Targeted lipid nanobubbles carrying exosomes and their preparation method and application

Abstract

The present invention relates to the field of medical technology and relates to targeted exosome-carrying lipid nanobubbles, as well as a preparation method and application thereof. The exosome-carrying targeted lipid nanobubbles utilize mechanical oscillation and membrane fusion methods to connect exosomes to lipid nanobubbles to obtain exosome-carrying lipid nanobubbles, which are then covalently linked to MYH6 antibodies using a phospholipid coupling method to prepare targeted exosome-carrying lipid nanobubbles. On the one hand, the MYH6 antibody is used to target hiPSC-CMs cardiomyocytes. On the other hand, ultrasonic directed blasting technology can be used to further achieve targeted delivery and release of exosomes, thereby achieving visual dual-targeted delivery of therapeutic factor exosomes to hiPSC-CMs cardiomyocytes in vivo and sustained biological efficacy, providing a novel drug delivery system with dual-targeted and efficient drug delivery capabilities for exosome therapy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and is directed to targeted lipid nanobubbles carrying exosomes, as well as a preparation method and application thereof. Background Art

[0002] Exosomes (Exo) are small vesicles with a lipid bilayer membrane structure that are actively secreted by cells. They are approximately 30nm to 120nm in diameter and contain proteins, mRNA, miRNA and other substances. They play an important role in regulating cell behavior and establishing cell communication.

[0003] Studies have shown that exosomes (Exo) synthesized and secreted by stem cells exert a cardioprotective effect by regulating cell apoptosis, inflammation, and functional integration by delivering protective factors to damaged myocardium. However, natural, unmodified exosomes (Exo) lack targeting and are easily rapidly taken up by non-target cells or interact with cell membranes once they enter the body, resulting in the exosomes being unable to exert their sustained therapeutic effects. Therefore, exploring how to achieve sustained and precisely targeted delivery of exosomes (Exo) in vivo is a key issue that needs to be addressed urgently.

[0004] Some scholars have used liposomes as carriers to fuse exosome (Exo) membranes with liposomes to achieve in vivo targeted delivery of exosomes (Exo). However, the targeting of Exo-liposome hybrids is poor, mainly relying on the exosomes (Exo) themselves or the targeting substances they carry, and they cannot be visualized in real time in vivo, which limits the application of Exo-liposomes in treatment.

[0005] With the development of molecular imaging, ultrasound microbubbles can be used not only as contrast agents, but also as carriers to carry genes or drugs. They rupture under ultrasound stimulation and release genes or drugs in a targeted manner, thereby achieving targeted imaging and targeted treatment effects in the treatment area.

[0006] Currently, there are no reports on the preparation of targeted lipid nanobubbles (NBs) carrying exosomes using lipid nanobubbles (NBs) as carriers and MYH6 antibody (a specific surface marker of pluripotent stem cell-derived cardiomyocytes hiPSC-CMs) as a target. Summary of the Invention

[0007] The present invention provides a targeted lipid nanobubble carrying exosomes, as well as a preparation method and application thereof, which overcomes the shortcomings of the above-mentioned prior art. The targeted lipid nanobubble carrying exosomes of the present invention has good stability, obvious targeting and high affinity, good in vitro development, and good biosafety. It can be used to prepare nanoscale ultrasound contrast agents with cardiomyocyte targeting, providing a new targeted delivery platform for precise targeted therapy of exosomes.

[0008] One of the technical solutions of the present invention is achieved by the following measures: a targeted lipid nanobubble carrying exosomes is obtained by the following method:

[0009] In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in PBS buffer containing glycerol and incubated, and then exosomes are added and mixed to obtain a mixed solution;

[0010] In the second step, the mixture was placed in a perfluoropropane gas atmosphere for oscillation and allowed to stand, and then PBS buffer was added to obtain a suspension. After the suspension was centrifuged, the upper layer was discarded to obtain lipid nanobubbles carrying exosomes;

[0011] In the third step, the MYH6 antibody is phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody, and the phospholipid-coupled antibody is added to the lipid nanobubbles for incubation to obtain an incubation solution. The incubation solution is centrifuged, and the precipitate is washed with PBS buffer. After further centrifugation, the upper layer is discarded to obtain targeted lipid nanobubbles carrying exosomes.

[0012] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions:

[0013] In the first step, the mass ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, and exosomes is 5:(1.5 to 2.5):(1.25 to 5.00).

[0014] In the third step, the mass concentration of the phospholipid-coupled antibody is 0.1 mg / mL, and 0.20 mL to 0.30 mL of the phospholipid-coupled antibody is added to each mL of lipid nanobubbles.

[0015] In the first step, the mass concentration of glycerol in the PBS buffer containing glycerol is 8% to 12%.

[0016] In the first step, during incubation, the incubation time is 25 min to 35 min, and the incubation temperature is 55° C. to 65° C.

[0017] In the second step, when oscillating, the oscillation speed is 4000 rpm to 4200 rpm, the oscillation time is 2 min to 3 min, and when standing, the standing time is 3 min to 5 min.

[0018] In the second step, centrifugation includes low-speed centrifugation and high-speed centrifugation. Specifically, low-speed centrifugation is centrifugation at 750 rpm to 850 rpm for 1 to 2 minutes, and specifically, high-speed centrifugation is centrifugation at 5000 rpm to 5200 rpm for 3 to 5 minutes.

[0019] In the third step, the incubation temperature is room temperature and the incubation time is 4.0 h to 4.5 h.

[0020] In the third step, during centrifugation, the rotation speed is 5000 rpm to 5200 rpm, and the centrifugation time is 5 min to 10 min.

[0021] The second technical solution of the present invention is achieved by the following measures: A method for preparing targeted lipid nanobubbles carrying exosomes is carried out as follows:

[0022] In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in PBS buffer containing glycerol and incubated, and then exosomes are added and mixed to obtain a mixed solution;

[0023] In the second step, the mixture was placed in a perfluoropropane gas atmosphere for oscillation and allowed to stand, and then PBS buffer was added to obtain a suspension. After the suspension was centrifuged, the upper layer was discarded to obtain lipid nanobubbles carrying exosomes;

[0024] In the third step, the MYH6 antibody is phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody, and the phospholipid-coupled antibody is added to the lipid nanobubbles for incubation to obtain an incubation solution. The incubation solution is centrifuged, and the precipitate is washed with PBS buffer. After further centrifugation, the upper layer is discarded to obtain targeted lipid nanobubbles carrying exosomes.

[0025] The third technical solution of the present invention is achieved through the following measures: an application of targeted lipid nanobubbles carrying exosomes in the preparation of nanoscale ultrasound contrast agents that can carry exosomes and have cardiomyocyte targeting.

[0026] The present invention specifically uses mechanical oscillation and membrane fusion methods to connect exosomes to lipid nanobubbles to obtain lipid nanobubbles carrying exosomes, and then uses phospholipid coupling covalent method to connect MYH6 antibodies to prepare targeted lipid nanobubbles carrying exosomes. The targeted lipid nanobubbles carrying exosomes of the present invention, on the one hand, use MYH6 antibodies to target hiPSC-CMs cardiomyocytes, and on the other hand, can use ultrasonic directed blasting technology to further complete the targeted delivery and release of exosomes, thereby achieving dual-targeted delivery of therapeutic factor exosomes to hiPSC-CMs cardiomyocytes under in vivo visualization and continuous biological efficacy, providing a new drug delivery system with dual-targeted and efficient drug delivery capabilities for exosome therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the morphology of the targeted lipid nanobubbles carrying exosomes of the present invention.

[0028] Figure 2Figure 3 is a diagram showing the particle size and potential distribution of the targeted lipid nanobubbles carrying exosomes of the present invention.

[0029] Figure 3 The particle size changes of the targeted lipid nanobubbles carrying exosomes of the present invention are observed after being placed at room temperature for different periods of time.

[0030] Figure 4 This is a laser confocal microscopy image of the targeted lipid nanobubbles carrying exosomes of the present invention.

[0031] Figure 5 This is the fluorescence spectrum of the targeted lipid nanobubbles carrying exosomes of the present invention.

[0032] Figure 6 This is a graph of the in vitro imaging capabilities of the MEB-NBs prepared in the present invention.

[0033] Figure 7 This is the biosafety detection graph of the targeted lipid nanobubbles carrying exosomes of the present invention.

[0034] Figure 8 This is a graph showing the changes in loading rate and loading amount of exosome-loaded targeted lipid nanobubbles prepared with different exosome addition amounts.

[0035] Figure 9 This is an IVIS detection graph of the ability of the targeted lipid nanobubbles carrying exosomes of the present invention to target hiPSC-CMs cells in vitro.

[0036] Figure 10 This is a laser confocal microscopy image showing the ability of the exosome-carrying targeted lipid nanobubbles of the present invention to target hiPSC-CMs cells in vitro.

[0037] Figure 11 This is a flow cytometry detection graph of the ability of the targeted lipid nanobubbles carrying exosomes of the present invention to target hiPSC-CMs cells in vitro. DETAILED DESCRIPTION

[0038] The present invention is not limited to the following embodiments, and specific implementation methods can be determined based on the technical solutions of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals commonly known in the prior art; unless otherwise specified, the percentages in the present invention are all percentages by mass; unless otherwise specified, the solutions in the present invention are all aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous hydrochloric acid solution; normal temperature and room temperature in the present invention generally refer to temperatures between 15°C and 25°C, and are generally defined as 25°C.

[0039] The present invention will be further described below in conjunction with the embodiments:

[0040] Example 1: The targeted lipid nanobubbles carrying exosomes are obtained by the following method:

[0041] In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in PBS buffer containing glycerol and incubated, and then exosomes are added and mixed to obtain a mixed solution;

[0042] In the second step, the mixture was placed in a perfluoropropane gas atmosphere for oscillation and allowed to stand, and then PBS buffer was added to obtain a suspension. After the suspension was centrifuged, the upper layer was discarded to obtain lipid nanobubbles carrying exosomes;

[0043] In the third step, the MYH6 antibody is phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody, and the phospholipid-coupled antibody is added to the lipid nanobubbles for incubation to obtain an incubation solution. The incubation solution is centrifuged, and the precipitate is washed with PBS buffer. After further centrifugation, the upper layer is discarded to obtain targeted lipid nanobubbles carrying exosomes.

[0044] Example 2: As an optimization of the above example, in the first step, the mass ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol and exosomes is 5: (1.5 to 2.5): (1.25 to 5.00).

[0045] Example 3: As an optimization of the above example, in the third step, the mass concentration of the phospholipid-coupled antibody is 0.1 mg / mL, and 0.20 mL to 0.30 mL of the phospholipid-coupled antibody is added to each mL of lipid nanobubbles.

[0046] Example 4: As an optimization of the above example, in the first step, the mass concentration of glycerol in the glycerol-containing PBS buffer solution is 8% to 12%.

[0047] Example 5: As an optimization of the above example, in the first step, during incubation, the incubation time is 25 min to 35 min, and the incubation temperature is 55°C to 65°C.

[0048] Example 6: As an optimization of the above example, in the second step, during oscillation, the oscillation speed is 4000 rpm to 4200 rpm, the oscillation time is 2 min to 3 min, and during standing, the standing time is 3 min to 5 min.

[0049] Example 7: As an optimization of the above example, in the second step, centrifugation includes low-speed centrifugation and high-speed centrifugation. The low-speed centrifugation is specifically centrifugation at 750 rpm to 850 rpm for 1 min to 2 min, and the high-speed centrifugation is specifically centrifugation at 5000 rpm to 5200 rpm for 3 min to 5 min.

[0050] Example 8: As an optimization of the above example, in the third step, during incubation, the incubation temperature is room temperature, and the incubation time is 4.0 h to 4.5 h.

[0051] Example 9: As an optimization of the above example, in the third step, during centrifugation, the rotation speed is 5000 rpm to 5200 rpm, and the centrifugation time is 5 min to 10 min.

[0052] Example 10: Application of the targeted lipid nanobubbles carrying exosomes in the preparation of nanoscale ultrasound contrast agents that can carry exosomes and are targeted to cardiomyocytes.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention uses distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials, and adopts mechanical oscillation method and membrane fusion technology to successfully prepare targeted lipid nanobubbles carrying exosomes. The exosomes are spherical and uniform in size, with an average particle size of (597.1±38.9) nm, an average surface potential of (-11.70±0.17) mV, and an average concentration of (2.43±0.33)×10 7 / mL and good stability. In this invention, distearoylphosphatidylethanolamine-polyethylene glycol (PEG) with long hydrophilic groups is used as a stabilizer, resulting in excellent stability of the prepared exosome-carrying targeted lipid nanobubbles at room temperature for 2 hours. Laser confocal microscopy and fluorescence FRET analysis confirmed that the exosome-carrying targeted lipid nanobubbles of the present invention were effectively loaded with exosomes (Exo), and the exosome-carrying targeted lipid nanobubbles (MEB-NBs) of the present invention had an exosome-carrying efficiency of 73.8%. Laser confocal microscopy and flow cytometry results showed that the exosome-carrying targeted lipid nanobubbles effectively targeted hiPSC-CM cardiomyocytes, demonstrating strong targeting and high affinity. Furthermore, the exosome-carrying targeted lipid nanobubbles (MEB-NBs) of the present invention exhibited good in vitro visualization and good biosafety. Therefore, the targeted lipid nanobubbles (MEB-NBs) carrying exosomes of the present invention are stable in nature, have excellent ultrasound imaging capabilities, and can precisely target hiPSC-CMs cardiomyocytes. They can be used to prepare nanoscale ultrasound contrast agents with cardiomyocyte targeting, providing a new targeted delivery platform for precise exosome targeted therapy.

[0055] Example 11:

[0056] The targeted lipid nanobubbles carrying exosomes are obtained according to the following method:

[0057] In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol were dissolved in PBS buffer containing 10% glycerol. After incubation at 55°C for 25 minutes, exosomes were added and mixed to obtain a mixed solution. The mass ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, and exosomes was 5:2:1.25.

[0058] In the second step, the mixture was placed in a perfluoropropane gas atmosphere and shaken at 4000 rpm for 2 minutes. After standing for 3 minutes, PBS buffer was added to obtain a suspension. The suspension was first centrifuged at 750 rpm for 1 minute, and then at 5000 rpm for 3 minutes. The upper layer was discarded to obtain lipid nanobubbles carrying exosomes (EB-NBs);

[0059] In the third step, the MYH6 antibody was phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody with a mass concentration of 0.1 mg / mL. 0.20 mL of phospholipid-coupled antibody with a mass concentration of 0.1 mg / mL was added to each milliliter of lipid nanobubbles and incubated at room temperature for 4.0 hours to obtain an incubation solution. The incubation solution was centrifuged at 5000 rpm for 5 minutes. The precipitate was rinsed once with PBS buffer and then centrifuged at 5000 rpm for 5 minutes. The upper layer was discarded to obtain targeted lipid nanobubbles carrying exosomes (MEB-NBs).

[0060] Example 12:

[0061] The targeted lipid nanobubbles carrying exosomes are obtained according to the following method:

[0062] In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol were dissolved in PBS buffer containing 10% glycerol. After incubation at 65°C for 35 minutes, exosomes were added and mixed to obtain a mixed solution. The mass ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, and exosomes was 5:2:5.

[0063] In the second step, the mixture was placed in a perfluoropropane gas atmosphere and shaken at 4200 rpm for 3 minutes. After standing for 5 minutes, PBS buffer was added to obtain a suspension. The suspension was first centrifuged at 850 rpm for 2 minutes and then at 5200 rpm for 5 minutes. The upper layer was discarded to obtain lipid nanobubbles carrying exosomes (EB-NBs).

[0064] In the third step, the MYH6 antibody was phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody with a mass concentration of 0.1 mg / mL. 0.30 mL of phospholipid-coupled antibody with a mass concentration of 0.1 mg / mL was added to each milliliter of lipid nanobubbles and incubated at room temperature for 4.5 hours to obtain an incubation solution. The incubation solution was centrifuged at 5200 rpm for 5 minutes. The precipitate was rinsed once with PBS buffer and then centrifuged at 5200 rpm for 5 minutes. The upper layer was discarded to obtain targeted lipid nanobubbles carrying exosomes (MEB-NBs).

[0065] Example 13:

[0066] The targeted lipid nanobubbles carrying exosomes are obtained according to the following method:

[0067] In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol were dissolved in PBS buffer containing 10% glycerol. After incubation at 60°C for 30 minutes, exosomes were added and mixed to obtain a mixed solution. The mass ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, and exosomes was 5:2:1.25.

[0068] In the second step, the mixture was placed in a perfluoropropane gas atmosphere and shaken at 4200 rpm for 2 minutes. After standing for 3 minutes, PBS buffer was added to obtain a suspension. The suspension was first centrifuged at 800 rpm for 1 minute, and then at 5000 rpm for 3 minutes. The upper layer was discarded to obtain lipid nanobubbles carrying exosomes (EB-NBs);

[0069] In the third step, the MYH6 antibody was phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody with a mass concentration of 0.1 mg / mL. 0.25 mL of phospholipid-coupled antibody with a mass concentration of 0.1 mg / mL was added to each milliliter of lipid nanobubbles and incubated at room temperature for 4.0 hours to obtain an incubation solution. The incubation solution was centrifuged at 5000 rpm for 5 minutes. The precipitate was rinsed once with PBS buffer and then centrifuged at 5000 rpm for 5 minutes. The upper layer was discarded to obtain targeted lipid nanobubbles carrying exosomes (MEB-NBs).

[0070] Comparative Example:

[0071] The lipid nanobubble is obtained by the following method:

[0072] In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol were dissolved in PBS buffer containing 10% glycerol and incubated at 60°C for 30 minutes to obtain a mixed solution. The mass ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol to exosomes was 5:2:1.25.

[0073] In the second step, the mixed solution was placed in a perfluoropropane gas atmosphere, oscillated at 4200 rpm for 2 minutes, and then allowed to stand for 3 minutes. PBS buffer was then added to obtain a suspension. The suspension was first centrifuged at 800 rpm for 1 minute, and then at 5000 rpm for 5 minutes. The upper layer was discarded to obtain lipid nanobubbles (NBs).

[0074] The following mainly examines the physicochemical properties, in vitro imaging capability, biosafety evaluation, Exo loading capacity, and in vitro targeting ability of hiPSC-CMs cells of the targeted lipid nanobubbles carrying exosomes of the present invention.

[0075] Experimental materials: 1,2-Dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphoethanolamine-PEG2000 (DSPE-PEG2000), phospholipid-coupled antibody kit, perfluoropropane C3F8 and Dil fluorescent dye were all from Xi'an Ruixi Biotechnology Co., Ltd.; MYH6 polyclonal antibody was from Proteintech, USA; PKH67 fluorescent dye was from Shanghai Yumeibo Biotechnology Co., Ltd.; Hoechst33342 cell nuclear stain and CCK-8 kit were both from Shanghai Biyuntian Biotechnology Co., Ltd.; hiPSC-CMs cells were from Beijing Saibei Biotechnology Co., Ltd.; exosomes were human bone marrow mesenchymal stem cell exosomes (Exo) from Guangzhou Saiye Biotechnology Co., Ltd.

[0076] Experimental instruments: Malvern Panalytical Ltd. (Malvern Instruments, UK); optical microscope (DMIL LED Fluo) (Leica, Germany); transmission electron microscope (TF20) (FEI, USA); UV-visible absorption spectrometer (TU-1810) (Beijing Puxi General Instrument Co., Ltd.); multifunctional microplate reader (Infinite E Plex) (Tecan, Switzerland); flow cytometer (NovoCyte2040R) (Agilent, USA); full-band near-infrared confocal microscope (FV3000) (Olympus, Japan); small animal in vivo imaging system (IVIS® Lumina III) (Perkin Elmer, USA); fluorescence spectrometer (F-4600) (Hitachi, Japan); refrigerated high-speed centrifuge (TGL-16A) (Hunan Pingfan Technology Co., Ltd.); silver-mercury blender (Hangzhou Yinya New Materials Co., Ltd.); ultrasound diagnostic instrument (Vivid7) (GE, USA).

[0077] Test sample:

[0078] PKH67 fluorescent dye-labeled exosomes were performed according to the instructions of the PKH67 dye kit to obtain a PKH67 fluorescently labeled exosome aqueous solution with a mass concentration of 5 mg / mL;

[0079] Phospholipid-coupled antibodies were prepared according to the instructions of the phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody with a mass concentration of 0.1 mg / mL;

[0080] The steps for labeling lipid nanobubbles (NBs) with Dil fluorescent dye, lipid nanobubbles carrying exosomes (EB-NBs), and targeted lipid nanobubbles carrying exosomes (MEB-NBs) are as follows:

[0081] S1. Preparation of phospholipid solution: Weigh 1 mg of DiI and dissolve it in 3.3 mL of methanol to obtain a 0.3 mg / mL DiI methanol solution, and take 1.5 mL for later use. Weigh 15 mg of distearoylphosphatidylcholine and 6 mg of distearoylphosphatidylethanolamine-polyethylene glycol and dissolve them in 1.5 mL of DiI methanol solution. Take 0.5 mL of the mixed solution and place it in three 2 mL centrifuge tubes. Vacuum dry to remove methanol. Each tube contains: 5 mg of distearoylphosphatidylcholine, 2 mg of distearoylphosphatidylethanolamine-polyethylene glycol, and 0.15 mg of DiI fluorescent dye; each tube is labeled Tube 1, Tube 2, and Tube 3.

[0082] Add 0.8 mL of PBS buffer containing 10% glycerol to tube 1 and incubate at 60°C for 30 min. Pass perfluoropropane gas through tube 1 for 30 min and then seal. Oscillate tube 1 at 4200 rpm for 2 min using a silver-mercury blender. After standing for 3 min, add 0.8 mL of PBS buffer to tube 1 and centrifuge at 800 rpm for 1 min to remove the white bubbles on the upper layer. Centrifuge again at 5000 rpm for 3 min, and discard the upper layer to obtain lipid nanobubbles (NBs) labeled with the Dil fluorescent dye.

[0083] 0.8 mL of PBS buffer containing 10% glycerol was added to each tube 2 and tube 3. The mixture was incubated at 60°C for 30 min. After cooling, 0.25 mL of a 5 mg / mL aqueous solution of PKH67 fluorescently labeled exosomes was added to each tube. Perfluoropropane gas was passed through tubes 2 and 3 for 30 min and then sealed. Tubes 2 and 3 were shaken at 4200 rpm for 2 min using a silver mercury blender. After standing for 3 min, 0.55 mL of PBS buffer was added to each tube 2 and tube 3. The mixture was centrifuged at 800 rpm for 1 min to remove the white bubbles in the upper layer. After centrifugation at 5000 rpm for 3 min, the upper layer was discarded to obtain Dil fluorescent dye-labeled exosome-carrying lipid nanobubbles (EB-NBs).

[0084] Add 0.25 mL of 0.1 mg / mL phospholipid-coupled antibody to the exosome-carrying lipid nanobubbles (EB-NBs) labeled with Dil fluorescent dye obtained in tube 3 and incubate at room temperature for 4.0 h. Then centrifuge at 5000 rpm for 5 min. Rinse the precipitate once with PBS buffer and centrifuge at 5000 rpm for 5 min. Discard the upper layer to obtain Dil fluorescent dye-labeled targeted lipid nanobubbles (MEB-NBs) carrying exosomes.

[0085] Experiment 1:

[0086] The physicochemical properties of the targeted lipid nanobubbles carrying exosomes of the present invention were investigated.

[0087] Experimental method: The lipid nanobubbles prepared in the comparative example of the present invention were labeled as NBs, the lipid nanobubbles carrying exosomes prepared in Example 13 of the present invention were labeled as EB-NBs, and the targeted lipid nanobubbles carrying exosomes prepared in Example 13 of the present invention were labeled as MEB-NBs. The physicochemical properties of NBs, EB-NBs, and MEB-NBs were investigated, including morphology, particle size, average surface potential, average concentration, particle size changes after different storage times at room temperature, and exosome (hereinafter referred to as Exo) loading in EB-NBs.

[0088] The morphology of MEB-NBs was observed using an inverted microscope and a transmission electron microscope. The particle size and average surface potential of each group were analyzed using a Malvern particle size analyzer. The average concentration of each group was calculated using a hemocytometer. Laser confocal microscopy was used to examine the Exo loading of MEB-NBs. NBs were labeled with the fluorescent dye Dil, and Exo were labeled with the fluorescent dye PKH67, following the same method used for labeling the samples with the fluorescent dyes Dil and PKH67. Fluorescence resonance energy transfer (FRET) was also used to further examine whether MEB-NBs were effectively loaded with Exo.

[0089] Experimental results: The morphology of MEB-NBs prepared by the present invention is as follows Figure 1 As shown, Figure 1 A is an inverted microscope image of MEB-NBs, with a scale bar of 50 μm; B is a transmission electron microscope image of the MEB-NBs group, with a scale bar of 500 nm. Figure 1 It can be seen that the MEB-NBs group has a regular spherical shape with uniform size and good dispersion;

[0090] The particle size and potential distribution of MEB-NBs prepared by the present invention are shown in FIG. Figure 2 As shown in Table 1, A is the particle size distribution of MEB-NBs prepared in the present invention, B is the potential distribution of MEB-NBs prepared in the present invention, the particle size, average surface potential and average concentration of NBs prepared in the comparative example, EB-NBs prepared in the present invention and MEB-NBs are shown in Table 1. Figure 2 As can be seen from Table 1, the particle size of the MEB-NBs prepared in the present invention was (597.1±38.9) nm, and the average surface potential was (-11.70±0.17) mV. There was no statistically significant difference in the average concentrations among the groups (P>0.05). Compared with the NBs group, the particle size of the MEB-NBs group was larger, and the average surface potential was more negative, and the difference between the two groups was statistically significant (P<0.05).

[0091] The particle size changes of MEB-NBs prepared by the present invention after being placed at room temperature for different time periods are shown in FIG. Figure 3 As shown, from Figure 3 It can be seen that the MEB-NBs prepared in the present invention did not show any significant change in particle size when placed at room temperature for 2 h;

[0092] The laser confocal microscopy images of MEB-NBs prepared by the present invention are as follows: Figure 4 As shown, Dil fluorescent dye labels NBs, showing red fluorescence; PKH67 fluorescent dye labels Exo, showing green fluorescence; the fluorescence of the two modifications overlaps and shows yellow. Figure 4 It can be seen that the MEB-NBs prepared in the present invention successfully connected to Exo;

[0093] The fluorescence spectrum of MEB-NBs prepared by the present invention is shown in FIG. Figure 5 As shown, Figure 5 The blank group, NBs group, Exo group, EB-NBs group and MEB-NBs group were included. Figure 5 It can be seen that the fluorescence FRET analysis results show that when 430 nm is selected as the excitation wavelength, the EB-NBs group and the MEB-NBs group have strong emission bands around 550 nm to 650 nm, compared with the Exo group around 513 nm, indicating that the EB-NBs and MEB-NBs prepared in the present invention are both effectively loaded with Exo.

[0094] Experiment 2:

[0095] The in vitro imaging ability of the targeted lipid nanobubbles carrying exosomes of the present invention was investigated.

[0096] Experimental method: GE vivid7 ultrasound instrument was used to evaluate the MEB-NBs prepared in Example 13 of the present invention at different concentrations (10 3 , 10 4 , 10 5 , 10 6 , 10 7 The probe frequency was set to 14.0 MHz, and the imaging effect of MEB-NBs was observed in contrast-enhanced mode.

[0097] Experimental results: The in vitro imaging ability of MEB-NBs prepared by the present invention is as follows Figure 6 As shown, A is the in vitro ultrasound imaging effect diagram of MEB-NBs at different concentrations, B is the in vitro ultrasound imaging effect diagram of MEB-NBs at different times, C is the semi-quantitative analysis diagram of in vitro ultrasound imaging of MEB-NBs at different concentrations, and D is the semi-quantitative analysis diagram of in vitro ultrasound imaging of MEB-NBs at different times. Figure 6 It can be seen that the concentration of MEB-NBs prepared by the present invention is 1×10 6 / mL, the imaging intensity was (31.82±1.23) au. The results showed that when the concentration of MEB-NBs was less than 1×10 6 / mL, the ultrasonic imaging intensity gradually increased with the increase of concentration ( Figure 6 A), the differences between the groups were statistically significant ( P <0.05); and 1×10 6 / mL and 1×10 7 There was no statistically significant difference between the groups (P >0.05) ( Figure 6 C). The imaging intensity of MEB-NBs after 45 minutes and 60 minutes was approximately (32.95±0.70) au and (28.13±0.50) au, respectively. The difference between the two groups was statistically significant ( P <0.05) ( Figure 6 B and 6D).

[0098] Experiment 3:

[0099] The biosafety evaluation of the targeted lipid nanobubbles carrying exosomes of the present invention was investigated.

[0100] Experimental Method: The biosafety evaluation of the targeted lipid nanobubbles carrying exosomes prepared in Example 13 of the present invention was performed. The biosafety evaluation included a cytotoxicity test and a hemolysis test. The specific method is as follows:

[0101] Cytotoxicity experiment: The CCK-8 kit was used to detect the cytotoxicity of the MEB-NBs prepared by the present invention on hiPSC-CMs cells. hiPSC-CMs cells were seeded with different concentrations of MEB-NBs prepared by the present invention (0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) in a 96-well plate and incubated for 24 hours. A control group without MEB-NBs and a blank group without MEB-NBs and hiPSC-CMs cells were set up. 100 μL of CCK-8 solution was added to each well, incubated at 37°C in the dark for 30 minutes, and the absorbance at 450 nm was detected using a microplate reader. Wherein, cell viability (%) = [(A 实验 -A 空白 ) / (A 对照 -A 空白 )]×100%.

[0102] Hemolysis experiment: Purified mouse erythrocytes were obtained by extracting orbital blood from BALB / c mice (female, 7 weeks old). Different concentrations of MEB-NBs (25, 50, 100, and 200 μg / mL) were added to centrifuge tubes. Pure water and saline were used as positive and negative controls, respectively. Each group was mixed with 20 μL of mouse erythrocytes and incubated for 12 hours. Subsequently, the tubes were centrifuged at 3500 rpm for 5 minutes and photographed for preservation. The absorbance at 540 nm was measured using a microplate reader. Hemolysis rate (%) = [(A MEB-NBs组 -A 阴性对照组 ) / (A 阳性对照组 -A 阴性对照组 )]×100%.

[0103] Experimental results: The biosafety test results of the targeted lipid nanobubbles carrying exosomes of the present invention are as follows Figure 7 As shown, A is the cell viability after co-incubation of MEB-NBs with hiPSC-CMs at different concentrations for 24 hours, and B is the hemolysis rate after co-incubation of MEB-NBs with 2% mouse red blood cells at different concentrations for 12 hours. Figure 7 It can be seen that within 24 h, with the increase of MEB-NBs concentration, there was no statistically significant difference in the cell activity of hiPSC-CMs cells ( P >0.05), until the MEB-NBs concentration reached 200 μg / mL, the activity of hiPSC-CMs cells was lower than that of the control group ( P <0.05) ( Figure 7 A); The solution containing different concentrations of MEB-NBs was almost transparent. Quantitative results showed that the hemolysis rate of MEB-NBs at different concentrations was not significantly different from that of the negative control group ( P >0.05) ( Figure 7 B).

[0104] Experiment 4:

[0105] The ability of the targeted lipid nanobubbles carrying exosomes of the present invention to load Exo was investigated.

[0106] Experimental Method: UV-visible absorption spectroscopy was used to quantify the Exo loading and loading rate. Following the method for preparing MEB-NBs in Example 13 of the present invention, 0.20 mL, 0.25 mL, 0.334 mL, 0.50 mL, and 1.0 mL of a 5 mg / mL aqueous solution of PKH67 fluorescently labeled exosomes were added to prepare MEB-NBs with varying Exo loading. The absorbance of the supernatant at 485 nm was measured using a UV-visible absorption spectrometer to calculate the Exo loading and loading rate. Here, drug loading (%) = drug mass / carrier mass × 100%; loading rate (%) = drug mass / total drug mass × 100%.

[0107] Experimental results: The curves of the loading rate and loading capacity of MEB-NBs obtained by adding different Exo dosages are shown in Figure 2. Figure 8 As shown, from Figure 8 It can be seen that when the amount of Exo added is 0.25 ml (that is, the amount of Exo added is 0.25 ml*5 mg / mL=1.25 mg), the loading rate is the highest. When the amount added is 1.0 ml (that is, the amount of Exo added is 1.0 ml*5 mg / mL=5 mg), the loading rate and drug loading capacity both reach the highest level, both of which are 73.8%.

[0108] Experiment 5:

[0109] The ability of the exosome-carrying targeted lipid nanobubbles of the present invention to target hiPSC-CMs cells in vitro was investigated.

[0110] Experimental Methods: To verify the targeting ability of the MEB-NBs of the present invention, four experimental groups were designed: NBs prepared in the comparative example, EB-NBs prepared in Example 13 of the present invention, and MEB-NBs were used as experimental groups (NBs group, EB-NBs group, and MEB-NBs group), respectively. A negative control group containing only PBS buffer was also used. hiPSC-CMs were added to each group and incubated for 30 minutes. NBs and Exosomes were labeled with Dil and PKH67 fluorescent dyes, respectively. The fluorescence intensity of the Dil and PKH67 fluorescent dyes was measured using an in vivo imaging system (IVIS), a confocal microscope, and a flow cytometer.

[0111] Experimental results: The IVIS results of the ability of the targeted lipid nanobubbles carrying exosomes of the present invention to target hiPSC-CMs cells in vitro are as follows: Figure 9 As shown, A is the fluorescence intensity of hiPSC-CMs cells in the microplate detected by the small animal in vivo imaging system (IVIS), B is the quantitative analysis of the fluorescence intensity of Dil fluorescent dye, and C is the quantitative analysis of the fluorescence intensity of PKH67 fluorescent dye. Figure 9 It can be seen that the results of the small animal in vivo imaging system (IVIS) showed that the MEB-NBs group had the strongest fluorescence expression;

[0112] Laser confocal microscopy images of the ability of the exosome-carrying targeted lipid nanobubbles of the present invention to target hiPSC-CMs cells in vitro, as shown in Figure 10 As shown, from Figure 10 It can be seen under laser confocal microscopy that the EB-NBs group and the MEB-NBs group adhered to the periphery of the hiPSC-CMs cells and were taken up into the cells. Yellow fluorescent signals accumulated around and inside the hiPSC-CMs cells in the EB-NBs and MEB-NBs groups, and the density was higher in the MEB-NBs group.

[0113] The flow cytometry results of the targeting ability of the exosome-carrying targeted lipid nanobubbles of the present invention to target hiPSC-CMs cells in vitro are as follows: Figure 11 As shown, from Figure 11 It can be seen that the targeted uptake rates of the NBs group, EB-NBs group, and MEB-NBs group were 0.79%, 82.3%, and 99.6%, respectively.

[0114] The results of experiments 1 to 5 show that the present invention successfully prepared targeted lipid nanobubbles (MEB-NBs) carrying exosomes using mechanical oscillation and membrane fusion technology. The MEB-NBs were spherical and uniform in size, with an average particle size of (597.1±38.9) nm, an average surface potential of (-11.70±0.17) mV, and an average concentration of (2.43±0.33)×10 7 / mL and good stability. Laser confocal microscopy and fluorescence FRET analysis confirmed that the targeted exosome-carrying lipid nanobubbles (MEB-NBs) of the present invention were all effectively loaded with exosomes (Exo); the loading rate of the targeted exosome-carrying lipid nanobubbles (MEB-NBs) of the present invention was 73.8%. Laser confocal microscopy and flow cytometry results showed that the targeted exosome-carrying lipid nanobubbles could effectively target hiPSC-CMs cardiomyocytes, showing significant targeting and high affinity. Furthermore, the targeted exosome-carrying lipid nanobubbles (MEB-NBs) of the present invention developed well in vitro and had good biosafety.

[0115] In summary, the present invention uses mechanical oscillation and membrane fusion methods to connect exosomes to lipid nanobubbles to obtain lipid nanobubbles carrying exosomes, and then uses phospholipid coupling covalent method to connect MYH6 antibodies to prepare targeted lipid nanobubbles carrying exosomes. The targeted lipid nanobubbles carrying exosomes of the present invention, on the one hand, use MYH6 antibodies to target hiPSC-CMs cardiomyocytes, and on the other hand, can use ultrasonic directed blasting technology to further complete the targeted delivery and release of exosomes, thereby achieving dual-targeted delivery of therapeutic factor exosomes to hiPSC-CMs cardiomyocytes under in vivo visualization and continuous biological efficacy, providing a new drug delivery system with dual-targeted and efficient drug delivery capabilities for exosome therapy.

[0116] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

[0117]

Claims

1. A targeted lipid nanobubble carrying exosomes, characterized in that Obtained as follows: In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in PBS buffer containing glycerol and incubated, and then exosomes are added and mixed to obtain a mixed solution; In the second step, the mixture was placed in a perfluoropropane gas atmosphere for oscillation and allowed to stand, and then PBS buffer was added to obtain a suspension. After the suspension was centrifuged, the upper layer was discarded to obtain lipid nanobubbles carrying exosomes; In the third step, the MYH6 antibody is phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody, and the phospholipid-coupled antibody is added to the lipid nanobubbles for incubation to obtain an incubation solution. The incubation solution is centrifuged, and the precipitate is washed with PBS buffer. After further centrifugation, the upper layer is discarded to obtain targeted lipid nanobubbles carrying exosomes.

2. The targeted lipid nanobubble carrying exosomes according to claim 1, characterized in that In the first step, the mass ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, and exosomes is 5:1.5 to 2.5:1.25 to 5.

00.

3. The exosome-carrying targeted lipid nanobubbles according to claim 1 or 2, characterized in that In the third step, the mass concentration of the phospholipid-coupled antibody is 0.1 mg / mL, and 0.20 mL to 0.30 mL of the phospholipid-coupled antibody is added to each mL of lipid nanobubbles.

4. The exosome-carrying targeted lipid nanobubbles according to claim 1 or 2, characterized in that In the first step, the mass concentration of glycerol in the PBS buffer containing glycerol is 8% to 12%.

5. The targeted lipid nanobubble carrying exosomes according to claim 3, characterized in that In the first step, the mass concentration of glycerol in the PBS buffer containing glycerol is 8% to 12%.

6. The exosome-carrying targeted lipid nanobubbles according to claim 1, 2 or 5, characterized in that In the first step, during incubation, the incubation time is 25 minutes to 35 minutes, and the incubation temperature is 55° C. to 65° C.

7. The targeted lipid nanobubble carrying exosomes according to claim 3, characterized in that In the first step, during incubation, the incubation time is 25 minutes to 35 minutes, and the incubation temperature is 55° C. to 65° C.

8. The targeted lipid nanobubble carrying exosomes according to claim 4, characterized in that In the first step, during incubation, the incubation time is 25 minutes to 35 minutes, and the incubation temperature is 55° C. to 65° C.

9. The exosome-carrying targeted lipid nanobubbles according to claim 1, 2, 5, 7, or 8, characterized in that In the second step, when oscillating, the oscillation speed is 4000 rpm to 4200 rpm, the oscillation time is 2 min to 3 min, and when standing, the standing time is 3 min to 5 min.

10. The targeted lipid nanobubble carrying exosomes according to claim 6, characterized in that In the second step, when oscillating, the oscillation speed is 4000 rpm to 4200 rpm, the oscillation time is 2 min to 3 min, and when standing, the standing time is 3 min to 5 min.

11. The exosome-carrying targeted lipid nanobubble according to claim 1 or 2 or 5 or 7 or 8 or 10, characterized in that In the second step, centrifugation includes low-speed centrifugation and high-speed centrifugation. Specifically, low-speed centrifugation is centrifugation at 750 rpm to 850 rpm for 1 to 2 minutes, and specifically, high-speed centrifugation is centrifugation at 5000 rpm to 5200 rpm for 3 to 5 minutes.

12. The targeted lipid nanobubble carrying exosomes according to claim 9, characterized in that In the second step, centrifugation includes low-speed centrifugation and high-speed centrifugation. Specifically, low-speed centrifugation is centrifugation at 750 rpm to 850 rpm for 1 to 2 minutes, and specifically, high-speed centrifugation is centrifugation at 5000 rpm to 5200 rpm for 3 to 5 minutes.

13. The exosome-carrying targeted lipid nanobubble according to claim 1 or 2 or 5 or 7 or 8 or 10 or 12, characterized in that In the third step, during incubation, the incubation temperature is room temperature, the incubation time is 4.0 hours to 4.5 hours, and during centrifugation, the rotation speed is 5000 rpm to 5200 rpm, and the centrifugation time is 5 minutes to 10 minutes.

14. A method for preparing targeted lipid nanobubbles carrying exosomes according to any one of claims 2 to 13, characterized in that Proceed as follows: In the first step, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol are dissolved in PBS buffer containing glycerol and incubated, and then exosomes are added and mixed to obtain a mixed solution; In the second step, the mixture was placed in a perfluoropropane gas atmosphere for oscillation and allowed to stand, and then PBS buffer was added to obtain a suspension. After the suspension was centrifuged, the upper layer was discarded to obtain lipid nanobubbles carrying exosomes; In the third step, the MYH6 antibody is phospholipid-coupled using a phospholipid-coupled antibody kit to obtain a phospholipid-coupled antibody, and the phospholipid-coupled antibody is added to the lipid nanobubbles for incubation to obtain an incubation solution. The incubation solution is centrifuged, and the precipitate is washed with PBS buffer. After further centrifugation, the upper layer is discarded to obtain targeted lipid nanobubbles carrying exosomes.

15. Use of the exosome-carrying targeted lipid nanobubbles according to any one of claims 1 to 13 in the preparation of a cardiomyocyte-targeted nanoscale ultrasound contrast agent that can carry exosomes.

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