Engineered exosome as well as preparation method and application thereof

Abstract

The invention relates to an engineered exosome as well as a preparation method and application thereof. According to the invention, an integrated platform technology is developed, composite nanoparticles with specific structures and composition are designed, and an exosome preparation process is developed based on the composite nanoparticles, so that four steps of an engineered exosome production process are expected to be improved at the same time, efficient preparation of the engineered exosome is realized, and the obtained exosome has good stability, can efficiently load drugs, and has good application prospects. The medicine can be applied to treatment of cranial nerve diseases, respiratory system diseases and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an engineered exosome, its preparation method, and its application. Background Technology

[0002] Extracellular vesicles (EVs), such as exosomes, have shown great potential as emerging cell therapies and nanomedicines. Compared to other types of EVs, such as microvesicles and apoptotic bodies, exosomes (commonly ranging in diameter from 50 to 150 nm) are considered more suitable for therapeutic use because their biogenesis involves unique intracellular regulatory processes that may determine their composition and function, resulting in more controlled therapeutic effects.

[0003] However, the therapeutic effects of exosomes alone are often insufficient to generate clinical application value; therefore, "engineered exosomes" have been developed, which are combined with drugs (including small molecules, nucleic acids, and protein drugs with therapeutic effects) to endow them with enhanced and / or additional functions. For example, CN116271097A discloses an engineered exosome based on a metal-organic framework, which uses tumor homing peptide-modified exosomes as the shell, metal-organic framework ZIF-8 as the core, and loads superparamagnetic iron oxide nanoparticles and doxorubicin as a multifunctional tumor diagnostic and therapeutic reagent.

[0004] Currently, the clinical translation of engineered exosomes faces numerous challenges in production, including insufficient quantity and purity, difficulty in efficiently loading high-volume cargo, low efficiency, poor scalability, the need for expensive separation equipment, poor storage stability, and high overall cost. To improve the production of engineered exosomes, various technologies have been developed, such as starving source cells to stimulate exosome production; using tangential flow filtration to separate exosomes to improve separation scalability; mechanically treating exosomes to temporarily open the exosome membrane to increase cargo loading; and using cryoprotectants to reduce the stability loss of exosomes during freeze-thaw cycles during lyophilized storage. However, typically each of these technologies can only improve one of the four steps in the engineered exosome manufacturing process (or, in very rare cases, two steps): exosome biosynthesis (secretion), cargo loading, separation, and storage. This is insufficient to meet the increasing demands for efficiency and quality in exosome preparation for clinical translation.

[0005] In conclusion, developing efficient methods for preparing high-quality exosomes is of great significance for the application of exosomes. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an engineered exosome, its preparation method, and its application, with the aim of efficiently preparing high-quality exosomes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a composite nanoparticle, the composite nanoparticle comprising nanoparticles and exosome biosynthesis stimulating ligands coupled thereto; the nanoparticles comprising self-assembling proteins and superparamagnetic iron oxide nanoparticles.

[0008] This invention designs specific structural composite nanoparticles that can serve as a link to significantly improve the efficiency of all major steps in the engineered exosome production process (including secretion, drug loading, separation, and storage), thereby achieving efficient preparation of high-quality exosomes.

[0009] Optionally, the exosome biosynthesis-stimulating ligand includes a membrane-penetrating peptide.

[0010] Optionally, the membrane-penetrating peptide includes Tat membrane-penetrating peptide.

[0011] Optionally, the amino acid sequence of the Tat membrane-penetrating peptide includes the sequence shown in SEQ ID NO.1.

[0012] Optionally, the protein includes any one or a combination of at least two of the following: bovine serum albumin, human serum albumin, egg white lysozyme, bovine lactalbumin, zein, β-casein, ovalbumin, β-lactoglobulin, or lactoferrin.

[0013] Optionally, the composite nanoparticles may further contain a drug and / or a detection agent, the drug and / or detection agent being loaded onto the nanoparticles.

[0014] Optionally, the drug comprises at least one of small molecules, nucleic acids, or peptides.

[0015] Optionally, the detection agent includes a molecular imaging probe.

[0016] In a second aspect, the present invention provides a method for preparing the composite nanoparticles described in the first aspect, comprising: Dissolve the protein solution in an aqueous solution to prepare a protein solution; Superparamagnetic iron oxide nanoparticles were dissolved in an organic solvent to prepare a nanocrystalline solution; The nanocrystal solution was mixed with the protein solution, and the mixture was incubated at room temperature to obtain an incubated mixture. The incubated mixture was centrifuged, the supernatant was removed, and the precipitate was collected to obtain nanoparticles. The nanoparticles were coupled with exosome biosynthesis stimulating ligands to obtain the composite nanoparticles.

[0017] Alternatively, other functional nanoparticles, such as fluorescent quantum dots, can be added while using superparamagnetic iron oxide nanoparticles to give engineered exosomes more functions.

[0018] Thirdly, the present invention provides the application of the composite nanoparticles described in the first aspect in the preparation of exosomes.

[0019] Fourthly, the present invention provides a method for preparing exosomes, the method comprising: The exosome-synthetic cells were mixed and incubated with the composite nanoparticles described in the first aspect, and the exosomes were magnetically separated to obtain exosomes.

[0020] Optionally, the magnetic separation method includes: A modular magnet array in the shape of a cuboid is assembled using permanent magnets; the modular magnet array is placed inside a separation container, and a driving magnet is set outside the separation container. A driving motor and a transmission device are linked with the driving magnet, so that the modular magnet array inside the separation container moves slowly at a set speed, capturing exosomes containing composite nanoparticles onto the surface of the modular magnet array; then, the exosomes are detached and harvested from the surface of the modular magnet array by assisted flushing with physical ultrasound or chemical dissociation fluid (brief flushing).

[0021] Fifthly, the present invention provides an engineered exosome, which is prepared by the method for preparing exosomes described in the third aspect.

[0022] In a sixth aspect, the present invention provides the use of the engineered exosomes described in the fifth aspect in the preparation of medicaments for treating diseases, said diseases including at least one of neurological diseases, respiratory diseases, skin diseases, cardiovascular diseases, reproductive diseases, cancer, metabolic diseases, immune system diseases, or ophthalmic diseases.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects: This invention develops an integrated platform technology, designs composite nanoparticles with specific structures and compositions, and develops an exosome preparation process based on them. The aim is to simultaneously improve the four steps of the engineered exosome production process, achieve efficient preparation of engineered exosomes, and obtain exosomes with good stability, which can efficiently load drugs and can be applied to the treatment of neurological diseases, respiratory diseases, etc. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of an integrated production platform.

[0025] Figure 2 The figure shows the results of comparing the number of exosomes secreted in BMSC cells by different methods.

[0026] Figure 3Figure 1 shows the characterization results of drug and nanoparticle loading tests. Figure A shows the dynamic light scattering (DLS) analysis of Supraparticle and Supraparticle-Tat; Figure B shows the transmission electron microscopy (TEM) image of Supraparticle-Tat; scale bar: 20 nm; Figure C shows the Zeta potential analysis of Supraparticle-Tat; Figure D shows the thermogravimetric analysis (TGA) of Supraparticle; Figure E shows the superconducting quantum interference device (SQUID) analysis of Supraparticle; Figure F shows the drug loading of Supraparticle (drug: curcumin); Figure G shows the colloidal stability of CUR-loaded Supraparticle-Tat in PBS at 4 °C determined by DLS; Figure H shows the quantitative analysis of the amount of Supraparticle loaded in each BMSC exosome using two different methods (TEM and ICP-OES); Figure I shows the biological transmission electron microscopy used to observe Supraparticle-Tat in BMSC exosomes. The exocytosis process; Figure J shows the percentage of Supraparticle-Tat encapsulated in exosomes out of total exocytraparticle-Tat for BMSCs (total exocytraparticle-Tat includes two parts: Supraparticle-Tat encapsulated in exosomes and free Supraparticle-Tat in the cell culture medium); Figure K shows the percentage of Supraparticle-Tat-loaded exosomes out of total exosomes for BMSCs (total exosomes include two parts: Supraparticle-Tat-loaded exosomes and unloaded exosomes); Figure L shows the percentage of Supraparticle-Tat encapsulated in exosomes out of total exocytraparticle-Tat for MCF-7 cells (total exocytraparticle-Tat includes two parts: Supraparticle-Tat encapsulated in exosomes and unloaded exosomes). (and free Supraparticle-Tat in cell culture medium); Figure M shows the key sorting ratio of BMSCs for MCF-7 cells, quantifying the following: the percentage of Supraparticle-Tat-loaded exosomes to total exosomes (total exosomes consist of two parts, namely Supraparticle-Tat-loaded exosomes and unloaded Supraparticle-Tat exosomes).

[0027] Figure 4A The figure shows the separation efficiency of separating supraparticles in PBS using a conventional magnetic separation design with different time lengths. The magnet used was an N35 neodymium magnet (27.5 mm × 17.5 mm × 7.5 mm), and the container was a beaker with a bottom diameter of 6 cm. The magnetic poles of the magnet were in contact with the outer surface of the container.

[0028] Figure 4B The graph shows the variation of magnetic field gradients with the distance between magnet tips for different neodymium magnets (including N35 (27.5 mm × 17.5 mm × 7.5 mm and 20 × 5 × 5 mm), N45 (20 mm × 5 mm × 5 mm), and N33SH (20 mm × 5 mm × 5 mm, 5 mm × 5 mm × 5 mm, and an assembly consisting of four 5 mm × 5 mm × 5 mm magnets). The data were measured by a magnetometer.

[0029] Figure 4C This is a schematic diagram of the overall design of the novel magnetic separation system.

[0030] Figure 4D The diagram shows a magnet used in the novel magnetic separation, where (1) the novel magnetic separation magnet is composed of multiple cubic magnet units. The diagram shows a photograph and a top view of the magnet (in this example, it is composed of 4 cubic magnet units), (2) is a three-dimensional magnetic field distribution simulation of the novel magnetic separation magnet, and (3) is a top view magnetic field gradient distribution of the novel magnetic separation magnet. White indicates zero magnetic field gradient.

[0031] Figure 4E The diagram shows the instrument used in the novel magnetic separation process. (1) is the sample loading area, (2) is the instrument appearance, (3) is the internal structure of the instrument, and (4) is the three-dimensional model of the internal structure. ①-Aluminum shell, ②-Portable handle, ③-Sample container (beaker), ④-Power supply, ⑤-Polytetrafluoroethylene coated trapping magnet, ⑥-Support platform, ⑦-Drive magnet, ⑧-Drive motor, ⑨-Transmission gear.

[0032] Figure 4F The figure shows a direct comparison of the isolation efficiency between the traditional magnet design and the novel magnet design. For direct comparison, both the traditional and novel magnetic separation methods used the same magnet, namely a component consisting of 4 cubic magnet units. The separation time was 1 hour, and the container used was a beaker with a diameter of 6 cm.

[0033] Figure 4G The results of the experiment demonstrate the unique scalability (separate scale-up) of the novel magnet design compared to the traditional magnet design.

[0034] Figure 5 To utilize the novel magnetic separation design, the recovery efficiency of Supraparticle and encapsulated Supraparticle exosomes was assessed (recovery was performed using brief sonication).

[0035] Figure 6 Transmission electron microscopy (TEM) images of engineered exosome products. White arrows point to exosomes. Red arrows point to Supraparticle-Tat (encapsulated within exosomes).

[0036] Figure 7 To test the enhancing effect of Supraparticle on the stability of exosomes in engineered exosome products, the following figures are presented: Figure A shows the particle number concentration changes of different exosomes under different storage conditions (-80℃ and lyophilization): conventional exosomes and Supraparticle-encapsulated exosomes, storage time: 1 month; Figures B and C show the particle size distribution changes of different exosomes under different storage conditions (-80℃ and lyophilization): conventional exosomes (Figure B) and Supraparticle-encapsulated exosomes (Figure C), storage time: 1 month; Figure D shows the results of the first and second lyophilization cycles of different exosomes. The changes in particle number concentration after the first and second freeze-drying cycles: traditional exosomes and exosomes encapsulated with Supraparticle; Figure E shows the changes in average particle size of different exosomes after the first and second freeze-drying cycles: traditional exosomes and exosomes encapsulated with Supraparticle; Figure F shows the changes in particle number concentration of different exosomes (traditional exosomes and exosomes encapsulated with Supraparticle) after stress treatment (atomization) during application; Figures G and H show the changes in particle size distribution of different exosomes (traditional exosomes (Figure G) and exosomes encapsulated with Supraparticle (Figure H)) after stress treatment (atomization).

[0037] Figure 8The figures show the efficacy evaluation results of engineered exosome products in the treatment of neurological diseases. Figure A is a schematic diagram of the experimental procedure; Figure B shows representative movement trajectories of mice in open-field behavioral analysis; Figures C and D show the quantitative analysis of Figure B; Figure E shows the quantitative analysis results of the rotating rod behavior experiment; Figure F shows hematoxylin-eosin (H&E) staining in the substantia nigra region of the mouse brain, with black arrows indicating the nuclei of normal neurons and red arrows indicating condensed nuclei in diseased neurons (scale bar: 100 µm); Figure G shows Nissl staining in the substantia nigra region of the mouse brain. Black arrows indicate positive staining of Nissl bodies (rough endoplasmic reticulum and ribosomes) in neurons; damaged or dying neurons may show a lack of Nissl bodies (scale bar: 100 µm). Figure H shows the quantitative analysis of Nissl staining results; Figure I shows the immunohistochemical staining of tyrosine hydroxylase (TH) in the substantia nigra region of mouse brain; black arrows indicate TH-positive dopaminergic neurons; scale bar: 100 µm; Figure J shows the quantitative analysis of TH-positive dopaminergic neurons in the substantia nigra region of mouse brain.

[0038] Figure 9Figure A shows the efficacy evaluation results of engineered exosomes in treating idiopathic pulmonary fibrosis (IPF) in a mouse model. Different mouse groups include: sham-operated group (normal mouse control group treated with PBS), IPF group (IPF mice treated with PBS), Supraparticle-Cur group (IPF mice treated with Supraparticle-Tat loaded with curcumin), Exosome group (IPF mice treated with MSC-derived exosomes without Supraparticle or curcumin), Supraparticle-Exosome group (IPF mice treated with MSC-derived exosomes encapsulated in Supraparticle-Tat without curcumin), and Supraparticle-Exosome-Cur group (IPF mice treated with MSC-derived exosomes encapsulated in Supraparticle-Tat loaded with curcumin). Figure A is a schematic diagram of the experimental procedure; Figure B shows the H&E of lung tissue (including areas near and away from the bronchi). Staining assessment: White arrows: alveoli; Yellow arrows: bronchial fibrosis; Figure C shows quantitative analysis of Ashcroft score based on H&E staining results; Figure D shows Masson staining assessment of lung tissue: Green arrows: blue staining of fibrotic tissue; Scale bar: 100 µm; Figure E shows quantitative analysis of Masson staining results: AOD: mean optical density; Figure F shows immunohistochemical staining of α-SMA in lung tissue: Black arrows: α-SMA positive cells; Scale bar: 100 µm; Figure G shows quantitative analysis of α-SMA immunohistochemical staining results; Figure H shows immunohistochemical staining of type I collagen in lung tissue: Black arrows: type I collagen positive cells; Scale bar: 100 µm; Figure I shows quantitative analysis of type I collagen (Collagen I) immunohistochemical staining results.

[0039] Figure 10 For proteomics analysis, Venn diagrams were used to compare BMSC-derived cells, engineered BMSC exosomes (produced by BMSC cells stimulated with Supraparticle-Tat), and conventional BMSC exosomes (produced by BMSC cells stimulated with starvation). The results showed that after BMSCs were stimulated with Supraparticle-Tat, the total number of proteins in the exosomes increased from 831 to 1061. Of the 1862 proteins detected in BMSCs, starvation-stimulated exosomes contained 34.9%, while Supraparticle-Tat-stimulated exosomes increased this percentage to 43.4%.

[0040] Figure 11The figure shows the results of evaluating the efficacy of engineered exosomes in promoting wound healing in a mouse model. The initial wound was located on the skin of the mouse's back and measured 0.6 cm in size. The mice were divided into the following groups: PBS group (wounded mice were injected with PBS only), Supraparticle-Cur group (wounded mice were injected with Supraparticle-Tat containing curcumin), Exosome group (wounded mice were injected with MSC-derived exosomes without Supraparticle-Tat or curcumin), Supraparticle-Exosome group (wounded mice were injected with MSC-derived exosomes encapsulated in Supraparticle-Tat without curcumin), and Supraparticle-Exosome-Cur group (wounded mice were injected with MSC-derived exosomes encapsulated in Supraparticle-Tat containing curcumin). Figure A shows a schematic diagram of the experimental procedure; Figure B shows wound images after different treatments; Figure C shows a quantitative analysis of wound healing based on the images in Figure B; Figure D shows H&E staining images of mouse skin 11 days after different treatments; Figure E shows a quantitative analysis of scar length based on H&E staining results, with a scale bar of 400 μm; Figure F shows Masson staining of mouse skin 11 days after different treatments, with a scale bar of 20 μm; Figure G shows a quantitative analysis of collagen content based on Masson staining results; Figure H shows immunohistochemical staining of mouse skin 11 days after different treatments for CD31 (also known as PECAM-1, a biomarker of angiogenesis), with a scale bar of 400 μm; Figure I shows a quantitative analysis of the CD31 immunohistochemical staining results.

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0043] This invention develops an integrated platform technology to simultaneously improve the four steps of the engineered exosome production process (exosome biosynthesis (secretion), cargo loading, separation, and storage). It designs composite nanoparticles with specific structures and compositions, which load magnetic nanoparticles and drugs, and are coated with exosome biosynthesis stimulating ligands. Source cells (cells capable of synthesizing exosomes) are co-incubated with the composite nanoparticles, utilizing the exosome biosynthesis stimulating ligands to increase the number of exosomes produced by each source cell. The natural cellular uptake process of the composite nanoparticles allows for simultaneous loading of drugs and nanoparticles during exosome biosynthesis. Utilizing the magnetism of the magnetic nanoparticles loaded within the exosomes, the engineered exosomes (exosomes loaded with drugs and nanoparticles) formed thereby can be separated using a magnet. Due to the presence of structurally stable nanoparticles (magnetic nanoparticles) within the exosomes, the storage stability of the engineered exosomes is improved compared to natural exosomes.

[0044] Optionally, the composite nanoparticles include nanoparticles and exosome biosynthesis stimulating ligands cross-linked therewith. The nanoparticles are formed by self-assembly of albumin and magnetic nanoparticles (named Suparaparticle). The nanoparticles can be loaded with drugs. The exosome biosynthesis stimulating ligand is a membrane-penetrating peptide (CPP), which can be named Suparaparticle-CPP, or alternatively, a Tat membrane-penetrating peptide, which can be named Suparaparticle-Tat.

[0045] In this invention, the composite nanoparticles formed by cross-linking membrane-penetrating peptides and nanoparticles, after being incubated with source cells, can not only significantly stimulate the generation of exosomes, but also significantly enhance the endocytosis of source cells. Furthermore, a considerable portion of the endocytosed composite nanoparticles will be secreted by the source cells, and almost all of the secreted composite nanoparticles are present in the exosomes. Moreover, the nanoparticles can amplify the stimulatory effect of the ligands on exosome biosynthesis, thereby significantly enhancing the stimulatory effect of the membrane-penetrating peptides.

[0046] Optionally, the method for preparing the nanoparticles (Suparaparticles) includes: 1) Dissolve the protein solution in protein buffer to prepare a protein solution; 2) Dissolve oil-soluble nanoparticles in an organic solvent to prepare a nanocrystal solution; 3) Mix the nanocrystal solution with the protein solution and shake well; 4) Incubate the above mixture at room temperature to obtain the incubated mixture; 5) Centrifuge the incubated mixture to remove the supernatant; the precipitate is Suparaparticle.

[0047] Optionally, the protein is selected from at least one of bovine serum albumin, human serum albumin, egg white lysozyme, bovine lactalbumin, zein, β-casein, ovalbumin, β-lactoglobulin, or lactoferrin; the oil-soluble nanoparticles include superparamagnetic iron oxide nanoparticles (SPIONs) and / or quantum dots (QDs); the concentration of the protein solution is 2 mg / mL, and the concentration of the nanocrystal solution is 1 mg / mL; in step 4), the incubation time is more than three hours; the organic solvent is tetrahydrofuran.

[0048] In this invention, nanoparticles with a specific composition and a physical diameter of 50-100 nm are used, which can efficiently load drug molecules (such as small molecules, nucleic acids, peptides, etc. with therapeutic functions) and have excellent structural and colloidal stability.

[0049] Specifically, the process of separating exosomes using magnets can employ a novel magnetic separation method, including: A modular magnet array in the shape of a cuboid is assembled using permanent magnets; the modular magnet array is placed inside a separation container, and a driving magnet is set outside the separation container. A driving motor and a transmission device are linked with the driving magnet, so that the modular magnet array inside the separation container moves slowly at a set speed, capturing exosomes containing composite nanoparticles onto the surface of the modular magnet array; then, the exosomes are detached and harvested from the surface of the modular magnet array by assisted flushing with physical ultrasound or chemical dissociation fluid (brief flushing).

[0050] This novel magnetic separation method is easy to scale up and can almost completely separate exosomes from the extracellular medium in a short time. The time required to achieve near-complete separation is basically the same for different sized separation containers.

[0051] In summary, based on the above design scheme, an exosome preparation process was developed, such as... Figure 1 As shown, engineered exosomes exhibit superior performance in all four production steps. Compared to natural exosomes, engineered exosomes, due to the presence of nanoparticles and drugs within them, possess enhanced and / or additional functions in biomedical applications, particularly in imaging, delivery, and therapy: the presence of SPIONs and / or QDs in exosomes enables imaging and tracking of exosomes via magnetic resonance imaging (MRI), rotating disk confocal fluorescence microscopy, and transmission electron microscopy (TEM); the ultra-high drug loading capacity of supraparticles and the magnetically responsive motion of SPIONs, combined with the barrier-crossing capabilities of exosomes, can enhance drug delivery; the presence of a large amount of drug within exosomes can enhance the therapeutic effect between the drug and the exosome.

[0052] Example 1 This embodiment prepares composite nanoparticles.

[0053] The composite nanoparticles include supraparticles and cross-linked transmembrane peptides.

[0054] The membrane-penetrating peptide is Tat membrane-penetrating peptide, and its amino acid sequence is shown in SEQ ID NO.1.

[0055] SEQ ID NO.1: YGRKKRRQRRR.

[0056] Specific preparation methods include: A solution of hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) (200 μL, 1 mg / mL, THF) was added to a bovine serum albumin (BSA) solution (3 mL, 2 mg / mL, PBS) and magnetically stirred (1000 rpm). The mixture was incubated in a 25°C water bath for at least 3 hours to form supraparticles. The formation of supraparticles is a self-assembly process driven by strong hydrophobic interactions between the large hydrophobic surface of the SPIONs and the hydrophobic regions of the BSA molecules. To purify the supraparticle product, centrifugation (13000 rpm, 4°C, 45 min) was performed. The precipitate (pure supraparticles) was redispersed in double-distilled water for physicochemical characterization.

[0057] After the superparticles were formed, they were coupled with Tat membrane-penetrating peptides using SMCC [succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate] coupling chemistry to prepare composite nanoparticles Supraparticle-Tat.

[0058] The hydrodynamic diameter of Supraparticle-Tat, measured by dynamic light scattering (DLS), was 108.5 ± 1.5 nm, with a polydispersity index (PDI) of 0.226 ± 0.047, only slightly larger than the uncoupled Tat particle size (104.7 ± 0.9 nm, PDI 0.197 ± 0.039). Transmission electron microscopy (TEM) results showed that each Supraparticle contained multiple electron-dense nanoparticles with a physical diameter of 54.6 nm ± 1.8 nm. During the coupling reaction, the addition of a greater number of Tat transmembrane peptides to each Supraparticle led to an increase in the surface charge of Supraparticle-Tat, manifested as an increase in the zeta potential, due to the positive charge of the Tat peptides. As the number of Tat peptides added to each supraparticle gradually increased from 2000 to 20000, the zeta potential value also gradually changed from -28.21±0.47 mV to -6.54±0.32 mV.

[0059] Example 2 This embodiment tests the effect of the composite nanoparticles prepared in Example 1 on the secretion of exosomes by cells.

[0060] The specific experimental procedure includes: BMSCs (a type of mesenchymal stem cell commonly used in regenerative medicine) were incubated for 8 hours in complete cell culture medium containing Supraparticle-Tat (endocytosis phase). After removing the culture medium and washing three times with phosphate-buffered saline (PBS), the BMSCs were incubated for 16 hours in culture medium without Supraparticle-Tat and serum (efflux phase). The culture medium containing extracellular vesicles (EVs) secreted by the BMSCs was collected at the end of the efflux phase.

[0061] In addition, two control experiments were conducted: a starvation stimulation group and a free transmembrane peptide group. In the starvation stimulation group, BMSCs were cultured in serum-free cell culture medium for 24 hours, and the culture medium containing extracellular vesicles (EVs) secreted by BMSCs was collected. In the free transmembrane peptide group, BMSCs were incubated for 8 hours in complete cell culture medium containing free transmembrane peptides (the same number of peptide molecules as in the Supraparticle-Tat group) (endocytosis phase); after removing the culture medium and washing three times with phosphate-buffered saline (PBS), BMSCs were incubated for 16 hours in culture medium without free transmembrane peptides and serum (efflux phase), and the culture medium at the end of the efflux phase, containing extracellular vesicles (EVs) secreted by BMSCs, was collected.

[0062] The number of exosomes in the culture medium, i.e., the number of microscopic objects with a diameter range of 50-150 nm, was measured by nanoparticle tracking analysis (NTA).

[0063] The results are as follows Figure 2 As shown, free transmembrane peptides have a certain stimulatory effect on the number of exosomes secreted by BMSCs (approximately 3 times higher than the conventional starvation method). However, after the transmembrane peptides are crosslinked onto the nanoparticle Supraparticle, the stimulatory effect on the number of exosomes secreted is significantly enhanced (nearly 20 times higher than the conventional starvation method).

[0064] It is evident that when multiple transmembrane peptide molecules are crosslinked onto a single nanoparticle (each Supraparticle nanoparticle contains approximately 2000 crosslinked peptide molecules), they work synergistically, significantly enhancing their binding force with the cell membrane. Subsequently, after entering the cell via endocytosis, Supraparticle-Tat is encapsulated in an exosome and expelled from the cell through the Secretory Autophagy process. This enhanced binding force with the cell membrane leads to an enhancement of the downstream process (secretory autophagy).

[0065] Example 3 In this embodiment, drug-loaded composite nanoparticles were prepared and tested. After co-culturing with BMSC cells using Supraparticle-Tat, drug and nanoparticle loading on exosomes were achieved, along with characterization and analysis of the loading.

[0066] In the process of loading curcumin (CUR) onto Supraparticle, superparamagnetic iron oxide nanoparticles (SPION, 1 mg / mL, 200 μL, dissolved in tetrahydrofuran) were first mixed with curcumin (CUR, 2.5 mg / mL, 50 μL, dissolved in dimethylformamide), and then mixed with bovine serum albumin (BSA) solution (3 mL, 2 mg / mL, dissolved in phosphate buffer) to form a BSA-SPION-CUR co-assembly. The mixture was then incubated in a 25°C water bath for at least 3 hours and purified by centrifugation at 13,500 rpm for 45 minutes.

[0067] To determine drug loading and encapsulation efficiency, an "indirect analytical method" was employed, using an Agilent Cary 100 UV-Vis spectrophotometer to measure the absorbance of the drug not encapsulated in the supraparticle (supernatant after centrifugation). For curcumin (CUR), the absorption peak wavelength used was 425 nm. Alternatively, a "direct analytical method" was used to directly determine the drug content in the supraparticle. In this method, the supraparticle loaded with curcumin was resuspended in DMF and centrifuged (13,500 rpm, 45 min) to extract the drug-loaded curcumin from the supraparticle. The supernatant was collected and analyzed using high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity, USA, C18 column, mobile phase composition 50:50 acetonitrile:water, flow rate 1 mL / min, detection wavelength 425 nm). The results obtained by the two analytical methods (direct and indirect methods) were found to be almost identical.

[0068] The following formulas are used to calculate drug loading and encapsulation efficiency (W: weight): ; .

[0069] Test characterization analysis results as follows Figure 3 As shown, after the formation of curcumin-loaded supraparticles, they were coupled to the Tat transmembrane peptide using the SMCC [succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate] coupling chemistry. The hydrodynamic diameter of the supraparticle-Tat was measured by dynamic light scattering (DLS) to be 108.5 ± 1.5 nm, and the polydispersity index (PDI) was 0.226 ± 0.047, only slightly larger than the diameter of Tat before coupling (104.7 ± 0.9 nm, PDI 0.197 ± 0.039). Figure 3(Figure A). Transmission electron microscopy (TEM) images of Supraparticle-Tat show that each Supraparticle molecule contains multiple electron-dense nanoparticles with a physical diameter of 54.6 nm ± 1.8 nm. Figure 3 (See Figure B). Although it is difficult to determine the exact number of SPIONs in each supraparticle, TEM images show that the number is quite large: 10-20 SPIONs can be observed in each supraparticle on just one imaging focal plane. Figure 3 (See Figure B). In the coupling reaction, adding a greater number of Tat transmembrane peptides to each Supraparticle leads to an increase in the surface charge of the Supraparticle-Tat, which is reflected in a higher zeta potential value due to the positive charge of the Tat peptides. Figure 3 (See Figure C). As the number of Tat peptides added to each Supraparticle gradually increased from 2000 to 20000, the zeta potential value also gradually changed from -28.21 ± 0.47 mV to -6.54 ± 0.32 mV. Figure 3 (See Figure C). Thermogravimetric analysis (TGA) results of the Supraparticle, compared with those of SPIONs and BSA, indicate that the mass percentage of SPIONs in the Supraparticle is approximately 81.87% (see Figure C). Figure 3 (Figure D). Hysteresis loops measured by a superconducting quantum interference device (SQUID) indicate that both supraparticles and SPIONs exhibit superparamagnetism (zero remanence and zero coercivity) at 300 K. Figure 3 (See Figure E). The saturation magnetization of supraparticles and SPIONs, measured using a superconducting quantum interference device (SQUID), was 39.08 emu / g and 53.14 emu / g, respectively. Figure 3 (See Figure E). The ratio of the two is 73.7%, which is very close to the mass percentage of SPIONs in the Supraparticle measured by thermogravimetric analysis (TGA), indicating that the interaction between the large number of SPIONs in the Supraparticle is small (if any) and insufficient to significantly affect its magnetism.

[0070] Curcumin (CUR) was used as a model drug. Curcumin is a compound widely found in food and has been reported to have anti-inflammatory, antioxidant, and neuroprotective activities. Curcumin was loaded into the Supraparticle using the drug loading method described above. With increasing curcumin content, the drug loading and encapsulation efficiency also increased, with a maximum drug loading of approximately 53% and an encapsulation efficiency of approximately 90%. Figure 3 (Figure F). After two months of storage in PBS buffer at 4°C, the hydrodynamic diameter of Supraparticle-Tat loaded with curcumin showed minimal change, indicating its excellent colloidal stability. Figure 3 (China G map).

[0071] Two different methods, transmission electron microscopy (TEM) and inductively coupled plasma optical emission spectrometry (ICP-OES), were used to analyze the average number of supraparticles loaded per exosome in the product (after magnetic separation). The results from the two methods were similar, indicating that each exosome in the product (after magnetic separation) carried an average of approximately 1.5 supraparticles. Figure 3 Figures H and I are transmission electron microscopy images used to observe the exocytosis of Supraparticle-Tat in exosomes derived from BMSCs. This indicates that each exosome in the product (after magnetic separation) is loaded with either one or two Supraparticles, and the number of exosomes loaded with one Supraparticle is approximately equal to the number loaded with two Supraparticles. Given that the maximum CUR loading capacity of each Supraparticle is approximately 53% ( Figure 3 (See Figure F in the middle). It can be calculated that using Supraparticle-Tat can reduce the maximum value to approximately 1.0275 × 10⁻⁶. 6 (That is, about 1 million) CUR molecules are loaded into each exosome.

[0072] Several key sorting ratios in cell transport associated with Supraparticle-Tat loading into exosomes were quantitatively analyzed, including: (1) the percentage of Supraparticle-Tat encapsulated in exosomes relative to total exocytosolic Supraparticle-Tat (total exocytosolic Supraparticle-Tat consists of two parts, namely Supraparticle-Tat encapsulated in exosomes and free Supraparticle-Tat in the extracellular medium, see schematic diagram). Figure 3 (2) Percentage of Supraparticle-Tat-loaded exosomes to total exosomes (total exosomes include two parts, namely Supraparticle-Tat-loaded exosomes and unloaded Supraparticle-Tat exosomes, see schematic diagram). Figure 3(3) The percentage of exocytotic Supraparticle-Tat to endocytotic Supraparticle-Tat (i.e., the percentage of Supraparticle-Tat released from the cell to the total amount of Supraparticle-Tat entering the cell).

[0073] First, the percentage of Supraparticle-Tat encapsulated in extracellular vesicles within the total Supraparticle-Tat exocytosis of BMSCs was analyzed (the first key sorting ratio). Figure 3 (See Figure J). Fluorescent colocalization analysis was performed on the cell culture medium after the exocytosis stage (before magnetic separation) to quantify the percentage of colocalization between green (supraparticle-Tat) and red (vesicle dye). Results showed that this percentage was >90% in BMSCs. Figure 3 (Figure J in Chinese). Secondly, the percentage of extracellular vesicles loaded with Supraparticle-Tat in the total exosomes exocytotic by BMSCs was investigated (the second key sorting ratio). Figure 3 (See figure 1). Fluorescent colocalization analysis was performed on the cell culture medium after the exocytosis stage (before magnetic separation) to quantify the percentage of colocalization between red (vesicle dye) and green (supraparticle-Tat content). It is important to note that this measurement was performed before magnetic separation, thus preserving the portion of exosomes unloaded with Supraparticle-Tat for measurement. Through colocalization analysis, we found that the percentage of Supraparticle-Tat-loaded exosomes in the total exosomes derived from BMSCs was approximately 20-40%, and this percentage was significantly correlated with the Tat content per Supraparticle. Figure 3 (Middle K-plot). When the number of Tats per supraparticle is between 2,000 and 20,000, this percentage is higher in BMSCs ( Figure 3 (Middle K chart).

[0074] Furthermore, it was found that the sorting of Supraparticle-Tat during cell transport was cell-type dependent. In MCF-7 cells, only about 5% of the total exocytotic Supraparticle-Tat was contained in extracellular vesicles; and the percentage of extracellular vesicles carrying Supraparticle-Tat was also only about 5% of the total exocytotic Supraparticle-Tat (the number of Tat on each Supraparticle molecule was between 2000 and 20000). Figure 3 The middle L diagram and Figure 3(M-Figure). Both percentages of MCF-7 cells were significantly lower than those of BMSCs cells ( Figure 3 China J map and Figure 3 (Middle K chart).

[0075] Example 4 This embodiment describes the separation of exosomes prepared using composite nanoparticles.

[0076] The novel magnetic separation process for the resulting engineered exosomes includes: Centrifuge the cell supernatant at 2000 g for 10 minutes to remove dead cells and cell debris, and transfer the supernatant to a magnetic separation container (e.g., a beaker). Separate the exosomes containing magnetic nanoparticles by slowly rotating (e.g., 5 rpm) a magnet inside the container. Transfer the magnet (with exosomes attached to its surface) to another container containing PBS buffer. Perform a brief (10-second) sonication to release the exosomes into the buffer.

[0077] Because supraparticles are very small, and the core of an exosome can only hold a small number (close to one) of supraparticles, magnetically separating supraparticle-encapsulated exosomes from the extracellular medium requires overcoming a key challenge: the design of the magnetic device. We need a magnetic device design that is significantly different from existing devices. This requirement can be seen by analyzing the fundamental equations for magnetic isolation of magnetic particles in fluids: Among them, F mag Represents magnetic force, µ0 is the free permeability (4π×10⁻⁶). -7 H∙m -1 ), r is the radius of the magnetic particle (half its diameter), M p The saturation magnetization is The external magnetic field gradient is used. Because the magnetic particles in our technique are encapsulated within extracellular bodies, the physical diameter of the particles must be smaller than the diameter of the extracellular bodies (50-150 nm, 1-2 orders of magnitude smaller than the cells and magnetic particles typically used for extracellular body separation). Therefore, the magnetic force is subject to this fundamental limitation described by the equation above. In fact, our experimental results show that after 1 hour of separation using a conventional magnetic separation device (i.e., contacting a magnet (here, a neodymium magnet N35, 27.5 mm × 17.5 mm × 7.5 mm) with the outer surface of a container (here, a beaker with a diameter of 6 cm) containing the Supraparticle dispersion), the separation efficiency of Supraparticle (measured by NTA) is only about 1%. Figure 4AFurthermore, even with stronger magnets, the magnetic field gradient still drops to near zero very close to the edge of the magnet (approximately 10 mm). Figure 4B Therefore, traditional magnetic separation devices perform poorly when scaled up experiments: larger sample containers lead to significantly longer separation times. Figure 4G This embodiment uses Supraparticle as a convenient alternative to exosomes encapsulated by Supraparticle to evaluate the magnetic separation effect, because... Figure 3 As shown in the H-figure, each exosome encapsulated by a Supraparticle contains an average of about 1.5 Supraparticle molecules.

[0078] To achieve efficient separation, a feasible approach is to significantly enhance the magnetic field gradient in the above equation by using other magnetic devices. This invention proposes a novel design to solve this problem.

[0079] The novel design enhances the magnetic field gradient at the magnetic particles by significantly reducing the distance between the magnet and the magnetic particles. As mentioned above, the magnetic field gradient decreases rapidly with increasing distance from the magnet (Figure 4B). The magnet can only generate a high magnetic field gradient near its surface. In the novel design, the distance is reduced by: (1) placing the magnet in the magnetic particle dispersion; (2) rotating the magnet. With the novel design, almost every exosome encapsulated by the Supraparticle can be brought to a position very close to the magnet surface. In the novel design, a separation cycle consists of two phases: a capture phase and a release phase. Figure 4C During the capture phase, the magnet is slowly rotated in the extracellular medium (e.g., 5 rpm), capturing exosomes wrapped in supraparticles along its path. During the release phase, after the medium is replaced with PBS, the magnet is briefly sonicated to release the captured supraparticle-wrapped exosomes into the PBS. During the capture phase, the magnet's rotation speed must be very slow to minimize liquid flow opposite to the direction of magnetic capture motion.

[0080] In the new design, the capturing magnet consists of multiple cubic magnet units, each coated with a thin layer of inert polytetrafluoroethylene (PTEE, also known as Teflon) to prevent corrosion. Figure 4D (1) Figure 4D Figure (2) shows the magnetic field distribution around the magnet, which was obtained by finite element simulation using Ansys Maxwell software. Figure 4DAs shown in (3), in the magnetic field gradient distribution of the novel magnet trapping design, the magnetic field gradient near the magnet is significantly higher than that far from the magnet. Crucially, the maximum magnetic field gradient exists at all connections between adjacent cubic magnet units. Figure 4D (3) Therefore, when the novel design trap magnet is slowly rotated in a dispersion of magnetic objects, these maximum values ​​distributed along the entire length of the trap magnet can generate a strong magnetic attraction at almost every location in the dispersion. For magnetic isolation of larger-scale dispersion systems, a trap magnet composed of a large number of cubic magnet units can be simply used to ensure that each magnetic object in the dispersion system is close to the magnet.

[0081] Figure 4F The results show a comparison of the separation efficiency between the novel and conventional designs using the same permanent magnet (composed of 4 cubic magnet units). After 1 hour, the novel design achieved a separation efficiency exceeding 95% for both Supraparticles and their encapsulated exosomes. In stark contrast, the conventional design achieved a separation efficiency of only about 1% for Supraparticles after the same time (1 hour). Furthermore, a key advantage of the novel design lies in its unique scalability (separation scale-up): using more cubic magnet units and larger sample containers, near-complete separation can be achieved with essentially the same separation time. Scalability is a critical challenge for applying magnetic separation technology to production, as production typically requires a larger scale than analytical applications. To verify the scalability of the novel design, Figure 4G The figure shows the separation efficiency results of the novel and conventional designs after 1 hour of separation of Supraparticle dispersions in five different container sizes (beaker diameters of 1 cm, 2.5 cm, 4 cm, 6 cm, and 10 cm). The results show that the novel design achieved a separation efficiency exceeding 95% after 1 hour for all five container sizes. In contrast, the separation efficiency of the conventional design decreased significantly with increasing container size after 1 hour. In the largest container tested (10 cm), the conventional design achieved a separation efficiency of only ~1% after 1 hour. Furthermore, the novel design exhibited superior repeatability in terms of separation efficiency: the standard deviation of three independent separation experiments was less than 1.5%, far superior to the conventional design, especially in larger containers. During the separation cycle, the release phase could be completed in as little as 10 seconds; through ultrasonic treatment and additional rinsing of the trapping magnet, we were able to recover more than 90% of the trapped Supraparticles or exosomes encapsulated by Supraparticles (using NTA testing results). Figure 5 Transmission electron microscopy (TEM) Figure 6The results, including the similar number of exosomes encapsulated in Supraparticle before and after treatment, indicate that the exosomes encapsulated in Supraparticle remained intact during the separation process.

[0082] Example 5 This embodiment tests the enhancing effect of encapsulating Supraparticles on the stability of exosomes in engineered exosome products.

[0083] The testing and experimental process includes: First, an exosome dispersion was tested using NTA (Nanoparticle Trajectory Tracking Analysis) to obtain the number and size distribution of exosomes. Then, the exosome dispersion was subjected to a certain degree of mechanical destructive treatment. Finally, the exosome dispersion was tested again using NTA to obtain the number and size distribution of exosomes. By comparing the NTA test results before and after the mechanical destructive treatment, the stability of the exosome dispersion to the mechanical destructive treatment can be determined.

[0084] Our tests show that the presence of supraparticles in supraparticle-encapsulated exosomes significantly improves the stability of exosomes during storage and under mechanically destructive handling conditions encountered in certain applications in our engineered exosome products. To evaluate the potential stability-enhancing effect during storage, we examined two commonly used exosome storage conditions: -80°C (typically used for short-term storage) and lyophilization (typically used for long-term storage). Mechanical damage caused by phase transitions under these storage conditions is the main cause of decreased stability. Nanoparticle tracking analysis (NTA) showed that for exosomes not encapsulated with supraparticles (conventional exosomes, also known as traditional exosomes), the number of exosomes (microscopic particles of exosome size) was significantly reduced (approximately 30%) after storage at -80°C for one month or after lyophilization to form a lyophilized powder. Figure 7 (Figure A). In contrast, for exosomes encapsulated by Supraparticle (separated and purified using our novel magnetic separation design), the number of exosomes was not significantly reduced. Figure 7 (Figure A). Further analysis of the particle size distribution using nanoparticle tracking analysis (NTA) revealed that for unencapsulated supraparticle exosomes, the maximum particle size increased from 159 nm (before storage) to 206 nm (after 1 month of storage at -80°C), and then further to 219 nm (after 1 month of freeze-dried storage). Figure 7(Figure B). The control group (exosomes not encapsulated with Supraparticle) was prepared as follows: BMSCs were stimulated with starvation (serum-free culture), and the cell supernatant was centrifuged at 2,000 g for 10 minutes to remove dead cells and cell debris. Then, it was ultracentrifuged at 100,000 g for 70 minutes, washed with PBS, and ultracentrifuged again at 100,000 g for 70 minutes (Beckman Optima L-XP ultracentrifuge). In contrast, the particle size variation of the Supraparticle-encapsulated exosomes (isolated and purified using our novel magnetic separation design) was minimal: the maximum particle size fraction increased only from 123 nm (before storage) to 125 nm (after 1 month of storage at -80°C), and then to 131 nm (after 1 month of lyophilized storage). Figure 7 (See Figure C). Next, we focused our investigation on freeze-thaw cycles, as these are the primary cause of decreased exosome stability under low-temperature storage or lyophilization conditions. We found that the first freeze-thaw cycle significantly reduced the number of unencapsulated supraparticle exosomes and increased the average particle size measured by NTA (…). Figure 7 Figures D and E in the middle. Conversely, for exosomes encapsulated with Supraparticle (separated and purified using our novel magnetic separation design), after one freeze-thaw cycle, there was no significant change in the number of exosomes and the average particle size measured by NTA (see Figures D and E). Figure 7 Figures D and E in the middle. After the second freeze-thaw cycle, the number of exosomes encapsulated by Supraparticle (separated and purified using our novel magnetic separation design) remained unchanged, but the average particle size measured by NTA increased significantly. Figure 7 (Figures D and E in the middle).

[0085] The stability of exosomes under mechanical damage conditions in several application scenarios was further evaluated. The focus here is on the mechanical damage condition of nebulized inhalation in the application scenario of inhaled drug delivery (a commonly used method of pulmonary drug administration). Experimental results show that, as determined by nanoparticle tracking analysis (NTA), the number and particle size distribution of exosomes encapsulated with Supraparticle (separated and purified using our novel magnetic separation design) did not change significantly, while exosomes without Supraparticle encapsulation showed a decrease in exosome number and the appearance of a new NTA peak with a particle size larger than that of the exosomes. Figure 7 (Middle FH diagram).

[0086] Example 6 This embodiment demonstrates the application of engineered exosomes in the treatment of neurological diseases.

[0087] In this example, the exosomes were mesenchymal stem cell (MSC)-derived exosomes loaded with curcumin (CUR). They were prepared using either CUR-loaded Supraparticle-Tat or CUR-free Supraparticle-Tat, with conventional methods serving as a control. The disease model used was a Parkinson's disease (PD) mouse model: mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin. PD mice were treated via tail vein injection with the following formulations: curcumin-loaded Supraparticle-Tat, MSC-derived exosomes (without CUR or Supraparticle), MSC-derived exosomes encapsulated in Supraparticle-Tat (without CUR), or CUR-loaded Supraparticle-Tat encapsulated in MSC-derived exosomes. For the Exosomes group: BMSCs were stimulated with starvation (serum-free culture), and the cell supernatant was centrifuged at 2,000 g for 10 minutes to remove dead cells and cell debris. Then, it was ultracentrifuged at 100,000 g for 70 minutes, washed with PBS, and then ultracentrifuged at 100,000 g for 70 minutes (Beckman Optima L-XP ultracentrifuge).

[0088] The dosage for each injection is 1×10 9 One pellet per mouse, injected every 3 days for a total of 30 days. Figure 8 (Figure A). Negative control mice were injected with PBS. For the CUR-containing formulation, the CUR loading in Supraparticle-Tat was 53%. Magnets were placed on the skull of all mice treated via tail vein injection for 3 hours to enhance drug delivery. We used behavioral analysis and neuronal staining to assess the therapeutic effect in mice. The results of these methods consistently demonstrated that engineered mesenchymal stem cell (MSC)-derived exosomes had a significant therapeutic effect on Parkinson's disease (PD), and that the CUR-loaded exosome formulation had better therapeutic effects than the CUR-free Supraparticle exosome formulation or the CUR-loaded Supraparticle-Tat formulation. Figure 8(Figure AJ). Literature reports that MSC-derived exosomes can treat Parkinson's disease (PD) through regenerative and immunomodulatory effects; on the other hand, curcumin has been reported to treat PD through anti-inflammatory effects and by regulating α-synuclein aggregation and toxicity. Therefore, our results indicate that curcumin-loaded mesenchymal stem cell-derived exosomes can combine the therapeutic effects of both mesenchymal stem cell-derived exosomes and curcumin, resulting in better efficacy compared to using either alone. After treatment with the most effective formulation (curcumin-loaded Supraparticle-Tat-encapsulated mesenchymal stem cell-derived exosomes), Parkinson's disease mice showed near-complete recovery in both behavioral analysis and neuronal staining results. Figure 8 (Image of BJ in China).

[0089] Example 7 This embodiment demonstrates the application of engineered exosomes in the treatment of respiratory diseases.

[0090] The exosomes were mesenchymal stem cell (MSC)-derived exosomes, loaded with curcumin (CUR). The disease model used was a mouse idiopathic pulmonary fibrosis (IPF) model, induced by a single high-dose intratracheal injection of bleomycin. On day 7, when pulmonary fibrosis began to appear, mice received nebulized inhalation treatment every 2 days for 14 days. The nebulized formulations included: MSC-derived exosomes encapsulated with Supraparticle-Tat containing CUR, Supraparticle-Tat containing curcumin, MSC-derived exosomes (without curcumin and Supraparticle-Tat), MSC-derived exosomes encapsulated with Supraparticle-Tat (without curcumin), or an equal volume of PBS buffer, with each inhalation dose containing 1 × 10⁻⁶ CUR. 9 Particles (except for the PBS group) Figure 9 (See Figure A). For the curcumin-containing formulation, the curcumin loading in Supraparticle was 53%. For the Exosomes group (MSC-derived exosomes (without curcumin and Supraparticle-Tat)): BMSCs were stimulated with starvation (serum-free culture), and the cell supernatant was centrifuged at 2,000 g for 10 minutes to remove dead cells and cell debris. Then, the cells were ultracentrifuged at 100,000 g for 70 minutes, washed with PBS, and then ultracentrifuged at 100,000 g for 70 minutes (Beckman Optima L-XP ultracentrifuge).

[0091] We assessed the degree of fibrosis and collagen deposition in lung tissue using hematoxylin and eosin (H&E) staining, Masson staining, and immunostaining for α-SMA and type I collagen (Collagen I). The results showed that MSC-derived exosomes loaded with CUR were more effective than MSC-derived exosomes (without CUR) or CUR-loaded supraparticle-Tat formulations in reducing fibrosis and collagen deposition, thus supporting the therapeutic value of combined exosome and CUR therapy. Figure 9 (See figure BI). Prussian blue staining showed that, after inhalation, engineered exosomes were able to reach the bronchi and alveoli. Interestingly, MSC-derived exosomes encapsulated with Supraparticle-Tat without CUR appeared to have better therapeutic effects than MSC-derived exosomes without Supraparticle-Tat (Ashcroft Score and Masson staining results were statistically significant, while α-SMA and type I collagen immunostaining results were not statistically significant). Figure 9 (See BI diagram). This could be because supraparticle encapsulation enhances the stability of exosomes under inhalation stress. Another reason could be that supraparticle-Tat-stimulated BMSC-derived exosomes have an increased number of bioactive molecules compared to conventional exosomes (BMSCs stimulated by starvation), as shown in the omics analysis. Figure 10 Finally, the best-performing formulation (MSC-derived exosomes encapsulated with curcumin in Supraparticle-Tat) reversed pulmonary fibrosis and collagen deposition to levels close to those in healthy mice. Figure 9 (Chinese BI picture).

[0092] Example 8 This embodiment demonstrates the application of engineered exosomes in the treatment of skin diseases.

[0093] We investigated the therapeutic effect in a mouse model of skin wound healing. A 0.6 cm incision was made in the skin on the back of the mouse. The mice were administered a subcutaneous injection (1 × 10⁻⁶ mg / L) every 3 days. 9 (Except for the sham surgery group), the injection contents include: MSC-derived exosomes encapsulated in Supraparticle-Tat loaded with curcumin (CUR), Supraparticle-Tat loaded with curcumin, MSC-derived exosomes (without curcumin or Supraparticle-Tat), MSC-derived exosomes encapsulated in Supraparticle-Tat (without curcumin), or an equal volume of PBS ( Figure 11(See Figure A). For the curcumin-loaded formulation, the curcumin loading in Supraparticle was 53%. For the Exosomes group (MSC-derived exosomes (without curcumin and Supraparticle-Tat)): BMSCs were stimulated with starvation (serum-free culture), and the cell supernatant was centrifuged at 2,000 g for 10 min to remove dead cells and cell debris. Then, the cells were ultracentrifuged at 100,000 g for 70 min, washed with PBS, and then ultracentrifuged at 100,000 g for 70 min (Beckman Optima L-XP ultracentrifuge).

[0094] Treatment efficacy was evaluated using visual observation (to assess wound healing rate), H&E staining (to assess scar length), Masson staining (to assess collagen content), and CD31 immunostaining (angiogenesis marker). Results showed that, compared to MSC-derived exosomes without CUR or Supraparticle-Tat, or CUR-loaded Supraparticle-Tat formulations, CUR-loaded Supraparticle-Tat-encapsulated MSC-derived exosomes exhibited better wound healing in terms of scar length, collagen content, and angiogenesis, thus supporting the therapeutic value of combined exosome and CUR application. Figure 11 (Chinese BI picture).

[0095] In summary, this invention develops an integrated platform technology, designs composite nanoparticles with specific structures and compositions, and develops an exosome preparation process based on them. The aim is to simultaneously improve the four steps of the engineered exosome production process, achieve efficient preparation of engineered exosomes, and obtain exosomes with good stability, capable of efficiently loading drugs, and applicable to the treatment of neurological diseases, respiratory diseases, etc.

[0096] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A composite nanoparticle, characterized in that, The composite nanoparticles include nanoparticles and exosome biosynthesis stimulating ligands coupled thereto. The nanoparticles include self-assembling proteins and superparamagnetic iron oxide nanoparticles.

2. The composite nanoparticles according to claim 1, characterized in that, The exosome biosynthesis-stimulating ligands include membrane-penetrating peptides; Optionally, the membrane-penetrating peptide includes Tat membrane-penetrating peptide; Optionally, the amino acid sequence of the Tat membrane-penetrating peptide includes the sequence shown in SEQ ID NO.

1.

3. The composite nanoparticles according to claim 1 or 2, characterized in that, The protein includes at least one of bovine serum albumin, human serum albumin, egg white lysozyme, bovine lactalbumin, zein, β-casein, ovalbumin, β-lactoglobulin, or lactoferrin.

4. The composite nanoparticles according to any one of claims 1-3, characterized in that, The composite nanoparticles also contain a drug and / or a detection agent, wherein the drug and / or detection agent are loaded onto the nanoparticles; Optionally, the drug comprises at least one of small molecules, nucleic acids, or peptides; Optionally, the detection agent includes a molecular imaging probe.

5. The method for preparing the composite nanoparticles according to claim 1, characterized in that, The preparation method includes: Dissolve the protein solution in an aqueous solution to prepare a protein solution; Superparamagnetic iron oxide nanoparticles were dissolved in an organic solvent to prepare a nanocrystalline solution; The nanocrystal solution was mixed with the protein solution, and the mixture was incubated at room temperature to obtain an incubated mixture. The incubated mixture was centrifuged, the supernatant was removed, and the precipitate was collected to obtain nanoparticles. The nanoparticles were coupled with exosome biosynthesis stimulating ligands to obtain the composite nanoparticles.

6. The use of the composite nanoparticles according to any one of claims 1-4 in the preparation of exosomes.

7. A method for preparing exosomes, characterized in that, The preparation method includes: The exosome-synthetic cells were mixed and incubated with the composite nanoparticles described in any one of claims 1-4, and the exosomes were magnetically separated to obtain exosomes.

8. The method for preparing exosomes according to claim 7, characterized in that, The magnetic separation method includes: A modular magnet array in the shape of a cuboid is assembled using permanent magnets; the modular magnet array is placed inside a separation container, and a driving magnet is set outside the separation container. A driving motor and a transmission device are linked with the driving magnet, so that the modular magnet array inside the separation container moves slowly at a set speed, capturing exosomes containing composite nanoparticles onto the surface of the modular magnet array. Then, the exosomes are dissociated and harvested from the surface of the modular magnet array by rinsing with physical ultrasound or chemical dissociation fluid.

9. An engineered exosome, characterized in that, The engineered exosomes are prepared by the method for preparing exosomes as described in claim 7 or 8.

10. The use of the engineered exosomes according to claim 9 in the preparation of medicaments for treating diseases, characterized in that, The diseases mentioned include at least one of the following: neurological diseases, respiratory diseases, skin diseases, cardiovascular diseases, reproductive diseases, cancer, metabolic diseases, immune system diseases, or ophthalmic diseases.

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