CN115825425A

CN115825425A - Method for eliminating protein corona on surface of nano-particles

Info

Publication number: CN115825425A
Application number: CN202211162306.8A
Authority: CN
Inventors: 王国伟; 黄品同; 江依凡; 张超; 林涛
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2023-03-21

Abstract

The invention relates to a method for eliminating protein crowns on the surface of nanoparticles. Specifically, the present invention provides a method for eliminating the protein corona of a protein corona-modified nanoparticle, the method comprising the steps of: and carrying out ultrasonic irradiation treatment on the nanoparticle modified by the protein corona so as to eliminate the protein corona of the nanoparticle modified by the protein corona, wherein the nanoparticle carries perfluoro-n-pentane. The ultrasonic irradiation can eliminate the protein corona on the surface of the nano-particles, overcome the covering effect of the protein corona on the ligand modified on the surface of the nano-particles and avoid the protein corona from hindering the combination of the ligand modified on the surface of the nano-particles and a cell receptor.

Description

Method for eliminating protein corona on surface of nano-particles

Technical Field

The invention relates to the field of medicines, in particular to a method for eliminating protein crowns on the surfaces of nanoparticles.

Background

Ligand/receptor mediated drug-loaded nanoparticles such as nanoparticles and liposomes are a strategy for improving the anti-tumor effect of drugs, particularly for low-Permeability solid tumors such as liver cancer or pancreatic cancer, because the vascular endothelial cells of tumors of the low-Permeability solid tumors are good in tissue and tightly packed, and the gaps among the vascular endothelial cells are small, even if the drugs are delivered to the blood vessels of the tumors, the drugs are hindered by the vascular endothelial cells of the tumors which are good in tissue and tightly packed, and are difficult to effectively penetrate through the gaps among the vascular endothelial cells to reach the microenvironment of the tumor part, so that when the anti-tumor drugs are used for treating the tumors with low vascular Permeability, the anti-tumor drugs cannot effectively penetrate from the gaps among the tumor blood vessels to the tumor part through the traditional tumor Permeability and Retention effect (EPR effect), and further cannot effectively exert the anti-tumor effect. For low-permeability solid tumors, the ligand modified on the surface of the drug-loaded nanoparticles is specifically combined with the surface receptor of tumor vascular endothelial cells, and the ligand/receptor combination mediates the endocytosis and exocytosis of the tumor vascular endothelial cells to the drug-loaded nanoparticles to promote the drug-loaded nanoparticles to permeate to tumor sites from blood so as to enhance the anti-tumor effect.

However, the surface of the nanoparticle in a protein-containing environment such as a serum-containing culture medium or a body fluid such as blood can adsorb protein to form protein corona (protein corona), which masks the ligand on the surface of the nanoparticle like a fluid biological barrier, thereby preventing the ligand on the surface of the nanoparticle from binding with the ligand on the surface of a cell such as a tumor vascular endothelial cell or a tumor cell, and thus cannot realize the endocytosis and exocytosis of the nanoparticle by the ligand/receptor-mediated cell. Also, in experiments for screening or identifying potential ligands targeted to cells by modifying the ligand to be tested on the surface of the nanoparticle, the ligand to be tested modified nanoparticle often needs to be incubated with cells under the condition containing protein (such as serum-containing culture medium) to screen and identify the ligand to cells, however, because the protein corona formed by adsorbing the protein on the nanoparticle under the condition containing protein (such as serum-containing culture medium) covers the ligand to be tested on the surface of the nanoparticle, the ligand to be tested can not be effectively and accurately screened and identified under the condition containing protein in vitro (such as serum-containing culture medium), and particularly, false negative results are easy to occur under the condition that the amount of the ligand to be tested is low, thereby limiting the development of the ligand.

In addition, the existing drug-loaded nanoparticles have many disadvantages, for example, low biological safety, easy retention in lysosomes, degradation of the nanoparticles and the drug loaded therein by various enzymes in the lysosomes, thereby reducing the therapeutic effect, and the like.

Therefore, there is a need in the art to develop a method for eliminating the protein corona on the surface of nanoparticles to overcome the masking effect of the protein corona on the ligand modified on the surface of nanoparticles, thereby effectively realizing the targeted therapy of ligand-modified nanoparticles or the screening, identification and development of potential ligands in protein-containing conditions (such as serum-containing medium).

Disclosure of Invention

The invention aims to provide a nanoparticle, and the ultrasonic irradiation can effectively eliminate a protein corona on the surface of the nanoparticle, so that the covering effect of the protein corona on a ligand modified on the surface of the nanoparticle is overcome.

In a first aspect of the invention, there is provided a nanoparticle comprising perfluoro-n-pentane.

Preferably, the nanoparticle is a nanoparticle or a liposome.

Preferably, the nanoparticles encapsulate perfluoro-n-pentane.

Preferably, the nanoparticles comprise a nanomaterial.

Preferably, the nanomaterial comprises an amphiphilic material.

Preferably, the nanomaterial comprises a nanomaterial of nanoparticles and/or a lipid material of liposomes.

Preferably, the amphiphilic material comprises amphiphilic material of nanoparticles and/or lipid material of liposomes.

Preferably, the liposomes comprise a lipid material.

Preferably, the lipid material comprises one or more of 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), soya lecithin, phosphatidylcholine (PC, lecithin), cholesterol, phosphatidylethanolamine (PE, cephalin), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), dicetyl phosphate (DCP), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC) and Dioleoylphosphatidylcholine (DOPC).

Preferably, the lipid material comprises 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

Preferably, said distearoylphosphatidylethanolamine-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.

Preferably, the DPPC is 1 to 10 parts by weight, preferably 2 to 8 parts by weight, more preferably 4 to 6 parts by weight, most preferably 3 parts by weight.

Preferably, the DSPE-PEG is 0.5 to 8 parts by weight, preferably 1 to 5 parts by weight, more preferably 1 to 3 parts by weight, most preferably 2 parts by weight.

Preferably, the perfluoro-n-pentane is present in an amount of 0.01 to 0.5 parts by weight, preferably 0.02 to 0.2 parts by weight, more preferably 0.05 to 0.15 parts by weight, more preferably 0.08 to 0.12 parts by weight, most preferably 0.1 parts by weight.

Preferably, the weight ratio of DPPC to DSPE-PEG is 0.2-8:1, preferably 0.5 to 5:1, more preferably 1-2:1, more preferably 1.3 to 1.7:1, optimally 1.5. .

Preferably, the volume to weight ratio (ml: mg) of said perfluoro-n-pentane to said DPPC is from 1:30.

preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the nanoparticle comprises a drug-loaded nanoparticle or a drug-loaded liposome.

Preferably, the amount of the drug is 0.5 to 8 parts by weight, preferably 1 to 5 parts by weight, more preferably 1 to 3 parts by weight, and most preferably 2 parts by weight.

Preferably, the weight ratio of the DPPC to the medicament is 0.2-8:1, preferably 0.5 to 5:1, more preferably 1-2:1, more preferably 1.3 to 1.7:1, optimally 1.5.

Preferably, the drug comprises a drug that is unstable in the lysosome of the cell.

Preferably, the drug comprises a drug that is retained and/or degraded by the lysosomes of the cells.

Preferably, said degradation comprises degradation by lysosomal enzymes.

Preferably, the drug comprises a drug that is degraded by a lysosomal enzyme of the cell.

Preferably, the target of action of the drug is in the cytoplasm or in the nucleus.

Preferably, the drug comprises a gene or a protein.

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

Preferably, the drug comprises an anti-cancer drug.

Preferably, the anti-cancer drug comprises a chemical drug.

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

Preferably, the drug comprises a free drug form or a prodrug form.

Preferably, the drug comprises a prodrug.

Preferably, the prodrug comprises a prodrug formed by modifying a free drug on a prodrug carrier.

Preferably, the prodrug comprises a free drug chemically linked to a prodrug carrier.

Preferably, the drug comprises a hydrophobic drug or a hydrophilic drug.

Preferably, the free drug comprises a hydrophobic drug or a hydrophilic drug.

Preferably, the free drug comprises an anti-cancer drug.

Preferably, the prodrug carrier comprises a hydrophobic carrier or a hydrophilic carrier.

Preferably, the prodrug carrier comprises a higher fatty acid carrier or a higher fatty alcohol carrier.

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

Preferably, the prodrug carrier comprises a higher aliphatic alcohol containing 12 to 26 (preferably 14 to 22, more preferably 16 to 20) carbon atoms.

Preferably, the higher fatty acid carrier is selected from the group consisting of: palmitic acid (hexadecanoic acid), pearlescent fatty acid (heptadecanoic acid), stearic acid (octadecanoic acid), oleic acid (octadecenoic acid), linoleic acid (octadecadienoic acid), linolenic acid (octadecatrienoic acid), arachidic acid (eicosanoic acid), eicosapentaenoic acid, behenic acid (docosanoic acid), DHA (docosahexaenoic acid), lignoceric acid (tetracosanoic acid), or combinations thereof.

Preferably, said oleic acid comprises elaidic acid.

Preferably, the higher aliphatic alcohol carrier is selected from the group consisting of: palmitol, stearyl alcohol, oleyl alcohol, linoleyl alcohol, linolenyl alcohol, arachidyl alcohol, eicosapentaenoic alcohol, behenyl alcohol, docosahexaenoic alcohol, or combinations thereof.

Preferably, the prodrug comprises an amphiphilic prodrug.

Preferably, the amphiphilic prodrug is provided as a nanomaterial of nanoparticles.

Preferably, the amphiphilic prodrug is used as a nano material of nanoparticles.

Preferably, the amphiphilic prodrug serves as the lipid material of the liposome.

Preferably, the amphiphilic prodrug acts as a lipid bilayer.

Preferably, the amphiphilic prodrug comprises a pharmaceutically active ingredient as a hydrophilic moiety and a prodrug carrier as a hydrophobic moiety.

Preferably, the amphiphilic prodrug comprises a pharmaceutically active ingredient as a hydrophobic moiety and a prodrug carrier as a hydrophilic moiety;

preferably, the prodrug comprises:

D-C

wherein, "D" is the active pharmaceutical ingredient, "C" is the prodrug carrier, "-" linkage.

Preferably, the pharmaceutically active ingredient comprises a drug that is unstable in the lysosomes of cells.

Preferably, the pharmaceutically active ingredient comprises a hydrophobic pharmaceutically active ingredient or a hydrophilic pharmaceutically active ingredient.

Preferably, the pharmaceutically active ingredient comprises a pharmaceutically active ingredient that is retained and/or degraded by lysosomes of cells.

Preferably, said degradation comprises degradation by lysosomal enzymes.

Preferably, the pharmaceutically active ingredient comprises a pharmaceutically active ingredient that is degraded by a lysosomal enzyme of the cell.

Preferably, the active ingredient is targeted in the cytoplasm or nucleus.

Preferably, the pharmaceutically active ingredient comprises a gene or a protein.

Preferably, the pharmaceutically active ingredient comprises an anti-cancer drug.

Preferably, the prodrug comprises:

wherein R is an anticancer drug, and the anticancer drug comprises gemcitabine, cytarabine, doxorubicin, fluorouracil, or a combination thereof.

Preferably, the drug comprises gemcitabine elaidic acid ester.

Preferably, the prodrug comprises gemcitabine elaidic acid ester.

Preferably, the gemcitabine elaidic acid ester has the following structure:

preferably, the nanoparticles further comprise water, a buffer solution and/or perfluoro-n-pentane.

Preferably, the nanoparticles encapsulate water, buffer solution and/or perfluoro-n-pentane.

Preferably, the lipid bilayer of the liposome encapsulates water, buffer solution and/or perfluoro-n-pentane.

Preferably, the buffer solution comprises a phosphate buffer containing glycerol.

Preferably, the glycerol-containing phosphate buffer has a glycerol volume fraction of 5-15%, preferably 8-12%, more preferably 10%.

Preferably, the phosphate buffer is present in a concentration of 5 to 15mM, preferably 8 to 12mM, more preferably 10mM, in terms of phosphate concentration.

Preferably, the phosphate buffer containing glycerol has a pH of 7.2 to 7.6, preferably 7.4.

Preferably, the lipid bilayer encapsulates perfluoron-pentane and/or phosphate buffer containing glycerol.

Preferably, the drug-loaded nanoparticles have an encapsulation efficiency of 90% or more, preferably 95% or more, preferably 99% or more, most preferably 100%.

Preferably, the drug-loaded nanoparticles have a drug loading of 8-15wt%, preferably 9-11wt%.

In a second aspect of the present invention, there is provided a method of preparing a nanoparticle according to the first aspect of the present invention, said method comprising the steps of:

(1) Dissolving the nano material in an organic solvent, and removing the organic solvent to obtain a nano particle film;

(2) And (3) soaking perfluoro-n-pentane into the nanoparticle membrane, adding a buffer solution to hydrate the nanoparticle membrane, and stirring to obtain the nanoparticles.

Preferably, the nanoparticle is a drug-loaded nanoparticle, and the preparation method of the drug-loaded nanoparticle comprises the following steps:

(1) Dissolving the nano material and the medicine in an organic solvent, and removing the organic solvent to obtain a nano particle membrane;

Preferably, the nanoparticle is a liposome, and the preparation method of the liposome comprises the following steps:

(1) Dissolving lipid material in organic solvent, and removing organic solvent to obtain lipid membrane;

(2) And (3) soaking perfluoro-n-pentane into the lipid membrane, adding a buffer solution to hydrate the lipid membrane, and stirring to obtain the liposome.

Preferably, the nanoparticle is a drug-loaded liposome, and the preparation method of the drug-loaded liposome comprises the following steps:

(1) Dissolving lipid material and medicine in organic solvent, and removing organic solvent to obtain lipid membrane;

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

Preferably, in the step (1), the weight to volume ratio (mg: ml) of the DPPC to the organic solvent is 1.

Preferably, the volume ratio of the perfluoro-n-pentane to the buffer solution is 1.

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

preferably, in the step (1), the organic solvent is removed by rotary reduced pressure evaporation.

Preferably, in the step (1), the organic solvent is removed by rotary evaporation at 35-40 ℃ under reduced pressure.

Preferably, in the step (2), perfluoro-n-pentane is impregnated into the lipid membrane at a low temperature.

Preferably, in the step (2), the hydration is performed at a low temperature.

Preferably, in the step (2), the stirring comprises the steps of:

stirring is carried out at low temperature and then at elevated temperature.

Preferably, the low temperature is 2-10 ℃, preferably 2-6 ℃, and most preferably 4 ℃.

Preferably, the stirring time at low temperature is 0.2 to 0.8h, preferably 0.4 to 0.6h, more preferably 0.5h.

Preferably, the elevated temperature is from 20 to 40 ℃, preferably from 25 to 35 ℃, more preferably from 28 to 32 ℃.

Preferably, the stirring time after the temperature is raised is 0.5 to 1.5 hours, preferably 0.8 to 1.2 hours, more preferably 1 hour.

Preferably, the agitation comprises stirring while the vessel is open.

Preferably, the agitation removes unencapsulated perfluoro-n-pentane.

Preferably, the stirring comprises magnetic stir bar stirring.

Preferably, in the stirring after the temperature is raised, the container for placing the stirring liquid is in an open state.

Preferably, the elevated temperature is followed by agitation to remove any unencapsulated perfluoro-n-pentane.

Preferably, the liposomes are in the form of liposomal nanodroplets.

Preferably, the preparation method of the liposome comprises the steps of:

(1) Dissolving DPPC and DSPE-PEG in organic solvent in a round-bottom flask, removing the organic solvent by rotary reduced pressure evaporation, and forming a lipid membrane in the round-bottom flask;

(2) Cooling the lipid membrane to low temperature, adding perfluoro-n-pentane to immerse the lipid membrane, adding buffer solution to hydrate, stirring at 2-6 deg.C for 0.2-0.8h, and stirring in round-bottomed flask at 25-35 deg.C for 0.8-1.2h to obtain liposome.

Preferably, the preparation method of the liposome comprises the steps of:

(1) Dissolving 2.8-3.2mg of DPPC and 1.8-2.2mg of DSPE-PEG in the organic solvent in the round-bottomed flask, and evaporating under reduced pressure to remove the organic solvent to form a lipid membrane in the round-bottomed flask;

(2) Cooling the lipid membrane to 2-6 ℃, adding 90-110 mu L of perfluoro-n-pentane to immerse the lipid membrane, then adding 4.5-5.5mL of buffer solution for hydration, stirring for 0.3-0.7h at 2-6 ℃, then stirring for 0.8-1.2h in a water bath kettle at 28-32 ℃ under the open condition in a round bottom flask to obtain the liposome.

Preferably, the preparation method of the drug-loaded liposome comprises the steps of:

(1) Dissolving DPPC, DSPE-PEG and the drug in an organic solvent in a round-bottom flask, and performing rotary reduced pressure evaporation to remove the organic solvent to form a lipid membrane in the round-bottom flask;

(2) Cooling the lipid membrane to low temperature, adding perfluoro-n-pentane to immerse the lipid membrane, adding buffer solution to hydrate, stirring at 2-6 deg.C for 0.2-0.8h, and stirring in round-bottomed flask at 25-35 deg.C for 0.8-1.2h to obtain drug-loaded liposome.

(1) Dissolving 2.8-3.2mg of DPPC, 1.8-2.2mg of DSPE-PEG and 1.8-2.2mg of drug in an organic solvent in a round-bottomed flask, and evaporating under reduced pressure to remove the organic solvent to form a lipid film in the round-bottomed flask;

(2) Cooling the lipid membrane to 2-6 ℃, adding 90-110 mu L of perfluoro-n-pentane to immerse the lipid membrane, then adding 4.5-5.5mL of buffer solution for hydration, stirring for 0.3-0.7h at 2-6 ℃, then stirring for 0.8-1.2h in a water bath kettle at 28-32 ℃ under the open condition in a round bottom flask to obtain the drug-loaded liposome.

In a third aspect of the invention, there is provided a ligand-modified nanoparticle comprising a nanoparticle according to the first aspect of the invention; and a ligand.

Preferably, the ligand-modified nanoparticle comprises a ligand-modified nanoparticle or a ligand-modified liposome.

Preferably, the ligand comprises a targeting ligand.

Preferably, the surface of the nanoparticle contains a ligand.

Preferably, said surface comprises an outer surface.

Preferably, the surface of the nanoparticle comprises the outer surface of the nanoparticle.

Preferably, the outer surface of the nanoparticle contains a ligand.

Preferably, the ligand is modified on the nanomaterial.

Preferably, the ligand is modified on the nanomaterial of the nanoparticle.

Preferably, the ligand is modified on the surface of the nanoparticle.

Preferably, the modification comprises a physical modification and/or a chemical modification.

Preferably, the modification comprises physisorption, chemisorption and/or coupling.

Preferably, the ligand is adsorbed on the surface of the nanoparticle.

Preferably, the adsorption comprises physisorption and/or chemisorption.

Preferably, the ligand is coupled to the nanomaterial on the surface of the nanoparticle.

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

Preferably, the ligand comprises a ligand that targets tumor vascular cells and/or tumor cells.

Preferably, the ligand comprises a polypeptide or protein ligand.

Preferably, the ligand comprises an RGD polypeptide and/or an NGR polypeptide.

Preferably, the lipid material comprises a ligand-modified lipid material.

Preferably, the ligand is coupled to the nanomaterial of the nanoparticle.

Preferably, the ligand is coupled to a lipid material.

Preferably, said coupling comprises chemical coupling.

Preferably, the ligand is coupled to distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) to form a DSPE-PEG-ligand.

Preferably, the ligand is coupled to a portion of the nanomaterial (e.g., lipid material).

Preferably, the DSPE-PEG-ligand comprises DSPE-PEG-RGD and/or DSPE-PEG-NGR.

Preferably, the ligand is coupled to the lipid material and comprises DSPE-PEG-RGD and/or DSPE-PEG-NGR.

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 combinations thereof.

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 their combination.

Preferably, the DSPE-PEG-ligand is present in an amount of 1 to 10 parts by weight, preferably 2 to 8 parts by weight, more preferably 4 to 6 parts by weight, most preferably 3 parts by weight.

Preferably, the weight ratio of DSPE-PEG-ligand to DPPC is 1.

Preferably, the ligand-modified nanoparticle has a particle size of 120-260nm, preferably 160-210nm, more preferably 170-200nm, more preferably 180-200nm.

Preferably, the ligand-modified nanoparticle has a potential of-2 to-18 mV, preferably-2 to-15 mV, more preferably-5 to-12 mV.

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

Preferably, the ligand comprises a ligand that targets a tumor vascular cell surface receptor.

Preferably, the ligand comprises a ligand that targets a tumor cell surface receptor.

Preferably, said surface comprises an outer surface.

Preferably, the cell surface receptor comprises a cell membrane surface receptor.

Preferably, the cell surface receptor comprises an outer cell membrane surface receptor.

Preferably, the receptor comprises a protein receptor, a lipoprotein receptor or a glycoprotein receptor.

Preferably, the ligand comprises a ligand that mediates cellular uptake of the ligand-modified nanoparticle.

Preferably, the ligand comprises a ligand that mediates endocytosis of the ligand-modified nanoparticle by a cell.

Preferably, the ligand comprises a ligand that mediates endocytosis and exocytosis of the ligand-modified nanoparticle by a cell.

Preferably, the ligand is capable of mediating ligand-modified nanoparticle penetration from the tumor vasculature to the tumor site.

Preferably, the ligand is capable of mediating ligand-modified nanoparticle penetration from the tumor vasculature to the tumor site by endocytosis and exocytosis.

Preferably, the ligand targets vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of the ligand-modified nanoparticle by vascular endothelial cells.

Preferably, the ligand is capable of mediating endocytosis, post-endocytosis and exocytosis of the ligand-modified nanoparticle in blood by tumor vasculature (e.g., tumor tissue microenvironment).

In a fourth aspect of the invention, there is provided a method of preparing a ligand-modified nanoparticle according to the third aspect of the invention, the method comprising:

modifying said ligand on said nanoparticle to obtain a ligand-modified nanoparticle.

Preferably, the method for preparing the ligand-modified nanoparticle comprises:

(1) Dissolving the ligand modified nano material in an organic solvent, and removing the organic solvent to obtain a nano particle membrane;

(2) And (3) soaking perfluoro-n-pentane into the nanoparticle membrane, adding a buffer solution to hydrate the nanoparticle membrane, and stirring to obtain the ligand-modified nanoparticle.

Preferably, the preparation method of the ligand-modified nanoparticle comprises:

(1) Dissolving the nano material containing ligand modification and the medicine in an organic solvent, and removing the organic solvent to obtain a nano particle membrane;

Preferably, the ligand modification-containing nanomaterial comprises one or more of DPPC, DSPE-PEG and DSPE-PEG-ligand.

Preferably, the ligand-modified nanomaterial comprises one or more of DPPC, DSPE-PEG-RGD and DSPE-PEG-NGR.

Preferably, the method for preparing the ligand-modified liposome comprises the steps of:

(1) Dissolving DPPC, DSPE-PEG-ligand and DSPE-PEG in organic solvent in a round-bottom flask, performing rotary reduced pressure evaporation to remove the organic solvent, and forming a lipid membrane in the round-bottom flask;

(2) Cooling the lipid membrane to low temperature, adding perfluoro-n-pentane to immerse the lipid membrane, adding a buffer solution to hydrate, stirring at 2-6 ℃ for 0.2-0.8h, and stirring in a round-bottom flask at 25-35 ℃ for 0.8-1.2h to obtain the ligand-modified liposome.

(1) Dissolving 2.8-3.2mg DPPC, 2.8-3.2mg DSPE-PEG-ligand and 1.8-2.2mg DSPE-PEG in organic solvent in round-bottomed flask, evaporating under reduced pressure to remove organic solvent to form lipid membrane in round-bottomed flask;

(2) Cooling the lipid membrane to 2-6 ℃, adding 90-110 mu L of perfluoro-n-pentane to immerse the lipid membrane, then adding 4.5-5.5mL of buffer solution for hydration, stirring at 2-6 ℃, then stirring in a round bottom flask under an open condition in a water bath kettle at 28-32 ℃ to obtain the ligand modified liposome.

(1) Dissolving DPPC, DSPE-PEG-ligand, DSPE-PEG and drug in organic solvent in round-bottom flask, rotary vacuum evaporating to remove organic solvent to form lipid membrane in round-bottom flask;

(1) Dissolving 2.8-3.2mg DPPC, 2.8-3.2mg DSPE-PEG-ligand, 1.8-2.2mg DSPE-PEG and 1.8-2.2mg drug in organic solvent in round-bottomed flask, evaporating under rotary reduced pressure to remove organic solvent, and forming lipid membrane in round-bottomed flask;

Preferably, step (1) is as described above in relation to the second aspect of the invention.

Preferably, step (2) is as described above in relation to the second aspect of the invention.

In a fifth aspect of the invention, there is provided a composition comprising a nanoparticle according to the first aspect of the invention and/or a ligand-modified nanoparticle according to the third aspect of the invention.

Preferably, the composition is a pharmaceutical composition.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

Preferably, the composition is a solid, liquid or semi-solid formulation.

Preferably, the composition is an injection preparation, an oral preparation or an external preparation.

Preferably, the injection preparation is an intravascular injection preparation.

Preferably, the injection preparation is intravenous injection preparation, arterial injection preparation, intratumoral injection preparation or tumor microenvironment injection preparation.

Preferably, the intravenous formulation is an upper extremity intravenous formulation or a lower extremity intravenous formulation.

In a sixth aspect of the invention, there is provided a use of a nanoparticle according to the first aspect of the invention and/or a ligand-modified nanoparticle according to the third aspect of the invention for the preparation of a composition for the prevention and/or treatment of a disease.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the disease is an indication disease of the drug.

Preferably, the drug comprises an anti-cancer drug.

Preferably, the medicament is as described in the first aspect of the invention.

Preferably, the disease comprises a tumor.

Preferably, the tumor comprises a human tumor (e.g., a human tumor) or a non-human mammalian tumor.

Preferably, the tumor comprises a low permeability tumor.

Preferably, the tumor comprises a tumor with low permeability of tumor vessels.

Preferably, the tumor comprises a solid tumor.

Preferably, the tumor comprises a solid tumor of low tumor vascular permeability.

Preferably, the tumor comprises liver cancer.

Preferably, the tumor comprises human liver cancer.

Preferably, the cancer cells of liver cancer include Huh7 cells and/or HepG2 cells.

Preferably, the tumor comprises pancreatic cancer.

Preferably, the pancreatic cancer comprises pancreatic adenocarcinoma.

Preferably, the pancreatic cancer comprises an in situ pancreatic cancer.

Preferably, the pancreatic cancer comprises orthotopic pancreatic adenocarcinoma.

Preferably, the pancreatic cancer comprises pancreatic ductal adenocarcinoma.

Preferably, the pancreatic cancer comprises human pancreatic ductal adenocarcinoma.

Preferably, the cancer cells of pancreatic cancer include BxPC-3 cells.

Preferably, the tumor comprises a tumor with poor tumor Permeability and Retention (EPR effect).

Preferably, said low permeability of tumor vessels comprises low drug penetration from tumor vessels to the tumor site.

Preferably, said low permeability of tumor vasculature comprises low drug penetration from the tumor vascular intercellular space to the tumor site.

Preferably, said tumor vessels of low tumor vascular permeability comprise one or more characteristics selected from the group consisting of:

(a) The tumor vascular cell tissues are well and closely packed; and/or

(b) The tumor has small vascular intercellular spaces.

Preferably, the vascular cells comprise vascular endothelial cells.

Preferably, the composition is a pharmaceutical composition.

Preferably, the composition is in the form of a solid, liquid or semisolid preparation.

Preferably, the composition is in the form of injection, oral preparation or external preparation.

Preferably, the intravenous formulation is an upper limb intravenous formulation or a lower limb intravenous formulation.

Preferably, said treatment comprises inhibition, alleviation, remission, reversal or eradication.

In a seventh aspect of the invention, there is provided a system or device for treating a disease, said system or device comprising a nanoparticle according to the first aspect of the invention and/or a ligand-modified nanoparticle according to the third aspect of the invention; and an ultrasonic device.

Preferably, the system or apparatus further comprises instructions or labels describing:

in the treatment of a disease by administering to a subject in need thereof nanoparticles according to the first aspect of the invention and/or ligand-modified nanoparticles according to the third aspect of the invention, the focal site (e.g. a tumour site) is subjected to ultrasound irradiation.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the ultrasound device comprises an ultrasound apparatus.

Preferably, the subject comprises a human or non-human mammal.

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

Preferably, the disease is an indication disease of a drug.

Preferably, the drug comprises an anti-cancer drug.

Preferably, the disease comprises a tumor.

Preferably, the tumour is as described in the sixth aspect of the invention.

Preferably, the administration is injection, oral or topical.

Preferably, the administration by injection is intravenous administration, arterial administration, intratumoral administration or intratumoral administration.

Preferably, the injection administration is intravascular injection administration.

Preferably, the intravenous administration is intravenous administration to the upper limb or intravenous administration to the lower limb.

In an eighth aspect of the present invention, there is provided a method for preventing and/or treating a disease by administering the nanoparticle according to the first aspect of the present invention and/or the ligand-modified nanoparticle according to the third aspect of the present invention to a subject in need thereof, thereby preventing and/or treating a disease.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the subject comprises a human or non-human mammal.

Preferably, the disease is an indication disease of a drug.

Preferably, the disease comprises a tumor.

Preferably, the drug comprises an anti-cancer drug.

Preferably, the tumour is as described in the sixth aspect of the invention.

Preferably, the focal site (e.g. tumor site) is subjected to ultrasound irradiation after administration of the nanoparticles according to the first aspect of the invention and/or the ligand-modified nanoparticles according to the third aspect of the invention to a subject in need thereof.

Preferably, the administration is injection, oral or topical.

Preferably, the injection administration is intravascular administration.

In a ninth aspect of the invention, there is provided a corona protein modified nanoparticle comprising a nanoparticle according to the first aspect of the invention and/or a ligand modified nanoparticle according to the third aspect of the invention; and a protein corona.

Preferably, the nanoparticles are loaded with perfluoro-n-pentane.

Preferably, the protein of the protein corona comprises serum protein, plasma protein and/or tissue protein.

Preferably, the serum, plasma and/or tissue proteins comprise human or non-human mammalian serum, plasma and/or tissue proteins.

Preferably, the serum, plasma and/or tissue proteins comprise serum, plasma and/or tissue proteins isolated or isolated ex vivo.

Preferably, the bovine comprises fetal bovine.

Preferably, the serum and/or plasma comprises fetal bovine serum and/or fetal bovine plasma.

Preferably, the protein corona modified nanoparticle comprises an isolated or ex vivo protein corona modified nanoparticle.

Preferably, said protein corona modification is on a nanoparticle according to the first aspect of the invention and/or a ligand-modified nanoparticle according to the third aspect of the invention.

Preferably, the protein corona is modified at the surface of the nanoparticle according to the first aspect of the invention and/or the ligand-modified nanoparticle according to the third aspect of the invention.

Preferably, said surface comprises an outer surface.

Preferably, the modification comprises adsorption.

In a tenth aspect of the present invention, there is provided a method for preparing a protein corona-modified nanoparticle according to the ninth aspect of the present invention, the method comprising the steps of:

incubating the nanoparticle according to the first aspect of the invention and/or the ligand-modified nanoparticle according to the third aspect of the invention with a protein, isolating the protein corona-modified nanoparticle,

preferably, the method is an in vitro method or an in vivo method.

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

Preferably, the incubation is in vitro or in vivo.

Preferably, said incubation comprises incubation in conditions comprising a protein.

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

Preferably, said incubation comprises incubation in a culture medium.

Preferably, the culture medium comprises a liquid culture medium.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the medium comprises a protein.

Preferably, the protein comprises serum protein, plasma protein and/or tissue protein.

Preferably, the culture medium comprises serum proteins, plasma proteins and/or tissue proteins.

Preferably, the culture medium comprises a serum, plasma or and/or tissue protein containing medium.

Preferably, the medium comprises a serum-containing medium.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

Preferably, the serum is present in the serum-containing medium in a volume fraction of 5-15%, preferably 8-12%, more preferably 10%.

Preferably, said culturing comprises in vitro culturing.

Preferably, said incubation comprises incubation in blood, serum or plasma.

Preferably, the blood, serum or plasma comprises ex vivo or isolated blood, serum or plasma.

Preferably, the blood, serum or plasma comprises human or non-human mammalian blood, serum or plasma.

Preferably, the bovine comprises fetal bovine.

Preferably, the incubation time is 0.25-6h, preferably 0.25-4h, more preferably 0.25-2h, preferably 0.25-1h, preferably 0.25-0.5h, e.g.0.5-1 h.

Preferably, the separation comprises gel chromatography.

Preferably, the gel comprises a dextran gel.

Preferably, the separation comprises size exclusion chromatography.

In an eleventh aspect of the present invention, there is provided a method for eliminating a protein corona from a protein corona-modified nanoparticle, the method comprising the steps of:

and carrying out ultrasonic irradiation treatment on the nanoparticle modified by the protein corona so as to eliminate the protein corona of the nanoparticle modified by the protein corona.

Preferably, the nanoparticles are loaded with perfluoro-n-pentane.

Preferably, the method comprises an in vitro method or an in vivo method.

Preferably, the method comprises a non-therapeutic and/or non-diagnostic method.

Preferably, the protein corona modified nanoparticle is as described in the ninth aspect of the invention.

Preferably, said elimination comprises reduction or removal.

Preferably, said reduction comprises a reduction in protein content.

Preferably, the protein corona-modified nanoparticle comprises a protein corona-modified nanoparticle in a protein-free condition.

Preferably, the protein-free conditions include saline, PBS buffer, or serum-free medium.

Preferably, said protein corona-modified nanoparticle comprises a protein corona-modified nanoparticle in a protein-containing condition.

Preferably, the protein corona modified nanoparticle comprises a protein corona modified nanoparticle in blood, serum or plasma.

Preferably, the protein corona-modified nanoparticle comprises a protein corona-modified nanoparticle in a culture medium.

Preferably, the bovine comprises fetal bovine.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

Preferably, the culture medium comprises a liquid culture medium.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the culture medium comprises a protein-containing medium.

Preferably, the medium contains a protein.

Preferably, the culture medium comprises a medium comprising serum, plasma and/or tissue proteins.

Preferably, the medium comprises a serum-containing medium.

Preferably, the cells comprise tumor cells and/or tumor vascular cells.

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

Preferably, the cells include cells that need to be cultured or grown in conditions containing the protein.

Preferably, the cells include cells that require culture or growth in a serum-containing medium.

Preferably, the cells include cells that require culturing in a serum-containing medium.

Preferably, said culturing comprises in vitro culturing.

Preferably, the sound intensity (acoustic intensity) of the ultrasonic irradiation is 0.1-40W/cm ² Preferably 0.1-20W/cm ² More preferably 0.2 to 15W/cm ² More preferably 0.5-10W/cm ² More preferably 1 to 5W/cm ² More preferably 1 to 3W/cm ² Most preferably 1.5-2.5W/cm ² More preferably 1.8-2.2W/cm ² More preferably 2.0W/cm ² 。

Preferably, the frequency (frequency) of the ultrasonic irradiation is 0.02 to 30MHz, preferably 0.1 to 20MHz, more preferably 0.2 to 15MHz, more preferably 0.5 to 10MHz, more preferably 1 to 8MHz, more preferably 1 to 5MHz, most preferably 2 to 4MHz, more preferably 2.5 to 3.5MHz, more preferably 2.8 to 3.2MHz, more preferably 3MHz.

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

Preferably, the time of the ultrasonic irradiation is more than 2min, preferably more than 5min, preferably more than 10min, preferably more than 15min, for example 15-20min.

In a twelfth aspect of the invention, there is provided a method of screening for or identifying potential ligands that target cells or cell surface receptors, said method comprising the steps of:

(I) Modifying a ligand on the nanoparticle to obtain a ligand-modified nanoparticle;

(II) incubating the cell or cell surface receptor with the ligand-modified nanoparticle of step (I), performing a sonication treatment, and determining the binding of the ligand-modified nanoparticle of step (I) or the ligand-modified nanoparticle 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.

Preferably, the nanoparticles are loaded with perfluoro-n-pentane.

Preferably, the nanoparticles are as described in the first aspect of the invention.

Preferably, the ligand-modified nanoparticle of step (I) is a ligand-modified nanoparticle according to the third aspect of the invention.

Preferably, the ligand of step (I) comprises a ligand to be tested.

Preferably, the step (II) includes:

(II) incubating the cell or cell surface receptor with the ligand-modified nanoparticle of step (I), performing a sonication treatment, and determining whether the ligand of the ligand-modified nanoparticle of step (I) or the ligand-modified nanoparticle of step (I) binds 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.

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

Preferably, in step (II), if the ligand of the ligand-modified nanoparticle or ligand-modified nanoparticle does not bind to a cell or cell surface receptor, the ligand of step (I) is not a potential ligand for targeting a cell or cell surface receptor.

Preferably, the method further comprises providing a control group comprising nanoparticles without ligand modification, and determining binding of the nanoparticles without ligand modification to the cell or cell surface receptor.

Preferably, the method further comprises providing a control comprising nanoparticles without ligand modification and otherwise identical to the ligand-modified nanoparticles, and determining binding of the nanoparticles without ligand modification to the cells or cell surface receptors.

Preferably, if the binding force B1 of the ligand-modified nanoparticle or ligand-modified nanoparticle to a cell or cell surface receptor is greater than the binding force B0 of the nanoparticle without ligand modification to a cell or cell surface receptor, it indicates that the ligand of step (I) is a potential ligand for targeting a cell or cell surface receptor.

Preferably, the step (II) includes:

(II-1) in the test group, incubating cells or cell surface receptors with the ligand-modified nanoparticles of step (I), performing ultrasonic irradiation treatment, and determining the binding force B1 of the ligand-modified nanoparticles or ligand-modified nanoparticles of step (I) to the cells or cell surface receptors; setting a control group, wherein the control group comprises the nanoparticles without ligand modification and other determination conditions are the same as those of the test group, and determining the binding force B0 of the nanoparticles without ligand modification and cells or cell surface receptors;

(II-2) if the binding force B1 of the ligand-modified nanoparticle or ligand-modified nanoparticle of step (I) to a cell or cell surface receptor is greater than the binding force B0 of the nanoparticle without ligand modification to a cell or cell surface receptor, it indicates that the ligand of step (I) is a potential ligand for targeting a cell or cell surface receptor.

Preferably, if the binding force B1 of the ligand-modified nanoparticle or ligand-modified nanoparticle of step (I) to a cell or cell surface receptor is similar to the binding force B0 of the nanoparticle without ligand modification to a cell or cell surface receptor, it indicates that the ligand of step (I) is not a potential ligand for targeting a cell or cell surface receptor.

Preferably, the targeting comprises specific targeting or non-specific targeting.

Preferably, the term "greater than" includes significantly greater than.

Preferably, the term "greater than" includes significantly greater than and statistically significant.

Preferably, the term "greater than" refers to the ratio (B1/B0) of the binding force B1 of the ligand-modified nanoparticle or ligand-modified nanoparticle to the cell or cell surface receptor to the binding force B0 of the nanoparticle without ligand modification to the cell or cell surface receptor is greater than 1.0, preferably greater than or equal to 1.2, more preferably greater than or equal to 1.5, more preferably greater than or equal to 2, more preferably greater than or equal to 3, more preferably greater than or equal to 5, more preferably greater than or equal to 10, more preferably greater than or equal to 15, more preferably greater than or equal to 20, more preferably greater than or equal to 30, more preferably greater than or equal to 50, more preferably greater than or equal to 80, more preferably greater than or equal to 100, more preferably greater than or equal to 80, more preferably greater than or equal to 150, more preferably greater than or equal to 200, more preferably greater than or equal to 500, more preferably greater than or equal to 1000, more preferably greater than or equal to 5000, more preferably greater than or equal to 10000.

Preferably, B1/B0 is 1.5 to 10000, preferably 2 to 500, more preferably 2 to 200, more preferably 2 to 100, more preferably 2 to 50, more preferably 5 to 30.

Preferably, the term "greater than" means that the binding force B1 of the ligand-modified nanoparticles or ligand-modified nanoparticles in the test group with biological repeats to the cell or cell surface receptor is greater than the binding force B0 of the nanoparticles without ligand modification to the cell or cell surface receptor in the control group with biological repeats, and the P value thereof is less than 0.05 by t-test.

Preferably, the ligand comprises a polypeptide or protein ligand.

Preferably, said binding comprises affinity.

Preferably, the binding capacity includes affinity.

Preferably, the ligand comprises a latent ligand.

Preferably, the ligand-modified nanoparticle comprises an ex vivo or isolated ligand-modified nanoparticle.

Preferably, the cell or cell surface receptor comprises an ex vivo or isolated cell or cell surface receptor.

Preferably, the method comprises an in vitro method or an in vivo method.

Preferably, the incubation is in vitro or in vivo.

Preferably, the body comprises the body of a human or non-human mammal.

Preferably, the incubation comprises incubation in conditions comprising a protein.

Preferably, said incubation comprises incubation of the cells or cell surface receptors with the ligand-modified nanoparticles of step (I) in protein-containing conditions.

Preferably, the conditions comprise in vivo conditions or in vitro conditions.

Preferably, the cells include cells that require culturing or growth in conditions containing a protein.

Preferably, said culturing comprises in vitro culturing.

Preferably, the protein-containing conditions include blood, serum, plasma, cellular tissue microenvironment, or culture medium.

Preferably, the serum, plasma and/or tissue proteins comprise serum, plasma and/or tissue proteins ex vivo or isolated.

Preferably, the cattle are fetal cattle.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

Preferably, the culture medium comprises a liquid culture medium.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the culture medium comprises a protein-containing medium.

Preferably, the medium contains a protein.

Preferably, the medium comprises a serum-containing medium.

Preferably, said incubation comprises incubation of cells or cell surface receptors with the ligand-modified nanoparticles of step (I) in a medium containing serum, plasma and/or tissue proteins.

Preferably, said incubation comprises incubation of the cells or cell surface receptors with the ligand-modified nanoparticles of step (I) in a serum-containing medium.

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

Preferably, the cells comprise tumor cells and/or tumor vascular cells.

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor comprises a low permeability tumor.

Preferably, the tumor comprises a tumor with low permeability of tumor vessels.

Preferably, the tumor comprises a solid tumor.

Preferably, the tumor comprises a solid tumor of low permeability of tumor vessels.

Preferably, the tumor comprises liver cancer.

Preferably, the tumor comprises human liver cancer.

Preferably, the tumor comprises pancreatic cancer.

Preferably, the pancreatic cancer comprises pancreatic adenocarcinoma.

Preferably, the pancreatic cancer comprises an in situ pancreatic cancer.

Preferably, the pancreatic cancer comprises an orthotopic pancreatic adenocarcinoma.

Preferably, the pancreatic cancer comprises pancreatic ductal adenocarcinoma.

Preferably, the cancer cells of pancreatic cancer include BxPC-3 cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

Preferably, said low tumor vascular permeability comprises low drug penetration from the tumor vascular intercellular spaces to the tumor site.

Preferably, said tumor vasculature of low tumor vasculature permeability comprises one or more characteristics selected from the group consisting of:

(a) The tumor vascular cells are well organized and closely packed; and/or

(b) The tumor has small vascular intercellular spaces.

Preferably, the vascular cells comprise vascular endothelial cells.

Preferably, the ligand is capable of binding to a cell or cell surface receptor.

Preferably, the binding comprises specific binding or non-specific binding.

Preferably, the receptor comprises a receptor on the outer surface of a cell membrane.

Preferably, the bound assay comprises an isotope lost assay, a fluorescein assay, a flow cytometry assay and/or a transwell migration assay.

Preferably, said nanoparticle and/or said ligand is labelled with an isotope and/or fluorescein.

Preferably, the Fluorescein comprises FITC (Fluorescein isothiocyanate isomer), cyanine 5 (Cy 5) and/or Cyanine 5.5 (Cy5.5).

Preferably, the binding mediates uptake of the ligand-modified nanoparticle by the cell.

Preferably, the binding mediates endocytosis of the ligand-modified nanoparticle by the cell.

Preferably, the binding mediates endocytosis and exocytosis of the ligand-modified nanoparticle by the cell.

Preferably, the method of measuring binding comprises measuring the uptake efficiency of the ligand-modified nanoparticle of step (I) by the cell.

Preferably, the cells do not have uptake capacity for nanoparticles in the control that are not modified with a ligand.

Preferably, said screening or identifying whether the ligand of step (I) is a potential ligand for targeting a cell or cell surface receptor comprises:

(ii) indicating that the ligand of step (I) is a potential ligand for targeting a cell or cell surface receptor if the cellular uptake efficiency of the ligand-modified nanoparticle of step (I) is greater than the cellular uptake efficiency of the nanoparticle without ligand modification in the control group.

Preferably, the method of measuring binding comprises measuring the endocytosis capacity of the cells to the ligand-modified nanoparticle of step (I).

Preferably, the cells are not endocytosed with respect to the nanoparticles in the control group that are not modified with the ligand.

if the endocytosis capacity of the cell to the ligand-modified nanoparticle of step (I) is greater than the endocytosis capacity of the cell to the nanoparticle without ligand modification in the control group, then the ligand of step (I) is a potential ligand for targeting a cell or cell surface receptor.

Preferably, the method of measuring binding comprises measuring the endocytosis and exocytosis of the ligand-modified nanoparticle of step (I) by a cell.

Preferably, the cells do not have endocytosis and exocytosis capacity for the nanoparticles without ligand modification in the control group.

if the endocytosis and exocytosis capacity of the cell to the ligand-modified nanoparticle of step (I) is greater than the endocytosis and exocytosis capacity of the cell to the nanoparticle without ligand modification in the control group, the ligand of step (I) is a potential ligand for targeting a cell or cell surface receptor.

Preferably, the ligand of step (I) comprises a ligand that mediates endocytosis of the ligand-modified nanoparticle by a cell.

Preferably, the ligand of the ligand-modified nanoparticle or the ligand-modified nanoparticle is capable of mediating endocytosis of the ligand-modified nanoparticle by a cell after binding to the cell or a cell surface receptor.

after the ligand of the ligand-modified nanoparticle or the ligand-modified nanoparticle is bound to a cell or a cell surface receptor, the ligand can mediate the cell to perform endocytosis on the ligand-modified nanoparticle, and the ligand in the step (I) is a potential ligand targeting the cell or the cell surface receptor.

Preferably, the ligand of step (I) comprises a ligand that mediates endocytosis and exocytosis of the ligand-modified nanoparticle by a cell.

Preferably, the ligand of the ligand-modified nanoparticle or the ligand-modified nanoparticle is capable of mediating endocytosis and exocytosis of the ligand-modified nanoparticle by a cell after binding to a cell or a cell surface receptor.

After the ligand of the ligand-modified nanoparticle or the ligand-modified nanoparticle is bound to a cell or a cell surface receptor, the ligand can mediate the cell to perform endocytosis and exocytosis on the ligand-modified nanoparticle, and the ligand in the step (I) is a potential ligand targeting the cell or the cell surface receptor.

Preferably, after the ligand of the ligand-modified nanoparticle or the ligand-modified nanoparticle is bound to the tumor vascular cell or a receptor on the surface of the tumor vascular cell, the ligand-modified nanoparticle in blood can be mediated by the tumor vascular cell to perform endocytosis, endocytosis and exocytosis to the outside of the tumor vascular cell (such as a tumor tissue microenvironment).

after the ligand of the ligand-modified nanoparticle or the ligand of the ligand-modified nanoparticle is combined with a tumor vascular cell or a tumor vascular cell surface receptor, the ligand-modified nanoparticle in blood can be mediated by the tumor vascular cell to carry out endocytosis on the ligand-modified nanoparticle in blood, and the ligand-modified nanoparticle can be exocytosid to the outside of the tumor vascular cell (such as a tumor tissue microenvironment), so that the ligand in the step (I) is a potential ligand of a target cell or a cell surface receptor.

Preferably, the method step of determining the binding or binding force of the ligand-modified nanoparticle or ligand-modified nanoparticle of step (I) to a cell or cell surface receptor comprises:

the ligand can bind to a cell or a cell surface receptor if the tumor vasculature cells are capable of endocytosing, and exocytosis the blood ligand-modified nanoparticle outside the tumor vasculature (e.g., tumor tissue microenvironment).

Preferably, the tumor vascular cells are incapable of endocytosis of the nanoparticle in blood circulation without ligand modification.

Preferably, said surface comprises an outer surface.

Preferably, the ligand targets vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of the ligand-modified nanoparticle by the vascular endothelial cells.

Preferably, the ligand comprises a ligand that mediates uptake of the ligand-modified nanoparticle by a cell.

Preferably, the sound intensity (acoustic intensity) of the ultrasonic irradiation is 0.1-40W/cm ² Preferably 0.1-20W/cm ² More preferably 0.2 to 15W/cm ² More preferably 0.5 to 10W/cm ² More preferably 1 to 5W/cm ² More preferably 1 to 3W/cm ² Most preferably 1.5-2.5W/cm ² More preferably 1.8 to 2.2W/cm ² More preferably 2.0W/cm ² 。

Preferably, the frequency (frequency) of the ultrasonic irradiation is from 0.02 to 30MHz, preferably from 0.1 to 20MHz, more preferably from 0.2 to 15MHz, more preferably from 0.5 to 10MHz, more preferably from 1 to 8MHz, more preferably from 1 to 5MHz, more preferably from 2 to 4MHz, more preferably from 2.5 to 3.5MHz, more preferably from 2.8 to 3.2MHz, more preferably 3MHz.

In a thirteenth aspect of the invention, there is provided a use of the nanoparticle according to the first aspect of the invention for the preparation of a vector for screening or identifying potential ligands targeting cells or cell surface receptors.

Preferably, the method of screening or identifying potential ligands that target a cell or cell surface receptor comprises the steps of:

Preferably, the method of screening or identifying potential ligands that target cells or cell surface receptors is as described in the twelfth aspect of the invention.

In a fourteenth aspect of the present invention, there is provided a method of inhibiting a cell in vitro, said method comprising the steps of:

the cells are incubated with the nanoparticles according to the first aspect of the invention or the ligand-modified nanoparticles according to the third aspect of the invention in a culture medium and treated with ultrasound irradiation to inhibit the cells.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the method comprises a method of enhancing the inhibition of cells in vitro by ligand-modified nanoparticles.

Preferably, the methods include non-diagnostic and non-therapeutic methods.

Preferably, the ligand comprises an RGD polypeptide and/or an NGR polypeptide.

Preferably, the cells comprise tumor cells and/or tumor vascular cells.

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

Preferably, the drug comprises a cytostatic agent.

Preferably, the drug comprises an anti-tumor drug.

Preferably, the tumour is as described in the sixth aspect of the invention.

Preferably, the bovine comprises fetal bovine.

Preferably, the protein-containing conditions are as described in the twelfth aspect of the invention.

Preferably, the culture medium comprises a liquid culture medium.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the medium contains a protein.

Preferably, the medium comprises a serum-containing medium.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

Preferably, said culturing comprises in vitro culturing.

Preferably, the incubation is in vitro incubation.

Preferably, the nanoparticle surface contains protein corona in the culture medium under the condition of no ultrasonic irradiation.

Preferably, the protein corona is adsorbed on the surface of the nanoparticle.

Preferably, the ligand is capable of mediating uptake of the ligand-modified nanoparticle by a cell upon binding to a cell or cell surface receptor.

Preferably, the ligand is capable of mediating endocytosis of the ligand-modified nanoparticle by a cell upon binding to a cell or cell surface receptor.

Preferably, the cell surface receptor comprises a cell membrane outer surface receptor

In a fifteenth aspect of the present invention, there is provided the use of an ultrasound apparatus for the manufacture of a device for one or more uses selected from the group consisting of:

(a) Eliminating the protein corona of the protein corona-modified nanoparticle by ultrasonic irradiation;

(b) For screening or identifying potential ligands that target cells or cell surface receptors;

(c) Treatment of disease by enhancing administration of ligand-modified nanoparticles by ultrasound irradiation of lesions (e.g., tumors); and/or

(d) Improved retention and/or degradation of nanoparticles, ligand-modified nanoparticles, and/or protein corona-modified nanoparticles by cell lysosomes by ultrasound irradiation.

Preferably, the nanoparticles are loaded with perfluoro-n-pentane.

Preferably, the ligand-modified nanoparticle is as described in the third aspect of the invention.

Preferably, the method of eliminating the protein corona of the protein corona modified nanoparticle is as described in the eleventh aspect of the present invention.

Preferably, the (a) comprises eliminating the protein corona of the protein corona-modified nanoparticle by ultrasonic irradiation to improve the efficacy of the nanoparticle.

Preferably, the (a) eliminating the protein corona of the protein corona-modified nanoparticle by ultrasonic irradiation comprises:

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

Preferably, said (d) improving the retention and/or degradation of the nanoparticles, ligand-modified nanoparticles and/or protein corona-modified nanoparticles by cell lysosomes by ultrasound irradiation comprises:

after the nanoparticles, the ligand modified nanoparticles and/or the protein corona modified nanoparticles are incubated with cells, the cells are subjected to ultrasonic irradiation, so that the retention and/or degradation of the nanoparticles, the ligand modified nanoparticles and/or the protein corona modified nanoparticles by lysosomes of the cells are improved.

Preferably, the method comprises an in vitro method or an in vivo method.

Preferably, the incubation is in vivo or in vitro.

Preferably, said incubation comprises incubation in a serum-containing medium.

Preferably, the incubation is as described in the twelfth aspect of the invention.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the disease is an indication disease of a drug.

Preferably, the cells comprise tumor cells and/or tumor vascular cells.

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

Preferably, the disease comprises a tumor.

Preferably, the tumour is as described in the sixth aspect of the invention.

Preferably, the administration is injection, oral or topical.

Preferably, the improvement comprises avoidance or overcoming.

Preferably, said degradation comprises degradation by lysosomal enzymes.

In a sixteenth aspect of the invention, there is provided a use of a system or device according to the seventh aspect of the invention for the manufacture of a device for treating a disease.

Preferably, the apparatus further comprises instructions or labels which recite:

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the subject comprises a human or non-human mammal.

Preferably, the disease is an indication disease of a drug.

Preferably, the drug comprises an anti-cancer drug.

Preferably, the disease comprises a tumor.

Preferably, the tumour is as described in the sixth aspect of the invention.

Preferably, the administration is injection, oral or topical.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows LPGL liposome nanoparticles incubated in PBS 7.4 buffer, plasma, and then irradiated with ultrasound (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50 percent; time: 5 min) processed cryo-transmission electron microscope (cryo-TEM) images.

FIG. 2 shows the PGL liposome nanoparticles incubated in plasma and irradiated with ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration 5 min) frozen transmission electron micrographs before and after treatment.

FIG. 3 is a frozen transmission electron microscope image of GL, LGL and PGL liposome nanoparticle dispersions.

Fig. 4 shows the total protein concentration in the protein corona obtained after the protein corona modified PGL or LPGL liposome nanoparticles are treated with different ultrasonic irradiation times, wherein the ultrasonic irradiation conditions are as follows: sound intensity: 2W/cm ² Frequency: 3MHz, duty cycle: 50 percent.

FIG. 5 is ultrasonic irradiation for eliminating protein corona on the surface of the nanoparticles. (5A) Performing SDS-PAGE analysis on protein corona solution in liposome nanoparticles obtained by separating a Sephadex G200 chromatographic layer after GL, LGL, PGL and LPGL liposome nanoparticle and plasma incubation mixture are subjected to or without ultrasonic irradiation, wherein (-US) is a group in which the liposome nanoparticle and plasma incubation mixture is not subjected to ultrasonic irradiation treatment, and (+ US) is a group in which the liposome nanoparticle and plasma incubation mixture is subjected to ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, time: 5 min) treatment group. (5B) Incubation mixtures of GL, LGL, PGL or LPGL liposomal nanoparticles and plasma with or without ultrasound irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% duration 5 min), HPLC-MS assay on liposomal nanoparticlesTotal protein content.

Fig. 6 shows that after PGL or LPGL liposome nanoparticle and plasma incubation mixture is processed by different ultrasonic powers, HPLC-MS determines total protein content on the liposome nanoparticle, wherein ultrasonic frequency: 3MHz, duty cycle: 50%, duration: and 5min.

Figure 7 is a graph of cellular uptake, trans-endothelial cell transport, endocytic pathways, and intercellular transcytosis of liposomal nanoparticles. (7A) Cy 5-labeled GL, LGL, PGL and LPGL liposomal nanoparticles (all equivalent to fluorescent lipid 60. Mu.g/mL, 20. Mu.L) were premixed with 1mL of fresh serum-free medium or medium containing 10% FBS (fetal bovine serum), respectively, for 30min, and the resulting mixture was mixed with BxPC3 cells, with or without ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% for 5 min), continuously incubating 1h, average fluorescence intensity of Cy 5-labeled different liposome nanoparticles taken up by BxPC3 cells, wherein 'serum-free medium' represents average fluorescence intensity of Cy 5-labeled different liposome nanoparticles taken up by 1h, bxPC3 cells, continuously incubating without ultrasonic irradiation after mixing the mixture of Cy 5-labeled different liposome nanoparticles and the serum-free medium with the BxPC3 cells; "serum medium" represents the mean fluorescence intensity of Cy 5-labeled different liposome nanoparticles taken up by BxPC3 cells after mixing a mixture of Cy 5-labeled different liposome nanoparticles and a medium containing 10% fbs (fetal bovine serum) with BxPC3 cells, without ultrasonic irradiation, and continuing incubation for 1h; "serum medium + sonication" represents the mean fluorescence intensity of Cy 5-labeled different liposome nanoparticles taken up by BxPC3 cells after mixing a mixture of Cy 5-labeled different liposome nanoparticles and a medium containing 10% fbs (fetal bovine serum) with BxPC3 cells, followed by ultrasonic irradiation, and further incubation for 1h. (7B) To study the transcellular transport of liposomal nanoparticles through ECDHCC vessels, two types of transwell models were designed (modes I or II): the ultrasonic irradiation treatment is carried out in a centrifuge tube (I) or a top compartment (II). (7C) Flow cytometry assay determination of plasma or serum-free Medium Pre-incubation mixtures containing different Cy 5-labeled Liposomal nanoparticles added to Transwell containing serum-free Medium Mean Fluorescence Intensity (MFI) of Cy5 within BxPC3 cells in transwell after sonication of plasma pre-incubation mixtures containing different Cy 5-labeled liposomal nanoparticles in apical compartment and without sonication treatment under non-contact (mode I in fig. 7B) or contact (mode II in fig. 7B) conditions. (7D) After pretreatment of BxPC3 with different endocytosis inhibitors, flow cytometry was used to determine the mean fluorescence intensity of Cy5 in BxPC3 cells with or without ultrasonic irradiation treatment after incubation of the serum-containing medium or serum-free culture mixture supplemented with Cy 5-labeled LPGL liposomal nanoparticles with BxPC3 cells. (7E) Adding Cy 5-labeled LPGL liposome nanoparticle serum-containing medium or serum-free medium mixture into BxPC3 cells without pretreatment of BxPC3 cells or with exocytosis inhibitor EXO1, and mixing with BxPC3 cells without ultrasonic irradiation or ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), and at different culture time points after the addition of new BxPC3 cells, the CLSM pattern of the newly added BxPC3 cells, scale bar =50 μm, wherein "+ ultrasonic irradiation" means treatment with ultrasonic irradiation and "-ultrasonic irradiation" means no ultrasonic irradiation. (7F) The new BxPC3 cells were added to the culture dish as shown in fig (7E), and after culturing for 120min, the Mean Fluorescence Intensity (MFI) of Cy5 in the newly added BxPC3 cells was quantitatively determined by flow cytometry, wherein "serum" represents the incubation of the serum-containing medium mixture with LPGL liposome nanoparticles with the BxPC3 cells; "ultrasound" means ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min) treatment; "EXO1" represents EXO1 pretreated BxPC3 cells; "+" represents selection or adoption; "-" means unselected or not employed.

Fig. 8 shows subcellular distribution of Cy 5-labeled LPGL liposomal nanoparticle with and without serum pre-incubation mixture with BxPC3 cells, with and without sonication treatment. (8A) Serum-free pre-incubation mixture of Cy 5-labeled LPGL liposome nanoparticles mixed with BxPC3 cells, and then irradiated with or without ultrasound (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% and lasting for 5 min), continuously incubating for 1hSM showed subcellular distribution of Cy 5-labeled LPGL liposomal nanoparticles in BxPC3 cells, with Cy 5-labeled LPGL liposomal nanoparticles appearing red and lysosomes appearing green. (8B) The image analysis software Cellprofiler v2.2.0 was used to analyze the Mander Overlap Coefficient (MOC) of Cy 5-labeled LPGL liposome nanoparticles (red) with lysosomes (green) in graph (8A), calculated as:

the MOC ranges from 0 to 1, where 1 means complete overlap (co-localization) and 0 means none. R _i，coloc Representing the pixel intensity, R, of a red pixel overlapping a green pixel _i Representing the total pixel intensity of the red pixels. Scale bar =25 μm.

FIG. 9 shows that the serum-containing medium mixture supplemented with Cy 5-labeled GL, LGL or PGL liposome nanoparticles was mixed with BxPC3 cells and incubated, followed by ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min) treatment, CLSM map of newly added BxPC3 cells at different culture time points after addition of new BxPC3 cells, scale bar =50 μm.

Figure 10 is the in vitro cytotoxicity and penetration of free GEM and different liposomal nanoparticles on 3D tumor spheres under ultrasound irradiation. (10A) LPGL, PGL, LGL, GL or Gemcitabine (GEM) and a serum-containing culture medium mixture (with the GEM concentration of 0-10 mu M) and BxPC3 or Huh7 three-dimensional (3D) multicellular tumor 3D spheres are incubated, then treated by ultrasonic irradiation, and cultured for 72h, and the Gemcitabine (GEM) and different liposome nanoparticles have the inhibiting effect on the activity of the BxPC3 tumor 3D spheres. (10B) IC of Gemcitabine (GEM) and different liposomal nanoparticles on BxPC3 or Huh7 tumor 3D spheres ₅₀ (50%) inhibition concentration value. (10C) Observation of apoptosis before and after 3D multicellular tumor spheroids of BxPC3 treated with LPGL, PGL, LGL, GL liposomal nanoparticles and Gemcitabine (GEM) by light microscopy and TUNEL staining, the operation of the free GEM and different liposomal nanoparticles to treat 3D multicellular tumor spheroids of BxPC3 was as follows: serum-containing medium (all equivalent to 0.1. Mu.M GEM equivalent dose) with free GEM and different liposomal nanoparticles was added with BxPC at 37 ℃ 3 tumor 3D sphere mixing and ultrasonic irradiation (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% duration 5 min), after 72h of culture, the morphology of the BxPC3 tumor 3D spheres of the different treatment groups was observed by light microscopy and TUNEL staining with scale bar =500 μm. (10D) The mixture of different Cy 5-labeled liposome nanoparticles and serum-containing medium was added to 3D multicellular tumor spheroids of BxPC3 pretreated or not with exocytosis inhibitor EXO1, irradiated with ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration 5 min) followed by incubation for 6h, CLSM plot of permeation characteristics of 3D multicellular tumor spheres of different Cy 5-labeled liposomal nanoparticles to BxPC3, scale bar =500 μm. (10E) The 75 μm and 100 μm level mean Integrated Optical Density (IOD) of the 3D spheres of BxPC3 in the graph (10D) was analyzed using Image-pro Plus 6.0 software to assess the deep penetration capability of the liposomal nanoparticles.

FIG. 11 shows the CP4126 blood levels at various time points after tail vein administration of GL, LGL, PGL or LPGL liposomal nanoparticles.

FIG. 12 is the biodistribution, tumor accumulation and infiltration of different Cy 5-labeled liposome nanoparticles in BALB/c nude mice subcutaneously loaded with human PDA tumor. (12A) For the ultrasonic instrument of tumor ultrasonic irradiation, ultrasonic irradiation (sound intensity: 2W/cm) is carried out on the subcutaneous human PDA-loaded tumor on the right side of the mouse after the Cy 5-labeled liposome nano-particles are injected into the vein ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min) while the left subcutaneous human PDA-loaded tumor was not irradiated with ultrasound. (12B) Injecting different Cy 5-labeled liposome nanoparticles into the tail vein of a BALB/c nude mouse loaded with the human PDA tumor, and performing ultrasonic irradiation (acoustic intensity: 2W/cm) on the human PDA tumor loaded subcutaneously on the right side ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min), carrying out no ultrasonic irradiation on the human PDA tumor loaded subcutaneously on the left side, and carrying out ultrasonic irradiation on BALB/c nude mice and separated tissues (1 is the tumor which is subjected to and not subjected to ultrasonic irradiation, 2 is the heart, 3 is the liver, 4 is the spleen, 5 is the lung, and 6 is the tissue which is subjected to ultrasonic irradiation and loaded with the human PDA tumor subcutaneously on the left and right sides by a Caliper IVIS Lumina II fluorescence spectrum imager 12h after injection and administrationKidney, 7 small intestine) for in vivo and in vitro fluorescence imaging. (12C) By Living

The software quantitated the fluorescence intensity of the isolated tumors, heart, liver, spleen, lung, kidney and small intestine in FIG (12B), with and without ultrasound irradiation. (12D) CLSM images of penetration of different Cy 5-labeled liposomal nanoparticles at the ultrasonically irradiated tumor sites were performed as follows: injecting different Cy 5-labeled liposome nanoparticles into the tail vein of a BALB/c nude mouse loaded with the human PDA tumor, and performing ultrasonic irradiation (acoustic intensity: 2W/cm) on the human PDA tumor loaded subcutaneously on the right side ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min), left-side subcutaneously-loaded human-derived PDA tumors were not irradiated with ultrasound, mice were injected with FITC-labeled tomato lectin (FITC-LEL) to stain blood vessels for 5min 12h after injection dosing, then cardiac perfusion was performed, the ultrasonically-irradiated tumors were excised, frozen in Tissue-Tek OCT embedding medium and cut into 10 μm thick sections and imaged using CLSM, scale bar =100 μm. (12E) Mean fluorescence intensity gradient from tumor vessels to deep tumor region indicated by white arrows in fig. (12D).

FIG. 13 is the in vivo real-time extravasation of blood vessels and tumor aggregation of different Cy 5-labeled liposomal nanoparticles in human-derived PDA tumor-loaded BALB/c nude mice. (13A) Ultrasound and CLSM instruments are used to study in vivo real-time extravascular infiltration and tumor aggregation. (13B) After injecting different Cy 5-labeled liposome nanoparticles into tail vein, the tumor site was treated by ultrasonic irradiation (acoustic intensity: 2W/cm) ² 3MHz, duty cycle: 50%, duration: 20 min), 10min, 30min and 60min after tail vein injection of different Cy 5-labeled liposomal nanoparticles, the CLSM observed Cy 5-labeled liposomal nanoparticles extravasated from PDA tumor vessels into the PDA tumor area in real time, scale bar =200 μm. (13C) The mean fluorescence integrated optical density of the selected region (circled) of figure (13B) was quantitatively analyzed at 10min, 30min and 60min of tail vein injection of Cy 5-labeled liposomal nanoparticles. (13D) Transmission Electron Microscope (TEM) Observation and analysis of the Tail vein injection of FIG. 13B Ultrastructure of tumor vessels 60min after ejection of different Cy 5-labeled liposome nanoparticles, arrows show trans-endothelial cell transporters of vesicles, scale bar =500nm.

FIG. 14 is the antitumor activity of free GEM and different liposomal nanoparticles in BALB/c nude mice subcutaneously loaded with human-derived PDA tumors. (14A) Construction, experimental schedule and tumor treatment scheme of BALB/c nude mouse animal model loaded with human PDA tumor. Immediately after intravenous administration of GEM, GL, LGL, PGL, LPGL, LPL or PBS 7.4, all mice were subjected to ultrasonic irradiation treatment (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min). (14B) tumor volume changes in groups of mice during treatment. (14C) photographs of the groups of mice at the end of the experiment for 36 days. (14D) Photographs of tumor resection of each group of mice were taken at the end of the 36 day experiment. (14E) The average tumor weight of excised tumors in each group of mice was terminated at 36 days. (14F) weight change of mice in each group during treatment. (14G) The 36 day experiment ended the White Blood Cell (WBC) values in the blood of each group of mice. (14H) The 36 day experiment ended the Platelet (PLT) value in blood of each group of mice. (14I) H of tumor tissue of each group of mice at the end of the experiment &E staining, IHC staining for Ki67 and TUNEL staining, scale bar =100 μm. ^* :P<0.05, ^** :P<0.01, ^*** :P<0.001。

Fig. 15 is the average integrated optical density of positive tumor cells in Ki67 immunohistochemical staining of tumor tissue from each group of mice at the end of the 36 day experiment, P <0.05 and P <0.01.

Fig. 16 is the ultrasonic irradiation to eliminate uptake of liposome nanoparticles by protein corona and cells on the surface of nanoparticles. (16A) After incubation of NGR ligand-modified LPGL and NGR ligand-modified LGL liposome nanoparticles with mouse plasma for 30min, the cells were irradiated without ultrasound irradiation (-US) or with ultrasound irradiation (+ US, acoustic intensity: 2W/cm ² Frequency: 3MHz, duty cycle: 50% for 5 min), the concentration of total protein in the protein corona on NGR ligand-modified LPGL and NGR ligand-modified LGL liposomal nanoparticles. (16B) Serum-free medium or serum-containing medium mixture supplemented with Cy 5-labeled NGR-modified LGL and Cy 5-labeled NGR-modified LPGL liposomal nanoparticles withAfter mixing the Huh7 cells, ultrasonic irradiation (sound intensity: 2W/cm) was performed or not ² Frequency: 3MHz, duty cycle: 50% and the duration is 5 min), continuing to incubate the average fluorescence intensity of Cy 5-labeled different liposome nanoparticles taken up by 1h and Huh7 cells, wherein the term "serum-free" refers to the average fluorescence intensity in Huh7 cells after incubation for 1h without ultrasonic irradiation treatment after the serum-free medium mixture of the Cy 5-labeled NGR-modified LGL and the Cy 5-labeled NGR-modified LPGL liposome nanoparticles is added and the Huh7 cells are incubated; "serum" refers to the average fluorescence intensity in Huh7 cells after 1h incubation without ultrasonic irradiation treatment after incubation of the serum-containing medium mixture supplemented with Cy 5-labeled NGR-modified LGL and Cy 5-labeled NGR-modified LPGL liposome nanoparticles with Huh7 cells; "serum + sonication" means that a serum-containing medium mixture supplemented with Cy 5-labeled NGR-modified LGL and Cy 5-labeled NGR-modified LPGL liposome nanoparticles was incubated with Huh7 cells and then treated with ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), mean fluorescence intensity in Huh7 cells after 1h incubation.

FIG. 17 shows the antitumor activity of GEM, GL, NGR-modified LGL, PGL, NGR-modified LPGL, or PBS 7.4 in BALB/c nude mice subcutaneously loaded with human HCC tumor, wherein the doses (in terms of GEM) of GEM, GL, NGR-modified LGL, PGL, and NGR-modified LPGL in each group were the same. (17A) Treatment schedules and tumor treatment protocols for BALB/c nude mouse animal models bearing human HCC tumors by treating the tumor sites of mice with ultrasound irradiation (sound intensity: 2W/cm) after intravenous injection of GEM, GL, NGR-modified LGL, PGL, NGR-modified LPGL, or PBS ₂ Frequency: 3MHz, duty cycle: 50%, duration: 20 min). (17B) Cumulative concentrations of gemm gemcitabine triphosphate active metabolite dFdCTP in mice tumors of each group 24h after 1 st intravenous injection of GEM, GL, NGR modified LGL, PGL, NGR modified LPGL liposome nanoparticles. (17C) weight change of mice in each group during treatment. (17D) tumor volume changes in groups of mice during treatment. (17E) Photographs of tumor resection of each group of mice were taken at the end of the experiment on day 34. (17F) At day 34 the experiment was completed and groups of mice had mean tumor weights of excised tumors. (17G) Groups at the end of the experiment Mice excised tumor tissue H&E staining and IHC staining of Ki67, scale bar =100 μm. ^* :P<0.05, ^** :P<0.01, ^*** :P<0.001。

Detailed Description

The present inventors have developed a nanoparticle that entraps perfluoro-n-pentane. In addition, the nano-particles can be effectively used as carriers for screening or identifying potential ligands targeted to cells or cell surface receptors under ultrasonic irradiation treatment, and the nano-particles can have excellent lysosome escape and lysosome degradation prevention capacity through ultrasonic irradiation, and the lysosome escape can effectively protect the nano-particles and the loaded drugs from being degraded and degraded by lysosomes, so that the stability of the drugs loaded by the nano-particles in the cells is enhanced, and the therapeutic effect of the drugs is improved.

Term(s) for

Unless defined otherwise, 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 belongs.

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-ended definitions. In other words, the term includes "consisting of 8230; \8230; composition;" consisting essentially of 8230; \8230; composition 8230).

As used herein, "cancer," "tumor," and "neoplasm" are used interchangeably.

As used herein, "cryo-TEM" refers to a cryo-transmission electron microscope.

As used herein, "DSPE-PEG" is distearoylphosphatidylethanolamine-polyethylene glycol, known by the English name Distearoylphosphatidylethanolamine-PEG.

As used herein, "DSPE-PEG2000" refers to distearoylphosphatidylethanolamine-polyethylene glycol 2000, known by the English name Distearoylphosphoethanolamine-PEG 2000.

As used herein, "DPPC" refers to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, also known as: dipalmitoylphosphatidylcholine, known as 1, 2-dipalmitoyl-sn-glycerol-3-phosphorylcholine.

As used herein, "FBS" refers to fetal bovine serum, known under the English name total bone serum.

As used herein, "GEM" refers to gemcitabine, gemcitabine.

As used herein, "Cy5" refers to Cyanine 5.

As used herein, "cy5.5" refers to cy anine 5.5.

As used herein, "TUNEL" refers to a TdT-mediated dUTP nick end tag, and is TdT-mediated dUTP nickend labeling in English.

As used herein, "CLSM" refers to a laser scanning Confocal microscope, which is a Confocal laser scanning microscope.

As used herein, the term "IC ₅₀ "refers to the half inhibitory concentration (50% inhibition concentration), i.e., the concentration of inhibitor at which 50% inhibition is achieved.

As used herein, the terms "gemcitabine prodrug CP4126" and "CP4126" are used interchangeably, and the structural formula of gemcitabine prodrug CP4126 is as follows:

as used herein, the term "glycerophosphate buffer" refers to a phosphate buffer containing glycerol.

As used herein, the terms "IC50" and "IC ₅₀ "used interchangeably refers to the half inhibitory concentration (50% inhibition concentration), i.e., the concentration of inhibitor at which 50% inhibition is achieved.

As used herein, the term "+ US" refers to treatment with ultrasonic radiation.

As used herein, the term "-US" means without ultrasonic irradiation treatment.

As used herein, the terms "PBS", "phosphate buffer" and "PBS buffer" are used interchangeably to refer to an aqueous phosphate buffered solution.

As used herein, the term "RPMI 1640 medium" refers to the Roswell Park mental Institute 1640 medium.

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

As used herein, the terms "RGD", "RGD polypeptide", "RGD targeting peptide" and "RGD ligand" are used interchangeably and have the amino acid sequence Cys (cysteine) -Arg (arginine) -Gly (glycine) -Asp (aspartic acid) -Lys (lysine) -Gly (glycine) -Pro (proline) -Asp (aspartic acid) -Cys (cysteine).

As used herein, the terms "NGR," "NGR polypeptide," "NGR targeting peptide," and "NGR ligand" are used interchangeably and have the amino acid sequences Gly (glycine) -Cys (cysteine) -Asn (asparagine) -Gly (glycine) -Arg (arginine) -Cys (cysteine).

As used herein, the term "DSPE-PEG-ligand" refers to a ligand coupled to DSPE-PEG, e.g., DSPE-PEG2000-RGD (distearoylphosphatidylethanolamine-polyethylene glycol 2000-RGD) refers to RGD coupled to DSPE-PEG 2000; DSPE-PEG2000-NGR (distearoylphosphatidylethanolamine-polyethylene glycol 2000-NGR) means that NGR is coupled on DSPE-PEG 2000.

As used herein, the term "perfluoro-n-pentane" is used in english as perfluoropentane.

As used herein, the terms "ultrasonic irradiation" and "ultrasonic stimulation" are used interchangeably.

As used herein, "LPGL" and "LPGL liposomal nanoparticles" are used interchangeably.

As used herein, "LGL" is used interchangeably with "LGL liposomal nanoparticles".

As used herein, "PGL" is used interchangeably with "PGL liposome nanoparticle".

As used herein, "GL" is used interchangeably with "GL liposome nanoparticle".

As used herein, "LPL" is used interchangeably with "LPL liposome nanoparticle".

As used herein, acoustic intensity refers to acoustics intensity.

As used herein, frequency refers to frequency.

As used herein, duty cycle refers to a duty cycle

In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and/or its attendant symptoms or protecting a subject from acquiring a disease.

In the present invention, the term "treating" includes inhibiting, reducing, alleviating, reversing or eradicating the progression of the disease, and does not require 100% inhibition, eradication or reversal. In some embodiments, the drug-loaded nanoparticles of the invention reduce, inhibit and/or reverse the associated disease (e.g., tumor) and its complications, e.g., by 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%, as compared to the levels observed in the absence of the drug-loaded nanoparticles of the invention.

In the present invention, the term "elimination" includes reduction or removal, and 100% removal is not required. In some embodiments, sonication of the protein corona modified nanoparticle reduces the protein content of the protein corona modified nanoparticle, for example, by at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or about 100%, for example, 80-90%, as compared to the protein corona modified nanoparticle prior to sonication.

Tumor(s)

The tumor of the present invention may include, but is not limited to, a human tumor (e.g., a human tumor) or a non-human mammalian tumor.

In a preferred embodiment of the invention, the tumor comprises a low permeability tumor.

Preferably, the tumor comprises a tumor with low permeability of tumor vessels.

Preferably, the tumor comprises a solid tumor.

In a preferred embodiment of the present invention, the tumor comprises liver cancer.

Preferably, the tumor comprises human liver cancer.

In a preferred embodiment of the present invention, the tumor comprises pancreatic cancer.

Preferably, the pancreatic cancer comprises pancreatic adenocarcinoma.

Preferably, the pancreatic cancer comprises an in situ pancreatic cancer.

Preferably, the pancreatic cancer comprises pancreatic ductal adenocarcinoma.

Preferably, the cancer cells of pancreatic cancer include BxPC-3 cells.

In a preferred embodiment of the present invention, the tumor comprises a tumor with poor Permeability and Retention (EPR effect).

In a preferred embodiment of the present invention, the tumor vessels of low tumor vessel permeability comprise one or more characteristics selected from the group consisting of:

(a) The tumor vascular cell tissues are well and closely packed; and/or

(b) The tumor has small vascular intercellular spaces.

Preferably, the vascular cells comprise vascular endothelial cells.

Cells

The cells of the present invention may include, but are not limited to, tumor cells and/or tumor vascular cells, and the like.

In a preferred embodiment of the invention, the cells comprise tumor cells.

Preferably, the tumor cells comprise one or more of Huh7 cells, hepG2 cells and/or BxPC-3 cells.

In a preferred embodiment of the present invention, the cells comprise tumor vascular cells

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

In a preferred embodiment of the invention, the cells comprise cells that are desired to be cultured or grown in conditions comprising a protein.

Preferably, the serum comprises fetal bovine serum.

Ligands

The ligands of the present invention may include, but are not limited to, polypeptide or protein ligands.

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

Preferably, said surface comprises an outer surface.

In a preferred embodiment of the invention, the ligand comprises a ligand that mediates cellular uptake of the ligand-modified nanoparticle.

In a preferred embodiment of the invention, the ligand comprises a ligand that mediates endocytosis of the ligand-modified nanoparticle by a cell.

In a preferred embodiment of the invention, the ligand comprises a ligand that mediates endocytosis and exocytosis of the ligand-modified nanoparticle by cells.

In a preferred embodiment of the invention, the ligand is capable of mediating ligand-modified nanoparticle penetration from the tumor vasculature to the tumor site.

In a preferred embodiment of the invention, the ligand can mediate the penetration of the ligand-modified nanoparticle from the tumor vessel to the tumor site through endocytosis and exocytosis.

In a preferred embodiment of the invention, the ligand is targeted to vascular endothelial cells and the ligand is capable of mediating endocytosis and exocytosis of the ligand-modified nanoparticle by the vascular endothelial cells.

In a preferred embodiment of the present invention, the ligand can mediate tumor vascular cells to perform endocytosis, post-endocytosis and exocytosis of the ligand-modified nanoparticle in blood to outside of the tumor vascular vessels (e.g., tumor tissue microenvironment).

Preferably, the ligand comprises an RGD polypeptide and/or an NGR polypeptide.

Medicine

The drug of the present invention is not particularly limited, and may include, but is not limited to, an antitumor drug.

In a preferred embodiment of the invention, the drug comprises a drug that is unstable in the lysosomes of cells.

Preferably, said degradation comprises degradation by lysosomal enzymes.

In a preferred embodiment of the invention, the target of action of the drug is in the cytoplasm or in the nucleus.

In a preferred embodiment of the present invention, the drug comprises a gene or a protein.

Preferably, the gene includes, but is not limited to, DNA, RNA, or a combination thereof.

In a preferred embodiment of the present invention, the drug comprises an anticancer drug

Preferably, the anti-cancer drug comprises a chemical drug.

Typically, the anti-cancer drugs include (but are not limited to): gemcitabine, cytarabine, doxorubicin, fluorouracil, or combinations thereof.

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

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

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

Preferably, the free drug comprises a hydrophobic drug or a hydrophilic drug.

Preferably, the free drug comprises an anti-cancer drug.

Typically, the higher fatty acid carriers include (but are not limited to): palmitic acid (hexadecanoic acid), pearlescent aliphatic acid (heptadecanoic acid), stearic acid (octadecanoic acid), oleic acid (octadecenoic acid), linoleic acid (octadecadienoic acid), linolenic acid (octadecatrienoic acid), arachidic acid (eicosanoic acid), eicosapentaenoic acid, behenic acid (behenic acid), DHA (docosahexaenoic acid), lignoceric acid (lignoceric acid), or combinations thereof.

Preferably, said oleic acid comprises elaidic acid.

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

Preferably, the amphiphilic prodrug is present as a lipid bilayer.

typically, the prodrugs include:

D-C

Preferably, said degradation comprises degradation by lysosomal enzymes.

Preferably, the active ingredient is targeted in the cytoplasm or nucleus.

Typically, the prodrugs include:

wherein R is an anticancer drug.

Preferably, R is an anticancer drug comprising gemcitabine, cytarabine, doxorubicin, fluorouracil, or a combination thereof.

Typically, the drug comprises gemcitabine elaidic acid ester.

Typically, the prodrug comprises gemcitabine elaidic acid ester.

Preferably, the gemcitabine elaidic acid ester has the following structure:

non-human mammal

Non-human mammals of the invention can include, but are not limited to, mice, rats, rabbits, monkeys, cows, horses, sheep, dogs, cats, chimpanzees, or baboons.

Preferably, the bovine comprises fetal bovine.

Ultrasonic irradiation

The sound intensity, frequency, duty ratio and time of the ultrasonic irradiation can be determined according to specific requirements.

Preferably, the time of the ultrasonic irradiation is more than 2min, preferably more than 5min, preferably more than 10min, preferably more than 15min, such as 15-20min or 25-35min.

Conditions containing protein

In the present invention, the conditions containing the protein may include, but are not limited to, blood, serum, plasma, tissue microenvironment or culture medium.

In a preferred embodiment of the present invention, the blood, serum or plasma comprises isolated or separated blood, serum or plasma.

In a preferred embodiment of the invention, the protein comprises serum protein, plasma protein and/or tissue protein.

In a preferred embodiment of the present invention, the serum protein, the plasma protein and/or the tissue protein comprises serum protein, plasma protein and/or tissue protein of human or non-human mammal.

In a preferred embodiment of the present invention, the serum protein, the plasma protein and/or the tissue protein comprises serum protein, plasma protein and/or tissue protein isolated or separated in vitro.

Preferably, the bovine comprises fetal bovine.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

In a preferred embodiment of the present invention, the culture medium comprises a liquid culture medium.

In a preferred embodiment of the present invention, the culture medium comprises a cell culture medium.

In a preferred embodiment of the present invention, the culture medium comprises a protein-containing culture medium.

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

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

In a preferred embodiment of the present invention, the culture medium according to the present invention comprises a culture medium containing serum, plasma and/or tissue protein.

Preferably, the medium comprises a serum-containing medium.

Nanoparticles and method for preparing same

The nano particles in the invention refer to nano-scale microscopic particles.

In a preferred embodiment of the invention, the nanoparticles are loaded with perfluoro-n-pentane.

In a preferred embodiment of the invention, the nanoparticles encapsulate perfluoro-n-pentane.

In a preferred embodiment of the present invention, the nanoparticle is a nanoparticle or a liposome.

In a preferred embodiment of the present invention, the Nanoparticles of the present invention are Nanoparticles (nanoparticules). The nano-particle is colloid particle made of natural or synthetic high molecular material and with the particle size of nano-order (0.1-100 nm)

In a preferred embodiment of the present invention, the nanoparticle of the present invention is a liposome. Liposomes are particles with a bilayer structure, resembling a cell membrane.

In a preferred embodiment of the present invention, the nanoparticles comprise a nanomaterial.

Preferably, the nanomaterial comprises an amphiphilic material.

Preferably, the liposomes comprise a lipid material.

In a preferred embodiment of the invention, the lipid material comprises one or more of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), soya lecithin, phosphatidylcholine (PC, lecithin), cholesterol, phosphatidylethanolamine (PE, cephalin), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), dicetyl phosphate (DCP), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC) and Dioleoylphosphatidylcholine (DOPC).

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

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

In a preferred embodiment of the present invention, the perfluoro-n-pentane is 0.01 to 0.5 parts by weight, preferably 0.02 to 0.2 parts by weight, more preferably 0.05 to 0.15 parts by weight, more preferably 0.08 to 0.12 parts by weight, most preferably 0.1 parts by weight.

in a preferred embodiment of the present invention, the nanoparticles comprise drug-loaded nanoparticles.

The present invention also provides a method for preparing the nanoparticle of the present invention, the method comprising the steps of:

In a preferred embodiment of the present invention, the nanoparticle is a drug-loaded nanoparticle, and the method for preparing the drug-loaded nanoparticle comprises the steps of:

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

In a preferred embodiment of the present invention, in the step (1), the weight/volume ratio (mg: ml) of the DPPC to the organic solvent is 1.

In a preferred embodiment of the present invention, in the step (1), the weight-to-volume ratio (mg: ml) of the drug to the organic solvent is 1:2-5, preferably 1:1-2, more specifically 1:1.3-1.7, more specifically 1:1.5.

Preferably, in the step (2), the hydration is performed at a low temperature.

Preferably, in the step (2), the stirring comprises the steps of:

stirring is carried out at low temperature and then at elevated temperature.

Preferably, the stirring time after the temperature is raised is 0.5 to 1.5h, preferably 0.8 to 1.2h, more preferably 1h.

Preferably, the stirring comprises magnetic stir bar stirring.

Preferably, the increased temperature is followed by agitation to remove unencapsulated perfluoro-n-pentane.

Preferably, the liposomes are in the form of liposomal nanodroplets.

Typically, the method of preparing the liposome comprises the steps of:

(1) DPPC and DSPE-PEG are dissolved in an organic solvent in a round-bottom flask, the organic solvent is removed by rotary reduced pressure evaporation, and a lipid film is formed in the round-bottom flask;

Typically, the method of preparing the liposomes comprises the steps of:

Typically, the method of preparing the drug-loaded liposome comprises the steps of:

Ligand-modified nanoparticles and methods of making the same

The invention provides a ligand-modified nanoparticle comprising a nanoparticle according to the invention; and a ligand.

The ligands of the invention may include targeting ligands.

In a preferred embodiment of the present invention, the surface of the nanoparticle contains a ligand.

Preferably, said surface comprises an outer surface.

Preferably, the outer surface of the nanoparticle contains a ligand.

Preferably, the ligand is modified on the nanomaterial.

In a preferred embodiment of the present invention, the ligand is modified on the nanomaterial of the nanoparticle.

Preferably, the ligand is modified on the surface of the nanoparticle.

Preferably, the ligand is adsorbed on the surface of the nanoparticle.

Preferably, the adsorption comprises physisorption and/or chemisorption.

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

The ligands of the invention may include polypeptide or protein ligands.

Preferably, the ligand comprises an RGD polypeptide and/or an NGR polypeptide.

In a preferred embodiment of the present invention, the lipid material comprises a ligand-modified lipid material.

Preferably, the ligand is coupled to the nanomaterial of the nanoparticle.

Preferably, the ligand is coupled to a lipid material.

Preferably, said coupling comprises chemical coupling.

In a preferred embodiment of the invention, the ligand is coupled to distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) to form a DSPE-PEG-ligand.

In a preferred embodiment of the invention, the ligand is coupled to a portion of the nanomaterial (e.g., lipid material).

In a preferred embodiment of the invention, the DSPE-PEG-ligand comprises DSPE-PEG-RGD and/or DSPE-PEG-NGR.

In a preferred embodiment of the present invention, the DSPE-PEG-ligand is present in an amount of 1 to 10 parts by weight, preferably 2 to 8 parts by weight, more preferably 4 to 6 parts by weight, most preferably 3 parts by weight.

In a preferred embodiment of the invention, the weight ratio of DSPE-PEG-ligand to DPPC is 1.

In a preferred embodiment of the present invention, the particle size of the ligand-modified nanoparticle is 120-260nm, preferably 160-210nm, more preferably 170-200nm, and still more preferably 180-200nm.

In a preferred embodiment of the invention, the ligand-modified nanoparticle has a potential of-2 to-18 mV, preferably-2 to-15 mV, more preferably-5 to-12 mV.

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

Preferably, said surface comprises an outer surface.

Preferably, the ligand is capable of mediating tumor vasculature to endocytose, post-endocytosis and exocytosis of the blood ligand-modified nanoparticle (e.g., tumor tissue microenvironment).

The present invention also provides a method of preparing the ligand-modified nanoparticle of the present invention, the method comprising:

In a preferred embodiment of the present invention, the method for preparing the ligand-modified nanoparticle comprises:

Typically, the method of preparing the ligand-modified nanoparticle comprises:

Typically, the method of preparing the ligand-modified liposome comprises the steps of:

(2) Cooling the lipid membrane to low temperature, adding perfluoro-n-pentane to immerse the lipid membrane, adding a buffer solution to hydrate, stirring for 0.2-0.8h at 2-6 ℃, and then stirring for 0.8-1.2h in a round-bottom flask under the conditions of an open mouth and 25-35 ℃ to obtain the ligand modified liposome.

Protein crown modified nano-particles and preparation method thereof

The invention provides a protein corona modified nanoparticle, which comprises the nanoparticle and/or the ligand modified nanoparticle; and a protein corona.

Preferably, the nanoparticles are loaded with perfluoro-n-pentane.

In a preferred embodiment of the present invention, the protein of the protein corona comprises serum protein, plasma protein and/or tissue protein.

Preferably, the bovine comprises fetal bovine.

The protein corona modified nanoparticle of the present invention may comprise an isolated or ex vivo protein corona modified nanoparticle.

Preferably, said protein corona modification is on a nanoparticle according to the invention and/or a ligand-modified nanoparticle according to the invention.

Preferably, the protein corona is modified on the surface of the nanoparticle according to the invention and/or the ligand-modified nanoparticle according to the invention.

Preferably, said surface comprises an outer surface.

Preferably, the modification comprises adsorption.

The present invention also provides a method for preparing the protein corona modified nanoparticle according to the present invention, the method comprising the steps of:

contacting the nanoparticle according to the invention and/or the ligand-modified nanoparticle according to the invention with a protein, and separating to obtain a protein corona-modified nanoparticle,

preferably, the method is a non-diagnostic and non-therapeutic method.

Preferably, the contacting is in vitro or in vivo.

Preferably, said contacting comprises contacting in a protein-containing condition.

In a preferred embodiment of the invention, said contacting comprises contacting in a culture medium.

Preferably, the culture medium comprises a liquid culture medium.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the medium comprises a protein.

Preferably, the medium comprises a serum-containing medium.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

Preferably, said culturing comprises in vitro culturing.

In a preferred embodiment of the invention, said contacting comprises contacting in blood, serum or plasma.

Preferably, the bovine comprises fetal bovine.

Preferably, the contact time is from 0.25 to 6 hours, preferably from 0.25 to 4 hours, more preferably from 0.25 to 2 hours, preferably from 0.25 to 1 hour, preferably from 0.25 to 0.5 hours, for example from 0.5 to 1 hour.

Preferably, the separation comprises gel chromatography.

Preferably, the gel comprises a dextran gel.

Preferably, the separation comprises size exclusion chromatography.

Method for eliminating protein corona of protein corona modified nanoparticles

The invention provides a method for eliminating the protein corona of the protein corona modified nanoparticle, which comprises the following steps:

Preferably, the nanoparticles are loaded with perfluoro-n-pentane.

The nanoparticle surface protein corona is eliminated through ultrasonic irradiation, the masking effect of the protein corona on the nanoparticle surface modified ligand is overcome, the binding of the nanoparticle surface modified ligand and a receptor of a target cell (such as a tumor vascular cell or a tumor cell) is recovered, and the uptake of the ligand modified nanoparticle of the invention by a cell can be enhanced.

Preferably, the method comprises an in vitro method or an in vivo method.

In a preferred embodiment of the invention, said elimination comprises a reduction or elimination.

Preferably, said reduction comprises a reduction in protein content.

In a preferred embodiment of the invention, the protein corona modified nanoparticle comprises a protein corona modified nanoparticle in a protein-containing condition.

Preferably, the bovine comprises fetal bovine.

Preferably, the culture medium comprises a liquid culture medium.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the culture medium comprises a protein-containing medium.

Preferably, the medium contains a protein.

Preferably, the medium comprises a serum-containing medium.

Preferably, the cells comprise tumor cells and/or tumor vascular cells.

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

Preferably, said culturing comprises in vitro culturing.

The ultrasonic irradiation can effectively remove the protein corona of the nanoparticle modified by the protein corona, and for cells needing to be cultured in a culture medium containing protein (such as a serum-containing culture medium), the ultrasonic irradiation can overcome the masking effect of the protein corona on the ligand modified on the surface of the nanoparticle, and avoid the protein corona from obstructing the incubation of the ligand modified on the surface of the nanoparticle and the cells under the condition containing the protein (such as the serum-containing culture medium), so that whether the ligand to be detected modified on the surface of the nanoparticle can be combined with the cells or cell surface receptors or not can be accurately determined, the potential ligand targeted on the cells or cell surface receptors can be accurately screened or identified, and the occurrence of false negative results (particularly under the condition that the dosage of the ligand to be detected is low) can be avoided.

Methods of screening or identifying potential ligands that target cells or cell surface receptors

The present invention provides a method of screening for or identifying potential ligands that target a cell or cell surface receptor, said method comprising the steps of:

Preferably, the nanoparticles are loaded with perfluoro-n-pentane.

Preferably, the nanoparticles are as described above.

Preferably, the ligand-modified nanoparticle is a ligand-modified nanoparticle as described above.

Preferably, the ligand of step (I) comprises a ligand to be tested.

Preferably, the step (II) includes:

In a preferred embodiment of the present invention, the method further comprises providing a control group comprising nanoparticles without ligand modification, and determining the binding of the nanoparticles without ligand modification to the cells or cell surface receptors.

Preferably, the method further comprises providing a control group comprising nanoparticles that are not modified with a ligand and otherwise identical to the ligand-modified nanoparticles, and determining binding of the nanoparticles that are not modified with a ligand to a cell or cell surface receptor.

In a preferred embodiment of the present invention, the step (II) includes:

Preferably, the term "greater than" includes significantly greater than.

In a preferred embodiment of the invention, the ligand comprises a polypeptide or protein ligand.

In a preferred embodiment of the invention, the receptor comprises a protein receptor, a lipoprotein receptor or a glycoprotein receptor.

In a preferred embodiment of the invention, said binding comprises affinity.

Preferably, the binding capacity includes affinity.

Preferably, the ligand comprises a latent ligand.

In a preferred embodiment of the invention, the cell or cell surface receptor comprises an ex vivo or isolated cell or cell surface receptor.

In a preferred embodiment of the invention, the method comprises an in vitro method or an in vivo method.

In a preferred embodiment of the invention, the method comprises a non-therapeutic and/or non-diagnostic method.

In a preferred embodiment of the present invention, the incubation is in vitro incubation or in vivo incubation.

In a preferred embodiment of the invention, the body comprises a human or non-human mammal.

In a preferred embodiment of the invention, said incubation comprises incubation in protein-containing conditions.

Preferably, the conditions comprise in vivo conditions or in vitro conditions.

Preferably, said culturing comprises in vitro culturing.

In a preferred embodiment of the invention, the protein-containing conditions include blood, serum, plasma, tissue microenvironment or culture medium.

Preferably, the bovine comprises fetal bovine.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the culture medium comprises a protein-containing medium.

Preferably, the medium contains a protein.

In a preferred embodiment of the present invention, the culture medium comprises a serum-containing culture medium.

In a preferred embodiment of the invention, said incubation comprises incubation of cells or cell surface receptors with the ligand-modified nanoparticles of step (I) in a medium containing serum, plasma and/or tissue proteins.

In a preferred embodiment of the invention, said incubation comprises incubation of cells or cell surface receptors with the ligand-modified nanoparticles of step (I) in a serum-containing medium.

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

In a preferred embodiment of the present invention, the cells comprise tumor cells and/or tumor vascular cells.

In a preferred embodiment of the present invention, the tumor vascular cells comprise tumor vascular endothelial cells.

In a preferred embodiment of the invention, the ligand is capable of binding to a cell or cell surface receptor.

In a preferred embodiment of the invention, said binding comprises specific binding or non-specific binding.

In a preferred embodiment of the invention, the receptor comprises a receptor on the outer surface of a cell membrane.

In a preferred embodiment of the invention, the bound assay comprises an isotope-loss assay, a fluorescein assay, a flow cytometry assay and/or a transwell migration assay.

In a preferred embodiment of the invention, the binding mediates uptake of the ligand-modified nanoparticle by the cell.

In a preferred embodiment of the invention, the binding mediates endocytosis of the ligand-modified nanoparticle by the cell.

In a preferred embodiment of the invention, the binding mediates endocytosis and exocytosis of the ligand-modified nanoparticle by a cell.

In a preferred embodiment of the present invention, the method for measuring binding comprises measuring the uptake efficiency of the ligand-modified nanoparticle of step (I) by a cell.

In a preferred embodiment of the present invention, the method for measuring binding comprises measuring the endocytosis capacity of the cells for the ligand-modified nanoparticle of step (I).

if the endocytosis capacity of the cell for the ligand-modified nanoparticle of step (I) is greater than the endocytosis capacity of the cell for the nanoparticle without ligand modification in the control group, the ligand of step (I) is a potential ligand for targeting a cell or a cell surface receptor.

In a preferred embodiment of the invention, the method for measuring binding comprises measuring the endocytosis and exocytosis of the ligand-modified nanoparticle of step (I) by a cell.

if the endocytosis and exocytosis capacity of the cell for the ligand-modified nanoparticle of step (I) is greater than the endocytosis and exocytosis capacity of the cell for the nanoparticle without ligand modification in the control group, the ligand of step (I) is a potential ligand for targeting a cell or cell surface receptor.

In a preferred embodiment of the present invention, the ligand of step (I) comprises a ligand that mediates endocytosis of the ligand-modified nanoparticle by cells.

In a preferred embodiment of the present invention, the ligand of step (I) comprises a ligand that mediates endocytosis and exocytosis of the ligand-modified nanoparticle by cells.

In a preferred embodiment of the invention, the ligand comprises a ligand that targets tumor vascular cell surface receptors.

Preferably, the ligand comprises a ligand that mediates endocytosis and exocytosis of the ligand-modified nanoparticle by cells.

Use of

The invention provides a use of the nanoparticle according to the invention and/or the ligand-modified nanoparticle according to the invention for the preparation of a composition for the prevention and/or treatment of a disease.

In a preferred embodiment of the present invention, the disease is an indication disease of the drug.

In a preferred embodiment of the invention, the disease comprises a tumor.

Preferably, the composition is a pharmaceutical composition.

The present invention also provides a method for preventing and/or treating a disease by administering the nanoparticle according to the present invention and/or the ligand-modified nanoparticle according to the present invention to a subject in need thereof, thereby preventing and/or treating a disease.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the subject comprises a human or non-human mammal.

Preferably, the disease is an indication disease of a drug.

Preferably, the disease comprises a tumor.

Preferably, after administering the nanoparticles according to the invention and/or the ligand-modified nanoparticles according to the invention to a subject in need thereof, the focal site (e.g. tumor site) is subjected to ultrasound irradiation.

Preferably, the administration is injection, oral or topical.

The invention provides a use of the nanoparticle according to the invention for the preparation of a vector for screening or identifying potential ligands targeting cells or cell surface receptors.

In a preferred embodiment of the present invention, the method for screening or identifying potential ligands targeting cells or cell surface receptors comprises the steps of:

Preferably, the method of screening or identifying potential ligands that target cells or cell surface receptors is as described above.

The invention also provides the use of an ultrasound apparatus for the manufacture of a device for one or more uses selected from the group consisting of:

(b) For screening or identifying potential ligands that target cells or cell surface receptors; and/or

Preferably, the nanoparticles are as described above.

Preferably, the ligand-modified nanoparticle is as described above.

In a preferred embodiment of the present invention, the protein corona modified nanoparticle is as described above.

In a preferred embodiment of the present invention, the method for eliminating the protein corona of the protein corona-modified nanoparticle is as described above.

In a preferred embodiment of the present invention, the step (a) of eliminating the protein corona of the protein corona-modified nanoparticle by ultrasonic irradiation comprises:

In a preferred embodiment of the invention, the method for screening or identifying potential ligands that target cells or cell surface receptors is as described above.

after the nanoparticles, the ligand modified nanoparticles and/or the protein corona modified nanoparticles are contacted with cells, ultrasonic irradiation is carried out on the cells, so that retention and/or degradation of the nanoparticles, the ligand modified nanoparticles and/or the protein corona modified nanoparticles by cell lysosomes are improved.

Preferably, the method comprises an in vitro method or an in vivo method.

Preferably, the contacting is in vivo contacting or in vitro contacting.

Preferably, said contacting comprises contacting in a serum-containing medium.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the disease is an indication disease of a drug.

Preferably, the cells comprise tumor cells and/or tumor vascular cells.

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

Preferably, the disease comprises a tumor.

Preferably, the tumor is as described above.

In a preferred embodiment of the present invention, the administration is injection administration, oral administration or external administration.

Preferably, the improvement comprises avoidance or overcoming.

Preferably, said degradation comprises degradation by lysosomal enzymes.

Method for inhibiting cells in vitro

The invention provides a method for inhibiting cells in vitro, which can be used for researching the inhibition mechanism of an inhibitor by inhibiting the cells in vitro.

The in vitro method for inhibiting cells comprises the following steps:

contacting cells with the nanoparticles according to the invention or the ligand-modified nanoparticles according to the invention in a culture medium, and performing ultrasonic irradiation treatment, thereby inhibiting the cells.

In a preferred embodiment of the invention, the method comprises a method of enhancing the in vitro inhibition of cells by ligand-modified nanoparticles.

Preferably, the methods include non-diagnostic and non-therapeutic methods.

Preferably, the ligand comprises an RGD polypeptide and/or an NGR polypeptide.

Preferably, the tumor vascular cells comprise tumor vascular endothelial cells.

Preferably, the tumor vascular cells comprise ECDHCC cells.

In a preferred embodiment of the present invention, the drug comprises a cytostatic agent.

In a preferred embodiment of the present invention, the drug comprises an anti-tumor drug.

Preferably, the medicament is as described above.

Preferably, the tumor is as described above.

In a preferred embodiment of the invention, the protein-containing conditions include blood, serum, plasma and/or culture medium.

Preferably, the bovine comprises fetal bovine.

In a preferred embodiment of the present invention, the protein-containing conditions are as described above.

Preferably, the culture medium comprises a cell culture medium.

Preferably, the medium contains a protein.

Preferably, the medium comprises a serum-containing medium.

Preferably, the serum comprises fetal bovine serum.

Preferably, the plasma comprises fetal bovine plasma.

Preferably, said culturing comprises in vitro culturing.

Preferably, the contacting is in vitro.

In a preferred embodiment of the present invention, the nanoparticle surface contains protein corona in the culture medium under the condition of no ultrasonic irradiation.

Preferably, the protein corona is adsorbed on the surface of the nanoparticle.

In a preferred embodiment of the present invention, the ligand is capable of mediating endocytosis of the ligand-modified nanoparticle by a cell after binding to the cell or a cell surface receptor.

In a preferred embodiment of the invention, the cell surface receptor comprises a cell membrane surface receptor.

Systems or arrangements

The present invention provides a system or device for treating a disease, said system or device comprising a nanoparticle of the invention and/or a ligand-modified nanoparticle of the invention; and an ultrasonic device.

In a preferred embodiment of the present invention, the system or apparatus further comprises a description or label describing:

In the course of treating a disease by administering the nanoparticles according to the invention and/or the ligand-modified nanoparticles according to the invention to a subject in need thereof, the focal site (e.g. tumor site) is subjected to ultrasound irradiation.

Preferably, the nanoparticles comprise drug-loaded nanoparticles.

Preferably, the ultrasound device comprises an ultrasound apparatus.

In a preferred embodiment of the invention, the subject comprises a human or non-human mammal.

Preferably, the disease is an indication disease of a drug.

Preferably, the drug comprises an anti-cancer drug.

Preferably, the disease comprises a tumor.

Preferably, the administration is injection, oral or topical.

Composition comprising a metal oxide and a metal oxide

The present invention provides a composition including, but not limited to, a pharmaceutical composition.

The compositions of the present invention may also include a pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of acceptable carrier parts of the pharmaceutically acceptable carrier include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): injection, oral administration or topical administration.

The composition or the preparation of the invention is in the form of oral preparation, external preparation or injection preparation. Typically, solid dosage forms for oral administration or 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 ingredients: (a) Fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) Disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary amine compounds; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

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 nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration or administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The composition is administered by applying a safe and effective amount of the nanoparticles or liposomes of the invention to human or non-human animals (e.g., rats, mice, dogs, cats, cows, sheep, chickens, ducks, etc.) in need of treatment, wherein the dosage is a pharmaceutically acceptable and effective dosage. The term "safe and effective amount" as used herein, refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. It will be understood by those skilled in the art that the safe and effective amount may vary with the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and the combination with other drugs. For example, for a human of 60kg body weight, the daily dose is usually 0.1 to 1000mg, preferably 1 to 600mg, more preferably 2 to 300mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main excellent technical effects of the invention comprise:

1. the invention develops a method for eliminating the protein corona on the surface of a nanoparticle through ultrasonic irradiation, overcomes the masking effect of the protein corona on a ligand modified on the surface of the nanoparticle, recovers the binding of the ligand modified on the surface of the nanoparticle and a receptor of a target cell (such as tumor vascular cells or tumor cells), can enhance the uptake of the ligand modified nanoparticle by the cell, can be used for promoting the endocytosis and exocytosis of the nanoparticle by tumor vascular endothelial cells mediated by the binding of the ligand on the surface of drug-loaded nanoparticle and the receptor on the surface of the tumor vascular endothelial cells, promotes the ligand modified nanoparticle to permeate to a tumor part from blood through tumor blood vessels (particularly tumor blood vessels with low permeability of the tumor blood vessels), and can promote the uptake of the nanoparticle by the tumor cells through the endocytosis mediated by the ligand/receptor, thereby improving the anticancer effect of an antitumor drug.

2. After the nano-particles are intravenously administered, the surface ligand of the nano-particles can be remarkably recovered and enhanced to be combined with a receptor on the surface of a tumor vascular endothelial cell under the ultrasonic irradiation of a tumor part, the ligand-modified nano-particles are promoted to permeate into the tumor part from tumor blood vessels (especially the blood vessels of tumors with low tumor blood vessel permeability), and the uptake of the nano-particles by tumor cells through ligand/receptor-mediated endocytosis can be promoted, so that the anticancer effect of an antitumor drug is improved. In addition, under ultrasonic irradiation, the nano-particles have excellent lysosome escape and lysosome degradation prevention capability, and the lysosome escape can effectively protect the nano-particles and the loaded drugs from being degraded and degraded by lysosomes, so that the stability of the nano-particles and the loaded drugs in cells is enhanced, and the treatment effect of the drugs is improved. In addition, the nanoparticle disclosed by the invention has the advantages of excellent blood clearance half-life, good biocompatibility, high safety and small side effect.

3. The ultrasonic irradiation can eliminate the protein corona of the nanoparticle modified by the ligand to be detected under the condition containing protein (such as serum-containing culture medium), overcome the masking effect of the protein corona on the ligand to be detected modified on the surface of the nanoparticle, and avoid the protein corona from hindering the contact of the ligand to be detected modified on the surface of the nanoparticle and cells under the condition containing protein (such as serum-containing culture medium), so that whether the ligand to be detected modified on the surface of the nanoparticle can be combined with cells or cell surface receptors which need to be cultured or grown under the condition containing protein (such as serum-containing culture medium) or not can be accurately determined, and the method is further used for accurately screening or identifying potential ligands targeted at the cells or cell surface receptors, and avoiding the occurrence of false negative results.

4. The invention develops a method for screening or identifying potential ligands targeted to cells or cell surface receptors, which can incubate cells or cell surface receptors and nanoparticles modified by ligands to be detected under the condition of containing protein (such as serum-containing culture medium), can effectively eliminate protein crowns modified on the surfaces of the nanoparticles under ultrasonic irradiation treatment, overcome the masking effect of the protein crowns on the ligands to be detected modified on the surfaces of the nanoparticles, thereby avoiding the protein crowns from obstructing the contact of the ligands modified on the surfaces of the nanoparticles and the cells, and further can efficiently and accurately determine whether the ligands to be detected on the nanoparticles can be combined with the cells or cell surface receptors which need to be cultured or grown under the condition of containing protein (such as serum-containing culture medium), and further screen or identify whether the ligands to be detected are the potential ligands targeted to the cells or cell surface receptors. The method for screening or identifying the potential ligand targeting the cell or the cell surface receptor is simple and convenient, can effectively eliminate the protein corona modified on the surface of the nanoparticle through ultrasonic irradiation, overcomes the masking effect of the protein corona on the ligand to be detected modified on the surface of the nanoparticle, can efficiently and accurately determine whether the ligand to be detected modified on the surface of the nanoparticle can be combined with the cell or the cell surface receptor, is particularly used for screening or identifying whether the ligand to be detected is targeted to cells needing to be cultured or grown under the condition containing protein (such as serum-containing culture medium), further accurately screens or identifies the potential ligand targeting the cell or the cell surface receptor, and avoids the false negative result (particularly under the condition of less using amount of the ligand to be detected) caused by the masking effect of the protein corona on the ligand to be detected modified on the surface of the nanoparticle.

5. The invention also provides the use of an ultrasound apparatus for overcoming the protein corona of protein corona-modified nanoparticles by ultrasound irradiation for screening or identifying potential ligands targeting cells or cell surface receptors, for improving the retention and/or degradation of nanoparticles and protein corona-modified nanoparticles by cell lysosomes and for the treatment of diseases by ligand-modified nanoparticles administered with enhanced ultrasound irradiation of lesions such as tumors.

The invention will be further illustrated with reference to the following specific examples. It should be understood that the following specific examples are provided to illustrate the detailed embodiments and specific procedures, but the scope of the present invention is not limited to these examples.

Example 1

1. Materials and instruments

DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine) and DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) were purchased from Avanti Lipids Inc.

DSPE-PEG2000 (DSPE-PEG 200) marked by DSPE-PEG2000-RGD and Cyanine 5 (Cy 5) ^Cy5 ) And Cyanine 5.5 (Cy5.5) labeled DSPE-PEG2000 (DSPE-PEG 2000) ^Cy5.5 ) Purchased from Xian Ruixi Biotechnology Ltd, DSPE-PEG2000-RGD is distearoylphosphatidylethanolamine-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)。

Chlorpromazine and cytochalasin D were purchased from Santa Cruz Biotechnology.

RPMI 1640 medium, DMEM medium, fetal Bovine Serum (FBS) and 0.25% trypsin solution were purchased from GIBCO (USA).

The serum-free culture medium mainly comprises water, glucose, amino acids and inorganic salts, and does not contain any protein component.

The serum-containing medium is a medium containing 10% by volume fetal bovine serum.

Alamar Blue Cell visual Reagent, hoechst 33342, and

green DND 26 was purchased from Thermo Fisher Scientific Inc.

Ki67 antibodies were purchased from Proteintech Group.

TUNEL apoptosis assay kit was purchased from roche.

Enhanced BCA Protein detection Kit (Enhanced BCA Protein Assay Kit) and Golgi-Tracker Green were purchased from Beyotime Biotechnology.

Matrigel basement membrane matrix (Matrigel based membrane matrix) was purchased from BD Biosciences.

EXO1 and gemcitabine prodrug CP4126 were purchased from Med-chem Express. Gemcitabine prodrug CP4126 is abbreviated "CP4126" and gemcitabine prodrug CP4126 is gemcitabine elaidic acid ester, and the structure is as follows:

the ultrasonic instrument is Mettler Sonifier-740.

GEM is short for Gemcitabine (Gemcitabine).

The acoustic intensity refers to the acoustic intensity.

Frequency refers to frequency.

The duty cycle is referred to as a duty cycle.

2. Cell and animal model

Human-derived Pancreatic Ductal Adenocarcinoma (PDA) cell line BxPC3, hepatocellular carcinoma (HCC) cell line Huh7, hepatocellular carcinoma vascular endothelial cell line ECDHCC (hepatocellular carcinoma cardiovascular-derived endethiral cells) were purchased from American Type Culture Collection (ATCC).

Male BALB/c nude mice (6-8 weeks old) and male NOD/SCID mice (6-8 weeks old) were provided by the university of medicine laboratory animal, zhejiang. Mice were housed in animal care facilities on a 12h light/dark cycle, on free diet, and animal experiments were approved by the animal ethics committee. The use of tumor samples for clinical PDA and HCC patients was approved by the ethics committee for human research at the second hospital affiliated with the university of zhejiang medical college.

The construction of a subcutaneous human PDA or HCC tumor-loaded BALB/c nude mouse animal model: clinical PDA or HCC tumor samples were taken from patients with PDA or HCC tumors without any prior radiation or chemotherapy. Immediately after the tumor specimen was washed with PBS and cut into small pieces (1X 1 mm) under sterile conditions, the tumor pieces were immersed in Matrigel basement membrane matrix, which were then transplanted subcutaneously into the right side of NOD/SCID mice, and when the subcutaneous tumor diameter reached 6-8mm, the NOD/SCID mice were sacrificed and the tumor tissue was excised. Then, tumor tissues were immediately washed with PBS and cut into small pieces (1X 1 mm) for being transplanted subcutaneously at the right and/or left side of BALB/c nude mice or beside abdominal blood vessels, thereby constructing a BALB/c nude mouse animal model loaded with human-derived PDA or HCC tumor subcutaneously.

3. Preparation of liposomal nanoparticles and characterization thereof

3.1 preparation of Liposomal nanoparticles

3.1.1 Preparation of LPGL liposome nanoparticle dispersion liquid:

(1) 3.0mg DPPC, 3.0mg DSPE-PEG2000-RGD, 2.0mg DSPE-PEG2000 and 2mg gemcitabine prodrug CP4126 were dissolved in 3mL chloroform in a 10mL round bottom flask, and the organic solvent was removed by rotary evaporation at 40 ℃ under reduced pressure to form a lipid film in the round bottom flask.

(2) The lipid film was cooled to 4 ℃, 100 μ L of perfluoro-n-pentane (perfluoropentane) was added to impregnate the lipid film, then 5mL of a phosphate buffer solution containing glycerol (phosphate concentration: 10mm, ph =7.4, volume fraction of glycerol: 10% (v/v)) was added for hydration, and after stirring with a magnetic stirrer bar at 4 ℃ for 30min, the mixture was then stirred with a magnetic stirrer bar in a 30 ℃ water bath under open conditions in a round-bottomed flask for 1h to obtain LPGL liposome nanoparticle dispersion.

3.1.2 Preparation of LGL liposome nanoparticle dispersion:

(2) The lipid film was cooled to 4 ℃, 5mL of a phosphate buffer containing glycerol (phosphate concentration of 10mm, ph =7.4, volume fraction of glycerol of 10% (v/v)) was added for hydration, and after stirring with a magnetic stirrer at 4 ℃ for 30min, the round-bottomed flask was then stirred with a magnetic stirrer in a 30 ℃ water bath under open conditions for 1h, to obtain an LGL liposome nanoparticle dispersion.

3.1.3 Preparation of PGL Liposome nanoparticle Dispersion

(1) 4.8mg DPPC, 3.2mg DSPE-PEG2000 and 2mg gemcitabine prodrug CP4126 were dissolved in 3mL chloroform in a 10mL round bottom flask, the organic solvent was removed by rotary evaporation at 40 ℃ under reduced pressure, and a lipid film was formed in the round bottom flask.

(2) The lipid film was cooled to 4 ℃, 100 μ L of perfluoro-n-pentane (perfluoropentane) was added to impregnate the lipid film, then 5mL of a phosphate buffer solution containing glycerol (phosphate concentration: 10mm, ph =7.4, volume fraction of glycerol: 10% (v/v)) was added for hydration, and after stirring with a magnetic stirrer bar at 4 ℃ for 30min, the round-bottomed flask was then stirred with a magnetic stirrer bar in a 30 ℃ water bath under open conditions for 1h to obtain a PGL liposome nanoparticle dispersion.

3.1.4 Preparation of GL liposome nanoparticle dispersion liquid

(2) The lipid membrane was cooled to 4 ℃, 5mL of a phosphate buffer containing glycerol (phosphate concentration: 10mm, ph =7.4, volume fraction of glycerol: 10% (v/v)) was added for hydration, and after stirring with a magnetic stirrer at 4 ℃ for 30min, the round-bottomed flask was then stirred with a magnetic stirrer in a 30 ℃ water bath under open conditions for 1h to obtain a GL liposome nanoparticle dispersion.

3.1.5 Preparation of LPL blank liposome nanoparticle dispersion liquid

(1) 5.0mg of DPPC, 3.0mg of DSPE-PEG2000-RGD and 2.0mg of DSPE-PEG2000 were dissolved in 3mL of chloroform in a 10mL round-bottomed flask, and the organic solvent was evaporated under reduced pressure at 40 ℃ to form a lipid film in the round-bottomed flask.

(2) The lipid film was cooled to 4 ℃, 100 μ L of perfluoro-n-pentane (perfluoropentane) was added to impregnate the lipid film, then 5mL of a phosphate buffer solution containing glycerol (phosphate concentration: 10mm, ph =7.4, volume fraction of glycerol: 10% (v/v)) was added for hydration, and after stirring with a magnetic stirrer bar at 4 ℃ for 30min, the flask was then stirred with a magnetic stirrer bar in a 30 ℃ water bath under open conditions for 1h to obtain an LPL blank liposome nanoparticle dispersion.

3.1.6 Preparation of Cy5 or Cy5.5 labeled Liposomal nanoparticle Dispersion

The preparation method of Cy5 or Cy5.5 labeled liposome nanoparticle dispersion liquid is the same as that of each liposome nanoparticle dispersion liquid, except that 0.3mg of DSPE-PEG2000 is replaced by 0.3mg of Cy5 labeled DSPE-PEG2000 (DSPE-PEG 2000) ^Cy5 ) Or 0.3mg Cy5.5-labeled DSPE-PEG2000 (DSPE-PEG 2000) ^Cy5.5 ) And obtaining Cy5 or Cy5.5 labeled LPGL, LGL, PGL, GL or LPL liposome nanoparticle dispersion liquid.

3.2 epilography and characterization of Liposomal nanoparticles

The particle size and zeta potential of the liposomal nanoparticles were measured using a dynamic light scattering analyzer (Nano-ZS 90, malvern), with the refractive index of the liposomal nanoparticles selected to be 1.59 to determine particle size by percent intensity (% intensity).

The morphology of the liposomal nanoparticles was imaged by cryo-transmission electron microscopy (cryo-TEM) (Talos F200C 200kv, feiinc.) on a carbon-coated 200 mesh copper TEM grid.

Fluorescence spectra and fluorescence intensities of Cy 5-or Cy5.5-labeled liposome nanoparticles were measured with a microplate reader (SpectraMaxM 5, molecular devices).

The liposome nanoparticle dispersion (1 mL) was dialyzed (Mw cutoff 3.5 kDa) in 100mL PBS buffer (containing 5v/v% glycerol) for 24h, and the concentration of CP4126 (GEM molar equivalent) in the dialysate was determined by high performance liquid chromatography. The Entrapment Efficiency (EE) and GEM loading efficiency (LR) were calculated using the following formulas (1) and (2), respectively. The Loading (LC) of perfluoro-n-pentane was determined using gas chromatography-mass spectrometry and calculated using equation (3):

EE = mass GEM in liposomes/mass of total GEM x 100% (formula 1);

LR = GEM mass in liposome/total mass (lipid + CP 4126) × 100% (formula 2);

LC = volume of perfluoro-n-pentane/(volume of perfluoro-n-pentane + volume of PBS) × 100% (formula 3).

The encapsulation rate of the liposome nano-particles is more than 99.5 percent and the GEM load rate is more than 9.5 percent.

cryo-TEM images of the prepared LPGL liposome nanoparticles under different treatment conditions are shown in FIG. 1, and the particle sizes and potentials of the prepared GL, LGL, PGL and LPGL liposome nanoparticle dispersions after dilution in PBS 7.4 buffer or mouse plasma are shown in Table 1. As can be seen from table 1, the GL, LGL, PGL and LPGL liposomal nanoparticles increased in particle size and decreased zeta potential after incubation in plasma, indicating that the GL, LGL, PGL and LPGL liposomal nanoparticles adsorb plasma proteins. The prepared PGL liposome nanoparticles are incubated in plasma and then irradiated by ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% duration 5 min) before and after the treatment as shown in fig. 2, it can be further seen from the cryo-TEM images of fig. 1 and 2 that plasma proteins were able to form protein corona (protein corona) on the surface of the liposome nanoparticles after the plasma was incubated with the liposome nanoparticles, thereby further confirming that the plasma proteins were adsorbed on the surface of the liposome nanoparticles. And it can be seen from the cryo-TEM images of FIGS. 1 and 2 that the incubates of LPGL and PGL liposome nanoparticles and plasma were irradiated with ultrasound (sound intensity) Degree: 2W/cm ² Frequency: 3MHz, duty cycle: 50%, time: 5 min), the protein corona around the surface of the liposome nanoparticles disappeared.

cryo-TEM in FIG. 1 shows LPGL liposomal nanoparticles with a uniform monolayer lipid membrane in PBS 7.4 and plasma, with perfluoron-pentane loading showing a pronounced icecloud shade within the liposomes and a perfluoron-pentane Loading (LC) of 0.16vol%.

The cryo-transmission electron microscopy of GL, LGL and PGL liposome nanoparticle dispersions is shown in fig. 3, and it can be seen from fig. 1 and 3 that the inside of GL liposomes and LGL liposomes not loaded with perfluoro-n-pentane exhibit distinct empty particles, while the inside of PGL liposomes exhibit distinct shade of ice cloud of perfluoro-n-pentane and have a uniform monolayer lipid membrane, compared to LPGL liposomes.

TABLE 1 particle size and potential of GL, LGL, PGL and LPGL liposomal nanoparticles in PBS 7.4 or plasma

The operation of incubation of each liposomal nanoparticle in plasma was as follows: the GL, LGL, PGL or LPGL liposomal nanoparticle dispersions were mixed with mouse plasma at a lipid/protein mass ratio of 1/50, respectively, and incubated in a shaker (60 rmp) at 37 ℃ for 30min.

4. Preparation and obtaining of protein coronas

Blood was collected from the mice through the orbital venous plexus, then mixed with heparin solution (1 mg/m,50 μ L), and plasma was separated by centrifugation at 5,000rpm for 5min at 4 deg.C and centrifuged at 20,000g for 30min before use to remove any aggregated proteins. Plasma protein concentration was measured to be 61mg/mL using the enhanced BCA protein assay kit, using Bovine Serum Albumin (BSA) as a control standard. The Cy 5-labeled GL, LGL, PGL or LPGL liposomal nanoparticle dispersions were mixed with plasma at a lipid/protein mass ratio of 1/50, respectively, and after incubation in a shaker (60 rmp) at 37 ℃ the mixture was sampled for 1mL and irradiated with or without ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50 percent ofDuration 5 min) and then immediately poured onto Sephadex G200 chromatography and eluted with 5 volumes of PBS buffer for separation. The eluate was lyophilized using a lyophilizer and then re-dissolved in 200 μ L RIPA lysis buffer to give a protein corona solution in liposome nanoparticles, and the protein concentration in the protein corona solution was determined using the enhanced BCA protein assay kit with BSA as a control standard.

Further, a Cy 5-labeled PGL or LPGL liposome nanoparticle dispersion liquid and plasma were mixed at a lipid/protein mass ratio of 1/50, followed by incubation in a shaker (60 rmp) at 37 ℃ for 15min to obtain a mixture, PGL or LPGL liposome nanoparticles containing a protein corona separated by Sephadex G200 chromatography without ultrasonic irradiation were added to a PBS7.4 buffer solution, and ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%) of the protein, then pouring the mixture into Sephadex G200 chromatography, eluting the mixture by using PBS7.4 buffer solution with 5 times of volume for separation, using a freeze dryer to freeze-dry the eluent, then re-dissolving the eluent in 200 mu L of RIPA lysis buffer solution to obtain protein corona solution, using an enhanced BCA protein assay kit to determine the protein concentration in the protein corona solution by taking BSA as a standard, and processing the protein corona modified PGL or LPGL liposome nanoparticles at different ultrasonic irradiation times to obtain the total protein concentration in the protein corona as shown in figure 4, wherein the ultrasonic irradiation can remove more than 90% of the protein corona on the surface of the PGL or LPGL liposome nanoparticles as shown in figure 4.

5. Determination of protein coronas by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

For SDS-PAGE determination of protein corona, GL, LGL, PGL or LPGL liposome nanoparticles were incubated with mouse plasma for 15min, and the resulting incubation mixture was irradiated with or without ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% for 5 min), sephadex G200 chromatographic separation to obtain 20 μ L protein corona solution, mixing with 4 μ L SDS-PAGE sample buffer solution, and applying to Tris-Gly protein gel (Beyogel) ^TM Plus Precast PAGE Gel), pre-stained with a colored Protein Standard (Prestained Color Protein Standard Marker, 10-18)0kDa,) as molecular markers, run at 140V in Tris-Glycine running buffer for 60min. The gel was then stained with simprolyblue SafeStain and analyzed using Azure c600 Imager.

Incubating GL, LGL, PGL or LPGL liposome nanoparticles with plasma for 15min to obtain an incubation mixture, and optionally subjecting the incubation mixture to ultrasonic irradiation (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% for 5 min), and an SDS-PAGE analysis chart of a protein corona solution in the liposome nanoparticles obtained by Sephadex G200 chromatographic separation is shown in FIG. 5A of FIG. 5, and as can be seen from FIG. 5A, after the liposome nanoparticles are incubated with plasma for 15min, the surfaces of GL, LGL, PGL and LPGL liposome nanoparticles absorb a large amount of protein (molecular weight is from 25kDa to 180 kDa), while ultrasonic irradiation can significantly reduce the protein content on the surfaces of PGL and LPGL liposome nanoparticles, thereby eliminating the protein corona on the surfaces of PGL and LPGL liposome nanoparticles, while the protein content on the surfaces of GL and LGL liposome nanoparticles has no significant change, thereby indicating that ultrasonic irradiation can eliminate the protein corona on the surfaces of PGL or LPGL liposome nanoparticles.

6. High performance liquid chromatography-mass spectrometry (HPLC-MS) for determining protein crown

Firstly, after GL, LGL, PGL or LPGL liposome nanoparticles and mouse plasma are incubated for 15min, the obtained incubation mixture is irradiated with or without ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% for 5 min), a protein corona solution sample in the liposome nanoparticles is obtained by Sephadex G200 chromatographic separation, the protein corona solution sample is mixed with trypsin in a 1/50 mass ratio (enzyme/protein) and then digested, then diluted to 5-fold volume with 0.1% formic acid aqueous solution, and 100 fmol/. Mu.l Hi3 EColi standard is added for absolute quantification, and the protein sample is quantitatively analyzed using high performance liquid coupled mass spectrometry (HPLC-MS).

After GL, LGL, PGL or LPGL liposome nanoparticle and plasma incubation mixture is subjected to or without ultrasonic irradiation, HPLC-MS determines total content of protein on the liposome nanoparticle as shown in fig. 5B of fig. 5, as can be seen from fig. 5B, after the liposome nanoparticle and plasma are incubated for 15min, a large amount of protein is absorbed on the surface of the GL, LGL, PGL and LPGL liposome nanoparticle, and the total content of protein corona reaches 21-24 μ g/mg lipid, however, ultrasonic irradiation can significantly reduce the protein content on the surface of the PGL and LPGL liposome nanoparticle, and the protein content on the surface of the LPGL and PGL liposome nanoparticle is reduced by at least 80%, while the protein content on the surface of the GL and LGL liposome nanoparticle is not significantly changed, so that the surface ultrasonic irradiation can effectively eliminate the protein corona on the surface of the PGL or LPGL liposome nanoparticle.

After being incubated with the plasma of the mouse, the LPGL liposome nanoparticles are not subjected to ultrasonic irradiation treatment, protein crown solution samples obtained by Sephadex G200 chromatographic layer separation are subjected to HPLC-MS determination after being treated, and the protein in the protein crowns is identified by a Uniprot database, wherein the results are shown in Table 2:

TABLE 2 qualitative and quantitative analysis of proteins in the corona of LPGL liposomes without ultrasonic irradiation treatment for the first 10 plasma proteins with the highest content in the corona of proteins on the surface of LPGL liposomes nanoparticles using HPLC-MS and UniProt database

In addition, after the PGL or LPGL liposome nanoparticles and plasma are incubated for 15min and treated by ultrasonic irradiation (frequency: 3MHz, duty ratio: 50%, duration 5 min) with different sound intensities, a protein corona solution sample in the liposome nanoparticles is obtained by Sephadex G200 chromatographic layer separation, and the total content of protein on the liposome nanoparticles is measured by HPLC-MS after treatment as shown in FIG. 6, and it can be seen from FIG. 6 that the increase of the ultrasonic irradiation intensity can enhance the capability of eliminating the protein corona on the PGL or LPGL liposome nanoparticles.

7. Cellular uptake

BxPC3 cells (1X 10) ⁵ Individual cells/mL, 1 mL) were seeded in 12-well plates and cultured for 24h. Cy 5-labeled GL, LGL, PGL and LPGL liposome nanoparticle dispersions (all equivalent to 60. Mu.g/mL fluorescent lipid, 20. Mu.L) were mixed with 1mL of fresh serum-free medium or medium containing 10% FBS (fetal bovine serum) for 30min, and the wells were washed with PBS BxPC3 cells in the plate are followed by the addition of a mixture of different liposome nanoparticles and serum-free medium or medium containing 10% FBS (fetal bovine serum) and irradiation with or without ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% for 5 min) and incubation is continued for 1h. Cells were washed 3 times with heparin sodium solution (2 mg/ml), digested, collected, and analyzed using flow cytometry to determine the mean fluorescence intensity of Cy 5-labeled different liposomal nanoparticles taken up by BxPC3 cells.

The efficiency of uptake of liposomal nanoparticles by BxPC3 cells under different conditions is shown in fig. 7A of fig. 7, and it can be seen from fig. 7A that the efficiency of uptake of RGD ligand-modified LGL and LPGL liposomal nanoparticles by BxPC3 cells is significantly higher than that of GL and PGL liposomal nanoparticles under the condition of mixing liposomal nanoparticles and serum-free medium, however, the efficiency of uptake of RGD ligand-modified LGL and LPGL liposomal nanoparticles by BxPC3 cells was significantly reduced without ultrasonic irradiation when the liposomal nanoparticles and a mixture containing 10% fbs (fetal bovine serum) were mixed and incubated with BxPC3 cells, and similar to the efficiency of uptake of GL and PGL liposomal nanoparticles, indicating that the protein corona adsorbed on the surface of liposomal nanoparticles can significantly inhibit recognition of RGD ligands for BxPC3 cell receptors, thereby reducing the efficiency of uptake of RGD ligand-modified LGL and GL liposomal nanoparticles by BxPC3 cells. However, the mixture of liposome nanoparticles and a medium containing 10% FBS (fetal bovine serum) was subjected to ultrasonic irradiation (sound intensity: 2W/cm) while being incubated in admixture with BxPC3 cells ² Frequency: 3MHz, duty cycle: 50% for 5 min) treatment was able to enhance again the uptake efficiency of the RGD ligand-modified LPGL liposomal nanoparticles by the BxPC3 cells and the uptake efficiency was restored to substantially the same order of magnitude as in the mixing with serum-free medium, whereas the mixture of LGL, GL and PGL liposomal nanoparticles and 10% fbs (fetal bovine serum) -containing medium was subjected to ultrasonic irradiation (sound intensity: 2W/cm ² Frequency: 3MHz, duty cycle: 50% for 5 min), the cellular uptake efficiency of the BxPC3 cells on LGL, GL and PGL liposome nanoparticles remained low, indicating that ultrasonic irradiation was able to eliminate LPGThe protein corona on the surface of the L liposome nanoparticle has the effect of covering ligand RGD on the surface of the liposome nanoparticle, and the effect of enhancing the ligand RGD mediated cell uptake on the surface of the liposome nanoparticle is recovered.

8. Subcellular distribution

BxPC3 cells (1X 10) ⁵ Individual cells/mL, 1 mL) were cultured in a confocal dish for 24h, nuclei were stained with Hoechst 33342 for 20min, and lysosomes were used

Green DND26 (0.2. Mu.L) was stained for 30min. Cy 5-labeled LPGL liposome nanoparticles (equivalent to 60. Mu.g/mL of fluorescent lipid, 20. Mu.L) were mixed with 1mL of fresh serum-free medium or medium containing 10% FBS (fetal bovine serum) for 30min, respectively, to give a preincubation mixture. BxPC3 cells were washed twice with PBS and 1mL of the preincubation mixture was added, with or without ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% for 5 min), incubation is continued for 1h, and subcellular distribution maps are taken using a laser scanning confocal microscope (CLSM) with 405nm, 488nm and 640nm wavelength channels. The operator overlap factor of LPGL liposome nanoparticles with lysosomes after incubation was analyzed using the image analysis software Cellprofiler v2.2.0.

After mixing the pre-incubated mixture of Cy 5-labeled LPGL liposomal nanoparticles with and without serum with BxPC3 cells, the subcellular distribution in the presence and absence of ultrasonic irradiation treatment is shown in fig. 8, and as can be seen in fig. 8A and 8B, the pre-incubated mixture of LPGL liposomal nanoparticles and serum-free medium after mixing and incubating with BxPC3 cells, without ultrasonic irradiation treatment, found that LPGL liposomal nanoparticles are mainly distributed in the cytoplasm and rarely distributed in the lysosome, whereas the pre-incubated mixture of LPGL liposomal nanoparticles and medium containing 10 fbs (fetal bovine serum) after mixing and incubating with BxPC3 cells, found that more than 65% of the intracellular LPGL liposomal nanoparticles are distributed in the lysosome, without ultrasonic irradiation treatment. When a pre-incubation mixture of LPGL liposome nanoparticles and a medium containing 10% FBS (fetal bovine serum) was mixed with BxPC3 cells and incubated, the mixture was subjected to ultrasonic irradiation treatment (sound intensity: 2W/cm) ² Frequency:3MHz, duty cycle: 50% and lasting for 5 min), the LPGL liposome nanoparticles are recovered and mainly distributed in cytoplasm, therefore, ultrasonic irradiation enables the LPGL liposome nanoparticles containing protein crowns to have excellent lysosome escape and lysosome degradation prevention capability, the lysosome escape can effectively prevent the drug-loaded nanoparticles and the drugs loaded by the drug-loaded nanoparticles from being damaged and degraded by degradation enzymes in lysosomes, the stability of the drug-loaded nanoparticles and the drugs loaded by the drug-loaded nanoparticles in cells is improved, and the treatment effect of the drugs on diseases is improved.

9. Transvascular endothelial cell transport

The Transwell system was used to study transcellular transport of liposomes in vascular endothelial cells. ECDHC vascular endothelial cells (5X 10) ⁵ Individual cells/mL, 1 mL) were incubated in the apical compartment for 4 days to form a dense cell layer. BxPC3 cells (1X 10) ⁵ Individual cells/mL, 1 mL) were inoculated into the basolateral compartment for 12h incubation. The apical compartment was placed on the basolateral compartment for an acclimatization of 6 h. Cy 5-labeled GL, LGL, PGL or LPGL liposome nanoparticle dispersions (both equivalent to 1.2mg/mL total lipid, 2.5 mL) were mixed with mouse plasma (2.5 mL) at a lipid/protein mass ratio of 1/50 or serum-free medium (2.5 mL), respectively, followed by incubation in a shaker (60 rmp) at 37 ℃ for 30min to give a plasma or serum-free medium pre-incubation mixture containing Cy 5-labeled liposome nanoparticles, plasma or serum-free medium pre-incubation mixture containing Cy 5-labeled liposome nanoparticles was added to the apical compartment of a Transwell containing serum-free medium, without ultrasonic irradiation treatment, cultured continuously for 3h in the Transwell, the fluorescence intensity of the substrate-lateral medium was measured using a microplate reader, flow-type BxPC3 cells were collected and the Cy5 fluorescence intensity was determined by cytometry. Furthermore, in order to distinguish between the ultrasound irradiation induced transport through the gaps between endothelial cells or ligand/receptor mediated transport across vascular endothelial cells, two procedures were designed (as shown in fig. 7B): (mode I) plasma pre-incubation mixture (equivalent to 60. Mu.g/mL of fluorescent lipid, 20. Mu.L) containing Cy 5-labeled liposome nanoparticles was first subjected to ultrasonic irradiation in a centrifuge tube (ultrasonic irradiation has no effect on vascular endothelial cells ECDCC, and sound intensity is 2W/cm ² Frequency: 3MHz, duty cycle: 50% duration: 5 min) and then added to the apical compartment (non-contact condition); (mode II) the plasma pre-incubation mixture (equivalent to 60. Mu.g/mL of fluorescent lipid, 20. Mu.L) containing Cy 5-labeled liposomal nanoparticles was first added to the apical compartment, followed by ultrasonic irradiation in the apical compartment (ultrasonic irradiation produces a cavitation effect on vascular endothelial cells ECDCC, with a sound intensity of 2W/cm ² Frequency: 3MHz, duty cycle: 50% with duration: 5 min) pretreatment (contact conditions); then, the culture was continued for 3 hours in a Transwell, the fluorescence intensity of the substrate-outside medium was measured using a microplate reader, bxPC3 cells were collected and the Cy5 fluorescence intensity was measured by flow cytometry.

Trans-vascular endothelial cell ECDHCC transport of different liposomal nanoparticles under different conditions as shown in fig. 7C of fig. 7, it can be seen from fig. 7C that LGL and LPGL liposomal nanoparticles both exhibited the highest trans-vascular endothelial cell ECDHCC transport capacity under the serum-free medium pre-incubation mixture of liposomal nanoparticles and the non-ultrasonic irradiation condition, whereas the trans-vascular endothelial cell transport capacity of LGL and LPGL liposomal nanoparticles was significantly reduced under the plasma pre-incubation mixture of liposomal nanoparticles and the non-ultrasonic irradiation condition, and the trans-vascular endothelial cell transport capacity of GL and PGL was low and did not significantly change under the serum-free medium pre-incubation mixture of GL and PGL, or the plasma pre-incubation mixture and the non-ultrasonic irradiation condition. Under the conditions of a liposome nanoparticle plasma pre-incubation mixture and ultrasonic irradiation, the trans-vascular endothelial cell transport efficiency of PGL and LPGL liposome nanoparticles is remarkably changed, and different trans-vascular endothelial cells are expressed along with different treatment methods, which are specifically as follows: compared with the efficiency of trans-vascular endothelial cell transport under the condition of no ultrasonic irradiation of the plasma pre-incubation mixture of the liposome nanoparticles, the plasma pre-incubation mixture of the Cy 5-labeled liposome nanoparticles uses ultrasonic irradiation (non-contact condition) in a centrifuge tube, so that the trans-vascular endothelial cell transport efficiency of the LPGL liposome nanoparticles is remarkably enhanced and is recovered to the same trans-vascular endothelial cell transport level as that of the serum-free culture medium incubation mixture, while the transport efficiency of GL, LGL and PGL liposome nanoparticles is not remarkably changed, thereby indicating that the ultrasonic irradiation can overcome the masking effect of protein corona on the surface of the LPGL liposome nanoparticles on ligands (RGD) on the surface of the liposome nanoparticles, and the ultrasonic irradiation recovers the effect of the RGD ligands of LPGL and vascular endothelial cell receptor mediated trans-vascular endothelial cell transport. However, the trans-vascular endothelial cell transport capacity of LPGL liposomal nanoparticles was significantly higher than that of non-contact conditions and was about 3-4 times higher than that of GL, LGL and PGL liposomal nanoparticles when the plasma pre-incubation mixture of Cy 5-labeled liposomal nanoparticles was added to apical compartment for ultrasound irradiation (contact conditions), indicating that ultrasound irradiation was more able to significantly enhance the trans-vascular endothelial cell transport of LPGL liposomal nanoparticles under contact conditions.

10. Endocytic pathway of tumor cells

BxPC3 cells (1X 10) ⁵ cells/mL, 1 mL) were cultured in 12-well plates for 24h, then the medium was changed to 1mL fresh serum-free medium, chlorpromazine (50 μ M, clathrin-mediated endocytosis inhibitor), genistein (genistein, 200 μ M, pit-mediated endocytosis inhibitor), wortmannin (wortmannin, 5 μ M, phosphatidylinositol 3-kinase-mediated macropinocytosin inhibitor) or cytochalasin D (cytochalasin D,5 μ M, actin polymerization inhibitor) endocytosis inhibitor were added, respectively, incubated with BxPC3 cells for 2h, while a group of BxPC3 cells not treated with any endocytosis inhibitor was selected as a blank for 2h. Then, serum-containing medium or serum-free medium mixture supplemented with Cy 5-labeled LPGL liposome nanoparticles (equivalent to 60. Mu.g/mL of fluorescent lipid, 20. Mu.L) was added to the above-mentioned wells containing BxPC3 cells, respectively, followed by irradiation with or without ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), after incubation for 3h, the cells were washed, digested and collected in a centrifuge tube and the Mean Fluorescence Intensity (MFI) was detected by flow cytometry.

After pretreatment of BxPC3 with different endocytosis inhibitors, flow cytometry was used to determine the mean fluorescence intensity of Cy5 in BxPC3 cells with or without sonication after incubation of the serum-containing medium or serum-free culture mixture supplemented with Cy 5-labeled LPGL liposomal nanoparticles with BxPC3 cells as shown in fig. 7D, and as can be seen from fig. 7D, under the conditions of BxPC3 cell incubation with Cy 5-labeled LPGL liposomal nanoparticle serum-free mixture and without sonication, the inhibition rates of chlorpromazine, genistein, wortmannin, and cytochalasin D on BxPC3 endocytosis LPGL by BxPC3 cells were 21.1%, 57.6%, 13.4%, and 19.9%, respectively, indicating that the uptake of BxPC3 cellular LPGL liposomal gl nanoparticles was mainly driven by pit-mediated endocytosis. However, under the conditions of incubation of BxPC3 cells with a serum-containing mixture of Cy 5-labeled LPGL liposome nanoparticles and no ultrasonic irradiation, the protein corona on the surface of LPGL liposome nanoparticles significantly affected the rate of inhibition of the endocytosis of LPGL liposome nanoparticles, and compared with the blank control group of BxPC3 cells which were not treated with the endocytosis inhibitor, the rates of inhibition of the endocytosis of BxPC3 cells by chlorpromazine, genistein, wortmannin and cytochalasin D were 35.9%, 28.9%, 23.5% and 10.2%, respectively, indicating that the uptake of LPGL liposome nanoparticles by BxPC3 cells under serum conditions is a mixed uptake pathway of clathrin-mediated endocytosis, crypt-mediated endocytosis and endocytosis. However, in the case of BxPC3 cells incubated with the serum-containing mixture with Cy 5-labeled LPGL liposome nanoparticles and with ultrasonic irradiation, inhibition rates of chlorpromazine, genistein, wortmannin, and cytochalasin D on the internalization of LPGL by BxPC3 cells were 24.4%, 52.5%, 24.8%, and 17.0%, respectively, compared to the blank control group without the addition of the endocytosis inhibitor to the BxPC3 cells, thereby indicating that ultrasonic irradiation was able to remove the protein corona on the surface of the liposome nanoparticles.

11. Transcytosis transport between tumor cells

BxPC3 cells (1X 10) ⁵ Individual cells/mL, 1 mL) were cultured in confocal culture dishes for 24h, then the medium was replaced with 1mL of fresh serum-free medium. Then, serum-containing medium or serum-free medium mixture supplemented with Cy 5-labeled GL, LGL, PGL or LPGL liposomal nanoparticles (equivalent to 60. Mu.g/mL of fluorescent lipid, 20. Mu.L) was added to the confocal dishFollowed by irradiation with or without ultrasound (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), after 3h incubation, the dishes were washed twice with PBS and 1mL contained new 1X 10 ⁵ Serum-free medium of individual BxPC3 cells was added to the culture dish and trans-cellular transport of liposomes from previously added cells to newly added cells was photographed at different culture time points using CLSM with 640nm excitation wavelength. Finally, the newly added cells are easily washed away, collected in a centrifuge tube, and tested with a flow cytometer. Meanwhile, after pretreatment of the previously added BxPC3 cells with exocytosis inhibitor EXO1 (20. Mu.M) was measured using the same method, cy 5-labeled LPGL liposome nanoparticles and a serum-containing medium or serum-free medium mixture were added, followed by irradiation with or without ultrasonic waves (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), then, 1mL of a solution containing new 1X 10 ⁵ Serum-free medium of individual BxPC3 cells was added to the culture dish and trans-cellular transport of liposomes from previously added cells to newly added cells was photographed at different culture time points using CLSM with 640nm excitation wavelength.

Under the condition that BxPC3 cells are not pretreated or are pretreated by exocytosis inhibitor EXO1, a serum-containing culture medium or a serum-free culture medium mixture added with Cy5 labeled GL, LGL, PGL or LPGL liposome nano-particles is mixed with the BxPC3 cells and then is not subjected to or is subjected to ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), and at different culture time points after adding new BxPC3 cells, the CLSM profile of the newly added BxPC3 cells is shown in FIG. 7E and FIG. 9; the Mean Fluorescence Intensity (MFI) of Cy5 in the newly added BxPC3 cells was quantitatively determined by flow cytometry after the new BxPC3 cells were added to the culture dish for 2h as shown in fig. 7F. As can be seen from FIGS. 7E, 7F, and 9, the GL, LGL, and PGL liposome nanoparticles were difficult to transport to the newly added BxPC3 cells when the serum-containing medium mixture to which the GL, LGL, and PGL liposome nanoparticles were added was mixed with BxPC3 cells and then treated with ultrasonic irradiation, but the serum-containing medium mixture to which the LPGL liposome nanoparticles were added was difficult to transport to the newly added BxPC3 cells The LPGL liposome nanoparticles can be effectively transported to newly added BxPC3 cells (the trend is also inhibited by an exocytosis inhibitor of EXO 1) by mixing the culture medium mixture with the BxPC3 cells and the transport capacity is similar to the capacity of the serum-free culture medium mixture added with the LPGL liposome nanoparticles and the BxPC3 cells without ultrasonic irradiation treatment after mixing, thereby indicating that the ultrasonic irradiation can eliminate the masking effect of a protein crown on the surface of the LPGL liposome nanoparticles on ligands (RGD) on the surface of the liposome nanoparticles and restore the ligand (RGD) mediated intercellular transport on the surface of the liposome nanoparticles.

Liposomal nanoparticles can be transported from one cell to another under different conditions. GL, LGL and PGL were hardly transported to newly added BxPC3 cells, while LPGL could be efficiently transported to newly added cancer cells after co-incubation (fig. 7E and 7F and 9). In particular, in the absence of ultrasonic irradiation, the intercellular transport of LPGL was significantly reduced, and this tendency was also inhibited by exocytosis inhibitors of EXO1, indicating that LPGL was transported between cancer cells by transcellular action. In addition, under ultrasonic irradiation, the intercellular transport of LPGL can reach a level similar to that in serum-free medium, confirming that ultrasonic irradiation can effectively eliminate the protein corona on the surface of liposome nanoparticles and restore the ligand-mediated intercellular transport of LPGL.

12. Cytotoxicity and apoptosis assays

Cytotoxicity of different liposomal nanoparticles was tested on three-dimensional (3D) multicellular tumor spheroids of BxPC3 and Huh7, which were established using the hanging drop method. LPGL, PGL, LGL, GL or Gemcitabine (GEM) and serum-containing medium were mixed in advance for 30min, and then added to a well plate containing BxPC3 or Huh7 three-dimensional (3D) multicellular tumor 3D spheres at a GEM concentration of 0 to 10. Mu.M and irradiated with ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% for 5 min), and culturing for 72h. Meanwhile, LPL blank liposome nanoparticles in an equimolar ratio to LPGL were used as a control. Then, the medium was replaced with a mixture of 90. Mu.L of fresh medium and 10. Mu.L of Alamar Blue Cell Viability Reagent, and the culture was continued for 12h. Then using a plate reader at an excitation wavelength of 530nmAnd 590nm emission wavelength to obtain a fluorescence intensity reading. In addition, liposome nanoparticle-induced apoptosis in 3D multicellular tumor spheroids was further determined by the TdT-mediated dUTP nick-end labeling (TUNEL) method, and positive TUNEL-stained cells were detected by CLSM.

The inhibitory effect of LPGL, PGL, LGL, GL or Gemcitabine (GEM) and serum-containing media mixture (GEM concentration 0-10 μ M) on BxPC3 or Huh7 three-dimensional (3D) multicellular tumor 3D spheroids after incubation with ultrasonic irradiation treatment, cultured for 72h, gemcitabine (GEM) and different liposomal nanoparticles on BxPC3 tumor 3D spheroid viability as shown in fig. 10A and 10B of fig. 10, it can be seen from fig. 10A and 10B that both free GEM and liposomal nanodroplets show dose-dependent cytotoxicity on BxPC3 and Huh7 three-dimensional (3D) multicellular tumor spheroids, LPGL being the most cytotoxic, while GL, LGL and PGL have low cytotoxicity. Fig. 10C shows the apoptosis induced by LPGL, PGL, LGL, GL liposomal nanoparticles and Gemcitabine (GEM) on BxPC 3D multicellular tumor spheroids by light microscopy and TUNEL staining, and it can be seen from fig. 10C that LPGL liposomal nanoparticles cause the tumor perimeter of the spherically smooth BxPC3 tumor spheroids to become irregularly collapsed morphology, and the BxPC3 tumor spheroid center also erodes, apoptotic cells are distributed throughout the tumor spheroid, while free Gemcitabine (GEM) and the BxPC3 tumor spheroids before and after PGL, LGL, GL liposomal nanoparticle treatment do not significantly change in morphology and there are fewer apoptotic cells distributed around the tumor spheroid, indicating that LPGL liposomal nanoparticles have significantly superior tumor penetration capacity compared to PGL, LGL, GL liposomal nanoparticles.

13. Penetration of tumor spheres

BxPC 3D multicellular tumor spheroids without or pre-treated with exocytosis inhibitor EXO1 (20 μ M) were transferred to confocal culture dishes containing fresh serum-free medium. Cy 5-labeled different liposome nanoparticles (equivalent to 60. Mu.g/mL of fluorescent lipid, 30. Mu.L) and a mixture containing serum medium were added, followed by ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration 5 min), incubate for 6h. Washing with PBSThereafter, CLSM assay was performed (images were taken from apex to equator at 25 μm intervals in XYZ-3D-stack).

The mixture of different Cy 5-labeled liposome nanoparticles and serum-containing medium was added to 3D multicellular tumor spheroids of BxPC3 pretreated with exocytosis inhibitor EXO1 or not, and irradiated with ultrasound (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50% duration 5 min) and permeation of 3D multicellular tumor spheres of BxPC3 by different Cy 5-labeled liposomal nanoparticles as shown in fig. 10D and 10E of fig. 10, it can be seen from fig. 10D that GL, LGL and PGL liposomal nanoparticles are mainly distributed at the periphery of the 3D spheres of BxPC3, while LPGL liposomal nanoparticles can penetrate deeply into the 3D spheres of BxPC3 and are distributed throughout the spheres, and furthermore, fig. 10E shows that the permeation of LPGL liposomal nanoparticles through the 3D multicellular tumor spheres of BxPC3 is about 2.7-10 times as high as GL, LGL and PGL liposomal nanoparticles by measuring the average Integrated Optical Density (IOD) of 75 μm and 100 μm thick layers of the 3D spheres of BxPC3 to evaluate the permeation capability of liposomal nanoparticles, indicating that LPGL liposomal nanoparticles have excellent tumor permeation capability. However, LPGL liposomal nanoparticles were confined to the periphery of the spheres after EXO1 pretreatment of 3D multicellular tumor spheres of BxPC3, LPGL significantly decreased the mean Integrated Optical Density (IOD) of 75 μm and 100 μm thick layers of 3D spheres of BxPC3, indicating that exocytosis inhibitor EXO1 was able to significantly inhibit the ability of LPGL liposomal nanoparticles to penetrate 3D spheres of BxPC 3. Therefore, the permeation result of the LPGL lipidosome nano-particles in the 3D sphere of BxPC3 shows that the deep permeation of LPGL depends on an RGD ligand/receptor mediated cell transfer pathway, the ultrasonic irradiation can eliminate the protein corona on the surfaces of the LPGL lipidosome nano-particles, the covering effect of the protein corona on the surfaces of the LPGL lipidosome nano-particles on the ligand (RGD) on the surfaces of the lipidosome nano-particles is overcome, and the ligand (RGD) mediated intercellular transfer on the surfaces of the lipidosome nano-particles is recovered.

14. Blood clearance

BALB/c nude mice inoculated with human PDA tumor subcutaneously and injected with GL, LGL, PGL or LPGL liposome nanoparticle dispersion liquid (the dose is equivalent to GEM 10mg/kg, 3 mice in each group)Mouse) followed by ultrasonic irradiation of the tumor site (sound intensity: 2W/cm ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min). Blood samples (50 μ L) were collected at 2min, 0.5h, 1h, 2h, 4h, 6h, 8h and 12h after tail intravenous injection administration by mixing with heparin solution (1 mg/mL,50 μ L) at orbital venous plexus of mice, separating the supernatant from the blood by centrifugation at 5,000rpm for 5min at 4 ℃, then diluting the supernatant with acetonitrile (900 μ L) to give a mixture, vortexing, sonicating and centrifuging at 5,000rpm for 5min. The supernatant was then taken and the CP4126 content was calculated by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) determination and analyzed for pharmacokinetic parameters.

After tail vein injection of GL, LGL, PGL or LPGL liposome nanoparticles, the blood content of CP4126 at different time points is shown in fig. 11, and it can be seen from fig. 11 that GL, LGL, PGL or LPGL liposome nanoparticles have excellent blood clearance half-life, wherein GL and LGL liposome nanoparticles show similar blood clearance curve, and the elimination half-life is 1.33h, which is longer than that of PGL liposome nanoparticles (elimination half-life is 1.02 h) and LPGL liposome nanoparticles (elimination half-life is 1.17 h).

15. Biodistribution, infiltration and in vivo imaging

The aggregation and penetration of liposome nanoparticles in tumors were studied by BALB/c nude mice subcutaneously loaded with human-derived PDA tumors. In vivo fluorescence imaging and biodistribution of liposome nanoparticles were performed in BALB/c nude mice loaded with PDA tumors on both subcutaneous sides, respectively. Injecting Cy 5-labeled GL, LGL, PGL or LPGL liposome nanoparticle dispersion liquid (the dose is equivalent to GEM 10mg/kg, 3 mice in each group) into tail vein of BALB/c nude mouse loaded with human PDA tumor, and performing ultrasonic irradiation (sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min), the left subcutaneous-loaded human PDA tumor is not subjected to ultrasonic irradiation, the whole body of the mouse is imaged by a Caliper IVIS Lumina II fluorescence spectrum imager equipped with a fluorescence filter set (excitation/emission wavelength is 640/670 nm) 12h after injection administration, and then FI is injected into the tail vein of each mouseTC (fluorescein isothiocyanate) -labeled tomato Lectin (FITC-Lectin, 0.05mg per mouse) and perfused with 2% glutaraldehyde solution 5min after injection into the heart, followed by collection of tumors, heart, liver, spleen, lung, kidney and small intestine with and without ultrasonic irradiation, imaging by photographing with a Caliper IVIS Lumina II fluorescence spectrometer and Living

The software performs fluorescence quantification of each Tissue isolated, the ultrasonically irradiated tumor is frozen in Tissue OCT-Freeze, after cutting into 10 μm thick sections, the images of the penetration of the liposomal nanoparticles in the ultrasonically irradiated tumor site are taken using CLSM and the fluorescence intensity gradient from tumor vessels to the deep tumor region is quantified using Image J software.

Biodistribution, tumor accumulation and infiltration of different Cy 5-labeled liposome nanoparticles in BALB/c nude mice subcutaneously loaded with human-derived PDA tumors are shown in fig. 12. The ultrasonic irradiation device shown in FIG. 12A is used to study the biological distribution, tumor accumulation and penetration of liposome nanoparticles in BALB/c nude mice loaded with human PDA tumor on the left and right sides, and ultrasonic irradiation is performed on the human PDA tumor loaded on the right side of the subcutaneous layer after injecting liposome nanoparticles into tail vein (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min), carrying human PDA tumor on the left subcutaneous side without ultrasonic irradiation. Biodistribution of different liposome nanoparticles 12h after tail vein injection as shown in fig. 12B and 12C, it can be seen from fig. 12B and 12C that LPGL liposome nanoparticles showed higher fluorescence intensity in the ultrasonically irradiated tumor than other liposome nanoparticles at 12h of liposome nanoparticle intravenous injection, the fluorescence intensity of LPGL liposome nanoparticles in the ultrasonically irradiated tumor was 3.2-5.8 times of that of GL, LGL and PGL liposome nanoparticles in the ultrasonically irradiated tumor, however, in the tumor without ultrasonic irradiation treatment, there was no significant difference in fluorescence intensity of LPGL and LGL liposome nanoparticles, and the fluorescence intensity of LPGL liposome nanoparticles in the ultrasonically irradiated tumor was LPGL liposome nanoparticle The fluorescence intensity of the particles in the tumor without ultrasonic irradiation is 5.11 times, thereby indicating that the ultrasonic irradiation of the tumor can remarkably enhance the targeted aggregation of LPGL at the tumor site. Meanwhile, the fluorescence intensity of the LGL liposome nanoparticle in the tumor subjected to or not subjected to ultrasonic irradiation is only 1.4-1.6 times of that of the GL liposome nanoparticle in the tumor subjected to or not subjected to ultrasonic irradiation, which indicates that effective aggregation of the liposome nanoparticle in the tumor part is difficult to promote by ligand modification due to the fact that a protein corona on the surface of the liposome nanoparticle covers ligand (RGD). Based on the fluorescence intensity of GL liposome nanoparticles in the tumor subjected to ultrasonic irradiation, compared with the fluorescence intensity increase of LGL and PGL liposome nanoparticles in the tumor subjected to ultrasonic irradiation, the ligand/receptor mediated cell transfer function restarted by ultrasonic irradiation accounts for more than 70% of the fluorescence intensity increase of the LPGL liposome nanoparticles in the tumor subjected to ultrasonic irradiation, namely the ligand modification enhances the LPGL tumor accumulation mainly because the ligand/receptor mediated cell transfer is restarted by ultrasonic irradiation, so that the ultrasonic irradiation can eliminate the protein corona on the surface of the LPGL liposome nanoparticles, overcome the masking effect of the protein corona on the ligand (RGD) on the surface of the liposome nanoparticles, and recover the ligand (RGD) mediated endocytosis transfer on the surface of the liposome nanoparticles.

Before cardiac perfusion, tumor vessels were stained with FITC-Lectin simultaneously, and in vivo penetration of liposome nanoparticles in PDA tumors irradiated with ultrasound was analyzed by co-localization of blood vessels and liposome nanoparticles (as shown in fig. 12D and 12E), and it can be seen from fig. 12D that GL liposome nanoparticles are located substantially at tumor vessels and hardly penetrated from tumor vessels to PDA tumors, and PGL and LGL liposome nanoparticles are mainly distributed around tumors beside tumor vessels, however, LPGL liposome nanoparticles can be effectively distanced from tumor vessels and deeply aggregated into tumor parenchyma, and it can be seen from fig. 12E that fluorescence intensity of PGL and LGL liposome nanoparticles is rapidly attenuated from tumor vessels to tumor sites under the condition of ultrasound irradiation of tumors, fluorescence intensity of PGL and LGL liposome nanoparticles is hardly detectable from tumor vessels to tumor sites over 50 μm, however, fluorescence signal of LPGL liposome nanoparticles is strong even if it exceeds 100 μm from tumor vessels to tumor sites, thus indicating that LPGL liposome nanoparticles have excellent tumor permeability from tumor vessels to tumor sites under the condition of ultrasound irradiation.

16. Real-time extravasation of blood vessels in vivo under ultrasonic irradiation

Human PDA tumor is loaded under the skin beside abdominal blood vessel of BALB/c nude mouse, and animal model of loaded tumor is constructed. The tumor size reaches 100mm ³ When the tumor was fixed on a microscope slide glass using a dorsal skin wrinkle cell (APJ tracing Co. Inc.), an ultrasonic probe was mounted above the dorsal skin wrinkle cell, a coupling agent was applied, and after a Cy 5-labeled GL, LGL, PGL or LPGL liposome nanoparticle dispersion (dose equivalent to GEM 10 mg/kg) was injected into the tail vein, the tumor site was treated with ultrasonic irradiation (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min), tumor areas were imaged using CLSM 10min, 30min and 60min after tail vein injection of different Cy 5-labeled liposome nanoparticle dispersions, and the corresponding tumor fluorescence intensities were calculated using Image J software. Mice were heart perfused 60min after tail vein injection of Cy 5-labeled liposomal nanoparticle dispersion, tumors were isolated and fixed in 10 volumes of perfusion solution at 4 ℃ for at least 24 hours, then tumors were cut into small pieces (about 2 × 2 × 2 mM), immersed in 7% agarose solution, cut into sections 0.25-1 mM thick, sections washed with sodium cacodylate buffer (100 mM), then fixed for 2h with 1% osmic acid, stained with uranyl acetate at 37 ℃ for 48h, acetone gradient dehydrated, tumors embedded in epoxy resin, sections (60-80 nm), stained with lead citrate, and samples were observed with Transmission Electron Microscope (TEM).

Real-time in vivo extravasation of vessels and tumor aggregation in human PDA tumor-loaded BALB/c nude mice with different Cy5 labeled liposomal nanoparticles is shown in figure 13. Real-time extravascular extravasation and tumor accumulation in vivo of liposomal nanoparticles was further studied in BALB/c nude mice bearing human PDA tumors (as shown in figure 13A) by using CLSM and ultrasound device, and it can be seen from figure 13A that the tumor was fixed in the dorsal cutaneous rugose chamber with obvious tumor vessels around the tumor. Tumor sites were treated with ultrasonic irradiation after tail vein injection of different Cy 5-labeled liposomal nanoparticles, tumor extravasation of Cy 5-labeled liposomal nanoparticles 10min, 30min and 60min after tail vein injection was shown in fig. 13B and 13C, and it can be seen from fig. 13B and 13C that GL liposomal nanoparticles hardly exude from capillaries and fluorescence signals in the tumor interior region are negligible, which also confirms that PDA is a low permeability tumor (with low level of EPR effect), LGL and PGL liposomal nanoparticles slightly exude from tumor vessels and are both mainly located around tumor vessels, while LPGL liposomal nanoparticles are effectively exuded from tumor vessels and distributed to tumor parenchyma, with fluorescence intensity at the tumor site significantly higher than GL, PGL and LGL liposomal nanoparticles, thus demonstrating that ultrasonic irradiation can effectively promote the exudation of LPGL liposomal nanoparticles from tumor vessels and at the tumor parenchyma.

After injecting different Cy 5-labeled liposome nanoparticles into the tail vein, performing ultrasonic irradiation treatment on a tumor part, and after 60min of tail vein injection administration, observing the collected vascular structure of the tumor by a Transmission Electron Microscope (TEM) (as shown in FIG. 13D), wherein the PDA tumor is a low-permeability solid tumor, the endothelial cells in the vascular structure are good in tissue and are closely arranged, and as can be seen from FIG. 13D, in all tumor vascular TEM images after liposome nanoparticle treatment, there are no large number of leakage gaps between vascular endothelial cells, and there is only one gap (shown by an arrow) in the wall of the tumor vessel treated by PGL liposome nanoparticles, thereby indicating that GL, PGL, LGL and LPGL liposome nanoparticles are difficult to leak out through the gaps between the tumor vascular endothelial cells (i.e. are difficult to leak out by the conventional EPR effect). Unexpectedly, typical transcellular transporter vesicles (as indicated by arrows) are very abundant in the vessel wall of LPGL liposome nanoparticle-treated tumors, while GL, PGL and LGL liposome nanoparticle-treated tumors have few vesicles distributed on the vessel wall, and the vesicles are the only transporters transported across the vascular endothelium, thus indicating that ultrasonic irradiation can effectively promote the exudation of LPGL liposome nanoparticles from tumor vessels to tumors through ligand/receptor-mediated transcellular transport pathways, while ultrasonic irradiation has difficulty in promoting the exudation of GL, PGL and LGL liposome nanoparticles from tumor vessels to tumors through ligand/receptor-mediated transcellular transport pathways.

17. In vivo antitumor Activity

After human-derived PDA tumor cells were subcutaneously inoculated into BALB/c nude mice, incubation was performed for 16 days, and the obtained BALB/c nude mice bearing human-derived PDA tumors were randomly divided into 7 groups of 6 mice each, each group was separately administered with LPGL liposome nanoparticle dispersion (administration dose equivalent to GEM 10 mg/kg), PGL liposome nanoparticle dispersion (administration dose equivalent to GEM 10 mg/kg), LGL liposome nanoparticle dispersion (administration dose equivalent to GEM 10 mg/kg), GL liposome nanoparticle dispersion (administration dose equivalent to GEM 10 mg/kg), PBS7.4 dispersion of GEM (administration dose equivalent to GEM 10 mg/kg), LPL blank liposome nanoparticle dispersion or PBS7.4 buffer, day 16 was administered intravenously for the first time, administered once every two days, and after administration by injection, the tumor site was subjected to ultrasonic treatment (sound intensity: 2W/cm ² Frequency: 3MHz, duty cycle: 50%, duration: 20 min), 4 times in total. The width and length of the tumor and the body weight of the mice were measured during the treatment period. At the end of the experiment at 36 days, mice were euthanized after blood draw, and tumors were isolated and weighed. The efficacy of the treatment was assessed by comparing the tumor size of the experimental and control groups. Tumor inhibition =100% × (PBS group mean tumor weight-experimental group mean tumor weight)/PBS group mean tumor weight.

Tumors isolated after the end of the experiment on day 36 were fixed with 4% neutral paraformaldehyde buffer and embedded in paraffin, and 5 μm thick tissue sections were fixed on glass slides and stained with hematoxylin-eosin (H & E) and examined by light microscopy. Ki67 immunohistochemical staining study the percentage of proliferating tumor cells positively stained in the examination area. Tissue sections were subjected to Ki67 staining analysis using a Ki67 antibody assay kit (Ki 67-antibodyAssayKit). Apoptotic cells were identified using the TUNEL Apoptosis detection Kit (TUNEL Apoptosis Assay Kit) and examined using CLSM.

The anti-tumor activity of free GEM and different liposome nanoparticles in BALB/c nude mice loaded with human PDA tumors subcutaneously is shown in fig. 14, and fig. 14A shows the construction, experimental schedule and tumor treatment protocol of the BALB/c nude mice animal model loaded with human PDA tumors. The change in tumor volume over time after treatment of the different groups is shown in fig. 14B, from fig. 14B it can be seen that a sustained increase in tumor volume was observed in mice treated with PBS 7.4 buffer and LPL blank liposome nanoparticles, that in mice treated with free GEM, GL, LGL and PGL liposome nanoparticles, tumor growth was first delayed, but after cessation of the administration, tumor growth resumed to increase, whereas in mice treated with LPGL liposome nanoparticles, tumor volume decreased more and more from the beginning of the administration to the end of the administration, and tumor growth continued to be significantly inhibited, and as can be seen from the photographs of the groups of mice at the end of the 36-day experiment (as shown in fig. 14C) and the isolated tumor photographs (as shown in fig. 14D), the tumors of the LPGL liposome nanoparticle-treated mice were significantly suppressed and half of the LPGL liposome nanoparticle-treated tumors were completely eradicated, as compared to the other treatment groups, and the average tumor weight of the mice at the end of the 36-day experiment, from which the LPGL liposome nanoparticles were resected, was as high as 98.3%, which was significantly higher than GEM, GL, LGL and PGL, as shown in fig. 14E. In addition, as can be seen from the body weight change of each group of mice during the treatment period (as in fig. 14F) and the blood leukocyte value (as in fig. 14G) and the blood platelet value (as in fig. 14H) of each group of mice at the end of the 36-day experiment, the free GEM treatment resulted in significant weight loss of the mice and significantly reduced leukocyte and platelet values, causing hematologic damage, while each group of liposomal nanoparticle treatment had no significant effect on the body weight, leukocyte and platelet of the mice, thus indicating that the liposomal nanoparticles had no significant side effects and high safety, thus indicating that the liposomal nanoparticles had good biocompatibility and biosafety.

H & E staining, IHC staining of Ki67 and TUNEL staining of tumors isolated at the end of the 36 day experiment are shown in fig. 14I and fig. 15 to study the mechanism of antitumor activity of liposomal nanoparticles. As can be seen from fig. 14I and fig. 15, hematoxylin-eosin (H & E) staining showed a significant decrease in cell density of free GEM, GL, LGL, PGL and LPGL liposome nanoparticle treated tumors compared to LPL liposome nanoparticle and PBS buffer treated tumors, tumor ablation occurred in the LPGL liposome nanoparticle treated group, exhibiting a large number of apoptotic cells, a large number of contracted nuclei, and a wide intercellular space. LPGL liposomal nanoparticles can significantly reduce the percentage of Ki 67-positive tumor cells compared to free GEM, GL, LGL, and PGL liposomal nanoparticles, indicating a better prognosis for LPGL liposomal nanoparticles even after short-term treatment. DNA fragments were tested in situ using TUNEL method to assess drug-induced apoptosis, and compared to other groups, LPGL liposomal nanoparticle treated tumors showed a large number of apoptotic cells (green fluorescence), and apoptotic cells in LPGL liposomal nanoparticle treated tumors were distributed almost throughout the tumor parenchyma, while apoptotic cells in GEM, GL, LGL and PGL liposomal nanoparticle treated tumors were present only around the tumor. The results indicate that LPGL liposomal nanoparticles are able to aggregate and penetrate deeply in PDA tumors and transport the active drug throughout the tumor parenchyma, thereby inducing apoptosis.

Example 2

The lipid nanoparticle of this example 2 and the study of its properties and antitumor activity were the same as in example 1, except that the LPGL liposome nanoparticle and the LGL liposome nanoparticle were prepared by replacing DSPE-PEG2000-RGD with DSPE-PEG2000-NGR, wherein DSPE-PEG2000-NGR was distearoylphosphatidylethanolamine-polyethylene glycol 2000-NGR targeting peptide, and the amino acid sequence of the NGR targeting peptide was Gly (glycine) -Cys (cysteine) -Asn (asparagine) -Gly (glycine) -Arg (arginine) -Cys (cysteine), and the amino acid sequence was GGCNGRC (SEQ ID NO: 2).

NGR modified liposome nanoparticles and their properties and tumor aggregation and anti-tumor activity in BALB/c nude mice loaded with human HCC tumors subcutaneously are shown in FIG. 16, FIG. 17 and Table 3.

Table 3 particle size and potential of NGR modified LGL and NGR modified LPGL liposomal nanoparticles in PBS or plasma

The procedure for dilution of each liposomal nanoparticle in plasma was as follows: the NGR-modified LGL or LPGL liposomal nanoparticle dispersions were mixed with mouse plasma at a lipid/protein mass ratio of 1/50, respectively, and incubated in a shaker (60 rmp) at 37 ℃ for 30min.

As can be seen from FIG. 16A, after incubation with mouse plasma for 30min, the total content of protein in the corona proteolics on the surface of NGR-modified LPGL and PGL liposome nanoparticles was as high as about 28. Mu.g/mg lipid, upon ultrasonic irradiation (+ US; sound intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), the total protein content in the protein corona on the surface of the NGR ligand-modified LPGL liposome nanoparticle is reduced by 74.9% remarkably, while the total protein content on the surface of the NGR ligand-modified LGL liposome nanoparticle is kept unchanged, thereby showing that the ultrasonic irradiation can effectively remove the protein corona on the surface of the NGR ligand-modified LPGL liposome nanoparticle. As can be seen from FIG. 16B, the serum-free medium mixture of Cy 5-labeled NGR-modified LGL and NGR-modified LPGL liposomal nanoparticles exhibited good Huh7 cell uptake efficiency when incubated with Huh7 cells, but when the serum-containing medium mixture of Cy 5-labeled NGR-modified LGL and NGR-modified LPGL liposomal nanoparticles was incubated with Huh7 cells and not subjected to ultrasonic irradiation treatment, the uptake capacity of the Huh7 cells to the NGR-modified LGL and LPGL liposomal nanoparticles was significantly reduced, indicating that the protein corona on the surface of the NGR-modified LGL and LPGL liposomal nanoparticles prevented NGR ligand/receptor-mediated cellular uptake, when the serum-containing medium mixture of Cy 5-labeled NGR-modified LGL and NGR-modified LPGL liposomal nanoparticles was incubated with Huh7 cells and subjected to ultrasonic irradiation (acoustic intensity: 2W/cm) ² Frequency: 3MHz, duty cycle: 50%, duration: 5 min), uptake efficiency of NGR modified LPGL liposomal nanoparticles by Huh7 cells was restored and the results of the serum-free medium mixture incubated with NGR modified LPGL liposomal nanoparticles were similar and not statistically different from the results of Huh7 cells incubated without ultrasound irradiation, whereas NGR modified LGL liposomal nanoparticles by Huh7 cells were not statistically different from the results of Huh7 cellsThe uptake efficiency of the nanoparticles is still very low, thus indicating that the ultrasonic irradiation can eliminate the masking effect of the protein corona on the surface of LPGL liposome nanoparticles on the ligands (such as NGR) on the surface of the liposome nanoparticles and restore the cell uptake enhancement effect mediated by the ligands (such as NGR) on the surface of the liposome nanoparticles.

The antitumor activities of GEM, GL, NGR-modified LGL, PGL, NGR-modified LPGL and PBS 7.4 on BALB/c nude mice subcutaneously loaded with human HCC tumors are shown in FIGS. 17A-17G.

After subcutaneous inoculation of human HCC tumor cells into BALB/c nude mice, after 20 days of incubation, the resulting human HCC tumor-loaded BALB/c nude mice were randomly divided into 6 groups (n =9, 3 of them were used for tumor determination of cumulative concentration of gemc triphosphate active metabolite dFdCTP of GEM in tumors taken 24h after the 1 st i.v. administration). As with the effect observed in the PDA animal model in example 1, the tail vein injection of ngl-modified LPGL, NGR-modified LGL liposome nanoparticles, PGL liposome nanoparticles, GL liposome nanoparticles, GEM-free PBS 7.4 buffer and PBS 7.4 buffer into BALB/c nude mice bearing human HCC tumor of subcutaneous origin, the ultrasonic irradiation of the tumor sites of each group of mice, 24h after the first vein injection, the drug concentration of gemcitabine triphosphate active metabolite dFdCTP of GEM in tumors isolated from each group of mice as shown in fig. 17B, since GEM is rapidly phosphorylated in tumor tissues to its active metabolite dFdCTP, dFdCTP is widely used as a quantitative index for GEM, so that it can be seen in fig. 17B that the dFdCTP content in 24h, NGR-modified LPGL nanoparticle-treated tumors after the first vein injection is about free GEM, liposome GL nanoparticles, NGR-modified lgr nanoparticles and PGL nanoparticles 2.9 times as high as the tumor targeting capability of liposome-modified LPGL nanoparticles to lgr 2.8.

Fig. 17C, 17D, 17E and 17F show the tumor size and weight change after iv injection of nglb/C nude mice with human HCC tumors loaded with NGR modified LPGL, NGR modified LGL liposome nanoparticles, PGL liposome nanoparticles, GL liposome nanoparticles, free GEM and PBS 7.4 buffer treatment, as can be seen in fig. 17C, 17D, 17E and 17F, the weight of GEM treated mice gradually decreased, while the weights of GL, NGR modified LGL, PGL and NGR modified LPGL could be restored to normal levels, indicating negligible side effects and high safety without counting these liposome nanoparticles. In addition, at the end of the 34-day experiment, each group was bled and serum alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), creatinine (CRE) and Blood Urea Nitrogen (BUN) were measured and the results are shown in table 4 to assess liver and kidney toxicity in vivo, and from table 4, it can be seen that serum content values of AST, ALT, BUN and CRE in mice after all liposome nanoparticle treatments remained within normal limits, while the GEM group exceeded the maximum of the normal limits, indicating that the liposome nanoparticles have good biosafety and biocompatibility. As can be seen from fig. 17D, during the treatment period, the tumor growth of the mice treated with GL, PGL, NGR modified LGL and NGR modified LPGL liposomal nanoparticles was significantly inhibited compared to the PBS or GEM treated mice, but only the tumors treated with NGR modified LPGL liposomal nanoparticles were still reduced and regressed when the administration was stopped, unexpectedly, the tumors of the mice treated with NGR modified LPGL liposomal nanoparticles were significantly inhibited and more than half of the tumors treated with NGR modified LPGL liposomal nanoparticles were completely eradicated compared to the other treatment groups, thereby indicating that the NGR modified LPGL liposomal nanoparticles had excellent anti-tumor efficiency and were capable of eradicating cancer, and as can be seen from fig. 17E and 17F, the tumor elimination rate of the NGR modified LPGL liposomal nanoparticles was 96.8% better than that of the free GEM, GL liposomal LGL nanoparticles, NGR modified LPGL liposomal nanoparticles and PGL nanoparticles, indicating that the NGR modified LPGL nanoparticles had a general anti-tumor activity against solid tumors. The H & E staining of fig. 17G, IHC staining of Ki67 also showed that NGR-modified LPGL liposomal nanoparticles can significantly lead to a large number of apoptotic cells, leading to a better prognosis even after short-term treatment (fig. 17G).

TABLE 4 Biochemical analysis of mouse serum after Liposomal nanoparticle treatment

While the invention has been described in terms of a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A method of eliminating protein corona from a protein corona-modified nanoparticle, said method comprising the steps of:

performing ultrasonic irradiation treatment on the nanoparticle modified by the protein corona, so as to eliminate the protein corona of the nanoparticle modified by the protein corona;

wherein the nanoparticles entrap perfluoro-n-pentane.

2. The method of claim 1, wherein the protein corona-modified nanoparticle comprises a protein corona-modified nanoparticle in a protein-containing condition.

3. The method of claim 1, wherein the protein-containing conditions comprise blood, serum, plasma, and/or culture medium;

the blood, serum or plasma comprises isolated or separated blood, serum or plasma;

the culture medium comprises a culture medium containing serum, plasma and/or tissue protein.

4. A method of screening for or identifying potential ligands that target a cell or cell surface receptor, said method comprising the steps of:

(I) Modifying a ligand on a nanoparticle to obtain a ligand-modified nanoparticle, wherein the nanoparticle comprises perfluoro-n-pentane;

5. The method of claim 4, wherein said incubating comprises incubating in conditions comprising a protein.

6. The method of claim 4, wherein step (II) comprises:

7. Use of a nanoparticle for the preparation of a vector for screening or identifying potential ligands for targeting a cell or cell surface receptor, wherein said nanoparticle comprises perfluoron-pentane.

8. The use of claim 7, wherein the method of screening or identifying potential ligands that target cells or cell surface receptors comprises the steps of:

9. A system or device for treating a disease, comprising nanoparticles and/or ligand-modified nanoparticles; and an ultrasonic device;

the nanoparticles are coated with perfluoro-n-pentane;

the nanoparticles comprise drug-loaded nanoparticles.

10. Use of an ultrasound machine for the preparation of a device for one or more uses selected from the group consisting of:

(c) Enhancing treatment of disease by ultrasound irradiation of lesions (e.g., tumors) with administered ligand-modified nanoparticles; and/or

(d) Improving the retention and/or degradation of nanoparticles, ligand-modified nanoparticles, and/or protein corona-modified nanoparticles by cell lysosomes by ultrasound irradiation;

wherein the nanoparticles entrap perfluoro-n-pentane.