CN111557926A

CN111557926A - Targeting phase-change nano-drug system and preparation method and application thereof

Info

Publication number: CN111557926A
Application number: CN202010387824.4A
Authority: CN
Inventors: 曾粒; 凡奎; 钟玲; 王志刚; 冉海涛; 郝兰
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2020-08-21
Anticipated expiration: 2040-05-09
Also published as: CN111557926B

Abstract

The invention relates to the technical field of biological medicines, in particular to a targeting phase-change nano-drug system and a preparation method and application thereof. The targeting phase-change nano-drug system comprises a main body, wherein liquid fluorocarbon is wrapped in the main body, a drug is loaded on the main body, and BMS-470539 is covalently connected to the main body. The medicine system can solve the problems of toxic and side effects and weak targeting when the immune-related kidney diseases are treated by immunosuppression in the prior art. The targeting phase-change nano-drug system can be applied to the preparation of related drugs for treating immune nephropathy.

Description

Targeting phase-change nano-drug system and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a targeting phase-change nano-drug system and a preparation method and application thereof.

Background

Immune nephropathy is a group of chronic glomerular diseases with the same immunopathological characteristics, which are caused by multiple etiologies. Immune nephropathy includes purpuric nephritis, lupus nephritis, IgA nephropathy, etc. due to the dysfunction of the patient's immune system, the produced immune complex is deposited in the kidney to damage the inherent cells of the kidney and induce inflammation reaction, etc. to destroy the normal function of the inherent cells of the kidney, so that the patient may have symptoms of nephropathy, such as proteinuria, hematuria, edema, etc.

Immune-related kidney diseases such as Membranous Nephropathy (MN) are one of the common pathological types of nephrotic syndrome, the annual incidence rate of MN is 1.2/10 ten thousand worldwide, and most of MN is Idiopathic Membranous Nephropathy (IMN). IMN can develop varying degrees of impairment of renal function 5-10 years from disease onset, especially with the potential for renal venous thrombosis (up to 40% -50%), which reduces renal survival, and therefore immunosuppressive therapy is recommended for patients at risk of progression or suffering from severe and persistent nephritic syndrome to obtain complete or partial remission over a long period of time.

The immunosuppressive treatment is carried out by using the immunosuppressant, and the problems of strong toxic and side effects, lack of drug targeting and the like of the drug still exist while the benefit is obtained. For example, the risks associated with intravenous cyclophosphamide (CTX/CP) use include: bone marrow suppression, liver function impairment, hemorrhagic cystitis, gonadal suppression, gastrointestinal reactions, hair loss, and the like. At present, the toxic and side effects of the immunosuppressant for treatment by intravenous administration or oral administration can not be effectively avoided. And the medicine cannot target tissues, so that the toxic and side effects of the medicine are further aggravated.

Disclosure of Invention

The invention aims to provide a targeting phase-change nano-drug system to solve the problems of toxic and side effects and weak targeting when immunosuppressive therapy is adopted for immune-related renal diseases in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the targeting phase-change nano-drug system comprises a main body, wherein liquid fluorocarbon is wrapped in the main body, a drug is loaded on the main body, and BMS-470539 is covalently connected to the main body.

By adopting the technical scheme, the technical principle is as follows: the kidney lesion part is targeted at a fixed point by the compound BMS-470539, then LIFU is irradiated at the kidney lesion part by low-power focused ultrasound, and the phase change property of liquid fluorocarbon is utilized to explode the main body due to volume change, so that the fixed-point drug release is realized, the effect of targeted therapy is achieved, and the side effect caused by the release of the drug at other parts is avoided.

The invention has the beneficial effects that:

(1) the compound BMS-470539 (abbreviated as BMS-alpha) is used as a targeting ligand, and only activates the melanocortin 1 receptor MC1R highly expressed in the podocytes of the kidney of the patient with the idiopathic membranous nephropathy, but not activates any other subtype melanophore, so the compound has the characteristics of high efficiency and high selectivity and can play a role in navigation; meanwhile, after the compound BMS-470539 is combined with melanocortin 1 receptor MC1R, the cAMP level is increased, signal conduction is realized through a cAMP-PKA pathway, a nuclear transcription factor is regulated, the effect of improving catalase activity, reducing podocyte active oxygen level and stabilizing and repairing cytoskeleton of podocytes is achieved, and therefore the effect of repairing and treating the diseased region of the kidney of a patient is achieved.

(2) By coating the liquid fluorocarbon with the acoustic phase change in the main body, the liquid fluorocarbon has good stability in a liquid state, and after being coated in the main body, the liquid fluorocarbon can generate liquid-gas phase change under the irradiation of low-power focused ultrasound LIFU, so that the volume of the liquid fluorocarbon is increased, the internal pressure of the main body is increased, and the main body can be exploded when the internal pressure is increased to a certain value; therefore, by utilizing the change process, when the main body reaches the pathological change part of the kidney, the liquid fluorocarbon in the main body generates liquid-gas phase change by irradiating the main body outside the tissue through low-power focused ultrasound LIFU, so that the pathological change part is blasted, and the drug release is realized.

Further, the main body is a lipid nano-microsphere.

By adopting the technical scheme, the liposome has the advantages of similar structure with the cell membrane of an organism, longer in-vivo circulation time, capability of carrying hydrophilic and hydrophobic medicaments, low toxicity and high tissue uptake efficiency.

Further, the liquid fluorocarbon is perfluoro-n-pentane.

By adopting the technical scheme, the perfluoro-n-pentane (PFP) is one of the commonly used liquid fluorocarbons, has the boiling point of 29 ℃, is liquid at normal temperature, can generate liquid-gas phase transformation when the external pressure is reduced to the gasification pressure threshold value or the temperature is increased to be higher than the boiling point, and can be used for ultrasonic imaging.

Further, the drug is dexamethasone.

By adopting the technical scheme, the dexamethasone is mainly suitable for allergic and autoimmune inflammatory diseases and is a common immunosuppressant. The inventor wraps dexamethasone in the nano-drug system, and wants to reduce the side effect of dexamethasone on organisms through the targeting function and the site-specific release function of the system, and the toxicity of dexamethasone can also be reduced by wrapping the dexamethasone by a lipid body and realizing the transmission in the organisms. The inventor unexpectedly finds that dexamethasone has a promoting effect on the proliferation of podocytes after being encapsulated in the drug system and surface-linked with BMS-alpha to form DEX/PFP @ Lips-BMS-alpha, and the dexamethasone has positive significance on promoting the stability of the podocytes and relieving immune-related nephropathy (experimental example 4).

Further, the preparation method of the targeting phase-change nano-drug system comprises the following steps:

(1) using distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl and BMS-470539 as raw materials to synthesize DSPE-PEG-COOH-BMS-alpha;

(2) dissolving lipid and a medicine by using a mixed solvent, wherein the lipid consists of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, cholesterol and DSPE-PEG-COOH-BMS-alpha, and then evaporating the mixed solvent to dryness to obtain a lipid film;

(3) the lipid membrane is hydrated by using a buffer solution, then perfluoro-n-pentane is added, and then ultrasonic treatment is carried out to obtain a drug system DEX/PFP @ LIPs-BMS-alpha.

According to the technical scheme, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl, cholesterol and a compound BMS-470539 are used as raw materials, distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl and the compound BMS-470539 are reacted through a carbodiimide method to obtain a condensed compound, then a double-emulsion method is adopted to prepare a nano main body, and finally the nano main body is reacted with perfluoro-n-pentane PFP to obtain the medicine system. The preparation method is mild in condition and simple in operation, and can quickly and successfully prepare the nano main body of the targeting phase change compound BMS-470539 with uniform size, regular shape, good dispersibility and stable property. The nanoparticles have good biocompatibility, have good targeting capability on glomeruli through the compound BMS-470539, and are combined with low-intensity focused ultrasound to carry out fixed-point release on the drugs, so that the treatment specificity is improved, and the side effect of treatment is avoided.

Further, in the step (1), distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl and BMS-470539 are condensed by a carbodiimide method to obtain DSPE-PEG-COOH-BMS-alpha.

By adopting the technical scheme, the carboxyl in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl and the amino in the BMS-470539 can be condensed under certain conditions.

Further, in the step (2), the molar ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, DSPE-PEG-COOH-BMS-alpha, and cholesterol was 69:8:8: 15.

By adopting the technical scheme, a stable lipid film can be formed, and DSPE-PEG-COOH-BMS-alpha with targeting and therapeutic functions is fully loaded.

Further, in the step (2), the mixed solvent is composed of chloroform and methanol.

By adopting the technical scheme, the mixed solvent consisting of the trichloromethane and the methanol can fully dissolve the lipid substances and uniformly mix the lipid substances.

Further, in the step (2), the medicine is dexamethasone, and the mass ratio of the dexamethasone to the lipid is 1: 2.

by adopting the technical scheme, the dexamethasone is mainly suitable for allergic and autoimmune inflammatory diseases and is a common immunosuppressant. And the medicine can be stably loaded on a medicine system by adopting the mass ratio.

Further, the targeting phase transition nano-drug system is applied to the preparation of drugs for treating immune nephropathy.

By adopting the technical scheme, the BMS-470539 has the functions of podocyte targeting and podocyte skeleton stabilization, and can be applied to the treatment of immune nephropathy.

Drawings

FIG. 1 is a confocal microscope observation (. times.800) of DEX/PFP @ LIPs-BMS-. alpha.in example 1 of the present invention;

FIG. 2 shows the results of analysis of the particle size distribution and stability of DEX/PFP @ LIPs-BMS-. alpha.in example 1 of the present invention;

FIG. 3 is a graph showing potential distributions of the drug systems (DEX/PFP @ LIPs-BMS- α, PFP @ LIPs and Dex/PFP @ LIPs) according to examples 1, 3 and 4 of the present invention;

FIG. 4 is a transmission electron microscope observation result of DEX/PFP @ LIPs-BMS-alpha in example 1 of the present invention;

FIG. 5 shows confocal microscope observation results of Experimental example 1 of the present invention (PA treatment 144h, recovery 24h, from left to right: merge channel, DAPI channel and FITC (Alexa)

488) A channel);

FIG. 6 shows the results of confocal microscopy observations of Experimental example 1 of the present invention (DSPE-PEG-COOH-BMS- α treatment for 144h, recovery for 24h, merge channel, DAPI channel and FITC (Alexa) from left to right

488) A channel);

FIG. 7 shows confocal microscope observation results of Experimental example 1 of the present invention (PA treatment 72h, recovery 96h, from left to right: merge channel, DAPI channel andFITC(Alexa

488) a channel);

FIG. 8 shows confocal microscope observation results of Experimental example 1 of the present invention (PBS treatment 168h, from left to right in this order: merge channel, DAPI channel and FITC (Alexa)

488) A channel);

FIG. 9 shows the results of fluorescence semiquantitative analysis in Experimental example 1 of the present invention;

FIG. 10 shows confocal microscope observation results (blank control, merge channel) of Experimental example 2 of the present invention;

FIG. 11 shows the observation results of confocal microscopy in Experimental example 2 of the present invention (negative control group 1, TRTIC (DiI) channel);

FIG. 12 shows the confocal microscope observation results (negative control group 2, TRTIC (DiI) channel) in Experimental example 2 of the present invention;

FIG. 13 shows confocal microscope observation results (negative control group 3, TRTIC (DiI) channel) in Experimental example 2 of the present invention;

FIG. 14 shows the observation results of confocal microscopy in Experimental example 2 (Experimental group 1, TRTIC (DiI) channel);

FIG. 15 shows the observation results of confocal microscopy in Experimental example 2 (Experimental group 2, TRTIC (DiI) channel);

FIG. 16 shows the observation results of confocal microscope (Experimental group 3, TRTIC (DiI) channel) in Experimental example 2 of the present invention;

FIG. 17 shows the confocal microscope observation results of Experimental example 3 of the present invention (control group, TRTIC (rhodamine-labeled Phalloidin/TRITC Phalloidin) black-and-white channel);

FIG. 18 shows the observation result of confocal microscope (Experimental group 1, TRTIC (rhodamine-labeled Phalloidin/TRITC Phalloidin) black-and-white channel) in Experimental example 3 of the present invention;

FIG. 19 shows the confocal microscope observation results of Experimental example 3 (Experimental group 2, TRTIC (rhodamine-labeled Phalloidin/TRITC Phalloidin) black-and-white channel);

FIG. 20 shows the confocal microscope observation results of Experimental example 3 of the present invention (Experimental group 3, TRTIC (rhodamine-labeled Phalloidin/TRITC Phalloidin) black-and-white channel);

FIG. 21 shows the confocal microscope observation results of Experimental example 3 (Experimental group 4, TRTIC (rhodamine-labeled Phalloidin/TRITC Phalloidin) black-and-white channel);

FIG. 22 shows the observation result of confocal microscope (Experimental group 5, TRTIC (rhodamine-labeled Phalloidin/TRITC Phalloidin) black-and-white channel) in Experimental example 3 of the present invention;

FIG. 23 shows the results of fluorescence semiquantitative analysis in Experimental example 3 of the present invention;

FIG. 24 shows the results of the cytotoxicity test in Experimental example 4 of the present invention;

FIG. 25 shows the results of the ultraviolet absorbance test in Experimental example 5 of the present invention;

FIG. 26 is a graph of the LIFU induced phase transition B-mode in experimental example 6 of the present invention;

FIG. 27 is a CEUS-mode graph showing LIFU induced phase transition in experimental example 6 of the present invention;

FIG. 28 shows the results of the drug release test (4 ℃ C.) in Experimental example 7 of the present invention;

FIG. 29 shows the result of the drug release test (37 ℃) in Experimental example 7 of the present invention;

FIG. 30 shows the results of a drug release test (37 ℃ in combination with LIFU) in Experimental example 7 of the present invention;

Detailed Description

The following is further detailed by way of specific embodiments:

example 1: preparation of targeting phase change nano-drug system (DEX/PFP @ LIPs-BMS-alpha)

(1) Synthesis of DSPE-PEG-COOH-BMS-alpha

The condensed compound (distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl-BMS-alpha, DSPE-PEG-COOH-BMS-alpha) is prepared by taking distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl (DSPE-PEG (2000) -COOH) and a compound BMS-470539 (BMS-alpha for short) as raw materials through a carbodiimide method.

80mg (0.027mmol) of DSPE-PEG (2000) -COOH were dissolved in 20ml of Dimethylformamide (DMF); adding 3.8mg (0.027mmol) of anhydrous 1-Hydroxybenzotriazole (HOBT); then 3.5mg (0.027mmol) of N, N-Diisopropylcarbodiimide (DIC) is added, and the mixture is shaken for 0.5 h; then adding 15mg (0.025mmol) of compound BMS-470539 dissolved in 2ml of DMF, and shaking for 24h at normal temperature; and purifying by HPLC to obtain a target product DSPE-PEG-COOH-BMS-alpha. And finally, detecting the obtained target product by Mass Spectrometry and HPLC (high Performance liquid chromatography), and determining that the target product is DSPE-PEG-COOH-BMS-alpha.

(2) Preparation of lipid film

Dipalmitoylphosphatidylcholine (DPPC), Dipalmitoylphosphatidylglycerol (DPPG), the compound produced in step (1) (DSPE-PEG-COOH-BMS- α) and Cholesterol (CHO) were placed in a round bottom flask in a molar ratio of 69:8:8:15, and 4mg Dexamethasone (DEX) was added per 10mg of lipid (the lipid includes four substances of DPPC, DPPG, DSPE-PEG-COOH-BMS- α and CHO), followed by addition of 5ml of chloroform and 2ml of methanol for complete dissolution. Rotary evaporating at 55 deg.C and negative pressure of-0.1 MPa for 30min to obtain lipid film.

(3) Preparation of the Nanoparticulate System DEX/PFP @ LIPs-BMS-alpha

The lipid film obtained in step (2) was hydrated and sufficiently eluted by adding 2ml of PBS buffer (PH 7.4) per 10mg of lipid. 200ul of perfluoro-n-pentane PFP was then added per 10mg of lipid and sonicated using a cryosonicator (1000w 5%, 20HZ, 5s/5s on/off, 5 min). And centrifuging at 4 deg.C (8000 rpm for 5min), removing supernatant to obtain lower precipitate, i.e. DEX/PFP @ LIPs-BMS-alpha.

The DEX/PFP @ LIPs-BMS- α prepared in this example is a targeting phase-change nano-drug system of this scheme, and includes a main body lipid microsphere, in which perfluoro-n-pentane PFP (one of liquid fluorocarbons) that undergoes phase change by sound is wrapped, a drug dexamethasone DEX is embedded in the main body, and the main body is connected with a compound BMS-470539 for targeting glomerulus through an amide bond. The finished DEX/PFP @ LIPs-BMS-. alpha.prepared in this example was milky in appearance. The morphology of DEX/PFP @ LIPs-BMS-. alpha.prepared in this example was observed under a confocal microscope (. times.800), and the results are shown in FIG. 1. Transmission electron microscope observation is carried out on DEX/PFP @ LIPs-BMS-alpha prepared by the scheme, and the result is shown in figure 4, wherein the DEX/PFP @ LIPs-BMS-alpha is in a spherical shape. Therefore, the shape of the nano lipid microsphere MP/PFP @ LIPs-BMS-470539 is spherical, the size is uniform, the shape is regular, and the dispersibility is good.

The DEX/PFP @ LIPs-BMS-. alpha.prepared in this example was analyzed for particle size distribution and stability using a Malvern particle size analyzer (shown in FIG. 2): DEX/PFP @ LIPs-BMS- α was allowed to stand for 0, 7 and 14 days, respectively, and then the particle size of DEX/PFP @ LIPs-BMS- α was measured. The particle size of the particles is (197.9 + -3.9) nm at day 0, the particle size is (207.5 + -11.5) nm at day 7, and the particle size is (218.9 + -16.3) nm at day 14. The comparison difference of all time points has no statistical significance (the anova analysis is carried out on the particle size data of the three groups, p is larger than 0.1, and the three groups of data have no significant difference), which indicates that the drug system has good stability.

Potential analysis was performed on DEX/PFP @ LIPs-BMS-. alpha.prepared in this example, and the average Zeta potential was (-53.8. + -. 1.7) mV, as shown in FIG. 3. Dex/PFP @ LIPs-BMS- α showed significant statistical significance compared to PFP/LIPs, Dex/PFP @ LIPs, respectively, where p is < 0.05 and p is < 0.01.

Example 2: preparation of targeting phase change nano-drug system (PFP @ LIPs-BMS-alpha)

The preparation method of this example is different from that of example 1 in the step (2), specifically as follows:

(2) preparation of lipid film

Dipalmitoylphosphatidylcholine (DPPC), Dipalmitoylphosphatidylglycerol (DPPG), the compound produced in step (1) (DSPE-PEG-COOH-BMS- α) and Cholesterol (CHO) were placed in a round bottom flask in a molar ratio of 69:8:8:15 and Dil was added simultaneously. Dil is used in an amount of 0.5mg per 10mg of lipid (including four substances DPPC, DPPG, DSPE-PEG-COOH-BMS-alpha and CHO). Then, 5ml of chloroform and 2ml of methanol were added thereto to sufficiently dissolve the chloroform. Rotary evaporating at 55 deg.C and negative pressure of-0.1 MPa for 30min to obtain lipid film.

Example 3: preparation of phase-change Nanoparticulate systems (PFP @ LIPs)

This embodiment is basically the same as embodiment 2, except that: instead of using DSPE-PEG-COOH-BMS- α (i.e., without (1) synthesis of DSPE-PEG-COOH-BMS- α), DSPE-PEG-COOH was used in place of DSPE-PEG-COOH-BMS- α in the synthesis steps (2) and (3). Potential analysis of PFP @ LIPs prepared in this example showed that the average Zeta potential was (-42.3. + -. 0.9) mV, as shown in FIG. 3.

Example 4: preparation of phase-change Nanoparticulate systems (DEX/PFP @ LIPs)

This embodiment is basically the same as embodiment 1 except that: instead of using DSPE-PEG-COOH-BMS- α (i.e., without (1) synthesis of DSPE-PEG-COOH-BMS- α), DSPE-PEG-COOH was used in place of DSPE-PEG-COOH-BMS- α in the synthesis steps (2) and (3). Potential analysis was performed on DEX/PFP @ LIPs prepared in this example, and the result is shown in FIG. 3, where the average Zeta potential was (-47.6. + -. 1.2) mV.

Experimental example 1: human kidney podocyte MC1R induction high expression and verification

Inoculating human kidney podocyte (product number: BNCC340460, Beijing Beinana Biotechnology Limited) into a sterile culture bottle, adding McCoy's 5A culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placing the culture bottle at 37 deg.C and 5% CO₂The podocytes were inoculated in T25 cell culture flasks after eight days of culture in a cell culture incubator, 4ml of medium containing 5ug/ml puromycin aminonucleoside (PA; MCE, NSC 3056) was added to experimental group 1 for 144h and 24h recovery, 4ml of medium containing 10nM DSPE-PEG-COOH-BMS- α (prepared in example 1) was added to experimental group 2 for 144h and 24h recovery, 4ml of medium containing 5ug/ml PA was added to experimental group 3 for 72h and 96h recovery, and an equivalent amount of 1 × PBS was added to control group for 168 h.

Two days before the experiment, each group of cells was seeded on a confocal dish (radius r 15mm) and cultured. When the cells in the confocal dish have proper density, washing the cells twice by PBS at 37 ℃; fixing with 4% paraformaldehyde-containing PBS at room temperature for 15min, and washing with PBS at 37 deg.C for three times; the cells were permeabilized with 0.1% polyoxyethylene octylphenyl ether with PBS (Triton X-100) for 5min at room temperature and washed three times with PBS; 1% Bovine Serum Albumin (BSA) containing PBS was treated at room temperature for 1h, and washed three times with PBS; 100ul of rabbit anti-MC 1R antibody (abcam, ab236734, dilution 1: 130) was added, placed in a refrigerator at 4 ℃ overnight, and then washed three times with PBS; adding into100ul goat anti-rabbit IgG H&L(Alexa

488. abcam, ab150077, dilution ratio 1: 1000) incubating at room temperature for 1h, and washing with PBS for three times; 4', 6-diamidino-2-phenylindole DAPI was counterstained for 10min, washed twice with PBS and observed under a confocal microscope.

Confocal microscope observation results are shown in FIGS. 7-10, and goat anti-rabbit IgG H&The results of the semiquantitative analysis of fluorescence are shown in FIG. 9, wherein P is represented by P < 0.05, the results of the above experiment show that both normal human renal podocytes and PA-induced human renal podocytes express MC1R, PA promotes MC1R expression and is related to induction time, the decrease in fluorescence intensity after DSPE-PEG-COOH-BMS- α treatment is related to the decrease in MC1R expression after DSPE-PEG-COOH-BMS- α treatment

488 green fluorescence, the DAPI channel displays the DAPI blue fluorescence, FITC (Alexa)

488) The channel shows Alexa

488 (only the merge channel is shown in figures 5-8, the remaining two channels are not shown).

Experimental example 2: PFP @ LIPs-BMS-alpha targeting human kidney podocyte verification

Inoculating human kidney podocytes into a sterile culture flask, adding McCoy's 5A medium containing 10% fetal calf serum and 1% penicillin-streptomycin, and placing the culture flask at 37 deg.C and 5% CO₂Culturing for seven days in a cell culture box, inoculating podocytes into a T25 culture flask, adding a culture medium containing 5ug/ml Puromycin Aminonucleoside (PA) into the cells, treating for 72h with 4ml, and recovering for 120 h.

Two days before the following experiment, cells were seeded on a confocal dish (radius r 15mm) and cultured. When the cells in the confocal dish have proper density, washing the cells twice by PBS at 37 ℃; adding 1ml culture medium (McCoy's 5A, 10% fetal calf serum and 1% penicillin-streptomycin) into a confocal dish, adding PBS (40ul) into a blank control group, and incubating for 120 min; adding PFP @ LIPs (2.5mg/ml, 40ul) into the negative control group 1, and incubating for 30 min; adding PFP @ LIPs (2.5mg/ml, 40ul) into the negative control group 2, and incubating for 60 min; adding PFP @ LIPs (2.5mg/ml, 40ul) into the negative control group 3, and incubating for 120 min; PFP @ LIPs-BMS-alpha (2.5mg/ml, 40ul) was added to the experimental group 1 and incubated for 30 min; PFP @ LIPs-BMS-alpha (2.5mg/ml, 40ul) was added to the experimental group 2 and incubated for 60 min; experiment group 3 was incubated for 120min with PFP @ LIPs-BMS-. alpha. (2.5mg/ml, 40 ul). The procedure for the preparation of PFP @ LIPs-BMS-. alpha.was the same as in example 2, and DiI fluorescent dye was added during the preparation.

All groups were treated according to the following conditions after incubation under the above conditions: washing the cells with 37 deg.C PBS for three times, 5min each time; fixing with 4% paraformaldehyde-containing PBS at room temperature for 15min, and washing with 37 deg.C PBS for three times; DAPI was counterstained for 10min, washed twice with PBS and observed under a confocal microscope.

The observation result of the confocal microscope is shown in fig. 10-16, a DiI fluorescent dye is added in the preparation process of the nano lipid microsphere PFP @ LIPs-BMS-alpha, and the nano lipid microsphere PFP @ LIPs-BMS-alpha emits co-color fluorescence under the excitation of laser; DAPI stains the nucleus of the cell and emits blue fluorescence after laser excitation. Cells were treated under the above conditions in each group, and the TRTIC channel of the blank control group showed no red fluorescence but only blue fluorescence emitted by DAPI (shown by the arrow in FIG. 10); in the negative control group, as the incubation time of PFP @ LIPs-BMS-alpha and cells is prolonged, scattered and gradually enhanced red fluorescence can be seen in the TRTIC channel (fig. 11-13, bright spots and parts with colors lighter than the background in the figure are red fluorescence emitted by the DiI fluorescent dye on the nanoparticles); experimental groups (FIGS. 14-16) the TRTIC channel fluorescence intensity was gradually increased with increasing PFP @ LIPs-BMS-alpha and cell incubation times. When the incubation time is short, the fluorescence signal is scattered around the cell membrane, the incubation time is prolonged, red fluorescence is generated around the cell membrane and in the cell, and the nano lipid microsphere PFP @ LIPs-BMS-alpha can effectively target the human kidney podocyte. In this example, the sample was observed using four channels, the merge channel shows red fluorescence emitted from Dil and blue fluorescence emitted from DAPI, the DAPI channel shows blue fluorescence emitted from DAPI, the trtic (dii) channel shows red fluorescence emitted from Dil, and the TD channel shows a black-and-white image of the merge channel (fig. 11 to 16 show only a part of the channels).

Experimental example 3: human kidney podocyte stabilization and repair validation

Inoculating human kidney podocytes into a sterile culture flask, adding McCoy's 5A medium containing 10% fetal calf serum and 1% penicillin-streptomycin, and placing the culture flask at 37 deg.C and 5% CO₂Culturing for seven days in a cell culture box, inoculating podocytes into a T25 flask, adding 4ml of medium containing 100nM BMS- α to culture medium in experimental group 1 for 72h and recovering for 96h, adding 4ml of medium containing DSPE-PEG-COOH-BMS- α (prepared in example 1 and calculated as 100nM by BMS- α equivalent) to culture medium in experimental group 1 for 72h, adding 4ml of medium containing PA (5ug/ml) to culture group 3 for 72h, adding 4ml of medium containing PA (5ug/ml) to culture medium in experimental group 4 for 72h, adding 4ml of medium containing BMS- α (100nM) to culture medium in experimental group 3 for 72h, recovering for 24h, adding 4ml of medium containing PA (5ug/ml) to culture medium in experimental group 5 for 72h, adding 4ml of medium containing DSPE-PEG-COOH-BMS- α (calculated as 100nM) to culture medium for 72h, adding PBS in experimental group 5 for 24 min, adding PBS (5ug/ml) to culture medium containing PBS for 72h, washing cells in experimental group 5min, washing the experimental group for 24 min, adding PBS for 24 min, washing cells in PBS for 5min, adding PBS for 1 min, washing cells in PBS containing 20min, incubating after incubating, adding 1 min, adding PBS for 1 min, adding PBS containing formaldehyde, washing the PBS for 1 mm, incubating, and incubating the cells, wherein the cells are washed for 1 mm after incubating.

The results of confocal microscopy are shown in FIGS. 17-22, and the results of fluorescence semiquantitative analysis are shown in FIG. 23. The experimental results show that: incubating rhodamine-labeled Phalloidin (TRITC Phalloidin) with each group of cells for 30min, emitting red fluorescence under laser, and enabling cytoskeleton to be in a red filamentous shape; DAPI stains the nuclei and emits blue fluorescence upon laser excitation (not shown). The control group and the

experimental groups

1 and 2 have the strongest fluorescence, and the cytoskeletons are clear and ordered; the cytoskeleton is visible when the fluorescence of the

experimental groups

4 and 5 is inferior; experimental group 3 was the weakest, cytoskeleton disturbed, ruptured. In the present experimental example, the merge channel shows red fluorescence of rhodamine-labeled phalloidin and blue fluorescence of DAPI under a fluorescence microscope, the TRTIC channel shows red fluorescence of rhodamine-labeled phalloidin under a fluorescence microscope, and the TRTIC channel black and white converts an image displayed by the TRTIC channel into a black and white image (only the TRTIC channel black and white is shown in fig. 17 to 22). The control group (PBS 168h) was subjected to fluorescence semiquantitative analysis with respect to experiment group 1 (BMS-. alpha.72 h) and experiment group 2 (DSPE-BMS-. alpha.72 h), and no significant difference was observed (P > 0.05). The experiment group 3 and the rest groups have obvious difference (P is less than 0.05) by fluorescence semi-quantitative analysis. BMS-alpha and DSPE-PEG-COOH-BMS-alpha do not influence the cytoskeleton arrangement of normal renal podocytes, PA can cause the breakage and disorder of the cytoskeleton of the renal podocytes, BMS-alpha and DSPE-PEG-COOH-BMS-alpha have the function of nondifferential repair of the broken and disordered cytoskeleton of the renal podocytes, and DSPE-PEG (2000) -COOH and BMS-alpha are covalently connected, so that the skeleton repair function of BMS-alpha is not influenced.

Experimental example 4: cytotoxicity test

Experimental studies on the cytotoxicity of DEX/PFP @ LIPs-BMS- α with Dex human kidney podocytes were inoculated into sterile flasks and McCoy's 5A medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added, the flasks were placed at 37 ℃ and 5% CO₂Culturing for about 13 days in a cell culture box, inoculating 10000 cells per well in a 96-well plate for 24h, then adding 100ul Dex/PFP @ LIPs-BMS- α with the concentration of 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0mg/ml respectively in the drug system group, and repeating each concentration for 5 timesg/ml, each concentration was repeated 5 times, and the cells were further cultured for 24 hours after addition, and the cytotoxicity was measured according to the CCK-8 protocol, the results of the experiment are shown in FIG. 24, where Dex has no significant toxicity to the cells, DEX/PFP @ LIPs-BMS- α has the effect of promoting cell proliferation with increasing concentration, and DEX/PFP @ LIPs-BMS- α has the strong effect of promoting proliferation at 0.8 and 1.0 mg/ml.

Experimental example 5: ultraviolet absorbance photometric analysis

UV absorbance analysis was performed on the drug systems prepared in example 1 and example 2 (DEX/PFP @ LIPs-BMS-alpha and PFP @ LIPs-BMS-alpha, respectively) and the results are shown in FIG. 25, indicating that Dex was successfully loaded in the drug system of example 1.

Experimental example 6: LIFU phase transition experiment

DEX/PFP @ LIPs-BMS- α nano-sized lipid microspheres (original concentration 2.5mg/ml) from example 1 were diluted 32-fold and loaded in agar wells at LIFU power of 0.8w/cm²、1.6w/cm²、2.4w/cm²The ultrasonic wave is acted for 0-5min under the condition of 5s work/5 s pause, the B-mode and the CEUS-mode are subjected to imaging observation, the experimental result is shown in figure 26 and figure 27, and the DEX/PFP @ LIPs-BMS- α has the ultrasonic imaging function, and the LIFU can cause the nano microspheres to release the drug in a phase change manner at 2.4w/cm²The phase change is most obvious when the powder is acted for 3 min.

Example 7: drug release test

DEX/PFP @ LIPs-BMS- α nano-liposome microspheres (prepared in example 1) were dialyzed at 37 ℃ and 37 ℃ in combination with LIFU at 4 ℃ and 2.4w/cm, and DEX release rate was measured by UV spectrophotometry, PBS (0.01mol/L, pH 7.4) was added with 0.5% Tween-80(V/V) to prepare release solution, 3 dialysis bags with a molecular cut-off of 3000Da were placed in each dialysis bag, 2ml of release solution was added, Dex/PFP @ LIPs-BMS- α 1mg was added, the dialysis bags were soaked in 60ml of release solution, placed in 4 ℃, 37 ℃ and 37 ℃ for 120 rpm and continuously stirred, 2ml of release solution was added in 2h, 4h, 6h, 8h, 10h, 12h, 24h and 48h, and 2ml of release solution was added, 37 ℃ to LIFU was added to 8h, 12h LIFU 2.4w/cm, 12h²3min (5s work/5 pause). The absorbance of the released solution at 242nm was measured spectrophotometrically. The test results are shown in FIG. 28-30, and the light absorption value at the same time is 37-LIFU > 37 ℃ and > 4 ℃, the surface temperature rise is beneficial to the release of Dex drugs, and LIFU can promote the release of drugs.

Example 8: in vivo experiments

Dex/PFP @ LIPs-BMS-alpha nano lipid microspheres are targeted to a passive type Heimann nephritis (PHN) model mouse glomerulus. The PHN model was prepared using rabbit Anti-Fx1A antibody (Probetex Inc.) in healthy male SD rats at 140-160 g, and Anti-Fx1A IgG antibody was first administered at 1.5ml and added at 0.5ml after 1 week. And evaluating and screening successful animal models through indexes such as urine protein and the like in 14 days. DiI-labeled Dex/PFP @ LIPs (2.5mg/ml,500ul) were administered to the tail vein of the non-target group (n ═ 5), and kidney tissues were taken at 4h, 8h, 16h, 24h, and 48h after injection, respectively, and were frozen and visualized at confocal. DiI-labeled Dex/PFP @ LIPs-BMS-alpha (2.5mg/ml,500ul) was administered to the tail vein of the target group (n ═ 5), and kidney tissues were taken at 4h, 8h, 16h, 24h, and 48h after injection, respectively, and were frozen and visualized under confocal conditions. The experimental results show that: DiI-labeled Dex/PFP @ LIPs and Dex/PFP @ LIPs-BMS-alpha are injected into tail veins in equal amount (2.5mg), and the kidney is frozen section and observed by a confocal microscope, wherein the blue color is cell nucleus stained by DAPI, the blue color and the dark red color are self-luminous of kidney tissues, and the granular orange color is a DiI-labeled targeted nano-drug system and a DiI-labeled non-targeted nano-drug system. The increase of the drug system marked on glomeruli 8h after Dex/PFP @ LIPs injection is not significant at 16h and 24h, and the enrichment of the drug system is reduced at 48h compared with that of the prodrug system. After Dex/PFP @ LIPs-BMS-alpha injection, glomeruli are almost all targeted, a labeled drug system appears at 4h, and the significant enrichment of the drug system can be observed at 16h, 24h and 48 h.

The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

1. The targeting phase-change nano-drug system is characterized in that: comprises a main body, liquid fluorocarbon is wrapped in the main body, a medicine is loaded on the main body, and BMS-470539 is covalently connected to the main body.

2. The targeted phase change nano-drug system of claim 1, wherein: the main body is lipid nano-microspheres.

3. The targeted phase change nano-drug system of claim 2, wherein: the liquid fluorocarbon is perfluoro-n-pentane.

4. The targeted phase change nano-drug system of claim 3, wherein: the drug is dexamethasone.

5. The method for preparing the targeted phase transition nano-drug system of claim 4, comprising the steps of:

6. The method of claim 5, wherein in step (1), distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl group and BMS-470539 are condensed by a carbodiimide method to obtain DSPE-PEG-COOH-BMS- α.

7. The method according to claim 6, wherein in the step (2), the molar ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, DSPE-PEG-COOH-BMS- α, and cholesterol is 69:8:8: 15.

8. The production method according to claim 7, wherein in the step (2), the mixed solvent is composed of chloroform and methanol.

9. The preparation method according to claim 7, wherein in the step (2), the medicament is dexamethasone, and the mass ratio of the dexamethasone to the lipid is 1: 2.

10. use of the targeted phase transition nano-drug system according to any one of claims 1-4 for the preparation of a medicament for the treatment of immune nephropathy.