Exosome-based targeted complex nanoparticulate drug particles and methods of preparation

Abstract

The application belongs to the technical field of biological medicine, and relates to a targeted composite nano-drug particle based on exosomes and a preparation method. The targeted composite nano-drug particle based on exosomes comprises a poly-exosome core and a multi-shell structure; the poly-exosome core is spherical, and comprises a plurality of exosome monomers and a plurality of poly-arginine cationic materials which are adsorbed by positive and negative charges; and the multi-shell structure comprises VEGF plasmid, poly-arginine cationic material and platelet membrane vesicle which are wrapped layer by layer. The poly-arginine cationic material is used for polymerizing a plurality of exosome monomers, so that the drug loading capacity is improved; the drug particle size is less than 400 nm through the contraction and polymerization of the platelet membrane, so that the drug can be targeted to the myocardial ischemic area in blood circulation without causing embolism, and the delivery efficiency is improved. The various materials used in the application can be derived from active organisms in vivo, so that the tissue utilization is improved, and the application has a good clinical transformation application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a targeted composite nanoparticle based on exosomes and its preparation method. Background Technology

[0002] Patients with acute myocardial infarction have a high probability of complications and late-stage mortality. Myocardial ischemia has a long-term negative impact on mitochondrial ATP production, leading to damage to the mitochondrial electron transport chain in cardiomyocytes, a decreased rate of oxidative phosphorylation, and an increase in reactive oxygen species. Exosomes have low immunogenicity and good tissue bioavailability, but their short half-life and susceptibility to phagocytosis by the immune system during in vivo circulation limit their effective delivery to target sites, resulting in limited therapeutic efficacy. Currently, exosome therapy is unsatisfactory in terms of both drug loading and delivery efficiency. Existing methods to increase exosome drug loading involve multiple injections, requiring multiple invasive procedures; while platelet membranes improve in vivo delivery efficiency through fusion and encapsulation, resulting in large drug particle sizes that easily form embolisms in the bloodstream. Summary of the Invention

[0003] This invention proposes a targeted composite nanoparticle based on exosomes. It utilizes polyarginine to construct an aggregate of multiple exosomes as the core; the outer shell consists of a VEGF plasmid system, a polyarginine cationic material, and a platelet membrane nanosystem. This nanoparticle mainly comprises two parts: a polyexosome core and a multi-shell structure encapsulating the core, achieving a dual improvement in drug loading capacity and in vivo delivery efficiency.

[0004] To achieve the above objectives, the present invention is implemented using the following technical solution: A targeted composite nanoparticle based on exosomes includes a polyexosome core and a multi-shell structure encapsulating the polyexosome core; the polyexosome core is spherical and includes several exosome monomers adsorbed with positive and negative charges and several polyarginine cationic materials; the multi-shell structure includes VEGF plasmids, polyarginine cationic materials and platelet membrane vesicles encapsulated layer by layer, and the outermost part of the multi-shell structure is a platelet membrane vesicle with targeting properties to myocardial ischemia areas.

[0005] The preparation method of the exosome-based targeted composite nanoparticles includes the following steps: (1) After the mice were sacrificed, bone marrow was collected and primary mesenchymal stem cells were isolated and cultured for expansion. When the cell confluence reached 80-90%, the cells were passaged. The cells passaged to the third generation were washed with phosphate buffer and then cultured in a culture medium in which exosomes and serum were removed. After the culture was completed, the cells were centrifuged to remove impurities. The supernatant was filtered through a sterile membrane and then centrifuged twice to obtain exosome precipitate. PBS buffer was added to disperse the precipitate and the exosome suspension was frozen for later use.

[0006] (2) Take whole blood from mice, centrifuge to obtain supernatant, add EDTA solution and phosphate buffer of prostaglandin E1, centrifuge twice to obtain platelet precipitate; after repeated freezing and thawing several times, sonicate and centrifuge a third time to obtain platelet membrane, add phosphate buffer of PMSF to resuspend, centrifuge a fourth time and wash, resuspend again with phosphate buffer and squeeze through several polycarbonate porous membranes for 15-20 cycles to obtain platelet membrane nanovesicles.

[0007] (3) The exosome suspension (3-4 g / L) was adsorbed with a sufficient amount of positively charged poly-L-arginine hydrochloride to obtain substance A.

[0008] (4) Centrifuge and resuspend substance A, add exosome suspension twice and centrifuge and resuspend twice, then add poly-L-arginine hydrochloride, let stand and centrifuge and resuspend. (5) Add VEGF plasmid, let stand, centrifuge and resuspend, then add poly-L-arginine hydrochloride again, let stand and centrifuge and resuspend, then add platelet membrane nanovesicles, let stand and centrifuge again, and finally resuspend with pre-cooled PBS and place at low temperature.

[0009] Preferably, in step (1), after the mice are euthanized, the bone marrow is taken from the femur and tibia of the hind limb. Then, primary mesenchymal stem cells are separated by density gradient centrifugation. The third-generation cells are washed with phosphate buffer and cultured in a culture medium in which exosomes and serum have been removed for 70-80 hours. After the culture is completed, the cells are centrifuged at 4°C at 200-400 rpm for 10-15 minutes, at 2000-4000 rpm for 15-25 minutes, and at 8000-12000 rpm for 25-35 minutes. The second centrifugation program is centrifugation at 110000g for 80-100 minutes at 4°C. The resulting exosome resuspended suspension is stored at -75-85°C.

[0010] Preferably, in step (2), the whole blood centrifugation program is 4℃, 1000-1500rpm for 15-25min, repeated 1-2 times. After each centrifugation, the supernatant is taken, and then 1mM EDTA solution and 2μM prostaglandin E1 phosphate buffer are added. The mixture is then centrifuged a second time at room temperature, 3000-3500rpm for 15-25min. The mixture is then frozen at -75-85℃ for 10-15min, then thawed at room temperature. This freeze-thaw cycle is repeated 6-8 times, followed by sonication. The mixture is then centrifuged a third time at 7000-9000rpm for 4-6min. The concentration of PMSF in the phosphate buffer is 0.5-1.5M. The fourth centrifugation is performed at 7000-9000rpm for 4-6min. The pore sizes of the polycarbonate porous membranes are 19mm, 800nm, 400nm, 200nm, and 100nm, respectively.

[0011] Preferably, in step (3), the mass ratio of exosome suspension to poly-L-arginine hydrochloride is 1:(100-200).

[0012] As a preferred option, in step (4), the mass ratio of substance A: exosome suspension: poly-L-arginine hydrochloride is (1-2): (0.04-0.1): (2-4).

[0013] As a preferred option, the mass ratio of VEGF plasmid: poly-L-arginine hydrochloride: platelet membrane nanovesicles in step (5) is (0.01-0.02): (4-8): (8-16).

[0014] This invention integrates a multi-shell structure encapsulating the core with a polyexosome core using the principle of positive and negative charge adsorption to form a single unit. The multi-shell structure encapsulating the core adheres tightly to the outer layer of the polyexosome core. Through the contraction and polymerization function of platelet membrane vesicles, the polyexosome nanoparticles have a small particle size, facilitating injection into the body via the tail vein without causing embolism. This ensures both the safety of the product's blood circulation and targeted delivery to areas of myocardial ischemia. This structure increases the drug loading capacity and cardiac targeting of the polyexosome core, improves the therapeutic efficacy of myocardial ischemia injury, and reduces the incidence of pulmonary embolism and cerebral embolism. Furthermore, the multi-shell structure encapsulating the core contains a VEGF plasmid system with therapeutic efficacy and a polyarginine cationic material with anti-inflammatory effects, which can effectively enhance the therapeutic efficacy against areas of myocardial ischemia.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. This invention improves drug loading by polymerizing several exosome monomers using polyarginine cationic materials. The drug particle size is kept within 400nm by platelet membrane contraction polymerization, which facilitates blood circulation and targets the ischemic area of ​​myocardium without causing embolism, thereby improving delivery efficiency.

[0016] 2. The various materials used in the preparation of the drug in this invention can all be derived from living organisms, which improves tissue utilization and has good prospects for clinical translational application. Attached Figure Description

[0017] Figure 1 This is a TEM image of exosomes.

[0018] Figure 2 The image shows the TEM characterization of PL-Exo nanoparticles. Detailed Implementation

[0019] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0020] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0021] In the following examples, some raw material manufacturers and product models are listed below: C57BL / 6 mice: Jinan Xingkang Biotechnology.

[0022] High-glucose DMEM medium: BasalMedia L 110KJ.

[0023] Low-glucose DMEM medium: Zhongke Maichen CM10014.

[0024] Fetal bovine serum: Pronos, 164210.

[0025] Penicillin-streptomycin mixture: Solarbio, P1400.

[0026] Phosphate-buffered saline (PBS): Xavier Biotech, G4202.

[0027] Poly-L-arginine hydrochloride (PLA): Shanghai Yuanye Biotechnology, JS275375.

[0028] Exosome-free fetal bovine serum: Shanghai Umibio, UR50202.

[0029] Exosome fluorescent dye (DIR): Umibio, UR21017.

[0030] Whole blood: C57BL / 6 mice.

[0031] 2% Uranium acetate: SPI, America.

[0032] CD36 antibody: Diagbio, db14457.

[0033] CD62P antibody: Diagbio, db12727.

[0034] EDTA solution: Solarbio, E1170.

[0035] Prostaglandin E1: Xinhengyan, 745-65-3.

[0036] HE dye: Cisco, EE0012.

[0037] In the following examples, unless otherwise specified, the percentage content of each substance is a mass fraction. Example 1

[0038] This embodiment provides a process for preparing targeted composite nanomedicine particles based on exosomes.

[0039] (1) Preparation and identification of exosomes C57BL / 6 mice (4-5 weeks old) were humanely euthanized, and under aseptic conditions, the bone marrow was flushed from one end of the femur and tibia using a syringe filled with 1 mL of DMEM complete medium. The other end of the syringe was placed directly above a 10 cm culture dish to collect the bone marrow flushing fluid. The flushing fluid was filtered through a 70 μm filter and then centrifuged at 1000 rpm for 5 minutes at room temperature to isolate primary mesenchymal stem cells. These cells were then expanded in 10 mL of complete medium (high glucose DMEM medium supplemented with an equimolar mixture of 10% fetal bovine serum and 1% penicillin-streptomycin). When the cell confluence reached 85%, the cells were passaged. Third-generation (P3) bone marrow mesenchymal stem cells (BMSCs) were used in the experiment to ensure phenotypic stability. To prepare exosomes, confluent P3 BMSCs were washed twice with pre-chilled phosphate-buffered saline (PBS) at 4°C, and then cultured in high-glucose DMEM medium supplemented with exosome-free serum for 72 hours. After culture, the cells were centrifuged sequentially at 300 × g (10 min), 3,000 × g (20 min), and 10,000 × g (30 min) at 4°C to remove cells, apoptotic bodies, and large vesicles. The resulting supernatant was centrifuged at 110,000 × g for 90 min at 4°C. The resulting exosome pellet (BMSC-Exo) was washed once with 10 mL of PBS buffer at 4°C, and then resuspended in 100 μL of PBS buffer at 4°C to obtain an exosome suspension for characterization and use, and stored at −80°C for later use.

[0040] Exosomes were observed using a Hitachi HT7800 transmission electron microscope, and the results are as follows: Figure 1 The exosomes varied in size, mostly exhibiting a saucer-like structure, with a particle size ranging from 100 to 200 nm. The particle size distribution and concentration were determined using nanoparticle tracking analysis (NTA), revealing a particle size range of 20-500 nm, predominantly 100-150 nm. Western blotting experiments confirmed the presence of markers CD9, CD63, and TSG101 in the exosomes prepared in this embodiment.

[0041] (2) Preparation and identification of platelet nanovesicles 10 mL of whole blood from C57BL / 6 mice was collected in EDTA tubes and immediately centrifuged at 1200 rpm for 20 min at 4°C. The supernatant was centrifuged again at 1200 rpm for 20 min, and the supernatant was transferred to a new 15 mL centrifuge tube. To inhibit platelet activation, 5 mL of phosphate buffer containing 1 mM EDTA and 2 μM prostaglandin E1 was added, and the mixture was centrifuged again at 3400 rpm for 20 min at room temperature to obtain platelet pellet. The platelet pellet was frozen at -80°C for 10 min, then thawed rapidly at room temperature for 5 min. This freeze-thaw treatment was repeated 7 times. The pellet was then sonicated for 5 min and centrifuged at 8000 rpm for 5 min at room temperature to obtain platelet membrane pellet. The platelet membrane pellet was resuspended in phosphate buffer containing 1 mM PMSF, centrifuged at 8000 rpm for 5 min at room temperature, and washed 3 times, each time with 3 mL of phosphate buffer containing 1 mM PMSF. The platelet membrane pellet was then collected. Platelet membrane precipitate was resuspended in 2 mL of sterile phosphate buffer. The resulting membrane was immediately extruded sequentially through a liposome extruder using 19 mm, 800 nm, 400 nm, 200 nm, and 100 nm polycarbonate porous membranes for 20 cycles to obtain platelet membrane nanovesicles. Western blotting confirmed the presence of platelet membrane markers CD36 and CD62P in the platelet membrane nanovesicles prepared in this embodiment.

[0042] (3) Preparation and phenotypic characterization of polyexosome nanoparticle drugs 1) BMSC-Exo resuspension with a particle size smaller than 220 nm was screened using a 0.22 μm pore size filter. 100 μg of the resuspension was added to 12.5 mg of positively charged PLA for adsorption. The mixture was centrifuged at 110,000 × g for 70 minutes at 4°C. After resuspension, 400 μg of BMSC-Exo suspension was added, and the mixture was thoroughly mixed. The mixture was centrifuged again at 110,000 × g for 70 minutes at 4°C. 25 mg of PLA was added, and the mixture was centrifuged again at 110,000 × g for 70 minutes at 4°C. 100 μg of VEGF plasmid (provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army; the preparation method is detailed in the PLA Medical Journal DOI: 10.11855 / j.issn.0577-7402.2019.02.02) was added, and the mixture was centrifuged again at 110,000 × g for 4°C. Centrifuge for 70 minutes, mix thoroughly, and centrifuge again at 110,000 × g for 70 minutes at 4°C. Add 25-50 mg of PLA and centrifuge at 110,000 × g for 70 minutes at 4°C. Finally, add 50-100 mg of platelet membrane nanovesicles and centrifuge at 110,000 × g for 70 minutes at 4°C. Resuspend in 50 μL of PBS buffer to obtain a nanodrug suspension (PL-Exo) with BMSC-Exo aggregated nanoparticles as the core and platelet nanovesicles with cardiotargeting function as the shell (2 μg / μL, note: the concentration is calculated by dividing the mass of the innermost exosome suspension (100 μg) by the volume of PBS buffer added for resuspension (50 μL).

[0043] 2) After negative staining with 2% uranium acetate, the PL-Exo nanoparticle suspension was observed using a transmission electron microscope (TEM). The results are as follows: Figure 2 ,from Figure 2 The successful construction of the multilayer film structure is evident. Quantitative analysis of particle size distribution and concentration was performed using nanoparticle tracking analysis (NTA). The PL-Exo particles were found to have a size range of 100-500 nm, predominantly 200-300 nm. The surface zeta potential was measured using a BeNano 180 Zeta Pro instrument via dynamic light scattering (DLS). The PLA layer was found to have a positive charge of approximately +6 mV. The structure of each nanoparticle layer met expectations and was suitable for tail vein injection in animals. Fourier transform infrared (FTIR) spectroscopy (wavenumber range: 500-4000 cm⁻¹) was also performed. -1 This confirmed its lipid-protein composition. Example 2

[0044] 1. Biological distribution analysis To evaluate the organ-specific targeting ability of PL-Exo, 2 mg of BMSC-Exo was modified into a PL-Exo suspension by layer-by-layer adsorption according to the instructions for using the exosome fluorescent dye DIR, following the method described in Example 1. C57BL / 6 mice underwent myocardial infarction surgery, and immediately after surgery, 100 μL of a DIR-labeled PL-Exo nanoparticle suspension at a concentration of 2 μg / μL was injected via the tail vein. 72 h after injection, whole-body fluorescence images and organ-specific signals were acquired using an IVIS imaging system. The results showed that the mouse heart exhibited the strongest fluorescence intensity (especially in the left ventricle at the site of myocardial infarction), and the exosomes were most concentrated.

[0045] 2. Biosafety testing of exosome-based targeted composite nanoparticles Unless otherwise specified, the PL-Exo suspension used in the following tests was prepared using the method in Example 1.

[0046] At the cellular level: When H9C2 cells (OriCell®, China) proliferated to 70-80%, 25 μL of PL-Exo nanoparticle suspension (50 μg BMSC-Exo / 1*10) was added. 6 Cells were co-incubated for 24 hours, and then cytotoxicity was detected using the CCK8 kit (Vazyme, Nanjing, China) according to the kit's instructions. The results indicated that PL-Exo had good biocompatibility.

[0047] Animal studies using short-term and long-term myocardial infarction models: Short-term: C57BL / 6 mice underwent myocardial infarction surgery, with six mice in each group. Each mouse was then injected with 100 μL of PL-Exo suspension via the tail vein. Three days post-surgery, liver and kidney function were assessed using kits (Suzhou Greens Biotechnology), specifically G0423W, G0424W, G1201W, and G1204W. The results showed that in the short-term myocardial infarction model, PL-Exo nanoparticles did not damage liver and kidney function, demonstrating biocompatibility.

[0048] Long-term: C57BL / 6 mice underwent myocardial infarction surgery, with six mice in each group. One month later, each mouse was injected with 100 μL of PL-Exo suspension via the tail vein. HE staining was used to identify morphological changes in the liver, kidneys, spleen, and lungs. Results indicated that no significant morphological changes were observed in the liver, kidneys, spleen, and lungs of the PL-Exo nanoparticle group, demonstrating the biocompatibility of the nanoparticles.

[0049] 3. Functional evaluation of exosome-based targeted composite nanoparticles (1) Cellular level Cellular ROS detection: When H9C2 cells (OriCell®, China) proliferated to 70-80%, 25 μL of the PL-Exo suspension prepared in Example 1 was added, and the cells were incubated in a hypoxic, low-nutrient simulated myocardial infarction microenvironment (a 1:1 mixture of low-glucose DMEM and RAW264.7 cell culture supernatant with 1% penicillin and streptomycin added, in a three-gas incubator set to 1% O2, 94% N2, and 5% CO2) for 24 hours. The ROS level of the cells was then detected using a ROS kit (Beyotime, s0033) according to its instructions. The results showed that PL-Exo nanoparticles significantly improved the oxidative stress level of cells.

[0050] (2) Animal level Cardiac function: C57BL / 6 mice underwent myocardial infarction surgery, and each mouse was injected with 100 μL of PL-Exo suspension via the tail vein. Echocardiographic assessment was performed using a Vevo 2100 imaging system on days 3 and 30. Parasternal long-axis images were acquired, and left ventricular diameter, volume, left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were quantified using M-mode image analysis. The results suggest that PL-Exo nanoparticles significantly improved cardiac function in mice with myocardial infarction.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A targeted composite nanoparticle based on exosomes, characterized in that, It includes a polyexosome core and a multi-shell structure enclosing the polyexosome core; the polyexosome core is spherical and includes several exosome monomers adsorbed with positive and negative charges and several polyarginine cationic materials; the multi-shell structure includes VEGF plasmids, polyarginine cationic materials and platelet membrane vesicles wrapped layer by layer, and the outermost part of the multi-shell structure is a platelet membrane vesicle with myocardial ischemia-targeting ability.

2. The method for preparing exosome-based targeted composite nanoparticles according to claim 1, characterized in that, The steps are as follows: (1) After the mice were sacrificed, bone marrow was taken and primary mesenchymal stem cells were isolated and cultured for expansion. When the cell confluence reached 80-90%, the cells were passaged. The cells passaged to the third generation were washed with phosphate buffer and then cultured in a culture medium in which exosomes and serum were removed. After the culture was completed, the cells were centrifuged to remove impurities. The supernatant was filtered through a sterile membrane and then centrifuged twice to obtain exosome precipitate. PBS buffer was added to disperse the precipitate and the exosome suspension was frozen and stored for later use. (2) Take whole blood from mice, centrifuge to obtain supernatant, add EDTA solution and phosphate buffer of prostaglandin E1, centrifuge twice to obtain platelet precipitate; after repeated freezing and thawing several times, sonicate and centrifuge a third time to obtain platelet membrane, add phosphate buffer of PMSF to resuspend, centrifuge a fourth time and wash, resuspend again with phosphate buffer and squeeze through several polycarbonate porous membranes for 15-20 cycles to obtain platelet membrane nanovesicles; (3) The exosome suspension was adsorbed with a sufficient amount of positively charged poly-L-arginine hydrochloride to obtain substance A; (4) Centrifuge and resuspend substance A, add exosome suspension twice and centrifuge and resuspend twice, then add poly-L-arginine hydrochloride, let stand and centrifuge and resuspend. (5) Then add VEGF plasmid, let stand, centrifuge and resuspend, then add poly-L-arginine hydrochloride again, let stand and centrifuge and resuspend, then add platelet membrane nanovesicles, let stand and centrifuge again, and finally resuspend with pre-cooled PBS and place at low temperature.

3. The method for preparing exosome-based targeted composite nanoparticles according to claim 2, characterized in that, In step (1), after the mice were euthanized, the bone marrow was taken from the femur and tibia of the hind limb. Then, primary mesenchymal stem cells were separated by density gradient centrifugation. The third-generation cells were washed with phosphate buffer and cultured in a culture medium in which exosomes and serum were removed for 70-80 h. After the culture was completed, the cells were centrifuged at 200-400 rpm for 10-15 min, 2000-4000 rpm for 15-25 min, and 8000-12000 rpm for 25-35 min at 4 °C. The second centrifugation program was centrifugation at 110000g for 80-100 min at 4 °C. The resulting exosomes were resuspended and the resulting suspension was stored at -75-85 °C.

4. The method for preparing exosome-based targeted composite nanoparticles according to claim 2, characterized in that, In step (2), the whole blood centrifugation program is as follows: centrifuge at 4℃ and 1000-1500 rpm for 15-25 min, repeat 1-2 times. After each centrifugation, take the supernatant, add 1 mM EDTA solution and 2 μM prostaglandin E1 phosphate buffer, and centrifuge a second time at room temperature and 3000-3500 rpm for 15-25 min. Freeze at -75-85℃ for 10-15 min, then thaw at room temperature. Repeat the freeze-thaw cycle 6-8 times, then sonicate, and centrifuge a third time at 7000-9000 rpm for 4-6 min. The concentration of PMSF in the phosphate buffer is 0.5-1.5 M. The fourth centrifugation rate is 7000-9000 rpm, and the centrifugation time is 4-6 min. The pore sizes of the extruded polycarbonate porous membranes are 19 mm, 800 nm, 400 nm, 200 nm, and 100 nm, respectively.

5. The method for preparing exosome-based targeted composite nanoparticles according to claim 2, characterized in that, In step (3), the mass ratio of exosomes to poly-L-arginine hydrochloride is 1:(100-200).

6. The method for preparing exosome-based targeted composite nanoparticles according to claim 2, characterized in that, In step (4), the mass ratio of substance A: exosomes: poly-L-arginine hydrochloride is (1-2): (0.04-0.1): (2-4).

7. The method for preparing exosome-based targeted composite nanoparticles according to claim 2, characterized in that, In step (5), the mass ratio of VEGF plasmid: poly-L-arginine hydrochloride: platelet membrane nanovesicles is (0.01-0.02): (4-8): (8-16).

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