Double-layer wrapped drug cascade delivery carrier and preparation method thereof

Abstract

The invention provides a double-layer wrapped drug cascade delivery carrier and a preparation method thereof, and the double-layer wrapped drug cascade delivery carrier is prepared by performing specific fluorination modification on drug-loaded nanoparticles, so that the drug-loaded nanoparticles can be efficiently dispersed in fluorine phase liquid; and specific lipid, an emulsifier and surface charge are modified and matched with specific components, so that the obtained liquid drops can smoothly wrap and disperse nano-particles in fluorine-phase liquid through an emulsification process, and finally, the double-layer wrapped drug cascade delivery carrier with efficient drug loading and ultrasonic response is obtained. After the double-layer wrapped drug cascade delivery carrier provided by the invention is injected into a body through veins, outer-layer liquid drops can realize blasting at a targeted part under the action of ultrasound to release the nano-particles, and cell-penetrating peptides modified on the surfaces of the inner-layer nano-particles can ensure that a load can smoothly penetrate through cells to reach the interiors of the cells, so that the delivery effect of the double-layer wrapped drug cascade delivery carrier is improved. The precise delivery of the medicine into the cells is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the structural design of a double-layer wrapped drug cascade delivery carrier and a new method for realizing the structural design. Background Art

[0002] Currently, in the field of drug delivery, molecular targeted drug delivery technology has attracted widespread attention because of its ability to specifically recognize antibodies in the body. However, after the molecularly specifically modified drug carrier is injected into the body through the vein, due to the complexity of the blood environment, it is easy to adhere to the surface of the drug carrier to form a layer of protein corona, which greatly reduces or even loses its excellent targeting performance in in vitro cell experiments; at the same time, the heterogeneity of blood flow rate makes it difficult for the molecularly modified drug carrier to have sufficient time and recognition of the targeting ligand, thereby enriching at the target location. Therefore, the development of an exogenous stimulation targeting technology is currently crucial for the targeted delivery of drugs.

[0003] Liquid droplets with phase change function have unique advantages in the field of ultrasound-responsive drug delivery because they can achieve liquid-gas phase change under ultrasound stimulation. Currently, nano droplets formed by phospholipids, proteins, polymers and other materials as membrane materials to wrap fluorine-phase liquid can be used to load water-soluble or oil-soluble drugs. After intravenous injection into the body, the droplets can be stimulated by external ultrasound to explode at the target site and release the loaded drugs.

[0004] However, since the core of the current nanodroplets is a single perfluorocarbon liquid, the solubility of general water-soluble drugs or oil-soluble drugs in the fluorocarbon liquid is very low. Therefore, the drugs are generally only loaded in the shell of the nanodroplets, resulting in a decrease in drug loading efficiency; in addition, the stability of the nanodroplets is significantly reduced due to the presence of drugs in the shell. More importantly, some drugs, such as RNA drugs, are only released in the blood. They are difficult to enter the cells efficiently, so they will be quickly degraded and cannot be expressed efficiently to exert therapeutic effects. Therefore, for the development of drug carriers, there is an urgent need for new cascade drug delivery systems that can achieve efficient drug loading and delivery into cells. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned problems existing in current nano droplets and provide a double-layer wrapped drug cascade delivery carrier and a preparation method thereof. The double-layer wrapped drug delivery system structure and preparation method proposed in the present invention can make the obtained droplets have higher drug loading efficiency and more accurate drug delivery effect, can achieve accurate delivery of drugs into cells, and can better meet the needs of different types of drugs for delivery carriers in clinical and scientific research.

[0006] The inventors of the present invention have discovered that by subjecting drug-loaded nanoparticles to specific fluorination modification, the drug-loaded nanoparticles can be efficiently dispersed in a fluorine-phase liquid, and then specific lipids, emulsifiers and surface charge modifications are combined with specific components. Through an emulsification process, the resulting droplets can smoothly encapsulate the nanoparticles dispersed in the fluorine-phase liquid, thereby obtaining a double-layer encapsulated drug cascade delivery carrier with efficient drug loading and ultrasonic response.

[0007] Therefore, the first aspect of the present invention provides a double-layer encapsulated drug cascade delivery structure, the drug carrier is composed of droplets encapsulating drug-loaded nanoparticles, relative to the nanoparticles with a particle size of 1, the particle size range of the droplets is 5-50; wherein the nanoparticles in the droplets are dispersed in a fluorine phase liquid; wherein the nanoparticles are fluorinated inorganic nanoparticles or organic nanoparticles containing fluorine surfactants, and the surface modification may be modified with cell-penetrating peptides; the particle size of the nanoparticles is 10-800 nm, more preferably 50-500 nm; the drug may be one or more of chemical drugs, biological drugs, antibody drugs, and nucleic acid drugs.

[0008] The nanoparticles described in the present invention are inorganic nanoparticles that have been fluorinated or organic nanoparticles containing fluorine-based surfactants. Preferably, the inorganic nanoparticles are particles containing iron, silicon, gold, and silver elements, and the particles are specifically selected from one or more of ferroferric oxide nanoparticles, mesoporous silicon nanoparticles, nanogold particles, or nanosilver particles. The inorganic nanoparticles need to be fluorinated to achieve the purpose of being dispersed in the fluorine phase liquid. Preferably, the fluorine-based reagent used in the fluorination treatment is a fluorine-containing silane, preferably tridecafluorooctyltriethoxysilane and tetrafluorosilane. The content of the fluorinated group on the surface of the inorganic nanoparticles is determined by infrared spectroscopy, wherein the content of the fluorinated carbon chain or the fluorinated silicon chain is 20-200 parts by weight relative to 100 parts by weight of the inorganic non-metallic nanoparticles. Preferably, the organic nanoparticles are nanoparticles composed of dendritic macromolecules, lipid molecules, amphiphilic polymer molecules, and bioprotein molecules, and the particles are selected from one or more of micelles, liposomes, lipid nanoparticles, polymer nanoparticles, and exosomes. The organic nanoparticles need to be mixed with fluorine-based surfactants to achieve the purpose of dispersion in the fluorine phase liquid. Preferably, the fluorine-based surfactant is selected from one or more of fluorocarbon surfactants, fluoroalkane surfactants, fluoropolymer surfactants, fluorosilicone surfactants, and fluoroalkoxyphosphates.

[0009] The inventors of the present invention have also found that by introducing a targeting peptide or a cell-penetrating peptide into the surface of the nanoparticle, the therapeutic effect of the drug in the cell can be improved, and the targeting peptide or the cell-penetrating peptide is related to a specific target site, preferably, the targeting peptide or the cell-penetrating peptide is selected from one or more of a transcriptional transactivator protein, VP22, transportan, a model amphipathic peptide (MAP), a signal transduction peptide, and an arginine-rich sequence peptide.

[0010] In the present invention, the double-layer wrapped drug cascade delivery structure also contains drugs, and the drugs can be drugs required in various practical treatments. For example, the drugs include but are not limited to imatinib, imatinib, cyclophosphamide, doxorubicin, vinorelbine, prednisone, tamoxifen, anastrozole, trastuzumab, etc. All drugs that can be used in contrast agents in the art can be used in the present invention, and those skilled in the art can select specific substances and dosages as needed.

[0011] In the present invention, the droplets in the double-layer encapsulated drug cascade delivery structure are mainly composed of lipids, emulsifiers and surface charge modifiers. Relative to 100 parts by weight of the lipids, the content of the emulsifier is 20-50 parts by weight, and the content of the surface charge modifier is 10-35 parts by weight; wherein the surface charge modifier is a polyelectrolyte.

[0012] The specific selection and ratio of the above-mentioned lipids, emulsifiers and surface charge modifiers of the present invention can achieve better effects. In order to further improve the comprehensive performance of the contrast agent, preferably, relative to 100 parts by weight of the lipids, the content of the emulsifier is 25-45 parts by weight, and the content of the surface charge modifier is 15-30 parts by weight; more preferably, relative to 100 parts by weight of the lipids, the content of the emulsifier is 30-40 parts by weight, and the content of the surface charge modifier is 22-28 parts by weight.

[0013] In the present invention, the lipid is a phospholipid, the polar head group of the phospholipid is PC (acylcholine), PE (acylethanolamine), PG (sodium acylglycerate), PS (acylserine) and PA (glycerophosphatidic acid), and the non-polar polar group is a long carbon chain with a length of 16-28, selected from 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), diarachidonylphosphatidylcholine (DAPC) , 1,2-bis(diphenylphosphino)ethane (DPPE), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) One or more.

[0014] In the present invention, the emulsifier may be an emulsifier conventionally used in the art. Preferably, the emulsifier is selected from one or more of polyethylene glycol 4000 (PEG4000), polyethylene glycol 40s (PEG40s), polyoxypropylene polyoxyethylene block polyether (Pluronic), polyethylene glycol 1400 (PEG1400) and polysorbate 80.

[0015] In the present invention, the term "polyelectrolyte" is a conventional concept in the art, that is, a long-chain polymer with ionizable groups. This type of polymer will be ionized in a polar solvent, so that the polymer chain is charged.

[0016] The present invention can use various polyelectrolytes that can provide positive or negative charges as surface charge modifiers. In a preferred case, in order to better cooperate with other components, the surface charge modifier is selected from one or more of hyaluronic acid, chitosan, sodium hydroxymethylcellulose, carbomer, sodium alginate, polyamine, and hard amine; more preferably, the surface charge modifier is selected from one or more of sodium alginate, polyethyleneimine, and polyallylamine hydrochloride.

[0017] In the present invention, the nanoparticles in the droplets are dissolved in a fluorine-phase liquid that can change phases, wherein the term "phase-changeable liquid biocompatible material" refers to a substance that can be in liquid state under normal temperature and pressure conditions or under certain environmental conditions at normal temperature, but can change to gaseous state under certain environmental or excitation conditions at normal temperature, such as perfluorocarbon; and perfluorocarbon also has the characteristics of high molecular weight, stable properties, and good biosafety. The contrast agent obtained with liquid perfluorocarbon as the content has the unique performance of liquid-gas phase change, and can be excited to form gaseous state under conditions such as ultrasound and light, thereby forming a microbubble contrast agent; the microbubble can undergo inertial cavitation when it breaks, so that it can load drugs and burst at a fixed point in the target area and release drugs locally, exerting the therapeutic effect of targeted drug delivery; microbubble cavitation generates microflow and shear force locally, stimulating the opening of the pores of the endothelial cell membrane and the channels between cells, and promoting the delivery of therapeutic drugs into cells. Therefore, the contrast agent obtained with liquid perfluorocarbon as the content can not only achieve similar effects as traditional microbubble ultrasound contrast agents, but also its liquid core allows the nanodroplets to have a smaller particle size and higher stability, thereby providing huge application prospects for tissue imaging and targeted precision treatment. Therefore, it is specially studied in the present invention. Therefore, according to a preferred specific embodiment of the present invention, the content is liquid perfluorocarbon.

[0018] Preferably, the perfluorocarbon is selected from one or more of perfluoropropane, perfluorobutane, perfluoropentane and perfluorohexane. When the perfluorocarbon is perfluoropentane and / or perfluorohexane, since these two substances are in liquid state at room temperature and pressure, no special operation is required in the process of preparing the contrast agent film-forming agent and the contrast agent; and when the perfluorocarbon is perfluoropropane and / or perfluorobutane, since these two substances are in gaseous state at room temperature and pressure, the temperature and / or pressure need to be controlled in the process of preparing the contrast agent film-forming agent and the contrast agent to keep them in liquid state.

[0019] The contrast agent film-forming agent composition of the present invention may also contain other conventional additives in the art. As long as they do not adversely affect the performance of other components, those skilled in the art can select them, and the content of these other additives can refer to the conventional content in the art.

[0020] The second aspect of the present invention provides a method for double-layer packaging of a drug cascade delivery carrier, the method comprising the following steps: (1) preparing drug-loaded fluorinated inorganic nanoparticles or organic nanoparticles containing fluorinated surfactants, and mixing them with an organic fluorine phase liquid to obtain a solution A; (2) preparing a liposome and a lipid membrane solution, and dissolving the liposome and the lipid membrane solution in an aqueous solvent to obtain a solution B; (3) Take an appropriate amount of solution A and add it to solution B. Use an emulsification process to dissolve solution A and evenly disperse it in solution B. The liposome then changes its membrane structure and wraps the evenly dispersed solution A. Centrifugal reselection is performed to remove the unstructured membrane material, and a double-layer wrapped drug cascade delivery carrier can be obtained.

[0021] In step (1), there is no particular restriction on the amount of the fluorine phase liquid, as long as it can evenly disperse the fluorinated inorganic nanoparticles or the organic nanoparticles containing a fluorine-based surfactant. Preferably, the amount of the fluorine phase liquid is such that the concentration of the nanoparticles in the solution is 5-80 mg / ml, preferably 10-50 mg / ml, and most preferably 20-40 mg / ml.

[0022] In step (2), the aqueous solution may be a hydration solution commonly used in the art; preferably, the hydration solution contains glycerol, propylene glycol and phosphate; more preferably, the volume ratio of glycerol, propylene glycol and phosphate is 1:(0.5-3):(5-12), and further preferably 1:(1-2):(7-10). There is no particularly strict restriction on the amount used, as long as the liposome or lipid membrane in the present invention can be evenly dispersed. Preferably, the concentration of the lipid in the solution is 0.5-8 mg / mL, preferably 1-5 mg / mL, and most preferably 2-4 mg / mL.

[0023] In step (3), the appropriate amount of solution A is added to solution B. For every 100 parts by volume of solution B, the volume of solution A is 1-40 parts by volume, preferably 2-15 parts by volume, and most preferably 3-8 parts by volume.

[0024] In step (3), the emulsification process is a high-energy emulsification method, specifically one or more of mechanical oscillation, ultrasonic cavitation, and homogenization. The purpose of the emulsification process is to change the reorganization mode of liposomes or lipid membranes, thereby encapsulating the evenly dispersed A solution to form droplets loaded with nanoparticles.

[0025] The mechanical oscillation method is often applicable to fluorine-phase gases that need to be liquefied under high pressure, such as perfluoropropane or perfluorobutane. Generally, the above-mentioned A and B mixed evenly solution is placed in a sealed vial, and the upper layer is filled with perfluorocarbon gas. By continuously adding perfluorocarbon gas, the vial is pressurized to liquefy the fluorocarbon gas. Generally, the pressure is controlled at 10-500PSI, preferably 50-150PSI; the mechanical oscillation method generally has an oscillation frequency of 2000-10000 rpm, preferably 3500-5500 rpm, and an oscillation time of 20-180 s, preferably 30-80 s. The droplet size can be regulated by adjusting the oscillation frequency and oscillation time.

[0026] Ultrasonic cavitation is often applicable to fluorine-phase liquids that are liquid at room temperature, such as perfluoropentane, perfluorohexane, perfluorooctane bromide, etc. The volume of the solution is relatively small, generally 1ml-50ml; during the action, the above-mentioned A and B mixed evenly solution needs to be placed in a vessel, and the vessel is placed in a constant temperature device to eliminate the effect of the ultrasonic cavitation head stimulating heat generation on the solution temperature, and the temperature is generally controlled at -5-25 ℃; the power of ultrasonic cavitation is generally related to the volume, the power range is 10-1000 w, and the ultrasonic cavitation time is 0.5-10 min, preferably 1-5 min; The homogenization method is often applicable to fluorine-phase liquids that are liquid at room temperature, such as perfluoropentane, perfluorohexane, perfluorooctane bromide, etc. The volume of the reaction solution is relatively large, generally 10-5000 ml, preferably 50-500 ml; during the reaction, the above-mentioned A and B mixed evenly solution needs to be placed in a container, and the container uses a constant temperature device to control the temperature to eliminate the effect of the heat generated by the homogenizer head on the solution temperature. The temperature is generally controlled at -5-25 ℃; the homogenization speed is 3000-50000 rpm, and the homogenization time is related to the solution volume, generally 1-120 min.

[0027] Through the above technical solution, the present invention has at least the following advantages compared with the prior art: The double-layer wrapped drug cascade delivery carrier prepared by the present invention is applied to different types of drugs, which are water-soluble or oil-soluble, and have a higher drug loading capacity; The double-layer wrapped drug cascade delivery carrier prepared by the present invention can be vaporized under the activation of an ultrasound imaging probe, thereby obtaining a significantly enhanced echo signal in the ultrasound image, and can realize the targeted release of the drug under the planning of high-energy ultrasound; The double-layer encapsulated drug cascade delivery carrier prepared by the present invention can further target specific cells after ultrasound-targeted release through specific modification of drug-loaded nanoparticles, thereby achieving accurate and efficient intracellular drug delivery; The contrast agent of the present invention can be used not only as an ultrasound contrast agent, but also as a photoacoustic contrast agent and a multimodal contrast agent in a preferred embodiment, and has a broader application prospect.

[0028] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is the image of the drug carrier I1 obtained in Example 3 under an optical microscope Figure 2 Ultrasonic images of the drug carrier I1 obtained in Example 3 before and after in vivo ultrasonic activation DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The embodiments described in the present invention are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the materials used in the following examples were all commercially available analytically pure. Example 1

[0032] (1) mixing fluorinated mesoporous silica nanoparticles loaded with doxorubicin with perfluoropentane to obtain solution A, so that the final concentration of silica is 20 mg / ml, the content of doxorubicin is 2 mg / ml, the fluorinated group is tridecafluorooctyltriethoxysilane, and the content of the fluorinated group is 5 mg / ml; (2) DSPC, DSPE, CHO, and PEG40s were weighed and mixed in a weight ratio of 100:35:25:25, and a liposome solution was prepared by a thin film emulsification method, which was dissolved in an aqueous solvent (mixed with glycerol, propylene glycol, and PBS buffer in a volume ratio of 1:1.5:8.5) to obtain a solution B, so that the final concentration of DSPC was 4 mg / ml; (3) Take 500 μl of solution A and add it to a beaker containing 10,000 μl of solution B. Place the beaker in a water bath and control the water bath temperature to 4°C. Use ultrasonic cavitation to dissolve solution A and evenly disperse it in solution B. The liposomes then change their membrane structure and wrap the evenly dispersed solution A. The power of ultrasonic cavitation is 240 W, the ultrasonic time is 1 min, and the duty cycle is 50%. After ultrasound, centrifuge the lower layer of liquid at 4000 rpm for 10 min, and then remove the upper layer of solution again. Remove the unstructured membrane material to obtain a double-layer wrapped drug cascade delivery carrier, which is recorded as I1.

[0033] The image structure of the droplet under the electron microscope is as follows: Figure 1 As shown, from Figure 1 It can be seen that the droplet has a double-layer wrapped structure. Example 2

[0034] (1) Take paclitaxel-loaded liposomes containing a fluorinated surfactant FSL, mix the liposomes with perfluorohexane to obtain a solution A, so that the final concentration of the liposomes is 20 mg / ml, the content of paclitaxel is 2 mg / ml, the fluorinated surfactant is 1H, 2H, 2H, 1H-perfluorooctanethiol, and the content of the fluorinated surfactant is 3 mg / ml; (2) DPPC, DSPG, CHO, and PEG4000 were weighed and mixed in a weight ratio of 100:35:25:25, and liposome and lipid membrane solution were prepared by thin film emulsification method, and dissolved in an aqueous solvent (glycerol, propylene glycol, and PBS buffer were mixed in a volume ratio of 1:1.5:8.5) to obtain solution B, so that the final concentration of DSPC was 4 mg / ml; (3) Take 30 μl of solution A and add it to 1000 μl of solution B. Use ultrasonic cavitation to dissolve solution A and evenly disperse it in solution B. The liposomes then change their membrane structure and wrap the evenly dispersed solution A. The power of ultrasonic cavitation is 120w, the ultrasonic time is 1min, and the duty cycle is 50%. After ultrasound, the lower layer of liquid is centrifuged at 4000rpm for 10min, and the upper layer of solution is sucked off again. The unstructured membrane material is removed to obtain a double-layer wrapped drug cascade delivery carrier, which is recorded as I2. Example 3

[0035] Prepare nanoparticles of fluorinated dendrimer PAMAM-G4 loaded with siRNA-PLK1, mix them with perfluoropentane to obtain solution A, so that the final concentration of PAMAM-G4 is 20 mg / ml, the content of siRNA-PLK1 is 2 mg / ml, the fluorinated group is perfluorodecanoic acid, and the content of the fluorinated group is 2 mg / ml; (2) DSPS, DSPE, CHO, and PEG40s were weighed and mixed in a weight ratio of 100:35:25:25, and liposome and lipid membrane solution were prepared by thin film emulsification method, which was dissolved in an aqueous solvent (mixed with glycerol, propylene glycol and PBS buffer in a volume ratio of 1:1.5:8.5) to obtain solution B, so that the final concentration of DSPC was 4 mg / ml; (3) Take 100 μl of solution A and add it to 1000 μl of solution B. Use ultrasonic cavitation to dissolve solution A and evenly disperse it in solution B. The liposome then changes its membrane structure and wraps the evenly dispersed solution A. The power of ultrasonic cavitation is 120w, the ultrasonic time is 1min, and the duty cycle is 50%. After ultrasonication, the lower layer of liquid is centrifuged at 4000rpm for 10min, and the upper layer of solution is sucked off again. The unstructured membrane material is removed to obtain a double-layer wrapped drug cascade delivery carrier, which is recorded as I3. Example 4

[0036] (1) preparing MC3 liposomes loaded with siRNA-PLK1 and containing a fluorinated surfactant, and mixing the MC3 liposomes with perfluoropentane to obtain a solution A, so that the final concentration of the MC3 lipid is 20 mg / ml, wherein the content of siRNA-PLK1 is 2 mg / ml, the fluorinated surfactant is 1H, 2H, 2H, 1H-perfluorooctanethiol, and the content of the fluorinated surfactant is 3 mg / ml; (2) DSPS, DSPE, CHO, and PEG40s were weighed and mixed in a weight ratio of 100:35:25:25, and liposome and lipid membrane solution were prepared by thin film emulsification method, which was dissolved in an aqueous solvent (mixed with glycerol, propylene glycol and PBS buffer in a volume ratio of 1:1.5:8.5) to obtain solution B, so that the final concentration of DSPC was 4 mg / ml; (3) Take 80 μl of solution A and add it to 1000 μl of solution B. Use ultrasonic cavitation to dissolve solution A and evenly disperse it in solution B. The liposome then changes its membrane structure and wraps the evenly dispersed solution A. The power of ultrasonic cavitation is 120w, the ultrasonic time is 1min, and the duty cycle is 50%. After ultrasound, the lower layer of liquid is centrifuged at 4000rpm for 10min, and the upper layer of solution is sucked off again. The unstructured membrane material is removed to obtain a double-layer wrapped drug cascade delivery carrier, which is recorded as I4. Example 5

[0037] Refer to the method of Example 1, except that in step 3, 5 ml of solution A is added to 50 ml of solution B, and the homogenization method is used to dissolve A and evenly disperse it in solution B. The liposome then changes the membrane structure and wraps the evenly dispersed solution A. The temperature is controlled at 20°C; the homogenization speed is 15000 rpm, and the homogenization time is 10 min. After homogenization, the lower layer liquid is centrifuged at a speed of 4000 rpm for 10 minutes, the upper layer solution is sucked off again, and the unstructured membrane material is removed to obtain a double-layer wrapped drug cascade delivery carrier, which is recorded as I5. Example 6

[0038] Referring to the method of Example 3, the difference is that the surface of the PAMAM particles is modified with a cell-penetrating peptide PEp-1, and the content of the cell-penetrating peptide is 0.5 mg / ml, to obtain a double-layer coated drug cascade delivery carrier, which is denoted as I6.

[0039] Comparative Example 1 Referring to the method of Example 1, the difference is that the mesoporous silica particles used do not contain fluorinated groups, and finally the droplets are obtained, which are recorded as D1; Comparative Example 2 Referring to the method of Example 2, the difference is that the mesoporous silica particles used do not contain fluorinated groups, and finally droplets are obtained, which are recorded as D2; Comparative Example 3 The method of Example 3 was followed, except that the PAMAM particles used did not contain fluorinated groups, and droplets were finally obtained, which were recorded as D3; Comparative Example 4 The method of Example 3 was followed, except that the liposome particles used did not contain a fluorinated surfactant, and droplets were finally obtained, which were recorded as D4; Comparative Example 5 The method of Example 3 was followed, except that only liposome particles were used, which were designated as D5; Test Case Droplet size and uniformity The average particle size (number average particle size, unit nm) and polydispersity coefficient of the nanodroplets were measured by a particle size analyzer based on the DLS principle (Malvern, Zatesizer pro), and the results are recorded in Table 1; Droplet stability The stability of contrast agents in the body is reflected by their half-life. The longer the half-life, the higher the stability. The specific test method includes: using Japanese long-eared white rabbits as experimental subjects, injecting drug-loaded nanodroplets through the rabbit's ear vein, and adjusting the concentration of the drug-loaded droplets to 1×10 11 / ml, the injection dose was 0.1ml / kg, blood was drawn from long-eared white rabbits at different times, and the phospholipid content in the blood was monitored by high performance liquid chromatography, and the time corresponding to the time when the phospholipid concentration in the blood was half of the initial injection concentration was set as the half-life of the contrast agent.

[0040] The half-life results of the contrast agents of the examples and comparative examples are respectively recorded in Table 1.

[0041] Droplet drug loading assay The drug content in the droplets was detected by liquid chromatography-mass spectrometry (LC-MS). The specific test method is as follows: After obtaining the absorption peak and concentration curve of the standard drug using LC-MS, the droplets were dissolved in pure acetonitrile to obtain a clear and transparent solution of the drug, the solution was diluted to an appropriate multiple, and the sample was tested to obtain the drug concentration, and finally the specific drug content in the droplets was calculated. The drug loading was calculated using the following formula: Drug-loading capacity = [drug content in nanodroplets / (drug content in nanodroplets + membrane material)] * 100% The drug loading results of the contrast agents of the examples and comparative examples are respectively recorded in Table 1.

[0042] Experimental determination of the effect of ultrasound activation in vivo under ultrasound activation The droplet I1 prepared in Example 1 was used as an example for testing. Specifically: Japanese long-eared white rabbits were used as experimental subjects, and a peripheral venous channel was established in the left ear of the rabbit through the ear vein. A three-way tube was connected to the end of the catheter, one of which was used to inject the drug-loaded ultrasound contrast agent prepared by the present invention, and one of which was used to inject normal saline. The Japanese long-eared white rabbits were intraperitoneally anesthetized with 3% (40 mg / kg) sodium pentobarbital. After the rabbit was completely anesthetized, the right waist was depilated to facilitate kidney imaging. The concentration of the drug-loaded ultrasound contrast agent I1 prepared in Example 1 was adjusted to 1*1011 / ml, and then a bolus injection of 0.1 ml / kg was given through the ear vein, followed by 1 ml of normal saline to flush the pipeline. The B mode of the Vearsonics ultrasound imaging system was used for observation. The center frequency of the ultrasound probe was 7.8 MHz, the mechanical index of the imaging ultrasound pulse before activation was MI=0.4, and the mechanical index of the activated ultrasound pulse was 1.5 (the safety threshold value for the human body is 1.9). The real-time ultrasound images before and during the ultrasound activation were observed as follows: Figure 2 (a) and Figure 2 (b). Figure 2 It can be seen that before activation, the nanodroplets are not vaporized and appear as dark signals in the ultrasound image. After activation, the nanodroplets are vaporized and appear as bright signals in the ultrasound image. Therefore, the nanodroplets have the ability of controllable imaging in vivo.

[0043] Table 1 Examples Average particle size (nm) Polydispersity coefficient Half-life (h) Drug loading Ultrasound activation threshold I1 800 0.30 6 50% 1.0 I2 850 0.28 5.5 40% 1.4 I3 900 0.20 6 30% 1.0 I4 930 0.24 5 45% 1.0 I5 910 0.26 5.5 50% 1.0 I6 800 0.3 6 50% 1.0 D1 780 0.35 5 5% 1.0 D2 850 0.27 5 3% 1.4 D3 900 0.21 5 1% 1.0 D4 900 0.22 5 2% 1 D5 280 0.19 0.3 5% —— It can be seen from Table 1 that, compared with the prior art, the drug delivery system prepared by the present invention has and possesses a higher drug loading capacity, can be vaporized under the activation of an ultrasonic imaging probe, thereby obtaining a significantly enhanced echo signal in the ultrasonic image, and can achieve targeted drug release under the planning of high-energy ultrasound. Through the specific modification of the drug-loaded nanoparticles, after targeted release via ultrasound, it can further target specific cells, thereby achieving accurate and efficient intracellular drug delivery.

[0044] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A double-layer packaged drug cascade delivery carrier, characterized in that: The drug carrier is composed of nanoparticles wrapped in outer layer droplets, while the inner layer nanoparticles are loaded with targeted drugs; the nanoparticles in the droplets are dispersed in a fluorine phase liquid; relative to the nanoparticles with a particle size of 1, the particle size range of the droplets is 5-50.

2. The double-layer wrapped drug cascade delivery carrier according to claim 1, characterized in that: The nanoparticles are fluorinated inorganic nanoparticles or organic nanoparticles containing fluorinated surfactants; Preferably, the inorganic particles are particles containing iron, silicon, gold, and silver elements, and the particles are selected from one or more of magnetic particles, mesoporous silica particles, nano-gold particles, and nano-silver particles; Preferably, the organic nanoparticles are nanoparticles composed of dendritic macromolecules, lipid molecules, amphiphilic polymer molecules and bioprotein molecules, and the particles are selected from one or more of micelles, liposomes, lipid nanoparticles, polymer nanoparticles and exosomes; The particle size of the nanoparticles is 10-800 nm, more preferably 50-500 nm.

3. The double-layer wrapped drug cascade delivery carrier according to claim 1, characterized in that: The targeted drug is one or more of chemical drugs, biological drugs, antibody drugs, and nucleic acid drugs; Preferably, the chemical drug is a first-line anticancer intravenous drug, selected from one or more of imatinib, imatinib, cyclophosphamide, doxorubicin, vinorelbine, prednisone, tamoxifen, anastrozole, trastuzumab, paclitinib, cisplatin, etoposide, gefitinib, erbetinib, xeloda, fluorouracil, folinic acid, oxaliplatin, ofalini, docetaxel, carboplatin, and paclitaxel; Preferably, the antibody drug is for intravenous injection, and is selected from one or more of trolizumab, bevacizumab, pembrolizumab, nabril, invitro zumab, oclaurum, imatinib, rituximab, tocilizumab, abacizumab, and ketorumab; Preferably, the biological drug is for intravenous injection, and is selected from one or more of pembrolizumab, nabril, trolizumab, invitro lumab, adalimumab, oclaudomide, imatinib, rituximab, tocilizumab, and enoxolone; Preferably, the nucleic acid drug is selected from one or more of recombinant interferon, siRNA, mRNA, anti-SENSE oligonucleotide, and oligonucleotide antibody.

4. The double-layer wrapped drug cascade delivery carrier according to claim 1-2, characterized in that: The fluorination treatment is to modify the fluorocarbon chain or fluorosilicone chain on the surface of the inorganic non-metallic particles by grafting, substitution and other reactions; wherein the content of the fluorocarbon chain or fluorosilicone chain is 20-200 parts by weight relative to 100 parts by weight of the inorganic non-metallic nanoparticles; wherein the organic nanoparticles are doped with a fluorine-based surfactant; preferably, the fluorine-based surfactant is selected from one or more of a fluorocarbon surfactant, a fluoroalkyl surfactant, a fluoropolymer surfactant, a fluorosilicone surfactant, and a fluoroalkyloxyphosphate.

5. The double-layer wrapped drug cascade delivery carrier according to claim 1, characterized in that: The droplets are composed of lipids, emulsifiers and surface charge modifiers; wherein the content of the emulsifier is 20 to 80 parts by weight relative to 100 parts by weight of the lipids, and the component of the surface charge modifier is 5 to 20 parts by weight; Preferably, the polar head group of the phospholipid is PC (acylcholine), PE (acylethanolamine), PG (sodium acylglycerate), PS (acylserine) and PA (glycerophosphatidic acid), and the non-polar head group is a long carbon chain with a length of 16-28, selected from 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), diarachidonylphosphatidylcholine (DAPC), 1,2 - one or more of bis(diphenylphosphino)ethane (DPPE), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG); Preferably, the emulsifier is selected from one or more of polyethylene glycol 4000 and polyethylene glycol 40s; Preferably, the surface charge modifier is selected from one or more of hyaluronic acid, chitosan, sodium hydroxymethylcellulose, carbomer, sodium alginate, polyamine and hard amine.

6. The double-layer wrapped drug cascade delivery carrier according to claims 1-5, characterized in that: The fluorine-phase liquid is a phase-changeable liquid organic fluorocarbon compound; preferably a fluorocarbon compound with a carbon chain length greater than or equal to 3.

7. A method for preparing a double-layer wrapped drug cascade delivery carrier, characterized in that: The method comprises the following steps: Mixing drug-loaded fluorinated inorganic nanoparticles or organic nanoparticles containing fluorinated surfactants with an organic fluorine phase liquid to obtain a solution A; (2) preparing liposomes and dissolving them in an aqueous solvent to obtain solution B; (3) Take an appropriate amount of solution A and add it to solution B. Use an emulsification process to dissolve solution A and evenly disperse it in solution B. The liposome then changes its membrane structure and encapsulates the evenly dispersed solution A, ultimately obtaining a double-layer encapsulated drug cascade delivery carrier.

8. The method according to claim 7, characterized in that In step (2), the aqueous solution contains, in addition to water, one or more of sodium chloride, glycerol, propylene glycol and phosphate; in step (3), the content of solution B is 100 to 1000 parts by volume relative to 10 parts by volume of solution A.

9. The method according to claim 7, characterized in that: In step (3), the emulsification process is produced by a high-energy emulsification method, and the specific implementation method of the high-energy emulsification method is one or more of mechanical oscillation, ultrasonic cavitation, and high-pressure homogenization.

Images