includes a receptor on the outer surface of the cell membrane.

[0460] Preferably, the assay method for the binding includes isotope disappearance assay, fluorescein assay, flow cytometry assay and / or transwell migration assay.

[0461] Preferably, the nanoparticles and / or the ligands are labeled with isotopes and / or fluoresceins.

[0462] Preferably, the fluorescein includes FITC (Fluorescein isothiocyanate isomer), Cyanine 5 (Cy5), and / or Cyanine 5.5 (Cy5.5).

[0463] Preferably, the binding mediates the uptake of ligand-modified nanoparticles by cells.

[0464] Preferably, the binding mediates endocytosis of ligand-modified nanoparticles by cells.

[0465] Preferably, the binding mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[0466] Preferably, the method for determining the binding includes determining the uptake efficiency of cells of the ligand-modified nanoparticles of step (I).

[0467] Preferably, the cells do not have the ability to take up nanoparticles without ligand modification in the control group.

[0468] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[0469] If the cell’s uptake efficiency of the ligand-modified nanoparticles in step (I) is greater than the cell’s uptake efficiency of the unmodified nanoparticles in the control group, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[0470] Preferably, the method for determining the binding includes determining the endocytosis capacity of cells for the ligand-modified nanoparticles of step (I).

[0471] Preferably, the cells in the control group do not have the ability to endocytose nanoparticles without ligand modification.

[0472] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[0473] If the cell's endocytosis capacity for the ligand-modified nanoparticles of step (I) is greater than the cell's endocytosis capacity for the unmodified nanoparticles in the control group, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[0474] Preferably, the method for determining the binding includes measuring the endocytosis and exocytosis capacity of cells for the ligand-modified nanoparticles of step (I).

[0475] Preferably, the cells in the control group do not have endocytosis or exocytosis capabilities for the nanoparticles without ligand modification.

[0476] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[0477] If the cell's ability to endocytose and exocytose the ligand-modified nanoparticles of step (I) is greater than the cell's ability to endocytose and exocytose the unmodified nanoparticles in the control group, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[0478] Preferably, the ligand in step (I) includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[0479] Preferably, the ligand-modified nanoparticles or the ligands of the ligand-modified nanoparticles bind to cells or cell surface receptors, thereby mediating endocytosis of the ligand-modified nanoparticles by the cells.

[0480] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[0481] If a ligand-modified nanoparticle or a ligand of a ligand-modified nanoparticle binds to a cell or a cell surface receptor and can mediate endocytosis of the ligand-modified nanoparticle by the cell, then the ligand in step (I) is a potential ligand for targeting the cell or cell surface receptor.

[0482] Preferably, the ligand in step (I) includes a ligand that mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[0483] Preferably, the ligand-modified nanoparticles or the ligands of the ligand-modified nanoparticles bind to cells or cell surface receptors, thereby mediating endocytosis and exocytosis of the ligand-modified nanoparticles by the cells.

[0484] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[0485] If ligand-modified nanoparticles or ligands of ligand-modified nanoparticles bind to cells or cell surface receptors and can mediate endocytosis and exocytosis of cells, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[0486] Preferably, the ligand-modified nanoparticles or the ligands of the ligand-modified nanoparticles bind to tumor vascular cells or receptors on the surface of tumor vascular cells, thereby mediating endocytosis of the ligand-modified nanoparticles in the blood by the tumor vascular cells, and after endocytosis, they can be exocytodated outside the tumor blood vessels (such as in the tumor tissue microenvironment).

[0487] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[0488] If ligand-modified nanoparticles or ligands of ligand-modified nanoparticles bind to tumor vascular cells or tumor vascular cell surface receptors, and can mediate endocytosis of ligand-modified nanoparticles in the blood by tumor vascular cells, and can be exocytodated outside the tumor blood vessels (such as the tumor tissue microenvironment), then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[0489] Preferably, the method for determining the binding or binding force between the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles and the cell or cell surface receptor in step (I) includes:

[0490] If tumor vascular cells can endocytose ligand-modified nanoparticles in the blood and then exocytose them outside the tumor blood vessels (such as in the tumor tissue microenvironment), then the ligands can bind to cells or cell surface receptors.

[0491] Preferably, the tumor vascular cells cannot perform endocytosis on unmodified nanoparticles in the blood circulation.

[0492] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor vascular cells.

[0493] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor cells.

[0494] Preferably, the surface includes an outer surface.

[0495] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site.

[0496] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site via endocytosis and exocytosis.

[0497] Preferably, the ligand targets vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of ligand-modified nanoparticles by vascular endothelial cells.

[0498] Preferably, the ligand includes a ligand that mediates the uptake of ligand-modified nanoparticles by cells.

[0499] Preferably, the ligand includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[0500] Preferably, the ligand includes a ligand that mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[0501] Preferably, the ligand can mediate the endocytosis of ligand-modified nanoparticles in the blood by tumor vascular cells, and after endocytosis, it can be exocytodated outside the tumor blood vessels (such as the tumor tissue microenvironment).

[0502] Preferably, the acoustic intensity of the ultrasonic irradiation is 0.1-40 W / cm². 2 The optimal concentration is 0.1-20 W / cm². 2 More preferably 0.2-15W / cm 2 More preferably 0.5-10W / cm 2 Better 1-5W / cm 2 Better 1-3W / cm 2 The optimal concentration is 1.5-2.5 W / cm². 2 Optimal 1.8-2.2W / cm 2 Better 2.0W / cm 2 .

[0503] Preferably, the frequency of the ultrasonic irradiation is 0.02-30MHz, more preferably 0.1-20MHz, even more preferably 0.2-15MHz, even more preferably 0.5-10MHz, even more preferably 1-8MHz, even more preferably 1-5MHz, most preferably 2-4MHz, even more preferably 2.5-3.5MHz, even more preferably 2.8-3.2MHz, and even more preferably 3MHz.

[0504] Preferably, the duty cycle of the ultrasonic irradiation is 10-80%, more preferably 20-80%, more preferably 30-70%, more preferably 35-65%, more preferably 40-60%, most preferably 45-55%, more preferably 48-52%, and more preferably 50%.

[0505] In a thirteenth aspect of the present invention, there is a use of nanoparticles as described in the first aspect of the present invention for preparing carriers for screening or identifying potential ligands targeting cell or cell surface receptors.

[0506] Preferably, the method for screening or identifying potential ligands targeting cell or cell surface receptors includes the following steps:

[0507] (I) Ligands are modified onto nanoparticles to obtain ligand-modified nanoparticles;

[0508] (II) Incubate the cell or cell surface receptor with the ligand-modified nanoparticles of step (I), subject them to ultrasonic irradiation, and determine the binding of the ligand-modified nanoparticles of step (I) or the ligand of the ligand-modified nanoparticles to the cell or cell surface receptor, thereby screening or identifying whether the ligand of step (I) is a potential ligand for targeting the cell or cell surface receptor.

[0509] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[0510] Preferably, the cell surface receptor includes extracellular receptors.

[0511] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[0512] Preferably, the method for screening or identifying potential ligands targeting cell or cell surface receptors is as described in the twelfth aspect of the present invention.

[0513] According to a fourteenth aspect of the present invention, a method for inhibiting cells in vitro is provided, the method comprising the steps of:

[0514] Cells are incubated in a culture medium with nanoparticles as described in the first aspect of the present invention or ligand-modified nanoparticles as described in the third aspect of the present invention, and then subjected to ultrasonic irradiation to inhibit cell growth.

[0515] Preferably, the nanoparticles include drug-loaded nanoparticles.

[0516] Preferably, the method includes a method for enhancing the in vitro cell inhibition of ligand-modified nanoparticles.

[0517] Preferably, the method includes non-diagnostic and non-therapeutic methods.

[0518] Preferably, the ligand comprises a receptor that targets a cell or the cell surface.

[0519] Preferably, the ligand includes a ligand that mediates the uptake of ligand-modified nanoparticles by cells.

[0520] Preferably, the ligand includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[0521] Preferably, the ligand comprises RGD peptide and / or NGR peptide.

[0522] Preferably, the cells include tumor cells and / or tumor vascular cells.

[0523] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[0524] Preferably, the tumor vascular cells include ECDHCC cells.

[0525] Preferably, the drug comprises a cell inhibitor.

[0526] Preferably, the drug includes an antitumor drug.

[0527] Preferably, the drug is as described in the first aspect of the present invention.

[0528] Preferably, the tumor is as described in the sixth aspect of the present invention.

[0529] Preferably, the cells include cells that need to be cultured or grown in conditions containing proteins.

[0530] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[0531] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[0532] Preferably, the protein-containing conditions include blood, serum, plasma, and / or culture medium.

[0533] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[0534] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[0535] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0536] Preferably, the cattle include fetal cattle.

[0537] Preferably, the conditions for containing the protein are as described in the twelfth aspect of the present invention.

[0538] Preferably, the culture medium comprises a liquid culture medium.

[0539] Preferably, the culture medium includes a cell culture medium.

[0540] Preferably, the culture medium contains protein.

[0541] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[0542] Preferably, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0543] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[0544] Preferably, the culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

[0545] Preferably, the culture medium includes a serum-containing culture medium.

[0546] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[0547] Preferably, the serum comprises fetal bovine serum.

[0548] Preferably, the plasma includes fetal bovine plasma.

[0549] Preferably, the culture includes in vitro culture.

[0550] Preferably, the incubation is in vitro incubation.

[0551] Preferably, in the culture medium without ultrasonic irradiation, the surface of the nanoparticles contains a protein crown.

[0552] Preferably, the protein crown is adsorbed on the surface of the nanoparticles.

[0553] Preferably, the ligand, after binding to a cell or a cell surface receptor, can mediate the uptake of ligand-modified nanoparticles by the cell.

[0554] Preferably, the ligand, after binding to a cell or a cell surface receptor, can mediate endocytosis of the ligand-modified nanoparticles by the cell.

[0555] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[0556] Preferably, the cell surface receptor includes extracellular receptors.

[0557] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[0558] According to a fifteenth aspect of the present invention, there is an application of an ultrasonic instrument for manufacturing a device, said device being used for one or more uses selected from the group consisting of:

[0559] (a) Elimination of protein crowns in nanoparticles modified with protein crowns by ultrasonic irradiation;

[0560] (b) Used to screen or identify potential ligands that target cell or cell surface receptors;

[0561] (c) Treatment of disease by ligand-modified nanoparticles applied in conjunction with ultrasound irradiation of lesions (e.g., tumors); and / or

[0562] (d) Improve the retention and / or degradation of nanoparticles, ligand-modified nanoparticles and / or protein crown-modified nanoparticles by lysosomes through ultrasonic irradiation.

[0563] Preferably, the nanoparticles are loaded with perfluoropentane.

[0564] Preferably, the nanoparticles are as described in the first aspect of the present invention.

[0565] Preferably, the ligand-modified nanoparticles are as described in the third aspect of the present invention.

[0566] Preferably, the protein crown-modified nanoparticles are as described in the ninth aspect of the present invention.

[0567] Preferably, the method for eliminating the protein crown of the protein crown-modified nanoparticles is as described in the eleventh aspect of the present invention.

[0568] Preferably, (a) includes improving the efficacy of nanoparticles by eliminating the protein crown of the protein crown-modified nanoparticles through ultrasonic irradiation.

[0569] Preferably, the protein crown of the nanoparticles modified with protein crown by ultrasonic irradiation includes:

[0570] The protein crown-modified nanoparticles are subjected to ultrasonic irradiation to eliminate the protein crown of the nanoparticles.

[0571] Preferably, the method for screening or identifying potential ligands targeting cell or cell surface receptors is as described in the twelfth aspect of the present invention.

[0572] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[0573] Preferably, the cell surface receptor includes extracellular receptors.

[0574] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[0575] Preferably, (d) improving the retention and / or degradation of nanoparticles, ligand-modified nanoparticles, and / or protein crown-modified nanoparticles by lysosomes via ultrasonic irradiation includes:

[0576] After the nanoparticles, ligand-modified nanoparticles, and / or protein crown-modified nanoparticles are incubated with cells, the cells are subjected to ultrasonic irradiation, thereby improving the retention and / or degradation of the nanoparticles, ligand-modified nanoparticles, and / or protein crown-modified nanoparticles by cell lysosomes.

[0577] Preferably, the method includes an in vitro method or an in vivo method.

[0578] Preferably, the method includes non-therapeutic and / or non-diagnostic methods.

[0579] Preferably, the incubation is in vivo incubation or in vitro incubation.

[0580] Preferably, the incubation includes incubation in conditions containing protein.

[0581] Preferably, the incubation is performed in a serum-containing culture medium.

[0582] Preferably, the incubation is as described in the twelfth aspect of the present invention.

[0583] Preferably, the cells include cells that need to be cultured or grown in conditions containing proteins.

[0584] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[0585] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[0586] Preferably, the conditions for containing the protein are as described in the twelfth aspect of the present invention.

[0587] Preferably, the nanoparticles include drug-loaded nanoparticles.

[0588] Preferably, the drug is as described in the first aspect of the present invention.

[0589] Preferably, the disease is an indication for the drug.

[0590] Preferably, the cells include tumor cells and / or tumor vascular cells.

[0591] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[0592] Preferably, the tumor vascular cells include ECDHCC cells.

[0593] Preferably, the disease includes a tumor.

[0594] Preferably, the tumor is as described in the sixth aspect of the present invention.

[0595] Preferably, the application is by injection, oral administration, or topical application.

[0596] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[0597] Preferably, the injection is administered via intravascular injection.

[0598] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[0599] Preferably, the improvement includes avoiding or overcoming.

[0600] Preferably, the degradation includes degradation by lysosomal enzymes.

[0601] In a sixteenth aspect, the present invention provides the use of a system or apparatus as described in a seventh aspect for preparing a device for treating a disease.

[0602] Preferably, the device further includes an instruction manual or label, which states:

[0603] In the process of treating a disease by applying nanoparticles as described in the first aspect of the present invention and / or ligand-modified nanoparticles as described in the third aspect of the present invention to a desired object, ultrasound irradiation is performed on the lesion site (such as a tumor site).

[0604] Preferably, the nanoparticles include drug-loaded nanoparticles.

[0605] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[0606] Preferably, the object includes a human or a non-human mammal.

[0607] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0608] Preferably, the disease is an indication for the drug.

[0609] Preferably, the drug includes an anticancer drug.

[0610] Preferably, the drug is as described in the first aspect of the present invention.

[0611] Preferably, the disease includes a tumor.

[0612] Preferably, the tumor is as described in the sixth aspect of the present invention.

[0613] Preferably, the application is by injection, oral administration, or topical application.

[0614] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[0615] Preferably, the injection is administered via intravascular injection.

[0616] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[0617] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0618] Figure 1 LPGL liposome nanoparticles were incubated in PBS 7.4 buffer, plasma, and then subjected to ultrasonic irradiation (sound intensity: 2 W / cm²). 2 Cryo-transmission electron microscopy (cryo-TEM) image of the processed material (frequency: 3MHz, duty cycle: 50%; time: 5min).

[0619] Figure 2 PGL liposome nanoparticles were incubated in plasma and then irradiated with ultrasound (sound intensity: 2 W / cm²). 2 Cryo-transmission electron microscopy images before and after treatment (frequency: 3MHz, duty cycle: 50%, duration: 5min).

[0620] Figure 3 Cryo-transmission electron microscopy images of dispersions of GL, LGL and PGL liposome nanoparticles.

[0621] Figure 4 The total protein concentration in the protein corona was obtained after treating protein corona-modified PGL or LPGL liposome nanoparticles with different ultrasound irradiation times. The ultrasound irradiation conditions were as follows: sound intensity: 2 W / cm². 2 Frequency: 3MHz, Duty Cycle: 50%.

[0622] Figure 5To eliminate the protein crown on the surface of nanoparticles by ultrasonic irradiation. (5A) SDS-PAGE analysis of the protein crown solution obtained by Sephadex G200 chromatography after incubation of GL, LGL, PGL, LPGL liposome nanoparticles and plasma with or without ultrasonic irradiation. Among them, (-US) represents the liposome nanoparticle and plasma incubation mixture without ultrasonic irradiation, and (+US) represents the liposome nanoparticle and plasma incubation mixture after ultrasonic irradiation (sound intensity: 2W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Time: 5min) Treatment group. (5B) A mixture of GL, LGL, PGL or LPGL liposome nanoparticles and plasma incubated with or without ultrasonic irradiation (sound intensity: 2W / cm²). 2 After HPLC-MS determination of the total protein content on liposome nanoparticles (frequency: 3MHz, duty cycle: 50%, duration: 5min), the protein content on the liposome nanoparticles was determined.

[0623] Figure 6 After incubating a mixture of PGL or LPGL liposome nanoparticles and plasma with different ultrasonic powers, the total protein content on the liposome nanoparticles was determined by HPLC-MS. The ultrasonic frequency was 3 MHz, the duty cycle was 50%, and the duration was 5 min.

[0624] Figure 7 To investigate the cellular uptake, transendothelial cell transport, endocytosis, and intercellular endocytosis of liposome nanoparticles. (7A) Cy5-labeled GL, LGL, PGL, and LPGL liposome nanoparticles (each equivalent to 60 μg / mL of fluorescent lipid, 20 μL) were premixed with 1 mL of fresh serum-free culture medium or culture medium containing 10% FBS (fetal bovine serum) for 30 min. The resulting mixture was then mixed with BxPC3 cells and subjected to or without ultrasonic irradiation (sound intensity: 2 W / cm²). 2(Frequency: 3MHz, Duty Cy: 50%, Duration: 5min), incubated for another 1h. The average fluorescence intensity of different Cy5-labeled liposome nanoparticles taken up by BxPC3 cells was recorded. "Serium-free medium" represents the average fluorescence intensity of different Cy5-labeled liposome nanoparticles taken up by BxPC3 cells after mixing with a mixture of different Cy5-labeled liposome nanoparticles and serum-free medium, without ultrasonic irradiation, and incubated for another 1h. "Serium medium" represents the average fluorescence intensity of different Cy5-labeled liposome nanoparticles taken up by BxPC3 cells. After mixing BxPC3 cells with a mixture of Cy5-labeled liposome nanoparticles and a medium containing 10% FBS (fetal bovine serum), the cells were incubated for 1 hour without sonication. The average fluorescence intensity of the Cy5-labeled liposome nanoparticles taken up by the BxPC3 cells was measured. "Serium medium + sonication" represents the average fluorescence intensity of the Cy5-labeled liposome nanoparticles taken up by the BxPC3 cells after mixing BxPC3 cells with a mixture of Cy5-labeled liposome nanoparticles and a medium containing 10% FBS (fetal bovine serum), followed by sonication and incubation for 1 hour. (7B) To study the transcellular transport of liposome nanoparticles through ECDHCC vessels, two types of transwell models (mode I or II) were designed: sonication treatment was performed in centrifuge tubes (I) or top compartments (II). (7C) Flow cytometry assay: Plasma or serum-free culture medium pre-incubation mixtures containing liposome nanoparticles with different Cy5 labels were added to the top compartment of a Transwell containing serum-free culture medium without ultrasonic irradiation. Plasma pre-incubation mixtures containing liposome nanoparticles with different Cy5 labels were incubated under non-contact conditions. Figure 7 Method I in B) or contact condition ( Figure 7 (7D) Mean fluorescence intensity (MFI) of Cy5 in BxPC3 cells in transwell after ultrasonic irradiation under different endocytosis inhibitors. Flow cytometry was used to determine the mean fluorescence intensity of Cy5 in BxPC3 cells after incubation with serum-containing or serum-free culture media containing Cy5-labeled LPGL liposome nanoparticles, with or without ultrasonic irradiation. (7E) BxPC3 cells were incubated with serum-containing or serum-free culture media containing Cy5-labeled LPGL liposome nanoparticles, with or without ultrasonic irradiation (sound intensity: 2 W / cm²) after pretreatment with the exocytosis inhibitor EXO1 and without ultrasonic irradiation. 2(Frequency: 3MHz, duty cycle: 50%, duration: 5min) after treatment, and then adding new BxPC3 cells at different culture time points. CLSM diagram of the newly added BxPC3 cells, scale bar = 50μm, where "+ultrasound irradiation" represents ultrasonic irradiation treatment, and "-ultrasound irradiation" represents no ultrasonic irradiation treatment. (7F) As shown in Figure (7E), new BxPC3 cells were added to a culture dish and cultured for 120min. The mean fluorescence intensity (MFI) of Cy5 in the newly added BxPC3 cells was quantitatively measured by flow cytometry. Wherein, "serum" represents incubation of BxPC3 cells with a serum-containing culture medium mixture containing LPGL liposome nanoparticles; "ultrasound" represents ultrasonic irradiation (sound intensity: 2W / cm). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 5min) Processing; “EXO1” represents EXO1 pretreatment of BxPC3 cells; “+” represents selection or use; “-” represents no selection or use.

[0625] Figure 8 Subcellular distribution of Cy5-labeled LPGL liposome nanoparticles with and without serum pre-incubation mixture with BxPC3 cells under ultrasound irradiation. (8A) Subcellular distribution of Cy5-labeled LPGL liposome nanoparticles with and without serum pre-incubation mixture with BxPC3 cells under ultrasound irradiation (sound intensity: 2 W / cm²). 2 At a frequency of 3 MHz, a duty cycle of 50%, and a duration of 5 min, the cells were incubated for another 1 h. CLSM showed the subcellular distribution of Cy5-labeled LPGL liposome nanoparticles in BxPC3 cells, with Cy5-labeled LPGL liposome nanoparticles appearing red and lysosomes appearing green. (8B) The Mander overlap coefficient (MOC) of Cy5-labeled LPGL liposome nanoparticles (red) and lysosomes (green) in Figure (8A) was analyzed using Cellprofiler V2.2.0 image analysis software. The calculation formula is as follows: MOC ranges from 0 to 1, where 1 indicates complete overlap (co-location) and 0 indicates none. i，coloc R represents the pixel intensity of the red pixel that overlaps with the green pixel. i This represents the total pixel intensity of the red pixels. Scale bar = 25μm.

[0626] Figure 9 A serum-containing culture medium mixture containing Cy5-labeled GL, LGL, or PGL liposome nanoparticles was incubated with BxPC3 cells and then subjected to ultrasonic irradiation (sound intensity: 2 W / cm²). 2(Frequency: 3MHz, Duty Cycle: 50%, Duration: 5min) Treatment, followed by the addition of new BxPC3 cells at different culture time points, CLSM plot of the newly added BxPC3 cells, scale bar = 50μm.

[0627] Figure 10 To investigate the in vitro cytotoxicity and permeability of free GEM and different liposome nanoparticles to 3D tumor spheres under ultrasound irradiation. (10A) Incubation of BxPC3 or Huh7 three-dimensional (3D) multicellular tumor spheres with a mixture of LPGL, PGL, LGL, GL or gemcitabine (GEM) and serum-containing culture medium (GEM concentration 0–10 μM) followed by ultrasound irradiation and culture for 72 h, and the inhibitory effect of gemcitabine (GEM) and different liposome nanoparticles on the viability of BxPC3 tumor 3D spheres. (10B) IC50 of gemcitabine (GEM) and different liposome nanoparticles on BxPC3 or Huh7 tumor 3D spheres. 50 (50% inhibiting concentration) value. (10C) Apoptosis of BxPC3 3D multicellular tumor spheroids before and after treatment with LPGL, PGL, LGL, GL liposome nanoparticles and gemcitabine (GEM) was observed by optical microscopy and TUNEL staining. The operation of treating BxPC3 3D multicellular tumor spheroids with free GEM and different liposome nanoparticles was as follows: Under 37℃, serum-containing culture medium (all equivalent to 0.1 μM GEM equivalent dose) containing free GEM and different liposome nanoparticles was mixed with BxPC3 tumor 3D spheroids and irradiated with ultrasound (sound intensity: 2 W / cm). 2 After treatment with (frequency: 3MHz, duty cycle: 50%, duration: 5min), and cultured for 72h, the morphology of BxPC3 tumor 3D spheroids in different treatment groups was observed by optical microscopy and TUNEL staining (scale bar = 500μm). (10D) A mixture of different Cy5-labeled liposome nanoparticles and serum-containing culture medium was added to 3D multicellular tumor spheroids of BxPC3 pretreated or untreated with the exocytosis inhibitor EXO1, and subjected to ultrasound irradiation (sound intensity: 2W / cm²). 2 (Frequency: 3 MHz, duty cycle: 50%, duration: 5 min) After incubation for 6 h, CLSM plots of the permeation characteristics of different Cy5-labeled liposome nanoparticles to 3D multicellular tumor spheres of BxPC3 were obtained, scale bar = 500 μm. (10E) The average integrated optical density (IOD) at 75 μm and 100 μm levels of the 3D spheres of BxPC3 in plot (10D) was analyzed using Image-pro Plus 6.0 software to evaluate the depth permeation capability of the liposome nanoparticles.

[0628] Figure 11Blood CP4126 levels at different time points after tail vein injection of GL, LGL, PGL or LPGL liposome nanoparticles.

[0629] Figure 12 To investigate the biodistribution, tumor accumulation, and penetration of different Cy5-labeled liposome nanoparticles in BALB / c nude mice with subcutaneously loaded human PDA tumors. (12A) An ultrasound instrument for tumor ultrasound irradiation, in which subcutaneous human PDA tumors on the right side of mice were irradiated with ultrasound after intravenous injection of Cy5-labeled liposome nanoparticles (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min), while the left subcutaneous human PDA-loaded tumor was not subjected to ultrasound irradiation. (12B) After intravenous injection of different Cy5-labeled liposome nanoparticles into the tail vein of BALB / c nude mice loaded with human PDA tumors, the right subcutaneous human PDA-loaded tumor was subjected to ultrasound irradiation (sound intensity: 2W / cm). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min) Human PDA tumors loaded subcutaneously on the left side were not irradiated with ultrasound. 12h after injection, in vivo and in vitro fluorescence imaging was performed on BALB / c nude mice and isolated tissues (1: tumors irradiated and unirradiated, 2: heart, 3: liver, 4: spleen, 5: lung, 6: kidney, 7: small intestine) loaded subcutaneously with human PDA tumors on both sides using a Caliper IVIS Lumina II fluorescence spectroscopy imaging system. (12C) Living The software quantitatively analyzed the fluorescence intensity of tumors, heart, liver, spleen, lung, kidney, and small intestine separated by ultrasound irradiation and those not shown in Figure (12B). (12D) CLSM images of different Cy5-labeled liposome nanoparticles penetrating ultrasound-irradiated tumor sites, as follows: After intravenous injection of different Cy5-labeled liposome nanoparticles into the tail vein of BALB / c nude mice loaded with human PDA tumors, the right subcutaneous loaded human PDA tumor was subjected to ultrasound irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min) Human PDA tumors loaded in the left subcutaneous layer were not irradiated with ultrasound. Twelve h after injection, mice were injected with FITC-labeled tomato lectin (FITC-LEL) to stain blood vessels for 5 min, followed by cardiac perfusion. The ultrasound-irradiated tumors were excised, frozen in Tissue-Tek OCT embedding medium, and cut into 10 μm thick sections. CLSM imaging was performed, scale bar = 100 μm. (12E) The average fluorescence intensity gradient from tumor vessels to deep tumor regions is indicated by white arrows in Figure (12D).

[0630] Figure 13 Real-time in vivo extravasation and tumor aggregation of liposomes labeled with different Cy5 in BALB / c nude mice loaded with human PDA tumors. (13A) Ultrasound and CLSM instruments were used to study real-time in vivo extravasation and tumor aggregation. (13B) After injecting different Cy5-labeled liposomes into the tail vein, the tumor site was subjected to ultrasound irradiation (sound intensity: 2 W / cm²). 2 (3MHz, duty cycle: 50%, duration: 20min) At 10min, 30min and 60min after tail vein injection of different Cy5-labeled liposome nanoparticles, CLSM was used to observe in real time the infiltration of Cy5-labeled liposome nanoparticles from PDA tumor vessels into the PDA tumor region. Scale bar = 200μm. (13C) At 10min, 30min and 60min after tail vein injection of Cy5-labeled liposome nanoparticles, the average fluorescence integrated density of the selected area (circled) in Figure (13B) was quantitatively analyzed. (13D) Transmission electron microscopy (TEM) was used to observe and analyze the ultrastructure of tumor vessels 60min after tail vein injection of different Cy5-labeled liposome nanoparticles in Figure (13B). Arrows indicate transendothelial transporters of vesicles. Scale bar = 500nm.

[0631] Figure 14 To investigate the antitumor activity of free GEM and different liposomal nanoparticles in BALB / c nude mice subcutaneously loaded with human PDA tumors. (14A) Construction of the BALB / c nude mouse animal model loaded with human PDA tumors, experimental schedule, and tumor treatment regimen. Immediately after intravenous injection of GEM, GL, LGL, PGL, LPGL, LPL, or PBS 7.4, the tumor sites of all mice were subjected to ultrasound irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min). (14B) Tumor volume changes in each group of mice during treatment. (14C) Photographs of each group of mice at the end of the 36-day experiment. (14D) Photographs of tumors removed from each group of mice at the end of the 36-day experiment. (14E) Average tumor weight removed from each group of mice at the end of the 36-day experiment. (14F) Weight changes in each group of mice during treatment. (14G) White blood cell (WBC) count in the blood of each group of mice at the end of the 36-day experiment. (14H) Platelet (PLT) count in the blood of each group of mice at the end of the 36-day experiment. (14I) H&E staining, Ki67 IHC staining, and TUNEL staining of tumor tissue in each group of mice at the end of the experiment, scale bar = 100μm. * :P<0.05, ** :P<0.01, *** :P<0.001.

[0632] Figure 15 The mean integrated optical density of positive tumor cells in Ki67 immunohistochemical staining of tumor tissues from mice in each group at the end of the 36-day experiment is *: P < 0.05, **: P < 0.01.

[0633] Figure 16 To eliminate the protein crown on the surface of nanoparticles and the uptake of liposome nanoparticles by cells by ultrasonic irradiation. (16A) NGR ligand-modified LPGL and NGR ligand-modified LGL liposome nanoparticles were incubated with mouse plasma for 30 min, and then subjected to ultrasonic irradiation (-US) or (+US, sound intensity: 2 W / cm) to eliminate the protein crown on the surface of nanoparticles and the uptake of liposome nanoparticles by cells. 2 (Frequency: 3MHz, duty cycle: 50%, duration: 5min) Concentration of total protein in the protein crowns of NGR ligand-modified LPGL and NGR ligand-modified LGL liposome nanoparticles. (16B) Serum-free or serum-containing medium mixtures containing Cy5-labeled NGR-modified LGL and Cy5-labeled NGR-modified LPGL liposome nanoparticles were mixed with Huh7 cells and subjected to or without ultrasonic irradiation (sound intensity: 2W / cm²). 2 (Frequency: 3MHz, Duty Cy: 50%, Duration: 5min), and incubated for another 1h. The average fluorescence intensity of Cy5-labeled liposome nanoparticles taken up by Huh7 cells was measured. "Serium-free" refers to the average fluorescence intensity in Huh7 cells after incubation for 1h with a serum-free culture medium mixture containing Cy5-labeled NGR-modified LGL and Cy5-labeled NGR-modified LPGL liposome nanoparticles without ultrasonic irradiation. "Serium" refers to the average fluorescence intensity in Huh7 cells after incubation for 1h with a serum-containing culture medium mixture containing Cy5-labeled NGR-modified LGL and Cy5-labeled NGR-modified LPGL liposome nanoparticles without ultrasonic irradiation. "Serium + Ultrasound" refers to the average fluorescence intensity in Huh7 cells after incubation for 1h with a serum-containing culture medium mixture containing Cy5-labeled NGR-modified LGL and Cy5-labeled NGR-modified LPGL liposome nanoparticles followed by ultrasonic irradiation (sound intensity: 2W / cm²). 2 (Frequency: 3MHz, duty cycle: 50%, duration: 5min), mean fluorescence intensity in Huh7 cells after 1h incubation.

[0634] Figure 17The antitumor activity of GEM, GL, NGR-modified LGL, PGL, NGR-modified LPGL, or PBS on subcutaneously loaded human HCC tumors in BALB / c nude mice was investigated. The dosage (based on GEM) was the same in each group of GEM, GL, NGR-modified LGL, PGL, and NGR-modified LPGL. (17A) Treatment schedule and tumor treatment regimen for BALB / c nude mouse animal models loaded with human HCC tumors: After intravenous injection of GEM, GL, NGR-modified LGL, PGL, NGR-modified LPGL, or PBS, the tumor sites of mice were subjected to ultrasonic irradiation (sound intensity: 2 W / cm2, frequency: 3 MHz, duty cycle: 50%, duration: 20 min). (17B) 24 h after the first intravenous injection of GEM, GL, NGR-modified LGL, PGL, and NGR-modified LPGL liposome nanoparticles, the cumulative concentration of gemcitabine triphosphate active metabolite dFdCTP of GEM in the tumors of mice in each group. (17C) Changes in body weight of mice in each group during treatment. (17D) Changes in tumor volume of mice in each group during treatment. (17E) Photographs of tumors removed from mice in each group at the end of the experiment on day 34. (17F) Average tumor weight of mice in each group after tumor removal at the end of the experiment on day 34. (17G) H&E staining and Ki67 IHC staining of tumor tissue removed from mice in each group at the end of the experiment, scale bar = 100 μm. * :P<0.05, ** :P<0.01, *** :P<0.001. Detailed Implementation

[0635] The inventors have developed a nanoparticle loaded with perfluoropentane. Ultrasonic irradiation can eliminate the protein crown on the nanoparticle surface, overcoming the masking effect of the protein crown on the ligands modified on the nanoparticle surface, thereby restoring the binding of the ligands modified on the nanoparticle surface to receptors on target cells (such as tumor vascular cells or tumor cells). Furthermore, the nanoparticles of this invention can be effectively used as carriers for screening or identifying potential ligands targeting cells or cell surface receptors under ultrasonic irradiation treatment. Ultrasonic irradiation also enables the nanoparticles of this invention to possess excellent lysosomal escape and lysosomal degradation prevention capabilities. Lysosomal escape effectively protects the nanoparticles and their loaded drugs from lysosomal degradation, thereby enhancing the stability of the drug loaded on the nanoparticles within cells and improving the therapeutic effect.

[0636] the term

[0637] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0638] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0639] As used in this article, “cancer”, “tumor” and “carcinoma” can be used interchangeably.

[0640] As used in this article, “cryo-TEM” refers to cryo-transmission electron microscope.

[0641] As used in this article, "DSPE-PEG" stands for disteaaroyl phosphoethanolamine-PEG.

[0642] As used in this article, "DSPE-PEG2000" refers to disteaaroyl phosphoethanolamine-PEG2000.

[0643] As used in this article, "DPPC" refers to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, also known as: dipalmitoylphosphatidylcholine, with the English name 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.

[0644] As used in this article, "FBS" refers to fetal bovine serum.

[0645] As used in this article, “GEM” refers to gemcitabine.

[0646] As used in this article, "Cy5" refers to Cyanine 5.

[0647] As used in this article, "Cy5.5" refers to Cyanine 5.5.

[0648] As used in this article, "TUNEL" refers to TdT-mediated dUTP nick end labeling.

[0649] As used in this article, "CLSM" refers to a confocal laser scanning microscope.

[0650] As used in this article, the term "IC" 50 "50% inhibiting concentration" refers to the concentration of the inhibitor that achieves a 50% inhibitory effect.

[0651] As used in this article, the terms "gemcitabine prodrug CP4126" and "CP4126" are used interchangeably. The structural formula of gemcitabine prodrug CP4126 is as follows:

[0652]

[0653] As used herein, the term "glycerol phosphate buffer" refers to a phosphate buffer containing glycerol.

[0654] As used herein, the terms "IC50" and "IC" are distinct. 50 "Interchangeable" refers to half-inhibiting concentration, which is the concentration of the inhibitor that achieves a 50% inhibitory effect.

[0655] As used in this article, the term "+US" refers to ultrasonic irradiation.

[0656] As used in this article, the term "-US" refers to treatment without ultrasonic irradiation.

[0657] As used herein, the terms “PBS,” “phosphate buffer,” and “PBS buffer” are used interchangeably and refer to an aqueous solution of phosphate buffer.

[0658] As used herein, the term "RPMI 1640 medium" refers to Roswell Park Memorial Institute 1640 medium.

[0659] As used herein, the term "DMEM medium" refers to Dulbecco's Modified Eagle Medium.

[0660] As used herein, the terms “RGD”, “RGD polypeptide”, “RGD targeting peptide” and “RGD ligand” are used interchangeably, and their amino acid sequence is Cys(cysteine)-Arg(arginine)-Gly(glycine)-Asp(aspartic acid)-Lys(lysine)-Gly(glycine)-Pro(proline)-Asp(aspartic acid)-Cys(cysteine).

[0661] As used herein, the terms “NGR”, “NGR polypeptide”, “NGR targeting peptide” and “NGR ligand” are used interchangeably, and their amino acid sequence is Gly-Gly-Cys-Asn-Gly-Arg-Cys.

[0662] As used herein, the term "DSPE-PEG-ligand" refers to a ligand coupled to DSPE-PEG. For example, DSPE-PEG2000-RGD (distearylphosphatidylethanolamine-polyethylene glycol 2000-RGD) means that RGD is coupled to DSPE-PEG2000; DSPE-PEG2000-NGR (distearylphosphatidylethanolamine-polyethylene glycol 2000-NGR) means that NGR is coupled to DSPE-PEG2000.

[0663] As used in this article, the term "perfluoropentane" is translated as perfluoropentane.

[0664] As used in this article, the terms “ultrasound irradiation” and “ultrasound stimulation” are used interchangeably.

[0665] As used in this article, “LPGL” and “LPGL liposome nanoparticles” are used interchangeably.

[0666] As used in this article, “LGL” and “LGL liposome nanoparticles” are used interchangeably.

[0667] As used in this article, “PGL” and “PGL liposome nanoparticles” are used interchangeably.

[0668] As used in this article, "GL" and "GL liposome nanoparticles" are used interchangeably.

[0669] As used in this article, “LPL” and “LPL liposome nanoparticles” are used interchangeably.

[0670] As used in this article, acoustic intensity refers to acoustic intensity.

[0671] As used in this article, frequency refers to frequency.

[0672] As used in this article, duty cycle refers to the duty cycle.

[0673] In this invention, the term "prevention" means a method of preventing the onset of a disease and / or its accompanying symptoms or protecting a subject from acquiring a disease.

[0674] In this invention, the term "treatment" includes inhibiting, reducing, alleviating, reversing, or eradicating the progression of a disease, and does not require 100% inhibition, eradication, or reversal. In some embodiments, the drug-loaded nanoparticles of this invention reduce, inhibit, and / or reverse the associated disease (such as tumor) and its complications by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 100%, compared to levels observed in the absence of the drug-loaded nanoparticles of this invention.

[0675] In this invention, the term "elimination" includes reduction or removal, and does not necessarily require 100% removal. In some embodiments, ultrasonic treatment of protein crown-modified nanoparticles reduces the protein content of the protein crown by, for example, at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or about 100%, such as 80-90%, compared to protein crown-modified nanoparticles before ultrasonic treatment.

[0676] tumor

[0677] The tumors described in this invention may include, but are not limited to, human tumors (such as human tumors) or non-human mammalian tumors.

[0678] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0679] In a preferred embodiment of the invention, the tumor comprises a low-permeability tumor.

[0680] Preferably, the tumor includes tumors with low vascular permeability.

[0681] Preferably, the tumor includes a solid tumor.

[0682] Preferably, the tumor includes a solid tumor with low vascular permeability.

[0683] In a preferred embodiment of the invention, the tumor includes liver cancer.

[0684] Preferably, the tumor includes human liver cancer.

[0685] Preferably, the liver cancer cells include Huh7 cells and / or HepG2 cells.

[0686] In a preferred embodiment of the invention, the tumor includes pancreatic cancer.

[0687] Preferably, the pancreatic cancer includes pancreatic adenocarcinoma.

[0688] Preferably, the pancreatic cancer includes pancreatic cancer in situ.

[0689] Preferably, the pancreatic cancer includes pancreatic adenocarcinoma in situ.

[0690] Preferably, the pancreatic cancer includes pancreatic ductal adenocarcinoma.

[0691] Preferably, the pancreatic cancer includes human pancreatic ductal adenocarcinoma.

[0692] Preferably, the pancreatic cancer cells include BxPC-3 cells.

[0693] In a preferred embodiment of the invention, the tumor includes tumors with poor enhanced permeability and retention (EPR effect).

[0694] Preferably, the low permeability of the tumor vessels includes low drug penetration from the tumor vessels to the tumor site.

[0695] Preferably, the low permeability of the tumor vessels includes low drug penetration from the intercellular spaces of the tumor vessels to the tumor site.

[0696] In a preferred embodiment of the invention, the tumor vessels with low permeability include one or more features selected from the group consisting of:

[0697] (a) The tumor vascular cells are well-formed and densely packed; and / or

[0698] (b) The tumor vascular cells have small intercellular spaces.

[0699] Preferably, the vascular cells include vascular endothelial cells.

[0700] cell

[0701] The cells described in this invention may include, but are not limited to, tumor cells and / or tumor vascular cells, etc.

[0702] In a preferred embodiment of the invention, the cells include tumor cells.

[0703] Preferably, the tumor cells include one or more of Huh7 cells, HepG2 cells, and / or BxPC-3 cells.

[0704] In a preferred embodiment of the invention, the cells include tumor vascular cells.

[0705] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[0706] Preferably, the tumor vascular cells include ECDHCC cells.

[0707] In a preferred embodiment of the invention, the cells include cells that need to be cultured or grown in conditions containing proteins.

[0708] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[0709] Preferably, the serum comprises fetal bovine serum.

[0710] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[0711] ligands

[0712] The ligands described in this invention may include, but are not limited to, polypeptide or protein ligands.

[0713] In a preferred embodiment of the invention, the ligand comprises a ligand that targets a cell surface receptor.

[0714] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[0715] Preferably, the cell surface receptor includes extracellular receptors.

[0716] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor vascular cells.

[0717] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor cells.

[0718] Preferably, the surface includes an outer surface.

[0719] In a preferred embodiment of the invention, the ligand includes a ligand that mediates the uptake of ligand-modified nanoparticles by cells.

[0720] In a preferred embodiment of the invention, the ligand includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[0721] In a preferred embodiment of the invention, the ligand includes a ligand that mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[0722] In a preferred embodiment of the invention, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site.

[0723] In a preferred embodiment of the invention, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site via endocytosis and exocytosis.

[0724] In a preferred embodiment of the invention, the ligand targets vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of ligand-modified nanoparticles by vascular endothelial cells.

[0725] In a preferred embodiment of the present invention, the ligand is capable of mediating endocytosis of ligand-modified nanoparticles in the blood by tumor vascular cells, and after endocytosis, can be exocytodated outside the tumor blood vessels (such as the tumor tissue microenvironment).

[0726] Preferably, the ligand comprises RGD peptide and / or NGR peptide.

[0727] drug

[0728] The drugs described in this invention are not particularly limited and may include, but are not limited to, anti-tumor drugs.

[0729] In a preferred embodiment of the invention, the drug includes a drug that is unstable in lysosomes.

[0730] Preferably, the drug includes drugs that are retained and / or degraded by lysosomes.

[0731] Preferably, the degradation includes degradation by lysosomal enzymes.

[0732] Preferably, the drug comprises a drug that is degraded by lysosomal enzymes.

[0733] In a preferred embodiment of the present invention, the target site of the drug is in the cytoplasm or in the cell nucleus.

[0734] In a preferred embodiment of the invention, the drug comprises a gene or a protein.

[0735] Preferably, the gene includes (but is not limited to) DNA, RNA, or a combination thereof.

[0736] In a preferred embodiment of the present invention, the drug includes an anticancer drug.

[0737] Preferably, the anticancer drug includes a chemical drug.

[0738] Representatively, the anticancer drugs include (but are not limited to): gemcitabine, cytarabine, doxorubicin, fluorouracil, or combinations thereof.

[0739] In a preferred embodiment of the invention, the drug comprises a free drug form or a prodrug form.

[0740] In a preferred embodiment of the invention, the drug comprises a prodrug.

[0741] Preferably, the prodrug comprises a prodrug formed by modifying a prodrug carrier with free drug.

[0742] Preferably, the prodrug comprises a free drug and a prodrug carrier connected by chemical bonds.

[0743] In a preferred embodiment of the present invention, the drug comprises a hydrophobic drug or a hydrophilic drug.

[0744] Preferably, the free drug includes a hydrophobic drug or a hydrophilic drug.

[0745] Preferably, the free drug includes an anticancer drug.

[0746] Preferably, the prodrug carrier includes a hydrophobic carrier or a hydrophilic carrier.

[0747] Preferably, the prodrug carrier includes a higher fatty acid carrier or a higher fatty alcohol carrier.

[0748] Preferably, the prodrug carrier comprises a higher fatty acid containing 12-26 (preferably 14-22, more preferably 16-20) carbon atoms.

[0749] Preferably, the prodrug carrier comprises a higher fatty alcohol containing 12-26 (preferably 14-22, more preferably 16-20) carbon atoms.

[0750] Representatively, the higher fatty acid carriers include (but are not limited to): palmitic acid (hexadecanoic acid), pearlitic acid (heptadecanoic acid), stearic acid (octadecanoic acid), oleic acid (octadecenoic acid), linoleic acid (octadecadienoic acid), linolenic acid (octadecanetrienoic acid), arachidic acid (eicosanoic acid), eicosapentaenoic acid, benzanoic acid (docosahexaenoic acid), DHA (docosahexaenoic acid), ligninic acid (tetracosanoic acid), or combinations thereof.

[0751] Preferably, the oleic acid includes trans-oleic acid.

[0752] Preferably, the higher fatty alcohol carrier is selected from the group consisting of: palmitol, stearyl alcohol, oleyl alcohol, linoleyl alcohol, linolenic acid alcohol, arachidonic acid alcohol, eicosapentaenoic acid, betaine alcohol, docosahexaenoic acid, or combinations thereof.

[0753] In a preferred embodiment of the invention, the prodrug comprises an amphiphilic prodrug.

[0754] Preferably, the amphiphilic prodrug is used as a nanomaterial of nanoparticles.

[0755] Preferably, the amphiphilic prodrug is used as a nanomaterial of nanoparticles.

[0756] Preferably, the amphiphilic prodrug is used as the lipid material of the liposome.

[0757] Preferably, the amphiphilic prodrug is a lipid bilayer.

[0758] Preferably, the amphiphilic prodrug comprises a pharmaceutical active ingredient as a hydrophilic portion and a prodrug carrier as a hydrophobic portion.

[0759] Preferably, the amphiphilic prodrug comprises a pharmaceutical active ingredient as a hydrophobic portion and a prodrug carrier as a hydrophilic portion;

[0760] Typically, the prodrug includes:

[0761] DC

[0762] Wherein, "D" represents the active pharmaceutical ingredient, "C" represents the prodrug carrier, and "-" is the connecting bond.

[0763] Preferably, the active pharmaceutical ingredient includes a drug that is unstable in lysosomes.

[0764] Preferably, the active pharmaceutical ingredient includes a hydrophobic active pharmaceutical ingredient or a hydrophilic active pharmaceutical ingredient.

[0765] Preferably, the active pharmaceutical ingredient includes active pharmaceutical ingredients that are retained and / or degraded by lysosomes.

[0766] Preferably, the degradation includes degradation by lysosomal enzymes.

[0767] Preferably, the active pharmaceutical ingredient includes an active pharmaceutical ingredient that has been degraded by lysosomal enzymes.

[0768] Preferably, the target site of the active pharmaceutical ingredient is in the cytoplasm or the cell nucleus.

[0769] Preferably, the active pharmaceutical ingredient includes a gene or a protein.

[0770] Preferably, the gene is selected from the group consisting of DNA, RNA, or a combination thereof.

[0771] Preferably, the active pharmaceutical ingredient includes an anticancer drug.

[0772] Typically, the prodrug includes:

[0773]

[0774] Where R represents an anticancer drug.

[0775] Preferably, R is an anticancer drug, including gemcitabine, cytarabine, doxorubicin, fluorouracil, or combinations thereof.

[0776] Typically, the drugs mentioned include gemcitabine transoleate.

[0777] Typically, the prodrugs include gemcitabine trans oleate.

[0778] Preferably, the gemcitabine trans oleate has the following structure:

[0779]

[0780] Non-human mammals

[0781] The non-human mammals described in this invention may include, but are not limited to, mice, rats, rabbits, monkeys, cattle, horses, sheep, dogs, cats, orangutans, or baboons.

[0782] Preferably, the cattle include fetal cattle.

[0783] Ultrasonic irradiation

[0784] The acoustic intensity, frequency, duty cycle, and time of the ultrasonic irradiation described in this invention can be determined according to specific requirements.

[0785] Preferably, the acoustic intensity of the ultrasonic irradiation is 0.1-40 W / cm². 2 The optimal concentration is 0.1-20 W / cm². 2 More preferably 0.2-15W / cm 2 More preferably 0.5-10W / cm 2 Better 1-5W / cm 2 Better 1-3W / cm 2 The optimal concentration is 1.5-2.5 W / cm². 2 Optimal 1.8-2.2W / cm 2 Better 2.0W / cm 2 .

[0786] Preferably, the frequency of the ultrasonic irradiation is 0.02-30MHz, more preferably 0.1-20MHz, even more preferably 0.2-15MHz, even more preferably 0.5-10MHz, even more preferably 1-8MHz, even more preferably 1-5MHz, most preferably 2-4MHz, even more preferably 2.5-3.5MHz, even more preferably 2.8-3.2MHz, and even more preferably 3MHz.

[0787] Preferably, the duty cycle of the ultrasonic irradiation is 10-80%, more preferably 20-80%, more preferably 30-70%, more preferably 35-65%, more preferably 40-60%, most preferably 45-55%, more preferably 48-52%, and more preferably 50%.

[0788] Preferably, the ultrasonic irradiation time is greater than 2 minutes, more preferably greater than 5 minutes, more preferably greater than 10 minutes, more preferably greater than 15 minutes, for example 15-20 minutes or 25-35 minutes.

[0789] Conditions containing protein

[0790] In this invention, the conditions containing proteins may include, but are not limited to, blood, serum, plasma, cell tissue microenvironment, or culture medium.

[0791] In a preferred embodiment of the invention, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[0792] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[0793] In a preferred embodiment of the invention, the proteins include serum proteins, plasma proteins, and / or tissue proteins.

[0794] In a preferred embodiment of the invention, the serum proteins, plasma proteins and / or tissue proteins include serum proteins, plasma proteins and / or tissue proteins of humans or non-human mammals.

[0795] In a preferred embodiment of the present invention, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0796] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0797] Preferably, the cattle include fetal cattle.

[0798] Preferably, the serum comprises fetal bovine serum.

[0799] Preferably, the plasma includes fetal bovine plasma.

[0800] In a preferred embodiment of the invention, the culture medium comprises a liquid culture medium.

[0801] In a preferred embodiment of the invention, the culture medium comprises a cell culture medium.

[0802] In a preferred embodiment of the invention, the culture medium comprises a protein-containing culture medium.

[0803] In a preferred embodiment of the invention, the culture medium contains protein.

[0804] In a preferred embodiment of the invention, the culture medium comprises serum proteins, plasma proteins, and / or tissue proteins.

[0805] In a preferred embodiment of the present invention, the culture medium of the present invention comprises a culture medium containing serum, plasma and / or tissue proteins.

[0806] Preferably, the culture medium includes a serum-containing culture medium.

[0807] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[0808] Nanoparticles and their preparation methods

[0809] The nanoparticles mentioned in this invention refer to microscopic particles at the nanometer scale.

[0810] In a preferred embodiment of the present invention, the nanoparticles are loaded with perfluoropentane.

[0811] In a preferred embodiment of the present invention, the nanoparticles are coated with perfluoropentane.

[0812] In a preferred embodiment of the present invention, the nanoparticles are nanoparticles or liposomes.

[0813] In a preferred embodiment of the present invention, the nanoparticles described herein are nanoparticles. Nanoparticles are colloidal microparticles with a particle size on the nanometer scale (0.1–100 nm) made from natural or synthetic polymer materials.

[0814] In a preferred embodiment of the present invention, the nanoparticles described herein are liposomes. Liposomes are particles with a bilayer structure, similar to cell membranes.

[0815] In a preferred embodiment of the present invention, the nanoparticles comprise nanomaterials.

[0816] Preferably, the nanomaterials include amphiphilic materials.

[0817] Preferably, the nanomaterials include nanoparticles and / or lipid materials of liposomes.

[0818] Preferably, the amphiphilic material includes amphiphilic nanoparticles and / or lipid materials of liposomes.

[0819] Preferably, the liposomes comprise lipid materials.

[0820] In a preferred embodiment of the present invention, the lipid material comprises one or more of the following: 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPE), soybean lecithin, phosphatidylcholine (PC, lecithin), cholesterol, phosphatidylethanolamine (PE, cephalin), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), dicetrin (DCP), dimyristicoylphosphatidylcholine (DMPC), distearylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), and dioleoylphosphatidylcholine (DOPC).

[0821] Preferably, the lipid material comprises 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0822] Preferably, the distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) is selected from the group consisting of: DSPE-PEG600, DSPE-PEG800, DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG4000, DSPE-PEG6000, or combinations thereof.

[0823] In a preferred embodiment of the present invention, the DPPC is 1-10 parts by weight, more preferably 2-8 parts by weight, more preferably 4-6 parts by weight, and most preferably 3 parts by weight.

[0824] In a preferred embodiment of the present invention, the DSPE-PEG is 0.5-8 parts by weight, more preferably 1-5 parts by weight, more preferably 1-3 parts by weight, and most preferably 2 parts by weight.

[0825] In a preferred embodiment of the present invention, the perfluoropentane is 0.01-0.5 parts by weight, more preferably 0.02-0.2 parts by weight, more preferably 0.05-0.15 parts by weight, more preferably 0.08-0.12 parts by weight, and most preferably 0.1 parts by weight.

[0826] Preferably, the weight ratio of DPPC to DSPE-PEG is 0.2-8:1, more preferably 0.5-5:1, even more preferably 1-2:1, even more preferably 1.3-1.7:1, and most preferably 1.5:1.

[0827] Preferably, the volume weight ratio (ml:mg) of the perfluoropentane to the DPPC is 1:20-40, more preferably 1:25-35, even more preferably 1:27-32, and most preferably 1:30.

[0828] In a preferred embodiment of the invention, the nanoparticles include drug-loaded nanoparticles.

[0829] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[0830] Preferably, the drug is in the form of 0.5-8 parts by weight, more preferably 1-5 parts by weight, even more preferably 1-3 parts by weight, and most preferably 2 parts by weight.

[0831] Preferably, the weight ratio of DPPC to the drug is 0.2-8:1, more preferably 0.5-5:1, even more preferably 1-2:1, even more preferably 1.3-1.7:1, and most preferably 1.5:1.

[0832] Preferably, the nanoparticles further include water, a buffer solution, and / or perfluoropentane.

[0833] Preferably, the nanoparticles encapsulate water, a buffer solution, and / or perfluoropentane.

[0834] Preferably, the lipid bilayer of the liposome encapsulates water, a buffer solution, and / or perfluoropentane.

[0835] Preferably, the buffer solution comprises a phosphate buffer containing glycerol.

[0836] Preferably, the glycerol-containing phosphate buffer contains 5-15% glycerol by volume, more preferably 8-12%, and even more preferably 10%.

[0837] Preferably, the concentration of the phosphate buffer is 5-15 mM, more preferably 8-12 mM, and even more preferably 10 mM, based on the concentration of phosphate.

[0838] Preferably, the pH of the glycerol-containing phosphate buffer solution is 7.2-7.6, more preferably 7.4.

[0839] Preferably, the lipid bilayer is coated with perfluoropentane and / or a phosphate buffer containing glycerol.

[0840] Preferably, the encapsulation efficiency of the drug-loaded nanoparticles is ≥90%, more preferably ≥95%, more preferably ≥99%, and most preferably 100%.

[0841] Preferably, the drug loading of the drug-loaded nanoparticles is 8-15 wt%, more preferably 9-11 wt%.

[0842] The present invention also provides a method for preparing the nanoparticles described herein, the method comprising the steps of:

[0843] (1) Dissolve the nanomaterials in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0844] (2) After immersing the nanoparticle membrane in perfluoropentane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain nanoparticles.

[0845] In a preferred embodiment of the present invention, the nanoparticles are drug-loaded nanoparticles, and the method for preparing the drug-loaded nanoparticles includes the following steps:

[0846] (1) Dissolve nanomaterials and drugs in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0847] (2) After immersing the nanoparticle membrane in perfluoropentane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain nanoparticles.

[0848] Preferably, the nanoparticles are liposomes, and the method for preparing the liposomes includes the following steps:

[0849] (1) Dissolve the lipid material in an organic solvent, remove the organic solvent, and obtain a lipid membrane;

[0850] (2) After immersing the lipid membrane in perfluoropentane, add a buffer solution to hydrate the lipid membrane and stir to obtain liposomes.

[0851] Preferably, the nanoparticles are drug-loaded liposomes, and the method for preparing the drug-loaded liposomes includes the following steps:

[0852] (1) The lipid material and the drug are dissolved in an organic solvent, and the organic solvent is removed to obtain a lipid membrane;

[0853] (2) After immersing the lipid membrane in perfluoropentane, add a buffer solution to hydrate the lipid membrane and stir to obtain liposomes.

[0854] In a preferred embodiment of the present invention, in step (1), the organic solvent is selected from the group consisting of chloroform, dichloromethane, or combinations thereof.

[0855] In a preferred embodiment of the present invention, in step (1), the weight-to-volume ratio (mg:ml) of the DPPC to the organic solvent is 1:0.2-5, more preferably 1:0.5-2, more preferably 1:0.5-1.5, even more preferably 1:0.8-1.2, and most preferably 1:1.

[0856] Preferably, the volume ratio of the perfluoropentane to the buffer solution is 1:30-70, more preferably 1:40-60, even more preferably 1:45-55, and even more preferably 1:48-52.

[0857] In a preferred embodiment of the present invention, in step (1), the weight-to-volume ratio (mg:ml) of the drug to the organic solvent is 1:2-5, more preferably 1:1-2, more preferably 1:1.3-1.7, and more preferably 1:1.5.

[0858] Preferably, in step (1), the organic solvent is removed by rotary vacuum evaporation.

[0859] Preferably, in step (1), the organic solvent is removed by rotary vacuum evaporation at 35-40°C.

[0860] Preferably, in step (2), perfluoropentane is immersed in the lipid membrane at low temperature.

[0861] Preferably, in step (2), the hydration is carried out at a low temperature.

[0862] Preferably, in step (2), the stirring includes the following steps:

[0863] First stir at a low temperature, then stir again after raising the temperature.

[0864] Preferably, the low temperature is 2-10°C, more preferably 2-6°C, and most preferably 4°C.

[0865] Preferably, the stirring time at low temperature is 0.2-0.8h, more preferably 0.4-0.6h, and even more preferably 0.5h.

[0866] Preferably, the temperature rise is 20-40°C, more preferably 25-35°C, and even more preferably 28-32°C.

[0867] Preferably, the stirring time after the temperature is increased is 0.5-1.5h, more preferably 0.8-1.2h, and even more preferably 1h.

[0868] Preferably, the stirring includes stirring with a magnetic stir bar.

[0869] Preferably, during the stirring process after the temperature is raised, the container holding the stirring liquid is in an open state.

[0870] Preferably, the stirring after raising the temperature can remove unencapsulated perfluoropentane.

[0871] Preferably, the liposomes are in the form of liposome nanodroplets.

[0872] Typically, the method for preparing the liposomes includes the following steps:

[0873] (1) DPPC and DSPE-PEG are dissolved in the organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0874] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain liposomes.

[0875] Typically, the method for preparing the liposomes includes the steps of:

[0876] (1) 2.8-3.2 mg DPPC and 1.8-2.2 mg DSPE-PEG were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0877] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃ for 0.3-0.7h, and then stir in a round-bottom flask in a water bath at 28-32℃ for 0.8-1.2h under open conditions to obtain liposomes.

[0878] Typically, the method for preparing the drug-loaded liposomes includes the following steps:

[0879] (1) DPPC, DSPE-PEG and the drug are dissolved in an organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0880] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain drug-loaded liposomes.

[0881] Typically, the method for preparing the drug-loaded liposomes includes the steps of:

[0882] (1) 2.8-3.2 mg DPPC, 1.8-2.2 mg DSPE-PEG and 1.8-2.2 mg of drug were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation, forming a lipid film in the round-bottom flask;

[0883] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃ for 0.3-0.7h, and then stir in a round-bottom flask in a water bath at 28-32℃ for 0.8-1.2h under open conditions to obtain drug-loaded liposomes.

[0884] Ligand-modified nanoparticles and their preparation methods

[0885] The present invention provides ligand-modified nanoparticles, wherein the ligand-modified nanoparticles include nanoparticles as described in the present invention; and ligands.

[0886] Preferably, the ligand-modified nanoparticles include ligand-modified nanoparticles or ligand-modified liposomes.

[0887] The ligands described in this invention may include targeted ligands.

[0888] In a preferred embodiment of the invention, the surface of the nanoparticles contains ligands.

[0889] Preferably, the surface includes an outer surface.

[0890] Preferably, the surface of the nanoparticles includes the outer surface of the nanoparticles.

[0891] Preferably, the outer surface of the nanoparticles contains ligands.

[0892] Preferably, the ligand modification is performed on nanomaterials.

[0893] In a preferred embodiment of the present invention, the ligand is modified on the nanomaterial of the nanoparticles.

[0894] Preferably, the ligand is modified on the surface of the nanoparticles.

[0895] Preferably, the modification includes physical modification and / or chemical modification.

[0896] Preferably, the modification includes physical adsorption, chemisorption, and / or coupling.

[0897] Preferably, the ligand is adsorbed on the surface of the nanoparticles.

[0898] Preferably, the adsorption includes physical adsorption and / or chemical adsorption.

[0899] Preferably, the ligand is coupled to the nanomaterial on the surface of the nanoparticles.

[0900] In a preferred embodiment of the invention, the ligand comprises a receptor that targets a cell or the cell surface.

[0901] Preferably, the ligand includes a ligand that targets tumor vascular cells and / or tumor cells.

[0902] The ligands described in this invention may include polypeptide or protein ligands.

[0903] Preferably, the ligand comprises RGD peptide and / or NGR peptide.

[0904] In a preferred embodiment of the present invention, the lipid material comprises a ligand-modified lipid material.

[0905] Preferably, the ligand is coupled to the nanomaterial of the nanoparticles.

[0906] Preferably, the ligand is coupled to a lipid material.

[0907] Preferably, the coupling includes chemical coupling.

[0908] In a preferred embodiment of the invention, the ligand is coupled to distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) to form a DSPE-PEG-ligand.

[0909] In a preferred embodiment of the invention, the ligand is coupled to a portion of a nanomaterial (such as a lipid material).

[0910] In a preferred embodiment of the invention, the DSPE-PEG-ligand comprises DSPE-PEG-RGD and / or DSPE-PEG-NGR.

[0911] Preferably, the ligand coupling on the lipid material includes DSPE-PEG-RGD and / or DSPE-PEG-NGR.

[0912] Preferably, the DSPE-PEG-RGD is selected from the group consisting of: DSPE-PEG600-RGD, DSPE-PEG800-RGD, DSPE-PEG1000-RGD, DSPE-PEG2000-RGD, DSPE-PEG4000-RGD, DSPE-PEG6000-RGD, or a combination thereof.

[0913] Preferably, the DSPE-PEG-NGR is selected from the group consisting of: DSPE-PEG600-NGR, DSPE-PEG800-NGR, DSPE-PEG1000-NGR, DSPE-PEG2000-NGR, DSPE-PEG4000NGR, DSPE-PEG6000-NGR, or a combination thereof.

[0914] In a preferred embodiment of the present invention, the DSPE-PEG-ligand is 1-10 parts by weight, more preferably 2-8 parts by weight, more preferably 4-6 parts by weight, and most preferably 3 parts by weight.

[0915] In a preferred embodiment of the present invention, the weight ratio of the DSPE-PEG-ligand to the DPPC is 1:0.2-5, more preferably 1:0.5-2, more preferably 1:0.5-1.5, even more preferably 1:0.8-1.2, and most preferably 1:1.

[0916] In a preferred embodiment of the present invention, the particle size of the ligand-modified nanoparticles is 120-260 nm, more preferably 160-210 nm, more preferably 170-200 nm, and even more preferably 180-200 nm.

[0917] In a preferred embodiment of the present invention, the potential of the ligand-modified nanoparticles is -2 to -18 mV, more preferably -2 to -15 mV, and even more preferably -5 to -12 mV.

[0918] Preferably, the ligand comprises a ligand that targets a cell surface receptor.

[0919] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor vascular cells.

[0920] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor cells.

[0921] Preferably, the surface includes an outer surface.

[0922] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[0923] Preferably, the cell surface receptor includes extracellular receptors.

[0924] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[0925] Preferably, the ligand includes a ligand that mediates the uptake of ligand-modified nanoparticles by cells.

[0926] Preferably, the ligand includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[0927] Preferably, the ligand includes a ligand that mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[0928] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site.

[0929] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site via endocytosis and exocytosis.

[0930] Preferably, the ligand targets vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of ligand-modified nanoparticles by vascular endothelial cells.

[0931] Preferably, the ligand can mediate the endocytosis of ligand-modified nanoparticles in the blood by tumor vascular cells, and after endocytosis, it can be exocytodated outside the tumor blood vessels (such as the tumor tissue microenvironment).

[0932] The present invention also provides a method for preparing the ligand-modified nanoparticles described herein, the method comprising:

[0933] The ligand is modified onto the nanoparticles to obtain ligand-modified nanoparticles.

[0934] In a preferred embodiment of the present invention, the method for preparing the ligand-modified nanoparticles includes:

[0935] (1) Dissolve the ligand-modified nanomaterial in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0936] (2) After immersing perfluoropentane into the nanoparticle membrane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain ligand-modified nanoparticles.

[0937] Typically, the preparation method of the ligand-modified nanoparticles includes:

[0938] (1) Dissolve the ligand-modified nanomaterial and drug in an organic solvent, remove the organic solvent, and obtain a nanoparticle film;

[0939] (2) After immersing perfluoropentane into the nanoparticle membrane, a buffer solution is added to hydrate the nanoparticle membrane, and then the mixture is stirred to obtain ligand-modified nanoparticles.

[0940] Preferably, the ligand-modified nanomaterial includes one or more of DPPC, DSPE-PEG, and DSPE-PEG-ligand.

[0941] Preferably, the ligand-modified nanomaterial includes one or more of DPPC, DSPE-PEG, DSPE-PEG-RGD, and DSPE-PEG-NGR.

[0942] Typically, the method for preparing the ligand-modified liposomes includes the following steps:

[0943] (1) DPPC, DSPE-PEG-ligand and DSPE-PEG are dissolved in the organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0944] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain ligand-modified liposomes.

[0945] Typically, the method for preparing the ligand-modified liposomes includes the steps of:

[0946] (1) 2.8-3.2 mg DPPC, 2.8-3.2 mg DSPE-PEG-ligand and 1.8-2.2 mg DSPE-PEG were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0947] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃, and then stir in a round-bottom flask in a water bath at 28-32℃ under open conditions to obtain ligand-modified liposomes.

[0948] Typically, the method for preparing the ligand-modified liposomes includes the following steps:

[0949] (1) DPPC, DSPE-PEG-ligand, DSPE-PEG and the drug are dissolved in an organic solvent in a round-bottom flask, and the organic solvent is removed by rotary vacuum evaporation to form a lipid film in the round-bottom flask.

[0950] (2) Cool the lipid membrane to a low temperature, add perfluoropentane to immerse the lipid membrane, then add a buffer solution for hydration, stir at 2-6℃ for 0.2-0.8h, and then stir in a round-bottom flask at 25-35℃ for 0.8-1.2h to obtain ligand-modified liposomes.

[0951] Typically, the method for preparing the ligand-modified liposomes includes the steps of:

[0952] (1) 2.8-3.2 mg DPPC, 2.8-3.2 mg DSPE-PEG-ligand, 1.8-2.2 mg DSPE-PEG and 1.8-2.2 mg drug were dissolved in an organic solvent in a round-bottom flask, and the organic solvent was removed by rotary vacuum evaporation, forming a lipid film in the round-bottom flask;

[0953] (2) Cool the lipid membrane to 2-6℃, add 90-110μL of perfluoron-pentane to immerse the lipid membrane, then add 4.5-5.5mL of buffer solution for hydration, stir at 2-6℃, and then stir in a round-bottom flask in a water bath at 28-32℃ under open conditions to obtain ligand-modified liposomes.

[0954] Protein crown modified nanoparticles and their preparation methods

[0955] This invention provides a protein crown-modified nanoparticle, wherein the protein crown-modified nanoparticle includes the nanoparticles as described in this invention and / or the ligand-modified nanoparticles as described in this invention; and a protein crown.

[0956] Preferably, the nanoparticles are loaded with perfluoropentane.

[0957] In a preferred embodiment of the invention, the proteins of the protein crown include serum proteins, plasma proteins, and / or tissue proteins.

[0958] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[0959] In a preferred embodiment of the present invention, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0960] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0961] Preferably, the cattle include fetal cattle.

[0962] Preferably, the serum and / or plasma includes fetal bovine serum and / or fetal bovine plasma.

[0963] The protein crown modified nanoparticles of the present invention may include isolated or in vitro protein crown modified nanoparticles.

[0964] Preferably, the protein crown modification is performed on nanoparticles as described in this invention and / or ligand-modified nanoparticles as described in this invention.

[0965] Preferably, the protein crown modification is applied to the surface of nanoparticles as described in this invention and / or ligand-modified nanoparticles as described in this invention.

[0966] Preferably, the surface includes an outer surface.

[0967] Preferably, the modification includes physical modification and / or chemical modification.

[0968] Preferably, the modification includes physical adsorption, chemisorption, and / or coupling.

[0969] Preferably, the modification includes adsorption.

[0970] This invention also provides a method for preparing protein crown-modified nanoparticles as described herein, the method comprising the steps of:

[0971] By contacting the nanoparticles as described in this invention and / or the ligand-modified nanoparticles as described in this invention with the protein, protein crown-modified nanoparticles are obtained.

[0972] Preferably, the method is a non-diagnostic and non-therapeutic method.

[0973] Preferably, the contact is external or internal.

[0974] Preferably, the contact includes contact in conditions containing protein.

[0975] Preferably, the protein-containing conditions include blood, serum, plasma, and / or culture medium.

[0976] In a preferred embodiment of the invention, the contact includes contact in a culture medium.

[0977] Preferably, the culture medium comprises a liquid culture medium.

[0978] Preferably, the culture medium includes a cell culture medium.

[0979] Preferably, the culture medium contains protein.

[0980] Preferably, the proteins include serum proteins, plasma proteins, and / or tissue proteins.

[0981] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[0982] Preferably, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[0983] Preferably, the culture medium includes a culture medium containing serum, plasma, and / or tissue protein.

[0984] Preferably, the culture medium includes a serum-containing culture medium.

[0985] Preferably, the serum comprises fetal bovine serum.

[0986] Preferably, the plasma includes fetal bovine plasma.

[0987] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[0988] Preferably, the serum, plasma, and / or tissue proteins include serum, plasma, and / or tissue proteins from humans or non-human mammals.

[0989] Preferably, the serum and / or plasma includes fetal bovine serum and / or fetal bovine plasma.

[0990] Preferably, the culture includes in vitro culture.

[0991] In a preferred embodiment of the invention, the contact includes contact in blood, serum, or plasma.

[0992] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[0993] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[0994] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0995] Preferably, the cattle include fetal cattle.

[0996] Preferably, the contact time is 0.25-6h, more preferably 0.25-4h, even more preferably 0.25-2h, more preferably 0.25-1h, more preferably 0.25-0.5h, for example 0.5-1h.

[0997] Preferably, the separation includes gel chromatography separation.

[0998] Preferably, the gel comprises a dextran gel.

[0999] Preferably, the separation includes size exclusion chromatography.

[1000] Methods for eliminating protein crowns in protein crown-modified nanoparticles

[1001] This invention provides a method for eliminating the protein crown of the protein crown-modified nanoparticles described herein, the method comprising the steps of:

[1002] The protein crown-modified nanoparticles are subjected to ultrasonic irradiation to eliminate the protein crown of the nanoparticles.

[1003] Preferably, the nanoparticles are loaded with perfluoropentane.

[1004] By eliminating the protein crown on the surface of the nanoparticles described in this invention through ultrasonic irradiation, the masking effect of the protein crown on the ligands modified on the nanoparticle surface is overcome, and the binding of the ligands modified on the nanoparticle surface to the receptors of target cells (such as tumor vascular cells or tumor cells) is restored, which can enhance the uptake of the ligand-modified nanoparticles by cells.

[1005] Preferably, the method includes an in vitro method or an in vivo method.

[1006] Preferably, the method includes non-therapeutic and / or non-diagnostic methods.

[1007] In a preferred embodiment of the invention, the elimination includes reduction or removal.

[1008] Preferably, the reduction includes a reduction in protein content.

[1009] Preferably, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in protein-free conditions.

[1010] Preferably, the protein-free conditions include physiological saline, PBS buffer, or serum-free culture medium.

[1011] In a preferred embodiment of the invention, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in conditions containing protein.

[1012] Preferably, the protein-containing conditions include blood, serum, plasma, and / or culture medium.

[1013] Preferably, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in blood, serum, or plasma.

[1014] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[1015] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[1016] Preferably, the protein crown-modified nanoparticles include protein crown-modified nanoparticles in a culture medium.

[1017] Preferably, the proteins in the protein crown include serum proteins, plasma proteins, and / or tissue proteins.

[1018] In a preferred embodiment of the present invention, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[1019] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[1020] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[1021] Preferably, the cattle include fetal cattle.

[1022] Preferably, the serum and / or plasma includes fetal bovine serum and / or fetal bovine plasma.

[1023] Preferably, the culture medium comprises a liquid culture medium.

[1024] Preferably, the culture medium includes a cell culture medium.

[1025] Preferably, the culture medium includes a protein-containing culture medium.

[1026] Preferably, the culture medium contains protein.

[1027] Preferably, the proteins include serum proteins, plasma proteins, and / or tissue proteins.

[1028] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[1029] Preferably, the culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

[1030] Preferably, the culture medium includes a serum-containing culture medium.

[1031] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[1032] Preferably, the cells include tumor cells and / or tumor vascular cells.

[1033] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[1034] Preferably, the tumor vascular cells include ECDHCC cells.

[1035] Preferably, the cells include cells that need to be cultured or grown in conditions containing proteins.

[1036] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[1037] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[1038] Preferably, the culture includes in vitro culture.

[1039] Ultrasonic irradiation can effectively remove the protein crown of protein-corona-modified nanoparticles. For cells that need to be cultured in protein-containing media (such as serum-containing media), ultrasonic irradiation can overcome the masking effect of protein crowns on ligands modified on the surface of nanoparticles, preventing protein crowns from hindering the incubation of ligands modified on the surface of nanoparticles with cells under protein-containing conditions (such as serum-containing media). This allows for accurate determination of whether the test ligands modified on the surface of nanoparticles can bind to cells or cell surface receptors, thereby accurately screening or identifying potential ligands targeting cells or cell surface receptors and avoiding false negative results (especially under conditions where the amount of test ligand is low).

[1040] Methods for screening or identifying potential ligands that target cell or cell surface receptors

[1041] This invention provides a method for screening or identifying potential ligands targeting cell or cell surface receptors, the method comprising the steps of:

[1042] (I) Ligands are modified onto nanoparticles to obtain ligand-modified nanoparticles;

[1043] (II) Incubate the cell or cell surface receptor with the ligand-modified nanoparticles of step (I), subject them to ultrasonic irradiation, and determine the binding of the ligand-modified nanoparticles of step (I) or the ligand of the ligand-modified nanoparticles to the cell or cell surface receptor, thereby screening or identifying whether the ligand of step (I) is a potential ligand for targeting the cell or cell surface receptor.

[1044] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[1045] Preferably, the cell surface receptor includes extracellular receptors.

[1046] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor cells.

[1047] Preferably, the nanoparticles are loaded with perfluoropentane.

[1048] Preferably, the nanoparticles are nanoparticles as described above.

[1049] Preferably, the ligand-modified nanoparticles are ligand-modified nanoparticles as described above.

[1050] Preferably, the ligand in step (I) includes the ligand to be tested.

[1051] Preferably, step (II) includes:

[1052] (II) Incubate the cell or cell surface receptor with the ligand-modified nanoparticles of step (I), subject them to ultrasonic irradiation, and determine whether the ligand-modified nanoparticles of step (I) or the ligand of the ligand-modified nanoparticles bind to the cell or cell surface receptor, thereby screening or identifying whether the ligand of step (I) is a potential ligand for targeting the cell or cell surface receptor.

[1053] Preferably, in step (II), if the ligand-modified nanoparticle or the ligand of the ligand-modified nanoparticle binds to a cell or a cell surface receptor, then the ligand in step (I) is a potential ligand targeting the cell or cell surface receptor.

[1054] Preferably, in step (II), if the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles do not bind to the cell or cell surface receptor, then the ligand in step (I) is not a potential ligand for targeting the cell or cell surface receptor.

[1055] In a preferred embodiment of the present invention, the method further includes setting up a control group comprising unmodified nanoparticles, and measuring the binding of the unmodified nanoparticles to cells or cell surface receptors.

[1056] Preferably, the method further includes setting up a control group, which comprises unmodified nanoparticles and other conditions are the same as those for ligand-modified nanoparticles, and measuring the binding of unmodified nanoparticles to cells or cell surface receptors.

[1057] Preferably, if the binding force B1 between the ligand-modified nanoparticle or the ligand of the ligand-modified nanoparticle and the cell or cell surface receptor is greater than the binding force B0 between the unmodified nanoparticle and the cell or cell surface receptor, then the ligand in step (I) is a potential ligand for targeting the cell or cell surface receptor.

[1058] In a preferred embodiment of the present invention, step (II) includes:

[1059] (II-1) In the test group, cells or cell surface receptors were incubated with ligand-modified nanoparticles from step (I) and subjected to ultrasonic irradiation. The binding force B1 between the ligand-modified nanoparticles from step (I) or the ligands of the ligand-modified nanoparticles and the cells or cell surface receptors was measured. A control group was set up, which included nanoparticles without ligand modification and other measurement conditions were the same as those in the test group. The binding force B0 between the nanoparticles without ligand modification and the cells or cell surface receptors was measured.

[1060] (II-2) If the binding force B1 between the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles in step (I) and the cell or cell surface receptor is greater than the binding force B0 between the unmodified nanoparticles and the cell or cell surface receptor, then the ligand in step (I) is a potential ligand for targeting the cell or cell surface receptor.

[1061] Preferably, if the binding force B1 between the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles in step (I) and the cell or cell surface receptor is similar to the binding force B0 between the unmodified nanoparticles and the cell or cell surface receptor, then the ligand in step (I) is not a potential ligand for targeting the cell or cell surface receptor.

[1062] Preferably, the targeting includes specific targeting or non-specific targeting.

[1063] Preferably, "greater than" includes significantly greater than.

[1064] Preferably, "greater than" includes significantly greater than and statistically significant.

[1065] Preferably, "greater than" means that the ratio (B1 / B0) of the binding force B1 between the ligand of the ligand-modified nanoparticle or the ligand of the ligand-modified nanoparticle and the cell or cell surface receptor to the binding force B0 between the unmodified nanoparticle and the cell or cell surface receptor is >1.0, more preferably ≥1.2, more preferably ≥1.5, more preferably ≥2, more preferably ≥3, more preferably ≥5, more preferably ≥10, more preferably ≥15, more preferably ≥20, more preferably ≥30, more preferably ≥50, more preferably ≥80, more preferably ≥100, more preferably ≥80, more preferably ≥150, more preferably ≥200, more preferably ≥500, more preferably ≥1000, more preferably ≥5000, more preferably ≥10000.

[1066] Preferably, B1 / B0 is 1.5-10000, more preferably 2-500, even more preferably 2-200, even more preferably 2-100, even more preferably 2-50, and even more preferably 5-30.

[1067] Preferably, "greater than" means that the binding force B1 of the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles to the cell or cell surface receptor in the test group with biological reproducibility is greater than the binding force B0 of the unmodified nanoparticles to the cell or cell surface receptor in the control group with biological reproducibility, and the P value is less than 0.05 after t-test.

[1068] In a preferred embodiment of the invention, the ligand comprises a polypeptide or protein ligand.

[1069] In a preferred embodiment of the present invention, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[1070] In a preferred embodiment of the invention, the binding includes affinity.

[1071] Preferably, the binding force includes affinity.

[1072] Preferably, the ligand includes a potential ligand.

[1073] In a preferred embodiment of the present invention, the cell or cell surface receptor includes in vitro or isolated cell or cell surface receptors.

[1074] In a preferred embodiment of the invention, the method includes an in vitro method or an in vivo method.

[1075] In a preferred embodiment of the invention, the method includes non-therapeutic and / or non-diagnostic methods.

[1076] In a preferred embodiment of the invention, the incubation is either in vitro incubation or in vivo incubation.

[1077] In a preferred embodiment of the invention, the body refers to the body of a human or non-human mammal.

[1078] In a preferred embodiment of the invention, the incubation includes incubation in conditions containing protein.

[1079] Preferably, the incubation involves incubating the cell or cell surface receptor with the ligand-modified nanoparticles of step (I) in a protein-containing environment.

[1080] Preferably, the conditions include in vivo conditions or in vitro conditions.

[1081] In a preferred embodiment of the invention, the cells include cells that need to be cultured or grown in conditions containing proteins.

[1082] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[1083] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[1084] Preferably, the culture includes in vitro culture.

[1085] In a preferred embodiment of the invention, the conditions containing the protein include blood, serum, plasma, cellular tissue microenvironment, or culture medium.

[1086] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[1087] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[1088] In a preferred embodiment of the invention, the proteins include serum proteins, plasma proteins, and / or tissue proteins.

[1089] In a preferred embodiment of the present invention, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[1090] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[1091] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[1092] Preferably, the cattle include fetal cattle.

[1093] Preferably, the serum comprises fetal bovine serum.

[1094] Preferably, the plasma includes fetal bovine plasma.

[1095] In a preferred embodiment of the invention, the culture medium comprises a liquid culture medium.

[1096] Preferably, the culture medium includes a cell culture medium.

[1097] Preferably, the culture medium includes a protein-containing culture medium.

[1098] Preferably, the culture medium contains protein.

[1099] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[1100] Preferably, the culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

[1101] In a preferred embodiment of the invention, the culture medium comprises a serum-containing culture medium.

[1102] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[1103] In a preferred embodiment of the invention, the incubation comprises incubating the cell or cell surface receptor with the ligand-modified nanoparticles of step (I) in a culture medium containing serum, plasma and / or tissue proteins.

[1104] In a preferred embodiment of the invention, the incubation comprises incubating the cell or cell surface receptor with the ligand-modified nanoparticles of step (I) in a serum-containing culture medium.

[1105] In a preferred embodiment of the invention, the ligand includes a ligand that targets a cell or a cell surface receptor.

[1106] In a preferred embodiment of the invention, the cells include tumor cells and / or tumor vascular cells.

[1107] In a preferred embodiment of the present invention, the tumor vascular cells include tumor vascular endothelial cells.

[1108] In a preferred embodiment of the invention, the ligand is capable of binding to cells or cell surface receptors.

[1109] In a preferred embodiment of the invention, the binding includes specific binding or non-specific binding.

[1110] In a preferred embodiment of the invention, the receptor comprises a receptor on the outer surface of the cell membrane.

[1111] In a preferred embodiment of the invention, the determination method for the binding includes isotope disappearance assay, fluorescein assay, flow cytometry assay, and / or transwell migration assay.

[1112] Preferably, the nanoparticles and / or the ligands are labeled with isotopes and / or fluoresceins.

[1113] Preferably, the fluorescein includes FITC (Fluorescein isothiocyanate isomer), Cyanine 5 (Cy5), and / or Cyanine 5.5 (Cy5.5).

[1114] In a preferred embodiment of the invention, the binding mediates the uptake of ligand-modified nanoparticles by cells.

[1115] In a preferred embodiment of the invention, the binding mediates endocytosis of ligand-modified nanoparticles by cells.

[1116] In a preferred embodiment of the invention, the binding mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[1117] In a preferred embodiment of the invention, the method for determining the binding includes determining the uptake efficiency of cells of the ligand-modified nanoparticles of step (I).

[1118] Preferably, the cells do not have the ability to take up nanoparticles without ligand modification in the control group.

[1119] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[1120] If the cell’s uptake efficiency of the ligand-modified nanoparticles in step (I) is greater than the cell’s uptake efficiency of the unmodified nanoparticles in the control group, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[1121] In a preferred embodiment of the invention, the method for determining the binding includes measuring the endocytic capacity of cells for the ligand-modified nanoparticles of step (I).

[1122] Preferably, the cells in the control group do not have the ability to endocytose nanoparticles without ligand modification.

[1123] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[1124] If the cell's endocytosis capacity for the ligand-modified nanoparticles of step (I) is greater than the cell's endocytosis capacity for the unmodified nanoparticles in the control group, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[1125] In a preferred embodiment of the invention, the method for determining the binding includes measuring the endocytosis and exocytosis capacity of cells for the ligand-modified nanoparticles of step (I).

[1126] Preferably, the cells in the control group do not have endocytosis or exocytosis capabilities for the nanoparticles without ligand modification.

[1127] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[1128] If the cell's ability to endocytose and exocytose the ligand-modified nanoparticles of step (I) is greater than the cell's ability to endocytose and exocytose the unmodified nanoparticles in the control group, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[1129] In a preferred embodiment of the present invention, the ligand in step (I) includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[1130] Preferably, the ligand-modified nanoparticles or the ligands of the ligand-modified nanoparticles bind to cells or cell surface receptors, thereby mediating endocytosis of the ligand-modified nanoparticles by the cells.

[1131] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[1132] If a ligand-modified nanoparticle or a ligand of a ligand-modified nanoparticle binds to a cell or a cell surface receptor and can mediate endocytosis of the ligand-modified nanoparticle by the cell, then the ligand in step (I) is a potential ligand for targeting the cell or cell surface receptor.

[1133] In a preferred embodiment of the present invention, the ligand in step (I) includes a ligand that mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[1134] Preferably, the ligand-modified nanoparticles or the ligands of the ligand-modified nanoparticles bind to cells or cell surface receptors, thereby mediating endocytosis and exocytosis of the ligand-modified nanoparticles by the cells.

[1135] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[1136] If ligand-modified nanoparticles or ligands of ligand-modified nanoparticles bind to cells or cell surface receptors and can mediate endocytosis and exocytosis of cells, then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[1137] Preferably, the ligand-modified nanoparticles or the ligands of the ligand-modified nanoparticles bind to tumor vascular cells or receptors on the surface of tumor vascular cells, thereby mediating endocytosis of the ligand-modified nanoparticles in the blood by the tumor vascular cells, and after endocytosis, they can be exocytodated outside the tumor blood vessels (such as in the tumor tissue microenvironment).

[1138] Preferably, whether the ligand in the screening or identification step (I) is a potential ligand for a cell or cell surface receptor includes:

[1139] If ligand-modified nanoparticles or ligands of ligand-modified nanoparticles bind to tumor vascular cells or tumor vascular cell surface receptors, and can mediate endocytosis of ligand-modified nanoparticles in the blood by tumor vascular cells, and can be exocytodated outside the tumor blood vessels (such as the tumor tissue microenvironment), then the ligand in step (I) is a potential ligand for targeting cells or cell surface receptors.

[1140] Preferably, the method for determining the binding or binding force between the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles and the cell or cell surface receptor in step (I) includes:

[1141] If tumor vascular cells can endocytose ligand-modified nanoparticles in the blood and then exocytose them outside the tumor blood vessels (such as in the tumor tissue microenvironment), then the ligands can bind to cells or cell surface receptors.

[1142] Preferably, the tumor vascular cells cannot perform endocytosis on unmodified nanoparticles in the blood circulation.

[1143] In a preferred embodiment of the invention, the ligand comprises a ligand that targets receptors on the surface of tumor vascular cells.

[1144] Preferably, the ligand includes a ligand that targets receptors on the surface of tumor cells.

[1145] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site.

[1146] Preferably, the ligand is capable of mediating the infiltration of ligand-modified nanoparticles from tumor blood vessels into the tumor site via endocytosis and exocytosis.

[1147] Preferably, the ligand targets vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of ligand-modified nanoparticles by vascular endothelial cells.

[1148] Preferably, the ligand includes a ligand that mediates the uptake of ligand-modified nanoparticles by cells.

[1149] Preferably, the ligand includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[1150] Preferably, the ligand includes a ligand that mediates endocytosis and exocytosis of ligand-modified nanoparticles by cells.

[1151] Preferably, the ligand can mediate the endocytosis of ligand-modified nanoparticles in the blood by tumor vascular cells, and after endocytosis, it can be exocytodated outside the tumor blood vessels (such as the tumor tissue microenvironment).

[1152] use

[1153] This invention provides the use of nanoparticles as described in this invention and / or ligand-modified nanoparticles as described in this invention for preparing compositions for the prevention and / or treatment of diseases.

[1154] In a preferred embodiment of the invention, the nanoparticles include drug-loaded nanoparticles.

[1155] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[1156] In a preferred embodiment of the present invention, the disease is an indication of the drug.

[1157] In a preferred embodiment of the present invention, the drug includes an anticancer drug.

[1158] In a preferred embodiment of the invention, the disease includes a tumor.

[1159] Preferably, the composition is a pharmaceutical composition.

[1160] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[1161] Preferably, the dosage form of the composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[1162] Preferably, the dosage form of the composition is an injectable formulation, an oral formulation, or a topical formulation.

[1163] Preferably, the injectable formulation is an intravascular injection formulation.

[1164] Preferably, the injectable formulation is an intravenous injection formulation, an arterial injection formulation, an intratumoral injection formulation, a tumor intravascular injection formulation, or a tumor microenvironment injection formulation.

[1165] Preferably, the treatment includes inhibition, reduction, relief, reversal, or eradication.

[1166] The present invention also provides a method for preventing and / or treating diseases by applying nanoparticles as described in the present invention and / or ligand-modified nanoparticles as described in the present invention to a desired object, thereby preventing and / or treating diseases.

[1167] Preferably, the nanoparticles include drug-loaded nanoparticles.

[1168] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[1169] Preferably, the object includes a human or a non-human mammal.

[1170] Preferably, the disease is an indication for the drug.

[1171] Preferably, the disease includes a tumor.

[1172] Preferably, after applying nanoparticles as described in this invention and / or ligand-modified nanoparticles as described in this invention to the desired object, the lesion site (such as a tumor site) is subjected to ultrasound irradiation.

[1173] Preferably, the application is by injection, oral administration, or topical application.

[1174] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[1175] Preferably, the injection is administered via intravascular injection.

[1176] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[1177] This invention provides a use of the nanoparticles described herein for preparing carriers for screening or identifying potential ligands targeting cell or cell surface receptors.

[1178] In a preferred embodiment of the present invention, the method for screening or identifying potential ligands targeting cell or cell surface receptors includes the steps of:

[1179] (I) Ligands are modified onto nanoparticles to obtain ligand-modified nanoparticles;

[1180] (II) Incubate the cell or cell surface receptor with the ligand-modified nanoparticles of step (I), subject them to ultrasonic irradiation, and determine the binding of the ligand-modified nanoparticles of step (I) or the ligand of the ligand-modified nanoparticles to the cell or cell surface receptor, thereby screening or identifying whether the ligand of step (I) is a potential ligand for targeting the cell or cell surface receptor.

[1181] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[1182] Preferably, the cell surface receptor includes extracellular receptors.

[1183] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[1184] Preferably, the method for screening or identifying potential ligands targeting cell or cell surface receptors is as described above.

[1185] The present invention also provides an use of an ultrasonic instrument for manufacturing a device, said device being used for one or more uses selected from the group consisting of:

[1186] (a) Elimination of protein crowns in nanoparticles modified with protein crowns by ultrasonic irradiation;

[1187] (b) Used for screening or identifying potential ligands targeting cell or cell surface receptors; and / or

[1188] (c) Treatment of disease by ligand-modified nanoparticles applied in conjunction with ultrasound irradiation of lesions (e.g., tumors); and / or

[1189] (d) Improve the retention and / or degradation of nanoparticles, ligand-modified nanoparticles and / or protein crown-modified nanoparticles by lysosomes through ultrasonic irradiation.

[1190] Preferably, the nanoparticles are as described above.

[1191] Preferably, the ligand-modified nanoparticles are as described above.

[1192] In a preferred embodiment of the present invention, the protein crown-modified nanoparticles are as described above.

[1193] In a preferred embodiment of the present invention, the method for eliminating the protein crown of the protein crown modified nanoparticles is as described above.

[1194] Preferably, (a) includes improving the efficacy of nanoparticles by eliminating the protein crown of the protein crown-modified nanoparticles through ultrasonic irradiation.

[1195] In a preferred embodiment of the present invention, (a) the protein crown of the protein crown-modified nanoparticles eliminated by ultrasonic irradiation comprises:

[1196] The protein crown-modified nanoparticles are subjected to ultrasonic irradiation to eliminate the protein crown of the nanoparticles.

[1197] Preferably, the cell surface receptor includes a cell membrane surface receptor.

[1198] Preferably, the cell surface receptor includes extracellular receptors.

[1199] In a preferred embodiment of the invention, the method for screening or identifying potential ligands targeting cell or cell surface receptors is as described above.

[1200] Preferably, (d) improving the retention and / or degradation of nanoparticles, ligand-modified nanoparticles, and / or protein crown-modified nanoparticles by lysosomes via ultrasonic irradiation includes:

[1201] After the nanoparticles, ligand-modified nanoparticles, and / or protein crown-modified nanoparticles come into contact with cells, the cells are subjected to ultrasonic irradiation, thereby improving the retention and / or degradation of the nanoparticles, ligand-modified nanoparticles, and / or protein crown-modified nanoparticles by cell lysosomes.

[1202] Preferably, the method includes an in vitro method or an in vivo method.

[1203] Preferably, the method includes non-therapeutic and / or non-diagnostic methods.

[1204] Preferably, the contact is in vivo or in vitro.

[1205] Preferably, the contact includes contacting under conditions containing protein.

[1206] Preferably, the contact includes contact in a serum-containing culture medium.

[1207] Preferably, the cells include cells that need to be cultured or grown in conditions containing proteins.

[1208] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[1209] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[1210] Preferably, the nanoparticles include drug-loaded nanoparticles.

[1211] Preferably, the disease is an indication for the drug.

[1212] Preferably, the cells include tumor cells and / or tumor vascular cells.

[1213] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[1214] Preferably, the tumor vascular cells include ECDHCC cells.

[1215] Preferably, the disease includes a tumor.

[1216] Preferably, the tumor is as described above.

[1217] In a preferred embodiment of the present invention, the application is by injection, oral administration, or topical application.

[1218] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[1219] Preferably, the injection is administered via intravascular injection.

[1220] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[1221] Preferably, the improvement includes avoiding or overcoming.

[1222] Preferably, the degradation includes degradation by lysosomal enzymes.

[1223] Methods to inhibit cells in vitro

[1224] This invention provides a method for inhibiting cells in vitro. Inhibiting cells in vitro can be used to study the inhibitory mechanism of inhibitors.

[1225] The in vitro cell inhibition method of the present invention includes the following steps:

[1226] Cells are contacted in a culture medium with nanoparticles as described in this invention or ligand-modified nanoparticles as described in this invention, and then subjected to ultrasonic irradiation to inhibit cell growth.

[1227] In a preferred embodiment of the invention, the nanoparticles include drug-loaded nanoparticles.

[1228] In a preferred embodiment of the invention, the method includes a method for enhancing the in vitro cell inhibition of ligand-modified nanoparticles.

[1229] Preferably, the method includes non-diagnostic and non-therapeutic methods.

[1230] Preferably, the ligand comprises a receptor that targets a cell or the cell surface.

[1231] Preferably, the ligand includes a ligand that mediates the uptake of ligand-modified nanoparticles by cells.

[1232] Preferably, the ligand includes a ligand that mediates endocytosis of ligand-modified nanoparticles by cells.

[1233] Preferably, the ligand comprises RGD peptide and / or NGR peptide.

[1234] In a preferred embodiment of the invention, the cells include tumor cells and / or tumor vascular cells.

[1235] Preferably, the tumor vascular cells include tumor vascular endothelial cells.

[1236] Preferably, the tumor vascular cells include ECDHCC cells.

[1237] In a preferred embodiment of the invention, the drug comprises a cell inhibitor.

[1238] In a preferred embodiment of the present invention, the drug includes an antitumor drug.

[1239] Preferably, the drug is as described above.

[1240] Preferably, the tumor is as described above.

[1241] In a preferred embodiment of the invention, the cells include cells that need to be cultured or grown in conditions containing proteins.

[1242] Preferably, the cells include cells that need to be cultured or grown in a serum-containing culture medium.

[1243] Preferably, the cells include cells that need to be cultured in a serum-containing culture medium.

[1244] In a preferred embodiment of the invention, the protein-containing conditions include blood, serum, plasma, and / or culture medium.

[1245] Preferably, the blood, serum, or plasma includes isolated or separated blood, serum, or plasma.

[1246] Preferably, the blood, serum, or plasma includes the blood, serum, or plasma of a human or non-human mammal.

[1247] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[1248] Preferably, the cattle include fetal cattle.

[1249] In a preferred embodiment of the present invention, the conditions for containing the protein are as described above.

[1250] In a preferred embodiment of the invention, the culture medium comprises a liquid culture medium.

[1251] Preferably, the culture medium includes a cell culture medium.

[1252] Preferably, the culture medium contains protein.

[1253] Preferably, the culture medium contains serum proteins, plasma proteins, and / or tissue proteins.

[1254] Preferably, the serum proteins, plasma proteins and / or tissue proteins include in vitro or isolated serum proteins, plasma proteins and / or tissue proteins.

[1255] Preferably, the serum proteins, plasma proteins, and / or tissue proteins include serum proteins, plasma proteins, and / or tissue proteins of humans or non-human mammals.

[1256] Preferably, the culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

[1257] Preferably, the culture medium includes a serum-containing culture medium.

[1258] Preferably, in the serum-containing culture medium, the volume fraction of the serum is 5-15%, more preferably 8-12%, and even more preferably 10%.

[1259] Preferably, the serum comprises fetal bovine serum.

[1260] Preferably, the plasma includes fetal bovine plasma.

[1261] Preferably, the culture includes in vitro culture.

[1262] Preferably, the contact is an external contact.

[1263] In a preferred embodiment of the invention, the nanoparticles contain a protein crown on their surface in the culture medium without ultrasonic irradiation.

[1264] Preferably, the protein crown is adsorbed on the surface of the nanoparticles.

[1265] Preferably, the ligand, after binding to a cell or a cell surface receptor, can mediate the uptake of ligand-modified nanoparticles by the cell.

[1266] In a preferred embodiment of the present invention, the binding of the ligand to a cell or a cell surface receptor mediates endocytosis of the ligand-modified nanoparticles by the cell.

[1267] In a preferred embodiment of the present invention, the cell surface receptor includes a cell membrane surface receptor.

[1268] Preferably, the cell surface receptor includes extracellular receptors.

[1269] Preferably, the receptor includes a protein receptor, a lipoprotein receptor, or a glycoprotein receptor.

[1270] System or device

[1271] The present invention provides a system or apparatus for treating diseases, the system or apparatus comprising the nanoparticles described in the present invention and / or the ligand-modified nanoparticles described in the present invention; and an ultrasound device.

[1272] In a preferred embodiment of the present invention, the system or apparatus further includes a specification or label, which states:

[1273] In the process of treating a disease by applying nanoparticles as described in this invention and / or ligand-modified nanoparticles as described in this invention to a desired object, ultrasound irradiation is performed on the lesion site (such as a tumor site).

[1274] Preferably, the nanoparticles include drug-loaded nanoparticles.

[1275] Preferably, the nanoparticles include drug-loaded nanoparticles or drug-loaded liposomes.

[1276] Preferably, the ultrasonic device includes an ultrasonic transducer.

[1277] In a preferred embodiment of the invention, the object includes a human or a non-human mammal.

[1278] Preferably, the disease is an indication for the drug.

[1279] Preferably, the drug includes an anticancer drug.

[1280] Preferably, the disease includes a tumor.

[1281] Preferably, the application is by injection, oral administration, or topical application.

[1282] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[1283] Preferably, the injection is administered via intravascular injection.

[1284] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[1285] Composition

[1286] The present invention provides a composition, which includes, but is not limited to, pharmaceutical compositions.

[1287] The compositions of the present invention may further include pharmaceutically acceptable carriers. "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances suitable for human use and possessing sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and be mixed with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[1288] There are no particular limitations on the method of application of the composition of the present invention. Representative methods of application include (but are not limited to): injection, oral administration or topical application.

[1289] Preferably, the injection is administered via intravascular injection.

[1290] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[1291] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[1292] The dosage forms of the compositions or preparations described in this invention are oral preparations, topical preparations, or injectable preparations. Representatively, solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[1293] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[1294] Dosage forms of the compounds of the present invention for topical application or administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as needed.

[1295] When administering the composition, a safe and effective amount of the nanoparticles or liposomes described in this invention is applied to humans or non-human animals (such as rats, mice, dogs, cats, cattle, sheep, chickens, ducks, etc.) requiring treatment, wherein the dosage administered is a pharmaceutically acceptable effective dosage. As used herein, the term "safe and effective amount" refers to an amount that produces function or activity in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "safe and effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs. For example, for a person weighing 60 kg, the daily dosage is typically 0.1–1000 mg, preferably 1–600 mg, and more preferably 2–300 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are within the scope of the skill of a skilled physician.

[1296] The main superior technical effects of this invention include:

[1297] 1. This invention develops a method for eliminating the protein crown on the surface of nanoparticles by ultrasonic irradiation, overcoming the masking effect of the protein crown on the ligands modified on the nanoparticle surface, restoring the binding of the ligands modified on the nanoparticle surface to receptors on target cells (such as tumor vascular cells or tumor cells), enhancing the uptake of ligand-modified nanoparticles by cells, and promoting the endocytosis and exocytosis of nanoparticles by tumor vascular endothelial cells mediated by the binding of ligands on the surface of drug-loaded nanoparticles to receptors on the surface of tumor vascular endothelial cells, promoting the penetration of ligand-modified nanoparticles from the blood through tumor blood vessels (especially blood vessels of tumors with low permeability) to the tumor site, and promoting the uptake of nanoparticles by tumor cells through ligand / receptor-mediated endocytosis, thereby improving the anticancer effect of antitumor drugs.

[1298] 2. This invention develops a nanoparticle. After intravenous administration, under ultrasound irradiation of the tumor site, the nanoparticle significantly restores and enhances the binding of surface ligands to receptors on the surface of tumor vascular endothelial cells. This promotes the penetration of the ligand-modified nanoparticles from tumor vessels (especially those of tumors with low permeability) into the tumor site. Furthermore, it promotes the uptake of the nanoparticles by tumor cells through ligand / receptor-mediated endocytosis, thereby improving the anticancer effect of antitumor drugs. In addition, under ultrasound irradiation, the nanoparticles of this invention exhibit excellent lysosomal escape and resistance to lysosomal degradation. Lysosomal escape effectively protects the nanoparticles and their loaded drugs from lysosomal degradation, thereby enhancing the intracellular stability of the nanoparticles and their loaded drugs and improving the therapeutic effect. Moreover, the nanoparticles of this invention have excellent blood clearance half-life, good biocompatibility, high safety, and few side effects.

[1299] 3. Ultrasonic irradiation can eliminate the protein crown of nanoparticles modified with the test ligand under protein-containing conditions (such as serum-containing culture medium), overcoming the masking effect of the protein crown on the test ligand modified on the nanoparticle surface. This avoids the protein crown hindering the contact between the test ligand modified on the nanoparticle surface and the cells under protein-containing conditions (such as serum-containing culture medium), thereby efficiently and accurately determining whether the test ligand modified on the nanoparticle surface can bind to cells or cell surface receptors that need to be cultured or grown under protein-containing conditions (such as serum-containing culture medium). This can then be used to accurately screen or identify potential ligands targeting cells or cell surface receptors, avoiding false negative results.

[1300] 4. This invention develops a method for screening or identifying potential ligands targeting cell or cell surface receptors. The method involves incubating cells or cell surface receptors with nanoparticles modified with the target ligand under protein-containing conditions (such as serum-containing culture medium). Ultrasonic irradiation effectively eliminates the protein crown on the nanoparticle surface, overcoming the masking effect of the protein crown on the target ligand. This prevents the protein crown from hindering the contact between the ligand and the cell, thus enabling efficient and accurate determination of whether the target ligand on the nanoparticle can bind to cells or cell surface receptors that need to be cultured or grown under protein-containing conditions (such as serum-containing culture medium). This allows for the screening or identification of whether the target ligand is a potential ligand targeting cell or cell surface receptor. Therefore, the nanoparticles described in this invention, under ultrasonic irradiation conditions, can be effectively used as a carrier for screening or identifying potential ligands targeting cell or cell surface receptors. The method for screening or identifying potential ligands targeting cell or cell surface receptors described in this invention is simple and convenient. Ultrasonic irradiation effectively eliminates the protein crown modified on the surface of nanoparticles, thereby overcoming the masking effect of the protein crown on the test ligands modified on the nanoparticle surface. This allows for efficient and accurate determination of whether the test ligands modified on the nanoparticle surface can bind to cells or cell surface receptors. It is particularly useful for screening or identifying whether the test ligands are targeted to cells that require culture or growth in protein-containing conditions (such as serum-containing medium), thus accurately screening or identifying potential ligands targeting cells or cell surface receptors and avoiding false negative results due to the masking effect of the protein crown on the test ligands modified on the nanoparticle surface (especially when the amount of test ligand is small).

[1301] 5. The present invention also provides the use of an ultrasound device to overcome the protein crown of protein crown-modified nanoparticles by ultrasound irradiation, for screening or identifying potential ligands targeting cell or cell surface receptors, improving the retention and / or degradation of nanoparticles and protein crown-modified nanoparticles by cell lysosomes, and for the treatment of diseases by ligand-modified nanoparticles applied to lesions (such as tumors) with enhanced ultrasound irradiation.

[1302] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following specific embodiments are based on the present technical solution and provide detailed implementation methods and specific operation processes, but the scope of protection of the present invention is not limited to these embodiments.

[1303] Example 1

[1304] 1. Materials and Instruments

[1305] DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) and DSPE-PEG2000 (distearate-phosphatidylethanolamine-polyethylene glycol 2000) were purchased from Avanti Lipids Inc.

[1306] DSPE-PEG2000-RGD, Cyanine 5 (Cy5) labeled DSPE-PEG2000 (DSPE-PEG200) Cy5 DSPE-PEG2000 (DSPE-PEG2000) labeled with Cyanine 5.5 (Cy5.5) Cy5.5 Purchased from Xi'an Ruixi Biotechnology Co., Ltd., DSPE-PEG2000-RGD is a distearate phosphatidylethanolamine-polyethylene glycol 2000-RGD targeting peptide. The amino acid sequence of the RGD targeting peptide is Cys (cysteine)-Arg (arginine)-Gly (glycine)-Asp (aspartic acid)-Lys (lysine)-Gly (glycine)-Pro (proline)-Asp (aspartic acid)-Cys (cysteine), and the amino acid sequence is CRGDKGPDC (SEQ ID NO:1).

[1307] Chlorpromazine and cytochalasin D were purchased from Santa Cruz Biotechnology.

[1308] RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS) and 0.25% trypsin solution were purchased from GIBCO (USA).

[1309] Serum-free culture medium mainly consists of water, glucose, amino acids, and inorganic salts, and contains no protein components.

[1310] The serum-containing culture medium is a culture medium containing 10% fetal bovine serum by volume.

[1311] Alamar Blue Cell Viability Reagent, Hoechst 33342 and GreenDND 26 was purchased from Thermo Fisher Scientific Inc.

[1312] Ki67 antibody was purchased from Proteintech Group.

[1313] The TUNEL apoptosis assay kit was purchased from Roche.

[1314] The Enhanced BCA Protein Assay Kit and Golgi-Tracker Green were purchased from Beyotime Biotechnology.

[1315] The Matrigel basement membrane matrix was purchased from BDBiosciences.

[1316] EXO1 and gemcitabine prodrug CP4126 were purchased from Med-chem Express. Gemcitabine prodrug CP4126 is abbreviated as "CP4126". Gemcitabine prodrug CP4126 is gemcitabine trans oleate, with the following structure:

[1317]

[1318] The ultrasound machine was a Mettler Sonicator-740.

[1319] GEM is short for gemcitabine.

[1320] Sound intensity refers to acoustic intensity.

[1321] Frequency refers to the frequency of a frequency.

[1322] Duty cycle refers to the period of operation.

[1323] 2. Cell and animal models

[1324] Human pancreatic ductal adenocarcinoma (PDA) cell line BxPC3, hepatocellular carcinoma (HCC) cell line Huh7, and hepatocellular carcinoma-derived endothelial cells (ECDHCC) were purchased from the American Type Culture Collection (ATCC).

[1325] Male BALB / c nude mice (6-8 weeks old) and male NOD / SCID mice (6-8 weeks old) were provided by the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine. Mice were housed in animal housing facilities with a 12-hour light / dark cycle and free access to food. Animal experiments were approved by the Animal Ethics Committee. The use of tumor samples from clinical PDA and HCC patients was approved by the Human Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine.

[1326] Construction of a subcutaneous BALB / c nude mouse model of human PDA or HCC tumors: Clinical PDA or HCC tumor samples were obtained from patients with PDA or HCC tumors who had not undergone any radiation or chemotherapy before surgery. Under sterile conditions, tumor samples were immediately washed with PBS and cut into small pieces (1×1×1 mm). The tumor pieces were then immersed in Matrigel basement membrane matrix and transplanted subcutaneously to the right side of NOD / SCID mice. When the subcutaneous tumor diameter reached 6-8 mm, the NOD / SCID mice were sacrificed and the tumor tissue was removed. The tumor tissue was then immediately washed with PBS and cut into small pieces (1×1×1 mm) for transplantation into the right and / or left side of BALB / c nude mice, or subcutaneously next to abdominal blood vessels, thereby constructing a subcutaneous BALB / c nude mouse model of human PDA or HCC tumors.

[1327] 3. Preparation, characterization, and properties of liposome nanoparticles

[1328] 3.1 Preparation of liposome nanoparticles

[1329] 3.1.1 Preparation of LPGL liposome nanoparticle dispersion:

[1330] (1) 3.0 mg DPPC, 3.0 mg DSPE-PEG2000-RGD, 2.0 mg DSPE-PEG2000 and 2 mg gemcitabine prodrug CP4126 were dissolved in 3 mL chloroform in a 10 mL round-bottom flask. The organic solvent was removed by rotary vacuum evaporation at 40 °C, and a lipid film was formed in the round-bottom flask.

[1331] (2) Cool the lipid membrane to 4°C, add 100 μL of perfluoropentane to immerse the lipid membrane, and then add 5 mL of phosphate buffer containing glycerol (phosphate concentration of 10 mM, pH = 7.4, glycerol volume fraction of 10% (v / v)) for hydration. Stir with a magnetic stir bar at 4°C for 30 min, and then stir with a magnetic stir bar in a round bottom flask at 30°C for 1 h under open conditions to obtain LPGL liposome nanoparticle dispersion.

[1332] 3.1.2 Preparation of LGL liposome nanoparticle dispersion:

[1333] (1) 3.0 mg DPPC, 3.0 mg DSPE-PEG2000-RGD, 2.0 mg DSPE-PEG2000 and 2 mg gemcitabine prodrug CP4126 were dissolved in 3 mL chloroform in a 10 mL round-bottom flask. The organic solvent was removed by rotary vacuum evaporation at 40 °C, and a lipid film was formed in the round-bottom flask.

[1334] (2) The lipid membrane was cooled to 4°C and 5 mL of phosphate buffer containing glycerol (phosphate concentration of 10 mM, pH = 7.4, glycerol volume fraction of 10% (v / v)) was added for hydration. After stirring with a magnetic stir bar at 4°C for 30 min, the round bottom flask was stirred with a magnetic stir bar in a 30°C water bath for 1 h under open conditions to obtain LGL liposome nanoparticle dispersion.

[1335] 3.1.3 Preparation of PGL liposome nanoparticle dispersion

[1336] (1) 4.8 mg DPPC, 3.2 mg DSPE-PEG2000 and 2 mg gemcitabine prodrug CP4126 were dissolved in 3 mL chloroform in a 10 mL round-bottom flask. The organic solvent was removed by rotary vacuum evaporation at 40 °C, and a lipid film was formed in the round-bottom flask.

[1337] (2) Cool the lipid membrane to 4°C, add 100 μL of perfluoropentane to immerse the lipid membrane, and then add 5 mL of phosphate buffer containing glycerol (phosphate concentration of 10 mM, pH = 7.4, glycerol volume fraction of 10% (v / v)) for hydration. Stir with a magnetic stir bar at 4°C for 30 min, and then stir with a magnetic stir bar in a round bottom flask at 30°C for 1 h under open conditions to obtain PGL liposome nanoparticle dispersion.

[1338] 3.1.4 Preparation of GL liposome nanoparticle dispersion

[1339] (1) 4.8 mg DPPC, 3.2 mg DSPE-PEG2000 and 2 mg gemcitabine prodrug CP4126 were dissolved in 3 mL chloroform in a 10 mL round-bottom flask. The organic solvent was removed by rotary vacuum evaporation at 40 °C, and a lipid film was formed in the round-bottom flask.

[1340] (2) The lipid membrane was cooled to 4°C and 5 mL of phosphate buffer containing glycerol (phosphate concentration of 10 mM, pH = 7.4, glycerol volume fraction of 10% (v / v)) was added for hydration. After stirring with a magnetic stir bar at 4°C for 30 min, the round bottom flask was stirred with a magnetic stir bar in a 30°C water bath for 1 h under open conditions to obtain GL liposome nanoparticle dispersion.

[1341] 3.1.5 Preparation of LPL blank liposome nanoparticle dispersion

[1342] (1) 5.0 mg DPPC, 3.0 mg DSPE-PEG2000-RGD and 2.0 mg DSPE-PEG2000 were dissolved in 3 mL chloroform in a 10 mL round-bottom flask. The organic solvent was removed by rotary vacuum evaporation at 40 °C, and a lipid film was formed in the round-bottom flask.

[1343] (2) Cool the lipid membrane to 4°C, add 100 μL of perfluoropentane to immerse the lipid membrane, and then add 5 mL of phosphate buffer containing glycerol (phosphate concentration of 10 mM, pH = 7.4, glycerol volume fraction of 10% (v / v)) for hydration. Stir with a magnetic stir bar at 4°C for 30 min, and then stir with a magnetic stir bar in a round bottom flask at 30°C for 1 h under open conditions to obtain LPL blank liposome nanoparticle dispersion.

[1344] 3.1.6 Preparation of Cy5 or Cy5.5 labeled liposome nanoparticle dispersions

[1345] The preparation method of Cy5 or Cy5.5 labeled liposome nanoparticle dispersions is the same as that of the above-mentioned liposome nanoparticle dispersions, except that 0.3 mg DSPE-PEG2000 is replaced with 0.3 mg Cy5 labeled DSPE-PEG2000. Cy5 ) or 0.3 mg Cy5.5-labeled DSPE-PEG2000 (DSPE-PEG2000) Cy5.5 ), to obtain dispersions of Cy5 or Cy5.5 labeled LPGL, LGL, PGL, GL or LPL liposome nanoparticles.

[1346] 3.2 Evidence and properties of liposome nanoparticles

[1347] The particle size and zeta potential of the liposome nanoparticles were measured using a dynamic light scattering analyzer (Nano-ZS90, Malvern). The refractive index of the liposome nanoparticles was selected to be 1.59, and the particle size was determined by intensity percentage (intensity%).

[1348] The morphology of liposome nanoparticles was imaged on a carbon-coated 200-mesh copper TEM grid using cryo-transmission electron microscopy (cryo-TEM) (Talos F200C 200kv, FEIInc.).

[1349] The fluorescence spectra and fluorescence intensity of Cy5 or Cy5.5 labeled liposome nanoparticles were detected using a microplate reader (SpectraMax M5, MolecularDevices).

[1350] The liposome nanoparticle dispersion (1 mL) was dialyzed in 100 mL PBS buffer (containing 5 v / v % glycerol) for 24 h (Mw molecular weight cutoff 3.5 kDa). The concentration of CP4126 (GEM molar equivalent) in the dialysate was determined by high performance liquid chromatography. The encapsulation efficiency (EE) and GEM loading (LR) were calculated using the following equations (1) and (2), respectively. The loading of perfluoro-n-pentane (LC) was determined by gas chromatography-mass spectrometry and calculated using equation (3):

[1351] EE = mass of GEM in liposomes / mass of total GEM × 100% (Equation 1);

[1352] LR = mass of GEM in liposomes / total mass (lipids + CP4126) × 100% (Equation 2);

[1353] LC = Volume of perfluoropentane / (Volume of perfluoropentane + Volume of PBS) × 100% (Equation 3).

[1354] Calculations show that the encapsulation efficiency of the liposome nanoparticles is >99.5%, and the GEM loading rate is >9.5%.

[1355] Cryo-TEM images of the prepared LPGL liposome nanoparticles under different treatment conditions are shown below. Figure 1 As shown in Table 1, the particle size and zeta potential of the prepared GL, LGL, PGL, and LPGL liposome nanoparticle dispersions after dilution in PBS 7.4 buffer or mouse plasma are as follows. Table 1 shows that the particle size of GL, LGL, PGL, and LPGL liposome nanoparticles increased and the zeta potential decreased after incubation in plasma, indicating that the GL, LGL, PGL, and LPGL liposome nanoparticles adsorbed plasma proteins. The prepared PGL liposome nanoparticles were incubated in plasma and then subjected to ultrasonic irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 5min) Cryo-transmission electron microscopy images before and after treatment are shown below. Figure 2 As shown, from Figure 1 and Figure 2 The cryo-TEM images further show that after incubation with liposome nanoparticles, plasma proteins can form protein coronas on the surface of the liposome nanoparticles, thus further confirming that plasma proteins are adsorbed onto the surface of the liposome nanoparticles. Furthermore, from... Figure 1 and Figure 2The cryo-TEM images show that the incubation of LPGL and PGL liposome nanoparticles and plasma was subjected to ultrasonic irradiation (sound intensity: 2 W / cm²). 2 After 3 MHz, 50% duty cycle, and 5 min time, the protein crown around the surface of the liposome nanoparticles disappeared.

[1356] Figure 1 Cryo-TEM showed that LPGL liposome nanoparticles had a uniform monolayer lipid membrane in PBS 7.4 and plasma. The loaded perfluoropentane showed obvious ice cloud shadows in the liposomes, with a perfluoropentane loading (LC) of 0.16 vol.

[1357] Cryo-transmission electron microscopy images of GL, LGL, and PGL liposome nanoparticle dispersions are shown below. Figure 3 As shown, from Figure 1 and Figure 3 As can be seen, compared with LPGL liposomes, GL liposomes and LGL liposomes without perfluoropentane loading show obvious empty particles inside, while PGL liposomes show obvious perfluoropentane ice cloud shadows and have a uniform monolayer lipid membrane.

[1358] Table 1. Particle size and potential of GL, LGL, PGL and LPGL liposome nanoparticles in PBS 7.4 or plasma.

[1359]

[1360] The incubation of each liposome nanoparticle in plasma was performed as follows: GL, LGL, PGL or LPGL liposome nanoparticle dispersions were mixed with mouse plasma at a lipid / protein ratio of 1 / 50 and incubated in a shaker (60 rpm) at 37°C for 30 min.

[1361] 4. Preparation and Acquisition of Protein Crowns

[1362] Mouse blood was collected via the orbital venous plexus and mixed with heparin solution (1 mg / mL, 50 μL). Plasma was obtained by centrifugation at 5,000 rpm for 5 min at 4°C. Before use, the plasma was centrifuged at 20,000 g for 30 min to remove any aggregated proteins. Using bovine serum albumin (BSA) as a control, the plasma protein concentration was determined to be 61 mg / mL using an enhanced BCA protein assay kit. Cy5-labeled GL, LGL, PGL, or LPGL liposome nanoparticle dispersions were mixed with plasma at a lipid / protein ratio of 1 / 50. After incubation at 37°C on a shaker (60 rpm), a 1 mL sample was taken and subjected to ultrasound irradiation (with or without ultrasound intensity: 2 W / cm²). 2The mixture was processed under the conditions of 3 MHz frequency, 50% duty cycle, and 5 min duration, and then immediately poured into a Sephadex G200 chromatography system for elution with 5 volumes of PBS buffer. The eluent was lyophilized and then redissolved in 200 μL of RIPA lysis buffer to obtain a protein crown solution in liposome nanoparticles. The protein concentration in the protein crown solution was determined using an enhanced BCA protein assay kit with BSA as a control standard.

[1363] In addition, a dispersion of Cy5-labeled PGL or LPGL liposome nanoparticles was mixed with plasma at a lipid / protein ratio of 1 / 50, and then incubated in a shaker (60 rpm) at 37°C for 15 min to obtain a mixture. PGL or LPGL liposome nanoparticles containing protein coronas, separated by Sephadex G200 chromatography without ultrasonic irradiation, were added to PBS 7.4 buffer solution and subjected to ultrasonic irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, duty cycle: 50%) After different irradiation times, the mixture was poured into a Sephadex G200 chromatography system and eluted with 5 volumes of PBS 7.4 buffer for separation. The eluent was lyophilized and then redissolved in 200 μL of RIPA lysis buffer to obtain a protein corona solution. The protein concentration in the protein corona solution was determined using an enhanced BCA protein assay kit with BSA as the standard. The total protein concentration in the protein corona was obtained after treating protein corona-modified PGL or LPGL liposome nanoparticles with different ultrasonic irradiation times. Figure 4 As shown, from Figure 4 As can be seen, ultrasonic irradiation can remove up to 90% of the protein crowns on the surface of PGL or LPGL liposome nanoparticles.

[1364] 5. Determination of protein crown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[1365] For SDS-PAGE assays of protein crowns, GL, LGL, PGL, or LPGL liposome nanoparticles were co-incubated with mouse plasma for 15 min, and the resulting incubation mixture was subjected to ultrasonic irradiation (sound intensity: 2 W / cm²) with or without. 2 After chromatography (frequency: 3 MHz, duty cycle: 50%, duration: 5 min), 20 μL of the protein crown solution was obtained by Sephadex G200 chromatography and mixed with 4 μL of SDS-PAGE loading buffer, and then applied to Tris-Gly protein gel (BeyoGel). TMPlus Precast PAGE Gel, using prestained color protein standard (10-180 kDa) as molecular markers, was electrophoresed at 140 V for 60 min in Tris-Glycine run buffer. The gel was then stained with SimplyBlue SafeStain and analyzed using an Azure c600 Imager.

[1366] The incubation mixture obtained by co-incubating GL, LGL, PGL or LPGL liposome nanoparticles with plasma for 15 min was subjected to ultrasonic irradiation (with or without sound intensity: 2 W / cm²). 2 After (frequency: 3MHz, duty cycle: 50%, duration: 5min), the protein crown solution in the liposome nanoparticles was separated by Sephadex G200 chromatography, and the SDS-PAGE analysis chromatogram is shown below. Figure 5 of Figure 5 As shown in A, from Figure 5 As shown in Figure A, after 15 min of co-incubation with plasma, GL, LGL, PGL, and LPGL liposome nanoparticles absorbed a large amount of protein (molecular weight from 25 kDa to 180 kDa) on their surfaces. Ultrasonic irradiation significantly reduced the protein content on the surface of PGL and LPGL liposome nanoparticles, thereby eliminating the protein crown on their surfaces. However, the protein content on the surface of GL and LGL liposome nanoparticles did not change significantly, indicating that ultrasonic irradiation can eliminate the protein crown on the surface of PGL or LPGL liposome nanoparticles.

[1367] 6. Determination of protein crown by high performance liquid chromatography-mass spectrometry (HPLC-MS)

[1368] First, GL, LGL, PGL, or LPGL liposome nanoparticles were co-incubated with mouse plasma for 15 min. The resulting incubation mixture was then subjected to ultrasonic irradiation (with or without ultrasound intensity: 2 W / cm²). 2 After separation by Sephadex G200 chromatography (frequency: 3MHz, duty cycle: 50%, duration: 5min), the protein crown solution sample in the liposome nanoparticles was obtained. The protein crown solution sample was digested with trypsin at a mass ratio of 1 / 50 (enzyme / protein), then diluted to 5 times the volume with 0.1% formic acid aqueous solution, and 100 fmol / μl Hi3 EColi standard was added for absolute quantification. The protein sample was quantitatively analyzed by high performance liquid chromatography-coupled mass spectrometry (HPLC-MS).

[1369] After incubation of GL, LGL, PGL, or LPGL liposome nanoparticles and plasma with or without ultrasonic irradiation, the total protein content on the liposome nanoparticles was determined by HPLC-MS. Figure 5 of Figure 5 As shown in B, from Figure 5 As shown in Figure B, after liposome nanoparticles were co-incubated with plasma for 15 min, the surfaces of GL, LGL, PGL, and LPGL liposome nanoparticles absorbed a large amount of protein, with the total protein crown content reaching 21-24 μg / mg lipid. However, ultrasonic irradiation could significantly reduce the protein content on the surface of PGL and LPGL liposome nanoparticles, with the protein content on the surface of LPGL and PGL liposome nanoparticles decreasing by at least 80%, while the protein content on the surface of GL and LGL liposome nanoparticles did not change significantly. Thus, surface ultrasonic irradiation can effectively eliminate the protein crown on the surface of PGL or LPGL liposome nanoparticles.

[1370] LPGL liposome nanoparticles were co-incubated with mouse plasma without ultrasonic irradiation. Protein corona solution samples were obtained by Sephadex G200 chromatography, processed, and analyzed by HPLC-MS. Proteins in the protein corona were identified using the UniProt database. The results are shown in Table 2.

[1371] Table 2 shows the top 10 most abundant plasma proteins in the protein corona of LPGL liposome nanoparticles without ultrasonic irradiation treatment. Qualitative and quantitative analysis of the proteins in the protein corona was performed using HPLC-MS and the UniProt database.

[1372]

[1373] Furthermore, after co-incubating PGL or LPGL liposome nanoparticles with plasma for 15 min, and then treating them with ultrasonic irradiation of different intensities (frequency: 3 MHz, duty cycle: 50%, duration: 5 min), the protein corona solution sample of the liposome nanoparticles was obtained by Sephadex G200 chromatography. After further processing, the total protein content on the liposome nanoparticles was determined by HPLC-MS. Figure 6 As shown, from Figure 6 It can be seen that increasing the intensity of ultrasonic irradiation can enhance the ability to eliminate the protein crown on PGL or LPGL liposome nanoparticles.

[1374] 7. Cellular uptake

[1375] BxPC3 cells (1×10) 5BxPC3 cells were seeded in 12-well plates (1 mL per cell) and cultured for 24 h. Dispersions of Cy5-labeled GL, LGL, PGL, and LPGL liposomes (each equivalent to 60 μg / mL fluorescent lipid, 20 μL) were mixed with 1 mL of fresh serum-free medium or medium containing 10% FBS (fetal bovine serum) for 30 min. After washing the BxPC3 cells in the wells with PBS, a mixture of different liposomes and serum-free or 10% FBS medium was added, followed by ultrasound irradiation (sound intensity: 2 W / cm²). 2 Cells were treated under the following conditions: frequency: 3 MHz, duty cycle: 50%, duration: 5 min, and incubated for another 1 h. Cells were washed three times with heparin sodium solution (2 mg / ml), digested, collected, and analyzed by flow cytometry to determine the average fluorescence intensity of different Cy5-labeled liposome nanoparticles taken up by BxPC3 cells.

[1376] The uptake efficiency of liposome nanoparticles by BxPC3 cells under different conditions is as follows: Figure 7 of Figure 7 As shown in A, from Figure 7 As shown in Figure A, under the condition of mixing liposome nanoparticles and serum-free culture medium, the uptake efficiency of RGD ligand-modified LGL and LPGL liposome nanoparticles by BxPC3 cells was significantly higher than that of GL and PGL liposome nanoparticles. However, when BxPC3 cells were incubated with a mixture of liposome nanoparticles and culture medium containing 10% FBS (fetal bovine serum) without ultrasonic irradiation, the uptake efficiency of RGD ligand-modified LGL and LPGL liposome nanoparticles by BxPC3 cells was significantly reduced, and similar to that of GL and PGL liposome nanoparticles. This indicates that the protein crown adsorbed on the surface of the liposome nanoparticles can significantly inhibit the recognition of RGD ligands by BxPC3 cell receptors, thereby reducing the uptake efficiency of RGD ligand-modified LGL and LPGL liposome nanoparticles by BxPC3 cells. However, when BxPC3 cells were incubated with a mixture of liposome nanoparticles and culture medium containing 10% FBS (fetal bovine serum) and ultrasonic irradiation (sound intensity: 2 W / cm²), the uptake efficiency was significantly reduced. 2 Treatment with a frequency of 3 MHz, a duty cycle of 50%, and a duration of 5 min (5 min) again enhanced the uptake efficiency of RGD ligand-modified LPGL liposome nanoparticles by BxPC3 cells, and the uptake efficiency was essentially restored to the same order of magnitude as that in serum-free medium. Furthermore, when BxPC3 cells were incubated with a mixture of LGL, GL, and PGL liposome nanoparticles and a medium containing 10% FBS (fetal bovine serum), ultrasonic irradiation (sound intensity: 2 W / cm²) was applied. 2(Frequency: 3MHz, Duty Cycle: 50%, Duration: 5min) The cellular uptake efficiency of BxPC3 cells for LGL, GL and PGL liposome nanoparticles remained at a low level, indicating that ultrasound irradiation can eliminate the masking effect of the protein crown on the surface of LPGL liposome nanoparticles on the ligand RGD on the surface of liposome nanoparticles, and restore the enhanced cellular uptake effect mediated by ligand RGD on the surface of liposome nanoparticles.

[1377] 8. Subcellular distribution

[1378] BxPC3 cells (1×10) 5 Cells were cultured in confocal culture dishes (1 mL, 1 cell / mL) for 24 h. Cell nuclei were stained with Hoechst 33342 for 20 min, and lysosomes were stained with... Green DND26 (0.2 μL) staining for 30 min. Cy5-labeled LPGL liposome nanoparticles (equivalent to 60 μg / ml fluorescent lipid, 20 μL) were mixed with 1 mL of fresh serum-free medium or medium containing 10% FBS (fetal bovine serum) for 30 min to obtain a pre-incubation mixture. BxPC3 cells were washed twice with PBS, and then 1 mL of the pre-incubation mixture was added. Ultrasonic irradiation (2 W / cm²) was applied with or without irradiation. 2 Cells were treated with a frequency of 3 MHz, a duty cycle of 50%, and a duration of 5 min, and incubated for another 1 h. Subcellular distribution maps were then captured using a laser scanning confocal microscope (CLSM) with wavelength channels of 405 nm, 488 nm, and 640 nm. The Mander overlap coefficient between LPGL liposome nanoparticles and lysosomes after incubation was analyzed using Cellprofiler V2.2.0 image analysis software.

[1379] Subcellular distribution of Cy5-labeled LPGL liposome nanoparticles pre-incubated with and without serum and then mixed with BxPC3 cells, with and without ultrasound irradiation treatment, is as follows: Figure 8 As shown, from Figure 8 A and Figure 8 As shown in Figure B, after incubation of BxPC3 cells with a pre-incubated mixture of LPGL liposome nanoparticles and serum-free medium without ultrasonic irradiation, the LPGL liposome nanoparticles were mainly distributed in the cytoplasm and rarely in lysosomes. However, after incubation of BxPC3 cells with a pre-incubated mixture of LPGL liposome nanoparticles and medium containing 10% FBS (fetal bovine serum) without ultrasonic irradiation, over 65% of the intracellular LPGL liposome nanoparticles were distributed in lysosomes. When the pre-incubated mixture of LPGL liposome nanoparticles and medium containing 10% FBS (fetal bovine serum) was incubated with BxPC3 cells and then subjected to ultrasonic irradiation (sound intensity: 2 W / cm²), the LPGL liposome nanoparticles were found to be mainly distributed in the cytoplasm and rarely in lysosomes. 2(Frequency: 3MHz, Duty Cycle: 50%, Duration: 5min) LPGL liposome nanoparticles regained their main distribution within the cytoplasm. Therefore, ultrasound irradiation enabled LPGL liposome nanoparticles containing protein crowns to exhibit excellent lysosomal escape and avoidance of lysosomal degradation. Lysosomal escape can effectively prevent drug-loaded nanoparticles and their loaded drugs from being destroyed and degraded by degrading enzymes in lysosomes, thereby improving the stability of drug-loaded nanoparticles and their loaded drugs in cells and thus improving the therapeutic effect of drugs on diseases.

[1380] 9. Transendothelial transport

[1381] The Transwell system was used to study transcellular transport of liposomes in vascular endothelial cells. ECDHCC vascular endothelial cells (5 × 10⁶) were used. 5 BxPC3 cells (1 × 10⁻⁶ cells / mL, 1 mL) were incubated in the top compartment for 4 days to form a dense cell layer. 5 Cells / mL (1 mL) were seeded into the outer compartment of the transwell and incubated for 12 h. The top compartment was placed on the outer compartment of the transwell for 6 h of adaptation incubation. Cy5-labeled GL, LGL, PGL, or LPGL liposome nanoparticle dispersions (each equivalent to 1.2 mg / mL total lipids, 2.5 mL) were mixed with mouse plasma (2.5 mL) at a lipid / protein ratio of 1 / 50 or serum-free medium (2.5 mL), respectively, and then incubated at 37 °C (60 rpm) for 30 min to obtain a pre-incubation mixture of plasma or serum-free medium containing Cy5-labeled liposome nanoparticles. This pre-incubation mixture of plasma or serum-free medium containing Cy5-labeled liposome nanoparticles was added to the top compartment of a Transwell containing serum-free medium. No sonication was performed. The cells were cultured continuously in the Transwell for 3 h. The fluorescence intensity of the outer compartment of the transwell was measured using a microplate reader. BxPC3 cells were collected and the Cy5 fluorescence intensity was measured by flow cytometry. In addition, to distinguish between ultrasound-induced transvascular transport across the intercellular space and ligand / receptor-mediated transvascular endothelial transport, the following two methods were designed (e.g. Figure 7 (As shown in B): (Method I) The plasma pre-incubation mixture containing Cy5-labeled liposome nanoparticles (equivalent to 60 μg / mL fluorescent lipid, 20 μL) was first subjected to ultrasonic irradiation in a centrifuge tube (ultrasonic irradiation has no effect on vascular endothelial cells ECDHCC, sound intensity: 2 W / cm²). 2(Method II) Pretreatment with Cy5-labeled liposome nanoparticles pre-incubated with plasma (equivalent to 60 μg / mL fluorescent lipid, 20 μL) was first added to the top compartment, followed by ultrasonic irradiation in the top compartment (ultrasonic irradiation induces cavitation in vascular endothelial cells ECDHCC, sound intensity: 2 W / cm²). 2 Pretreatment (contact conditions): frequency: 3MHz, duty cycle: 50%, duration: 5min; then cultured continuously in Transwell for 3h, the fluorescence intensity of the medium on the outside of the substrate was measured using a microplate reader, BxPC3 cells were collected and the fluorescence intensity of Cy5 was measured by flow cytometry.

[1382] Transvascular endothelial cell ECDHCC transport of different liposome nanoparticles under different conditions, such as Figure 7 of Figure 7 As shown in C, from Figure 7As shown in Figure C, both LGL and LPGL liposome nanoparticles exhibited the highest transendothelial ECDHCC transport capacity under serum-free culture medium pre-incubation mixture and ultrasound-free conditions. However, under plasma pre-incubation mixture and ultrasound-free conditions, the transendothelial transport capacity of LGL and LPGL liposome nanoparticles decreased significantly. The transendothelial transport capacity of GL and PGL under serum-free culture medium pre-incubation mixture or plasma pre-incubation mixture and ultrasound-free conditions was low and did not change significantly. Under both plasma pre-incubation and ultrasound irradiation conditions, the transendothelial cell transport efficiency of PGL and LPGL liposome nanoparticles changed significantly, exhibiting different transendothelial cell transport efficiencies depending on the treatment method. Specifically, compared to the transendothelial cell transport efficiency of plasma pre-incubation mixtures of liposome nanoparticles without ultrasound irradiation, ultrasound irradiation (non-contact condition) of the plasma pre-incubation mixture of Cy5-labeled liposome nanoparticles in centrifuge tubes significantly enhanced the transendothelial cell transport efficiency of LPGL liposome nanoparticles, restoring it to the same level as the serum-free culture medium incubation mixture. The transport efficiencies of GL, LGL, and PGL liposome nanoparticles did not change significantly. This indicates that ultrasound irradiation can overcome the masking effect of the protein crown on the surface of LPGL liposome nanoparticles on the ligands (RGD) of the liposome nanoparticles, and ultrasound irradiation restores the transendothelial cell transport effect mediated by the RGD ligands and endothelial cell receptors of LPGL. However, when a plasma pre-incubation mixture of Cy5-labeled liposome nanoparticles was added to the top compartment for ultrasound irradiation (contact conditions), the transendothelial transport capacity of LPGL liposome nanoparticles was significantly higher than that under non-contact conditions, and was approximately 3 to 4 times that of GL, LGL, and PGL liposome nanoparticles. This indicates that ultrasound irradiation significantly enhances the transendothelial transport of LPGL liposome nanoparticles under contact conditions.

[1383] 10. The endocytic pathway of tumor cells

[1384] BxPC3 cells (1×10) 5BxPC3 cells were cultured in 12-well plates for 24 h with 1 mL of serum-free medium (1 cell / mL). The medium was then replaced with 1 mL of fresh serum-free medium, and chlorpromazine (50 μM, clathrin-mediated endocytosis inhibitor), genistein (200 μM, pituitary-mediated endocytosis inhibitor), wortmannin (5 μM, phosphatidylinositol 3-kinase-mediated macropinocytosis inhibitor), or cytochalasin D (5 μM, actin polymerization inhibitor) were added to each well. The cells were incubated with these endocytosis inhibitors for 2 h. A control group of BxPC3 cells without any endocytosis inhibitors was also incubated for 2 h. Then, a mixture of serum-containing or serum-free medium supplemented with Cy5-labeled LPGL liposome nanoparticles (equivalent to 60 μg / mL fluorescent lipid, 20 μL) was added to the wells containing the BxPC3 cells. The cells were then subjected to ultrasound irradiation (2 W / cm²) with or without the ultrasound. 2 The cells were treated under the following conditions: frequency: 3MHz, duty cycle: 50%, duration: 5min. After incubation for 3h, the cells were washed, digested and collected into centrifuge tubes, and the mean fluorescence intensity (MFI) was detected by flow cytometry.

[1385] After pretreatment with different endocytosis inhibitors, flow cytometry was used to determine the average fluorescence intensity of Cy5 in BxPC3 cells after incubation with serum-containing or serum-free culture mixtures containing Cy5-labeled LPGL liposome nanoparticles, with and without ultrasonic irradiation. Figure 7 As shown in D, from Figure 7As shown in Figure D, under serum-free incubation of BxPC3 cells with Cy5-labeled LPGL liposome nanoparticles and without ultrasound irradiation, compared with the blank control group without endocytosis inhibitors, the inhibition rates of LPGL endocytosis in BxPC3 cells by chlorpromazine, genistein, womanpem, and cytochalasin D were 21.1%, 57.6%, 13.4%, and 19.9%, respectively. This indicates that the uptake of LPGL liposome nanoparticles by BxPC3 cells is mainly driven by pit-mediated endocytosis. However, when BxPC3 cells were incubated with a serum-containing mixture containing Cy5-labeled LPGL liposome nanoparticles under conditions without ultrasound irradiation, the protein crown on the surface of the LPGL liposome nanoparticles significantly affected the endocytosis inhibition rate of the LPGL liposome nanoparticles. Compared with the blank control group of BxPC3 cells without endocytosis inhibitors, the inhibition rates of LPGL endocytosis by chlorpromazine, genistein, woumacil, and cytochalasin D in BxPC3 cells were 35.9%, 28.9%, 23.5%, and 10.2%, respectively. This indicates that under serum conditions, the uptake of LPGL liposome nanoparticles by BxPC3 cells is a mixed uptake pathway involving clathrin-mediated endocytosis, pit-mediated endocytosis, and macrocytosis. However, when BxPC3 cells were incubated with a serum-containing mixture containing Cy5-labeled LPGL liposome nanoparticles and under ultrasound irradiation, compared with the blank control group without endocytosis inhibitors, chlorpromazine, genistein, womanpem, and cytochalasin D inhibited LPGL endocytosis in BxPC3 cells by 24.4%, 52.5%, 24.8%, and 17.0%, respectively, indicating that ultrasound irradiation can remove the protein crown on the surface of liposome nanoparticles.

[1386] 11. Transcytosis and transport between tumor cells

[1387] BxPC3 cells (1×10) 5 Cells / mL (1 mL) were cultured in confocal culture dishes for 24 h, then the medium was replaced with 1 mL of fresh serum-free medium. Next, a mixture of serum-containing or serum-free medium supplemented with Cy5-labeled GL, LGL, PGL, or LPGL liposome nanoparticles (equivalent to 60 μg / mL fluorescent lipid, 20 μL) was added to the confocal culture dishes, followed by ultrasound irradiation (with or without ultrasound intensity: 2 W / cm²). 2 Pretreatment was performed under the following conditions: frequency: 3MHz, duty cycle: 50%, duration: 5min. After incubation for 3 hours, the culture dish was washed twice with PBS, and 1 mL of the solution containing 1×10⁻⁶ new PBS was added. 5Serum-free medium containing BxPC3 cells was added to culture dishes, and transcellular transport of liposomes from previously added cells to newly added cells was imaged at different time points using a CLSM with an excitation wavelength of 640 nm. Finally, newly added cells were easily washed away, collected in centrifuge tubes, and analyzed by flow cytometry. Simultaneously, BxPC3 cells were pretreated with the exocytosis inhibitor EXO1 (20 μM) using the same method, followed by the addition of a mixture of serum-containing or serum-free medium containing Cy5-labeled LPGL liposome nanoparticles, and then subjected to ultrasound irradiation (2 W / cm²). 2 The process involved processing (frequency: 3MHz, duty cycle: 50%, duration: 5min), followed by adding 1 mL of a new 1×10⁻⁶ ppm solution. 5 Serum-free medium was added to a culture dish for BxPC3 cells, and transcellular transport of liposomes from previously added cells to newly added cells was captured using a CLSM with an excitation wavelength of 640 nm at different culture time points.

[1388] BxPC3 cells were pretreated with the exocytosis inhibitor EXO1 or without pretreatment, and then mixed with serum-containing or serum-free culture media containing Cy5-labeled GL, LGL, PGL, or LPGL liposome nanoparticles, followed by ultrasound irradiation (2 W / cm²) without or with the ultrasound intensity. 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 5min) The CLSM plots of the newly added BxPC3 cells at different culture time points after treatment with 3MHz frequency, 50% duty cycle, and 5min duration are shown below. Figure 7 E and Figure 9 As shown; newly added BxPC3 cells were added to a culture dish and cultured for 2 hours. The mean fluorescence intensity (MFI) of Cy5 in the newly added BxPC3 cells was quantitatively measured by flow cytometry. Figure 7 As shown in F. From Figure 7 E, Figure 7 F, and Figure 9As can be seen, after mixing serum-containing culture medium containing GL, LGL, and PGL liposome nanoparticles with BxPC3 cells and then treating with ultrasound, the GL, LGL, and PGL liposome nanoparticles were difficult to transport to newly added BxPC3 cells. However, after mixing serum-containing culture medium containing LPGL liposome nanoparticles with BxPC3 cells and then treating with ultrasound, the LPGL liposome nanoparticles could be effectively transported to newly added BxPC3 cells (this trend was also inhibited by the EXO1 exocytosis inhibitor), and the transport capacity was similar to that of serum-free culture medium containing LPGL liposome nanoparticles mixed with BxPC3 cells without ultrasound treatment. This indicates that ultrasound irradiation can eliminate the masking effect of the protein crown on the surface of LPGL liposome nanoparticles on the ligands (RGD) on the surface of liposome nanoparticles, and restore the intercellular transport mediated by the ligands (RGD) on the surface of liposome nanoparticles.

[1389] Liposome nanoparticles can be transported from one cell to another under different conditions. GL, LGL, and PGL are difficult to transport into newly introduced BxPC3 cells, while LPGL can be effectively transported into newly introduced cancer cells after co-incubation. Figure 7 E and Figure 7 F and Figure 9 In particular, in the absence of ultrasound irradiation, intercellular transport of LPGL was significantly reduced, and this trend was inhibited by the EXO1 exocytosis inhibitor, indicating that LPGL is transported between cancer cells via transcellular interactions. Furthermore, under ultrasound irradiation, intercellular transport of LPGL reached levels similar to that in serum-free culture medium, confirming that ultrasound irradiation can effectively eliminate the protein crown on the surface of liposome nanoparticles and restore ligand-mediated intercellular transport of LPGL.

[1390] 12. Cytotoxicity and Apoptosis Assays

[1391] The cytotoxicity of different liposomal nanoparticles was tested on three-dimensional (3D) multicellular tumor spheroids containing BxPC3 and Huh7. Tumor spheroids were constructed using the hanging drop method. LPGL, PGL, LGL, GL, or gemcitabine (GEM) were premixed with serum-containing culture medium for 30 min, and then added at a GEM concentration of 0–10 μM to well plates containing three-dimensional (3D) multicellular tumor spheroids of BxPC3 or Huh7 and irradiated with ultrasound (sound intensity: 2 W / cm²). 2The culture medium was set at 3 MHz (frequency, duty cycle, 50%, duration, 5 min) and cultured for 72 h. Simultaneously, blank liposome nanoparticles (LPPL) in an equimolar ratio with LPGL were used as a control. The culture medium was then replaced with a mixture of 90 μL fresh medium and 10 μL Alamar Blue Cell Viability Reagent, and cultured for another 12 h. The sample plates were then analyzed using a plate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm to obtain fluorescence intensity readings. Furthermore, liposome-induced apoptosis in 3D multicellular tumor spheroids was further determined by TdT-mediated dUTP nick-end labeling (TUNEL) assay, and TUNEL-positive cells were detected by CLSM.

[1392] A mixture of LPGL, PGL, LGL, GL, or gemcitabine (GEM) and serum-containing culture medium (GEM concentration 0–10 μM) was incubated with three-dimensional (3D) multicellular tumor spheroids of BxPC3 or Huh7, followed by ultrasonic irradiation and culture for 72 h. The inhibitory effects of gemcitabine (GEM) and different liposome nanoparticles on the viability of BxPC3 tumor 3D spheroids were as follows: Figure 10 of Figure 10 A and Figure 10 B, from Figure 10 A and Figure 10 As shown in Figure B, both free GEM and liposome nanodroplets exhibited dose-dependent cytotoxicity against BxPC3 and Huh7 three-dimensional (3D) multicellular tumor spheroids, with LPGL showing the strongest cytotoxicity, while GL, LGL, and PGL exhibited low cytotoxicity. Figure 10 C represents the observation of apoptosis induced by LPGL, PGL, LGL, GL liposome nanoparticles and gemcitabine (GEM) in BxPC3 3D multicellular tumor spheroids using optical microscopy and TUNEL staining. Figure 10 As shown in Figure C, LPGL liposome nanoparticles caused the smooth, spherical BxPC3 tumor spheres to become irregularly collapsed around the tumor periphery, and the center of the BxPC3 tumor spheres was also eroded. Apoptotic cells were distributed throughout the tumor spheres. In contrast, the BxPC3 tumor spheres treated with free gemcitabine (GEM) and PGL, LGL, and GL liposome nanoparticles did not show significant morphological changes and had fewer apoptotic cells around the tumor spheres. This indicates that compared with PGL, LGL, and GL liposome nanoparticles, LPGL liposome nanoparticles have significantly superior tumor penetration ability.

[1393] 13. Infiltration of tumor spheroids

[1394] 3D multicellular tumor spheroids of BxPC3, either untreated or pretreated with the exocytosis inhibitor EXO1 (20 μM), were transferred to confocal culture dishes containing fresh serum-free medium. A mixture of Cy5-labeled liposome nanoparticles (equivalent to 60 μg / mL fluorescent lipids, 30 μL) and serum-containing medium was added, followed by ultrasonic irradiation (sound intensity: 2 W / cm²). 2 Frequency: 3MHz, duty cycle: 50%, duration: 5min), incubated for 6h. After washing with PBS, CLSM measurements were performed (images were acquired in the XYZ-3D-stack at 25μm intervals from the vertex to the equator).

[1395] A mixture of liposomes labeled with different Cy5 labels and serum-containing culture medium was added to 3D multicellular tumor spheroids of BxPC3 pretreated or untreated with the exocytosis inhibitor EXO1 and subjected to ultrasound irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, duty cycle: 50%, duration: 5min) followed by incubation for 6h. The permeability of different Cy5-labeled liposome nanoparticles to BxPC3 3D multicellular tumor spheroids was as follows: Figure 10 of Figure 10 D and Figure 10 As shown in E, from Figure 10 As shown in Figure D, GL, LGL, and PGL liposome nanoparticles are mainly distributed on the periphery of the 3D spheres of BxPC3, while LPGL liposome nanoparticles can penetrate deep into the 3D spheres of BxPC3 and be distributed throughout the entire sphere. Furthermore, Figure 10E. The penetration ability of liposome nanodroplets was assessed by measuring the average integrated optical density (IOD) of 75 μm and 100 μm thick layers of BxPC3 3D spheres. It was found that the average integrated optical density (IOD) of LPGL liposome nanoparticles penetrating into the 3D multicellular tumor spheres of BxPC3 was approximately 2.7–10 times that of GL, LGL, and PGL liposome nanoparticles, indicating that LPGL liposome nanoparticles possess excellent tumor penetration ability. However, after pretreatment with EXO1, the LPGL liposome nanoparticles were confined to the periphery of the 3D multicellular tumor spheres of BxPC3. The average integrated optical density (IOD) of LPGL in the 75 μm and 100 μm thick layers of BxPC3 3D spheres was significantly reduced, indicating that the exocytosis inhibitor EXO1 can significantly inhibit the penetration ability of LPGL liposome nanoparticles into the 3D spheres of BxPC3. Therefore, the penetration results of LPGL liposome nanoparticles into the 3D spheres of BxPC3 indicate that the deep penetration of LPGL depends on the RGD ligand / receptor-mediated cell transport pathway. Ultrasonic irradiation can eliminate the protein crown on the surface of LPGL liposome nanoparticles, overcome the masking effect of the protein crown on the ligand (RGD) on the surface of liposome nanoparticles, and restore the ligand (RGD)-mediated intercellular transport on the surface of liposome nanoparticles.

[1396] 14. Blood removal

[1397] BALB / c nude mice with subcutaneous inoculation of human PDA tumors were injected intravenously via the tail vein with GL, LGL, PGL, or LPGL liposome nanoparticle dispersions (dose equivalent to GEM 10 mg / kg, 3 mice per group), followed by ultrasound irradiation of the tumor site (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min). Blood samples (50μL) were collected from the orbital venous plexus of mice at 2min, 0.5h, 1h, 2h, 4h, 6h, 8h, and 12h after intravenous injection into the tail vein. The samples were mixed with heparin solution (1mg / mL, 50μL), centrifuged at 5,000rpm for 5min at 4°C to separate the supernatant. The supernatant was then diluted with acetonitrile (900μL) to obtain a mixture, which was vortexed, sonicated, and then centrifuged at 5,000rpm for 5min. The supernatant was then used to determine the CP4126 content and analyze pharmacokinetic parameters by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[1398] Following tail vein injection of GL, LGL, PGL, or LPGL liposome nanoparticles, the CP4126 blood content at different time points was as follows: Figure 11 As shown, from Figure 11As can be seen, GL, LGL, PGL or LPGL liposome nanoparticles have excellent blood clearance half-lives. Among them, GL and LGL liposome nanoparticles show similar blood clearance curves with an elimination half-life of 1.33 h, which is longer than that of PGL liposome nanoparticles (elimination half-life of 1.02 h) and LPGL liposome nanoparticles (elimination half-life of 1.17 h).

[1399] 15. Biodistribution, osmosis, and in vivo imaging

[1400] The aggregation and penetration of liposome nanoparticles in BALB / c nude mice subcutaneously loaded with human PDA tumors were studied. In vivo fluorescence imaging and biodistribution of the liposome nanoparticles were performed on both sides of the BALB / c nude mice subcutaneously loaded with PDA tumors. After intravenous injection of Cy5-labeled GL, LGL, PGL, or LPGL liposome nanoparticle dispersions (dose equivalent to GEM 10 mg / kg, 3 mice per group) into the tail vein of BALB / c nude mice loaded with human PDA tumors, the right subcutaneously loaded human PDA tumor was irradiated with ultrasound (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min) Human PDA tumors loaded subcutaneously on the left side were not irradiated with ultrasound. Twelve hours after injection, whole-body imaging of mice was performed using a Caliper IVIS Lumina II fluorescence spectroscopy imager equipped with a fluorescence filter set (excitation / emission wavelengths of 640 / 670nm). Then, each mouse was injected intravenously with FITC-labeled tomato lectin (FITC-Lectin, 0.05mg per mouse). Five minutes after injection, the heart was perfused with 2% glutaraldehyde solution. Tumors, hearts, livers, spleens, lungs, kidneys, and small intestines, both irradiated and unirradiated, were collected and imaged using the Caliper IVIS Lumina II fluorescence spectroscopy imager. The software performed quantitative fluorescence analysis on the isolated tissues. The ultrasound-irradiated tumors were frozen in Tissue OCT-Freeze and cut into 10 μm thick slices. The CLSM was used to capture images of the permeation of liposome nanoparticles in the ultrasound-irradiated tumor sites, and ImageJ software was used to quantitatively analyze the fluorescence intensity gradient from tumor blood vessels to deep tumor regions.

[1401] Biodistribution, tumor accumulation, and penetration of different Cy5-labeled liposome nanoparticles in BALB / c nude mice subcutaneously loaded with human PDA tumors, such as... Figure 12 As shown. Using... Figure 12The ultrasound irradiation device shown in Figure A was used to study the biodistribution, tumor accumulation, and penetration of liposome nanoparticles in BALB / c nude mice subcutaneously loaded with human PDA tumors on both sides. After tail vein injection of liposome nanoparticles, the right subcutaneous mouse loaded with human PDA tumor was subjected to ultrasound irradiation (sound intensity: 2 W / cm²). 2 (Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min), subcutaneous left-sided human PDA-loaded tumor was not subjected to ultrasound irradiation. Biodistribution of different liposome nanoparticles 12h after tail vein injection is shown below. Figure 12 B and Figure 12 C, from Figure 12 B and Figure 12 As shown in Figure C, 12 hours after intravenous injection of liposome nanoparticles, LPGL liposome nanoparticles exhibited higher fluorescence intensity in ultrasound-irradiated tumors than other liposome nanodroplets. The fluorescence intensity of LPGL liposome nanoparticles in ultrasound-irradiated tumors was 3.2–5.8 times that of GL, LGL, and PGL liposome nanoparticles in ultrasound-irradiated tumors. However, in tumors without ultrasound irradiation, there was no significant difference in fluorescence intensity between LPGL and LGL liposome nanoparticles, and the fluorescence intensity of LPGL liposome nanoparticles in ultrasound-irradiated tumors was 5.11 times that of LPGL liposome nanoparticles in unirradiated tumors. This indicates that ultrasound irradiation of tumors can significantly enhance the targeted aggregation of LPGL at the tumor site. Meanwhile, the fluorescence intensity of LGL liposome nanoparticles in tumors irradiated or unirradiated by ultrasound was only 1.4 to 1.6 times that of GL liposome nanoparticles in tumors irradiated or unirradiated by ultrasound, indicating that ligand modification is difficult to promote the effective aggregation of liposome nanoparticles at the tumor site. This is due to the protein crown (RGD) on the surface of the liposome nanoparticles. Using the fluorescence intensity of LPGL liposome nanoparticles in ultrasound-irradiated tumors as a benchmark, and comparing the increased fluorescence intensity of LGL and PGL liposome nanoparticles in ultrasound-irradiated tumors, ultrasound irradiation restarted ligand / receptor-mediated cell transport, accounting for more than 70% of the increase in fluorescence intensity of LPGL liposome nanoparticles in ultrasound-irradiated tumors. This indicates that the enhanced tumor accumulation of LPGL due to ligand modification is mainly due to the restart of ligand / receptor-mediated cell transport by ultrasound irradiation. This further suggests that ultrasound irradiation can eliminate the protein crown on the surface of LPGL liposome nanoparticles, overcome the masking effect of the protein crown on the ligand (RGD) on the surface of liposome nanoparticles, and restore ligand (RGD)-mediated endocytic transport on the surface of liposome nanoparticles.

[1402] Before cardiac perfusion, tumor vessels were simultaneously stained with FITC-Lectin. Co-localization analysis of blood vessels and liposome nanoparticles was used to assess the in vivo penetration of liposome nanoparticles into ultrasound-irradiated PDA tumors (e.g., Figure 12 D and Figure 12 As shown in E), from Figure 12 As shown in Figure D, GL liposome nanoparticles are primarily located at the tumor blood vessels, making it almost impossible for them to penetrate into the PDA tumor from within the blood vessels. PGL and LGL liposome nanoparticles are mainly distributed around the tumor near the blood vessels. However, LPGL liposome nanoparticles can effectively move away from the tumor blood vessels, deeply penetrating the tumor parenchyma and accumulating there. Figure 12 As shown in Figure E, under ultrasound irradiation of tumors, the fluorescence intensity of PGL and LGL liposome nanoparticles rapidly decreases from the tumor vessels to the tumor site. Beyond 50 μm from the tumor vessels towards the tumor, the fluorescence intensity of PGL and LGL liposome nanoparticles is almost undetectable. However, the fluorescence signal of LPGL liposome nanoparticles remains strong even beyond 100 μm from the tumor vessels towards the tumor. This indicates that under ultrasound irradiation of tumors in vivo, LPGL liposome nanoparticles have excellent penetration ability from the tumor vessels to the tumor site and excellent deep tumor penetration.

[1403] 16. Real-time extravasation of blood vessels in vivo under ultrasound irradiation

[1404] A tumor-loaded animal model was constructed by subcutaneously loading human PDA tumors near abdominal blood vessels in BALB / c nude mice. The tumor size reached 100 mm. 3 During the procedure, the tumor was fixed onto a microscope slide using a dorsal skin fold chamber (APJ Trading Co., Inc.). An ultrasound probe was mounted above the chamber, coated with a coupling agent, and Cy5-labeled GL, LGL, PGL, or LPGL liposome nanoparticle dispersions (dose equivalent to 10 mg / kg GEM) were injected via the tail vein. The tumor site was then subjected to ultrasound irradiation (sound intensity: 2 W / cm²). 2(Frequency: 3MHz, Duty Cy: 50%, Duration: 20min) Tumor regions were imaged using CLSM at 10min, 30min, and 60min after tail vein injection of different Cy5-labeled liposome nanoparticle dispersions, and the corresponding tumor fluorescence intensity was calculated using ImageJ software. Sixty min after tail vein injection of Cy5-labeled liposome nanoparticle dispersions, mice underwent cardiac perfusion, tumors were isolated, and fixed in 10 volumes of perfusion fluid at 4°C for at least 24 hours. The tumors were then cut into small fragments (approximately 2×2×2mm), immersed in 7% agarose solution, and cut into sections 0.25–1mm thick. The sections were washed with sodium cacodylic buffer (100mM), fixed with 1% osmium tetroxide for 2h, stained with uranium acetate at 37°C for 48h, dehydrated with acetone gradients, embedded in epoxy resin, sectioned (60–80nm), stained with lead citrate, and observed using transmission electron microscopy (TEM).

[1405] Real-time in vivo extravasation and tumor aggregation of liposomes labeled with different Cy5 in BALB / c nude mice loaded with human PDA tumors, such as... Figure 13 As shown. Using a CLSM and ultrasound device, the in vivo real-time extravasation of liposome nanoparticles and tumor accumulation (e.g., ...) were further investigated in BALB / c nude mice carrying human PDA tumors. Figure 13 As shown in A), from Figure 13 As shown in Figure A, the tumor is fixed in a small chamber within the skin folds on the back, and obvious tumor vessels are visible around the tumor. After tail vein injection of different Cy5-labeled liposome nanoparticles followed by ultrasound irradiation of the tumor site, the extravasation of the Cy5-labeled liposome nanoparticles into the tumor vessels at 10, 30, and 60 minutes post-tail vein injection is as follows: Figure 13 B and Figure 13 As shown in C, from Figure 13 B and Figure 13 As shown in Figure C, GL liposome nanoparticles can hardly leak out of the capillaries, and the fluorescence signal in the tumor region is negligible. This confirms that PDA is a low-permeability tumor (with a low level of EPR effect). LGL and PGL liposome nanoparticles leak slightly from the tumor vessels and are mainly located around the tumor vessels. LPGL liposome nanoparticles can effectively leak out of the tumor vessels and distribute into the tumor parenchyma. The fluorescence intensity of LPGL liposome nanoparticles accumulated in the tumor site is significantly higher than that of GL, PGL, and LGL liposome nanoparticles. This indicates that ultrasound irradiation can effectively promote the leakage of LPGL liposome nanoparticles from the tumor vessels and their accumulation in the tumor parenchyma.

[1406] Tumor sites were subjected to ultrasound irradiation after tail vein injection of liposomes labeled with different Cy5 labels. Transmission electron microscopy (TEM) was used to observe the vascular structures of the collected tumors 60 minutes after tail vein injection. Figure 13 As shown in D), the PDA tumor is a low-permeability solid tumor. Within the tumor's vascular structure, the endothelial cells are well-formed and tightly packed. Figure 13 As shown in Figure D, in the TEM images of tumor vessels after treatment with liposome nanoparticles, there were no large number of leakage gaps between vascular endothelial cells. Only one gap was observed in the tumor vessel wall treated with PGL liposome nanoparticles (indicated by the arrow), indicating that GL, PGL, LGL, and LPGL liposome nanoparticles have difficulty leaking through the gaps between tumor vascular endothelial cells (i.e., difficult extravasation due to the traditional EPR effect). Unexpectedly, typical transcellular transport protein vesicles (as indicated by the arrow) were abundant in the tumor vessel wall treated with LPGL liposome nanoparticles, while vesicles were scarce in the tumor vessel walls treated with GL, PGL, and LGL liposome nanoparticles. These vesicles are the only transporters for transendothelial transport, indicating that ultrasound irradiation can effectively promote the leakage of LPGL liposome nanoparticles from tumor vessels into the tumor via ligand / receptor-mediated transcellular transport, while ultrasound irradiation has difficulty promoting the leakage of GL, PGL, and LGL liposome nanoparticles from tumor vessels into the tumor via ligand / receptor-mediated transcellular transport.

[1407] 17. In vivo antitumor activity

[1408] Human PDA tumor cells were subcutaneously inoculated into BALB / c nude mice and incubated for 16 days. The resulting BALB / c nude mice bearing human PDA tumors were randomly divided into 7 groups of 6 mice each. Each group received a tail vein injection of either LPGL liposome nanoparticle dispersion (equivalent to 10 mg / kg GEM), PGL liposome nanoparticle dispersion (equivalent to 10 mg / kg GEM), LGL liposome nanoparticle dispersion (equivalent to 10 mg / kg GEM), GL liposome nanoparticle dispersion (equivalent to 10 mg / kg GEM), GEM in PBS 7.4 dispersion (equivalent to 10 mg / kg GEM), LPL blank liposome nanoparticle dispersion, or PBS 7.4 buffer. The first intravenous administration was on day 16, with subsequent intravenous injections every two days. After administration, the tumor site was treated with ultrasound irradiation (sound intensity: 2 W / cm²). 2(Frequency: 3MHz, Duty Cycle: 50%, Duration: 20min), administered 4 times. Tumor width and length, as well as mouse weight, were measured during treatment. At the end of the experiment at 36 days, mice were euthanized after blood collection, and tumors were dissected and weighed. Treatment efficacy was assessed by comparing tumor size between the experimental and control groups. Tumor inhibition rate = 100% × (mean tumor weight in the PBS group - mean tumor weight in the experimental group) / mean tumor weight in the PBS group.

[1409] Tumors isolated at the end of the experiment on day 36 were fixed in 4% neutral paraformaldehyde buffer and embedded in paraffin. 5 μm thick tissue sections were fixed onto glass slides and stained with hematoxylin and eosin (H&E), then examined under a light microscope. Ki67 immunohistochemical staining was used to investigate the percentage of positively stained proliferative tumor cells in the examined areas. Ki67 staining analysis of tissue sections was performed using the Ki67-antibodyAssay Kit. Apoptotic cells were identified using the TUNEL Apoptosis Assay Kit and examined using CLSM.

[1410] Antitumor activity of free GEM and different liposome nanoparticles in BALB / c nude mice subcutaneously loaded with human PDA tumors, such as Figure 14 As shown, Figure 14 Figure A shows the construction, experimental schedule, and tumor treatment regimen of a BALB / c nude mouse model loaded with human PDA tumors. Tumor volume changes over time in different drug administration groups are also shown. Figure 14 As shown in B, from Figure 14 As shown in Figure B, continuous tumor volume growth was observed in mice treated with PBS 7.4 buffer and LPL blank liposome nanoparticles. In mice treated with free GEM, GL, LGL, and PGL liposome nanoparticles, tumor growth was initially delayed, but resumed after drug administration was stopped. In mice treated with LPGL liposome nanoparticles, tumor volume decreased progressively from the start of administration to the end of administration, and tumor growth was significantly and continuously inhibited. The photographs of mice in each group at the end of the 36-day experiment (e.g., [images would be inserted here]) further illustrate this. Figure 14 (as shown in C) and its isolated tumor photographs (e.g. Figure 14 As shown in Figure D, compared with other treatment groups, the tumors of mice treated with LPGL liposome nanoparticles were significantly inhibited, and half of the tumors treated with LPGL liposome nanoparticles were completely eradicated. At the end of the 36-day experiment, the average tumor weight removed from each group of mice was as follows: Figure 14 As shown in E, from Figure 14As shown in Figure E, the tumor inhibition rate of LPGL liposome nanoparticles reached 98.3%, significantly higher than that of GEM, GL, LGL, and PGL. Furthermore, changes in body weight in mice during treatment (e.g., ...) were observed. Figure 14 F) and the white blood cell count of each group of mice at the end of the 36-day experiment (e.g., F) Figure 14 G) Blood platelet count (e.g.) Figure 14 H) It can be seen that treatment with free GEM resulted in a significant reduction in the weight of mice and a significant decrease in the values ​​of white blood cells and platelets, causing hematological damage. However, the treatment with liposome nanoparticles in each group had no significant effect on the weight, white blood cells, and platelets of mice, thus indicating that liposome nanoparticles have no obvious side effects and are highly safe. This shows that liposome nanodroplets have good biocompatibility and biosafety.

[1411] Tumors isolated after 36 days of experimentation were stained with H&E, Ki67 IHC, and TUNEL. Figure 14 I and Figure 15 As shown, this study aimed to investigate the antitumor activity mechanism of liposome nanoparticles. From Figure 14 I and Figure 15 As can be seen from the results, hematoxylin and eosin (H&E) staining showed that compared with tumors treated with LPL liposome nanoparticles and PBS buffer, the tumor cell density treated with free GEM, GL, LGL, PGL, and LPGL liposome nanoparticles was significantly reduced. The LPGL liposome nanoparticle treatment group showed tumor ablation, exhibiting a large number of apoptotic cells, significant nuclear shrinkage, and extensive intercellular spaces. Compared with free GEM, GL, LGL, and PGL liposome nanoparticles, LPGL liposome nanoparticles significantly reduced the percentage of Ki67-positive tumor cells, indicating a better prognosis even after short-term treatment. In situ detection of DNA fragments using the TUNEL assay to assess drug-induced apoptosis showed a large number of apoptotic cells (green fluorescence) in tumors treated with LPGL liposome nanoparticles compared to other groups. Furthermore, the apoptotic cells in LPGL liposome nanoparticle-treated tumors were almost distributed throughout the entire tumor parenchyma, while the apoptotic cells in tumors treated with GEM, GL, LGL, and PGL liposome nanoparticles were only present around the tumor. The results showed that LPGL liposome nanoparticles could aggregate and penetrate deeply into PDA tumors, delivering active drugs to the entire tumor parenchyma and thereby inducing apoptosis.

[1412] Example 2

[1413] The lipid nanoparticles and their properties and antitumor activity in Example 2 are studied in the same way as in Example 1, except that DSPE-PEG2000-RGD is replaced with DSPE-PEG2000-NGR to prepare LPGL liposome nanoparticles and LGL liposome nanoparticles. DSPE-PEG2000-NGR is a distearate phosphatidylethanolamine-polyethylene glycol 2000-NGR targeting peptide. The amino acid sequence of the NGR targeting peptide is Gly-Gly-Cys-Asn-Gly-Arg-Cys (SEQ ID NO:2).

[1414] NGR-modified liposome nanoparticles and their properties, as well as their tumor aggregation and antitumor activity in BALB / c nude mice subcutaneously loaded with human HCC tumors, such as... Figure 16 , Figure 17 As shown in Table 3.

[1415] Table 3. Particle size and potential of NGR-modified LGL and NGR-modified LPGL liposome nanoparticles in PBS or plasma.

[1416]

[1417] The dilution of each liposome nanoparticle in plasma was performed as follows: NGR-modified LGL or LPGL liposome nanoparticle dispersions were mixed with mouse plasma at a lipid / protein ratio of 1 / 50 and incubated in a shaker (60 rpm) at 37°C for 30 min.

[1418] from Figure 16 As shown in Figure A, after incubation with mouse plasma for 30 min, the total protein content in the protein crowns on the surface of NGR-modified LPGL and PGL liposome nanoparticles reached approximately 28 μg / mg lipid. This was further enhanced by ultrasonic irradiation (+US; sound intensity: 2 W / cm²). 2 After ultrasonic irradiation (frequency: 3 MHz, duty cycle: 50%, duration: 5 min), the total protein content in the protein crown on the surface of NGR ligand-modified LPGL liposome nanoparticles decreased significantly by 74.9%, while the total protein content on the surface of NGR ligand-modified LGL liposome nanoparticles remained unchanged, indicating that ultrasonic irradiation can effectively remove the protein crown on the surface of NGR ligand-modified LPGL liposome nanoparticles. Figure 16As shown in Figure B, a serum-free culture mixture of Cy5-labeled NGR-modified LGL and NGR-modified LPGL liposome nanoparticles exhibited good uptake efficiency by Huh7 cells. However, when a serum-containing culture mixture of Cy5-labeled NGR-modified LGL and NGR-modified LPGL liposome nanoparticles was incubated with Huh7 cells without ultrasonic irradiation, the uptake capacity of Huh7 cells for NGR-modified LGL and LPGL liposome nanoparticles was significantly reduced. This indicates that the protein crown on the surface of NGR-modified LGL and LPGL liposome nanoparticles hinders NGR ligand / receptor-mediated cell uptake. Furthermore, when a serum-containing culture mixture of Cy5-labeled NGR-modified LGL and NGR-modified LPGL liposome nanoparticles was incubated with Huh7 cells and subjected to ultrasonic irradiation (sound intensity: 2 W / cm²), the uptake efficiency of Huh7 cells was significantly reduced. 2 Treatment with a frequency of 3 MHz, a duty cycle of 50%, and a duration of 5 min restored the uptake efficiency of NGR-modified LPGL liposome nanoparticles by Huh7 cells. The results of serum-free culture medium mixtures containing NGR-modified LPGL liposome nanoparticles and Huh7 cells incubated without ultrasonic irradiation were similar and showed no statistical difference. However, the uptake efficiency of NGR-modified LPGL liposome nanoparticles by Huh7 cells remained very low. This indicates that ultrasonic irradiation can eliminate the masking effect of the protein crown on the surface of LPGL liposome nanoparticles on the ligands (such as NGR) on the surface of liposome nanoparticles, and restore the enhanced cell uptake effect mediated by ligands (such as NGR) on the surface of liposome nanoparticles.

[1419] The antitumor activity of GEM, GL, NGR-modified LGL, PGL, NGR-modified LPGL, and PBS 7.4 against subcutaneously loaded human HCC tumors in BALB / c nude mice was as follows: Figure 17 As shown in A-17G.

[1420] Human HCC tumor cells were subcutaneously inoculated into BALB / c nude mice and incubated for 20 days. The resulting BALB / c nude mice carrying human HCC tumors were randomly divided into 6 groups (n=9, with 3 mice used for tumor analysis 24 hours after the first intravenous injection to determine the cumulative concentration of gemcitabine triphosphate active metabolite dFdCTP in the tumor). Similar to the results observed in the PDA animal model in Example 1, BALB / c nude mice carrying human HCC tumors were injected subcutaneously via tail vein with NGR-modified LPGL, NGR-modified LGL liposome nanoparticles, PGL liposome nanoparticles, GL liposome nanoparticles, free GEM in PBS 7.4 buffer, and PBS 7.4 buffer. The tumor sites of each group of mice were then subjected to ultrasound irradiation. 24 hours after the first intravenous injection, the drug concentrations of gemcitabine triphosphate active metabolite dFdCTP in the isolated tumors of each group of mice were as follows: Figure 17 As shown in Figure B, because GEM is rapidly phosphorylated to its active metabolite dFdCTP in tumor tissue, dFdCTP is widely used as a quantitative indicator of GEM. Figure 17 As shown in Figure B, 24 hours after the first intravenous injection, the dFdCTP content in tumors treated with NGR-modified LPGL liposome nanoparticles was approximately 2.9 to 8.7 times that of free GEM, GL liposome nanoparticles, NGR-modified LGL liposome nanoparticles, and PGL liposome nanoparticles, indicating that NGR-modified LPGL liposome nanoparticles have superior tumor-targeting aggregation ability in HCC tumors.

[1421] Figure 17 C Figure 17 D、 Figure 17 E and Figure 17 F shows changes in tumor size and body weight in BALB / c nude mice loaded with human HCC tumors after intravenous treatment with NGR-modified LPGL, NGR-modified LGL liposome nanoparticles, PGL liposome nanoparticles, GL liposome nanoparticles, free GEM, and PBS 7.4 buffer. Figure 17 C Figure 17 D、 Figure 17 E and Figure 17 As shown in Figure F, the body weight of mice treated with GEM gradually decreased, while the body weight of GL, NGR-modified LGL, PGL, and NGR-modified LPGL recovered to normal levels, indicating that the side effects of these liposome nanoparticles were negligible and their safety was high. Furthermore, at the end of the 34-day experiment, blood samples were collected from each group, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and blood urea nitrogen (BUN) were measured. The results are shown in Table 4 to assess in vivo hepatotoxicity and nephrotoxicity. Table 4 shows that the serum levels of AST, ALT, BUN, and CRE in mice treated with all liposome nanoparticles remained within the normal limits, while the GEM group exceeded the maximum normal limits, indicating that the liposome nanoparticles have good biosafety and biocompatibility. Figure 17As shown in Figure D, during the treatment period, compared with mice treated with PBS or GEM, the tumor growth of mice treated with GL, PGL, NGR-modified LGL, and NGR-modified LPGL liposome nanoparticles was significantly inhibited. However, only the tumors treated with NGR-modified LPGL liposome nanoparticles continued to shrink and decline upon discontinuation of administration. Unexpectedly, compared with other treatment groups, the tumors of mice treated with NGR-modified LPGL liposome nanoparticles were significantly inhibited, and more than half of the tumors treated with NGR-modified LPGL liposome nanoparticles were completely eradicated. This indicates that NGR-modified LPGL liposome nanoparticles have excellent anti-tumor efficiency and can eradicate cancer. Figure 17 E and Figure 17 As can be seen from F, the tumor elimination rate of NGR-modified LPGL liposome nanoparticles was 96.8%, which was superior to that of free GEM, GL liposome nanoparticles, NGR-modified LGL liposome nanoparticles and PGL liposome nanoparticles, indicating that NGR-modified LPGL liposome nanoparticles have a general and effective anti-tumor activity against solid tumors. Figure 17 H&E staining of G and IHC staining of Ki67 also showed that NGR-modified LPGL liposome nanoparticles could significantly induce a large number of apoptotic cells, resulting in better prognosis even after short-term treatment. Figure 17 G).

[1422] Table 4 shows the serum biochemical analysis of mice after liposome nanoparticle treatment.

[1423]

[1424] The above description is an implementation scheme designed for one case of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a product for eliminating protein crown-modified nanoparticles, characterized in that, The method includes the following steps: The protein crown-modified nanoparticles are subjected to ultrasonic irradiation to eliminate the protein crown of the protein crown-modified nanoparticles. The nanoparticles are loaded with perfluoron-n-pentane. The nanoparticles are liposomes; The liposomes were prepared by the following method: (1) The lipid material is dissolved in an organic solvent, and the organic solvent is removed to obtain a lipid membrane, wherein the lipid material includes 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine and distearate phosphatidylethanolamine-polyethylene glycol; (2) After immersing the lipid membrane in perfluoropentane, a buffer solution is added to hydrate the lipid membrane, and then the mixture is stirred to obtain liposomes; The acoustic intensity of the ultrasonic irradiation was 2 W / cm². 2 ; The frequency of the ultrasonic irradiation is 3 MHz; The duty cycle of the ultrasonic irradiation is 50%. The ultrasonic irradiation time is 5 minutes; The protein crown-modified nanoparticles are protein crown-modified nanoparticles in conditions containing protein. The protein-containing conditions include blood, serum, plasma, and / or culture medium.

2. The method as described in claim 1, characterized in that, The method described is an in vitro method.

3. The method as described in claim 1, characterized in that, The blood, serum, or plasma mentioned includes blood, serum, or plasma extracted from or separated from the body.

4. The method as described in claim 1, characterized in that, The culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

5. The method as described in claim 1, characterized in that, The culture medium includes a serum-containing culture medium.

6. A method for screening or identifying potential ligands targeting cell or cell surface receptors, characterized in that, The method includes the following steps: (I) A ligand is modified onto nanoparticles to obtain ligand-modified nanoparticles, wherein the nanoparticles are loaded with perfluoro-n-pentane; (II) Incubate the cell or cell surface receptor with the ligand-modified nanoparticles of step (I), subject them to ultrasonic irradiation, and determine the binding of the ligand-modified nanoparticles of step (I) or the ligand of the ligand-modified nanoparticles to the cell or cell surface receptor, thereby screening or identifying whether the ligand of step (I) is a potential ligand for targeting the cell or cell surface receptor. The nanoparticles are liposomes; The liposomes were prepared by the following method: (1) The lipid material is dissolved in an organic solvent, and the organic solvent is removed to obtain a lipid membrane, wherein the lipid material includes 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine and distearate phosphatidylethanolamine-polyethylene glycol; (2) After immersing the lipid membrane in perfluoropentane, a buffer solution is added to hydrate the lipid membrane, and then the mixture is stirred to obtain liposomes; The acoustic intensity of the ultrasonic irradiation was 2 W / cm². 2 ; The frequency of the ultrasonic irradiation is 3 MHz; The duty cycle of the ultrasonic irradiation is 50%. The ultrasonic irradiation time is 5 minutes; The incubation is carried out under conditions containing protein; The conditions containing proteins include blood, serum, plasma, or culture medium.

7. The method as described in claim 6, characterized in that, The method described is an in vitro method.

8. The method as described in claim 6, characterized in that, The ligand in step (I) includes the test ligand.

9. The method as described in claim 6, characterized in that, Step (II) includes: (II-1) In the test group, cells or cell surface receptors are incubated with ligand-modified nanoparticles from step (I) and subjected to ultrasonic irradiation. The binding force B1 between the ligand-modified nanoparticles from step (I) or the ligand of the ligand-modified nanoparticles and the cells or cell surface receptors is measured. A control group is set up, which includes nanoparticles without ligand modification and other measurement conditions are the same as those in the test group. The binding force B0 between nanoparticles without ligand modification and cells or cell surface receptors is measured. The incubation is carried out under conditions containing protein. (II-2) If the binding force B1 between the ligand-modified nanoparticles or the ligand of the ligand-modified nanoparticles in step (I) and the cell or cell surface receptor is greater than the binding force B0 between the unmodified nanoparticles and the cell or cell surface receptor, then the ligand in step (I) is a potential ligand for targeting the cell or cell surface receptor.

10. The method as described in claim 6, characterized in that, The culture medium includes a culture medium containing serum, plasma and / or tissue proteins.

11. The method as described in claim 6, characterized in that, The culture medium includes a serum-containing culture medium.

12. The method as described in claim 6, characterized in that, The cells mentioned include tumor cells and / or tumor vascular cells.

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