CN117137886A

CN117137886A - Bionic targeted drug-loaded nanoparticle modified by fusion cell membrane and used for heart failure treatment, and preparation method and application thereof

Info

Publication number: CN117137886A
Application number: CN202311111460.7A
Authority: CN
Inventors: 王媛; 杜杰; 李煜钰; 于佳琪
Original assignee: BEIJING INSTITUTE OF HEART LUNG AND BLOOD VESSEL DISEASES
Current assignee: BEIJING INSTITUTE OF HEART LUNG AND BLOOD VESSEL DISEASES
Priority date: 2023-08-31
Filing date: 2023-08-31
Publication date: 2023-12-01

Abstract

The application discloses a bionic targeted drug-loaded nanoparticle modified by fusion cell membranes for heart failure treatment, and a preparation method and application thereof. The application provides application of a drug-loaded nanoparticle modified by fusion cell membranes in preparation of drugs; the drug-loaded nanoparticle modified by the fusion cell membrane comprises the fusion cell membrane and a drug-loaded nanoparticle inner core wrapped in the fusion cell membrane; the fusion cell membrane is formed by fusion of a platelet membrane and an erythrocyte membrane; the drug-loaded nanoparticles are JQ 1-loaded PLGA nanoparticles; the medicament is a medicament for treating heart failure and/or myocardial infarction. The drug-loaded nano-particles modified by the fusion cell membrane can target and identify myofibroblasts and collagen, adhere to the collagen part and can prevent immunophagy. The drug-loaded nano-particles modified by the fusion cell membrane have the advantages of high efficiency, targeting and low toxicity when used as heart failure treatment drugs.

Description

Bionic targeted drug-loaded nanoparticle modified by fusion cell membrane and used for heart failure treatment, and preparation method and application thereof

Technical Field

The application belongs to the field of biological medicine, and relates to a bionic targeted drug-loaded nanoparticle modified by fusion cell membranes for heart failure treatment, and a preparation method and application thereof.

Background

Heart failure is the final stage of various heart diseases, with poor prognosis. Fibroblast activation and secretion of a large amount of collagen cause heart fibrosis, and are main pathological factors of heart failure. Prevention and treatment of cardiac fibrosis is critical for suppressing development of heart failure and improving prognosis, wherein targeted inhibition of activation and secretion of collagen by cardiac fibroblasts is an important point for development of anti-cardiac fibrosis treatment strategies, and it is expected that heart failure treatment will be achieved through nanocarrier targeting sites. However, the process of releasing the drug from intravenous injection to cardiac fibroblasts by nano-drug carriers is complex, and various barriers such as phagocytosis of macrophages, incapacity of targeted enrichment to cardiac sites and the like exist, so that the treatment effect is affected. Although various nano-drug carriers are reported at home and abroad, research on nano-drug carriers for heart failure treatment with clinical application prospect is still a serious challenge.

The main reasons for failure of nano-drug targeted fibrotic cardiac delivery are the rapid clearance of the immune system and stability in the blood circulatory system. There is a need to develop new nano-drug targeted drugs.

Disclosure of Invention

The application aims to provide a bionic targeted drug-loaded nanoparticle modified by fusion cell membranes for heart failure treatment, and a preparation method and application thereof.

The application provides application of a drug-loaded nanoparticle modified by fusion cell membranes in preparation of drugs; the medicament is a medicament for treating heart failure and/or myocardial infarction.

The application also provides application of the drug-loaded nano-particles modified by the fusion cell membrane in preparation of drugs; the medicament is used for inhibiting cardiac fibrosis and/or inhibiting myocardial cell hypertrophy. Specifically, the cardiac fibrosis is cardiac fibrosis caused by heart failure. Specifically, the cardiomyocyte hypertrophy is a cardiomyocyte hypertrophy caused by myocardial infarction.

The application also provides application of the drug-loaded nano-particles modified by the fusion cell membrane in preparation of drugs; the medicine is used for inhibiting heart injury after heart failure and/or heart injury after myocardial infarction. Specifically, the cardiac injury is cardiac functional injury.

Specifically, any of the above heart failure is chronic heart failure.

Specifically, any one of the above myocardial infarction is an acute myocardial infarction.

The drug-loaded nanoparticle modified by the fusion cell membrane comprises the fusion cell membrane and a drug-loaded nanoparticle inner core wrapped in the fusion cell membrane; the fusion cell membrane is formed by fusing a platelet membrane and an erythrocyte membrane; the drug-loaded nanoparticles are JQ 1-loaded PLGA nanoparticles; the fusion cell membrane is formed by fusing 4 parts by mass of platelet membrane and 1 part by mass of erythrocyte membrane.

The preparation method of the drug-loaded nanoparticle modified by the fusion cell membrane comprises the following steps: the drug-loaded nanoparticles are coated in the fusion cell membrane.

The fusion cell membrane plays a targeting and protecting role. The fusion cell membrane can target and identify myofibroblasts and collagen and adhere to the collagen site and can prevent immunophagia.

The nanoparticles exert supporting and drug-carrying effects, and may also exert structural modification effects.

PLGA: polylactic acid-glycolic acid copolymer.

Specifically, the PLGA is PLGA (75:25).

PLGA (75:25) was polymerized from 75% polylactic acid (PLA) and 25% polyglycolic acid (PGA).

In any one of the above fusion cell membranes, the mass ratio of the platelet membrane to the erythrocyte membrane is 4:1. the mass ratio is calculated by the protein meter.

The fusion cell membrane is formed by fusing 4 parts by mass of platelet membrane and 1 part by mass of erythrocyte membrane. The mass parts are calculated by protein.

Specifically, the platelet membrane is obtained by crushing platelets and collecting the crushed platelets.

In particular, the platelets are isolated from blood.

Specifically, the erythrocyte membrane is obtained by crushing and collecting erythrocytes.

In particular, the erythrocytes are isolated from blood.

Specifically, the blood is human blood.

Specifically, the blood is whole blood of type O obtained from a healthy person.

Specifically, the blood is O-type blood whole blood of a healthy person collected by an EDTA anticoagulation blood collection tube, and the blood is used within 16 hours after blood drawing.

The method for coating the drug-loaded nano-particles in the fusion cell membrane comprises the following steps: and (3) carrying out ultrasonic treatment on a fused cell membrane liquid phase (the adding amount of the fused cell membrane is n in the amount of platelets used for preparing the fused cell membrane), then adding drug-loaded nano-particles (the adding amount of the drug-loaded nano-particles is m in the amount of PLGA raw materials added in the amount of the drug-loaded nano-particles), and carrying out ultrasonic treatment to obtain the drug-loaded nano-particles modified by the fused cell membrane.

Specifically, when n is 10 ⁷ -10 ¹¹ M is 1mg when used.

Specifically, when n is 10 ⁸ -10 ¹⁰ M is 1mg when used.

Specifically, when n is 3×10 ⁹ M is 1mg when used.

The sonication may in particular be a plurality of sonications, for example three times.

Specifically, the ultrasonic probe parameters of ultrasonic treatment are: 42kHz, 100W, 5min.

When the drug-loaded nano-particles are prepared, the mass ratio of JQ1 to PLGA can be specifically 1:10.

the drug-loaded nanoparticles can be prepared by any method known in the art.

Specifically, the method for preparing the drug-loaded nanoparticle comprises the following steps: and (3) dissolving JQ1 and PLGA in DMSO, dripping double distilled water, transferring into a dialysis bag for dialysis, collecting liquid phase in the dialysis bag, and freeze-drying to obtain the drug-loaded nano-particles. The molecular weight cut-off of the dialysis bag may specifically be 3500Da.

Specifically, the method for preparing the drug-loaded nanoparticle comprises the following steps: 1.5mg of JQ1 and 15mg of PLGA are dissolved in 1mL of DMSO, then 4mL of double distilled water is added dropwise while slowly stirring, then the whole system is transferred into a dialysis bag (the molecular cut-off is 3500 Da), the dialysis bag is placed into the double distilled water for dialysis, then the liquid phase in the dialysis liquid is collected, and freeze drying is carried out, so that dry matters are obtained, namely JQ1-loaded PLGA nano particles, and the JQ1 NPs are used for representing.

Specifically, the method for preparing the fusion cell membrane is as follows: mixing the erythrocyte membrane suspension and the platelet membrane suspension, and performing ultrasonic treatment to obtain a fused cell membrane liquid phase. The sonication may specifically be three times of sonication. Specifically, the parameters of the ultrasonic probe for each ultrasonic treatment are as follows: 42kHz, 100W, 5min.

Specifically, the method for preparing the fusion cell membrane is as follows: mixing 1 part by volume of erythrocyte membrane suspension (with the protein concentration of 5 mu g/mu L) and 4 parts by volume of platelet membrane suspension (with the protein concentration of 5 mu g/mu L), and carrying out ultrasonic treatment three times (the parameters of an ultrasonic probe are 42kHz, 100W and 5min in each ultrasonic treatment), so as to obtain a fused cell membrane liquid phase.

Specifically, the erythrocyte membrane suspension is obtained by suspending erythrocyte membranes in a solvent. The solvent may be a buffer, such as a PBS buffer.

Specifically, the platelet membrane suspension is obtained by suspending a platelet membrane in a solvent. The solvent may be a buffer, such as a PBS buffer.

Any prior art method may be used to obtain erythrocytes and to prepare erythrocyte membranes.

The preparation of erythrocyte membrane comprises the following steps: red blood cells are isolated from whole blood and disrupted by hypotonic treatment to collect red blood cell membranes.

Illustratively, the method of preparing an erythrocyte membrane comprises the steps of:

(1) erythrocytes were isolated from whole blood and washed 2 times with PBS buffer containing 1mM EDTA, 50. Mu.M Leupeptin and 1. Mu.g/mL Aprotin (centrifugation at 720g for 10min at 4℃after each wash), the supernatant was discarded, and the pellet was retained).

(2) After the completion of step (1), suspending the pellet with 4 volumes of hypotonic solution relative to the pellet, incubating for 60min, centrifuging at 4 ℃ for 20min at 20000g, discarding the supernatant, and retaining the pellet; hypotonic solution: contains 0.2mM EDTA-2Na, and the balance is deionized water.

(3) After completion of step (2), the pellet was washed with PBS buffer containing 50. Mu.M Leupeptin and 1. Mu.g/mL Aprotin, and then centrifuged at 20000g for 20 minutes at 4℃to discard the supernatant, leaving the pellet (pellet is erythrocyte membrane).

The erythrocyte membrane is preserved at 4 ℃ and used within 6 hours after preparation.

Any of the methods known in the art may be used to obtain platelets and prepare platelet membranes.

Illustratively, the method of preparing a platelet membrane comprises the steps of: platelets are separated from whole blood, broken by freeze thawing treatment, and platelet membranes are collected.

Illustratively, the method of preparing a platelet membrane comprises the steps of: platelets were isolated from whole blood, resuspended in PBS buffer containing EDTA (1 mM) and protease inhibitor tablets (1 tablet/50 mL), and the pellet was repeatedly freeze-thawed 3 times (-80 ℃ C. Freeze, room temperature thawing), then centrifuged at 4 ℃ C., 4000g for 3min, the supernatant was discarded, and the pellet was retained (the pellet was the platelet membrane).

The platelet membrane was stored at 4℃and used within 6 hours after preparation.

Illustratively, a method for separating red blood cells from whole blood includes the steps of: whole blood was centrifuged at 4℃and 100g for 20min, the lower layer was collected, and after centrifugation at 720g for 10min at 4℃the supernatant was discarded, and the pellet (the pellet was red blood cells) was retained.

Illustratively, a method for separating platelets from whole blood includes the steps of: centrifuging whole blood at 4deg.C and 100g for 20min, collecting upper layer, centrifuging at 4deg.C and 100g for 20min, discarding bottom precipitate, adding prostaglandin E containing 1mM EDTA and 2mM into the rest liquid phase ₁ Is centrifuged at 800g for 20min at 4 ℃,the supernatant was discarded and the pellet (the pellet being the platelet) was retained.

The platelet membrane surface has GPIba, GPIIbIIIa and GPVI proteins, and the GPIba, GPIIbIIIa and GPVI proteins can target and identify myofibroblasts and collagen and adhere to the collagen part. The surface of erythrocyte membrane has CD47 protein, which can prevent immunophagy (preventing phagocytosis by macrophage), and erythrocyte membrane has the function of prolonging residence time in blood. The drug-loaded nano-particles modified by the fusion cell membrane specifically target the heart and prevent the activation and secretion of collagen by heart fibroblasts, so that the effect of treating heart fibrosis and further treating heart failure is better exerted.

The preparation method of the drug-loaded nanoparticle modified by the fusion cell membrane is simple to operate, mild in reaction condition and free from environmental pollution. The drug-loaded nano-particles modified by the fusion cell membrane are used as drugs, and have the advantages of good stability, strong specificity, low side effect and high safety.

The drug-loaded nanoparticle modified by the fusion cell membrane provided by the application has the advantages of high efficiency, targeting and low toxicity when used as a heart failure treatment drug, and has a considerable application prospect in the research and development fields of drugs with the indications of heart failure and heart fibrosis and the treatment fields of heart failure and heart fibrosis.

Drawings

Fig. 1 is a photograph of the transmission electron microscope observation in example 1.

FIG. 2 shows the results of the potentiometric measurement in example 1.

FIG. 3 is a graph showing fluorescence spectra at the optimum ratio in example 2.

Fig. 4 is a photograph of laser confocal co-location observation in example 3.

FIG. 5 is a photograph of confocal microscopy when detecting the degree of enrichment of myofibroblasts in example 4.

FIG. 6 is a confocal microscope photograph of example 4 showing the degree of phagocytosis by macrophages.

FIG. 7 is a photograph showing fluorescence distribution of an isolated organ in example 5.

FIG. 8 shows the results of the ejection fraction and the short axis shortening ratio in example 6.

FIG. 9 is a quantitative analysis of the heart fibrosis region line and myocardial cell cross-sectional area in example 6.

FIG. 10 shows the results of the ejection fraction and the short axis shortening ratio in example 7.

FIG. 11 is a graph showing the results of quantitative analysis of the heart fibrosis region line and myocardial cell cross-sectional area in example 7.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged. In an example, BCA kit was used to detect protein concentration. The whole blood used in the examples was O-type blood of a healthy person collected by an EDTA anticoagulation blood collection tube, and was used within 16 hours of blood collection. The PBS buffers used in the examples were PBS buffers at pH7.4 unless otherwise specified. Platelets, english, platlet. Red blood cells, english, are erythrocyte. Platelet membrane, english Platelet Membrane, is denoted by PM. Erythrocyte membranes, english Erythrocyte Membrane, are denoted by EM.

Leupeptin (Leupeptin is effective in inhibiting serine, cysteine and threonine proteases): the MCE company has a product catalog number of HY-18234.Aprotinin (Aprotinin is a serine protease inhibitor): MCE company, product catalog number is HY-P0017. Prostaglandin E ₁ : sigma Aldrich, cat# P8908. Protease inhibitor tablet, collectively Pierce Protease Inhibitor tables: thermo Fisher company, catalog number A32963.Ang II (angiotensin II): sigma Aldrich, cat# A9525.DiO (green fluorescent probe for cell membrane): beyotime corporation, catalog number C1038.DiI (cell membrane red fluorescent probe): beyotime corporation, catalog number C1036.DiD (cell membrane far infrared fluorescent probe): beyotime corporation, catalog number C1039. The PLGA used in the examples was PLGA (75:25), which was polymerized from 75% polylactic acid (PLA) and 25% polyglycolic acid (PGA); PLGA (75:25) has CAS number 34346-01-5; PLGA (75:25): the MCE company has a product catalog number of HY-B2247A. JQ1 (BET bromodomain inhibitor) CAS number: 1268524-69-1; JQ1: sigma Aldrich, cat# SML0974.

Example 1 preparation of bionic targeting drug-loaded nanoparticle

The bionic targeting drug-loaded nanoparticle is the drug-loaded nanoparticle modified by fusion cell membranes.

1. Preparation of fusion cell membranes

1. Whole blood was centrifuged at 4 ℃ for 20min at 100g and then separated into three layers (the upper layer is platelet rich plasma layer, the middle layer is leukocyte layer, and the lower layer is erythrocyte layer), the upper layer was collected first, the middle layer was discarded, and the lower layer was collected.

2. Preparation of erythrocyte membranes

(1) Taking the lower layer (erythrocyte layer) obtained in the step 1, centrifuging at 4 ℃ and 720g for 10min, removing the supernatant, and reserving the sediment (the sediment is erythrocyte).

(2) After completion of step (1), the pellet was washed 2 times with PBS buffer containing 1mM EDTA, 50. Mu.M Leupeptin and 1. Mu.g/mL Aprotin (after each washing, centrifugation was performed at 4 ℃ C., 720g for 10min, the supernatant was discarded, and the pellet was retained).

(3) After completion of step (2), the pellet was suspended with 4 volumes of hypotonic solution relative to the pellet, incubated for 60min, then centrifuged at 4℃and 20000g for 20min, the supernatant discarded and the pellet was retained.

Hypotonic solution: contains 0.2mM EDTA-2Na, and the balance is deionized water.

(4) After completion of step (3), the pellet was washed thoroughly with PBS buffer containing 50. Mu.M Leupeptin and 1. Mu.g/mL Aprotin, and then centrifuged at 20000g for 20min at 4℃and the supernatant was discarded, leaving the pellet (pellet is erythrocyte membrane). The erythrocyte membrane is preserved at 4 ℃ and used within 6 hours after preparation.

In use, the erythrocyte membrane is suspended in PBS buffer to obtain erythrocyte membrane suspension, and then used.

3. Preparation of platelet membrane

(1) Taking the upper layer (platelet rich plasma layer) obtained in step 1, centrifuging at 4deg.C for 20min (red blood cells are at bottom after centrifugation), discarding the red blood cells at bottom, adding prostaglandin E containing 1mM EDTA and 2mM to the rest liquid phase ₁ Is centrifuged at 800g for 20min at 4 ℃, the supernatant is discarded, and the precipitate (the precipitate is the platelet) is retained. Samples were taken and platelet counts were performed.

(2) After the completion of the step (1), the pellet was resuspended in PBS buffer containing EDTA (1 mM) and protease inhibitor tablets (1 tablet/50 mL), and freeze-thawed repeatedly 3 times (-80 ℃ C. Freeze, room temperature thawing), and centrifuged at 4 ℃ C., 4000g for 3min, the supernatant was discarded, and the pellet was retained (the pellet was a platelet membrane). The platelet membrane was stored at 4℃and used within 6 hours after preparation.

In use, the platelet membrane is suspended with PBS buffer to give a platelet membrane suspension, which is then used.

4. Preparation of fusion cell membranes

Mixing 1 part by volume of erythrocyte membrane suspension (with the protein concentration of 5 mu g/mu L) and 4 parts by volume of platelet membrane suspension (with the protein concentration of 5 mu g/mu L), and carrying out ultrasonic treatment three times (the parameters of an ultrasonic probe are 42kHz, 100W and 5min in each ultrasonic treatment), so as to obtain a fused cell membrane liquid phase.

That is, the mass ratio (based on the amount of protein) of the erythrocyte membrane to the platelet membrane was 1:4.

2. Preparation of drug-loaded nanoparticles

1.5mg of JQ1 and 15mg of PLGA are dissolved in 1mL of DMSO, then 4mL of double distilled water is added dropwise while slowly stirring, then the whole system is transferred into a dialysis bag (the molecular cut-off is 3500 Da), the dialysis bag is placed into the double distilled water for dialysis, and then the liquid phase in the dialysate is collected, namely the JQ1 NPs liquid phase (JQ1 NPs represents JQ1-loaded PLGA nano particles). And (3) performing liquid-phase freeze drying on the JQ1 NPs to obtain a dry substance, namely the JQ1 NPs.

3. Preparation of drug-loaded nanoparticles modified by fusion cell membrane

Taking the fused cell membrane liquid phase prepared in the first step (the adding amount of the fused cell membrane is the platelet amount when preparing the fused cell membrane, the platelet raw material amount is n), carrying out ultrasonic treatment for three times (the ultrasonic probe parameters are 42kHz, 100W and 5min when carrying out ultrasonic treatment each time), then adding JQ1 NPs (the adding amount of the JQ1 NPs is the PLGA amount when preparing the JQ1 NPs, the PLGA raw material amount is m), carrying out ultrasonic treatment for three times (the ultrasonic probe parameters are 42kHz, 100W and 5min when carrying out ultrasonic treatment each time), and obtaining PM&EM/JQ1 NPs liquid phase (PM&EM/JQ1 NPs represent fusion cell membrane modified JQ1 NPs). When n is 3×10 ⁹ M is 1mg when used.

4. Preparation of PM vesicles and EM vesicles

And (3) taking an erythrocyte membrane suspension (2 preparation in the first step, wherein the protein concentration is 5 mug/mug), and performing ultrasonic treatment for three times (the parameters of an ultrasonic probe are 42kHz, 100W and 5min during each ultrasonic treatment), so as to obtain the EM vesicle.

And (3) taking a platelet membrane suspension (3 preparation in the first step, wherein the protein concentration is 5 mug/mu L), and performing ultrasonic treatment for three times (the parameters of an ultrasonic probe are 42kHz, 100W and 5min during each ultrasonic treatment), so as to obtain PM vesicles.

5. Transmission electron microscope observation

Test article: JQ1 NPs and PM & EM/JQ1 NPs.

The results of the transmission electron microscope observation of the test substance are shown in FIG. 1. In FIG. 1, the left plot is JQ1 NPs and the right plot is PM & EM/JQ1 NPs. Compared with JQ1 NPs, PM & EM/JQ1 NPs are more regular in appearance and better in stability. The particle size of PM & EM/JQ1 NPs was about 200 nm.

6. Potentiometric measurement

Test article: PM vesicles, EM vesicles, JQ1 NPs and PM & EM/JQ1 NPs.

The results of the potential measurement of the test substance are shown in FIG. 2. In FIG. 2, four columns correspond to PM vesicles, EM vesicles, JQ1 NPs and PM & EM/JQ1 NPs in order from left to right. The surface of PM & EM/JQ1 NPs is electronegative, and the potential is about-30 mv.

7. Membrane protein detection and Western Blot protein identification

Test article: PM vesicles, EM vesicles, JQ1 NPs and PM & EM/JQ1 NPs. The test substance was taken, RIPA cell lysate was added and lysed on ice for 30min, followed by centrifugation at 13000g for 15min, and the supernatant was taken.

The supernatants were subjected to SDS-PAGE, then transferred to NC membrane, and then stained with ponceau stain. The electrophoretogram shows: the protein band in PM & EM/JQ1 NPs lane is the sum of the protein band in PM vesicle lane and the band protein band in EM vesicle lane, and the protein types are the same.

The supernatants were subjected to SDS-PAGE, then transferred to NC membrane, and then to Western Blot. The targets detected by Western Blot are GPIbα, GPIIbIIIa, GPVI and CD47, respectively. GPIbα, GPIIbIIIa and GPVI are all marker proteins of platelet membrane. CD47 is a marker protein of the erythrocyte membrane. GPIbα, GPIIbIIIa, GPVI are all detectable in PM vesicles and PM & EM/JQ1 NPs, and CD47 is detectable in EM vesicles and PM & EM/JQ1 NPs.

Example 2 detection of formation of fused cell membranes Using FRET

DiO and DiI are FRET dye pairs, diI is a donor fluorescent molecule (DiI has an emission wavelength of 555 nm), and DiO is an acceptor fluorescent molecule.

1. Step 1 is the same as step 1 of example 1.

2. Step 2 of example 1.

3. Step 3 as in example 1.

4. Taking the platelet membrane suspension prepared in the step 3 (the protein concentration is 5 mug/mug L), adding DiO and DiI (the adding mass of DiO and DiI is 0.1% of the platelet membrane protein mass), centrifuging for 15min at 18000g, discarding the supernatant, and reserving the sediment. The pellet was washed 3 times with PBS buffer containing protease inhibitor tablets (1 tablet/50 mL) and centrifuged at 18000g for 15min each time, the supernatant was discarded and the pellet was retained.

5. After the step 4 is completed, the erythrocyte membrane suspension prepared in the step 2 is added for three times of ultrasonic treatment (the parameters of an ultrasonic probe are 42kHz, 100W and 5min in each ultrasonic treatment). Different addition amounts of erythrocyte membrane suspensions are set to realize different mass ratios (all based on protein) of erythrocyte membranes and platelet membranes.

6. After completion of step 4, measurement (platelet membrane) was performed using a fluorescence spectrometer. After completion of step 5, measurement was performed using a fluorescence spectrometer (platelet membrane & erythrocyte membrane). The fluorescence spectrometer measures the excitation wavelength of 450nm and the receiving wavelength of 500nm to 650nm.

The recovery of fluorescence intensity at 555nm indicates a decrease in fluorescence resonance energy transfer efficiency, indicating that the two dyes gradually become on one platelet membrane to the two cell membranes. When the mass ratio (based on the protein amount) of the erythrocyte membrane to the platelet membrane is 1:4, the energy transfer efficiency is highest, and the fluorescence intensity of 555nm is not recovered, which indicates that the erythrocyte membrane and the platelet membrane are fused optimally. The results show that a mass ratio (based on protein amount) of the erythrocyte membrane to the platelet membrane of 1:4 is the optimal ratio for preparing the fusion cell membrane. The fluorescence spectrum diagram under the optimal proportioning is shown in figure 3.

Example 3 observation of the formation of fused cell membranes Using laser confocal Co-localization

1. Step 1 is the same as step 1 of example 1.

2. Step 2 of example 1.

3. Step 3 as in example 1.

4. DiD-labeled erythrocyte membranes were prepared.

Taking the erythrocyte membrane suspension prepared in the step 2 (the protein concentration is 5 mug/mug), adding DiD (the adding mass of the DiD is 0.1% of the erythrocyte membrane protein mass), centrifuging for 15min at 18000g, removing the supernatant, and reserving the sediment. The pellet was washed 3 times with PBS buffer containing protease inhibitor tablets (1 tablet/50 mL) and centrifuged at 18000g for 15min each time, the supernatant was discarded and the pellet was retained.

5. DiO-labeled platelet membranes were prepared.

Taking the platelet membrane suspension prepared in the step 3, adding DiO (the adding mass of DiO is 0.1% of the platelet membrane protein mass), centrifuging for 15min at 18000g, discarding the supernatant, and reserving the precipitate. The pellet was washed 3 times with PBS buffer containing protease inhibitor tablets (1 tablet/50 mL) and centrifuged at 18000g for 15min each time, the supernatant was discarded and the pellet was retained.

6. DiI NPs were prepared.

1.5mg of DiI and 15mg of PLGA are dissolved in 1mL of DMSO, then 4mL of double distilled water is added dropwise while slowly stirring, then the whole system is transferred into a dialysis bag (the molecular cut-off is 3500 Da), the dialysis bag is placed into the double distilled water for dialysis, and then the liquid phase in the dialysis liquid is collected, namely DiI NPs liquid phase (DiI NPs represent DiI-loaded PLGA nano particles). And (3) performing liquid-phase freeze drying on the DiI NPs to obtain a dry substance, namely the DiI NPs.

7. DiD-labeled erythrocyte membranes, diO-labeled platelet membranes, and DiI NPs were directly mixed (ratio of the three was the same as in step 8), and observed under a laser confocal microscope, the results are shown in the left panel of FIG. 4.

8. DiD-labeled erythrocyte membranes and DiO-labeled platelet membranes were prepared as fused cell membranes (method see step one, 4, example 1), and then fused cell membrane-modified DiI-loaded nanoparticles were prepared along with DiI NPs (method see step three, example 1), and observed under a laser confocal microscope, the results shown in the right panel of FIG. 4.

The results in fig. 4 show that the fused cell membrane modified DiI-loaded nanoparticle shows yellow fluorescence under a laser confocal microscope, which represents that erythrocyte membrane, platelet membrane and DiI NPs are fluorescence after co-localization, indicating that the preparation of the fused membrane-coated nanoparticle is successful.

Example 4 in vitro targeting and immune evasion of fusion membrane-coated nanoparticles (cell experiments)

1. Preparation of fusion cell membranes

Step one of example 1.

2. Preparation of DiI-loaded PLGA nanoparticles

3. Preparation of DiI-loaded nanoparticles modified by fusion cell membranes

The procedure three of example 1 was followed, substituting DiI NPs for JQ1 NPs, to obtain PM & EM/DiI NPs liquid phase (PM & EM/DiI NPs represent fused cell membrane modified DiI NPs).

4. In vitro targeting of fibroblasts

3T3-L1 cells (mouse embryonic fibroblasts): ATCC number CL-173.

1. Inoculating 3T3-L1 cells to a 24-well plate, culturing by adopting a DMEM culture medium until the cell density reaches 60%, adding Ang II to make the concentration of Ang II be 1 mu mol/L, and culturing for 12-24 hours to obtain myofibroblasts.

2. After the step 1 was completed, the culture supernatant in the wells was aspirated, the wells of the 24-well plate were divided into three groups, the first group was added with PBS buffer (addition amount was 500. Mu.l/well), the second group was added with DiI NPs prepared in the step two (addition amount was 500. Mu.l/well), the third group was added with PM & EM/DiI NPs liquid phase prepared in the step three (addition amount was 500. Mu.l/well), and then incubated for 24 hours. The second and third groups contained the same amount of DiI NPs in 500 μl.

3. After step 2 was completed, DAPI staining and collagen staining were performed, and then the extent of enrichment of DiI NPs and PM & EM/DiI NPs to myofibroblasts was examined using a confocal microscope.

The results are shown in FIG. 5. In fig. 5, the first behavior is a first group, the second behavior is a second group, and the third behavior is a third group. PM & EM/DiI NPs have significantly increased adhesion to myofibroblasts and collagen compared to DiI NPs.

5. Immune evasion against macrophages.

1. RAW264.7 cells were seeded into 24-well plates and cultured with DMEM medium until the cell density reached 60%.

3. After step 2 was completed, DAPI staining and lysosomal staining were performed, and then the extent of phagocytosis of DiI NPs and PM & EM/DiI NPs by macrophages was examined using confocal microscopy.

The results are shown in FIG. 6. In fig. 6, the left diagram is the first group, the middle diagram is the second group, and the right diagram is the third group. The amount of PM & EM/DiI NPs phagocytosed by macrophages is significantly reduced compared to DiI NPs.

Example 5 in vivo targeting experiments of fusion film-coated nanoparticles (Small animal in vivo imaging experiments)

Test animals: 8 week old male C57BL/6J mice.

The aortic arch constriction (TAC) method is used to model chronic heart failure. The specific method comprises the following steps: open chest surgery was performed after anesthetizing the mice with pentobarbital, the aortic arch was contracted between the left common carotid artery and the brachiocephalic trunk artery of the mice using 7-0 silk and 27 gauge needle, and then sutured.

The test animals were randomly divided into 3 groups of 6 animals, and the grouping treatment method was as follows:

PM & EM/DiI NPs group: on test day 1, a chronic heart failure model is established; on test day 18, PM & EM/DiI NPs prepared in example 4 were injected tail vein (100. Mu.l injection volume for single test animals; 50mg/kg body weight for single test animals, diI amount for dosing, and physiological saline for concentration adjustment).

DiI NPs group: on test day 1, a chronic heart failure model is established; on test day 18, diI NPs prepared in example 4 were injected tail vein (100 μl of injection volume per test animal; 50mg/kg body weight per test animal, diI amount, and physiological saline as solvent).

Control group: on test day 1, a chronic heart failure model is established; on test day 18, saline was injected into the tail vein (100 μl of injection volume per test animal).

On day 45 of the experiment, animals were sacrificed and individual organs were isolated, and the fluorescence distribution of the isolated organs was examined and recorded by photographing.

The photograph is shown in FIG. 7. In FIG. 7, the left column corresponds to the Control group, the middle column corresponds to the DiI NPs group, and the right column corresponds to the PM & EM/DiI NPs group. Compared with DiI NPs, PM & EM/DiI NPs can target to the activation and secretion collagen sites of heart fibroblasts, and reach fewer organs such as liver, kidney and the like, so that the fusion cell membrane is suitable for serving as a nanoparticle outer layer targeting coating to deliver functional drugs.

Example 6 therapeutic action against TAC model

Test animals: 8 week old male C57BL/6J mice.

The method for establishing the chronic heart failure model adopts an aortic arch constriction (TAC) method, and comprises the following specific steps: open chest surgery was performed after anesthetizing the mice with pentobarbital, the aortic arch was contracted between the left common carotid artery and the brachiocephalic trunk artery of the mice using 7-0 silk and 27 gauge needle, and then sutured. The false operation method comprises the following steps: mice were anesthetized with pentobarbital, then chest opened and then directly sutured.

The test animals were randomly divided into 5 groups of 6 animals, and the grouping treatment method was as follows:

PM & EM/JQ1 NPs group: on test day 1, a chronic heart failure model is established; PM & EM/JQ1 NPs were injected once daily on days 18 to 42 of the trial (100 μl of single-trial animals per injection volume; 50mg/kg body weight per dose of single-trial animals per dose, JQ1 meter, and physiological saline adjusted concentration). PM & EM/JQ1 NPs solution was prepared in step three of example 1.

JQ1 NPs group: on test day 1, a chronic heart failure model is established; on days 18 to 42 of the test, JQ1 NPs were injected once daily (100 μl of single test animals was injected into the tail vein, 50mg/kg body weight was administered to the single test animals, JQ1 amount was used as the dose, and physiological saline was used as the solvent). JQ1 NPs were prepared in step two of example 1.

Free JQ1 group: on test day 1, a chronic heart failure model is established; on days 18 to 42 of the test, JQ1 was injected once daily (the injection volume of a single test animal was 100 μl; the dose of a single test animal was 50mg/kg body weight, the dose was JQ1 and the solvent was physiological saline).

TAC-Con group: on test day 1, a chronic heart failure model is established; physiological saline was injected once per tail vein on days 18 to 42 of the trial (single trial animal injection volume was 100 μl).

Sham group: on test day 1, performing a sham operation; physiological saline was injected once per tail vein on days 18 to 42 of the trial (single animal injection volume was 100 μl).

On test day 45, the heart of the mice is dehaired, isoflurane is inhaled for anesthesia, the supine position is placed on a physiological information detection platform, echocardiography detection is carried out, ejection fraction (ejection fraction) and short axis shortening rate (fractional shortening) are obtained, and the result is shown in fig. 8. In the left and right diagrams of FIG. 8, 5 columns correspond to the Sham group, TAC-Con group, free JQ1 group, JQ1 NPs group, PM & EM/JQ1 NPs group in order from left to right. Free JQ1, JQ1 NPs, PM & EM/JQ1 NPs treatment all improved cardiac function and left ventricular contractility compared to TAC-Con group mice. Comparison of three treatment groups: PM & EM/JQ1 NPs significantly improved the ejection fraction and left ventricular short axis shortening rate of TAC mice; compared to the therapeutic effects of Free JQ1 and JQ1 NPs, the post-PM & EM/JQ1 NPs treatment ejection fraction increased from 49.0% ± 5.16% and 48.2% ± 4.83% to 58.5% ± 5.50%, and the left ventricular short axis shortening rate increased from 24.25% ± 3.09% and 23.88 ± 2.90% to 30.37% ± 3.44%. This suggests that PM & EM/JQ1 NPs can significantly improve TAC-induced heart failure prognosis compared to Free JQ1 and JQ1 NPs.

On day 45 of the experiment, mice were sacrificed, hearts were taken, washed with PBS buffer, and paraffin sections were made. Paraffin sections were taken, sirius red stained and the heart fibrosis area was quantitatively analyzed using ImageJ. Paraffin sections were taken and fluorescent staining of Wheat Germ Agglutinin (WGA) was performed and the cardiomyocyte cross-sectional area was quantitatively analyzed using ImageJ. The results are shown in FIG. 9. In the left and right panels of FIG. 9, 5 columns correspond in order from left to right to the Sham group, TAC-Con group, free JQ1 group, JQ1 NPs group, PM & EM/JQ1 NPs group. The results indicate that PM & EM/JQ1 NPs are effective in reducing the degree of fibrosis in the heart over FreeJQ1 and JQ1 NPs. Since hypertrophy of cardiomyocytes is often accompanied during cardiac fibrosis, hypertrophy of cardiomyocytes also often means a poorer prognosis. Compared with the Sham group, the myocardial cell size of the TAC-Con group was increased by about 1.84+ -0.23 times, and the myocardial cell size of the PM & EM/JQ1 NPs treatment group was increased by only 1.15+ -0.02 times. The results show that PM & EM/JQ1 NPs are more effective than Free JQ1 and JQ1 NPs in reducing the extent of cardiomyocyte hypertrophy.

Example 7 therapeutic Effect on myocardial infarction model (MI model)

Test animals: 8 week old male C57BL/6J mice.

The method for establishing the post-MI heart failure model comprises the following steps: after anesthetizing the mice with isoflurane, an open chest procedure was performed using 7-0 silk to permanently ligate the proximal left anterior coronary artery descending (LAD) at the left atrial margin, followed by suturing. The false operation method comprises the following steps: mice were anesthetized with isoflurane and then thoracotomy was performed, followed by direct suturing.

PM & EM/JQ1 NPs group: on test day 1, establishing a heart failure model after MI; PM & EM/JQ1 NPs are injected once daily from day 6 to day 14 of the test, wherein the injection volume of a single test animal is 100 mu l, the administration dose of the single test animal is 25mg/kg body weight, the administration dose is calculated by JQ1, and the concentration is adjusted by physiological saline; PM & EM/JQ1 NPs were injected once daily on the tail vein from day 15 to day 27 (single test animals injected at a volume of 100 μl; single test animals administered at a dose of 50mg/kg body weight, based on JQ1, and the concentration was adjusted with physiological saline). PM & EM/JQ1 NPs solution was prepared in step three of example 1.

JQ1 NPs group: on test day 1, establishing a heart failure model after MI; on test days 6 to 14, JQ1 NPs are injected once daily by tail vein (the injection volume of a single test animal is 100 [ mu ] l; the administration dose of the single test animal is 25mg/kg body weight, the administration dose is calculated by JQ1, and the solvent is normal saline); on the 15 th to 27 th days of the test, JQ1 NPs were injected once daily (the injection volume of a single test animal was 100. Mu.l; the dose of the single test animal was 50mg/kg body weight, the dose was based on JQ1, and the solvent was physiological saline). JQ1 NPs were prepared in step two of example 1.

Free JQ1 group: on test day 1, establishing a heart failure model after MI; on test days 6 to 14, JQ1 is injected once per tail vein (the injection volume of a single test animal is 100 mu l; the administration dose of the single test animal is 25mg/kg body weight, the administration dose is calculated by JQ1, and the solvent is normal saline); on the 15 th to 27 th days of the test, JQ1 was injected once per tail vein (the injection volume of a single test animal was 100. Mu.l; the dose of the single test animal was 50mg/kg body weight, the dose was calculated as JQ1, and the solvent was physiological saline).

MI-Con group: on test day 1, establishing a heart failure model after MI; physiological saline was injected once per tail vein on days 6 to 27 of the test (single animal injection volume was 100 μl).

Sham group: on test day 1, performing a sham operation; physiological saline was injected once per tail vein on days 6 to 27 of the test (single animal injection volume was 100 μl).

On test day 30, the heart of the mice was dehaired, then inhaled with isoflurane for anesthesia, and placed in supine position on a physiological information detection platform for echocardiography detection to obtain ejection fraction (ejection fraction) and short axis shortening rate (fractional shortening), and the result is shown in fig. 10. In the left and right panels of FIG. 10, 5 columns correspond in order from left to right to the Sham group, MI-Con group, free JQ1 group, JQ1 NPs group, PM & EM/JQ1 NPs group. All three treatment groups (Free JQ1 group, JQ1 NPs group and PM & EM/JQ1 NPs group) can improve the heart function and left ventricular contractility of mice after myocardial infarction, and the PM & EM/JQ1 NPs treatment group has the most obvious treatment effect.

On test day 30, mice were sacrificed, hearts were taken, washed with PBS buffer, and paraffin sections were made. Paraffin sections were taken, masson stained and the heart fibrosis area was quantitatively analyzed using ImageJ. Paraffin sections were taken and fluorescent staining of Wheat Germ Agglutinin (WGA) was performed and the cardiomyocyte cross-sectional area was quantitatively analyzed using ImageJ. The results are shown in FIG. 11. All three treatment groups (Free JQ1 group, JQ1 NPs group and PM & EM/JQ1 NPs group) can improve heart failure after large-area anterior wall myocardial infarction, and the PM & EM/JQ1 NPs treatment group has the most obvious treatment effect.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. Application of fusion cell membrane modified drug-loaded nano-particles in preparation of drugs;

the drug-loaded nanoparticle modified by the fusion cell membrane comprises the fusion cell membrane and a drug-loaded nanoparticle inner core wrapped in the fusion cell membrane; the drug-loaded nanoparticles are JQ 1-loaded PLGA nanoparticles; the fusion cell membrane is formed by fusing 4 parts by mass of platelet membrane and 1 part by mass of erythrocyte membrane;

the medicament is a medicament for treating heart failure and/or myocardial infarction.

2. Application of fusion cell membrane modified drug-loaded nano-particles in preparation of drugs;

the medicament is used for inhibiting cardiac fibrosis and/or inhibiting myocardial cell hypertrophy.

3. Application of fusion cell membrane modified drug-loaded nano-particles in preparation of drugs;

the medicine is used for inhibiting heart injury after heart failure and/or heart injury after myocardial infarction.